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.

野生动物监测项目对于评估物种、栖息地状况以及管理影响它们的因素至关重要。这对于淡水生态系统中的物种（如两栖动物）尤为重要，因为它们的估计灭绝率高于陆地物种。我们开发并验证了两种物种特异性的环境DNA（eDNA）方法，并将其应用于实地，以检测哈扎拉激流蛙（Allopaa hazarensis）和穆里山蛙（Nanorana vicina）。此外，我们将eDNA调查与目视相遇调查进行了比较，并估算了位点占有率。对于哈扎拉激流蛙和穆里山蛙，eDNA调查得出的出现概率均高于目视相遇调查。使用eDNA的检测概率对两种物种均更高，尤其对于哈扎拉激流蛙。目视相遇调查的排名最高检测模型包含年份和温度对两种物种的影响，而排名最高的占有率模型包含海拔和年份的影响。eDNA数据的排名最高检测模型是空模型，而排名最高的占有率模型包含海拔、年份和湿地类型的影响。据我们所知，这是首次在喜马拉雅地区使用eDNA调查来监测两栖动物物种。