Validation of environmental DNA (eDNA) as a detection tool for at-risk freshwater pearly mussel species (Bivalvia: Unionidae)

1. Documenting the occurrence and habitat occupancy of rare aquatic species is an ongoing challenge for conservation. Characterization of environmental DNA (eDNA) from bulk water samples has emerged as a powerful tool to infer species presence or absence without the need to observe or handle organisms. 2. Previous eDNA studies have yet to develop species-specific markers that target taxa with many potentially sympatric confamilials. Forty-one freshwater pearly mussel species (Unionidae) are found in southern Ontario, Canada, with many of these listed as threatened, endangered, or of conservation concern; however, locating populations for protection can be challenging owing to morphological crypsis and species scarcity. 3. Species-specific eDNA markers were developed to target four unionid species. Following in silico and in vitro validation, markers were validated in the field by comparing eDNA results from water samples to detections based on quadrat sampling. 4. Target species were detected by eDNA sampling at all sites where they had previously been located by quadrat sampling. 5. The paired sampling design showed that species-specific markers can be designed even within speciose families, and that eDNA detection of mussels is at least as sensitive as quadrat sampling. Furthermore, detection probabilities were not affected by sampling depth, and eDNA concentrations were positively correlated with mussel densities. 6. These findings confirm that eDNA assays are a valuable complement to traditional methods for locating and managing imperilled unionid populations.