Assessing the Seasonal and Spatial Dynamics of Zooplankton through DNA Metabarcoding in a Temperate Estuary.

Zooplankton are key components of estuarine trophic networks. However, routine monitoring is hindered by the difficulty of morphology-based identification. DNA-based methods allow us to circumvent some of these hurdles, providing precise species identifications regardless of the taxonomic expertise of the investigator or the developmental stage of the specimens. However, the process is dependent on the completeness of the reference libraries. In this study, we sought to evaluate the potential of DNA metabarcoding to assess the seasonal (summer, autumn, and early spring) and spatial dynamics of zooplankton (four locations spanning ca. 6 km) in the Lima estuary (NW Portugal). Two genetic markers were used: the cytochrome c oxidase subunit I and the V4 hypervariable region of the ribosomal 18S rRNA genes. Overall, 327 species were recovered, and both markers displayed minute overlap (7% were detected with both markers). Species richness, composition, and taxonomic distinctness were majorly influenced by the season, with a declining tendency from summer (highest number of exclusive species, n = 74) to spring. Second to season, the taxa composition was influenced by spatial variation where the most downstream site displayed the highest number of exclusive species, n = 53. A total of 16 non-indigenous species were detected using metabarcoding, but only one (Austrominus modestus) has been documented out in the estuary. In conclusion, both the seasonal and spatial gradients influenced the recovered richness, composition, and taxonomic distinctness, confirming the great aptitude of DNA metabarcoding for providing higher density monitoring and shedding new light on the composition and dynamics of complex zooplankton communities.

Hurdles and opportunities in implementing marine biosecurity systems in data-poor regions.

Managing marine nonindigenous species (mNIS) is challenging, because marine environments are highly connected, allowing the dispersal of species across large spatial scales, including geopolitical borders. Cross-border inconsistencies in biosecurity management can promote the spread of mNIS across geopolitical borders, and incursions often go unnoticed or unreported. Collaborative surveillance programs can enhance the early detection of mNIS, when response may still be possible, and can foster capacity building around a common threat. Regional or international databases curated for mNIS can inform local monitoring programs and can foster real-time information exchange on mNIS of concern. When combined, local species reference libraries, publicly available mNIS databases, and predictive modeling can facilitate the development of biosecurity programs in regions lacking baseline data. Biosecurity programs should be practical, feasible, cost-effective, mainly focused on prevention and early detection, and be built on the collaboration and coordination of government, nongovernment organizations, stakeholders, and local citizens for a rapid response.

Current status and topical issues on the use of eDNA-based targeted detection of rare animal species.

Animal detection through DNA present in environmental samples (eDNA) is a valuable tool for detecting rare species, that are difficult to observe and monitor. eDNA-based tools are underpinned by molecular evolutionary principles, key to devising tools to efficiently single out a targeted species from an environmental sample. Here, we present a comprehensive review of the use of eDNA-based methods for the detection of targeted animal species, such as rare, endangered, or invasive species, through the analysis of 549 publications (2008-2022). Aquatic ecosystems have been the most surveyed, in particular, freshwaters (74 %), and to a less extent marine (14 %) and terrestrial systems (10 %). Vertebrates, in particular, fish (38 %), and endangered species, have been the focus of most of these studies, and Cytb and COI are the most employed markers. Among invertebrates, assays have been mainly designed for Mollusca and Crustacea species (21 %), in particular, to target invasive species, and COI the most employed marker. Targeted molecular approaches, in particular qPCR, have been the most adopted (75 %), while eDNA metabarcoding has been rarely used to target single or few species (approx. 6 %). However, less attention has been given in these studies to the effects of environmental factors on the amount of shed DNA, the differential amount of shed DNA among species, or the sensitivity of the markers developed, which may impact the design of the assays, particularly to warrant the required detection level and avoid false negatives and positives. The accuracy of the assays will also depend on the availability of genetic data and vouchered tissue or DNA samples from closely related species to assess both marker and primers' specificity. In addition, eDNA-based assays developed for a particular species may have to be refined for use in a new geographic area taking into account site-specific populations, as well as any intraspecific variation.

