Spatial patterns and environmental associations of plankton communities in the Karst free-flowing Chishui river: Insights from eDNA metabarcoding and network analysis

Understanding the mechanisms governing plankton community assembly and spatial heterogeneity is fundamental for freshwater ecosystem conservation, yet such knowledge remains scarce for free - flowing rivers in Karst regions facing compounding anthropogenic pressures. Here, we investigated the spatial patterns, community – environment relationships, and co-occurrence network architecture of phytoplankton and zooplankton communities along the mainstem of the Chishui River, the last undammed tributary of the upper Yangtze River and a biodiversity hotspot of global conservation significance. Integrating environmental DNA (eDNA) metabarcoding with ecological network analysis, we quantified the relative contributions of environmental variables and spatial factors to community structuring across 15 sites along the river continuum. Our results revealed striking trophic-level divergence in community-environment relationships: zooplankton showed significantly stronger associations with measured environmental variables (∼22% explained variance) than phytoplankton, which exhibited weak spatial and environmental structuring, However, large unexplained fraction in both groups precludes assigning this differential pattern to deterministic versus stochastic assembly without formal null-model frameworks. Along the longitudinal gradient, we observed a descriptive trend of increasing network modularity and internal connectivity in the downstream reach, where organic pollution indicators (COD and BOD 5 ) were relatively elevated. We tentatively term this pattern of network densification as a hypothesis consistent with observed topology, but its functional implications remain to be empirically tested. Our findings provide a descriptive and hypothesis generating basis for understanding multi-trophic community structuring in Karst free - flowing rivers, and offer empirical reference points for bio-assessment and adaptive management of riverine ecosystems facing combined anthropogenic stressors in comparable systems.