Development of a ddPCR assay for the detection of the Smoky Madtom ( Noturus baileyi ) from eDNA in stream water samples

The Smoky Madtom Noturus baileyi is a federally endangered species, whose native distribution includes lower Abrams Creek in Great Smoky Mountains National Park (GRSM) and Citico Creek in nearby Cherokee National Forest. Due to challenges for bio-monitoring posed by its nocturnality and cryptic life history, an environmental DNA (eDNA)-based approach for detection would be useful to complement existing electrofishing and seining efforts to better understand the distribution of this species. We developed a probe-based droplet digital PCR (ddPCR) assay to detect Smoky Madtoms from non-invasively collected water samples. The assay was specific to N. baileyi and did not amplify concentrated genomic DNA of 16 co-occurring or regional fish species, including the yellowfin madtom N. flavipinnis and stonecat N. flavus . The assay limit of detection (LOD) was determined to be 4.18 copies (95% CI: 3.95, 4.41). Several 2 L water samples collected from throughout various streams in GRSM in 2016 and 2017 were tested for the presence of N. baileyi using the ddPCR assay. N. baileyi was detected at two different sites in 2016 and 2017 within Abrams Creek previously known to contain N. baileyi , but no novel detections in other sampled streams were observed. This assay should prove useful for continued surveys of N. baileyi in GRSM.