Network Publications
[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.
[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. 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.
[Wilson, J. J., Landry, J.-F., Janzen, D. H., Hallwachs, W., Nazari, V., Hajibabaei, M. & Hebert, P. D. N. 2010. ZooKeys. 40 4160.]
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.
[LeGall, L., & Saunders, G. W. 2010. Journal of Phycology. 46(2) 374 - 389.]
Previous studies have established that the 5' end of the mitochondrial gene COI (cytochrome oxidase subunit I) is useful for rapid and reliable identification of red algal species and have demonstrated that our understanding of red algal biodiversity and biogeography is fragmentary. In this context, we are completing a thorough sampling along the Canadian coast and using the DNA barcode for the assignment of collections to genetic species to explore algal diversity in the Canadian flora. In the present study, we provide results regarding diversity of members of the red algal family Phyllophoraceae. We have analyzed 354 individuals from the Arctic, Atlantic, and Pacific coasts of Canada, as well as 26 specimens from the USA, Europe, and Australia, resolving 29 species based on the analyses of the DNA barcode. Twenty-three of these genetic species were present in Canada where only 18 species are currently recognized, including Ceratocolax hartzii Rosenv., which was in the same genetic species group as its host Coccotylus truncatus (Pall.) M. J. Wynne et N. J. Heine and is thus transferred to Coccotylus, C. hartzii (Rosenv.) comb. nov., but retained as a distinct species owing to its unique habit and phenology. Our results revealed the presence of cryptic diversity within the genera Coccotylus, Mastocarpus, Ozophora, and Stenogramme, for which we resurrect Coccotylus brodiei (Turner) Kütz. and describe Mastocarpus pachenicus sp. nov., Ozophora lanceolata sp. nov., and Stenogramme bamfieldiensis sp. nov., leaving a multitude of unnamed Mastocarpus spp. in need of further taxonomic study. In addition, we report range extensions into British Columbia of Besa papillaeformis Setch., previously known only from its type and nearby localities in California; Gymnogongrus crenulatus (Turner) J. Agardh, recorded only from the Atlantic; and Stenogramme cf. rhodymenioides Joly et Alveal, previously only known from South America. Finally, the phylogenetic affinities of the Canadian species of Phyllophoraceae characterized in this study were investigated using LSU rDNA, RUBISCO LSU (rbcL), and combined analyses.
[Victor, B. C. 2010. Journal of the Ocean Science Foundation. 3 .]
A new micro-endemic goby, Elacatinus rubrigenis, is described from Utila in the Bay Islands of the Gulf of Honduras (Western Atlantic). The new species is similar to the Greenbanded Goby, E. multifasciatus, but differs in having a prominent red stripe across the cheek, more-numerous green bars on the body, and 11 second-dorsal-fin elements (vs. equal numbers of 11 and 12). The new species, the Redcheek Goby, replaces the Greenbanded Goby on the island of Utila and has not been sighted at any other location, potentially one of the smallest ranges reported for a Caribbean reef fish. The COI barcode mtDNA sequence for the Redcheek Goby is 11.2% divergent from the original type population of the Greenbanded Goby from the U. S. Virgin Islands. However, Panamanian Greenbanded Gobies, with no red cheek stripe, show a similarly large 11.3% genetic distance from the type population (within-population sequence variation is less than 1%). Despite the prominent marking difference, there is only a 3.3% sequence difference between Redcheek Gobies and Panamanian Greenbanded Gobies. These results highlight the lack of concordance between genetic and phenotypic divergence among cryptic lineages of reef fishes. The Panamanian population has some small meristic differences from the type population and is (reluctantly) described here as the new species Elacatinus panamensis. An unexpected 4.3% sequence difference between the adjacent Puerto Rican and Virgin Islands populations indicates that the Greenbanded Goby is likely to break up into inconveniently numerous discrete genetic lineages, presumably in allopatry. These sequence differences are generally greater than those separating the Elacatinus cleaning-goby species in the Caribbean and greater than the differences found among most genera of reef fishes. The remarkably deep DNA-sequence divergence among these allopatric cryptic species and lineages raises important and difficult questions about genetic structure, speciation processes, and species definitions among some coral reef fishes.
[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.
[Locke, S. A., McLaughlin, J. D., Dayanandan, S., & Marcogliese, D. J 2010. Int J Parasitol. 40(3) 333-343.]
In this study, sequences from the barcode region of cytochrome c oxidase I (COI) were used to distinguish Diplostomum spp. in a sample of 497 metacercariae collected from diverse fishes of the St. Lawrence River, Canada and findings were corroborated with internal transcribed spacer (ITS) regions of rDNA. Twelve species were detected based on sequences and metacercarial specificity for hosts and tissues. Although this is an unusually high diversity, additional species are likely to exist in the study area. Two species were indistinguishable with ITS data and there is evidence that they may be undergoing hybridization and/or have recently diverged. The ITS sequences of another species are similar to those of Diplostomum pseudospathaceum from Europe, but ITS data are insufficient to show that they are conspecific. Diplostomum spp. that infect tissues other than the lens are more host-specific than species inhabiting the lenses of fishes, which is attributed to the enhanced immunological privilege of the lens site compared with other tissues. Overall, COI sequences were superior to more commonly used ITS markers for delineating species of this important and taxonomically difficult pathogen.
[Clarkston, B. E., & Saunders, G. W 2010. Botany. 88 119-131.]
Accurate identification of many red algae to the species level using only morphological characters can be difficult. The emerging field of “molecular-assisted alpha taxonomy” can greatly alleviate this issue. In this approach, a large number of specimens are sequenced for a standard DNA marker as a first step to genetic species assignment, followed by detailed morphological observations. Regions of both the mitochondrial cytochromec oxidase I gene (COI-5P) and the plastid 23S rRNA gene (UPA) have been proposed as DNA barcode markers to accomplish this task. We compared the utility of each marker as a species identification tool using members of the marine red algal family Kallymeniaceae from British Columbia, Canada. Our results indicate that COI-5P is a more sensitive marker for delimiting species, but that it can be difficult to acquire clean amplification products for many isolates of Kallymeniaceae, owing to biological contamination. This problem can be overcome by using specific primers. UPA, on the other hand, has universal primers that work in diverse lineages (e.g., red, brown, and green algae), but lower interspecific sequence variation, which has the potential to underestimate species diversity, although this was not observed in our study. During our survey, we uncovered a new species of the Kallymeniaceae, Euthora timburtonii Clarkston et G.W.Saunders sp. nov., which we describe here.
[Moniz, M. B., & Kaczmarska, I. 2010. Protist. 161 7-34.]
DNA-barcoding is based on the premise that the divergence of a small DNA fragment coincides with biological separation of species. If true, it offers an additional tool for worldwide consistent species recognition even in cases of semi-cryptic species. Our study includes 618 sequences representing 114 diatom species belonging to the two most species-rich classes of diatoms (Mediophyceae and Bacillariophyceae). A 99.5% success rate in separating biologically defined species and a 91% success rate in separating all species tested was obtained when using the proposed barcode starting at the 5' end of 5.8S and ending in the conserved motif of helix III of ITS2 (300 to 400bp). Including the whole 5.8S+ITS2 region did not significantly improve species resolution. We tested our barcode on 17 unidentified, misidentified or contaminated strains derived mostly from a culture collection, and these were correctly flagged as erroneous by their ITS sequences. We conclude that the proposed barcode represents for the Mediophyceae and Bacillariophyceae a robust, economical, and rapid way to recognize and identify most species (when a reference sequence is available) that is as good as or better than other molecular markers thus far proposed.
[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.
[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.
[deWaard, J.R., Landry, J.-F., Schmidt, B.C., Derhousoff, J., McLean, J.A. and Humble, L.M. 2009. Biodiversity and Conservation. 18(14) 3825-3839.]
To facilitate future assessments of diversity following disturbance events, we conducted a first level inventory of nocturnal Lepidoptera in Stanley Park, Vancouver, Canada. To aid the considerable task, we employed high-throughput DNA barcoding for the rough sorting of all material and for tentative species identifications, where possible. We report the preliminary species list of 190, the detection of four new exotic species (Argyresthia pruniella, Dichelia histrionana, Paraswammerdamia lutarea, and Prays fraxinella), and the potential discovery of two cryptic species. We describe the magnitude of assistance that barcoding presents for faunal inventories, from reducing specialist time to facilitating the detection of native and exotic species at low density.
[Xu, S., Hebert, P. D. N., Kotov, A. A., & Cristescu, M. E. 2009. Molecular Ecology. 18(24) 5161-5179.]
A major question in our understanding of eukaryotic biodiversity is whether small bodied taxa have cosmopolitan distributions or consist of geographically localized cryptic taxa. Here, we explore the global phylogeography of the freshwater cladoceran Polyphemus pediculus (Linnaeus, 1761) (Crustacea, Onychopoda) using two mitochondrial genes, cytochrome c oxidase subunit I and 16s ribosomal RNA, and one nuclear marker, 18s ribosomal RNA. The results of neighbour-joining and Bayesian phylogenetic analyses reveal an exceptionally pronounced genetic structure at both inter- and intra-continental scales. The presence of well-supported, deeply divergent phylogroups across the Holarctic suggests that P. pediculus represents an assemblage of at least nine, largely allopatric cryptic species. Interestingly, all phylogenetic analyses support the reciprocal paraphyly of Nearctic and Palaearctic clades. Bayesian inference of ancestral distributions suggests that P. pediculus originated in North America or East Asia and that European lineages of Polyphemus were established by subsequent intercontinental dispersal events from North America. Japan and the Russian Far East harbour exceptionally high levels of genetic diversity at both regional and local scales. In contrast, little genetic subdivision is apparent across the formerly glaciated regions of Europe and North America, areas that historical demographic analyses suggest that were recolonized just 5500201324 000 years ago.
[Hausler, D., O'Brien, S. J., Ryder, O. A., & 58 co-authors including Hebert, P. D. N. 2009. J Heredity. 100(6) 659-74.]
The human genome project has been recently complemented by whole-genome assessment sequence of 32 mammals and 24 nonmammalian vertebrate species suitable for comparative genomic analyses. Here we anticipate a precipitous drop in costs and increase in sequencing efficiency, with concomitant development of improved annotation technology and, therefore, propose to create a collection of tissue and DNA specimens for 10,000 vertebrate species specifically designated for whole-genome sequencing in the very near future. For this purpose, we, the Genome 10K Community of Scientists (G10KCOS), will assemble and allocate a biospecimen collection of some 16,203 representative vertebrate species spanning evolutionary diversity across living mammals, birds, nonavian reptiles, amphibians, and fishes (ca. 60,000 living species). In this proposal, we present precise counts for these 16,203 individual species with specimens presently tagged and stipulated for DNA sequencing by the G10KCOS. DNA sequencing has ushered in a new era of investigation in the biological sciences, allowing us to embark for the first time on a truly comprehensive study of vertebrate evolution, the results of which will touch nearly every aspect of vertebrate biological enquiry.
[De Prins, J., Mozuraitis, R., Lopez-Vaamonde, C., & Rougerie, R. 2009. Zootaxa. 2281 53-67.]
The sex attractant for Phyllonorycter melanosparta (Meyrick, 1912) has been determined as (10E)-dodec-10-en-1-yl acetate and (10E)-dodec-10-en-1-ol combined in a ratio 10:1. The distribution of this species in Eastern Africa is updated and its presence in Kenya is recorded for the first time. We discuss the taxonomic status of P. melanosparta with reference to three character sets: semiochemicals, morphological and molecular characters (DNA barcodes). This combination of characters is also proposed as a new approach to study the diversity and phylogeny of Phyllonorycter in the Afrotropical region.
