Environmental DNA Assays for Conservation: Tools to Monitor Endangered and Range‐Expanding Turtle Populations

ABSTRACT Over half of all turtle species globally are at risk of extinction. In New South Wales (NSW), Australia, two endemic freshwater turtles—the Manning River helmeted turtle ( Myuchelys purvisi Wells & Wellington, 1985) and the Bell's turtle ( Myuchelys bellii Gray 1844)—are listed as Endangered. While vulnerable in some Australian states, the widespread Macquarie River turtle ( Emydura macquarii Gray, 1831) is locally abundant in NSW as translocations are expanding its distribution beyond its natural range. E. macquarii is now detectable in microsympatry with range‐restricted Myuchelys spp., raising concerns about its potential to displace, compete or hybridize with the endemic species, potentially threatening the survival and genetic integrity of endemic turtle populations. To improve the detection of shifting species distributions and inform conservation, we developed three species‐specific environmental DNA (eDNA) assays targeting M. purvisi , M. bellii , and E. macquarii . All assays were validated in silico , i, and ex situ , demonstrating high specificity and sensitivity (Limit of Detection or LOD at 4 × 10 −3 ng/reaction for both M. purvisi and M. bellii and a LOD at 4 × 10 −4 ng/reaction for E. macquarii ) . While the E. macquarii assay exhibited cross‐amplification of non‐target M. purvisi and M. bellii gDNA at later cycle thresholds (Ct 27–38 vs. target Ct 16–17), unique qPCR melt curve profiles and confirmatory sequencing provide robust secondary validation, ensuring high specificity for environmental monitoring. Additional in situ field validation for M. purvisi across five sites demonstrated the effectiveness of eDNA sampling in a natural riverine environment. These assays provide a non‐invasive, highly sensitive tool for monitoring native populations and early detection of incursions by E. macquarii . When integrated with traditional surveys or citizen‐science programs, these assays will enhance conservation outcomes and help monitor range shifts and hybridisation risks as freshwater turtle's distributions change across the landscape.