Revision and annotation of DNA barcode records for marine invertebrates: Report of the 8th iBOL conference hackathon

The accuracy of specimen identification through DNA barcoding and metabarcoding relies on reference libraries containing records with reliable taxonomy and sequence quality. The considerable growth in barcode data requires stringent data curation, especially in taxonomically difficult groups such as marine invertebrates. A major effort in curating marine barcode data in the Barcode of Life Data Systems (BOLD) was undertaken during the 8th International Barcode of Life Conference (Trondheim, Norway, 2019). Major taxonomic groups (crustaceans, echinoderms, molluscs, and polychaetes) were reviewed to identify those which had disagreement between Linnaean names and Barcode Index Numbers (BINs). The records with disagreement were annotated with four tags: A) MIS-ID (misidentified, mislabeled, or contaminated records), b) AMBIG (ambiguous records unresolved with the existing data), c) COMPLEX (species names occurring in multiple BINs), and d) SHARE (barcodes shared between species). A total of 83,712 specimen records corresponding to 7,576 species were reviewed and 39% of the species were tagged (7% MIS-ID, 17% AMBIG, 14% COMPLEX, and 1% SHARE). High percentages (>50%) of AMBIG tags were recorded in gastropods, whereas COMPLEX tags dominated in crustaceans and polychaetes. The high proportion of tagged species reflects either flaws in the barcoding workflow (e.g., misidentification, cross-contamination) or taxonomic difficulties (e.g., synonyms, undescribed species). Although data curation is essential for barcode applications, such manual attempts to examine large datasets are unsustainable and automated solutions are extremely desirable.The hackathon was organized with financial support from the European Union COST Action DNAqua-Net (CA 15219 https://dnaqua.net/) in the scope of the 8th International Barcode of Life Conference in Trondheim, Norway on 16 June 2019. DNAqua-Net is acknowledged for the funding provided and the local conference organizers for all the logistical support that ensured a successful event. Tyler Elliot and the rest of the BOLD team are acknowledged for their help with data queries and analytics. The authors also thank the hackathon participants for vibrant discussions during and after the event: Berry van der Hoorn, Katrine Konsghavn, Guy Paz, Mouna Rifi, Malin Strand, Anne Helene Tandberg, Adam Wall, and Endre Willassen. Marcos A. L. Teixeira was supported by a PhD grant from the Portuguese Foundation for Science and Technology (FCT I.P.) co-financed by ESF (SFRH/BD/131527/2017). Financial support granted by FCT to Sofia Duarte (CEECIND/00667/2017) and to Pedro E. Vieira (project NIS-DNA, PTDC/BIA-BMA/29754/2017) is also acknowledged. Sanna Majaneva was financially supported by the Norwegian Taxonomy Initiative (project no. 70184235). The authors thank the five reviewers who provided valuable input into the earlier version of the manuscript

A reference library for Canadian invertebrates with 1.5 million barcodes, voucher specimens, and DNA samples

