Leveraging the double uniparental inheritance of freshwater mussels to detect male spawning activity through eDNA

Sampling aquatic habitats for environmental DNA (eDNA) has emerged as a valuable population monitoring tool, particularly for species that are difficult to survey with conventional methods. However, eDNA approaches are limited in their ability to provide demographic information, resulting in most applications focusing on documenting species presence and distributions. Advancing molecular detection methods to assess reproductive phenology could significantly improve conservation outcomes by informing management efforts. In this study, we leveraged the unique double uniparental mitochondrial inheritance system of one of the most imperiled taxonomic groups, freshwater mussels, to enable detection of reproductive activity. We developed separate qPCR assays for eDNA detection of male mitotypes, found almost exclusively in male gametic tissue, and female mitotypes, found in female gametes and somatic cells of both sexes, of the endangered spectaclecase, Cumberlandia monodonta (Say, 1829). Following assay development and laboratory validation, we verified their utility in situ by detecting male and female mitotypes from captive populations in a controlled mesocosm and from natural populations in streams at sites with known and previously unconfirmed occupancy. Male mitotype eDNA was detected almost exclusively during the March–May spawning window, with concentrations often an order of magnitude higher than female mitotype concentrations. Our findings suggest targeting male mitotype DNA and strategically timing eDNA sampling during spawning periods can improve detection probabilities and aid efforts to conserve at-risk freshwater mussels.