Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France

Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available.We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes.     

Identification of North Sea molluscs with DNA barcoding

Sequence‐based specimen identification, known as DNA barcoding, is a common method complementing traditional morphology‐based taxonomic assignments. The fundamental resource in DNA barcoding is the availability of a taxonomically reliable sequence database to use as a reference for sequence comparisons. Here, we provide a reference library including 579 sequences of the mitochondrial cytochrome c oxidase subunit I for 113 North Sea mollusc species. We tested the efficacy of this library by simulating a sequence‐based specimen identification scenario using Best Match, Best Close Match (BCM) and All Species Barcode (ASB) criteria with three different threshold values. Each identification result was compared with our prior morphology‐based taxonomic assignments. Our simulation resulted in 87.7% congruent identifications (93.8% when excluding singletons). The highest number of congruent identifications was obtained with BCM and ASB and a 0.05 threshold. We also compared identifications with genetic clustering (Barcode Index Numbers, BINs) computed by the Barcode of Life Datasystem (BOLD). About 68% of our morphological identifications were congruent with BINs created by BOLD. Forty‐nine sequences were clustered in 16 discordant BINs, and these were divided in two classes: sequences from different species clustered in a single BIN and conspecific sequences divided in more BINs. Whereas former incongruences were probably caused by BOLD entries in need of a taxonomic update, the latter incongruences regarded taxa requiring further investigations. These include species with amphi‐Atlantic distribution, whose genetic structure should be evaluated over their entire range to produce a reliable sequence‐based identification system.     

应用DNA条形码对植物标本的核查

DNA条形码主要目的是物种鉴定和新物种或隐存种的发现,而DNA条形码参考数据库是物种快速鉴定的重要基础。目前中国维管植物DNA条形码参考数据库正在建设之中,借助于公共数据库（NCBI）和初步建立的中国植物DNA条形码参考数据库,运用DNA条形码数据开展了植物标本鉴定的核查工作：（1）比较DNA序列信息与标本鉴定信息,从科、属、种级水平查找鉴定错误的标本;（2）基于有较好研究基础的DNA条形码参考数据库,开展未知标本的鉴定;（3）通过对标本核查的总结,提出DNA条形码参考数据库建设过程中的几点建议。     

Identifying gastropod spawn from DNA barcodes: possible but not yet practicable

Identifying life stages of species with complex life histories is problematic as species are often only known and/or described from a single stage. DNA barcoding has been touted as an important tool for linking life-history stages of the same species. To test the current efficacy of DNA barcodes for identifying unknown mollusk life stages, 24 marine gastropod egg capsules were collected off the Philippines in deep water and sequenced for partial fragments of the COI, 16S and 12S mitochondrial genes. Two egg capsules of known shallow-water Mediterranean species were used to calibrate the method. These sequences were compared to those available in GenBank and the Barcode of Life Database (BOLD). Using COI sequences alone, only a single Mediterranean egg capsule was identified to species, and a single Philippine egg capsule was identified tentatively to genus; all other COI sequences recovered matches between 76% and 90% with sequences from BOLD and GenBank. Similarity-based identification using all three markers confirmed the Mediterranean specimens' identifications. A phylogenetic approach was also implemented to confirm similarity-based identifications and provide a higher-taxonomic identification when species-level identifications were not possible. Comparison of available GenBank sequences to the diversity curve of a well-sampled coral reef habitat in New Caledonia highlights the poor taxonomic coverage achieved at present in existing genetic databases, emphasizing the need to develop DNA barcoding projects for megadiverse and often taxonomically challenging groups such as mollusks, to fully realize its potential as an identification and discovery tool.     

The Hemiptera (Insecta) of Canada: Constructing a Reference Library of DNA Barcodes

DNA barcode reference libraries linked to voucher specimens create new opportunities for high-throughput identification and taxonomic re-evaluations. This study provides a DNA barcode library for about 45% of the recognized species of Canadian Hemiptera, and the publically available R workflow used for its generation. The current library is based on the analysis of 20,851 specimens including 1849 species belonging to 628 genera and 64 families. These individuals were assigned to 1867 Barcode Index Numbers (BINs), sequence clusters that often coincide with species recognized through prior taxonomy. Museum collections were a key source for identified specimens, but we also employed high-throughput collection methods that generated large numbers of unidentified specimens. Many of these specimens represented novel BINs that were subsequently identified by taxonomists, adding barcode coverage for additional species. Our analyses based on both approaches includes 94 species not listed in the most recent Canadian checklist, representing a potential 3% increase in the fauna. We discuss the development of our workflow in the context of prior DNA barcode library construction projects, emphasizing the importance of delineating a set of reference specimens to aid investigations in cases of nomenclatural and DNA barcode discordance. The identification for each specimen in the reference set can be annotated on the Barcode of Life Data System (BOLD), allowing experts to highlight questionable identifications; annotations can be added by any registered user of BOLD, and instructions for this are provided.     

Meta-Fish-Lib: A generalised,dynamic DNA reference library pipeline for metabarcoding of fishes

The accuracy and reliability of DNA metabarcoding analyses depend on the breadth and quality of the reference libraries that underpin them. However, there are limited options available to obtain and curate the huge volumes of sequence data that are available on public repositories such as NCBI and BOLD. Here, we provide a pipeline to download, clean and annotate mitochondrial DNA sequence data for a given list of fish species. Features of this pipeline include (a) support for multiple metabarcode markers; (b) searches on species synonyms and taxonomic name validation; (c) phylogeny assisted quality control for identification and removal of misannotated sequences; (d) automatically generated coverage reports for each new GenBank release update; and (e) citable, versioned DOIs. As an example we provide a ready-to-use curated reference library for the marine and freshwater fishes of the U.K. To augment this reference library for environmental DNA metabarcoding specifically, we generated 241 new MiFish-12S sequences for 88 U.K. marine species, and make available new primer sets useful for sequencing these. This brings the coverage of common U.K. species for the MiFish-12S fragment to 93%, opening new avenues for scaling up fish metabarcoding across wide spatial gradients. The Meta-Fish-Lib reference library and pipeline is hosted at https://github.com/genner-lab/meta-fish-lib .     

