A reliable DNA barcode reference library for the identification of the North European shelf fish fauna

Valid fish species identification is an essential step both for fundamental science and fisheries management. The traditional identification is mainly based on external morphological diagnostic characters, leading to inconsistent results in many cases. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I (COI) for a valid identification of 93 North Atlantic fish species originating from the North Sea and adjacent waters, including many commercially exploited species. Neighbour‐joining analysis based on K2P genetic distances formed nonoverlapping clusters for all species with a ≥99% bootstrap support each. Identification was successful for 100% of the species as the minimum genetic distance to the nearest neighbour always exceeded the maximum intraspecific distance. A barcoding gap was apparent for the whole data set. Within‐species distances ranged from 0 to 2.35%, while interspecific distances varied between 3.15 and 28.09%. Distances between congeners were on average 51‐fold higher than those within species. The validation of the sequence library by applying BOLDs barcode index number (BIN) analysis tool and a ranking system demonstrated high taxonomic reliability of the DNA barcodes for 85% of the investigated fish species. Thus, the sequence library presented here can be confidently used as a benchmark for identification of at least two‐thirds of the typical fish species recorded for the North Sea.     

A DNA barcode library for Korean Chironomidae (Insecta: Diptera) and indexes for defining barcode gap

Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified larvae. The interspecies and intraspecies COI sequence variations were analyzed. Sophisticated indexes were developed in order to properly evaluate indistinct barcode gaps that are created by insufficient sampling on both the interspecies and intraspecies levels and by variable mutation rates across taxa. In a variety of insect datasets, these indexes were useful for re-evaluating large barcode datasets and for defining COI barcode gaps. The COI-based DNA barcode library will provide a rapid and reliable tool for the molecular identification of Korean chironomid species. Furthermore, this reverse-taxonomic approach will be improved by the continuous addition of other speceis’ sequences to the library.     

Untangling taxonomy: a DNA barcode reference library for Canadian spiders

Approximately 1460 species of spiders have been reported from Canada, 3% of the global fauna. This study provides a DNA barcode reference library for 1018 of these species based upon the analysis of more than 30 000 specimens. The sequence results show a clear barcode gap in most cases with a mean intraspecific divergence of 0.78% vs. a minimum nearest‐neighbour (NN) distance averaging 7.85%. The sequences were assigned to 1359 Barcode index numbers (BINs) with 1344 of these BINs composed of specimens belonging to a single currently recognized species. There was a perfect correspondence between BIN membership and a known species in 795 cases, while another 197 species were assigned to two or more BINs (556 in total). A few other species (26) were involved in BIN merges or in a combination of merges and splits. There was only a weak relationship between the number of specimens analysed for a species and its BIN count. However, three species were clear outliers with their specimens being placed in 11–22 BINs. Although all BIN splits need further study to clarify the taxonomic status of the entities involved, DNA barcodes discriminated 98% of the 1018 species. The present survey conservatively revealed 16 species new to science, 52 species new to Canada and major range extensions for 426 species. However, if most BIN splits detected in this study reflect cryptic taxa, the true species count for Canadian spiders could be 30–50% higher than currently recognized.     

A minimalist barcode can identify a specimen whose DNA is degraded

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.     

Limited performance of DNA barcoding in a diverse community of tropical butterflies

DNA 'barcoding' relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5' half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet.     

A DNA barcode reference library for the native woody seed plants of Japan

DNA barcode databases are increasingly available for a range of organisms, facilitating the wide application of DNA barcode-based studies. Here we announce the development of a comprehensive DNA barcode reference library of Japanese native woody seed plants representing 43 orders, 99 families, 303 genera and 834 species, and comprising 77.3% of the genera and 72.2% of the species of native woody seed plants in Japan. A total of 6216 plant specimens were collected from 223 sites across the subtropical, temperate, boreal and alpine biomes in Japan with most species represented by multiple accessions. This reference library utilized three chloroplast DNA regions (rbcL, trnH-psbA and matK) and consists of 14,403 barcode sequences. Individual regions varied in their identification rates, with species-level and genus-level rates for rbcL, trnH-psbA and matK based on blast being 57.4%/96.2%, 78.5%/99.1% and 67.8%/98.1%, respectively. Identification rates were higher using region combinations, with total species-level rates for two region combinations (rbcL & trnH-psbA, rbcL & matK and trnH-psbA & matK) ranging between 90.6% and 95.8%, and for all three regions being equal to 98.6%. Genus-level identification rates were even higher, ranging between 99.7% and 100% for two region combinations and being 100% for the three regions. These results indicate that this DNA barcode reference library is an effective resource for investigations of native woody seed plants in Japan using DNA barcodes and provides a useful template for the development of libraries for other components of the Japanese flora.     

Accelerating plant DNA barcode reference library construction using herbarium specimens: improved experimental techniques

A well‐covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self‐primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four‐sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400–500 bp without bringing or inducing any sequence errors. About one‐third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well‐covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA‐labelled next‐generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality.     

