| California red-legged frog (Rana draytonii, CRLF) is a visually evasive species living at low densities, which results in low detection probability using standard field survey methods such as traps or visual encounter surveys. The loss of historical habitat for CRLF has resulted in extirpations or serious declines throughout its former range. Management actions are sub-optimal if current distribution and occupancy information is unreliable. Enhancing survey method sensitivity would improve compliance monitoring and recovery planning for the species under the Endangered Species Act. We designed and validated a species-specific diagnostic qPCR assay for identifying a 61bp fragment of the Cytochrome B (CytB) mitochondrial gene of CRLF. The CytB CRLF qPCR assay specificity was shown to reliably detect CRLF DNA and not amplify or cross react with DNA from other species of Rana, including R. luteiventris, R. pretiosa, R. aurora, and R. cascadae. We tested several environmental DNA (eDNA) samples from sites with documented CRLF populations. Samples from sites with known CRLF occupation tested positive for CRLF DNA. Samples from sites with no documented CRLF and negative field controls were negative for CRLF DNA. The CytB CRLF qPCR-based DNA species specific detection coupled with eDNA sampling methods provide a means to obtain critical population metrics from this otherwise cryptic and hard to study organism. |