Development and validation of an eDNA protocol for monitoring endemic Asian spiny frogs in the Himalayan region of Pakistan.

Wildlife monitoring programs are instrumental for the assessment of species, habitat status, and for the management of factors affecting them. This is particularly important for species found in freshwater ecosystems, such as amphibians, as they have higher estimated extinction rates than terrestrial species. We developed and validated two species-specific environmental DNA (eDNA) protocols and applied them in the field to detect the Hazara Torrent Frog (Allopaa hazarensis) and Murree Hills Frog (Nanorana vicina). Additionally, we compared eDNA surveys with visual encounter surveys and estimated site occupancy. eDNA surveys resulted in higher occurrence probabilities for both A. hazarensis and N. vicina than for visual encounter surveys. Detection probability using eDNA was greater for both species, particularly for A. hazarensis. The top-ranked detection model for visual encounter surveys included effects of both year and temperature on both species, and the top-ranked occupancy model included effects of elevation and year. The top-ranked detection model for eDNA data was the null model, and the top-ranked occupancy model included effects of elevation, year, and wetland type. To our knowledge, this is the first time an eDNA survey has been used to monitor amphibian species in the Himalayan region.