Comprehensive DNA barcoding of the herpetofauna of Germany

We present the first comprehensive DNA barcoding study of German reptiles and amphibians representing likewise the first on the European herpetofauna. A total of 248 barcodes for all native species and subspecies in the country and a few additional taxa were obtained in the framework of the projects ‘Barcoding Fauna Bavarica’ (BFB) and ‘German Barcode of Life’ (GBOL). In contrast to many invertebrate groups, the success rate of the identification of mitochondrial lineages representing species via DNA barcode was almost 100% because no cases of Barcode Index Number (BIN) sharing were detected within German native reptiles and amphibians. However, as expected, a reliable identification of the hybridogenetic species complex in the frog genus Pelophylax was not possible. Deep conspecific lineages resulting in the identification of more than one BIN were found in Lissotriton vulgaris, Natrix natrix and the hybridogenetic Pelophylax complex. A high variety of lineages with different BINs was also found in the barcodes of wall lizards (Podarcis muralis), confirming the existence of many introduced lineages and the frequent occurrence of multiple introductions. Besides the reliable species identification of all life stages and even of tissue remains, our study highlights other potential applications of DNA barcoding concerning German amphibians and reptiles, such as the detection of allochthonous lineages, monitoring of gene flow and also noninvasive sampling via environmental DNA. DNA barcoding based on COI has now proven to be a reliable and efficient tool for studying most amphibians and reptiles as it is already for many other organism groups in zoology.     

An update on DNA barcoding: low species coverage and numerous unidentified sequences

DNA barcoding was proposed in 2003, the Consortium for the Barcode of Life was established in 2004, and the movement has since attracted more than $80 million funding. Here we investigate how many species of multicellular animals have been barcoded. We compare the numbers in a public database (GenBank as of January 2012) with those in the Barcode of Life Database (BOLD) and find that GenBank contains COI (cytochrome c oxidase subunit 1) sequences for ca. 60 000 species while BOLD reports barcodes for ca. 150 000 species. The discrepancy is likely due to a large amount of unpublished data in BOLD. Overall, the species coverage remains sparse, growth rates are low, and the barcode accumulation curve for Metazoa is linear with only 4788 species having been added in 2011. In addition, the vast majority of species in the public database (73%) were barcoded by projects that are unlikely to be related to the DNA barcoding movement. Particularly surprising was the large number of DNA barcodes in GenBank that were not identified to species (Jan 2012: 74%), with insect barcodes often being identified only to order. Of these several hundred thousand have since been suppressed by NCBI because they did not satisfy the iBOL/GenBank early release agreement. Species coverage is considerably better for target taxa of DNA barcoding campaigns (e.g. birds, fishes, Lepidoptera), although it also falls short of published campaign targets. © The Willi Hennig Society 2012     

The campaign to DNA barcode all fishes, FISH-BOL

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.     

DNA barcode analysis of butterfly species from Pakistan points towards regional endemism

DNA barcodes were obtained for 81 butterfly species belonging to 52 genera from sites in north‐central Pakistan to test the utility of barcoding for their identification and to gain a better understanding of regional barcode variation. These species represent 25% of the butterfly fauna of Pakistan and belong to five families, although the Nymphalidae were dominant, comprising 38% of the total specimens. Barcode analysis showed that maximum conspecific divergence was 1.6%, while there was 1.7–14.3% divergence from the nearest neighbour species. Barcode records for 55 species showed <2% sequence divergence to records in the Barcode of Life Data Systems (BOLD), but only 26 of these cases involved specimens from neighbouring India and Central Asia. Analysis revealed that most species showed little incremental sequence variation when specimens from other regions were considered, but a threefold increase was noted in a few cases. There was a clear gap between maximum intraspecific and minimum nearest neighbour distance for all 81 species. Neighbour‐joining cluster analysis showed that members of each species formed a monophyletic cluster with strong bootstrap support. The barcode results revealed two provisional species that could not be clearly linked to known taxa, while 24 other species gained their first coverage. Future work should extend the barcode reference library to include all butterfly species from Pakistan as well as neighbouring countries to gain a better understanding of regional variation in barcode sequences in this topographically and climatically complex region.     

