DNA barcoding Australia's fish species

Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (barcoded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (cox1). Most species were represented by multiple specimens, and 754 sequences were generated. The GC content of the 143 species of teleosts was higher than the 61 species of sharks and rays (47.1% versus 42.2%), largely due to a higher GC content of codon position 3 in the former (41.1% versus 29.9%). Rays had higher GC than sharks (44.7% versus 41.0%), again largely due to higher GC in the 3rd codon position in the former (36.3% versus 26.8%). Average within-species, genus, family, order and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18% and 23.27%, respectively. All species could be differentiated by their cox1 sequence, although single individuals of each of two species had haplotypes characteristic of a congener. Although DNA barcoding aims to develop species identification systems, some phylogenetic signal was apparent in the data. In the neighbour-joining tree for all 754 sequences, four major clusters were apparent: chimaerids, rays, sharks and teleosts. Species within genera invariably clustered, and generally so did genera within families. Three taxonomic groups-dogfishes of the genus Squalus, flatheads of the family Platycephalidae, and tunas of the genus Thunnus-were examined more closely. The clades revealed after bootstrapping generally corresponded well with expectations. Individuals from operational taxonomic units designated as Squalus species B through F formed individual clades, supporting morphological evidence for each of these being separate species. We conclude that cox1 sequencing, or 'barcoding', can be used to identify fish species.     

DNA barcoding discriminates echinoderm species

DNA barcode sequences (a 657-bp segment of the mtDNA cytochrome oxidase I gene, COI) were collected from 191 species (503 specimens) of Echinodermata. All five classes were represented: Ophiuroidea, Asteroidea, Echinoidea, Holothuroidea and Crinoidea. About 30% of sequences were collected specifically for this study, the remainder came from GenBank. Fifty-one species were represented by multiple samples, with a mean intraspecific divergence of 0.62%. Several possible instances of cryptic speciation were noted. Thirty-two genera were represented by multiple species, with a mean congeneric divergence of 15.33%. One hundred and eighty-seven of the 191 species (97.9%) could be distinguished by their COI barcodes. Those that could not were from the echinoid genus Amblypneustes. Neighbour-joining trees of COI sequences generally showed low bootstrap support for anything other than shallow splits, although with very rare exceptions, members of the same class clustered together. Two ophiuran species, in both nucleotide and amino acid neighbour-joining trees, grouped loosely as sister taxa to Crinoidea rather than Ophiuroidea; sequences of these two species appear to have evolved very quickly. Results suggest that DNA barcoding is likely to be an effective, accurate and useful method of species diagnosis for all five classes of Echinodermata.     

An integrative approach to species discovery in odonates: from character‐based DNA barcoding to ecology

Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision‐making. Such an approach can enhance both species identification and species discovery. The character‐based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA‐barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.     

Biodiversity studies in Phaseolus species by DNA barcoding

The potential of DNA barcoding was tested as a system for studying genetic diversity and genetic traceability in bean germplasm. This technique was applied to several pure lines of Phaseolus vulgaris L. belonging to wild, domesticated, and cultivated common beans, along with some accessions of Phaseolus coccineus L., Phaseolus lunatus L., and Vigna unguiculata (L.) Walp. A multilocus approach was exploited using three chloroplast genic regions (rbcL, trnL, and matK), four intergenic spacers (rpoB-trnC, atpBrbcL, trnT-trnL, and psbA-trnH), and nuclear ITS1 and ITS2 rDNA sequences. Our main goals were to identify the markers and SNPs that show the best discriminant power at the variety level in common bean germplasm, to examine two methods (tree based versus character based) for biodiversity analysis and traceability assays, and to evaluate the overall utility of chloroplast DNA barcodes for reconstructing the origins of modern Italian varieties. Our results indicate that the neighbor-joining method is a powerful approach for comparing genetic diversity within plant species, but it is relatively uninformative for the genetic traceability of plant varieties. In contrast, the character-based method was able to identify several distinct haplotypes over all target regions corresponding to Mesoamerican or Andean accessions; Italian accessions originated from both gene pools. On the whole, our findings raise some concerns about the use of DNA barcoding for intraspecific genetic diversity studies in common beans and highlights its limitations for resolving genetic relationships between landraces and varieties.     

DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.     

断眼天牛DNA条码试剂盒的研制

【目的】为了加强对检疫口岸截获木材中的断眼天牛种类进行准确、快速的鉴定,克服单纯依赖于形态学方法为主的局限性,我们研发了断眼天牛属DNA条码试剂盒检测技术。【方法】本研究基于线粒体COⅠ基因(线粒体细胞色素氧化酶亚基Ⅰ),采用加拿大条码中心开发的昆虫及植物DNA提取方法,对基因序列分析,获得碱基位点规律。【结果】断眼天牛属中的9个常截获种类取得了标记性碱基位点。【结论】检疫中常截获的断眼天牛属中的9个种通过标记性碱基位点得到区分;研发了断眼天牛属DNA条码试剂盒检测技术,为进出境口岸的检疫工作提供一种新的鉴定手段。     

生物分类学的新动向--DNA条形编码

过去的一年中 ,DNA条形编码 (DNABarcoding)成为生物分类学中引人注目的新方向。DNA条形编码 ,根据对一个统一的目标基因DNA序列的分析 ,达到物种鉴定的目的 ,它操作的简便性和高效性将以我们无法想象的速度加快物种鉴定和进化历史研究的步伐 ,但国际上对此的争论也不少。本文综述了DNA条形编码的原理、操作过程及最新进展 ,讨论了其可能存在的问题     

DNA barcoding of eight North American coregonine species

Coregonine fishes have a circumpolar distribution in the Arctic and sub-Arctic Northern Hemisphere. This subfamily of Salmonidae consists of three genera: Prosopium, Stenodus and Coregonus, including over 30 species. Many species overlap spatially and are difficult to distinguish based on morphological characteristics, especially as larvae or juveniles. Here we present a method for rapid and cost-effective species identification for representatives of the three genera based on sequence variation at the mitochondrial cytochrome c oxidase subunit I gene (COI). We examined eight species common to North America with distributional overlap in Alaska. Mean pairwise sequence divergence for all eight species was 7.04% and ranged from 0.46% to 14.23%. This sequence variation was used to develop a genetic assay based on restriction fragment length polymorphism. In a blind test, this assay provided correct species assignment for 48 of 49 individuals representing all eight species. The single incorrect assignment may reflect hybridization between two closely related species. This DNA barcode-based assay promises to aid fishery managers and researchers by providing a cost-effective alternative to large-scale sequence analysis for identification of North American coregonine fishes.     

Use of DNA barcoding to detect invertebrate invasive species from diapausing eggs

The global transhipment of ballast water and associated flora and fauna by cargo vessels has increased dramatically in recent decades. Invertebrate species are frequently carried in ballast water and sediment, although identification of diapausing eggs can be extremely problematic. Here we test the application of DNA barcoding using mitochondrial cytochrome c oxidase subunit I and 16S rDNA to identify species from diapausing eggs collected in ballast sediment of ships. The accuracy of DNA barcoding identification was tested by comparing results from the molecular markers against each other, and by comparing barcoding results to traditional morphological identification of individuals hatched from diapausing eggs. Further, we explored two public genetic databases to determine the broader applicability of DNA barcodes. Of 289 diapausing eggs surveyed, sufficient DNA for barcoding was obtained from 96 individuals (33%). Unsuccessful DNA extractions from 67% of eggs in our study were most likely due to degraded condition of eggs. Of 96 eggs with successful DNA extraction, 61 (64%) were identified to species level, while 36% were identified to possible family/order level. Species level identifications were always consistent between methodologies. DNA barcoding was suitable for a wide range of taxa, including Branchiopoda, Copepoda, Rotifera, Bryozoa and Ascidia. Branchiopoda and Copepoda were respectively the best and worst represented groups in genetic databases. Though genetic databases remain incomplete, DNA barcoding resolved nearly double the number of species identified by traditional taxonomy (19 vs. 10). Notorious invaders are well represented in existing databases, rendering these NIS detectable using molecular methods. DNA barcoding provides a rapid and accurate approach to identification of invertebrate diapausing eggs that otherwise would be very difficult to identify.     

