Publications

Barcoding Life to Conserve Biological Diversity: Beyond the Taxonomic Imperative - Jul 13, 2010 ()
[Vernooy, R., Haribabu, E., Muller, M. R., Vogel, J. H., Hebert, P. D. N., Schindel, D. E., Shimura, J. & Singer, G. A. C. 2010. PLoS Biol. 8(7 e1000417.]

Barcoding scientists aspire to adhere to the objectives of the Convention on Biological Diversity by promoting conservation, sustainability, and the equitable sharing of benefits arising from use of genetic resources.

Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2 - Jul 06, 2010 ()
[Gao, T., Yao, H., Song, J., Liu, C., Zhu, Y., Ma, X., Pang, X., Xu, H. & Chen, S. 2010. J Ethnopharmacol. 130(1 116-121.]

AIM OF THE STUDY: To test whether the ITS2 region is an effective marker for use in authenticating of the family Fabaceae which contains many important medicinal plants. MATERIALS AND METHODS: The ITS2 regions of 114 samples in Fabaceae were amplified. Sequence assembly was assembled by CodonCode Aligner V3.0. In combination with sequences from public database, the sequences were aligned by Clustal W, and genetic distances were computed using MEGA V4.0. The intra- vs. inter-specific variations were assessed by six metrics, wilcoxon two-sample tests and "barcoding gaps". Species identification was accomplished using TaxonGAP V2.4, BLAST1 and the nearest distance method. RESULTS: ITS2 sequences had considerable variation at the genus and species level. The intra-specific divergence ranged from 0% to 14.4%, with an average of 1.7%, and the inter-specific divergence ranged from 0% to 63.0%, with an average of 8.6%. Twenty-four species found in the Chinese Pharmacopoeia, along with another 66 species including their adulterants, were successfully identified based on ITS2 sequences. In addition, ITS2 worked well, with over 80.0% of species and 100% of genera being correctly differentiated for the 1507 sequences derived from 1126 species belonging to 196 genera. CONCLUSIONS: Our findings support the notion that ITS2 can be used as an efficient and powerful marker and a potential barcode to distinguish various species in Fabaceae.

Discrimination of Culicoides obsoletus and Culicoides scoticus, potential bluetongue vectors, by morphometrical and mitochondrial cytochrome oxidase subunit I analysis - Jul 01, 2010 ()
[Augot, D., Sauvage, F., Jouet, D., Simphal, E., Veuille, M., Couloux, A., Kaltenbach, M. L. & Depaquit, J. 2010. Infect Genet Evol. 629-637 10(5).]

Biting midges of the Culicoides obsoletus Meigen species complex (Diptera: Ceratopogonidae) are increasingly suspected as vectors of the recent emergence of bluetongue virus in Europe. Within this complex, identification of the C. obsoletus and Culicoides scoticus females is considered as difficult or sometimes not possible while the identification of males is easy, based on genitalia observation. Nolan et al. (2007) concluded that the distinction of C. obsoletus and C. scoticus females is not possible according to morphology but require molecular analyses. In 2010, the identification of biting midges is done under a stereomicroscope without specific identification within the C. obsoletus species complex. However, such a specific identification distinguishing C. obsoletus s. str. and C. scoticus s. str. is crucial to identify the European competent vectors of the virus, their relative abundances and then accurately assess the risk. We performed morphometric analyses of head, genitalia and thorax of females combined with sequencing of the cytochrome oxidase I barcode fragment of mitochondrial DNA on 88 specimens in order to have a molecular identification of our sampled species. As we knew the actual species of individuals thanks to molecular results, we explored the discriminant power of 15 morphometric variables to distinguish the females according to their species. Multivariate analyses were performed on the morphometric measurements to identify and validate a combination of variables leading to an accurate species identification. It appears that females of C. obsoletus and C. scoticus can be accurately distinguished based on only four variables: width between chitinous plates, length and width of spermathecae1 and length of spermatheca2. This approach should improve the accuracy of morphologically-based species identification.

Molecular phylogeny and DNA barcoding in the meadow-spittlebug Philaenus spumarius (Hemiptera, Cercopidae) and its related species - Jul 01, 2010 ()
[Seabra, S. G., Pina-Martins, F., Marabuto, E., Yurtsever, S., Halkka, O., Quartau, J. A. and Paulo, O. S 2010. Mol Phylogenet Evol. 56(1) 462-467.]

Philaenus spumarius, widely studied for its colour/pattern polymorphism, is a widespread species across the Holartic. The patterns of haplotype divergence at the mitochondrial gene cytochrome oxidase I (COI) found in this study suggest a postglacial western Europe (Iberian and Italian peninsulas to Britain) and a eastern (from Near East to Finland) south-to-north colonization. The haplotypes found in North America are most likely derived from the British haplotypes. The barcode fragment used here allowed the distinction of the species within genus Philaenus and questioned some taxonomic identifications of sequences present in Genbank.

DNA barcoding of arbuscular mycorrhizal fungi - Jul 01, 2010 ()
[Stockinger, H., Kruger, M., & Schussler, A. 2010. New Phytol. 187(2) 461-474.]

Summary *Currently, no official DNA barcode region is defined for the Fungi. The COX1 gene DNA barcode is difficult to apply. The internal transcribed spacer (ITS) region has been suggested as a primary barcode candidate, but for arbuscular mycorrhizal fungi (AMF; Glomeromycota) the region is exceptionably variable and does not resolve closely related species. *DNA barcoding analyses were performed with datasets from several phylogenetic lineages of the Glomeromycota. We tested a c. 1500 bp fragment spanning small subunit (SSU), ITS region, and large subunit (LSU) nuclear ribosomal DNA for species resolving power. Subfragments covering the complete ITS region, c. 800 bp of the LSU rDNA, and three c. 400 bp fragments spanning the ITS2, the LSU-D1 or LSU-D2 domains were also analysed. *Barcode gap analyses did not resolve all species, but neighbour joining analyses, using Kimura two-parameter (K2P) distances, resolved all species when based on the 1500 bp fragment. The shorter fragments failed to separate closely related species. *We recommend the complete 1500 bp fragment as a basis for AMF DNA barcoding. This will also allow future identification of AMF at species level based on 400 or 1000 bp amplicons in deep sequencing approaches.

Barcoding bushmeat: molecular identification of Central African and South American harvested vertebrates - Jun 01, 2010 ()
[Eaton, M. J., Meyers, G. L., Kolokotronis, S.-O., Leslie, M. S., Martin, A. P., & Amato, G. 2010. Conservation Genetics. 11(4) 1389-1404.]

The creation and use of a globally available database of DNA sequences from a standardized gene region has been proposed as a tool for species identification, assessing genetic diversity and monitoring the legal and illegal trade in wildlife species. Here, we contribute to the Barcode of Life Data System and test whether a short region of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene would reliably distinguish among a suite of commonly hunted African and South American mammal and reptile species. We used universal primers to generate reference barcode sequences of 645 bp for 23 species from five vertebrate families (Crocodilidae, Alligatoridae, Bovidae, Suidae and Cercopithecidae). Primer cocktails yielded high quality barcode sequences for 179 out of 204 samples (87.7%) from all species included in the study. For most taxa, we sequenced multiple individuals to estimate intraspecific sequence variability and document fixed diagnostic characters for species identification. Polymorphism in the COX1 fragment was generally low (mean = 0.24%), while differences between congeneric species averaged 9.77%. Both fixed character differences and tree-based maximum likelihood distance methods unambiguously identified unknown and misidentified samples with a high degree of certainty. Barcode sequences also differentiated among newly identified lineages of African crocodiles and identified unusually high levels of genetic diversity in one species of African duiker. DNA barcoding offers promise as an effective tool for monitoring poaching and commercial trade in endangered species, especially when investigating semi-processed or morphologically indistinguishable wildlife products. We discuss additional benefits of barcoding to ecology and conservation

The integrative future of taxonomy - May 25, 2010 ()
[Padial, J. M., Miralles, A., De la Riva, I., & Vences, M. 2010. Front Zool. 7 16.]

ABSTRACT: BACKGROUND: Taxonomy is the biological discipline that identifies, describes, classifies and names extant and extinct species and other taxa. Nowadays, species taxonomy is confronted with the challenge to fully incorporate new theory, methods and data from disciplines that study the origin, limits and evolution of species. RESULTS: Integrative taxonomy has been proposed as a framework to bring together these conceptual and methodological developments. Here we review perspectives for an integrative taxonomy that directly bear on what species are, how they can be discovered, and how much diversity is on Earth. CONCLUSIONS: We conclude that taxonomy needs to be pluralistic to improve species discovery and description, and to develop novel protocols to produce the much-needed inventory of life in a reasonable time. To cope with the large number of candidate species revealed by molecular studies of eukaryotes, we propose a classification scheme for those units that will facilitate the subsequent assembly of data sets for the formal description of new species under the Linnaean system, and will ultimately integrate the activities of taxonomists and molecular biologists.

Identity of the ailanthus webworm moth (Lepidoptera, Yponomeutidae), a complex of two species: evidence from DNA barcoding, morphology and ecology - May 01, 2010 ()
[Wilson, J. J., Landry, J.-F., Janzen, D. H., Hallwachs, W., Nazari, V., Hajibabaei, M. & Hebert, P. D. N. 2010. ZooKeys. 40 41–60.]

During extensive ongoing campaigns to inventory moths of North America and Area de Conservacion Guanacaste (ACG), northwestern Costa Rica, we discovered that morphologically similar yponomeutid moths were assigned two different names, Atteva ergatica Walsingham in Costa Rica and A. punctella (Stoll) in North America, but had identical DNA barcodes. Combining DNA barcoding, morphology and food plant records also revealed a complex of two sympatric species that are diagnosable by their DNA barcodes and their facies in Costa Rica. However, neither of the names could be correctly applied to either species, as A. ergatica is a junior synonym and A. punctella a junior homonym. By linking our specimens to type material through morphology and DNA barcoding, we determined that the ACG dry forest species, distributed from Costa Rica to southern Quebec and Ontario, should be called A. aurea, whereas the similar and marginally sympatric ACG rain forest species found in Central America should be called A. pustulella. Neotypes are designated for Phalaena Tinea  punctella Stoll, 1781 and Deiopeia aurea Fitch, 1857. Atteva floridana has identical barcodes to A. aurea and provisionally maintained as a synonym.

Assessing the value of DNA barcodes and other priority gene regions for molecular phylogenetics of Lepidoptera - May 01, 2010 ()
[Wilson, J. J. 2010. PLoS One. 5(5) e10525.]

BACKGROUND: Despite apparently abundant amounts of observable variation and species diversity, the order Lepidoptera exhibits a morphological homogeneity that has provided only a limited number of taxonomic characters and led to widespread use of nucleotides for inferring relationships. This study aims to characterize and develop methods to quantify the value of priority gene regions designated for Lepidoptera molecular systematics. In particular, I assess how the DNA barcode segment of the mitochondrial COI gene performs across a broad temporal range given its number one position of priority, most sequenced status, and the conflicting opinions on its phylogenetic performance. METHODOLOGY/PRINCIPAL FINDINGS: Gene regions commonly sequenced for lepidoptera phylogenetics were scored using multiple measures across three categories: practicality, which includes universality of primers and sequence quality; phylogenetic utility; and phylogenetic signal. I found that alternative measures within a category often appeared correlated, but high scores in one category did not necessarily translate into high scores in another. The DNA barcode was easier to sequence than other genes, and had high scores for utility but low signal above the genus level. CONCLUSIONS/SIGNIFICANCE: Given limited financial resources and time constraints, careful selection of gene regions for molecular phylogenetics is crucial to avoid wasted effort producing partially informative data. This study introduces an approach to assessing the value of gene regions prior to the initiation of new studies and presents empirical results to help guide future selections.

DNA barcodes provide new evidence of a recent radiation in the genus Sporophila (Aves: Passeriformes) - May 01, 2010 ()
[Campagna, L., Lijtmaer, D. A., Kerr, K. C. R., Barreira, A. S., Hebert, P. D. N., Lougheed, S. C., & Tubaro, P. 2010. Molecular Ecology Resources. 10(3) 449-458.]

The capuchinos are a group of birds in the genus Sporophila that has apparently radiated recently, as evidenced by their lack of mitochondrial genetic diversity. We obtained cytochrome c oxidase I (COI) sequences (or DNA barcodes) for the 11 species of the group and various outgroups. We compared the patterns of COI variability of the capuchinos with those of the largest barcode data set from neotropical birds currently available (500 species representing 51% of avian richness in Argentina), and subjected COI sequences to neighbour-joining, maximum parsimony and Bayesian phylogenetic analyses as well as statistical parsimony network analysis. A clade within the capuchinos, the southern capuchinos, showed higher intraspecific and lower interspecific divergence than the remaining Argentine species. As most of the southern capuchinos shared COI haplotypes and pairwise distances within species were in many cases higher than distances between them, the phylogenetic affinities within the group remained unresolved. The observed genetic pattern is consistent with both incomplete lineage sorting and gene flow between species. The southern capuchinos constitute the only large group of species among the neotropical birds barcoded so far that are inseparable when using DNA barcodes, and one of few multispecies avian groups known to lack reciprocal monophyly. Extending the analysis to rapidly evolving nuclear and mitochondrial markers will be crucial to understanding this radiation. Apart from giving insights into the evolution of the capuchinos, this study shows how DNA barcoding can rapidly flag species or groups of species worthy of deeper study.

DNA barcoding is a powerful tool to uncover algal diversity: A case study of the Phyllophoraceae (Gigartinales, Rhodophyta) in the Canadian flora - Apr 01, 2010 ()
[LeGall, L., & Saunders, G. W. 2010. Journal of Phycology. 46(2) 374 - 389.]

Previous studies have established that the 5' end of the mitochondrial gene COI (cytochrome oxidase subunit I) is useful for rapid and reliable identification of red algal species and have demonstrated that our understanding of red algal biodiversity and biogeography is fragmentary. In this context, we are completing a thorough sampling along the Canadian coast and using the DNA barcode for the assignment of collections to genetic species to explore algal diversity in the Canadian flora. In the present study, we provide results regarding diversity of members of the red algal family Phyllophoraceae. We have analyzed 354 individuals from the Arctic, Atlantic, and Pacific coasts of Canada, as well as 26 specimens from the USA, Europe, and Australia, resolving 29 species based on the analyses of the DNA barcode. Twenty-three of these genetic species were present in Canada where only 18 species are currently recognized, including Ceratocolax hartzii Rosenv., which was in the same genetic species group as its host Coccotylus truncatus (Pall.) M. J. Wynne et N. J. Heine and is thus transferred to Coccotylus, C. hartzii (Rosenv.) comb. nov., but retained as a distinct species owing to its unique habit and phenology. Our results revealed the presence of cryptic diversity within the genera Coccotylus, Mastocarpus, Ozophora, and Stenogramme, for which we resurrect Coccotylus brodiei (Turner) Kütz. and describe Mastocarpus pachenicus sp. nov., Ozophora lanceolata sp. nov., and Stenogramme bamfieldiensis sp. nov., leaving a multitude of unnamed Mastocarpus spp. in need of further taxonomic study. In addition, we report range extensions into British Columbia of Besa papillaeformis Setch., previously known only from its type and nearby localities in California; Gymnogongrus crenulatus (Turner) J. Agardh, recorded only from the Atlantic; and Stenogramme cf. rhodymenioides Joly et Alveal, previously only known from South America. Finally, the phylogenetic affinities of the Canadian species of Phyllophoraceae characterized in this study were investigated using LSU rDNA, RUBISCO LSU (rbcL), and combined analyses.

DNA barcoding for conservation and management of Amazonian commercial fish - Apr 01, 2010 ()
[Ardura, A., Linde, A. R., Moreira, J. C., & Garcia-Vazquez, E. 2010. Biological Conservation. 143(6) 1438-1443.]

DNA barcoding is used to assign a biological specimen to a species. DNA-based procedure has become the preferred forensic tool for criminal prosecution in cases involving the sale of incorrectly identified food. The aim of this work was to develop a DNA-based marker for allowing an accurate and reliable identification of Amazonian fish species of commercial interest. For this purpose, we extracted DNA from fish directly purchased in local markets and identified de visu by local experts. We PCR amplified the mitochondrial 12S rRNA and cytochrome oxidase I (COI) genes. Twenty-nine commercial species accounting for most commercial landings in the River Amazon markets were unambiguously identified based on their DNA for the first time. Phylogenetic trees reconstructed based on the sequences of the two mitochondrial genes clustered species in concordance with their taxonomic classification. We illustrated the utility of DNA barcoding demonstrating that the group of fish generically sold as "Acará" includes seven different species, which are being exploited together as a single species, thus estimation of exploitation rates was not possible until now. Application of genetic markers for species authentication in markets and control of commercial landings will contribute to recognition of the real fishing targets and to the conservation of fish resources in the Amazon basin.

Barcoding of arrow worms (Phylum Chaetognatha) from three oceans: genetic diversity and evolution within an enigmatic phylum - Apr 01, 2010 ()
[Jennings, R. M., Bucklin, A., & Pierrot-Bults, A. 2010. PLoS One. 5(4) e9949.]

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The Redcheek Paradox: the mismatch between genetic and phenotypic divergence among deeply-divided mtDNA lineages in a coral-reef goby, with the description of two new cryptic species from the Caribbean Sea - Apr 01, 2010 ()
[Victor, B. C. 2010. Journal of the Ocean Science Foundation. 3 .]

A new micro-endemic goby, Elacatinus rubrigenis, is described from Utila in the Bay Islands of the Gulf of Honduras (Western Atlantic). The new species is similar to the Greenbanded Goby, E. multifasciatus, but differs in having a prominent red stripe across the cheek, more-numerous green bars on the body, and 11 second-dorsal-fin elements (vs. equal numbers of 11 and 12). The new species, the Redcheek Goby, replaces the Greenbanded Goby on the island of Utila and has not been sighted at any other location, potentially one of the smallest ranges reported for a Caribbean reef fish. The COI barcode mtDNA sequence for the Redcheek Goby is 11.2% divergent from the original type population of the Greenbanded Goby from the U. S. Virgin Islands. However, Panamanian Greenbanded Gobies, with no red cheek stripe, show a similarly large 11.3% genetic distance from the type population (within-population sequence variation is less than 1%). Despite the prominent marking difference, there is only a 3.3% sequence difference between Redcheek Gobies and Panamanian Greenbanded Gobies. These results highlight the lack of concordance between genetic and phenotypic divergence among cryptic lineages of reef fishes. The Panamanian population has some small meristic differences from the type population and is (reluctantly) described here as the new species Elacatinus panamensis. An unexpected 4.3% sequence difference between the adjacent Puerto Rican and Virgin Islands populations indicates that the Greenbanded Goby is likely to break up into inconveniently numerous discrete genetic lineages, presumably in allopatry. These sequence differences are generally greater than those separating the Elacatinus cleaning-goby species in the Caribbean and greater than the differences found among most genera of reef fishes. The remarkably deep DNA-sequence divergence among these allopatric cryptic species and lineages raises important and difficult questions about genetic structure, speciation processes, and species definitions among some coral reef fishes.

DNA Barcodes for Marine Biodiversity: Moving Fast Forward? - Mar 23, 2010 ()
[Radulovici, A. E., Archambault, P., & Dufresne, F. 2010. Diversity. 2(4) 450-472.]

‘Biodiversity’ means the variety of life and it can be studied at different levels (genetic, species, ecosystem) and scales (spatial and temporal). Last decades showed that marine biodiversity has been severely underestimated at all levels. In order to investigate diversity patterns and underlying processes, there is a need to know what species live in the marine environment. An emerging tool for species identification, DNA barcoding can reliably assign unknown specimens to known species, also flagging potential cryptic species and genetically distant populations. This paper will review the role of DNA barcoding for the study of marine biodiversity at the species level.

Poles Apart: The "Bipolar" Pteropod Species Limacina helicina Is Genetically Distinct Between the Arctic and Antarctic Oceans - Mar 23, 2010 ()
[Hunt, B., Strugnell, J., Bednarsek, N., Linse, K., Nelson, R. J., Pakhomov, E., Seibel, B., Steinke, D., & Wurzberg, L. 2010. PLoS ONE. e9835.]

The shelled pteropod (sea butterfly) Limacina helicina is currently recognised as a species complex comprising two sub-species and at least five “forma”. However, at the species level it is considered to be bipolar, occurring in both the Arctic and Antarctic oceans. Due to its aragonite shell and polar distribution L. helicina is particularly vulnerable to ocean acidification. As a key indicator of the acidification process, and a major component of polar ecosystems, L. helicina has become a focus for acidification research. New observations that taxonomic groups may respond quite differently to acidification prompted us to reassess the taxonomic status of this important species. We found a 33.56% (±0.09) difference in cytochrome c  oxidase subunit I (COI) gene sequences between L. helicina collected from the Arctic and Antarctic oceans. This degree of separation is sufficient for ordinal level taxonomic separation in other organisms and provides strong evidence for the Arctic and Antarctic populations of L. helicina differing at least at the species level. Recent research has highlighted substantial physiological differences between the poles for another supposedly bipolar pteropod species, Clione limacina. Given the large genetic divergence between Arctic and Antarctic L. helicina populations shown here, similarly large physiological differences may exist between the poles for the L. helicina species group. Therefore, in addition to indicating that L. helicina is in fact not bipolar, our study demonstrates the need for acidification research to take into account the possibility that the L. helicina species group may not respond in the same way to ocean acidification in Arctic and Antarctic ecosystems.

Population genetics of ecological communities with DNA barcodes: An example from New Guinea Lepidoptera - Mar 16, 2010 ()
[Craft, K. J., Pauls, S. U., Darrow, K., Miller, S. E., Hebert, P. D. N., Helgen, L. E., Novotny, V. & Weiblen, G. D. 2010. Proc Natl Acad Sci U S A. 107(11) 5041-6.]

Comparative population genetics of ecological guilds can reveal generalities in patterns of differentiation bearing on hypotheses regarding the origin and maintenance of community diversity. Contradictory estimates of host specificity and beta diversity in tropical Lepidoptera (moths and butterflies) from New Guinea and the Americas have sparked debate on the role of host-associated divergence and geographic isolation in explaining latitudinal diversity gradients. We sampled haplotypes of mitochondrial cytochrome c oxidase I from 28 Lepidoptera species and 1,359 individuals across four host plant genera and eight sites in New Guinea to estimate population divergence in relation to host specificity and geography. Analyses of molecular variance and haplotype networks indicate varying patterns of genetic structure among ecologically similar sympatric species. One-quarter lacked evidence of isolation by distance or host-associated differentiation, whereas 21% exhibited both. Fourteen percent of the species exhibited host-associated differentiation without geographic isolation, 18% showed the opposite, and 21% were equivocal, insofar as analyses of molecular variance and haplotype networks yielded incongruent patterns. Variation in dietary breadth among community members suggests that speciation by specialization is an important, but not universal, mechanism for diversification of tropical Lepidoptera. Geographically widespread haplotypes challenge predictions of vicariance biogeography. Dispersal is important, and Lepidoptera communities appear to be highly dynamic according to the various phylogeographic histories of component species. Population genetic comparisons among herbivores of major tropical and temperate regions are needed to test predictions of ecological theory and evaluate global patterns of biodiversity.

Lutzomyia sand fly diversity and rates of infection by wolbachia and an exotic leishmania species on barro colorado island, panama - Mar 09, 2010 ()
[Azpurua, J., De La Cruz, D., Valderama, A., & Windsor, D. 2010. PLoS Negl Trop Dis. 4(3) e627.]

BACKGROUND: Sand flies (Diptera, Psychodidae, Phlebotominae) in the genus Lutzomyia are the predominant vectors of the protozoan disease leishmaniasis in the New World. Within the watershed of the Panama Canal, the cutaneous form of leishmaniasis is a continuous health threat for residents, tourists and members of an international research community. Here we report the results of screening a tropical forest assemblage of sand fly species for infection by both Leishmania and a microbe that can potentially serve in vector population control, the cytoplasmically transmitted rickettsia, Wolbachia pipientis. Knowing accurately which Lutzomyia species are present, what their evolutionary relationships are, and how they are infected by strains of both Leishmania and Wolbachia is of critical value for building strategies to mitigate the impact of this disease in humans. METHODOLOGY AND FINDINGS: We collected, sorted and then used DNA sequences to determine the diversity and probable phylogenetic relationships of the Phlebotominae occurring in the understory of Barro Colorado Island in the Republic of Panama. Sequence from CO1, the DNA barcoding gene, supported 18 morphology-based species determinations while revealing the presence of two possible "cryptic" species, one (Lu. sp. nr vespertilionis) within the Vespertilionis group, the other (Lu. gomezi) within the Lutzomyia-cruciata series. Using ITS-1 and "minicircle" primers we detected Leishmania DNA in 43.3% of Lu. trapidoi, 26.3% of Lu. gomezi individuals and in 0% of the other 18 sand fly species. Identical ITS-1 sequence was obtained from the Leishmania infecting Lu. trapidoi and Lu. gomezi, sequence which was 93% similar to Leishmania (viannia) naiffi in GenBank, a species previously unknown in Panama, but recognized as a type of cutaneous leishmaniasis vectored broadly across northern and central South America. Distinct strains of the intracellular bacterium Wolbachia were detected in three of 20 sand fly species, including Lu. trapidoi, in which it frequently co-occurred with Leishmania. CONCLUSIONS: Both morphological and molecular methods were used to examine an assemblage of 20 sand fly species occurring in the forests of the Panama Canal area. Two of these species, members of separate clades, were found to carry Leishmania at high frequency and hence are likely vectors of leishmaniasis to humans or other mammal species. A single Leishmania species, identified with high confidence as Le. naiffi, was carried by both species. That Le. naiffi is known to cause cutaneous lesions in South America but has hitherto not been reported or implicated in Panama opens the possibility that its range has recently expanded to include the Isthmus or that it occurs as a recent introduction. The occurrence of Leishmania and Wolbachia in Lu. trapidoi identifies one important vector of the disease as a potential target for gene introductions using Wolbachia population sweeps.

Mitochondrial DNA barcoding detects some species that are real, and some that are not - Mar 01, 2010 ()
[Dasmahapatra, K. K., Elias, M., Hill, R. I., Hoffman, J. I., & Mallet, J. 2010. Molecular Ecology Resources. 10(2) 264-273.]

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.

Diversity and specificity in Diplostomum spp. metacercariae in freshwater fishes revealed by cytochrome c oxidase I and internal transcribed spacer sequences - Mar 01, 2010 ()
[Locke, S. A., McLaughlin, J. D., Dayanandan, S., & Marcogliese, D. J 2010. Int J Parasitol. 40(3) 333-343.]

In this study, sequences from the barcode region of cytochrome c oxidase I (COI) were used to distinguish Diplostomum spp. in a sample of 497 metacercariae collected from diverse fishes of the St. Lawrence River, Canada and findings were corroborated with internal transcribed spacer (ITS) regions of rDNA. Twelve species were detected based on sequences and metacercarial specificity for hosts and tissues. Although this is an unusually high diversity, additional species are likely to exist in the study area. Two species were indistinguishable with ITS data and there is evidence that they may be undergoing hybridization and/or have recently diverged. The ITS sequences of another species are similar to those of Diplostomum pseudospathaceum from Europe, but ITS data are insufficient to show that they are conspecific. Diplostomum spp. that infect tissues other than the lens are more host-specific than species inhabiting the lenses of fishes, which is attributed to the enhanced immunological privilege of the lens site compared with other tissues. Overall, COI sequences were superior to more commonly used ITS markers for delineating species of this important and taxonomically difficult pathogen.

Taxonomy and the Mediocrity of DNA Barcoding – Some Remarks on PACKER et al. 2009: DNA Barcoding and the Mediocrity of Morphology - Feb 28, 2010 ()
[Holynski, R. B. 2010. Arthropod Systematics & Phylogeny. 68(1) 143-150.]

The paper is a reaction to that published by PACKER et al. (2009, Molecular Ecology Resources 9, Suppl.1: 42–50), depreciating the value of traditional – especially morphological – data in taxonomical studies as “mediocre” and boosting instead the simplistic ‘barcoding’ procedures as “obviously effi cient”. Having explicitly stated my – as a ‘traditional’ taxonomist – ‘decalogue’, I show that accusation of“lust for monopolization of knowledge” and “vociferous hostility” towards the adherents of an alternate approach is glaringly misdirected by PACKER et al. and in fact fi ts much better the attitude of ‘barcoders’ themselves; while point-by-point evaluation of the arguments and examples set forth by them allowed to refute both their main claims and confi rm once again that morphological data, far from being accusable of “mediocrity”, still usually (some special situations excepted) provide the most reliable source of evidence for taxonomic conclusions, whereas simplistic ‘barcoding’ is obviously inefficient in basic research (as opposed to some practical applications) and thence unqualifi ed for the role of anything more than occasional preliminary ‘proxy’.

A comparison of two DNA barcode markers for species discrimination in the red algal family Kallymeniaceae (Gigartinales, Florideophyceae), with a description of Euthora timburtonii sp. nov. - Feb 01, 2010 ()
[Clarkston, B. E., & Saunders, G. W 2010. Botany. 88 119-131.]

Accurate identification of many red algae to the species level using only morphological characters can be difficult. The emerging field of “molecular-assisted alpha taxonomy” can greatly alleviate this issue. In this approach, a large number of specimens are sequenced for a standard DNA marker as a first step to genetic species assignment, followed by detailed morphological observations. Regions of both the mitochondrial cytochromec oxidase I gene (COI-5P) and the plastid 23S rRNA gene (UPA) have been proposed as DNA barcode markers to accomplish this task. We compared the utility of each marker as a species identification tool using members of the marine red algal family Kallymeniaceae from British Columbia, Canada. Our results indicate that COI-5P is a more sensitive marker for delimiting species, but that it can be difficult to acquire clean amplification products for many isolates of Kallymeniaceae, owing to biological contamination. This problem can be overcome by using specific primers. UPA, on the other hand, has universal primers that work in diverse lineages (e.g., red, brown, and green algae), but lower interspecific sequence variation, which has the potential to underestimate species diversity, although this was not observed in our study. During our survey, we uncovered a new species of the Kallymeniaceae, Euthora timburtonii Clarkston et G.W.Saunders sp. nov., which we describe here.

Barcoding of Diatoms: Nuclear Encoded ITS Revisited - Jan 01, 2010 ()
[Moniz, M. B., & Kaczmarska, I. 2010. Protist. 161 7-34.]

DNA-barcoding is based on the premise that the divergence of a small DNA fragment coincides with biological separation of species. If true, it offers an additional tool for worldwide consistent species recognition even in cases of semi-cryptic species. Our study includes 618 sequences representing 114 diatom species belonging to the two most species-rich classes of diatoms (Mediophyceae and Bacillariophyceae). A 99.5% success rate in separating biologically defined species and a 91% success rate in separating all species tested was obtained when using the proposed barcode starting at the 5' end of 5.8S and ending in the conserved motif of helix III of ITS2 (300 to 400bp). Including the whole 5.8S+ITS2 region did not significantly improve species resolution. We tested our barcode on 17 unidentified, misidentified or contaminated strains derived mostly from a culture collection, and these were correctly flagged as erroneous by their ITS sequences. We conclude that the proposed barcode represents for the Mediophyceae and Bacillariophyceae a robust, economical, and rapid way to recognize and identify most species (when a reference sequence is available) that is as good as or better than other molecular markers thus far proposed.

Barcoding cryptic bumblebee taxa: B. lucorum, B. crytarum and B. magnus, a case study - Dec 15, 2009 ()
[Bertsch, A. 2009. Beitr. Ent. 59(2) 287-310.]

Spring queens of five taxa of the subgenus Bombus sensu stricto (Bombus sporadicus, B. terrestris, B. lucorum, B. cryptarum and B. magnus) were collected from different localities throughout Europe to rear artificial colonies. The mitochondrial cytochrome oxidase subunit I of 40 specimens was sequenced (partial sequence of 1005 bp length). Interspecific sequence divergence was about 30 to 60 base substitutions and the Tamura-Nei genetic distance was approximately 0.05 to 0.25, whereas the intraspecific sequence divergence was only 1 to 6 base substitutions and the Tamura-Nei genetic distance was about 0.002 to 0.007. In addition to the B. sporadicus and B. terrestris cluster, three clusters were obtained in the phylogenetic tree: cluster α for B. lucorum, cluster β for B. cryptarum and cluster γ for B. magnus. The three clusters α, β and γ, which represent taxa of the so-called lucorum-complex, were well separated, with low variability, no intergrading and no terminal units of unclear position. As there are no gaps in the alignments of the cytochrome oxidase subunit I sequences single nucleotide sites can be used as positional homologies. Each taxon is characterised by about 8 to 12 substitutions, which are unique (“private”) and can be used as diagnostic characters to define and identify that taxon. Using the classical tools of cladistics, a tree was built on the basis of these diagnostic characters. The Barcode engine successfully identified all critical specimens. The topological position of GenBank sequences of misidentified specimens and sequences with potentially degraded DNA is discussed. Museum specimens of three Asiatic taxa of the lucorum-complex with unknown relationships were sequenced to investigate the possibility of identifying specimens with degraded DNA by diagnostic positions. Identification based on morphological and molecular characters is discussed and the identification of critical specimens by tree building (= genetic distance) and by diagnostic characters is compared.

Filling the gap - COI barcode resolution in eastern Palearctic birds - Dec 09, 2009 ()
[Kerr, K., Birks, S., Kalyakin, M., Red'kin, Y., Koblik, E., & Hebert, P. D. N. 2009. Frontiers in Zoology. 6(1) 29.]

BACKGROUND:The Palearctic region supports relatively few avian species, yet recent molecular studies have revealed that cryptic lineages likely still persist unrecognized. A broad survey of cytochrome c oxidase I (COI) sequences, or DNA barcodes, can aid on this front by providing molecular diagnostics for species assignment. Barcodes have already been extensively surveyed in the Nearctic, which provides an interesting comparison to this region; faunal interchange between these regions has been very dynamic. We explored COI sequence divergence within and between species of Palearctic birds, including samples from Russia, Kazakhstan, and Mongolia. As of yet, there is no consensus on the best method to analyze barcode data. We used this opportunity to compare and contrast three different methods routinely employed in barcoding studies: clustering-based, distance-based, and character-based methods. RESULTS:We produced COI sequences from 1,674 specimens representing 398 Palearctic species. These were merged with published COI sequences from North American congeners, creating a final dataset of 2,523 sequences for 599 species. Ninety-six percent of the species analyzed could be accurately identified using one or a combination of the methods employed. Most species could be rapidly assigned using the cluster-based or distance-based approach alone. For a few select groups of species, the character-based method offered an additional level of resolution. Of the five groups of indistinguishable species, most were pairs, save for a larger group comprising the herring gull complex. Up to 44 species exhibited deep intraspecific divergences, many of which corresponded to previously described phylogeographic patterns and endemism hotspots. CONCLUSIONS:COI sequence divergence within eastern Palearctic birds is largely consistent with that observed in birds from other temperate regions. Sequence variation is primarily congruent with taxonomic boundaries; deviations from this trend reveal overlooked biological patterns, and in some cases, overlooked species. More research is needed to further refine the taxonomic status of some Palearctic birds, but large genetic surveys such as this may facilitate this effort. DNA barcodes are a practical means for rapid species assignment, although efficient analytical methods will likely require a two-tiered approach to differentiate closely related pairs of species.

Towards a comprehensive barcode library for arctic life-Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Canada - Dec 09, 2009 ()
[Zhou, X., Adamowicz, S., Jacobus, L., DeWalt, R., & Hebert, P. D. N. 2009. Frontiers in Zoology. 6(1) 30.]

BACKGROUND:This study reports progress in assembling a DNA barcode reference library for Ephemeroptera, Plecoptera, and Trichoptera ("EPTs") from a Canadian subartic site, which is the focus of a comprehensive biodiversity inventory using DNA barcoding. These three groups of aquatic insects exhibit a moderate level of species diversity, making them ideal for testing the feasibility of DNA barcoding for routine biotic surveys. We explore the correlation between the morphological species delineations, DNA barcode-based haplotype clusters delimited by a sequence threshold (2%), and a threshold-free approach to biodiversity quantification-phylogenetic diversity.RESULTS:A DNA barcode reference library is built for 112 EPT species from the focal region, consisting of 2272 COI sequences. Close correspondence was found between EPT morphospecies and haplotype clusters as designated using a standard threshold value. Similarly, the shapes of taxon accumulation curves based upon haplotype clusters were very similar to those generated using phylogenetic diversity accumulation curves, but were much more computationally efficient.CONCLUSION:The results of this study will facilitate other lines of research on northern EPTs and also bode well for rapidly conducting initial biodiversity assessments in unknown EPT faunas.

DNA barcodes provide new evidence of a recent radiation in the genus Sporophila (Aves: Passeriformes) - Dec 09, 2009 ()
[Campagna, L., Lijtmaer, D. A., Kerr, K. C. R., Barreira, A. S., Hebert, P. D. N., Lougheed, S. C. & Tubaro, P. L. 2009. Molecular Ecology Resources. Online Early .]

The capuchinos are a group of birds in the genus Sporophila that has apparently radiated recently, as evidenced by their lack of mitochondrial genetic diversity. We obtained cytochrome c oxidase I (COI) sequences (or DNA barcodes) for the 11 species of the group and various outgroups. We compared the patterns of COI variability of the capuchinos with those of the largest barcode data set from neotropical birds currently available (500 species representing 51% of avian richness in Argentina), and subjected COI sequences to neighbour-joining, maximum parsimony and Bayesian phylogenetic analyses as well as statistical parsimony network analysis. A clade within the capuchinos, the southern capuchinos, showed higher intraspecific and lower interspecific divergence than the remaining Argentine species. As most of the southern capuchinos shared COI haplotypes and pairwise distances within species were in many cases higher than distances between them, the phylogenetic affinities within the group remained unresolved. The observed genetic pattern is consistent with both incomplete lineage sorting and gene flow between species. The southern capuchinos constitute the only large group of species among the neotropical birds barcoded so far that are inseparable when using DNA barcodes, and one of few multispecies avian groups known to lack reciprocal monophyly. Extending the analysis to rapidly evolving nuclear and mitochondrial markers will be crucial to understanding this radiation. Apart from giving insights into the evolution of the capuchinos, this study shows how DNA barcoding can rapidly flag species or groups of species worthy of deeper study.

DNA-based identification of forensically important Australian Sarcophagidae (Diptera) - Dec 07, 2009 ()
[Meiklejohn, K. A., Wallman, J. F., & Dowton, M. 2009. International Journal of Legal Medicine. Online Early.]

The utility of the forensically important Sarcophagidae (Diptera) for time since death estimates has been severely limited, as morphological identification is difficult and thermobiological histories are inadequately documented. A molecular identification method involving the sequencing of a 658-bp ‘barcode’ fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene from 85 specimens, representing 16 Australian species from varying populations, was evaluated. Nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model and a neighbour-joining phylogenetic tree generated. All species were resolved as reciprocally monophyletic, except Sarcophaga dux. Intraspecific and interspecific variation ranged from 0.000% to 1.499% (SE = 0.044%) and 6.658% to 8.983% (SE = 0.653%), respectively. The COI ‘barcode’ sequence was found to be suitable for the molecular identification of the studied Australian Sarcophagidae: 96.5% of the examined specimens were assigned to the correct species. Given that the sarcophagid fauna is poorly described, it is feasible that the few incorrectly assigned specimens represent cryptic species. The results of this research will be instrumental for implementation of the Australian Sarcophagidae in forensic entomology.

In the dark in a large urban park: DNA barcodes illuminate cryptic and introduced moth species - Dec 01, 2009 ()
[deWaard, J.R., Landry, J.-F., Schmidt, B.C., Derhousoff, J., McLean, J.A. and Humble, L.M. 2009. Biodiversity and Conservation. 18(14) 3825-3839.]

To facilitate future assessments of diversity following disturbance events, we conducted a first level inventory of nocturnal Lepidoptera in Stanley Park, Vancouver, Canada. To aid the considerable task, we employed high-throughput DNA barcoding for the rough sorting of all material and for tentative species identifications, where possible. We report the preliminary species list of 190, the detection of four new exotic species (Argyresthia pruniella, Dichelia histrionana, Paraswammerdamia lutarea, and Prays fraxinella), and the potential discovery of two cryptic species. We describe the magnitude of assistance that barcoding presents for faunal inventories, from reducing specialist time to facilitating the detection of native and exotic species at low density.

The noncosmopolitanism paradigm of freshwater zooplankton: insights from the global phylogeography of the predatory cladoceran Polyphemus pediculus (Linnaeus, 1761) (Crustacea, Onychopoda) - Nov 11, 2009 ()
[Xu, S., Hebert, P. D. N., Kotov, A. A., & Cristescu, M. E. 2009. Molecular Ecology. 18(24) 5161-5179.]

A major question in our understanding of eukaryotic biodiversity is whether small bodied taxa have cosmopolitan distributions or consist of geographically localized cryptic taxa. Here, we explore the global phylogeography of the freshwater cladoceran Polyphemus pediculus (Linnaeus, 1761) (Crustacea, Onychopoda) using two mitochondrial genes, cytochrome c oxidase subunit I and 16s ribosomal RNA, and one nuclear marker, 18s ribosomal RNA. The results of neighbour-joining and Bayesian phylogenetic analyses reveal an exceptionally pronounced genetic structure at both inter- and intra-continental scales. The presence of well-supported, deeply divergent phylogroups across the Holarctic suggests that P. pediculus represents an assemblage of at least nine, largely allopatric cryptic species. Interestingly, all phylogenetic analyses support the reciprocal paraphyly of Nearctic and Palaearctic clades. Bayesian inference of ancestral distributions suggests that P. pediculus originated in North America or East Asia and that European lineages of Polyphemus were established by subsequent intercontinental dispersal events from North America. Japan and the Russian Far East harbour exceptionally high levels of genetic diversity at both regional and local scales. In contrast, little genetic subdivision is apparent across the formerly glaciated regions of Europe and North America, areas that historical demographic analyses suggest that were recolonized just 5500201324 000 years ago.

Genome 10K: a proposal to obtain whole-genome sequence for 10,000 vertebrate species - Nov 05, 2009 ()
[Hausler, D., O'Brien, S. J., Ryder, O. A., & 58 co-authors including Hebert, P. D. N. 2009. J Heredity. 100(6) 659-74.]

The human genome project has been recently complemented by whole-genome assessment sequence of 32 mammals and 24 nonmammalian vertebrate species suitable for comparative genomic analyses. Here we anticipate a precipitous drop in costs and increase in sequencing efficiency, with concomitant development of improved annotation technology and, therefore, propose to create a collection of tissue and DNA specimens for 10,000 vertebrate species specifically designated for whole-genome sequencing in the very near future. For this purpose, we, the Genome 10K Community of Scientists (G10KCOS), will assemble and allocate a biospecimen collection of some 16,203 representative vertebrate species spanning evolutionary diversity across living mammals, birds, nonavian reptiles, amphibians, and fishes (ca. 60,000 living species). In this proposal, we present precise counts for these 16,203 individual species with specimens presently tagged and stipulated for DNA sequencing by the G10KCOS. DNA sequencing has ushered in a new era of investigation in the biological sciences, allowing us to embark for the first time on a truly comprehensive study of vertebrate evolution, the results of which will touch nearly every aspect of vertebrate biological enquiry.

Sex attractant, distribution and DNA barcodes for the Afrotropical leaf-mining moth Phyllonorycter melanosparta (Lepidoptera: Gracillariidae) - Nov 04, 2009 ()
[De Prins, J., Mozuraitis, R., Lopez-Vaamonde, C., & Rougerie, R. 2009. Zootaxa. 2281 53-67.]

The sex attractant for Phyllonorycter melanosparta (Meyrick, 1912) has been determined as (10E)-dodec-10-en-1-yl acetate and (10E)-dodec-10-en-1-ol combined in a ratio 10:1. The distribution of this species in Eastern Africa is updated and its presence in Kenya is recorded for the first time. We discuss the taxonomic status of P. melanosparta with reference to three character sets: semiochemicals, morphological and molecular characters (DNA barcodes). This combination of characters is also proposed as a new approach to study the diversity and phylogeny of Phyllonorycter in the Afrotropical region.

Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama - Nov 03, 2009 ()
[Kress, W. J., Erickson, D. L., Jones, F. A., Swenson, N. G., Perez, R., Sanjur, O. and Bermingham, E. 2009. Proc Natl Acad Sci U S A. 106(44) 18621-6.]

The assembly of DNA barcode libraries is particularly relevant within species-rich natural communities for which accurate species identifications will enable detailed ecological forensic studies. In addition, well-resolved molecular phylogenies derived from these DNA barcode sequences have the potential to improve investigations of the mechanisms underlying community assembly and functional trait evolution. To date, no studies have effectively applied DNA barcodes sensu strictu in this manner. In this report, we demonstrate that a three-locus DNA barcode when applied to 296 species of woody trees, shrubs, and palms found within the 50-ha Forest Dynamics Plot on Barro Colorado Island (BCI), Panama, resulted in >98% correct identifications. These DNA barcode sequences are also used to reconstruct a robust community phylogeny employing a supermatrix method for 281 of the 296 plant species in the plot. The three-locus barcode data were sufficient to reliably reconstruct evolutionary relationships among the plant taxa in the plot that are congruent with the broadly accepted phylogeny of flowering plants (APG II). Earlier work on the phylogenetic structure of the BCI forest dynamics plot employing less resolved phylogenies reveals significant differences in evolutionary and ecological inferences compared with our data and suggests that unresolved community phylogenies may have increased type I and type II errors. These results illustrate how highly resolved phylogenies based on DNA barcode sequence data will enhance research focused on the interface between community ecology and evolution.

Hypobapta tachyhalotaria spec. nov. from Tasmania – an example of a new species revealed by DNA barcoding - Nov 01, 2009 ()
[Hausmann, A., Sommerer, M., Rougerie, R., & Hebert, P. D. N 2009. SPIXIANA. 32(2) 161-166.]

In Tasmanian Hypobapta percomptaria Guenée, 1858, slightly bigger and clearer grey specimens without a rosy tinged underside were hitherto deemed to reflect intraspecific variation. However, clear-cut differences in the mtDNA sequences (COI; 5' barcoding fragment; 648 bp) support the assumption of a separate species beside H. percomptaria: H. tachyhalotaria spec. nov. is diagnosed and figured. The original type specimen of H. percomptaria, for which a DNA barcode was successfully obtained, is included in the tree-diagram illustrating the sequence similarities/ differences of all specimens of Hypobapta species that were barcoded in the “Australia” campaign of the All-Leps project. The potential for rapid biodiversity assessment is exemplified by the discovery of this new species hitherto hidden under H. percomptaria.

Identification of cryptic species of Culicoides (Diptera: Ceratopogonidae) in the subgenus Culicoides and development of species-specific PCR assays based on barcode regions - Nov 01, 2009 ()
[Pagès, N., Muñoz-Muñoz, F., Talavera, S., Sarto, V., Lorca, C. and Núñez, J. I. 2009. Veterinary Parasitology. 165(3-4) 298-310.]

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of important diseases affecting wild and domestic animals. During the last decade they have played a major role in the epidemiology of the largest bluetongue epizootic ever recorded in Europe, the disease is transmitted between hosts almost exclusively by bites of Culicoides midges and affects both domestic and wild ruminants however severe disease usually occurs in certain breeds of sheep and some species of deer. An accurate vector identification is of major importance in arthropod borne diseases surveillance, as great differences in vectorial capacity are found even between close species. Unfortunately, specialized taxonomic knowledge of Culicoides identification is rarely available in routine surveillance, mainly based on wing morphology. Recently, some European species of Culicoides belonging to the subgenus Avaritia Fox, 1955 and Culicoides Latreille, 1809 have been described as new bluetongue virus vectors. In the present study, by using a fragment of the barcode region (COI gene) we report the presence of up to 11 species within the subgenus Culicoides in Catalonia (NE Spain), a region recently affected by a bluetongue epizootic. The molecular analysis revealed new non-described cryptic species which were grouped in three complexes of morphologically similar species, two in the Pulicaris complex resembling Culicoides pulicaris, two in the Fagineus complex resembling Culicoides fagineus and three in the Newsteadi complex resembling Culicoides newsteadi. The phylogenetic relationships among them showed that cryptic species detected in both Pulicaris and Fagineus complexes were closely related, whereas those in the Newsteadi complex were more distant. Accurate analysis of all species using morphological and molecular approaches resulted in the detection of diagnostic metric traits for cryptic species and the design of several new species-specific single and multiplex PCR assays to identify unambiguously all the species, most of them still lacking a specific molecular diagnosis.

Molecular systematics and phylogeny of the 'Marbled Whites' (Lepidoptera: Nymphalidae, Satyrinae, Melanargia Meigen) - Sep 28, 2009 ()
[Nazari, V., Hagen, W. T., & Bozano, G. C 2009. Systematic Entomology. Online Early .]

We investigated genetic divergence and phylogenetic relationships amongst all known species of Palaearctic butterflies of the genus Melanargia using sequence information from three genes [mitochondrial cox1 barcode region (658 bp), ribosomal 16S rRNA (c. 518 bp), and nuclear wg (404 bp)]. Results show a lack of DNA divergence among several poorly characterized taxa, as well as deep divergences within and between others. We corroborated the molecular information with morphological and genitalic characters as well as with geographic data. We revise the taxonomy of Melanargia, and propose a new systematic scheme for the group. We revive some previous synonymies (M. lucasi meadwaldoistat. rev., M. ines fathmestat. rev., M. ines jahandiezistat. rev., M. meridionalis tapaishanensisstat. rev.), revise the status of some subspecies into species (M. transcaspicastat. nov., M. lucidastat. nov., M. wiskottistat. nov.) and of several species into subspecies of other taxa (M. evartianae sadjadiistat. nov., M. larissa hylatastat. nov., M. larissa grumistat. nov., M. larissa syriacastat. nov., M. larissa titeastat. nov., M. lugens montanastat. nov., M. epimede ganymedesstat. nov.), revise the status of subspecies and transfer them to other species (M. larissa lorestanensisstat. nov., M. larissa iranicastat. nov., M. larissa karabagistat. rev., M. larissa kocakistat. nov., M. transcaspica ebertistat. nov.), and propose new synonymies (M. larissa titea = M. titea standfussisyn. nov. = M. titea titaniasyn. nov., M. leda leda = M. leda yunnanasyn. nov., M. lugens lugens = M. lugens ahyouisyn. nov., M. lugens hengshanensis = M. lugens hoeneisyn. nov., M. halimede halimede = M. halimede gratianisyn. nov., M. asiatica asiatica = M. asiatica dejeanisyn. nov., = M. asiatica elisasyn. nov., = M. asiatica sigbertisyn. nov.).

Revision of the Australian Oenochroma vinaria Guenée, 1858 species-complex (Lepidoptera: Geometridae, Oenochrominae): DNA barcoding reveals cryptic diversity and assesses status of type specimen without dissection - Sep 24, 2009 ()
[Hausmann, A., Hebert, P. D. N., Mitchell, A., Rougerie, R., Sommerer, M., Edwards, T., & Young, C. J. 2009. Zootaxa. 2239 1-21.]

The assembly of a DNA barcode library for Australian Lepidoptera revealed that Oenochroma vinaria Guenée, 1858, as currently understood, is actually a mix of two different species. By analyzing DNA barcodes from recently collected specimens and the 150 year-old female lectotype of O. vinaria, we propose a reliable assignment of the name vinaria to one of these two species. A lectotype is designated for Monoctenia decora, a confirmed synonym of O. vinaria, and a new species, Oenochroma barcodificata sp. nov. is described. This species is only known from Tasmania and New South Wales; its biology and immature stages are described in detail.

DNA barcoding of commercially important salmon and trout species (Oncorhynchus and Salmo) from North America - Sep 23, 2009 ()
[Rasmussen, R. S., Morrissey, M. T., & Hebert, P. D. N. 2009. J Agric Food Chem. 57(18) 8379-8385.]

The present study investigated the ability of DNA barcoding to reliably identify the seven commercially important salmon and trout species (genera Oncorhynchus and Salmo ) in North America. More than 1000 salmonid reference samples were collected from a wide geographic range. DNA extracts from these samples were sequenced for the standard 650 bp barcode region of the cytochrome c oxidase subunit I gene (COI). DNA barcodes showed low intraspecies divergences (mean, 0.26%; range, 0.04-1.09%), and the mean congeneric divergence was 32-fold greater, at 8.22% (range, 3.42-12.67%). The minimum interspecies divergence was always greater than the maximum intraspecies divergence, indicating that these species can be reliably differentiated using DNA barcodes. Furthermore, several shorter barcode regions (109-218 bp), termed "mini-barcodes", were identified in silico that can differentiate all eight species, providing a potential means for species identification in heavily processed products.

Disentangling Vector-Borne Transmission Networks: A Universal DNA Barcoding Method to Identify Vertebrate Hosts from Arthropod Bloodmeals - Sep 21, 2009 ()
[Alcaide, M., Rico, C., Ruiz, S., Soriguer, R., Muñoz, J., & Figuerola, J. 2009. PLoS ONE. 4(9) e7092.]

Emerging infectious diseases represent a challenge for global economies and public health. About one fourth of the last pandemics have been originated by the spread of vector-borne pathogens. In this sense, the advent of modern molecular techniques has enhanced our capabilities to understand vector-host interactions and disease ecology. However, host identification protocols have poorly profited of international DNA barcoding initiatives and/or have focused exclusively on a limited array of vector species. Therefore, ascertaining the potential afforded by DNA barcoding tools in other vector-host systems of human and veterinary importance would represent a major advance in tracking pathogen life cycles and hosts. Here, we show the applicability of a novel and efficient molecular method for the identification of the vertebrate host's DNA contained in the midgut of blood-feeding arthropods. To this end, we designed a eukaryote-universal forward primer and a vertebrate-specific reverse primer to selectively amplify 758 base pairs (bp) of the vertebrate mitochondrial Cytochrome <italic>c</italic> Oxidase Subunit I (COI) gene. Our method was validated using both extensive sequence surveys from the public domain and Polymerase Chain Reaction (PCR) experiments carried out over specimens from different Classes of vertebrates (Mammalia, Aves, Reptilia and Amphibia) and invertebrate ectoparasites (Arachnida and Insecta). The analysis of mosquito, culicoid, phlebotomie, sucking bugs, and tick bloodmeals revealed up to 40 vertebrate hosts, including 23 avian, 16 mammalian and one reptilian species. Importantly, the inspection and analysis of direct sequencing electropherograms also assisted the resolving of mixed bloodmeals. We therefore provide a universal and high-throughput diagnostic tool for the study of the ecology of haematophagous invertebrates in relation to their vertebrate hosts. Such information is crucial to support the efficient management of initiatives aimed at reducing epidemiologic risks of arthropod vector-borne pathogens, a priority for public health.

Polyommatus (Aricia) crassipunctus varicolor ssp. n., a new subspecies from Iran (Lepidoptera: Lycaenidae) - Sep 14, 2009 ()
[ten Hagen, W., & Schurian, K. G 2009. Nachrichten des Entomologischen Vereins Apollo. 30(1/2) 9-17.]

From the central Zagros Mountains in Iran a new subspecies of the lycaenid Polyommarus (Aricia) crassipunctus (CHRISTOPH, 1893) is described: varicolor ssp. n.  The butterflies are similar to the nominotypical subspecies from eastern Anatolia (Kasikoparan, Agri), but differ by the greenish tint of the blue ground color on the upperside of ♂♂ as well as by the more intensive colouration of maculae and ocelli on the underside of the wings, producing their own appearance.  The blue butterfly is polyvoltine and could be found from May to July at 2450 m above sea level.  Ab-ovo breeding was successful until pupation.  At the type locality (prov. Esfahan, Fereydoun Shahr) larvae feed on Geranium persicum SCHÖNBECK-TEMERSY (Geraniaceae).  Preimaginal instars and genitalia did not show significant differences to other members of the anteros-crassipunctus group.  First prelimery results of DNA barcoding justify the subspecific level.

DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification - Sep 01, 2009 ()
[Lukhtanov, V. A., Sourakov, A., Zakharov, E. V., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(5) 1302-1310.]

DNA barcoding employs short, standardized gene regions (5' segment of mitochondrial cytochrome oxidase subunit I for animals) as an internal tag to enable species identification. Prior studies have indicated that it performs this task well, because interspecific variation at cytochrome oxidase subunit I is typically much greater than intraspecific variation. However, most previous studies have focused on local faunas only, and critics have suggested two reasons why barcoding should be less effective in species identification when the geographical coverage is expanded. They suggested that many recently diverged taxa will be excluded from local analyses because they are allopatric. Second, intraspecific variation may be seriously underestimated by local studies, because geographical variation in the barcode region is not considered. In this paper, we analyse how adding a geographical dimension affects barcode resolution, examining 353 butterfly species from Central Asia. Despite predictions, we found that geographically separated and recently diverged allopatric species did not show, on average, less sequence differentiation than recently diverged sympatric taxa. Although expanded geographical coverage did substantially increase intraspecific variation reducing the barcoding gap between species, this did not decrease species identification using neighbour-joining clustering. The inclusion of additional populations increased the number of paraphyletic entities, but did not impede species-level identification, because paraphyletic species were separated from their monophyletic relatives by substantial sequence divergence. Thus, this study demonstrates that DNA barcoding remains an effective identification tool even when taxa are sampled from a large geographical area.

Barcoding bushmeat: molecular identification of Central African and South American harvested vertebrates - Sep 01, 2009 (pdf)
[Eaton, MJ; Meyers, GL; Kolokotronis, S; Leslie, MS; Martin, AP; Amato, G 2009. Conservation Genetics. .]

A new hidden species of the Cymothoe caenis-complex (Lepidoptera: Nymphalidae) from western Africa - Aug 13, 2009 (pdf)
[Van Velzen, R., Larsen, T.B., & Bakker, F.T. 2009. Zootaxa 2197. 53-63.]
Barcoding of Plants and Fungi - Aug 07, 2009 ()
[Chase, M. W., & Fay, M. F. 2009. Science. 325 682-683.]

DNA barcodes for soil animal taxonomy - Aug 01, 2009 ()
[Rougerie, R., Decaëns, T., Deharveng, L., Porco, D., James, S. W., Chang, C.-H., Richard, B., Potapov, M., Suhardjono, Y& Hebert, P. D. N. 2009. Pesquisa Agropecuária Brasileira. 44(8) 789-801.]

The biodiversity of soil communities remains very poorly known and understood. Soil biological sciences are strongly affected by the taxonomic crisis, and most groups of animals in that biota suffer from a strong taxonomic impediment. The objective of this work was to investigate how DNA barcoding - a novel method using a microgenomic tag for species identification and discrimination - permits better evaluation of the taxonomy of soil biota. A total of 1,152 barcode sequences were analyzed for two major groups of animals, collembolans and earthworms, which presented broad taxonomic and geographic sampling. Besides strongly reflecting the taxonomic impediment for both groups, with a large number of species-level divergent lineages remaining unnamed so far, the results also highlight a high level (15%) of cryptic diversity within known species of both earthworms and collembolans. These results are supportive of recent local studies using a similar approach. Within an impeded taxonomic system for soil animals, DNA-assisted identification tools can facilitate and improve biodiversity exploration and description. DNA-barcoding campaigns are rapidly developing in soil animals and the community of soil biologists is urged to embrace these methods.

A DNA barcode for land plants - Jul 30, 2009 ()
[Hollingsworth, P. M., Forrest, L. L., Spouge, J. L., Hajibabaei, M., Ratnasingham, S., van der Bank, M., Chase, M. W., Cowan, R. S., Erickson, D. L., Fazekas, A. J., Graham, S. W., James, K. E., Kim, K.-J., Kress, W. J., Schneider, H., van AlphenStahl, J. 2009. Proceedings of the National Academy of Sciences. Online Early .]

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions ( spacer, gene, gene, gene, gene, spacer, and spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+ matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

Prospects for using DNA barcoding to identify spiders in species-rich genera - Jul 29, 2009 ()
[Robinson, E. A., Blagoev, G. A., Hebert, P. D. N., & Adamowicz, S. J. 2009. ZooKeys. 27-46.]

While previous research has indicated the utility of DNA barcoding in identifying spider species sampled from a localized region, the effectiveness of this method over a broader geographic scale and with denser taxon sampling has not yet been extensively considered. Using both new and published data from 1801 individuals belonging to 361 morphospecies, this study examined intra- and interspecific divergences for 19 genera that were each represented by at least 10 morphospecies. We particularly focused on increasing species-level sampling in order to better characterize levels of interspecific divergence within species-rich genera and to examine the prevalence of a “barcode gap” (discontinuity between intra- and interspecific divergences). Overall, the mean intraspecific divergence value was found to be 2.15%, the average maximum intraspecific divergence was 3.16%, while the mean divergence between nearest interspecific neighbours was 6.77%, demonstrating the typical presence of a barcode gap. Of the 66% of morphospecies that formed monophyletic sequence clusters, the majority (92.5%) possessed a barcode gap. We also examine possible biological explanations for the large proportion of paraphyletic and polyphyletic clusters and discuss the need for further taxonomic investigations. The overlap between intra- and interspecific divergences was not unexpected for some ‘species’, such as Pardosa groenlandica, since prior morphological studies have suggested that it is an example of a species complex. However, other cases of high intraspecific divergences may reflect cryptic species diversity, indicating the need for a taxonomic approach that combines both morphological and molecular methods. The list of the species, COI sequences, and source references used in the analysis is published as a dataset under doi: 10.3897/zookeys.16.239.app.A.ds. The list of analyzed species, mean and maximum intraspecific divergences, distances to the nearest neighbouring species in its genus, general localities, and lifestyle characteristics is published as a dataset under doi: 10.3897/zookeys.16.239.app.B.ds.

Barcoding Nemo: DNA-Based Identifications for the Ornamental Fish Trade - Jul 21, 2009 ()
[Steinke, D., Zemlak, T. S., & Hebert, P. D. N. 2009. http://www.plosone.org. 4(7) e6300.]

Background
Trade in ornamental fishes represents, by far, the largest route for the importation of exotic vertebrates. There is growing pressure to regulate this trade with the goal of ensuring that species are sustainably harvested and that their point of origin is accurately reported. One important element of such regulation involves easy access to specimen identifications, a task that is currently difficult for all but specialists because of the large number of species involved. The present study represents an important first step in making identifications more accessible by assembling a DNA barcode reference sequence library for nearly half of the ornamental fish species imported into North America.

Methodology/Principal Findings
Analysis of the cytochrome c oxidase subunit I (COI) gene from 391 species from 8 coral reef locations revealed that 98% of these species exhibit distinct barcode clusters, allowing their unambiguous identification. Most species showed little intra-specific variation (adjusted mean = 0.21%), but nine species included two or three lineages showing much more divergence (2.19–6.52%) and likely represent overlooked species complexes. By contrast, three genera contained a species pair or triad that lacked barcode divergence, cases that may reflect hybridization, young taxa or taxonomic over-splitting.

Conclusions/Significance
Although incomplete, this barcode library already provides a new species identification tool for the ornamental fish industry, opening a realm of applications linked to collection practices, regulatory control and conservation.

Mitochondrial and microsatellite DNA markers reveal a Balkan origin for the highly invasive horse-chestnut leaf miner Cameraria ohridella (Lepidoptera, Gracillariidae) - Jul 21, 2009 ()
[Valade, R., Kenis, M., Hernandez-Lopez, A., Augustin, S., Mari Mena, N., Magnoux, E., Rougerie, R., Lakatos, F., Roques, A. and Lopez-Vaamonde, C. 2009. Molecular Ecology. Online Early .]

Abstract Biological invasions usually start with a small number of founder individuals. These founders are likely to represent a small fraction of the total genetic diversity found in the source population. Our study set out to trace genetically the geographical origin of the horse-chestnut leafminer, Cameraria ohridella, an invasive microlepidopteran whose area of origin is still unkown. Since its discovery in Macedonia 25 years ago, this insect has experienced an explosive westward range expansion, progressively colonizing all of Central and Western Europe. We used cytochrome oxidase I sequences (DNA barcode fragment) and a set of six polymorphic microsatellites to assess the genetic variability of C. ohridella populations, and to test the hypothesis that C. ohridella derives from the southern Balkans (Albania, Macedonia and Greece). Analysis of mtDNA of 486 individuals from 88 localities allowed us to identify 25 geographically structured haplotypes. In addition, 480 individuals from 16 populations from Europe and the southern Balkans were genotyped for 6 polymorphic microsatellite loci. High haplotype diversity and low measures of nucleotide diversities including a significantly negative Tajima's D indicate that C. ohridella has experienced rapid population expansion during its dispersal across Europe. Both mtDNA and microsatellites show a reduction in genetic diversity of C. ohridella populations sampled from artificial habitats (e.g. planted trees in public parks, gardens, along roads in urban or sub-urban areas) across Europe compared with C. ohridella sampled in natural stands of horse-chestnuts in the southern Balkans. These findings suggest that European populations of C. ohridella may indeed derive from the southern Balkans.

Multigene phylogeny and DNA barcoding indicate that the Sandwich tern complex (Thalasseus sandvicensis, Laridae, Sternini) comprises two species - Jul 01, 2009 ()
[Efe, M. A., Tavares, E. S., Baker, A. J., & Bonatto, S. L 2009. Molecular Phylogenetics and Evolution. 52(1) 263-267.]

DNA barcoding of animal and plant species as an approach for their molecular identification and describing of diversity - Jul 01, 2009 ()
[Shneyer, V.S. 2009. Zh Obshch Biol. 70(4) 296-315.]

DNA barcoding was recently developed as a method of species identification across a broad range of eucaryotes taxa by sequencing a standardized short DNA fragment. Due to modern technologies, it is possible to do this with a tiny piece of any tissue taken from an organism at any developmental phase, often without damaging it. A variable 5' half of mitochondial gene CO1 is suggested as a standard region for most of animals; it is not identified yet for fungi and plants. "The Barcode of Life Initiative" implies creating and developing the barcode library for all the species on Earth to facilitate both assigning of newly obtained specimens to the known species and for discovering new and cryptic species or at least their provisional recognition. This approach has a great potential for the use in global biodiversity studies, especially in the case of poorly investigated taxa and environments. The initiative in question involves accomplish of a new web-based sequence database with rigorous rules for taxonomic information on the specimens and records of their storage as well as for standards of sequence quality and their entry. Critical objections of opponents to DNA barcoding are reviewed as well as limitations of the approach, the problems to be taken into consideration, and the fields where it can be used. Numerous recent studies on different animal groups convincingly demonstrate the efficacy of DNA barcoding and its potentials. The latter depends on availability of comprehensive and unbiased reference database implying correct identification of the source specimens and adequate knowledge of intraspecies variation, so the Barcode Initiative would be more successful as a part of the integrative analysis of the taxs being barcoded.

Towards barcode markers in Fungi: an intron map of Ascomycota mitochondria. - Jun 16, 2009 ()
[Santamaria, M., Vicario, S., Pappada, G., Scioscia, G., Scazzocchio, C., & Saccone, C. 2009. BMC Bioinformatics. 10(Suppl 6) S15.]

BACKGROUND: A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality. In particular the potential use of mitochondrial DNA species markers has been taken in account. Unfortunately, a serious difficulty in the PCR and bioinformatic surveys is due to the presence of mobile introns in almost all the fungal mitochondrial genes. The aim of this work is to verify the incidence of this phenomenon in Ascomycota, testing, at the same time, a new bioinformatic tool for extracting and managing sequence databases annotations, in order to identify the mitochondrial gene regions where introns are missing so as to propose them as species markers.

METHODS: The general trend towards a large occurrence of introns in the mitochondrial genome of Fungi has been confirmed in Ascomycota by an extensive bioinformatic analysis, performed on all the entries concerning 11 mitochondrial protein coding genes and 2 mitochondrial rRNA (ribosomal RNA) specifying genes, belonging to this phylum, available in public nucleotide sequence databases. A new query approach has been developed to retrieve effectively introns information included in these entries.

RESULTS: After comparing the new query-based approach with a blast-based procedure, with the aim of designing a faithful Ascomycota mitochondrial intron map, the first method appeared clearly the most accurate. Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals.

CONCLUSION: The development of the new query system described here would answer the pressing requirement to improve drastically the bioinformatics support to the DNA Barcode Initiative. The large scale investigation of Ascomycota mitochondrial introns performed through this tool, allowing to exclude the introns-rich sequences from the barcode candidates exploration, could be the first step towards a mitochondrial barcoding strategy for these organisms, similar to the standard approach employed in metazoans.

iBarcode.org: web-based molecular biodiversity analysis - Jun 16, 2009 ()
[Singer, G., & Hajibabaei, M 2009. BMC Bioinformatics. 10(Suppl 6) S14.]

BACKGROUND: DNA sequences have become a primary source of information in biodiversity analysis. For example, short standardized species-specific genomic regions, DNA barcodes, are being used as a global standard for species identification and biodiversity studies. Most DNA barcodes are being generated by laboratories that have an expertise in DNA sequencing but not in bioinformatics data analysis. Therefore, we have developed a web-based suite of tools to help the DNA barcode researchers analyze their vast datasets.

RESULTS: Our web-based tools, available at http://www.ibarcode.org, allow the user to manage their barcode datasets, cull out non-unique sequences, identify haplotypes within a species, and examine the within- to between-species divergences. In addition, we provide a number of phylogenetics tools that will allow the user to manipulate phylogenetic trees generated by other popular programs.

CONCLUSION: The use of a web-based portal for barcode analysis is convenient, especially since the WWW is inherently platform-neutral. Indeed, we have even taken care to ensure that our website is usable from handheld devices such as PDAs and smartphones. Although the current set of tools available at iBarcode.org were developed to meet our own analytic needs, we hope that feedback from users will spark the development of future tools. We also welcome user-built modules that can be incorporated into the iBarcode framework.

Migratory Canada geese cause crash of US Airways Flight 1549 - Jun 08, 2009 ()
[Marra, P. P., Dove, C. J., Dolbeer, R., Dahlan, N. F., Heacker, M., Whatton, J. F, Diggs, N. E, France, C. & Henkes, G. A. 2009. Frontiers in Ecology and the Environment. Online Early .]

In the United States alone, over 7400 bird–aircraft collisions (birdstrikes) were reported in 2007. Most of these strikes occurred during takeoff or landing of the flight, and it is during these flight phases that aircraft experience their highest risk of substantial damage after colliding with birds. Birdstrikes carry enormous potential costs in terms of lives and money. Using feather remains and other tissue samples collected from the engines of US Airways Flight 1549, which crash landed in the Hudson River in New York City on 15 January 2009 after a birdstrike, we apply molecular tools and stable hydrogen isotopes to demonstrate that migratory Canada geese were responsible for the crash. Determining whether the geese involved in this birdstrike event were resident or migratory is essential to the development of management techniques that could reduce the risk of future collisions. Currently, the US civil aviation industry is not required to report birdstrikes, yet information on frequency, timing, and species involved, as well as the geographic origin of the birds, is critical to reducing the number of birdstrikes. Integrating this information with bird migration patterns, bird-detecting radar, and bird dispersal programs at airports can minimize the risk of such collisions in the future.

On the utility of DNA barcoding for species differentiation among brown macroalgae (Phaeophyceae) including a novel extraction protocol - Jun 01, 2009 ()
[McDevit, D. C. and G. W. Saunders 2009. Phycological Research. 57(2) 131-141.]

The generation of a species-rich DNA barcode database in combination with rapid and affordable sequencing techniques will dramatically change specimen identification in ecological, biogeographical and taxonomic applications. Though cytochrome c oxidase 1 has been shown to be a useful tool for differentiating some groups of marine algae, its wide application in the Phaeophyceae has yet to be studied. The presence of polymerase chain reaction (PCR) inhibiting compounds in members of the Fucales, Laminariales and Tilopteridales, that are often co-extracted with DNA, has hampered the rapid processing associated with barcode projects. Polyphenolics and polysaccharides are present in concentrations such that DNA extraction methods typically include extensive series of washes, organelle extractions and/or cesium columns. In this paper we examine the utility of cytochrome c oxidase 1 for barcoding the Phaeophyceae and present a method for extracting PCR friendly DNA from brown macroalgae in about 2 h, dramatically reducing the time required from previous methods, some of which take days. This method is easily adapted to a 96 well, high-throughput format and may have applications in other organisms where the presence of similar PCR inhibiting compounds hinders molecular analyses. We extracted DNA from 106 isolates representing 29 species from 20 genera in nine families from five orders of Phaeophyceae. We were able to amplify the barcode marker (cytochrome c oxidase 1) from all samples and a nuclear marker (internal transcribed spacer region) from 54 selected samples. Cytochrome c oxidase 1 was able to differentiate clearly among species, showing within species divergence of 0.00–0.46%, with the exception of one previously studied genus, and between species divergences of greater than 3%.

Ethnobotany Genomics study reveals three new species from the Velliangiri Holy Hills in the Nilgiri Biosphere Reserve, Western Ghats, India - Jun 01, 2009 ()
[Newmaster, S. G., Murugesan, M., Ragupathy, S., Nallasamy, N., & Balasubramaniam, V 2009. Ethnobotany. 21 2-24.]

