One Assay to Rule Them All: Development of a Global Environmental DNA Tool to Support Range-Wide Surveys of an...

Environmental DNA (eDNA) has emerged as a powerful tool to support the conservation of threatened species. The sawfishes are one of the most imperiled vertebrate groups, with all five species classified as Critically Endangered on the IUCN Red List of Threatened Species. Owing to their toothy rostra, sawfishes are easily entangled in a wide range of fishing gear, with mortalities in fisheries and loss of habitat driving global declines. The Largetooth Sawfish, Pristis pristis, historically had a circumtropical range, but has experienced severe declines in geographic range and abundance. Viable populations are thought to be confined to northern Australia, but a handful of other nations may harbor remnant populations. To identify promising locations where populations may persist, a novel species-specific Droplet Digital PCR (ddPCR) assay was designed capable of detecting a single copy of DNA (Limit of Detection 0.07 copies/μL), with in silico and in vitro validation experiments indicating functionality across the species' historical range. Water samples were then collected in 2022 at 99 sites in the Eastern Tropical Pacific from the Gulf of Chiriquí (N = 48) and Darién (N = 18) regions of Panama, and the Chocó region (N = 33) of Colombia, but P. pristis DNA was not detected at any sampling sites. Habitat Suitability Index modeling revealed that sampling sites were located within, or in proximity to, basins with highly suitable habitats for sawfish. Detecting rare species in remote data-poor regions presents major challenges, and additional spatially and temporally comprehensive eDNA surveys using this novel ddPCR eDNA assay are recommended. These surveys should focus on locations where the presence of P. pristis remains uncertain to identify priority locations for global conservation efforts.