DNA barcode assessment of Mediterranean mayflies (Ephemeroptera), benchmark data for a regional reference library for rapid biomonitoring of freshwaters

Accurate identification of aquatic species is fundamental to freshwater research. In this paper, we targeted Ephemeroptera, a key taxonomic insect group for biomonitoring of water bodies and present an overview on the efficacy of the DNA barcoding approach to document species identity in the Mediterranean region. We sequenced the mitochondrial cytochrome c oxidase (COI) in 39 nominal species. Sample discrimination and species identification were investigated by evaluating haplotype identity and similarity, intra-/interspecific genetic distances, optimal identification of barcoding gap thresholds, estimates of species monophyly and comparative species matches on available reference libraries. The resolving power of the obtained data was discussed in the light of statistical tools such as Spider R-package and Poisson Tree Processes. High levels of species identification were achieved with all the used methodologies, and the occurrence of cryptic species was suggested. We conclude that DNA barcoding is a powerful tool for taxonomic research in Mediterranean mayflies, with great promise to ameliorate biodiversity inventories of freshwater ecosystems and to provide the necessary accuracy for water quality assessment programs. Our results further indicated we need to upgrade the current regional mayfly diversity knowledge. The development of a Mediterranean reference library could integrate this new information system.     

DNA barcodes as a tool in biodiversity research: testing pre-existing taxonomic hypotheses in Delphic Apollo butterflies (Lepidoptera,Papilionidae)

Numerous studies have demonstrated that DNA barcoding is an effective tool for detecting DNA clusters, which can be viewed as operational taxonomic units (OTUs), useful for biodiversity research. Frequently, the OTUs in these studies remained unnamed, not connected with pre-existing taxonomic hypotheses, and thus did not really contribute to feasible estimation of species number and adjustment of species boundaries. For the majority of organisms, taxonomy is very complicated with numerous, often contradictory interpretations of the same characters, which may result in several competing checklists using different specific and subspecific names to describe the same sets of populations. The highly species-rich genus Parnassius (Lepidoptera: Papilionidae) is but one example, such as several mutually exclusive taxonomic systems have been suggested to describe the phenotypic diversity found among its populations. Here we provide an explicit flow chart describing how the DNA barcodes can be combined with the existing knowledge of morphology-based taxonomy and geography (sympatry versus allopatry) of the studied populations in order to support, reject or modify the pre-existing taxonomic hypotheses. We then apply this flow chart to reorganize the taxa within the Parnassius delphius species group, solving long-standing taxonomic problems.     

Method for quick DNA barcode reference library construction

DNA barcoding has become one of the most important techniques in plant species identification. Successful application of this technology is dependent on the availability of reference database of high species coverage. Unfortunately, there are experimental and data processing challenges to construct such a library within a short time. Here, we present our solutions to these challenges. We sequenced six conventional DNA barcode fragments (ITS1, ITS2, matK1, matK2, rbcL1, and rbcL2) of 380 flowering plants on next‐generation sequencing (NGS) platforms (Illumina Hiseq 2500 and Ion Torrent S5) and the Sanger sequencing platform. After comparing the sequencing depths, read lengths, base qualities, and base accuracies, we conclude that Illumina Hiseq2500 PE250 run is suitable for conventional DNA barcoding. We developed a new “Cotu” method to create consensus sequences from NGS reads for longer output sequences and more reliable bases than the other three methods. Step‐by‐step instructions to our method are provided. By using high‐throughput machines (PCR and NGS), labeling PCR, and the Cotu method, it is possible to significantly reduce the cost and labor investments for DNA barcoding. A regional or even global DNA barcoding reference library with high species coverage is likely to be constructed in a few years.     

Integrating a comprehensive DNA barcode reference library with a global map of yews (Taxus L.) for forensic identification

Rapid and accurate identification of endangered species is a critical component of biosurveillance and conservation management, and potentially policing illegal trades. However, this is often not possible using traditional taxonomy, especially where only small or preprocessed parts of plants are available. Reliable identification can be achieved via a comprehensive DNA barcode reference library, accompanied by precise distribution data. However, these require extensive sampling at spatial and taxonomic scales, which has rarely been achieved for cosmopolitan taxa. Here, we construct a comprehensive DNA barcode reference library and generate distribution maps using species distribution modelling (SDM), for all 15 Taxus species worldwide. We find that trnL‐trnF is the ideal barcode for Taxus: It can distinguish all Taxus species and in combination with ITS identify hybrids. Among five analysis methods tested, NJ was the most effective. Among 4,151 individuals screened for trnL‐trnF, 73 haplotypes were detected, all species‐specific and some population private. Taxonomical, geographical and genetic dimensions of sampling strategy were all found to affect the comprehensiveness of the resulting DNA barcode library. Maps from SDM showed that most species had allopatric distributions, except T. mairei in the Sino‐Himalayan region. Using the barcode library and distribution map data, two unknown forensic samples were identified to species (and in one case, population) level and another was determined as a putative interspecific hybrid. This integrated species identification system for Taxus can be used for biosurveillance, conservation management and to monitor and prosecute illegal trade. Similar identification systems are recommended for other IUCN‐ and CITES‐listed taxa.     