Our research utilized Traditional Tribal Knowledge (TK) and Scientific Knowledge (SK) to explore the relationship between scientific and tribal systems of botanical classification and the corresponding valorisation(s) of biological diversity in the Western Ghats of southern India. We worked with two tribal communities namely ‘Irulas’ and ‘Malasars’ of the Nilgiri Biosphere Reserve with an objective of evaluating the ability of different knowledge systems (SK and TK) to distinguish species belonging to the genus Biophytum. We discovered that the tribal informants identified three ethnotaxa representing three new species namely, Biophytum velliangirianum, B. coimbatorense, B. tamilnadense, which we confimed using quantitative morphology and DNA evidence. The new taxa were confirmed by DNA barcoding and a morphometric analysis of the taxonomic evidence including comparisons with several closely related taxa: Biophytum insignis Gamble, B. longipedunculatum Govind. and Biophytum proliferum (Arn.) Wight. The recognition of these taxa has several consequences for conservation of plant diversity in the Nilgiri Biosphere and possible applications to society-at-large given the ethnobiological importance of these new taxa to the local tribals.

Morphological and molecular characterization of renal ciliates infecting farmed snails in Spain - Jun 01, 2009 ()
[Segade, P., Kher, C. P., Lynn, D. H., & Iglesias, R. 2009. Parasitology. 136(7) 771-782.]

Renal infections by parasitic ciliates were studied in adult snails of Helix aspersa aspersa and Helix aspersa maxima collected from 2 mixed rearing system-based heliciculture farms located in Galicia (NW Spain). The occurrence of ciliates was also examined in slugs (Deroceras reticulatum) invading the greenhouses where first growing and fattening of snails is carried out. Histological examinations revealed a severe destruction of the renal epithelium in heavily infected hosts. Three ciliate isolates, one from each host species, were obtained and grown in axenic cultures. Cultured and parasitic ciliates were characterized morphologically and morphometrically. In addition, the encystment behaviour, the occurrence of autogamy, and the sequences of the mitochondrial cytochrome-c oxidase subunit 1 (cox1) and the small subunit ribosomal RNA (SSU rRNA) genes were also studied in the 3 isolates. A polymorphic life cycle involving resting and reproductive cysts, together with the morphological and morphometrical characteristics and the confirmation that autogamy occurs within cysts, demonstrate that our ciliates belong to the species Tetrahymena rostrata (Kahl, 1926) Corliss, 1952. The 3 isolates formed a well-supported clade using both genetic markers, and were clearly separate from the strain ATCC(R) 30770, which has been identified as Tetrahymena rostrata. We argue that our Spanish isolates should be regarded as Tetrahymena rostrata, and that the ATCC isolate should be regarded as a misidentification as neither cytological nor cytogenetical support for its identity has been presented.

A new mouse-eared bat (Mammalia: Chiroptera: Vespertilionidae) from Vietnam - Jun 01, 2009 ()
[Borisenko, A. V., Kruskop, S. V., & Ivanova, N. V. 2009. Russian Journal of Theriology. 7(2) 57-69.]

A new mouse-eared bat (Mammalia: Chiroptera: Vespertilionidae) from the Myotis “siligorensis” species group is being described from the Hon Ba Mountain, ca. 30 km WSW of Nha Trang, Khanh Hoa Province, Vietnam (12.1113° N, 108.953° E, 1250 m ASL), based on a set of morphological and genetic characters. The new species is essentially similar to M. siligorensis alticraniatus, differing in slightly larger size, morphometrics, fine cranial and bacular traits. 12S rDNA demonstrates ca. 2% sequence divergence between the new species and its nearest neighbour, suggesting a history of genetic isolation. Provisional bat survey data from the Bi Doup-Hon Ba massif suggest that, although the new species co-occurs with M. siligorensis in the southern part of the Vietnam Central Highlands area, they are separated by an altitudinal gradient and habitat preferences, the former occupying mature forest at higher elevations and the latter confined to disturbed foothill areas.

Species on the menu of a generalist predator, the eastern red bat (Lasiurus borealis): using a molecular approach to detect arthropod prey - Jun 01, 2009 ()
[Clare, E. L., Fraser, E. E., Braid, H. E., Fenton, M. B., & Hebert, P. D. N. 2009. Molecular Ecology. 18(11) 2532-2542.]

One of the most difficult interactions to observe in nature is the relationship between a predator and its prey. When direct observations are impossible, we rely on morphological classification of prey remains, although this is particularly challenging among generalist predators whose faeces contain mixed and degraded prey fragments. In this investigation, we used a polymerase chain reaction and sequence-based technique to identify prey fragments in the guano of the generalist insectivore, the eastern red bat (Lasiurus borealis), and evaluate several hypotheses about prey selection and prey defences. The interaction between bats and insects is of significant evolutionary interest because of the adaptive nature of insect hearing against echolocation. However, measuring the successes of predator tactics or particular prey defences is limited because we cannot normally identify these digested prey fragments beyond order or family. Using a molecular approach, we recovered sequences from 89% of the fragments tested, and through comparison to a reference database of sequences, we were able to identify 127 different species of prey. Our results indicate that despite the robust jaws of L. borealis, most prey taxa were softer-bodied Lepidoptera. Surprisingly, more than 60% of the prey species were tympanate, with ears thought to afford protection against these echolocating bats. Moths of the family Arctiidae, which employ multiple defensive strategies, were not detected as a significant dietary component. Our results provide an unprecedented level of detail for the study of predator2013prey relationships in bats and demonstrate the advantages which molecular tools can provide in investigations of complex ecological systems and food-web relationships.

Loss of all plastid ndh genes in Gnetales and conifers: extent and evolutionary significance for the seed plant phylogeny - Jun 01, 2009 ()
[Braukmann, T. W. A., Kuzmina, M., & Stefanovic, S. 2009. Current Genetics. 55(3) 323-337.]

The exact phylogenetic position of Gnetales, a small, highly modified group of gymnosperms with an accelerated rate of molecular evolution, is one of the most challenging issues for seed plant systematics. Recent results from entire plastid genome (ptDNA) sequencing revealed the absence of the entire suite of plastid ndh genes in several species of Gnetales and the pine family (Pinaceae) potentially highlighting a major structural feature linking these two groups—concerted loss of all plastid genes for the NADH dehydrogenase complex. However, the precise extent of ndh gene loss in gymnosperms has not been surveyed. Using a slot-blot hybridization method, we probed all 11 ndh genes in 162 species from 70 of 85 gymnosperm genera. We find that all ndh genes are absent across Gnetales and Pinaceae, but not in any other group of gymnosperms. This feature represents either a major synapomorphy for a clade consisting of these two lineages or, less likely, a convergent loss. Our survey substantially extends previous inferences based on more limited sampling and, if the former evolutionary interpretation is correct, it provides additional support for the contentious “gnepine” hypothesis, which places Gnetales as sister to Pinaceae.

Multiple copies of cytochrome oxidase 1 in species of the fungal genus Fusarium - May 01, 2009 ()
[Gilmore, S. R., Grafenhan, T., Louis-Seize, G., & Seifert, K. A. 2009. Molecular Ecology Resources. 9(s1) 90-98.]

Using data from published mitochondrial or complete genomes, we developed and tested primers for amplification and sequencing of the barcode region of cytochrome oxidase 1 (COX1) of the fungal genus Fusarium, related genera of the order Hypocreales, and degenerate primers for fungi in the subdivision Pezizomycotina. The primers were successful for amplifying and sequencing COX1 barcodes from 13 genera of Hypocreales (Acremonium, Beauveria, Clonostachys, Emericellopsis, Fusarium, Gliocladium, Hypocrea, Lanatonectria, Lecanicillium, Metarhizium, Monocillium, Neonectria and Stilbella), 22 taxa of Fusarium, and two genera in other orders (Arthrosporium, Monilochaetes). Parologous copies of COX1 occurred in several strains of Fusarium. In some, copies of the same length were detected either by heterozygous bases in otherwise clean sequences or in different replicates of amplification and sequencing events; this may indicate multiple transcribed copies. Other strains included one or two introns. Two intron insertion sites had at least two nonhomologous intron sequences among Fusarium species. Irrespective of whether the multiple copy issue could be resolved by sequencing RNA transcripts, developing a precise COX1-based barcoding system for Fusarium may not be feasible. The overall divergence among homologous COX1 sequences obtained so far is rather low, with many species sharing identical sequences.

Evaluation of mitochondrial genes as DNA barcode for Basidiomycota - May 01, 2009 ()
[Vialle, A., Feau, N., Allaire, M., Didukh, M., Martin, F., Moncalvo, J.-M., & Hamelin, R. C. 2009. Molecular Ecology Resources. 9(s1) 99-113.]

Our study evaluated in silico the potential of 14 mitochondrial genes encoding the subunits of the respiratory chain complexes, including cytochrome c oxidase I (CO1), as Basidiomycota DNA barcode. Fifteen complete and partial mitochondrial genomes were recovered and characterized in this study. Mitochondrial genes showed high values of molecular divergence, indicating a potential for the resolution of lower-level relationships. However, numerous introns occurred in CO1 as well as in six other genes, potentially interfering with polymerase chain reaction amplification. Considering these results and given the minimal length of 600-bp that is optimal for a fungal barcode, the genes encoding for the ATPase subunit 6, the cytochrome oxidase subunit 3 and the NADH dehydrogenase subunit 6 have the most promising characteristics for DNA barcoding among the mitochondrial genes studied. However, biological validation on two fungal data sets indicated that no single mitochondrial gene gave a better taxonomic resolution than the ITS, the region already widely used in fungal taxonomy.

A high density COX1 barcode oligonucleotide array for identification and detection of species of Penicillium subgenus Penicillium - May 01, 2009 ()
[Chen, W., Seifert, K. A., & Levesque, C. A. 2009. Molecular Ecology Resources. 9(s1) 114-129.]

We developed a COX1 barcode oligonucleotide array based on 358 sequences, including 58 known and two new species of Penicillium subgenus Penicillium, and 12 allied species. The array was robotically spotted at near microarray density on membranes. Species and clade-specific oligonucleotides were selected using the computer programs SigOli and Array Designer. Robotic spotting allowed 768 spots with duplicate sets of perfect match and the corresponding mismatch and positive control oligonucleotides, to be printed on 2 × 6 cm2 nylon membranes. The array was validated with hybridizations between the array and digoxigenin (DIG)-labelled COX1 polymerase chain reaction amplicons from 70 pure DNA samples, and directly from environmental samples (cheese and plants) without culturing. DNA hybridization conditions were optimized, but undesired cross-reactions were detected frequently, reflecting the relatively high sequence similarity of the COX1 gene among Penicillium species. Approximately 60% of the perfect match oligonucleotides were rejected because of low specificity and 76 delivered useful group-specific or species-specific reactions and could be used for detecting certain species of Penicillium in environmental samples. In practice, the presence of weak signals on arrays exposed to amplicons from environmental samples, which could have represented weak detections or weak cross reactions, made interpretation difficult for over half of the oligonucleotides. DNA regions with very few single nucleotide polymorphisms or lacking insertions/deletions among closely related species are not ideal for oligonucleotide-based diagnostics, and supplementing the COX1-based array with oligonucleotides derived from additional genes would result in a more robust hierarchical identification system.

Are plant species inherently harder to discriminate than animal species using DNA barcoding markers? - May 01, 2009 ()
[Fazekas, A. J., Kesanakurti, P. R., Burgess, K. S., Percy, D. M., Graham, S. W., Barrett, S. C. H., Newmaster, S. G., Hajibabaei, M., & Husband, B. C. 2009. Molecular Ecology Resources. 9(s1) 130-139.]

The ability to discriminate between species using barcoding loci has proved more difficult in plants than animals, raising the possibility that plant species boundaries are less well defined. Here, we review a selection of published barcoding data sets to compare species discrimination in plants vs. animals. Although the use of different genetic markers, analytical methods and depths of taxon sampling may complicate comparisons, our results using common metrics demonstrate that the number of species supported as monophyletic using barcoding markers is higher in animals (&gt; 90%) than plants (~70%), even after controlling for the amount of parsimony-informative information per species. This suggests that more than a simple lack of variability limits species discrimination in plants. Both animal and plant species pairs have variable size gaps between intra- and interspecific genetic distances, but animal species tend to have larger gaps than plants, even in relatively densely sampled genera. An analysis of 12 plant genera suggests that hybridization contributes significantly to variation in genetic discontinuity in plants. Barcoding success may be improved in some plant groups by careful choice of markers and appropriate sampling; however, overall fine-scale species discrimination in plants relative to animals may be inherently more difficult because of greater levels of gene-tree paraphyly.

Plant DNA barcodes and species resolution in sedges (Carex, Cyperaceae) - May 01, 2009 ()
[Starr, J. R., Naczi, R. F. C., & Chouinard, B. N. 2009. Molecular Ecology Resources. 9(s1) 151-163.]

We investigate the species discriminatory power of a subset of the proposed plant barcoding loci (matK, rbcL, rpoC1, rpoB, trnH-psbA) in Carex, a cosmopolitan genus that represents one of the three largest plant genera on earth (c. 2000 species). To assess the ability of barcoding loci to resolve Carex species, we focused our sampling on three of the taxonomically best-known groups in the genus, sections Deweyanae (6/8 species sampled), Griseae (18/21 species sampled), and Phyllostachyae (10/10 species sampled). Each group represents one of three major phylogenetic lineages previously identified in Carex and its tribe Cariceae, thus permitting us to evaluate the potential of DNA barcodes to broadly identify species across the tribe and to differentiate closely related sister species. Unlike some previous studies that have suggested that plant barcoding could achieve species identification rates around 90%, our results suggest that no single locus or multilocus barcode examined will resolve much greater than 60% of Carex species. In fact, no multilocus combination can significantly increase the resolution and statistical support (i.e., 2265 70% bootstrap) for species than matK alone, even combinations involving the second most variable region, trnH-psbA. Results suggest that a matK barcode could help with species discovery as 47% of Carex taxa recently named or resolved within cryptic complexes in the past 25 years also formed unique species clusters in upgma trees. Comparisons between the nrDNA internal transcribed spacer region (ITS) and matK in sect. Phyllostachyae suggest that matK not only discriminates more species (50201360% vs. 25%), but it provides more resolved phylogenies than ITS. Given the low levels of species resolution in rpoC1 and rpoB (0201313%), and difficulties with polymerase chain reaction amplification and DNA sequencing in rbcL and trnH-psbA (alignment included), we strongly advocate that matK should be part of a universal plant barcoding system. Although identification rates in this study are low, they can be significantly improved by a regional approach to barcoding.

Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity. - May 01, 2009 ()
[Janzen, D. H., Hallwachs, W., Blandin, P., Burns, J. M., Cadiou, J.-M., Chacon, I., et al. 2009. Molecular Ecology Resources. 9(s1) 1-26.]

Inventory of the caterpillars, their food plants and parasitoids began in 1978 for today's Area de Conservacion Guanacaste (ACG), in northwestern Costa Rica. This complex mosaic of 120 000 ha of conserved and regenerating dry, cloud and rain forest over 020132000 m elevation contains at least 10 000 species of non-leaf-mining caterpillars used by more than 5000 species of parasitoids. Several hundred thousand specimens of ACG-reared adult Lepidoptera and parasitoids have been intensively and extensively studied morphologically by many taxonomists, including most of the co-authors. DNA barcoding 2014 the use of a standardized short mitochondrial DNA sequence to identify specimens and flush out undisclosed species 2014 was added to the taxonomic identification process in 2003. Barcoding has been found to be extremely accurate during the identification of about 100 000 specimens of about 3500 morphologically defined species of adult moths, butterflies, tachinid flies, and parasitoid wasps. Less than 1% of the species have such similar barcodes that a molecularly based taxonomic identification is impossible. No specimen with a full barcode was misidentified when its barcode was compared with the barcode library. Also as expected from early trials, barcoding a series from all morphologically defined species, and correlating the morphological, ecological and barcode traits, has revealed many hundreds of overlooked presumptive species. Many but not all of these cryptic species can now be distinguished by subtle morphological and/or ecological traits previously ascribed to 'variation' or thought to be insignificant for species-level recognition. Adding DNA barcoding to the inventory has substantially improved the quality and depth of the inventory, and greatly multiplied the number of situations requiring further taxonomic work for resolution.

Routine DNA barcoding of Canadian Gracilariales (Rhodophyta) reveals the invasive species Gracilaria vermiculophylla in British Columbia - May 01, 2009 ()
[Saunders, G. W. 2009. Molecular Ecology Resources. 9(s1) 140-150.]

As part of an extensive DNA-based floristic survey of marine macroalgae in Canadian waters, an unexpected sequence for a Gracilaria sp. was generated from British Columbia. Before further molecular analyses and corresponding morphological/anatomical observations this mystery sequence was temporarily entered into our database as Gracilaria BCsp. Continued sampling uncovered this species from four additional locations. A timely collaboration with international colleagues introduced sequences from the invasive Gracilaria vermiculophylla into our cytochrome c oxidase I alignments 2014 these a perfect match to BCsp indicating that this species occurs in British Columbia. A discussion of the origin of this taxon in Canadian waters, whether natural or introduced, is provided.

Progress towards DNA barcoding of fungi - May 01, 2009 ()
[Seifert, K. A. 2009. Molecular Ecology Resources. 9(s1) 83-89.]

The use of DNA sequences for identifying fungi and fungus-like organisms predates the DNA barcoding movement by at least 10 years. A brief overview of the mycological shift from phenotypic to molecular taxonomy is provided. Exploration of the animal barcode marker, cytochrome oxidase 1, by Canadian mycologists has been fruitful for some fungi, but intron issues and lack of resolution in other taxa prevent its universal application. The momentum established by 15 years of research on the fungal nuclear ribosomal internal transcribed spacer (ITS) sequences will lead to a proposal to the Consortium for the Barcode of Life on the adoption of this marker as the fungal barcode. Existing mycological research networks should facilitate the rapid development of DNA barcoding of fungi once the marker issue is settled. Some available online fungal identification databases are briefly described.

Biological agent detection technologies - May 01, 2009 ()
[Jakupciak, J. P., & Colwell, R. R 2009. Molecular Ecology Resources. 9(s1) 51-57.]

The challenge for first responders, physicians in the emergency room, public health personnel, as well as for food manufacturers, distributors and retailers is accurate and reliable identification of pathogenic agents and their corresponding diseases. This is the weakest point in biological agent detection capability today. There is intense research for new molecular detection technologies that could be used for very accurate detection of pathogens that would be a concern to first responders. These include the need for sensors for multiple applications as varied as understanding the ecology of pathogenic micro-organisms, forensics, environmental sampling for detect-to-treat applications, biological sensors for 'detect to warn' in infrastructure protection, responses to reports of 'suspicious powders', and customs and borders enforcement, to cite a few examples. The benefits of accurate detection include saving millions of dollars annually by reducing disruption of the workforce and the national economy and improving delivery of correct countermeasures to those who are most in need of the information to provide protective and/or response measures.

Express barcodes: racing from specimen to identification - May 01, 2009 ()
[Ivanova, N. V., Borisenko, A. V., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(s1) 35-41.]

Although devices combining microfluidic and advanced sequencing technologies promise a future where one can generate a DNA barcode in minutes, current analytical regimes typically involve workflows that extend over 2 days. Here we describe simple protocols enabling the advance from a specimen to barcode-based identification in less than 2 h. The protocols use frozen or lyophilized reagents that can be prepackaged into 'kits' and support barcode analysis across the animal kingdom. The analytical procedure allows 5 min for DNA extraction, 25 min for polymerase chain reaction amplification of the barcode region, 25 min for cycle-sequencing, 10 min for cleanup, 45 min for capillary sequencing and 5 min for trace file analysis to complete DNA-based identification. This study involved the comparison of varied DNA preservation and extraction methods, and evaluated Taq polymerases with high processivity and resistance to inhibitors.

DNA barcoding and the mediocrity of morphology - May 01, 2009 ()
[Packer, L., Gibbs, J., Sheffield, C., & Hanner, R. 2009. Molecular Ecology Resources. 9(s1) 42-50.]

A small but vocal community of critics has questioned the epistemological value of DNA barcoding by suggesting that either it 'cannot work' for the identification or discovery of species or that it ignores the 'richness' inherent in traditional approaches. We re-examine these arguments through a comparison of DNA barcoding and morphological taxonomy in terms of their accuracy and diversity of characters employed. We conclude that morphology often does not work and that it is often nowhere near as 'rich' as has been argued. Morphology is particularly poor in numerous important situations, such as the association of larvae with adults and discrimination among cryptic species. The vehemence of some of the criticisms is surprising given that morphology alone is known to be inadequate to the task of species-level identification in many instances.

The front-end logistics of DNA barcoding: challenges and prospects - May 01, 2009 ()
[Borisenko, A. V., Sones, J. E., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(s1) 27-34.]

Building a global library of DNA barcodes will require efficient logistics of pre-laboratory specimen processing and seamless interfacing with molecular protocols. If not addressed properly, the task of aggregating specimens may become the biggest bottleneck in the analytical chain. Three years of experience in developing a collection management system to facilitate high-throughput DNA barcoding have allowed the Canadian Centre for DNA Barcoding to recognize and resolve the most common logistical obstacles. Dealing with these challenges on a larger scale will be an important step towards building a solid collection-based foundation for the international DNA barcoding effort.

DNA barcoding discriminates a new cryptic grass species revealed in an ethnobotany study by the hill tribes of the Western Ghats in southern India - May 01, 2009 ()
[Ragupathy, S., Newmaster, S. G., Murugesan, M., & Balasubramaniam, V. 2009. Molecular Ecology Resources. 9(s1) 164-171.]

Our research brought together traditional aboriginal knowledge (TK) and scientific knowledge (SK) to explore the relationship between scientific and aboriginal systems of botanical classification and the corresponding valorization(s) of biological diversity in the Western Ghats of southern India. We worked with two aboriginal cultures namely 'Irulas' and 'Malasars' of the Nilgiri Biosphere Reserve with an objective of evaluating the ability of different knowledge systems (SK and TK) to distinguish grass species belonging to the genus Tripogon, and assess the ability of DNA barcoding to discriminate a new cryptic species 'Tripogon cope' as deciphered by the hill tribes. We discovered that the aboriginal informants identified a common ethnotaxa 'Sunai pul', which is a cryptic species of grass not recognized by the SK classification.'sunai pul' is very important to both aboriginal cultures with ritualistic and economic utility. Morphometric analysis confirms the cryptic nature of this new species, which was validated using DNA barcoding. DNA barcode regions matK and trnH-psbA showed distinct sequence variations among the closely related ethnotaxa. Given the cryptic nature of ethnotaxa, we propose that a DNA barcode may be a reliable tool to identify ethnotaxa. We have initiated further studies in other cultures to develop theoretically sophisticated insights concerning the encounter between 'local' and 'scientific' approaches to the use of biodiversity knowledge. Furthermore, the research will add to a unifying global effort to speed up the documentation and understanding of the planet's natural diversity, while simultaneously respecting the cultural heterogeneity as a vital component of biological diversity.

Barcoding diatoms: Is there a good marker? - May 01, 2009 ()
[Moniz, M. B. J., & Kaczmarska, I. 2009. Molecular Ecology Resources. 9(s1) 65-74.]

The promise of DNA barcoding is based on a small DNA fragment divergence coinciding with biological species separation. Here we evaluated the performance of three markers as diatom barcodes, the small ribosomal subunit (1600 bp), a 5' end fragment of cytochrome c oxidase subunit 1 (430 bp), and the second internal transcribed spacer region combined with the 5.8S gene (5.8S + ITS-2, 3002013400 bp). Forty-four sequences per marker representing 28 species from all diatom classes were analysed. Sequence alignment of the three genetic markers and uncorrected genetic distances (P) were calculated at the intra- and heterospecific level. All three markers correctly separated the species examined and had advantages which contribute to their feasibility as a DNA barcode. Small ribosomal subunit had the largest GenBank data set, its success rate in amplification and sequencing was assumed to be the highest of all three and was readily aligned. However, it required a long fragment to recover divergence sufficient for species separation and small genetic distances increased the potential for misidentifications. Cytochrome c oxidase subunit 1 demonstrated a substantial heterospecific divergence level and was also readily alignable, but it showed very low amplification and sequencing success rates with currently existing primers. 5.8S + ITS-2 was amplified and sequenced with high success rate and was the most variable of the three markers, but its secondary structure was needed to aid in alignment. However, since it has been recently suggested that ITS-2 may provide insight into sexual compatibility, this marker offers an additional advantage. We therefore propose that the 5.8S + ITS-2 fragment is the best candidate as a diatom DNA barcode.

Efficient algorithms for the discovery of DNA oligonucleotide barcodes from sequence databases - May 01, 2009 ()
[Zahariev, M., Dahl, V., Chen, W., & Levesque, C. A. 2009. Molecular Ecology Resources. 9(s1) 58-64.]

Efficient design of barcode oligonucleotides can lead to significant cost reductions in the manufacturing of DNA arrays. Previous methods are based on either a preliminary alignment, which reduces their efficiency for intron-rich regions, or on a brute force approach, not feasible for large-scale problems or on data structures with very poor performance in the worst case. One of the algorithms we propose uses 'oligonucleotide sorting' for the discovery of oligonucleotide barcodes of given sizes, with good asymptotic performance. Specific barcode oligonucleotides with at least one base difference from other sequences in a database are found for each individual sequence. With another algorithm, specific oligonucleotides can also be found for groups or clades in the database, which have 100% homology for all oligonucleotide sequences within the group or clade while having differences with the rest of the data. By re-organizing the sequences/groups in the database, oligonucleotides for different hierarchical levels can be found. The oligonucleotides or polymorphism locations identified as species or clade specific by the new algorithm are refined and screened further for hybridization thermodynamic properties with third party software.

Development of primers for the mitochondrial cytochrome c oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths - May 01, 2009 ()
[Moszczynska, A., Locke, S. A., McLaughlin, J. D., Marcogliese, D. J., & Crease, T. J. 2009. Molecular Ecology Resources. 9(s1) 75-82.]

The phylum Platyhelminthes is a diverse group of flatworms that includes parasites with serious impacts on human health, animal husbandry, aquaculture and wildlife management. Here we present degenerate primers for the barcode region of the mitochondrial cytochrome c oxidase I (COI) gene in flatworms. Although amplicons were obtained from a wide taxonomic range in the Cestoda and Trematoda, COI fragments from many taxa in these classes did not amplify. Primers specific to trematodes in the family Diplostomidae were also developed. Amplification success was much higher with diplostomid-specific primers and sequences were obtained from 504 of 585 specimens of Diplostomum and Tylodelphys. Sequences from the barcode region resolved all specimens to the species level, with mean divergence between congeners of 19% (3.9201325%). Because many of our specimens were small, we initially amplified part of the nuclear small subunit ribosomal (r) RNA gene to evaluate the quality and quantity of DNA in our specimens. Short sequences (~380 nt) of this gene were recovered from most specimens and can be used to distinguish specimens at the family level and often the generic level. We suggest that rRNA genes could be used to screen samples of completely unknown taxonomy, after which specific COI primers could be used to obtain species-level identifications.

DNA barcodes to identify species and explore diversity in the Adelgidae (Insecta: Hemiptera: Aphidoidea) - May 01, 2009 ()
[Foottit, R. G., Maw, H. E. L., Havill, N. P., Ahern, R. G., & Montgomery, M. E. 2009. Molecular Ecology Resources,. 9(s1) 188-195.]

The Adelgidae are relatively small, cryptic insects, exhibiting complex life cycles with parthenogenetic reproduction. Due to these characteristics, the taxonomy of the group is problematic. Here, we test the effectiveness of the standard 658-bp barcode fragment from the 5'-end of the mitochondrial cytochrome c oxidase 1 gene (COI) in differentiating among 17 species of Adelgidae, in associating life-cycle stages, and in assessing patterns of geographical variation in selected species. Species of Adelgidae are well-differentiated by DNA barcodes, enabling the identification of different morphological forms, immature stages and individuals on different hosts and at different periods of the life cycle. DNA barcodes have uncovered cryptic diversity within taxa and, in other cases, a lack of sequence divergence in species pairs previously separated by life-cycle characteristics, indicating a need for further taxonomic analysis.

Countering criticisms of single mitochondrial DNA gene barcoding in birds - May 01, 2009 ()
[Baker, A. J., Tavares, E. S., & Elbourne, R. F 2009. Molecular Ecology Resources. 9(s1) 257-268.]

General criticisms of a single mtDNA gene barcodes include failure to identify newly evolved species, use of species-delimitation thresholds, effects of selective sweeps and chance occurrence of reciprocal monophyly within species, inability to deal with hybridization and incomplete lineage sorting, and superiority of multiple genes in species identification. We address these criticisms in birds because most species are known and thus provide an ideal test data set, and we argue with selected examples that with the exception of thresholds these criticisms are not problematic for avian taxonomy. Even closely related sister species of birds have distinctive COI barcodes, but it is not possible to universally apply distance thresholds based on ratios of within-species and among-species variation. Instead, more rigorous methods of species delimitation should be favoured using coalescent-based techniques that include tests of chance reciprocal monophyly, and times of lineage separation and sequence divergence. Incomplete lineage sorting is also easily detected with DNA barcodes, and usually at a younger time frame than a more slowly evolving nuclear gene. Where DNA barcodes detect divergent reciprocally monophyletic lineages, the COI sequences can be combined with multiple nuclear genes to distinguish between speciation or population subdivision arising from high female philopatry or regional selective sweeps. Although selective sweeps are increasingly invoked to explain patterns of shallow within-species coalescences in COI gene trees, caution is warranted in this conjecture because of limited sampling of individuals and the reduced power to detect additional mtDNA haplotypes with one gene.

Identifying sharks with DNA barcodes: assessing the utility of a nucleotide diagnostic approach - May 01, 2009 ()
[Wong, E. H.-K., Shivji, M. S., & Hanner, R. H. 2009. Molecular Ecology Resources. 9(s1) 243-256.]

Shark fisheries worldwide are mostly unmanaged, but the burgeoning shark fin industry in the last few decades has made monitoring catch and trade of these animals critical. As a tool for molecular species identification, DNA barcoding offers significant potential. However, the genetic distance-based approach towards species identification employed by the Barcode of Life Data Systems may oftentimes lack the specificity needed for regulatory or legal applications that require unambiguous identification results. This is because such specificity is not typically realized by anything less than a 100% match of the query sequence to an entry in the reference database using genetic distance. Although various divergence thresholds have been proposed to define acceptable levels of intraspecific variation, enough exceptions exist to cast reasonable doubt on many less than exact matches using a distance-based approach for the identification of unknowns. An alternative approach relies on the identification of discrete molecular characters that can be used to unambiguously diagnose species. The objective of this study was to assess the performance differences between these competing approaches by examining more than 1000 DNA barcodes representing nearly 20% of all known elasmobranch species. Our results demonstrate that a character-based, nucleotide diagnostic (ND) approach to barcode identification is feasible and also provides novel insights into the structure of haplotype diversity among closely related species of sharks. Considerations for the use of NDs in applied fields are also explored.

High-level genetic diversity but no population structure inferred from nuclear and mitochondrial markers of the peritrichous ciliate Carchesium polypinum in the Grand River basin (North America) - May 01, 2009 ()
[Gentekaki, E., & Lynn, D. H. 2009. Appl Environ Microbiol,. 75(10) 3187-3195.]

Studies that assess intraspecific genetic variation in ciliates are few and quite recent. Consequently, knowledge of the subject and understanding of the processes that underlie it are limited. We sought to assess the degree of intraspecific genetic variation in Carchesium polypinum (Ciliophora: Peritrichia), a cosmopolitan, freshwater ciliate. We isolated colonies of C. polypinum from locations in the Grand River basin in Southwestern Ontario, Canada. We then used the nuclear markers--ITS1, ITS2, and the hypervariable regions of the large subunit rRNA--and an 819-bp fragment of the mitochondrial cytochrome c oxidase I gene (cox-1) to investigate the intraspecific genetic variation of C. polypinum and the degree of resolution of the above-mentioned markers at the population level. We also sought to determine whether the organism demonstrated any population structure that mapped onto the geography of the region. Our study shows that there is a high degree of genetic diversity at the isolate level, revealed by the mitochondrial markers but not the nuclear markers. Furthermore, our results indicate that C. polypinum is likely not a single morphospecies as previously thought.

A new cleptoparasitic Lasioglossum (Hymenoptera, Halictidae) from Africa - May 01, 2009 ()
[Gibbs, J. 2009. Journal of Hymenoptera Research. 18 74-79.]

Ethnobotany genomics – use of DNA barcoding to explore cryptic diversity in economically important plants - May 01, 2009 ()
[Newmaster, S. G., & Ragupathy, S. 2009. Indian Journal of Science and Technology. 2(5) 2-8.]

The ethnobotany genomics concept is founded on the idea of ‘assemblage’ of biodiversity knowledge. This includes a coming together of different ways of knowing and valorizing species variation in a novel approach seeking to add value to both traditional knowledge (TK) and scientific knowledge (SK). Ethnobotany genomics is defined as exploring the variation in genomic sequences from many species, and here we present some of our recent work that demonstrates the potential benefits of this approach for ethnobotanical research with economic implications. DNA barcoding was used to identify Acacia and nutmeg taxa that are economically important to society-at-large. Furthermore we identified considerable variation that is recognized by several indigenous cultures. The impacts of ethnobotany genomics will extend well beyond biodiversity science. Explorations of the genomic properties across the expanse of life are now possible using DNA barcoding to assemble sequence information for a standard portion of the genome from large assemblages of species. Perhaps the most important contribution is major barcode projects will leave an important legacy; a comprehensive repository of highquality DNA extracts that will facilitate future genomic investigations.

DNA barcoding reveals overlooked marine fishes - May 01, 2009 ()
[Zemlak, T. S., Ward, R. D., Connell, A. D., Holmes, B. H., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(s1) 237-242.]

With more than 15 000 described marine species, fishes are a conspicuous, diverse and increasingly threatened component of marine life. It is generally accepted that most large-bodied fishes have been described, but this conclusion presumes that current taxonomic systems are robust. DNA barcoding, the analysis of a standardized region of the cytochrome c oxidase 1 gene (COI), was used to examine patterns of sequence divergence between populations of 35 fish species from opposite sides of the Indian Ocean, chosen to represent differing lifestyles from inshore to offshore. A substantial proportion of inshore species showed deep divergences between populations from South African and Australian waters (mean = 5.10%), a pattern which also emerged in a few inshore/offshore species (mean = 0.84%), but not within strictly offshore species (mean = 0.26%). Such deep divergences, detected within certain inshore and inshore/offshore taxa, are typical of divergences between congeneric species rather than between populations of a single species, suggesting that current taxonomic systems substantially underestimate species diversity. We estimate that about one third of the 1000 fish species thought to bridge South African and Australian waters actually represent two taxa.

Testing plant barcoding in a sister species complex of pantropical Acacia (Mimosoideae, Fabaceae) - May 01, 2009 ()
[Newmaster, S. G., & Ragupathy, S. 2009. Molecular Ecology Resources,. 9(s1) 172-180.]

Acacia species are quite difficult to differentiate using morphological characters. Routine identification of Acacia samples is important in order to distinguish invasive species from rare species or those of economic importance, particularly in the forest industry. The genus Acacia is quite abundant and diverse comprising approximately 1355 species, which is currently divided into three subgenera: subg. Acacia (c. 161 species), subg. Aculiferum (c. 235 species), and subg. Phyllodineae (c. 960 species). It would be prudent to utilize DNA barcoding in the accurate and efficient identification of acacias. The objective of this research is to test barcoding in discriminating multiple populations among a sister-species complex in pantropical Acacia subg. Acacia, across three continents. Based on previous research, we chose three cpDNA regions (rbcL, trnH-psbA and matK). Our results show that all three regions (rbcL, matK and trnH-psbA) can distinguish and support the newly proposed genera of Vachellia Wight & Arn. from Acacia Mill., discriminate sister species within either genera and differentiate biogeographical patterns among populations from India, Africa and Australia. A morphometric analysis confirmed the cryptic nature of these sister species and the limitations of a classification based on phenetic data. These results support the claim that DNA barcoding is a powerful tool for taxonomy and biogeography with utility for identifying cryptic species, biogeograhic patterns and resolving classifications at the rank of genera and species.

DNA barcoding a regional bee (Hymenoptera: Apoidea) fauna and its potential for ecological studies - May 01, 2009 ()
[Sheffield, C. S., Hebert, P. D. N., Kevan, P. G., & Packer, L. 2009. Molecular Ecology Resources. 9(s1) 196-207.]

DNA barcoding has been evaluated for many animal taxa and is now advocated as a reliable and rapid means for species-level identification. The coming-to-light of this identification tool is timely as we are now facing perhaps the greatest rate of species loss in recent millennia. This study contributes to an ever-increasing number of published accounts of DNA barcoding successfully and accurately distinguishing animal taxa, in this instance, the bee fauna of Nova Scotia, Canada. Most members of this well-known fauna were resolved with particular clarity; the average intraspecific divergence was less than 0.5%, and COI sequences from over 75% of the province's species are now in the Barcodes of Life Data System. DNA barcoding also revealed some surprises within this fauna, including the possible recognition of two undescribed genetically unique species, one in the genus Ceratina (subgenus Zadontomerus), the second in the genus Andrena (subgenus Larandrena); both are presently receiving further taxonomic study. In addition, DNA barcoding has allowed sex-associations among two pairs of cleptoparasitic species. The resulting utility of DNA barcoding for ecological studies of bee communities is discussed.

Identification of Nearctic black flies using DNA barcodes (Diptera: Simuliidae) - May 01, 2009 ()
[Rivera, J., & Currie, D. C. 2009. Molecular Ecology Resources. 9(s1) 224-236.]

DNA barcoding has gained increased recognition as a molecular tool for species identification in various groups of organisms. In this preliminary study, we tested the efficacy of a 615-bp fragment of the cytochrome c oxidase I (COI) as a DNA barcode in the medically important family Simuliidae, or black flies. A total of 65 (25%) morphologically distinct species and sibling species in species complexes of the 255 recognized Nearctic black fly species were used to create a preliminary barcode profile for the family. Genetic divergence among congeners averaged 14.93% (range 2.83201315.33%), whereas intraspecific genetic divergence between morphologically distinct species averaged 0.72% (range 020133.84%). DNA barcodes correctly identified nearly 100% of the morphologically distinct species (87% of the total sampled taxa), whereas in species complexes (13% of the sampled taxa) maximum values of divergence were comparatively higher (max. 4.5820136.5%), indicating cryptic diversity. The existence of sibling species in Prosimulium travisi and P. neomacropyga was also demonstrated, thus confirming previous cytological evidence about the existence of such cryptic diversity in these two taxa. We conclude that DNA barcoding is an effective method for species identification and discovery of cryptic diversity in black flies.

DNA barcode accumulation curves for understudied taxa and areas - May 01, 2009 ()
[Smith, M. A., Fernandez-Triana, J., Roughley, R., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(s1) 208-216.]

Frequently, the diversity of umbrella taxa is invoked to predict patterns of other, less well-known, life. However, the utility of this strategy has been questioned. We tested whether a phylogenetic diversity (PD) analysis of CO1 DNA barcodes could act as a proxy for standard methods of determining sampling efficiency within and between sites, namely that an accumulation curve of barcode diversity would be similar to curves generated using morphology or nuclear genetic markers. Using taxa at the forefront of the taxonomic impediment 2014 parasitoid wasps (Ichneumonidae, Braconidae, Cynipidae and Diapriidae), contrasted with a taxon expected to be of low diversity (Formicidae) from an area where total diversity is expected to be low (Churchill, Manitoba), we found that barcode accumulation curves based on PD were significantly different in both slope and scale from curves generated using names based on morphological data, while curves generated using nuclear genetic data were only different in scale. We conclude that these differences clearly identify the taxonomic impediment within the strictly morphological alpha-taxonomy of these hyperdiverse insects. The absence of an asymptote within the barcode PD trend of parasitoid wasps reflects the as yet incomplete sampling of the site (and more accurately its total diversity), while the morphological analysis asymptote represents a collision with the taxonomic impediment rather than complete sampling. We conclude that a PD analysis of standardized DNA barcodes can be a transparent and reproducible triage tool for the management and conservation of species and spaces.

http://www3.interscience.wiley.com/cgi-bin/fulltext/122342777/HTMLSTART

DNA barcoding of marine crustaceans from the Estuary and Gulf of St Lawrence: a regional-scale approach - May 01, 2009 ()
[Radulovici, A. E., Sainte-Marie, B., & Dufresne, F. 2009. Molecular Ecology Resources. 9(s1) 181-187.]

Marine crustaceans are known as a group with a high level of morphological and ecological diversity but are difficult to identify by traditional approaches and usually require the help of highly trained taxonomists. A faster identification method, DNA barcoding, was found to be an effective tool for species identification in many metazoan groups including some crustaceans. Here we expand the DNA barcode database with a case study involving 80 malacostracan species from the Estuary and Gulf of St Lawrence. DNA sequences for 460 specimens grouped into clusters corresponding to known morphological species in 95% of cases. Genetic distances between species were on average 25 times higher than within species. Intraspecific divergence was high (3.78201313.6%) in specimens belonging to four morphological species, suggesting the occurrence of cryptic species. Moreover, we detected the presence of an invasive amphipod species in the St Lawrence Estuary. This study reconfirms the usefulness of DNA barcoding for the identification of marine crustaceans.

Combining DNA barcoding and morphological analysis to identify specialist floral parasites (Lepidoptera: Coleophoridae: Momphinae: Mompha) - May 01, 2009 ()
[Emery, V. J., Landry, J.-F., & Eckert, C. G. 2009. Molecular Ecology Resources. 9(s1) 217-223.]

Close interactions between insects and plants have played a major role in the evolution of both these diverse groups of organisms. Studying these interactions, however, can be difficult because many insects, especially parasites, impinge most strongly on plants during larval stages when they are morphologically difficult to identify, and many belong to diverse groups for which most species remain undescribed. We used DNA barcoding to identify nondescript lepidopteran larvae that regularly parasitize flower buds of the coastal dune endemic Camissoniopsis cheiranthifolia (Onagraceae). We obtained cytochrome oxidase 1 mitochondrial DNA sequences from 201 parasite specimens from across the host geographical range. The Barcode of Life Database Identification System combined with Bayesian analysis grouped all 15 parasite haplotypes in a distinct, monophyletic clade within the genus Mompha (Lepidoptera: Coleophoridae: Momphinae), a group known to be host specialists on plants of the Onagraceae. Species identity and phylogenetic affinities within Mompha could not be confirmed because few barcode sequences exist from this diverse and poorly known group of moths. However, morphological analysis, including detailed dissection of genitalia for a subsample of 23 reared adults and comparison with known species of Mompha, also indicated that the larvae parasitizing C. cheiranthifolia constitute a distinct and undescribed species within this genus. Knowing that floral parasitism of C. cheiranthifolia involves a single, putatively host-specific microlepidopteran greatly facilitates formulating and testing hypotheses concerning how floral parasitism has promoted the evolution of striking floral diversity within this species. More generally, DNA barcoding combined with morphological analysis can greatly hasten identification of problematic specimens and enhance our understanding of the diversity, ecology and evolution of plant2013insect interactions.

Molecular Ecology Resources: Special Issue on Barcoding Life - Apr 23, 2009 ()
[http://www3.interscience.wiley.com/journal/122342767/issue 2009. Molecular Ecology Resources. Volume 9 Issue S1 1-268.]

The Canadian Barcode of Life Network has made a substantial contribution to the literature on DNA barcoding with the new release of  a  special issue of Molecular Ecology Resources that is entirely dedicated to barcoding. This volume stems from our Network's  Scientific Symposium held at the ROM last spring and represents a major milestone for our national network.

This collection of 27 papers is accessible online: http://www3.interscience.wiley.com/journal/122342767/issue

A botanical renaissance: state-of-the-art DNA bar coding facilitates an Automated Identification Technology system for plants - Apr 01, 2009 ()
[Newmaster, S. G., Ragupathy, S., & Janovec, J. 2009. International Journal of Computer Applications in Technology. 35(1) 50-60.]

Traditional taxonomic practices are insufficient on their own to cope with the growing need for accurate identifications. The recent development of DNA barcoding has been applied to plants. The next step is the development of a high-throughput Automated Identification Technology (AIT) system. Our research indicates that the efficacy of an AIT system equates with savings in time and funding. Given the potential interconnectivity of web-based applications, we suggest an AIT system for plants that uses several existing systems and suggest several applications where AIT could serve as a tool for biologists and for society at large.

Public Health Response to Puffer Fish (Tetrodotoxin) Poisoning from Mislabeled Product - Apr 01, 2009 ()
[Cohen, N. J., Deeds, J. R., Wong, E. S., Hanner, R. J., Yancy, H. F., White, K. D., Thompson, T. M., Wahl, M., Pham, T. D., Guichard, F. M., Huh, I., Austion, C., Dizikes, G., & Gerber, S. I. 2009. Journal of Food Protection. 72(4) 810-817.]

Tetrodotoxin is a neurotoxin that occurs in select species of the family Tetraodontidae (puffer fish). It causes paralysis and potentially death if ingested in sufficient quantities. In 2007, two individuals developed symptoms consistent with tetrodotoxin poisoning after ingesting home-cooked puffer fish purchased in Chicago. Both the Chicago retailer and the California supplier denied having sold or imported puffer fish but claimed the product was monkfish. However, genetic analysis and visual inspection determined that the ingested fish and others from the implicated lot retrieved from the supplier belonged to the family Tetraodontidae. Tetrodotoxin was detected at high levels in both remnants of the ingested meal and fish retrieved from the implicated lot. The investigation led to a voluntary recall of monkfish distributed by the supplier in three states and placement of the supplier on the U.S. Food and Drug Administration’s Import Alert for species misbranding. This case of tetrodotoxin poisoning highlights the need for continued stringent regulation of puffer fish importation by the U.S. Food and Drug Administration, education of the public regarding the dangers of puffer fish consumption, and raising awareness among medical providers of the diagnosis and management of foodborne toxin ingestions and the need for reporting to public health agencies.

Population genetic structure of the salmon louse, Lepeophtheirus salmonis (Krøyer) on wild and farmed salmonids around the Pacific coast of Canada - Mar 09, 2009 ()
[Boulding, E. G., deWaard, J. R., Ang, K. P., & Hebert, P. D. N. 2009. Aquaculture Research. 40(8) 973-979.]
Integrative taxonomy identifies new (and old) species in the Lasioglossum (Dialictus) tegulare (Robertson) species group (Hymenoptera, Halictidae) - Mar 01, 2009 ()
[Gibbs, J. 2009. Zootaxa. 2032 1-38.]

An integrative taxonomic approach that utilizes the DNA barcode region of cytochrome c oxidase subunit 1 in conjunction with traditional morphological approaches identifies five distinct species previously recognized as Lasioglossum (Dialictus) tegulare (Robertson). Differences in DNA sequences and congruent, albeit minor, morphological variation support separation of L. tegulare into five species. Unique nucleotide substitution patterns for each species allows for character-based diagnostics using DNA barcodes. The names L. ellisiae (Sandhouse) and L. lepidii (Graenicher) are removed from synonymy. Two new species, L. puteulanum Gibbs sp. n. and L. carlinvillense Gibbs sp. n., are described. A key is provided, which permits the identification of both males and females. The utility of the DNA barcode region as part of an integrative taxonomic framework is discussed.

Letters to the Editor - Mar 01, 2009 ()
[Wilson, John J., Floyd, R., Hanner, R. H., & Castle, D. 2009. Isis. 100(1) 117.]

DNA Barcodes and Insect Biodiversity - Mar 01, 2009 ()
[Floyd, R., Wilson, J. J., & Hebert, P. D. N. 2009. In R. G. Foottit & P. H. Adler (Eds.), Insect Biodiversity: Science and Society. Oxford, UK: Blackwell Publishing. 417-431.]

Fungal pathogen (mis-) identifications: A case study with DNA barcodes on Melampsora rusts of aspen and white poplar - Feb 26, 2009 ()
[Feau, N., Vialle, A., Allaire, M., Tanguay, P., Joly, D. L., Frey, P., Callan, B. E., & Hamelin, R. C 2009. Mycological Research. 113(6-7) 713-724.]

Wide variation and overlap in morphological characters have led to confusion in species identification within the fungal rust genus Melampsora. The Melampsora species with uredinial–telial stages on white poplar and aspens are especially prone to misidentification. This group includes the Melampsora populnea species complex and the highly destructive pine twisting rust, Melampsora pinitorqua, which alternates between hosts in Populus section Populus and Pinus. Our objective was to compare morphologically based identification to genetic material extracted from Melampsora species pathogenic to aspen and white poplar. We compared morphometric traits and DNA barcodes obtained from internal transcribed spacer (ITS), large ribosomal RNA subunit (28S), and mitochondrial cytochrome oxidase 1 (CO1) sequences to delimit within this taxonomically difficult group. Eight different Melampsora species were initially defined based on host specificity and morphometric data. DNA barcodes were then overlaid on these initial species definitions. The DNA barcodes, specifically those defined on ITS and 28S sequences, provided a highly accurate means of identifying and resolving Melampsora taxa. We highlighted species misidentification in specimens from Canadian herbaria related to either Melampsora medusae f. sp. tremuloidae or Melampsora aecidioides. Finally, we evidenced that the north-American species found on Populus alba, M. aecidioides is closely related but distinct from the four species of the M. populnea complex (Melampsora larici-tremulae, Melampsora magnusiana, Melampsora pinitorqua, and Melampsora rostrupii) found in Eurasia.

How Many Loci Does it Take to DNA Barcode a Crocus? - Feb 25, 2009 ()
[Seberg, O., & Petersen, G. 2009. PLoS ONE. 4(2) e4598.]

Background

DNA barcoding promises to revolutionize the way taxonomists work, facilitating species identification by using small, standardized portions of the genome as substitutes for morphology. The concept has gained considerable momentum in many animal groups, but the higher plant world has been largely recalcitrant to the effort. In plants, efforts are concentrated on various regions of the plastid genome, but no agreement exists as to what kinds of regions are ideal, though most researchers agree that more than one region is necessary. One reason for this discrepancy is differences in the tests that are used to evaluate the performance of the proposed regions. Most tests have been made in a floristic setting, where the genetic distance and therefore the level of variation of the regions between taxa is large, or in a limited set of congeneric species.

Methodology and Principal Findings

Here we present the first in-depth coverage of a large taxonomic group, all 86 known species (except two doubtful ones) of crocus. Even six average-sized barcode regions do not identify all crocus species. This is currently an unrealistic burden in a barcode context. Whereas most proposed regions work well in a floristic context, the majority will – as is the case in crocus – undoubtedly be less efficient in a taxonomic setting. However, a reasonable but less than perfect level of identification may be reached – even in a taxonomic context.

Conclusions/Significance

The time is ripe for selecting barcode regions in plants, and for prudent examination of their utility. Thus, there is no reason for the plant community to hold back the barcoding effort by continued search for the Holy Grail. We must acknowledge that an emerging system will be far from perfect, fraught with problems and work best in a floristic setting.

Probing Evolutionary Patterns in Neotropical Birds through DNA Barcodes - Feb 05, 2009 ()
[Kerr, K. C. R., Lijtmaer, D. A., Barreira, A. S., Hebert, P. D. N., & Tubaro, P. L. 2009. PLoS ONE. 4(2) e4379.]

Background

The Neotropical avifauna is more diverse than that of any other biogeographic region, but our understanding of patterns of regional divergence is limited. Critical examination of this issue is currently constrained by the limited genetic information available. This study begins to address this gap by assembling a library of mitochondrial COI sequences, or DNA barcodes, for Argentinian birds and comparing their patterns of genetic diversity to those of North American birds.

Methodology and Principal Findings

Five hundred Argentinian species were examined, making this the first major examination of DNA barcodes for South American birds. Our results indicate that most southern Neotropical bird species show deep sequence divergence from their nearest-neighbour, corroborating that the high diversity of this fauna is not based on an elevated incidence of young species radiations. Although species ages appear similar in temperate North and South American avifaunas, patterns of regional divergence are more complex in the Neotropics, suggesting that the high diversity of the Neotropical avifauna has been fueled by greater opportunities for regional divergence. Deep genetic splits were observed in at least 21 species, though distribution patterns of these lineages were variable. The lack of shared polymorphisms in species, even in species with less than 0.5M years of reproductive isolation, further suggests that selective sweeps could regularly excise ancestral mitochondrial polymorphisms.

Conclusions

These findings confirm the efficacy of species delimitation in birds via DNA barcodes, even when tested on a global scale. Further, they demonstrate how large libraries of a standardized gene region provide insight into evolutionary processes.

DNA barcoding for ecologists - Feb 01, 2009 (pdf)
[Valentini, A., Pompanon, F. and P. Taberlet 2009. Trends in Ecology and Evolution. 24(2) 110-117.]

DNA barcoding  taxon identification using a standardized DNA region  has received much attention recently, and is being further developed through an international initiative. We anticipate that DNA barcoding techniques will be increasingly used by ecologists. They will be able to not only identify a single species from a specimen or an organism's remains but also determine the species composition of environmental samples. Short DNA fragments persist in the environment and might allow an assessment of local biodiversity from soil or water. Even DNA-based diet composition can be estimated using fecal samples. Here we review the new avenues offered to ecologists by DNA barcoding, particularly in the context of new sequencing technologies.

The campaign to DNA barcode all fishes, FISH-BOL - Jan 29, 2009 ()
[Ward, R. D., Hanner, R., & Hebert, P. D. N. 2009. Journal of Fish Biology. 74(2) 329-356.]

FISH-BOL, the Fish Barcode of Life campaign, is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes. Analysis is targeting a 648 base pair region of the mitochondrial cytochrome c oxidase I (COI) gene. More than 5000 species have already been DNA barcoded, with an average of five specimens per species, typically vouchers with authoritative identifications. The barcode sequence from any fish, fillet, fin, egg or larva can be matched against these reference sequences using BOLD; the Barcode of Life Data System (http://www.barcodinglife.org). The benefits of barcoding fishes include facilitating species identification, highlighting cases of range expansion for known species, flagging previously overlooked species and enabling identifications where traditional methods cannot be applied. Results thus far indicate that barcodes separate c. 98 and 93% of already described marine and freshwater fish species, respectively. Several specimens with divergent barcode sequences have been confirmed by integrative taxonomic analysis as new species. Past concerns in relation to the use of fish barcoding for species discrimination are discussed. These include hybridization, recent radiations, regional differentiation in barcode sequences and nuclear copies of the barcode region. However, current results indicate these issues are of little concern for the great majority of specimens.

Species identification of North American guinea worms (Nematoda: Dracunculus) with DNA barcoding - Jan 28, 2009 ()
[Elsasser, S. C., Floyd, R., Hebert, P. D. N., & Schulte-Hostedde, A. I. 2009. Molecular Ecology Resources. Online Early .]

Dracunculus insignis is a nematode parasite that infects the subcutaneous tissues of mammals such as raccoon (Procyon lotor), mink (Neovison vison) and fisher (Martes pennanti). D. lutrae, a morphologically similar species, has only been recovered from the otter (Lontra canadensis). Species identification of these two North American guinea worms has only been achieved by morphology of males and host identity. As a result, where only female specimens are present, accurate identifications are not possible. To date, specimens recovered from otter have been assumed to be D. lutrae, while those from all other hosts are assumed to be D. insignis. This study uses DNA barcoding to differentiate between these two North American dracunculoids. Our results show that D. insignis is a 'true' generalist, showing little sequence divergence regardless of host association, although our studies did validate its occurrence in a new host 2014 the otter. Interestingly, specimens of the host specialist, D. lutrae, showed some sequence divergence, although it was low. The finding of D. insignis in otter substantiates the need to supplement morphology-based methods in providing species identifications for certain dracunculoids.

On the Morphology and Mitochondrial DNA Barcoding of the Flesh Fly Sarcophaga (Liopygia) Argyrostoma (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) — An Important Species in Forensic Entomology - Jan 18, 2009 ()
[Draber-Mo&#324;ko, A., Malewski, T., Pomorski, J., &#321;o&#347;, M., & &#346;lipi&#324;ski, P. 2009. Annales Zoologici. 59(4) 465-493.]

Descriptions of the developmental stages of Sarcophaga (Liopygia) argyrostoma (R.-D.) are given. Scanning electron microscope images of most of its immature stages are presented for the first time. The sequence of mitochondrial cytochrome c oxidase subunit I (COI) gene fulfilling DNA barcoding standards is presented for the first time.

Identification of shark and ray fins using DNA barcoding - Jan 14, 2009 ()
[Holmes, B. H., Steinke, D., & Ward, R. D. 2009. Fisheries Research. 95(2-3) 280-288.]

Fisheries managers and scientists worldwide are struggling with a lack of basic information for many shark and ray species. One factor hampering the data collection is inaccurate identification of many chondrichthyan species and their body parts. Morphologically similar species, and specimens which are poorly preserved or have had key diagnostic features removed, can be difficult to identify. This study examined DNA barcoding as a method to identify shark species from dried fins, confiscated from a vessel fishing illegally in Australian waters. 211 left pectoral fins were examined. 18 either did not provide a sequenceable product or yielded a microbial sequence, while 193 fins (91.5%) provided a chondrichthyan sequence. All of these could be matched to reference specimens in a DNA barcode database, and so were able to be identified. 27 species were detected, 20 species of sharks and seven species of rays The most abundant species (22% of fins) was Carcharhinus dussumieri. Many of these species are listed on the World Conservation Union (IUCN) Red List and include one, Anoyxpristis cuspidata (3%), rated as critically endangered. Fishing authorities can use DNA barcoding to gather data on which chondrichthyan species are targeted by illegal fishers, information that will greatly assist in management and conservation.

Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda) - Jan 07, 2009 ()
[Ferri, E., Barbuto, M., Bain, O., Galimberti, A., Uni, S., Guerrero, R. A., Ferte, H., Bandi, C., Martin, C., & Casiraghi, M. 2009. Front Zool. 6(1) 1.]

ABSTRACT: BACKGROUND: We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (coxI and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms. RESULTS: DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species. CONCLUSIONS: An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.

Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes - Jan 01, 2009 ()
[Valdez-Moreno, M., Ivanova, N. V., Elías-Gutiérrez, M., Contreras-Balderas, S., & Hebert, P. D. N. 2009. Journal of Fish Biology. 74(2) 377-402.]

The freshwater fish fauna of Mexico and Guatemala is exceptionally diverse with &gt;600 species, many endemic. In this study, patterns of sequence divergence were analysed in representatives of this fauna using cytochrome c oxidase subunit 1 (COI) DNA barcodes for 61 species in 36 genera. The average divergence among conspecific individuals was 0·45%, while congeneric taxa showed 5·1% divergence. Three species of Poblana, each occupying a different crater lake in the arid regions of Central Mexico, have had a controversial taxonomic history but are usually regarded as endemics to a single lake. They possess identical COI barcodes, suggesting a very recent history of isolation. Representatives of the Cichlidae, a complex and poorly understood family, were well discriminated by barcodes. Many species of Characidae seem to be young, with low divergence values (&lt;2%), but nevertheless, clear barcode clusters were apparent in the Bramocharax2013Astyanax complex. The symbranchid, Opisthernon aenigmaticum, has been regarded as a single species ranging from Guatemala to Mexico, but it includes two deeply divergent barcode lineages, one a possible new endemic species. Aside from these special cases, the results confirm that DNA barcodes will be highly effective in discriminating freshwater fishes from Central America and that a comprehensive analysis will provide new important insights for understanding diversity of this fauna.

Rapid Range Expansion of the Wool-Carder Bee, Anthidium manicatum (Linnaeus) (Hymenoptera: Megachilidae), in North America - Jan 01, 2009 ()
[Gibbs, J. & Sheffield, C. S. 2009. Journal of the Kansas Entomological Society. 82(1) 21-29.]

Anthidium manicatum (L.) is an adventive species of European origin first recorded in North America in the late 1960’s; from that point until 2001 its range on the continent was restricted to the northeast central USA and central Canada (Ontario, more recently Que´bec). In 2005, this species was reported from Nova Scotia, a rapid and wide increase in its distribution. In this paper, we document a similar rapid spread of A. manicatum into western North America, including British Columbia and Idaho, and discuss the potential risks of this species in eastern Canada. In addition, the potential of DNA barcoding as a rapid and reliable means of recognizing adventive bee species is advocated.

The Use of Mean Instead of Smallest Interspecific Distances Exaggerates the Size of the "Barcoding Gap" and Leads to Misidentification - Jan 01, 2009 ()
[Meier, R., Zhang, G., & Ali, F. 2009. Systematic Biology. 57(5) 809-813.]

Uncorrected nucleotide bias in mtDNA can mimic the effects of positive Darwinian selection - Dec 01, 2008 ()
[Albu, M., Min, X. J., Hickey, D., & Golding, B. 2008. Mol Biol Evol. 25(12) 2521-2524.]

The relative rates of nucleotide substitution at synonymous and nonsynonymous sites within protein-coding regions have been widely used to infer the action of natural selection from comparative sequence data. It is known, however, that mutational and repair biases can affect rates of evolution at both synonymous and nonsynonymous sites. More importantly, it is also known that synonymous sites are particularly prone to the effects of nucleotide bias. This means that nucleotide biases may affect the calculated ratio of substitution rates at synonymous and nonsynonymous sites. Using a large data set of animal mitochondrial sequences, we demonstrate that this is, in fact, the case. Highly biased nucleotide sequences are characterized by significantly elevated dN/dS ratios, but only when the nucleotide frequencies are not taken into account. When the analysis is repeated taking the nucleotide frequencies at each codon position into account, such elevated ratios disappear. These results suggest that the recently reported differences in dN/dS ratios between vertebrate and invertebrate mitochondrial sequences could be explained by variations in mitochondrial nucleotide frequencies rather than the effects of positive Darwinian selection.

Integrating DNA barcoding into the mycological sciences - Dec 01, 2008 ()
[Seifert, K. A. 2008. Persoonia. 21 162-166.]
Phylogenetic analysis of freshwater sponges provide evidence for endemism and radiation in ancient lakes - Dec 01, 2008 ()
[Meixner, M. J., Luter, C., Eckert, C., Itskovich, V., Janussen, D., von Rintelen, T., Bohne, A. V., Meixner, J. M., and Hess, W. R. 2008. Mol Phylogenet Evol. 45(3) 875-886.]

Morphologic and phylogenetic analysis of freshwater sponges endemic to lakes in Central Sulawesi, Siberia and South-East Europe is presented. We also analyzed several cosmopolitan sponge species from Eurasia and North America and included sponge sequences from public databases. In agreement with previous reports [Addis, J.S., Peterson, K.J., 2005. Phylogenetic relationships of freshwater sponges (Porifera, Spongillina) inferred from analyses of 18S rDNA, COI mtDNA, and ITS2 rDNA sequences. Zool. Scr. 34, 549-557], the metaniid sponge Corvomeyenia sp. was the most deeply branching species within a monophyletic lineage of the suborder Spongillina. Pachydictyum globosum (Malawispongiidae) and Nudospongilla vasta (Spongillidae), two morphologically quite distinct species from Sulawesi were found in a joint clade with Trochospongilla (Spongillidae) rendering Trochospongilla paraphyletic. Furthermore, Ochridaspongia sp., another Malawispongiidae, clustered far away from that clade, together with Ephydatia fluviatilis, making the latter family polyphyletic. The Lubomirskiidae endemic to Lake Baikal, Lubomirskia abietina, Baikalospongia bacillifera, B. intermedia, and Swartschewskia papyracea formed a well-supported clade that was most closely linked to the genus Ephydatia (99.9% identity over a total length of 2169 concatenated nucleotide positions). Our study indicates the frequent and independent origin of sponge species endemic to different freshwater ecosystems from a few cosmopolitan founder species. The highly specific primer sets newly developed here facilitate work on the molecular phylogeny and DNA barcoding of sponges.

DNA Barcoding of Lepetodrilus Limpets Reveals Cryptic Species - Dec 01, 2008 ()
[Johnson, S. B., Warén, A., & Vrijenhoek, R. C. 2008. Journal of Shellfish Research. 27(1) 43-51.]

Lepetodrilid limpets are common inhabitants of deep-sea hydrothermal vents worldwide, but the frequent occurrence of morphologically cryptic species makes their identification very difficult. To facilitate these identifications, we provide DNA barcodes based on 1,000 bp of cytochrome-c-oxidase subunit I (COI), for 20 taxa within the genus Lepetodrilus. The method was also used to identify lepetodrilids that were found living on vent decapods. A preliminary phylogenetic analysis resolved relationships among members of several cryptic species complexes; however, COI sequences alone were unable to resolve higher-level systematic relationships caused by saturation of synonymous nucleotide substitutions.

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes - Dec 01, 2008 ()
[Chantangsi, C., & Lynn, D. H. 2008. Mol Phylogenet Evol. 49(3) 979-987.]

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three "classical" morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.

Descriptions of two new species of Hemileucinae (Lepidoptera: Saturniidae) from the region of Muzo in Colombia—evidence from morphology and DNA barcodes - Nov 01, 2008 ()
[Decaens, T. & R. Rougerie 2008. Zootaxa. 1944 34-52.]

Two new species of Hemileucinae are described from the region of Muzo (Boyaca department) in the Eastern Cordillera of Colombia. Leucanella bonillensis, new species, is a small greyish species whose closest relatives are L. newmani (Lemaire) and L. acutissima (Walker). It can be distinguished from those two species by several subtle differences in wing pattern and coloration as well as a few characters of the male genitalia, which are overall very conserved within the genus. Cerodirphia zulemae, new species, belongs to the very uniform species-group of C. speciosa (Cramer), characterised by a pink ground colour and the presence of a “Y”-shaped discal mark on the forewing. Based on its male genitalia, the new species is related to C. brunnea (Draudt) and C. apunctata Dias & Lemaire. It may be distinguished from the former by its more vivid ground colour, but detailed examination of the male genitalia are necessary to differentiate it from C. apunctata. Colour pictures of the habitus of the new species and their relatives are provided, and their genital structures are figured as well, including both sexes for C. zulemae. We also provide additional support to these descriptions based on genetic data obtained in the context of a global DNA barcoding campaign recently initiated for saturniid moths. Both L. bonillensis and C. zulemae are unambiguously distinguished from closest relatives based on genetic distances (no intraspecific distances in either case; interspecific distance ranges 5.6–6.6% and 6.7–12.5%, respectively) and inference of phylogenetic hypotheses based on partial sequences of the COI mitochondrial gene. These results emphasize the potential of DNA barcoding to support taxonomic work in species-groups considered difficult to address through morphology.

Analysis of the Pythium ultimum transcriptome using Sanger and Pyrosequencing approaches - Nov 01, 2008 ()
[Cheung, F., Win, J., Lang, J.M., Hamilton, J., Vuong, H., Leach, J.E., Kamoun, S., Lévesque, C.A., Tisserat, N., and Buell, C.R. 2008. BMC Genomics. 9 542.]

BACKGROUND: Pythium species are an agriculturally important genus of plant pathogens, yet are not understood well at the molecular, genetic, or genomic level. They are closely related to other oomycete plant pathogens such as Phytophthora species and are ubiquitous in their geographic distribution and host rage. To gain a better understanding of its gene complement, we generated Expressed Sequence Tags (ESTs) from the transcriptome of Pythium ultimum DAOM BR144 (= ATCC 200006 = CBS 805.95) using two high throughput sequencing methods, Sanger-based chain termination sequencing and pyrosequencing-based sequencing-by-synthesis. RESULTS: A single half-plate pyrosequencing (454 FLX) run on adapter-ligated cDNA from a normalized cDNA population generated 90,664 reads with an average read length of 190 nucleotides following cleaning and removal of sequences shorter than 100 base pairs. After clustering and assembly, a total of 35,507 unique sequences were generated. In parallel, 9,578 reads were generated from a library constructed from the same normalized cDNA population using dideoxy chain termination Sanger sequencing, which upon clustering and assembly generated 4,689 unique sequences. A hybrid assembly of both Sanger- and pyrosequencing-derived ESTs resulted in 34,495 unique sequences with 1,110 sequences (3.2%) that were solely derived from Sanger sequencing alone. A high degree of similarity was seen between P. ultimum sequences and other sequenced plant pathogenic oomycetes with 91% of the hybrid assembly derived sequences > 500 bp having similarity to sequences from plant pathogenic Phytophthora species. An analysis of Gene Ontology assignments revealed a similar representation of molecular function ontologies in the hybrid assembly in comparison to the predicted proteomes of three Phytophthora species, suggesting a broad representation of the P. ultimum transcriptome was present in the normalized cDNA population. P. ultimum sequences with similarity to oomycete RXLR and Crinkler effectors, Kazal-like and cystatin-like protease inhibitors, and elicitins were identified. Sequences with similarity to thiamine biosynthesis enzymes that are lacking in the genome sequences of three Phytophthora species and one downy mildew were identified and could serve as useful phylogenetic markers. Furthermore, we identified 179 candidate simple sequence repeats that can be used for genotyping strains of P. ultimum. CONCLUSION: Through these two technologies, we were able to generate a robust set (approximately 10 Mb) of transcribed sequences for P. ultimum. We were able to identify known sequences present in oomycetes as well as identify novel sequences. An ample number of candidate polymorphic markers were identified in the dataset providing resources for phylogenetic and diagnostic marker development for this species. On a technical level, in spite of the depth possible with 454 FLX platform, the Sanger and pyro-based sequencing methodologies were complementary as each method generated sequences unique to each platform.

Morphological and molecular evidence for a new species of longnose skate (Rajiformes: Rajidae: Dipturus) from Argentinean waters based on DNA barcoding - Nov 01, 2008 ()
[De Astarloa, J. M. D., Mabragana, E., Hanner, R., & Figueroa, D. E 2008. Zootaxa. 1921 35-46.]

A new species of Dipturus is described from ten specimens collected off Patagonia, Argentina. Morphological and molecular approaches were used to compare among specimens of recognized Dipturus species. By comparing morphometric, meristic and mitochondrial cytochrome c oxidase I (COI) sequence data, specimens referred to as longnose skate and originally regarded as D. chilensis were shown to be a discrete species as distinguished from both the Yellownose skate, D. chilensis and the Roughskin skate, D. trachyderma. Dipturus argentinensis n. sp. can be distinguished from all other southwestern Atlantic longnose skate species by its color pattern, lack of squamation on both upper and lower surfaces of the disc, and a long, thin tail that is approximately half the total length. The new species has one median row of 10 to 24 small caudal thorns, one or two interdorsal thorns and 35 to 40, and 34 to 43 tooth rows on upper and lower jaws, respectively. The 648 base pair COI mitochondrial DNA “barcodes” derived from specimens of D. argentinensis are identical to each other and exhibit greater than 3% sequence divergence from all other Dipturus species similarly characterized to date. Taken together, these independent morphological and molecular observations serve to corroborate one another and thus provide strong evidence for the recognition of D. argentinensis as a new species

Morphology and DNA barcoding reveal three cryptic species within the Xylophanes neoptolemus and loelia species-groups (Lepidoptera: Sphingidae) - Nov 01, 2008 ()
[Vaglia, T. Haxaire, J. Kitching, I. J. Meusnier, I. & Rougerie, R 2008. Zootaxa. 1923 18-36.]

Two species complexes within the genus Xylophanes are addressed using a combination of morphological study and analysis of DNA barcode sequences. The existence of two and three cryptic species respectively within the X. loelia and X. neoptolemus complexes is revealed following consideration of both adult habitus and genital morphology, and the results of a phylogenetic analysis of partial COI sequences—DNA barcodes—for 38 specimens. The taxonomic status of the available names is discussed and to clarify and stabilize the confused nomenclature of this group, a neotype for Sphinx neoptolemus Cramer, 1780, and lectotypes for Choerocampa loelia Druce, 1878 and Chaerocampa trilineata Walker, [1865], are designated. We describe three new species: X. lolita n. sp. Vaglia and Haxaire; X. balcazari n. sp. Haxaire and Vaglia; and X. cthulhu n. sp. Haxaire and Vaglia. The first is endemic to southeastern Brazil and closely allied to X. loelia; the second two are relatives of X. neoptolemus, of which the first is known only from Guerrero and Michoacán states in Mexico while the second is widely distributed in lowland forests of Central America.

Species identification of aphids (Insecta: Hemiptera: Aphididae) through DNA barcodes - Nov 01, 2008 ()
[Foottit, R. G., Maw, H. E. L., Von Dohlen, C. D., & Hebert, P. D. N. 2008. Molecular Ecology Resources. 8(6) 1189-1201.]

A 658-bp fragment of mitochondrial DNA from the 5' region of the mitochondrial cytochrome c oxidase 1 (COI) gene has been adopted as the standard DNA barcode region for animal life. In this study, we test its effectiveness in the discrimination of over 300 species of aphids from more than 130 genera. Most (96%) species were well differentiated, and sequence variation within species was low, averaging just 0.2%. Despite the complex life cycles and parthenogenetic reproduction of aphids, DNA barcodes are an effective tool for identification.

Phylogeography and genetic diversity of a widespread Old World butterfly, Lampides boeticus (Lepidoptera: Lycaenidae) - Oct 30, 2008 (pdf)
[David J. Lohman, Djunijanti Peggi, Naomi E. Pierce, Rudolf Meier 2008. BioMed Central Evolutionary Biology. 8: 301 .]

