Assembly and co-occurrence networks of nitrogen-fixing bacteria associated with epiphyllous liverworts in fragmented tropical forests

Understanding the spatial dynamics of plant-associated microbial communities is increasingly urgent in the context of habitat loss and the biodiversity crisis. However, the influence of reduced habitat size and connectivity on the assembly mechanisms underlying microbial associations is fundamental to advancing microbial ecology and conservation. In the Brazilian Amazon, we investigated nitrogen-fixing (diazotrophic) bacterial communities associated with two epiphyllous liverworts, <i>Cololejeunea surinamensis</i> and <i>Radula flaccida</i>, across 11 forest sites within the Biological Dynamics of Forest Fragments Project landscape. Using amplicon sequencing targeting the nitrogenase gene (<i>nif</i>H), we characterized diazotroph community diversity, inferred assembly mechanisms through null models, and analyzed co-occurrence network structure. Host-specific associations were evident: <i>C. surinamensis</i> predominantly hosted <i>Hassallia</i>, while <i>R. flaccida</i> was primarily associated with <i>Fischerella</i>. Despite habitat fragmentation, diazotrophic richness and composition remained similar across habitats of different sizes, consistent with strong homogenizing dispersal. Network analyses revealed that smaller fragments harbored more modular communities with fewer module hubs, pronounced shifts in key species relative abundance, and reduced network robustness. Our findings underscore the influence of habitat size on the stability of liverwort-associated diazotrophs, with smaller fragments exhibiting lower key species specificity and disruption of microbe-microbe interactions. Our results emphasize the importance of conserving large, connected forest habitats to maintain the functional integrity of phyllosphere N-fixing microbiota.