FISH-BOL and seafood identification: Geographically dispersed case studies reveal systemic market substitution across Canada

BackgroundAll-inclusive DNA-barcoding libraries in the storage and analysis platform of the BOLD (Barcode of Life Data) system are essential for the study of the marine biodiversity and are pertinent for regulatory purposes, including ecosystem monitoring and assessment, such as in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). Here, we investigate knowledge gaps in the lists of DNA barcoded organisms within two inventories, Cnidaria (Anthozoa and Hydrozoa) and Ascidiacea from the reference libraries of the European Register of Marine Species (ERMS) dataset (402 ascidians and 1200 cnidarian species). ERMS records were checked species by species, against publicly available sequence information and other data stored in BOLD system. As the available COI barcode data adequately cover just a small fraction of the ERMS reference library, it is of importance to employ quality control on existing data, to close the knowledge gaps and purge errors off BOLD.ResultsResults revealed that just 22.9% and 29.2% of the listed ascidians and cnidarians species, respectively, are BOLD barcodes of which 58.4% and 52.3% of the seemingly barcoded species, respectively, were noted to have complete BOLD pages. Thus, only 11.44% of the tunicate and 17.07% of the cnidarian data in the ERMS lists are of high quality. Deep analyses revealed seven common types of gaps in the list of the barcoded species in addition to a wide range of discrepancies and misidentifications, discordances, and errors primarily in the GenBank mined data as with the BINs assignments, among others.ConclusionsGap knowledge in barcoding of important taxonomic marine groups exists, and in addition, quality management elements (quality assurance and quality control) were not employed when using the list for national monitoring projects, for regulatory compliance purposes and other purposes. Even though BOLD is the most trustable DNA-barcoding reference library, worldwide projects of DNA barcoding are needed to close these gaps of mistakes, verifications, missing data, and unreliable sequencing labs. Tight quality control and quality assurance are important to close the knowledge gaps of Barcoding of the European recommended ERMS reference library.

DNA barcoding as a regulatory tool for seafood authentication in Canada

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.

DNA barcoding is one of the best funded and most visible large-scale initiatives in systematic biology and has generated both much interest and controversy. DNA barcoding has also attracted significant support from foundations that had previously shown little interest in systematics. Yet, the project is controversial because many systematists feel that currently the conceptual foundation of DNA barcoding remains weak. This problem can only be alleviated through additional research that can lead to improved tools and concepts. Here, we scrutinize a key concept of DNA barcoding, the so-called barcoding gap (Meyer and Paulay, 2005), and use empirical data to document that it needs to be computed based on the smallest instead of the mean interspecific distances. In the literature on DNA barcoding, the “barcoding gap” (Meyer and Paulay, 2005) refers to the separation between mean intraand interspecific sequence variability for congeneric COI sequences. The barcoding gap is so essential to barcoding that a widely cited publication was dedicated to documenting these gaps across major metazoan taxa (Hebert et al., 2003b). It is also regularly mentioned in articles promoting barcoding to a broader audience (Check, 2005; Cognato and Caesar, 2006; Dasmahapatra and Mallet, 2006) and is one of the few metrics included in the Web-based identification system BOLD, “The Barcode of Life Data System,” which is a major identification tool for the DNA barcoding community (http://www.barcodinglife.org; Ratnasingham and Hebert, 2007). Large barcoding gaps are routinely used to predict DNA-barcoding success for the taxon under study (Hebert et al., 2003a, 2003b, 2004a, 2004b; Hogg and Hebert, 2004; Powers, 2004; Zehner et al., 2004; Armstrong and Ball, 2005; Ball et al., 2005; Barrett and Hebert, 2005; Lorenz et al., 2005; Saunders, 2005; Smith et al., 2005, 2006; Ward et al., 2005; Cywinska et al., 2006; Hajibabaei et al., 2006a, 2006b; Lefebure et al., 2006; Clare et al., 2007; Seifert et al., 2007). However, here we argue and document that barcoding gaps are currently incorrectly computed and that the values reported in the barcoding literature are misleading. The main problem is that the barcoding gap is generally quantified as the difference between intraspecific and mean interspecific, congeneric distances, whereas we will argue here that for species identification only the smallest interspecific distance should be used. Other authors have also pointed out that the use of smallest interspecific distances would be more appropriate (see Sperling, 2003; Moritz and Cicero, 2004; Vences et al., 2005a, 2005b; Cognato, 2006; Meier et al., 2006; Meyer and Paulay, 2005; Roe and Sperling, 2007), but currently we lack a comparative study that documents that the two measures yield different results. Here we provide evidence based on 43,137 COI sequences from 12,459 Metazoan species that barcoding gaps based on mean interspecific distances are artificially inflated and that only smallest interspecific distances correctly reflect that species identification gets more difficult as more species are sampled. Using DNA barcodes for species identification is analogous to identifying an unidentified specimen by comparing it to a reference collection of identified specimens. Initially one may compare an unidentified specimen to all identified material in the same genus, but ultimately the identification problem pares down to deciding whether a specimen belongs to one of a few, very similar, congeneric species. Determining an unidentified specimen to species is straightforward if the intraspecific variability is small—i.e., the unidentified specimen is a good match to a referenced species—and the differences between the best-matching species and the next best match is large—i.e., the specimen is a good match to only one of the referenced species. Analogously, the ease with which a query sequence can be identified to species is only dependent on how different it is from the most similar allospecific sequence, whereas its distinctness from a hypothetical “average” congeneric species does not matter (see Sperling, 2003; Moritz and Cicero, 2004; Vences et al., 2005a, 2005b; Cognato, 2006; Meier et al., 2006; Meyer and Paulay, 2005; Roe and Sperling, 2007). Yet, DNA barcoding publications and BOLD continue to report the mean instead of the smallest interspecific distances for congeneric species.

