Multiplex qPCR assays for detection of 2 imperiled anuran species, Anaxyrus californicus and Spea hammondii, from environmental DNA

We developed a species-specific, quantitative polymerase chain reaction assay multiplex for the detection of 2 threatened and endangered anuran species, Arroyo Toad (Anaxyrus californicus Camp, 1915) and Western Spadefoot (Spea hammondii Baird, 1859), from environmental DNA (eDNA). Both species have experienced range wide declines over the last century, mostly because of habitat loss. More accurate methods to detect and monitor both species are needed given their declines and cryptic life histories. We tested species-specificity and sensitivity of eDNA assays in the laboratory and validated the multiplexed assays with field-collected eDNA samples. Both assays were species-specific and sensitive and effectively detected the target species from streams, rivers, and ponds, although detection was imperfect. These assays provide an efficient and economical tool to detect, monitor, manage, and conserve these at-risk species.