Freshwater unionid mussels are a taxa‐rich group of bivalves that play a critical role in maintaining freshwater ecosystems, but are heavily imperilled due to anthropogenic influences. Environmental DNA (eDNA) provides potential benefits for the monitoring of unionids through higher detection sensitivity, lower costs, less intrusion on the environment, and added advantages for sampling challenging and remote habitats. We compare an extensive mussel rescue survey conducted as part of the demolition of a dam with the detection of mussels using eDNA metabarcoding. The unique mussel rescue survey provided a rare opportunity for an in‐depth analysis of unionid eDNA detection across both small sampling cells and a large sampling region. Environmental DNA detected 22 of the 24 (91.67%) species sampled in the rescue survey, with the two absent species found as only single individuals. A species missed by the rescue survey was detected with eDNA (Potamilus alatus), along with hidden cryptic diversity within the genus Pyganodon. Both survey methods detected the presence of two federally listed species from multiple sampling cells (Plethobasus cyphyus and Theliderma cylindrica). Furthermore, eDNA provided similar estimates of relative mussel abundances across the sampling region. Estimates of mussel diversity were similar between the two methods, and eDNA successfully detected the two federally listed species present. These results contribute to the growing knowledge of eDNA‐based biological assessments and provide valuable insight into comparing sampling effort and detection rates from conventional and eDNA survey methods. As eDNA collection can widen the traditional sampling season and improve the ability to sample difficult or dangerous environments, these results demonstrate the practicality of eDNA metabarcoding as a supplemental sampling method to describe diverse mussel communities and improve the detection of threatened and endangered species.

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Declines in population and diversity of North American bats are rapidly occurring due to habitat loss, incidental take from various industry projects, and lethal White-nose Syndrome disease. It is critical to accurately census habitat for appropriate conservation measures, yet traditional sampling methodology, such as mist netting and acoustic recordings, can be time-intensive and biased. Instead, a passive sampling tool that does not rely on the a priori knowledge of bat roosts may provide crucial information on bat communities. In the water-limited habitats of forested uplands of the Appalachian Plateau, water-filled road-ruts are important resources for bats. Therefore, we developed an environmental DNA (eDNA) protocol to sample isolated road-ruts that may have the presence of sloughed cellular material from actively drinking bats. The detection of bat eDNA was investigated from a positive control experiment, and across 47 water samples collected in Kentucky and Ohio. Water samples were analyzed using both species-specific quantitative polymerase chain reaction (qPCR) and community metabarcoding methodologies. Using qPCR analysis, we detected eDNA from big brown bat (Eptesicus fuscus) and eastern red bat (Lasiurus borealis) from water-filled road-ruts. While the community metabarcoding approach failed to detect any bat eDNA, many non-target amphibians, birds, and mammals were identified. These results suggest eDNA found within road-ruts provides an additional detection tool for surveying biodiversity across upland forests. Additionally, the use of qPCR increased the detection of rare eDNA targets, which will be crucial for properly implementing future eDNA applications for improving bat conservation efforts across the landscape.Article impact statementEnvironmental DNA provides detection of bats from drinking sources offering a novel survey method for management and conservation efforts