DNA barcoding detects market substitution in North American seafood - Oct 01, 2008 (pdf)
[Wong, E.H.-K. & Hanner, H. R. 2008. Food Research International. 41(8) 828-837.]

Seafood authentication and food safety concerns are a growing issue in today’s global marketplace, although traditional morphology-based identification keys and existing molecular approaches have limitations for species identification. Recently, DNA barcoding has gained support as a rapid, cost-effective and broadly applicable molecular diagnostic technique for this purpose. However, the maturity of the barcode database as a tool for seafood authentication has yet to be tested using real market samples. The present case study was undertaken for this reason. Though the database is undergoing continual development, it was able to provide species matches of >97% sequence similarity for 90 of 91 samples tested. Twenty-five percent of the samples were potentially mislabeled, demonstrating that DNA barcodes are already a powerful tool for the identification of seafood to the species level. We conclude that barcodes have broad applicability for authenticity testing and the phylogeographic patterning of genetic diversity can also inform aspects of traceability.

Rapid high-quality imaging of fishes using a flat-bed scanner - Oct 01, 2008 (pdf)
[Steinke, D., Hanner, R., & Hebert, P. D. N. 2008. Ichthyological Research. Online Early .]

Development of COS genes as universally amplifiable markers for phylogenetic reconstructions of closely related plant species - Oct 01, 2008 ()
[Li, M., Wunder, J., Bissoli, G., Scarponi, E., Gazzani, S., Barbaro, E., Saedler, H., and C. Varotto 2008. Cladistics. 24(5) 727-745.]

With the aim of developing widely applicable gene markers for phylogenetic reconstructions at low taxonomic level, we tested the low copy nuclear Conserved Ortholog Set (COS) genes. Most of the 15 genes tested provided good amplification efficiency (as compared with rbcL) from a set of 67 representative angiosperm families. Nine selected COS markers were further characterized at both intra- and interfamilial level on a test set, including 25 species representative of 15 different families. While four of the COS led to incongruent results, the remaining five improved the phylogenetic reconstructions of closely related species as illustrated in the case of Orobanchaceae species. They were found to be highly informative in phylogenetic reconstruction of congeneric species, where introns provide a higher proportion of parsimony informative sites in comparison with traditional phylogenetic markers such as ITS and matK. At higher phylogenetic distance, where only coding regions could be aligned, the polymorphism levels of the COS ranged between those of ndhF and matK. On the basis of these results, the success rate in developing universally amplifiable low copy nuclear markers based on COS genes is about 30%. We report the successful development of five pCOS that, together with a few other well characterized genes, such as Rpb2 and GbssI, can be considered the closest approximation to low-copy "universally" amplifiable markers for phylogeny in plants at present. The possible pitfalls of universally amplifiable COS marker development and their range of applicability at different taxonomic levels in comparison with traditional phylogenetic molecular markers are discussed. © The Willi Hennig Society 2008.

Molecular analysis of Southern Ocean skates (Bathyraja) reveals a new species of Antarctic skate - Oct 01, 2008 ()
[Smith, P. J., Steinke, D., Mcveagh, S. M., Stewart, A. L., Struthers, C. D., & Roberts, C. D. 2008. Journal of Fish Biology. 73(5) 1170-1182.]

Two regions of mtDNA, cytochrome b and cytochrome c oxidase subunit 1, were sequenced in nine species of Bathyraja from the Southern Ocean and New Zealand. Based on sequence divergence, the species that has been referred to as Bathyraja eatonii from the Antarctic continental shelf and slope is a species distinct from B. eatonii from the Kerguelen Plateau (the type locality) and is a new and undescribed species Bathyraja sp. (cf. eatonii). There was no sequence divergence among samples of Bathyraja sp. (dwarf) from the Ross Sea and the South Atlantic. However, for both Bathyraja sp. (cf. eatonii) and Bathyraja maccaini in the Ross Sea and the South Atlantic Ocean, the DNA sequence divergences indicate differentiation among ocean basins and within Bathyraja sp. (cf. eatonii) divergences are similar to those among recognized species of Bathyraja in the North Pacific Ocean.

Tripogon cope (Poaceae: Chloridoideae), a New Species Supported by Morphometric Analysis and a Synopsis of Tripogon in India - Oct 01, 2008 ()
[Newmaster, S. G., Balasubramaniam, V., Murugesan, M. & Ragupathy, S. 2008. Systematic Botany. 33(4) 695-701.]

Tripogon cope Newmaster S. G., V. Balalasubramaniam, M. Murugesan, & S. Ragupathy a new species from South India, is described and illustrated. A key for the identification of all Indian Tripogon species is included. A detrended correspondence analysis identified 21 groups of taxa including the sp. novum from the 48 samples, analyzing 36 morphological characters. A discriminant function analysis was used to rigorously test the classification of specimens provided in the cluster analysis. This study provides preliminary evidence of morphometric variation within and among species of Tripogon, which allows further development of hypothesis concerning species boundaries. Discussions concerning ecological data and distribution are presented in the context of conservation initiatives of rare and endemic Tripogon taxa within India.

Bar Code of Life: DNA Tags Help Classify Animals - Oct 01, 2008 (pdf)
[Stoeckle, M. Y. & P. D. N. Hebert 2008. Scientific American. 299(4) 66-71.]

Inspired by commercial barcodes, DNA tags could provide a quick inexpensive way to identify species.

Speciation and DNA barcodes: testing the effects of dispersal on the formation of discrete sequence clusters - Sep 27, 2008 ()
[Papadopoulou, A., Bergsten, J., Fujisawa, T., Monaghan, M. T., Barraclough, T. G., & Vogler, A. P. 2008. Philos Trans R Soc Lond B Biol Sci. 363(1506) 2987-2996.]

Large-scale sequencing of short mtDNA fragments for biodiversity inventories ('DNA barcoding') indicates that sequence variation in animal mtDNA is highly structured and partitioned into discrete genetic clusters that correspond broadly to species-level entities. Here we explore how the migration rate, an important demographic parameter that is directly related to population isolation, might affect variation in the strength of mtDNA clustering among taxa. Patterns of mtDNA variation were investigated in two groups of beetles that both contain lineages occupying habitats predicted to select for different dispersal abilities: predacious diving beetles (Dytiscidae) in the genus Bidessus from lotic and lentic habitats across Europe and darkling beetles (Tenebrionidae) in the genus Eutagenia from sand and other soil types in the Aegean Islands. The degree of genetic clustering was determined using the recently developed 'mixed Yule coalescent' (MYC) model that detects the transition from between-species to within-population branching patterns. Lineages from presumed stable habitats, and therefore displaying lower dispersal ability and migration rates, showed greater levels of mtDNA clustering and geographical subdivision than their close relatives inhabiting ephemeral habitats. Simulations of expected patterns of mtDNA variation under island models showed that MYC clusters are only detected when the migration rates are much lower than the value of Nm=1 typically used to define the threshold for neutral genetic divergence. Therefore, discrete mtDNA clusters provide strong evidence for independently evolving populations or species, but their formation is suppressed even under very low levels of dispersal.

A universal DNA mini-barcode for biodiversity analysis - Sep 17, 2008 (pdf)
[Isabelle Meusnier, Gregory AC Singer, Jean-Francois Landry, Donal A Hickey, Paul DN Hebert and Mehrdad Hajibabaei 2008. BioMed Central. 9:214 .]

Background: The goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed).

Results: We used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens.

Conclusion: In this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.

An aberrant bee of the species Lasioglossum (Dialictus) disparile (Cresson) with brief taxonomic notes on the species - Sep 10, 2008 ()
[Gibbs, J. 2008. Journal of the Kansas Entomological Society. In Press 12pp.]

Molecular evolution of mitochondrial ribosomal DNA in the fungal genus Tricholoma: barcoding implications - Sep 01, 2008 ()
[Mouhamadou, B., Carriconde, F., Gryta, H., Jargeat, P., Manzi, S., & Gardes, M. 2008. Fungal Genet Biol. 45(9) 1219-1226.]

The molecular evolution of the V6 and V9 domains of the mitochondrial SSU-rDNA was investigated to evaluate the use of these sequences for DNA barcodes in the Basidiomycota division. The PCR products from 27 isolates belonging to 11 Tricholoma species were sequenced. Both domains in the isolates belonging to the same species had identical sequences. All the species possess distinctive V9 sequences due to point mutations and insertion/deletion events. Secondary structures revealed that the insertion-deletion events occurred in regions not directly involved in the maintenance of the standard SSU-rRNA structure. The inserted sequences possess conserved motifs that enable their alignment among phylogenetically distant species. Hence, the V9 domain by displaying identical sequences within species, an adequate divergence level, easy amplification, and alignment represents an alternative molecular marker for the Basidiomycota division and opens the way for this sequence to be used as specific molecular markers of the fungal kingdom.

Assembling DNA barcodes: analytical protocols - Sep 01, 2008 ()
[deWaard, J. R. I, vanova, N. V., Hajibabaei, M., and P. D. N. Hebert 2008. Methods in Molecular Biology: Environmental Genetics. C. Martin. Totowa, Humana Press. 275-293.]

Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified - Sep 01, 2008 ()
[Song, H., Buhay, J. E., Whiting, M. F., & Crandall, K. A. 2008. Proc Natl Acad Sci U S A. 105(36) 13486-13491.]

Nuclear mitochondrial pseudogenes (numts) are nonfunctional copies of mtDNA in the nucleus that have been found in major clades of eukaryotic organisms. They can be easily coamplified with orthologous mtDNA by using conserved universal primers; however, this is especially problematic for DNA barcoding, which attempts to characterize all living organisms by using a short fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Here, we study the effect of numts on DNA barcoding based on phylogenetic and barcoding analyses of numt and mtDNA sequences in two divergent lineages of arthropods: grasshoppers and crayfish. Single individuals from both organisms have numts of the COI gene, many of which are highly divergent from orthologous mtDNA sequences, and DNA barcoding analysis incorrectly overestimates the number of unique species based on the standard metric of 3% sequence divergence. Removal of numts based on a careful examination of sequence characteristics, including indels, in-frame stop codons, and nucleotide composition, drastically reduces the incorrect inferences of the number of unique species, but even such rigorous quality control measures fail to identify certain numts. We also show that the distribution of numts is lineage-specific and the presence of numts cannot be known a priori. Whereas DNA barcoding strives for rapid and inexpensive generation of molecular species tags, we demonstrate that the presence of COI numts makes this goal difficult to achieve when numts are prevalent and can introduce serious ambiguity into DNA barcoding.

Effects of transgenic hybrid aspen overexpressing polyphenol oxidase on rhizosphere diversity. - Sep 01, 2008 ()
[Oliver, K. L., Hamelin, R. C., & Hintz, W. E. 2008. Appl Environ Microbiol,. 74(17) 5340-5348.]

This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.

Assigning morphological variants of Fucus (Fucales, Phaeophyceae) in Canadian waters to recognized species using DNA barcoding - Sep 01, 2008 ()
[Kucera, H. and G. W. Saunders 2008. Botany. 86(9) 1065-1079.]

The intertidal brown algal genus Fucus (Phaeophyceae) consists of individuals with a generally dichotomously branched habit. Morphological variability within species, combined with morphological similarity between species, renders field identification difficult. In light of recent taxonomic revisions, which reduced 10 taxa traditionally recognized in Canada to four species, we tested the utility of the DNA barcode (mitochondrial cytochrome oxidase 1, 5′) for assigning individuals to these species. We sequenced the DNA barcode for 125 specimens representing all morphologies recognized. We confirmed our results by sequencing the internal transcribed spacer region for 66 specimens. This is the first study to establish that the DNA barcode successfully assigns different morphologies of brown algae to known species as well as other single-gene molecular markers currently used. Furthermore, the results uncovered substantial phenotypic plasticity in Pacific Fucus distichus, from moss-like fragments embedded in estuarine mud, strap-like morphs on exposed rocky coasts, to “spiralis”-like morphs in the upper intertidal whereas phenotypic expression for this species was more restricted in the Atlantic.

Editorial for Molecular and Cell Science JFB - Sep 01, 2008 ()
[Maclean, N. 2008. Journal of Fish Biology,. 73(5) 1091-1095.]

Widespread decoupling of mtDNA variation and species integrity in Grammia tiger moths (Lepidoptera: Noctuidae). - Sep 01, 2008 ()
[Schmidt, B. C., & Sperling, F. A. H. 2008. Systematic Entomology. 33(4) 613-634.]

We investigate the diversity of the North American tiger moth genus Grammia Rambur (Lepidoptera: Noctuidae) by comparing mitochondrial DNA (mtDNA) 'barcode' fragments of cytochrome oxidase I with non-molecular characters such as morphology, ecology, behaviour and distribution. Mitochondrial DNA genealogy is strikingly at odds with morpho-species taxonomy for most of the 28 sampled species, as haplotypic polyphyly not only is taxonomically widespread, but involves multiple shared haplotypes among two to four species. Morpho-ecological traits show that those species sharing haplotypes are often not closely related. Furthermore, high mtDNA divergences occur within species. Haplotypic variation is highly discordant with species taxonomy, but variation at a continental scale reveals significant geographic structuring of haplogroups, transcending morpho-species boundaries. A nested clade analysis and comparison of non-molecular with mtDNA data indicate that most discordance between mtDNA and taxonomy in Grammia is explained best by taxonomically and geographically widespread ongoing hybridization events resulting in mtDNA introgression. We hypothesize that broad areas of sympatry, interspecifically compatible genitalic structure, and species overlap in pheromone components facilitate hybridization, with disparate interspecies abundances promoting mitochondrial introgression. The molecular evolution of Grammia challenges the view that interspecific gene exchange occurs rarely and is restricted to recently diverged species. These results show the value of mtDNA in detecting cryptic hybridization, while highlighting the inherent dangers of drawing taxonomic conclusions based solely on mtDNA.

Pythium solare sp. nov., a new pathogen of green beans in Spain - Sep 01, 2008 ()
[de Cock, A. W., Levesque, C. A., Melero-Vara, J. M., Serrano, Y., Guirado, M. L., & Gomez, J. 2008. Mycological Research. 112(Pt9) 1115-1121.]

A new disease causing wilt and death of adult plants of Phaseolus vulgaris was discovered in plastic-house crops of southeast Spain in 2004. The causal agent was shown to be a Pythium species with a unique type of oogonium ornamentation different from any of the described species. Zoospores were not observed, but globose or subglobose hyphal swellings, intercalary or terminal, were frequently found. Moreover, the ribosomal ITS region showed a unique sequence, significantly different (>14%) from any other known species of Pythium. This paper describes and illustrates the morphology of the new Pythium species and its pathogenicity to green beans. Its taxonomic position and phylogenetic relationships with other Pythium species are discussed.

Four years of DNA barcoding: Current advances and prospects - Sep 01, 2008 ()
[Frezal, L. & R. Leblois 2008. Infection, Genetics and Evolution. 8(5) 727-36.]

Research using cytochrome c oxidase barcoding techniques on zoological specimens was initiated by Hebert et al. [Hebert, P.D.N., Ratnasingham, S., deWaard, J.R., 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B 270, S96-S99]. By March 2004, the Consortium for the Barcode of Life started to promote the use of a standardized DNA barcoding approach, consisting of identifying a specimen as belonging to a certain animal species based on a single universal marker: the DNA barcode sequence. Over the last 4 years, this approach has become increasingly popular and advances as well as limitations have clearly emerged as increasing amounts of organisms have been studied. Our purpose is to briefly expose DNA Barcode of Life principles, pros and cons, relevance and universality. The initially proposed Barcode of life framework has greatly evolved, giving rise to a flexible description of DNA barcoding and a larger range of applications.

Description and DNA barcoding of three new species of Leohumicola from South Africa and the United States - Aug 27, 2008 ()
[Nguyen, H. D. T. and K. A. Seifert 2008. Persoonia. 21 57-69.]

Three new species of Leohumicola (anamorphic Leotiomycetes) are described using morphological characters and phylogenetic analyses of DNA barcodes. Leohumicola levissima and L. atra were isolated from soils collected after forest fires in Crater Lake National Park, United States. Leohumicola incrustata was isolated from burned fynbos from the Cape of Good Hope Nature Reserve, South Africa. The three species exhibit characteristic Leohumicola morphology but are morphologically distinct based on conidial characters. Two DNA barcode regions, the Internal Transcribed Spacer (ITS) nuclear rDNA region and the cytochrome oxidase subunit I (Cox1) mitochondrial gene, were sequenced. Single gene parsimony, dual-gene parsimony and dual-gene Bayesian inference phylogenetic analyses support L. levissima, L. atra, L. incrustata as distinct phylogenetic species. Both ITS and Cox1 barcodes are effective for the molecular identification of Leohumicola species.

Extreme diversity of tropical parasitoid wasps exposed by iterative integration of natural history, DNA barcoding, morphology, and collections. - Aug 26, 2008 ()
[Smith, M. Alex, Rodriguez, J. J., Whitfield, J. B., Deans, A. R., Janzen, D. H., Hallwachs, W., and Hebert, P. D. N. 2008. PNAS. 105:34 12359-12364.]

We DNA barcoded 2,597 parasitoid wasps belonging to 6 microgastrine braconid genera reared from parapatric tropical dry forest, cloud forest, and rain forest in Area de Conservacio´n Guanacaste (ACG) in northwestern Costa Rica and combined these data with records of caterpillar hosts and morphological analyses. We asked whether barcoding and morphology discover the same provisional species and whether the biological entities revealed by our analysis are congruent with wasp host specificity. Morphological analysis revealed 171 provisional species, but barcoding exposed an additional 142 provisional species; 95% of the total is likely to be undescribed. These 313 provisional species are extraordinarily host specific; more than 90% attack only 1 or 2 species of caterpillars out of more than 3,500 species sampled. The most extreme case of overlooked diversity is the morphospecies Apanteles leucostigmus. This minute black wasp with a distinctive white wing stigma was thought to parasitize 32 species of ACG hesperiid caterpillars, but barcoding revealed 36 provisional species, each attacking one or a very few closely related species of caterpillars. When host records and/or within-ACG distributions suggested that DNA barcoding had missed a species-pair, or when provisional species were separated only by slight differences in their barcodes, we examined nuclear sequences to test hypotheses of presumptive species boundaries and to further probe host specificity. Our iterative process of combining morphological analysis, ecology, and DNA barcoding and reiteratively using specimens maintained in permanent collections has resulted in a much more fine-scaled understanding of parasitoid diversity and host specificity than any one of these elements could have produced on its own.


Full text: http://www.pnas.org/content/105/34/12359.full

DNA barcode information for the sugar cane moth borer Diatraea saccharalis - Aug 14, 2008 ()
[Bravo, J. P. Silva, J. L. Munhoz, R. E. & M. A. Fernandez 2008. Genet Mol Res. 7(3) 741-8.]

We reviewed the use and relevance of barcodes for insect studies and investigated the barcode sequence of Diatraea saccharalis. This sequence has a high level of homology (99%) with the barcode sequence of the Crambidae (Lepidoptera). The sequence data can be used to construct relationships between species, allowing a multidisciplinary approach for taxonomy, which includes morphological, molecular and distribution data, all of which are essential for the understanding of biodiversity. The D. saccharalis barcode is a previously undescribed sequence that could be used to analyze Lepidoptera biology.

DNA barcodes for Cladocera and Copepoda from Mexico and Guatemala, highlights and new discoveries - Aug 01, 2008 ()
[Elias-Gutierrez, M., Jeronimo, F.M., Ivanova, N.V., Valdez-Moreno, M., and P. D. N. Hebert 2008. Zootaxa. 1839 1-42.]

DNA barcoding, based on sequence diversity in the mitochondrial COI gene, has proven an excellent tool for identifying species in many animal groups. Here, we report the first barcode studies for freshwater zooplankton from Mexico and Guatemala and discuss the taxonomic and biological implications of this work. Our studies examined 61 species of Cladocera and 21 of Copepoda, about 40% of the known fauna in this region. Sequence divergences among conspecific individuals of cladocerans and copepods averaged 0.82%and 0.79%, respectively, while sequence divergences among congeneric taxa were on average 15-20 times as high. Barcodes were successful in discriminating all species in our study, but sequences for Mexican Daphnia exilis overlapped with those of D. spinulata from Argentina. Our barcode data revealed evidence of many species overlooked by current classification systems —for example, based on COI genotypes the Diapahanosoma birgei group appears to include 5 species, while Ceriodaphnia cf. rigaudi, Moina cf. micrura, Mastigodiaptomus albuquerquensis and Mastigodiaptomus reidae all include 2–3 taxa. The barcode results support recent taxonomic revisions, such as recognition of the genus Leberis, and the presence of several species in the D. birgei and Chydorus sphaericus complexes. The present results indicate that DNA barcoding will provide powerful new insights into both the incidence of cryptic species and a better understanding of zooplankton distributions, aiding evaluation of the factors influencing competitive outcomes, and the colonization of aquatic environments.

A role for barcoding in the study of African fish diversity and conservation : review article - Aug 01, 2008 ()
[Swartz, E.R., Mwale, M. and R. Hanner 2008. South African Journal of Science. 104(7&8) 293-298.]

Africa has a rich diversity of marine and freshwater fishes, but very little taxonomic expertise or funding to describe it. New approaches to using modern technology, such as DNA barcoding, can facilitate collaboration between field biologists, reference collections and sequencing facilities to speed up the process of species identification and diversity assessments, provided specimen vouchers, tissues, photographs of the specimen and DNA sequences (barcodes) are clearly linked. The FISH-BOL project in Africa aims to establish a collaborative Pan-African regional working group to facilitate barcoding of fish across the continent and the surrounding FAO marine regions. This is being established through existing African biodiversity networks and global biodiversity programmes that are already in place. Barcoding is expected to inform African fisheries management and conservation through more accurate identification of species and their different life-history stages, by speeding up biodiversity assessments. Barcoding is an important development, contributing towards an evolutionary history perspective on which to base Africa's conservation strategies.

DNA barcoding shared fish species from the North Atlantic and Australasia: minimal divergence for most taxa but a likely two species for both Zeus faber (John dory) and Lepidopus caudatus (silver scabbardfish) - Aug 01, 2008 ()
[Ward, R.D., Costa, F.O., Holmes, B.H., & D. Steinke 2008. Aquatic Biology. 3 71-78.]

Fifteen fish species, totalling 149 specimens, were cytochrome c oxidase I sequenced—barcoded—from Northern (Atlantic and Mediterranean) and Southern (Australasian) Hemisphere waters. Thirteen species showed no significant evidence of spatial genetic differentiation for this gene, although small sample sizes reduced statistical power. For marine fish, barcodes collected in one part of a species range are likely to be useful as identifiers in all other parts of its range. Two species did show striking north–south differentiation, with FST values of 0.84 and 0.96 (both p << 0.001). One of these, the silver scabbardfish Lepidopus caudatus, showed 2.75% genetic distance between northern and southern clades. The other, John dory Zeus faber, showed 7.44% differentiation between northern and southern clades. All specimens of these 2 species fell correctly into the northern or southern clade. We suggest that both taxa conceal a currently unrecognised, cryptic species, and recommend further taxonomic and genetic investigation.

An integrative method for delimiting cohesion species: finding the population-species interface in a group of californian trapdoor spiders with extreme genetic divergence and geographic structuring - Aug 01, 2008 ()
[Bond, J. E., & Stockman, A. K 2008. Systematic Biology. 57(4) 628-646.]

Here we present an objective, repeatable approach to delineating species when populations are divergent and highly structured geographically using the Californian trapdoor spider species complex Aptostichus atomarius Simon as a model system. This system is particularly difficult because under strict criteria of geographical concordance coupled with estimates of genetic divergence, an unrealistic number of population lineages would qualify as species (20 to 60). Our novel phylogeographic approach, which is generally applicable but particularly relevant to highly structured systems, uses genealogical exclusivity to establish a topological framework to examine lineages for genetic and ecological exchangeability in an effort to delimit cohesion species. Both qualitative assessments of habitat and niche-based distribution modeling are employed to evaluate selective regime and ecological interchangeability among genetic lineages; adaptive divergence among populations is weighted more heavily than simple geographical concordance. Based on these analyses we conclude that five cohesion species should be recognized, three of which are new to science.

Molecular phylogenetic and scanning electron microscopical analyses places the Choanephoraceae and the Gilbertellaceae in a monophyletic group within the Mucorales (Zygomycetes, Fungi) - Jul 31, 2008 ()
[Voigt, K. and Olsson, L. 2008. Acta Biologica Hungarica. 59(3) .]

A multi-gene genealogy based on maximum parsimony and distance analyses of the exonic genes for actin (act) and translation elongation factor 1 alpha (tef), the nuclear genes for the small (18S) and large (28S) subunit ribosomal RNA (comprising 807, 1092, 1863, 389 characters, respectively) of all 50 genera of the Mucorales (Zygomycetes) suggests that the Choanephoraceae is a monophyletic group. The monotypic Gilbertellaceae appears in close phylogenetic relatedness to the Choanephoraceae. The monophyly of the Choanephoraceae has moderate to strong support (bootstrap proportions 67 % and 96 % in distance and maximum parsimony analyses, respectively), whereas the monophyly of the Choanephoraceae-Gilbertellaceae clade is supported by high bootstrap values (100 % and 98 %). This suggests that the two families can be joined into one family, which leads to the elimination of the Gilbertellaceae as a separate family. In order to test this hypothesis single-locus neighbor-joining analyses were performed on nuclear genes of the 18S, 5.8S, 28S and internal transcribed spacer (ITS) 1 ribosomal RNA and the translation elongation factor 1 alpha (tef) and beta tubulin (tub) nucleotide sequences. The common monophyletic origin of the Choanephoraceae-Gilbertellaceae clade could be confirmed in all gene trees and by investigation of their ultrastructure. Sporangia with persistent, sutured walls splitting in half at maturity and ellipsoidal sporangiospores with striated ornamentations and polar ciliate appendages arising from spores in persistent sporangia and dehiscent sporangiola represent synapomorphic characters of this group. We discuss our data in the context of the historical development of their taxonomy and physiology and propose a reduction of the two families to one family, the Choanephoraceae sensu lato comprising species which are facultative plant pathogens and parasites, especially in subtropical to tropical regions.

Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers - Jul 08, 2008 ()
[Tedersoo, L., Jairus, T., Horton, B. M., Abarenkov, K., Suvi, T., Saar, I., & Koljalg, U. 2008. The New Phytologist. 180(2) 479-490.]

* Ectomycorrhizal (ECM) symbiosis is a widespread plant nutrition strategy in Australia, especially in semiarid regions. This study aims to determine the diversity, community structure and host preference of ECM fungi in a Tasmanian wet sclero-phyll forest. * Ectomycorrhizal fungi were identified based on anatomotyping and rDNA internal transcribed spacer (ITS)-large subunit (LSU) sequence analysis using taxon-specific primers. Host tree roots were identified based on root morphology and length differences of the chloroplast trnL region. * A total of 123 species of ECM fungi were recovered from root tips of Eucalyptus regnans (Myrtaceae), Pomaderris apetala (Rhamnaceae) and Nothofagus cunninghamii (Nothofagaceae). The frequency of two thirds of the most common ECM fungi from several lineages was significantly influenced by host species. The lineages of Cortinarius, Tomentella-Thelephora, Russula-Lactarius, Clavulina, Descolea and Laccaria prevailed in the total community and their species richness and relative abundance did not differ by host species. * This study demonstrates that strongly host-preferring, though not directly specific, ECM fungi may dominate the below-ground community. Apart from the richness of Descolea, Tulasnella and Helotiales and the lack of Suillus-Rhizopogon and Amphinema-Tylospora, the ECM fungal diversity and phylogenetic community structure is similar to that in the Holarctic realm.

Microbial diversity across a Canadian sub-Arctic, isostatically rebounding, soil transect - Jul 02, 2008 ()
[Trevors, J. T., Kevan, P.G., & L. Tam 2008. Polar Science. Online Early .]

Seacoast to inland soil transects of 1 and 2 km were researched over 2 years to understand the previous termmicrobial diversitynext term in previous termanext term post ice age, isostatically, rebounding, soil environment. Community level substrate utilization analysis and 16S rDNA eubacterial previous termdiversitynext term were employed. The community level substrate analysis demonstrated that regardless of the location along the transect from seacoast to forest, sandy or peat soil, the previous termmicrobial diversitynext term (Shannon previous termdiversitynext term index about 3) was virtually the same. Shannon previous termdiversitynext term indexes based on PCR-DGGE analysis yielded values between about 0.6 and about 2 depending on the sand or peat soil type and the year the samples were collected and analyzed (2002 and 2003). Regardless of the genetic previous termdiversity,next term the soils exhibited similar metabolic capabilities. This is previous termanext term good example of redundant, functional, physiology regardless of the species present at each location along the transects.

Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well - Jul 01, 2008 ()
[Fazekas, A. J. Burgess, K. S. Kesanakurti, P. R. Graham, S. W. Newmaster, S. G. Husband, B. C. Percy, D. M. Hajibabaei, M. & S.C. Barrett 2008. PLoS ONE. 3(7) e2802.]

A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF-atpH, and psbK-psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85-100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK-psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69-71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems.

DNA barcoding plants in biodiversity hot spots: progress and outstanding questions - Jul 01, 2008 ()
[Hollingsworth, P. M. 2008. Heredity. 101(1) 1-2.]

Discrimination of Cricotopus species (Diptera: Chironomidae) by DNA barcoding - Jul 01, 2008 ()
[Sinclair, C. S. and S. E. Gresens 2008. Bull Entomol Res. 1-9.]

Chironomids (Diptera) typically comprise the most abundant group of macroinvertebrates collected in water quality surveys. Species in the genus Cricotopus display a wide range of tolerance for manmade pollutants, making them excellent bioindicators. Unfortunately, the usefulness of Cricotopus is overshadowed by the difficulty of accurately identifying larvae using current morphological keys. Molecular approaches are now being used for identification and taxonomic resolution in many animal taxa. In this study, a sequence-based approach for the mitochondrial gene, cytochrome oxidase I (COI), was developed to facilitate identification of Cricotopus species collected from Baltimore area streams. Using unique COI sequence variations, we developed profiles for seven described Cricotopus sp., four described Orthocladius sp., one described Paratrichocladius sp. and one putative species of Cricotopus. In addition to providing an accurate method for identification of Cricotopus, this method will make a useful contribution to the development of keys for Nearctic Cricotopus.

A DNA barcode examination of the red algal family Dumontiaceae in Canadian waters reveals substantial cryptic species diversity. 1. The foliose Dilsea–Neodilsea complex and Weeksia - Jul 01, 2008 ()
[Saunders, G. W. 2008. Botany. 86(7) 773-789.]

The field of DNA barcoding is working towards generating a genetic system for the quick and accurate identification of eukaryotic species. For the more systematic minded, however, DNA barcoding offers a new approach towards screening and uniting large numbers of biological specimens in genetic groups as a first step towards assigning them to species and genera in an approach best termed “molecular-assisted alpha taxonomy”. This approach is particularly amenable in organisms with simple morphologies, a propensity for convergence, extensive phenotypic plasticity, and life histories with an alternation of heteromorphic generations. It is hard to imagine a group of organisms better defined by all of these traits than the marine macroalgae. In an effort to assess the utility of the DNA barcode (COI-5′) for testing the current concepts of biodiversity of marine macroalgae in Canada, a study to assess species diversity in the red algal family, Dumontiaceae, was initiated. Through this work I confirm the presence in Canadian waters of Dilsea californica (J. Agardh) Kuntze, Dilsea integra (Kjellman) Rosenvinge, and Neodilsea borealis (I.A. Abbott) Lindstrom of the Dilsea–Neodilsea complex, and Weeksia coccinea (Harvey) Lindstrom for the genus Weeksia. However, our work has uncovered two additional species of the former complex, Dilsea lindstromiae Saunders sp. nov. and Dilsea pygmaea (Setchell) Setchell, and an additional species of the latter, Weeksia reticulata Setchell, effectively doubling representation of these foliose dumontiacean genera in Canadian waters.

Morphological, biological, and molecular characteristics of the diatom Pseudo-nitzschia delicatissima from the Canadian Maritimes - Jul 01, 2008 ()
[Kaczmarska, I., Reid, C., Martin, J. L., & Moniz, M. B. J. 2008. Botany. 86(7) 763-772.]

Forty-six monoclonal cultures of the diatom Pseudo-nitzschia delicatissima (Cleve) Heiden were isolated from coastal waters of Eastern Canada. Of these, 12 clones were successfully sexualized. The range of their morphological and genetic divergence was used as a reference for clones whose sexual identity remains unknown. All characters that were examined, including valve morphology, the nuclear internal transcribed spacer (ITS) region, and mitochondrial cytochrome c oxidase (cox1) sequences, showed a high degree of similarity within and between mating and nonsexualized clones. Within the 638 bp long aligned fragment in the ITS region, only five variable sites were found and just two were found within the 576 bp fragment of cox1 near the 5′ terminus of the gene. Our own data and those retrieved from GenBank suggest that the northern North Atlantic is populated by a single metapopulation of genetically very similar P. delicatissima, as determined using the ITS sequence of the epitype of the species. The ITS region of our clones was distinct from ITS-types present in isolates that we will refer to as P. delicatissima-like diatoms from the Mediterranean Sea and other low latitude Atlantic sites, thereby providing a means to discriminate between otherwise morphologically indistinguishable (cryptic) species. Such a distribution pattern suggests different physiological and environmental requirements for mating optima. This work furthers our understanding of the relationship between biological, molecular, and morphological species boundaries in diatoms and their ecology, and contributes to evaluation of the utility of ITS and cox1 sequences in DNA barcoding of diatoms.

First Report of Amylostereum areolatum, the Fungal Symbiont of Sirex noctilio, on Pinus spp. in Canada - Jul 01, 2008 ()
[Bergeron, M. J., Hamelin, R. C., Leal, I., Davis, C., & de Groot, P. 2008. Plant Disease. 92(7) 1138.]

Amylostereum areolatum (Fr.) Boidin (Russulales: Stereaceae) is a white rot fungus that has a symbiotic relationship with several woodwasps including Sirex noctilio Fabricius (Hymenoptera: Siricidae). The vectored fungus together with a phytotoxic mucus, both injected during oviposition by the female S. noctilio, rapidly weaken the host tree, rendering it susceptible to larval development (3). Host trees of A. areolatum include species of Pinus (mainly), Abies, Larix, and Picea and Cryptomeria japonica and Pseudotsuga menziesii (Fungal Databases [online]; USDA). The siricid woodwasp is native to Eurasia and North Africa and has been introduced into New Zealand, Australia, South America, and South Africa (1). In July of 2005, the first established North American population of S. noctilio was reported in Oswego, NY. Prompted by this initial discovery, a trap survey of Ontario counties located along the Canada-U.S. border, close to Upstate New York, was conducted in September and October of 2005. S. noctilio females were captured in four locations in southern Ontario. Two additional locations for S. noctilio were also reported in a survey conducted independently (2). In September and October of 2006, logs of Scots pines showing current Sirex oviposition sites were harvested from the Ontario area bordered by Lakes Huron, Erie, and Ontario to determine the presence of the species-specific fungal symbiont of S. noctilio, A. areolatum. Fungal isolates were obtained by surface sterilizing wood chips showing decay columns followed by incubation at 20°C on 2% malt extract agar. Cultures with morphological characteristics typical of A. areolatum–presence of clamp connections and arthrospores–were used for DNA analysis to confirm species identification. DNA sequences of the internal transcribed spacer (ITS) of the ribosomal RNA gene were queried against the NCBI GenBank database. There was a 99 to 100% match between the ITS sequences from the Ontario isolates and sequences from European and Asian A. areolatum isolates (GenBank Accession Nos. EU249343 and EU249344 versus AF454428, AF506405, AY781245, and AF218389). Matches with A. chailletii (Pers.) Boidin, a native related species, were around 97%. These results confirmed the presence of A. areolatum in the infested material. Cultures were deposited in the National Mycological Herbarium of Canada (DAOM 239280–DAOM 239295). To our knowledge, this represents the first report of A. areolatum in Canada. In its natural range, the insect-fungal complex exists in equilibrium with its host trees and parasites, thus, few negative impacts are observed. However, in the Southern Hemisphere where it has been introduced, it has become a major pest, attacking many important commercial North American species planted as exotics (1). Conifer forests in Canada are threatened by the spread of the S. noctilio/A. areolatum complex, particularly plantations and stands of Pinus banksiana, P. contorta, P. ponderosa, P. resinosa, P. strobus, and P. sylvestris. A survey of Eastern Canada to detect the presence of S. noctilio is on going, and genetics work is being conducted to determine the origin of the introduction of A. areolatum.

Testing candidate plant barcode regions in the Myristicaceae - Jun 28, 2008 (pdf)
[Newmaster, S.G., A.J. Fazekas, R.A.D. Steeves, and J. Janovec 2008. Molecular Ecology Resources. 8(3) 480-490.]

The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon — UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH-psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a more variable region (trnH-psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis.

Fruiting body and soil rDNA sampling detects complementary assemblage of Agaricomycotina (Basidiomycota, Fungi) in a hemlock-dominated forest plot in southern Ontario - Jun 28, 2008 ()
[Porter, T. M. Skillman, J. E. & J. -M. Moncalvo 2008. Molecular Ecology. 17(13) 3037-3050.]

This is the first study to assess the diversity and community structure of the Agaricomycotina in an ectotrophic forest using above-ground fruiting body surveys as well as soil rDNA sampling. We recovered 132 molecular operational taxonomic units, or 'species', from fruiting bodies and 66 from soil, with little overlap. Fruiting body sampling primarily recovered fungi from the Agaricales, Russulales, Boletales and Cantharellales. Many of these species are ectomycorrhizal and form large fruiting bodies. Soil rDNA sampling recovered fungi from these groups in addition to taxa overlooked during the fruiting body survey from the Atheliales, Trechisporales and Sebacinales. Species from these groups form inconspicuous, resupinate and corticioid fruiting bodies. Soil sampling also detected fungi from the Hysterangiales that form fruiting bodies underground. Generally, fruiting body and soil rDNA samples recover a largely different assemblage of fungi at the species level; however, both methods identify the same dominant fungi at the genus-order level and ectomycorrhizal fungi as the prevailing type. Richness, abundance, and phylogenetic diversity (PD) identify the Agaricales as the dominant fungal group above- and below-ground; however, we find that molecularly highly divergent lineages may account for a greater proportion of total diversity using the PD measure compared with richness and abundance. Unless an exhaustive inventory is required, the rapidity and versatility of DNA-based sampling may be sufficient for a first assessment of the dominant taxonomic and ecological groups of fungi in forest soil.

Molecular identification of vertebrate species by oligonucleotide microarray in food and forensic samples - Jun 28, 2008 ()
[Teletchea, F. Bernillon, J. Duffraisse, M. Laudet, V. & C. Hänni 2008. Journal of Applied Ecology. 45(3) 967-975.]

Molecular identification of animal or plant species in fresh and degraded products (e.g. food, faeces, hair and other organic remains) has become a very important issue in both conservation biology and food science. In this proof-of-concept study, we developed a microarray-based method using cytochrome b-derived probes to identify the main commercial and/or endangered vertebrate species in both food and forensic samples. This method allowed the unambiguous identification of 71 out of 77 species tested. In the remaining six cases, identification was hampered due to false sequences deposited in GenBank and high intraspecific variability. Our evaluation of this DNA chip for routine control demonstrated its effectiveness for the simultaneous identification of at least five species, and that its sensitivity varied according to the type of sample analysed. Synthesis and applications. Taken together, our results suggest that cytb-based microarray is a reliable and powerful identification tool for vertebrates, and more generally highlights the significant role of both molecular and traditional taxonomy in the development of molecular identification methods.

Working together to put molecules on the map - Jun 18, 2008 ()
[Field, D., Morrison, N., Glockner, F. O., Kottmann, R., Cochrane, G., Vaughan, R., Garrity, G., Cole, J., Hirschman, L., Schriml, L., Schindel, D., Miller, S., Hebert, P., Ratnasingham, S., Hanner, R., Amaral-Zettler, L., Sogin, M., Ashburner, M., Lewis, 2008. Nature. 453 978.]
DNA barcoding of fish of the Antarctic Scotia Sea indicate priority groups for taxonomic and systematics focus - Jun 01, 2008 ()
[Rock, J. Costa, F. O. Hutchinson, W. Walker, D. & G.R., Carvalho 2008. Antarctic Science. 20(3) 253-262.]

We analysed cytochrome oxidase I (COI) barcodes for 35 putative fish species collected in the Scotia Sea, and compared the resultant molecular data with field-based morphological identifications, and additional sequence data obtained from GenBank and the Barcode of Life Data System (BOLD). There was high congruence between morphological and molecular classification, and COI provided effective species-level discrimination for nearly all putative species. No effect of geographic sampling was observed for COI sequence variation. For two families, including the Liparidae and Zoarcidae, for which morphological field identification was unable to resolve taxonomy, DNA barcoding revealed significant species-level divergence. However, the dataset lacked sufficient sensitivity for resolving species within the Bathydraco and Artedidraco genera. Analysis of cytochrome b for these two genera also failed to resolve taxonomic identity. The data are discussed in relation to emergent priorities for additional taxonomic studies. We emphasize the utility of DNA barcoding in providing a valuable taxonomic framework for fundamental population studies through assigning life history stages or other morphologically ambiguous samples to parental species.

DNA barcode discovers two cryptic species and two geographical radiations in the invasive drosophilid Zaprionus indianus - Jun 01, 2008 ()
[Yassin, A., Capy, P., Madi-Ravazzi, L., Ogereau, D., and David, J. R 2008. Molecular Ecology Resources. 8(3) 491-501.]

Comparing introduced to ancestral populations within a phylogeographical context is crucial in any study aiming to understand the ecological genetics of an invasive species. Zaprionus indianus is a cosmopolitan drosophilid that has recently succeeded to expand its geographical range upon three continents (Africa, Asia and the Americas). We studied the distribution of mitochondrial DNA (mtDNA) haplotypes for two genes (CO-I and CO-II) among 23 geographical populations. mtDNA revealed the presence of two well-supported phylogenetic lineages (phylads), with bootstrap value of 100%. Phylad I included three African populations, reinforcing the African-origin hypothesis of the species. Within phylad II, a distinct phylogeographical pattern was discovered: Atlantic populations (from the Americas and Madeira) were closer to the ancestral African populations than to Eastern ones (from Madagascar, Middle East and India). This means that during its passage from endemism to cosmopolitanism, Z. indianus exhibited two independent radiations, the older (the Eastern) to the East, and the younger (the Atlantic) to the West. Discriminant function analysis using 13 morphometrical characters was also able to discriminate between the two molecular phylads (93.34 ± 1.67%), although detailed morphological analysis of male genitalia using scanning electron microscopy showed no significant differences. Finally, crossing experiments revealed the presence of reproductive barrier between populations from the two phylads, and further between populations within phylad I. Hence, a bona species status was assigned to two new, cryptic species: Zaprionus africanus and Zaprionus gabonicus, and both were encompassed along with Z. indianus and Zaprionus megalorchis into the indianus complex. The ecology of these two species reveals that they are forest dwellers, which explains their restricted endemic distribution, in contrast to their relative cosmopolitan Z. indianus, known to be a human-commensal. Our results reconfirm the great utility of mtDNA at both inter- and intraspecific analyses within the frame of an integrated taxonomical project.

Parasite misidentifications in GenBank: how to minimize their number? - Jun 01, 2008 ()
[Valkiunas, G. Atkinson, C. T. Bensch, S. Sehgal, R. N. & R.E. Ricklefs 2008. Trends in parasitology. 24(6) 247-8.]
Gut content identification of larvae of the Anopheles gambiae complex in western Kenya using a barcoding approach - Jun 01, 2008 ()
[Garros, C. Ngugi, N. Githeko, A. E. Tuno, N. & G. Yan 2008. Molecular Ecology Resources. 8(3) 512-518.]

Although larvae feeding and food source are vital to the development, survival and population regulation of African malaria vectors, the prey organisms of Anopheles gambiae larvae in the natural environment have not been well studied. This study used a molecular barcoding approach to investigate the natural diets of Anopheles gambiae s.l. larvae in western Kenya. Gut contents from third- and fourth-instar larvae from natural habitats were dissected and DNA was extracted. The 18S ribosomal DNA gene was amplified, the resulting clones were screened using a restriction fragment length polymorphism method and nonmosquito clones were sequenced. Homology search and phylogenetic analyses were then conducted using the sequences of non-mosquito clones to identify the putative microorganisms ingested. The phylogenetic analyses clustered ingested microorganisms in four clades, including two clades of green algae (Chlorophyta, Chlorophyceae Class, Chlamydomonadales and Chlorococcales families), one fungal clade, and one unknown eukaryote clade. In parallel, using the same approach, an analysis of the biodiversity present in the larval habitats was carried out. This present study demonstrated the feasibility of the barcoding approach to infer the natural diets of Anopheles gambiae larvae. Our analysis suggests that despite the wide range of microorganisms available in natural habitats, mosquito larvae fed on specific groups of algae. The novel tools developed from this study can be used to improve our understanding of the larval ecology of African malaria vectors and to facilitate the development of new mosquito control tools.

DNA barcoding in surveys of small mammal communities: a field study in Suriname - Jun 01, 2008 (pdf)
[Borisenko, A. V., Lim, B. K., Ivanova, N. V., Hanner, R. H., and Hebert, P. D. N. 2008. Molecular Ecology Notes. 8(3) 471-479.]

The performance of DNA barcoding as a tool for fast taxonomic verification in ecological assessment projects of small mammals was evaluated during a collecting trip to a lowland tropical rainforest site in Suriname. We also compared the performance of tissue sampling onto FTA CloneSaver cards vs. liquid nitrogen preservation. DNA barcodes from CloneSaver cards were recovered from 85% of specimens, but DNA degradation was apparent, because only 36% of sequence reads were long (over 600 bp). In contrast, cryopreserved tissue delivered 99% barcode recovery (97% > 600 bp). High humidity, oversampling or tissue type may explain the poor performance of CloneSaver cards. Comparison of taxonomic assignments made in the field and from barcode results revealed inconsistencies in just 3.4% of cases and most of the discrepancies were due to field misidentifications (3%) rather than sampling/analytical error (0.5%). This result reinforces the utility of DNA barcoding as a tool for verification of taxonomic identifications in ecological surveys, which is especially important when the collection of voucher specimens is not possible.

Identifying Canadian Freshwater Fishes through DNA Barcodes - Jun 01, 2008 (pdf)
[Hubert, N., Hanner, R., Holm, E., Mandrak, N.E., Taylor, E., Burridge, M., Watkinson, D., Dumont, P., Curry, A., Bentzen, P., Zhang, J., April, J., & Bernatchez, L. 2008. PLoS ONE. 3(6) e2490.]

Background

DNA barcoding aims to provide an efficient method for species-level identifications using an array of species specific molecular tags derived from the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene. The efficiency of the method hinges on the degree of sequence divergence among species and species-level identifications are relatively straightforward when the average genetic distance among individuals within a species does not exceed the average genetic distance between sister species. Fishes constitute a highly diverse group of vertebrates that exhibit deep phenotypic changes during development. In this context, the identification of fish species is challenging and DNA barcoding provide new perspectives in ecology and systematics of fishes. Here we examined the degree to which DNA barcoding discriminate freshwater fish species from the well-known Canadian fauna, which currently encompasses nearly 200 species, some which are of high economic value like salmons and sturgeons.

Methodology/Principal Findings

We bi-directionally sequenced the standard 652 bp "barcode" region of COI for 1360 individuals belonging to 190 of the 203 Canadian freshwater fish species (95%). Most species were represented by multiple individuals (7.6 on average), the majority of which were retained as voucher specimens. The average genetic distance was 27 fold higher between species than within species, as K2P distance estimates averaged 8.3% among congeners and only 0.3% among concpecifics. However, shared polymorphism between sister-species was detected in 15 species (8% of the cases). The distribution of K2P distance between individuals and species overlapped and identifications were only possible to species group using DNA barcodes in these cases. Conversely, deep hidden genetic divergence was revealed within two species, suggesting the presence of cryptic species.

Conclusions/Significance

The present study evidenced that freshwater fish species can be efficiently identified through the use of DNA barcoding, especially the species complex of small-sized species, and that the present COI library can be used for subsequent applications in ecology and systematics.

A mitochondrial-DNA-based phylogeny for some evolutionary-genetic model species of Colias butterflies (Lepidoptera, Pieridae) - Jun 01, 2008 ()
[Wheat, C. W. and W. B. Watt 2008. Molecular phylogenetics and evolution. 47(3) 893-902.]

We study the phylogenetic relationships among some North American Colias ("sulfur") butterflies, using mitochondrial gene sequences (ribosomal RNA, cytochrome oxidase I+II) totaling about 20% of the mitochondrial genome. We find that (1) the lowland species complex shows a branching order different from earlier views; (2) several montane and northern taxa may be more distinct than in earlier views; (3) one morphologically conservative Holarctic assemblage, C. hecla, is differentiated at the molecular-genetic level into at least three taxa which occupy distinct positions in the phylogeny and are sisters to diverse other taxa. These conclusions, constituting phylogenetic hypotheses, are supported by parsimony, maximum-likelihood, and Bayesian reconstruction algorithms. They are tested formally, by interior branch tests and paired-site tests, against alternative hypotheses derived from conventional species and subspecies naming combinations. In all cases our hypotheses are supported by these tests and the conventional alternatives are rejected. The "barcoding" subset of cytochrome oxidase I sequence identifies only some of the taxa supported by our full data set. Comparison of genetic divergence values among Colias taxa with those among related Pierid butterflies suggests that species radiations within Colias are comparatively younger. This emerging Colias phylogeny facilitates comparisons of genetic polymorphism and other adaptive mechanisms among taxa, thereby connecting micro- and macro-evolutionary processes.

Molecular characterization of freshwater snails in the genus Bulinus: a role for barcodes? - Jun 01, 2008 ()
[Kane, R. A., Stothard, J. R., Emery, A. M., & Rollinson, D. 2008. Parasit Vectors. 1(1) 15.]

ABSTRACT: BACKGROUND: Reliable and consistent methods are required for the identification and classification of freshwater snails belonging to the genus Bulinus (Gastropoda, Planorbidae) which act as intermediate hosts for schistosomes of both medical and veterinary importance. The current project worked towards two main objectives, the development of a cost effective, simple screening method for the routine identification of Bulinus isolates and the use of resultant sequencing data to produce a model of relationships within the group. RESULTS: Phylogenetic analysis of the DNA sequence for a large section (1009 bp) of the mitochondrial gene cytochrome oxidase subunit 1 (cox1) for isolates of Bulinus demonstrated superior resolution over that employing the second internal transcribed spacer (its2) of the ribosomal gene complex. Removal of transitional substitutions within cox1 because of saturation effects still allowed identification of snails at species group level. Within groups, some species could be identified with ease but there were regions where the high degree of molecular diversity meant that clear identification of species was problematic, this was particularly so within the B. africanus group. CONCLUSION: The sequence diversity within cox1 is such that a barcoding approach may offer the best method for characterization of populations and species within the genus from different geographical locations. The study has confirmed the definition of some accepted species within the species groups but additionally has revealed some unrecognized isolates which underlines the need to use molecular markers in addition to more traditional methods of identification. A barcoding approach based on part of the cox1 gene as defined by the Folmer primers is proposed.

Taxonomy and why history of science matters for science: a case study - Jun 01, 2008 ()
[Hamilton, A., & Wheeler, Q. D. 2008. Isis. 99(2) 331-340.]

The history of science often has difficulty connecting with science at the lab-bench level, raising questions about the value of history of science for science. This essay offers a case study from taxonomy in which lessons learned about particular failings of numerical taxonomy (phenetics) in the second half of the twentieth century bear on the new movement toward DNA barcoding. In particular, it argues that an unwillingness to deal with messy theoretical questions in both cases leads to important problems in the theory and practice of identifying taxa. This argument makes use of scientific and historical considerations in a way that the authors hope leads to convincing conclusions about the history of taxonomy as well as about its present practice.

A revision of Malagasy species of Anochetus mayr and Odontomachus latreille (Hymenoptera: Formicidae) - May 01, 2008 ()
[Fisher, B. L., & Smith, M. A. 2008. PLoS ONE. 3(5) e1787.]

Species inventories are essential for documenting global diversity and generating necessary material for taxonomic study and conservation planning. However, for inventories to be immediately relevant, the taxonomic process must reduce the time to describe and identify specimens. To address these concerns for the inventory of arthropods across the Malagasy region, we present here a collaborative approach to taxonomy where collectors, morphologists and DNA barcoders using cytochrome c oxidase 1 (CO1) participate collectively in a team-driven taxonomic process. We evaluate the role of DNA barcoding as a tool to accelerate species identification and description. This revision is primarily based on arthropod surveys throughout the Malagasy region from 1992 to 2006. The revision is based on morphological and CO1 DNA barcode analysis of 500 individuals. In the region, five species of Anochetus (A. boltonisp. nov., A. goodmanisp. nov., A. grandidieri, and A. madagascarensis from Madagascar, and A. pattersonisp. nov. from Seychelles) and three species of Odontomachus (O. coquereli, O. troglodytes and O. simillimus) are recognized. DNA barcoding (using cytochrome c oxidase 1 (CO1)) facilitated caste association and type designation, and highlighted population structure associated with reproductive strategy, biogeographic and evolutionary patterns for future exploration. This study provides an example of collaborative taxonomy, where morphology is combined with DNA barcoding. We demonstrate that CO1 DNA barcoding is a practical tool that allows formalized alpha-taxonomy at a speed, detail, precision, and scale unattainable by employing morphology alone.

Full text: http://dx.doi.org/10.1371/journal.pone.0001787

A new species of clawed frog (genus Xenopus) from the Itombwe Massif, Democratic Republic of the Congo: implications for DNA barcodes and biodiversity conservation - May 01, 2008 ()
[Evans, B. J., Carter, T. F., Tobias, M. L., Kelley, D. B., Hanner, R., & Tinsley, R. C. 2008. Zootaxa. 1780 55-68.]

Here we describe a new octoploid species of clawed frog from the Itombwe Massif of South Kivu Province, Democratic Republic of the Congo. This new species is the sister taxon of Xenopus wittei, but is substantially diverged in morphology, male vocalization, and mitochondrial and autosomal DNA. Analysis of mitochondrial “DNA barcodes” in polyploid clawed frogs demonstrates that they are variable between most species, but also reveals limitations of this type of information for distinguishing closely related species of differing ploidy level. The discovery of this new species highlights the importance of the Itombwe Massif for conservation of African biodiversity south of the Sahara.

Application of PDF417 symbology for 'DNA Barcoding' - May 01, 2008 ()
[Kumar, N. P., Rajavel, A. R., & Jambulingam, P. 2008. Computer methods and programs in biomedicine. 90(2) 187-189.]

DNA sequences consisting of about 600 base pairs of the 5' region of the cytochrome c oxidase subunit 1 (COI) gene has been proposed as DNA Barcodes for taxonomical identification of species in different animals. We evaluated the application of two-dimensional barcodes for 'DNA Barcoding'. 'PDF417' symbology was applied to convert DNA Barcode sequences already proposed [N. Pradeep Kumar, A.R. Rajavel, R. Natarajan, P. Jambulingam, DNA Barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae). J. Med. Entomol. 77 (2007) 1-7.] for 10 different species of mosquitoes prevalent in India. Decoding of these digital images using 2-D scanner and a suitable software reproduced the input DNA sequences unchanged. This analysis indicated the utility of PDF417 for 'DNA Barcoding', which could be of definite use for taxonomic documentation of animals.

Resurrecting the red algal genus Grania within the order Acrochaetiales (Florideophyceae, Rhodophyta) - May 01, 2008 ()
[Clayden, S. L., & Saunders, G. W. 2008. European Journal of Phycology. 43(2) 151-160.]

An isolate of the filamentous red alga Acrochaetium efflorescens from the NE Atlantic was followed in culture through a triphasic sexual life history. Morphology of each phase matched previous descriptions for this alga. However, inclusion of A. efflorescens in the genus Acrochaetium is problematic. In current taxonomic treatments this genus includes acrochaetioid algae with a stellate plastid and a large central pyrenoid. Acrochaetium efflorescens, on the other hand, has consistently been reported to possess spiral to ribbon-shaped plastids. To determine the phylogenetic affinities of A. efflorescens, we sequenced large subunit ribosomal DNA and resolved full support (Bayesian posterior probabilities; maximum likelihood, neighbour joining, and parsimony bootstrap percentages) for inclusion of A. efflorescens within the order Acrochaetiales. However, it does not associate closely with any of the existing genera in this order, showing only a weak affinity to the genus Rhodochorton. The genus Grania, originally established by Kylin, is currently available for this species. Given its distinct anatomy (ribbon-shaped plastids in combination with seriate carposporangia), unique molecular signature, and lack of congruence with other current generic concepts within the Acrochaetiales, we advocate resurrecting the genus Graniafor G. efflorescens.

Semi-automated, Membrane-Based Protocol for DNA Isolation from Plants - May 01, 2008 ()
[Ivanova, N. V. Fazekas, A. J. & P.D.N. Hebert 2008. Plant Mol. Biol. Rep. 26(3) 186-198.]

Many plant species are considered difficult for DNA isolation due to their high concentrations of secondary metabolites such as polysaccharides and polyphenols. Several protocols have been developed to overcome this problem, but they are typically time-consuming, not scalable for high throughput and not compatible with automation. Although a variety of commercial kits are available for plant DNA isolation, their cost is high and these kits usually have limited taxonomic applicability. In a previous study we developed an inexpensive automation-friendly protocol for DNA extraction from animal tissues. Here we demonstrate that a similar protocol allows DNA isolation from plants.

Exophiala spinifera and its allies: diagnostics from morphology to DNA barcoding - May 01, 2008 ()
[Zeng, J. S., & De Hoog, G. S. 2008. Med Mycol. 46(3) 193-208.]

Diagnostic features of morphology, physiology, serology and genetics of species belonging to the Exophiala spinifera clade (including 11 species: Exophiala oligosperma, E. spinifera, E. xenobiotica, E. jeanselmei, E. exophialae, E. nishimurae, E. bergeri, E. nigra, Rhinocladiella similis, Ramichloridium basitonum and Phaeoannellomyces elegans), comprising a large number of human-associated Exophiala species, are summarized. Several species have closely similar morphological characters and physiological profiles. Taxonomy is therefore primarily based on sequence diversity of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA). Multilocus sequencing has shown that ITS is reliable for identification of the species in this clade, and is a therefore a good candidate for barcoding species of Exophiala. Species-specific fragments were searched in the ITS region of species in the Exophiala spinifera clade and can be used to design probes for diagnosis by hybridization

DNA barcodes and cryptic species of skipper butterflies in the genus Perichares in Area de Conservacion Guanacaste, Costa Rica. - Apr 29, 2008 ()
[Burns, J. M., Janzen, D. H., Hajibabaei, M., Hallwachs, W., and P. D. N. Hebert 2008. Proc Natl Acad Sci U S A. 105(17) 6350-5.]

DNA barcodes can be used to identify cryptic species of skipper butterflies previously detected by classic taxonomic methods and to provide first clues to the existence of yet other cryptic species. A striking case is the common geographically and ecologically widespread neotropical skipper butterfly Perichares philetes (Lepidoptera, Hesperiidae), described in 1775, which barcoding splits into a complex of four species in Area de Conservacion Guanacaste (ACG) in northwestern Costa Rica. Three of the species are new, and all four are described. Caterpillars, pupae, and foodplants offer better distinguishing characters than do adults, whose differences are mostly average, subtle, and blurred by intraspecific variation. The caterpillars of two species are generalist grass-eaters; of the other two, specialist palm-eaters, each of which feeds on different genera. But all of these cryptic species are more specialized in their diet than was the morphospecies that held them. The four ACG taxa discovered to date belong to a panneotropical complex of at least eight species. This complex likely includes still more species, whose exposure may require barcoding. Barcoding ACG hesperiid morphospecies has increased their number by nearly 10%, an unexpectedly high figure for such relatively well known insects.

Diagnosing mitochondrial DNA diversity: applications of a sentinel gene approach - Apr 01, 2008 (pdf)
[Clare, E. L., Kerr, K. C., von Konigslow, T. E., Wilson, J. J., & Hebert, P. D.N. 2008. Journal of Molecular Evolution. 66(4) 362-367.]

Mitochondrial genomes show wide variation in their GC content. This study examines the correlations between mitochondrial genome-wide shifts in this feature and a fragment of the cytochrome c oxidase subunit I (COI) gene in animals, plants, and fungi. Because this approach utilizes COI as a sentinel, analyzing sequences from repositories such as GenBank and the Barcode of Life Data System (BOLD) can provide rapid insights into nucleotide usage. With this approach we probe nucleotide composition in a variety of taxonomic groups and establish the degree to which mitochondrial GC content varies among them. We then focus on two groups in particular, the classes Insecta and Aves, which possess the highest and lowest GC content, respectively. We establish that the sentinel approach provides strong indicators of mitochondrial GC content within divergent phyla (R values = 0.86-0.95, p < 0.001, in test cases) and provide evidence that selective pressures acting on GC content extend to noncoding regions of the plant and fungal mitochondrial genomes. We demonstrate that there is considerable variation in GC content of the mitochondrial genome within phyla and at each taxonomic level, leading to a substantial overlap zone in GC content between chordates and invertebrates. Our results provide a novel insight into the mitochondrial genome composition of animals, plants, and fungi and advocate this sentinel technique for the detection of rapid alterations in nucleotide usage as a measure of mitochondrial genome biodiversity.

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Single-molecule DNA sequencing of a viral genome - Apr 01, 2008 ()
[Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, Braslavsky I, Causey M, Colonell J, Dimeo J, Efcavitch JW, Giladi E, Gill J, Healy J, Jarosz M, Lapen D, Moulton K, Quake SR, Steinmann K, Thayer E, Tyurina A, Ward R, Weiss H, Xie Z. 2008. Science. 320(5872) 106-9.]

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.

Testing the reliability of genetic methods of species identification via simulation - Apr 01, 2008 ()
[Ross, H. A., Murugan, S., & Li, W. L. 2008. Systematic Biology. 57(2) 216-30.]

Although genetic methods of species identification, especially DNA barcoding, are strongly debated, tests of these methods have been restricted to a few empirical cases for pragmatic reasons. Here we use simulation to test the performance of methods based on sequence comparison (BLAST and genetic distance) and tree topology over a wide range of evolutionary scenarios. Sequences were simulated on a range of gene trees spanning almost three orders of magnitude in tree depth and in coalescent depth; that is, deep or shallow trees with deep or shallow coalescences. When the query's conspecific sequences were included in the reference alignment, the rate of positive identification was related to the degree to which different species were genetically differentiated. The BLAST, distance, and liberal tree-based methods returned higher rates of correct identification than did the strict tree-based requirement that the query was within, but not sister to, a single-species clade. Under this more conservative approach, ambiguous outcomes occurred in inverse proportion to the number of reference sequences per species. When the query's conspecific sequences were not in the reference alignment, only the strict tree-based approach was relatively immune to making false-positive identifications. Thresholds affected the rates at which false-positive identifications were made when the query's species was unrepresented in the reference alignment but did not otherwise influence outcomes. A conservative approach using the strict tree-based method should be used initially in large-scale identification systems, with effort made to maximize sequence sampling within species. Once the genetic variation within a taxonomic group is well characterized and the taxonomy resolved, then the choice of method used should be dictated by considerations of computational efficiency. The requirement for extensive genetic sampling may render these techniques inappropriate in some circumstances.

Detection of cranberry fruit rot fungi using DNA array hybridization - Apr 01, 2008 ()
[Robideau, G.P., Caruso, F.L., Oudemans, P.V., McManus, P.S., Renaud, M.A., Auclair, M.E., Bilodeau, G.J., Yee, D., Désaulniers, N.L., DeVerna, J.W., and C. A. Lévesque 2008. Canadian Journal of Plant Pathology. 30 226-240.]

A PCR-based DNA macroarray hybridization technique (also called reverse dot blot hybridization) was developed for cranberry fruit rot (CFR) fungal pathogens, and its detection capability was compared with that of the traditional isolation plating method for CFR isolation and identification from over 2000 field samples. DNA array hybridization results correlated well with detection by isolation when cranberry fruit samples had calyces removed. It also provided detection of CFR fungi not recovered by isolation. When calyces were not removed, the number of cranberry samples where a species was isolated but not detected on the array increased. Isolation without array detection was also correlated with using greater amounts of berry mass for DNA extraction. This was due to the complexity of DNA template mixtures and the presence of some fungal species at very low concentrations. Multiple PCR reactions may be necessary to accurately detect the diversity of fungal pathogens in such situations. Overall, the use of DNA array hybridization for CFR fungi detection is a rapid, sensitive, and cost-effective technique that shows great potential for future CFR research

Inferring species membership using DNA sequences with back-propagation neural networks - Apr 01, 2008 ()
[Zhang, A. B., Sikes, D. S., Muster, C., & Li, S. Q 2008. Systematic biology. 57(2) 202-15.]

DNA barcoding as a method for species identification is rapidly increasing in popularity. However, there are still relatively few rigorous methodological tests of DNA barcoding. Current distance-based methods are frequently criticized for treating the nearest neighbor as the closest relative via a raw similarity score, lacking an objective set of criteria to delineate taxa, or for being incongruent with classical character-based taxonomy. Here, we propose an artificial intelligence-based approach - inferring species membership via DNA barcoding with back-propagation neural networks (named BP-based species identification) - as a new advance to the spectrum of available methods. We demonstrate the value of this approach with simulated data sets representing different levels of sequence variation under coalescent simulations with various evolutionary models, as well as with two empirical data sets of COI sequences from East Asian ground beetles (Carabidae) and Costa Rican skipper butterflies. With a 630-to 690-bp fragment of the COI gene, we identified 97.50% of 80 unknown sequences of ground beetles, 95.63%, 96.10%, and 100% of 275, 205, and 9 unknown sequences of the neotropical skipper butterfly to their correct species, respectively. Our simulation studies indicate that the success rates of species identification depend on the divergence of sequences, the length of sequences, and the number of reference sequences. Particularly in cases involving incomplete lineage sorting, this new BP-based method appears to be superior to commonly used methods for DNA-based species identification.

Molecular evidence for long distance dispersal across the Southern Hemisphere in the Ganoderma applanatum-australe species complex (Basidiomycota) - Apr 01, 2008 ()
[Moncalvo, J. M., & Buchanan, P. K. 2008. Mycological Research. 112(Pt4) 425-36.]

We examined phylogeographic relationships in the cosmopolitan polypore fungus Ganoderma applanatum and allies, and conservatively infer a possible age of origin for these fungi. Results indicate that it is very unlikely that members of this species complex diversified before the break-up of Gondwana from Laurasia ca 120M years ago, and also before the final separation of the Gondwanan landmasses from each other that was achieved about 66M years ago. An earliest possible age of origin of 30M years was estimated from nucleotide substitution rates in the 18S rDNA gene. Phylogenetic reconstruction of a worldwide sampling of ITS rDNA sequences reveals at least eight distinct clades that are strongly correlated with the geographic origin of the strains, and also correspond to mating groups. These include one Southern Hemisphere clade, one Southern Hemisphere-Eastern Asia clade, two temperate Northern Hemisphere clades, three Asian clades, and one neotropical clade. Geographically distant collections from the Southern Hemisphere shared identical ITS haplotypes, and an ITS recombinant was noted. Nested clade analysis of a parsimony network among isolates of the Southern Hemisphere clade indicated restricted gene flow with isolation-by-distance among the New Zealand, Australia-Tasmania, Chile-Argentine, and South Africa populations, suggesting episodic events of long-distance dispersal within the Southern Hemisphere. This study indicates that dispersal bias plays a more important role than generally admitted to explain the Southern Hemisphere distribution of many taxa, at least for saprobic fungi.

Rapid, one-step DNA extraction for insect pest identification by using DNA barcodes - Apr 01, 2008 ()
[Ball, S. L., & Armstrong, K. F. 2008. Journal of Economic Entomology. 101(2) 523-532.]

Early detection of economically important insects is critical to preventing their establishment as serious pests. To accomplish this, tools for rapid and accurate species identification are needed. DNA barcoding, using short DNA sequences as species "genetic identification tags," has already shown large potential as a tool for rapid and accurate detection of economically important insects. DNA extraction is the critical first step in generating DNA barcodes and can be a rate-limiting step in very large barcoding studies. Consequently, a DNA extraction method that is rapid, easy to use, cost-effective, robust enough to cope with range of qualities and quantities of tissue, and can be adapted to robotic systems will provide the best method for high-throughput production of DNA barcodes. We tested the performance of a new commercial kit (prepGEM), which uses a novel, streamlined approach to DNA extraction, and we compared it with two other commercial kits (ChargeSwitch and Aquapure), which differ in their method of DNA extraction. We compared performance of these kits by measuring percentage of polymerase chain reaction (PCR) success and mean PCR product yield across a variety of arthropod taxa, whichincluded freshly collected, ethanol-preserved, and dried specimens of different ages. ChargeSwitch and prepGEM performed equally well, but they outperformed Aquapure. prepGEM was much faster, easier to use, and cheaper than ChargeSwitch, but ChargeSwitch performed slightly better for older (> 5-yr-old) dried insect specimens. Overall, prepGEM may provide a highly streamlined method of DNA extraction for fresh, ethanol-preserved, and young, dried specimens, especially when adapted for high-throughput, robotic systems.

On the genetic diversity in the mitochondrial 12S rRNA gene of Platymantis frogs from Western New Guinea (Anura: Ceratobatrachidae) - Mar 18, 2008 ()
[Köhler, F., Schultze, K.-J., Günther, R., & Plötner, J. 2008. Journal of Zoological Systematics and Evolutionary Research. 46(2) 177-185.]

Platymantis is a group of neobatrachian frogs that occurs from the Philippines to New Guinea – an area situated at the interface between the Australian and Asian biogeographical region that is highly fragmented by stretches of open sea. Partial sequences of the mitochondrial 12S rRNA gene are herein used to infer the relationships of species from the Indonesian part of New Guinea (Papua and West Papua Province). The phylogenetic trees reveal a deep bifurcation between the Asian and Western New Guinean clades being consistent with phylogeographic patterns observed in various other faunal groups. While most species are well differentiated in the examined locus, low interspecific genetic distances between one and three percent were observed in the New Guinean species Platymantis papuensis and P. cryptotis as well as P. pelewensis from Palau. Platymantis papuensis and P. pelewensis are geographically separated from each other by a 1100 km stretch of open sea. The minor degree of genetic differentiation between both species points to a recent event of transmarine dispersal as causation for the occurrence of P. pelewensis on Palau. The low genetic differentiation between P. cryptotis and the sympatric P. papuensis, two species that are bioacoustically and morphologically distinct, may indicate its possibly recent evolutionary origin or, alternatively, yet undetected hybridization between the two species. The same may also hold true for frogs from Yapen that exhibit calls different from the sympatric P. papuensis. Tentatively referred to as Platymantis spec., these frogs are also genetically not well differentiated. It is furthermore concluded that the partly low genetic differentiation of the New Guinean Platymantis species render this group one of the cases in which DNA barcoding would likely fail to produce reliable results.

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Identifying earthworms through DNA barcodes: Pitfalls and promise - Mar 10, 2008 ()
[Chang, C.-H., Rougerie, R., & Chen, J.-H. 2008. Pedobiologia. 52(3) 171-180.]

This paper re-evaluated the use of DNA barcodes in earthworm species identification by re-analyzing sequence data for the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. This analysis unveiled and confirmed taxonomic inconsistencies which significantly affect data interpretation. When considering synonymy and misidentification in published records, our results revealed no shared COI haplotypes between morphologically distinct species and higher interspecific than intraspecific divergence in most cases, with interspecific and intraspecific distances averaging 18.7% and 1.3% respectively. However, a few earthworm species endemic to Taiwan have deep intraspecific divergences which may represent potential cases of cryptic diversity, although incomplete lineage sorting cannot be ruled out without further study. We recognize the potential of DNA barcoding for earthworm taxonomy, but have identified several issues regarding the evolution of the COI gene in these organisms which remain to be further elucidated.

Single mitochondrial gene barcodes reliably identify sister-species in diverse clades of birds - Mar 09, 2008 ()
[Tavares, E. S., & Baker, A. J. 2008. BMC Evol Biol. 8 81.]

BACKGROUND: DNA barcoding of life using a standardized COI sequence was proposed as a species identification system, and as a method for detecting putative new species. Previous tests in birds showed that individuals can be correctly assigned to species in ~94% of the cases and suggested a threshold of 10x mean intraspecific difference to detect potential new species. However, these tests were criticized because they were based on a single maternally inherited gene rather than multiple nuclear genes, did not compare phylogenetically identified sister species, and thus likely overestimated the efficacy of DNA barcodes in identifying species. RESULTS: To test the efficacy of DNA barcodes we compared ~650 bp of COI in 60 sister-species pairs identified in multigene phylogenies from 10 orders of birds. In all pairs, individuals of each species were monophyletic in a neighbor-joining (NJ) tree, and each species possessed fixed mutational differences distinguishing them from their sister species. Consequently, individuals were correctly assigned to species using a statistical coalescent framework. A coalescent test of taxonomic distinctiveness based on chance occurrence of reciprocal monophyly in two lineages was verified in known sister species, and used to identify recently separated lineages that represent putative species. This approach avoids the use of a universal distance cutoff which is invalidated by variation in times to common ancestry of sister species and in rates of evolution. CONCLUSION: Closely related sister species of birds can be identified reliably by barcodes of fixed diagnostic substitutions in COI sequences, verifying coalescent-based statistical tests of reciprocal monophyly for taxonomic distinctiveness. Contrary to recent criticisms, a single DNA barcode is a rapid way to discover monophyletic lineages within a metapopulation that might represent undiscovered cryptic species, as envisaged in the unified species concept. This identifies a smaller set of lineages that can also be tested independently for species status with multiple nuclear gene approaches and other phenotypic characters.

