Mapping the northernmost transnational non-native population of Xenopus laevis using pooled eDNA sampling

Early-detection-rapid response (EDRR) programs are invaluable for managing incipient invasions, but require monitoring strategies that maximize search coverage and minimize costs. While environmental DNA (eDNA)-based monitoring techniques are now widely used for monitoring aquatic invasions, the large monitoring efforts in EDRR programs demand further improvements in sampling strategies. This need is exemplified by the recently discovered African clawed frog Xenopus laevis (Daudin, 1802) invasion along the French-Belgian border region – its northernmost introduced population – where it was first detected in France in 2018 near the Belgian border. In response to the risk of its permanent establishment and cross-boundary spread, we employed quantitative eDNA barcoding analyses in the context of an EDRR program. We (i) developed and validated a novel cost-efficient sampling strategy that involves pooling water from multiple sites allowing for broader area coverage, (ii) delineated the spatial extent of the invasion, and (iii) assessed whether lotic systems serve as cross-boundary dispersal corridors. In 2020, 2022, and 2023, we determined the presence and eDNA concentrations of X. laevis from a total of 426 sites by pooling water from 366 sites in 83 samples, and sampling 59 additional sites individually. We found that this pooled approach can accurately determine X laevis presence and approximate its average eDNA concentration across pooled sites. We detected X. laevis in 26 samples, revealing an interconnected population spanning an area of 103 km 2 , contradicting prior assumptions of an early invasion stage. We provide evidence that the river Lys and the Douvebeek facilitate cross-boundary movement. The pooled sampling technique presented here is a cost-effective method for providing timely, actionable data to inform management decisions; however, further validation is needed to confirm its reliability and broader applicability. The X. laevis invasion along the French-Belgian border underscores the complexities of managing biological invasions across national and administrative boundaries, providing valuable insights for other transboundary invasions.