A conservation planning assessment of basin wide Unionid mussel assemblages using environmental DNA

Conservation planning for rare, threatened, and endangered species requires basic information for distribution and abundance. Often this information is lacking due to the nature of traditional survey methods which can be time and labor intensive and thus costly. Environmental DNA (eDNA) metabarcoding offers a promising approach for monitoring freshwater mussel assemblages, a taxonomic group that is both highly imperiled and difficult to survey using traditional methods. We evaluated the performance of eDNA metabarcoding across 30 km of Fish Creek, in Ohio and Indiana, U.S.. We compared results to visual surveys conducted at the same sites. eDNA detected 25 mussel species, including four species not observed alive visually, while visual surveys detected 22 live species. Both methods confirmed the presence of three federally protected species, and eDNA uniquely detected Simpsonaias ambigua, a species rarely encountered in conventional surveys. Incorporating detection repeatability improved congruence between methods: high-repeatability detections strongly aligned with visual presence, whereas moderate and low repeatability detections likely represented reach-scale occupancy. Overall, eDNA metabarcoding offers an efficient and sensitive tool for assessing mussel assemblages and can substantially enhance monitoring programs when integrated with species ecology and hydrological context.