CCDB Publications
Researchers and staff at the Canadian Centre for DNA Barcoding have made many contributions to the body of scientific literature in this growing field. Browse the following list to access DNA barcoding publications that CCDB personnel have co-authored.
[Vernooy, R., Haribabu, E., Muller, M. R., Vogel, J. H., Hebert, P. D. N., Schindel, D. E., Shimura, J. & Singer, G. A. C. 2010. PLoS Biol. 8(7 e1000417.]
Barcoding scientists aspire to adhere to the objectives of the Convention on Biological Diversity by promoting conservation, sustainability, and the equitable sharing of benefits arising from use of genetic resources.
[Wilson, J. J. 2010. PLoS One. 5(5) e10525.]
BACKGROUND: Despite apparently abundant amounts of observable variation and species diversity, the order Lepidoptera exhibits a morphological homogeneity that has provided only a limited number of taxonomic characters and led to widespread use of nucleotides for inferring relationships. This study aims to characterize and develop methods to quantify the value of priority gene regions designated for Lepidoptera molecular systematics. In particular, I assess how the DNA barcode segment of the mitochondrial COI gene performs across a broad temporal range given its number one position of priority, most sequenced status, and the conflicting opinions on its phylogenetic performance. METHODOLOGY/PRINCIPAL FINDINGS: Gene regions commonly sequenced for lepidoptera phylogenetics were scored using multiple measures across three categories: practicality, which includes universality of primers and sequence quality; phylogenetic utility; and phylogenetic signal. I found that alternative measures within a category often appeared correlated, but high scores in one category did not necessarily translate into high scores in another. The DNA barcode was easier to sequence than other genes, and had high scores for utility but low signal above the genus level. CONCLUSIONS/SIGNIFICANCE: Given limited financial resources and time constraints, careful selection of gene regions for molecular phylogenetics is crucial to avoid wasted effort producing partially informative data. This study introduces an approach to assessing the value of gene regions prior to the initiation of new studies and presents empirical results to help guide future selections.
[Campagna, L., Lijtmaer, D. A., Kerr, K. C. R., Barreira, A. S., Hebert, P. D. N., Lougheed, S. C., & Tubaro, P. 2010. Molecular Ecology Resources. 10(3) 449-458.]
The capuchinos are a group of birds in the genus Sporophila that has apparently radiated recently, as evidenced by their lack of mitochondrial genetic diversity. We obtained cytochrome c oxidase I (COI) sequences (or DNA barcodes) for the 11 species of the group and various outgroups. We compared the patterns of COI variability of the capuchinos with those of the largest barcode data set from neotropical birds currently available (500 species representing 51% of avian richness in Argentina), and subjected COI sequences to neighbour-joining, maximum parsimony and Bayesian phylogenetic analyses as well as statistical parsimony network analysis. A clade within the capuchinos, the southern capuchinos, showed higher intraspecific and lower interspecific divergence than the remaining Argentine species. As most of the southern capuchinos shared COI haplotypes and pairwise distances within species were in many cases higher than distances between them, the phylogenetic affinities within the group remained unresolved. The observed genetic pattern is consistent with both incomplete lineage sorting and gene flow between species. The southern capuchinos constitute the only large group of species among the neotropical birds barcoded so far that are inseparable when using DNA barcodes, and one of few multispecies avian groups known to lack reciprocal monophyly. Extending the analysis to rapidly evolving nuclear and mitochondrial markers will be crucial to understanding this radiation. Apart from giving insights into the evolution of the capuchinos, this study shows how DNA barcoding can rapidly flag species or groups of species worthy of deeper study.
[Wilson, J. J., Landry, J.-F., Janzen, D. H., Hallwachs, W., Nazari, V., Hajibabaei, M. & Hebert, P. D. N. 2010. ZooKeys. 40 41–60.]
