Improved Detection Sensitivity Using An Optimal Edna Preservation And Extraction Workflow And Its Application To Threatened Sawfishes

Pressures on coastal ecosystems are increasing and aquatic species that are restricted to these habitats are facing the threat of extinction. However, the true extent of many threatened and rare aquatic species, especially elasmobranchs, remains unclear due to high levels of data deficiency and poor efficacy of traditional survey methods. Sawfishes (Pristidae), a family of shark-like rays, are among the most threatened and rare elasmobranch species and are difficult to detect in turbid, coastal habitats. Reliable cost-effective tools to detect these species are urgently needed to increase their conservation potential.Characterization of environmental DNA (eDNA) extracted from water samples has garnered significant appeal for detection of rare and threatened species. To assist conservation and monitoring efforts for sawfishes using eDNA, species-specific TaqMan quantitative polymerase chain reaction assays were developed and validated to detect 1.25-5 copies of a 12S rRNA gene fragment. Filter samples were collected in Northern Territory, Australia to assess the utility of the developed eDNA assays and compare the efficacy of preservation and extraction workflows for detecting rare species.Dwarf sawfish (Pristis clavata) were detected in three of 20 sites, and there was a significant effect of preservation and extraction workflow on total eDNA yield and subsequent detection success. Longmire's preserved samples extracted using glycogen-aided precipitation yielded a significantly higher concentration of total eDNA (n = 60; beta = 1.27, t(95) = 8.172, P < 0.0001) and yielded positive P. clavata eDNA detections compared to ethanol preserved samples extracted using QIAGEN DNeasy kit, which did not yield any positive detections.The optimized eDNA assays were developed to support monitoring efforts for endangered sawfishes. Importantly, this study demonstrates that choice of preservation and extraction workflow requires careful consideration, especially when detection of rare or threatened species can have important management and conservation outcomes.