Abstract
Fish are essential to the functioning of aquatic ecosystems, but changes in land use and increasing hydropower development are placing freshwater ecosystems under stress. Bhutan supports relatively pristine mountain river ecosystems, but information on fish diversity is still limited. Fish sampling is largely based on conventional methods and may underestimate the presence of small or rare species. Environmental DNA (eDNA) metabarcoding offers an effective approach for determining species composition over a large river segment. This study assessed current knowledge on fish composition by comparing eDNA metabarcoding data with checklist data from three river basins in Bhutan. The match between the checklist and the eDNA detections was high in the Punatsangchhu and Wangchhu basins, and low in the Drangmechhu basin, where the eDNA metabarcoding detected the genus
Amblypharyngodon
, which may represent a new occurrence in the country. Overall, eDNA reliably reflects spatial patterns of diversity, with decreasing species richness as the elevation and river segment steepness increase and different fish assemblages in isolated basins. These findings demonstrate the effectiveness of eDNA metabarcoding, where knowledge of the distribution of fish is still limited. Repeated sampling and improvements in reference databases are needed to improve taxonomic resolution and the accuracy of the detection.