eDNA reveals extraordinary fish diversity in the Urauchi River, Iriomote Island, Japan, a UNESCO World Heritage Site

Robust biodiversity monitoring is essential for the management of UNESCO World Natural Heritage Sites; yet, systematic assessments of ichthyofaunal communities remain limited. Here, we apply environmental DNA (eDNA) metabarcoding to surface water samples collected in the Urauchi River (Iriomote Island, Japan), a United Nations Educational, Scientific and Cultural Organization (UNESCO) World Heritage site and one of Japan’s most fish-diverse rivers. Surface water samples were collected at 10–11 sites along the 18.8 km stretch in May 2021 and November 2024. Using MiFish primers, high-throughput Illumina sequencing, and QIIME 2 pipeline, we obtained 35.7 million quality-filtered reads, from which 332 operational taxonomic units (OTUs) belonging to Actinopterygii and Chondrichthyes were assigned to 254 species, 64 genera, and 11 families, along with three unannotated OTUs due to low taxonomic resolution. Species richness peaked in the mid-to-lower estuary within dense mangrove zones, with α-diversity indices (Shannon, Simpson) increasing downstream. β-Diversity analyses [Bray–Curtis, Non-metric Multidimensional Scaling (NMDS), distance-based Redundancy Analysis (dbRDA), and Permutational Multivariate Analysis of Variance (PERMANOVA)] revealed clear community structuring primarily driven by salinity, temperature, vegetation type, sampling year, and spatial distance (all p < 0.05). eDNA detected 16 conservation-priority taxa listed on the Japan Red List [Critically Endangered (CR): 4, Endangered (EN): 4, Vulnerable (VU): 6, and Near Threatened (NT): 2] and six International Union for Conservation of Nature (IUCN) Red List species (EN:2, VU:4), including Anguilla japonica and Cheilinus undulatus . Unexpected detections of deep-sea taxa likely reflect tidal transport or diel migrations. Compared with previous checklists by Suzuki & Seno (2005), this study detected additional taxa but not all recorded species, indicating that fish biodiversity in the Urauchi River system remains incompletely characterized and highlighting the importance of integrating eDNA with conventional surveys for a comprehensive assessment. Nevertheless, our study provides the first molecular baseline of fish diversity in the Urauchi River, demonstrating a complementary, non-invasive biodiversity assessment in dynamic mangrove estuaries. Routine eDNA monitoring, combined with multi-seasonal and depth-stratified sampling, provides a practical early warning system to detect ecological shifts and inform targeted conservation and management actions in this globally significant watershed.