DNA barcoding of fishes from Zhoushan coastal waters using mitochondrial COI and 12S rRNA genes

Accurate species identification is a key component of biodiversity research. DNA barcoding is an effective molecular method used for fish species identification. We aimed to study the DNA barcoding of fish in Zhoushan coastal waters, explore the differences and applicability of two gene fragments (12S rRNA and COI) of DNA barcoding in fish species identification, and established a comprehensive fish barcoding reference database. Two hundred and eighty-seven captured fish samples from Zhoushan coastal waters were identified using morphological characteristics and DNA barcoding. A total of 264 12S rRNA sequences (belonging to eight orders, 31 families, 55 genera, and 66 species) and 188 COI sequences (belonging to seven orders, 30 families, 48 genera, and 58 species) were obtained. The lengths of the 12S rRNA sequences ranged from 165 to 178 bp, and the guanine-cytosine (GC) content was 45.37%. The average 12S rRNA interspecific and intraspecific genetic distances (K2P) were 0.10% and 26.66%, respectively. The length of the COI sequence ranged 574-655 bp, and the content of GC was 45.97%. The average 12S rRNA interspecific and intraspecific genetic distances (K2P) were 0.16% and 27.45%, respectively. The minimum interspecific genetic distances of 12S rRNA and COI (1.23% and 1.86%) were both greater than their maximum intraspecific genetic distances (2.42% and 8.66%). Three molecular analyses (NJ tree, ABGD, and GMYC) were performed to accurately identify and delineate species. Clustering errors occurred when the 12S rRNA sequences were delimited using the NJ tree method, and the delimitation results of ABGD and GMYC are consistent with the final species identification results. Our results demonstrate that DNA barcoding based on 12S rRNA and COI can be used as an effective tool for fish species identification, and 12S rRNA has good application prospects in the environmental DNA (eDNA) metabarcoding of marine fish.