Haplotype-level analysis of environmental DNA metabarcoding revealed the biogeography and phylogeography of freshwater fishes in Korean Peninsula

Environmental DNA (eDNA) metabarcoding is a cost-effective, sensitive, and minimally invasive tool for monitoring fish diversity in freshwater ecosystems. To evaluate its suitability for assessing fish biogeography and phylogeography in Korean streams, we analyzed fish assemblages in three streams—Gilan, Seom, and Oshipcheon—located in different Korean biogeographical subdistricts. Over two sampling campaigns at nine sites, a total of 107 amplicon sequence variants (ASVs) were identified, representing 20 families, 46 genera, and 76 species. Among the 76 detected species, 29 were endemic to freshwater habitats in South Korea and played a significant role in shaping fish communities across the surveyed streams. We identified potential sources of six translocated species to the eastern region by examining their ASVs. After excluding these potential translocations, no endemic fish species were simultaneously detected in all three streams, and the phylogeography of endemic fish species Odontobutis platycephala, Coreoperca herzi, Microphysogobio yaluensis, Coreoleuciscus splendidus, Nipponocypris koreanus, and Koreocobitis rotundicaudata was clearly observed across the three biogeographical regions. Additionally, analysis of relative abundance and presence-absence data of fish communities yielded comparable β-diversity metrics reflecting spatiotemporal variation. The fish communities in the streams exhibited distinct groups, with Gilan showing a closer relationship to Seom than to Oshipcheon, consistent with well-known allopatric studies of freshwater fishes in the Korean Peninsula. Interestingly, we did not find any differences in fish assemblages among the three mesohabitat types (riffle, run, and pool), regardless of the sampling site. Our results highlight the potential of water-derived eDNA metabarcoding for detecting endemic fish, unraveling the biogeography and phylogeography of allopatric populations of freshwater fishes, and providing genetic forensic tools to estimate the original sources of translocated species.