To Be, or Not to Be: That Is the Hamletic Question of Cryptic Evolution in the Eastern Atlantic and Mediterranean Raja miraletus Species Complex.

Despite a high species diversity, skates (Rajiformes) exhibit remarkably conservative morphology and ecology. Limited trait variations occur within and between species, and cryptic species have been reported among sister and non-sister taxa, suggesting that species complexes may be subject to stabilising selection. Three sibling species are currently recognised in the Raja miraletus complex: (i) R. miraletus occurring along the Portuguese and Mediterranean coasts, (ii) R. parva in the Central-Eastern Atlantic off West Africa and (iii) R. ocellifera in the Western Indian Ocean off South Africa. In the present study, the genetic variation at mitochondrial and nuclear markers was estimated in the species complex by analysing 323 individuals sampled across most of its geographical distribution area to test the hypothesis that restricted gene flow and genetic divergence within species reflect known climate and bio-oceanographic discontinuities. Our results support previous morphological studies and confirm the known taxonomic boundaries of the three recognised species. In addition, we identified multiple weakly differentiated clades in the Northeastern Atlantic Ocean and Mediterranean, at least two additional cryptic taxa off Senegal and Angola, a pronounced differentiation of ancient South African clades. The hidden genetic structure presented here may represent a valuable support to species' conservation action plans.

Molecular diversity within the genus Laeonereis (Annelida, Nereididae) along the west Atlantic coast: paving the way for integrative taxonomy.

The polychaete genus Laeonereis (Annelida, Nereididae) occurs over a broad geographic range and extends nearly across the entire Atlantic coast of America, from the USA to Uruguay. Despite the research efforts to clarify its diversity and systematics, mostly by morphological and ecological evidence, there is still uncertainty, mainly concerning the species Laeonereis culveri, which constitutes an old and notorious case of taxonomic ambiguity. Here, we revised the molecular diversity and distribution of Laeonereis species based on a multi-locus approach, including DNA sequence analyses of partial segments of the cytochrome c oxidase subunit I (COI), 16S rRNA, and 28S rRNA genes. We examined Laeonereis specimens collected from 26 sites along the American Atlantic coast from Massachusetts (USA) to Mar del Plata (Argentina). Although no comprehensive morphological examination was performed between different populations, the COI barcodes revealed seven highly divergent MOTUs, with a mean K2P genetic distance of 16.9% (from 6.8% to 21.9%), which was confirmed through four clustering algorithms. All MOTUs were geographically segregated, except for MOTUs 6 and 7 from southeastern Brazil, which presented partially overlapping ranges between Rio de Janeiro and São Paulo coast. Sequence data obtained from 16S rRNA and 28S rRNA markers supported the same MOTU delimitation and geographic segregation as those of COI, providing further evidence for the existence of seven deeply divergent lineages within the genus. The extent of genetic divergence between MOTUs observed in our study fits comfortably within the range reported for species of polychaetes, including Nereididae, thus providing a strong indication that they might constitute separate species. These results may therefore pave the way for integrative taxonomic studies, aiming to clarify the taxonomic status of the Laeonereis MOTUs herein reported.

Status and prospects of marine NIS detection and monitoring through (e)DNA metabarcoding.

