Global efforts to conserve declining amphibian populations have necessitated the development of rapid, reliable, and targeted survey methods. Environmental DNA surveys offer alternative or complementary methods to traditional amphibian survey techniques. The California tiger salamander Ambystoma californiense (CTS) is endemic to California, where it breeds in vernal pools. In the past 25 y, CTS has faced a 21% loss of known occurrences, largely through habitat loss, and it is threatened by hybridization with an introduced congener. Protecting and managing remaining CTS populations rely on accurately monitoring changes in their spatial distribution. Current monitoring practices typically use dip-net surveys that are time-consuming and prone to false negative errors. To provide a new resource for monitoring and surveying larval CTS, we designed an assay and tested it on water samples collected from 29 vernal pools at two locations in California. We compared environmental DNA results to contemporaneous dip-net surveying results and found the assay agreed with positive dip-net results in 100% of cases. In several instances, we also detected the presence of CTS genetic material in the early spring before larvae hatched, potentially offering a new, earlier detection option for this imperiled species. This assay provides a valuable, noninvasive molecular tool for monitoring the spatial distribution of the CTS in vernal pools.

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