During extensive ongoing campaigns to inventory moths of North America and Area de Conservacion Guanacaste (ACG), northwestern Costa Rica, we discovered that morphologically similar yponomeutid moths were assigned two different names, Atteva ergatica Walsingham in Costa Rica and A. punctella (Stoll) in North America, but had identical DNA barcodes. Combining DNA barcoding, morphology and food plant records also revealed a complex of two sympatric species that are diagnosable by their DNA barcodes and their facies in Costa Rica. However, neither of the names could be correctly applied to either species, as A. ergatica is a junior synonym and A. punctella a junior homonym. By linking our specimens to type material through morphology and DNA barcoding, we determined that the ACG dry forest species, distributed from Costa Rica to southern Quebec and Ontario, should be called A. aurea, whereas the similar and marginally sympatric ACG rain forest species found in Central America should be called A. pustulella. Neotypes are designated for Phalaena Tinea punctella Stoll, 1781 and Deiopeia aurea Fitch, 1857. Atteva floridana has identical barcodes to A. aurea and provisionally maintained as a synonym.
[Radulovici, A. E., Archambault, P., & Dufresne, F. 2010. Diversity. 2(4) 450-472.]
‘Biodiversity’ means the variety of life and it can be studied at different levels (genetic, species, ecosystem) and scales (spatial and temporal). Last decades showed that marine biodiversity has been severely underestimated at all levels. In order to investigate diversity patterns and underlying processes, there is a need to know what species live in the marine environment. An emerging tool for species identification, DNA barcoding can reliably assign unknown specimens to known species, also flagging potential cryptic species and genetically distant populations. This paper will review the role of DNA barcoding for the study of marine biodiversity at the species level.
[Hunt, B., Strugnell, J., Bednarsek, N., Linse, K., Nelson, R. J., Pakhomov, E., Seibel, B., Steinke, D., & Wurzberg, L. 2010. PLoS ONE. e9835.]
The shelled pteropod (sea butterfly) Limacina helicina is currently recognised as a species complex comprising two sub-species and at least five “forma”. However, at the species level it is considered to be bipolar, occurring in both the Arctic and Antarctic oceans. Due to its aragonite shell and polar distribution L. helicina is particularly vulnerable to ocean acidification. As a key indicator of the acidification process, and a major component of polar ecosystems, L. helicina has become a focus for acidification research. New observations that taxonomic groups may respond quite differently to acidification prompted us to reassess the taxonomic status of this important species. We found a 33.56% (±0.09) difference in cytochrome c oxidase subunit I (COI) gene sequences between L. helicina collected from the Arctic and Antarctic oceans. This degree of separation is sufficient for ordinal level taxonomic separation in other organisms and provides strong evidence for the Arctic and Antarctic populations of L. helicina differing at least at the species level. Recent research has highlighted substantial physiological differences between the poles for another supposedly bipolar pteropod species, Clione limacina. Given the large genetic divergence between Arctic and Antarctic L. helicina populations shown here, similarly large physiological differences may exist between the poles for the L. helicina species group. Therefore, in addition to indicating that L. helicina is in fact not bipolar, our study demonstrates the need for acidification research to take into account the possibility that the L. helicina species group may not respond in the same way to ocean acidification in Arctic and Antarctic ecosystems.
[Craft, K. J., Pauls, S. U., Darrow, K., Miller, S. E., Hebert, P. D. N., Helgen, L. E., Novotny, V. & Weiblen, G. D. 2010. Proc Natl Acad Sci U S A. 107(11) 5041-6.]
