| Summary: | Abstract Environmental DNA (eDNA) detection has been shown to be an effective biosurveillance tool for freshwater fishes, but further research is needed to apply eDNA detection tools to small and rare fishes in large rivers. We developed an eDNA surveillance assay and protocol for monitoring the presence of the pygmy madtom (Noturus stanauli), a federally protected freshwater fish endemic to the Clinch and Duck rivers in Tennessee (United States, North America). Noturus stanauli is a diminutive fish that is exceedingly rare throughout its range; it is currently known only from a 115‐river‐km section of the Duck River and a 5‐river‐km section of the Clinch River. The aim of this research was to develop an eDNA assay to detect the presence of N. stanauli in both the Duck and Clinch rivers. We used this newly developed eDNA protocol to assess detection as a function of water depth and to further delineate the distribution of N. stanauli in both the Duck and Clinch rivers. Field sampling was performed to delineate the extent of N. stanauli's range in both rivers. Our results indicated that samples collected from three areas within the water column, as well as a sediment core samples, yielded equal detection rates. Our assay detected the presence of N. stanauli at a previously unknown site outside the current distribution in the Clinch River, located approximately 2.5‐river‐km downstream from the Tennessee‐Virginia state line. We demonstrated that eDNA detection is a promising tool for delineating the distribution of N. stanauli; however, further research is needed to assess environmental and life history variables that influence eDNA detection probability of small fish in large rivers.
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