[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.
[Efe, M. A., Tavares, E. S., Baker, A. J., & Bonatto, S. L 2009. Molecular Phylogenetics and Evolution. 52(1) 263-267.]
[Segade, P., Kher, C. P., Lynn, D. H., & Iglesias, R. 2009. Parasitology. 136(7) 771-782.]
Renal infections by parasitic ciliates were studied in adult snails of Helix aspersa aspersa and Helix aspersa maxima collected from 2 mixed rearing system-based heliciculture farms located in Galicia (NW Spain). The occurrence of ciliates was also examined in slugs (Deroceras reticulatum) invading the greenhouses where first growing and fattening of snails is carried out. Histological examinations revealed a severe destruction of the renal epithelium in heavily infected hosts. Three ciliate isolates, one from each host species, were obtained and grown in axenic cultures. Cultured and parasitic ciliates were characterized morphologically and morphometrically. In addition, the encystment behaviour, the occurrence of autogamy, and the sequences of the mitochondrial cytochrome-c oxidase subunit 1 (cox1) and the small subunit ribosomal RNA (SSU rRNA) genes were also studied in the 3 isolates. A polymorphic life cycle involving resting and reproductive cysts, together with the morphological and morphometrical characteristics and the confirmation that autogamy occurs within cysts, demonstrate that our ciliates belong to the species Tetrahymena rostrata (Kahl, 1926) Corliss, 1952. The 3 isolates formed a well-supported clade using both genetic markers, and were clearly separate from the strain ATCC(R) 30770, which has been identified as Tetrahymena rostrata. We argue that our Spanish isolates should be regarded as Tetrahymena rostrata, and that the ATCC isolate should be regarded as a misidentification as neither cytological nor cytogenetical support for its identity has been presented.
[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.
[McDevit, D. C. and G. W. Saunders 2009. Phycological Research. 57(2) 131-141.]
The generation of a species-rich DNA barcode database in combination with rapid and affordable sequencing techniques will dramatically change specimen identification in ecological, biogeographical and taxonomic applications. Though cytochrome c oxidase 1 has been shown to be a useful tool for differentiating some groups of marine algae, its wide application in the Phaeophyceae has yet to be studied. The presence of polymerase chain reaction (PCR) inhibiting compounds in members of the Fucales, Laminariales and Tilopteridales, that are often co-extracted with DNA, has hampered the rapid processing associated with barcode projects. Polyphenolics and polysaccharides are present in concentrations such that DNA extraction methods typically include extensive series of washes, organelle extractions and/or cesium columns. In this paper we examine the utility of cytochrome c oxidase 1 for barcoding the Phaeophyceae and present a method for extracting PCR friendly DNA from brown macroalgae in about 2 h, dramatically reducing the time required from previous methods, some of which take days. This method is easily adapted to a 96 well, high-throughput format and may have applications in other organisms where the presence of similar PCR inhibiting compounds hinders molecular analyses. We extracted DNA from 106 isolates representing 29 species from 20 genera in nine families from five orders of Phaeophyceae. We were able to amplify the barcode marker (cytochrome c oxidase 1) from all samples and a nuclear marker (internal transcribed spacer region) from 54 selected samples. Cytochrome c oxidase 1 was able to differentiate clearly among species, showing within species divergence of 0.00–0.46%, with the exception of one previously studied genus, and between species divergences of greater than 3%.
[Newmaster, S. G., Murugesan, M., Ragupathy, S., Nallasamy, N., & Balasubramaniam, V 2009. Ethnobotany. 21 2-24.]
Our research utilized Traditional Tribal Knowledge (TK) and Scientific Knowledge (SK) to explore the relationship between scientific and tribal systems of botanical classification and the corresponding valorisation(s) of biological diversity in the Western Ghats of southern India. We worked with two tribal communities namely ‘Irulas’ and ‘Malasars’ of the Nilgiri Biosphere Reserve with an objective of evaluating the ability of different knowledge systems (SK and TK) to distinguish species belonging to the genus Biophytum. We discovered that the tribal informants identified three ethnotaxa representing three new species namely, Biophytum velliangirianum, B. coimbatorense, B. tamilnadense, which we confimed using quantitative morphology and DNA evidence. The new taxa were confirmed by DNA barcoding and a morphometric analysis of the taxonomic evidence including comparisons with several closely related taxa: Biophytum insignis Gamble, B. longipedunculatum Govind. and Biophytum proliferum (Arn.) Wight. The recognition of these taxa has several consequences for conservation of plant diversity in the Nilgiri Biosphere and possible applications to society-at-large given the ethnobiological importance of these new taxa to the local tribals.
[Vialle, A., Feau, N., Allaire, M., Didukh, M., Martin, F., Moncalvo, J.-M., & Hamelin, R. C. 2009. Molecular Ecology Resources. 9(s1) 99-113.]
Our study evaluated in silico the potential of 14 mitochondrial genes encoding the subunits of the respiratory chain complexes, including cytochrome c oxidase I (CO1), as Basidiomycota DNA barcode. Fifteen complete and partial mitochondrial genomes were recovered and characterized in this study. Mitochondrial genes showed high values of molecular divergence, indicating a potential for the resolution of lower-level relationships. However, numerous introns occurred in CO1 as well as in six other genes, potentially interfering with polymerase chain reaction amplification. Considering these results and given the minimal length of 600-bp that is optimal for a fungal barcode, the genes encoding for the ATPase subunit 6, the cytochrome oxidase subunit 3 and the NADH dehydrogenase subunit 6 have the most promising characteristics for DNA barcoding among the mitochondrial genes studied. However, biological validation on two fungal data sets indicated that no single mitochondrial gene gave a better taxonomic resolution than the ITS, the region already widely used in fungal taxonomy.
[Gilmore, S. R., Grafenhan, T., Louis-Seize, G., & Seifert, K. A. 2009. Molecular Ecology Resources. 9(s1) 90-98.]
Using data from published mitochondrial or complete genomes, we developed and tested primers for amplification and sequencing of the barcode region of cytochrome oxidase 1 (COX1) of the fungal genus Fusarium, related genera of the order Hypocreales, and degenerate primers for fungi in the subdivision Pezizomycotina. The primers were successful for amplifying and sequencing COX1 barcodes from 13 genera of Hypocreales (Acremonium, Beauveria, Clonostachys, Emericellopsis, Fusarium, Gliocladium, Hypocrea, Lanatonectria, Lecanicillium, Metarhizium, Monocillium, Neonectria and Stilbella), 22 taxa of Fusarium, and two genera in other orders (Arthrosporium, Monilochaetes). Parologous copies of COX1 occurred in several strains of Fusarium. In some, copies of the same length were detected either by heterozygous bases in otherwise clean sequences or in different replicates of amplification and sequencing events; this may indicate multiple transcribed copies. Other strains included one or two introns. Two intron insertion sites had at least two nonhomologous intron sequences among Fusarium species. Irrespective of whether the multiple copy issue could be resolved by sequencing RNA transcripts, developing a precise COX1-based barcoding system for Fusarium may not be feasible. The overall divergence among homologous COX1 sequences obtained so far is rather low, with many species sharing identical sequences.
[Chen, W., Seifert, K. A., & Levesque, C. A. 2009. Molecular Ecology Resources. 9(s1) 114-129.]
We developed a COX1 barcode oligonucleotide array based on 358 sequences, including 58 known and two new species of Penicillium subgenus Penicillium, and 12 allied species. The array was robotically spotted at near microarray density on membranes. Species and clade-specific oligonucleotides were selected using the computer programs SigOli and Array Designer. Robotic spotting allowed 768 spots with duplicate sets of perfect match and the corresponding mismatch and positive control oligonucleotides, to be printed on 2 × 6 cm2 nylon membranes. The array was validated with hybridizations between the array and digoxigenin (DIG)-labelled COX1 polymerase chain reaction amplicons from 70 pure DNA samples, and directly from environmental samples (cheese and plants) without culturing. DNA hybridization conditions were optimized, but undesired cross-reactions were detected frequently, reflecting the relatively high sequence similarity of the COX1 gene among Penicillium species. Approximately 60% of the perfect match oligonucleotides were rejected because of low specificity and 76 delivered useful group-specific or species-specific reactions and could be used for detecting certain species of Penicillium in environmental samples. In practice, the presence of weak signals on arrays exposed to amplicons from environmental samples, which could have represented weak detections or weak cross reactions, made interpretation difficult for over half of the oligonucleotides. DNA regions with very few single nucleotide polymorphisms or lacking insertions/deletions among closely related species are not ideal for oligonucleotide-based diagnostics, and supplementing the COX1-based array with oligonucleotides derived from additional genes would result in a more robust hierarchical identification system.
[Saunders, G. W. 2009. Molecular Ecology Resources. 9(s1) 140-150.]
As part of an extensive DNA-based floristic survey of marine macroalgae in Canadian waters, an unexpected sequence for a Gracilaria sp. was generated from British Columbia. Before further molecular analyses and corresponding morphological/anatomical observations this mystery sequence was temporarily entered into our database as Gracilaria BCsp. Continued sampling uncovered this species from four additional locations. A timely collaboration with international colleagues introduced sequences from the invasive Gracilaria vermiculophylla into our cytochrome c oxidase I alignments 2014 these a perfect match to BCsp indicating that this species occurs in British Columbia. A discussion of the origin of this taxon in Canadian waters, whether natural or introduced, is provided.
[Ragupathy, S., Newmaster, S. G., Murugesan, M., & Balasubramaniam, V. 2009. Molecular Ecology Resources. 9(s1) 164-171.]
Our research brought together traditional aboriginal knowledge (TK) and scientific knowledge (SK) to explore the relationship between scientific and aboriginal systems of botanical classification and the corresponding valorization(s) of biological diversity in the Western Ghats of southern India. We worked with two aboriginal cultures namely 'Irulas' and 'Malasars' of the Nilgiri Biosphere Reserve with an objective of evaluating the ability of different knowledge systems (SK and TK) to distinguish grass species belonging to the genus Tripogon, and assess the ability of DNA barcoding to discriminate a new cryptic species 'Tripogon cope' as deciphered by the hill tribes. We discovered that the aboriginal informants identified a common ethnotaxa 'Sunai pul', which is a cryptic species of grass not recognized by the SK classification.'sunai pul' is very important to both aboriginal cultures with ritualistic and economic utility. Morphometric analysis confirms the cryptic nature of this new species, which was validated using DNA barcoding. DNA barcode regions matK and trnH-psbA showed distinct sequence variations among the closely related ethnotaxa. Given the cryptic nature of ethnotaxa, we propose that a DNA barcode may be a reliable tool to identify ethnotaxa. We have initiated further studies in other cultures to develop theoretically sophisticated insights concerning the encounter between 'local' and 'scientific' approaches to the use of biodiversity knowledge. Furthermore, the research will add to a unifying global effort to speed up the documentation and understanding of the planet's natural diversity, while simultaneously respecting the cultural heterogeneity as a vital component of biological diversity.
[Starr, J. R., Naczi, R. F. C., & Chouinard, B. N. 2009. Molecular Ecology Resources. 9(s1) 151-163.]