Comparative population genetics of ecological guilds can reveal generalities in patterns of differentiation bearing on hypotheses regarding the origin and maintenance of community diversity. Contradictory estimates of host specificity and beta diversity in tropical Lepidoptera (moths and butterflies) from New Guinea and the Americas have sparked debate on the role of host-associated divergence and geographic isolation in explaining latitudinal diversity gradients. We sampled haplotypes of mitochondrial cytochrome c oxidase I from 28 Lepidoptera species and 1,359 individuals across four host plant genera and eight sites in New Guinea to estimate population divergence in relation to host specificity and geography. Analyses of molecular variance and haplotype networks indicate varying patterns of genetic structure among ecologically similar sympatric species. One-quarter lacked evidence of isolation by distance or host-associated differentiation, whereas 21% exhibited both. Fourteen percent of the species exhibited host-associated differentiation without geographic isolation, 18% showed the opposite, and 21% were equivocal, insofar as analyses of molecular variance and haplotype networks yielded incongruent patterns. Variation in dietary breadth among community members suggests that speciation by specialization is an important, but not universal, mechanism for diversification of tropical Lepidoptera. Geographically widespread haplotypes challenge predictions of vicariance biogeography. Dispersal is important, and Lepidoptera communities appear to be highly dynamic according to the various phylogeographic histories of component species. Population genetic comparisons among herbivores of major tropical and temperate regions are needed to test predictions of ecological theory and evaluate global patterns of biodiversity.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.]
[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.
[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.
[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.
[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.
[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.
[Steinke, D., Hanner, R., & Hebert, P. D. N. 2008. Ichthyological Research. Online Early .]
[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.
[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
[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.
[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.
[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.
[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.
[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. |  |
[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.
[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.
[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. |  |
[Smith, M.A. 2008. Zootaxa. 1691 67-68.]
[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.
[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. |  |
[Lissovsky, A. A., Ivanova, N. V. and Borisenko, A. V. 2007. Journal of Mammalogy. 88(5) .]
[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.
[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. |  |
[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. |  |
[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. |  |
[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. |  |
[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. |  |
[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. |  |
[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. |  |
[Costa F.O., deWaard, J. R., Boutillier, J., Ratnasingham S., Dooh R., Hajibabaei M., Hebert, P.D.N. 2007. Canadian Journal of Fisheries and Aquatic Sciences. 64(2) 272-295.]
The ability of a 650 base pair section of the mitochondrial cytochrome c oxidase I (COI) gene to provide species-level identifications has been demonstrated for large taxonomic assemblages of animals such as insects, birds and fishes, but not for the subphylum Crustacea, one of the most diverse groups of arthropods. In this study we test the ability of COI to provide identifications in this group, examining two disparate levels in the taxonomic hierarchy – orders and species. The first phase of our study involved the development of a sequence profile for 23 dominant crustacean orders, based upon the analysis of 150 species, each belonging to a different family. The COI amino acid data placed these taxa into cohesive assemblages whose membership coincided with currently accepted boundaries at the order, superorder and subclass levels. Species-level resolution was subsequently examined in an assemblage of Decapoda, and in representatives of the genera Daphnia (Cladocera) and Gammarus (Amphipoda). These studies revealed that levels of nucleotide sequence divergence were from 19 to 48 times greater between congeneric species than between individuals of a species. We conclude that sequence variation in the COI barcode region will be very effective for discriminating species of Crustacea.
[Burns, J.M., Janzen, D.H., Hajibabaei, M., Hallwachs, W. and Hebert, P.D.N. 2006. Journal of the LepidopteristsÂ’ Society. 61(3) 138-153.]
Unlike most species of Lepidoptera whose DNA barcodes have been examined, closely related taxa in each of three pairs of hesperiids (Polyctor cleta and P. polyctor, Cobalus virbius and C. fidicula, Neoxeniades luda and N. pluviasilva Burns, new species) seem indistinguishable by their barcodes; but that is when some of the cytochrome c oxidase I (COI) sequences are short and sample sizes are small. These skipper butterflies are unquestionably distinct species, as evidenced by genitalic and facies differences and by ecologic segregation, i.e., one species of each pair in dry forest, the other in adjacent rain forest in Area de Conservación Guanacaste in northwestern Costa Rica. This national park is the source of the specimens used in this study, all of which were reared. Larval foodplants are of no or problematic value in distinguishing these species. Large samples of individuals whose barcodes are acceptably long reveal slight interspecific differentiation (involving just one to three nucleotides) in all three pairs of skippers. Clearly, the chronic practice of various taxonomists of setting arbitrary levels of differentiation for delimiting species is unrealistic.
