Integrating environmental DNA, diving surveys and DNA barcoding to track endangered twaite shad (Alosa fallax) spawning sites.

The twaite shad (A. fallax), an anadromous fish species belonging to the Clupeidae family, has experienced population declines throughout Europe. This decline is attributable to a range of anthropogenic pressures, including migration barriers, environmental pollution, habitat degradation, predation by invasive species and unsustainable fishing practices. These factors disrupt the species' lifecycle and ecological balance, leading to significant conservation concerns. Typically, spawning occurs from late spring to early summer, which is when the migration might be up to 400 km upstream from the sea. The demersal eggs hatch relatively quickly, and the larvae start a planktonic life in the river. Traditional monitoring methods have often proved inadequate due to the cryptic nature of A. fallax. With the goal of improving the efficacy of current biomonitoring designs, we tested an environmental DNA (eDNA) approach based on 12S rRNA gene Illumina sequencing to monitor A. fallax migration and spawning sites in the Po and Taro rivers in Northern Italy. This molecular approach was supplemented by scuba diving observations to visually confirm fish presence and habitat conditions at the same sites. Additionally, the collected larvae were morphologically identified and analysed with DNA barcoding to assess the correct taxonomic assignment of the species. This multi-level framework confirmed the upstream migration and identified specific spawning sites, as well as the presence of larvae, highlighting the effectiveness of eDNA in detecting A. fallax populations. These findings underline the potential of eDNA as a powerful conservation tool for monitoring anadromous fish populations and guiding management strategies.