Fine-scale aquatic eDNA sampling reveals significant within- and across-site variation during the expected breeding season of Cryptobranchus alleganiensis alleganiensis

Abstract Environmental DNA (eDNA) analysis is a powerful tool for monitoring wildlife populations. While seasonal peaks in eDNA concentration during breeding seasons of target aquatic species are well documented, fine-scale temporal changes in eDNA are yet to be extensively examined. We used C. a. alleganiensis , an aquatic salamander whose breeding season is characterized by high eDNA concentrations, as a model species to test three hypotheses: eDNA concentration would remain high throughout the expected breeding season, eDNA signatures would reflect synchronous breeding among local populations, and eDNA concentration changes would correspond to seasonal stream-temperature changes. We sampled five tributaries of the Susquehanna River in Pennsylvania, USA, daily from August to October to examine how eDNA concentrations associated with breeding activities changed. We found eDNA concentrations were highly variable within and across sites, but four of the five tributaries showed a predicted eDNA concentration peak in the middle of the sampling period. Temporal changes in eDNA concentrations were site-specific and asynchronous among sites. Despite notable variation across sites, water temperature and its quadratic term were significant predictors of eDNA profiles. Our results revealed the importance of a careful spatiotemporal sampling design for eDNA characterization of breeding seasons in aquatic organisms.