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Highlights The microbial network associations were strengthened along the AMD-impacted river. Community assembly processes shifted from the headwaters to downstream. Relative modularity was essential biotic factor in shaping community composition. Strong succession in prokaryotic association networks and community assembly mechanisms in an acid mine drainage-impacted riverine ecosystem Mengmeng Wanga,#, Xiaonan Wanga,#, Sining Zhoua, Zifeng Chena, Mengyun Chena, Shiwei Fenga, Jintian Lia, Wensheng Shua, Baichuan Caoa,* aInstitute of Ecological Science and Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China. #These authors contributed equally: Mengmeng Wang, Xiaonan Wang *To whom correspondence may be addressed. E-mail: baichuanc@; Phone: +86-020 Fax: +86-020Running title: Microbial community succession in an AMD-impacted river Keywords: microbial community succession, acidic mine drainage, community assembly, microbial network Type of article: Research article Abstract Acid mine drainage (AMD) serves as an ideal model system for investigating microbial ecology, interaction, and assembly mechanism in natural environments. While previous studies have explored the structure and function of microbial communities in AMD, the succession patterns of microbial association networks and underlying assembly mechanisms during natural attenuation processes remain elusive. Here, we investigated prokaryotic microbial diversity and community assembly along an AMD-impacted river, from the extremely acidic, heavily polluted headwaters to the nearly neutral downstream sites. Microbial diversity was increased along the river, and microbial community composition shifted from acidophile-dominated to freshwater taxa-dominated communities. The complexity and relative modularity of the microbial networks were also increased, indicating greater network stability during