Environmental DNA reveals the distinct genetic diversity and evolutionary pathways of the Chinese Minnow Rhynchocypris oxycephalus in Korean freshwater systems

Establishing an environmental DNA (eDNA) reference library at regional and local scales is essential not only for accurate biodiversity assessment but also for comprehensive long-term monitoring. To date, genetic diversity studies of the Chinese minnow (Rhynchocypris oxycephalus) have largely been restricted to China, leaving substantial knowledge gaps across its broader distribution, including South Korea. Hence, the present study identified suitable regions for guiding eDNA surveys and non-invasive sampling, based on documented occurrences retrieved from the IUCN Geospatial Conservation Assessment Tool (GeoCAT). The newly designed primer pairs successfully amplified long mitochondrial fragments (~ 1 kb) of the cytochrome b (Cytb) and 16S ribosomal RNA (16S rRNA) genes. The generated sequences revealed 29 haplotypes from 41 Cytb sequences and 13 haplotypes from 21 16S rRNA sequences, corresponding to high intraspecific genetic diversity (5.57% for Cytb and 2.46% for 16S rRNA), thereby indicating potential cryptic diversity of R. oxycephalus in South Korea. The phylogenetic analyses, combined with multiple species delimitation methods, resolved several putative molecular operational taxonomic units and highlighted a distinct genetic clade in the Seomjin River basin, likely driven by microhabitat-specific evolutionary processes. In addition, the shared haplotypes across catchments of different river basins indicate either ongoing gene flow or anthropogenic influences contributing to genetic admixture of R. oxycephalus. The time-calibrated phylogenetic analyses also suggest that historical geographic changes and ancient river networks, from the Early Miocene to the Late Pliocene, likely facilitated the diversification of R. oxycephalus across China, the Korean Peninsula, and Japan. Overall, this study represents the first eDNA-based assessment of R. oxycephalus diversity in South Korea, while also providing new evolutionary insights from a broader geographic context in China and Japan. Given the complexity of multiple river networks in South Korea, further investigations using multiple genetic markers are recommended to enhance understanding of this cyprinid species phylogeography in the region.