Ecological water replenishment reshapes the river microbiome along an urban-rural gradient: Enhanced diversity but compromised stability

Yongding River Basin, a critical ecological corridor in the Beijing-Tianjin-Hebei region of northern China, characterized by distinct urban-rural gradients. This study investigated the multi-scale impacts of Ecological Water Replenishment (EWR) on riverine bacterial communities along an urban-rural gradient (Rural-RS, Urban-US, Estuarine-ES). We systematically evaluated changes in microbial diversity, community assembly mechanisms, and molecular ecological network structure before and after EWR implementation. EWR universally enhanced bacterial alpha diversity (RS:156%, US:43%, ES:71%), reversing the pre-EWR "urban-highest" pattern to "rural-highest". It significantly altered community composition and source contributions, increasing stochasticity in assembly, particularly shifting the urban section from deterministic to stochastic dominance.While EWR increased network complexity, it concurrently reduced stability, especially in the urban section. Rural sections exhibited the greatest increase in keystone species (270%), whereas urban sections showed minimal gains. These findings demonstrate that EWR achieves optimal restoration in rural zones with preserved natural conditions, while enhancing diversity at the cost of stability in urbanized stretches, highlighting the need for integrated basin management. • EWR increased bacterial diversity but reversed its urban-rural spatial pattern. • EWR shifted microbial assembly to stochastic dominance, notably in urban rivers. • EWR raised network complexity but decreased its stability and robustness. • Rural sites showed the largest gain in keystone taxa after water replenishment.