Abstract
Globally, amphibian species have suffered drastic population declines over the past 40 years. Hundreds of species are now listed as Critically Endangered, with many of these considered “possibly extinct”. Most of these species are stream-dwelling frogs inhabiting remote, montane areas, where traditional surveys limit our ability to find remnant populations. Environmental DNA (eDNA) monitoring could revolutionize surveys for missing and endangered amphibian populations by enabling sampling of downstream sections to screen the vast upstream catchments. However, this is dependent on quantifying downstream detection probability and distances. Here we tested this in two endangered stream frogs (Litoria nannotis and L. lorica) that co-occur in a remote stream catchment, and for which we know precise downstream distributional limits from traditional surveys. Importantly, the last populations of L. lorica persist in this catchment; one small (~1,000 frogs) and one very small (~100 frogs). We conducted eDNA screening at set distances downstream from the populations (up to 22.8 km) using precipitation from two water volumes and via filtering, during moderately high and low flow conditions. During high flow, we reliably detected both species as far as 22.8 km downstream using the larger water volumes and filtering techniques. We could only detect the very small population of L. lorica immediately downstream. Detection success was higher at the sites further downstream for both species during high flow compared to low flow. Our downstream detection distances (> 20 km) suggest eDNA is a valuable tool for detecting rare stream amphibians. We provide recommendations on optimal survey techniques.
Highlights
Amphibians contain a greater proportion of Critically Endangered and Endangered species than any other Class of animal (IUCN, 2020)
Samples were considered putative positive detections if: (1) the amplification curve crossed the common fluorescence threshold within 50 cycles; (2) the amount of environmental DNA (eDNA) was above the limit of detection (LOD); and (3) the melt curve analysis showed a dissociation temperature peak at 78.52 C (±0.62–99% confidence interval) for L. lorica and 79.66 C (±0.75–99% confidence interval) for L. nannotis
Detection frequencies and downstream detection distances for the three methods All positive detections satisfied the following conditions: (1) amplification occurred within 50 cycles; (2) amount of eDNA was above the LOD; (3) the melt curve peak corresponded to that of each species; and (4) amplicon sequences from positive detections had >97% and >99% match with targeted c oxidase subunit I (COI) regions for L. lorica and L. nannotis, respectively
Summary
Amphibians contain a greater proportion of Critically Endangered and Endangered species than any other Class of animal (IUCN, 2020). Of particular concern is that 587 amphibians are listed as Critically Endangered, which is double the number of Critically Endangered mammals, birds or reptiles (IUCN, 2020) These species are on the brink of extinction; the persistence of many is uncertain (Scheele et al, 2019; Stuart et al, 2004). Hundreds of species are listed as Critically Endangered, with many of these considered “possibly extinct” Most of these species are stream-dwelling frogs inhabiting remote, montane areas, where remnant populations are hard to find using traditional surveys. Environmental DNA (eDNA) could revolutionize surveys for ‘missing’ and endangered amphibian populations by screening water samples from downstream sections to assess presence in the upstream catchments. Methods: Here we tested downstream detection distances in two endangered stream frogs (Litoria lorica and L. nannotis) that co-occur in a remote stream catchment in north-east Australia, and for which we know precise downstream distributional limits from traditional surveys. We demonstrate that eDNA from a small population of approximately 1,000 frogs can be detected as far as 22.8 km downstream from the
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