Identification of 'extinct' freshwater mussel species using DNA barcoding

Freshwater mollusks are highly imperiled, with 70% of the North American species extinct, endangered, or at risk of extinction. Impoundments and other human impacts on the Coosa River of Alabama, Georgia and Tennessee of the southeastern USA alone are believed to have caused 50 mollusk species extinctions, but uncertainty over boundaries among several putatively closely related species makes this number preliminary. Our examination of freshwater mussels collected during an extensive survey of the upper-drainage basin, DNA barcoding and molecular phylogenetic analyses confirm the rediscovery of four morphospecies in the genus Pleurobema (Unionidae) previously thought to be extinct from the upper Coosa basin. A fifth 'extinct' form was found in an adjoining basin. Molecular data show that the Coosa morphologies represent at least three species-level taxa: Pleurobema decisum, P. hanleyianum and P. stabile. Endemism is higher than currently recognized, both at the species level and for multispecies clades. Prompt conservation efforts may preserve some of these taxa and their ecosystem.     

A likelihood ratio test for species membership based on DNA sequence data

DNA barcoding as an approach for species identification is rapidly increasing in popularity. However, it remains unclear which statistical procedures should accompany the technique to provide a measure of uncertainty. Here we describe a likelihood ratio test which can be used to test if a sampled sequence is a member of an a priori specified species. We investigate the performance of the test using coalescence simulations, as well as using the real data from butterflies and frogs representing two kinds of challenge for DNA barcoding: extremely low and extremely high levels of sequence variability.     

DNA barcoding and phylogeny in neotropical species of the genus Spondias

The genus Spondias belongs to the Anacardiaceae family, with about 18 species, having significant economic and social importance and with some species used in the agricultural industry, however, problems are encountered when trying to identify phylogenetic relationships among the species. The use of DNA barcoding is of importance to this group, allowing species identification at the molecular level and in determining the phylogenetic relationships within the group. The objective of this study is to obtain DNA barcoding and to determine the phylogenetic relationships among the species. For this, DNA from six species of the genus was extracted and amplified by PCR using sequences from the rbcL and matK genes and the trnH-psbA spacer gene, followed by sequencing using the Sanger method. The results show that the matK and rbcL genes cannot be used for DNA barcoding, because their discriminatory level between species is low. On the other hand, trnH-psbA shows a high level of discrimination, allowing most of the species to be identified. However it is not possible to separate Spondias venulosa and Spondias tuberosa. Phylogenetic analysis shows that Spondias mombim and S. tuberosa are distinct “umbucajá” clades, suggesting a non-hybrid origin for “umbucajá”.     

DNA barcoding of Scandinavian birds reveals divergent lineages in trans-Atlantic species