In coastal ecosystems, non-indigenous species (NIS) are recognized as a major threat to biodiversity, ecosystem functioning and socio-economic activities. Here we present a systematic review on the use of metabarcoding for NIS surveillance in marine and coastal ecosystems, through the analysis of 42 publications. Metabarcoding has been mainly applied to environmental DNA (eDNA) from water samples, but also to DNA extracted from bulk organismal samples. DNA extraction kits have been widely used and the 18S rRNA and the COI genes the most employed markers, but less than half of the studies targeted more than one marker loci. The Illumina MiSeq platform has been used in >50% of the publications. Current weaknesses include potential occurrence of false negatives due to the primer-biased or faulty DNA amplification and the incompleteness of reference libraries. This is particularly concerning in the case of NIS surveillance, where proficiency in species level detection is critical. Until these weaknesses are resolved, ideally NIS metabarcoding should be supported by complementary approaches, such as morphological analysis or more targeted molecular approaches (e.g. qPCR, ddPCR). Even so, metabarcoding has already proved to be a highly sensitive tool to detect small organisms or undifferentiated life stages across a wide taxonomic range. In addition, it also seems to be very effective in ballast water management and to improve the spatial and temporal sampling frequency of NIS surveillance in marine and coastal ecosystems. Although specific protocols may be required for species-specific NIS detection, for general monitoring it would be vital to settle on a standard protocol able to generate comparable results among surveillance campaigns and regions of the globe, seeking the best approach for detecting the broadest range of species, while minimizing the chances of a false positive or negative detection.

BAGS: An automated Barcode, Audit & Grade System for DNA barcode reference libraries.

Biodiversity studies greatly benefit from molecular tools, such as DNA metabarcoding, which provides an effective identification tool in biomonitoring and conservation programmes. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries. The role of these libraries is to support species identification, but accidental errors in the generation of the barcodes may compromise their accuracy. Here, we present an R-based application, Barcode, Audit & Grade System (BAGS) (https://github.com/tadeu95/BAGS), that performs automated auditing and annotation of cytochrome c oxidase subunit I (COI) sequences libraries, for a given taxonomic group of animals, available in the Barcode of Life Data System (BOLD). This is followed by implementing a qualitative ranking system that assigns one of five grades (A to E) to each species in the reference library, according to the attributes of the data and congruency of species names with sequences clustered in barcode index numbers (BINs). Our goal is to allow researchers to obtain the most useful and reliable data, highlighting and segregating records according to their congruency. Different tests were performed to perceive its usefulness and limitations. BAGS fulfils a significant gap in the current landscape of DNA barcoding research tools by quickly screening reference libraries to gauge the congruence status of data and facilitate the triage of ambiguous data for posterior review. Thereby, BAGS has the potential to become a valuable addition in forthcoming DNA metabarcoding studies, in the long term contributing to globally improve the quality and reliability of the public reference libraries.

How oogenesis analysis combined with DNA barcode can help to elucidate taxonomic ambiguities: a polychaete study-based approach / Como análises de oogênese combinadas com DNA barcode podem elucidar ambiguidades taxonômicas: uma abordagem baseada em estudos com poliquetas

Abstract: Polychaetes are common in coastal and estuarine environments worldwide and constitute one of the most complex groups of marine invertebrates. The morpho-physiology of the female reproductive system (FRS) can be understood by using histological tools to describe reproductive cycle and gametogenesis paths and, among other purposes, aiming to identify and differentiate polychaete species. However, this histology-based approach is rarely combined with molecular tools, which is known to accurately delimitate species. In the same way, the description and understanding of oogenesis and vitellogenesis paths within polychaetes are lacking for most families, narrowing the range of its utility. Therefore, the present study aims to describe the oogenesis in three polychaete species common and abundant on the South American Atlantic coast (Laeonereis culveri, Scolelepis goodbodyi and Capitella biota) and investigate the utility of reproductive features and gametogenesis as a relevant associate knowledge to discriminate species, particularly useful for putative cryptic species, integrated with morphological and molecular data. In a first attempt, the results obtained herein allow the authors to describe two new subtypes of oogenesis, dividing it in extraovarian oogenesis type I and II and intraovarian type I and II. The results also demonstrate that the following histological characters of the FRS can be relevant for the separation of related species: a) oogenesis type, b) occurrence or absence of a true ovary, c) ovary tissue organization, d) type of accessory cells present, and e) oocyte morphology. Additionally, these histological features of FRS, when compared with correlated species studied under this scope, converge with the genetic data. The analysis of cytochrome oxidase I (COI) barcode sequences differentiates between North and South American Atlantic populations of L. culveri (16.78% genetic distance), while in S. goodbodyi and C. biota it discriminates them from their congeneric species. These results highlight the importance of multi-tool approach and shows that both FRS histology and histo-physiology, and DNA barcoding can be used to identify and discriminate cryptic species, which is usually not possible when using morphological characters. Besides, these characters may also be useful in differentiating related species, and/or geographically distinct populations among polychaetes.