We investigate the species discriminatory power of a subset of the proposed plant barcoding loci (matK, rbcL, rpoC1, rpoB, trnH-psbA) in Carex, a cosmopolitan genus that represents one of the three largest plant genera on earth (c. 2000 species). To assess the ability of barcoding loci to resolve Carex species, we focused our sampling on three of the taxonomically best-known groups in the genus, sections Deweyanae (6/8 species sampled), Griseae (18/21 species sampled), and Phyllostachyae (10/10 species sampled). Each group represents one of three major phylogenetic lineages previously identified in Carex and its tribe Cariceae, thus permitting us to evaluate the potential of DNA barcodes to broadly identify species across the tribe and to differentiate closely related sister species. Unlike some previous studies that have suggested that plant barcoding could achieve species identification rates around 90%, our results suggest that no single locus or multilocus barcode examined will resolve much greater than 60% of Carex species. In fact, no multilocus combination can significantly increase the resolution and statistical support (i.e., 2265 70% bootstrap) for species than matK alone, even combinations involving the second most variable region, trnH-psbA. Results suggest that a matK barcode could help with species discovery as 47% of Carex taxa recently named or resolved within cryptic complexes in the past 25 years also formed unique species clusters in upgma trees. Comparisons between the nrDNA internal transcribed spacer region (ITS) and matK in sect. Phyllostachyae suggest that matK not only discriminates more species (50201360% vs. 25%), but it provides more resolved phylogenies than ITS. Given the low levels of species resolution in rpoC1 and rpoB (0201313%), and difficulties with polymerase chain reaction amplification and DNA sequencing in rbcL and trnH-psbA (alignment included), we strongly advocate that matK should be part of a universal plant barcoding system. Although identification rates in this study are low, they can be significantly improved by a regional approach to barcoding.
[Seifert, K. A. 2009. Molecular Ecology Resources. 9(s1) 83-89.]
The use of DNA sequences for identifying fungi and fungus-like organisms predates the DNA barcoding movement by at least 10 years. A brief overview of the mycological shift from phenotypic to molecular taxonomy is provided. Exploration of the animal barcode marker, cytochrome oxidase 1, by Canadian mycologists has been fruitful for some fungi, but intron issues and lack of resolution in other taxa prevent its universal application. The momentum established by 15 years of research on the fungal nuclear ribosomal internal transcribed spacer (ITS) sequences will lead to a proposal to the Consortium for the Barcode of Life on the adoption of this marker as the fungal barcode. Existing mycological research networks should facilitate the rapid development of DNA barcoding of fungi once the marker issue is settled. Some available online fungal identification databases are briefly described.
[Fazekas, A. J., Kesanakurti, P. R., Burgess, K. S., Percy, D. M., Graham, S. W., Barrett, S. C. H., Newmaster, S. G., Hajibabaei, M., & Husband, B. C. 2009. Molecular Ecology Resources. 9(s1) 130-139.]
The ability to discriminate between species using barcoding loci has proved more difficult in plants than animals, raising the possibility that plant species boundaries are less well defined. Here, we review a selection of published barcoding data sets to compare species discrimination in plants vs. animals. Although the use of different genetic markers, analytical methods and depths of taxon sampling may complicate comparisons, our results using common metrics demonstrate that the number of species supported as monophyletic using barcoding markers is higher in animals (> 90%) than plants (~70%), even after controlling for the amount of parsimony-informative information per species. This suggests that more than a simple lack of variability limits species discrimination in plants. Both animal and plant species pairs have variable size gaps between intra- and interspecific genetic distances, but animal species tend to have larger gaps than plants, even in relatively densely sampled genera. An analysis of 12 plant genera suggests that hybridization contributes significantly to variation in genetic discontinuity in plants. Barcoding success may be improved in some plant groups by careful choice of markers and appropriate sampling; however, overall fine-scale species discrimination in plants relative to animals may be inherently more difficult because of greater levels of gene-tree paraphyly.
[Moniz, M. B. J., & Kaczmarska, I. 2009. Molecular Ecology Resources. 9(s1) 65-74.]
The promise of DNA barcoding is based on a small DNA fragment divergence coinciding with biological species separation. Here we evaluated the performance of three markers as diatom barcodes, the small ribosomal subunit (1600 bp), a 5' end fragment of cytochrome c oxidase subunit 1 (430 bp), and the second internal transcribed spacer region combined with the 5.8S gene (5.8S + ITS-2, 3002013400 bp). Forty-four sequences per marker representing 28 species from all diatom classes were analysed. Sequence alignment of the three genetic markers and uncorrected genetic distances (P) were calculated at the intra- and heterospecific level. All three markers correctly separated the species examined and had advantages which contribute to their feasibility as a DNA barcode. Small ribosomal subunit had the largest GenBank data set, its success rate in amplification and sequencing was assumed to be the highest of all three and was readily aligned. However, it required a long fragment to recover divergence sufficient for species separation and small genetic distances increased the potential for misidentifications. Cytochrome c oxidase subunit 1 demonstrated a substantial heterospecific divergence level and was also readily alignable, but it showed very low amplification and sequencing success rates with currently existing primers. 5.8S + ITS-2 was amplified and sequenced with high success rate and was the most variable of the three markers, but its secondary structure was needed to aid in alignment. However, since it has been recently suggested that ITS-2 may provide insight into sexual compatibility, this marker offers an additional advantage. We therefore propose that the 5.8S + ITS-2 fragment is the best candidate as a diatom DNA barcode.
[Ivanova, N. V., Borisenko, A. V., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(s1) 35-41.]
Although devices combining microfluidic and advanced sequencing technologies promise a future where one can generate a DNA barcode in minutes, current analytical regimes typically involve workflows that extend over 2 days. Here we describe simple protocols enabling the advance from a specimen to barcode-based identification in less than 2 h. The protocols use frozen or lyophilized reagents that can be prepackaged into 'kits' and support barcode analysis across the animal kingdom. The analytical procedure allows 5 min for DNA extraction, 25 min for polymerase chain reaction amplification of the barcode region, 25 min for cycle-sequencing, 10 min for cleanup, 45 min for capillary sequencing and 5 min for trace file analysis to complete DNA-based identification. This study involved the comparison of varied DNA preservation and extraction methods, and evaluated Taq polymerases with high processivity and resistance to inhibitors.
[Newmaster, S. G., & Ragupathy, S. 2009. Molecular Ecology Resources,. 9(s1) 172-180.]
Acacia species are quite difficult to differentiate using morphological characters. Routine identification of Acacia samples is important in order to distinguish invasive species from rare species or those of economic importance, particularly in the forest industry. The genus Acacia is quite abundant and diverse comprising approximately 1355 species, which is currently divided into three subgenera: subg. Acacia (c. 161 species), subg. Aculiferum (c. 235 species), and subg. Phyllodineae (c. 960 species). It would be prudent to utilize DNA barcoding in the accurate and efficient identification of acacias. The objective of this research is to test barcoding in discriminating multiple populations among a sister-species complex in pantropical Acacia subg. Acacia, across three continents. Based on previous research, we chose three cpDNA regions (rbcL, trnH-psbA and matK). Our results show that all three regions (rbcL, matK and trnH-psbA) can distinguish and support the newly proposed genera of Vachellia Wight & Arn. from Acacia Mill., discriminate sister species within either genera and differentiate biogeographical patterns among populations from India, Africa and Australia. A morphometric analysis confirmed the cryptic nature of these sister species and the limitations of a classification based on phenetic data. These results support the claim that DNA barcoding is a powerful tool for taxonomy and biogeography with utility for identifying cryptic species, biogeograhic patterns and resolving classifications at the rank of genera and species.
[Borisenko, A. V., Sones, J. E., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(s1) 27-34.]
Building a global library of DNA barcodes will require efficient logistics of pre-laboratory specimen processing and seamless interfacing with molecular protocols. If not addressed properly, the task of aggregating specimens may become the biggest bottleneck in the analytical chain. Three years of experience in developing a collection management system to facilitate high-throughput DNA barcoding have allowed the Canadian Centre for DNA Barcoding to recognize and resolve the most common logistical obstacles. Dealing with these challenges on a larger scale will be an important step towards building a solid collection-based foundation for the international DNA barcoding effort.
[Janzen, D. H., Hallwachs, W., Blandin, P., Burns, J. M., Cadiou, J.-M., Chacon, I., et al. 2009. Molecular Ecology Resources. 9(s1) 1-26.]
Inventory of the caterpillars, their food plants and parasitoids began in 1978 for today's Area de Conservacion Guanacaste (ACG), in northwestern Costa Rica. This complex mosaic of 120 000 ha of conserved and regenerating dry, cloud and rain forest over 020132000 m elevation contains at least 10 000 species of non-leaf-mining caterpillars used by more than 5000 species of parasitoids. Several hundred thousand specimens of ACG-reared adult Lepidoptera and parasitoids have been intensively and extensively studied morphologically by many taxonomists, including most of the co-authors. DNA barcoding 2014 the use of a standardized short mitochondrial DNA sequence to identify specimens and flush out undisclosed species 2014 was added to the taxonomic identification process in 2003. Barcoding has been found to be extremely accurate during the identification of about 100 000 specimens of about 3500 morphologically defined species of adult moths, butterflies, tachinid flies, and parasitoid wasps. Less than 1% of the species have such similar barcodes that a molecularly based taxonomic identification is impossible. No specimen with a full barcode was misidentified when its barcode was compared with the barcode library. Also as expected from early trials, barcoding a series from all morphologically defined species, and correlating the morphological, ecological and barcode traits, has revealed many hundreds of overlooked presumptive species. Many but not all of these cryptic species can now be distinguished by subtle morphological and/or ecological traits previously ascribed to 'variation' or thought to be insignificant for species-level recognition. Adding DNA barcoding to the inventory has substantially improved the quality and depth of the inventory, and greatly multiplied the number of situations requiring further taxonomic work for resolution.
[Zahariev, M., Dahl, V., Chen, W., & Levesque, C. A. 2009. Molecular Ecology Resources. 9(s1) 58-64.]
Efficient design of barcode oligonucleotides can lead to significant cost reductions in the manufacturing of DNA arrays. Previous methods are based on either a preliminary alignment, which reduces their efficiency for intron-rich regions, or on a brute force approach, not feasible for large-scale problems or on data structures with very poor performance in the worst case. One of the algorithms we propose uses 'oligonucleotide sorting' for the discovery of oligonucleotide barcodes of given sizes, with good asymptotic performance. Specific barcode oligonucleotides with at least one base difference from other sequences in a database are found for each individual sequence. With another algorithm, specific oligonucleotides can also be found for groups or clades in the database, which have 100% homology for all oligonucleotide sequences within the group or clade while having differences with the rest of the data. By re-organizing the sequences/groups in the database, oligonucleotides for different hierarchical levels can be found. The oligonucleotides or polymorphism locations identified as species or clade specific by the new algorithm are refined and screened further for hybridization thermodynamic properties with third party software.
[Jakupciak, J. P., & Colwell, R. R 2009. Molecular Ecology Resources. 9(s1) 51-57.]
The challenge for first responders, physicians in the emergency room, public health personnel, as well as for food manufacturers, distributors and retailers is accurate and reliable identification of pathogenic agents and their corresponding diseases. This is the weakest point in biological agent detection capability today. There is intense research for new molecular detection technologies that could be used for very accurate detection of pathogens that would be a concern to first responders. These include the need for sensors for multiple applications as varied as understanding the ecology of pathogenic micro-organisms, forensics, environmental sampling for detect-to-treat applications, biological sensors for 'detect to warn' in infrastructure protection, responses to reports of 'suspicious powders', and customs and borders enforcement, to cite a few examples. The benefits of accurate detection include saving millions of dollars annually by reducing disruption of the workforce and the national economy and improving delivery of correct countermeasures to those who are most in need of the information to provide protective and/or response measures.
[Moszczynska, A., Locke, S. A., McLaughlin, J. D., Marcogliese, D. J., & Crease, T. J. 2009. Molecular Ecology Resources. 9(s1) 75-82.]
