eDNA‐based detection of a vulnerable crocodile newt (Tylototriton uyenoi) to influence government policy and raise public awareness.

Aim: Although at least five Tylototriton species were recorded in Thailand, only Tylototriton verrucosus is registered as a protected species under the Wildlife Preservation and Protection Act in Thailand. Populations of T. uyenoi are now severely declining, caused by anthropogenic activities. As intense human pressure is having profound effects on the diminishment in T. uyenoi populations, a conservation plan is needed. Information such as the abundance and distribution of a species is necessary. Yet, current established survey methods are either time‐consuming or labour‐intensive. Here, eDNA‐based detection was developed for tracking the presence of the T. uyenoi. Location: Thailand. Methods: We target the surveillance of T. uyenoi using eDNA. Primers and a probe specific to T. uyenoi were designed and tested for their specificity and sensitivity. Water samples were collected once a month from August to January at three sites in Doi Suthep and at three extra sites within the range of the species. Three hundred ml samples of water were collected and filtered. Environmental DNA was extracted and then subjected to qPCR assay in an attempt to detect T. uyenoi. Results: The qPCR assay was found to be species‐specific to T. uyenoi. Both PCR and qPCR did not result in any positive detection of three congeners or other non‐target species. The LOD and the LOQ of the assay were determined by an analysis of the standard curve, and it was found that the LOD and the LOQ were 7.99 and 9.0 copies per reaction, respectively. Environmental DNA was detected in water samples from all sites where T. uyenoi has been known to occur, but detection varied among sites and sampling times. In addition, low amounts of eDNA were detected in three sites with unknown occupancy of newts, but within the species' range. Conclusions: Our findings suggest that eDNA survey is a powerful tool for tracking T. uyenoi throughout the year regardless of the sampling site conditions. Similar to other amphibians, T. uyenoi is severely declining due to anthropogenic factors. In order to have an effective conservation plan, knowledge of a species' distribution is needed. To our knowledge, this was the first study that used eDNA to track the crocodile newt in Thailand. [ABSTRACT FROM AUTHOR]