Resumo: Os poliquetas são comuns em ambientes costeiros e estuarinos em todo o mundo e constituem um dos grupos mais complexos de invertebrados marinhos. A morfo-fisiologia do sistema reprodutor feminino (FRS) pode ser compreendida por meio de ferramentas histológicas para identificar e diferenciar estes anelídeos. No entanto, essa abordagem histológica raramente é combinada com ferramentas moleculares, amplamente conhecidas por delimitar espécies congenéricas ou crípticas com maior precisão. Do mesmo modo, a descrição e o entendimento da oogênese e vitelogênese dentre os poliquetas, para a maioria das famílias, é ainda limitado. Portanto, o presente estudo tem como objetivo descrever a oogênese em três espécies de poliquetas comuns e abundantes na costa sul-americana (Laeonereis culveri, Scolelepis goodbodyi e Capitella biota) e investigar a utilidade das características reprodutivas e da gametogênese como um conhecimento associado relevante para discriminar espécies, particularmente útil para espécies crípticas putativas, integradas a dados morfológicos e moleculares. Os resultados aqui obtidos permitiram descrever dois novos subtipos de oogênese, dividindo-a em oogênese extra-ovariana dos tipos I e II e intra-ovariana dos tipos I e II. Os resultados também demonstram que os seguintes caracteres histológicos do FRS podem ser relevantes para a separação de espécies relacionadas: a) tipo de oogênese, b) presença ou ausência de um ovário verdadeiro, c) organização tissular ovariana, d) tipo de células acessórias presentes e, e) morfologia do ovócito. Além disso, essas características histológicas do FRS, quando comparadas às espécies correlatas estudadas sob esse escopo, convergem com os dados genéticos separando espécies putativas e congenéricas. As análises com DNA barcode demonstraram que em L. culveri é possível diferenciar as populações atlânticas Norte e Sul-americanas (16,78% de distância genética), enquanto para S. goodbodyi e C. biota fica evidente sua distinção com espécies congenéricas. Esses resultados destacam a importância da abordagem com múltiplas ferramentas e mostram que tanto a histologia quanto a histo-fisiologia do FRS e o DNA barcode podem ser usados para identificar e discriminar espécies crípticas e potencialmente crípticas, o que geralmente não é possível quando se utilizam apenas caracteres morfológicos. Além disso, esses caracteres também podem ser úteis na diferenciação de espécies relacionadas e / ou populações geograficamente distintas desses poliquetas.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

Deep segregation in the open ocean: Macaronesia as an evolutionary hotspot for low dispersal marine invertebrates.