The phylum Platyhelminthes is a diverse group of flatworms that includes parasites with serious impacts on human health, animal husbandry, aquaculture and wildlife management. Here we present degenerate primers for the barcode region of the mitochondrial cytochrome c oxidase I (COI) gene in flatworms. Although amplicons were obtained from a wide taxonomic range in the Cestoda and Trematoda, COI fragments from many taxa in these classes did not amplify. Primers specific to trematodes in the family Diplostomidae were also developed. Amplification success was much higher with diplostomid-specific primers and sequences were obtained from 504 of 585 specimens of Diplostomum and Tylodelphys. Sequences from the barcode region resolved all specimens to the species level, with mean divergence between congeners of 19% (3.9201325%). Because many of our specimens were small, we initially amplified part of the nuclear small subunit ribosomal (r) RNA gene to evaluate the quality and quantity of DNA in our specimens. Short sequences (~380 nt) of this gene were recovered from most specimens and can be used to distinguish specimens at the family level and often the generic level. We suggest that rRNA genes could be used to screen samples of completely unknown taxonomy, after which specific COI primers could be used to obtain species-level identifications.
[Gibbs, J. 2009. Journal of Hymenoptera Research. 18 74-79.]
[Rivera, J., & Currie, D. C. 2009. Molecular Ecology Resources. 9(s1) 224-236.]
DNA barcoding has gained increased recognition as a molecular tool for species identification in various groups of organisms. In this preliminary study, we tested the efficacy of a 615-bp fragment of the cytochrome c oxidase I (COI) as a DNA barcode in the medically important family Simuliidae, or black flies. A total of 65 (25%) morphologically distinct species and sibling species in species complexes of the 255 recognized Nearctic black fly species were used to create a preliminary barcode profile for the family. Genetic divergence among congeners averaged 14.93% (range 2.83201315.33%), whereas intraspecific genetic divergence between morphologically distinct species averaged 0.72% (range 020133.84%). DNA barcodes correctly identified nearly 100% of the morphologically distinct species (87% of the total sampled taxa), whereas in species complexes (13% of the sampled taxa) maximum values of divergence were comparatively higher (max. 4.5820136.5%), indicating cryptic diversity. The existence of sibling species in Prosimulium travisi and P. neomacropyga was also demonstrated, thus confirming previous cytological evidence about the existence of such cryptic diversity in these two taxa. We conclude that DNA barcoding is an effective method for species identification and discovery of cryptic diversity in black flies.
[Emery, V. J., Landry, J.-F., & Eckert, C. G. 2009. Molecular Ecology Resources. 9(s1) 217-223.]
Close interactions between insects and plants have played a major role in the evolution of both these diverse groups of organisms. Studying these interactions, however, can be difficult because many insects, especially parasites, impinge most strongly on plants during larval stages when they are morphologically difficult to identify, and many belong to diverse groups for which most species remain undescribed. We used DNA barcoding to identify nondescript lepidopteran larvae that regularly parasitize flower buds of the coastal dune endemic Camissoniopsis cheiranthifolia (Onagraceae). We obtained cytochrome oxidase 1 mitochondrial DNA sequences from 201 parasite specimens from across the host geographical range. The Barcode of Life Database Identification System combined with Bayesian analysis grouped all 15 parasite haplotypes in a distinct, monophyletic clade within the genus Mompha (Lepidoptera: Coleophoridae: Momphinae), a group known to be host specialists on plants of the Onagraceae. Species identity and phylogenetic affinities within Mompha could not be confirmed because few barcode sequences exist from this diverse and poorly known group of moths. However, morphological analysis, including detailed dissection of genitalia for a subsample of 23 reared adults and comparison with known species of Mompha, also indicated that the larvae parasitizing C. cheiranthifolia constitute a distinct and undescribed species within this genus. Knowing that floral parasitism of C. cheiranthifolia involves a single, putatively host-specific microlepidopteran greatly facilitates formulating and testing hypotheses concerning how floral parasitism has promoted the evolution of striking floral diversity within this species. More generally, DNA barcoding combined with morphological analysis can greatly hasten identification of problematic specimens and enhance our understanding of the diversity, ecology and evolution of plant2013insect interactions.
[Zemlak, T. S., Ward, R. D., Connell, A. D., Holmes, B. H., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(s1) 237-242.]
With more than 15 000 described marine species, fishes are a conspicuous, diverse and increasingly threatened component of marine life. It is generally accepted that most large-bodied fishes have been described, but this conclusion presumes that current taxonomic systems are robust. DNA barcoding, the analysis of a standardized region of the cytochrome c oxidase 1 gene (COI), was used to examine patterns of sequence divergence between populations of 35 fish species from opposite sides of the Indian Ocean, chosen to represent differing lifestyles from inshore to offshore. A substantial proportion of inshore species showed deep divergences between populations from South African and Australian waters (mean = 5.10%), a pattern which also emerged in a few inshore/offshore species (mean = 0.84%), but not within strictly offshore species (mean = 0.26%). Such deep divergences, detected within certain inshore and inshore/offshore taxa, are typical of divergences between congeneric species rather than between populations of a single species, suggesting that current taxonomic systems substantially underestimate species diversity. We estimate that about one third of the 1000 fish species thought to bridge South African and Australian waters actually represent two taxa.
[Wong, E. H.-K., Shivji, M. S., & Hanner, R. H. 2009. Molecular Ecology Resources. 9(s1) 243-256.]
Shark fisheries worldwide are mostly unmanaged, but the burgeoning shark fin industry in the last few decades has made monitoring catch and trade of these animals critical. As a tool for molecular species identification, DNA barcoding offers significant potential. However, the genetic distance-based approach towards species identification employed by the Barcode of Life Data Systems may oftentimes lack the specificity needed for regulatory or legal applications that require unambiguous identification results. This is because such specificity is not typically realized by anything less than a 100% match of the query sequence to an entry in the reference database using genetic distance. Although various divergence thresholds have been proposed to define acceptable levels of intraspecific variation, enough exceptions exist to cast reasonable doubt on many less than exact matches using a distance-based approach for the identification of unknowns. An alternative approach relies on the identification of discrete molecular characters that can be used to unambiguously diagnose species. The objective of this study was to assess the performance differences between these competing approaches by examining more than 1000 DNA barcodes representing nearly 20% of all known elasmobranch species. Our results demonstrate that a character-based, nucleotide diagnostic (ND) approach to barcode identification is feasible and also provides novel insights into the structure of haplotype diversity among closely related species of sharks. Considerations for the use of NDs in applied fields are also explored.
[Baker, A. J., Tavares, E. S., & Elbourne, R. F 2009. Molecular Ecology Resources. 9(s1) 257-268.]
General criticisms of a single mtDNA gene barcodes include failure to identify newly evolved species, use of species-delimitation thresholds, effects of selective sweeps and chance occurrence of reciprocal monophyly within species, inability to deal with hybridization and incomplete lineage sorting, and superiority of multiple genes in species identification. We address these criticisms in birds because most species are known and thus provide an ideal test data set, and we argue with selected examples that with the exception of thresholds these criticisms are not problematic for avian taxonomy. Even closely related sister species of birds have distinctive COI barcodes, but it is not possible to universally apply distance thresholds based on ratios of within-species and among-species variation. Instead, more rigorous methods of species delimitation should be favoured using coalescent-based techniques that include tests of chance reciprocal monophyly, and times of lineage separation and sequence divergence. Incomplete lineage sorting is also easily detected with DNA barcodes, and usually at a younger time frame than a more slowly evolving nuclear gene. Where DNA barcodes detect divergent reciprocally monophyletic lineages, the COI sequences can be combined with multiple nuclear genes to distinguish between speciation or population subdivision arising from high female philopatry or regional selective sweeps. Although selective sweeps are increasingly invoked to explain patterns of shallow within-species coalescences in COI gene trees, caution is warranted in this conjecture because of limited sampling of individuals and the reduced power to detect additional mtDNA haplotypes with one gene.
[Sheffield, C. S., Hebert, P. D. N., Kevan, P. G., & Packer, L. 2009. Molecular Ecology Resources. 9(s1) 196-207.]
DNA barcoding has been evaluated for many animal taxa and is now advocated as a reliable and rapid means for species-level identification. The coming-to-light of this identification tool is timely as we are now facing perhaps the greatest rate of species loss in recent millennia. This study contributes to an ever-increasing number of published accounts of DNA barcoding successfully and accurately distinguishing animal taxa, in this instance, the bee fauna of Nova Scotia, Canada. Most members of this well-known fauna were resolved with particular clarity; the average intraspecific divergence was less than 0.5%, and COI sequences from over 75% of the province's species are now in the Barcodes of Life Data System. DNA barcoding also revealed some surprises within this fauna, including the possible recognition of two undescribed genetically unique species, one in the genus Ceratina (subgenus Zadontomerus), the second in the genus Andrena (subgenus Larandrena); both are presently receiving further taxonomic study. In addition, DNA barcoding has allowed sex-associations among two pairs of cleptoparasitic species. The resulting utility of DNA barcoding for ecological studies of bee communities is discussed.
[Smith, M. A., Fernandez-Triana, J., Roughley, R., & Hebert, P. D. N. 2009. Molecular Ecology Resources. 9(s1) 208-216.]
Frequently, the diversity of umbrella taxa is invoked to predict patterns of other, less well-known, life. However, the utility of this strategy has been questioned. We tested whether a phylogenetic diversity (PD) analysis of CO1 DNA barcodes could act as a proxy for standard methods of determining sampling efficiency within and between sites, namely that an accumulation curve of barcode diversity would be similar to curves generated using morphology or nuclear genetic markers. Using taxa at the forefront of the taxonomic impediment 2014 parasitoid wasps (Ichneumonidae, Braconidae, Cynipidae and Diapriidae), contrasted with a taxon expected to be of low diversity (Formicidae) from an area where total diversity is expected to be low (Churchill, Manitoba), we found that barcode accumulation curves based on PD were significantly different in both slope and scale from curves generated using names based on morphological data, while curves generated using nuclear genetic data were only different in scale. We conclude that these differences clearly identify the taxonomic impediment within the strictly morphological alpha-taxonomy of these hyperdiverse insects. The absence of an asymptote within the barcode PD trend of parasitoid wasps reflects the as yet incomplete sampling of the site (and more accurately its total diversity), while the morphological analysis asymptote represents a collision with the taxonomic impediment rather than complete sampling. We conclude that a PD analysis of standardized DNA barcodes can be a transparent and reproducible triage tool for the management and conservation of species and spaces.
http://www3.interscience.wiley.com/cgi-bin/fulltext/122342777/HTMLSTART
[Gentekaki, E., & Lynn, D. H. 2009. Appl Environ Microbiol,. 75(10) 3187-3195.]
Studies that assess intraspecific genetic variation in ciliates are few and quite recent. Consequently, knowledge of the subject and understanding of the processes that underlie it are limited. We sought to assess the degree of intraspecific genetic variation in Carchesium polypinum (Ciliophora: Peritrichia), a cosmopolitan, freshwater ciliate. We isolated colonies of C. polypinum from locations in the Grand River basin in Southwestern Ontario, Canada. We then used the nuclear markers--ITS1, ITS2, and the hypervariable regions of the large subunit rRNA--and an 819-bp fragment of the mitochondrial cytochrome c oxidase I gene (cox-1) to investigate the intraspecific genetic variation of C. polypinum and the degree of resolution of the above-mentioned markers at the population level. We also sought to determine whether the organism demonstrated any population structure that mapped onto the geography of the region. Our study shows that there is a high degree of genetic diversity at the isolate level, revealed by the mitochondrial markers but not the nuclear markers. Furthermore, our results indicate that C. polypinum is likely not a single morphospecies as previously thought.
[Packer, L., Gibbs, J., Sheffield, C., & Hanner, R. 2009. Molecular Ecology Resources. 9(s1) 42-50.]
