Multiplex qPCR assays for detection of two imperiled anuran species,Anaxyrus californicusandSpea hammondii, from environmental DNA

We developed a species-specific, quantitative PCR assay multiplex for the detection of two threatened and endangered anuran species, arroyo toad ( Anaxyrus californicus) and western spadefoot ( Spea hammondii ), from environmental DNA (eDNA). Both species have experienced range wide declines over the last century, mostly as a result of habitat loss. Given these declines and their cryptic life histories, improved tools for detecting and monitoring both species are needed. Species-specificity and sensitivity of assays were empirically tested in the lab, and the multiplexed assays were validated with field-collected eDNA samples. Both assays were species-specific, sensitive, and effectively detected the target species from streams, rivers and ponds, although detection was imperfect. These assays provide an efficient and economical tool that will aid in the detection, monitoring, management, and conservation of these at-risk species.