Diversification and speciation of terrestrial organisms are anticipated in oceanic islands such as Macaronesia, a group of Atlantic islands that have remained unconnected to continental landmasses. Hitherto, the diversification of marine organisms in oceanic islands, especially those with low vagility, has received little direct empirical analysis using molecular markers. Here, we focus on such a case study, through applying a multilocus molecular approach to investigate the diversity and evolution of a group that lacks a planktonic larval stage, the isopod genus Dynamene, in Macaronesia and Northeast Atlantic. Sequences of two mitochondrial (cytochrome c oxidase subunit I and 16S rRNA) and two nuclear (18S rRNA and 28S rRNA) loci were obtained from specimens of Dynamene edwardsi (Lucas, 1849), Dynamene magnitorata Holdich, 1968 and Dynamene bidentata (Adams, 1800) collected along the Northeast Atlantic and Macaronesia. Although no major phylogeographic structure was detected in D. bidentata and D. magnitorata, from five to nine deeply divergent lineages were evident within D. edwardsi. The divergent lineages displayed genetic distances comparable to those found among established species of peracarids. D. edwardsi exhibits a long, rich and complex phylogeographic history in Macaronesia, where the geodynamics of the islands possibly associated with founder effects and subsequent lack of gene flow among populations confounds patterns based on geographic proximity of targeted populations. Our findings collectively suggest a much larger role of oceanic islands in the diversification of marine invertebrates than previously anticipated. The work provides insights into the origins and dynamics of ongoing geographic segregation and associated deep divergence among sister evolutionary lineages in Macaronesia.

Priming of a DNA metabarcoding approach for species identification and inventory in marine macrobenthic communities.

In marine and estuarine benthic communities, the inventory and estimation of species richness are often hampered by the need for broad taxonomic expertise across several phyla. The use of DNA metabarcoding has emerged as a powerful tool for the fast assessment of species composition in a diversity of ecological communities. Here, we tested the amplification success of five primer sets targeting different COI-5P regions by 454 pyrosequencing to maximize the recovery of two simulated macrobenthic communities containing 21 species (SimCom1 and SimCom 2). Species identification was first performed against a compiled reference library of macrobenthic species. Reads with similarity results to reference sequences between 70% and 97% were then submitted to GenBank and BOLD to attempt the identification of concealed species in the bulk sample. The combination of at least three primer sets was able to recover more species than any primer set alone, achieving 85% of represented species in SimCom1 and 76% in SimCom2. Our approach was successful to detect low-frequency specimens, as well as concealed species, in the bulk sample, indicating the potential for the application of this approach on marine bioassessment and inventory, including the detection of "hidden" biodiversity that would hardly be possible based on morphology only.

Contrasting morphological and DNA barcode-suggested species boundaries among shallow-water amphipod fauna from the southern European Atlantic coast.

In this study we compared DNA barcode-suggested species boundaries with morphology-based species identifications in the amphipod fauna of the southern European Atlantic coast. DNA sequences of the cytochrome c oxidase subunit I barcode region (COI-5P) were generated for 43 morphospecies (178 specimens) collected along the Portuguese coast which, together with publicly available COI-5P sequences, produced a final dataset comprising 68 morphospecies and 295 sequences. Seventy-five BINs (Barcode Index Numbers) were assigned to these morphospecies, of which 48 were concordant (i.e., 1 BIN = 1 species), 8 were taxonomically discordant, and 19 were singletons. Twelve species had matching sequences (<2% distance) with conspecifics from distant locations (e.g., North Sea). Seven morphospecies were assigned to multiple, and highly divergent, BINs, including specimens of Corophium multisetosum (18% divergence) and Dexamine spiniventris (16% divergence), which originated from sampling locations on the west coast of Portugal (only about 36 and 250 km apart, respectively). We also found deep divergence (4%-22%) among specimens of seven species from Portugal compared to those from the North Sea and Italy. The detection of evolutionarily meaningful divergence among populations of several amphipod species from southern Europe reinforces the need for a comprehensive re-assessment of the diversity of this faunal group.

Distribution and species identification in the crustacean isopod genus Dynamene Leach, 1814 along the North East Atlantic-Black Sea axis.