CO1 DNA barcoding amphibians: take the chance, meet the challenge - Mar 07, 2008 (pdf)
[Smith, M.A., Poyarkov, N.A., and P.D.N. Hebert 2008. Molecular Ecology Resources. 8 235-246.]

Although a mitochondrial DNA barcode has been shown to be of great utility for species identification and discovery in an increasing number of diverse taxa, caution has been urged with its application to one of the most taxonomically diverse vertebrate groups - the amphibians. Here, we test three of the perceived shortcomings of a CO1 DNA barcode's utility with a group of Holarctic amphibians: primer fit, sequence variability and overlapping intra- and interspecific variability. We found that although the CO1 DNA barcode priming regions were variable, we were able to reliably amplify a CO1 fragment from degenerate primers and primers with G-C residues at the 3' end. Any overlap between intra- and interspecific variation in our taxonomic sampling was due to introgressive hybridization (Bufo/Anaxyrus), complex genetics (Ambystoma) or incomplete taxonomy (Triturus). Rates of hybridization and species discovery are not expected to be greater for amphibians than for other vertebrate groups, and thus problems with the utility of using a single mitochondrial gene for species identification will not be specific to amphibians. Therefore, we conclude that there is greater potential for a CO1 barcode's use with amphibians than has been reported to date. A large-scale effort to barcode the amphibians of the world, using the same primary barcode region of CO1, will yield important findings for science and conservation.

DNA barcodes: Genes, genomics, and bioinformatics - Feb 26, 2008 (pdf)
[W. John Kress and David L. Erickson 2008. PNAS. Vol. 105. No. 8 3761-3762.]

DNA barcoding the floras of biodiversity hotspots - Feb 26, 2008 ()
[Lahaye, R., van der Bank, M., Bogarin, D., Warner, J., Pupulin, F., Gigot, G., Maurin, O., Duthoit, S., Barraclough, T. G., and V. Savolainen 2008. Proc Natl Acad Sci U S A. 105(8) 2923-8.]

DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a "DNA barcoding gap" is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes.

Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata - Feb 07, 2008 ()
[Rach, J., Desalle, R., Sarkar, I. N., Schierwater, B., & Hadrys, H. 2008. Proc Biol Sci. 275(1632) 237-47.]

DNA barcoding has become a promising means for identifying organisms of all life stages. Currently, phenetic approaches and tree-building methods have been used to define species boundaries and discover 'cryptic species'. However, a universal threshold of genetic distance values to distinguish taxonomic groups cannot be determined. As an alternative, DNA barcoding approaches can be 'character based', whereby species are identified through the presence or absence of discrete nucleotide substitutions (character states) within a DNA sequence. We demonstrate the potential of character-based DNA barcodes by analysing 833 odonate specimens from 103 localities belonging to 64 species. A total of 54 species and 22 genera could be discriminated reliably through unique combinations of character states within only one mitochondrial gene region (NADH dehydrogenase 1). Character-based DNA barcodes were further successfully established at a population level discriminating seven population-specific entities out of a total of 19 populations belonging to three species. Thus, for the first time, DNA barcodes have been found to identify entities below the species level that may constitute separate conservation units or even species units. Our findings suggest that character-based DNA barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii) complementing taxonomic identification systems.

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DNA barcoding reveals a likely second species of Asian sea bass (barramundi) (Lates calcarifer) - Feb 01, 2008 ()
[Ward, R. D., Holmes, B. H., and Yearsley, G. K. 2008. Journal of Fish Biology. 72(2) 458–463.]

DNA barcoding - the sequencing of a c. 650 base pair region of the mitochondrial cytochrome c oxidase I gene - strongly suggests that barramundi (Lates calcarifer) from Australia and from Myanmar are different species (Kimura 2 parameter distance of c. 9·5%). Cytochrome b sequence data support this conclusion (distance c. 11.3%). Further examination, both genetic and morphological, of L. calcarifer throughout its range is recommended.

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DNA barcoding for the identification of smoked fish products - Feb 01, 2008 ()
[Smith, P. J., McVeagh, S. M., and Steinke, D. 2008. Journal of Fish Biology. 72(2) 464-471.]

DNA barcoding was applied to the identification of smoked products from fish in 10 families in four orders and allowed identification to the species level, even among closely related species in the same genus. Barcoding is likely to become a standard tool for identification of fish specimens and products.

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Predispersal seed predation by a coleophorid on the threatened Gulf of St. Lawrence aster - Feb 01, 2008 ()
[Steeves, R., Nazari, V., Landry, J.-F., & Lacroix, C. R. 2008. The Canadian Entomologist. 140(3) 297-305.]

The Gulf of St. Lawrence aster, Symphyotrichum laurentianum (Fernald) G.L. Nesom (Asteraceae), a small annual halophyte endemic to disturbed and highly transient habitats in the Gulf of St. Lawrence, is classified as “threatened” by the Committee on the Status of Endangered Wildlife in Canada. Lepidopteran larvae that are predispersal seed predators of the Gulf of St. Lawrence aster are reported for the first time from populations in Prince Edward Island National Park. DNA barcoding was used to identify the seed predators tentatively as larvae of the casebearing moth Coleophora triplicis McDunnough (Lepidoptera: Coleophoridae), which is typically associated with a related halophyte, Solidago sempervirens L. (Asteraceae). These larvae were found to consume a large proportion of seeds from one of two aster populations in Prince Edward Island National Park and may be yet another risk to the survival of this threatened species.

Widespread occurrence and phylogenetic placement of a soil clone group adds a prominent new branch to the fungal tree of life. - Feb 01, 2008 ()
[Porter, T. M., Schadt, C. W., Rizvi, L., Martin, A. P., Schmidt, S. K., Scott-Denton, L., Vilgalys, R., & Moncalvo, J. M. 2008. Molecular Phylogenetics and Evolution. 46(2) 635-644.]

Fungi are one of the most diverse groups of Eukarya and play essential roles in terrestrial ecosystems as decomposers, pathogens and mutualists. This study unifies disparate reports of unclassified fungal sequences from soils of diverse origins and anchors many of them in a well-supported clade of the Ascomycota equivalent to a subphylum. We refer to this clade as Soil Clone Group I (SCGI). We expand the breadth of environments surveyed and develop a taxon-specific primer to amplify 2.4 kbp rDNA fragments directly from soil. Our results also expand the known range of this group from North America to Europe and Australia. The ancient origin of SCGI implies that it may represent an important transitional form among the basal Ascomycota groups. SCGI is unusual because it currently represents the only major fungal lineage known only from sequence data. This is an important contribution towards building a more complete fungal phylogeny and highlights the need for further work to determine the function and biology of SCGI taxa.

Slow mitochondrial COI sequence evolution at the base of the metazoan tree and its implications for DNA barcoding - Feb 01, 2008 ()
[Huang, D., Meier, R., Todd, P. A., & Chou, L. M. 2008. Journal of Molecular Evolution. 66(2) 167-74.]

The evolution rates of mtDNA in early metazoans hold important implications for DNA barcoding. Here, we present a comprehensive analysis of intra- and interspecific COI variabilities in Porifera and Cnidaria (separately as Anthozoa, Hydrozoa, and Scyphozoa) using a data set of 619 sequences from 224 species. We found variation within and between species to be much lower in Porifera and Anthozoa compared to Medusozoa (Hydrozoa and Scyphozoa), which has divergences similar to typical metazoans. Given that recent evidence has shown that fungi also exhibit limited COI divergence, slow-evolving mtDNA is likely to be plesiomorphic for the Metazoa. Higher rates of evolution could have originated independently in Medusozoa and Bilateria or been acquired in the Cnidaria + Bilateria clade and lost in the Anthozoa. Low identification success and substantial overlap between intra- and interspecific COI distances render the Anthozoa unsuitable for DNA barcoding. Caution is also advised for Porifera and Hydrozoa because of relatively low identification success rates as even threshold divergence that maximizes the "barcoding gap" does not improve identification success.

Parallel tagged sequencing on the 454 platform - Jan 31, 2008 ()
[Meyer, M., Stenzel, U., & Hofreiter, M 2008. Nature Protocols. 3(2) 267-78.]

Parallel tagged sequencing (PTS) is a molecular barcoding method designed to adapt the recently developed high-throughput 454 parallel sequencing technology for use with multiple samples. Unlike other barcoding methods, PTS can be applied to any type of double-stranded DNA (dsDNA) sample, including shotgun DNA libraries and pools of PCR products, and requires no amplification or gel purification steps. The method relies on attaching sample-specific barcoding adapters, which include sequence tags and a restriction site, to blunt-end repaired DNA samples by ligation and strand-displacement. After pooling multiple barcoded samples, molecules without sequence tags are effectively excluded from sequencing by dephosphorylation and restriction digestion, and using the tag sequences, the source of each DNA sequence can be traced. This protocol allows for sequencing 300 or more complete mitochondrial genomes on a single 454 GS FLX run, or twenty-five 6-kb plasmid sequences on only one 16th plate region. Most of the reactions can be performed in a multichannel setup on 96-well reaction plates, allowing for processing up to several hundreds of samples in a few days

Using DNA barcodes to assess identity and diversity of Dendropsophus minutus: Failure? - Jan 29, 2008 ()
[Smith, M.A. 2008. Zootaxa. 1691 67-68.]

Read the publication here.

DNA barcoding Australasian chondrichthyans: results and potential uses in conservation - Jan 25, 2008 ()
[Ward, R. D., Holmes, B. H., William, T. W., and Last, P. R. 2008. Marine and Freshwater Research. 59(1) 57-71.]

DNA barcoding – sequencing a region of the mitochondrial cytochrome c oxidase 1 gene (cox1) – promises a rapid and accurate means of species identification, and of any life history stage. For sharks and rays, it may offer a ready means of identifying legal or illegal shark catches, including shark fins taken for the profitable shark fin market. Here it is shown that an analysis of sequence variability in a 655 bp region of cox1 from 945 specimens of 210 chondrichthyan species from 36 families permits the discrimination of 99.0% of these species. Only the two stingarees Urolophus sufflavus and U. cruciatus could not be separated, although these could be readily distinguished from eight other congeners. The average Kimura 2 parameter distance separating individuals within species was 0.37%, and the average distance separating species within genera was 7.48%. Two specimens that clustered with congeners rather than with their identified species-cluster were noted: these could represent instances of hybridisation (although this has not be documented for chondrichthyans), misidentification or mislabelling. It is concluded that cox1 barcoding can be used to identify shark and ray species with a very high degree of accuracy. The sequence variability characteristics of individuals of five species (Aetomylaeus nichofii, Dasyatis kuhlii, Dasyatis leylandi, Himantura gerrardi and Orectolobus maculatus) were consistent with cryptic speciation, and it is suggested that these five taxa be subjected to detailed taxonomic examination to confirm or refute this suggestion. The present barcoding study holds out great hope for the ready identification of sharks, shark products and shark fins, and also highlights some taxonomic issues that need to be investigated further.

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DNA barcoding and the documentation of alien species establishment on sub-Antarctic Marion Island - Jan 11, 2008 ()
[Chown, S. L., Sinclair, B. J., and Jansen van Vuuren, B. 2008. Polar Biology. .]

Invasive alien species constitute a substantial conservation challenge in the terrestrial sub-Antarctic. Management plans, for many of the islands in the region, call for the prevention, early detection, and management of such alien species. However, such management may be confounded by difficulties of identification of immatures, especially of holometabolous insects. Here we show how a DNA barcoding approach has helped to overcome such a problem associated with the likely establishment of an alien moth species on Marion Island. The discovery of unidentifiable immatures of a noctuid moth species, 5 km from the research station, suggested that a new moth species had colonized the island. Efforts to identify the larvae by conventional means or by rearing to the adult stage failed. However, sequencing of 617 bp of the mitochondrial cytochrome oxidase subunit I gene, and comparison of the sequence data with sequences on GENBANK and the barcoding of life database enabled us to identify the species as Agrotis ipsilon (Hufnagel), a species of which adults had previously been found regularly at the research station. Discovery of immatures of this species, some distance from the research station, suggests that a population may have established. It is recommended that steps to be taken to eradicate the species from Marion Island.

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DNA barcoding of animal species-response to DeSalle - Jan 01, 2008 ()
[Waugh, J.,Huynen, L., Millar, C., and D. Lambert 2008. Bioessays. 30(1) 92-3.]

A novel, high-throughput technique for species identification reveals a new species of tsetse-transmitted trypanosome related to the Trypanosoma brucei subgenus, Trypanozoon - Jan 01, 2008 ()
[Hamilton, P. B. , Adams, E. R., Malele, I.I. and W. C. Gibson 2008. Infect Genet Evol. 8(1) 26-33.]

We describe a novel method of species identification, fluorescent fragment length barcoding, based on length variation in regions of the 18S and 28Salpha ribosomal DNA. Fluorescently tagged primers, designed in conserved regions of the 18S and 28Salpha ribosomal DNA, were used to amplify fragments with inter-species size variation, and sizes determined accurately using an automated DNA sequencer. By using multiple regions and different fluorochromes, a barcode unique to each species was generated. The technique was developed for the identification of African tsetse-transmitted trypanosomes and validated using DNA from laboratory isolates representing known species, subspecies and subgroups. To test the methodology, we examined 91 trypanosome samples from infected tsetse fly midguts from Tanzania, most of which had already been identified by species-specific and generic PCR tests. Identifications were mainly in agreement, but the presence of an unknown trypanosome in several samples was revealed by its unique barcode. Phylogenetic analyses based on 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase gene sequences confirmed that this trypanosome is a new species and it is within the Trypanosoma brucei clade, as a sister group of subgenus Trypanozoon. The overall identification rate of trypanosome-infected midgut samples increased from 78 to 96% using FFLB instead of currently available PCR tests. This was due to the high sensitivity of FFLB as well as its capacity to identify previously unrecognised species. FFLB also allowed the identification of multiple species in mixed infections. The method enabled high-throughput and accurate species identification and should be applicable to any group of organisms where there is length variation in regions of rDNA.

Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects - Jan 01, 2008 ()
[Hunter, S.J., T.I. Goodall, K.A. Walsh, R. Owen and J.C. Day 2008. Molecular Ecology Resources. 8(1) 56-61.]

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.

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The missing fungi - New insights from culture-independent molecular studies of soil. - Jan 01, 2008 ()
[Schmidt, S. K., Wilson, K. L., Meyer, A. F., Porter, T. M., Schadt, C. W., & Moncalvo, J. M. 2008. In K. Zengler (Ed.), Acessing Uncultivated Microorganisms. Washington, DC: American Society for Microbiology Press 55-66.]

Potential use of DNA barcodes in regulatory science: applications of the Regulatory Fish Encyclopedia - Jan 01, 2008 ()
[Yancy, H. F., Zemlak, T. S., Mason, J. A., Washington, J. D., Tenge, B. J., Nguyen, N. L., Barnett, J. D., Savary, W. E., Hill, W. E., Moore, M. M., Fry, F. S., Randolph, S. C., Rogers, P. L., & Hebert, P. D.N. 2008. J Food Prot. 71(1) 210-217.]

The use of a DNA-based identification system (DNA barcoding) founded on the mitochondrial gene cytochrome c oxidase subunit I (COI) was investigated for updating the U.S. Food and Drug Administration Regulatory Fish Encyclopedia (RFE; http://www.cfsan.fda.gov/-frf/rfe0.html). The RFE is a compilation of data used to identify fish species. It was compiled to help regulators identify species substitution that could result in potential adverse health consequences or could be a source of economic fraud. For each of many aquatic species commonly sold in the United States, the RFE includes high-resolution photographs of whole fish and their marketed product forms and species-specific biochemical patterns for authenticated fish species. These patterns currently include data from isoelectric focusing studies. In this article, we describe the generation of DNA barcodes for 172 individual authenticated fish representing 72 species from 27 families contained in the RFE. These barcode sequences can be used as an additional identification resource. In a blind study, 60 unknown fish muscle samples were barcoded, and the results were compared with the RFE barcode reference library. All 60 samples were correctly identified to species based on the barcoding data. Our study indicates that DNA barcoding can be a powerful tool for species identification and has broad potential applications.

Undisciplined thinking: morphology and Hennig’s unfinished revolution - Jan 01, 2008 ()
[Wheeler, Q.D. 2008. Systematic Entomology. 33(1) 2-7.]
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Redescription of Coryphopterus tortugae (Jordan) and a new allied species Coryphopterus bol (Perciformes: Gobiidae: Gobiinae) from the tropical western Atlantic Ocean - Jan 01, 2008 ()
[Victor, B. C. 2008. Journal of the Ocean Science Foundation. 1 1-19.]

A re-examination of the holotype and mtDNA barcoding confirms Garzon-Ferreira and Acero’s separation of Coryphopterus tortugae from C. glaucofraenum. However, specimens matching the markings of their Santa Marta variant of C. tortugae comprise a distinct clade about 10% sequence divergent from true C. tortugae and C. glaucofraenum. The variant is described here as a new species, the sand canyon goby Coryphopterus bol, from specimens collected in Puerto Rico, the US Virgin Islands, and the Atlantic coast of Panama. The new species is abundant and widespread in the region and has not been recognized as distinct from the bridled goby, C. glaucofraenum. C. bol can be distinguished from C. tortugae by markings: a dark oval spot on the lower third of the pectoral-fin base, a chain-link pattern of melanophores on the top of the head, and a thick C-shaped basicaudal mark and scale-edges outlined in lines of tiny melanophores on well-marked individuals. Putative bridled gobies from three different reef types were sampled: a wide and clearly-zoned shelf in the Greater Antilles (off La Parguera, Puerto Rico), a narrower mixed-zone island of the Lesser Antilles (St. Thomas, US Virgin Islands), and narrow fringing continental reefs in the Southern Caribbean (the Atlantic coast of Panama near Portobelo). In all three locations, C. bol was found in deeper and more offshore reef areas with strong currents, i.e. in the channels of the buttress-canyon zone just inshore of the drop-off in Puerto Rico, around exposed rocky points in St. Thomas, and on wave-swept reefs just offshore of the sediment influenced coastline in Panama.

Disentangling taxonomy within the Rhabditis (Pellioditis) marina (Nematoda, Rhabditidae) species complex using molecular and morhological tools. - Jan 01, 2008 ()
[Derycke, S., Fonseca, G., Vierstraete, A., Vanfleteren, J., Vincx, M., & Moens, T. 2008. Zoological Journal of the Linnean Society,. 152(1) 1-15.]

Correct taxonomy is a prerequisite for biological research, but currently it is undergoing a serious crisis, resulting in the neglect of many highly diverse groups of organisms. In nematodes, species delimitation remains problematic due to their high morphological plasticity. Evolutionary approaches using DNA sequences can potentially overcome the problems caused by morphology, but they are also affected by theoretical flaws. A holistic approach with a combination of morphological and molecular methods can therefore produce a straightforward delimitation of species. The present study investigates the taxonomic status of some highly divergent mitochondrial haplotypes in the Rhabditis (Pellioditis) marina species complex by using a combination of molecular and morphological tools. We used three molecular markers (COI, ITS, D2D3) and performed phylogenetic analyses. Subsequently, morphometric data from nearly all lineages were analysed with multivariate techniques. We included R. (P.) mediterranea and R. (R.) nidrosiensis to infer species status of the observed lineages. The results showed that highly divergent genotypic clusters were accompanied by morphological differences, and we created a graphical polytomous key for future identifications. This study indisputably demonstrates that R. (P.) marina and R. (P.) mediterranea belong to a huge species complex and that biodiversity in free-living marine nematodes may be seriously underestimated.

MtDNA COI barcodes reveal cryptic diversity in the Baetis vernus group (Ephemeroptera, Baetidae) - Jan 01, 2008 ()
[Stahls, G., and Savolainen, E. 2008. Molecular phylogenetics and evolution. 46(1) 82-87.]

Partial mitochondrial COI sequences (barcoding fragment) were explored for the understanding of the species boundaries of Baetis vernus group taxa (Ephemeroptera, Baetidae) in northern Europe. We sampled all species of this group occurring in Finland, but focused on taxa for which morphological and taxonomical confusion have been most apparent. The sequence matrix comprised 627 nucleotides for 96 specimens, and was analysed using parsimony. Results provided strong evidence that Baetis macani Kimmins and B. vernus Curtis comprise morphologically cryptic but molecularly distinct taxa, as intraspecific uncorrected divergences within haplogroups ranged between 0.3% and 1.4% and interspecific divergences were from 13.1% to 16.5%. These interesting findings prompt for further taxonomic studies of B. vernus taxa using more extensive specimen sampling from the known distributional areas in the Palaearctic/Holarctic region for better understanding of haplotype distributions. We stress the importance of integration of morphological and molecular data, and the necessity to employ additional nuclear DNA sequence data.

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DNA barcoding reveals cryptic diversity in marine hydroids (Cnidaria, Hydrozoa) from coastal and deep-sea environments - Jan 01, 2008 ()
[Moura, C. J., Harris, D. J., Cunha, M. R., and Rogers, A. D. 2008. Zoologica Scripta. 37(1) 93-108.]

Fifty-six sequences of the mitochondrial 16S RNA gene were generated for hydroids, belonging to six nominal families — Eudendriidae, Lafoeidae, Haleciidae, Sertulariidae, Plumulariidae and Aglaopheniidae — collected from bathyal environments of the Gulf of Cadiz (22 haplotypes), Greenland (1 haplotype), Azores (1 haplotype), the shallow waters of the UK (17 haplotypes) and Portugal (2 haplotypes). When combined and analysed with 68 additional sequences published in GenBank, corresponding to 63 nominal species of these families (nine species in common between the GenBank sequences and those presented by the authors), cryptic species were detected (e.g. two species of Nemertesia and other of Lafoea), as well as apparent cases of conspecificity (e.g. Nemertesia antennina and N. perrieri and Aglaophenia octodonta, A. pluma and A. tubiformis). Other taxonomic inconsistencies were found in the data including cases where species from different genera clustered together (e.g. Sertularia cupressina, Thuiaria thuja, Abietinaria abietina and Ab. filicula). The mitochondrial 16S rRNA proved to be a useful DNA ‘barcode’ gene for hydroids, not only allowing discrimination of species, but also in some cases of populations, genera and families, and their intra- or interphylogenetic associations. Although still under-represented in public data bases, the 16S rRNA gene is starting to be used frequently in the study of hydroids. These data provide powerful complementary evidence for advancing our understanding of hydrozoan systematics.

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Feeding ecology of Xenoturbella bocki (phylum Xenoturbellida) revealed by genetic barcoding - Jan 01, 2008 ()
[Bourlat, S.J., H. Nakano, M. Akerman, M.J. Telford, M.C. Thorndyke and M. Obst 2008. Molecular Ecology Resources. 8(1) 18-22.]

The benthic marine worm Xenoturbella is frequently contaminated with molluscan DNA, which had earlier caused confusion resulting in a suggested bivalve relationship. In order to find the source of the contaminant, we have used molluscan sequences derived from Xenoturbella and compared them to barcodes obtained from several individuals of the nonmicroscopic molluscs sharing the same environment as Xenoturbella. Using cytochrome oxidase 1, we found the contaminating sequences to be 98% similar to the bivalve Ennucula tenuis. Using the highly variable D1–D2 region of the large ribosomal subunit in Xenoturbella, we found three distinct species of contaminating molluscs, one of which is 99% similar to the bivalve Abra nitida, one of the most abundant bivalves in the Gullmarsfjord where Xenoturbella was found, and another 99% similar to the bivalve Nucula sulcata. These data clearly show that Xenoturbella only contains molluscan DNA originating from bivalves living in the same environment, refuting former hypotheses of a bivalve relationship. In addition, these data suggest that Xenoturbella feeds specifically on bivalve prey from multiple species, possibly in the form of eggs and larvae.

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An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa - Dec 31, 2007 ()
[Meldal, B. H., Debenham, N. J., De Ley, P., De Ley, I. T., Vanfleteren, J. R., Vierstraete, A. R., Bert, W., Borgonie, G., Moens, T., Tyler, P. A., Austen, M. C., Blaxter, M. L., Rogers, A. D., & P.J. Lambshead 2007. Mol. Phylogenet. Evol.. 42(3) 622-36.]

Phylogenetic reconstructions of relations within the phylum Nematoda are inherently difficult but have been advanced with the introduction of large-scale molecular-based techniques. However, the most recent revisions were heavily biased towards terrestrial and parasitic species and greater representation of clades containing marine species (e.g. Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, Enoplida, and Monhysterida) is needed for accurate coverage of known taxonomic diversity. We now add small subunit ribosomal DNA (SSU rDNA) sequences for 100 previously un-sequenced species of nematodes, including 46 marine taxa. SSU rDNA sequences for >200 taxa have been analysed based on Bayesian inference and LogDet-transformed distances. The resulting phylogenies provide support for (i) the re-classiWcation of the Secernentea as the order Rhabditida that derived from a common ancestor of chromadorean orders Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, and Monhysterida and (ii) the position of Bunonema close to the Diplogasteroidea in the Rhabditina. Other, previously controversial relationships can now be resolved more clearly: (a) Alaimus, Campydora, and Trischistoma belong in the Enoplida, (b) Isolaimium is placed basally to a big clade containing the Axonolaimidae, Plectidae, and Rhabditida, (c) Xyzzors belongs in the Desmodoridae, (d) Comesomatidae and Cyartonema belongs in the Monhysterida, (e) Globodera belongs in the Hoplolaimidae and (f) Paratylenchus dianeae belongs in the Criconematoidea. However, the SSU gene did not provide signiWcant support for the class Chromadoria or clear evidence for the relationship between the three classes, Enoplia, Dorylaimia, and Chromadoria. Furthermore, across the whole phylum, the phylogenetically informative characters of the SSU gene are not informative in a parsimony analysis, highlighting the short-comings of the parsimony method for large-scale phylogenetic modelling.

Lumping lumpsuckers: molecular and morphological insights into the taxonomic status of Eumicrotremus spinosus (Fabricius, 1776) and E. eggvinii Koefoed, 1956 (Teleostei: Cyclopteridae) - Dec 31, 2007 ()
[Byrkjedal I., Rees D. and E. Willassen 2007. Journal of Fish Biology. 71 (Suppl A) 111-131.]

The genus Eumicrotremus comprises 16 lumpsucker species distributed in the Arctic and northern Atlantic and Pacific Oceans. The most common species in the North Atlantic is Eumicrotremus spinosus, described in 1776, and characterized partly by numerous bony tubercles on the head and body. Another Atlantic species, Eumicrotremus eggvinii, described in 1956, remained known only from a single specimen until additional specimens were recently recovered. To reassess the status of E. eggvinii, 21 meristic and 32 morphometric characters were analysed for a total of 83 specimens of E. spinosus and E. eggvinii. Mitochondrial (COI, COII and cyt-b) and nuclear (Tmo-4C4) genes were also sequenced for both species, along with Eumicrotremus derjugini. The results indicate that although E. spinosus and E. eggvinii are clearly separated by a considerable number of morphological characters, they in fact constitute a single, sexually dimorphic species. Thirteen specimens of E. eggvinii (including the holotype) and 59 E. spinosus could be sexed; all individuals of E. eggvinii turned out to be males and all E. spinosus were females. Identical DNA sequences were found in all E. eggvinii and E. spinosus for COI, COII and Tmo-4C4, and a single shared synonymous substitution found in cyt-b. In contrast, E. spinosus, E. eggvinii and E. derjugini differed by 5.9% for COI and COII, 1.2% for Tmo-4C4 and 8.3% for cyt-b.

DNA barcoding in Greek freshwater fish: the cases of Doirani and Volvi lakes - Dec 31, 2007 ()
[Triantafyllidis, A., Bobori, D.C. and C. Koliamitra 2007. 11th Congress of The European Society for Evolutionary Biology, Uppsala, Sweden, August 20-25. 432.]

The scope of the present research was to initiate and apply DNA barcoding in Greek freshwater fish species aiming to reveal new approaches on their protection and sustainable management as well as unmask look-alikes to prevent falsification. In the present study DNA barcoding was carried out in a total of 141 individuals, representing 18 fish species from both lakes Doirani and Volvi (Northern Greece). A 655bp region of the mitochondrial cytochrome oxidase subunit I (cox1) was sequenced using universal primers. Average within-species, -family and -order Kimura two parameter (K2P) distances were 0.41%, 14.9% and 15.6% respectively. All species could be differentiated by their cox1 sequence. Barcoded common species from both lakes had lake-specific haplotypes, indicating that location-based discrimination of species is possible. After constructing neighbor joining phylogenetic trees, the clades revealed generally corresponded well with expectations. Our study supports previous studies on the conclusion that cox1 sequencing, or ‘barcoding’, can be used to identify fish species.

Mitochondrial DNA phylogeography and mating compatibility reveal marked genetic structuring and speciation in the NE Atlantic bryozoan Celleporella hyalina - Dec 31, 2007 ()
[Gómez A., Hughes Roger N., Wright Peter J., Carvalho Gary R., Lunt, David H. 2007. Molecular Ecology. 16(10) 2173-2188.]

The marine bryozoan Celleporella hyalina is a species complex composed of many highly divergent and mostly allopatric genetic lineages that are reproductively isolated but share a remarkably similar morphology. One such lineage commonly encrusts macroalgae throughout the NE Atlantic coast. To explore the processes leading to geographical diversification, reproductive isolation and speciation in this taxon, we (i) investigated NE Atlantic C. hyalina mitochondrial DNA phylogeography, and (ii) used breeding trials between geographical isolates to ascertain reproductive isolation. We find that haplotype diversity is geographically variable and there is a strong population structure, with significant isolation by distance. NE Atlantic C. hyalina is structured into two main parapatric lineages that appear to have had independent Pleistocene histories. Range expansions have resulted in two contact zones in Spain and W Ireland. Lineage 1 is found from Ireland to Spain and has low haplotype diversity, with closely related haplotypes, suggesting a recent population expansion into the Irish Sea, S Ireland, S England and Spain. Lineage 2 is found from Iceland to Spain and has high haplotype diversity. Complete reproductive isolation was found between some geographical isolates representing both lineages, whereas it was incomplete or asymmetric between others, suggesting these latter phylogeographical groups probably represent incipient species. The phylogeographical distribution of NE Atlantic C. hyalina does not fall easily into a pattern of southern refugia, and we discuss likely differences between terrestrial and marine system responses to Pleistocene glacial cycles.

A new handfish, Brachionichthys australis sp. nov. (Lophiiformes:Brachionichthyidae), with a redescription of the critically endangered spotted handfish, B. hirsutus (Lacepède) - Dec 19, 2007 ()
[Last, P. R., Gledhill, D. C., and Holmes, B. H. 2007. Zootaxa. 1666 53-68.]

The micro-endemic Spotted Handfish, Brachionichthys hirsutus (Lacepède), which was discovered off Tasmania by the French explorer François Péron in the early 19th century, is now endangered. A second spotted handfish of the genus Brachionichthys was first identified in the early 1980’s and is formally described based on material from southern Australia. Brachionichthys australis sp. nov. differs from its close congener B. hirsutus, in having a larger eye, longer illicium with a smaller esca, longer first dorsal-fin ray, fewer second dorsal-fin rays, shorter second dorsal-fin base, and a more subtle colour pattern. It also has a sparse covering of long, thin streaks and stripes (rather than small spots or short streaks), and the caudal fin is sparsely spotted (densely covered with fine spots that usually form a dark submarginal bar in B. hirsutus). They can also be distinguished using molecular analysis. Brachionichthys australis, which has a much wider geographic distribution and depth range than B. hirsutus, occurs mainly on the continental shelf of southern Australia from Bermagui (New South Wales) to the western sector of the Great Australian Bight (Western Australia), including eastern Tasmania south to the D’Entrecasteaux Channel, at depths of 18–210 m (and possibly to 277 m). Brachionichthys hirsutus, which is endemic to southeastern Tasmania where it was once common in bays and estuaries at depths of 1–60 m, is now considered by the IUCN to be critically endangered. These species, known in the vernacular as the spotted handfishes, are otherwise similar in appearance and may have been confused in the past.

Molecular diagnostics of soilborne fungal pathogens and applications to disease management - Dec 01, 2007 ()
[Lévesque, C. A. 2007. In Z. K. Punja, S. DeBoer and H. Sanfaçon (Eds.), Biotechnology and Plant Disease Management. Wallingford, UK CABI Books 146-164.]

Deep genetic divergences in Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi): a widespread ‘mite harvestman’ defies DNA taxonomy - Dec 01, 2007 ()
[Boyer, S.L., Baker J.M. and Giribet, G. 2007. Molecular Ecology. 16(23) 4999-5016.]

Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi, Pettalidae), a widespread ‘mite harvestman’ endemic to the South Island of New Zealand, is found in leaf littler habitats throughout Nelson and Marlborough, and as far south as Arthur's Pass. We investigated the phylogeography and demographic history of A. denticulata in the first genetic population-level study within Opiliones. A total of 119 individuals from 17 localities were sequenced for 785 bp of the gene cytochrome c oxidase subunit I; 102 of these individuals were from the Aoraki subspecies A. denticulata denticulata and the remaining 17 were from the subspecies A. denticulata major. An extraordinarily high degree of genetic diversity was discovered in A. denticulata denticulata, with average uncorrected p-distances between populations as high as 19.2%. amova, average numbers of pairwise differences, and pairwise FST values demonstrated a significant amount of genetic diversity both within and between populations of this subspecies. Phylogenetic analysis of the data set revealed many well-supported groups within A. denticulata denticulata, generally corresponding to clusters of specimens from single populations with short internal branches, but separated by long branches from individuals from other populations. No haplotypes were shared between populations of the widespread small subspecies, A. denticulata denticulata. These results indicate a subspecies within which very little genetic exchange occurs between populations, a result consistent with the idea that Cyphophthalmi are poor dispersers. The highly structured populations and deep genetic divergences observed in A. denticulata denticulata may indicate the presence of cryptic species. However, we find a highly conserved morphology across sampling localities and large genetic divergences within populations from certain localities, equivalent to those typically found between populations from different localities. Past geological events may have contributed to the deep genetic divergences observed between sampling localities; additionally, the high divergence within populations of A. denticulata denticulata suggests that the rate of COI evolution may be accelerated in this taxon. In contrast, the larger subspecies A. denticulata major shows much less differentiation between and within sampling localities, suggesting that it may disperse more easily than its smaller counterpart. The fact that the remarkable genetic divergences within populations of A. denticulata denticulata from certain localities are equivalent to divergences between localities poses a challenge to the rapidly spreading practice of DNA taxonomy.

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Poplar leaf rusts: model pathogens for a model tree - Dec 01, 2007 ()
[Feau, N., Joly, D. L., & Hamelin, R. C. 2007. Canadian Journal of Botany. 85(12) 1127-1135.]

With the availability of the entire genome of the model tree Populus trichocarpa Torr. & A. Gray and the current genome sequencing project of its rust pathogen Melampsora larici-populina Kleb., rust–poplar interaction research has entered the genomic era. Recent genomics research on poplars has attempted to connect the genetic localizations of loci for qualitative and quantitative disease resistance with putative genes encoding resistance or signalling proteins. The interactions between these putative resistance genes and rust effectors remain unknown. Genomic resources developed for Melampsora spp. promise to contribute to our understanding of the molecular basis of pathogenicity by facilitating the isolation of pathogenicity genes. A multifaceted approach for the identification of such genes that relies largely on trimming and sequence data analysis has been developed. The strategy takes advantage of the resources available and combines EST libraries, bioinformatics data mining for extracellularly expressed secreted proteins, intra- and inter-specific comparative genomics, and testing for the presence of positive selection. It has resulted in the discovery of several putative candidate genes. In silico evidence for candidate genes will be further validated by robust experimental evidence through functional analyses.

Species delimitation: new approaches for discovering diversity - Dec 01, 2007 ()
[Wiens, J. J. 2007. Systematic Biology. 56(6) 875-8.]

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The European land leech: biology and DNA-based taxonomy of a rare species that is threatened by climate warming - Dec 01, 2007 ()
[Kutschera, U., Pfeiffer, I., & Ebermann, E. 2007. Naturwissenschaften. 94(12) 967-974.]

The European land leech Xerobdella lecomtei was discovered in 1868 and is one of the rarest animals on Earth. During the 1960s, several individuals of these approx. 40 mm long, cold-adapted terrestrial annelids that inhabit the moist soils of birch forests around Graz, Austria, were investigated. Only one original research paper has been published on the biology of this species. Between 2001 and 2005, we re-investigated the morphology of preserved specimens and searched for living individuals in their natural habitat that appeared to be intact. We found only one juvenile individual (length approx. 10 mm), indicating that this local leech population became largely extinct over the past four decades. The feeding behaviour of our 'lonesome George of the annelids' was studied and is described here in detail. After its death, the Xerobdella individual was used for chemical extraction and molecular studies (deoxyribonucleic acid [DNA] barcoding, based on one gene, the mitochondrial cytochrome c oxidase subunit I). In addition, novel DNA barcodes for a land leech from Madagascar and a recently discovered species from Europe were obtained. Our phylogenetic tree shows that X. lecomtei is not a member of the tropical land leeches (family Haemadipsidae), as previously thought, but represents a separate line of descent (family Xerobdellidae). The decline of the local leech population around Graz correlates with a rise in average summer temperatures of +3 degrees C between 1961 and 2004. This warming led to a drastic reduction in the moisture content of the soil where X. lecomtei lives. We suggest that human-induced climate change without apparent habitat destruction can lead to the extinction of populations of cold-adapted species that have a low colonization ability.

Species concepts and species delimitation - Dec 01, 2007 ()
[De Queiroz, K. 2007. Systematic Biology. 56(6) 879-86.]

The issue of species delimitation has long been confused with that of species conceptualization, leading to a half century of controversy concerning both the definition of the species category and methods for inferring the boundaries and numbers of species. Alternative species concepts agree in treating existence as a separately evolving metapopulation lineage as the primary defining property of the species category, but they disagree in adopting different properties acquired by lineages during the course of divergence (e.g., intrinsic reproductive isolation, diagnosability, monophyly) as secondary defining properties (secondary species criteria). A unified species concept can be achieved by treating existence as a separately evolving metapopulation lineage as the only necessary property of species and the former secondary species criteria as different lines of evidence (operational criteria) relevant to assessing lineage separation. This unified concept of species has several consequences for species delimitation, including the following: First, the issues of species conceptualization and species delimitation are clearly separated; the former secondary species criteria are no longer considered relevant to species conceptualization but only to species delimitation. Second, all of the properties formerly treated as secondary species criteria are relevant to species delimitation to the extent that they provide evidence of lineage separation. Third, the presence of any one of the properties (if appropriately interpreted) is evidence for the existence of a species, though more properties and thus more lines of evidence are associated with a higher degree of corroboration. Fourth, and perhaps most significantly, a unified species concept shifts emphasis away from the traditional species criteria, encouraging biologists to develop new methods of species delimitation that are not tied to those properties.

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Lepidoptera of Great Smoky Mountains National Park: Methods and Results of the Inventory - Dec 01, 2007 ()
[Scholtens, B.G. & Wagner, D.L. 2007. Southeastern Naturalist. Special Issue 1 193-206.]

As a part of an All Taxa Biodiversity Inventory, we documented 1843 Lepidoptera species from Great Smoky Mountains National Park. This total is based on previous survey efforts plus recent intensive sampling using bioblitzes. Various statistical estimators put the total number of species in the park between 1950 and 2340. The difference between actual and estimated numbers is mainly due to under-sampled Microlepidoptera groups. As a part of recent efforts, the mitochondrial COI gene has been sequenced from more than 940 species from the Park. DNA barcoding has already led to taxonomic insights in several groups, and is believed to be at least 95% accurate for identifications. Our samples include more than 20 undescribed species in the Park, including a park-endemic geometrid moth. Because of threats to their habitats, high-elevation species make up the largest group of species of special concern.

On the species-specificity of DNA: fifty years later - Dec 01, 2007 ()
[Shneyer, V. S. 2007. Biochemistry (Mosc). 72(12) 1377-84.]

Modern approaches in DNA-based species identification are considered. Long used methods of species identification in procaryotes (G+C ratio, 16S rRNA nucleotide sequence, DNA-DNA hybridization) have recently been supplemented by the method of multilocus sequence analysis based on comparison of nucleotide sequences of fragments of several genes. Species identification in eukaryotes also employs one or two standard short fragments of the genome (known as DNA-barcodes). Potential benefits of new approaches and some difficulties during their practical realization are discussed.

Delimiting Species without Monophyletic Gene Trees - Dec 01, 2007 ()
[Knowles, L. L., and Carstens, B. C. 2007. Systematic Biology. 56(6) 887-95.]

Genetic data are frequently used to delimit species, where species status is determined on the basis of an exclusivity criterium, such as reciprocal monophyly. Not only are there numerous empirical examples of incongruence between the boundaries inferred from such data compared to other sources like morphology - especially with recently derived species, but population genetic theory also clearly shows that an inevitable bias in species status results because genetic thresholds do not explicitly take into account how the timing of speciation influences patterns of genetic differentiation. This study represents a fundamental shift in how genetic data might be used to delimit species. Rather than equating gene trees with a species tree or basing species status on some genetic threshold, the relationship between the gene trees and the species history is modeled probabilistically. Here we show that the same theory that is used to calculate the probability of reciprocal monophyly can also be used to delimit species despite widespread incomplete lineage sorting. The results from a preliminary simulation study suggest that very recently derived species can be accurately identified long before the requisite time for reciprocal monophyly to be achieved following speciation. The study also indicates the importance of sampling, both with regards to loci and individuals. Withstanding a thorough investigation into the conditions under which the coalescent-based approach will be effective, namely how the timing of divergence relative to the effective population size of species affects accurate species delimitation, the results are nevertheless consistent with other recent studies (aimed at inferring species relationships), showing that despite the lack of monophyletic gene trees, a signal of species divergence persists and can be extracted. Using an explicit model-based approach also avoids two primary problems with species delimitation that result when genetic thresholds are applied with genetic data - the inherent biases in species detection arising from when and how speciation occurred, and failure to take into account the high stochastic variance of genetic processes. Both the utility and sensitivities of the coalescent-based approach outlined here are discussed; most notably, a model-based approach is essential for determining whether incompletely sorted gene lineages are (or are not) consistent with separate species lineages, and such inferences require accurate model parameterization (i.e., a range of realistic effective population sizes relative to potential times of divergence for the purported species). It is the goal (and motivation of this study) that genetic data might be used effectively as a source of complementation to other sources of data for diagnosing species, as opposed to the exclusion of other evidence for species delimitation, which will require an explicit consideration of the effects of the temporal dynamic of lineage splitting on genetic data.

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Rice Black Bugs; Taxonomy, Ecology and Management of Invasive Species - Dec 01, 2007 (pdf)
[Ravindra C. Joshi, Alberto T. Barrion, Leocadio S. Sebastian 2007. ]
Reconstructing the Tree of Life: Taxonomy and Systematics of Species Rich Taxa - Dec 01, 2007 ()
[Soltis, D. E. 2007. Evolution. 61(12) 3007–3011.]

Those not involved in evolutionary biology are often surprised that we are far from answering two fundamental questions concerning biological diversity—how many species are there? and how are these species related evolutionarily? The recent edited volume by Hodkinson and Parnell, Reconstructing the tree of life: taxonomy and systematics of species rich groups, overviews some of the many challenges involved in addressing these basic questions, with a special emphasis on the need "to tackle its species-rich groups." There are good reasons, they argue, for basing an entire book on species-rich taxa—large genera constitute a "large portion of the diversity we seek to describe." For example, more than 50 seed plant genera have > 500 species each; 20 have over 1000 each (Ronsted et al., p. 130). Yet, systematists tend to steer clear of many species-rich groups—particularly at the generic level. Such genera are too complex to break into "bite-sized" pieces; too much to give Ph.D. students (at least ones we like) as a discrete four to five year project; too risky to take on before we have tenure.

The chapters in this volume are divided into three general areas: (1) Introduction and general context, (2) Reconstructing and using the tree of life, (3) Overview of species-rich groups (case studies). I overview some of the major topics covered in this volume and discuss several broader issues involving species-rich groups. For example, how much research effort do we actually spend on species-rich groups? Should species-rich groups merit the lion's share of our research effort and funding?

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Revision of the genus Absidia (Mucorales, Zygomycetes) based on physiological, phylogenetic and morphological characters: Thermotolerant Absidia spp. form a coherent group, the Mycocladiaceae fam. - Nov 30, 2007 ()
[Hoffmann, K., Discher, S. and Voigt, K. 2007. Mycological Research. 111(10) 1169-1183.]

The genus Absidia comprise ubiquitously distributed soil fungi inhabiting different growth temperature optima ranging from 20 °C to 42 °C. While few of the mesophilic species imply biotechnological importance in biotransformation of steroids or as producers of rennin-like components, the species with higher growth temperature optima are of clinical relevance as opportunistic human pathogens. The aim of this study was to investigate the phylogenetic relationships between these species and to establish a revision of their systematics. For this purpose single and combined genealogies based on distance, maximum parsimony, maximum likelihood and Bayesian analyses of aligned nucleotide sequences of the nuclear-encoded genes for actin (act) and for the 5.8S ribosomal RNA flanked by the internal transcribed spacer (ITS) regions 1 and 2 (comprising 807 and 828 characters, respectively) of 16 Absidia species were reconstructed. The phylogenetic reconstructions suggest a trichotomy of the Absidia genus consisting of a mesophilic, a fast-growing thermotolerant and a slowly-growing mycoparasitic Absidia group. The trichotomous phylogenetic grouping is concordant with the morphology of the zygospores, which are zygotes resulting from sexual conjugation between two compatible mating partners. Whereas the mesophilic group comprises the majority of absidiaceaeous species forming sterile hair–like, mycelial appendages on the suspensors of their zygospores, the thermotolerant group is characterised by the formation of smooth-walled and the mycoparasitic group, namely Absidia parricida and A. zychae, by Mucor-like rough-walled zygospores. Based on the phylogenetic coherence of mesophilic and thermotolerant Absidia species, we propose to separate both groups into two distinct genera, Absidia for the mesophilic Absidia species resembling the Absidiaceae and Mycocladus for the thermotolerant species A. corymbifera, A. blakesleeana and A. hyalospora. Because Mycocladus is physiologically, phylogenetically and morphologically distinct from the Absidiaceae sensu stricto we suggest to classify them into a separate family, the Mycocladiaceae fam. nov., which comprises the three species Mycocladus corymbifer, M. blakesleeanus and M. hyalospora.

Back to the future: museum specimens in population genetics - Nov 04, 2007 ()
[Wandeler, P., Hoeck, P. E., and Keller, L. F. 2007. Trends in Ecology & Evolution. OnlineEarly .]

Museums and other natural history collections (NHC) worldwide house millions of specimens. With the advent of molecular genetic approaches these collections have become the source of many fascinating population studies in conservation genetics that contrast historical with present-day genetic diversity. Recent developments in molecular genetics and genomics and the associated statistical tools have opened up the further possibility of studying evolutionary change directly. As we discuss here, we believe that NHC specimens provide a largely underutilized resource for such investigations. However, because DNA extracted from NHC samples is degraded, analyses of such samples are technically demanding and many potential pitfalls exist. Thus, we propose a set of guidelines that outline the steps necessary to begin genetic investigations using specimens from NHC.

Application of DNA bar codes for screening of industrially important fungi: the haplotype of Trichoderma harzianum sensu stricto indicates superior chitinase formation - Nov 01, 2007 ()
[Nagy, V., Seidl, V., Szakacs, G., Komon-Zelazowska, M., Kubicek, C. P., & Druzhinina, I. S 2007. Applied and Environmental Microbiology. 73(21) 7048-58.]

Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed. The geographic origin of the strains was irrelevant for chitinase production. The improved chitinase production by strains containing this haplotype was not due to better growth on N-acetyl-beta-D-glucosamine or glucosamine. Isoenzyme electrophoresis showed that neither the isoenzyme profile of N-acetyl-beta-glucosaminidases or the endochitinases nor the intensity of staining of individual chitinase bands correlated with total chitinase in the culture filtrate. The superior chitinase producers did not exhibit similarly increased cellulase formation. Biolog Phenotype MicroArray analysis identified lack of N-acetyl-beta-D-mannosamine utilization as a specific trait of strains with the chitinase-overproducing haplotype. This observation was used to develop a plate screening assay for rapid microbiological identification of the strains. The data illustrate that desired industrial properties may be an attribute of certain populations within a species, and screening procedures should thus include a balanced mixture of all genotypes of a given species.

Fingerprint: visual depiction of variation in multiple sequence alignments - Nov 01, 2007 (pdf)
[Lou, M. and G.B. Golding 2007. Molecular Ecology Notes. 7(6) 908-914.]

There is a lack of programs available that focus on providing an overview of an aligned set of sequences such that the comparison of homologous sites becomes comprehensible and intuitive. Being able to identify similarities, differences, and patterns within a multiple sequence alignment is biologically valuable because it permits visualization of the distribution of a particular feature and inferences about the structure, function, and evolution of the sequences in question. We have therefore created a web server, fingerprint, which combines the characteristics of existing programs that represent identity, variability, charge, hydrophobicity, solvent accessibility, and structure along with new visualizations based on composition, heterogeneity, heterozygosity, dN/dS and nucleotide diversity. fingerprint is easy to use and globally accessible through any computer using any major browser. fingerprint is available at http://evol.mcmaster.ca/fingerprint/.

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Vouchering DNA-barcoded specimens: test of a nondestructive extraction protocol for terrestrial arthropods - Nov 01, 2007 ()
[Rowley, D.L., J.A. Coddington, M.W. Gates, A.L. Norrbom, R.A. Ochoa, N.J. Vandenberg, and M.H. Greenstone 2007. Molecular Ecology Notes. 7(6) 915-924.]

Morphology-based keys support accurate identification of many taxa. However, identification can be difficult for taxa that are either not well studied, very small, members of cryptic species complexes, or represented by immature stages. For such cases, DNA barcodes may provide diagnostic characters. Ecologists and evolutionary biologists deposit museum vouchers to document the species studied in their research. If DNA barcodes are to be used for identification, then both the DNA and the specimen from which it was extracted should be vouchered. We describe a protocol for the nondestructive extraction of DNA from terrestrial arthropods, using as examples members of the orders Acarina, Araneae, Coleoptera, Diptera, and Hymenoptera chosen to represent the ranges in size, overall sclerotization, and delicacy of key morphological characters in the group. We successfully extracted sequenceable DNA from all species after 1–4 h of immersion in extraction buffer. The extracted carcasses, processed and imaged using protocols standard for the taxon, were distinguishable from closely related species, and adequate as morphological vouchers. We provide links from the carcasses and DNA vouchers to image (MorphBank) and sequence (GenBank) databases.

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Species identification in cell culture: a two-pronged molecular approach - Nov 01, 2007 (pdf)
[Cooper, J. K., Sykes, G., King, S., Cottrill, K., Ivanova, N. V., Hanner, R., and Ikonomi, P. 2007. In vitro cellular & developmental biology. 43(10) 344-51.]

Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the "barcode region" by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.

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DNA barcoding in the cycadales: testing the potential of proposed barcoding markers for species identification of cycads - Nov 01, 2007 ()
[Sass, C., Little, D. P., Stevenson, D. W., and C. D. Specht 2007. PLoS ONE. 2(11) e1154.]

Barcodes are short segments of DNA that can be used to uniquely identify an unknown specimen to species, particularly when diagnostic morphological features are absent. These sequences could offer a new forensic tool in plant and animal conservation-especially for endangered species such as members of the Cycadales. Ideally, barcodes could be used to positively identify illegally obtained material even in cases where diagnostic features have been purposefully removed or to release confiscated organisms into the proper breeding population. In order to be useful, a DNA barcode sequence must not only easily PCR amplify with universal or near-universal reaction conditions and primers, but also contain enough variation to generate unique identifiers at either the species or population levels. Chloroplast regions suggested by the Plant Working Group of the Consortium for the Barcode of Life (CBoL), and two alternatives, the chloroplast psbA-trnH intergenic spacer and the nuclear ribosomal internal transcribed spacer (nrITS), were tested for their utility in generating unique identifiers for members of the Cycadales. Ease of amplification and sequence generation with universal primers and reaction conditions was determined for each of the seven proposed markers. While none of the proposed markers provided unique identifiers for all species tested, nrITS showed the most promise in terms of variability, although sequencing difficulties remain a drawback. We suggest a workflow for DNA barcoding, including database generation and management, which will ultimately be necessary if we are to succeed in establishing a universal DNA barcode for plants.

Underestimation of species richness in neotropical frogs revealed by mtDNA analyses - Oct 31, 2007 ()
[Fouquet, A., Gilles, A., Vences, M., Marty, C., Blanc, M., & Gemmell, N. J. 2007. PLoS ONE. 2(10) e1109.]

BACKGROUND: Amphibians are rapidly vanishing. At the same time, it is most likely that the number of amphibian species is highly underestimated. Recent DNA barcoding work has attempted to define a threshold between intra- and inter-specific genetic distances to help identify candidate species. In groups with high extinction rates and poorly known species boundaries, like amphibians, such tools may provide a way to rapidly evaluate species richness. METHODOLOGY: Here we analyse published and new 16S rDNA sequences from 60 frog species of Amazonia-Guianas to obtain a minimum estimate of the number of undescribed species in this region. We combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs. PRINCIPAL FINDINGS: In most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contrary to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species. CONCLUSIONS: Using this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia-Guianas. SIGNIFICANCE: As a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised.

Things fall apart: biological species form unconnected parsimony networks - Oct 22, 2007 ()
[Hart, M. W., & Sunday, J. 2007. Biol Lett. 3(5) 509-512.]

The generality of operational species definitions is limited by problematic definitions of between-species divergence. A recent phylogenetic species concept based on a simple objective measure of statistically significant genetic differentiation uses between-species application of statistical parsimony networks that are typically used for population genetic analysis within species. Here we review recent phylogeographic studies and reanalyse several mtDNA barcoding studies using this method. We found that (i) alignments of DNA sequences typically fall apart into a separate subnetwork for each Linnean species (but with a higher rate of true positives for mtDNA data) and (ii) DNA sequences from single species typically stick together in a single haplotype network. Departures from these patterns are usually consistent with hybridization or cryptic species diversity.

A Divergent mtDNA Lineage Among Mesoplodon Beaked Whales: Molecular Evidence for a New Species in the Tropical Pacific? - Oct 01, 2007 ()
[Dalebout, M.L., C.S. Baker, D. Steel, K. M. Robertson, S.J. Chivers, W.F. Perrin, J.G. Mead, R.V. Grace, and T.D. Schofield Jr 2007. Marine Mammal Science. 23(4) 954-966.]

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Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena. - Oct 01, 2007 ()
[Chantangsi C., Lynn D.H., Brandl M.T., Cole J.C., Hetrick N., and Ikonomi P. 2007. International Journal of Systematic and Evolutionary Microbiology. 57(2007) 2412-2423.]

The mitochondrial cytochrome-c oxidase subunit 1 (cox1) gene has been proposed as a DNA barcode to identify animal species. To test the applicability of the cox1 gene in identifying ciliates, 75 isolates of the genus Tetrahymena and three non-Tetrahymena ciliates that are close relatives of Tetrahymena, Colpidium campylum, Colpidium colpoda and Glaucoma chattoni, were selected. All tetrahymenines of unproblematic species could be identified to the species level using 689 bp of the cox1 sequence, with about 11 % interspecific sequence divergence. Intraspecific isolates of Tetrahymena borealis, Tetrahymena lwoffi, Tetrahymena patula and Tetrahymena thermophila could be identified by their cox1 sequences, showing <0.65 % intraspecific sequence divergence. In addition, isolates of these species were clustered together on a cox1 neighbour-joining (NJ) tree. However, strains identified as Tetrahymena pyriformis and Tetrahymena tropicalis showed high intraspecific sequence divergence values of 5.01 and 9.07 %, respectively, and did not cluster together on a cox1 NJ tree. This may indicate the presence of cryptic species. The mean interspecific sequence divergence of Tetrahymena was about 11 times greater than the mean intraspecific sequence divergence, and this increased to 58 times when all isolates of species with high intraspecific sequence divergence were excluded. This result is similar to DNA barcoding studies on animals, indicating that congeneric sequence divergences are an order of magnitude greater than conspecific sequence divergences. Our analysis also demonstrated low sequence divergences of <1.0 % between some isolates of T. pyriformis and Tetrahymena setosa on the one hand and some isolates of Tetrahymena furgasoni and T. lwoffi on the other, suggesting that the latter species in each pair is a junior synonym of the former. Overall, our study demonstrates the feasibility of using the mitochondrial cox1 gene as a taxonomic marker for 'barcoding' and identifying Tetrahymena species and some other ciliated protists.

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A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing - Oct 01, 2007 ()
[Parameswaran, P., Jalili, R., Tao, L., Shokralla, S., Gharizadeh, B., Ronaghi, M., and A. Z. Fire 2007. Nucleic Acids Res. 35(19) e130.]

Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward-reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n(2) independent libraries for each set of 'n' forward and 'n' reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.

DNA Barcoding is not enough: mismatch of taxonomy and genealogy in New Zealand grasshoppers (Orthoptera: Acrididae). - Oct 01, 2007 ()
[Trewick, S.A. 2007. Cladistics. OnlineEarly .]

DNA barcoding has been touted as a program that will efficiently and relatively cheaply inform on biological diversity; yet many exemplars purporting to demonstrate the efficacy of the method have been undertaken by its principal proponents. Critics of DNA barcoding identify insufficient within-taxon sampling coupled with the knowledge that levels of haplotypic paraphyly are rather high as key reasons to be sceptical of the value of an exclusively DNA-based taxonomic. Here I applied a DNA barcoding approach using mtDNA sequences from the cytochrome oxidase I gene to examine diversity in a group of endemic New Zealand grasshoppers belonging to the genus Sigaus. The mtDNA data revealed high genetic distances among individuals of a single morpho-species, but this diversity was geographically partitioned. Phylogenetic analysis supported at least four haplogroups within one species (Sigaus australis) but paraphyly of this species with respect to several others. In some instances two morphologically and ecologically distinct species shared identical mtDNA haplotypes. The mismatch of genealogy and taxonomy revealed in the Sigaus australis complex indicates that, if used in isolation, DNA barcoding data can be highly misleading about biodiversity. Furthermore, failure to take into account evidence from natural history and morphology when utilizing DNA barcoding will tend to conceal the underlying evolutionary processes associated with speciation.

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Making the most of mitochondrial genomes – Markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea) - Oct 01, 2007 ()
[Zarowiecki, M.Z., T. Huyse and D.T.J. Littlewood 2007. International Journal for Parasitology. 37(12) 1401-1418.]

An increasing number of complete sequences of mitochondrial (mt) genomes provides the opportunity to optimise the choice of molecular markers for phylogenetic and ecological studies. This is particularly the case where mt genomes from closely related taxa have been sequenced; e.g., within Schistosoma. These blood flukes include species that are the causative agents of schistosomiasis, where there has been a need to optimise markers for species and strain recognition. For many phylogenetic and population genetic studies, the choice of nucleotide sequences depends primarily on suitable PCR primers. Complete mt genomes allow individual gene or other mt markers to be assessed relative to one another for potential information content, prior to broad-scale sampling. We assess the phylogenetic utility of individual genes and identify regions that contain the greatest interspecific variation for molecular ecological and diagnostic markers. We show that variable characters are not randomly distributed along the genome and there is a positive correlation between polymorphism and divergence. The mt genomes of African and Asian schistosomes were compared with the available intraspecific dataset of Schistosoma mansoni through sliding window analyses, in order to assess whether the observed polymorphism was at a level predicted from interspecific comparisons. We found a positive correlation except for the two genes (cox1 and nad1) adjoining the putative control region in S. mansoni. The genes nad1, nad4, nad5, cox1 and cox3 resolved phylogenies that were consistent with a benchmark phylogeny and in general, longer genes performed better in phylogenetic reconstruction. Considering the information content of entire mt genome sequences, partial cox1 would not be the ideal marker for either species identification (barcoding) or population studies with Schistosoma species. Instead, we suggest the use of cox3 and nad5 for both phylogenetic and population studies. Five primer pairs designed against Schistosoma mekongi and Schistosoma malayensis were tested successfully against Schistosoma japonicum. In combination, these fragments encompass 20-27% of the variation amongst the genomes (average total length approximately 14,000bp), thus providing an efficient means of encapsulating the greatest amount of variation within the shortest sequence. Comparative mitogenomics provides the basis of a rational approach to molecular marker selection and optimisation.

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DNA barcoding: the social frontier - Oct 01, 2007 ()
[Larson, B.M.H. 2007. Frontiers in Ecology and the Environment. 5(8) 437-442.]

DNA barcoding has been promoted as the holy grail of biodiversity conservation. Its proponents envision a time when anyone will be able to use a portable Life Barcoder to identify a fragment of an organism to the species level within seconds. While several critics have questioned whether DNA barcoding will work technically, claims about its social benefits have not been scrutinized. Here, I focus on two prevalent assumptions about the Life Barcoder: that it will democratize access to biodiversity and that it will increase appreciation for it. I argue that neither of these assumptions is well supported, since a Life Barcoder will prioritize one way of knowing over others, and create a technological distance between people and organisms. Consequently, DNA barcoding may not benefit conservation as much as its proponents assume.

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Molecular phylogenetics and taxonomy of the subgenus Pika (Ochotona, Lagomorpha) - Oct 01, 2007 ()
[Lissovsky, A. A., Ivanova, N. V. and Borisenko, A. V. 2007. Journal of Mammalogy. 88(5) .]

Read the publication here.

Barcoding corals: limited by interspecific divergence, not intraspecific variation - Sep 23, 2007 ()
[Shearer, T.L. and Coffroth, M.A. 2007. Molecular Ecology Notes. OnlineEarly .]

The expanding use of DNA barcoding as a tool to identify species and assess biodiversity has recently attracted much attention. An attractive aspect of a barcoding method to identify scleractinian species is that it can be utilized on any life stage (larva, juvenile or adult) and is not influenced by phenotypic plasticity unlike morphological methods of species identification. It has been unclear whether the standard DNA barcoding system, based on cytochrome c oxidase subunit 1 (COI), is suitable for species identification of scleractinian corals. Levels of intra- and interspecific genetic variation of the scleractinian COI gene were investigated to determine whether threshold values could be implemented to discriminate conspecifics from other taxa. Overlap between intraspecific variation and interspecific divergence due to low genetic divergence among species (0% in many cases), rather than high levels of intraspecific variation, resulted in the inability to establish appropriate threshold values specific for scleractinians; thus, it was impossible to discern most scleractinian species using this gene.

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Limited performance of DNA barcoding in a diverse community of tropical butterflies - Sep 04, 2007 ()
[Elias, M., Hill, R.I., Willmott, K.R., Dasmahapatra,K.K, Brower, A.V.Z., Mallet, J. and Jiggins, C.D. 2007. Proceeding of the Royal Society B. OnlineEarly .]

DNA 'barcoding' relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5' half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet.

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Successful biological invasion despite a severe genetic load. - Sep 01, 2007 ()
[Zayed, A., Constantin, S. A., and Packer, L 2007. PLoS One. 2(9) e868.]

Understanding the factors that influence the success of ecologically and economically damaging biological invasions is of prime importance. Recent studies have shown that invasive populations typically exhibit minimal, if any, reductions in genetic diversity, suggesting that large founding populations and/or multiple introductions are required for the success of biological invasions, consistent with predictions of the propagule pressure hypothesis. Through population genetic analysis of neutral microsatellite markers and a gene experiencing balancing selection, we demonstrate that the solitary bee Lasioglossum leucozonium experienced a single and severe bottleneck during its introduction from Europe. Paradoxically, the success of L. leucozonium in its introduced range occurred despite the severe genetic load caused by single-locus complementary sex-determination that still turns 30% of female-destined eggs into sterile diploid males, thereby substantially limiting the growth potential of the introduced population. Using stochastic modeling, we show that L. leucozonium invaded North America through the introduction of a very small number of propagules, most likely a singly-mated female. Our results suggest that chance events and ecological traits of invaders are more important than propagule pressure in determining invasion success, and that the vigilance required to prevent invasions may be considerably greater than has been previously considered.

Towards a collaborative, global infrastructure for biodiversity assessment - Sep 01, 2007 ()
[Guralnick, R.P, A.W. Hill, and M. Lane 2007. Ecology Letters. 10(8) 663-672.]

Biodiversity data are rapidly becoming available over the Internet in common formats that promote sharing and exchange. Currently, these data are somewhat problematic, primarily with regard to geographic and taxonomic accuracy, for use in ecological research, natural resources management and conservation decision-making. However, web-based georeferencing tools that utilize best practices and gazetteer databases can be employed to improve geographic data. Taxonomic data quality can be improved through web-enabled valid taxon names databases and services, as well as more efficient mechanisms to return systematic research results and taxonomic misidentification rates back to the biodiversity community. Both of these are under construction. A separate but related challenge will be developing web-based visualization and analysis tools for tracking biodiversity change. Our aim was to discuss how such tools, combined with data of enhanced quality, will help transform today's portals to raw biodiversity data into nexuses of collaborative creation and sharing of biodiversity knowledge.

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Halichrysis corallinarius sp. nov. (Rhodymeniaceae, Rhodophyta) from Puerto Rico, Caribbean Sea - Sep 01, 2007 ()
[Ballantine, D.L., Saunders, G.W., and H. Ruiz 2007. Phycological Researh. 55(3) 240-248.]

A new species, Halichrysis corallinarius sp. nov. is described from coral reef habitats in southwest Puerto Rico as well as from Grand Cayman Island. The new species produces strap-shaped thalli supported above the substrata by abundant peg-like stipes. The lobed branches are frequently anastomosed with colonies measuring to 4.0 cm broad with individual axes measuring to 7.0 mm across. Algae possess up to five layers of medullary cells with frequent open spaces in the medulla. Tetrasporangia, measure to 19 × 25 μm, and are borne in sori limited to ventral surfaces. Gametophytes are monoecious and hemispherical carposporophytes are produced dorsally, measuring to 900 μm in diameter and 600 μm high.

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Evolutionary acceleration in the most endangered mammal of Canada: speciation and divergence in the Vancouver Island marmot (Rodentia, Sciuridae) - Sep 01, 2007 ()
[Cardini, A., R.W. Thorington Jr. and P.D. Polly 2007. Journal of Evolutionary Biology. 20(5) 1833-1846.]

The Vancouver Island marmot is the most endangered mammal of Canada. Factors which have brought this population to the verge of extinction have not yet been fully elucidated, but the effects of deforestation and habitat fragmentation on survival rates, as well as those of variation in rainfall, temperature, snowpack depth and snowmelt strongly suggest that marmots on the island are struggling to keep pace with environmental changes. Genetic analyses, however, seem to indicate that the Vancouver Island marmot may merely represent a melanistic population of its parental species on the mainland. Were it not for its black pelage colour, it is unlikely that it would have attracted much attention as a conservation priority. Our study uses three-dimensional coordinates of cranial landmarks to further assess phenotypic differentiation of the Vancouver Island marmot. A pattern of strong interspecific divergence and low intraspecific variation was found which is consistent with aspects of drift-driven models of speciation. However, the magnitude of shape differences relative to the putatively neutral substitutions in synonymous sites of cytochrome b is too large for being compatible with a simple neutral model. A combination of bottlenecks and selective pressures due to natural and human-induced changes in the environment may offer a parsimonious explanation for the large phenotypic differentiation observed in the species. Our study exemplifies the usefulness of a multidisciplinary approach to the study of biological diversity for a better understanding of evolutionary models and to discover aspects of diversity that may be undetected by using only a few genetic markers to characterize population divergence and uniqueness.

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Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes - Sep 01, 2007 ()
[Sevilla, R.G., Diez, A., Noren, M., Mouchel, O., Jerome, M., Verrez-Bagnis, V., Van Pelt, H., Favre-Krey, L, Krey, G., The Fishtrace Consortium, and Bautista, J. 2007. Molecular Ecology Notes. 7(5) 730-734.]

This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine fish species comprising the main Actinopterygii family groups. When used in phylogenetic analyses, its combination of two genes with different evolutionary rates serves to identify fish at the species level. We provide a flow diagram indicating our validated polymerase chain reaction amplification conditions for barcoding and species identification applications as well as population structure or haplotyping analyses, adaptable to high-throughput analyses.

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Mitochondrial-DNA evidence shows the Australian Painted Snipe is a full species, Rostratula australis - Aug 31, 2007 ()
[Baker, A. J., Pereira, S. L., Rogers, D. I., Elbourne, R., & Hassell, C. J. 2007. Emu. 107(3) 185-189.]

Despite its distinctive morphology, the taxonomy of the Australian Painted Snipe has been unsettled, with some authors treating it as a full species, Rostratula australis (Gould 1838), and others treating it as a subspecies of the Greater Painted Snipe, Rostratula benghalensis. We sequenced the DNA of five mitochondrial genes (Cyt b, ND5, ATP 6–8, COIII and COI) of Australian Painted Snipe, Greater Painted Snipe and South American Painted Snipe, Nycticryphes semicollaris. The sequences of Australian Painted Snipe were 10% different from those of Greater Painted Snipe from Africa and South-east Asia, which differed from one another by only 2%. Plumage and anatomical characters can also distinguish the Australian and the Greater Painted Snipes. Our results clearly indicate that the Australian Painted Snipe is a distinct species that diverged ~19 million years ago (mya) (95% credible interval 13.0, 27.4 mya).

Spider mite (Acari: Tetranychidae) mitochondrial COI phylogeny reviewed: host plant relationships, phylogeography, reproductive parasites and barcoding. - Aug 22, 2007 ()
[Ros, V. I., & Breeuwer, J. A. 2007. Experimental & Applied Acarology. 42(4) 239-262.]

The past 15 years have witnessed a number of molecular studies that aimed to resolve issues of species delineation and phylogeny of mites in the family Tetranychidae. The central part of the mitochondrial COI region has frequently been used for investigating intra- and interspecific variation. All these studies combined yield an extensive database of sequence information of the family Tetranychidae. We assembled this information in a single alignment and performed an overall phylogenetic analysis. The resulting phylogeny shows that important patterns have been overlooked in previous studies, whereas others disappear. It also reveals that mistakes were made in submitting the data to GenBank, which further disturbed interpretation of the data. Our total analysis clearly shows three clades that most likely correspond to the species T. urticae, T. kanzawai and T. truncatus. Intraspecific variation is very high, possibly due to selective sweeps caused by reproductive parasites. We found no evidence for host plant associations and phylogeographic patterns in T. urticae are absent. Finally we evaluate the application of DNA barcoding.

Cross-species transfer of nuclear microsatellite markers: potential and limitations - Aug 06, 2007 ()
[Barbara, T., C. Palma-Silva, G.M. Paggi, F. Bered, M.F. Fay, and C. Lexer 2007. Molecular Ecology. OnlineEarly .]

Molecular ecologists increasingly require 'universal' genetic markers that can easily be transferred between species. The distribution of cross-species transferability of nuclear microsatellite loci is highly uneven across taxa, being greater in animals and highly variable in flowering plants. The potential for successful cross-species transfer appears highest in species with long generation times, mixed or outcrossing breeding systems, and where genome size in the target species is small compared to the source. We discuss the implications of these findings and close with an outlook on potential alternative sources of cross-species transferable markers.

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DNA taxonomy and barcoding of monogenean parasites: lessons from Gyrodactylus - Aug 01, 2007 ()
[Hansen, H., Bakke, T. A., & Bachmann, L. 2007. Trends Parasitol. 23(8) 363-367.]

DNA taxonomy and barcoding use nucleotide sequence data to achieve comprehensive species descriptions that facilitate reliable species diagnostics and rapid assessment of biodiversity, both of which are of great importance for parasitologists. Such molecular approaches have been applied to the monogenean genus Gyrodactylus, in particular to G. salaris, the cause of serious gyrodactylosis on Atlantic salmon. Here, we discuss, using the example of G. salaris and related species, why DNA barcodes, although powerful for biodiversity assessment, are insufficient to appropriately characterize parasite species--from a parasitological point of view--in the absence of additional data on and infection biology and morphology.

Targeted high-throughput sequencing of tagged nucleic acid samples - Aug 01, 2007 ()
[Meyer, M., Stenzel, U., Myles, S., Prufer, K., & Hofreiter, M. 2007. Nucleic Acids Res. 35(15) e97.]

