Hanfling, B.; Griffiths, N. P.; Macarthur, J. A.; Morrisey, B.; Svobodova, D.; Pritchard, V. L.; Tree, A.; Gaywood, M. J.

1. Environmental DNA (eDNA) metabarcoding is an emerging tool for biodiversity assessment in freshwater systems, offering high-resolution insights into community composition. Here, we apply eDNA metabarcoding to evaluate the ecological impacts of Eurasian beaver (Castor fiber) activity within a semi-natural enclosure in the Scottish Highlands. 2. We collected seasonal water samples from nine sites, six influenced by beaver dams and three control sites with no evidence of beaver engineering, across a 40-hectare enclosure. Samples were analysed for vertebrate and macroinvertebrate diversity using established 12S and COI markers. 3. Vertebrate alpha diversity did not differ significantly between beaver and control sites, likely reflecting the small spatial scale and low species richness of upland Scottish streams. However, community composition differed significantly between treatments, especially for fish (PERMANOVA, R2 = 0.55, P < 0.001), with beaver-influenced sites dominated by three-spined stickleback and control sites by brown trout. Macroinvertebrate communities showed a 78% increase in gamma diversity in beaver-modified habitats relative to controls. Species composition varied strongly with beaver presence (PERMANOVA, R2 = 0.29, P < 0.001), likely due to the creation of lentic-lotic mosaics and associated microhabitat diversity. Seasonal variation was significant in both taxonomic groups, with the lowest species richness and highest community dispersion observed in summer, probably reflecting hydrological and temperature-driven dynamics in eDNA production and transport. 4. Our findings reinforce previous evidence that beaver dam-building activity enhances beta diversity in headwater systems. Additionally, we demonstrate that eDNA metabarcoding is a sensitive method for detecting spatial patterns in freshwater biodiversity associated with these activities at scales ranging from tens to hundreds of meters. These approaches could inform future monitoring strategies aligned with landscape-scale beaver management and reintroductions.