Molecular detection of a cryptic salamander: Development of an eDNA assay for the detection of the mud salamander (Pseudotriton montanus).

The mud salamander (Pseudotriton montanus) is a notoriously cryptic semi-aquatic Plethodontid found throughout much of the eastern United States, reports of decades passing between observations of this species in areas of known occurrence are common. Although it is listed as imperiled or in need of conservation throughout much of its range, with extirpation suspected in many areas, relatively little is known of its current distribution due to its secretive nature. We developed a species-specific qPCR assay for use in eDNA detection of Pseudotriton montanus. Primers and probe were designed based on cytochrome b sequences obtained from specimens collected in central and eastern KY, compared to published sequences throughout the species range, and screened in silico (twenty-seven species) and in vitro (seventeen species) for specificity against sympatric salamander species. The developed assay was field tested via the collection of water samples at sites known or suspected to contain P. montanus in Kentucky, Ohio, and Tennessee. Of the 68 samples collected, P. montanus eDNA was detected in eight, including all sites (six) in which P. montanus larvae were observed in the field. Sequencing of each environmentally-obtained amplicon confirmed detection of P. montanus diastictus. This work provides thoroughly vetted tools that should prove useful for future monitoring and range delineation of this threatened and cryptic species.