Are BOLD searches scientific? A response to Federhen (2011)

In 2011 Waugh et al. presented the results of a DNA barcoding study that identified avian species involved in birdstrikes. Federhen (2011) criticised this study by suggesting that some of the sequences on which identifications were based were not publicly available because access to them in the Barcode of Life database was restricted. Hence, according to Federhen (2011) the study is not repeatable. We disagree and discuss the role of databases in DNA barcoding generally.     

Taxonomic challenges in freshwater fishes: a mismatch between morphology and DNA barcoding in fish of the north‐eastern part of the Congo basin

This study evaluates the utility of DNA barcoding to traditional morphology‐based species identifications for the fish fauna of the north‐eastern Congo basin. We compared DNA sequences (COI) of 821 samples from 206 morphologically identified species. Best match, best close match and all species barcoding analyses resulted in a rather low identification success of 87.5%, 84.5% and 64.1%, respectively. The ratio ‘nearest‐neighbour distance/maximum intraspecific divergence’ was lower than 1 for 26.1% of the samples, indicating possible taxonomic problems. In ten genera, belonging to six families, the number of species inferred from mtDNA data exceeded the number of species identified using morphological features; and in four cases indications of possible synonymy were detected. Finally, the DNA barcodes confirmed previously known identification problems within certain genera of the Clariidae, Cyprinidae and Mormyridae. Our results underscore the large number of taxonomic problems lingering in the taxonomy of the fish fauna of the Congo basin and illustrate why DNA barcodes will contribute to future efforts to compile a reliable taxonomic inventory of the Congo basin fish fauna. Therefore, the obtained barcodes were deposited in the reference barcode library of the Barcode of Life Initiative.     

Using DNA Barcodes to Identify Bird Species Involved in Birdstrikes

Abstract: We determined effectiveness of using mitochondrial DNA barcodes (cytochrome c oxidase subunit 1 [CO1]) to identify bird-aircraft collision (birdstrike) cases that lacked sufficient feather evidence for morphological diagnosis. From September through December 2006, 821 samples from birdstrike events occurring in the United States were submitted for DNA analysis. We successfully amplified a CO1 DNA barcode product from 554 (67.5%) of the samples; 267 (32.5%) did not contain viable DNA and depended on morphological methods (microscopy) for Order or Family level identification. We deemed 19 cases inconclusive either because the DNA barcode recovered from the sample did not meet our 98% match criteria when compared to the Barcode of Life Database (BoLD) or because the DNA barcode matched to a set of ≥ 2 closely related species with overlapping barcodes, preventing complete species identification. Age of the sample (≤6 months) did not affect DNA viability, but initial condition of the sample and the collection method was critical to DNA identification success. The DNA barcoding approach has great potential in aiding in identification of birds (and wildlife) for airfield management practices, particularly in regions of the world that lack the vast research collections and individual expertise for morphologic identifications.     

Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast

Annelid polychaetes have been seldom the focus of dedicated DNA barcoding studies, despite their ecological relevance and often dominance, particularly in soft‐bottom estuarine and coastal marine ecosystems. Here, we report the first assessment of the performance of DNA barcodes in the discrimination of shallow water polychaete species from the southern European Atlantic coast, focusing on specimens collected in estuaries and coastal ecosystems of Portugal. We analysed cytochrome oxidase I DNA barcodes (COI‐5P) from 164 specimens, which were assigned to 51 morphospecies. To our data set from Portugal, we added available published sequences selected from the same species, genus or family, to inspect for taxonomic congruence among studies and collection location. The final data set comprised 290 specimens and 79 morphospecies, which generated 99 Barcode Index Numbers (BINs) within Barcode of Life Data Systems (BOLD). Among these, 22 BINs were singletons, 47 other BINs were concordant, confirming the initial identification based on morphological characters, and 30 were discordant, most of which consisted on multiple BINs found for the same morphospecies. Some of the most prominent cases in the latter category include Hediste diversicolor (O.F. Müller, 1776) (7), Eulalia viridis (Linnaeus, 1767) (2) and Owenia fusiformis (delle Chiaje, 1844) (5), all of them reported from Portugal and frequently used in ecological studies as environmental quality indicators. Our results for these species showed discordance between molecular lineages and morphospecies, or added additional relatively divergent lineages. The potential inaccuracies in environmental assessments, where underpinning polychaete species diversity is poorly resolved or clarified, demand additional and extensive investigation of the DNA barcode diversity in this group, in parallel with alpha taxonomy efforts.     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Molecular identification of mosquitoes (Diptera: Culicidae) in southeastern Australia

