Publications
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.).
[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.
[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.
[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.
[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.
[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.
[Eaton, MJ; Meyers, GL; Kolokotronis, S; Leslie, MS; Martin, AP; Amato, G 2009. Conservation Genetics. .]
[Van Velzen, R., Larsen, T.B., & Bakker, F.T. 2009. Zootaxa 2197. 53-63.]
[Chase, M. W., & Fay, M. F. 2009. Science. 325 682-683.]
[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.
[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.
[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.
[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.
[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.
[Efe, M. A., Tavares, E. S., Baker, A. J., & Bonatto, S. L 2009. Molecular Phylogenetics and Evolution. 52(1) 263-267.]
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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%.
[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.
[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.
[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.
[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.
[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 (> 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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[Gibbs, J. 2009. Journal of Hymenoptera Research. 18 74-79.]
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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
[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
[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.
[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.
[Boulding, E. G., deWaard, J. R., Ang, K. P., & Hebert, P. D. N. 2009. Aquaculture Research. 40(8) 973-979.]
[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.]
[Wilson, John J., Floyd, R., Hanner, R. H., & Castle, D. 2009. Isis. 100(1) 117.]
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[Meier, R., Zhang, G., & Ali, F. 2009. Systematic Biology. 57(5) 809-813.]
[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 >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 (<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.
[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.
[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.
[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.
[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.
[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.
[Seifert, K. A. 2008. Persoonia. 21 162-166.]
[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
[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.
[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.
[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.
[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.
[David J. Lohman, Djunijanti Peggi, Naomi E. Pierce, Rudolf Meier 2008. BioMed Central Evolutionary Biology. 8: 301 .]
[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.
[Steinke, D., Hanner, R., & Hebert, P. D. N. 2008. Ichthyological Research. Online Early .]
[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.
[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.
[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.
[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.
[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.
[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.
[Gibbs, J. 2008. Journal of the Kansas Entomological Society. In Press 12pp.]
[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.]
[Maclean, N. 2008. Journal of Fish Biology,. 73(5) 1091-1095.]
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[Hollingsworth, P. M. 2008. Heredity. 101(1) 1-2.]
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.]
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[Valkiunas, G. Atkinson, C. T. Bensch, S. Sehgal, R. N. & R.E. Ricklefs 2008. Trends in parasitology. 24(6) 247-8.]
[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.
[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.
[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.
[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.
[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.
[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
[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.
[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.
[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.
[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.
[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.
| [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. |  |
[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.
[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.
[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
| [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. |  |
[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.
[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.
[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.
[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.
[W. John Kress and David L. Erickson 2008. PNAS. Vol. 105. No. 8 3761-3762.]
| [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. |  |
[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.
[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.
[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.
| [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. |  |
| [Ward, R. D., Holmes, B. H., and Yearsley, G. K. 2008. Journal of Fish Biology. 72(2) 458463.] 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. |  |
[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
[Smith, M.A. 2008. Zootaxa. 1691 67-68.]
| [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. |  |
| [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. |  |
[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.
| [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. |  |
[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.
| [Wheeler, Q.D. 2008. Systematic Entomology. 33(1) 2-7.] |  |
| [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. |  |
[Waugh, J.,Huynen, L., Millar, C., and D. Lambert 2008. Bioessays. 30(1) 92-3.]
[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.
| [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. |  |
[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.
[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.]
| [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. |  |
[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.
[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.
[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.
[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.
[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.
[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.
[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.
| [Soltis, D. E. 2007. Evolution. 61(12) 30073011.] 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? |  |
| [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. |  |
| [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. |  |
[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.]
| [Wiens, J. J. 2007. Systematic Biology. 56(6) 875-8.] |  |
| [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. |  |
[Ravindra C. Joshi, Alberto T. Barrion, Leocadio S. Sebastian 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.
[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.
[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.
[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.
| [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/. |  |
| [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. |  |
| [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. |  |
[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.
[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.
[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.
[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.
| [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. |  |
[Lissovsky, A. A., Ivanova, N. V. and Borisenko, A. V. 2007. Journal of Mammalogy. 88(5) .]
| [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. |  |
| [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. |  |
[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%.
| [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.] |  |
| [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. |  |
| [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. |  |
| [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. |  |
| [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. |  |
| [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. |  |
[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.
| [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. |  |
| [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. |  |
[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).
[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.
| [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. |  |
[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.
[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.
[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.
| [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. |  |
| [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. |  |
| [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. |  |
| [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. |  |
[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.
| [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. |  |
[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.
| [Carew, M. E., Pettigrove, V., Cox, R. L., & Hoffmann, A. A. 2007. Journal of the North American Benthological Society. 26(4) 587600.] 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 |  |
| [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. |  |
[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.
| [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% |  |
[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.
| [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. |  |
| [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. |  |
| [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. |  |
| [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. |  |
[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.
| [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. |  |
| [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. |  |
| [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. |  |
| [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. |  |
| [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. |  |
[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.
| [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. |  |
| [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. |  |
| [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 |  |
| [Darren Bito 2007. Journal of Biogeography. 34(5) 769-778.] |  |
[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.
| [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. |  |
[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.
[Hoef-Emden, K., Kupper, F. C., & Andersen, R. A. 2007. Protist. 158(2) 135-137.]
[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.
| [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. |  |
| [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. |  |
| [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. |  |
[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.
| [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. |  |
| [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. |  |
[Scott E. Miller 2007. PNAS. 104(12) 4775-4776.]
| [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. |  |
| [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. |  |
[Wiemers, M. and K. Fiedler 2007. Frontiers in Zoology. 4(8) .]
[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.
| [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. |  |
| [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). |  |
| [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. |  |
| [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. |  |
| [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. |  |
| [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. |  |
[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.
| [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. |  |
| [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. |  |
| [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. |  |
| [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. |  |
[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.
[Dawnay N., R. Ogden, R. McEwing, G.R. Carvalho and R.S. Thorpe. 2007. Forensic Science International. .]
| [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 spec |