Testing for divergence in a plant symbiont across two natural environmental gradients

Microbes dominate the biological diversity found in natural landscapes. Yet, studies of microbial population differentiation across environmental gradients have been surprisingly scarce. In this study, we tested for phenotypic and genetic divergence in rhizobia bacteria (genus Sinorhizobium) associated with the legume Medicago lupulina across two environmental gradients: an elevation gradient in the Appalachian Mountains and an urbanization gradient in Philadelphia. Common garden experiments in Medicago sativa revealed that symbiotic traits differed significantly between high- and low-elevation rhizobia populations, but not between urban and suburban populations. Nitrogen supplementation erased the trait differences between low- and high-elevation populations, consistent with the hypothesis that these differences arise from the nitrogen that rhizobia provide. In contrast to symbiotic traits, free-living growth traits showed minimal differentiation across gradients. To test the hypothesis that greater gene flow constrains phenotypic divergence in the urbanization gradient, we compared the strength of isolation by distance in the core and accessory genomes across the two gradients. Contrary to our expectation, isolation by distance was significantly stronger in the urbanization gradient in both the core and accessory genomes. These results suggest that our focal elevation gradient imposed stronger divergent selection on rhizobia than our focal urbanization gradient. Together, this study highlights the need for a holistic understanding of microbial evolution with regard to the traits across life stages, molecular evolution across genomic levels, and evolutionary processes across environmental gradients.