Environmental DNA (eDNA) metabarcoding is revolutionising the study of aquatic ecosystems, enabling high-throughput analysis of biodiversity with minimal disturbance. Despite its potential to support fisheries management, species identification and downstream analysis reliability are hindered by the lack of standardisation in DNA fragment choice. This study compares the species discrimination power of three markers used in marine fish (e)DNA (meta)barcoding – 12S rRNA, 16S rRNA and cytochrome oxidase subunit I (COI) – as well as two amplicons for each. We analysed sequences from NCBI GenBank for 10 orders of Actinopterygii (ray-finned fishes), including mitochondrial genomes. We assessed species discrimination by determining the percentage of monophyletic species in Neighbour-Joining trees and calculating congeneric divergences for two datasets: one with genomic regions extracted from mitochondrial genomes (771 species) and another with independent sequences for each region (3,879 species). Amongst (meta)barcoding amplicons in the mitochondrial genomes’ dataset, the COI Folmer and Leray-Lobo regions had the highest discriminatory power, with 89.2% and 87.0% monophyletic species, respectively, while the 12S Teleo region had the lowest at 71.6%. Conversely, using independent sequences of these amplicons, Folmer and Leray-Lobo had the lowest percentages of monophyletic species, at 64.8% and 63.5%, respectively, while Actinopterygii 16S (Ac16S) had the highest at 83.0%. Species discrimination is influenced by the marker’s evolutionary rate, fragment length, target fish order and the quality of reference sequence data. We recommend considering species discriminatory power differences for amplicon selection, especially for species-level identifications. We advise a standard multi-marker approach under certain scenarios, particularly when the presence of close congeneric species is expected.

Benchmarking the discrimination power of commonly used markers and amplicons in marine fish (e)DNA (meta)barcoding