Integrating DNA barcode data and taxonomic practice: determination, discovery, and description

DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.     

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.     

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.     

A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding‐based biomonitoring

This study summarizes results of a DNA barcoding campaign on German Diptera, involving analysis of 45,040 specimens. The resultant DNA barcode library includes records for 2,453 named species comprising a total of 5,200 barcode index numbers (BINs), including 2,700 COI haplotype clusters without species‐level assignment, so called “dark taxa.” Overall, 88 out of 117 families (75%) recorded from Germany were covered, representing more than 50% of the 9,544 known species of German Diptera. Until now, most of these families, especially the most diverse, have been taxonomically inaccessible. By contrast, within a few years this study provided an intermediate taxonomic system for half of the German Dipteran fauna, which will provide a useful foundation for subsequent detailed, integrative taxonomic studies. Using DNA extracts derived from bulk collections made by Malaise traps, we further demonstrate that species delineation using BINs and operational taxonomic units (OTUs) constitutes an effective method for biodiversity studies using DNA metabarcoding. As the reference libraries continue to grow, and gaps in the species catalogue are filled, BIN lists assembled by metabarcoding will provide greater taxonomic resolution. The present study has three main goals: (a) to provide a DNA barcode library for 5,200 BINs of Diptera; (b) to demonstrate, based on the example of bulk extractions from a Malaise trap experiment, that DNA barcode clusters, labelled with globally unique identifiers (such as OTUs and/or BINs), provide a pragmatic, accurate solution to the “taxonomic impediment”; and (c) to demonstrate that interim names based on BINs and OTUs obtained through metabarcoding provide an effective method for studies on species‐rich groups that are usually neglected in biodiversity research projects because of their unresolved taxonomy.     

A DNA mini‐barcode for marine macrophytes

Studies focusing on marine macrophyte metabarcoding from environmental samples are scarce, due to the lack of a universal barcode for these taxa, and to their poor representation in DNA databases. Here, we searched for a short barcode able to identify marine macrophytes from tissue samples; then, we created a DNA reference library which was used to identify macrophytes in eDNA from coastal sediments. Barcoding of seagrasses, mangroves and marine macroalgae (Chlorophyta, Rhodophyta and Phaeophyceae) was tested using 18 primer pairs from six barcoding genes: the plant barcodes rbcL, matK and trnL, plus the genes ITS2, COI and 18S. The 18S gene showed the highest universality among marine macrophytes, amplifying 95%–100% of samples; amplification performance of the other barcodes was limited. Taxonomy was assigned using a phylogeny‐based approach to create an 18S DNA reference library. Macrophyte tissue sequences were accurately identified within their phyla (88%), order (76%), genus (71%) and species (23%). Nevertheless, out of 86 macrophytes tested, only 48% and 15% had a reference sequence at genus and at species level, respectively. Identification at these levels can be improved by more inclusive reference libraries. Using the 18S mini‐barcode and the reference library, we recovered eDNA from 21 marine macrophytes in sediments, demonstrating the barcode's ability to trace primary producers that contribute to blue carbon. We expect this barcode to also be useful for other ecological questions, such as tracing macro primary producers in marine food webs.     

DNA barcoding largely supports 250 years of classical taxonomy: identifications for Central European bees (Hymenoptera,Apoidea partim)

This study presents DNA barcode records for 4118 specimens representing 561 species of bees belonging to the six families of Apoidea (Andrenidae, Apidae, Colletidae, Halictidae, Megachilidae and Melittidae) found in Central Europe. These records provide fully compliant barcode sequences for 503 of the 571 bee species in the German fauna and partial sequences for 43 more. The barcode results are largely congruent with traditional taxonomy as only five closely allied pairs of species could not be discriminated by barcodes. As well, 90% of the species possessed sufficiently deep sequence divergence to be assigned to a different Barcode Index Number (BIN). In fact, 56 species (11%) were assigned to two or more BINs reflecting the high levels of intraspecific divergence among their component specimens. Fifty other species (9.7%) shared the same Barcode Index Number with one or more species, but most of these species belonged to a distinct barcode cluster within a particular BIN. The barcode data contributed to clarifying the status of nearly half the examined taxonomically problematic species of bees in the German fauna. Based on these results, the role of DNA barcoding as a tool for current and future taxonomic work is discussed.     

A DNA mini‐barcode for land plants

Small portions of the barcode region – mini‐barcodes – may be used in place of full‐length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini‐barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini‐barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30 472)]. PCR amplification for all mini‐barcodes, as estimated by validated electronic simulation, was successful for 90.2–99.8% of species. Overall Sanger sequence quality for mini‐barcodes was very low – the best mini‐barcode tested produced sequences of adequate quality (B₂₀ ≥ 0.5) for 74.5% of samples. The majority of mini‐barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini‐barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini‐barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini‐barcode D (F52/R193).