Advancing Environmental DNA Methods for Monitoring the Reproduction of Quagga and Zebra Mussels in Lakes

ABSTRACT Invasive alien mussels threaten freshwater ecosystems worldwide by disrupting food webs and nutrient cycling. The effective monitoring of these populations is therefore essential to assess their spread and ecological impacts. This study demonstrates the applicability of environmental DNA (eDNA) methods to monitor the reproductive periods of two such species, Dreissena polymorpha (zebra mussel) and Dreissena rostriformis bugensis (quagga mussel), whose morphologically indistinguishable larvae (veligers) are difficult to monitor accurately. We developed new primers and probes for multiplex droplet digital PCR (ddPCR), allowing differentiation between the two species. We compared three eDNA sampling methods in four deep peri‐Alpine lakes—integrated water samples (0‐ to 50‐m depth), sub‐surface water samples, and plankton bulk samples—with traditional microscopic counts of veliger larvae. All eDNA methods correlated positively with veliger counts. Integrated water and plankton bulk samples proved more effective in quantifying veligers and identifying reproductive periods compared to sub‐surface water samples. The bulk plankton method excelled in qualitative detection, only showing a positive eDNA signal when larvae were present, and was the only method to detect zebra mussels in Lake Geneva. However, it overestimated winter reproduction in one lake. The integrated water method reflected larval dynamics quantitatively well but sometimes gave false positives (eDNA detected despite no larvae observed). These methods revealed the reproductive dynamics of the two species in different lakes. Quagga mussels reproduce year‐round in peri‐Alpine lakes where they are the dominant species, whereas zebra mussels mainly reproduce during the warmer seasons. Notably, the study provided the first evidence of the presence of quagga mussels in Lakes Annecy and Aiguebelette. Although we suggest further methodological refinements for the eDNA approaches evaluated in this study, those approaches are nonetheless already applicable and can contribute to a better understanding of the ecology of these invasive species in lakes.