Madagascar harbors a rich marine biodiversity, yet detailed knowledge of its fish species remains limited. Of the 1689 species listed in 2018, only 22% had accessible cytochrome oxidase I (
COI) sequences in public databases. In response to growing pressure on fishery resources, this study aims to strengthen biodiversity monitoring tools. Its objectives were to enrich the
COI database for Malagasy marine fishes, create the first
12S reference library, and evaluate the taxonomic resolution of different
12S metabarcodes for eDNA analysis, namely MiFish, Teleo1, AcMDB, Ac12S, and 12SF1/R1. An integrated approach combining morphological, molecular, and phylogenetic analyses was applied for specimen identification of fish captured using various types of fishing gear in Toliara and Ranobe Bays from 2018 to 2023. The Malagasy
COI database now includes 2146 sequences grouped into 502 Barcode Index Numbers (BINs) from 82 families, with 14 BINs newly added to BOLD (The Barcode of Life Data Systems), and 133 cryptic species. The
12S library comprises 524 sequences representing 446 species from 78 families. Together, the genetic datasets cover 514 species from 84 families, with the most diverse being Labridae, Apogonidae, Gobiidae, Pomacentridae, and Carangidae. However, the two markers show variable taxonomic resolution: 67 species belonging to 35 families were represented solely in the
COI dataset, while 10 species from nine families were identified exclusively in the 12S dataset. For 319 species with complete
12S gene sequences associated with
COI BINs (Barcode Index Numbers),
12S primer sets were used to evaluate the taxonomic resolution of five
12S metabarcodes. The MiFish marker proved to be the most effective, with an optimal similarity threshold of 98.5%. This study represents a major step forward in documenting and monitoring Madagascar’s marine biodiversity and provides a valuable genetic reference for future environmental DNA (eDNA) applications.
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