Birds are a taxonomically well-described group of animals, yet DNA barcoding, i.e., the molecular characterization of species using a standardized genetic marker, has revealed unexpected patterns of genetic divergences among North American birds. We performed a comprehensive COI (cytochrome c oxidase subunit I) barcode survey of 296 species of Scandinavian birds, and compared genetic divergences among 78 trans-Atlantic species whose breeding ranges include both Scandinavia and North America. Ninety-four percent of the Scandinavian species showed unique barcode clusters; the remaining 6% had overlapping barcodes with one or more congeneric species, which may reflect incomplete lineage sorting or a single gene pool. Four species showed large intra-specific divergences within Scandinavia, despite no apparent morphological differentiation or indications of reproductive isolation. These cases may reflect admixture of previously isolated lineages, and may thus warrant more comprehensive phylogeographic analyses. Nineteen (24%) of 78 trans-Atlantic species exhibited divergent genetic clusters which correspond with regional subspecies. Three of these trans-Atlantic divergences were paraphyletic. Our study demonstrates the effectiveness of COI barcodes for identifying Scandinavian birds and highlights taxa for taxonomic review. The standardized DNA barcoding approach amplified the power of our regional studies by enabling independently obtained datasets to be merged with the established avian barcode library.     

A regional approach to plant DNA barcoding provides high species resolution of sedges (Carex and Kobresia, Cyperaceae) in the Canadian Arctic Archipelago

Previous research on barcoding sedges (Carex) suggested that basic searches within a global barcoding database would probably not resolve more than 60% of the world’s some 2000 species. In this study, we take an alternative approach and explore the performance of plant DNA barcoding in the Carex lineage from an explicitly regional perspective. We characterize the utility of a subset of the proposed protein-coding and noncoding plastid barcoding regions (matK, rpoB, rpoC1, rbcL, atpF-atpH, psbK-psbI) for distinguishing species of Carex and Kobresia in the Canadian Arctic Archipelago, a clearly defined eco-geographical region representing 1% of the Earth’s landmass. Our results show that matK resolves the greatest number of species of any single-locus (95%), and when combined in a two-locus barcode, it provides 100% species resolution in all but one combination (matK + atpFH) during unweighted pair-group method with arithmetic mean averages (UPGMA) analyses. Noncoding regions were equally or more variable than matK, but as single markers they resolve substantially fewer taxa than matK alone. When difficulties with sequencing and alignment due to microstructural variation in noncoding regions are also considered, our results support other studies in suggesting that protein-coding regions are more practical as barcoding markers. Plastid DNA barcodes are an effective identification tool for species of Carex and Kobresia in the Canadian Arctic Archipelago, a region where the number of co-existing closely related species is limited. We suggest that if a regional approach to plant DNA barcoding was applied on a global scale, it could provide a solution to the generally poor species resolution seen in previous barcoding studies.     

DNA barcoding of brown Parmeliae (Parmeliaceae) species: a molecular approach for accurate specimen identification,emphasizing species in Greenland

Warming of Arctic and alpine regions has a substantial impact on high-altitude/-latitude ecosystems. Shifting biomes due to climate change may lead to adjustments in species distributions and potential extinctions. Therefore, detailed monitoring is requisite to assess biologically meaningful shifts in community composition and species distributions. Some Arctic-alpine lichens have been shown to be particularly sensitive to climatic shifts associated with global change. However, accurate identification of lichenized fungal species remains challenging and may limit the effective use of lichens in climate change research. Given the inherent difficulties in accurate identification of lichenized fungi and the potential value of efficient identifications for bio-monitoring research, we investigated the utility of DNA barcode identification of the 13 brown Parmeliae (Ascomycota) species occurring in Greenland. For these species, we assessed monophyly and genetic distances using the nuclear ribosomal internal transcribed spacer region (ITS), the standard DNA barcode for fungi. We also compared intraspecific distance values to a proposed intra-interspecific threshold value for Parmeliaceae to identify nominal taxa potentially masking previously unrecognized diversity. Our results indicated that the 13 brown Parmeliae species occurring in Greenland can be successfully discriminated using the ITS region. All phenotypically circumscribed species were recovered as well-supported, monophyletic clades. Furthermore, our data supported a barcode gap among congeners for all brown Parmeliae species investigated here. However, high intraspecific genetic distances suggest the potential for previously unrecognized species-lineages in at least five species: Melanelia agnata, M. hepatizon, Montanelia disjuncta, M. panniformis, and M. tominii. Our research facilitates effective, long-term bio-monitoring of climate change in Greenland using lichens by providing accurate molecular identification of brown Parmeliae specimens.     