DNA barcoding is a modern species identification technique that can be used to distinguish morphologically similar species, and is particularly useful when using small amounts of starting material from partial specimens or from immature stages. In order to use DNA barcoding in a surveillance program, a database containing mosquito barcode sequences is required. This study obtained Cytochrome Oxidase I (COI) sequences for 113 morphologically identified specimens, representing 29 species, six tribes and 12 genera; 17 of these species have not been previously barcoded. Three of the 29 species ─ Culex palpalis, Macleaya macmillani, and an unknown species originally identified as Tripteroides atripes ─ were initially misidentified as they are difficult to separate morphologically, highlighting the utility of DNA barcoding. While most species grouped separately (reciprocally monophyletic), the Cx. pipiens subgroup could not be genetically separated using COI. The average conspecific and congeneric p‐distance was 0.8% and 7.6%, respectively. In our study, we also demonstrate the utility of DNA barcoding in distinguishing exotics from endemic mosquitoes by identifying a single intercepted Stegomyia aegypti egg at an international airport. The use of DNA barcoding dramatically reduced the identification time required compared with rearing specimens through to adults, thereby demonstrating the value of this technique in biosecurity surveillance. The DNA barcodes produced by this study have been uploaded to the ‘Mosquitoes of Australia–Victoria’ project on the Barcode of Life Database (BOLD), which will serve as a resource for the Victorian Arbovirus Disease Control Program and other national and international mosquito surveillance programs.     

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA‐trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non‐flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA‐trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non‐flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra‐ and inter‐specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non‐flowering seed plants. In addition, we compared the abilities of the five most‐recommended markers (psbA‐trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non‐flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non‐flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.     

Exploring species boundaries with multiple genetic loci using empirical data from non‐biting midges

Over the past decade, molecular approaches to species delimitation have seen rapid development. However, species delimitation based on a single locus, for example, DNA barcodes, can lead to inaccurate results in cases of recent speciation and incomplete lineage sorting. Here, we compare the performance of Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson tree processes (PTP), networks, generalized mixed Yule coalescent (GMYC) and Bayesian phylogenetics and phylogeography (BPP) models to delineate cryptic species previously detected by DNA barcodes within Tanytarsus (Diptera: Chironomidae) non‐biting midges. We compare the results from analyses of one mitochondrial (cytochrome c oxidase subunit I [COI]) and three nuclear (alanyl‐tRNA synthetase 1 [AATS1], carbamoyl phosphate synthetase 1 [CAD1] and 6‐phosphogluconate dehydrogenase [PGD]) protein‐coding genes. Our results show that species delimitation based on multiple nuclear DNA markers is largely concordant with morphological variation and delimitations using a single locus, for example, the COI barcode. However, ABGD, GMYC, PTP and network models led to conflicting results based on a single locus and delineate species differently than morphology. Results from BPP analyses on multiple loci correspond best with current morphological species concept. In total, 10 lineages of the Tanytarsus curticornis species complex were uncovered. Excluding a Norwegian population of Tanytarsus brundini which might have undergone recent hybridization, this suggests six hitherto unrecognized species new to science. Five distinct species are well supported in the Tanytarsus heusdensis species complex, including two species new to science.     