Sphaeromatid isopods, such as Dynamene, are common and abundant members of the invertebrate fauna of littoral and shallow sublittoral substrates. Six species of Dynamene occur in the northern hemisphere. Only two species exist outside this range, in Australia. The distribution of the various species in the NE Atlantic-Black Sea axis has been controversial due to the difficulty in the identification of the different species. This has led to inaccurate records of their distribution, ultimately generating uncertain or faulty assessments on the biodiversity of these habitats. An update and a clarification about the distribution of this genus is therefore in order. In this study, we describe the distribution of Dynamene species in the light of new records from the NE Atlantic Ocean and its associated islands, and the Mediterranean, Black and Red Seas, and from re-examination of museum and several authors' personal collections. Based on these observations, we extend the northern and southern limits of Dynamene bidentata (Adams); the western and southern limits of Dynamene magnitorata Holdich; the northern, eastern and western limits of Dynamene edwardsi (Lucas); and the eastern and western limits of Dynamene bifida Torelli. The range of Dynamene tubicauda Holdich is extended, but is still only known from the eastern Mediterranean. We also clarify the synonymy of Dynamene torelliae Holdich with Dynamene bicolor (Rathke), and the occurrence of Dynamene bicolor in the Black Sea. New distribution maps of the six Dynamene species are presented. Illustrated keys to the adult males and females of the northern hemisphere species are provided.

With a little help from DNA barcoding: investigating the diversity of Gastropoda from the Portuguese coast.

The Gastropoda is one of the best studied classes of marine invertebrates. Yet, most species have been delimited based on morphology only. The application of DNA barcodes has shown to be greatly useful to help delimiting species. Therefore, sequences of the cytochrome c oxidase I gene from 108 specimens of 34 morpho-species were used to investigate the molecular diversity within the gastropods from the Portuguese coast. To the above dataset, we added available COI-5P sequences of taxonomically close species, in a total of 58 morpho-species examined. There was a good match between ours and sequences from independent studies, in public repositories. We found 32 concordant (91.4%) out of the 35 Barcode Index Numbers (BINs) generated from our sequences. The application of a ranking system to the barcodes yield over 70% with top taxonomic congruence, while 14.2% of the species barcodes had insufficient data. In the majority of the cases, there was a good concordance between morphological identification and DNA barcodes. Nonetheless, the discordance between morphological and molecular data is a reminder that even the comparatively well-known European marine gastropods can benefit from being probed using the DNA barcode approach. Discordant cases should be reviewed with more integrative studies.

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.

DNA barcoding for species assignment: the case of Mediterranean marine fishes.

BACKGROUND: DNA barcoding enhances the prospects for species-level identifications globally using a standardized and authenticated DNA-based approach. Reference libraries comprising validated DNA barcodes (COI) constitute robust datasets for testing query sequences, providing considerable utility to identify marine fish and other organisms. Here we test the feasibility of using DNA barcoding to assign species to tissue samples from fish collected in the central Mediterranean Sea, a major contributor to the European marine ichthyofaunal diversity. METHODOLOGY/PRINCIPAL FINDINGS: A dataset of 1278 DNA barcodes, representing 218 marine fish species, was used to test the utility of DNA barcodes to assign species from query sequences. We tested query sequences against 1) a reference library of ranked DNA barcodes from the neighbouring North East Atlantic, and 2) the public databases BOLD and GenBank. In the first case, a reference library comprising DNA barcodes with reliability grades for 146 fish species was used as diagnostic dataset to screen 486 query DNA sequences from fish specimens collected in the central basin of the Mediterranean Sea. Of all query sequences suitable for comparisons 98% were unambiguously confirmed through complete match with reference DNA barcodes. In the second case, it was possible to assign species to 83% (BOLD-IDS) and 72% (GenBank) of the sequences from the Mediterranean. Relatively high intraspecific genetic distances were found in 7 species (2.2%-18.74%), most of them of high commercial relevance, suggesting possible cryptic species. CONCLUSION/SIGNIFICANCE: We emphasize the discriminatory power of COI barcodes and their application to cases requiring species level resolution starting from query sequences. Results highlight the value of public reference libraries of reliability grade-annotated DNA barcodes, to identify species from different geographical origins. The ability to assign species with high precision from DNA samples of disparate quality and origin has major utility in several fields, from fisheries and conservation programs to control of fish products authenticity.

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.

Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans.