A small but vocal community of critics has questioned the epistemological value of DNA barcoding by suggesting that either it 'cannot work' for the identification or discovery of species or that it ignores the 'richness' inherent in traditional approaches. We re-examine these arguments through a comparison of DNA barcoding and morphological taxonomy in terms of their accuracy and diversity of characters employed. We conclude that morphology often does not work and that it is often nowhere near as 'rich' as has been argued. Morphology is particularly poor in numerous important situations, such as the association of larvae with adults and discrimination among cryptic species. The vehemence of some of the criticisms is surprising given that morphology alone is known to be inadequate to the task of species-level identification in many instances.
[Newmaster, S. G., & Ragupathy, S. 2009. Indian Journal of Science and Technology. 2(5) 2-8.]
The ethnobotany genomics concept is founded on the idea of ‘assemblage’ of biodiversity knowledge. This includes a coming together of different ways of knowing and valorizing species variation in a novel approach seeking to add value to both traditional knowledge (TK) and scientific knowledge (SK). Ethnobotany genomics is defined as exploring the variation in genomic sequences from many species, and here we present some of our recent work that demonstrates the potential benefits of this approach for ethnobotanical research with economic implications. DNA barcoding was used to identify Acacia and nutmeg taxa that are economically important to society-at-large. Furthermore we identified considerable variation that is recognized by several indigenous cultures. The impacts of ethnobotany genomics will extend well beyond biodiversity science. Explorations of the genomic properties across the expanse of life are now possible using DNA barcoding to assemble sequence information for a standard portion of the genome from large assemblages of species. Perhaps the most important contribution is major barcode projects will leave an important legacy; a comprehensive repository of highquality DNA extracts that will facilitate future genomic investigations.
[Foottit, R. G., Maw, H. E. L., Havill, N. P., Ahern, R. G., & Montgomery, M. E. 2009. Molecular Ecology Resources,. 9(s1) 188-195.]
The Adelgidae are relatively small, cryptic insects, exhibiting complex life cycles with parthenogenetic reproduction. Due to these characteristics, the taxonomy of the group is problematic. Here, we test the effectiveness of the standard 658-bp barcode fragment from the 5'-end of the mitochondrial cytochrome c oxidase 1 gene (COI) in differentiating among 17 species of Adelgidae, in associating life-cycle stages, and in assessing patterns of geographical variation in selected species. Species of Adelgidae are well-differentiated by DNA barcodes, enabling the identification of different morphological forms, immature stages and individuals on different hosts and at different periods of the life cycle. DNA barcodes have uncovered cryptic diversity within taxa and, in other cases, a lack of sequence divergence in species pairs previously separated by life-cycle characteristics, indicating a need for further taxonomic analysis.
[Radulovici, A. E., Sainte-Marie, B., & Dufresne, F. 2009. Molecular Ecology Resources. 9(s1) 181-187.]
Marine crustaceans are known as a group with a high level of morphological and ecological diversity but are difficult to identify by traditional approaches and usually require the help of highly trained taxonomists. A faster identification method, DNA barcoding, was found to be an effective tool for species identification in many metazoan groups including some crustaceans. Here we expand the DNA barcode database with a case study involving 80 malacostracan species from the Estuary and Gulf of St Lawrence. DNA sequences for 460 specimens grouped into clusters corresponding to known morphological species in 95% of cases. Genetic distances between species were on average 25 times higher than within species. Intraspecific divergence was high (3.78201313.6%) in specimens belonging to four morphological species, suggesting the occurrence of cryptic species. Moreover, we detected the presence of an invasive amphipod species in the St Lawrence Estuary. This study reconfirms the usefulness of DNA barcoding for the identification of marine crustaceans.
[Newmaster, S. G., Ragupathy, S., & Janovec, J. 2009. International Journal of Computer Applications in Technology. 35(1) 50-60.]
Traditional taxonomic practices are insufficient on their own to cope with the growing need for accurate identifications. The recent development of DNA barcoding has been applied to plants. The next step is the development of a high-throughput Automated Identification Technology (AIT) system. Our research indicates that the efficacy of an AIT system equates with savings in time and funding. Given the potential interconnectivity of web-based applications, we suggest an AIT system for plants that uses several existing systems and suggest several applications where AIT could serve as a tool for biologists and for society at large.
[Cohen, N. J., Deeds, J. R., Wong, E. S., Hanner, R. J., Yancy, H. F., White, K. D., Thompson, T. M., Wahl, M., Pham, T. D., Guichard, F. M., Huh, I., Austion, C., Dizikes, G., & Gerber, S. I. 2009. Journal of Food Protection. 72(4) 810-817.]
Tetrodotoxin is a neurotoxin that occurs in select species of the family Tetraodontidae (puffer fish). It causes paralysis and potentially death if ingested in sufficient quantities. In 2007, two individuals developed symptoms consistent with tetrodotoxin poisoning after ingesting home-cooked puffer fish purchased in Chicago. Both the Chicago retailer and the California supplier denied having sold or imported puffer fish but claimed the product was monkfish. However, genetic analysis and visual inspection determined that the ingested fish and others from the implicated lot retrieved from the supplier belonged to the family Tetraodontidae. Tetrodotoxin was detected at high levels in both remnants of the ingested meal and fish retrieved from the implicated lot. The investigation led to a voluntary recall of monkfish distributed by the supplier in three states and placement of the supplier on the U.S. Food and Drug Administration’s Import Alert for species misbranding. This case of tetrodotoxin poisoning highlights the need for continued stringent regulation of puffer fish importation by the U.S. Food and Drug Administration, education of the public regarding the dangers of puffer fish consumption, and raising awareness among medical providers of the diagnosis and management of foodborne toxin ingestions and the need for reporting to public health agencies.
[Boulding, E. G., deWaard, J. R., Ang, K. P., & Hebert, P. D. N. 2009. Aquaculture Research. 40(8) 973-979.]
[Wilson, John J., Floyd, R., Hanner, R. H., & Castle, D. 2009. Isis. 100(1) 117.]
[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.]
[Gibbs, J. 2009. Zootaxa. 2032 1-38.]
An integrative taxonomic approach that utilizes the DNA barcode region of cytochrome c oxidase subunit 1 in conjunction with traditional morphological approaches identifies five distinct species previously recognized as Lasioglossum (Dialictus) tegulare (Robertson). Differences in DNA sequences and congruent, albeit minor, morphological variation support separation of L. tegulare into five species. Unique nucleotide substitution patterns for each species allows for character-based diagnostics using DNA barcodes. The names L. ellisiae (Sandhouse) and L. lepidii (Graenicher) are removed from synonymy. Two new species, L. puteulanum Gibbs sp. n. and L. carlinvillense Gibbs sp. n., are described. A key is provided, which permits the identification of both males and females. The utility of the DNA barcode region as part of an integrative taxonomic framework is discussed.
[Feau, N., Vialle, A., Allaire, M., Tanguay, P., Joly, D. L., Frey, P., Callan, B. E., & Hamelin, R. C 2009. Mycological Research. 113(6-7) 713-724.]
Wide variation and overlap in morphological characters have led to confusion in species identification within the fungal rust genus Melampsora. The Melampsora species with uredinial–telial stages on white poplar and aspens are especially prone to misidentification. This group includes the Melampsora populnea species complex and the highly destructive pine twisting rust, Melampsora pinitorqua, which alternates between hosts in Populus section Populus and Pinus. Our objective was to compare morphologically based identification to genetic material extracted from Melampsora species pathogenic to aspen and white poplar. We compared morphometric traits and DNA barcodes obtained from internal transcribed spacer (ITS), large ribosomal RNA subunit (28S), and mitochondrial cytochrome oxidase 1 (CO1) sequences to delimit within this taxonomically difficult group. Eight different Melampsora species were initially defined based on host specificity and morphometric data. DNA barcodes were then overlaid on these initial species definitions. The DNA barcodes, specifically those defined on ITS and 28S sequences, provided a highly accurate means of identifying and resolving Melampsora taxa. We highlighted species misidentification in specimens from Canadian herbaria related to either Melampsora medusae f. sp. tremuloidae or Melampsora aecidioides. Finally, we evidenced that the north-American species found on Populus alba, M. aecidioides is closely related but distinct from the four species of the M. populnea complex (Melampsora larici-tremulae, Melampsora magnusiana, Melampsora pinitorqua, and Melampsora rostrupii) found in Eurasia.
[Ward, R. D., Hanner, R., & Hebert, P. D. N. 2009. Journal of Fish Biology. 74(2) 329-356.]
FISH-BOL, the Fish Barcode of Life campaign, is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes. Analysis is targeting a 648 base pair region of the mitochondrial cytochrome c oxidase I (COI) gene. More than 5000 species have already been DNA barcoded, with an average of five specimens per species, typically vouchers with authoritative identifications. The barcode sequence from any fish, fillet, fin, egg or larva can be matched against these reference sequences using BOLD; the Barcode of Life Data System (http://www.barcodinglife.org). The benefits of barcoding fishes include facilitating species identification, highlighting cases of range expansion for known species, flagging previously overlooked species and enabling identifications where traditional methods cannot be applied. Results thus far indicate that barcodes separate c. 98 and 93% of already described marine and freshwater fish species, respectively. Several specimens with divergent barcode sequences have been confirmed by integrative taxonomic analysis as new species. Past concerns in relation to the use of fish barcoding for species discrimination are discussed. These include hybridization, recent radiations, regional differentiation in barcode sequences and nuclear copies of the barcode region. However, current results indicate these issues are of little concern for the great majority of specimens.
[Elsasser, S. C., Floyd, R., Hebert, P. D. N., & Schulte-Hostedde, A. I. 2009. Molecular Ecology Resources. Online Early .]
Dracunculus insignis is a nematode parasite that infects the subcutaneous tissues of mammals such as raccoon (Procyon lotor), mink (Neovison vison) and fisher (Martes pennanti). D. lutrae, a morphologically similar species, has only been recovered from the otter (Lontra canadensis). Species identification of these two North American guinea worms has only been achieved by morphology of males and host identity. As a result, where only female specimens are present, accurate identifications are not possible. To date, specimens recovered from otter have been assumed to be D. lutrae, while those from all other hosts are assumed to be D. insignis. This study uses DNA barcoding to differentiate between these two North American dracunculoids. Our results show that D. insignis is a 'true' generalist, showing little sequence divergence regardless of host association, although our studies did validate its occurrence in a new host 2014 the otter. Interestingly, specimens of the host specialist, D. lutrae, showed some sequence divergence, although it was low. The finding of D. insignis in otter substantiates the need to supplement morphology-based methods in providing species identifications for certain dracunculoids.
[Holmes, B. H., Steinke, D., & Ward, R. D. 2009. Fisheries Research. 95(2-3) 280-288.]
Fisheries managers and scientists worldwide are struggling with a lack of basic information for many shark and ray species. One factor hampering the data collection is inaccurate identification of many chondrichthyan species and their body parts. Morphologically similar species, and specimens which are poorly preserved or have had key diagnostic features removed, can be difficult to identify. This study examined DNA barcoding as a method to identify shark species from dried fins, confiscated from a vessel fishing illegally in Australian waters. 211 left pectoral fins were examined. 18 either did not provide a sequenceable product or yielded a microbial sequence, while 193 fins (91.5%) provided a chondrichthyan sequence. All of these could be matched to reference specimens in a DNA barcode database, and so were able to be identified. 27 species were detected, 20 species of sharks and seven species of rays The most abundant species (22% of fins) was Carcharhinus dussumieri. Many of these species are listed on the World Conservation Union (IUCN) Red List and include one, Anoyxpristis cuspidata (3%), rated as critically endangered. Fishing authorities can use DNA barcoding to gather data on which chondrichthyan species are targeted by illegal fishers, information that will greatly assist in management and conservation.
