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

Pair-wise COI-5P barcode distances (expressed in %; K2P model) of marine fish species from the reference barcode library, at different taxonomic levels.

<p>A. Values calculated using all 792 COI-5P barcodes representing 146 species from the reference library. B. Values calculated using the subset of 689 COI-5P barcodes (122 species) assigned to Grades A and B.</p><p>Pair-wise COI-5P barcode distances (expressed in %; K2P model) of marine fish species from the reference barcode library, at different taxonomic levels.</p

Sampling locations of marine fishes along Portuguese mainland coast.

<p>Yellow circle – 1–5 specimens, orange circle – 5–50 specimens, red circle – >50 specimens.</p

Pairwise average COI-5P barcode distances of marine fish species¹ using Kimura 2-Parameter model.

1<p>Values are estimated from 659 COI-5P barcodes of 102 species from Portugal.</p

Indicator vector correlations of the COI-5P data set (Klee diagram).

<p>The false-color representation depicts correlations among 447 COI-5P test sequences (y-axis) and 83 species-level indicator vectors (x-axis). In total, 430 (96.2%) of the test sequences showed highest correlation with their respective species indicator vector.</p

Detailed partitioning of the number of species, COI-5P sequences, and GenBank accession numbers among the BOLD projects examined in this study.

<p>Detailed partitioning of the number of species, COI-5P sequences, and GenBank accession numbers among the BOLD projects examined in this study.</p

Attribution of grades (A to E)¹ to DNA barcodes of 102 marine fish species from Portugal, according to the ranking system proposed in this study.

1<p>Grade A: External Concordance; Grade B: Internal Concordance; Grade C: Sub-optimal Concordance; Grade D: Insufficient Data; Grade E: Discordant Species Assignments. Information is detailed in the text.</p><p>°new addition to the global COI-5P library.</p

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

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.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. ML's 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