DNA barcoding in native plants of the Labiatae (Lamiaceae) family from Chios Island (Greece) and the adjacent Çeşme-Karaburun Peninsula (Turkey)

The plant family Labiatae (Lamiaceae) is known for its fine medicinal and aromatic herbs like lavender, mint, oregano, sage and thyme and is a rich source of essential oils for the food, pharmaceutical and cosmetic industry. Besides its great economic importance, the Labiatae family contributes significantly to the endemic flora of Greece and Turkey. Owing to its economic and biological significance and to the difficult identification based on morphological characters of several of its taxa, the Labiatae family is an ideal case for developing DNA barcodes. The purpose of this study is to evaluate the utility of DNA barcoding on a local scale in discriminating Labiatae species in Chios Island (Greece) and the adjacent Çe?me‐Karaburun Peninsula (Turkey). We chose three cpDNA regions (matK, rbcL, trnH‐psbA) that were proposed by previous studies and tested them either as single region or as multiregion barcodes based on the criteria determined by Consortium for the Barcode of Life (CBOL). Our results show that matK and trnH‐psbA taken as useful in discriminating species of the Labiatae, for the species we examined, as any multiregion combination. matK and trnH‐psbA could serve as single‐region barcodes for Labiatae species contributing to the conservation and the trade control of valuable plant resources.     

DNA barcodes successfully delimit morphospecies in a superdiverse insect genus

Polypedilum Kieffer (Diptera: Chironomidae), with 520 currently known species worldwide, can be extremely difficult to identify species level based on the morphology. We used 3,670 cytochrome c oxidase subunit I (COI) barcodes to explore the efficiency of the COI barcodes to differentiate between species in a superdiverse aquatic insect genus. The Barcode of Life Data System (BOLD) presented 286 BIN clusters in Polypedilum, representing 163 morphospecies, of which 93 were contributed from our laboratory. Molecular operational taxonomic units (OTUs) ranged from 158 to 345, based on Automatic Barcode Gap Discovery (ABGD), the Barcode Index Number (BIN), Bayesian Poisson tree processes (bPTP), generalized mixed Yule coalescent (GMYC), jMOTU, multi‐rate Poisson tree processes (mPTP), neighbor‐joining (NJ) tree and prethreshold clustering. In comparison, GMYC, bPTP, mPTP and BIN suggested more species than warranted by morphology, while ABGD, jMOTU, NJ, prethreshold clustering and ABGD yielded a conservative number of species when setting higher thresholds. Nine species complexes with deep intraspecific divergences indicated 18 potentially cryptic species, which require further taxonomic research including complete life histories and nuclear genetic data to be resolved. The discrimination of Polypedilum species by DNA barcodes proved to be successful in 94.4% of all studied morphological species.     

Testing the potential of DNA barcoding in vertebrate radiations: the case of the littoral cichlids (Pisces,Perciformes, Cichlidae) from Lake Tanganyika

We obtained 398 cytochrome c oxidase subunit I barcodes of 96 morphospecies of Lake Tanganyika (LT) cichlids from the littoral zone. The potential of DNA barcoding in these fishes was tested using both species identification and species delineation methods. The best match (BM) and best close match (BCM) methods were used to evaluate the overall identification success. For this, three libraries were analysed in which the specimens were categorized into Operational Taxonomic Units (OTU) in three alternative ways: (A) morphologically distinct, including undescribed, species, (B) valid species and (C) complexes of morphologically similar or closely related species. For libraries A, B and C, 73, 73 and 96% (BM) and 72, 70 and 94% (BCM) of the specimens were correctly identified. Additionally, the potential of two species delineation methods was tested. The General Mixed Yule Coalescent (GMYC) analysis suggested 70 hypothetical species, while the Automatic Barcode Gap Discovery (ABGD) method revealed 115 putative species. Although the ABGD method had a tendency to oversplit, it outperformed the GMYC analysis in retrieving the species. In most cases where ABGD suggested oversplitting, this was due to intraspecific geographical variation. The failure of the GMYC method to retrieve many species could be attributed to discrepancies between mitochondrial gene trees and the evolutionary histories of LT cichlid species. Littoral LT cichlids have complex evolutionary histories that include instances of hybridization, introgression and rapid speciation. Nevertheless, although the utility of DNA barcoding in identification is restricted to the level of complexes, it has potential for species discovery in cichlid radiations.     