[Gibbs, J. & Sheffield, C. S. 2009. Journal of the Kansas Entomological Society. 82(1) 21-29.]
Anthidium manicatum (L.) is an adventive species of European origin first recorded in North America in the late 1960’s; from that point until 2001 its range on the continent was restricted to the northeast central USA and central Canada (Ontario, more recently Que´bec). In 2005, this species was reported from Nova Scotia, a rapid and wide increase in its distribution. In this paper, we document a similar rapid spread of A. manicatum into western North America, including British Columbia and Idaho, and discuss the potential risks of this species in eastern Canada. In addition, the potential of DNA barcoding as a rapid and reliable means of recognizing adventive bee species is advocated.
[Chantangsi, C., & Lynn, D. H. 2008. Mol Phylogenet Evol. 49(3) 979-987.]
Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three "classical" morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.
[Seifert, K. A. 2008. Persoonia. 21 162-166.]
[Albu, M., Min, X. J., Hickey, D., & Golding, B. 2008. Mol Biol Evol. 25(12) 2521-2524.]
The relative rates of nucleotide substitution at synonymous and nonsynonymous sites within protein-coding regions have been widely used to infer the action of natural selection from comparative sequence data. It is known, however, that mutational and repair biases can affect rates of evolution at both synonymous and nonsynonymous sites. More importantly, it is also known that synonymous sites are particularly prone to the effects of nucleotide bias. This means that nucleotide biases may affect the calculated ratio of substitution rates at synonymous and nonsynonymous sites. Using a large data set of animal mitochondrial sequences, we demonstrate that this is, in fact, the case. Highly biased nucleotide sequences are characterized by significantly elevated dN/dS ratios, but only when the nucleotide frequencies are not taken into account. When the analysis is repeated taking the nucleotide frequencies at each codon position into account, such elevated ratios disappear. These results suggest that the recently reported differences in dN/dS ratios between vertebrate and invertebrate mitochondrial sequences could be explained by variations in mitochondrial nucleotide frequencies rather than the effects of positive Darwinian selection.
[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.
[De Astarloa, J. M. D., Mabragana, E., Hanner, R., & Figueroa, D. E 2008. Zootaxa. 1921 35-46.]
A new species of Dipturus is described from ten specimens collected off Patagonia, Argentina. Morphological and molecular approaches were used to compare among specimens of recognized Dipturus species. By comparing morphometric, meristic and mitochondrial cytochrome c oxidase I (COI) sequence data, specimens referred to as longnose skate and originally regarded as D. chilensis were shown to be a discrete species as distinguished from both the Yellownose skate, D. chilensis and the Roughskin skate, D. trachyderma. Dipturus argentinensis n. sp. can be distinguished from all other southwestern Atlantic longnose skate species by its color pattern, lack of squamation on both upper and lower surfaces of the disc, and a long, thin tail that is approximately half the total length. The new species has one median row of 10 to 24 small caudal thorns, one or two interdorsal thorns and 35 to 40, and 34 to 43 tooth rows on upper and lower jaws, respectively. The 648 base pair COI mitochondrial DNA “barcodes” derived from specimens of D. argentinensis are identical to each other and exhibit greater than 3% sequence divergence from all other Dipturus species similarly characterized to date. Taken together, these independent morphological and molecular observations serve to corroborate one another and thus provide strong evidence for the recognition of D. argentinensis as a new species
[Cheung, F., Win, J., Lang, J.M., Hamilton, J., Vuong, H., Leach, J.E., Kamoun, S., Lévesque, C.A., Tisserat, N., and Buell, C.R. 2008. BMC Genomics. 9 542.]
BACKGROUND: Pythium species are an agriculturally important genus of plant pathogens, yet are not understood well at the molecular, genetic, or genomic level. They are closely related to other oomycete plant pathogens such as Phytophthora species and are ubiquitous in their geographic distribution and host rage. To gain a better understanding of its gene complement, we generated Expressed Sequence Tags (ESTs) from the transcriptome of Pythium ultimum DAOM BR144 (= ATCC 200006 = CBS 805.95) using two high throughput sequencing methods, Sanger-based chain termination sequencing and pyrosequencing-based sequencing-by-synthesis. RESULTS: A single half-plate pyrosequencing (454 FLX) run on adapter-ligated cDNA from a normalized cDNA population generated 90,664 reads with an average read length of 190 nucleotides following cleaning and removal of sequences shorter than 100 base pairs. After clustering and assembly, a total of 35,507 unique sequences were generated. In parallel, 9,578 reads were generated from a library constructed from the same normalized cDNA population using dideoxy chain termination Sanger sequencing, which upon clustering and assembly generated 4,689 unique sequences. A hybrid assembly of both Sanger- and pyrosequencing-derived ESTs resulted in 34,495 unique sequences with 1,110 sequences (3.2%) that were solely derived from Sanger sequencing alone. A high degree of similarity was seen between P. ultimum sequences and other sequenced plant pathogenic oomycetes with 91% of the hybrid assembly derived sequences > 500 bp having similarity to sequences from plant pathogenic Phytophthora species. An analysis of Gene Ontology assignments revealed a similar representation of molecular function ontologies in the hybrid assembly in comparison to the predicted proteomes of three Phytophthora species, suggesting a broad representation of the P. ultimum transcriptome was present in the normalized cDNA population. P. ultimum sequences with similarity to oomycete RXLR and Crinkler effectors, Kazal-like and cystatin-like protease inhibitors, and elicitins were identified. Sequences with similarity to thiamine biosynthesis enzymes that are lacking in the genome sequences of three Phytophthora species and one downy mildew were identified and could serve as useful phylogenetic markers. Furthermore, we identified 179 candidate simple sequence repeats that can be used for genotyping strains of P. ultimum. CONCLUSION: Through these two technologies, we were able to generate a robust set (approximately 10 Mb) of transcribed sequences for P. ultimum. We were able to identify known sequences present in oomycetes as well as identify novel sequences. An ample number of candidate polymorphic markers were identified in the dataset providing resources for phylogenetic and diagnostic marker development for this species. On a technical level, in spite of the depth possible with 454 FLX platform, the Sanger and pyro-based sequencing methodologies were complementary as each method generated sequences unique to each platform.
[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.
[Newmaster, S. G., Balasubramaniam, V., Murugesan, M. & Ragupathy, S. 2008. Systematic Botany. 33(4) 695-701.]
Tripogon cope Newmaster S. G., V. Balalasubramaniam, M. Murugesan, & S. Ragupathy a new species from South India, is described and illustrated. A key for the identification of all Indian Tripogon species is included. A detrended correspondence analysis identified 21 groups of taxa including the sp. novum from the 48 samples, analyzing 36 morphological characters. A discriminant function analysis was used to rigorously test the classification of specimens provided in the cluster analysis. This study provides preliminary evidence of morphometric variation within and among species of Tripogon, which allows further development of hypothesis concerning species boundaries. Discussions concerning ecological data and distribution are presented in the context of conservation initiatives of rare and endemic Tripogon taxa within India.
[Isabelle Meusnier, Gregory AC Singer, Jean-Francois Landry, Donal A Hickey, Paul DN Hebert and Mehrdad Hajibabaei 2008. BioMed Central. 9:214 .]
Background: The goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed).
Results: We used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens.
Conclusion: In this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.
[Gibbs, J. 2008. Journal of the Kansas Entomological Society. In Press 12pp.]
[Oliver, K. L., Hamelin, R. C., & Hintz, W. E. 2008. Appl Environ Microbiol,. 74(17) 5340-5348.]
This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.
[de Cock, A. W., Levesque, C. A., Melero-Vara, J. M., Serrano, Y., Guirado, M. L., & Gomez, J. 2008. Mycological Research. 112(Pt9) 1115-1121.]
A new disease causing wilt and death of adult plants of Phaseolus vulgaris was discovered in plastic-house crops of southeast Spain in 2004. The causal agent was shown to be a Pythium species with a unique type of oogonium ornamentation different from any of the described species. Zoospores were not observed, but globose or subglobose hyphal swellings, intercalary or terminal, were frequently found. Moreover, the ribosomal ITS region showed a unique sequence, significantly different (>14%) from any other known species of Pythium. This paper describes and illustrates the morphology of the new Pythium species and its pathogenicity to green beans. Its taxonomic position and phylogenetic relationships with other Pythium species are discussed.
[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.
[Bergeron, M. J., Hamelin, R. C., Leal, I., Davis, C., & de Groot, P. 2008. Plant Disease. 92(7) 1138.]
Amylostereum areolatum (Fr.) Boidin (Russulales: Stereaceae) is a white rot fungus that has a symbiotic relationship with several woodwasps including Sirex noctilio Fabricius (Hymenoptera: Siricidae). The vectored fungus together with a phytotoxic mucus, both injected during oviposition by the female S. noctilio, rapidly weaken the host tree, rendering it susceptible to larval development (3). Host trees of A. areolatum include species of Pinus (mainly), Abies, Larix, and Picea and Cryptomeria japonica and Pseudotsuga menziesii (Fungal Databases [online]; USDA). The siricid woodwasp is native to Eurasia and North Africa and has been introduced into New Zealand, Australia, South America, and South Africa (1). In July of 2005, the first established North American population of S. noctilio was reported in Oswego, NY. Prompted by this initial discovery, a trap survey of Ontario counties located along the Canada-U.S. border, close to Upstate New York, was conducted in September and October of 2005. S. noctilio females were captured in four locations in southern Ontario. Two additional locations for S. noctilio were also reported in a survey conducted independently (2). In September and October of 2006, logs of Scots pines showing current Sirex oviposition sites were harvested from the Ontario area bordered by Lakes Huron, Erie, and Ontario to determine the presence of the species-specific fungal symbiont of S. noctilio, A. areolatum. Fungal isolates were obtained by surface sterilizing wood chips showing decay columns followed by incubation at 20°C on 2% malt extract agar. Cultures with morphological characteristics typical of A. areolatum–presence of clamp connections and arthrospores–were used for DNA analysis to confirm species identification. DNA sequences of the internal transcribed spacer (ITS) of the ribosomal RNA gene were queried against the NCBI GenBank database. There was a 99 to 100% match between the ITS sequences from the Ontario isolates and sequences from European and Asian A. areolatum isolates (GenBank Accession Nos. EU249343 and EU249344 versus AF454428, AF506405, AY781245, and AF218389). Matches with A. chailletii (Pers.) Boidin, a native related species, were around 97%. These results confirmed the presence of A. areolatum in the infested material. Cultures were deposited in the National Mycological Herbarium of Canada (DAOM 239280–DAOM 239295). To our knowledge, this represents the first report of A. areolatum in Canada. In its natural range, the insect-fungal complex exists in equilibrium with its host trees and parasites, thus, few negative impacts are observed. However, in the Southern Hemisphere where it has been introduced, it has become a major pest, attacking many important commercial North American species planted as exotics (1). Conifer forests in Canada are threatened by the spread of the S. noctilio/A. areolatum complex, particularly plantations and stands of Pinus banksiana, P. contorta, P. ponderosa, P. resinosa, P. strobus, and P. sylvestris. A survey of Eastern Canada to detect the presence of S. noctilio is on going, and genetics work is being conducted to determine the origin of the introduction of A. areolatum.
[Kaczmarska, I., Reid, C., Martin, J. L., & Moniz, M. B. J. 2008. Botany. 86(7) 763-772.]
