Publications
[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.
[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.
[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.
[Isabelle Meusneir, 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.
[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.
[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.
[M. Alex Smith, Josephine J. Rodriguez, James B. Whitfield, Andrew R. Deans, Daniel H. Janzen, Winnie Hallwachs, and Paul D.N. Hebert 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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[Valkiunas, G. Atkinson, C. T. Bensch, S. Sehgal, R. N. & R.E. Ricklefs 2008. Trends in parasitology. 24(6) 247-8.]
[Brian L. Fisher, M. Alex Smith 2008. PLoS one. 3:5 .]
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.
boltoni sp. nov., A. goodmani sp. nov., A. grandidieri, and A. madagascarensis from Madagascar, and A. pattersoni sp. 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.
[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.
[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.
[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.
| [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.
[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.
[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.
| [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. |  |
[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. |  |
| [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. |  |
[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. |  |
| [Wheeler, Q.D. 2008. Systematic Entomology. 33(1) 2-7.] |  |
| [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. |  |
| [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. |  |
[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.
| [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. |  |
| [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. |  |
[Gómez A., Hughes Roger N., Wright Peter J., Carvalho Gary R., Lunt, David H. 2007. Molecular Ecology. 16 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.
[Randrianiaina, R.-D., F. Glaw, M. Thomas, J. Glos, N. Raminosoa & M. Vences 2007. Zootaxa. 1401 53-61.]
We describe the larval stages of two Malagasy frog species of the genus Gephyromantis, based on specimens identified by DNA barcoding. The tadpoles of Gephyromantis ambohitra are generalized stream-living Orton type IV type larvae with two lateral small constrictions of the body wall at the plane of spiracle. Gephyromantis pseudoasper tadpoles are characterized by totally keratinised jaw sheaths with hypertrophied indentation, a reduced number of labial tooth rows, enlarged papillae on the oral disc, and a yellowish coloration of the tip of the tail in life. The morphology of the tadpole of G. pseudoasper agrees with that of G. corvus, supporting the current placement of these two species in a subgenus Phylacomantis, and suggesting that the larvae of G. pseudoasper may also have carnivorous habits as known in G. corvus. Identifying the tadpole of Gephyromantis ambohitra challenges current assumptions of the evolution of different developmental modes in Gephyromantis, since this species is thought to be related to G. asper, a species of supposedly endotrophic direct development.
[Triantafyllidis, A., Bobori, D.C. and C. Koliamitra 2007. ESEB XI Congress Uppsala Sweden. .]
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.
| [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? |  |
[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.
[Ravindra C. Joshi, Alberto T. Barrion, Leocadio S. Sebastian 2007. ]
| [Boyer, S.L., Baker J.M. and Giribet, G. 2007. Molecular Ecology. 16(23) 4999-5016.] Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi, Pettalidae), a widespread ‘mite harvestman’ endemic to the South Island of New Zealand, is found in leaf littler habitats throughout Nelson and Marlborough, and as far south as Arthur's Pass. We investigated the phylogeography and demographic history of A. denticulata in the first genetic population-level study within Opiliones. A total of 119 individuals from 17 localities were sequenced for 785 bp of the gene cytochrome c oxidase subunit I; 102 of these individuals were from the Aoraki subspecies A. denticulata denticulata and the remaining 17 were from the subspecies A. denticulata major. An extraordinarily high degree of genetic diversity was discovered in A. denticulata denticulata, with average uncorrected p-distances between populations as high as 19.2%. amova, average numbers of pairwise differences, and pairwise FST values demonstrated a significant amount of genetic diversity both within and between populations of this subspecies. Phylogenetic analysis of the data set revealed many well-supported groups within A. denticulata denticulata, generally corresponding to clusters of specimens from single populations with short internal branches, but separated by long branches from individuals from other populations. No haplotypes were shared between populations of the widespread small subspecies, A. denticulata denticulata. These results indicate a subspecies within which very little genetic exchange occurs between populations, a result consistent with the idea that Cyphophthalmi are poor dispersers. The highly structured populations and deep genetic divergences observed in A. denticulata denticulata may indicate the presence of cryptic species. However, we find a highly conserved morphology across sampling localities and large genetic divergences within populations from certain localities, equivalent to those typically found between populations from different localities. Past geological events may have contributed to the deep genetic divergences observed between sampling localities; additionally, the high divergence within populations of A. denticulata denticulata suggests that the rate of COI evolution may be accelerated in this taxon. In contrast, the larger subspecies A. denticulata major shows much less differentiation between and within sampling localities, suggesting that it may disperse more easily than its smaller counterpart. The fact that the remarkable genetic divergences within populations of A. denticulata denticulata from certain localities are equivalent to divergences between localities poses a challenge to the rapidly spreading practice of DNA taxonomy. |  |
| [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. |  |
| [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. |  |
[Hoffmann, K., Discher, S. and Voigt, K. 2007. Mycological Research. .]
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.
| [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. |  |
[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.
| [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. |  |
| [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/. |  |
| [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. |  |