High-throughput 454 DNA sequencing technology allows much faster and more cost-effective sequencing than traditional Sanger sequencing. However, the technology imposes inherent limitations on the number of samples that can be processed in parallel. Here we introduce parallel tagged sequencing (PTS), a simple, inexpensive and flexible barcoding technique that can be used for parallel sequencing any number and type of double-stranded nucleic acid samples. We demonstrate that PTS is particularly powerful for sequencing contiguous DNA fragments such as mtDNA genomes: in theory as many as 250 mammalian mtDNA genomes can be sequenced in a single GS FLX run. PTS dramatically increases the sequencing throughput of samples in parallel and thus fully mobilizes the resources of the 454 technology for targeted sequencing.

Phylogeography and environmental diversification of a highly adaptable marine amphipod, Gammarus duebeni. - Jul 31, 2007 ()
[Rock J., Ironside J., Potter T., Whiteley N.M., Lunt D.H. 2007. Heredity. 99(1) 102-11.]

Genetic diversity and phylogeographic population structure in the gammarid amphipod, Gammarus duebeni, were investigated across its broad latitudinal distribution in the NE and NW Atlantic by analysis of mitochondrial DNA sequence. Gammarus duebeni has exceptional tolerance of salinity change and inhabits environments ranging from marine to freshwater. The longstanding debate on whether there are distinct marine and freshwater subspecies was assessed by sampling populations from sites characterized by different salinities. Our sequence data demonstrates that there are two major lineages, with little internal geographic structuring. Evidence is provided to suggest a pre-glacial divergence of these two clades, involving segregation between a region historically associated with the freshwater form and the majority of the marine localities on both sides of the Atlantic. A modern contact zone between the marine and freshwater forms is proposed in western Britain.

Population structure and species boundary delimitation of cryptic Dioryctria moths: an integrative approach - Jul 17, 2007 ()
[Roe, A.D. and F.A.H. Sperling 2007. Molecular Ecology. OnlineEarly .]

Accurate delimitation of species boundaries is especially important in cryptic taxa where one or more character sources are uninformative or are in conflict. Rather than relying on a single marker to delimit species, integrative taxonomy uses multiple lines of evidence such as molecular, morphological, behavioural and geographic characters to test species limits. We examine the effectiveness of this approach by testing the delimitation of two cryptic Nearctic species of Dioryctria (Lepidoptera: Pyralidae) using three independent molecular markers [cytochrome c oxidase I (COI), second internal transcribed spacer unit (ITS2), and elongation factor 1α (EF1α)], forewing variation and larval host plant association. Although mitochondrial DNA (mtDNA) haplotypes do not form reciprocally monophyletic clades, restricted gene flow between COI haplotype groups, and concordance with ITS2 genotypes, forewing variation and host plant associations support delimitation of two Nearctic species: eastern Dioryctria reniculelloides and western Dioryctria pseudotsugella. Conversely, EF1α genotype variation was incongruent with the two previous markers. A case of discordance between COI and ITS2 was detected, suggesting either introgression due to hybridization or retained ancestral polymorphism due to incomplete coalescence. This study is consistent with other similar literature where molecular loci in closely related species progress from shared to fixed haplotypes/alleles, and from polyphyletic to reciprocally monophyletic relationships, although loci may vary in these characteristics despite maintenance of genomic integrity between distinct species. In particular, mtDNA in other studies generally showed a lower rate of fixation of differences than did X-linked or autosomal loci, reinforcing the need to use an integrative approach for delimiting species.

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An analysis of nucleotide and amino acid variability in the barcode region of cytochrome c oxidase I (cox1) in fishes - Jul 13, 2007 ()
[Ward, R.D. and B.H. Holmes 2007. Molecular Ecology Notes. 7(6) 899-907.]

The 655 bp cytochrome c oxidase subunit I barcode region of single specimens of 388 species of fishes (four Holocephali, 61 Elasmobranchii and 323 Actinopterygii) was examined. All but two (Urolophus cruciatus and Urolophus sufflavus) showed different cox1 nucleotide sequences (99.5% species discrimination); the two that could not be resolved are suspected to hybridize. Most of the power of cox1 nucleotide sequence analysis for species identification comes from the degenerate nature of the genetic code and the highly variable nature of the third codon position of amino acids. Variation at the third codon position is bimodally distributed, and the more variable mode is dominated by amino acids with four or six codons, while the less variable mode is dominated by amino acids with two codons. The ratio of nonsynonymous to synomymous changes is much less than one, indicating that this gene is subject to strong purifying selection. Consequently, cox1 amino acid sequence diversity is much less than nucleotide sequence diversity and has very poor species resolution power. Fourteen of the 16 amino acid residues recognized as having important functions in the region of cox1 sequenced were completely conserved over all 388 species (and the bovine cox1 sequence), with one fish species varying at one of these sites, and three fish at another site. No significant differences in amino acid conservation were observed between residues in helices, strands and turns. Patterns of nucleotide and amino acid variability were very similar between elasmobranchs and actinopterygians.

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DNA barcoding confirms polyphagy in a generalist moth, Homona mermerodes (Lepidoptera: Tortricidae) - Jul 01, 2007 (pdf)
[Hulcr, J., S.E. Miller, G.P. Setliff, K. Darrow, N.D. Mueller, P.D.N. Hebert, and G.D. Weiblen 2007. Molecular Ecology Notes. 7(4) 549-557.]

Recent DNA barcoding of generalist insect herbivores has revealed complexes of cryptic species within named species. We evaluated the species concept for a common generalist moth occurring in New Guinea and Australia, Homona mermerodes, in light of host plant records and mitochondrial cytochrome c oxidase I haplotype diversity. Genetic divergence among H. mermerodes moths feeding on different host tree species was much lower than among several Homona species. Genetic divergence between haplotypes from New Guinea and Australia was also less than interspecific divergence. Whereas molecular species identification methods may reveal cryptic species in some generalist herbivores, these same methods may confirm polyphagy when identical haplotypes are reared from multiple host plant families. A lectotype for the species is designated, and a summarized bibliography and illustrations including male genitalia are provided for the first time.

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Coryphopterus kuna, a new goby (Perciformes: Gobiidae: Gobiinae) from the western Caribbean, with the identification of the late larval stage and an estimate of the pelagic larval duration - Jul 01, 2007 ()
[Victor, B.C. 2007. Zootaxa. 1526 51-61.]

A new goby, Coryphopterus kuna is described from the Atlantic coasts of Panama and Mexico. The species is distinguished from other Coryphopterus spp. by the low median fin and pectoral fin ray counts and the morphology of the pelvic fin. The pelvic fins are fully joined with a rounded outline and have branched and longer innermost pelvic fin rays.  There is no frenum connecting the two pelvic fin spines and the fin is heavily speckled with black spots in the male holotype.  The late larval stage of C. kuna is identified by DNA sequence matching and is morphologically similar to other larval Coryphopterus spp. but has a distinct melanophore pattern. Examination of the otolith microstructure reveals a relatively long pelagic larval duration of 63 days with a narrowing of the later daily increments suggesting delayed metamorphosis. The species is the first vertebrate to include gene sequence barcoding under the Barcode of Life Data System (BOLD) in the species description.

Patterns of evolution of mitochondrial cytochrome c oxidase I and II DNA and implications for DNA barcoding. - Jul 01, 2007 ()
[Roe, A.D. and F.A.H. Sperling 2007. Molecular Phylogenetics and Evolution. 44(1) 325-345.]

DNA barcoding has focused increasing attention on the use of specific regions of mitochondrial cytochrome c oxidase I and II genes (COI–COII) to diagnose and delimit species. However, our understanding of patterns of molecular evolution within these genes is limited. Here we examine patterns of nucleotide divergence in COI–COII within species and between species pairs of Lepidoptera and Diptera using a sliding window analysis. We found that: (1) locations of maximum divergence within COI–COII were highly variable among taxa surveyed in this study; (2) there was major overlap in divergence within versus between species, including within individual COI–COII profiles; (3) graphical DNA saturation analysis showed variation in percent nucleotide transitions throughout COI–COII and only limited association with levels of DNA divergence. Ultimately, no single optimally informative 600 bp location was found within the 2.3kb of COI–COII, and the DNA barcoding region was no better than other regions downstream in COI. Consequently, we recommend that researchers should maximize sequence length to increase the probability of sampling regions of high phylogenetic informativeness, and to minimize stochastic variation in estimating total divergence.

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An Assessment of Potential Diatom ‘‘Barcode’’ Genes (cox1, rbcL, 18S and ITS rDNA) and their Effectiveness in Determining Relationships in Sellaphora (Bacillariophyta) - Jun 22, 2007 ()
[Evans, Katharine M., Alexandra H. Wortley, and David G. Mann 2007. Protist. Online Early .]

Due to limited morphological differentiation, diatoms can be very difficult to identify and cryptic speciation is widespread. There is a need for a narrower species concept if contentious issues such as diatom biodiversities and biogeographies are to be resolved. We assessed the effectiveness of several genes (cox1, rbcL, 18S and ITS rDNA) to distinguish cryptic species within the model ‘morphospecies’, Sellaphora pupula agg. This is the first time that the suitability of cox1 as an identification tool for diatoms has been assessed. A range of cox1 primers was tested on Sellaphora and various outgroup taxa. Sequences were obtained for 34 isolates belonging to 22 Sellaphora taxa and three others (Pinnularia, Eunotia and Tabularia). Intraspecific divergences ranged from 0 to 5 bp ( ¼ 0.8%) and interspecific levels were at least 18 bp ( ¼ c. 3%). Cox1 divergence was usually much greater than rbcL divergence and always much more variable than 18S rDNA. ITS rDNA sequences were more variable than cox1, but well-known problems concerning intragenomic variability caution against its use in identification. More information and less sequencing effort mean that cox1 can be a very useful aid in diatom identification. The usefulness of cox1 for determining phylogenetic relationships among Sellaphora species was also assessed and compared to rbcL. Tree topologies were very similar, although support values were generally lower for cox1.

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Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring - Jun 13, 2007 (pdf)
[Hajibabaei, M., G.A.C. Singer, E.L. Clare and P.D.N. Hebert 2007. BMC Biology. 5(24) .]

Background

The rapid and accurate identification of species is a critical component of large-scale biodiversity monitoring programs. DNA arrays (micro and macro) and DNA barcodes are two molecular approaches that have recently garnered much attention. Here, we compare these two platforms for identification of an important group, the mammals.

Results

Our analyses, based on the two commonly used mitochondrial genes cytochrome c oxidase I (the standard DNA barcode for animal species) and cytochrome b (a common species-level marker), suggest that both arrays and barcodes are capable of discriminating mammalian species with high accuracy. We used three different datasets of mammalian species, comprising different sampling strategies. For DNA arrays we designed three probes for each species to address intraspecific variation. As for DNA barcoding, our analyses show that both cytochrome c oxidase I and cytochrome b genes, and even smaller fragments of them (mini-barcodes) can successfully discriminate species in a wide variety of specimens.

Conclusion

This study showed that DNA arrays and DNA barcodes are valuable molecular methods for biodiversity monitoring programs. Both approaches were capable of discriminating among mammalian species in our test assemblages. However, because designing DNA arrays require advance knowledge of target sequences, the use of this approach could be limited in large scale monitoring programs where unknown haplotypes might be encountered. DNA barcodes, by contrast, are sequencing-based and therefore could provide more flexibility in large-scale studies.

DNA identification of urban Tanytarsini chironomids (Diptera:Chironomidae) - Jun 11, 2007 ()
[Carew, M. E., Pettigrove, V., Cox, R. L., & Hoffmann, A. A. 2007. Journal of the North American Benthological Society. 26(4) 587–600.]

DNA barcoding is becoming widely used to provide species identifications in a variety of invertebrate taxa, but there has been little application so far to environmental monitoring. Here we make this connection using a group of aquatic macroinvertebrates, chironomids of the tribe Tanytarsini. Tanytarsini larvae commonly collected in biological surveys can be difficult to identify to species because of high intraspecific variability and because not all larvae are linked to described adult life stages. We examined whether Tanytarsini larvae could be reliably identified to species with polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) using the mitochondrial cytochrome oxidase I (COI) gene. Tanytarsini were collected from lentic environments and consisted of 3 common genera (Cladotanytarsus, Paratanytarsus, and Tanytarsus). COI PCR-RFLP profiles could discriminate larvae, including morphologically similar larvae, and could be linked to described life stages. COI sequences identified the same species from different localities. DNA identification has potential for distinguishing Tanytarsini species and facilitating their use as biological indicators

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Local scale DNA barcoding of bivalves (Mollusca): a case study - Jun 07, 2007 ()
[Mikkelsen, N.T., C. Schander and E. Willassen 2007. Zoologica Scripta. OnlineEarly .]

Divergence in cytochrome c oxidase 1 (COI), the genetic marker proposed for DNA barcoding, was investigated in marine bivalves from the genera Ennucula, Nucula, Yoldiella and Thyasira. No overlap in levels of intra- and interspecific variation was found. The levels of divergence found suggest that barcodes from COI will be useful in distinguishing between the species investigated in this study. The insufficiency of blast searches in GenBank to assign many of the obtained sequences to correct phylum was noted and clearly demonstrates the need for better search strategies specifically targeted at identification using DNA barcodes.

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Genomic diversity research and the role of biorepositories - Jun 01, 2007 ()
[Hanner, R. H., & Gregory, T. R 2007. Cell Preservation Technology. 5(2) 93-103.]

Biodiversity repositories underpin the future of research in the life sciences and biotechnology. However, they represent an extremely heterogeneous assemblage of collections that are lacking a comprehensive index of available resources. A set of “best practices” for biospecimen characterization is proposed for repository biomaterials involving standardized species-level molecular genotyping (DNA barcoding) and the quantification of nuclear DNA content (genome size). This approach has implications for upstream sample collection and preservation methods, as well as downstream implications for highlighting biorepository specimens available for genetic and genomic research. The broad application of the approach here proposed will raise the profile of participating biodiversity repositories, facilitate the compilation of validated reference sequences for molecular species recognition, and drive a deeper understanding of the evolution of the genome.

Spatiotemporal analysis of population genetic structure in Geomonhystera disjuncta (Nematoda, Monhysteridae) reveals high levels of molecular diversity - Jun 01, 2007 ()
[Derycke, S., T. Backeljau, C. Vlaeminck, A. Vierstraete, J. Vanfleteren, M. Vincx and T. Moens 2007. Marine Biology. 151(5) 1799-1812.]

Species identification in the phylum Nematoda is complicated due to the paucity of easily obtainable diagnostic morphological features. Furthermore, the cosmopolitan distribution of several species despite low dispersal abilities makes cryptic diversity potentially substantial within this phylum. We conducted a population genetic survey in the marine nematode Geomonhystera disjuncta in Belgium and The Netherlands in two seasons. The mitochondrial cytochrome oxidase c subunit 1 (COI) gene was screened with the single-strand conformation polymorphism method in 759 individuals. The 43 haplotypes were grouped into five lineages, with low divergences within (<3%) and high divergences between lineages (>14%). Analysis of the nuclear ITS region yielded concordant tree topologies, indicating the presence of five cryptic taxa within G. disjuncta. Analysis of Molecular Variance (AMOVA) illustrated a significant structuring in all lineages and temporal fluctuations in haplotype frequencies within and between locations. Metapopulation dynamics and/or priority effects best explained this structuring. Finally, our data indicate that the COI gene may be useful for DNA barcoding purposes.

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A new species of Kerivoula (Chiroptera: Vespertilionidae) from peninsular Malaysia - Jun 01, 2007 (pdf)
[Francis, C.M., T. Kingston and A. Zubaid 2007. Acta Chiropterologica. 9(1) 1-12.]

A new species of small Kerivoula is described from peninsular Malaysia. It is similar in size and form to Kerivoula hardwickii Miller 1898 or K. intermedia Hill and Francis 1984, but is distinguished by its distinctive colouration — dorsal fur has extensive black bases with shiny golden tips, ventral fur has dark grey bases with whitish-buff tips — as well as several characters of dentition and skull shape. Sequence analysis of the first 648 base pairs of cytochrome oxidase I gene (DNA barcode) indicates a divergence of at least 11%

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DNA barcodes for species identification of euphausiids (Euphausiacea, Crustacea) - Jun 01, 2007 ()
[Bucklin, A., Wiebe, P.H., Smolenack, S.B., Copley, N.J., Beaudet, J.G., Bonner, K.G., Färber-Lorda, J. and J.J. Pierson 2007. Journal of Plankton Research. 29(6) 483-493.]

Many species of euphausiids (Euphausiacea, Crustacea) are distinguished by subtle or geographically variable morphological characters, and erroneous identification of euphausiid species may be more frequent than currently acknowledged. DNA barcodes (short DNA sequences that discriminate species and aid in recognition of unknown species) are of use for this group. A 650 bp region of mitochondrial cytochrome oxidase I (mtCOI) was sequenced for 40 species of 10 euphausiid genera: Bentheuphausia, Euphausia, Meganyctiphanes, Nematobrachion, Nematoscelis, Nyctiphanes, Stylocheiron, Tessarabrachion, Thyssanoessa and Thysanopoda. mtCOI sequence variation discriminated all species; pairwise differences averaged 16.4% (range 7–24%); mean generalized time reversible (GTR) genetic distance was 26.7%. mtCOI reliably identified euphausiid species: variation within species was typically < 1% and GTR distance was typically < 2%. Atlantic and Pacific Ocean populations of Euphausia brevis differed by 13% (GTR genetic distance = 28%) and may deserve status as distinct species. mtCOI gene trees were reconstructed for five genera using maximum parsimony, maximum likelihood and Bayesian algorithms; best-fit models of nucleotide evolution were determined for each genus. The mtCOI gene tree for 20 species of Euphausia reproduced one of three morphologically defined species groups. mtCOI resolved relationships among closely related species of most genera, usually in accord with morphological groupings. A comprehensive DNA barcode database for euphausiids will help ensure accurate species identification, recognition of cryptic species and evaluation of taxonomically meaningful geographic variation.

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A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnHpsbA Spacer Region - Jun 01, 2007 ()
[Kress, W.J. and Erickson, D.L. 2007. PLOS One. 6(e508) 1-10.]

A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level.

Identification and differentiation of Heterotardigrada and Eutardigrada species by riboprinting - May 21, 2007 ()
[Schill, R.O. and G. Steinbrück 2007. Journal of Zoological Systematics and Evolutionary Research. .]

In the last decades, the number of known tardigrade species has considerably increased to more than 960 species with new ones being discovered every year. However, the study of tardigrade species presents a general problem which is frequently encountered during the work with invertebrates: small size and remarkable degrees of phenotypic plasticity may sometimes not permit a definite identification of the species. In this investigation we have used riboprinting, a tool to study rDNA sequence variation, in order to distinguish tardigrade species from each other. The method combines a restriction site variation approach of ribotyping with amplified DNAs. In eight investigated species of heterotardigrades and eutardigrades we have amplified the genes for the small subunit ribosomal RNA (SSU; 18S) and subsequently sequenced the genes. Virtual riboprints were used for identification of restriction sites from ten already published 18S rDNA sequences and seven new 18S rDNA sequences. On the basis of the obtained sequences, diagnostic restriction fragment patterns can be predicted with only 11 restriction enzymes. The virtual digestion confirmed the obtained restriction fragment patterns and restriction sites of all amplified and digested tardigrade DNAs. We show that the variation in positions and number of restriction sites obtained by standard restriction fragment analysis on agarose gels can be used successfully for taxonomic identification at different taxonomic levels. The simple restriction fragment analysis provides a fast and convenient method of molecular barcoding for species identification in tardigrades.

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Double trouble for grasshopper molecular systematics: intra-individual heterogeneity of both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer ribosomal DNA sequences in Hesperotettix viridis (Orthoptera: Acrididae) - May 21, 2007 ()
[Sword, G.A., L.B. Senior, J.F. Gaskin,and A. Joern. 2007. Systematic Entomology. .]

Hesperotettix viridis grasshoppers (Orthoptera: Acrididae: Melanoplinae) exhibit intra-individual variation in both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer (ITS) ribosomal DNA sequences. These findings violate core assumptions underlying DNA sequence data obtained via polymerase chain reaction (PCR) amplification for use in molecular systematics investigations. Undetected intra-individual variation of this sort can confound phylogenetic analyses at a range of taxonomic levels. The use of a DNA extraction protocol designed to enrich mitochondrial DNA as well as an initial long PCR of approximately 40% of the grasshopper mitochondrial genome failed to control for the presence of paralogous mitochondrial DNA-like sequences within individuals. These findings constitute the first demonstration of intra-individual heterogeneity in mitochondrial DNA-like sequences in the grasshopper subfamily, Melanoplinae, and only the second report of intra-individual variation in nuclear ITS ribosomal DNA sequences in grasshoppers. The fact that intra-individual variation was detected in two independent DNA marker sets in the same organism strengthens the notion that the orthology of PCR-derived DNA sequences should be examined thoroughly prior to their use in molecular phylogenetic analyses or as DNA barcodes.

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Assessing the effect of varying sequence length on DNA barcoding of fungi - May 01, 2007 (pdf)
[Min, X.J. and Hickey, D.A. 2007. Molecular Ecology Notes. 7(3) 365-373.]

DNA barcoding shows enormous promise for the rapid identification of organisms at the species level. There has been much recent debate, however, about the need for longer barcode sequences, especially when these sequences are used to construct molecular phylogenies. Here, we have analysed a set of fungal mitochondrial sequences - of various lengths - and we have monitored the effect of reducing sequence length on the utility of the data for both species identification and phylogenetic reconstruction. Our results demonstrate that reducing sequence length has a profound effect on the accuracy of resulting phylogenetic trees, but surprisingly short sequences still yield accurate species identifications. We conclude that the standard short barcode sequences (~600 bp) are not suitable for inferring accurate phylogenetic relationships, but they are sufficient for species identification among the fungi.

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BOLD: The Barcode of Life Data System (http://www.barcodinglife.org) - May 01, 2007 (pdf)
[Ratnasingham, S. and Hebert, P.D.N. 2007. Molecular Ecology Notes. 7(3) 355-364.]

The Barcode of Life Data System BOLD is an informatics workbench aiding the acquisition, storage, analysis and publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a traditional bioinformatics chasm. BOLD is freely available to any researcher with interests in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well positioned to support projects that involve broad research alliances. This paper provides a brief introduction to the key elements of BOLD discusses their functional capabilities, and concludes by examining computational resources and future prospects.

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Yeasts Isolated from Neotropical Wood-Boring Beetles in SE Peru - May 01, 2007 ()
[Berkov, A., J. Feinstein, J. Small, M. Nkamany, and P. Centeno 2007. Biotropica. .]

Some temperate wood-boring cerambycid beetles harbor intracellular gut yeasts believed to augment host nutrition, but species belonging to the subfamily Lamiinae are thought to lack endosymbionts. Almost 49 percent of Neotropical cerambycid species are lamiines, therefore, comparatively few rain forest species would be expected to host symbiotic gut yeasts. This study reports the isolation of gut yeasts from closely related Neotropical lamiines. We investigated species that feed on trees in the Brazil nut family (Lecythidaceae), because host plant associations are relatively well known. Our objectives were to determine if gut yeasts were present and, if possible, infer their mode of transmission. We collected and dissected 18 beetle specimens from three tree species, including 17 cerambycids and one curculionid. Every insect specimen yielded a gut yeast. DNA sequence libraries were used for a rapid identification of the yeasts and their larval hosts. The cerambycids included five lamiine species and one cerambycine. Six ascomycete yeasts were isolated from their guts; we found no evidence of strict vertical transmission. Larval gut yeasts were genetically similar to yeasts previously isolated from insects associated with wood or fungi, implying potential habitat specificity. The yeasts have not yet been localized, and potential function is not known, but they may contribute to rapid nutrient cycling or serve as the first line of defense against plant toxins

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An alien in an archipelago: Spathodea campanulata and the geographic variability of its moth (Lepidoptera) communities in the New Guinea and Bismarck Islands - May 01, 2007 ()
[Darren Bito 2007. Journal of Biogeography. 34(5) 769-778.]

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ITS barcodes for Trichophyton tonsurans and T. equinum - May 01, 2007 ()
[Summerbell, R.C., M.K. Moore, M. Starink-Willemse and A. Van Iperen 2007. Medical Mycology. 45(3) 193-200.]

Early molecular biosystematic studies of dermatophytes created considerable confusion about the taxonomic status of the horse-associated Trichophyton equinum vis-à-vis the anthropophilic T. tonsurans. Though this matter has recently been clarified, routine identification of these species based on the commonly used ribosomal internal transcribed spacer (ITS) sequence has been impractical. This is because, in the available sequences attributed to the species in GenBank, a clear species-level distinction does not appear to exist. In the present study, resequencing the ITS regions of several anomalous isolates is shown to eliminate this problem, which was mainly based on read errors in older sequences. Newly generated sequences and recent GenBank additions are analysed to show that T. equinum appears to be uniform in ITS sequence worldwide, while T. tonsurans is also uniform, excepting a single-base change found in one otherwise typical strain. Analysis also reveals a distinct, as yet incompletely classified Asian genotype that may belong to one or the other of these species. Standard ITS 'barcode sequences' are proposed for T. tonsurans and T. equinum, and a taxonomic neotype is designated to anchor the latter species. T. equinum var. autotrophicum is further evidenced as very closely related to T. equinum var. equinum, and the anomaly of its plesiomorphous phenotype is discussed in a population genetics context.

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DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae). - May 01, 2007 ()
[Whitworth T.L., R.D Dawson, H. Magalon and E. Baudry. 2007. Proceeding of the Royal Society - Biological Sciences. 1-9.]

In DNA barcoding, a short standardized DNA sequence is used to assign unknown individuals to species and aid in the discovery of new species. A fragment of the mitochondrial gene cytochrome c oxidase subunit 1 is emerging as the standard barcode region for animals. However, patterns of mitochondrial variability can be confounded by the spread of maternally transmitted bacteria that cosegregate with mitochondria. Here, we investigated the performance of barcoding in a sample comprising 12 species of the blow fly genus Protocalliphora, known to be infected with the endosymbiotic bacteria Wolbachia. We found that the barcoding approach showed very limited success: assignment of unknown individuals to species is impossible for 60% of the species, while using the technique to identify new species would underestimate the species number in the genus by 75%. This very low success of the barcoding approach is due to the non-monophyly of many of the species at the mitochondrial level. We even observed individuals from four different species with identical barcodes, which is, to our knowledge, the most extensive case of mtDNA haplotype sharing yet described. The pattern of Wolbachia infection strongly suggests that the lack of within-species monophyly results from introgressive hybridization associated with Wolbachia infection. Given that Wolbachia is known to infect between 15 and 75% of insect species, we conclude that identification at the species level based on mitochondrial sequence might not be possible for many insects. However, given that Wolbachia-associated mtDNA introgression is probably limited to very closely related species, identification at the genus level should remain possible.

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Host-specificity and coevolution among pollinating and nonpollinating New World fig wasps - May 01, 2007 ()
[Marussich, W. A., & Machado, C. A. 2007. Molecular Ecology. 16(9) 1925-1946.]

Figs (Ficus spp., Moraceae) and their pollinating wasps (Hymenoptera, Agaonidae, Chalcidoidea) constitute a classic example of an obligate plant-pollinator mutualism, and have become an ideal system for addressing questions on coevolution, speciation, and the maintenance of mutualisms. In addition to pollinating wasps, figs host several types of nonpollinating, parasitic wasps from a diverse array of Chalcid subfamilies with varied natural histories and ecological strategies (e.g. competitors, gallers, and parasitoids). Although a few recent studies have addressed the question of codivergence between specific genera of pollinating and nonpollinating fig wasps, no study has addressed the history of divergence of a fig wasp community comprised of multiple genera of wasps associated with a large number of sympatric fig hosts. Here, we conduct phylogenetic analyses of mitochondrial DNA sequences (COI) using 411 individuals from 69 pollinating and nonpollinating fig wasp species to assess relationships within and between five genera of fig wasps (Pegoscapus, Idarnes, Heterandrium, Aepocerus, Physothorax) associated with 17 species of New World Urostigma figs from section Americana. We show that host-switching and multiple wasp species per host are ubiquitous across Neotropical nonpollinating wasp genera. In spite of these findings, cophylogenetic analyses (treemap 1.0, treemap 2.02β, and parafit) reveal evidence of codivergence among fig wasps from different ecological guilds. Our findings further challenge the classical notion of strict-sense coevolution between figs and their associated wasps, and mirror conclusions from detailed molecular studies of other mutualisms that have revealed common patterns of diffuse coevolution and asymmetric specialization among the participants.

A comprehensive DNA sequence library is essential for identification with DNA barcodes - May 01, 2007 ()
[Ekrem, T., Willassen, E., & Stur, E. 2007. Molecular Phylogenetics and Evolution. 43(2) 530-542.]

In this study we examine the possibility of utilising partial cox1 gene sequences as barcodes to identify non-biting midges (Diptera: Chironomidae). We analysed DNA from 97 specimens of 47 species in the genera Cladotanytarsus, Micropsectra, Parapsectra, Paratanytarsus, Rheotanytarsus, Tanytarsus and Virgatanytarsus with a main focus on Micropsectra, Parapsectra and Paratanytarsus. Our findings show that (1) cox1 is easily amplified from extracts from different life stages with the standard barcoding primers. (2) Although K2P-distances between con-specific sequences varied up to 4.9%, con-specifics clustered together with 91–100% bootstrap support in mximum parsimony analysis. This indicates that barcodes may be excellent tools to identify species that are already in a cox1 library. 3) Both neighbour joining and maximum parsimony failed to reconstruct monophyletic genera. Thus, if a well-matching cox1 sequence is not already available in the library, the prospects of approximately identifying an unknown taxon, even to the correct genus of subtribe Tanytarsina, are not good.

Identifying earthworms through DNA barcodes - May 01, 2007 ()
[Jian Huang, Qin Xu, Zhen Jun Sun, Gui Lan Tang and Zi You Su 2007. Pedobiologia. 51(4) 301-309.]

With almost 3000 species, earthworms provide important model systems for studying soil fauna. However, species identification of earthworms is difficult and therefore limiting. The use of DNA barcodes, which are short sequences from standardized regions of the genome, has been regarded as a promising approach to resolve this taxonomic dilemma. We evaluated sequence diversity in the mitochondrial cytochrome-c oxidase I (COI) gene as a tool for resolving differences among species of Chinese earthworms. Members of six genera and 28 species were examined, and species were successfully discriminated in all cases. Sequence divergence within species was generally less than 1%, whereas divergence between species was greater than 15% in all cases. Divergence among individuals of Eisenia fetida were much higher (up to 7.8%); however, this may represent the presence of unrecognized sibling species or subspecies. We conclude that although it cannot completely replace taxonomy, the DNA barcode is a powerful tool for identifying species of earthworms and provides a useful complement to traditional morphological taxonomy.

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Taxonomic review of the leek moth genus Acrolepiopsis (Lepidoptera: Acrolepiidae) in North America - May 01, 2007 (pdf)
[Landry, J.-F. 2007. The Canadian Entomologist. 139(3) 319-353.]

The North American species of Acrolepiopsis are reviewed and include six described species: A. assectella (Zeller), A. californica Gaedike, A. heppneri Gaedike, A. incertella (Chambers), A. leucoscia (Meyrick), and A. reticulosa (Braun). Acrolepiopsis liliivora Gaedike is considered a junior synonym of A. californica (new synonymy). Acrolepiopsis assectella, commonly known as the leek moth, is a recently invasive alien species in North America and a pest of the plant genus Allium, including leek, onion, garlic, and related cultivated plants. A key to species based on adults is provided, diagnostic characters including male and female genitalia are illustrated, and geographical distribution, host plants, and larval feeding pattern and damage (where known) are given. Diagnostics and illustrations are presented also for A. sapporensis (Matsumura); known as the Asiatic onion leafminer, it is very similar to A. assectella and is an invasive alien species present in Hawaii, though not in North America. Adult diagnostic characters of the genus Acrolepiopsis, the family Acrolepiidae, and the superfamily Yponomeutoidea are also provided and illustrated. DNA barcoding data (short sequences of the mitochondrial cytochrome c oxidase I gene) obtained for five of the six species revealed interspecific differences averaging 8.1%, whereas intraspecific variation was ≤ 0.16%, and provided unequivocal species separation matching morphology-based identifications.

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Parasitoids, predators and PCR: the use of diagnostic molecular markers in biological control of Arthropods - May 01, 2007 ()
[Gariepy,T.D., U. Kuhlmann, C. Gillott,and M. Erlandson 2007. Journal of Applied Entomology. 131(4) 225-240.]

The polymerase chain reaction (PCR) revolutionized the field of diagnostics, and today it has routine applications in medical, veterinary, forensic and botanical sciences. The fields of biological control and insect pest management have generally been slow to adopt PCR-based diagnostics in comparison with other fields of science. However, there has been increasing interest in the use of molecular diagnostic tools in arthropod biological control. In applied entomology, molecular techniques have generally been used for insect identification and systematics; however, PCR-based techniques are increasingly becoming recognized as valuable tools in ecological studies. Here, we review research that has used PCR-based techniques for parasitoid and predator/prey identification and detection, and place these studies in the context of their contributions to biological control of arthropods. The status and future directions of diagnostic molecular markers in applied entomology and insect pest management are also discussed.

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Identification of genes suitable for DNA barcoding of morphologically indistinguishable korean halichondriidae sponges. - Apr 30, 2007 ()
[Park M.H., C.J. Sim, J. Baek and G.S. Min. 2007. Molecules and Cells. 23(2) 220-227.]

The development of suitable genetic markers would be useful for defining species and delineating the species boundaries of morphologically indistinguishable sponges. In this study, genetic variation in the sequences of nuclear rDNA and the mitochondrial cytochrome c oxidase subunit 1 and 3 (CO1 and CO3) regions were compared in morphologically indistinguishable Korean Halichondriidae sponges in order to determine the most suitable species-specific molecular marker region. The maximal congeneric nucleotide divergences of Halichondriidae sponges in CO1 and CO3 are similar to those found among anthozoan cnidarians, but they are 2- to 8-fold lower than those found among genera of other triploblastic metazoans. Ribosomal internal transcribed spacer regions (ITS: ITS1 + ITS2) showed higher congeneric variation (17.28% in ITS1 and 10.29% in ITS2) than those of CO1 and CO3. Use of the guidelines for species thresholds suggested in the recent literature indicates that the mtDNA regions are not appropriate for use as species-specific DNA markers for the Halichondriidae sponges, whereas the rDNA ITS regions are suitable because ITS exhibits a low level of intraspecific variation and a relatively high level of interspecific variation. In addition, to test the reliability of the ITS regions for identifying Halichondriidae sponges by PCR, a species-specific multiplex PCR primer set was developed.

Molecular evidence of host-associated genetic divergence in the holly leafminer Phytomyza glabricola (Diptera: Agromyzidae): apparent discordance among marker systems - Apr 15, 2007 ()
[Scheffer, S.J. and D.J. Hawthorne 2007. Molecular Ecology. 16(3) 2627-2637.]

Host races play a central part in understanding the role of host plant mediated divergence and speciation of phytophagous insects. Of greatest interest are host-associated populations that have recently diverged; however, finding genetic evidence for very recent divergences is difficult because initially only a few loci are expected to evolve diagnostic differences. The holly leafminer Phytomyza glabricola feeds on two hollies, Ilex glabra and I. coriacea, that are broadly sympatric throughout most of their ranges. The leafminer is often present on both host plants and exhibits a dramatic life history difference on the two hosts, suggesting that host races may be present. We collected 1393 bp of mitochondrial cytochrome oxidase I (COI) sequence and amplified fragment length polymorphism (AFLP) data (45 polymorphic bands) from sympatric populations of flies reared from the two hosts. Phylogenetic and frequency analysis of mitochondrial COI sequence data uncovered considerable variation but no structuring by the host plant, and only limited differentiation among geographical locations. In contrast, analysis of AFLP frequency data found a significant effect with host plant, and a much smaller effect with geographical location. Likewise, neighbour-joining analysis of AFLP data resulted in clustering by host plant. The AFLP data indicate that P. glabricola is most likely comprised of two host races. Because there were no fixed differences in mitochondrial or AFLP data, this host-associated divergence is likely to have occurred very recently. P. glabricola therefore provides a new sympatric system for exploring the role of geography and ecological specialization in the speciation of phytophagous insects.

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From DNA taxonomy to barcoding - how a vague idea evolved into a biosystematic tool - Apr 05, 2007 ()
[Kohler, Frank 2007. Mitteilungen aus dem Museum für Naturkunde Berlin, Zoologische Reihe. 83(Supp.) 44-51.]

The paper briefly reviews the development of the concept of DNA barcoding, a technique that proposes to identify species by analysis of short DNA sequences. The differences between this method and the concept of a DNA taxonomy based on sequence data as a universal reference system in biology are illucidated. The promises, limitations and restrictions of the different approaches are discussed. Barcoding is a useful systematic tool especially in connection with classical methods. It represents a valuable complement to conventional practice when applied to problems beyond the reach of morphological studies, such as the determination of larval stages. DNA barcoding studies detached from the fundament of integrative systematic research, however, are problematical.

DNA microarray to detect and identify trichothecene and moniliformin producing Fusarium species - Apr 01, 2007 ()
[Kristensen, R., Gauthier, G., Berdal, K.G., Hamels, S., Remacle, J. & Holst-Jensen, A. 2007. J. Applied Microbiology. 102(4) 1060-70.]

AIMS: To develop a DNA microarray for easy and fast detection of trichothecene- and moniliformin-producing Fusarium species.

METHOD AND RESULTS: A DNA microarray was developed for detection and identification of 14 trichothecene- and moniliformin-producing species of the fungal genus Fusarium. The array could also differentiate between four species groups. Capture probes were designed based on recent phylogenetic analyses of translation elongation factor-1 alpha (TEF-1α) sequences. Particular emphasis was put on designing capture probes corresponding to groups or species with particular mycotoxigenic synthetic abilities. A consensus PCR amplification of a part of the TEF-1α is followed by hybridization to the Fusarium chip and the results are visualized by a colorimetric Silverquant detection method. We validated the Fusarium chip against five naturally infected cereal samples for which we also have morphological and chemical data. The limit of detection was estimated to be less than 16 copies of genomic DNA in spiked commercial wheat flour.

CONCLUSIONS: The current Fusarium chip proved to be a highly sensitive and fast microarray for detection and identification of Fusarium species. We postulate that the method also has potential for (semi-)quantification.

SIGNIFICANCE AND IMPACT OF THE STUDY: The Fusarium chip may prove to be a very valuable tool for screening of cereal samples in the food and feed production chain, and may facilitate detection of new or introduced Fusarium spp.
Monitoring of Biological Diversity: a Common-Ground Approach - Apr 01, 2007 ()
[Teder, T., M. Moora, E. Roosaluste, K. Zobel, M. Partel, U. Koljalg, and M. Zobel. 2007. Conservation Biology. 21(2) 313-317.]

Practical approaches to monitoring biological diversity vary widely among countries, and the accumulating data are frequently not generalizable at the international scale. Although many present monitoring schemes, especially in developed countries, produce highly complex data, there is often a lack of basic data about the level and spatial distribution of biodiversity. We augmented the general framework for improving biomonitoring, proposed by Green et al. (2005), and identified its core tasks and attributes. The first priority for a more unified biodiversity monitoring is to agree on a minimum set of core tasks and attributes, which will make it possible to build a standardized biomonitoring system even in countries with few resources. Our scheme has two main organizational levels—taxa and ecosystems. The basic elements of the biomonitoring system proposed are recording of presence and absence of taxa and ecosystems in a target area, mapping of their distribution in space, and assessment of their status. All the elements have to be repeated over time. Although these tasks are fundamental, they are frequently not considered in currently functioning biomonitoring programs. The whole system has to be hierarchical and additive: if more resources are available, new activities may be added to the basic routine. Agreeing on a common standard will facilitate aggregating measures of biodiversity status and trends into regional and global indices. This information will relate directly to several Convention on Biological Diversity indicators for assessing progress toward the 2010 Biodiversity Target.


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Meeting report: Sloan Foundation Workshop to resolve problems relating to the taxonomy of microorganisms and to culture collections arising from the barcoding initiatives; Portland ME, November 6-7, 2006. - Apr 01, 2007 ()
[Hoef-Emden, K., Kupper, F. C., & Andersen, R. A. 2007. Protist. 158(2) 135-137.]

Recognizing Dinoflagellate Species using its rDNA Sequences - Apr 01, 2007 ()
[Litaker, R.W, M.W. Vandersea, S.R. Kibler, K.S. Reece, N.A. Stokes, F.M. Lutzoni, B.A. Yonish, M.A. West, M.N.D. Black, and P.A. Tester 2007. Journal of Phycology. 43(2) 344-355.]

Dinoflagellate taxonomy is based primarily on morphology and morphometric data that can be difficult to obtain. In contrast, molecular data can be rapidly and cost-effectively acquired, which has led to a rapid accumulation of sequence data in GenBank. Currently there are no systematic criteria for utilizing taxonomically unassigned sequence data to identify putative species that could in turn serve as a basis for testable hypotheses concerning the taxonomy, diversity, distribution, and toxicity of these organisms. The goal of this research was to evaluate whether simple, uncorrected genetic distances (p) calculated using ITS1/5.8S/ITS2 (ITS region) rDNA sequences could be used to develop criteria for recognizing putative species before formal morphological evaluation and classification. The current analysis used sequences from 81 dinoflagellate species belonging to 14 genera. For this diverse assemblage of dinoflagellate species, the within-species genetic distances between ITS region copies (p=0.000–0.021 substitutions per site) were consistently less than those observed between species (p=0.042–0.580). Our results indicate that a between-species uncorrected genetic distance of p≥0.04 could be used to delineate most free-living dinoflagellate species. Recently evolved species, however, may have ITS p values <0.04 and would require more extensive morphological and genetic analyses to resolve. For most species, the sequence of the dominant ITS region allele has the potential to serve as a unique species-specific "DNA barcode" that could be used for the rapid identification of dinoflagellates in field and laboratory studies.

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Inferring historical introduction pathways with mitochondrial DNA: the case of introduced Argentine ants (Linepithema humile) into New Zealand - Mar 30, 2007 ()
[Corin, S.E., P.J. Lester, K.L. Abbott,and P.A. Ritchie 2007. Diversity and Distributions. .]

The threat imposed by invasive species and difficulties associated with control and management places more impetus on trying to prevent their introduction. The identification of introduction pathways is a vital component towards this goal. In this study, we use a genetic marker-based approach to retrospectively investigate the pathway of origin of the invasive Argentine ant (Linepithema humile) into New Zealand. We intensively sample the mitochondrial gene cytochrome b, from the entire known range of Argentine ants in New Zealand. No genetic variation was found in New Zealand. In order to identify likely introduction pathways, we use two alternative genetic analyses and suggest that a tcs approach that collapses identical haplotypes and calculates the probability of parsimony is superior to standard phylogenetic tree-building algorithms. A minimum spanning network allowed relationships to be examined among sequences collated from previous international studies. The cytochrome b sequence, when compared to a global database, matched that from an Australian population. That Australia is the potential source of Argentine ants is in agreement with the New Zealand interception record, as goods from Australia have the highest number of interception records of Argentine ants. Our approach can easily be duplicated for other organisms and the methodology can be more widely applied to help aid further efforts to identify the routes of transmission for other invasive species and allow us to efficiently direct our biosecurity monitoring effort.

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DNA Barcodes Provide a Quick Preview of Mitochondrial Genome Composition - Mar 28, 2007 (pdf)
[Min, X.J. and Hickey, D.A. 2007. PLoS ONE. 2(3) e325.]

DNA barcodes have achieved prominence as a tool for species-level identifications. Consequently, there is a rapidly growing database of these short sequences from a wide variety of taxa. In this study, we have analyzed the correlation between the nucleotide content of the short DNA barcode sequences and the genomes from which they are derived. Our results show that such short sequences can yield important, and surprisingly accurate, information about the composition of the entire genome. In other words, for unsequenced genomes, the DNA barcodes can provide a quick preview of the whole genome composition.

Identifying Rattus species using mitochondrial DNA - Mar 26, 2007 ()
[Robins, J.H., M. Hingston, E. Matisoo-Smith, and H.A. Ross 2007. Molecular Ecology Notes. .]

In recent years, research has shown that geographical variation in mitochondrial DNA of commensal rats provides a strong signal of human dispersal and migration. However, interpretation of genetic variation is complicated by the presence of multiple species of Rattus especially in Island Southeast Asia, by the occurrence of some of these Rattus sp. as subfossils in archaeological and natural sites, and by the difficulty of osteological identification of these remains. Amplification of DNA from ancient sources usually yields only small fragments (~200 bp). We assessed whether we could identify Rattus sp. reliably with DNA barcoding using cytochrome oxidase I (COI) sequences, or tree-based methods using D-loop, cytochrome b and COI sequences. Species forming well-differentiated clades in a molecular phylogeny were accurately identified by both methods, even when we used short DNA fragments. Identification was less accurate for paraphyletic and polyphyletic species. We suggest that taxonomic revisions that recognize cryptic or polytypic species will lead to even greater accuracy of DNA-based identification methods.

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Universal primer cocktails for fish DNA barcoding - Mar 21, 2007 (pdf)
[Ivanova, N.V., T.S. Zemlak, R.H. Hanner, and P.D.N. Hebert. 2007. Molecular Ecology Notes. 7(4) 544-548.]

Reliable recovery of the 5' region of the cytochrome c oxidase 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all fish species. In this study, we develop and test primer cocktails with a view towards increasing the efficiency of barcode recovery. Specifically, we evaluate the success of polymerase chain reaction amplification and the quality of resultant sequences using three primer cocktails on DNA extracts from representatives of 94 fish families. Our results show that M13-tailed primer cocktails are more effective than conventional degenerate primers, allowing barcode work on taxonomically diverse samples to be carried out in a high-throughput fashion.

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DNA barcoding and the renaissance of taxonomy - Mar 20, 2007 (pdf)
[Scott E. Miller 2007. PNAS. 104(12) 4775-4776.]

A molecular assessment of northeast Pacific Alaria species (Laminariales, Phaeophyceae) with reference to the utility of DNA barcoding - Mar 16, 2007 ()
[Lane, C.E., S.C. Lindstrom, and G.W. Saunders 2007. Molecular Phylogenetics and Evolution. OnlineEarly .]

Despite their relatively complex morphologies, species in the genus Alaria Greville are notoriously difficult to identify with certainty. Morphological characters, often influenced by environmental factors, make individuals in similar habitats artificially appear related. Species identification would, therefore, benefit greatly from the application of molecular tools. We applied DNA barcoding, using the 5′ end of the cytochrome c oxidase I (coxI-5′) gene from the mitochondrial genome, to define species limits and relationships in northeast Pacific populations of Alaria. This emerging technique is being employed to catalogue species diversity worldwide, particularly among animals, and it has been shown to be sensitive enough to discriminate between closely related species. However, the utility of this marker for identifying or categorizing the majority of life remains unclear. We compared the resolution obtained with this marker to two other molecular systems commonly used in algal research: the nuclear internal transcribed spacer (ITS) of the ribosomal cistron, and the plastid Rubisco operon spacer (rbcSp). In agreement with previous results, Alaria fistulosa Postels & Ruprecht, with its distinct morphological, ecological and molecular features, stands apart from the other species in the genus and we establish Druehlia gen. nov. to accommodate it. For the remaining isolates, distinct mitochondrial haplotypes resolved with the barcode data indicate a period of genetic isolation for at least three incipient species in the northeast Pacific, whereas unexpected levels and patterns of ITS variation, as well as the extreme morphological plasticity found among these isolates, have most probably resulted from a recent collapse in species barriers. The cloning of ITS amplicons revealed multiple ITS copies in several individuals, further supporting this hypothesis.

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DNA barcodes affirm that 16 species of apparently generalist tropical parasitoid flies (Diptera, Tachinidae) are not all generalists - Mar 14, 2007 ()
[Smith, M.A., D.M. Wood , D.H. Janzen , W. Hallwachs , and P.D.N. Hebert 2007. PNAS. .]

Many species of tachinid flies are viewed as generalist parasitoids because what is apparently a single species of fly has been reared from many species of caterpillars. However, an ongoing inventory of the tachinid flies parasitizing thousands of species of caterpillars in Area de Conservación Guanacaste, northwestern Costa Rica, has encountered >400 species of specialist tachinids with only a few generalists. We DNA-barcoded 2,134 flies belonging to what appeared to be the 16 most generalist of the reared tachinid morphospecies and encountered 73 mitochondrial lineages separated by an average of 4% sequence divergence. These lineages are supported by collateral ecological information and, where tested, by independent nuclear markers (28S and ITS1), and we therefore view these lineages as provisional species. Each of the 16 apparently generalist species dissolved into one of four patterns: (i) a single generalist species, (ii) a pair of morphologically cryptic generalist species, (iii) a complex of specialist species plus a generalist, or (iv) a complex of specialists with no remaining generalist. In sum, there remained 9 generalist species among the 73 mitochondrial lineages we analyzed, demonstrating that a generalist lifestyle is possible for a tropical caterpillar parasitoid fly. These results reinforce the emerging suspicion that estimates of global species richness are likely underestimates for parasitoids (which may constitute as much as 20% of all animal life) and that the strategy of being a tropical generalist parasitic fly may be yet more unusual than has been envisioned for tachinids.

Read the publication here.

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Does the DNA barcoding gap exist? - a case study in blue butterflies (Lepidoptera: Lycaenidae) - Mar 07, 2007 ()
[Wiemers, M. and K. Fiedler 2007. Frontiers in Zoology. 4(8) .]

Marine ecological genomics: when genomics meets marine ecology - Mar 05, 2007 ()
[Dupont, S., K. Wilson, M. Obst, H. Sköld, H. Nakano and M.C. Thorndyke 2007. Marine Ecology Progress Series. 332 257-273.]

Genomics, proteomics and metabolomics (the 'omic' technologies) have revolutionized the way we work and are able to think about working, and have opened up hitherto unimagined opportunities in all research fields. In marine ecology, while 'standard' molecular and genetic approaches are well known, the newer technologies are taking longer to make an impact. In this review we explore the potential and promise offered by genomics, genome technologies, expressed sequence tag (EST) collections, microarrays, proteomics and bar coding for modern marine ecology.  Methods are succinctly presented with both benefits and limitations discussed. Through examples from the literature, we show how these tools can be used to answer fundamental ecological questions, e.g. 'what is the relationship between community structure and ecological function in ecosystems?';'how can a species and the phylogenetic relationship between taxa be identified?'; 'what are the factors responsible for the limits of the ecological niche?'; or 'what explains the variations in life-history patterns among species?' The impact of ecological ideas and concepts on genomic science is also discussed.

High-throughput species identification: from DNA isolation to bioinformatics - Mar 01, 2007 ()
[Richardson, D.E., Vanwye, J.D., Exum, A. M., Cowen, R.K., & D.L. Crawford 2007. Molecular Ecology Notes. 7(2) 199-207.]

Ichthyoplankton collections provide a valuable means to study fish life histories. However, these collections are greatly underutilized, as larval fishes are frequently not identified to species due to their small size and limited morphological development. Currently, there is an effort underway to make species identification more readily available across a broad range of taxa through the sequencing of a standard gene. This effort requires the development of new methodologies to both rapidly produce and analyse large numbers of sequences. The methodology presented in this paper addresses these issues with a focus on the larvae of large pelagic fish species. All steps of the methodology are targeted towards high-throughput identification using small amounts of tissue. To accomplish this, DNA isolation was automated on a liquid-handling robot using magnetic bead technologies. Polymerase chain reaction and a unidirectional sequencing reaction followed standard protocols with all template cleanup and transferring also automated. Manual pipetting was thus reduced to a minimum. A character-based bioinformatics program was developed to handle the large sequence output. This program incorporates base-call quality scores in two types of sample to voucher sequence comparisons and provides suggested identifications and sequence information in an easily interpreted spreadsheet format. This technique when applied to tuna and billfish larvae collected in the Straits of Florida had an 89% success rate. A single species (Thunnus atlanticus) was found to dominate the catch of tuna larvae, while billfish larvae were more evenly divided between two species (Makaira nigricans and Istiophorus platypterus).

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Host-specificity and coevolution among pollinating and nonpollinating New World fig wasps - Mar 01, 2007 ()
[Marussich, W.A., and C.A. Machado 2007. Molecular Ecology. .]

Figs (Ficus spp., Moraceae) and their pollinating wasps (Hymenoptera, Agaonidae, Chalcidoidea) constitute a classic example of an obligate plant-pollinator mutualism, and have become an ideal system for addressing questions on coevolution, speciation, and the maintenance of mutualisms. In addition to pollinating wasps, figs host several types of nonpollinating, parasitic wasps from a diverse array of Chalcid subfamilies with varied natural histories and ecological strategies (e.g. competitors, gallers, and parasitoids). Although a few recent studies have addressed the question of codivergence between specific genera of pollinating and nonpollinating fig wasps, no study has addressed the history of divergence of a fig wasp community comprised of multiple genera of wasps associated with a large number of sympatric fig hosts. Here, we conduct phylogenetic analyses of mitochondrial DNA sequences (COI) using 411 individuals from 69 pollinating and nonpollinating fig wasp species to assess relationships within and between five genera of fig wasps (Pegoscapus, Idarnes, Heterandrium, Aepocerus, Physothorax) associated with 17 species of New World Urostigma figs from section Americana. We show that host-switching and multiple wasp species per host are ubiquitous across Neotropical nonpollinating wasp genera. In spite of these findings, cophylogenetic analyses (treemap 1.0, treemap 2.02β, and parafit) reveal evidence of codivergence among fig wasps from different ecological guilds. Our findings further challenge the classical notion of strict-sense coevolution between figs and their associated wasps, and mirror conclusions from detailed molecular studies of other mutualisms that have revealed common patterns of diffuse coevolution and asymmetric specialization among the participants.

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Using COI barcodes to identify forensically and medically important blowflies - Mar 01, 2007 ()
[Nelson, L.A., J.F. Wallman, and M. Dowton 2007. Medical and Veterinary Entomology. 21(1) 44-52.]

The utility of cytochrome oxidase I (COI) DNA barcodes for the identification of nine species of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae), from Australia, was tested. A 658-bp fragment of the COI gene was sequenced from 56 specimens, representing all nine Chrysomya species and three calliphorid outgroups. Nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model and a neighbour-joining (NJ) analysis was performed to provide a graphic display of the patterns of divergence among the species. All species were resolved as reciprocally monophyletic on the NJ tree. Mean intraspecific and interspecific sequence divergences were 0.097% (range 0–0.612%, standard error [SE] = 0.119%) and 6.499% (range 0.458–9.254%, SE = 1.864%), respectively. In one case, a specimen that was identified morphologically was recovered with its sister species on the NJ tree. The hybrid status of this specimen was established by sequence analysis of the second ribosomal internal transcribed spacer (ITS2). In another instance, this nuclear region was used to verify four cases of specimen misidentification that had been highlighted by the COI analysis. The COI barcode sequence was found to be suitable for the identification of Chrysomya species from the east coast of Australia.

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Four genes, morphology and ecology: distinguishing a new species of Acesta (Mollusca; Bivalvia) from the Gulf of Mexico - Mar 01, 2007 ()
[Johanna Järnegren, Christoffer Schander, Jon-Arne Sneli, Vera Rønningen and Craig M. Young 2007. Mar Biol. 152(1) 43-55.]

There is currently an active scientiWc debate about the most correct and eYcient way to identify species. To date, few studies in the marine realm have combined the available taxonomic methods. In this study we have used morphology, ecology and molecular analyses to identify a new species within the bivalve genus Acesta. All four genes studied (12S, 16S, Cytb, COI) suggested that a common cold-seep species in the Gulf of Mexico, A. bullisi, should be divided into two distinct species. This conclusion was supported by morphological traits and by observations of ecological distribution. A. oophaga Järnegren, Schander and Young n. sp. is described here, and A. bullisi Vokes (Tulane Stud Geol 2:75–92, 1963) is re-described. This study shows that DNA barcoding in combination with traditional morphological and ecological analyses may be an important tools to identify hidden biodiversity among deep-water organisms such as bivalves.

Molecular phylogeny of Pacific Island Colymbetinae: radiation of New Caledonian and Fijian species (Coleoptera, Dytiscidae) - Mar 01, 2007 ()
[Balke, M., G. Wewalka, Y. Alarie and I. Ribera 2007. Zoologica Scripta. 36(2) 173-200.]

We present a molecular phylogeny and taxonomic review of the Pacific island colymbetine diving beetles, focusing on the Fijian and New Caledonian faunas. Four new species are described: Rhantus monteithi and R. poellerbauerae from New Caledonia, and R. kini and R. bula from Fiji. We also describe the 3rd instar larvae of R. monteithi and R. poellerbauerae spp. nov., assigned to adults using mtDNA sequence data and discuss larval characters in the light of phylogeny. The phylogenetic hypotheses derived from both parsimony and Bayesian inference based on 3508 aligned nucleotides from a combination of mitochondrial (cox1, cob and rrnL-tRNALeu-Nad1) and nuclear genes (18S rRNA and H3) reveal a clade comprising R. novaecaledoniae, R. alutaceus, R. pseudopacificus, R. monteithi sp. nov. and R. poellerbauerae sp. nov., which agrees with the R. pacificus group sensu Balke (1993). Carabdytes upin was included within this clade, possibly indicating paraphyly of the genus Rhantus. Rhantus annectens, R. bacchusi, R. supranubicus, R. suturalis, R. simulans, and the Palearctic R. exsoletus, R. latitans and R. bistriatus formed a clade corresponding to the R. suturalis group sensu Balke (2001). Rhantus vitiensis, previously assigned to the R. pacificus group, was included in the R. suturalis clade. We find some support for a scenario where the Pacific was colonized out of the Northern hemisphere only during the past c. 12 million years, rejecting a Gondwanan origin of the morphologically isolated endemics. The new species are all characterized by mtDNA haplotype clusters, the degree of divergence between sister species pairs ranging from 1.3 to 7%, while R. novaecaledoniae individuals from all over New Caledonia apparently form one morphospecies, with moderate genetic diversity (up to 2.3% mtDNA divergence between populations). The sisters R. pollerbauerae sp. nov. + R. monteithi sp. nov. occur sympatrically on Mont Panié but appear ecologically segregated, while the sisters R. vitiensis + R. bula sp. nov. were encountered syntopically on Viti Levu. Comparing genetic and morphological data of Fijian Rhantus and Copelatus diving beetles, we here show that even in island radiations it is not per se possible to know if mitochondrial DNA barcoding would perform well (Rhantus: YES, Copelatus: NO). At the same time we show that fixed cutoff values, as sometimes used to discriminate between barcodes, thus species, might be meaningless. We underpin the importance of morphology for sustainable exploration of global diversity.

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Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species - Mar 01, 2007 (pdf)
[Pfenninger, M., C. Nowak, C. Kley, D. Steinke, and B. Streit. 2007. Molecular Ecology. .]

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.

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Bionomics and morphological and molecular characterization of Elasmus schmitti and Baryscapus elasmi (Hymenoptera: Chalcidoidea, Eulophidae), parasitoids associated with a paper wasp, Polistes dominulus (Vespoidea, Vespidae) - Mar 01, 2007 ()
[Gumovsky, A., L. Rusina and L. Firman 2007. Entomological Science. 10(1) 21-34.]

Elasmus schmitti and Baryscapus elasmi have been recorded in southern Ukraine as gregarious parasitoids in the nests of the paper wasps Polistes dominulus and Polistes nimphus. Polistes dominulus nests infested with E. schmitti were less productive than uninfested nests in only one year (2004) of the three years of the present study, when an increase in the host population size occurred. Females of E. schmitti are synovigenic, and they lay their eggs on the skins of P. dominulus last instar larvae, without paralyzing the host. Rather, the parasitoid larvae feed on young host pupae. The pupae of E. schmitti are isolated from the host remnants by a thin fecal partition as in Elasmus polistis and Elasmus japonicus, other paper wasp parasitoids. Baryscapus elasmi is a pupal endoparasitoid of E. schmitti. The females of B. elasmi emerge without mature eggs in their ovaries and mate with males. They penetrate the paper wasp's cells with their ovipositor and feed on the extracted hemolymph exudate. Pupation of B. elasmi occurs inside or outside the pupa of the host, E. schmitti. If inside, then the cranial end of the pupa and the adult emergence hole of B. elasmi are situated in the caudal ends of the pupae of their hosts. Comparative notes and illustrations on the morphology of adults are provided, and DNA sequences of three genes (nuclear 28S D2 rDNA, mitochondrial cytochrome oxidase subunit I, and mitochondrial cytochrome b) were obtained for both parasitoid species. The similarity of the 28S D2 sequences of E. schmitti and E. polistis relative to other available Elasmus sequences suggests a single origin of parasitism on paper wasps in this genus.

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Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case - Feb 28, 2007 (pdf)
[K.A. Seifert, R.A. Samson, J.R. deWaard, J. Houbraken, C.A. Lévesque, J.-M. Moncalvo, G. Louis-Seize, P.D.N. Hebert 2007. Proc. Natl. Acad. Sci. USA. .]

DNA barcoding systems employ a short, standardized gene region to identify species. A 648-bp segment of mitochondrial cytochrome c oxidase 1 (CO1) is the core barcode region for animals, but its utility has not been tested in fungi. This study began with an examination of patterns of sequence divergences in this gene region for 38 fungal taxa with full CO1 sequences. Because these results suggested that CO1 could be effective in species recognition, we designed primers for a 545-bp fragment of CO1 and generated sequences for multiple strains from 58 species of Penicillium subgenus Penicillium and 12 allied species. Despite the frequent literature reports of introns in fungal mitochondrial genomes, we detected introns in only 2 of 370 Penicillium strains. Representatives from 38 of 58 species formed cohesive assemblages with distinct CO1 sequences, and all cases of sequence sharing involved known species complexes. CO1 sequence divergences averaged 0.06% within species, less than for internal transcribed spacer nrDNA or {beta}-tubulin sequences (BenA). CO1 divergences between species averaged 5.6%, comparable to internal transcribed spacer, but less than values for BenA (14.4%). Although the latter gene delivered higher taxonomic resolution, the amplification and alignment of CO1 was simpler. The development of a barcoding system for fungi that shares a common gene target with other kingdoms would be a significant advance.

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DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. - Feb 19, 2007 (pdf)
[Hajibabaei M., G.A. Singer, P.D.N. Hebert,and D.A. Hickey. 2007. Trends in Genetics. .]

DNA barcoding aims to provide an efficient method for species-level identifications and, as such, will contribute powerfully to taxonomic and biodiversity research. As the number of DNA barcode sequences accumulates, however, these data will also provide a unique 'horizontal' genomics perspective with broad implications. For example, here we compare the goals and methods of DNA barcoding with those of molecular phylogenetics and population genetics, and suggest that DNA barcoding can complement current research in these areas by providing background information that will be helpful in the selection of taxa for further analyses.

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DNA barcoding of Neotropical bats: species identification and discovery within Guyana - Feb 19, 2007 (pdf)
[Clare, E.L., B.K. Lim, M.D. Engstrom, J.L. Eger, and P.D.N. Hebert. 2007. Molecular Ecology Notes. .]

Sequence diversity in the cytochrome c oxidase subunit 1 gene has been shown to be an effective tool for species identification and discovery in various groups of animals, but has not been extensively tested in mammals. We address this gap by examining the performance of DNA barcodes in the discrimination of 87 species of bats from Guyana. Eighty-one of these species showed both low intraspecific variation (mean = 0.60%), and clear sequence divergence from their congeners (mean = 7.80%), while the other six showed deeply divergent intraspecific lineages suggesting that they represent species complexes. Although further work is needed to examine patterns of sequence diversity at a broader geographical scale, the present study validates the effectiveness of barcoding for the identification of regional bat assemblages, even highly diverse tropical faunas.

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Comprehensive DNA barcode coverage of North American birds - Feb 19, 2007 (pdf)
[Kerr, K.C.R, M.Y. Stoeckle, C.J. Dove, L.A. Weigt, C.M. Francis and P.D.N. Hebert 2007. Molecular Ecology Notes. .]

DNA barcoding seeks to assemble a standardized reference library for DNA-based identification of eukaryotic species. The utility and limitations of this approach need to be tested on well-characterized taxonomic assemblages. Here we provide a comprehensive DNA barcode analysis for North American birds including 643 species representing 93% of the breeding and pelagic avifauna of the USA and Canada. Most (94%) species possess distinct barcode clusters, with average neighbour-joining bootstrap support of 98%. In the remaining 6%, barcode clusters correspond to small sets of closely related species, most of which hybridize regularly. Fifteen (2%) currently recognized species are comprised of two distinct barcode clusters, many of which may represent cryptic species. Intraspecific variation is weakly related to census population size and species age. This study confirms that DNA barcoding can be effectively applied across the geographical and taxonomic expanse of North American birds. The consistent finding of constrained intraspecific mitochondrial variation in this large assemblage of species supports the emerging view that selective sweeps limit mitochondrial diversity. 

Supplementary material may be found here.

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An evaluation of LSU rDNA D1-D2 sequences for their use in species identification - Feb 16, 2007 ()
[Sonnenberg, R., A.W., and D. Tautz 2007. Frontiers in Zoology. 4(6) .]

Identification of species via DNA sequences is the basis for DNA taxonomy and DNA barcoding. Currently there is a strong focus on using a mitochondrial marker for this purpose, in particular a fragment from the cytochrome oxidase I gene (COI). While there is ample evidence that this marker is indeed suitable across a broad taxonomic range to delineate species, it has also become clear that a complementation by a nuclear marker system could be advantageous. Ribosomal RNA genes could be suitable for this purpose, because of their global occurrence and the possibility to design universal primers. However, it has so far been assumed that these genes are too highly conserved to allow resolution at, or even beyond the species level. On the other hand, it is known that ribosomal gene regions harbour also highly divergent parts. We explore here the information content of two adjacent divergence regions of the large subunit ribosomal gene, the D1-D2 region.

Validation of the barcoding gene COI for use in forensic genetic species identification. - Feb 12, 2007 ()
[Dawnay N., R. Ogden, R. McEwing, G.R. Carvalho and R.S. Thorpe. 2007. Forensic Science International. .]
Statistical Tests for Taxonomic Distintiveness From Observations of Monophyly - Feb 06, 2007 ()
[Rosenberg, N.A. 2007. Evolution. 61(2) 317-323.]

The observation of monophyly for a specified set of genealogical lineages is often used to place the lineages into a distinctive taxonomic entity. However, it is sometimes possible that monophyly of the lineages can occur by chance as an outcome of the random branching of lineages within a single taxon. Thus, especially for small samples, an observation of monophyly for a set of lineages—even if strongly supported statistically—does not necessarily indicate that the lineages are from a distinctive group. Here I develop a test of the null hypothesis that monophyly is a chance outcome of random branching. I also compute the sample size required so that the probability of chance occurrence of monophyly of a specified set of lineages lies below a prescribed tolerance. Under the null model of random branching, the probability that monophyly of the lineages in an index group occurs by chance is substantial if the sample is highly asymmetric, that is, if only a few of the sampled lineages are from the index group, or if only a few lineages are external to the group. If sample sizes are similar inside and outside the group of interest, however, chance occurrence of monophyly can be rejected at stringent significance levels (P < 10-5) even for quite small samples (≈ 20 total lineages). For a fixed total sample size, rejection of the null hypothesis of random branching in a single taxon occurs at the most stringent level if samples of nearly equal size inside and outside the index group—with a slightly greater size within the index group—are used. Similar results apply, with smaller sample sizes needed, when reciprocal monophyly of two groups, rather than monophyly of a single group, is of interest. The results suggest minimal sample sizes required for inferences to be made about taxonomic distinctiveness from observations of monophyly.

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Considering evolutionary processes in the use of single-locus genetic data for conservation, with examples from the Lepidoptera - Feb 01, 2007 ()
[Forister, M. L., Nice, C. C., Fordyce, J. A., Gompert, Z., and Shapiro, A. M. 2007. Journal of Insect Conservation. 12(1) 37-51.]

The increasing popularity of molecular taxonomy will undoubtedly have a major impact on the practice of conservation biology. The appeal of such approaches is undeniable since they will clearly be an asset in rapid biological assessments of poorly known taxa or unexplored areas, and for discovery of cryptic biodiversity. However, as an approach for diagnosing units for conservation, some caution is warranted. The essential issue is that mitochondrial DNA variation is unlikely to be causally related to, and thus correlated with, ecologically important components of fitness. This is true for DNA barcoding, molecular taxonomy in general, or any technique that relies on variation at a single, presumed neutral locus. Given that natural selection operates on a time scale that is often much more rapid than the rates of mutation and allele frequency changes due to genetic drift, neutral genetic variation at a single locus can be a poor predictor of adaptive variation within or among species. Furthermore, reticulate processes, such as introgressive hybridization, may also constrain the utility of molecular taxonomy to accurately detect significant units for conservation. A survey of published genetic data from the Lepidoptera indicates that these problems may be more prevalent than previously suspected. Molecular approaches must be used with caution for conservation genetics which is best accomplished using large sample sizes over extensive geography in addition to data from multiple loci.

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Descriptions of the tadpoles of two species of Gephyromantis, with a discussion of the phylogenetic origin of direct development in mantellid frogs - Feb 01, 2007 ()
[Randrianiaina, R.-D., F. Glaw, M. Thomas, J. Glos, N. Raminosoa & M. Vences 2007. Zootaxa. 1401 53-61.]

We describe the larval stages of two Malagasy frog species of the genus Gephyromantis, based on specimens identified by DNA barcoding. The tadpoles of Gephyromantis ambohitra are generalized stream-living Orton type IV type larvae with two lateral small constrictions of the body wall at the plane of spiracle. Gephyromantis pseudoasper tadpoles are characterized by totally keratinised jaw sheaths with hypertrophied indentation, a reduced number of labial tooth rows, enlarged papillae on the oral disc, and a yellowish coloration of the tip of the tail in life. The morphology of the tadpole of G. pseudoasper agrees with that of G. corvus, supporting the current placement of these two species in a subgenus Phylacomantis, and suggesting that the larvae of G. pseudoasper may also have carnivorous habits as known in G. corvus. Identifying the tadpole of Gephyromantis ambohitra challenges current assumptions of the evolution of different developmental modes in Gephyromantis, since this species is thought to be related to G. asper, a species of supposedly endotrophic direct development.

A step toward barcoding life: a model-based, decision-theoretic method to assign genes to preexisting species groups - Feb 01, 2007 ()
[Abdo, Z. and G. B. Golding 2007. Syst Biol. 56(1) 44-56.]

A major part of the barcoding of life problem is assigning newly sequenced or sampled individuals to existing groups that are preidentified externally (by a taxonomist, for example). This problem involves evaluating the statistical evidence towards associating a sequence from a new individual with one group or another. The main concern of our current research is to perform this task in a fast and accurate manner. To accomplish this we have developed a model-based, decision-theoretic framework based on the coalescent theory. Under this framework, we utilized both distance and the posterior probability of a group, given the sequences from members of this group and the sequence from a newly sampled individual to assign this new individual. We believe that this approach makes efficient use of the available information in the data. Our preliminary results indicated that this approach is more accurate than using a simple measure of distance for assignment.

Biological identifications through DNA barcodes: the case of the Crustacea - Feb 01, 2007 (pdf)
[Costa F.O., deWaard, J. R., Boutillier, J., Ratnasingham S., Dooh R., Hajibabaei M., Hebert, P.D.N. 2007. Canadian Journal of Fisheries and Aquatic Sciences. 64(2) 272-295.]

The ability of a 650 base pair section of the mitochondrial cytochrome c oxidase I (COI) gene to provide species-level identifications has been demonstrated for large taxonomic assemblages of animals such as insects, birds and fishes, but not for the subphylum Crustacea, one of the most diverse groups of arthropods. In this study we test the ability of COI to provide identifications in this group, examining two disparate levels in the taxonomic hierarchy – orders and species. The first phase of our study involved the development of a sequence profile for 23 dominant crustacean orders, based upon the analysis of 150 species, each belonging to a different family. The COI amino acid data placed these taxa into cohesive assemblages whose membership coincided with currently accepted boundaries at the order, superorder and subclass levels. Species-level resolution was subsequently examined in an assemblage of Decapoda, and in representatives of the genera Daphnia (Cladocera) and Gammarus (Amphipoda). These studies revealed that levels of nucleotide sequence divergence were from 19 to 48 times greater between congeneric species than between individuals of a species. We conclude that sequence variation in the COI barcode region will be very effective for discriminating species of Crustacea.

Diagnostic markers for Planococcus ficus (Signoret) and Planococcus citri (Risso) by random amplification of polymorphic DNA-polymerase chain reaction and species-specific mitochondrial DNA primers - Feb 01, 2007 ()
[Demontis M. A., Ortu S., Cocco A., Lentini A. and Migheli Q. 2007. Journal of Applied Entomology. 131(1) 59–64.]

