Multi-source ecological water replenishment reshapes microbial community assembly and network stability in a water-scarce river

• EWR decreased microbial α diversity and presented β diversity heterogeneity. • Stochastic process dominated the microbial community assembly. • NH 4 + -N in water and pH in sediment governed ecological process changes. • Under the influence of EWR, the complexity of microbial networks supported stability. Ecological water replenishment (EWR) has been an essential strategy to alleviate water scarcity and restore ecological functions in degraded rivers. However, the ecological consequences of multi-source replenishment, particularly how diverse water inputs jointly reshape microbial communities, assembly processes and network stability, remain largely underexplored. Here, we investigated the Yongding River Basin, a representative water-scarce river system in northern China that receives inflows including natural surface runoff, inter-basin transfers from Yellow River and South-to-North Water Diversion Project (SNWD), and reclaimed municipal wastewater. Through integrated analysis of bacterioplankton and bacteriobenthos communities, we found a progressive decline in alpha diversity and significant shifts in beta diversity from upstream to downstream sections with increasing engineered inputs. Stochastic processes dominated the assembly of both bacterioplankton and bacteriobenthos, although their relative importance varied with replenishment source. Compared with natural runoff, the proportions of dispersal limitation and homogeneous selection for bacterioplankton decreased under Yellow River and mixed replenishment, whereas bacteriobenthos assembly exhibited little overall change. Network analysis further revealed that EWR reduced the resilience of bacterioplankton network but enhanced that of bacteriobenthos. Across the entire aquatic ecosystem, microbial network complexity was strongly and positively associated with ecological stability. More, community assembly process affected network stability not only directly but also indirectly through their regulation of microbial diversity. These findings advance the mechanistic understanding of how large-scale, multi-source EWR reshapes microbial ecology and network structure, providing valuable insights for optimizing water input allocation, enhancing the functional recovery of aquatic microbial ecosystems, and supporting the sustainable management of water resources.