DNA barcoding is a new approach in comparative genomics of plants

DNA barcoding was proposed as a method for recognition and identification of eukaryotic species through comparison of sequences of a standard short DNA fragment—DNA barcode—from an unknown specimen to a library of reference sequences from known species. This allows identifying an organism at any stage of development from a very small tissue sample, fresh or conserved many years ago. Molecular identification of plant samples can be used in various scientific and applied fields. It would also help to find new species, which is particularly important for cryptogamic plants. An optimal DNA barcode region is a small fragment presented in all species of a major taxonomic group, having invariable nucleotide sequence in all members of the same species, but with sufficient variation to discriminate among the species. This fragment should be flanked by low-variable regions for use of universal primers in PCR for amplification and sequencing. The DNA barcode that is well established in animals is a sequence of a fragment of the mitochondrial cytochrome c oxidase gene CO1. However, searching for DNA barcode in plants proved to be a more challenging task. No DNA region universally suitable for all plants and meeting all of the necessary criteria has been found. Apparently, a multilocus or two-stage approach should be applied for this purpose. Several fragments of the chloroplast genome (trnH-psbA, matK, rpoC, rpoB, rbcL) in combinations of two or three regions were suggested as candidate regions with highest potential, but more representative samples should be examined to choose the best candidate. The possibility is discussed to use as DNA barcode internal transcribed spacers (ITS) of nuclear rRNA genes, which are highly variable, widely employed in molecular phylogenetic studies at the species level, but also have some limitations.     

DNA barcoding of catfish: species authentication and phylogenetic assessment

As the global market for fisheries and aquaculture products expands, mislabeling of these products has become a growing concern in the food safety arena. Molecular species identification techniques hold the potential for rapid, accurate assessment of proper labeling. Here we developed and evaluated DNA barcodes for use in differentiating United States domestic and imported catfish species. First, we sequenced 651 base-pair barcodes from the cytochrome oxidase I (COI) gene from individuals of 9 species (and an Ictalurid hybrid) of domestic and imported catfish in accordance with standard DNA barcoding protocols. These included domestic Ictalurid catfish, and representative imported species from the families of Clariidae and Pangasiidae. Alignment of individual sequences from within a given species revealed highly consistent barcodes (98% similarity on average). These alignments allowed the development and analyses of consensus barcode sequences for each species and comparison with limited sequences in public databases (GenBank and Barcode of Life Data Systems). Validation tests carried out in blinded studies and with commercially purchased catfish samples (both frozen and fresh) revealed the reliability of DNA barcoding for differentiating between these catfish species. The developed protocols and consensus barcodes are valuable resources as increasing market and governmental scrutiny is placed on catfish and other fisheries and aquaculture products labeling in the United States.     

鲫属鱼类DNA条码及种与亚种划分

鲫属(Carassius)鱼类由于表型变异大,分布范围广,分类一直不完善。该属常被分为三个种:黑鲫(C.carassius)、白鲫(C.cuvieri)和鲫(C.auratus)。鲫又可分为多个亚种(包括金鱼),其中银鲫(C.auratus gibelio)亚种有时被视作一个独立的种。该文研究了线粒体细胞色素c氧化酶亚基I(COI)基因5’端651bp的片段在这些种和亚种(共计128尾标本)中的变异。结果表明,C.carassius、C.cuvieri和C.auratus均为有效种,同时欧亚大陆的与日本列岛的C.auratus有明显分化;而C.auratusgibelio和C.auratusauratus有一些共享的单倍型,C.auratus gibelio应被视为C.auratus的一个亚种,而不是一个有效物种。由于C.auratus auratus和C.auratus gibelio等类群中同时存在二倍体和三倍体,因此,倍性不宜作为种或亚种的划分标准。