Development of a 16S metabarcoding assay for the environmental DNA (eDNA) detection of aquatic reptiles across northern Australia

A severe lack of distribution data for aquatic reptiles in northern Australia leaves many taxa vulnerable to extirpation and extinction. Environmental DNA (eDNA) technologies offer sensitive and non-invasive genetic alternatives to trapping and visual surveys and are increasingly employed for the detection of aquatic and semi-aquatic reptiles. However, these eDNA approaches have largely applied species-specific primers that do not provide a cost-effective avenue for the simultaneous detection of multiple reptilian taxa. Here, we present a mitochondrial 16S rRNA metabarcoding assay for the broad detection of aquatic and semi-aquatic reptile species. This assay is tested on water samples collected at multiple sampling sites at two tropical locations, including 12 marine and estuarine sites in Roebuck Bay, Western Australia, and four estuarine sites in Cooktown, Queensland, Australia. In total, nine reptile taxa were detected from 10 of the 16 sampled sites, including marine and freshwater turtles, aquatic, semi-aquatic and terrestrial snakes, and terrestrial skinks. However, inconsistencies in the detection of previously observed aquatic reptiles at our sampled sites, such as saltwater crocodile and sea snakes, indicated that further research is required to assess the reliability, strengths and limitations of eDNA methods for aquatic reptile detection before it can be integrated as a broad-scale bioassessment tool.