Environmental DNA as an Indicator of Seasonal Reproductive Phenology in Freshwater Mussels

Unionid freshwater mussels exhibit a unique form of mitochondrial inheritance, termed doubly uniparental inheritance, in which a maternal and a paternal mitotype is transmitted uniparentally. The exclusive presence of a male mitotype in gonadal tissue and sperm cells suggests that environmental DNA (eDNA) could serve as a non-invasive method for monitoring freshwater mussel reproduction. Yet, the dynamics of male mitotype detection within the environment remain poorly understood. This study analyzed seasonal eDNA samples from two diverse mussel beds, detecting 24 mitochondrial operational taxonomic units (MOTUs) associated with the male mitotype. Peaks in male mitotype signal for mussels identifiable to the species level generally aligned with expected spawning periods based on female gravidity records (e.g., Pyganodon grandis , Lasmigona costata , Ortmaniana ligamentina ). Additionally, male mitotype detection was often sporadic compared to the consistently detected female mitotype, indicating that male signals may be tied to behavioral or reproductive events rather than continuous shedding. While elevated male signals may reflect spawning, alternative sources such as tissue decay, mitotype leakage, glochidia release, or post-spawning gamete clearance complicate interpretation. A male-to-female mitotype ratio is proposed as a more reliable proxy for identifying sperm release events, given the high concentration of male mitotypes that occurs within spermatozeugmata. Limitations in male mitotype reference databases hindered species-level resolution for many MOTUs, underscoring the need for expanded genomic resources. Overall, this work demonstrates that male mitotype eDNA likely provides valuable insights into mussel reproductive ecology, while emphasizing the importance of long-term monitoring and integrated gametogenesis studies to refine its application in conservation.