Applying Knowledge of Migration Behaviour to Improve eDNA Detection of a Threatened Fish, the Australian Grayling ( Prototroctes maraena )

ABSTRACT Detecting rare fish species across extensive river systems can be challenging due to their low abundance. However, detection probability may increase when individuals aggregate within localised areas during the spawning season. This study investigates whether environmental DNA (eDNA) detection probability can be improved for the threatened Australian grayling ( Prototroctes maraena ) by concentrating sampling effort in the putative spawning grounds (lower freshwater reaches) during the spawning season. Eight replicate water samples were collected from each of three sites in the Tuross and Deua rivers in New South Wales (NSW), where Australian grayling occur in very low abundance, and from three sites in the Bunyip River in Victoria (positive field control)—one of the last rivers supporting a relatively high abundance of this species. Sampling was conducted on six occasions between April and June 2023 to coincide with the species' downstream migration to spawning grounds. A species‐specific quantitative PCR (qPCR) assay targeting the Australian grayling COI gene was developed and applied to detect the species using six replicate qPCR reactions per sample. Australian grayling were detected or putatively detected in 78% of sampling occasions in the Tuross River and 100% of sampling occasions in the Deua and Bunyip rivers. The proportion of positive qPCR replicates was much higher in the Bunyip River, consistent with the species' higher abundance in this location. Nevertheless, there was a peak in detection probability in the NSW sites from May to early June, confirming the efficacy of a targeted sampling strategy coinciding with spawning aggregations in the lower reaches. The study's findings demonstrate the benefits of incorporating knowledge of behaviour into eDNA survey design for improved detection of low‐abundance species, which can assist in informing threatened fish recovery actions.