The synthesis of this dataset was enabled by funding from the Canada Foundation for Innovation, from Genome Canada through Ontario Genomics, from NSERC, and from the Ontario Ministry of Research, Innovation and Science in support of the International Barcode of Life project. It was also enabled by philanthropic support from the Gordon and Betty Moore Foundation and from Ann McCain Evans and Chris Evans. The release of the data on GGBN was supported by a GGBN – Global Genome Initiative Award and we thank G. Droege, L. Loo, K. Barker, and J. Coddington for their support. Our work depended heavily on the analytical capabilities of the Barcode of Life Data Systems (BOLD, www.boldsystems.org). We also thank colleagues at the CBG for their support, including S. Adamowicz, S. Bateson, E. Berzitis, V. Breton, V. Campbell, A. Castillo, C. Christopoulos, J. Cossey, C. Gallant, J. Gleason, R. Gwiazdowski, M. Hajibabaei, R. Hanner, K. Hough, P. Janetta, A. Pawlowski, S. Pedersen, J. Robertson, D. Roes, K. Seidle, M. A. Smith, B. St. Jacques, A. Stoneham, J. Stahlhut, R. Tabone, J.Topan, S. Walker, and C. Wei. For bioblitz-related assistance, we are grateful to D. Ireland, D. Metsger, A. Guidotti, J. Quinn and other members of Bioblitz Canada and Ontario Bioblitz. For our work in Canada’s national parks, we thank S. Woodley and J. Waithaka for their lead role in organizing permits and for the many Parks Canada staff who facilitated specimen collections, including M. Allen, D. Amirault-Langlais, J. Bastick, C. Belanger, C. Bergman, J.-F. Bisaillon, S. Boyle, J. Bridgland, S. Butland, L. Cabrera, R. Chapman, J. Chisholm, B. Chruszcz, D. Crossland, H. Dempsey, N. Denommee, T. Dobbie, C. Drake, J. Feltham, A. Forshner, K. Forster, S. Frey, L. Gardiner, P. Giroux, T. Golumbia, D. Guedo, N. Guujaaw, S. Hairsine, E. Hansen, C. Harpur, S. Hayes, J. Hofman, S. Irwin, B. Johnston, V. Kafa, N. Kang, P. Langan, P. Lawn, M. Mahy, D. Masse, D. Mazerolle, C. McCarthy, I. McDonald, J. McIntosh, C. McKillop, V. Minelga, C. Ouimet, S. Parker, N. Perry, J. Piccin, A. Promaine, P. Roy, M. Savoie, D. Sigouin, P. Sinkins, R. Sissons, C. Smith, R. Smith, H. Stewart, G. Sundbo, D. Tate, R. Tompson, E. Tremblay, Y. Troutet, K. Tulk, J. Van Wieren, C. Vance, G. Walker, D. Whitaker, C. White, R. Wissink, C. Wong, and Y. Zharikov. For our work near Canada’s ports in Vancouver, Toronto, Montreal, and Halifax, we thank R. Worcester, A. Chreston, M. Larrivee, and T. Zemlak, respectively. Many other organizations improved coverage in the reference library by providing access to specimens – they included the Canadian National Collection of Insects, Arachnids and Nematodes, Smithsonian Institution’s National Museum of Natural History, the Canadian Museum of Nature, the University of Guelph Insect Collection, the Royal British Columbia Museum, the Royal Ontario Museum, the Pacifc Forestry Centre, the Northern Forestry Centre, the Lyman Entomological Museum, the Churchill Northern Studies Centre, and rare Charitable Research Reserve. We also thank the many taxonomic specialists who identifed specimens, including A. Borkent, B. Brown, M. Buck, C. Carr, T. Ekrem, J. Fernandez Triana, C. Guppy, K. Heller, J. Huber, L. Jacobus, J. Kjaerandsen, J. Klimaszewski, D. Lafontaine, J-F. Landry, G. Martin, A. Nicolai, D. Porco, H. Proctor, D. Quicke, J. Savage, B. C. Schmidt, M. Sharkey, A. Smith, E. Stur, A. Tomas, J. Webb, N. Woodley, and X. Zhou. We also thank K. Kerr and T. Mason for facilitating collections at Toronto Zoo and D. Iles for servicing the trap at Wapusk National Park. This paper contributes to the University of Guelph’s Food from Thought research program supported by the Canada First Research Excellence Fund. The Barcode of Life Data System (BOLD; www.boldsystems.org)8 was used as the primary workbench for creating, storing, analyzing, and validating the specimen and sequence records and the associated data resources48. The BOLD platform has a private, password-protected workbench for the steps from specimen data entry to data validation (see details in Data Records), and a public data portal for the release of data in various formats. The latter is accessible through an API (http://www.boldsystems.org/index.php/resources/api?type=webservices) that can also be controlled through R75 with the package ‘bold’76.Peer reviewedPublisher PD

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequence

L'histoire de deux niveaux de biodiversité démontrée par le code-barre d'ADN chez les crustacés de l'Atlantique du Nord