[Ivanova, N.V., deWaard, J.R. and P.D.N. Hebert 2006. Molecular Ecology Notes. 6(4) 998-1002.]
Although commercial kits are available for automated DNA extraction, 'artisanal' protocols are not. In this study, we present a silica-based method that is sensitive, inexpensive and compliant with automation. The effectiveness of this protocol has now been tested on more than 5000 animal specimens with highly positive results. |  |
[Cywinska A., Hunter F.F., and Hebert P.D.N. 2006. Medical and Veterinary Entomology. 20(4) 413-424.]
A short fragment of mt DNA from the cytochrome c oxidase 1 (CO1) region was used to provide the first CO1 barcodes for 37 species of Canadian mosquitoes (Diptera: Culicidae) from the provinces Ontario and New Brunswick. Sequence variation was analysed in a 617-bp fragment from the 5' end of the CO1 region. Sequences of each mosquito species formed barcode clusters with tight cohesion that were usually clearly distinct from those of allied species. CO1 sequence divergences were, on average, nearly 20 times higher for congeneric species than for members of a species; divergences between congeneric species averaged 10.4% (range 0.2–17.2%), whereas those for conspecific individuals averaged 0.5% (range 0.0–3.9%). |  |
[Hajibabaei, M., Smith, M.A., Janzen, F.H., Rodriguez, J.J., Whitfield, J.B., and P.D.N. Hebert 2006. Molecular Ecology Notes. 6(4) 959-964.]
A DNA barcode based on 650 bp of mitochondrial gene cytochrome c oxidase I is proving to be highly functional in species identification for various animal groups. However, DNA degradation complicates the recovery of a full-length barcode from many museum specimens. Here we explore the use of shorter barcode sequences for identification of such specimens. We recovered short sequences — i.e. 100 bp — with a single PCR pass from more than 90% of the specimens in assemblages of moth and wasp museum specimens from which full barcode recovery was only 50%, and the latter were usually less than 8 years old. Short barcodes were effective in identifying specimens, confirming their utility in circumstances where full barcodes are too expensive to obtain and the identification comparisons are within a confined taxonomic group. |  |
[Witt, Jonathan D.S., Threloff, Doug L. and Hebert, Paul D.N. 2006. Molecular Ecology. 15(10) 3073 - 3082.]
DNA barcoding has revealed unrecognized species in several animal groups. In this study we have employed DNA barcoding to examine Hyalella, a taxonomically difficult genus of amphipod crustaceans, from sites in the southern Great Basin of California and Nevada, USA. We assessed the extent of species diversity using a species screening threshold (SST) set at 10 times the average intrapopulation cytochrome c oxidase subunit I (COI) haplotype divergence. Despite the fact that this threshold approach is more conservative in delineating provisional species than the phylogenetic species concept, our analyses revealed extraordinary levels of cryptic diversity and endemism. The SST discriminated two provisional species within Hyalella sandra, and 33 provisional species within Hyalella azteca. COI nucleotide divergences among these provisional species ranged from 4.4% to 29.9%. These results have important implications for the conservation of life in desert springs — habitats that are threatened as a result of groundwater over-exploitation. |  |
[Hajibabaei, M., Singer, G.A.C., and D.A. Hickey 2006. Genome. 49(7) 851-854.]