Only 10% of the earth’s biota has been described despite250 yearsoftaxonomicresearch(Wilson2000).Thisisinlargepart a reflection of the extent and complexity of biologicaldiversity,butitisalsotruethattraditionaltaxonomictechniquesare labourious and highly specialised, and taxonomic expertiseis very thinly spread across the myriad groups of life (Scotland

DNA barcoding is widely used to identify commercial fish species sold in the U.S and internationally. This method is based on DNA sequencing of a standardized region of DNA from a sample. Sequences can then be analyzed against the Barcode of Life Data system (BOLD) for species identification. Additional analysis of samples by a method called DNA mini‐barcoding can be used for samples that cannot be correctly identified to the species level by the traditional DNA barcoding method. While relatively high levels of seafood mislabeling have been reported for sushi dishes, few studies have investigated mislabeling of other raw, ready‐to‐eat (RTE) seafood such as poke and ceviche. The objective of this study was to evaluate the presence of species mislabeling in raw, RTE seafood products in Orange County, CA. DNA barcoding and mini‐barcoding were used to investigate 70 samples of sushi, ceviche, and poke collected from various locations in Orange County. All samples underwent DNA barcoding, followed by species identification using the BOLD database. Six samples underwent further analysis with mini‐barcoding. The FDA seafood list was used to identify common and acceptable market names. The results showed that 13 samples were substituted on the basis of species, and 20 samples did not have an acceptable market name. The overall mislabeling rate for all samples combined was 52.3% (33/63). These results indicate a need for greater scrutiny of labeling practices for raw, RTE seafood sold in Orange County, California.

DNA barcoding reflects the diversity and variety of brooding traits of fish species in the family Syngnathidae along China’s coast

DNA barcoding analysis is based on the comparison of genetic distances to identify species using a segment of Cytochrome C Oxidase I (COI) gene. Species identification through DNA barcoding challenges problems in groups with high genetic diversity as molluscs. Thus, our aim was to estimate intraspecific divergence in the Amazonian land snail Systrophia helicycloides (Gastropoda, Scolodontidae) and evaluate the use of DNA barcoding in molecular identification of this land snail. Nucleotide sequences were compared with Genbank and BOLD (Barcode of Life Data Systems) databases. We conducted distance analyses using the Neighbour Joining method. Systrophia helicycloides showed two groups of haplotypes and intraspecific genetic distances higher than 4%. We observed an overlap between intraspecific and interspecific distances. The high divergence may be related to rapid mutation rate in the snail mitochondrial genome, to population distribution that influences genetic isolation and differentiation, and to ancestral DNA polymorphisms. COI profiles uploaded in BOLD are the first records of this species and can identify Systrophia helcycloides from other species. These profiles corroborated the high variation in the land snail genome. Therefore, species identification in this group needs a combined analysis of genetic distances, informative sites, and conventional taxonomy.