RÉSUMÉ: La biodiversité est la variété de la vie et elle peut être étudiée à différents niveaux (génétique, espèces, écosystèmes) et à différents échelles (spatiale et temporelle). Les dernières décennies ont montré que la biodiversité marine avait été gravement sous-estimée. Afin d'étudier les caractéristiques de la grande diversité des espèces marines et les processus sous-jacents de l'évolution de ces dernières, il est évident et nécessaire de connaître les espèces. Nous sommes aujourd'hui confrontés aux taux les plus élevés d'extinction depuis la constitution de la société humaine (<<crise de la biodiversité ») et seule une fraction d'espèces a été officiellement décrite (1,9 millions sur 11 millions) , en raison, entre autres, d'une pénurie de taxonomistes formés et disponibles pour cet immense travail. Tous ces facteurs ont conduit à la proposition d'outils moléculaires pour permettre et faciliter l'identification des espèces et notamment le barcode moléculaire (le code-barres d'ADN). Il s'agit de séquencer un fragment d'ADN du gène mitochondrial cytochrome c oxydase 1 (COI) qui constitue alors un outil rapide, précis et rentable pour identifier les espèces. Ainsi, chaque espèce peut être définie par une étiquette d'identification unique et permanente qui ne sera pas changée par une éventuelle modification taxonomique. Outre l'attribution d'échantillons inconnus à des espèces identifiées a priori, les données fournies par le code-barres d'ADN seront très utiles pour des études phylogéographiques comparatives entre taxons multiples, pour clarifier les relations phylogénétiques à différents niveaux taxonomiques et pour élaborer des patrons évolutifs et de spéciation entre les groupes d'organismes. Le Chapitre 1 présente une mise en contexte du code-barres d'ADN par une revue des études qui ont été publiées sur le sujet, notamment en ce qui concerne l'identification des espèces marines. Le Chapitre 2 élabore une bibliothèque pour les crustacés marins de l'estuaire et du golfe du St. Laurent. Toutes les données (taxonomie, informations sur l'échantillonnage, images, séquences d'ADN et chromatogrammes), sont stockées en ligne dans le Barcode of Life Data Systems (BOLD) et sont disponibles pour un usage général. Les spécimens utilisés sont conservés comme 'vouchers' dans des institutions publiques pour des vérifications futures . Les résultats ont montré la présence d'un amphipode invasif dans l'estuaire (mentionné précédemment dans les Grands Lacs et à Montréal, avec des effets sur la faune indigène d'amphipodes), et l'existence d'espèces cryptiques potentielles chez les amphipodes, mysidacés et décapodes. Le Chapitre 3 est axé sur l'utilisation des séquences COI fournies par le code-barres d'ADN comme un outil complémentaire pour la taxonomie et la phylogénie des amphipodes de la famille Talitridae dans l'Atlantique du Nord. En effet, la distribution et la diversité actuelle des espèces est le résultat de processus d'évolution et d'interaction avec l'environnement à l'échelle d'une région géographique. Les études phylogénétiques permettent d'appréhender cette problématique en élaborant des scenarios évolutifs des relations entre taxons. Les résultats montrent l'existence d'espèces cryptiques chez trois espèces morphologiques. En outre, les genres anciens ne semblent pas être monophylétiques, suggérant la nécessité d'une révision taxonomique chez cette famille. Le Chapitre 4 aborde le thème de la diversité génétique qui permet la persistance des populations et des espèces dans le temps en permettant une adaptation continue aux changements environnementaux. À de grandes échelles spatiales, la diversité intraspécifique peut être structurée en généalogies en fonction de la géographie, définissant alors des patrons phylogéographiques, qui peuvent coïncider ou pas avec les divisions biogéographiques. Les séquences COI générées par le code-barres d'ADN ont été utilisées pour déduire des patrons phylogéographiques chez une espèce d'amphipode avec une distribution amphiAtlantique, Gammarus oceanicus. Cette espèce est très abondante et représente une partie importante des communautés intertidales et des réseaux trophiques côtiers. Les résultats ont montré une division profonde au sein de cette espèce avec deux groupes ayant une séparation latitudinale (la région tempérée du Canada Atlantique versus la région subarctique du Baie d'Hudson et l'Europe), indiquant la présence des deux espèces cryptiques potentielles. L'ensemble de ces travaux de recherche a montré que la biodiversité marine, notamment chez les crustacés marins de l'Atlantique du Nord, était sous-estimée. Des espèces cryptiques potentielles ont été