DNA barcoding has been recently promoted as a method for both assigning specimens to known species and for discovering new and cryptic species. Here we test both the potential and the limitations of DNA barcodes by analysing a group of well-studied organisms—the primates. Our results show that DNA barcodes provide enough information to efficiently identify and delineate primate species, but that they cannot reliably uncover many of the deeper phylogenetic relationships. Our conclusion is that these short DNA sequences do not contain enough information to build reliable molecular phylogenies or define new species, but that they can provide efficient sequence tags for assigning unknown specimens to known species. As such, DNA barcoding provides enormous potential for use in global biodiversity studies. |  |
[Smith, M.A., Woodley, N.E., Janzen, D.H., Hallwachs, W., and P.D.N. Hebert 2006. PNAS. 103(10) 3657-3662.]
Notes: DNA barcoding discriminates 17 highly host-specific morphospecies of Belvosia, and further raises the species count to 32 by revealing that each of the generalist species are in fact highly host-specific cryptic species complexes. |  |
[Hajibabaei, M., Janzen, D.H., Burns, J.M., Hallwachs, W., and P.D.N. Hebert 2006. Proc Natl Acad Sci U S A. 103(4) 968 -71.]
Although central to much biological research, the identification of species is often difficult. The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. However, the effectiveness of DNA barcoding for identifying specimens in species-rich tropical biotas is unknown. Here we show that cytochrome c oxidase I DNA barcodes effectively discriminate among species in three Lepidoptera families from Area de Conservación Guanacaste in northwestern Costa Rica. We found that 97.9% of the 521 species recognized by prior taxonomic work possess distinctive cytochrome c oxidase I barcodes and that the few instances of interspecific sequence overlap involve very similar species. We also found two or more barcode clusters within each of 13 supposedly single species. Covariation between these clusters and morphological and/or ecological traits indicates overlooked species complexes. If these results are general, DNA barcoding will significantly aid species identification and discovery in tropical settings. |  |
[Janzen, D. H., Hajibabaei, M., Burns, J. M., Hallwachs, W., Remigio, E. & Hebert, P. D. 2005. Philosophical Transactions of the Royal Society B: Biological Sciences. 360(1462) 1835-1845.]
By facilitating bioliteracy, DNA barcoding has the potential to improve the way the world relates to wild biodiversity. Here we describe the early stages of the use of cox1 barcoding to supplement and strengthen the taxonomic platform underpinning the inventory of thousands of sympatric species of caterpillars in tropical dry forest, cloud forest and rain forest in northwestern Costa Rica. The results show that barcoding a biologically complex biota unambiguously distinguishes among 97% of more than 1000 species of reared Lepidoptera. Those few species whose barcodes overlap are closely related and not confused with other species. Barcoding also has revealed a substantial number of cryptic species among morphologically defined species, associated sexes, and reinforced identification of species that are difficult to distinguish morphologically. For barcoding to achieve its full potential, (i) ability to rapidly and cheaply barcode older museum specimens is urgent, (ii) museums need to address the opportunity and responsibility for housing large numbers of barcode voucher specimens, (iii) substantial resources need be mustered to support the taxonomic side of the partnership with barcoding, and (iv) hand-held field-friendly barcorder must emerge as a mutualism with the taxasphere and the barcoding initiative, in a manner such that its use generates a resource base for the taxonomic process as well as a tool for the user. |
[Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R., and P.D.N. Hebert 2005. Phil. Trans. R. Soc. B. 360 (1462) 1847 - 1857.]
Notes: This paper discusses the barcoding process of 207 species of Australian fishes showing the cox1 sequence can be used for identification. |
[Smith, M.A., Fisher, B.L., P.D.N. Hebert 2005. Phil. Trans. R. Soc. B. 360 (1462) 1825 - 1834.]
