Environmental DNA survey does not detect additional populations of a critically endangered leaf frog, but reveal another threat to the species

Environmental DNA (eDNA) is a non-invasive approach that uses DNA traces left by organisms in the environment. It is being widely explored in ecological studies to describe the composition and distribution of biodiversity, especially to detect and monitor threatened or invasive species. We used an eDNA metabarcoding approach to survey for new populations of the microendemic and critically endangered treefrog species, Pithecopus rusticus. We collected water samples from 24 sites in the Subtropical Highland Grasslands of the Atlantic Forest in Southern Brazil. We detected for the first time the potential presence of the invasive North American Bullfrog, Aquarana catesbeiana, in this region. However, no DNA trace of our target treefrog was found, revealing an additional threat to the critically endangered target frog. Overall, 13 anuran species were detected, which are commonly found in open grassland fields of the region and use lentic water for reproduction. We highlight that eDNA surveys might be a straightforward approach that can provide rich data applied to conservation in the South Brazilian Grasslands.