Planococcus ficus (Signoret) and Planococcus citri (Risso) (Hom., Pseudococcidae) are important phytophagous components in different agroecosystems. The two species may coexist in the same environment and are most difficult to distinguish by morphological features. The aim of this study was to find genetic markers suitable for distinguishing P. ficus from P. citri, to assist in the rapid identification of field specimens. By using synthetic sex pheromone-baited traps, pure male populations of both species were collected from a vineyard and from a citrus orchard in northern Sardinia, Italy. Individual males of citrus and vine mealybugs were preliminarily examined by the random amplification of polymorphic DNA (RAPD) technique. Among twelve 10-mer random primers, the oligonucleotide OPL-12 generated several markers suitable for distinguishing between the two species. This primer was then used to characterize individual males and females of both mealybug species collected near pheromone-baited traps in vineyards and orange orchards from different geographic areas. Reference samples from other regions of southern Italy were also included. A clear differentiation of the two species was accomplished according to their pattern of amplification, thus confirming a high level of intra-specific genetic homogeneity. Consequently, two fragments of the cytochrome c oxidase I gene from P. citri and P. ficus were compared and two pairs of species-specific polymerase chain reaction (PCR) primers were developed based on diverging sequences. These primers allowed sensitive and reliable PCR identification of both males and females of P. citri and of P. ficus of different geographic origin.

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A New Poecilogonous Species of Sea Slug (Opisthobranchia: Sacoglossa) from California: Comparison with the Planktotrophic Congener Alderia Modesta - Feb 01, 2007 ()
[Krug, P.J., R.A. Ellingson, R. Burton and A. Valdes 2007. Journal of Molluscan Studies. 73(1) 29-38.]

Cryptic species are increasingly recognized as commonplace among marine gastropods, especially in taxa such as shell-less opisthobranchs that lack many discrete taxonomic characters. Most cases of poecilogony, the presence of variable larval development within a single species, have historically turned out to represent cryptic species, with each possessing a single canalized type of development. One well-characterized example of poecilogony was attributed to the sacoglossan opisthobranch Alderia modesta; in southern California, slugs resembling this member of a monotypic genus produce both long-lived, planktotrophic and short-lived, lecithotrophic larvae. Paradoxically, however, A. modesta is exclusively planktotrophic everywhere else in the northern Pacific and Atlantic Oceans. A recently completed molecular study found that slugs from poecilogonous populations south of Bodega Harbor, California, comprise an evolutionarily distinct lineage separate from northern, strictly planktotrophic slugs. We now describe the southern species as A. willowi n. sp., based on differences in morphology of the dorsum and radula, characteristics of the egg mass, larval development mode and nuclear and mitochondrial genetic markers. A DNA barcode is provided, based on 27 fixed differences in the cytochrome c oxidase subunit I gene that can reliably differentiate Pacific specimens of Alderia species. Genetic and morphological data are concordant with developmental evidence, confirming that A. willowi is a true case of poecilogony. An improved understanding of the ecological differences between these sister taxa may shed light on the selective pressures that drove the evolution of lecithotrophy in the southern species.

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A comparison of algorithms for the identification of specimens using DNA barcodes: examples from gymnosperms - Feb 01, 2007 ()
[Little, D.P. and D.W. Stevenson 2007. Cladistics. 23(1) 1-21.]

In order to use DNA sequences for specimen identification (e.g., barcoding, fingerprinting) an algorithm to compare query sequences with a reference database is needed. Precision and accuracy of query sequence identification was estimated for hierarchical clustering (parsimony and neighbor joining), similarity methods (BLAST, BLAT and megaBLAST), combined clustering/similarity methods (BLAST/parsimony and BLAST/neighbor joining), diagnostic methods (DNA–BAR and DOME ID), and a new method (ATIM). We offer two novel alignment-free algorithmic solutions (DOME ID and ATIM) to identify query sequences for the purposes of DNA barcoding. Publicly available gymnosperm nrITS 2 and plastid matK sequences were used as test data sets. On the test data sets, almost all of the methods were able to accurately identify sequences to genus; however, no method was able to accurately identify query sequences to species at a frequency that would be considered useful for routine specimen identification (42–71% unambiguously correct). Clustering methods performed the worst (perhaps due to alignment issues). Similarity methods, ATIM, DNA–BAR, and DOME ID all performed at approximately the same level. Given the relative precision of the algorithms (median = 67% unambiguous), the low accuracy of species-level identification observed could be ascribed to the lack of correspondence between patterns of allelic similarity and species delimitations. Application of DNA barcoding to sequences of CITES listed cycads (Cycadopsida) provides an example of the potential application of DNA barcoding to enforcement of conservation laws.

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DNA barcoding reveals hidden species diversity in Cymothoe (Nymphalidae) - Jan 31, 2007 (pdf)
[Velzen, R., Bakker, F.T., & J. Loon 2007. PROC. NETH. ENTOMOL. SOC. MEET.. 18 95-103.]

DNA barcoding has recently become a prominent tool for species identification and discovery. In this paper we assess whether DNA barcoding can be used to identify and match different life stages of Cymothoe butterflies. Our results showed that DNA barcode sequences cluster according to species and that interspecific divergence is higher than intraspecific variation. In three cases, high levels of intraspecific DNA sequence variation revealed sibling species in Cymothoe, which are supported by morphology and host-plant data. We conclude that DNA barcoding is a powerful tool for the identification of eggs and caterpillars. In addition, combined with morphology, ecology and biogeography, DNA barcoding can be valuable in revealing hidden species.

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Direct PCR for DNA barcoding in the genera Phytophopthora and Pythium - Jan 21, 2007 ()
[Calmin, G., L. Belbahri,and F. Lefort 2007. Biotechnology and Biotechnological Equipment. 21(1) 40-42.]

A protocol for direct polymerase chain reaction (DPCR) amplification of commonly used phylogenetic markers of the genera Pythium and Phytophthora from mycelia was developed. This has proven useful for direct PCR amplification from hypha, zoospores and infected plant material, which is quicker and simpler, than other proposed attempts.  Optimal reaction conditions are described allowing amplification of commonly used phylogenetic markers with a size of up to 1 kbp. This approach proved successful for amplification of selected markers from hundreds of Phytophthora, Pythium and fungal isolates and has been so far used in routine genotyping identification of oomycota and fungi in our laboratory.

DNA barcoding in animal species: progress, potential and pitfalls - Jan 16, 2007 ()
[Waugh , John 2007. BioEssays. 29(2) 188-197.]

Despite 250 years of work in systematics, the majority of species remains to be identified. Rising extinction rates and the need for increased biological monitoring lend urgency to this task. DNA sequencing, with key sequences serving as a barcode, has therefore been proposed as a technology that might expedite species identification. In particular, the mitochondrial cytochrome c oxidase subunit 1 gene has been employed as a possible DNA marker for species and a number of studies in a variety of taxa have accordingly been carried out to examine its efficacy. In general, these studies demonstrate that DNA barcoding resolves most species, although some taxa have proved intractable. In some studies, barcoding provided a means of highlighting potential cryptic, synonymous or extinct species as well as matching adults with immature specimens. Higher taxa, however, have not been resolved as accurately as species. Nonetheless, DNA barcoding appears to offer a means of identifying species and may become a standard tool.

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DNA barcoding using chitons (genus Mopalia) - Jan 12, 2007 ()
[Kelly, R.P., I.N. Sarkar, D.J. Eernisse and R. Desalle 2007. Molecular Ecology Notes. .]

Incorporating substantial intraspecific genetic variation for 19 species from 131 individual chitons, genus Mopalia (Mollusca: Polyplacophora), we present rigorous DNA barcodes for this genus as per the currently accepted approaches to DNA barcoding. We also have performed a second kind of analysis that does not rely on blast or the distance-based neighbour-joining approach as currently resides on the Barcode of Life Data Systems website. Our character-based approach, called characteristic attribute organization system, returns fast, accurate, character-based diagnostics and can unambiguously distinguish between even closely related species based on these diagnostics. Using statistical subsampling approaches with our original data matrix, we show that the method outperforms blast and is equally effective as the neighbour-joining approach. Our approach differs from the neighbour-joining approach in that the end-product is a list of diagnostic nucleotide positions that can be used in descriptions of species. In addition, the diagnostics obtained from this character-based approach can be used to design oligonucleotides for detection arrays, polymerase chain reaction drop off diagnostics, TaqMan assays, and design of primers for generating short fragments that encompass regions containing diagnostics in the cytochrome oxidase I gene.

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Exploitation of archived marine nematodes - a hot lysis DNA extraction protocol for molecular studies - Jan 01, 2007 ()
[Bhadury, P., Austen, M. C., Bilton, D. T., Lambshead, P. J. D., Rogers, A. D. and Smerdon, G. R. 2007. Zoologica Scripta. 36(1) 93-98.]

Museums and other research organizations around the world have large numbers of formalin-fixed marine invertebrates in their collections. These have the potential to be a valuable resource for molecular ecological studies, but the development of methodologies for the molecular analysis of formalin-fixed material has been slow. In this study, a hot lysis protocol accompanied by the use of a commercial DNA extraction kit has been employed for DNA recovery from archived marine nematodes, followed by PCR amplification and sequencing. In total, 25 specimens ranging from estuarine to deep sea environments were subjected to molecular analyses. Successful amplification and sequencing of the nuclear small subunit ribosomal RNA (18S rRNA) gene was achieved in all individuals. Additionally, some estuarine nematodes were tentatively identified to genus and species using a phylogenetic approach. In the future, this technique should prove to be profitable for the genetic study of a wide range of formalin-fixed marine invertebrates.

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Cytochrome b barcoding, molecular systematics and geographic differentiation in rabbitfishes (Siganidae) - Jan 01, 2007 ()
[Lemera S., Aurelle D., Vigliola L., Durand J.D., and Borsa P. 2007. Comptes Rendus Biologies. 330(1) 86-94.]

The fish genus Siganus (Siganidae) is widely distributed in the coastal habitats of all the tropical Indo-Pacific, with 28 nominal species recognized so far, based on general morphology and coloration patterns. A mitochondrial phylogeny of 16 Siganidae species, based on the partial nucleotide sequences of the cytochome b gene, was produced. Individual haplotypes of given nominal species generally clustered at the extremity of long branches, thus validating the current taxonomy. However, S. lineatus haplotypes formed a paraphyletic group including S. guttatus, while S. fuscescens haplotypes were apparently splitted in two groups, calling for further investigation. S. woodlandi and S. argenteus formed a monophyletic group, as expected from their close morphological relatedness, although they were separated by a substantial, 14.5–16.3% nucleotide distance. Among eight species sampled from different locations across the Indo-West Pacific, S. argenteus and S. spinus showed the lowest degree of geographic differentiation, a result that correlated well with their extended pelagic larval stage. Fixation index estimates were high in all six other species tested (S. doliatus, S. fuscescens, S. lineatus, S. puellus, S. punctatus, S. vulpinus). The cytochrome b gene fragment chosen here proved useful as a barcode in Siganidae.

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Molecular Advances in the Study of Geographic Variation and Speciation in Birds - Jan 01, 2007 ()
[Baker, A.J. 2007. Ornithological Monographs. 63 18-29.]

Problems in deciphering the patterns and causes of geographic variation and speciation in birds occupied Ned Johnson (e.g., Johnson 1980, Cicero and Johnson 1998, Johnson and Cicero 2002) and many other ornithologists for much of their lives, but the recent onslaught of molecular studies and associated analytical methods are providing breakthroughs in understanding these evolutionary phenomena. In particular, coalescent theory and Markov chain Monte Carlo (MCMC) applications have shown that bird species are sometimes strongly structured into well-differentiated populations by historical subdivision, high philopatry, and small effective population sizes, whereas other species that have recently recolonized parts of their range are effectively panmictic. These are the sorts of results that were impossible to obtain from studies of geographic variation in phenotypic characters alone. Recovery of well-supported species trees from gene trees is much more likely when multiple genes are sequenced, and provides the means for inferring divergence times and patterns and processes of evolution in birds. As in other vertebrates, patterns of cladogenesis in large clades of birds correlate with major paleoenvironmental changes and associated adaptive radiations, reminding us that much of current biodiversity on the planet had its genesis in the distant past.

The current status of species recognition and identification in Aspergillus - Jan 01, 2007 ()
[Geiser, D. M., Klich, M. A., Frisvad, J. C., Peterson, S. W., Varga, J., & Samson, R. A. 2007. Stud Mycol. 59 1-10.]

The species recognition and identification of aspergilli and their teleomorphs is discussed. A historical overview of the taxonomic concepts starting with the monograph of Raper & Fennell (1965) is given. A list of taxa described since 2000 is provided. Physiological characters, particularly growth rates and the production of extrolites, often show differences that reflect phylogenetic species boundaries and greater emphasis should be placed on extrolite profiles and growth characteristics in species descriptions. Multilocus sequence-based phylogenetic analyses have emerged as the primary tool for inferring phylogenetic species boundaries and relationships within subgenera and sections. A four locus DNA sequence study covering all major lineages in Aspergillus using genealogical concordance theory resulted in a species recognition system that agrees in part with phenotypic studies and reveals the presence of many undescribed species not resolved by phenotype. The use of as much data from as many sources as possible in making taxonomic decisions is advocated. For species identification, DNA barcoding uses a short genetic marker in an organism"s DNA to quickly and easily identify it to a particular species. Partial cytochrome oxidase subunit 1 sequences, which are used for barcoding animal species, were found to have limited value for species identification among black aspergilli. The various possibilities are discussed and at present partial beta-tubulin or calmodulin are the most promising loci for Aspergillus identification. For characterising Aspergillus species one application would be to produce a multilocus phylogeny, with the goal of having a firm understanding of the evolutionary relationships among species across the entire genus. DNA chip technologies are discussed as possibilities for an accurate multilocus barcoding tool for the genus Aspergillus.

The Barcode of Life Initiative: synopsis and prospective societal impacts of DNA barcoding of Fish - Jan 01, 2007 ()
[Costa, F. O. & G. R. Carvalho 2007. Genomics, Society and Policy. 3(2) 29-40.]

Almost 250 years after the publication of the taxonomy-founding work Systema Naturae, by Carl Linnaeus, the inventory and catalogue of the planet’s biodiversity is still far from complete: only ca 1.5 to 1.8 million of an estimated 10+ million species are so far described. Notwithstanding the remarkable merits of the Linnean system, the task is too vast ever to be completed using current conventional approaches. Such a staggering reality, and the customary difficulty that the scientific community and society in general experience to access taxonomic knowledge, has prompted the search for novel tools or approaches for species identification. Such a tool has been recently proposed in the form of a standardised short DNA sequence from an agreed-upon region of the genome, which is expected to ultimately provide a means of fast and robust identification of any species on the planet: the DNA barcode. Received with as much enthusiasm by some as skepticism by others, this novel tool was set in motion on a worldwide scale by means of an international consortium of organisations (the Consortium for the Barcoding of Life), thus becoming a large-scale horizontal genomics project. While anchored within the knowledge and principles of taxonomy, DNA barcoding possesses unique characteristics which anticipate a diverse scope of new applications and benefits for society. Notably, it places the completion of the biodiversity catalogue within the reach of a single generation, with the promise to assist greatly in the discovery of new species. Alongside long-term, ultimate goals, such as democratisation of access to taxonomic knowledge and assistance in writing the encyclopaedia of life, there are several more prosaic applications that may also impact society, not only in certain scientific fields, but also in a range of social and economic activities. Here, we will use DNA barcoding of fish as an example to illustrate foreseen applications, and as a basis to stimulate reflection on potential societal impacts of this horizontal genomics project.

DNA Barcodes Can Distinguish Species of Indian Mosquitoes (Diptera: Culicidae) - Jan 01, 2007 ()
[Kumar Pradeep, N., A.R. Rajavel, R. Natarajan and P. Jambulingam 2007. Journal of Medical Entomology. 44(1) 1-7.]

Species identification of mosquitoes (Diptera: Culicidae) based on morphological characteristics remains often difficult in field-collected mosquito specimens in vector-borne disease surveillance programs. The use of DNA barcodes has been proposed recently as a tool for identification of the species in many diverse groups of animals. However, the efficacy of this tool for mosquitoes remains unexplored. Hence, a study was undertaken to construct DNA barcodes for several species of mosquitoes prevalent in India, which included major vector species. In total, 111 specimens of mosquitoes belonging to 15 genera, morphologically identified to be 63 species, were used. This number also included multiple specimens for 22 species. DNA barcode approach based on DNA sequences of mitochondrial cytochrome oxidase gene sequences could identify 62 species among these, in confirmation with the conventional taxonomy. However, two closely related species, Ochlerotatus portonovoensis (Tiwari & Hiriyan) and Ochlerotatus wardi (Reinert) could not be identified as separate species based on DNA barcode approach, their lineages indicating negligible genetic divergence (Kimura two-parameter genetic distance = 0.0043).

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DNA barcodes of closely related (but morphologically and ecologically distinct) species of Skipper Butterflies (Hesperiidae) can differ by only one to three nucleotides - Nov 30, 2006 (pdf)
[Burns, J.M., Janzen, D.H., Hajibabaei, M., Hallwachs, W. and Hebert, P.D.N. 2006. Journal of the Lepidopterists’ Society. 61(3) 138-153.]

Unlike most species of Lepidoptera whose DNA barcodes have been examined, closely related taxa in each of three pairs of hesperiids (Polyctor cleta and P. polyctor, Cobalus virbius and C. fidicula, Neoxeniades luda and N. pluviasilva Burns, new species) seem indistinguishable by their barcodes; but that is when some of the cytochrome c oxidase I (COI) sequences are short and sample sizes are small. These skipper butterflies are unquestionably distinct species, as evidenced by genitalic and facies differences and by ecologic segregation, i.e., one species of each pair in dry forest, the other in adjacent rain forest in Area de Conservación Guanacaste in northwestern Costa Rica. This national park is the source of the specimens used in this study, all of which were reared. Larval foodplants are of no or problematic value in distinguishing these species. Large samples of individuals whose barcodes are acceptably long reveal slight interspecific differentiation (involving just one to three nucleotides) in all three pairs of skippers. Clearly, the chronic practice of various taxonomists of setting arbitrary levels of differentiation for delimiting species is unrealistic.

Seasonal dynamics of population genetic structure in cryptic taxa of the Pellioditis marina complex (Nematoda: Rhabditida) - Dec 31, 2006 ()
[Derycke, S., Backeljau, T., Vlaeminck, C., Vierstraete, A., Vandfletern, J., Vinckx, M. & Moens, T. 2006. Genetica. 128 307-321.]

The distribution patterns and genetic structure of the Pellioditis marina species complex in Belgium and The Netherlands were compared between four consecutive seasons. Different types of habitats (coast, estuary, semi-estuary and lake) with different degrees of connectivity were sampled. In addition, each habitat type was characterised by either temporal or permanent algal deposits. We screened 426 bp of the mitochondrial cytochrome oxidase c (COI) gene with the single-strand conformation polymorphism (SSCP) method in 1615 individuals of Pellioditis marina. The 51 haplotypes were divided into four (sympatric) lineages, with divergences ranging from 0.25 to 10.6%. Our results show that the lineages have different temporal dynamics, which may be linked to abiotic factors. Analysis of Molecular Variance (AMOVA) indicated a significant structuring in the PmI lineage, which correlated with habitat characteristics and which changed over time (Mantel, r = 0.51; p = 0.126). Intrapopulational diversity was similar in all locations, and temporal changes in haplotype frequencies were not higher in temporary than in permanent algal deposits. Instead, the results of the temporal survey indicated that (some) P. marina populations are characterised by a metapopulation structure. It is emphasized that a complete and correct interpretation of processes causing genetic structuring within species and of the genetic structure itself can only be done when analyses are performed at several time points.

Low variation in partial cytochrome oxidase subunit I (COI) mitochondrial sequences in the coralline demosponge Astrosclera willeyana across the Indo-Pacific - Dec 31, 2006 ()
[Wörheide, G. 2006. Marine Biology. 148(5) 907-912.]

Partial sequences of the mitochondrial DNA (mtDNA) gene cytochrome oxidase subunit 1 (COI) were analysed from individuals of the coralline demosponge Astrosclera willeyana sensu lato out of ten Indo- Pacific populations from the Red Sea to the central Pacific. This taxon is widely distributed in cryptic coral reef habitats of the Indo-Pacific and is regarded as a modern representative of long-extinct, formerly reefbuilding stromatoporoid-type sponges. The aims were to clarify phylogeographic and taxonomic relationships in this ‘‘living fossil’’ and to explore mitochondrial DNA sequence variation over a wide geographic range. Very low variability was observed across the Indo-Pacific, as only three COI haplotypes were identified, with a maximum p-distance of 0.418% and low nucleotide diversity (p=0.00049). Very low genetic structure was revealed among populations: Haplotype 1 was found in all specimens from nine Pacific populations (N=45), separated by distances of more than 7,000 km; haplotype 2 was restricted to the Red Sea population (N=4); haplotype 3 was only found in the Tuamoto specimens (N=7). COI data presented here do not support the hypothesis of at least two sibling species belonging to genus Astrosclera in the Pacific.
Considering the maximum geographic distance of more than 20,000 km between sampled populations, mtDNA COI sequence variation observed here is among the lowest reported to date for a diploblastic taxon and adds to the growing evidence of a general mtDNA conservation in sponges. It is argued that this low mtDNA variation in A. willeyana s.l. is due to a low rate of mtDNA evolution caused by a combination of long generation time and low metabolic rate.

Comparison of preservation methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for reliable DNA amplification by PCR - Dec 31, 2006 ()
[Mtambo, J., Van Bortel, W., Madder, M., Roelants, P. & Backeljau, T. 2006. Exp. Appl. Acarol.. 38(2-3) 189-199.]

Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 °C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.

Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP - Dec 31, 2006 ()
[Fliegerova, K., Mrázek, J., & K. Voigt 2006. Folia Microbiologica. 51(4) 273-277.]

The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology.

DESS: A versatile solution for preserving morphology and extractable DNA of nematodes - Dec 31, 2006 ()
[Yoder, M., De Ley, I. T., King, I., Mundo-Ocampo, M., Mann, J., Blaxter, M., Poiras, L., and P. De Ley 2006. Nematology. 8(3) 367-376.]

A solution containing dimethyl sulphoxide, disodium EDTA, and saturated NaCl (abbreviated here as DESS) was tested for various applications in the preservation of nematodes for combined morphological and molecular analyses. The solution can be used to preserve individual nematodes, nematode extracts, or entire soil/sediment samples. Preserved material can be easily stored for months at room temperature, shipped by mail, or carried in luggage. Morphological features are usually well preserved; specimen quality being comparable to formalin-based fixatives and much better than ethanol fixation. Specimens can be transferred to glycerin with little or no modification of traditional protocols. Unlike formalin-preserved material, routine PCR can be performed on individual specimens after any of these procedures with success rates and amplification sizes comparable to PCR of fresh specimens. At this point we have no data on long-term preservation quality. Nevertheless, DESS solution clearly enhances and simplifies a wide range of nematological studies due to its combined suitability for morphological and molecular analyses, as well as its less hazardous chemical properties.

Molecular systematics of the Carinarion complex (Mollusca: Gastropoda: Pulmonata): a taxonomic riddle caused by a mixed breeding system - Dec 31, 2006 ()
[Geenen, S., Jordaens, K. & Backeljau, T. 2006. Biol. J. Linn. Soc.. 89(4) 589-604.]

The original description of the slugs Arion (Carinarion) fasciatus, Arion (Carinarion) silvaticus and Arion (Carinarion) circumscriptus was based on subtle differences in body pigmentation and genital anatomy. However, body pigmentation in these slugs may be influenced by their diet, whereas the genital differences between the species could not be confirmed by subsequent multivariate morphometric analyses. Hence, the status of the three nominal morphospecies remains controversial, with electrophoretic studies based on albumen gland proteins and allozymes also providing conflicting results. These studies suggested that Carinarion species are difficult to reconcile with the biological species concept because there is evidence of interspecific hybridization in places where these predominantly self-fertilizing slugs apparently outcross. Therefore, in the present study, the three Carinarion species are evaluated under a phylogenetic species concept, using nucleotide sequences of the nuclear ribosomal internal transcribed spacer 1 (ITS-1) and the mitochondrial 16S rDNA. ITS-1 showed no species specific variation. However, 16S rDNA yielded five haplotype groups. Three of these grouped haplotypes by species, whereas the two others joined haplotypes of different species and included all haplotypes that were shared by species (22% of all haplotypes). Hence, the three nominal Carinarion species appear to be inconsistent with a phylogenetic species concept. The present data also confirmed that North American Carinarion populations are genetically impoverished and may be not sufficiently representative with respect to the taxonomy of Carinarion. In conclusion, we currently regard Carinarion as a single species-level taxon, whose taxonomically deceiving, correlated phenotypic and genetic intraspecific variation is caused by sustained self-fertilization.

Molecular detection of marine nematodes from environmental samples- overcoming eukaryotic interference - Dec 31, 2006 ()
[Bhadury P, Austen MC, Bilton DT, Lambshead PJD, Rogers AD, Smerdon GR 2006. Aquatic Microbial Ecology. 44 97-103.]

Nematodes form an important and dominant component of many benthic marine ecosystems, but are frequently neglected by marine ecologists because of the time-consuming nature of their identification. Molecular techniques provide powerful tools for the rapid assessment of biodiversity, although few attempts have been made to apply these to marine meiofauna. We evaluated the success of 2 primer sets in amplifying nematode 18S rRNA from DNA templates extracted directly from marine and estuarine sediments. PCR products were separated using denaturing gradient gel electrophoresis (DGGE), and some of the intense DGGE bands were excised, cloned and sequenced to confirm their nematode origin. Initially, other eukaryotic 18S rRNA regions co-amplified with those from nematodes, possibly as a result of the high relative abundance and biomass of other organisms in the studied sediments. These problems were overcome by designing and evaluating consensus primers that selectively amplified nematode ribosomal regions from environmental DNA. Approximately 10 to 12 taxa from each site were detected in the denaturing gel in this study. Tentative affiliations of some the DGGE bands re-amplified using nematode-specific primers were determined by comparing with known marine nematode 18S rRNA sequences in a phylogenetic tree. Our study demonstrates for the first time that PCR combined with DGGE can be used to explore the community composition of many meiofaunal groups, such as nematodes, from DNA extracted directly from environmental samples.

TaxMan: a taxonomic database manager - Dec 31, 2006 ()
[Jones, M. and M. Blaxter 2006. BMC Bioinformatics. 7 536.]

BACKGROUND:
Phylogenetic analysis of large, multiple-gene datasets, assembled from public sequence databases, is rapidly becoming a popular way to approach difficult phylogenetic problems. Supermatrices (concatenated multiple sequence alignments of multiple genes) can yield more phylogenetic signal than individual genes. However, manually assembling such datasets for a large taxonomic group is time-consuming and error-prone. Additionally, sequence curation, alignment and assessment of the results of phylogenetic analysis are made particularly difficult by the potential for a given gene in a given species to be unrepresented, or to be represented by multiple or partial sequences. We have developed a software package, TaxMan, that largely automates the processes of sequence acquisition, consensus building, alignment and taxon selection to facilitate this type of phylogenetic study.

RESULTS:
TaxMan uses freely available tools to allow rapid assembly, storage and analysis of large, aligned DNA and protein sequence datasets for user-defined sets of species and genes. The user provides GenBank format files and a list of gene names and synonyms for the loci to analyse.  Sequences are extracted from the GenBank files on the basis of annotation and sequence similarity. Consensus sequences are built automatically. Alignment is carried out (where possible, at the protein level) and aligned sequences are stored in a database. TaxMan can automatically determine the best subset of taxa to examine phylogeny at a given taxonomic level. By using the stored aligned sequences, large concatenated multiple sequence alignments can be generated rapidly for a subset and output in analysis-ready file formats. Trees resulting from phylogenetic analysis can be stored and compared with a reference taxonomy.

CONCLUSION:
TaxMan allows rapid automated assembly of a multigene datasets of aligned sequences for large taxonomic groups. By extracting sequences on the basis of both annotation and BLAST similarity, it ensures that all available sequence data can be brought to bear on a phylogenetic problem, but remains fast enough to cope with many thousands of records. By automatically assisting in the selection of the best subset of taxa to address a particular phylogenetic problem, TaxMan greatly speeds up the process of generating multiple sequence alignments for phylogenetic analysis. Our results indicate that an automated phylogenetic workbench can be a useful tool when correctly guided by user knowledge.

DNA barcodes: Evaluating the potential of COI to diffentiate closely related species of Elachista (Lepidoptera: Gelechioiea: Elachistidae) from Australia - Dec 31, 2006 ()
[Kaila, L. and S. Gunilla 2006. Zootaxa. 1170 1-26.]

We compared DNA barcoding to “traditional” taxonomic tools in clarifying relationships in complexes of closely related, putative “species” of Elachistinae moths (Gelechioidea: Elachistidae) occurring in Australia. A 705 bp fragment of the 3’-end of cytochrome c oxidase subunit I gene (COI) was used. This mtDNA fragment did not differentiate between all species-level taxa that could be defined by morphological and/or ecological differences. Different evolutionary rates of COI among closely related lineages were observed. Although our findings are based on the variability of the 3’ end of the COI gene and not the 5’ end barcode fragment, we are convinced that thorough exploration of traditional morphology and ecology is a prerequisite for exploring insufficiently known taxonomies by the barcode approach. The sole use of COI barcoding, whether considering COI-5’ or COI-3’ fragment, may fail to recognize closely related species. Our results discourage this approach for delimitation of closely related species, but its use is encouraged as an additional tool for exploring little known taxonomies or as an identification tool for previously thoroughly studied species complexes.

DNA Barcoding Korean Birds. - Dec 31, 2006 ()
[Yoo HS, Eah JY, Kim JS, Kim YJ, Min MS, Paek WK, Lee H,and Kim CB. 2006. Molecules and Cells. 22(3) 323-327.]

DNA barcoding, an inventory of DNA sequences from a standardized genomic region, provides a bio-barcode for identifying and discovering species. Several recent studies suggest that the sequence diversity in a 648 bp region of the mitochondrial gene for cytochrome c oxi- dase I (COI) might serve as a DNA barcode for identify- ing animal species such as North American birds, in- sects and fishes. The present study tested the effective- ness of a COI barcode in discriminating Korean bird species. We determined the 5&cent; terminus of the COI bar- code for 92 species of Korean birds and found that spe- cies identification was unambiguous; the genetic differ- ences between closely related species were, on average, 25 times higher than the differences within species. We identified only one misidentified species out of 239 specimens in a genetic resource bank, so confirming the accuracy of species identification in the banking system. We also identified two potential composite species, calling for further investigation using more samples. The finding of large COI sequence differences between species confirms the effectiveness of COI barcodes for identifying Korean bird species. To bring greater reliability to the identification of species, increased in- tra- and interspecies sampling, as well as supplementa- tion of the mitochondrial barcodes with nuclear ones, is needed.

Diversity and significance of mold species in Norwegian drinking water - Dec 31, 2006 ()
[Hageskal, G., Knutsen A.K., Gaustad, P., de Hoog G.S. & Skaar, I. 2006. Applied and Environmental Microbiology. 72(12) 7586-7593.]

In order to determine the occurrence, distribution and significance of mold species in ground and surface-derived drinking water in Norway, molds isolated from 273 water samples were identified. Samples of raw water, treated water, and water from private homes and hospital installations were analysed by incubation of 100 ml membrane-filtered samples on DG18 media. The total count (CFU per 100 ml) of fungal species, and the species diversity within each sample was determined. The identification of mold species was based on morphological and molecular methods. In total, 94 mold species belonging to 30 genera were identified. The mycobiota was dominated by species of Penicillium, Trichoderma and Aspergillus, with some of them occurring throughout the drinking water system. Several of the same species as isolated from water may have the potential to cause allergic reactions or disease in humans. Other species are common contaminants of food and beverages, and some may cause unwanted sensoric changes in water. Present results indicate that the mycobiota of water should be considered when microbiological safety and quality of drinking water is assessed. In fact, molds in drinking water should possibly be included in the Norwegian water supply and drinking water regulations.

Taxonomic Reliability of DNA Sequences in Public Sequence Databases: A Fungal Perspective - Dec 20, 2006 ()
[Nilsson R.H., Ryberg M., Kristiansson E., Abarenkov K., Larsson K. and Koljalg U. 2006. PLoS One. 1(e59) .]

Background
DNA sequences are increasingly seen as one of the primary information sources for species identification in many organism groups. Such approaches, popularly known as barcoding, are underpinned by the assumption that the reference databases used for comparison are sufficiently complete and feature correctly and informatively annotated entries.

Methodology/Principal Findings
The present study uses a large set of fungal DNA sequences from the inclusive International Nucleotide Sequence Database to show that the taxon sampling of fungi is far from complete, that about 20% of the entries may be incorrectly identified to species level, and that the majority of entries lack descriptive and up-to-date annotations.

Conclusions
The problems with taxonomic reliability and insufficient annotations in public DNA repositories form a tangible obstacle to sequence-based species identification, and it is manifest that the greatest challenges to biological barcoding will be of taxonomical, rather than technical, nature.

Molecular barcoding, DNA from snake venom, and toxinological research: Considerations and concerns. - Dec 15, 2006 ()
[Powell R.L., S.R. Reyes and D.I. Lannutti. 2006. Toxicon. 48(8) 1095-1097.]

The problem of species identification in toxinological research and solutions such as molecular barcoding and DNA extraction from venom samples are addressed. Molecular barcoding is controversial with both perceived advantages and inherent problems. A method of species identification utilizing mitochondrial DNA from venom has been identified. This method could result in deemphasizing the importance of obtaining detailed information on the venom source prior to analysis. Additional concerns include; a cost prohibitive factor, intraspecific venom variation, and venom processing issues. As researchers demand more stringent records and verification, venom suppliers may be prompted to implement improved methods and controls.

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Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding - Dec 14, 2006 ()
[Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, Valentini A, Vermat T, Corthier G, Brochmann C, and Willerslev E. 2006. Nucleic Acids Research. .]

DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254-767 bp) and of a shorter fragment of this intron (the P6 loop, 10-143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.

An inexpensive, automation-friendly protocol for recovering high-quality DNA - Dec 01, 2006 (pdf)
[Ivanova, N.V., deWaard, J.R. and P.D.N. Hebert 2006. Molecular Ecology Notes. 6(4) 998-1002.]

Although commercial kits are available for automated DNA extraction, 'artisanal' protocols are not. In this study, we present a silica-based method that is sensitive, inexpensive and compliant with automation. The effectiveness of this protocol has now been tested on more than 5000 animal specimens with highly positive results.

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DNA-based identification of Alaska skates (Amblyraja, Bathyraja and Raja: Rajidae) using cytochrome c oxidase subunit I (coI) variation. - Dec 01, 2006 ()
[Spies, I. B., Gaichas, S., Stevenson, D. E., Orr, J. W. and Canino, M. F. 2006. Journal of Fish Biology. 69(sb) 283-292.]

Variation at the mitochondrial cytochrome c oxidase subunit I (mt-COI) gene was examined in 15 species of North Pacific skates. Thirteen species had unique sequences, indicating that a DNA-based barcoding approach may be useful for species identification.

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A minimalist barcode can identify a specimen whose DNA is degraded - Dec 01, 2006 (pdf)
[Hajibabaei, M., Smith, M.A., Janzen, F.H., Rodriguez, J.J., Whitfield, J.B., and P.D.N. Hebert 2006. Molecular Ecology Notes. 6(4) 959-964.]

A DNA barcode based on 650 bp of mitochondrial gene cytochrome c oxidase I is proving to be highly functional in species identification for various animal groups. However, DNA degradation complicates the recovery of a full-length barcode from many museum specimens. Here we explore the use of shorter barcode sequences for identification of such specimens. We recovered short sequences — i.e. 100 bp — with a single PCR pass from more than 90% of the specimens in assemblages of moth and wasp museum specimens from which full barcode recovery was only 50%, and the latter were usually less than 8 years old. Short barcodes were effective in identifying specimens, confirming their utility in circumstances where full barcodes are too expensive to obtain and the identification comparisons are within a confined taxonomic group.

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Identifying fern gametophytes using DNA sequences. - Dec 01, 2006 ()
[Schneider, H. and Schuettpelz, E. 2006. Molecular Ecology Notes. 6(4) 989-991.]

Identification of fern gametophytes is generally hampered by low morphological complexity. Here we explore an alternative: DNA-based identification. We obtained a plastid rbcL sequence from a sterile gametophyte of unknown origin (cultivated for more than 30 years) and employed blast to determine its affinities. Using this approach, we identified the gametophyte as Osmunda regalis. To evaluate the robustness of this determination, and the usefulness of rbcL in differentiating among species, we conducted a phylogenetic analysis of osmundaceous fern sequences. Based on our results, it is evident that DNA-based identification has considerable potential in exploring the ecology of fern gametophytes.

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Identifying Canadian mosquito species through DNA barcodes - Dec 01, 2006 (pdf)
[Cywinska A., Hunter F.F., and Hebert P.D.N. 2006. Medical and Veterinary Entomology. 20(4) 413-424.]

A short fragment of mt DNA from the cytochrome c oxidase 1 (CO1) region was used to provide the first CO1 barcodes for 37 species of Canadian mosquitoes (Diptera: Culicidae) from the provinces Ontario and New Brunswick. Sequence variation was analysed in a 617-bp fragment from the 5' end of the CO1 region. Sequences of each mosquito species formed barcode clusters with tight cohesion that were usually clearly distinct from those of allied species. CO1 sequence divergences were, on average, nearly 20 times higher for congeneric species than for members of a species; divergences between congeneric species averaged 10.4% (range 0.2–17.2%), whereas those for conspecific individuals averaged 0.5% (range 0.0–3.9%).

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Integrative taxonomy of Iberian Merodon species (Diptera, Syrphidae) - Dec 01, 2006 ()
[Mengual, Ximo, Ståhls, Gunilla, Vujic&#769;, Ante & Marcos-Garci&#769;a, Ma 2006. Zootaxa. 1377 1-26.]

The genus Merodon Meigen, 1803 (Syrphidae, Diptera), with more than 50 European species, is primarily distributed in the Mediterranean region, there being 34 species that occur in the Iberian Peninsula. The morphological variation found within some species from the Iberian Peninsula prompted us to test their taxonomic status by integrating morphological and molecular data. We generated partial sequences of the mitochondrial, protein-coding gene cytochrome c oxidase subunit I (COI), the nuclear, internal transcribed spacer (ITS2) region, and the D2 region of the nuclear 28S rRNA gene. COI and ITS2 sequences were obtained for most included taxa.
The variability of the COI sequences showed great differences between the studied species groups, exhibiting an interspecific range from 0.29% to 12.5% between ingroup taxa. Closely related taxa of the aureus complex (e.g. M. quercetorum and M. legionensis) presented identical COI sequences. The obtained ITS2 sequences showed low intraspecific variability, and only a few taxa presented more than one genotype. Species status and delimitation were discussed for all taxa in the light of available morphological and molecular character information. Using the obtained sequence data for COI and 28S we inferred the phylogenetic relationships of the included taxa, using parsimony analysis. Separate analysis of the COI sequences identified four, quite wellsupported clades within Merodon, the desuturinus, albifrons, nigritarsis and aureus groups. Combined analysis of the COI and 28S genes produced a topology similar to the COI topology.

A genomic perspective on the shortcomings of mitochondrial DNA for "barcoding" identification - Nov 29, 2006 ()
[Rubinoff, D., Cameron, S., & K. Will 2006. The Journal of Heredity. 97(6) 581-94.]

Approximately 600-bp sequences of mitochondrial DNA (mtDNA) have been designated as "DNA barcodes" and have become one of the most contentious and animated issues in the application of genetic information to global biodiversity assessment and species identification. Advocates of DNA barcodes have received extensive attention and promotion in many popular and refereed scientific publications. However, we suggest that the utility of barcodes is suspect and vulnerable to technical challenges that are particularly pertinent to mtDNA. We review the natural history of mtDNA and discuss problems for barcoding which are particularly associated with mtDNA and inheritance, including reduced effective population size, maternal inheritance, recombination, inconsistent mutation rate, heteroplasmy, and compounding evolutionary processes. The aforementioned could significantly limit the application and utility of mtDNA barcoding efforts. Furthermore, global use of barcodes will require application and acceptance of a barcode-based species concept that has not been evaluated in the context of the extensive literature concerning species designation. Implementation of mtDNA barcodes in spite of technical and practical shortcomings we discuss may degrade the longstanding synthesis of genetic and organism-based research and will not advance studies ranging from genomic evolution to biodiversity assessment.

Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe. - Nov 26, 2006 ()
[Froslev TG, Jeppesen TS, Laessoe T, and Kjoller R. 2006. Molecular Phylogenetics and Evolution. .]

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.

Comparing the efficacy of morphologic and DNA-based taxonomy in the freshwater gastropod genus - Nov 23, 2006 ()
[Pfenninger, M., M. Cordellier, and B. Streit 2006. BMC Evolutionary Biology. 6 100.]

Background: Reliable taxonomic identification at the species level is the basis for many biological disciplines. In order to distinguish species, it is necessary that taxonomic characters allow for the separation of individuals into recognisable, homogeneous groups that differ from other such groups in a consistent way. We compared here the suitability and efficacy of traditionally used shell morphology and DNA-based methods to distinguish among species of the freshwater snail genus Radix (Basommatophora, Pulmonata).

Results: Morphometric analysis showed that shell shape was unsuitable to define homogeneous, recognisable entities, because the variation was continuous. On the other hand, the Molecularly defined Operational Taxonomic Units (MOTU), inferred from mitochondrial COI sequence variation, proved to be congruent with biological species, inferred from geographic distribution patterns, congruence with nuclear markers and crossing experiments. Moreover, it could be shown that the phenotypically plastic shell variation is mostly determined by the environmental conditions experienced.

Conclusion: Contrary to DNA-taxonomy, shell morphology was not suitable for delimiting and recognising species in Radix. As the situation encountered here seems to be widespread in invertebrates, we propose DNA-taxonomy as a reliable, comparable, and objective means for species identification in biological research

Global Advances in the Ecology and Management of Golden Apple Snails - Nov 06, 2006 ()
[Dr. Ravindra C. Joshi and Dr. Leocadio S. Sebastian 2006. Philippine Rice Research Institute. 600.]
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Discovery and Barcoding by Analysis of Spliced Leader RNA Gene Sequences of New Isolates of Trypanosomatidae from Heteroptera in Costa Rica and Ecuador - Nov 01, 2006 ()
[Maslov, D.A., Westernberger, S.J, Xu, X., Campbell, D.A, and Sturm, N.R. 2006. Journal of Eukaryotic Microbiology. 54(1) 57–65.]

Trypanosomatid diversity in Heteroptera was sampled using a culture-independent approach based on amplification and sequencing of Spliced Leader RNA gene repeats from environmental samples. By combining the data collected herein with that of previous work, the prevalence of parasites was found to be 22% - 23%. Out of approximately 170 host species investigated nearly 60 were found to harbor trypanosomatids. The parasites found were grouped by cluster analysis into 48 typing units. Most of these were well separated from the known groups and, therefore, likely represent new trypanosomatid species. The sequences for each typing unit serve as barcodes to facilitate their recognition in the future. As the sampled host species represent a minor fraction of potential hosts, the entire trypanosomatid diversity is far greater than described thus far. Investigations of trypanosomatid diversity, host-specificity, and biogeography have become feasible using the approach described herein.

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Mitochondrial Phylogeography of the Vegetable Pest Liriomyza trifolii (Diptera: Agromyzidae): Diverged Clades and Invasive Populations - Nov 01, 2006 ()
[Scheffer, Sonja J. and Lewis, Matthew L. 2006. Annals of the Entomological Society of America. 99(6) 991-998.]

The leafmining fly Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) is an important pest of vegetable and cut-flower crops. In recent decades, this species has become invasive, spreading from the Americas to the rest of the world. Despite substantial losses caused by Liriomyza leafminers, the systematics of these flies has remained poorly understood because of their small size and morphological homogeneity. Previous molecular research on other polyphagous Liriomyza pests has suggested that cryptic species may be present. Here, we use mitochondrial cytochrome oxidase I sequence variation to investigate phylogeographic structure within L. trifolii. Our results indicate that L. trifolii harbors distinct phylogenetic clades, suggesting the presence of cryptic species. There is also evidence of a recently derived, highly specialized pepper (Capsicum spp., Solanaceae)-feeding population within L. trifolii that may represent a host race or even a distinct species. Introduced populations from various locations contained a highly restricted subset of the mitochondrial variation present within L. trifolii, suggesting one or more bottlenecks during colonization.

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Lessons from leeches: a call for DNA barcoding in the lab. - Nov 01, 2006 ()
[Bely, A. E. and Weisblat, D. A. 2006. Evolution & Development. 8(6) 491-501.]

SUMMARY Many evolution of development labs study organisms that must be periodically collected from the wild. Whenever this is the case, there is the risk that different field collections will recover genetically different strains or cryptic species. Ignoring this potential for genetic variation may introduce an uncontrolled source of experimental variability, leading to confusion or misinterpretation of the results. Leeches in the genus Helobdella have been a workhorse of annelid developmental biology for 30 years. Nearly all early Helobdella research was based on a single isolate, but in recent years isolates from multiple field collections and multiple sites across the country have been used. To assess the genetic distinctness of different isolates, we obtained specimens from most Helobdella laboratory cultures currently or recently in use and from some of their source field sites. From these samples, we sequenced part of the mitochondrial gene cytochrome oxidase I (COI). Sequence divergences and phylogenetic analyses reveal that, collectively, the Helobdella development community has worked on five distinct species from two major clades. Morphologically similar isolates that were thought to represent the same species (H. robusta) actually represent three species, two of which coexist at the same locality. Another isolate represents part of a species complex (the "H. triserialis" complex), and yet another is an invasive species (H. europaea). We caution researchers similarly working on multiple wild-collected isolates to preserve voucher specimens and to obtain from these a molecular "barcode," such as a COI gene sequence, to reveal genetic variation in animals used for research.

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To boldly sequence - Nov 01, 2006 ()
[Godfray, H.J.C. 2006. Trends in Ecology & Evolution. 21(11) 603-604.]

Book Review: DNA Barcoding of Life is a collection of papers on the barcoding of life initiative, bringing together the leading enthusiasts in the field.

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Recent advances in DNA taxonomy - Nov 01, 2006 ()
[Vogler, A. P. and Monaghan, M. T. 2006. Journal of Zoological Systematics and Evolutionary Research. 45(1) 1-10.]

Large-scale DNA sequencing of living species holds great promise in taxonomy, but has been controversial. In this article, we review the recent advances that follow the dramatic increase in data generation. We distinguish DNA taxonomy from DNA barcoding, where the former directly concerns the circumscription and delineation of species using evolutionary species concepts and the latter is a means of identifying a priori entities by sequence similarity. A key finding from recent studies in animals is that variation in mitochondrial DNA (mtDNA) is partitioned as tight clusters of closely related genotypes, which group specimens largely according to traditionally recognized species limits, and which are congruent with nuclear markers. This finding provides confidence to use sequence variation as the primary information for species delimitation in poorly known groups. A number of recent, large-scale studies support the power of mtDNA in species recognition, and previous application of molecular techniques to taxonomically complicated cases has likely led to an overestimate of the proportion of species with polyphyletic mtDNA haplotypes. The continued development of DNA taxonomy will lead to more refined sampling strategies and data analyses than those that are presently used. Sophisticated statistical methods of grouping have already been developed based on sequence similarity; yet, the units defined in this way have largely unknown evolutionary relevance. In future, a standard DNA taxonomic analysis will include broad sampling of the target taxa across their geographic range, followed by large-scale sequencing of representative samples for a DNA profile of the group, and algorithmic procedures for delineating species limits. The taxonomic system will be derived from the data rather than expert opinion, and hypothesized species entities can be tested against morphology, biogeography and other data, providing an evolutionary justification of the procedures used for species delimitation. Discrepancies between DNA and other data are used to refine species delimitations via a feedback loop that incorporates new data. We argue, however, that the use of DNA methodology in taxonomy (including DNA barcoding) will remain controversial until it is better founded in existing theory of evolutionary biology and phylogenetics.

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Environmental Protection Using DNA Barcodes or Taxa? - Nov 01, 2006 ()
[Stribling, James B. 2006. BioScience. 56(11) 878-879.]
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Simultaneous detection and identification of trichothecene and moniliformin producing Fusarium species based on multiplex SNP analysis - Oct 31, 2006 ()
[Kristensen, R., Berdal, K.G. & Holst-Jensen, A. 2006. J Applied Microbiology. 102(4) 1071 - 1081.]

AIMS: To develop a DNA microarray for easy and fast detection of trichothecene-and moniliformin-producing Fusarium species.

METHOD AND RESULTS: A DNA microarray was developed for detection and identification of 14 trichothecene- and moniliformin-producing species of the fungal genus Fusarium. The array could also differentiate between four species groups. Capture probes were designed based on recent phylogenetic analyses of translation elongation factor-1 alpha (TEF-1a) sequences. Particular emphasis was put on designing capture probes corresponding to groups or species with particular mycotoxigenic synthetic abilities. A consensus PCR amplification of a part of the TEF-1a is followed by hybridization to the Fusarium chip and the results are visualized by a colorimetric Silverquant detection method. We validated the Fusarium chip against five naturally infected cereal samples for which we also have morphological and chemical data. The limit of detection was estimated to be less than 16 copies of genomic DNA in spiked commercial wheat flour.

CONCLUSIONS: The current Fusarium chip proved to be a highly sensitive and fast microarray for detection and identification of Fusarium species. We postulate that the method also has potential for (semi-)quantification.

SIGNIFICANCE AND IMPACT OF THE STUDY: The Fusarium chip may prove to be a very valuable tool for screening of cereal samples in the food and feed production chain, and may facilitate detection of new or introduced Fusarium spp.

A molecular phylogeny of Anopheles annulipes (Diptera: Culicidae) sensu lato: The most species-rich anopheline Complex - Oct 17, 2006 ()
[Foley, D. H., Wilkerson, R. C., Cooper, R. D., Volovsek, M. E. & Bryan, J. H 2006. Molecular Phylogenetics and Evolution. 43(1) 283-297.]

The Australasian Annulipes Complex is the most species-rich among Anopheles mosquitoes, with at least 15 sibling species suspected. Members of this complex are the most likely vectors of malaria in the past in southern Australia and are involved in the spread of myxomatosis among rabbits. In this, the first comprehensive molecular study of the Annulipes Complex, 23 ITS2 rDNA variants were detected from collections throughout Australia and Papua New Guinea, including diagnostic variants for the previously identified An. annulipes species A–G. Specimens of each ITS2 variant were sequenced for portions of the mitochondrial COI, COII and nuclear EF-1α genes. Partitioned Bayesian and Maximum Parsimony analyses confirmed the monophyly of the Annulipes Complex and revealed at least 17 clades that we designate species A–Q. These species belong to two major clades, one in the north and one mainly in the south, suggesting that climate was a driver of species radiation. We found that 65% (11) of the 17 sibling species recorded here had unique COI sequences, suggesting that DNA barcoding will be useful for diagnosing species within the Annulipes Complex. A comparison of the taxa revealed morphological characters that may be diagnostic for some species. Our results substantially increase the size of the subgenus Cellia in Australasia, and will assist species-level studies of the Annulipes Complex.

Mating trials validate the use of DNA barcoding to reveal cryptic speciation of a marine bryozoan taxon - Oct 11, 2006 ()
[Gomez A.,Wright P.J., Lunt D.H.,Cancino J.M.,Carvalho G.R., and Hughes R.N. 2006. Proceedings of the Royal Society B: Biological Sciences. .]

Despite increasing threats to the marine environment, only a fraction of the biodiversity of the oceans has been described, owing in part to the widespread occurrence of cryptic species. DNA-based barcoding through screening of an orthologous reference gene has been proposed as a powerful tool to uncover biological diversity in the face of dwindling taxonomic expertise and the limitations of traditional species identification. Although DNA barcoding should be particularly useful in the sea, given the prevalence of marine cryptic species, the link between taxa identified through DNA barcodes and reproductively isolated taxa (biological species) has rarely been explicitly tested. Here, we use an integrated framework comparing breeding compatibility, morphology and mitochondrial (cytochrome c oxidase 1) and nuclear (elongation factor-1-alpha) DNA sequence variation among globally distributed samples of the cosmopolitan marine bryozoan Celleporella hyalina (L.). Our results reveal that C. hyalina comprises numerous deep, mostly allopatric, genetic lineages that are reproductively isolated, yet share very similar morphology, indicating rampant cryptic speciation. The close correspondence between genetic lineages and reproductively isolated taxa in the context of minimal morphological change suggests that DNA barcoding will play a leading role in uncovering the hidden biodiversity of the oceans and that the sole use of morphologically based taxonomy would grossly underestimate the number of marine species.

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Comparative larval morphology in Madagascan frogs of the genus Guibemantis (Amphibia : Mantellidae). - Oct 05, 2006 ()
[Vejarano S, Thomas M, and Vences M. 2006. Zootaxa. 1329 39-57.]

We describe the tadpole morphology for four species of frogs classified in the endemic Madagascan genus Guibemantis, based on larval specimens identified by DNA barcoding. The tadpoles of Guibemantis kathrinae and G. tornieri are reported for the first time. The tadpole of G. kathrinae has a heterogeneous coloration, emarginated oral disc bordered with papillae and one row of submarginal papillae. Labial tooth row formula is 6(2–6)/3(1). Number of labial teeth per millimetre is variable in each row, ranging from 36 to 64. The tadpole of G. tornieri is very similar to that of G. timidus (previously considered conspecific with G. tornieri) except for the patched coloration of G. tornieri (vs. rather uniform in G. timidus). The tadpole of G. depressiceps is characterized by having a higher number of teeth per millimetre in all tooth rows than the other species of the group. The tadpole of Guibemantis liber differs from the other species by having a lower number of upper labial tooth rows (two, three or four vs. five or more). No morphological differences were found between larvae of G. liber from two separate localities, Ranomafana and Andasibe. In general, the Guibemantis larvae examined (except G. liber) are morphologically smilar to each other but several of the characters examined were highly variable within populations and species, highlighting the usefulness of molecular tools for their identification.

DNA Barcoding Evolves into the Familiar - Oct 01, 2006 ()
[Rubinoff, Daniel 2006. Conservation Biology. 20 (5) 1548-1549.]

Daniel Rubinoff's response to Rob DeSalle regarding "Species discovery versus species identification in DNA
barcoding efforts: response to Rubinoff."

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MtDNA barcode identification of fish larvae in the southern Great Barrier Reef, Australia - Oct 01, 2006 ()
[Pegg, GG., Sinclair, B., Briskey, L., and Aspden, WJ. 2006. Scientia Marina. 70(S2) 7-12.]

SUMMARY: Planktonic larvae were captured above a shallow coral reef study site on the Great Barrier Reef (GBR) around spring-summer new moon periods (October-February) using light trap or net capture devices. Larvae were identified to the genus or species level by comparison with a phylogenetic tree of tropical marine fish species using mtDNA HVR1 sequence data. Further analysis showed that within-species HVR1 sequence variation was typically 1-3%, whereas between-species variation for the same genus ranged up to 50%, supporting the suitability of HVR1 for species identification. Given the current worldwide interest in DNA barcoding and species identification using an alternative mtDNA gene marker (cox1), we also explored the efficacy of different primer sets for amplification of cox1 in reef fish, and its suitability for species identification. Of those tested, the Fish-F1 and -R1 primer set recently reported by Ward et al. (2005) gave the best results.

Molecular identification, description, and phylogenetic implications of the tadpoles of 11 species of Malagasy treefrogs, genus Boophis - Oct 01, 2006 ()
[Raharivololoniaina, L., Grosjean, S., Raminosoa, NR., Glaw, F, and Vences, M. 2006. Journal of Natural History. 40(23-24) 1449-1480.]

Based on specimens identified by DNA barcoding, we describe the tadpoles of 11 species of treefrogs (Boophis) in the Malagasy family Mantellidae. All tadpoles belong to species of the stream‐breeding clade within Boophis. Based on these and other published descriptions of Boophis tadpoles which develop in running water bodies, we tentatively distinguish three ecomorphological guilds for these larvae. Guild A, in which we describe the larvae of B. boehmei, B. reticulatus, B. pyrrhus, B. tasymena, and B. viridis which have few lotic adaptations, their oral disc width being 31–43% of body width, with a single row of 48–81 marginal papillae, and the first upper keratodont row having 58–144 keratodonts. Guild B, in which we describe the tadpoles of B. albilabris, B. madagascariensis, B. luteus, and of an undescribed species here named B. sp. aff. elenae, is intermediate, with an enlarged oral disc, an increasing number of keratodont rows and a lower height of the caudal fin. In these tadpoles, oral disc width is 43–63% of body width, they have one or two rows of 69–164 marginal papillae, and the first upper keratodont row has 164–238 keratodonts. Guild C contains tadpoles with a very large oral disc, living on submerged rocks and stones in stream sections of strong current. In this guild we describe the tadpoles of B. marojezensis and B. sibilans. Their oral disc width is 63–89% of body width, there are multiple rows of many marginal papillae, and the first upper keratodont row has many small keratodonts which are difficult to count, but consistently amount to over 200. In B. marojezensis, the dorsal gap in the marginal papillae rows, apparent in all other species, is closed. These larval morphologies show a rather good fit with recently published molecular phylogenetic data: species groups that were confirmed to be monophyletic in most cases have similar larval morphologies, and, in contrast, where species of the same group have disparate larval morphologies the monophyly of the group is questionable (e.g. the B. majori group). Nevertheless, some cases of convergent evolution are apparent, such as the highly specialized Guild C morphology, which may have evolved separately in the B. albipunctatus group, B. mandraka group, and in some species of the B. majori group.

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Species discovery versus species identification in DNA barcoding efforts: response to Rubinoff - Oct 01, 2006 ()
[DeSalle, R. 2006. Conservation Biology. 20 (5) 1545-1547.]

Rubinoff's (2006) essay in Conservation Biology raises some important issues relevant to the DNA barcoding initiative. Some of these issues are valid, others have been discussed recently in the DNA barcoding literature (Blaxter & Floyd 2003; Sperling 2003; Hebert et al. 2004; Moritz & Cicero 2004; Ebach & Holdrege 2005), and still others can be addressed by looking at DNA barcoding in a different light (DeSalle et al. 2005). Because papers in the DNA barcoding literature suggest that conservation biology is one of the major fields that will benefit from DNA barcoding, Rubinoff correctly argues that examining the validity of this claim is necessary.

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On the use of DNA sequences for determining the species limits of a polymorphic new species in the stink bug genus Halys (Heteroptera : Pentatomidae) from Pakistan - Oct 01, 2006 ()
[Memon, N., Meier, R., Manan, A., and Su, KFY 2006. Systematic Entomology. 31(4) 703-710.]

We describe a new species of Halys Fabricius (Pentatomidae: Pentatominae: Halyini) based on morphological and DNA sequence data, and demonstrate the value of DNA sequences for taxonomic problems that are difficult to resolve on the basis of morphology alone. Halys sindillus Memon, Meier & Manan, sp.n. varies with regard to characters that are usually constant within the genus (spermathecal bulb of females; blade of male clasper; ratio between the second and third antennomeres; length of labium). The surprising levels of variation raised the question as to how many species were represented in three series of specimens from Pakistan. Because the morphological variability was largely continuous, we hypothesized the presence of one new species, and confirm this result here using sequence data from two mitochondrial markers. The data reveal very little molecular variation within the newly described species (COI: 730 bp: 0–0.16%; COI/tRNALeu/COII: 563 bp: 0–0.36%), that is, morphology and DNA sequences show very different patterns of variability. The new species is compared with the closely related Halys sulcatus (Thunberg) whose sequences are distinctly different and whose spermathecal bulbs are largely invariable (I: 2.87–3.28%; II: 2.13–2.49%). We discuss the shortcomings of mitochondrial data in taxonomy and compare the genetic distances in Halys with frequency distributions of intra- and interspecific distances obtained for all 878 Hemiptera COI sequences in GenBank. We conclude that the observed distances for Halys are consistent with our taxonomic conclusions, thus demonstrating the usefulness of DNA sequences for Halys taxonomy. However, the observed overlap between intra- and interspecific sequence variability in Hemiptera is so wide that it questions the feasibility of approaches to taxonomy based predominantly on DNA sequences (e.g. DNA taxonomy, DNA barcoding).

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Blind population genetics survey of tropical rainforest trees - Oct 01, 2006 ()
[Duminil, J., Caron H., Scotti, I., Cazal, S., and Petit, R. 2006. Molecular Ecology. 15(12) 3505-3513.]

Rainforest tree species can be difficult to identify outside of their period of reproduction. Vascular tissues from Carapa spp. individuals were collected during a short field trip in French Guiana and analysed in the laboratory with nuclear and chloroplast markers. Using a Bayesian approach, > 90% of the samples could be assigned to one of two distinct clusters corresponding to previously described species, making it possible to estimate the genetic structure of each species and to identify cases of introgression. We argue that this blind procedure represents a first-choice rather than a fallback option whenever related taxa are investigated.

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Taxonomy: DNA barcodes: recent successes and future prospects - Oct 01, 2006 (pdf)
[Dasmahapatra, K.K. and J. Mallet 2006. Heredity. 97(4) 254–255.]

In 'DNA barcoding' a short section of DNA sequence is used to identify species. Neither the idea nor the technology behind DNA barcoding is novel. What is new and controversial is the idea of using just a small portion of a single gene to identify species from a wide taxonomic range, including animals such as birds, fish and insects (Hebert et al, 2004b; Ward et al, 2005; Hajibabaei et al, 2006). This recent usage, and its subsequent successes, has induced criticism and taxonomic debate.

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Who Will Actually Use DNA Barcoding and What Will It Cost? - Sep 30, 2006 ()
[Stephen Cameron, Daniel Rubinoff, Kipling Will 2006. Systematic Biology. 55(5) 844-847.]

Mid-Pleistocene divergence of Cuban and North American ivory-billed woodpeckers - Sep 22, 2006 ()
[Fleischer RC, Kirchman JJ, Dumbacher, JP., Bevier, L., Dove, C., Rotzel, NC., Edwards, S., Lammertink, M., Miglia, KJ.,and Moore, WS. 2006. Biology Letters. 2(3) 466-469.]

We used ancient DNA analysis of seven museum specimens of the endangered North American ivory-billed woodpecker (Campephilus principalis) and three specimens of the species from Cuba to document their degree of differentiation and their relationships to other Campephilus woodpeckers. Analysis of these mtDNA sequences reveals that the Cuban and North American ivory bills, along with the imperial woodpecker (Campephilus imperialis) of Mexico, are a monophyletic group and are roughly equidistant genetically, suggesting each lineage may be a separate species. Application of both internal and external rate calibrations indicates that the three lineages split more than one million years ago, in the Mid-Pleistocene. We thus can exclude the hypothesis that Native Americans introduced North American ivory-billed woodpeckers to Cuba. Our sequences of all three woodpeckers also provide an important DNA barcoding resource for identification of non-invasive samples or remains of these critically endangered and charismatic woodpeckers.

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How many species of shore fishes are there in the Tropical Eastern Pacific? - Sep 18, 2006 ()
[Zapata, F.A. and Robertson, D.R. 2006. Journal of Biogeography. .]

To assess whether the total richness of the shore-fish fauna of a discrete biogeographical region can be predicted, and to estimate how long it is likely to take to enumerate that fauna.

The Tropical Eastern Pacific (TEP), an isolated biogeographical region with a high level of endemism (72%) among its modestly rich, known fauna of shore fishes (1222 named + 58 known undescribed shallow-water species).

We used patterns in the long-term dynamics and accumulation curves of descriptions of new species, which began in 1758, correlates of these patterns, and the body size–frequency distributions of various ecological groups of the fauna to (1) try to predict the total richness of that fauna, (2) estimate how many species might be missing and what biological characteristics they might have, and (3) estimate how long their discovery and description will take to complete.

Accumulation curves for the entire fauna, for all TEP endemics or for reef and soft-bottom species (77.5% of the fauna) are not approaching asymptotes, and their description rates have remained fairly stable over the past century. However, curves for pelagic and multi-habitat species (22.5% of the fauna) may be nearing asymptotes, perhaps because these species are relatively accessible to collection. These curves clearly indicate that the total TEP fauna is substantially richer than the presently known fauna, but do not allow reliable prediction of its richness. Extrapolations from frequency distributions of the body size of different ecological groups of TEP fishes indicate that the entire fauna is at least 12–15% larger than the currently known fauna.

From recent description trends, undiscovered species will tend to be small, have limited geographic and depth ranges, and live in deeper water. Poorly known, priority areas for taxonomic investigation in the TEP include deeper reef habitats, two isolated island groups, and several continental areas with unusual environments. At current levels of traditional taxonomic activity, the description of known unnamed species will take c.15 years, and assessment of the richness of unknown species, which probably number in the hundreds, will take decades.

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Identification of conserved regions in the plastid genome: implications for DNA barcoding and biological function - Sep 01, 2006 ()
[Presting, Gernot G. 2006. Canadian Journal of Botany. 84(9) 1434-1443.]

All oligonucleotides of the sugarcane chloroplast genome that are conserved in one or more of 36 other completed plastid genomes have been identified by computer-assisted sequence comparison. These regions are of interest because they (i) are indicative of strong selection pressures to maintain specific nucleotide sequences that may yield insights into plastid biology and (ii) can be used as priming sites for amplifying intervening sequences that represent potential DNA barcodes for species identification. The majority of conserved sites are located in the inverted repeat (IR) region, but several sites in the single copy region (predominantly in tRNA and psa/psb genes) are conserved among chloroplasts of all higher plants examined here. Of particular interest are protein coding regions that have been conserved at the nucleotide level, as these may be involved in transcript regulation. This analysis also provides the basis for rational design of a DNA barcode for plastids, and several potential barcode regions have been identified. In particular, two oligonucleotides of length 33 and 25, and separated by approximately 362 nucleotides, are found in all cyanobacteria, red, brown and green algae, as well as diatoms, euglenids, apicomplexans and land plants that have been examined to date. Their widespread occurrence makes the intervening sequence a universal marker for all photosynthetic lineages. Analysis of 160 GenBank accessions illustrates that this region discriminates many algae at the species level, but lacks sufficient variation among the more recently diverged land plants to serve as a single DNA barcode for this taxon. However, this marker should be particularly useful for the DNA barcoding of algal lineages and lichens, as well as for environmental sampling. More rapidly evolving regions of the plastid genome also identified here serve as a starting point to design and test barcodes for more narrowly defined lineages, including the more recently diverged angiosperms.

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Integrating DNA data and traditional taxonomy to streamline biodiversity assessment: an example from edaphic beetles in the Klamath ecoregion, California, USA. - Sep 01, 2006 ()
[Caesar RM, Sorensson M, and Cognato AI. 2006. Diversity and Distributions. 12(5) 483-489.]

Conservation and land management decisions may be misguided by inaccurate or misinterpreted knowledge of biodiversity. Non-systematists often lack taxonomic expertise necessary for an accurate assessment of biodiversity. Additionally, there are far too few taxonomists to contribute significantly to the task of identifying species for specimens collected in biodiversity studies. While species level identification is desirable for making informed management decisions concerning biodiversity, little progress has been made to reduce this taxonomic deficiency. Involvement of non-systematists in the identification process could hasten species identification. Incorporation of DNA sequence data has been recognized as one way to enhance biodiversity assessment and species identification. DNA data are now technologically and economically feasible for most scientists to apply in biodiversity studies. However, its use is not widespread and means of its application has not been extensively addressed. This paper illustrates how such data can be used to hasten biodiversity assessment of species using a little-known group of edaphic beetles. Partial mitochondrial cytochrome oxidase I was sequenced for 171 individuals of feather-wing beetles (Coleoptera: Ptiliidae) from the Klamath ecoregion, which is part of a biodiversity hotspot, the California Floristic Province. A phylogram of these data was reconstructed via parsimony and the strict consensus of 28,000 equally parsimonious trees was well resolved except for peripheral nodes. Forty-two voucher specimens were selected for further identification from clades that were associated with many synonymous and non-synonymous nucleotide changes. A ptiliid taxonomic expert identified nine species that corresponded to monophyletic groups. These results allowed for a more accurate assessment of ptiliid species diversity in the Klamath ecoregion. In addition, we found that the number of amino acid changes or percentage nucleotide difference did not associate with species limits. This study demonstrates that the complementary use of taxonomic expertise and molecular data can improve both the speed and the accuracy of species-level biodiversity assessment. We believe this represents a means for non-systematists to collaborate directly with taxonomists in species identification and represents an improvement over methods that rely solely on parataxonomy or sequence data.

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High Diversity of Fungi Recovered from the Roots of Mature Tanoak (Lithocarpus densiflorus) in Northern California - Sep 01, 2006 ()
[Bergemann, S.E. & M. Garbelotto 2006. Canadian Journal of Botany. 84(9) 1380-1394.]

We collected mature tanoak (Lithocarpus densiflorus (Hook. & Arn.) Rehder) roots from five stands to characterize the relative abundance and taxonomic richness of root-associated fungi. Fungi were identified using polymerase chain reaction (PCR), cloning, and sequencing of internal transcribed spacer (ITS) and 28S rDNA. A total of 382 cloned PCR inserts were successfully sequenced and then classified into 119 taxa. Of these taxa, 82 were basidiomycetes, 33 were ascomycetes, and 4 were zygomycetes. Thirty-one of the ascomycete sequences were identified as Cenococcum geophilum Fr. with overall richness of 22 ITS types. Other ascomycetes that form mycorrhizal associations were identified including Wilcoxina and Tuber as well as endophytes such as Lachnum, Cadophora, Phialophora, and Phialocephela. The most abundant mycorrhizal groups were Russulaceae (Lactarius, Macowanites, Russula) and species in the Thelephorales (Bankera, Boletopsis, Hydnellum, Tomentella). Our study demonstrates that tanoak supports a high diversity of ectomycorrhizal fungi with comparable species richness to that observed in Quercus root communities.

Species of Tetrahymena Identical by Small Subunit rRNA Gene Sequences are Discriminated by Mitochondrial Cytochrome c Oxidase I Gene Sequences - Sep 01, 2006 (pdf)
[Lynn, D. H. and Strüder-Kypke, M. C. 2006. Journal of Eukaryotic Microbiology. 53 (5) 385-387.]

The mitochondrial cytochrome c oxidase 1 (CO1) genes of two isolates of each of the seven mating types of Tetrahymena thermophila were sequenced and found to differ byo1% in nucleotide sequence and to be identical by putative protein sequence. As this gene was highly conserved in this species, the CO1 gene sequence was determined for four pairs of Tetrahymena species identical in their small subunit rRNA gene sequences. The following pairs of species showed from 1% to 12% divergence at the nucleotide level, enabling discrimination of all these species: (1) Tetrahymena pyriformis strain T and Tetrahymena setosa strain HZ-1; (2) Tetrahymena canadensis strain UM1215 and Tetrahymena rostrata strain ID-3; (3) Tetrahymena pigmentosa strain UM1285 and Tetrahymena hyperangularis strain EN112; and (4) Tetrahymena tropicalis strain TC-105 and Tetrahymena mobilis. However, because of the synonymous nature of the majority of substitutions, the pairs of species were identical based on the putative protein sequence.

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DNA barcoding reveals extraordinary cryptic diversity in an amphipod genus: implications for desert spring conservation - Sep 01, 2006 (pdf)
[Witt, Jonathan D.S., Threloff, Doug L. and Hebert, Paul D.N. 2006. Molecular Ecology. 15(10) 3073 - 3082.]

DNA barcoding has revealed unrecognized species in several animal groups. In this study we have employed DNA barcoding to examine Hyalella, a taxonomically difficult genus of amphipod crustaceans, from sites in the southern Great Basin of California and Nevada, USA. We assessed the extent of species diversity using a species screening threshold (SST) set at 10 times the average intrapopulation cytochrome c oxidase subunit I (COI) haplotype divergence. Despite the fact that this threshold approach is more conservative in delineating provisional species than the phylogenetic species concept, our analyses revealed extraordinary levels of cryptic diversity and endemism. The SST discriminated two provisional species within Hyalella sandra, and 33 provisional species within Hyalella azteca. COI nucleotide divergences among these provisional species ranged from 4.4% to 29.9%. These results have important implications for the conservation of life in desert springs — habitats that are threatened as a result of groundwater over-exploitation.

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DNA-based identification of preys from non-destructive, total DNA extractions of predators using arthropod universal primers - Sep 01, 2006 (pdf)
[Joan Pons 2006. Molecular Ecology Notes. 6(3) 623-626.]

Here, I show that prey sequences can be detected from DNA of tiger beetles of the genus Rivacindela using whole specimens, nondestructive methods, and universal cytochrome b primers for arthropods. BLAST searches of the obtained sequences against public databases revealed that the diet of Rivacindela is mostly composed of flies but also termites and other beetles. Accurate determination of order, family and even genus was achieved in most cases but rarely to species level. Results suggest that stored DNA samples extracted from whole predatory specimens could be an alternative to dissected gut contents as starting source for DNA-based dietary studies.

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Molecular taxonomy in pholcid spiders (Pholcidae, Araneae): evaluation of species identification methods using CO1 and 16S rRNA. - Sep 01, 2006 ()
[Astrin, J. J., Huber, B. A., Misof, B. and Klütsch, C. F. C. 2006. Zoologica Scripta. 35 (5) 441-457.]