Forty-six monoclonal cultures of the diatom Pseudo-nitzschia delicatissima (Cleve) Heiden were isolated from coastal waters of Eastern Canada. Of these, 12 clones were successfully sexualized. The range of their morphological and genetic divergence was used as a reference for clones whose sexual identity remains unknown. All characters that were examined, including valve morphology, the nuclear internal transcribed spacer (ITS) region, and mitochondrial cytochrome c oxidase (cox1) sequences, showed a high degree of similarity within and between mating and nonsexualized clones. Within the 638 bp long aligned fragment in the ITS region, only five variable sites were found and just two were found within the 576 bp fragment of cox1 near the 5′ terminus of the gene. Our own data and those retrieved from GenBank suggest that the northern North Atlantic is populated by a single metapopulation of genetically very similar P. delicatissima, as determined using the ITS sequence of the epitype of the species. The ITS region of our clones was distinct from ITS-types present in isolates that we will refer to as P. delicatissima-like diatoms from the Mediterranean Sea and other low latitude Atlantic sites, thereby providing a means to discriminate between otherwise morphologically indistinguishable (cryptic) species. Such a distribution pattern suggests different physiological and environmental requirements for mating optima. This work furthers our understanding of the relationship between biological, molecular, and morphological species boundaries in diatoms and their ecology, and contributes to evaluation of the utility of ITS and cox1 sequences in DNA barcoding of diatoms.
[Saunders, G. W. 2008. Botany. 86(7) 773-789.]
The field of DNA barcoding is working towards generating a genetic system for the quick and accurate identification of eukaryotic species. For the more systematic minded, however, DNA barcoding offers a new approach towards screening and uniting large numbers of biological specimens in genetic groups as a first step towards assigning them to species and genera in an approach best termed “molecular-assisted alpha taxonomy”. This approach is particularly amenable in organisms with simple morphologies, a propensity for convergence, extensive phenotypic plasticity, and life histories with an alternation of heteromorphic generations. It is hard to imagine a group of organisms better defined by all of these traits than the marine macroalgae. In an effort to assess the utility of the DNA barcode (COI-5′) for testing the current concepts of biodiversity of marine macroalgae in Canada, a study to assess species diversity in the red algal family, Dumontiaceae, was initiated. Through this work I confirm the presence in Canadian waters of Dilsea californica (J. Agardh) Kuntze, Dilsea integra (Kjellman) Rosenvinge, and Neodilsea borealis (I.A. Abbott) Lindstrom of the Dilsea–Neodilsea complex, and Weeksia coccinea (Harvey) Lindstrom for the genus Weeksia. However, our work has uncovered two additional species of the former complex, Dilsea lindstromiae Saunders sp. nov. and Dilsea pygmaea (Setchell) Setchell, and an additional species of the latter, Weeksia reticulata Setchell, effectively doubling representation of these foliose dumontiacean genera in Canadian waters.
[Newmaster, S.G., A.J. Fazekas, R.A.D. Steeves, and J. Janovec 2008. Molecular Ecology Resources. 8(3) 480-490.]
The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon — UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH-psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a more variable region (trnH-psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis.
[Field, D., Morrison, N., Glockner, F. O., Kottmann, R., Cochrane, G., Vaughan, R., Garrity, G., Cole, J., Hirschman, L., Schriml, L., Schindel, D., Miller, S., Hebert, P., Ratnasingham, S., Hanner, R., Amaral-Zettler, L., Sogin, M., Ashburner, M., Lewis, 2008. Nature. 453 978.]
[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.
[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.
[Evans, B. J., Carter, T. F., Tobias, M. L., Kelley, D. B., Hanner, R., & Tinsley, R. C. 2008. Zootaxa. 1780 55-68.]
Here we describe a new octoploid species of clawed frog from the Itombwe Massif of South Kivu Province, Democratic Republic of the Congo. This new species is the sister taxon of Xenopus wittei, but is substantially diverged in morphology, male vocalization, and mitochondrial and autosomal DNA. Analysis of mitochondrial “DNA barcodes” in polyploid clawed frogs demonstrates that they are variable between most species, but also reveals limitations of this type of information for distinguishing closely related species of differing ploidy level. The discovery of this new species highlights the importance of the Itombwe Massif for conservation of African biodiversity south of the Sahara.
[Clayden, S. L., & Saunders, G. W. 2008. European Journal of Phycology. 43(2) 151-160.]
An isolate of the filamentous red alga Acrochaetium efflorescens from the NE Atlantic was followed in culture through a triphasic sexual life history. Morphology of each phase matched previous descriptions for this alga. However, inclusion of A. efflorescens in the genus Acrochaetium is problematic. In current taxonomic treatments this genus includes acrochaetioid algae with a stellate plastid and a large central pyrenoid. Acrochaetium efflorescens, on the other hand, has consistently been reported to possess spiral to ribbon-shaped plastids. To determine the phylogenetic affinities of A. efflorescens, we sequenced large subunit ribosomal DNA and resolved full support (Bayesian posterior probabilities; maximum likelihood, neighbour joining, and parsimony bootstrap percentages) for inclusion of A. efflorescens within the order Acrochaetiales. However, it does not associate closely with any of the existing genera in this order, showing only a weak affinity to the genus Rhodochorton. The genus Grania, originally established by Kylin, is currently available for this species. Given its distinct anatomy (ribbon-shaped plastids in combination with seriate carposporangia), unique molecular signature, and lack of congruence with other current generic concepts within the Acrochaetiales, we advocate resurrecting the genus Graniafor G. efflorescens.
| [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. |  |
[Moncalvo, J. M., & Buchanan, P. K. 2008. Mycological Research. 112(Pt4) 425-36.]
We examined phylogeographic relationships in the cosmopolitan polypore fungus Ganoderma applanatum and allies, and conservatively infer a possible age of origin for these fungi. Results indicate that it is very unlikely that members of this species complex diversified before the break-up of Gondwana from Laurasia ca 120M years ago, and also before the final separation of the Gondwanan landmasses from each other that was achieved about 66M years ago. An earliest possible age of origin of 30M years was estimated from nucleotide substitution rates in the 18S rDNA gene. Phylogenetic reconstruction of a worldwide sampling of ITS rDNA sequences reveals at least eight distinct clades that are strongly correlated with the geographic origin of the strains, and also correspond to mating groups. These include one Southern Hemisphere clade, one Southern Hemisphere-Eastern Asia clade, two temperate Northern Hemisphere clades, three Asian clades, and one neotropical clade. Geographically distant collections from the Southern Hemisphere shared identical ITS haplotypes, and an ITS recombinant was noted. Nested clade analysis of a parsimony network among isolates of the Southern Hemisphere clade indicated restricted gene flow with isolation-by-distance among the New Zealand, Australia-Tasmania, Chile-Argentine, and South Africa populations, suggesting episodic events of long-distance dispersal within the Southern Hemisphere. This study indicates that dispersal bias plays a more important role than generally admitted to explain the Southern Hemisphere distribution of many taxa, at least for saprobic fungi.
[Chang, C.-H., Rougerie, R., & Chen, J.-H. 2008. Pedobiologia. 52(3) 171-180.]
This paper re-evaluated the use of DNA barcodes in earthworm species identification by re-analyzing sequence data for the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. This analysis unveiled and confirmed taxonomic inconsistencies which significantly affect data interpretation. When considering synonymy and misidentification in published records, our results revealed no shared COI haplotypes between morphologically distinct species and higher interspecific than intraspecific divergence in most cases, with interspecific and intraspecific distances averaging 18.7% and 1.3% respectively. However, a few earthworm species endemic to Taiwan have deep intraspecific divergences which may represent potential cases of cryptic diversity, although incomplete lineage sorting cannot be ruled out without further study. We recognize the potential of DNA barcoding for earthworm taxonomy, but have identified several issues regarding the evolution of the COI gene in these organisms which remain to be further elucidated.
[Tavares, E. S., & Baker, A. J. 2008. BMC Evol Biol. 8 81.]
BACKGROUND: DNA barcoding of life using a standardized COI sequence was proposed as a species identification system, and as a method for detecting putative new species. Previous tests in birds showed that individuals can be correctly assigned to species in ~94% of the cases and suggested a threshold of 10x mean intraspecific difference to detect potential new species. However, these tests were criticized because they were based on a single maternally inherited gene rather than multiple nuclear genes, did not compare phylogenetically identified sister species, and thus likely overestimated the efficacy of DNA barcodes in identifying species. RESULTS: To test the efficacy of DNA barcodes we compared ~650 bp of COI in 60 sister-species pairs identified in multigene phylogenies from 10 orders of birds. In all pairs, individuals of each species were monophyletic in a neighbor-joining (NJ) tree, and each species possessed fixed mutational differences distinguishing them from their sister species. Consequently, individuals were correctly assigned to species using a statistical coalescent framework. A coalescent test of taxonomic distinctiveness based on chance occurrence of reciprocal monophyly in two lineages was verified in known sister species, and used to identify recently separated lineages that represent putative species. This approach avoids the use of a universal distance cutoff which is invalidated by variation in times to common ancestry of sister species and in rates of evolution. CONCLUSION: Closely related sister species of birds can be identified reliably by barcodes of fixed diagnostic substitutions in COI sequences, verifying coalescent-based statistical tests of reciprocal monophyly for taxonomic distinctiveness. Contrary to recent criticisms, a single DNA barcode is a rapid way to discover monophyletic lineages within a metapopulation that might represent undiscovered cryptic species, as envisaged in the unified species concept. This identifies a smaller set of lineages that can also be tested independently for species status with multiple nuclear gene approaches and other phenotypic characters.
[Porter, T. M., Schadt, C. W., Rizvi, L., Martin, A. P., Schmidt, S. K., Scott-Denton, L., Vilgalys, R., & Moncalvo, J. M. 2008. Molecular Phylogenetics and Evolution. 46(2) 635-644.]
Fungi are one of the most diverse groups of Eukarya and play essential roles in terrestrial ecosystems as decomposers, pathogens and mutualists. This study unifies disparate reports of unclassified fungal sequences from soils of diverse origins and anchors many of them in a well-supported clade of the Ascomycota equivalent to a subphylum. We refer to this clade as Soil Clone Group I (SCGI). We expand the breadth of environments surveyed and develop a taxon-specific primer to amplify 2.4 kbp rDNA fragments directly from soil. Our results also expand the known range of this group from North America to Europe and Australia. The ancient origin of SCGI implies that it may represent an important transitional form among the basal Ascomycota groups. SCGI is unusual because it currently represents the only major fungal lineage known only from sequence data. This is an important contribution towards building a more complete fungal phylogeny and highlights the need for further work to determine the function and biology of SCGI taxa.
[Steeves, R., Nazari, V., Landry, J.-F., & Lacroix, C. R. 2008. The Canadian Entomologist. 140(3) 297-305.]
The Gulf of St. Lawrence aster, Symphyotrichum laurentianum (Fernald) G.L. Nesom (Asteraceae), a small annual halophyte endemic to disturbed and highly transient habitats in the Gulf of St. Lawrence, is classified as “threatened” by the Committee on the Status of Endangered Wildlife in Canada. Lepidopteran larvae that are predispersal seed predators of the Gulf of St. Lawrence aster are reported for the first time from populations in Prince Edward Island National Park. DNA barcoding was used to identify the seed predators tentatively as larvae of the casebearing moth Coleophora triplicis McDunnough (Lepidoptera: Coleophoridae), which is typically associated with a related halophyte, Solidago sempervirens L. (Asteraceae). These larvae were found to consume a large proportion of seeds from one of two aster populations in Prince Edward Island National Park and may be yet another risk to the survival of this threatened species.
| [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.
