eDNA metabarcoding of freshwater communities in the Royal and Heathcote National Parks

Freshwater biodiversity underpins ecosystem function and human wellbeing, yet river systems are increasingly impacted by habitat modification, invasive species, and altered hydrology. Despite the conservation significance of the Royal National Park (RNP) and Heathcote National Park (HNP) near Sydney, aquatic communities within these protected areas remain poorly characterised. We used multispecies environmental DNA (eDNA) metabarcoding to survey major rivers and tributaries across both parks and establish a baseline of freshwater biodiversity. Community composition at the order level differed significantly between parks, although this pattern was partly influenced by differences in multivariate dispersion among sites. Within parks, rivers in RNP were more strongly differentiated, whereas assemblages in HNP were comparatively homogeneous. Elevational patterns in order richness were inconsistent among rivers, with a significant decline observed only in Kangaroo Creek, suggesting river-specific environmental drivers. Invertebrate assemblages were dominated by Diptera and Trichoptera, while widespread detection of Tubificida may indicate organic enrichment at some sites. Native freshwater fishes were broadly distributed across both parks. In contrast, Percalates (Australian bass) were widespread in HNP but restricted to downstream reaches in RNP, likely reflecting dispersal barriers such as Audley Weir. This study provides the first multi-taxa eDNA baseline for these parks and supports future monitoring and conservation management.