The rapid molecular diagnostics of adulterants in herbal medicine using DNA barcoding forms the core of this meticulously detailed review, based on two decades of data. With 80% of the world's population using some form of herbal medicine, authentication, quality control, and detection of adulterants warrant DNA barcoding. A combined group of keywords were used for literature review using the PubMed, the ISI Web of Knowledge, Web of Science (WoS), and Google Scholar databases. All the papers (N=210) returned by the search engines were downloaded and systematically analyzed. Detailed analysis of conventional DNA barcodes were based on retrieved sequences for internal transcribed spacer (ITS) (412,189), rbcL (251,598), matK (210,835), and trnH-psbA (141,846). The utility of databases such as The Barcode of Life Data System (BOLD), NCBI, GenBank, and Medicinal Materials DNA Barcode Database (MMDBD) has been critically examined for the identification of unknown species from known databases. The current review gives an overview of the ratio of adulterated to authentic drugs for some countries along with the state of the art technology currently being used in the identification of adulterated medicines. In this review, efforts were made to systematically analyze and arrange the research and reviews on the basis of technical progress. The review concludes with the future of DNA-based herbal medicine adulteration detection, forecasting the reliance on the metabarcoding technology. DNA barcoding technology for differentiating adulterated herbal medicine.

Event Abstract Back to Event Complementing morphological classification of Anguilliform leptocephali with DNA barcoding Alessandra Anibaldi1, Claudia Benassi Franciosi1, Fausto Tinti1, 2, Corrado Piccinetti1 and Giulia Riccioni1* 1 University of Bologna, Department of Biological, Geological and Environmental Sciences, Italy 2 University of Bologna, Department of Biological, Geological and Environmental Sciences, Italy DNA barcoding is a molecular tool that enables rapid and accurate identification of biological species by sequencing a short, standardized region of the mitochondrial gene cytochrome c oxidase I ( COI) as internal species tag. The aim of the present study was to use DNA barcoding in addition to classical taxonomy in order to obtain a more reliable species-identification of leptocephali, the characteristic larval forms of the superorder Elopomorpha, often difficult to identify and to match with their adult stage. Based on the examination of external morphology, meristics, and pigmentation, 2785 leptocephalus larvae collected in the Adriatic Sea between 2010 and 2012, were ascribed to 7 morphotypes, belonging to Anguilliform order (Ariosoma balearicum, Conger conger, Gnathophis mystax, Nettastoma melanurum, Dalophis imberbis, Chlopsis bicolor, Facciolella sp.) and 69 specimens were sequenced for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (COI) to confirm the previous morphological analysis. The highly consistent results obtained revealed a good performance of COI barcoding as a diagnostic method for the identification of these larvae, but the limited number of leptocephali species annotated in the reference databases for barcode (Barcode of Life Data Systems and GenBank) allowed to validate only partially the morphological analysis. Moreover two species, Gnathophis mystax and Facciolella sp., showed unexpected outcomes. The data obtained in this work represent the first results of a wider project aimed at the creation of a new barcode database for the assessment of leptocephali diversity in the Mediterranean Sea (Barcoding of the Adriatic Leptocephali [BAL]), contributing to the knowledge of these unusual larvae and of their adult forms. Keywords: barcoding, Leptocephali, Adriatic sea, COI, Identification of larvae Conference: XV European Congress of Ichthyology, Porto, Portugal, 7 Sep - 11 Sep, 2015. Presentation Type: Oral Presentation Topic: Phylogeny, Systematics and Genetics Citation: Anibaldi A, Benassi Franciosi C, Tinti F, Piccinetti C and Riccioni G (2015). Complementing morphological classification of Anguilliform leptocephali with DNA barcoding. Front. Mar. Sci. Conference Abstract: XV European Congress of Ichthyology. doi: 10.3389/conf.fmars.2015.03.00021 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 24 Oct 2015; Published Online: 04 Nov 2015. * Correspondence: PhD. Giulia Riccioni, University of Bologna, Department of Biological, Geological and Environmental Sciences, Fano, 61032, Italy, giulia.riccioni@unibo.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alessandra Anibaldi Claudia Benassi Franciosi Fausto Tinti Corrado Piccinetti Giulia Riccioni Google Alessandra Anibaldi Claudia Benassi Franciosi Fausto Tinti Corrado Piccinetti Giulia Riccioni Google Scholar Alessandra Anibaldi Claudia Benassi Franciosi Fausto Tinti Corrado Piccinetti Giulia Riccioni PubMed Alessandra Anibaldi Claudia Benassi Franciosi Fausto Tinti Corrado Piccinetti Giulia Riccioni Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

