Filipe O. Costa

Systematic and Evolutionary Insights Derived from mtDNA COI Barcode Diversity in the Decapoda (Crustacea: Malacostraca)

Background Decapods are the most recognizable of all crustaceans and comprise a dominant group of benthic invertebrates of the continental shelf and slope, including many species of economic importance. Of the 17635 morphologically described Decapoda species, only 5.4% are represented by COI barcode region sequences. It therefore remains a challenge to compile regional databases that identify and analyse the extent and patterns of decapod diversity throughout the world. Methodology/Principal Findings We contributed 101 decapod species from the North East Atlantic, the Gulf of Cadiz and the Mediterranean Sea, of which 81 species represent novel COI records. Within the newly-generated dataset, 3.6% of the species barcodes conflicted with the assigned morphological taxonomic identification, highlighting both the apparent taxonomic ambiguity among certain groups, and the need for an accelerated and independent taxonomic approach. Using the combined COI barcode projects from the Barcode of Life Database, we provide the most comprehensive COI data set so far examined for the Order (1572 sequences of 528 species, 213 genera, and 67 families). Patterns within families show a general predicted molecular hierarchy, but the scale of divergence at each taxonomic level appears to vary extensively between families. The range values of mean K2P distance observed were: within species 0.285% to 1.375%, within genus 6.376% to 20.924% and within family 11.392% to 25.617%. Nucleotide composition varied greatly across decapods, ranging from 30.8 % to 49.4 % GC content. Conclusions/Significance Decapod biological diversity was quantified by identifying putative cryptic species allowing a rapid assessment of taxon diversity in groups that have until now received limited morphological and systematic examination. We highlight taxonomic groups or species with unusual nucleotide composition or evolutionary rates. Such data are relevant to strategies for conservation of existing decapod biodiversity, as well as elucidating the mechanisms and constraints shaping the patterns observed.

The Barcode of Life Initiative: synopsis and prospective societal impacts of DNA barcoding of Fish

Almost 250 years after the publication of the taxonomy-founding work Systema Naturae, by Carl Linnaeus, the inventory and catalogue of the planets biodiversity is still far from complete: only ca 1.5 to 1.8 million of an estimated 10+ million species are so far described. Notwithstanding the remarkable merits of the Linnean system, the task is too vast ever to be completed using current conventional approaches. Such a staggering reality, and the customary difficulty that the scientific community and society in general experience to access taxonomic knowledge, has prompted the search for novel tools or approaches for species identification. Such a tool has been recently proposed in the form of a standardised short DNA sequence from an agreedupon region of the genome, which is expected to ultimately provide a means of fast and robust identification of any species on the planet: the DNA barcode. Received with as much enthusiasm by some as skepticism by others, this novel tool was set in motion on a worldwide scale by means of an international consortium of organisations (the Consortium for the Barcoding of Life), thus becoming a large-scale horizontal genomics project. While anchored within the knowledge and principles of taxonomy, DNA barcoding possesses unique characteristics which anticipate a diverse scope of new applications and benefits for society. Notably, it places the completion of the biodiversity catalogue within the reach of a single generation, with the promise to assist greatly in the discovery of new species. Alongside long-term, ultimate goals, such as democratisation of access to taxonomic knowledge and assistance in writing the encyclopaedia of life, there are several more prosaic applications that may also impact society, not only in certain scientific fields, but also in a range of social and economic activities. Here, we will use DNA barcoding of fish as an example to illustrate foreseen applications, and as a basis to stimulate reflection on potential societal impacts of this horizontal genomics project.

Probing marine Gammarus (Amphipoda) taxonomy with DNA barcodes

Abstract The genus Gammarus (Amphipoda) is one of the most speciose genera of Crustacea, yet much uncertainty remains concerning taxonomy and systematic relationships, particularly for brackish and marine forms. We used DNA barcode sequences from the mitochondrial cytochrome c oxidase I (COI) gene to probe the taxonomy of prominent members of marine and brackish water Gammarus of the North Atlantic, Baltic, Mediterranean and Black Seas. We investigated 16 putative Gammarus spp. at an average number of 9 specimens per species. This constitutes the most taxonomically and geographically comprehensive molecular study of marine Gammarus to date. Average between‐species sequence divergence (26.8%) was much higher than intraspecific distances (0.8%), enabling clear molecular species identification and highlighting several possible misidentifications from previously published studies. Specimens of Gammarus aequicauda and G. insensibilis from the Black Sea were at least 14% distant from their putative conspecifics elsewhere. Placing these findings in a geographic context provides strong indication of cryptic speciation. Further, we detected phylogeographic splits in G. oceanicus and G. duebeni. Our analyses also suggest phylogenetic positioning of G. marinus with members of the genus Echinogammarus, thus confirming its classification as Echinogammarus marinus. We have demonstrated that comprehensive analyses of taxonomically complex groups using DNA barcodes can result in a diversity of complementary data on taxonomy, phylogeography and phylogenetics. The combination of these results, with further morphological and ecological data, will enable significant progress in our understanding of this ecologically important group of crustaceans.

