Development of an environmental DNA assay for detecting multiple shark species involved in human–shark conflicts in Australia

Abstract The number of human–shark interactions has increased worldwide during the past decade resulting in injuries and fatalities. In Australia, the white shark (Carcharodon carcharias), tiger shark (Galeocerdo cuvier), and bull shark (Carcharhinus leucas) are responsible for the majority of fatal incidents. On the southeast coast of Australia, monitoring programs currently rely on SMART (Shark‐Management‐Alert‐in‐Real‐Time) drumlines and mesh nets to catch, tag, and monitor shark movement. However, these methods are laborious, costly, and involve the capture of only a fraction of the total shark population. Here, we develop a multiplex environmental DNA assay capable of detecting all three shark species simultaneously from water samples by targeting conserved but specific mitochondrial sequences that are characteristic of each species. The specificity of the assay was validated by testing for cross‐amplification across a range of non‐target but co‐occurring shark species from eastern Australia. We test the sensitivity of the assay on water samples collected from shark capture events and sites where these shark species are known to frequent, and undertake DNA sequencing on positive samples to confirm species haplotype authenticity. Samples collected from one of these sites also demonstrate that eDNA detections are dependent on shark activity in the area. This assay will allow for rapid detection of DNA from each shark species in water samples, providing a cost‐effective alternative for monitoring sharks along the east coast of Australia and potentially elsewhere.