The identification of species using molecular characters is a promising approach in alpha taxonomy and in any discipline depending on reliable assignment of specimens. Previous studies have shown the feasibility of the method, but considerable controversy persists. In this study, we use pholcid spiders in an effort to address two main issues. First, we evaluate and calibrate molecular species (re-)identification within a closely related group of organisms by using specimens that are morphologically unambiguously either conspecific or not. Species limits hypothesized a priori based on morphology were almost universally reconstructed by both mitochondrial markers used. Second, we focus on species identification methodology in a morphology-calibrated scenario, i.e. on how to assess the quality of a dataset and of the method used to obtain distance estimates (e.g. choice of markers, alignment strategy, type of distance data). We develop a number of statistical estimators permitting the measurement and communication of the clarity of species boundaries in a dataset and discuss their benefits and drawbacks. We propose that box plots rather than histograms are the superior tool for graphically illustrating taxonomic signal and that the median is a more appropriate measure of central tendency than the mean. Applying the suggested tools to our data, we propose that in molecular species identification, indel-related alignment uncertainties may often be even advantageous (by accentuating taxonomy-relevant information) and we conclude that — at least for our dataset — 16S is better suited to taxonomy than CO1.

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The Value of Barcoding - Sep 01, 2006 ()
[Darling, John. 2006. BioScience. 56(9) 710-711.]

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Phylogeny of Palearctic wheatears (genus Oenanthe) - congruence between morphometric and molecular data - Aug 31, 2006 ()
[Aliabadian A, Kaboli M, Prodon R, Nijman V, Vences M 2006. Mol. Phyl. Evol.. DOI 10.1016/ympev.2006.08.018.]

Wheatears of the genus Oenanthe are birds specialized to desert ecosystems in the Palearctic region from Morocco to China. Although they have been the subject of many morphological and ecological studies, no molecular data have been used to elucidate their phylogenetic relationships, and, their relationships are still debated. Here we use DNA sequences of 1180 bp of two mitochondrial genes, 16S rRNA and cytochrome oxidase subunit I, from 32 individuals from Middle East and North Africa, and Bayesian methods to derive a phylogeny for 11 species of Oenanthe. The resulting tree supported three major clades: (A) O. alboniger, O. chrysopygia, O. lugens, O. finschii, O. leucopyga, O. picata, O. moesta, (B) O. deserti and O. pleschanka; and (C) O. isabellina and O. oenanthe. These results largely differ from previous hypotheses based on analysis of ecomorphological and chromatic characters. However, the two clades (B) and (C) were also supported by a phenetic analysis of new morphometric data presented here, indicating that characters related to colouration and ecology in Oenanthe are more strongly influenced by homoplasy than those of body shape.

Development and evaluation of a DNA-barcoding approach for the rapid identification of nematodes - Aug 29, 2006 ()
[Bhadury, P., Austen, MC., Bilton, DT., Lambshead, PJD., Rogers, AD., and Smerdon, GR. 2006. Marine Ecology Progress Series. 320 1-9.]

Free-living nematodes are abundant in all marine habitats, are highly diverse, and can be useful for monitoring anthropogenic impacts on the environment. Despite such attributes, nematodes are effectively ignored by many marine ecologists because of their time-consuming taxonomy. Nematode diagnostics has traditionally relied on detailed comparison of morphological characters which, given their abundance, is difficult and laborious, meaning that the biodiversity of the group is typically underestimated. Molecular methods such as DNA-barcoding offer potentially efficient alternative approaches to studying the biodiversity of marine nematode communities, allowing these organisms to be more effectively exploited in ecological surveys and environmental assessments. In this study, a number of nuclear and mitochondrial genomic regions were evaluated as potential diagnostic loci for marine nematode species identification. Of these, the 18S ribosomal RNA gene amplified most reliably from a range of taxa, and was therefore evaluated as a DNA barcode. In a comparison of molecular and morphological identifications, over 97% of specimens sequenced were correctly assigned on the basis of a short stretch of 18S rRNA sequence (approximately 345 bp), making this a potentially useful marker for the rapid molecular assignment of unknown nematode species, and evaluation of nematode species richness during ecological surveys or environmental assessments. This study showed that a single marker approach based on amplification and sequencing may prove invaluable in the rapid identification of nematodes during ecological surveys and, indeed, other taxonomically challenging invertebrate taxa.

Estimating diversity of Indo-Pacific coral reef stomatopods through DNA barcoding of stomatopod larvae. - Aug 22, 2006 ()
[Barber, P. and Boyce, S. L. 2006. Proceedings of the Royal Society B: Biological Sciences. 273(1597) 2053 - 2061.]

There is a push to fully document the biodiversity of the world within 25 years. However, the magnitude of this challenge, particularly in marine environments, is not well known. In this study, we apply DNA barcoding to explore the biodiversity of gonodactylid stomatopods (mantis shrimp) in both the Coral Triangle and the Red Sea. Comparison of sequences from 189 unknown stomatopod larvae to 327 known adults representing 67 taxa in the superfamily Gonodactyloidea revealed 22 distinct larval operational taxonomic units (OTUs). In the Western Pacific, 10 larval OTUs were members of the Gonodactylidae and Protosquillidae where success of positive identification was expected to be 96.5%. However, only five OTUs could be identified to species and at least three OTUs represent new species unknown in their adult form. In the Red Sea where the identification rate was expected to be 75% in the Gonodactylidae, none of four larval OTUs could be identified to species; at least two represent new species unknown in their adult forms. Results indicate that the biodiversity in this well-studied group in the Coral Triangle and Red Sea may be underestimated by a minimum of 50% to more than 150%, suggesting a much greater challenge in lesser-studied groups. Although the DNA barcoding methodology was effective, its overall success was limited due to the newly discovered taxonomic limitations of the reference sequence database, highlighting the importance of synergy between molecular geneticists and taxonomists in understanding and documenting our world's biodiversity, both in marine and terrestrial environments.

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TrichOKEY v. 2 - a DNA oligonucliotide BarCode program for the identification of multiple sequences of Hypocrea and Trichoderma - Aug 20, 2006 ()
[Druzhinina, I.S., Kopchinskiy, A.G. 2006. 2006 Proceedings of the 8th International Mycological Congress, Cairns, Australia, MEDIMOND S.r.l., ISBN 88-7587-277-5. 53-59.]

The first version of the genus-specific fungal oligonucleotide BarCode for the identification of Hypocrea and Trichoderma species – TrichOKey v. 1, was developed in 2005. The program provides an on-line method for the quick molecular identification of an unknown isolate at the genus, clade and species levels based on a diagnostic combination of several oligonucleotides (hallmarks) specifically nested within the internal transcribed spacer 1 and 2 (ITS1 and 2) sequences of the rRNA gene cluster. In this manuscript we report on the newly integrated modules to increase the reliability of the result and present the second version of the program - TrichOKey v. 2, which is powered by simultaneous identification of multiple sequences, complementary similarity search tool and has an advanced graphic interface.

Microbial diversity in the deep sea and the underexplored "rare biosphere". - Aug 08, 2006 ()
[Sogin, M. L., Morrison, H. G., Huber, J. A., Welch, D. M., Huse, S. M., Neal, P. R., Arrieta, J. M. and Herndl, G. J 2006. Proceedings of the National Academy of Sciences of the United States of America. 102(22) 12115-12120.]

The evolution of marine microbes over billions of years predicts that the composition of microbial communities should be much greater than the published estimates of a few thousand distinct kinds of microbes per liter of seawater. By adopting a massively parallel tag sequencing strategy, we show that bacterial communities of deep water masses of the North Atlantic and diffuse flow hydrothermal vents are one to two orders of magnitude more complex than previously reported for any microbial environment. A relatively small number of different populations dominate all samples, but thousands of low-abundance populations account for most of the observed phylogenetic diversity. This "rare biosphere" is very ancient and may represent a nearly inexhaustible source of genomic innovation. Members of the rare biosphere are highly divergent from each other and, at different times in earth's history, may have had a profound impact on shaping planetary processes.

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Revision of Fijian Collinias Aczél (Diptera: Pipinculidae) - Aug 01, 2006 (pdf)
[Skevington, J.H. 2006. Bishop Museum Occasional Papers. 89 11-43.]

The Fijian species of Collinias Aczél are revised and include one described species, C. vitiensis Muir and 3 new species: C. croceus, n. sp., C. dolabratus, n. sp. and C. schlingeri, n. sp. A key to species is provided and diagnostic characters, including male and female genitalia, are illustrated. DNA barcoding data are provided for all Fijian species and several other, mostly undescribed, Collinias species from Australia and New Caledonia. A phylogeny for the genus is proposed in light of the barcoding data. Pipunculus imparilis Hardy, formerly unplaced to subgenus within Cephalops, is transferred to Collinias, n. comb.

Phylogeny of venus clams (Bivalvia: Venerinae) as inferred from nuclear and mitochondrial gene sequences. - Aug 01, 2006 ()
[Kappner, I. and Bieler, R. 2006. Molecular Phylogenetics and Evolution. 40(2) 317-331.]

Venerinae (Heterodonta: Veneridae) is a diverse, commercially important, and cosmopolitan marine bivalve subfamily. Recent workers synonymized it with the subfamily Chioninae, due to their overall morphological similarity. The use of traditional shell-based characters alone, however, is questionable for resolving phylogenetic relationships of this group. A phylogenetic study was carried out, based on nucleotide sequences of the mitochondrial large ribosomal subunit (16S), cytochrome oxidase subunit I (COI), and the nuclear protein-coding gene histone 3, to investigate the relationships and circumscription of Venerinae and the phylogenetic pattern of characters in this group. This study consists of a total of 55 taxa: 13 venerine genera, 24 chionine taxa, and 18 taxa of other venerid subfamilies. We analyzed the alignments using a Bayesian approach using Markov Chain Monte Carlo tree sampling and maximum parsimony methods. The resulting phylogenetic hypothesis suggests that Chioninae and Venerinae are actually discrete taxa, but that the circumscription suffered from misplacement of some genera. Our analysis showed that the former chionine genera Chamelea and Clausinella should be placed in Venerinae, as sister taxa to Venus. We re-analyzed morphological and anatomical features in light of the molecular data to describe monophyletic entities. Features of the hinge and internal shell as well as the degree of siphonal fusion are identified as characters to morphologically distinguish the two subfamilies. Of the three genes used in this study, only COI (commonly used as “barcoding” gene) posed substantial problems in obtaining sequence data from older museum material.

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Relationship between morphological taxonomy and molecular divergence within Crustacea: proposal of a molecular threshold to help species delimitation - Aug 01, 2006 ()
[Lefebure, T., Douady, C. J., Gouy, M. and Gibert, J. 2006. Molecular Phylogenetics and Evolution. 40(2) 435-447.]

With today’s technology for production of molecular sequences, DNA taxonomy and barcoding arose as a new tool for evolutionary biology and ecology. However, their validities still need to be empirically evaluated. Of most importance is the strength of the correlation between morphological taxonomy and molecular divergence and the possibility to define some molecular thresholds. Here, we report measurements of this correlation for two mitochondrial genes (COI and 16S rRNA) within the sub-phylum Crustacea. Perl scripts were developed to ensure objectivity, reproducibility, and exhaustiveness of our tests. Our analysis reveals a general correlation between molecular divergence and taxonomy. This correlation is particularly high for shallow taxonomic levels allowing us to propose a COI universal crustacean threshold to help species delimitation. At higher taxonomic levels this correlation decreases, particularly when comparing different families. Those results plead for DNA use in taxonomy and suggest an operational method to help crustacean species delimitation that is linked to the phylogenetic species definition. This pragmatic tool is expected to fine tune the present classification, and not, as some would have believed, to tear it apart.

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Mitochondrial DNA Haplotype Variation in the Parasitic Cirripede Sacculina Carcini Observed in the Cytochrome Oxidase Gene (COI). - Aug 01, 2006 ()
[Gurney R.H., Grewe P.M., and Thresher R.E. 2006. Journal of Crustacean Biology. 23(3) 326-330.]

Nucleotide sequence data of the mitochondrial cytochrome oxidase I (COI) gene, were compared among 17 specimens of Sacculina carcini parasitising three portunid hosts as a preliminary assessment of haplotype structure in a poorly understood species for which there are few morphological characters on which to base taxonomic analysis. S. carcini parasitising Carcinus maenas (from Sweden, England and Denmark) were compared with S. carcini parasitising Liocarcinus marmoreus (from Ireland) and Liocarcinus holsatus (from Wales). Specimens of three congeneric sacculinid species and one confamilial species were included in the comparison as outgroups. The comparison confirmed that specimens of S. carcini from different hosts and different regions are all the same species. The data also suggested geographically consistent sequence differences among sampled sites, high levels of similarity within sites and very large differences between species, all of which suggests that analysis of the COI gene sequence could be a useful method for resolving population genetics and taxonomy of rhizocephalans.

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Detection of Contarinia nasturtii (Diptera: Cecidomyiidae) in New York, a New Pest of Cruciferous Plants in the United States - Aug 01, 2006 ()
[Kikkert J.R., Hoepting C.A., Wu Q., Wang P., Baur R., Shelton A.M. 2006. Journal of Economic Entomology. 99 (4) 1310-1315.]

The midge Contarinia nasturtii Kieffer (Diptera: Cecidomyiidae) was first confirmed in North America in Ontario, Canada, in 2000. The insect is now distributed throughout many counties in the provinces of Ontario and Québec. Nearly 1,200 farms in the northeastern United States that grow cruciferous vegetables are at risk for C. nasturtii infestation if this insect were to spread to that region. Over a period of 3 yr (2002–2004), ≈3,000 ha of crops on 94 farms in western New York State was scouted for C. nasturtii, but none were found. In 2004, 42 experimental pheromone traps were placed in fields of cruciferous vegetables in eight counties. C. nasturtii males were captured at low levels (1–50 per trap/8 wk) on four farms in Niagara County, but not at any other site. C. nasturtii larvae were found in plant tissue at one of the four farms. Insect specimens were identified by morphological methods, molecular methods, or both. This is the first confirmation of C. nasturtii in the United States, which we believe was made possible by the combined use of pheromone traps, morphological characters of trapped adults, and molecular methods. The early detection in New York presents an opportunity to implement measures to limit the spread and establishment of C. nasturtii across the state and into other regions of the United States.

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Utility of Mitochondrial DNA Barcodes in Species Conservation - Aug 01, 2006 ()
[Rubinoff, D. 2006. Conservation Biology. 20(4) 1026-1033.]

Molecular tools are a standard part of many conservation studies and can be informative at many different levels of analysis, although there are inherent limitations and strengths of different genes or parts of genes to inform specific questions. Animal DNA barcodes, 600- to 800-base-pair segments of the mitochondrial gene cytochrome oxidase I, have been proposed as a means to quantify global biodiversity. Although mitochondrial (mt) DNA has a long history of use at the species level, recent analyses suggest that the use of a single gene, particularly mitochondrial, is unlikely to yield data that are balanced, universally acceptable, or sufficient in taxonomic scope to recognize many species lineages. Mitochondrial and nuclear genomes have different patterns of evolution and modes of inheritance, which can result in very different assessments of biodiversity. The ramifications of choosing a particular definition of species (species concept) need to be carefully considered because current efforts have designated DNA barcodes as the universal species concept without demonstrating its superiority over preexisting concepts. The results of such a barcoding paradigm may include a failure to recognize significant portions of biodiversity or nuclear/mitochondrial mixed lineages and could spuriously focus conservation resources on populations with relatively minor mtDNA divergence. DNA barcodes are most likely to provide potentially useful information for groups that are already well studied, and such taxa do not constitute the majority of biodiversity or those in most need of research attention. DNA barcode-length sequences are an important source of data but, when used alone or out of context, may offer only a fraction of the information needed to characterize species while taking resources from broader studies that could produce information essential to robust and informed conservation decisions.

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Assessing the use of the mitochondrial cox1 marker for use in DNA barcoding of red algae (Rhodophyta) - Aug 01, 2006 ()
[Robba, L., Russell, S. J., Barker, G. L. and Brodie, J. 2006. American Journal of Botany. 93(8) 1101-1108.]

The red algae, a remarkably diverse group of organisms, are difficult to identify using morphology alone. Following the proposal to use the mitochondrial cytochrome c oxidase subunit I (cox1) for DNA barcoding animals, we assessed the use of this gene in the identification of red algae using 48 samples plus 31 sequences obtained from GenBank. The data set spanned six orders of red algae: the Bangiales, Ceramiales, Corallinales, Gigartinales, Gracilariales and Rhodymeniales. The results indicated that species could be discriminated. Intraspecific variation was between 0 and 4 bp over 539 bp analyzed except in Mastocarpus stellatus (0–14 bp) and Gracilaria gracilis (0–11 bp). Cryptic diversity was found in Bangia fuscopurpurea, Corallina officinalis, G. gracilis, M. stellatus, Porphyra leucosticta and P. umbilicalis. Interspecific variation across all taxa was between 28 and 148 bp, except for G. gracilis and M. stellatus. A comparison of cox1 with the plastid Rubisco spacer for Porphyra species revealed that it was a more sensitive marker in revealing incipient speciation and cryptic diversity. The cox1 gene has the potential to be used for DNA barcoding of red algae, although a good taxonomic foundation coupled with extensive sampling of taxa is essential for the development of an effective identification system.

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Standard Percent DNA Sequence Difference for Insects Does Not Predict Species Boundaries - Aug 01, 2006 ()
[Cognato, Anthony, I. 2006. Journal of Economic Entomology. 99 (4) 1037-1045.]

Diagnosis and assessment of species boundaries of economically important insects are often problematic because of limited morphological and/or biological characters. DNA data can help to identify and revise species. Nonoverlapping intra- and interspecific sequence divergences are often used as evidence for species. Thus, the establishment of a standardized percent nucleotide divergence to predict species boundaries would aid in cases where species status is suspect. However, given variation in nucleotide mutation rates and species concepts, association between a standard percent sequence divergence and species is questionable. This review surveys the percent DNA sequence difference found between sister-species of economically important insects, to assess whether a standard divergence associates with all taxa. Sixty-two comparisons of intra- and interspecific pairwise DNA differences were made for mitochondrial and nuclear loci spanning families of Isoptera, Phthiraptera, Hemiptera, Coleoptera, Lepidoptera, Diptera, and Hymenoptera. Intra- and interspecific sequence divergences varied widely among insects, 0.04–26.0 and 1.0–30.7%, respectively. The ranges of intra- and interspecific sequence divergences overlapped in 28 of 62 comparisons. This implies that a standardized percent sequence divergence would fail to correctly diagnose species for 45% of the cases. Common occurrence of nonmonophyly among closely related species probably explains this observation. Nonmonophyly and overlap of intra- and interspecific divergences were significantly associated. The reviewed studies suggest that a standard percent sequence divergence does not predict species boundaries among economically important insects. DNA data can help best to predict species boundaries via its inclusion in nonphenetic phylogenetic analysis and subsequent systematic expert scrutiny.

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Gall morpho-type corresponds to seperate species of gall-inducing thrips (Thysanoptera: Phlaeothripidae) - Aug 01, 2006 ()
[Michael J. McLeish, Thomas W. Chapman and Laurence A. Mound 2006. Biological Journal of the Linnean Society. Volume 88 Issue 4 555-563.]

300,000 species to identify: problems, progress, and prospects in DNA barcoding of land plants. - Aug 01, 2006 ()
[Cowan, RS., Chase, MW., Kress, WJ., and Savolainen, V. 2006. Taxon. 55(3) 611-616.]

DNA barcodes have been successfully applied to a limited number of animal groups with the application of the mitochondrial gene, cytochrome c oxidase subunit 1. Recently two DNA regions, the plastid trnH-psbA spacer and nuclear ribosomal ITS region, have been shown to have potential as an identification barcode for land plants, although with some significant drawbacks. The ideal barcode should be relatively short in length (∼700 bp), more variable between than within species, and easily amplifiable with universal primers. Building on current success, ongoing investigations are searching for the best barcode to apply to all land plants. Once established, a plant barcode may be effectively used in biodiversity inventories, conservation assessments, and applied forensic investigations. Advances in sequencing technology and the completion of the DNA barcode library have the potential to provide the public with increased access to information about the natural world.

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Benchmarking DNA barcodes: an assessment using available primate sequences - Jul 21, 2006 (pdf)
[Hajibabaei, M., Singer, G.A.C., and D.A. Hickey 2006. Genome. 49(7) 851-854.]

DNA barcoding has been recently promoted as a method for both assigning specimens to known species and for discovering new and cryptic species. Here we test both the potential and the limitations of DNA barcodes by analysing a group of well-studied organisms—the primates. Our results show that DNA barcodes provide enough information to efficiently identify and delineate primate species, but that they cannot reliably uncover many of the deeper phylogenetic relationships. Our conclusion is that these short DNA sequences do not contain enough information to build reliable molecular phylogenies or define new species, but that they can provide efficient sequence tags for assigning unknown specimens to known species. As such, DNA barcoding provides enormous potential for use in global biodiversity studies.

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Ribosomal RNA as molecular barcodes: a simple correlation analysis without sequence alignment. - Jul 01, 2006 ()
[Chu, K. H., Li, C. P. and Qi, J. 2006. Bioinformatics. 22(14) 1690-1701.]

Motivation: We explored the feasibility of using unaligned rRNA gene sequences as DNA barcodes, based on correlation analysis of composition vectors (CVs) derived from nucleotide strings. We tested this method with seven rRNA (including 12, 16, 18, 26 and 28S) datasets from a wide variety of organisms (from archaea to tetrapods) at taxonomic levels ranging from class to species.

Result: Our results indicate that grouping of taxa based on CV analysis is always in good agreement with the phylogenetic trees generated by traditional approaches, although in some cases the relationships among the higher systemic groups may differ. The effectiveness of our analysis might be related to the length and divergence among sequences in a dataset. Nevertheless, the correct grouping of sequences and accurate assignment of unknown taxa make our analysis a reliable and convenient approach in analyzing unaligned sequence datasets of various rRNAs for barcoding purposes.

Availability: The newly designed software (CVTree 1.0) is publicly available at the Composition Vector Tree (CVTree) web server http://cvtree.cbi.pku.edu.cn

Contact: kahouchu@cuhk.edu.hk

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A revision of the interrelationships of Schistosoma including the recently described Schistosoma guineensis - Jul 01, 2006 (pdf)
[Webster, B. L., Southgate, V. R., & D.T. Littlewood 2006. The International Journal for Parasitology. 36(8) 947-55.]

In light of the recently described human schistosome Schistosoma guineensis and recent phylogenetic studies of the genus Schistosoma, a revision of the interrelationships of the members of this genus is needed. This paper adds to previous phylogenetic studies on the family Schistosomatidae and offers the most up to date and robust phylogeny of the group based on complete small and large nuclear subunit rRNA genes and partial mitochondrial cox1, incorporating most of the 21 species of Schistosoma. Our findings show that the group retains the same topology as that resolved in previous studies except Schistosoma margrebowiei was resolved as the sister taxon to all others in the Schistosoma haematobium species group and S. guineensis was placed as sister species to both Schistosoma bovis and Schistosoma curassoni. The S. haematobium species group contains eight species of which many are of significant medical and veterinary importance. Additionally, many of these species have been shown to hybridise both in the wild and experimentally, making the correct identification and recognition of species very important. A pairwise comparison of cox1 among Schistosoma species suggests this gene alone would fail as a reliable barcode for species identification. Phylogenetic results clearly treat Schistosoma intercalatum and S. guineensis as separate taxa with each more closely related evolutionarily to S. haematobium than to each other. The study also highlights the problems associated with wrongly attributed sequences on public databases such as GenBank.

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Problems with DNA barcodes for species delimitation: 'ten species' of Astraptes fulgerator reassessed (Lepidoptera:Hesperiidae) - Jun 07, 2006 (pdf)
[Andrew V.Z. Brower 2006. Systematics and Biodiversity. 4 127-132.]

Hebert and colleagues (2004) used a short region of the mitochondrial Cytochrome oxidase subunit I gene as a delimiter for ten putative species fromamong 466 individuals of the skipper butterfly currently known as Astraptes fulgeratorfrom Guanacaste, Costa Rica. Their data are reanalysed to assess clusterstability and clade support using Neighbor-Joining bootstrap, population aggregationanalysis and cladistic haplotype analysis. At least three, but not more than seven mtDNA clades that may correspond to cryptic species are supported by the evidence.Additional difficulties with Hebert et al.’s interpretation of the data are discussed.

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DNA identification and morphological description of the first confirmed larvae of Hetaeriinae (Coleoptera: Histeridae) - Jun 05, 2006 (pdf)
[Caterino, M.S., and A.K. Tishechkin 2006. Systematic Entomology. 31(3) 405-418.]

Using DNA sequences of nuclear ribosomal (18S) and mitochondrial (cytochrome oxidase I) genes (a modified DNA barcoding approach), we positively identify, for the first time, larvae of hetaeriine Histeridae. Species in this subfamily occur as obligate associates of social insect colonies, particularly those of neotropical army ants. Of several larval specimens collected from bivouacs of Eciton burchelli, we identify the two larval instars of Paratropinus scalptus, and discuss a quite different first instar larva near Euxenister. The larvae are described and illustrated, with attempts to homologize all chaetotaxy to other known histerid larvae. Phylogenetic trees for 18S and cytochrome oxidase I, for over twenty hetaeriine taxa, are compared with each other and with a previous hypothesis of relationships in the subfamily.

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Morphological, ecological and genetic evidence for distinguishing Anastrophyllum joergensenii Schiffn. and A-alpinum Steph. (Jungermanniopsida : Lophoziaceae) - Jun 02, 2006 ()
[Long, DG., Paton, JA., Squirrell, J., Woodhead, M., and Hollingsworth, PM 2006. Journal of Bryology. 28(2) 108-117.]

The liverwort Anastrophyllum joergensenii Schiffn., reported from Norway, Scotland, Alaska and the Sino-Himalaya is shown to consist of two distinct species, A. alpinum Steph. (treated before as a synonym of A. joergensenii) in the Himalaya, western China, Alaska and Scotland, and A. joergensenii Schiffn. s. str. in Norway, Scotland and western China. The two species are distinguished on genetic characters, size, leaf and perianth characters, and appear to have different ecological preferences. Anastrophyllum alpinum, although the more widespread of the two in Scotland, is there known only as non-fertile plants, whereas in the Sino-Himalaya fertile populations and sporophytes are not infrequent; in contrast, the rarer A. joergensenii can produce perianths in Scotland, Norway and Yunnan but androecia and sporophytes are unknown. The differences between the two are detailed and the ecology and distribution outlined.

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A novel molecular protocol for the rapid extraction of DNA from bryophytes and the utility of direct amplification of DNA from a single dwarf male - Jun 01, 2006 (pdf)
[Pedersen, Niklas; Russell, Stephen J.; Newton, Angela E.; Ansell, 2006. The Bryologist. 109(2) 257-264.]

A novel protocol for the rapid extraction of bryophyte DNA ispresented and tested on nine mosses and one liverwort. Amplification products and sequences of the rps4 gene were obtained for all the samples tested. Direct amplification and sequencing of DNA from a single dwarf male was found to be possible. By adding single dwarf males of Dicranum scoparium directly to a PCR, amplification products of the ITS regions were obtained for nine of the 11 dwarf malestested. To obtain different gene sequences from a single dwarf male,individual dwarf males were incubated in buffer at 60°C for different time periods and the resulting suspensions used for amplification of the chloroplast regions trnG and trnL-F. Amplification products of the trnG region were obtained for all the samples, but amplification of the trnL-F region was less successful. Clean DNA sequences were obtained from all the amplification products that were used in bi-directional sequencing. The rapid method presented has the potential to be a useful tool for screening high numbers of plants for specific genomic markers, such as in DNA barcoding. Direct amplification of DNA provides the opportunity for the first time to study genetic variation among moss dwarf males.

Two Views: Will DNA barcoding advance efforts to conserve biodiversity more efficiently than traditional taxonomic methods? - Jun 01, 2006 ()
[Anthony I Cognato, Ryan M Caesar, Mark Blaxter and Alfried P Vogler 2006. Frontiers in Ecology and the Environment. 4(5) 268-273.]

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DNA Barcoding: An Instance of Technology-driven Science? - Jun 01, 2006 ()
[Fitzhugh, Kirk. 2006. BioScience. 56(6) 462-463.]

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DNA Barcoding will often Fail to Discover New Animal Species over Broad Parameter Space - May 10, 2006 ()
[Michael J. Hickerson, Christopher P. Meyer, and Craig Moritz 2006. Systematic Biology. 55 729-739.]
Molecular divergence between Gryllus rubens and Gryllus texensis, sister species of field crickets (Orthoptera: Gryllidae) - May 01, 2006 ()
[Gray D.A., Barnfield P., Seifried M., Richards M.H. 2006. The Canadian Entomologist. 138 (3) 305-313.]

We assess the degree of sequence divergence in the maternally inherited mitochondrial cytochrome c oxidase I (COI) and cytochrome b (CytB) genes between two sister species of field crickets, Gryllus rubens Scudder, 1902 and Gryllus texensis Cade and Otte, 2000. We analyzed 1460 base pairs from 10 individuals of each species; individuals were sampled from areas of both allopatry and sympatry. Overall average pairwise mitochondrial sequence divergence between species was 1.4% ± 0.1% (mean ± SD); however, there was almost an order of magnitude more divergence in COI (2.59% ± 2.25%) than in CytB (0.35% ± 0.24%). Gryllus texensis appears to harbor a much greater level of genetic variation than does G. rubens. Phylogenetic trees constructed from these sequences show reasonable separation of species; however, sequences are not reciprocally monophyletic. Gene tree polyphyly may reflect recent species-level divergence and (or) interspecific gene flow. The pattern of sequence divergence and genetic variation in these taxa is consistent with allopatric or peripatric speciation in Pleistocene glacial refugia in the southeastern (G. rubens ancestral lineage) and southcentral United States (G. texensis ancestral lineage).

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Identification of Dioryctria (Lepidoptera: Pyralidae) in a Seed Orchard at Chico, California - May 01, 2006 ()
[Roe A.D., Stein J.D., Gillette N.E. and Sperling F.A.H. 2006. Annals of the Entomological Society of America. 99(3) 433-448.]

Species of Dioryctria Zeller (Lepidoptera: Pyralidae) are important pests of conifers, particularly in seed orchards, and accurate species identification is needed for effective monitoring and control. Variable forewing morphology and lack of species-specific genitalic features hinder identification, prompting the search for additional diagnostic characters. Mitochondrial DNA (mtDNA) sequences from the cytochrome c oxidase I and II genes (COI and COII) were obtained from specimens collected at lights, pheromone traps, and host plants in the Pacific Northwest, focusing on a U.S. Forest Service seed orchard in Chico, CA. A 475-bp fragment of COI was used to identify eight distinct genetic lineages from 180 Dioryctria specimens, and these were identified as eight described species. Comparisons among mtDNA variation, adult morphology, larval host association, and pheromone attraction were used to assign individuals to species groups and to identify diagnostic characters for species identification. A 2.3-kb fragment of COI-COII was sequenced for 14 specimens to increase resolution of phylogenetic relationships. Species groups were well resolved using both the 475-bp and “DNA barcode” subsets of the 2.3-kb sequences, with the 475-bp fragment generally showing lower divergences. The zimmermani and ponderosae species groups were sister groups and had similar male genitalic morphology and larval feeding habits. The pentictonella group was sister to the zimmermani + ponderosae group clade, and all species have raised scales and a Pinus sp. larval host (where known). Combining molecular characters with morphological and behavioral characters improved identification of Dioryctria species and supported previous species group relationships.

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A new species of Capis (Lepidoptera: Noctuidae) from Québec, Canada - May 01, 2006 ()
[L. Handfield, D. Handfield 2006. The Canadian Entomologist. 138 (3) 333-338.]

Capis archaia sp. nov., a new species of Noctuidae (Lepidoptera), is described from Québec, Canada. The species is included in the genus Capis Grote, 1882, a trifid genus in the subfamily Eustrotiinae. Adults and genitalia of this species are described and illustrated, as are those of Capis curvata Grote, 1882.

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Identifying units for conservation using molecular systematics: the cautionary tale of the Karner blue butterfly - Apr 13, 2006 (pdf)
[Zachariah Gompert, Chris C. Nice, James A. Foryce, Matthew L. Forister, Arthur M. Shapiro 2006. Molecular Ecology. 15 1759-1768.]

The federally endangered North American Karner blue butterfly (Lycaeides melissa samuelis) and the closely related Melissa blue butterfly (L. m. melissa) can be distinguished based on life history and morphology. Western populations of L. m. samuelis share mitochondrial haplotypes with L. m. melissa populations, while eastern populations of L. m. samuelis have divergent haplotypes. Here we test two hypotheses concerning the presence of L. m. melissa mitochondrial haplotypes in western L. m. samuelis populations: (i) mitochondrial introgression has occurred from L. m. melissa populations into western L. m. samuelis populations, or (ii) western populations of the nominal L. m. samuelis are more closely related to L. m. melissa than to eastern L. m. samuelis populations, yet are phenotypically similar to the latter. A Bayesian algorithm was used to cluster 190 L. melissa individuals based on 143 informative amplified fragment length polymorphism (AFLP) loci. This method clearly differentiated L. m. samuelis and L. m. melissa. Thus, genomic divergence was greater between western L. m. samuelis populations and L. m. melissa populations than it was between western and eastern populations of L. m. samuelis. This supports the hypothesis that the presence of L. m. melissa mitochondrial haplotypes in western L. m. samuelis populations is the result of mitochondrial introgression. These data provide valuable information for conservation and management plans for the endangered L. m. samuelis, and illustrate the risks of using data from a single locus for diagnosing significant units of biodiversity for conservation.

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Beyond barcodes: complex DNA taxonomy of a South Pacific Island radiation - Apr 07, 2006 ()
[Monaghan, M. T., Balke, M., Pons, J.and Vogler, A. P. 2006. Proceedings of the Royal Society B: Biological Sciences. 273(1588) 887 - 893.]

DNA barcodes can provide rapid species identification and aid species inventories in taxonomically unstudied groups. However, the approach may fail in recently diverged groups with complex gene histories, such as those typically found on oceanic islands. We produced a DNA-based inventory of taxonomically little known diving beetles (genus Copelatus) in the Fiji archipelago, where they are a dominant component of the aquatic invertebrate fauna. Sampling from 25 localities on five islands and analysis of sequences from one nuclear (328bp histone 3) and three mitochondrial (492bp rrnL, 786bp cox1, 333bp cob) gene regions revealed high haplotype diversity, mainly originated since the Pleistocene, and subdivided into three major phylogenetic lineages and 22 statistical parsimony networks. A traditional taxonomic study delineated 25 morphologically defined species that were largely incongruent with the DNA-based groups. Haplotype diversity and their spatial arrangement demonstrated a continuum of relatedness in Fijian Copelatus, with evidence for introgression at various hierarchical levels. The study illustrates the difficulties for formal classification in evolutionarily complex lineages, and the potentially misleading conclusions obtained from either DNA barcodes or morphological traits alone. However, the sequence profile of Fijian Copelatus provides an evolutionary framework for the group and a DNA-based reference system for the integration of ecological and other biodiversity data, independent of the Linnaean naming system.

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DNA Barcoding and Taxonomy in Diptera: A Tale of High Intraspecific Variability and Low Identification Success - Apr 07, 2006 ()
[Rudolf Meier, Kwong Shiyang, Gaurav Vaidya, and Peter K.C. NG 2006. Systematic Biology. 55 715-728.]

Testing molecular barcodes: Invariant mitochondrial DNA sequences vs the larval and adult morphology of West Palaearctic Pandasyopthalmus species (Diptera : Syrphidae : Paragini). - Apr 06, 2006 ()
[Rojo, S., G. Stahls, C. Perez-Banon, and M. A. Marcos-Garcia. 2006. European Journal of Entomology. 103(2) 443-458.]

The intra- and interspecific variability in the West Palaearctic tibialis-group species of the subgenus Pandasyopthalmus (Diptera: Syrphidae: Paragus) was analysed. Novel immature and molecular characters were studied and the traditionally used adult characters reviewed with the aim of establishing the status of the most widespread taxa of the tibialis-group in the Palaearctic region. Moreover, a review of the morphology of the larvae of the subgenus Pandasyopthalmus is also presented and includes the first description of the chaetotaxy of the larval head of Syrphidae. The larval morphology showed a continuum between two extremes. There is intraspecific variability in the male genitalia characters typically used for diagnostic species identification in this group. Molecular characters of the mitochondrial cytochrome c-oxidase subunit I (COI) was invariant for the West Palaearctic Pandasyopthalmus taxa analysed. Despite the fact that no great differences were found when compared with Afrotropical tibialis-group individuals (uncorrected pairwise divergence 0.17–0.35%), the divergences of the West Palaearctic vs. Nearctic and Austral-Oriental tibialis-group taxa varied between 1.15–2.75% (uncorrected pairwise divergence). Molecular characters of the nuclear ribosomal internal transcribed spacer region (ITS2) revealed several molecular haplotypes of a dinucleotide repeat that was not constrained to morphospecies or to populations of the same geographic origin. The closely related and morphologically similar species of the tibialis-group known from the West Palaearctic region are separable in most cases only by the shape and size of male postgonites. The results of this study support the presence of a single polymorphic taxon in the West Palaearctic region (or a very recent origin of the taxa studied). Moreover larval morphology and the lack of a clear relation between ITS2 haplotypes and the geographic distribution or adult morphology, support the taxonomic implications of barcode taxonomy based on mitochondrial DNA for this species-group of Syrphidae.

DNA barcoding: A molecular tool to identify Antarctic marine larvae - Apr 01, 2006 ()
[Webb, KE., Barnes, DKA., Clark, MS., and DA. Bowden 2006. Deep-Sea Res Part II-Tropical Stud Oceanogr. 53(8-10) 1053-1060.]

To begin to understand overall patterns and processes influencing marine populations, communities and ecosystems, it is important to determine the timing, duration, mode and dispersal of larvae. However, few studies of the spatial and temporal variation in abundance of larvae have been undertaken at any locality, other than for a few commercially important species. In Antarctic seas the abundance and species-richness of marine larvae are key to a number of concepts (such as the validity of Thorson's rule and ecological versus evolutionary success of brooders compared to spawning species). Traditionally, marine larval identification (using microscopy), even to order level, is a time-consuming, labour-intensive and inexact process. Ontogenic changes during larval life make identification difficult and require high levels of expertise, and identification is generally confirmed only by laboratory spawning experiments. New molecular genetic methods enable faster direct identification of marine larvae to a higher resolution. Our preliminary results show that it is possible to identify larvae of Antarctic species using DNA barcoding techniques, but that the resolution is currently limited by the availability of comparative adult sequences in the DNA sequence databases.

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Evolutionary rates, divergence dates, and the performance of mitochondrial genes in Bayesian phylogenetic analysis. - Apr 01, 2006 ()
[Mueller, R. L 2006. Systematic Biology. 55(2) 289-300.]

The mitochondrial genome is one of the most frequently used loci in phylogenetic and phylogeographic analyses, and it is becoming increasingly possible to sequence and analyze this genome in its entirety from diverse taxa. However, sequencing the entire genome is not always desirable or feasible. Which genes should be selected to best infer the evolutionary history of the mitochondria within a group of organisms, and what properties of a gene determine its phylogenetic performance? The current study addresses these questions in a Bayesian phylogenetic framework with reference to a phylogeny of plethodontid and related salamanders derived from 27 complete mitochondrial genomes; this topology is corroborated by nuclear DNA and morphological data. Evolutionary rates for each mitochondrial gene and divergence dates for all nodes in the plethodontid mitochondrial genome phylogeny were estimated in both Bayesian and maximum likelihood frameworks using multiple fossil calibrations, multiple data partitions, and a clock-independent approach. Bayesian analyses of individual genes were performed, and the resulting trees compared against the reference topology. Ordinal logistic regression analysis of molecular evolution rate, gene length, and the Γ-shape parameter α demonstrated that slower rate of evolution and longer gene length both increased the probability that a gene would perform well phylogenetically. Estimated rates of molecular evolution vary 84-fold among different mitochondrial genes and different salamander lineages, and mean rates among genes vary 15-fold. Despite having conserved amino acid sequences, cox1, cox2, cox3, and cob have the fastest mean rates of nucleotide substitution, and the greatest variation in rates, whereas rrnS and rrnL have the slowest rates. Reasons underlying this rate variation are discussed, as is the extensive rate variation in cox1 in light of its proposed role in DNA barcoding. [Divergence dates; DNA barcoding; mitochondrial genomes; molecular evolution; phylogenetic performance; salamander phylogeny.]

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DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach - Mar 31, 2006 (pdf)
[Newmaster, S.G., Fazekas, A.J., and S. Ragupathy 2006. Can. J. Bot.. 84 335-341.]

DNA barcoding based on the mitochondrial cytochrome c oxidase 1 (cox1) sequence is being employed for diverse groups of animals with demonstrated success in species identification and new species discovery. Applying barcoding systems to land plants will be a more challenging task as plant genome substitution rates are considerably lower than those observed in animal mitochondria, suggesting that a much greater amount of sequence data from multiple loci will be required to barcode plants. In the absence of an obvious well-characterized plant locus that meets all the necessary criteria, a key first step will be identifying candidate regions with the most potential. To meet the challenges with land plants, we are proposing the adoption of a tiered approach wherein highly variable loci are nested under a core barcoding gene. Analysis of over 10 000 rbcL sequences from GenBank demonstrate that this locus could serve well as the core region, with sufficient variation to discriminate among species in approximately 85% of congeneric pair-wise comparisons. Use of a secondary locus can be implemented when required and can vary from group to group if necessary. The implementation of a barcoding tool has multiple academic and practical applications. It will speed routine identifications and the detection of alien species, advance ecological and taxonomic inquiry, permit fast and accurate forensic analysis of plant fragments, and can function as an additional layer of quality control in the food industry.

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DNA barcoding applied to invasive leafminers (Diptera: Agromyzidae) in the Philipines - Mar 30, 2006 (pdf)
[Scheffer, S.J., Lewis, M.L., and R.C. Joshi 2006. Ann. Entomol. Soc. Am.. 99(2) 204-210.]

DNA barcoding involves the sequencing of a single gene region from all species to provide a means for identifying all of life. Although appealing to many scientists, this idea has caused considerable controversy among systematists. We applied a DNA barcoding approach to outbreak populations of invasive Liriomyza spp. leafminer pests in the Philippines to explore the use of barcoding in a relatively well studied, economically important group. We sequenced a 527-bp portion of mitochondrial cytochrome oxidase I (COI) from 258 leafminers from 26 plant host species in the Philippines. Neighbor-joining and parsimony analysis were used to compare COI sequences from the Philippines to an extensive database of COI sequences previously obtained from samples of the invasive leafminers Liriomyza huidobrensis (Blanchard), Liriomyza trifolii (Burgess), and Liriomyza sativae Blanchard from locations around the world. We conclude that although a DNA barcoding approach can provide rapid species identifications, in certain instances it is likely to either overestimate or underestimate the number of species present. Only when placed within the context of considerable other data can DNA barcoding be fully interpreted and used. For economically and medically important species, which can be well studied, DNA barcoding offers a powerful means for rapid identifications.

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CO1 phylogenies in diploblasts and the 'Barcoding of Life' - are we sequencing a suboptimal partition? - Mar 15, 2006 (pdf)
[Erpenbeck, D., J.N.A. Hooper and G. Wörheide 2006. Molecular Ecology Notes. 6 550-553.]

Partitions of the cytochrome oxidase subunit 1 (CO1) gene, especially the 5' end, are frequently recruited to infer lower level phylogenies in animals. In diploblasts, mitochondrial genes were found to evolve in a slower rate than their bilaterian counterparts. Therefore, diploblast CO1 gene trees repeatedly remained unresolved, which also raises doubts on the suitability of CO1 for DNA barcoding in these animals. The complete mitochondrial genome sequences from Anthozoa and recently from Porifera allow us to compare the resolution power of the 5' partition, which has also been proposed as the standard marker for DNA barcoding, with a less frequently used partition further downstream. We report on the finding of significantly different substitution patterns of the downstream partition opposed to the 5' partition. We discuss the consequences and potential in the light of diploblast phylogenetic reconstruction and DNA barcoding.

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Barcoding helps biodiversity fly. - Mar 14, 2006 ()
[Herre, E. A. 2006. PNAS. 103(11) 3949-3950.]

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DNA barcodes reveal cryptic host-specificity within the presumed polyphagous members of a genus of parasitoid flies (Diptera: Tachinidae) - Mar 07, 2006 ()
[Smith, M.A., Woodley, N.E., Janzen, D.H., Hallwachs, W., and P.D.N. Hebert 2006. PNAS. 103(10) 3657-3662.]

Notes:  DNA barcoding discriminates 17 highly host-specific morphospecies of Belvosia, and further raises the species count to 32 by revealing that each of the generalist species are in fact highly host-specific cryptic species complexes.

Read the publication here.

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Deeply divergent lineages of the widespread New Zealand amphipod Paracalliope fluviatilis revealed using allozyme and mitochondrial DNA analysesDeeply divergent lineages of the widespread New Zealand amphipod Paracalliope fluviatilis revealed using allozy - Feb 01, 2006 ()
[Hogg, I. D., M. I. Stevens, K. E. Schnabel, and M. A. Chapman 2006. Freshwater Biology. 51(2) 236-248.]

1. We evaluated the population genetic structure of the common New Zealand amphipod Paracalliope fluviatilis using eight allozyme loci, and the mitochondrial cytochrome oxidase c subunit I (COI) gene locus. Morphological analyses were also conducted to evaluate any phenotypic differences. Individuals belonging to P. fluviatilis were collected from a total of 14 freshwater fluvial habitats on the North and South Islands, New Zealand.

2. We found evidence for strong genetic differentiation among locations (Wright's FST > 0.25), and fixed differences (non-shared alleles) at two of the eight allozyme loci indicating the possibility of previously unknown species. Analysis of a 545-bp fragment of the COI locus was mostly congruent with the allozyme data and revealed the same deeply divergent lineages (sequence divergences up to 26%).

3. Clear genetic breaks were identified between North Island and South Island populations. North Island populations separated by <100 km also showed genetic differences between east and west draining watersheds (sequence divergence >12%). Accordingly, present-day dispersal among hydrologically isolated habitats appears minimal for this taxon.

4. Although population differences were clearly shown by allozyme and mtDNA analyses, individuals were morphologically indistinguishable. This suggests that, as in North American and European taxa (e.g. Hyalella and Gammarus), morphological conservatism may be prevalent among New Zealand's freshwater amphipods. We conclude that molecular techniques, particularly the COI gene locus, may be powerful tools for resolving species that show no distinctive morphological differences.

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DNA barcodes for insect pest identification: a test case with tussock moths (Lepidoptera : Lymantriidae) - Feb 01, 2006 ()
[Ball, S. L., and K. F. Armstrong 2006. Canadian Journal of Forest Research-Revue Canadienne De Recherche Forestiere. 36(2) 337-350.]

Reliable and rapid identification of exotic pest species is critical to biosecurity. However, identification of morphologically indistinct specimens, such as immature life stages, that are frequently intercepted at borders is often impossible. Several DNA-based methods have been used for species identification; however, a more universal and anticipatory identification system is needed. Consequently, we tested the ability of DNA "barcodes" to identify species of tussock moths (Lymantriidae), a family containing several important pest species. We sequenced a 617 base pair fragment of the mitochondrial gene cytochrome c oxidase 1 for 20 lymantriid species. We used these, together with other Noctuoidea species sequences from GenBank and the Barcoding of Life Database to create a "profile" or reference sequence data set. We then tested the ability of this profile to provide correct species identifications for 93 additional lymantriid specimens from a data set of mock unknowns. Of the unknowns, 100% were correctly identified by the cytochrome c oxidase 1 profile. Mean interspecific sequence (Kimura 2-parameter) divergence was an order of magnitude greater (14%) than mean intraspecific divergence (<1%). Four species showed deeper genetic divergences among populations. We conclude that DNA barcodes provide a highly accurate means of identifying lymantriid species and show considerable promise as a universal approach to DNA-based identification of pest insects.

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DNA barcoding in taxonomy and the perception of species in nature - Feb 01, 2006 ()
[Kunz, W. 2006. BioScience. 56(2) 93.]

Statistical approaches for DNA barcoding - Feb 01, 2006 (pdf)
[Neilsen, R. and M. Matz 2006. Syst. Biol.. 55(1) 162-169.]
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DNA barcodes distinguish species of tropical Lepidoptera - Jan 24, 2006 (pdf)
[Hajibabaei, M., Janzen, D.H., Burns, J.M., Hallwachs, W., and P.D.N. Hebert 2006. Proc Natl Acad Sci U S A. 103(4) 968 -71.]

Although central to much biological research, the identification of species is often difficult. The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. However, the effectiveness of DNA barcoding for identifying specimens in species-rich tropical biotas is unknown. Here we show that cytochrome c oxidase I DNA barcodes effectively discriminate among species in three Lepidoptera families from Area de Conservación Guanacaste in northwestern Costa Rica. We found that 97.9% of the 521 species recognized by prior taxonomic work possess distinctive cytochrome c oxidase I barcodes and that the few instances of interspecific sequence overlap involve very similar species. We also found two or more barcode clusters within each of 13 supposedly single species. Covariation between these clusters and morphological and/or ecological traits indicates overlooked species complexes. If these results are general, DNA barcoding will significantly aid species identification and discovery in tropical settings.


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DNA Barcoding of Blastocystis - Jan 20, 2006 (pdf)
[Scicluna, S.M., Tawari, B., and C.G. Clark 2006. Protist. 1-9.]

We have developed a simple method for subtyping the intestinalprotistan parasite Blastocystis using an approach equivalent to DNAbarcoding in animals. Amplification of a 600 bp region of the smallsubunit ribosomal RNA gene followed by single primer sequencing of thePCR product provides enough data to assign isolates to specificsubtypes unambiguously. We believe that this approach will prove useful in future epidemiological studies.

Development of microarray-based diagnostics of voles and shrews for use in biodiversity monitoring studies, and evaluation of mitochondrial cytochrome oxidase I vs. cytochrome b as genetic markers - Nov 30, 2005 ()
[M. Pfunder, O. Holzgang and J.E. Frey 2005. Molecular Ecology. Vol. 13, No. 5 1277-1286.]

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UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi - Dec 31, 2005 ()
[Kõljalg U, Larsson K-H, Abarenkov K, Nilsson RH, Alexander IJ, Eberhardt U, Erland S, Høiland K, Kjøller R, Larsson E, Pennanen T, Sen R, Taylor AFS, Tedersoo L, Vrålstad T and Ursing B.M. 2005. New Phytologist. 166 1063-1068.]

Identification of ectomycorrhizal (ECM) fungi is often achieved through comparisons of ribosomal DNA internal transcribed spacer (ITS) sequences with accessioned sequences deposited in public databases. A major problem encountered is that annotation of the sequences in these databases is not always complete or trustworthy. In order to overcome this deficiency, we report on UNITE, an open access database.
UNITE comprises well annotated fungal ITS sequences from well defined herbarium specimens that include full herbarium reference identification data, collector/source and ecological data. At present, UNITE contains 1680 ITS sequences from 824 species and 110 genera of ECM fungi [23 Jan, 2007].
UNITE can be searched by taxon name, via sequence similarity using blastn, and via phylogenetic sequence identification using galaxie. Following implementation, galaxie performs a phylogenetic analysis of the query sequence after alignment to either pre-existing generic alignments, or to matches retrieved from a blast search on the UNITE data. It should be noted that the current version of UNITE is dedicated to the reliable identification of ectomycorrhizal fungi.
The UNITE database is accessible through the URL: http://unite.ut.ee

Phylogeny and toxigenic potential is correlated in Fusarium species as revealed by partial translation elongation factor 1 alpha gene sequences - Dec 31, 2005 ()
[Kristensen, R., Torp, M., Kosiak, B. & Holst-Jensen, A. 2005. Mycol. Res.. 109(2) 173-186.]

Partial translation elongation factor 1 alpha (TEF-1[alpha]) gene and intron sequences are reported from 148 isolates of 11 species of the anamorph genus Fusarium; F. avenaceum (syn. F. arthrosporioides), F. cerealis, F. culmorum, F. equiseti, F. flocciferum, F. graminearum, F. lunulosporum, F. sambucinum, F. torulosum, F. tricinctum and F. venenatum. The sequences were aligned with TEF-1[alpha] sequences retrieved from 35 isolates of F. kyushuense, F. langsethiae, F. poae and F. sporotrichioides in a previous study, and 39 isolates of F. cerealis, F. culmorum, F. graminearum and F. pseudograminearum retrieved from sequence databases. The 222 aligned sequences were subjected to phylogenetic analyses using maximum parsimony and Bayesian Markov Chain Monte Carlo maximum likelihood statistics. Support for internal branching topologies was examined by Bremer support, bootstrap and posterior probability analyses. The resulting trees were largely congruent. The taxon groups included in the sections Discolor, Gibbosum and Sporotrichiella sensu Wollenweber & Reinking (1935) all appeared to be polyphyletic. All species were monophyletic except F. flocciferum that was paraphyletic, and one isolate classified as F. cfr langsethiae on the basis of morphology that grouped with F. sporotrichioides. Mapping of toxin profiles, host preferences and geographic origin onto the DNA based phylogenetic tree structure indicated that in particular the toxin profiles corresponded with phylogeny, i.e. phylotoxigenic relationships were inferred. A major distinction was observed between the trichothecene and non-trichothecene producers, and the trichothecene producers were grouped into one clade of strictly type A trichothecene producers, one clade of strictly type B trichothecene producers and one clade with both type A and type B trichothecene producers. Furthermore, production of the type A trichothecenes T-2/HT-2 toxins are associated with a lineage comprising F. langsethiae and F. sporotrichioides. The ability to produce zearalenone was apparently gained parallel to the ability to produce trichothecenes, and later lost in a derived sublineage. The ability to produce enniatins is a shared feature of the entire study group, with the exception of the strict trichothecene type B producers and F. equiseti. The ability to produce moniliformin seems to be an ancestral feature of members of the genus Fusarium which seems to have been lost in the clades consisting of trichothecene/zearalenone producers. The aims of the present study were to determine the phylogenetic relationships between the different species of Fusarium commonly occurring on Norwegian cereals and some of their closest relatives, as well as to reveal underlying patterns such as the ability to produce certain mycotoxins, geographic distribution and host preferences. Implications for a better classification of Fusarium are discussed and highlighted.

Molecular systematics of the endemic Leptaxini (Gastropoda : Pulmonata) on the Azores islands - Dec 31, 2005 ()
[Van Riel, P., Jordaens, K., Van Houtte, N., Frias Martins, A.M., Verhagen, R. & Backeljau, T. 2005. Mol. Phyl. Evol.. 37 132-143.]

The Azorean representatives of the Leptaxini (Pulmonata) are single island endemics, where a high-spired shell distinguishes the monotypic genus Helixena from two slightly different low-spired forms within Leptaxis (azorica and caldeirarum type). We studied the evolutionary history of putative taxa and the three shell-types using 12 allozyme loci and sequences of nuclear (ITS-1 and ITS-2) and mitochondrial DNA (COI and 16S rRNA). While little variation was found in both ITS genes, allozyme and mtDNA divergence was among the highest reported for pulmonate land snails. Generally, phylogeographic patterns are indicative of allopatric differentiation via the successive colonization of (younger) islands, while a major role for adaptive evolution is not supported. The azorica shell-type is monophyletic and has no common history with other sympatric shell-types on the same islands. The (ambiguous) position of Helixena sanctaemariae makes Leptaxis paraphyletic on the Azores and possibly also the caldeirarum shell-type. Helixena can therefore not be distinguished as a separate genus on the Azores. Following a lineage-based concept, representatives on all (ancient) islands should be considered distinct species.

Population genetics and identity of an introduced terrestrial slug: Arion subfuscus s.l. in the north-east USA (Gastropoda, Pulmonata, Arionidae) - Dec 31, 2005 ()
[Pinceel, J., Jordaens, K., Van Houtte, N., Bernon, G. & Backeljau, T. 2005. Genetica. 125(2-3) 155-171.]

Several European species of the terrestrial slug genus Arion have been introduced into North America. A case in point is the species complex A. subfuscus s.l. which has become one of the most abundant slug taxa in North America. In Europe this complex consists of at least two cryptic species, viz. A. fuscus and A. subfuscus s.s., the latter of which is further subdivided in five strongly divergent mtDNA lineages (A. subfuscus S1-S5). In order to determine which of these A. subfsucus s.l. taxa are present in the NE USA and in order to assess their population genetic structure, we compared mtDNA, nDNA and allozyme variation between populations from the NE USA and Europe. Our results show that (1) at least A. subfuscus S1 has become successfully established in the NE USA, (2) founder effects are the most likely explanation for the loss of a large amount of molecular genetic variation in populations from the NE USA (i.e. a loss of 96% of the 16S rDNA haplotypes, 67% of the ITS1 alleles and 46% of the alleles at polymorphic allozyme loci), and (3) part of the remaining genetic variation in NE USA populations was probably due to multiple introductions from the British Isles and the European mainland, and the hybrid structure of most of these source populations. Apparently, the extreme loss of molecular genetic variation in this introduced species has not prevented it from successfully establishing and spreading in novel environments.

Extreme mtDNA divergences in a terrestrial slug (Arionidae, Pulmonata, Gastropoda): accelerated evolution, allopatric divergence and secondary contact - Dec 31, 2005 ()
[Pinceel, J., Jordaens, K., & Backeljau, T. 2005. J. Evol. Biology. 18 1264-1280.]

Extremely high levels of intraspecific mtDNA differences in pulmonate gastropods have been reported repeatedly and several hypotheses to explain them have been postulated. We studied the phylogeny and phylogeography of 51 populations (n = 843) of the highly polymorphic terrestrial slug Arion subfuscus (Draparnaud, 1805) across its native distribution range in Western Europe. By combining the analysis of single stranded conformation polymorphisms (SSCP) and nucleotide sequencing, we obtained individual sequence data for a fragment of the mitochondrial 16S rDNA and a fragment of the nuclear ITS1. Additionally, five polymorphic allozyme loci were scored. Based on the 16S rDNA phylogeny, five monophyletic haplotype groups with sequence divergences of 9-21% were found. Despite this deep mitochondrial divergence, the haplotype groups were not monophyletic for the nuclear ITS1 fragment and haplotype group-specific allozyme alleles were not found.
Although there is evidence for an accelerated mtDNA clock, the divergence among the haplotype groups is older than the Pleistocene and their current allopatric ranges probably reflect allopatric divergence and glacial survival in separate refugia from which different post-glacial colonization routes were established. A range-overlap of two mtDNA groups (S1 and S2, 21% sequence divergence) stretched from Central France and Belgium up to the North of the British Isles. The nuclear data suggest that this secondary contact resulted in hybridization between the allopatrically diverged groups. Therefore, it seems that, at least for two of the groups, the deep mtDNA divergence was only partially accompanied by the formation of reproductive isolation.

Montane tadpoles in Madagascar: molecular identification and description of the larval stages of Mantidactylus elegans, Mantidactylus madecassus, and Boophis laurenti from the Andringitra Massif - Dec 31, 2005 ()
[Thomas, M., L. Raharivololoniaina, F. Glaw, M. Vences & D. R. Vieites 2005. Copeia. 2005 174-183.]

The larval stages of three species of frogs from montane habitats in the Andringitra Massif, southern central Madagascar at 2100–2500 m above sea level, were identified through mitochondrial DNA sequences and are described herein. The tadpoles of Boophis laurenti agree with the previously known tadpoles of the closely related Boophis microtympanum, whereas the tadpoles of Mantidactylus madecassus are similar to those of other species of the subgenus Brygoomantis that occur at lower altitudes. The tadpoles of Mantidactylus elegans are very large (up to 106 mm total length) and show mouthparts largely agreeing with those of species of the subgenus Guibemantis, with a relatively high number of upper labial tooth rows (one continuous and six interrupted). These tadpoles are uniformly blackish on the dorsum, indicating a possible general trend of high frequency of dark color and melanism in montane amphibians. Molecular identification provides a fast and very efficient tool to identify larval stages of amphibians, especially in cases of specialized tadpoles from remote areas in which rearing is difficult.

Combined morphological and molecular analysis of individual nematodes through short-term preservation in formalin - Dec 31, 2005 ()
[Bhadury P, Austen MC, Bilton DT, Lambshead PJD, Rogers AD, Smerdon GR 2005. Molecular Ecology Notes. 5 (4) 965-968.]

Small metazoans such as marine nematodes are increasingly identified using both molecular and morphological techniques. Formalin is the preferred fixative for morphological analysis but specimens become unsuitable for molecular study due to formalin-induced modification of DNA. Nematodes fixed in ethanol work well for molecular studies but become unsuitable for taxonomy due to shrinkage. Here we show for the first time that formalin can be used as a short-term fixative (≤ 7 days) for marine nematodes, allowing both morphological and molecular work to be conducted on the same individual. No sequence ambiguities were detected in polymerase chain reaction (PCR) amplifications of 18S ribosomal DNA (rDNA) following short-term formalin preservation.

Mitochondrial DNA phylogeography of the Mesoamerican spiny-tailed lizards (Ctenosaura quinquecarinata complex): historical biogeography, species status and conservation - Dec 31, 2005 ()
[Hasbun, C.R.; Gómez A.; Kohler, G. and Lunt, D.H. 2005. Molecular Ecology. 14(10) 3095-3107.]

Through the examination of past and present distributions of plants and animals, historical biogeographers have provided many insights on the dynamics of the massive organismal exchange between North and South America. However, relatively few phylogeographic studies have been attempted in the land bridge of Mesoamerica despite its importance to better understand the evolutionary forces influencing this biodiversity 'hotspot'. Here we use mitochondrial DNA sequence data from fresh samples and formalin-fixed museum specimens to investigate the genetic and biogeographic diversity of the threatened Mesoamerican spiny-tailed lizards of the Ctenosaura quinquecarinata complex. Species boundaries and their phylogeographic patterns are examined to better understand their disjunct distribution. Three monophyletic, allopatric lineages are established using mtDNA phylogenetic and nested clade analyses in (i) northern: México, (ii) central: Guatemala, El Salvador and Honduras, and (iii) southern: Nicaragua and Costa Rica. The average sequence divergence observed between lineages varied between 2.0% and 3.7% indicating that they do not represent a very recent split and the patterns of divergence support the recently established nomenclature of C. quinquecarinata, Ctenosaura flavidorsalis and Ctenosaura oaxacana. Considering the geological history of Mesoamerica and the observed phylogeographic patterns of these lizards, major evolutionary episodes of their radiation in Mesoamerica are postulated and are indicative of the regions' geological complexity. The implications of these findings for the historical biogeography, taxonomy and conservation of these lizards are discussed.

New species of Diamesa (Diptera: Chironomidae) from Tibet: conspecific males and females associated with mitochondrial DNA - Dec 31, 2005 ()
[Willassen E. 2005. Zootaxa. 1049 19-32.]

Undescribed females representing four morphological types were found in a collection of adult Diamesa from about 5000 m altitude in Rongbuk, Tibet. Short DNA sequences of cytochrome oxidase subunit 2 were used to associate two single males in the material with conspecific females. Diamesa solhoyi n.sp. and Diamesa aculeata n.sp. are described. The complete type material and additional specimens have been deposited in the Insect Collection at the Institute of Zoology, Academia Sinica, Beijing (IZAS). The sequences are deposited in Genbank with accession numbers AM051227–AM051233.

Diagnostic molecular markers and the genetic relationships among three species of the Cheilosia canicularis group (Diptera: Syrphidae) - Dec 31, 2005 ()
[Milankov, V., Stamenkovic, J., Ludoski, J., Ståhls, G. & A. Vujic 2005. European Journal of Entomology. 102 125-131.]

To re-evaluate the taxonomic status of Cheilosia canicularis (Panzer, 1801), C. himantopus (Panzer, 1798) and C. orthotricha Vujic & Claussen, 1994, variation in the mitochondrial DNA (mtDNA) sequence of the cytochrome c oxidase subunit I (COI) gene and 18 nuclear allozyme genes were surveyed in allopatric and sympatric populations from Serbia and Montenegro. Genetic relationships among five populations of these species from the Fruška Gora (Serbia), Kopaonik (Serbia) and Durmitor (Montenegro) mountains were analyzed. Seven allozyme loci (Aat, Aco, Fum, Idh-1, Idh-2, Mdh-2 and Sdh) were diagnostic for delineating C. orthotricha from the other two species, while only a low, but consistent, genetic differentiation was observed between C. canicularis and C. himantopus. Differentiating all three species was possible based solely on the species-specific alleles at the Est-? locus. Sequence comparisons of 738 bp of the COI gene from eleven specimens was consistent with the variability in nuclear allozymes.
Sequence data revealed variation in 5% of the nucleotide sites among C. orthotricha and the C. canicularis/C. himantopus pair, while less variation (0.68%) was observed within the pair C. canicularis/C. himantopus. However, the presence of one diagnostic allozyme locus and five consistently variable nucleotide sites in sympatric populations of C. canicularis and C. himantopus (Durmitor, Montenegro) suggest that these two species have separate gene pools.

Nematode-specific PCR primers for the 18S small subunit rRNA gene. - Dec 31, 2005 ()
[Floyd RM, Rogers AD, Lambshead PJD, Smith CR 2005. Molecular Ecology Notes. 5 (3) 611-612.]

A set of polymerase chain reaction primers were designed, which amplify a c. 1 kb fragment of the 18S ribosomal DNA gene, and are specific to the phylum Nematoda. These have proven useful in isolating nematode genes from samples mixed with other biological material, particularly with application to DNA barcoding. Optimal reaction conditions are described. These primers have successfully amplified the correct fragment from a wide phylogenetic range of nematodes, and have so far generated no sequences from any other organismal group.

Phylogenetic relationships between members of the crucifer pathogenic Leptosphaeria maculans species complex as shown by mating type (MAT1-2), actin and b-tubulin sequences - Dec 31, 2005 ()
[Voigt, K., Cozijnsen, A.J., Kroymann, J., Pöggeler, S. and Howlett, B.J. 2005. Molecular Phylogenetics and Evolution. 37(2) 541-557.]

The dothideomycetous fungus Leptosphaeria maculans comprises a complex of species differing in specificity and pathogenicity on Brassica napus. Twenty-eight isolates were investigated and compared to 20 other species of the Pleosporales order. Sequences of the mating type MAT1-2 (23), fragments of actin (48) and β-tubulin (45) genes were determined and used for phylogenetic analyses inferred by maximum parsimony, distance, maximum likelihood, and Bayesian approaches. These different approaches using single genes essentially confirmed findings using concatanated sequences. L. maculans formed a monophyletic group separate from Leptosphaeria biglobosa. The L. biglobosa clade encompasses five sub-clades; this finding is consistent with classification made previously on the basis of internal-transcribed sequences of the ribosomal DNA repeat. The propensity for purifying and neutral evolution of the three genes was determined using sliding window analysis, a technique not previously applied to genes of filamentous fungi. For members of the L. maculans species complex, this approach showed that in comparison to actin and β-tubulin, exonic sequences of MAT1-2 were more diverse and appeared to evolve at a faster rate. However, different regions of MAT1-2 displayed different degrees of sequence conservation. The more conserved upstream region (including the High Mobility Group domain) may be better suited for interspecies differentiation, while the more diverse downstream region is more appropriate for intraspecies comparisons.

Strikingly variable divergence times inferred across an Amazonian butterfly 'suture zone' - Dec 07, 2005 ()
[Alaine Whinnett, Marie Zimmermann, Keith R. Willmott, Nimiadina Herrera, Ricardo Mallarino, Fraser Simpson , Mathieu Joron A1, Gerardo Lamas, James Mallet 2005. Proceedings of the Royal Society B: Biological Sciences. Vol. 272, No. 1580 2525-2533.]

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DNA barcoding: error rates based on comprehensive sampling - Dec 01, 2005 (pdf)
[Meyer, C.P., and G. Paulay 2005. PLoS Biology. 3(12) 2229-2238.]

DNA barcoding has attracted attention with promises to aid in species identification and discovery; however, few well-sampled datasets are available to test its performance. We provide the first examination of barcoding performance in a comprehensively sampled, diverse group (cypraeid marine gastropods, or cowries). We utilize previous methods for testing performance and employ a novel phylogenetic approach to calculate intraspecific variation and interspecific divergence. Error rates are estimated for (1) identifying samples against a well-characterized phylogeny, and (2) assisting in species discovery for partially known groups. We find that the lowest overall error for species identification is 4%. In contrast, barcoding performs poorly in incompletely sampled groups. Here, species delineation relies on the use of thresholds, set to differentiate between intraspecific variation and interspecific divergence. Whereas proponents envision a “barcoding gap” between the two, we find substantial overlap, leading to minimal error rates of ~17% in cowries. Moreover, error rates double if only traditionally recognized species are analyzed. Thus, DNA barcoding holds promise for identification in taxonomically well-understood and thoroughly sampled clades. However, the use of thresholds does not bode well for delineating closely related species in taxonomically understudied groups. The promise of barcoding will be realized only if based on solid taxonomic foundations.

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Mitochondrial DNA sequences from dried snake venom: a DNA barcoding approach to the identification of venom samples - Dec 01, 2005 ()
[Pook, C.E.and McEwing, R. 2005. Toxicon. 46(7) 711-715.]

Outdated nomenclature and incorrect taxonomic characterisation of snake venoms in the current toxinological literature have serious implications for the replicability of results from snake venom toxin research. The situation has not improved, despite attempts to supply toxinologists with regular updates on snake systematics. Here, we demonstrate the successful extraction of DNA, and subsequent sequencing of the mitochondrial 12S gene, from dried snake venoms. This approach offers a new and potentially straightforward method for accurate species identification. Mitochondrial DNA (mtDNA) sequences isolated from snake venom can be used to clarify or validate snake species identification through comparison against existing sequences in the GenBank database, and through phylogenetic analyses with other sequences. Pooled venoms can also be screened a priori for the presence of multiple species, and the species names on the labels of commercial venoms verified. Moreover, if the species from which the venom sample has been taken is known, and the specimen is available as a voucher, the mtDNA sequence of the haplotype isolated from that species venom sample could serve as a sequence standard (or ‘DNA barcode’) for that species. Our new method of DNA barcoding venoms ensures the identification of venoms even after future taxonomic changes.

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Are Plant DNA barcodes a search for the Holy Grail? - Nov 14, 2005 ()
[Daniel Rubinoff, Stephen Cameron, and Kipling Will 2005. TRENDS in Ecology and Evolution. Vol. 21, Issue 1, 2006 1-2.]
Phylogenetic Utility of Tektin, a Novel Region for Inferring Systematic Relationships Among Lepidoptera - Nov 01, 2005 ()
[Whinnett, Alaine; Brower, Andrew V. Z.; Lee, Ming-Min; Willmott, Keith R.; Mallet, James 2005. Annals of the Entomological Society of America. Vol. 98, No. 6 873-886.]
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On the application of molecular barcodes in toxinological research. - Nov 01, 2005 ()
[Creer, S. 2005. Toxicon. 46(6) 709-710.]

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Deciphering amphibian diversity through DNA barcoding: chances and challenges. - Oct 29, 2005 ()
[Vences, M., Thomas, M., Bonett, R. M. and Vieites, D. R. 2005. Philosophical Transactions of the Royal Society B: Biological Sciences. 360(1462) 1859-1868.]

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7–14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.

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Applying DNA Barcoding to red macroalgae: a preliminary appraisal holds promise for future applications - Oct 29, 2005 (pdf)
[Saunders, G. 2005. Phil. Trans. R. Soc. B. 360 (1462) 1879 - 1888.]

Notes:  The applicability of DNA barcoding to indentify red macroalgae is discussed in this communication. The barcodes were all resolved accurately, the species identification was unequivocal, and the study raised some further taxonomic questions.

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Microcoding: the second step in DNA barcoding - Oct 29, 2005 ()
[Summerbell, R. C., Levesque, C. A., Seifert, K. A., Bovers, M., Fell, J. W., Diaz, M. R., Boekhout, T., de Hoog, G. S., Stalpers, J. and Crous, P. W. 2005. Philosophical Transactions of the Royal Society B: Biological Sciences. 360(1462) 1897-1903.]

After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide ‘microcodes’ of less than 25bp can be found that allow rapid identification of circa 100-200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytometer. When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs. Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present.

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DNA barcodes for biosecurity: invasive species identification - Oct 29, 2005 ()
[Armstrong, K. F. and Ball, S. L. 2005. Philosophical Transactions of the Royal Society B: Biological Sciences. 360(1462) 1813 - 1823.]

Biosecurity encompasses protecting against any risk through ‘biological harm’, not least being the economic impact from the spread of pest insects. Molecular diagnostic tools provide valuable support for the rapid and accurate identification of morphologically indistinct alien species. However, these tools currently lack standardization. They are not conducive to adaptation by multiple sectors or countries, or to coping with changing pest priorities. The data presented here identifies DNA barcodes as a very promising opportunity to address this. DNA of tussock moth and fruit fly specimens intercepted at the New Zealand border over the last decade were reanalysed using the cox1 sequence barcode approach. Species identifications were compared with the historical dataset obtained by PCR–RFLP of nuclear rDNA. There was 90 and 96% agreement between the methods for these species, respectively. Improvements included previous tussock moth ‘unknowns’ being placed to family, genera or species and further resolution within fruit fly species complexes. The analyses highlight several advantages of DNA barcodes, especially their adaptability and predictive value. This approach is a realistic platform on which to build a much more flexible system, with the potential to be adopted globally for the rapid and accurate identification of invasive alien species.

