Geographic and species-specific determinants of rhizosphere microbiome assembly in Theobroma cacao wild populations

Root-associated microbiome is a fundamental component of plant evolutionary success. Domestication has led to a loss of microbial diversity associated with plant roots. Studying this plant-microbiome relationship in natural ecosystems enhances our understanding of its original dynamics. We characterized bacterial and fungal communities in the rhizosphere and bulk soil of wild Theobroma and Herrania populations across two contrasting Colombian regions: the Amazon (Caquetá and Putumayo) and the Pacific (Chocó). Using 16S rRNA and ITS2 amplicon sequencing, we found that host species structured bacterial communities, while geography shaped fungal assemblages. Theobroma cacao rhizospheres clustered together across >500 km of separation, demonstrating stronger host filtering than wild congeners from identical locations. These communities were distinctively enriched with nitrogen-cycling taxa (Nitrosomonadaceae, Nitrosotalea, and Reyranella) identified as keystone species. Functional predictions showed pronounced enrichment of denitrification and nitrogen fixation genes in T. cacao rhizospheres. We propose T. cacao roots create spatial compartmentalization of nitrogen processes through oxygen consumption and radial oxygen loss, generating anoxic microsites within millimeters of root surfaces where nitrogen-fixers coexist with nitrifiers and denitrifiers. These patterns distinguish T. cacao from wild relatives and suggest evolutionary refinement of microbial recruitment strategies.