DNA barcodes of fish of the Scotia Sea, Antarctica indicate priority groups for taxonomic and systematics focus

Abstract We analysed cytochrome oxidase I (COI) barcodes for 35 putative fish species collected in the Scotia Sea, and compared the resultant molecular data with field-based morphological identifications, and additional sequence data obtained from GenBank and the Barcode of Life Data System (BOLD). There was high congruence between morphological and molecular classification, and COI provided effective species-level discrimination for nearly all putative species. No effect of geographic sampling was observed for COI sequence variation. For two families, including the Liparidae and Zoarcidae, for which morphological field identification was unable to resolve taxonomy, DNA barcoding revealed significant species-level divergence. However, the dataset lacked sufficient sensitivity for resolving species within the Bathydraco and Artedidraco genera. Analysis of cytochrome b for these two genera also failed to resolve taxonomic identity. The data are discussed in relation to emergent priorities for additional taxonomic studies. We emphasize the utility of DNA barcoding in providing a valuable taxonomic framework for fundamental population studies through assigning life history stages or other morphologically ambiguous samples to parental species.

Effects of Temperature and Salinity on Life History of the Marine Amphipod Gammarus locusta. Implications for Ecotoxicological Testing

The life history of Gammarus locusta was analysed in the laboratory under the following temperature and salinity combinations: 20 °C–33‰, 15 °C–20‰ and 15 °C–33‰ (reference condition). Life history analysis comprised survival, individual growth, reproductive traits and life table parameters. Compared to 15 °C, life history at 20 °C was characterised by at least a four-week reduction in the life-span, lower life expectancy, shorter generation time, faster individual growth, anticipation of age at maturity and higher population growth rate. These temperature effects constituted an acceleration and condensation of the life cycle, compared to the reference condition. Concerning salinity effects, with few exceptions, results show that overall this amphipod life history did not differ significantly between the salinity conditions tested. Regarding ecotoxicological testing implications, findings from this study indicate that the range of temperature and salinity conditions acceptable for testing was substantially expanded both for acute and chronic assays. A temperature of 20 °C or higher (for a salinity of 33‰) is suggested for routine chronic sediment toxicity testing with G. locusta, in order to reduce the life cycle and consequently improve cost-effectiveness and standardisation. Results also suggest that a multiple-response approach, including survival, growth and reproduction, should be applied in chronic toxicity tests.

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.

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

BackgroundBuilding 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.ResultsAmplification 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.ConclusionsThese 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.

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.

Investigating the taxonomy and systematics of marine wood borers (Bivalvia : Teredinidae) combining evidence from morphology, DNA barcodes and nuclear locus sequences

Abstract. Marine wood-boring teredinids, some of the most destructive wood borers in the sea, are a particularly difficult group to identify from morphological features. While in most bivalve species shell features are used as diagnostic characters, in the teredinids shell morphology shows high intraspecific variation and thus identification is based almost entirely on the morphology of the pallets. In the present study we aimed at improving ‘taxonomic resolution’ in teredinids by combining morphological evidence with mitochondrial and nuclear DNA sequences, respectively Cytochrome c oxidase subunit I and small subunit rRNA 18S gene, to generate more rigorous and accessible identifications. DNA barcodes of Atlantic and Mediterranean populations of Lyrodus pedicellatus diverged by ∼20%, suggesting cryptic species in the morphospecies L. pedicellatus. The low intraspecific divergence found in barcodes of specimens of Nototeredo norvagica (0.78%) confirms that Atlantic and Mediterranean forms of N. norvagica, the latter sometimes reported as Teredo utriculus, are the same species. Teredothyra dominicensis was found for the first time in the Mediterranean. A match was obtained between our 18S sequences and sequences of T. dominicensis from Netherlands Antilles, confirming that T. dominicensis in the Mediterranean is the same species that occurs in the Caribbean. There were differences in 18S sequences between Bankia carinata from the Mediterranean and Caribbean, which may indicate cryptic species.