Building a Local Multi-Marker eDNA Reference Database Reveals the Limitations of Public Repositories for Freshwater Fish Monitoring in the Three Gorges Reservoir

Environmental DNA (eDNA) metabarcoding has emerged as a powerful tool for biodiversity monitoring, yet its accuracy is fundamentally constrained by the completeness and taxonomic reliability of reference sequence databases. For the Three Gorges Reservoir (TGR), no integrated multi-marker eDNA reference library exists, hampering standardized fish conservation monitoring under the Yangtze River Ten-Year Fishing Ban. Here, we constructed a comprehensive, multi-marker eDNA reference database for the fish fauna of the TGR, encompassing mitochondrial 12S rRNA, 16S rRNA, and cytochrome c oxidase subunit I (COI) gene sequences from 173 specimens (120 species) collected between 2021 and 2024. After integrating publicly available sequences, the final database comprised 161 species. Then, we quantitatively compared species annotation performance between this local database and public repositories. Results showed that while public databases achieved higher nominal species coverage (94.67%), they exhibited critical deficiencies in annotation accuracy, correctly annotating only 77.97% (12S rRNA), 75.00% (16S rRNA), and 38.14% (COI) of sequences from shared species under controlled conditions. In contrast, the local database exhibited 92.37%, 93.10% and 100% annotation accuracy for the respective markers. Optimal interspecific Kimura 2-parameter (K2P) thresholds for species delimitation were 0.00448 (12S rRNA), 0.00531 (16S rRNA), and 0.00734 (COI). In addition, 15, 0, and 4 species pairs exhibited zero interspecific distance for 12S rRNA, 16S rRNA, and COI, respectively. These limitations reinforce the need for cautious interpretation of eDNA metabarcoding results and the integration of multiple markers or complementary nuclear loci. This study provides preliminary evidence that regionally curated, multi-marker reference libraries could improve taxonomic assignment reliability in eDNA metabarcoding compared to uncurated public repositories, providing a foundational resource for biodiversity conservation.