Species identification rates of commonly used Environmental DNA primers for Yangtze River fishes

eDNA metabarcoding has emerged as a powerful approach for assessing aquatic biodiversity; however, it remains constrained by uncertainties in OTU-clustering thresholds and challenges in taxonomic assignment. The Yangtze River, China’s longest river system, supports over 4,300 aquatic species, among which fish constitute a dominant taxonomic group within the freshwater ecosystem. Owing to their sensitivity to fluctuations in water quality, fish serve as robust ecological indicators for monitoring environmental change. In order to enhance the referential value of Environmental DNA (eDNA) in the study of fish species diversity in the Yangtze River basin. In this study, DNA sequences of 320 Yangtze River fish species were collected from the Mitofish and NCBI public database. The 5 commonly used eDNA metabarcoding fragments (12S-AcDBM07, 12S-Mifish-U, 12S-Tele02, 16S-Ac16s, COⅠ-PS1) were extracted from 3 gene fragments (12S RNA, 16S RNA, COⅠ) for genetic distance calculations, which were analyzed under different interspecies difference thresholds (0.03, 0.02, 0.01). This aimed to assess the identification efficiency of commonly used eDNA metabarcoding fragments for Yangtze River fish species and explore their application in the study of fish diversity. Research shows that when the interspecific difference thresholds are at 0.03, 0.02, and 0.01, there are 137, 108 and 56 fish species that cannot be identified by these 5 commonly used eDNA metabarcoding fragments. Among these, the protein-coding gene (COⅠ-PS1) exhibited the highest identification rate for fish species, reaching 53.44%, 60.63%, and 72.50% at different interspecies difference thresholds (0.03, 0.02, 0.01). Among the 5 eDNA metabarcoding fragments, 12S-Tele02 with 12S-Mifish-U had the highest number of shared in identifying Yangtze River fish species, attaining 98.54%, 100%, and 91.11% under different interspecies difference thresholds (0.03, 0.02, 0.01). When choosing combinations of two eDNA metabarcoding fragments for fish identification, the combination of "12S-Tele02" and " COⅠ-PS1" identified the most species at every interspecies difference thresholds (0.03, 0.02, 0.01), with 180, 206, and 257 species, and it is 3, 6, and 7 species that different from the combination using 5 eDNA metabarcoding fragments. In summary, this research demonstrates that the relationship between the choice of interspecies difference thresholds and the identification of closely related species should be carefully considered.in the application of eDNA technology fish diversity in the Yangtze River, to prevent false positives in OTU annotations. For species that are difficult to identify but have high conservation value, a targeted primer design can be employed when using eDNA technology for species diversity monitoring. Additionally, combining eDNA technology with traditional netting can provide a more comprehensive assessment of local waterbody biodiversity.