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.

The COI gene is widely used as a DNA barcode in animals that can assist in the identification of species. One of the widely used aggregators of DNA barcodes is the Barcode of Life Data System (BOLD Systems), which contains around 2500 sequences of Tetrigidae, an understudied orthopteran family with unresolved taxonomy and species that are difficult to identify. In this paper, we provide a summary of the metadata provided with the COI sequences and present a phylogenetic analysis of photographically vouchered sequences using Maximum Likelihood and Bayesian analysis. We found that (1) the subfamily Tetriginae is disproportionately represented in the dataset, (2) most of the records are not identified beyond the family level, (3) most regions, except for Costa Rica, are undersampled, (4) most of the sequences do not have photographic vouchers, and (5) the taxonomic backbone of BOLD is out of date. The phylogenetic analysis showed that the clusters of COI barcodes mostly correspond to species, but some clusters remain ambiguous. The deeper nodes in the phylogenetic trees are not well-supported, indicating that this gene has a very weak phylogenetic signal beyond the specific level.

DNA barcoding is a widely used tool for species identification, with its reliability heavily dependent on reference databases. While the quality of these databases has long been debated, a critical knowledge gap remains in their comprehensive evaluation and comparison at regional scales. Marine metazoan species in the western and central Pacific Ocean (WCPO), a region characterized by high biodiversity and limited sequencing efforts, are an example of this gap. This study developed a systematic workflow to assess mitochondrial cytochrome c oxidase subunit I (COI) barcode coverage and sequence quality in two commonly used reference databases for DNA barcoding: the nucleotide reference database from the National Center for Biotechnology Information (NCBI); and from the Barcode of Life Data System (BOLD). Comparative analyses across marine phyla and WCPO regions identified significant barcode gaps and quality problems, providing insights to guide future barcoding efforts. NCBI exhibited higher barcode coverage, but lower sequence quality compared to BOLD. Quality issues, including over- or under-represented species, short sequences, ambiguous nucleotides, incomplete taxonomic information, conflict records, high intraspecific distances, and low inter-specific distances were identified in both databases, likely resulting from contamination, cryptic species, sequencing errors, or inconsistent taxonomic assignment. The barcode identification number (BIN) system in BOLD demonstrated potential for identifying and addressing problematic records, highlighting the benefits of curated databases. Significant barcode deficiencies and quality issues were observed in the south temperate region of WCPO and phyla such as Porifera, Bryozoa, and Platyhelminthes. Additionally, the COI barcode showed limited species-level resolution for certain taxa, including Scombridae and Lutjanidae. Addressing barcode coverage gaps, improving taxonomic representation, and enhancing sequence quality will be essential for strengthening future barcoding initiatives and advancing biodiversity monitoring and conservation in the WCPO and beyond. This study highlights the need for standardized database curation and sequencing practices to improve the global reliability and applicability of DNA barcoding.

Wing geometric morphometrics and DNA barcoding to distinguish three closely related species of Armigeres mosquitoes (Diptera: Culicidae) in Thailand

An integrative approach to DNA barcoding, geometric morphometrics, and machine learning for field identification of Culex mosquitoes (Diptera: Culicidae), with implications for vector-borne disease surveillance.