Barcoding, types and the Hirudo files: using information content to critically evaluate the identity of DNA barcodes

Species identifications based on DNA barcoding rely on the correct identity of previously barcoded specimens, but little attention has been given to whether deposited barcodes include correspondence to the species' name-bearing type. The information content associated with COX1 sequences in the two most commonly used repositories of barcodes, GenBank and the Barcode of Life Data System (BOLD), is often insufficient for subsequent evaluation of the robustness of the identification procedure. We argue that DNA barcoding and taxonomy alike will benefit from more information content in the annotations of barcoded specimens as this will allow for validation and re-evaluation of the initial specimen identification. The aim should be to closely connect specimens from which reference barcodes are generated with the holotype through straight-forward taxonomy, and geographical and genetic correlations. Annotated information should also include voucher specimens and collector/identifier information. We examine two case studies based on empirical data, in which barcoding and taxonomy benefit from increased information content. On the basis of data from the first case study, we designate a barcoded neotype of the European medicinal leech, Hirudo medicinalis, on morphological and geographical grounds.     

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.     

Identification of gobies (Teleostei: Perciformes: Gobiidae) from the North and Baltic Seas combining morphological analysis and DNA barcoding

Gobies are difficult to identify, as they are very similar in appearance. Here, we identified (sub)adult specimens of 12 goby species from the North Sea and the Baltic Sea by carefully analysing meristic characters, coloration patterns, papillae row patterns and morphometric measurements. The results of the morphological identifications were congruent with those obtained with the analysis of COI DNA barcodes; sequences from morphological conspecific specimens were clustered together in clades with bootstrap values ≥ 99%. Mean intra‐ and interspecific distance (uncorrected p) was 0.37 and 18.97%, respectively. A gap between the maximum intraspecific distance and the distance to the nearest neighbour was apparent in every species and ranged from 2.35 to 16.11%. The Barcode Index Number (BIN) analysis performed on the Barcode of Life Data Systems (BOLD) web platform, assigned the DNA barcodes to 12 separate clusters corresponding to sequence‐ and morphology‐based identification. In 25% of the investigated species, the BIN clusters showed taxonomic discordances, as they contained sequences assigned to more than one species. This result demonstrates the importance of accurate morphological species identification at the beginning of the barcoding pipeline. © 2014 The Linnean Society of London     

DNA barcodes of the native ray‐finned fishes in Taiwan

Species identification based on the DNA sequence of a fragment of the cytochrome c oxidase subunit I gene in the mitochondrial genome, DNA barcoding, is widely applied to assist in sustainable exploitation of fish resources and the protection of fish biodiversity. The aim of this study was to establish a reliable barcoding reference database of the native ray‐finned fishes in Taiwan. A total of 2993 individuals, belonging to 1245 species within 637 genera, 184 families and 29 orders of ray‐finned fishes and representing approximately 40% of the recorded ray‐finned fishes in Taiwan, were PCR amplified at the barcode region and bidirectionally sequenced. The mean length of the 2993 barcodes is 549 bp. Mean congeneric K2P distance (15.24%) is approximately 10‐fold higher than the mean conspecific one (1.51%), but approximately 1.4‐fold less than the mean genetic distance between families (20.80%). The Barcode Index Number (BIN) discordance report shows that 2993 specimens represent 1275 BINs and, among them, 86 BINs are singletons, 570 BINs are taxonomically concordant, and the other 619 BINs are taxonomically discordant. Barcode gap analysis also revealed that more than 90% of the collected fishes in this study can be discriminated by DNA barcoding. Overall, the barcoding reference database established by this study reveals the need for taxonomic revisions and voucher specimen rechecks, in addition to assisting in the management of Taiwan's fish resources and diversity.