BACKGROUND: Building reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called "universal" primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms. RESULTS: Amplification tests conducted on a wide range of marine animal taxa, rendered possible the first-time sequencing of DNA barcodes from eight separated phyla (Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes), comprising a total of 14 classes, 28 orders, 57 families, 68 genus and 76 species. CONCLUSIONS: These primers demonstrated to be highly cost-effective, which is of key importance for DNA barcoding procedures, such as for building comprehensive DNA barcode libraries of marine communities, where the processing of a large numbers of specimens from a wide variety of marine taxa is compulsory.

A ranking system for reference libraries of DNA barcodes: application to marine fish species from Portugal.

BACKGROUND: The increasing availability of reference libraries of DNA barcodes (RLDB) offers the opportunity to the screen the level of consistency in DNA barcode data among libraries, in order to detect possible disagreements generated from taxonomic uncertainty or operational shortcomings. We propose a ranking system to attribute a confidence level to species identifications associated with DNA barcode records from a RLDB. Here we apply the proposed ranking system to a newly generated RLDB for marine fish of Portugal. METHODOLOGY/PRINCIPAL FINDINGS: Specimens (n = 659) representing 102 marine fish species were collected along the continental shelf of Portugal, morphologically identified and archived in a museum collection. Samples were sequenced at the barcode region of the cytochrome oxidase subunit I gene (COI-5P). Resultant DNA barcodes had average intra-specific and inter-specific Kimura-2-parameter distances (0.32% and 8.84%, respectively) within the range usually observed for marine fishes. All specimens were ranked in five different levels (A-E), according to the reliability of the match between their species identification and the respective diagnostic DNA barcodes. Grades A to E were attributed upon submission of individual specimen sequences to BOLD-IDS and inspection of the clustering pattern in the NJ tree generated. Overall, our study resulted in 73.5% of unambiguous species IDs (grade A), 7.8% taxonomically congruent barcode clusters within our dataset, but awaiting external confirmation (grade B), and 18.7% of species identifications with lower levels of reliability (grades C/E). CONCLUSION/SIGNIFICANCE: We highlight the importance of implementing a system to rank barcode records in RLDB, in order to flag taxa in need of taxonomic revision, or reduce ambiguities of discordant data. With increasing DNA barcode records publicly available, this cross-validation system would provide a metric of relative accuracy of barcodes, while enabling the continuous revision and annotation required in taxonomic work.

Multigene molecular systematics confirm species status of morphologically convergent Pagurus hermit crabs.

INTRODUCTION: In spite of contemporary morphological taxonomy appraisals, apparent high morphological similarity raises uncertainty about the species status of certain Pagurus hermit crabs. This is exemplified between two European species, Pagurus excavatus (Herbst, 1791) and Pagurus alatus (Fabricius 1775), whose species status is still difficult to resolve using morphological criteria alone. METHODOLOGY/PRINCIPAL FINDINGS: To address such ambiguities, we used combinations of Maximum Likelihood (ML) and Bayesian Inference (BI) methods to delineate species boundaries of P. alatus and P. excavatus and formulate an intermediate Pagurus phylogenetic hypothesis, based upon single and concatenated mitochondrial (cytochrome oxidase I [COI]) and nuclear (16S and 28s ribosomal RNA) gene partitions. The molecular data supported the species status of P. excavatus and P. alatus and also clearly resolved two divergent clades within hermit crabs from the Northeast Atlantic Ocean and the Mediterranean Sea. CONCLUSIONS/SIGNIFICANCE: Despite the abundance and prominent ecological role of hermit crabs, Pagurus, in North East Atlantic Ocean and Mediterranean Sea ecosystems, many important aspects of their taxonomy, biology, systematics and evolution remain poorly explored. The topologies presented here should be regarded as hypotheses that can be incorporated into the robust and integrated understanding of the systematic relationships within and between species of the genus Pagurus inhabiting the Northeast Atlantic Ocean and the Mediterranean Sea.