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DNA barcoding Australia's fish species - Oct 29, 2005 (pdf)
[Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R., and P.D.N. Hebert 2005. Phil. Trans. R. Soc. B. 360 (1462) 1847 - 1857.]

Notes:  This paper discusses the barcoding process of 207 species of Australian fishes showing the cox1 sequence can be used for identification.

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Defining operational taxonomic units using DNA barcode data. - Oct 29, 2005 (pdf)
[Blaxter, M., Mann, J., Chapman, T., Thomas, F., Whitton., Floyd, R., and Eyualem-Abebe 2005. Phil. Trans. R. Soc. B. 360 (1462) 1935 - 1943.]

The scale of diversity of life on this planet is a significant challenge for any scientific programme hoping to produce a complete catalogue, whatever means is used. For DNA barcoding studies, this difficulty is compounded by the realization that any chosen barcode sequence is not the gene ‘for’ speciation and that taxa have evolutionary histories. How are we to disentangle the confounding effects of reticulate population genetic processes? Using the DNA barcode data from meiofaunal surveys, here we discuss the benefits of treating the taxa defined by barcodes without reference to their correspondence to ‘species’, and suggest that using this non-idealist approach facilitates access to taxon groups that are not accessible to other methods of enumeration and classification. Major issues remain, in particular the methodologies for taxon discrimination in DNA barcode data.

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DNA barcoding for effective biodiversity assessment of a hyperdiverse arthopod group: the ants of Madagascar. - Oct 29, 2005 ()
[Smith, M.A., Fisher, B.L., P.D.N. Hebert 2005. Phil. Trans. R. Soc. B. 360 (1462) 1825 - 1834.]

The role of DNA barcoding as a tool to accelerate the inventory and analysis of diversity for hyperdiverse arthropods is tested using ants in Madagascar. We demonstrate how DNA barcoding helps address the failure of current inventory methods to rapidly respond to pressing biodiversity needs, specifically in the assessment of richness and turnover across landscapes with hyperdiverse taxa. In a comparison of inventories at four localities in northern Madagascar, patterns of richness were not significantly different when richness was determined using morphological taxonomy (morphospecies) or sequence divergence thresholds (Molecular Operational Taxonomic Unit(s); MOTU). However, sequence-based methods tended to yield greater richness and significantly lower indices of similarity than morphological taxonomy. MOTU determined using our molecular technique were a remarkably local phenomenon—indicative of highly restricted dispersal and/or long-term isolation. In cases where molecular and morphological methods differed in their assignment of individuals to categories, themorphological estimate was always more conservative than the molecular estimate. In those cases where morphospecies descriptions collapsed distinct molecular groups, sequence divergences of 16% (on average) were contained within the same morphospecies. Such high divergences highlight taxa for further detailed genetic, morphological, life history, and behavioral studies.

Read the publication here.

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Critical Factors for assembling a high volume of DNA barcodes - Oct 29, 2005 (pdf)
[Hajibabaei, M., deWaard, J.R., Ivanova, N.V., Ratnasingham, S., Dooh, R.T., Kirk, S.L., Mackie, P.M., and P.D.N. Hebert 2005. Phil. Trans. R. Soc. B. 360 (1462) 1959 - 1967.]

Notes:  The key steps in establishing high volume DNA barcoding facilities are discussed in this communication. Strong emphasis is placed in this article on developing alliances with the taxonomic community in order to provision specimens. The current difficulties of analysing museum collections are also discussed.

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A likelihood ratio test for species membership based on DNA sequence data. - Oct 29, 2005 ()
[Matz, M. V. and Nielsen, R. 2005. Philosophical Transactions of the Royal Society B: Biological Sciences. 360(1462) 1969-1974.]

DNA barcoding as an approach for species identification is rapidly increasing in popularity. However, it remains unclear which statistical procedures should accompany the technique to provide a measure of uncertainty. Here we describe a likelihood ratio test which can be used to test if a sampled sequence is a member of an a priori specified species. We investigate the performance of the test using coalescence simulations, as well as using the real data from butterflies and frogs representing two kinds of challenge for DNA barcoding: extremely low and extremely high levels of sequence variability.

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Reverse taxonomy: an approach towards determining the diversity of meiobenthic organisms based on ribosomal RNA signature sequences. - Oct 29, 2005 (pdf)
[Markmann, M. and D. Holtz 2005. Phil. Trans. R. Soc. B. 360 (1462) 1917 - 1924.]

Notes:  The benthic fauna represent a broad and diverse fauna that is an important part of the nutrient cycle, and this study uses DNA barcode techniques to identify a large number of species. As many of these species have been reached with out morphological classification the authors propose a 'reverse taxonomy'.

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Land plants and DNA barcodes: short-term and long-term goals. - Oct 29, 2005 (pdf)
[Chase, M.W., Salamin, N., Wilkinson, M., Dunwell, J.M., Kesanakurth, R.P., Haidar, N. and V. Savolainen 2005. Phil. Trans. R. Soc. B. 360 (1462) 1889 - 1895.]

 

Notes:  This study discusses the methods which should be developed in both the short-term and long-term in order to bring plant DNA barcoding up to speed with other organisms.

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An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. - Oct 29, 2005 (pdf)
[De Ley, P., De Ley, I.T., Morris, K., Abebe, E., Mundo-Ocampo, M., Yoder, M., Heras, J., Waumann, D., Rocha-Olivares, Burr, A.H.J., Baldwin, J.G., and K.W. Thomas. 2005. Phil. Trans. R. Soc. B. 360 (1462) 1945 - 1958.]

 

Notes:  The focus of this communication is the integration of video capture and editing microscopy with DNA sequence analysis as an intermediary technology to bridge the gap created by the difficulties of matching DNA barcodes to existing morphological data in nematodes.

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DNA-based species delineation in tropical beetles using mitochondrial and nuclear markersneation in tropical beetles using mitochondrial and nuclear markers. - Oct 29, 2005 (pdf)
[Monaghan, M.T., Balke, M., Gregory, T.R., and Vogler, A.P. 2005. Phil. Trans. R. Soc. B. 360 (1462) 1925 - 1933.]

Notes:  This study uses DNA sequences to delimit putative species within groups that lack an existing taxonomic framework. The focus of the work is on tropical water beetles and shows that even small fragments of mitochondrial DNA can portray largely accurate species boundaries within poorly classified groups.

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Towards writing the encyclopaedia of life: an introduction to DNA barcoding - Oct 29, 2005 (pdf)
[Savolainen, V., Cowan, R.S., Vogler, A.P., Roderick, G.K. and R. Lane 2005. Phil. Trans. R. Soc. B. 360 (1462) 1805 - 1811.]

Notes:  This paper provides an overview of the Barcode of Life initiative.

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Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding. - Oct 29, 2005 (pdf)
[Janzen, D. H., Hajibabaei, M., Burns, J. M., Hallwachs, W., Remigio, E. & Hebert, P. D. 2005. Philosophical Transactions of the Royal Society B: Biological Sciences. 360(1462) 1835-1845.]

By facilitating bioliteracy, DNA barcoding has the potential to improve the way the world relates to wild biodiversity. Here we describe the early stages of the use of cox1 barcoding to supplement and strengthen the taxonomic platform underpinning the inventory of thousands of sympatric species of caterpillars in tropical dry forest, cloud forest and rain forest in northwestern Costa Rica. The results show that barcoding a biologically complex biota unambiguously distinguishes among 97% of more than 1000 species of reared Lepidoptera. Those few species whose barcodes overlap are closely related and not confused with other species. Barcoding also has revealed a substantial number of cryptic species among morphologically defined species, associated sexes, and reinforced identification of species that are difficult to distinguish morphologically. For barcoding to achieve its full potential, (i) ability to rapidly and cheaply barcode older museum specimens is urgent, (ii) museums need to address the opportunity and responsibility for housing large numbers of barcode voucher specimens, (iii) substantial resources need be mustered to support the taxonomic side of the partnership with barcoding, and (iv) hand-held field-friendly barcorder must emerge as a mutualism with the taxasphere and the barcoding initiative, in a manner such that its use generates a resource base for the taxonomic process as well as a tool for the user.

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The unholy trinity: taxonomy, species delimitation, and DNA barcoding. - Oct 29, 2005 (pdf)
[De Salle, R., Egan, M.G. and M. Siddall 2005. Phil. Trans. R. Soc. B. 360 (1462) 1905 - 1916.]

Notes:  This paper discuss two main concerns about the direction of the global DNA barcoding initiative. 

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The problems and promise of DNA barcodes for species diagnosis of primate biomaterial. - Oct 29, 2005 (pdf)
[Lorenz, J.G., Jackson, W.E., Beck, C.J., and R. Hanner 2005. Phil. Trans. R. Soc. B. 360 (1462) 1869 - 1877.]

Notes:  This study discusses the implications and benefits that establishing a DNA barcode library for primates would have, especially for conservation and law enforcement.

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TaxI: a software tool for DNA barcoding using distance methods - Oct 29, 2005 ()
[Steinke, D., Vences, M., Salzburger, W. and A. Meyer 2005. Phil. Trans. R. Soc. B. 360 (1462) 1975 - 1980.]

DNA barcoding is a promising approach to the diagnosis of biological diversity in which DNA sequences serve as the primary key for information retrieval. Most existing software for evolutionary analysis of DNA sequences was designed for phylogenetic analyses and, hence, those algorithms do not offer appropriate solutions for the rapid, but precise analyses needed for DNA barcoding, and are also unable to process the often large comparative datasets. We developed a flexible software tool for DNA taxonomy, named TaxI. This program calculates sequence divergences between a query sequence (taxon to be barcoded) and each sequence of a dataset of reference sequences defined by the user. Because the analysis is based on separate pairwise alignments this software is also able to work with sequences characterized by multiple insertions and deletions that are difficult to align in large sequence sets (i.e. thousands of sequences) by multiple alignment algorithms because of computational restrictions. Here, we demonstrate the utility of this approach with two datasets of fish larvae and juveniles from Lake Constance and juvenile land snails under different models of sequence evolution. Sets of ribosomal 16S rRNA sequences, characterized by multiple indels, performed as good as or better than cox1 sequence sets in assigning sequences to species, demonstrating the suitability of rRNA genes for DNA barcoding. 

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Association of insect life stages using DNA sequences: the larvae of Philodytes umbrinus (Motschulsky) (Coleoptera: Dytiscidae) - Oct 01, 2005 ()
[Miller, K.B., Y. Alarie, G. W. Wolfe,and M.F. Whiting 2005. Systematic Entomology. 30(4) 499–509.]

Insect life stages are known imperfectly in many cases, and classifications are based often on only one or a few semaphoronts of a species. This is unfortunate as information in alternative life stages often is useful for scientific study. Although recent examples of DNA in taxonomy have emphasized the identification of indistinguishable species, such sequence data facilitate the association of life history stages and hold considerable promise in phylogenetic analysis, evolutionary studies, diagnostics, etc. These concepts are discussed here and an example is provided from diving beetles (Dytiscidae: Coleoptera). Three unknown larval specimens of an apparent species of Laccophilinae collected in Namibia were associated with the species Philodytes umbrinus (Motschulsky) using DNA sequence data. An 806-bp portion of the gene cytochrome oxidase I was sequenced from the unknown larvae. Several identified adult specimens of species of Laccophilinae from Namibia were also sequenced, including two P. umbrinus specimens and specimens from four Laccophilus Leach species. Additional species of Laccophilus from other areas of the world also were sequenced, as were specimens of Agabetes acuductus (Harris), Australphilus saltus Watts, Neptosternus boukali Hendrich & Balke and a species of Laccodytes Régimbart. Parsimony analysis resulted in two most parsimonious trees with the unknown larva unambiguously resolved in a group with both adult specimens of P. umbrinus (bootstrap value = 100%). The average pairwise p-distance between the unknown larva and adult P. umbrinus specimens averaged 0.09% (0–0.14%), compared with an average divergence between other conspecifics in the analysis of 0.24% (0–0.82%) and an overall average divergence between species of 13.49% (1.90–19.86%). Based on this, the unknown larvae were assigned to P. umbrinus. The larvae are diagnosed and described and their relationship with other Laccophilinae is discussed.

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The perils of DNA barcoding and the need for integrative taxonomy. - Oct 01, 2005 ()
[Will, K.W., Mishler, B.D., and Wheeler, Q.D. 2005. Systematic Biology. 54(5) 844 - 851.]
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DNA barcoding: perspectives from a Partnerships for Enhancing Expertise in Taxonomy (PEET) debate - Oct 01, 2005 ()
[Smith, V. S. 2005. Systematic Biology. 54(5) 841-4.]

What's in a name? Lepidoptera : Hesperiidae : Pyrginae : Telemiades Hubner 1819 Pyrdalus Mabille 1903 : New combinations Telemiades corbulo (Stoll) and Telemiades oiclus (Mabille)-and more - Oct 01, 2005 ()
[Burns, J. M., and Janzen, D. H. 2005. Proceedings of the Entomological Society of Washington. 107(4) 770-781.]

Both the Central American skipper butterfly Achlyodes oiclus Mabille and the South American Pyrdalus corbulo (Stoll) belong in Telemiades. Pyrdalus becomes a junior synonym of Telemiades. Pyrdalus corbulo cora Evans, which is really a species (not a subspecies), is a new synonym of Telemiades oiclus, new combination. Though differing sharply in wingshape and color pattern, T. oiclus and Telemiades corbulo, new combination, share a distinctive male secondary sex character and are, in both sexes, genitalically similar to each other and to T. nicomedes (Möschler). Grown caterpillars of T. oiclus and T. nicomedes resemble each other (and suggest slugs). DNA barcoding further supports the relationship of these species. With its brown-forewing/brown-and-yellow-hindwing adult color pattern, T. oiclus superficially resembles 13 other species of skippers reared in the Area de Conservación Guanacaste (ACG) of northwestern Costa Rica. Of these presumably mimetic species, one is raised from reduction to subspecific rank, and two are raised from synonymy, to gain reinstated status: T. gallius (Mabille), T. chrysorrhoea (Godman and Salvin), and Eracon lachesis (Dyar). The pupa of T. oiclus shares distinctive features with the pupae of other species of Telemiades. All eight species of Telemiades reared in the ACG feed only on leaves of plants in the family Fabaceae. Six eat various species of Inga and, in a relatively few cases, species in three other mimosoid genera, whereas T. oiclus and T. nicomedes each use two species in one papilionoid genus—Dioclea and Machaerium, respectively.

More Taxonomy, not DNA barcoding - Oct 01, 2005 ()
[Ebach, M.C., and C. Holdrege 2005. BioScience. 55(10) 822-823.]
The Promise of DNA Barcoding for Taxonomy - Oct 01, 2005 (pdf)
[Hebert, P.D.N. and T.R. Gregory 2005. Syst. Biol.. 54(5) 852-859.]

Notes:  The authors address a series of questions and clarify the rationale and potential impacts of DNA barcoding.

 

 

 

 

 

 

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Nomenclature and Nomenklatura. Taxonomy, Taxinomy or Systematics - Oct 01, 2005 ()
[Jolivet, P. 2005. Nouvelle Revue d'Entomologie. 22(4) 355-360.]

Zoological and botanical nomenclature, formerly codified by Linnaeus, 250 years ago, are actually threatened by new proposals from various specialists influenced by cladistics and molecular biology. Several people even seriously proposed a barcode which has been tested mostly in ornithology and lepidopterology. Feasibility of such extravaganza seems questionable and it remains difficult to sort out the species,as food items in a supermarket, based only on DNA structure. Certain biologists, as Dan Janzen, were enthusiastic about those new possibilities, but it seems difficult to codify the part of DNA to be selected. To redescribe 1.700.000 Linnean species seems also a challenge and finally no one among those innovators has really seriously threatened the old classical system. The actual codes, as they are written, show discrepancies and often allow some flexibility in their interpretation.

Barcoding generalist predators by polymerase chain reaction: carabids and spiders - Sep 14, 2005 ()
[M.H. Greenstone, D.L. Rowley, U. Heimbach, J.G. Lundgren, R.S. Pfannenstiel, S.A. Rehner 2005. Molecular Ecology. 14 3247-3266.]

An oligonucleotide barcode for species identification on Trichoderma and Hypocrea - Sep 09, 2005 ()
[Druzhinina, I., Kopchinskiy, A. G., Komon, M., Bissett, J., Szakacs, G., Kubicek, C.P. 2005. Fungal Genetics and Biology. 42(10) 813-828.]

One of the biggest obstructions to studies on Trichoderma has been the incorrect and confused application of species names to isolates used in industry, biocontrol of plant pathogens and ecological surveys, thereby making the comparison of results questionable. Here we provide a convenient, on-line method for the quick molecular identification of Hypocrea/Trichoderma at the genus and species levels based on an oligonucleotide barcode: a diagnostic combination of several oligonucleotides (hallmarks) specifically allocated within the internal transcribed spacer 1 and 2 (ITS1 and 2) sequences of the rRNA repeat. The barcode was developed on the basis of 979 sequences of 88 vouchered species which displayed in total 135 ITS1 and 2 alleles. Oligonucleotide sequences which are constant in all known ITS1 and 2 of Hypocrea/Trichoderma but different in closely related fungal genera, were used to define genus-specific hallmarks. The library of species-, clade- and genus-specific hallmarks is stored in the MySQL database and integrated in the TrichOKey v. 1.0 - barcode sequence identification program with the web interface located on www.isth.info. TrichOKey v. 1.0 identiWes 75 single species, 5 species pairs and 1 species triplet. Verification of the DNA-barcode was done by a blind test on 53 unknown isolates of Trichoderma, collected in Central and South America. The obtained results were in a total agreement with phylogenetic identification based on tef1 (large intron), NCBI BLAST of vouchered records and postum morphological analysis. We conclude that oligonucleotide barcode is a powerful tool for the routine identification of Hypocrea/Trichoderma species and should be useful as a complement to traditional methods.

Biological identifications of mayflies (Ephemeroptera) using DNA barcodes - Sep 01, 2005 (pdf)
[Ball, S.L., Hebert, P.D.N., Burian, S.K., and J.M. Webb. 2005. J. N. Am. Benthol. Soc.. 24(3) 508-524.]

Notes:  This study tests the efficacy of DNA barcoding in the identification of mayflies.

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Demography of coleopterists and their thoughts on DNA barcoding and the phylocode, with commentary. - Sep 01, 2005 ()
[Lobl, I., and R. A. B. Leschen. 2005. Coleopterists Bulletin. 59(3) 284-292.]

Much has been stated in journals and at roundtable discussions, conferences, and on the Internet about the taxonomic impediment, the Phylocode, and DNA barcoding. But community responses about these topics are lacking apart from position papers. We developed a simple easy-to-answer survey regarding these issues and submitted it to contributors to the Catalogue of Palaearctic Coleoptera. We also asked for demographic information (age, sex), and data about their productivity. The survey was sent to 154 expert coleopterists, mainly in Europe, and we obtained 103 responses. Over 60% of the respondents have PhD's, over 60% are professionals, and over 70% are currently active. Although most of the respondents are traditional taxonomists, 28% participate in molecular research and 78% regard DNA work as potentially useful. Nevertheless, 35% of the respondents regard DNA barcoding as useless (23% consider it useful and 39% have no opinion) and 50% regard the Phylocode also useless (40% have no opinion and 8% find it useful). Based on survey results, a large portion of practicing beetle taxonomists are not influenced by bar-coding and phylocode initiatives, either by ignorance or by having strong opposition. The adoption of new character systems, like molecules, is favored and demonstrates a real interest and concern by taxonomists to find answers to meaningful biological problems.

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The inferential basis of species hypotheses: the solution to defining the term 'species' - Sep 01, 2005 ()
[Fitzhugh, Kirk 2005. Marine Ecology. 26 (3,4) 155-165.]

A formal definition of the term 'species' is presented that is logically consistent with the inferential structures that lead to other taxonomic categories in biological systematics. A species name denotes an explanatory hypothesis that accounts for specified intrinsic or relational properties of organisms that can be accounted for by way of past tokogenetic, i.e., reproductive events. The inference of species hypotheses involves a form of non-deductive reasoning known as abduction. Hypotheses in other taxonomic categories, such as 'semaphoronts' (sensuW. Hennig, Phylogenetic Systematics, University of Illinois Press, Urbana, 1966), genera, families, etc., exhibit the same abductive form. The differences between semaphoront, species, and supraspecific hypotheses is in the causal theories applied to infer each - these theories being ontogenetic, tokogenetic, and phylogenetic, respectively. By formally defining the term species as an explanatory hypothesis accounting for properties of organisms by way of tokogeny, there are several distinct consequences: (1) species cannot have the ontological status of individuals, and it would be inaccurate to reduce them to class constructs; (2) acknowledging species as representative of explanatory hypotheses leaves available a variety of tokogenetic theories, such as asexual, sexual, and metagenetic reproductive processes to account for relevant organismal properties; (3) removal of species hypotheses from consideration, as in the 'least inclusive taxonomic unit' concept of Pleijel & Rouse [Proceedings of the Royal Society of London, Series B267 (2000) 627; Zoologica Scripta29 (2000) 157] is nihilistic; and (4) advocating the reduction of inferences of species hypotheses to one class of characters, e.g., 'DNA barcoding,' would be irrational.

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Losing the plot: DNA "barcodes" and taxonomy - Sep 01, 2005 ()
[Wheeler, Quentin D. 2005. Cladistics. 21(4) 405-407.]

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Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marinae - Aug 31, 2005 ()
[Derycke, S., Rémerie, T., Vierstraete, A., Backeljau, T., Vanfleteren, J. Vinckx, M. & Moens, T. 2005. Marine Ecology Progress Series. 300 91-103.]

An inverse correlation between dispersal ability and genetic differentiation among populations of a species is frequently observed in the marine environment. We investigated the population genetic structure of the free-living marine nematode Pellioditis marina. A total of 426 bp of the mitochondrial cytochrome oxidase subunit 1 (COI) gene were surveyed on a geographical scale of approximately 100 km during spring 2003. Nematodes were collected from 2 coastal locations in Belgium, and from 2 estuaries and a saltwater lake (Lake Grevelingen) in The Netherlands. Molecular variation-was assessed with the single-strand conformation polymorphism (SSCP) method. In total, 32 different haplotypes were observed, and sequence divergence among 452 individuals ranged from 0.2 to 10.6%. We discovered 4 distinct mitochondrial lineages, with low divergences within the lineages (0.2 to 1.6%) and high divergences between the lineages (5.1 to 10.6%). The nuclear ribosomal ITS (internal transcribed spacer) region showed concordant phylogenetic patterns, suggesting that nematode species diversity may be considerably underestimated.

DNA-BAR: distinguisher selection for DNA barcoding - Aug 15, 2005 ()
[DasGupta, B., Konwar, K. M., Mandoiu, II and Shvartsman, A. A 2005. Bioinformatics. 21(16) 3424-3426.]

Summary: DNA-BAR is a software package for selecting DNA probes (henceforth referred to as distinguishers) that can be used in genomic-based identification of microorganisms. Given the genomic sequences of the microorganisms, DNA-BAR finds a near-minimum number of distinguishers yielding a distinct hybridization pattern for each microorganism. Selected distinguishers satisfy user specified bounds on length, melting temperature and GC content, as well as redundancy and cross-hybridization constraints.

Availability: DNA-BAR can be used online through the web interface provided at http://dna.engr.uconn.edu/~software/DNA-BAR/. The open source C code, released under the GNU General Public License, is also available at the above address.

Contact:ion@engr.uconn.edu

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An oligonucleotide microarray for the identification and differentiation of trichothecene producing and non-producing Fusarium species occurring on cereal grain - Jul 31, 2005 ()
[Nicolaisen, M., Justesen, A.F., Thrane, U., Skouboe, P. Holmstrøm, K. 2005. Journal of Microbiological Methods. 62(1) 57-69.]

Cereal grain may be infected with a number of Fusarium species some of which are producers of highly toxic compounds such as the trichothecenes. Correct identification of these species is essential for risk assessment of cereal grain for human or animal consumption. Most of the available methods for identification are either time consuming or aimed at only one or a few target species. Microarray technology offers parallel analysis of a high number of DNA targets. In this study 57 capture oligonucleotides (CO) were designed based upon Fusarium ITS2 rDNA sequences, and used for microarray production. From this array COs could be selected that were able to hybridise specifically to labelled PCR products from the ITS region of Fusarium graminearum/Fusarium culmorum, Fusarium pseudograminearum, Fusarium poae, Fusarium sporotrichioides, Fusarium equiseti, Fusarium langsethiae and Fusarium tricinctum/Fusarium avenaceum. A few COs showed some cross hybridisation to non-target species. In a preliminary experiment it was shown that this cross hybridisation could be eliminated by increasing hybridisation stringency. The array could be used to detect individual Fusarium species in mixed samples and in environmental samples. This study demonstrates the feasibility of oligonucleotide microarrays for parallel detection of a number of Fusarium species.

Approaching the taxonomic affiliation of unidentified sequences in public databases – an example from the mycorrhizal fungi - Jul 18, 2005 ()
[Nilsson, R.H., Kristiansson, E., Ryberg, M., Larsson, K.-H. 2005. BMC Bioinformatics. 6 178.]

BACKGROUND: During the last few years, DNA sequence analysis has become one of the primary means of taxonomic identification of species, particularly so for species that are minute or otherwise lack distinct, readily obtainable morphological characters. Although the number of sequences available for comparison in public databases such as GenBank increases exponentially, only a minuscule fraction of all organisms have been sequenced, leaving taxon sampling a momentous problem for sequence-based taxonomic identification. When querying GenBank with a set of unidentified sequences, a considerable proportion typically lack fully identified matches, forming an ever-mounting pile of sequences that the researcher will have to monitor manually in the hope that new, clarifying sequences have been submitted by other researchers. To alleviate these concerns, a project to automatically monitor select unidentified sequences in GenBank for taxonomic progress through repeated local BLAST searches was initiated. Mycorrhizal fungi--a field where species identification often is prohibitively complex--and the much used ITS locus were chosen as test bed. RESULTS: A Perl script package called emerencia is presented. On a regular basis, it downloads select sequences from GenBank, separates the identified sequences from those insufficiently identified, and performs BLAST searches between these two datasets, storing all results in an SQL database. On the accompanying web-service http://emerencia.math.chalmers.se, users can monitor the taxonomic progress of insufficiently identified sequences over time, either through active searches or by signing up for e-mail notification upon disclosure of better matches. Other search categories, such as listing all insufficiently identified sequences (and their present best fully identified matches) publication-wise, are also available. DISCUSSION: The ever-increasing use of DNA sequences for identification purposes largely falls back on the assumption that public sequence databases contain a thorough sampling of taxonomically well-annotated sequences. Taxonomy, held by some to be an old-fashioned trade, has accordingly never been more important. emerencia does not automate the taxonomic process, but it does allow researchers to focus their efforts elsewhere than countless manual BLAST runs and arduous sieving of BLAST hit lists. The emerencia system is available on an open source basis for local installation with any organism and gene group as targets.

Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts - Jul 11, 2005 (pdf)
[Gregory D.D. Hurst and Francis M. Jiggins 2005. Proceedings of the Royal Society B. 272(1572) 1525-1534.]
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Use of DNA barcodes to identify flowering plants - Jun 07, 2005 (pdf)
[Kress, W.J., Wurdack, K.J., Zimmer, E.A., Weigt, L.A., and D.H. Janzen 2005. PNAS. 102 23 8369-8374.]

Notes:  This study proposes the plastid trnH-psbA intragenic spacers as the usable DNA marker for the identification of flowering plants. A number of trials have been done on diverse plants species with good results.

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Molecular and morphometric data pinpoint species boundaries in Halimeda section Rhipsalis (Bryopsidales, Chlorophyta) - Jun 01, 2005 ()
[Verbruggen, H., O. De Clerck, W. Kooistra, and E. Coppejans 2005. Journal of Phycology. 41(3) 606-621.]

Molecular systematic studies have changed the face of algal taxonomy. Particularly at the species level, molecular phylogenetic research has revealed the inaccuracy of morphology-based taxonomy: Cryptic and pseudo-cryptic species were shown to exist within many morphologically conceived species. This study focused on section Rhipsalis of the green algal genus Halimeda. This section was known to contain cryptic diversity and to comprise species with overlapping morphological boundaries. In the present study, species diversity within the section and identity of individual specimens were assessed using ITS1–5.8S–ITS2 (nrDNA) and rps3 (cpDNA) sequence data. The sequences grouped in a number of clear-cut genotypic clusters that were considered species. The same specimens were subjected to morphometric analysis of external morphological and anatomical structures. Morphological differences between the genotypic cluster species were assessed using discriminant analysis. It was shown that significant morphological differences exist between genetically delineated species and that allocation of specimens to species on the basis of morphometric variables is nearly perfect. Anatomical characters yielded better results than external morphological characters. Two approaches were offered to allow future morphological identifications: a probabilistic approach based on classification functions of discriminant analyses and the classical approach of an identification key.

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Reply to the comment by L Prendini on 'Identifying spiders through DNA barcodes' - May 20, 2005 (pdf)
[Hebert, P.D.N. and R.D.H. Barrett 2005. Can. J. Zool. 83 505-506.]

Note:  This communication is a response to claims that DNA barcoding is not scalable enough to cover all eukaryotic life. It is also makes reference to the possible automation of systems in the near future to further progress species discovery and indentification.


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DNA barcoding a useful tool for taxonomists - May 05, 2005 (pdf)
[Schindel, D. E. and Miller, S. E 2005. Nature. 435 17.]
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Bending to bar codes - May 01, 2005 ()
[Beardsley, S. 2005. Scientific American. 292(5) 26,28.]

DNA-barcoding evidence for widespread introductions of a leech from the South AmericanHelobdella triserialis complex - May 01, 2005 ()
[Siddall, Mark E. and Budinoff, Rebecca B. 2005. Conservation Genetics. 6(3) 467-472.]

Morphological examination and a molecular phylogenetic analysis of leeches from Australia, New Zealand, South Africa and Hawaii show that these specimens are members of a species in the South American Helobdella triserialis species complex. Though it has been seen before, this leech was not recognized as an invasive species. Rather, it was first described as Helobdella striata from Germany later renamed Helobdella europaea and then independently described as Helobdella papillornata from Australia. Because the appropriate name for this leech from its South American endemic locale, Helobdella (triserialis) lineata, is preoccupied by a North American species, we formally recognize H. europaea as the valid taxon name. Although this invader is not a bloodfeeder it may be expected to have an impact on native annelid and mollusk faunas.

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DNA barcoding does not compete with taxonomy - Apr 28, 2005 (pdf)
[Gregory, T. R. 2005. Nature. 434 1067.]

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Denaturing Gradient Gel Electrophoresis (DGGE) as a tool for the identification of marine nematodes - Apr 28, 2005 ()
[Cook, A.A., Bhadury, P., Debenham, N.J., Meldal, B.H.M., Blaxter, M.L., Smerdon, G.R., Austen, M.C., Lambshead, P.J.D. and Rogers, A.J. 2005. Mar. Ecol. Prog. Ser. 291 103-113.]

Many phyla of marine invertebrates are difficult to identify using conventional morphological taxonomy. Larvae of a wider set of phyla are also difficult to identify as a result of conservation of morphology between species or because morphological characters are destroyed during sampling and preservation. DNA sequence analysis has the potential for identification of marine organisms to the species level. However, sequence analysis of specimens is time-consuming and impractical when species diversity is very high and densities of individuals huge, as they are in many marine habitats. The effectiveness of the 18S rRNA gene sequences for identification of one species-rich marine group, the Nematoda, is analysed. Following identification of variable regions of the 18S rRNA gene, primers were designed to amplify a small segment of sequences suitable for denaturing gradient gel electrophoresis (DGGE). The effectiveness of DGGE for identifying individual species is analysed. DGGE analysis of natural communities of nematodes detected less than 2/3 of the species present. This fraction of the community probably represents the abundant species in the original samples. It is concluded that DGGE is not a useful tool for analysis of species richness in marine communities as it fails to detect rare species of which there are usually many in the marine benthic environment. However, DGGE may be a useful method for detecting changes in communities that influence the abundance of the most common taxa.

DNA Barcoding is no substitute for taxonomy - Apr 07, 2005 (pdf)
[Malte C. Ebach and Craig Holdrege 2005. Nature. 697.]

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What can biological barcoding do for marine biology? - Mar 30, 2005 (pdf)
[Schander, C. and E. Willassen 2005. Mar. Biol. Res. 1 79-83.]

The idea of using nucleotide sequences as barcodes for species identification has stirred up debates in the community oftaxonomists and systematists. We argue that barcodes are potentially extremely useful tools for taxonomy for severalreasons. Barcodes may, for example, help to identify cryptic and polymorphic species and give means to associate life historystages of unknown identity. Barcode systems would thus be particularly helpful in cases when morphology is ambiguous oruninformative and would provide tools for higher taxonomic resolution of disparate life forms. Comparative analysis ofshort DNA sequences may also represent heuristic access cards to a deeper understanding of evolutionary relationshipsbetween organisms. However, barcodes are the ‘‘essence’’ of species identities no more than taxonomic holotypes are ‘‘thespecies’’. It makes no sense to think that morphology and other biological information about organisms can be madeobsolete by barcode systems. The biological significance of matching or diverging nucleotide sequences will still have to bethe subject of taxonomic decisions that must be open for scrutiny. It is imperative, therefore, that barcodes are associatedwith specimen vouchers.

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Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians. - Mar 16, 2005 ()
[Vences, M., Thomas, M., van der Meijden, A., Chiari, Y. & Vieites, D. R. 2005. Frontiers in Zoology. 2 5.]

Background

Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals.

Results

Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%), with low degrees of pairwise haplotype divergence within populations (0–1%).

Conclusion

We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem

Comments on 'Identifying spiders through DNA barcodes' - Mar 01, 2005 ()
[Prendini, Lorenzo 2005. Canadian Journal of Zoology. 83(3) 498-504(7).]

R.D.H. Barrett and P.D.N Hebert have demonstrated that it is possible to identify members of a mostly local spider fauna using a short fragment of the mitochondrial gene coding for cytochrome c oxidase I. There are instances where DNA-based identification may be very useful, e.g., in identifying juvenile life stages of groups in which adults are required for morphological identification, or matching morphologically different sexes or life stages when those associations are unknown. DNA-based identification may be the easiest and most cost-effective way, or even the only feasible way, to address some of these questions. However, these are also the least challenging problems in taxonomy, and their solution is unlikely to relieve the "taxonomic impediment". Furthermore, to promote the utility of DNA barcoding as a global identification system, these authors must demonstrate that their approach works for distinguishing all the members of a speciose clade, wherever in the world they occur. Much of diversity occurs allopatrically and neither the study by R.D.H. Barrett and P.D.N. Hebert, nor any other presented to date, even begins to address the feasibility of DNA-based identification at this level of detail.

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Taxonomy. Will DNA bar codes breath life into classification? - Feb 18, 2005 ()
[Marshall, E. 2005. Science. 307(5712) 1037.]

Is a Large-Scale DNA-Based Inventory of Ancient Life Possible? - Feb 01, 2005 (pdf)
[Lambert, D.M., A. Baker, L. Huynen, O. Haddrath, P.D.N. Hebert and C.D. Millar 2005. J. Hered.. 96(3) 1-6.]

Notes:  The authors use a number of genes to recover the phylogeny of extinct ratite birds from New Zealand. Results of this study indicate that DNA barcoding may be able to detect other extinct animal species and that an inventory of ancient life is possible.
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Morphological, chemical and molecular differentiation of Fusarium equiseti isolated from Norwegian cereals. - Jan 31, 2005 ()
[Kosiak, E.B., Holst-Jensen, A., Rundberget, T., Gonzales, M.T.J. & Torp, M. 2005. Int. J. Food Microbiol.. 99(2) 195-206.]

AIMS: To develop a DNA microarray for easy and fast detection of trichothecene- and  oniliformin-producing Fusarium species.

METHOD AND RESULTS: A DNA microarray was developed for detection and identification of 14 trichothecene- and moniliformin-producing species of the fungal genus Fusarium. The array could also differentiate between four species groups. Capture probes were designed based on recent phylogenetic analyses of translation elongation factor-1 alpha (TEF-1α) sequences. Particular emphasis was put on designing capture probes corresponding to groups or species with particular mycotoxigenic synthetic abilities. A consensus PCR amplification of a part of the TEF-1α is followed by hybridization to the Fusarium chip and the results are visualized by a colorimetric Silverquant detection method. We validated the Fusarium chip against five naturally infected cereal samples for which we also have morphological and chemical data. The limit of detection was estimated to be less than 16 copies of genomic DNA in spiked commercial wheat flour.

CONCLUSIONS: The current Fusarium chip proved to be a highly sensitive and fast microarray for detection and identification of Fusarium species. We postulate that the method also has potential for (semi-)quantification.

SIGNIFICANCE AND IMPACT OF THE STUDY: The Fusarium chip may prove to be a very valuable tool for screening of cereal samples in the food and feed production chain, and may facilitate detection of new or introduced Fusarium spp.

Organismer med strekkoder – artsbestemming med DNA-sekvenser - Jan 01, 2005 ()
[Torbjørn Ekrem og Bjarte H. Jordal 2005. Naturen nr.. 4 154-160.]

Tenk deg at du er på tur i skogen, kanskje med en skoleklasse på slep, og finner en planteeller et dyr som du ikke har sett før. Du bryter av et fragment og putter dette i denmedbrakte artsanalysatoren. I løpet av få minutter er arten bestemt og du har trådløstilgang til et artsleksikon der arten er utførlig beskrevet med karakteristiske kjennetegn,utbredelse og biologi. Futuristisk drømmetekning? Ja, kanskje det, men denne fremtiden kan være nærmere enn vi er klar over. I denne artikkelen vil vi ta for oss hva DNAstrekkodinger, hvordan det kan brukes for å identifisere arter, og hvilke utfordringerdenne metoden står overfor.

DNA sequence variation at the mitochondrial cytochrome oxidase I subunit among pheromotypes of the sibling taxa Diachrysia chrysitis and D. tutti (Lepidoptera: Noctuidae) - Jan 01, 2005 ()
[Hille, A., Miller, M. A. and Erlacher, S. 2005. Zoologica Scripta. 34(1) 49-56.]

We surveyed variation in the mtDNA cytochrome oxidase subunit I (COI) gene in the noctuid sibling species Diachrysia chrysitis and D. tutti, whose taxonomic status has been queried. Taxonomically, these taxa are separated on the basis of wing pattern and time of flight period. Samples were field-collected from different geographical sites where pheromone traps were baited to attract males containing different mixtures of two blends of pheromone components: (Z)-5-decenyl acetate and (Z)-7-decenyl acetate. Most specimens were sequenced over a 709-bp segment of the COI gene. Single specimens each of D. chrysitis and D. tutti were sequenced over a region of 1.5 kilobases. mtDNA variation within and among D. chrysitis and D. tutti is most simply interpreted as DNA polymorphism within a complex of closely related, but well-differentiated pheromotypes. Maximal nucleotide difference per site among haplotypes was 0.28%, which is at the lower end of the range for interspecific mtDNA nucleotide diversity in Lepidoptera. Coefficient of differentiation Gst was c. 76.3% ± 11.7%, a typical value at the intraspecific level. Sequences revealed stable diagnostic differences between pheromotypes irrespective of geographical origin. Identification of pheromone-trapped males based on morphology remained vague and uncorrelated to mtDNA haplotypes. The survey illustrated the potential utility of direct DNA sequencing in assessing lineage structures or taxon limits among moths that have been previously found to be different using the pheromone mate recognition system, but which have not been subjected to DNA analysis. The results of mtDNA analyses presented here support recognition of chrysitis and tutti lineages as presented in previous allozyme studies.

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Molecular studies of nematode diversity; past, present and future - Dec 31, 2004 ()
[Cook AA, Bhadury P, Debenham NJ, Meldal BHM, Blaxter ML, Smerdon GR, Austen MC, Rogers AD, Lambshead PJD 2004. Cook, R. & Hunt, D.J. (Eds). Proceedings of the Fourth International Congress of Nematology, 8 - 13 June 2002, Tenerife, Spain. Nematology Monographs and Perspectives Vol. 2, Brill Academic Publishing. .]
Kohninia linnaeicola - a new genus and species of the Sclerotinaceae pathogenic to Linnea borealis L. - Dec 31, 2004 ()
[Holst-Jensen, A., Vrålstad, T. & Schumacher, T. 2004. Mycologia. 96(1) 136-143.]

A new genus and species is described to accommodate a newly discovered fungus pathogenic to Linnaea borealis. The fungus forms true sclerotia on stems and leaves of its host and apothecia arise singly or gregariously on the surface of ripe sclerotia. The new fungus was collected together with a stromatic conidiomal fungus that occurred on the same host. A putative teleomorph-anamorph connection of the observed taxa was ruled out by sequence comparison of the nuclear ribosomal internal transcribed spacer DNA sequences (ITS rDNA). Based on morphology and pathogenicity, the new fungus belongs in the family Sclerotiniaceae, Helotiales, Ascomycota. A phylogenetic analysis of ITS rDNA sequences from 26 taxa of the family Sclerotiniaceae was performed to conclude on the systematic position of the new fungus. The small tuberoid sclerotia, brownish subsessile to substipitate apothecia, four-spored asci, ellipsoid to isthmoid ascospores, inability to grow on PDA culture media and a number of ITS rDNA sequence autapomorphies characterize and distinguish the fungus from other taxa of the Sclerotiniaceae.

DNA taxonomy of a neglected animal phylum: an unexpected diversity of tardigrades - Dec 31, 2004 ()
[Blaxter, M., Elsworth, B., & J. Daub 2004. Proc Biol Sci. 271 Suppl 4 S189-92.]

A molecular survey technique was used to investigate the diversity of terrestrial tardigrades from three sites within Scotland. Ribosomal small subunit sequence was used to classify specimens into molecular operational taxonomic units (MOTU). Most MOTU were identified to the generic level using digital voucher photography. Thirty-two MOTU were defined, a surprising abundance given that the documented British fauna is 68 species. Some tardigrade MOTU were shared between the two rural collection sites, but no MOTU were found in both urban and rural sites, which conflicts with models of ubiquity of meiofaunal taxa. The patterns of relatedness of MOTU were particularly intriguing, with some forming clades with low levels of divergence, suggestive of taxon flocks. Some morphological taxa contained well-separated MOTU, perhaps indicating the existence of cryptic taxa. DNA sequence-based MOTU proved to be a revealing method for meiofaunal diversity studies.

On reliability. (Letter) - Dec 31, 2004 ()
[Holst-Jensen, A., Vrålstad, T. & Schumacher, T. 2004. New Phytologist. 161 11-13.]
Molecular phylogeny and biogeography of the genus Pseudomma (Peracarida: Mysida) - Dec 31, 2004 ()
[Meland, K. and E. Willassen 2004. Journal of Crustacean Biology. 24 541-557.]

We used DNA sequences from 18S rDNA (808 bp) and COI mtDNA (599 bp) to infer evolutionary history of northern groups of the deep-sea mysid genus Pseudomma. The V4-V7 regions of 18S show an average of 1.31 % sequence divergence between species. A secondary structure model is constructed and used in phylogenetic analyses to allow for different evolutionary rates in paired and unpaired nucleotide partitions. COI is observed as highly variable with uncorrected p-distance averaging 33%. Phylogenies for these sequences were estimated by maximum-likelihood, Bayesian, and maximum-parsimony analyses. More or less similar tree topologies were obtained for each gene with these methods. Pseudomma longisquamosum was placed in a basal clade, using Parapseudomma and Aniblyops as outgroups, but the exact relationship of other basal taxa is less clear when results from the two genes are compared. An ancient presence of Pseudomma in the Tethys Sea is suggested by phylogenetic structure, molecular clock considerations, and present distributions. A well-supported Atlantic clade may have diverged from Indo-Pacific groups in the Miocene because of the closure of the Gibraltar Strait. More recent speciation events are proposed in the Norwegian Sea, and an Arctic intrusion from the North Pacific across the Bering Strait is suggested for the circumpolar species Pseudomma truncatum.

Classical and molecular approaches as a powerful tool for the characterization of rumen polycentric fungi - Dec 31, 2004 ()
[Fliegerova, K., Hodrova, B., & K. Voigt 2004. Folia Microbiologica. 49(2) 157-164.]

Ribosomal ITS1 and ITS2 fragments from 8 isolates of polycentric rumen anaerobic fungi were PCR-amplified and sequenced; the sequences obtained were aligned with published data and phylogenetic analyses were performed. Analysis of the ITS1 fragment clearly differentiated between the two polycentric genera Orpinomyces and Anaeromyces and this classification is supported by morphological observation. A multi-order phylogram based on ITS2 sequences proved that anaerobic rumen fungi are separated from aerobic chytrids, which form a well-supported monophylum with the highest possible bootstrap proportion values of 100%. Sequence analysis of ITS regions is a powerful tool for classification of anaerobic fungi but morphological description of strains is still necessary because some genera of rumen fungi display a high genetic heterogeneity.

Hidden Floridian biodiversity: mitochondrial and nuclear gene trees reveal four cryptic species within the scorched mussel, Brachidontes exustus, species complex - Nov 01, 2004 ()
[T. Lee, D. O Foighil 2004. Molecular Ecology. 13(11) 3527-42.]

Ten Species in One: DNA Barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerater - Oct 12, 2004 ()
[Paul D.N. Hebert, Erin H. Penton, John M. Burns, Daniel H. Janzen, and Winnie Hallwachs 2004. Preceedings of the National Academy of Sciences. Vol. 101 No. 41 12-17.]
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DNA Barcoding: Promise & Pitfalls - Oct 01, 2004 (pdf)
[Craig Moritz, Carla Cicero 2004. PLoS Biol. 1529-1531.]

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Identification of Birds through DNA Barcodes - Sep 28, 2004 (pdf)
[Hebert, P.D.N, Stoekle, M., Zemlak, T., and C.M. Francis 2004. PLoS Biology. 2 1657-1668.]

 

Notes:  This work tested the effectiveness of the COI barcode in distinguishing between bird species. 260 species of North american birds were tested, and found that 4 may prove to be new species.

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The Expansion of Conservation Genetics - Aug 31, 2004 ()
[Rob DeSalle, George Amato 2004. Nature Reviews Genetics. 5 702-712.]

Differences in straggling rates between two genera of dove lice (Insect: Phthiraptera) reinforce population genetic and cophylogenetic patterns - Jun 01, 2004 (pdf)
[Whiteman, N.K., D. Santiago-Alarcon, K.P. Johnson and P.G. Parker 2004. Int. J. Parasitol. 34 1113-1119.]

Notes:  This study incorporates a DNA barcoding approach to investigate varying powers of dispersal among different genera of dove lice. Cophylogenetic patterns implicate differential dispersal abilities as a contributing factor in the disparate evolutionary patterns observed between these genera.

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Identifying spiders through DNA barcodes - May 20, 2004 (pdf)
[Barret, D.H. and P.D.N. Hebert 2004. Can. J. Zool. 83 481-491.]

Notes:  This study shows the potential for DNA barcoding to rapidly identify spiders, a group which has suffered previously from difficulties in species identification. All of the 204 species of arachnids tested were correctly assigned to their species, showing the efficacy of barcoding.

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Nematode molecular diagnostics: from bands to barcodes - May 14, 2004 ()
[Powers, T. 2004. Annual Review Phytopathol. 42 367-383.]

Nematodes are considered among the most difficult animals to identify. DNA-based diagnostic methods have already gained acceptance in applications ranging from quarantine determinations to assessments of biodiversity. Researchers are currently in an information-gathering mode, with intensive efforts applied to accumulating nucleotide sequence of 18S and 28S ribosomal genes, internally transcribed spacer regions, and mitochondrial genes. Important linkages with collateral data such as digitized images, video clips and specimen voucher web pages are being established on GenBank and NemATOL, the nematode-specific Tree of Life database. The growing DNA taxonomy of nematodes has lead to their use in testing specific short sequences of DNA as a "barcode" for the identification of all nematode species.

Biological identification of springtails (Collembola: Hexapoda) from the Canadian Arctic, using mitochondrial DNA barcodes - May 01, 2004 (pdf)
[Hogg, I.D., and P.D.N. Hebert 2004. Can. J. Zool. 82 749-754.]

Notes:  This study examines Arctic collembolans from 19 species across 13 genera.

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Taxonomic triage and the poverty of phylogeny - Apr 29, 2004 ()
[Quentin D. Wheeler 2004. Philosophical Transactions of the Royal Society. Vol. 359, No. 1444 571-583.]
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The promise of a DNA taxonomy - Apr 01, 2004 (pdf)
[Blaxter, M.L. 2004. Phil. Trans. R. Soc. Lond. B. 359(1444) 669-679.]

Not only is the number of described species a very small proportion of the estimated extant number of taxa, but it also appears that all concepts of the extent and boundaries of 'species' fail in many cases. Using conserved molecular sequences it is possible to define and diagnose molecular operational taxonomic units (MOTU) that have a similar extent to traditional 'species'. Use of a MOTU system not only allows the rapid and effective identification of most taxa, including those not encountered before, but also allows investigation of the evolution of patterns of diversity. A MOTU approach is not without problems, particularly in the area of deciding what level of molecular difference defines a biologically relevant taxon, but has many benefits. Molecular data are extremely well suited to re-analysis and meta-analysis, and data from multiple independent studies can be readily collated and investigated by using new parameters and assumptions. Previous molecular taxonomic efforts have focused narrowly. Advances in high-throughput sequencing methodologies, however, place the idea of a universal, multi-locus molecular barcoding system in the realm of the possible.

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Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification - Feb 01, 2004 ()
[Kipling W. Will and Daniel Rubinoff 2004. Cladistics. Vol. 20, Issue 1 47-55.]
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Molecular systematics: Counting angels with DNA - Dec 31, 2003 ()
[Blaxter, M. 2003. Nature. 421 (6919) 122-4.]

It is impossible to describe biological diversity with traditional approaches. Molecular methods are the way forward — especially, perhaps, in the form of DNA barcodes.

Heterobasidiomycetes form symbiotic associations with hepatics: Jungermanniales have a sebacinoid mycobionts while Aneura pinguis (Metzgeriales) is associated with a Tulasnella species - Dec 31, 2003 ()
[Kottke, I., Beiter, A., Weiss, M., Haug, I., Oberwinkler, F. and Nebel, M. 2003. Mycological Research. 107(8) 957-968.]

In order to evaluate substrate dependence of the symbiotic fungal associations in leafy liverworts (Jungermanniopsida), 28 species out of 12 families were investigated by transmission electron microscopy and molecular methods. Samples were obtained from the diverse substrates: from naked soil, from the forest floor on needle litter, from between peat moss, from rotten bark of standing trees, and from stumps and rotten wood. Associations with ascomycetes were found in most of the specimens independent from the substrate. Seven species sampled from soil were found to contain basidiomycete hyphae. Ultrastructure consistently showed dolipores with imperforate parenthesomes. Molecular phylogenetic studies revealed that three specimens belonging to the Jungermanniales were associated with members of Sebacinaceae, while Aneura pinguis (Metzgeriales) was associated with a Tulasnella species. These taxa are so far the only basidiomycetes known to be symbiotically associated with leafy liverworts. The probability that the associations with Sebacinaceae are evolutionary old, but the Tulasnella associations more derived is discussed. The sebacinoid mycobionts form a similar interaction type with the jungermannialian leafy liverworts as do the associated ascomycetes. The term ‘jungermannioid mycorrhiza’ is proposed for this distinctive symbiotic interaction type.

Oligonucleotide primers for the universal amplification of beta-tubulin genes facilitate phylogenetic analyses in the regnum Fungi - Dec 31, 2003 ()
[Einax, E. and Voigt, K. 2003. Organisms, Diversity and Evolution. 3 (3) 185-194.]

Among genes coding for proteins with basic structural functions in all eukaryotes, the highly conserved and functionally essential gene for beta-tubulin is receiving increasing attention in the reconstruction of phylogenies within a broad organismic range. We therefore constructed a set of twelve universally applicable primers that allow reliable amplification of beta-tubulin genes among all major eukaryotic kingdoms including fungi (Fungi), animals (Animalia) and green plants (Planta). For primer design, the amino acid sequences of 35 beta-tubulin genes from Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Animalia, Oophyta and Planta were aligned and used for the definition of four well-conserved regions. These are suitable priming sites in PCR amplification experiments. Out of these amino acid regions twelve primers were designed, which initiate especially the amplification of fungal beta-tubulin genes. In four pair-wise primer applications gene fragments of up to 1,500 bp in size could be isolated, which comprise nearly complete beta-tubulin genes from twelve representative species of the Fungi. The sequences of 7 beta-tubulin fragments were obtained from Allomyces moniliformis, A. neomoniliformis, Blastocladiella britannica, Chytridium confervae, Mortierella isabellina and Trametes versicolor. Reliable amplification of beta-tubulin over a broad spectrum of organisms provides a strong basis for the establishment of both deep level phylogenies and studies of complex species groups based on beta-tubulin gene trees.

Molecular taxonomics for biodiversity surveys: already a reality - Dec 31, 2003 ()
[Blaxter, M. L. and R. Floyd 2003. Trends in Ecology and Evolution. 18(6) 268-269.]

The biosphere is so extraordinarily diverse that methodical cataloguing of biodiversity by traditional methods might never provide a complete ‘species list’ for the planet [1]. This realization, coupled with real controversy over both how to define and how to diagnose ‘species’ [2], has led to proposals for a molecular taxonomy based on a defined part of the genome [3, 4 and 5]. We would like to emphasize, from our practical experience [6], some of the features of a molecular taxon system that we hope will dispel the fears of more morphologically inclined taxonomists.

Taxonomic and nomenclatural clarification of the onion neck rotting Botrytis species - Dec 31, 2003 ()
[Yohalem, D.S., Nielsen, K. and Nicolaisen, M. 2003. Mycotaxon. 85 175-182.]

Five species of Botrytis are recognized as being associated with neck rot of onions: three of them, B. aclada, B. byssoidea and B. allii, exclusively. Due to the difficulty of distinguishing them by morphological criteriaand lack of type material associated with B. aclada and B. allii, several synonomies have been proposed. We suggest that B. aclada and B. allii are both valid names. Species may be differentiated by conidial size, but the character is subtle, variable and there is some overlap. Both the smallest spored group (B. aclada) and the largest spored group (B. byssoidea) have 16 mitotic chromosomes, while the intermediate group (B. allii) has 32. Based on significant differences in Nei's coefficient of genetic differentiation derived from universally primed PCR (UP-PCR) fingerprints it was possible to recognize distinctions among the three exclusively neck rot-associated Botrytis spp. and B. cinerea and B. squamosa. Primers, designed from a sequence characterized UP-PCR fragment were used for direct sequencing of DNA from isolates of the 16 chromosome nomengroups. Because of apparent ambiguities in the UP-PCR fragment from the 32-chromosome group, it was cloned and re-sequenced. Sequence alignment and unrooted clustering show identity with the small-spored B. aclada and the large-spored B. byssoidea for the two cloned DNA fragments from the intermediate, B. allii. Further, the internally transcribed spacer rDNA (ITS) amplicons of B. aclada have 2 Sphl restriction sites; those of B. allii and B. byssoidea have 1 Sphl site. The cumulative data suggest that the three groups are genetically distinct and that isolates of B. aclada and B. byssoidea were the ancestors of the polyploid B. allii.

DNA barcoding of parasites and invertebrate disease vectors: what you don't know can hurt you - Dec 01, 2003 (pdf)
[Besansky, N.J., D.W. Severson and M.T. Ferdig 2003. Trends Parasitol. 19(12) 545-546.]

Notes:  This paper considers the implications of developing a DNA barcoding system for parasites.

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Studies on New Guinea moths. 2. Description of a new species of Xenothictis Meyrick (Lepidoptera: Tortricidae: Archipini) - Oct 01, 2003 (pdf)
[Brown, J.W., S.E. Miller and M. Horak 2003. Proc. Entomol. Soc. Wash. 105(4) 1043-1050.]

 

Notes: The first species description to include a DNA barcode.

Testing the utility of partial COI sequences for phylogenetic estimates of gastropod relationships - Oct 01, 2003 (pdf)
[Remigio, E.A. and P.D.N. Hebert 2003. Mol. Phylogenet. Evol. 29 641-647.]

Notes:  This study shows the utility of COI-5' sequence data in recovering deep taxonomic assignments in the largest class of molluscs.

 

 

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How to conserve wild plants? Give the world the power to read them. - Oct 01, 2003 (pdf)
[Janzen, D.H. (forward) 2003. Plant Conservation: A Natural History Approach (in press). Edited by G. Krupnick and J. Kress. .]

Notes:  The potential for barcoding to futher plant conservation is outlined in this book forward.

Now is the time - Sep 26, 2003 (pdf)
[Janzen, D.H. 2003. Philos. T. Roy. Soc. B. .]

Notes:  This commentary is a call to speed 'bioliteracy' through DNA-based taxonomic identification.

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The Molecularisation of Taxonomy - Sep 22, 2003 ()
[Michael S.Y. Lee 2003. Invertebrate Systematics. 18, 2004 1-6.]
Genetic diversity in potato field populations of Thanatephorus cucumeris AG-3, revealed by ITS polymorphism and RAPD markers - Jul 31, 2003 ()
[Justesen, A.F., Bay, A., Yohalem, D.S. and Nicolaisen, M. 2003. Mycological Research. 107 1322-1331.]

DNA sequence analysis of the internal transcribed spacer region 1 (ITS1) and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability in relation to agronomic and regional factors among 60 isolates of Thanatephorus cucumeris (anamorph Rhizoctonia solani) collected from lesions on potato stems or sclerotia of potato tubers. Based on comparative sequence analysis it was shown that all isolates belonged to anastomosis group 3 subgroup Potato Type (AG-3 PT). ITS1 sequence polymorphisms were found within 45 of the 60 isolates showing that different types of the ITS-region are present in individual isolates. Cloning and sequence analysis of the ITS1 region from three selected isolates with sequence polymorphism showed that two different ITS1-types were present in each isolate. RAPD analysis identified 51 RAPD-phenotypes among the 60 investigated isolates indicating a high level of diversity within the subgroup AG-3 PT. Putative clonal isolates with identical RAPD- and ITS1-types were identified within fields, and in one case the same phenotype was found in two different fields separated by several hundred kilometers. Population subdivision analysis based on phenotypic as well as genotypic diversities showed differentiation among populations from different fields when isolates were sampled from tubers, indicating restricted gene flow among soil populations. Low differentiation was seen among field populations sampled from stems, indicating that gene flow is taking place. The population structure was not influenced by the previous crop in the rotation nor by the two cultivars ‘Sava’ and ‘Bintje’.

Biological sample collection and processing for molecular epidemiological studies - Jun 01, 2003 ()
[Holland, N. T., Smith, M. T. , Eskenazi, B., and M. Bastaki 2003. Mutation Research. 543(3) 217-234.]

Molecular epidemiology uses biomarkers and advanced technology to refine the investigation of the relationship between environmental exposures and diseases in humans. It requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples, and minimizing future research costs by use of banked samples. Many factors, such as tissue type, time of collection, containers used, preservatives and other additives, transport means and length of transit time, affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for cytogenetic, immunological and biochemical analyses. Given the multiple uses of the samples in molecular epidemiology studies, appropriate informed consent must be obtained from the study subjects prior to sample collection. Use of barcoding and electronic databases allow more efficient management of large sample banks. Development of standard operating procedures and quality control plans is a safeguard of the samples' quality and of the validity of the analyses results. Finally, specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples, as well as communication of study results.Here, we focus on the factors affecting the quality and the potential future use of biological samples and some of the provisions that must be made during collection, processing, and storage of samples, based on our experience in the Superfund Basic Research Program and Children's Environmental Health Center, at the University of California, Berkeley.

The blind leading the blind: cryptic subterranean species and DNA taxonomy - Jun 01, 2003 ()
[Graham Proudlove, Paul J. Wood 2003. Trends in Ecology and Evolution. 18 (6) 272-3.]

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www.DNA-surveillance: applied molecular taxonomy for species conservation and discovery - May 31, 2003 ()
[C. Scott Baker, Merel L. Dalebout, Shane Lavery, Howard A. Ross 2003. Trends in Ecology and Evolution. 18 (6) 271-2.]

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Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species - May 13, 2003 (pdf)
[Hebert, P.D.N., S. Ratnasingham and J.R. deWaard. 2003. Proc. R. Soc. Lond. B. 270 suppliment 03BL0066 1-4.]

Notes:  Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species.

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Comparison of biological, molecular and morphological methods of species identification in a set of cultured Panagrolaimus isolates - Mar 01, 2003 ()
[Eyualem, A. and M. Blaxter 2003. Journal of Nematology. 35(1) 119-128.]

We have developed a molecular barcode system that uses the small subunit ribosomal RNA (SSU) sequence to define molecular operational taxonomic units (MOTU) of soil nematodes. Here we attempt to differentiate five cultured isolates of a taxonomically difficult genus, Panagrolaimus, using morphological, molecular, and biological (breeding) criteria. The results indicated that the five culture populations belonged to two reproductively isolated species. The available morphological criteria, including scanning electron microscopy (SEM), were insufficient to differentiate among them, and all five could be classified as one morphospecies. Within-culture variation of the morphometrical data did not discern between the two biological species. Sequence data clearly separated the populations into two groups that supported the breeding results. Given this study represented only five populations of one genus, we suggest a congruence of MOTU analysis with the biological species concept. This multifaceted approach is promising for future identification of nematodes as it is simple, comparable, and transferable.

Shortcuts in systematics? A commentary on DNA-based taxonomy - Feb 01, 2003 ()
[Ole Seberg, Chris J. Humphries, Sandy Knapp, Dennis Wm. Stevenson, Gitte Peterson, Nikolaj Scharff and Nils Moller Anderson 2003. TRENDS in Ecology and Evolution. Vol. 18, No. 2 63-65.]
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Taxonomy: Renaissance or Tower of Babel? - Feb 01, 2003 ()
[Mallet J. and Willmott K. 2003. Trends in Ecology and Evolution. 18(2) 57-59.]

Taxonomy, the science of naming and classifying organisms, is the original bioinformatics and a fundamental basis for all biology. Yet over the past few decades, teaching and funding of taxonomy has declined. Last year, taxonomy suddenly became fashionable again, and revolutionary approaches to taxonomy using DNA and Internet technology are now being contemplated. For examples, see the article by Tautz et al. in this issue of TREE, and a separate paper by Hebert et al. in Proc. R. Soc. Lond. Ser B. The new excitement about taxonomy is driven partly by advances in technology, and partly by newly perceived needs given the biodiversity crisis. To reform and build on what taxonomists have already accomplished, the biology community must now begin to seek consensus, and avoid fragmenting into vociferous subdisciplines with multiple, competing aims.

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The intellectual content of taxonomy: A comment on DNA taxonomy. - Feb 01, 2003 ()
[Lipscomb D., Platnick N., and Wheeler Q. 2003. Trends in Ecology & Evolution. 18(2) 65-66.]

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Biological identifications through DNA barcodes - Jan 08, 2003 (pdf)
[Hebert, P.D.N., A. Cywinska, S.L. Ball and J.R. deWaard 2003. Proc. R. Soc. Lond. B. 270 313-322.]

Notes:  This work establishes COI as a primary barcoding tool in the development of a global bioidentification system.

 

 

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Speciation in ancient cryptic species complexes: evidence from the molecular phylogeny of Brachionus plicatilis (rotifera) - Dec 31, 2002 ()
[Gómez A.; Serra, M.; Carvalho, G.R.; Lunt, D.H. 2002. Evolution. 56(7) 1431-1444.]

Continental lake-dwelling zooplanktonic organisms have long been considered cosmopolitan species with little geographic variation in spite of the isolation of their habitats. Evidence of morphological cohesiveness and high dispersal capabilities support this interpretation. However, this view has been challenged recently as many such species have been shown either to comprise cryptic species complexes or to exhibit marked population genetic differentiation and strong phylogeographic structuring at a regional scale. Here we investigate the molecular phylogeny of the cosmopolitan passively dispersing rotifer Brachionus plicatilis (Rotifera: Monogononta) species complex using nucleotide sequence variation from both nuclear (ribosomal internal transcribed spacer 1, ITS1) and mitochondrial (cytochrome c oxidase subunit I, COI) genes. Analysis of rotifer resting eggs from 27 salt lakes in the Iberian Peninsula plus lakes from four continents revealed nine genetically divergent lineages. The high level of sequence divergence, absence of hybridization, and extensive sympatry observed support the specific status of these lineages. Sequence divergence estimates indicate that the B. plicatilis complex began diversifying many millions of years ago, yet has showed relatively high levels of morphological stasis. We discuss these results in relation to the ecology and genetics of aquatic invertebrates possessing dispersive resting propagules and address the apparent contradiction between zooplanktonic population structure and their morphological stasis.

DNA points the way ahead in taxonomy - Aug 01, 2002 ()
[Tautz, D., Arctander, P., Minelli, A., Thomas, R. H. and Vogler, A. P. 2002. Nature. 418 479.]

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Phylogenetic and diagnostic analyses on microscopic fungi: molecular strategies for strain typing, barcoding and systematics of large fungal groups - Jul 31, 2002 ()
[Voigt, K. 2002. Habilitation thesis, Friedrich-Schiller-Universität Jena. .]

Molecular barcodes for soil nematode identification. - Apr 01, 2002 (pdf)
[Floyd, R., Abebe, E., Papert, A. and Blaxter, M. 2002. Molecular Ecology. 11(4) 839-850.]

Using a molecular barcode, derived from single-specimen polymerase chain reaction (PCR) and sequencing of the 5' segment of the small subunit ribosomal RNA (SSU) gene, we have developed a molecular operational taxonomic unit (MOTU) scheme for soil nematodes. Individual specimens were considered to belong to the same MOTU when the sequenced segment of 450 bases was > 99.5% identical. A Scottish upland Agrostis-Festuca grassland soil was sampled, using both culture-based and random selection methods. One hundred and sixty-six cultured isolates were sequenced, and clustered into five MOTU. From 74 randomly sampled individuals across the study site, 19 MOTU were defined. A subsequent sample of 18 individuals from a single subplot contained eight MOTU, four of which were unique to the single subplot sample. Interestingly, seven of these MOTU were not present in the culture-independent sampling. Overall, a total of 23 MOTU were defined from only 240 sequences. Many MOTU could readily be assigned to classical, morphologically defined taxonomic units using a database of SSU sequences from named nematode species. The MOTU technique allows a rapid assessment of nematode taxon diversity in soils. Correlation with a database of sequences from known species offers a route to application of the technique in ecological surveys addressing biological as well as genetic diversity.

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Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-1alpha genes - Dec 31, 2001 ()
[Voigt, K. and Wöstemeyer, J. 2001. Gene. 270(1-2) 113-120.]

True fungi (Eumycota) are heterotrophic eukaryotic microorganisms encompassing ascomycetes, basidiomycetes, chytridiomycetes and zygomycetes. The natural systematics of the latter group, Zygomycota, are very poorly understood due to the lack of distinguishing morphological characters. We have determined sequences for the nuclear-encoded genes actin (act) from 82 zygomycetes representing all 54 currently recognized genera from the two zygomycetous orders Mucorales and Mortierellales. We also determined sequences for translation elongation factor EF-1alpha (tef) from 16 zygomycetes (total of  96,837 bp). Phylogenetic analysis in the context of available sequence data (total 2,062 nucleotide positions per species) revealed that current classification schemes for the mucoralean fungi are highly unnatural at the family and, to a large extent, at the genus level. The data clearly indicate a deep, ancient and distinct dichotomy of the orders Mucorales and Mortierellales, which are recognized only in some zygomycete systems. Yet at the same time the data show that two genera — Umbelopsis and Micromucor — previously placed within the Mortierellales on the basis of their weakly developed columella (a morphological structure of the sporangiophore well-developed within all Mucorales) are in fact members of the Mucorales. Phylogenetic analyses of the encoded amino acid sequences in the context of homologues from eukaryotes and archaebacterial outgroups indicate that the Eumycota studied here are a natural group but provide little or no support for the monophyly of either zygomycetes, ascomycetes or basidiomycetes. The data clearly indicate that a complete revision of zygomycete natural systematics is necessary.

Phylogeny and PCR identification of clinically important zygomycetes based on nuclear ribosomal-DNA sequence data - Dec 31, 1999 ()
[Voigt, K., Cigelnik, E. and O´Donnell, K. 1999. Journal of Clinical Microbiology. 37 3957-3964.]

A molecular database for all clinically important Zygomycetes was constructed from nucleotide sequences from the nuclear small-subunit (18S) ribosomal DNA and domains D1 and D2 of the nuclear large-subunit (28S) ribosomal DNA. Parsimony analysis of the aligned 18S and 28S DNA sequences was used to investigate phylogenetic relationships among 42 isolates representing species of Zygomycetes reported to cause infections in humans and other animals, together with commonly cultured contaminants, with emphasis on members of the Mucorales. The molecular phylogeny provided strong support for the monophyly of the Mucorales, exclusive of Echinosporangium transversale and Mortierella spp., which are currently misclassified within the Mucorales. Micromucor ramannianus, traditionally classified within Mortierella, and Syncephalastrum racemosum represent the basal divergences within the Mucorales. Based on the 18S gene tree topology, Absidia corymbifera and Rhizomucor variabilis appear to be misplaced taxonomically. A. corymbifera is strongly supported as a sister group of the Rhizomucor miehei-Rhizomucor pusillus clade, while R. variabilis is nested within Mucor. The aligned 28S sequences were used to design 13 taxon-specific PCR primer pairs for those taxa most commonly implicated in infections. All of the primers specifically amplified DNA of the size predicted based on the DNA sequence data from the target taxa; however, they did not cross-react with phylogenetically related species. These primers have the potential to be used in a PCR assay for the rapid and accurate identification of the etiological agents of mucormycoses and entomophthoromycoses.

Mitochondrial DNA sequence, morphology and ecology yield contrasting conservation implications for two threatened buckmoths (Hemileuca: Saturniidae) - In Press ()
[Daniel Rubinoff, Felix A.H. Sperling 1999. Biological Conservation. Vol. 118 341-451.]
An Oligonucleotide Microarray Based Assay for Identification of Phytoplasma 16S Ribosomal Groups - In Press ()
[Nicolaisen, M. and Bertaccini, A. 1999. Plant Pathology. 56 332-336.]

A method using consensus PCR followed by oligonucleotide microarray hybridization was developed for identification of phytoplasma 16Sr ribosomal groups. The array consisted of 21– to 33-nt-long oligonucleotides which were designed to hybridize to individual 16Sr groups. Two oligonucleotides were designed to detect all phytoplasma groups. The array could efficiently identify samples from 16SrI, 16SrII, 16SrIII, 16SrV, 16SrVI, 16SrVII, 16SrIX, 16SrX and 16SrXII ribosomal groups. This microarray-based test represents a rapid method for detection of phytoplasmas in unknown samples and for identification of most 16Sr groups.

Confirmation of the species status of the blackfly Simulium galeratum in Britain using molecular taxonomy - In Press ()
[Day, J. C., Goodall, T. I., and R. J. Post 1999. Medical and Veterinary Entomology. 22(1) 55-61.]

Since 1920 Simulium reptans (Linnaeus) (Diptera: Simuliidae) has been reported as exhibiting two different larval morphotypes, a typical S. reptans and an atypical S. reptans var. galeratum, which differ in the markings of the larval head capsule. Inconsistent variation in adults and no apparent variation in the pupae have led taxonomists to conclude that these types in Britain are a single species. We investigated populations in Britain where either the typical form or var. galeratum is found, and one population where the two exist sympatrically. A phylogenetic study based upon a region of the mitochondrial cytochrome c oxidase 1 gene (DNA barcoding) produced a tree that delineated the morphotypes into two distinct monophyletic clades. The average Kimura-2-parameter distances within each clade (i.e. within each morphotype) were very low (0.67% and 0.78%), with the distances between morphotypes being 9-10-fold greater (mean 7.06%). This is concordant with differences within and between species in other taxa; based upon the strict correlation between the molecular variation and the morphotypes, we propose the re-instatement of S. galeratum to species status.

Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea - In Press ()
[Hijri, M., Redecker, D., MacDonald-Comber Petetot, J.A., Voigt, K., Wöstemeyer, J. and Sanders, I.R. 1999. Applied and Environmental Microbiology. 68(9) 4567-4573.]

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.

The power and perils of 'molecular taxonomy': a case study of eyeless and endangered Cicurina (Araneae: Dictynidae) from Texas caves - In Press ()
[P. Paquin and M. Hedin 1999. Molecular Ecology. Volume 13, No. 10 3239-3255.]
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Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water - In Press ()
[L.J. Aridgides, M.A. Doblin, T. Berke, F.C. Dobbs, D.O. Matson, L.A. Drake 1999. Marine Pollution Bulletin. 48 (2004) 1096-1101.]

An Ultrasensitive method for detection of single crab larvae (sesarma reticulatum) by PCR amplification of a highly repetitive DNA sequence - In Press ()
[A.L. Bilodeau, W.S. Landford, T.J. Kim, D.L. Felder and J. E. Neigel 1999. Molecular Ecology. 8 (4) 681-684.]
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