[Schmidt, S. K., Wilson, K. L., Meyer, A. F., Porter, T. M., Schadt, C. W., & Moncalvo, J. M. 2008. In K. Zengler (Ed.), Acessing Uncultivated Microorganisms. Washington, DC: American Society for Microbiology Press 55-66.]
[Feau, N., Joly, D. L., & Hamelin, R. C. 2007. Canadian Journal of Botany. 85(12) 1127-1135.]
With the availability of the entire genome of the model tree Populus trichocarpa Torr. & A. Gray and the current genome sequencing project of its rust pathogen Melampsora larici-populina Kleb., rust–poplar interaction research has entered the genomic era. Recent genomics research on poplars has attempted to connect the genetic localizations of loci for qualitative and quantitative disease resistance with putative genes encoding resistance or signalling proteins. The interactions between these putative resistance genes and rust effectors remain unknown. Genomic resources developed for Melampsora spp. promise to contribute to our understanding of the molecular basis of pathogenicity by facilitating the isolation of pathogenicity genes. A multifaceted approach for the identification of such genes that relies largely on trimming and sequence data analysis has been developed. The strategy takes advantage of the resources available and combines EST libraries, bioinformatics data mining for extracellularly expressed secreted proteins, intra- and inter-specific comparative genomics, and testing for the presence of positive selection. It has resulted in the discovery of several putative candidate genes. In silico evidence for candidate genes will be further validated by robust experimental evidence through functional analyses.
[Lévesque, C. A. 2007. In Z. K. Punja, S. DeBoer and H. Sanfaçon (Eds.), Biotechnology and Plant Disease Management. Wallingford, UK CABI Books 146-164.]
| [Lou, M. and G.B. Golding 2007. Molecular Ecology Notes. 7(6) 908-914.] There is a lack of programs available that focus on providing an overview of an aligned set of sequences such that the comparison of homologous sites becomes comprehensible and intuitive. Being able to identify similarities, differences, and patterns within a multiple sequence alignment is biologically valuable because it permits visualization of the distribution of a particular feature and inferences about the structure, function, and evolution of the sequences in question. We have therefore created a web server, fingerprint, which combines the characteristics of existing programs that represent identity, variability, charge, hydrophobicity, solvent accessibility, and structure along with new visualizations based on composition, heterogeneity, heterozygosity, dN/dS and nucleotide diversity. fingerprint is easy to use and globally accessible through any computer using any major browser. fingerprint is available at http://evol.mcmaster.ca/fingerprint/. |  |
| [Cooper, J. K., Sykes, G., King, S., Cottrill, K., Ivanova, N. V., Hanner, R., and Ikonomi, P. 2007. In vitro cellular & developmental biology. 43(10) 344-51.] Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the "barcode region" by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts. |  |
| [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. |  |
[Baker, A. J., Pereira, S. L., Rogers, D. I., Elbourne, R., & Hassell, C. J. 2007. Emu. 107(3) 185-189.]
Despite its distinctive morphology, the taxonomy of the Australian Painted Snipe has been unsettled, with some authors treating it as a full species, Rostratula australis (Gould 1838), and others treating it as a subspecies of the Greater Painted Snipe, Rostratula benghalensis. We sequenced the DNA of five mitochondrial genes (Cyt b, ND5, ATP 6–8, COIII and COI) of Australian Painted Snipe, Greater Painted Snipe and South American Painted Snipe, Nycticryphes semicollaris. The sequences of Australian Painted Snipe were 10% different from those of Greater Painted Snipe from Africa and South-east Asia, which differed from one another by only 2%. Plumage and anatomical characters can also distinguish the Australian and the Greater Painted Snipes. Our results clearly indicate that the Australian Painted Snipe is a distinct species that diverged ~19 million years ago (mya) (95% credible interval 13.0, 27.4 mya).
| [Francis, C.M., T. Kingston and A. Zubaid 2007. Acta Chiropterologica. 9(1) 1-12.] A new species of small Kerivoula is described from peninsular Malaysia. It is similar in size and form to Kerivoula hardwickii Miller 1898 or K. intermedia Hill and Francis 1984, but is distinguished by its distinctive colouration — dorsal fur has extensive black bases with shiny golden tips, ventral fur has dark grey bases with whitish-buff tips — as well as several characters of dentition and skull shape. Sequence analysis of the first 648 base pairs of cytochrome oxidase I gene (DNA barcode) indicates a divergence of at least 11% |  |
[Hanner, R. H., & Gregory, T. R 2007. Cell Preservation Technology. 5(2) 93-103.]
Biodiversity repositories underpin the future of research in the life sciences and biotechnology. However, they represent an extremely heterogeneous assemblage of collections that are lacking a comprehensive index of available resources. A set of “best practices” for biospecimen characterization is proposed for repository biomaterials involving standardized species-level molecular genotyping (DNA barcoding) and the quantification of nuclear DNA content (genome size). This approach has implications for upstream sample collection and preservation methods, as well as downstream implications for highlighting biorepository specimens available for genetic and genomic research. The broad application of the approach here proposed will raise the profile of participating biodiversity repositories, facilitate the compilation of validated reference sequences for molecular species recognition, and drive a deeper understanding of the evolution of the genome.
| [Min, X.J. and Hickey, D.A. 2007. Molecular Ecology Notes. 7(3) 365-373.] DNA barcoding shows enormous promise for the rapid identification of organisms at the species level. There has been much recent debate, however, about the need for longer barcode sequences, especially when these sequences are used to construct molecular phylogenies. Here, we have analysed a set of fungal mitochondrial sequences - of various lengths - and we have monitored the effect of reducing sequence length on the utility of the data for both species identification and phylogenetic reconstruction. Our results demonstrate that reducing sequence length has a profound effect on the accuracy of resulting phylogenetic trees, but surprisingly short sequences still yield accurate species identifications. We conclude that the standard short barcode sequences (~600 bp) are not suitable for inferring accurate phylogenetic relationships, but they are sufficient for species identification among the fungi. |  |
| [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. |  |
| [Landry, J.-F. 2007. The Canadian Entomologist. 139(3) 319-353.] The North American species of Acrolepiopsis are reviewed and include six described species: A. assectella (Zeller), A. californica Gaedike, A. heppneri Gaedike, A. incertella (Chambers), A. leucoscia (Meyrick), and A. reticulosa (Braun). Acrolepiopsis liliivora Gaedike is considered a junior synonym of A. californica (new synonymy). Acrolepiopsis assectella, commonly known as the leek moth, is a recently invasive alien species in North America and a pest of the plant genus Allium, including leek, onion, garlic, and related cultivated plants. A key to species based on adults is provided, diagnostic characters including male and female genitalia are illustrated, and geographical distribution, host plants, and larval feeding pattern and damage (where known) are given. Diagnostics and illustrations are presented also for A. sapporensis (Matsumura); known as the Asiatic onion leafminer, it is very similar to A. assectella and is an invasive alien species present in Hawaii, though not in North America. Adult diagnostic characters of the genus Acrolepiopsis, the family Acrolepiidae, and the superfamily Yponomeutoidea are also provided and illustrated. DNA barcoding data (short sequences of the mitochondrial cytochrome c oxidase I gene) obtained for five of the six species revealed interspecific differences averaging 8.1%, whereas intraspecific variation was ≤ 0.16%, and provided unequivocal species separation matching morphology-based identifications. |  |
[Min, X.J. and Hickey, D.A. 2007. PLoS ONE. 2(3) e325.]
DNA barcodes have achieved prominence as a tool for species-level identifications. Consequently, there is a rapidly growing database of these short sequences from a wide variety of taxa. In this study, we have analyzed the correlation between the nucleotide content of the short DNA barcode sequences and the genomes from which they are derived. Our results show that such short sequences can yield important, and surprisingly accurate, information about the composition of the entire genome. In other words, for unsequenced genomes, the DNA barcodes can provide a quick preview of the whole genome composition.
| [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. |  |
| [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. |  |
[Baker, A.J. 2007. Ornithological Monographs. 63 18-29.]
Problems in deciphering the patterns and causes of geographic variation and speciation in birds occupied Ned Johnson (e.g., Johnson 1980, Cicero and Johnson 1998, Johnson and Cicero 2002) and many other ornithologists for much of their lives, but the recent onslaught of molecular studies and associated analytical methods are providing breakthroughs in understanding these evolutionary phenomena. In particular, coalescent theory and Markov chain Monte Carlo (MCMC) applications have shown that bird species are sometimes strongly structured into well-differentiated populations by historical subdivision, high philopatry, and small effective population sizes, whereas other species that have recently recolonized parts of their range are effectively panmictic. These are the sorts of results that were impossible to obtain from studies of geographic variation in phenotypic characters alone. Recovery of well-supported species trees from gene trees is much more likely when multiple genes are sequenced, and provides the means for inferring divergence times and patterns and processes of evolution in birds. As in other vertebrates, patterns of cladogenesis in large clades of birds correlate with major paleoenvironmental changes and associated adaptive radiations, reminding us that much of current biodiversity on the planet had its genesis in the distant past.
| [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. |  |
| [Lynn, D. H. and Strüder-Kypke, M. C. 2006. Journal of Eukaryotic Microbiology. 53 (5) 385-387.] The mitochondrial cytochrome c oxidase 1 (CO1) genes of two isolates of each of the seven mating types of Tetrahymena thermophila were sequenced and found to differ byo1% in nucleotide sequence and to be identical by putative protein sequence. As this gene was highly conserved in this species, the CO1 gene sequence was determined for four pairs of Tetrahymena species identical in their small subunit rRNA gene sequences. The following pairs of species showed from 1% to 12% divergence at the nucleotide level, enabling discrimination of all these species: (1) Tetrahymena pyriformis strain T and Tetrahymena setosa strain HZ-1; (2) Tetrahymena canadensis strain UM1215 and Tetrahymena rostrata strain ID-3; (3) Tetrahymena pigmentosa strain UM1285 and Tetrahymena hyperangularis strain EN112; and (4) Tetrahymena tropicalis strain TC-105 and Tetrahymena mobilis. However, because of the synonymous nature of the majority of substitutions, the pairs of species were identical based on the putative protein sequence. |  |
| [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. |  |
| [Newmaster, S.G., Fazekas, A.J., and S. Ragupathy 2006. Can. J. Bot.. 84 335-341.] DNA barcoding based on the mitochondrial cytochrome c oxidase 1 (cox1) sequence is being employed for diverse groups of animals with demonstrated success in species identification and new species discovery. Applying barcoding systems to land plants will be a more challenging task as plant genome substitution rates are considerably lower than those observed in animal mitochondria, suggesting that a much greater amount of sequence data from multiple loci will be required to barcode plants. In the absence of an obvious well-characterized plant locus that meets all the necessary criteria, a key first step will be identifying candidate regions with the most potential. To meet the challenges with land plants, we are proposing the adoption of a tiered approach wherein highly variable loci are nested under a core barcoding gene. Analysis of over 10 000 rbcL sequences from GenBank demonstrate that this locus could serve well as the core region, with sufficient variation to discriminate among species in approximately 85% of congeneric pair-wise comparisons. Use of a secondary locus can be implemented when required and can vary from group to group if necessary. The implementation of a barcoding tool has multiple academic and practical applications. It will speed routine identifications and the detection of alien species, advance ecological and taxonomic inquiry, permit fast and accurate forensic analysis of plant fragments, and can function as an additional layer of quality control in the food industry. |  |
| [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. |  |
| [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. |
| [Saunders, G. 2005. Phil. Trans. R. Soc. B. 360 (1462) 1879 - 1888.] Notes: The applicability of DNA barcoding to indentify red macroalgae is discussed in this communication. The barcodes were all resolved accurately, the species identification was unequivocal, and the study raised some further taxonomic questions. |
| [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. |
| [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. |
| [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.
|  |