DNA Barcoding for Species Assignment: The Case of Mediterranean Marine Fishes

et al. (2014) DNA Barcoding for Species Assignment: The Case of Mediterranean Marine Fishes. PLoS ONE 9(9): e106135. doi:10.1371/journal.pone.0106135 DNA Barcoding for Species Assignment: The Case of Mediterranean Marine Fishes Monica Landi 0 Mark Dimech 0 Marco Arculeo 0 Girolama Biondo 0 Rogelia Martins 0 Miguel Carneiro 0 Gary Robert Carvalho 0 Sabrina Lo Brutto 0 Filipe O. Costa 0 Sean Rogers, University of Calgary, Canada 0 1 Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal, 2 Malta Centre for Fisheries Science (MCFS), Fort San Lucjan Marsaxlokk, Malta, 3 Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), University of Palermo , Palermo , Italy , 4 Modelling and Management Fishery Resources Division (DIV-RP), Instituto Portugues do Mar e da Atmosfera , Lisboa, Portugal, 5 Molecular Ecology and Fisheries Genetics Laboratory , School of Biological Sciences, Bangor University , Bangor , United Kingdom 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. Current address; Fisheries and Aquaculture Department; Food and Agriculture Organisation of the United Nations; Athens; Greece - Funding: Research carried out at University of Palermo was supported by "Fondi di Ateneo ex 60% Universita` di Palermo". This work was supported by FEDER through POFCCOMPETE and by national funds from "Fundacaopara a Ciencia e a Tecnologia (FCT)" in the scope of the grants, FCOMP-01-0124-FEDER-010596 and PEst-OE/BIA/UI4050/2014. MLs work was supported by the fellowship Ref: SFRH/BPD/45246/2008 from Fundacaopara a Ciencia e a Tecnologia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The Mediterranean Sea is a semi-enclosed basin that embraces the marine area from the North East Atlantic Ocean, at West, to the Aegean Sea, at East. The confluences of marine ichthyofauna migrating from the Atlantic Ocean through the Strait of Gibraltar, from the Red Sea and the Indian Ocean through the Suez Channel, and from the Sea of Marmara and Black Sea through the Dardanelles, depicts a picture of the Mediterranean marine biodiversity characterized by a high species richness and peculiarities, including tropical species as well as endemisms [1], [2], [3]. Hosting 7% of the global marine ichthyofauna [4] the Mediterranean Sea is a fascinating prosperous biodiversity hotspot [5], [6] that captured the interest of numerous marine scientists since ancient times (e.g. Aristoteles) [1]. Holding such richness, the Mediterranean Sea can be elected as a very important scientific cradle in marine sciences. The considerable natural variation driven by distinctive regional evolutionary histories and dynamic anthropogenic pressures [7] presents major challenges in local biodiversity monitoring programmes. Over the past decade, DNA barcoding has played a facilitatory role for accurate identification of marine ichthyiofauna, thanks to the integration of molecular and traditional taxonomic methods [8]. Such DNA-based method provides a robust and standardized approach for marine species identification, as witnessed by the remarkable boost of species identified [9], as well as its use for various applications [10], as for example fisheries and conservation programs [11]. DNA barcoding has been adopted in numerous studies illustrating its speed, reliability and accessibility [12], [13]. The possibility of compiling taxonomic and molecular data into a globally accessible public database (Barcode of Life Data System, BOLD, http://www.barcodinglife.org) [14], [15], comprising taxonomically diverse reference libraries (e.g. Costa et al. [16]), allows usage by a diverse community of scientists and end-users [12]. Such wide-scale adoption enables global comparisons of putative cosmopolitan species [17] facilitating opportunities for comparisons of different marine environments, as well as tackling issues relating to molecular evolution [18]. The availability of a detailed reference library comprising validated DNA barcodes [16] constitutes a robust platform against which to test query sequences, and it represents a valid tool for attributing species to unknown sequences [19]. The DNA barcoding methodology has been applied recently to identify 98 marine species inhabiting the eastern basin of the Mediterranean Sea [20]. Such studies are yet to be extended to marine ichthyofauna populating one of the highest biodiversity richness spots of the Mediterranean, namely the sea around Sicily and Malta. Here, we present an extensive account of DNA barcodes for Mediterranean fishes based on the mitochondrial cytochrome c oxidase subunit I (COI). We used a query dataset composed of 486 specimens identified morphologica (...truncated)