trouvées chez huit espèces morphologiques, sachant que seulement les espèces les plus communes ont été échantillonnées pour cette étude. Le taux de diversité augmentera certainement avec l'ajout d'échantillonnes de différents taxons, de divers types d'habitat et de régions marines distinctes. -- ABSTRACT: Biodiversity is the variety of life and can be studied at different levels (genetic, species, ecosystems) and at different scales (spatial and temporal). The past decades have shown that marine biodiversity has been severely underestimated. To study the characteristics of the great diversity of marine species and the underlying processes of formation and maintenance of marine biodiversity, it is obvious and necessary to know what lives out there. We are now faced with the highest extinction rates since the formation of the human society ("biodiversity crisis") and only a fraction of species was formally described (1.9 million of 11 million), because of a shortage of trained taxonomists available for this immense work, among other things. Ali these factors have led to the proposai of molecular tools to enable and facilitate the identification of species including DNA barcoding. This method uses a DNA fragment of the mitochondrial gene cytochrome C oxidase subunit 1 (COI) as a fast, accu rate and cost effective tool to identify species. Thus, each species can be defined by a unique identification tag that will not be changed during taxonomic revisions. In addition to the assignment of unknown specimens to species identified a priori by taxonomists, data generated through barcoding studies will be very useful for comparative phylogeographic studies of multiple taxa, phylogenetic studies at different taxonomic levels and for studies on evolutionary patterns between groups of organisms. Chapter 1 provides some background on DNA barcoding with a review on studies that were published on the subject, especially those focusing on the identification of marine species. Chapter 2 develops a reference library for marine crustaceans from the Estuary and the Gulf of St. Lawrence. Ali data (taxonomy, collection information, images, DNA sequences and chromatograms) are stored online in the Barcode of Life Data Systems (BOLD) and are available for general use. Specimens used for barcoding are kept as "vouchers" in public institutions for future use. The results showed the presence of an invasive amphipod in the estuary (mentioned previously in the Great Lakes and near Montreal, with impact on the native fauna of amphipods), and the existence of potential cryptic species in amphipods, mysids and decapods. Chapter 3 focuses on the use of COI sequences provided through DNA barcoding as a complementary tool for taxonomy and phylogeny of the amphipod family Talitridae in the North Atlantic. The current distribution and diversity of species is the result of evolutionary processes and interaction with the environment across a geographic region . Phylogenetic studies can investigate this issue by developing evolutionary scenarios on the relationships between taxa. The results show the existence of cryptic species in three morphological species. In addition, older genera do not cryptic species in three morphological species. In addition, older genera do not appear to be monophyletic, suggesting the need for taxonomie revisions in this family. Chapter 4 addresses the issue of genetic diversity which enables the persistence of populations and species over time, allowing continuous adaptation to environ mental changes. At large spatial scales, diversity within species can be structured in genealogies according to geography, thus defining phylogeographic patterns, which may coincide or not with biogeographic divisions. COI sequences generated by DNA barcoding were used to infer phylogeographic patterns in an amphipod species with amphi-Atlantic distribution , Gammarus oceanicus. This species is very abundant and an important part of the intertidal communities and coastal food webs. The results showed a deep division within this species with two divergent groups corresponding to a latitudinal segregation (temperate region of Atlantic Canada versus the subarctic Hudson Bay and Europe), indicating the presence of two potential cryptic species. This research showed that marine biodiversity, as seen in marine crustaceans from North Atlantic, was underestimated. Potential cryptic species were found in eight morphological species, knowing that only the most common species were sam pied for this study. The level of diversity will certainly increase with the addition of different taxa, different types of habitat and distinct marine regions