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.     

Peptizer, a tool for assessing false positive peptide identifications and manually validating selected results

False positive peptide identifications are a major concern in the field of peptidecentric, mass spectrometry-driven gel-free proteomics. They occur in regions where the score distributions of true positives and true negatives overlap. Removal of these false positive identifications necessarily involves a trade-off between sensitivity and specificity. Existing postprocessing tools typically rely on a fixed or semifixed set of assumptions in their attempts to optimize both the sensitivity and the specificity of peptide and protein identification using MS/MS spectra. Because of the expanding diversity in available proteomics technologies, however, these postprocessing tools often struggle to adapt to emerging technology-specific peculiarity. Here we present a novel tool named Peptizer that solves this adaptability issue by making use of pluggable assumptions. This research-oriented postprocessing tool also includes a graphical user interface to perform efficient manual validation of suspect identifications for optimal sensitivity recovery. Peptizer is open source software under the Apache2 license and is written in Java.     

Re-integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding

Species identification of earthworms is usually achieved by careful observation of morphological features, often sexual characters only present in adult specimens. Consequently, juveniles or cocoons are often impossible to identify, creating a possible bias in studies that aim to document species richness and abundance. DNA barcoding, the use of a short standardized DNA fragment for species identification, is a promising approach for species discrimination. When a reference library is available, DNA-based identification is possible for all life stages. In this study, we show that DNA barcoding is an unrivaled tool for high volume identification of juvenile earthworms. To illustrate this advance, we generated DNA barcodes for specimens of Lumbricus collected from three temperate grasslands in western France. The analysis of genetic distances between individuals shows that juvenile sequences unequivocally match DNA barcode clusters of previously identified adult specimens, demonstrating the potential of DNA barcoding to provide exhaustive specimen identification for soil ecological research.     

A DNA barcode library for Germany′s mayflies,stoneflies and caddisflies (Ephemeroptera,Plecoptera and Trichoptera)

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well‐curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost‐efficient and reliable DNA‐based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two‐thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution.     

A DNA barcode library for North American Ephemeroptera: progress and prospects

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional species of mayflies (Insecta: Ephemeroptera) from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some Caenis. Minimum interspecific distances ranged from 0.3-24.7% (mean: 12.5%), while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species.     

Barcoding Nemo: DNA-Based Identifications for the Ornamental Fish Trade

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.     

Barcoding and border biosecurity: identifying cyprinid fishes in the aquarium trade

Background

Poorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species.

Methodology/Principal Findings

We sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90–99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii.

Conclusions/Significance

We demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade.     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

MALDI-TOF-Based Dermatophyte Identification

MALDI-TOF MS has become increasingly popular for microorganism identification in the routine laboratory. Compared with conventional morphology-based techniques, MALDI-TOF is relatively inexpensive (per-unit identification), involves a rapid result turnaround time and yields more accurate results without the need for highly qualified staff. However, this technology has been technically difficult to implement for filamentous fungi identification. Identification of dermatophytes, a type of filamentous fungi, remains particularly challenging, partly due to the lack of clear species definition for some taxa or within some species complexes. Review of the ten studies published between 2008 and 2015 shows that the accuracy of MALDI-TOF MS-based identification varied between 13.5 and 100 % for dermatophytes. This variability was partly due to inconsistencies concerning critical steps of the routine clinical laboratory process. Use of both a complete formic acid-acetonitrile protein extraction step and a manufacturer library supplemented with homemade reference spectra is essential for an accurate species identification. This technique is conversely unaffected by variations in other routine clinical laboratory conditions such as culture medium type, incubation time and type of mass spectrometry instrument. Provided that a reference spectra library is adequate for dermatophyte identification, MALDI-TOF MS identification is more economical and offers an accuracy comparable to that of DNA sequencing. The technique also represents an advantageous alternative to the protracted and labor-intensive dermatophyte identification via macroscopic and microscopic morphology in the routine clinical laboratory.     

After 7 years and 1000 citations: comparative assessment of the DNA barcoding and the DNA taxonomy proposals for taxonomists and non-taxonomists

In 2003, two different approaches-DNA taxonomy and DNA barcoding-were simultaneously proposed to overcome some of the perceived intrinsic weaknesses of the traditional morphology-based taxonomical system, and to help non-taxonomists to resolve their crucial need for accurate and rapid species identification tools. After 7 years, it seems unlikely that a completely new taxonomical system based on molecular characters only (DNA taxonomy) will develop in the future. It is more likely that both morphological and molecular data will be simultaneously analyzed, developing what has been coined as "integrative taxonomy". Concerning DNA barcoding, it is now clear that it does not focus on building a tree-of-life nor to perform DNA taxonomy, but rather to produce a universal molecular identification key based on strong taxonomic knowledge that is collated in the barcode reference library. The indisputable success of the DNA barcoding project is chiefly due to the fact that DNA barcoding standards considerably enhance current practices in the molecular identification field, and standardization offers virtually endless applications for various users.     

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.     

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.