The role of DNA barcoding as a tool to accelerate the inventory and analysis of diversity for hyperdiverse arthropods is tested using ants in Madagascar. We demonstrate how DNA barcoding helps address the failure of current inventory methods to rapidly respond to pressing biodiversity needs, specifically in the assessment of richness and turnover across landscapes with hyperdiverse taxa. In a comparison of inventories at four localities in northern Madagascar, patterns of richness were not significantly different when richness was determined using morphological taxonomy (morphospecies) or sequence divergence thresholds (Molecular Operational Taxonomic Unit(s); MOTU). However, sequence-based methods tended to yield greater richness and significantly lower indices of similarity than morphological taxonomy. MOTU determined using our molecular technique were a remarkably local phenomenon—indicative of highly restricted dispersal and/or long-term isolation. In cases where molecular and morphological methods differed in their assignment of individuals to categories, themorphological estimate was always more conservative than the molecular estimate. In those cases where morphospecies descriptions collapsed distinct molecular groups, sequence divergences of 16% (on average) were contained within the same morphospecies. Such high divergences highlight taxa for further detailed genetic, morphological, life history, and behavioral studies. |
[Hajibabaei, M., deWaard, J.R., Ivanova, N.V., Ratnasingham, S., Dooh, R.T., Kirk, S.L., Mackie, P.M., and P.D.N. Hebert 2005. Phil. Trans. R. Soc. B. 360 (1462) 1959 - 1967.]
Notes: The key steps in establishing high volume DNA barcoding facilities are discussed in this communication. Strong emphasis is placed in this article on developing alliances with the taxonomic community in order to provision specimens. The current difficulties of analysing museum collections are also discussed. |
[Hebert, P.D.N. and T.R. Gregory 2005. Syst. Biol.. 54(5) 852-859.]
Notes: The authors address a series of questions and clarify the rationale and potential impacts of DNA barcoding.
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[Ball, S.L., Hebert, P.D.N., Burian, S.K., and J.M. Webb. 2005. J. N. Am. Benthol. Soc.. 24(3) 508-524.]
Notes: This study tests the efficacy of DNA barcoding in the identification of mayflies. |  |
[Gregory, T. R. 2005. Nature. 434 1067.]
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[Lambert, D.M., A. Baker, L. Huynen, O. Haddrath, P.D.N. Hebert and C.D. Millar 2005. J. Hered.. 96(3) 1-6.]
Notes: The authors use a number of genes to recover the phylogeny of extinct ratite birds from New Zealand. Results of this study indicate that DNA barcoding may be able to detect other extinct animal species and that an inventory of ancient life is possible. |  |
[Paul D.N. Hebert, Erin H. Penton, John M. Burns, Daniel H. Janzen, and Winnie Hallwachs 2004. Preceedings of the National Academy of Sciences. Vol. 101 No. 41 12-17.]
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[Hebert, P.D.N, Stoekle, M., Zemlak, T., and C.M. Francis 2004. PLoS Biology. 2 1657-1668.]
Notes: This work tested the effectiveness of the COI barcode in distinguishing between bird species. 260 species of North american birds were tested, and found that 4 may prove to be new species. |  |
[Barret, D.H. and P.D.N. Hebert 2004. Can. J. Zool. 83 481-491.]
Notes: This study shows the potential for DNA barcoding to rapidly identify spiders, a group which has suffered previously from difficulties in species identification. All of the 204 species of arachnids tested were correctly assigned to their species, showing the efficacy of barcoding. |  |
[Hogg, I.D., and P.D.N. Hebert 2004. Can. J. Zool. 82 749-754.]
Notes: This study examines Arctic collembolans from 19 species across 13 genera. |  |
[Remigio, E.A. and P.D.N. Hebert 2003. Mol. Phylogenet. Evol. 29 641-647.]
Notes: This study shows the utility of COI-5' sequence data in recovering deep taxonomic assignments in the largest class of molluscs.
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[Hebert, P.D.N., S. Ratnasingham and J.R. deWaard. 2003. Proc. R. Soc. Lond. B. 270 suppliment 03BL0066 1-4.]
Notes: Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. |  |
[Hebert, P.D.N., A. Cywinska, S.L. Ball and J.R. deWaard 2003. Proc. R. Soc. Lond. B. 270 313-322.]
Notes: This work establishes COI as a primary barcoding tool in the development of a global bioidentification system.
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