Looking back on a decade of barcoding crustaceans

Species identification represents a pivotal component for large-scale biodiversity studies and conservation planning but represents a challenge for many taxa when using morphological traits only. Consequently, alternative identification methods based on molecular markers have been proposed. In this context, DNA barcoding has become a popular and accepted method for the identification of unknown animals across all life stages by comparison to a reference library. In this review we examine the progress of barcoding studies for the Crustacea using the Web of Science data base from 2003 to 2014. All references were classified in terms of taxonomy covered, subject area (identification/library, genetic variability, species descriptions, phylogenetics, methods, pseudogenes/numts), habitat, geographical area, authors, journals, citations, and the use of the Barcode of Life Data Systems (BOLD). Our analysis revealed a total number of 164 barcoding studies for crustaceans with a preference for malacostracan crustaceans, in particular Decapoda, and for building reference libraries in order to identify organisms. So far, BOLD did not establish itself as a popular informatics platform among carcinologists although it offers many advantages for standardized data storage, analyses and publication

Amphipods in estuaries: the sibling species low salinity switch hypothesis

A novel low salinity switch hypothesis is proposed to account for the speciation of an obligate estuarine (oligohaline) amphipod, Orchestia aestuarensis, from a closely-related one, Orchestia mediterranea, found in both estuarine and marine conditions (euryhaline). The underlying genetic mechanisms could involve: 1. A dimorphic allele, or linked set of alleles, carried by the euryhaline amphipod which controls the ability to breed in low salinity conditions in estuaries and which is selected for in these conditions, producing the oligohaline amphipod. 2. A genetically-assimilated gene or genes, controlling the ability to breed in low salinity conditions in estuaries, which is/are “switched on” by low salinity conditions. 3. Allopatric speciation from a euryhaline to an oligohaline amphipod species where low salinity conditions is the selective switch. It is possible that other estuarine, sibling, amphipod pairs have evolved by salinity switching. In the North Atlantic coastal region, this could include: Gammarus tigrinus/G. daiberi and G. salinus/G. zaddachi (Amphipoda, Gammaridae)

Amphipods in estuaries: the sibling species low salinity switch hypothesis

A novel low salinity switch hypothesis is proposed to account for the speciation of an obligate estuarine (oligohaline) amphipod, Orchestia aestuarensis, from a closely-related one, Orchestia mediterranea, found in both estuarine and marine conditions (euryhaline). The underlying genetic mechanisms could involve: 1. A dimorphic allele, or linked set of alleles, carried by the euryhaline amphipod which controls the ability to breed in low salinity conditions in estuaries and which is selected for in these conditions, producing the oligohaline amphipod. 2. A genetically-assimilated gene or genes, controlling the ability to breed in low salinity conditions in estuaries, which is/are “switched on” by low salinity conditions. 3. Allopatric speciation from a euryhaline to an oligohaline amphipod species where low salinity conditions is the selective switch. It is possible that other estuarine, sibling, amphipod pairs have evolved by salinity switching. In the North Atlantic coastal region, this could include: Gammarus tigrinus/G. daiberi and G. salinus/G. zaddachi (Amphipoda, Gammaridae)

DNA Barcodes for Marine Biodiversity: Moving Fast Forward?

‘Biodiversity’ means the variety of life and it can be studied at different levels (genetic, species, ecosystem) and scales (spatial and temporal). Last decades showed that marine biodiversity has been severely underestimated at all levels. In order to investigate diversity patterns and underlying processes, there is a need to know what species live in the marine environment. An emerging tool for species identification, DNA barcoding can reliably assign unknown specimens to known species, also flagging potential cryptic species and genetically distant populations. This paper will review the role of DNA barcoding for the study of marine biodiversity at the species level