Gap-analysis and annotated reference library for supporting macroinvertebrate metabarcoding in Atlantic Iberia

DNA metabarcoding provides a rapid and effective identification tool of macroinvertebrate species. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries, which, due to incompleteness, are currently a recognized limitation for metabarcoding applications. In this study, we assembled a comprehensive reference library of DNA barcodes for Atlantic Iberia marine macroinvertebrate species, assessed gaps in species coverage and examined data ambiguities. Initially, an Iberian species checklist for the three dominant groups of marine macroinvertebrates was compiled, comprising 2827 species (926 Annelida, 638 Crustacea and 1263 Mollusca). A total of 18162 DNA sequences of the cytochrome c oxidase subunit I barcode region (COI-5P) matching the species checklist were compiled in a BOLD dataset, where taxonomic discordances were evaluated and cases of deep intraspecific divergence flagged. Gap-analysis showed that 63% of the Iberian macroinvertebrate species still lack a DNA barcode. Coverage gaps varied considerably across taxonomic groups with Mollusca displaying the highest sequence representation in the dataset (427 species, 49% of the total number of sequences), and Crustacea the highest species coverage with 338 species barcoded (53% of the checklist). In contrast, Polychaeta displayed the lower levels of completion (288 species, 16% of the total number of sequences). In total, 1545 Barcode Index Numbers (BINs) were assigned to 1053 barcoded species, of which 66% were taxonomically concordant, 26% displayed multiple BINs and 8% were discordant. Overall, results show that there is still a large portion of marine invertebrate taxa in this region of Europe pending barcode coverage, even considering only the dominant groups. However, the most notable finding was the relevant proportion of species flagged for significant intraspecific divergence and possible hidden diversity. The annotated reference library and gap-analysis here provided can there

This study was supported by the project The NextSea [NORTE-01-0145-FEDER-000032], under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was supported by the "Contrato-Programa'' UIDB/04050/2020 funded by national funds through the FCT I.P. SD and PEV work was supported by national funds through the Portuguese Foundation for Science and Technology (FCT, I.P.) in the scope of the project NIS-DNA [PTDC/BIA-BMA/29754/2017]. BRL benefitted from an FCT fellowship PD/BD/127994/2016. MALT benefitted from an FCT fellowship co-financed by European Social Fund (ESF) SFRH/BD/131527/2017.