Microbial mutualists provide substantial benefits to hosts that feed back to enhance the fitness of the associated microbes. In many systems, beneficial microbes colonize symbiotic organs, specialized host structures that house symbionts and mediate resources exchanged between parties. Mutualisms are characterized by net benefits exchanged among members of different species, however, inequalities in the magnitude of these exchanges could result in evolutionary conflict, destabilizing the mutualism. We investigated joint fitness effects of root nodule formation, the symbiotic organ of legumes that house nitrogen-fixing rhizobia in planta. We quantified host and symbiont fitness parameters dependent on the number of nodules formed using near-isogenic Lotus japonicus and Mesorhizobium loti mutants, respectively. Empirically estimated fitness functions suggest that legume and rhizobia fitness is aligned as the number of nodules formed increases from zero until the host optimum is reached, a point where aligned fitness interests shift to diverging fitness interests between host and symbiont. However, fitness conflict was only inferred when analyzing wild-type hosts along with their mutants dysregulated for control over nodule formation. These data demonstrate that to avoid conflict, hosts must tightly regulate investment into symbiotic organs maximizing their benefit to cost ratio of associating with microbes.

Legumes preferentially associate with and reward beneficial rhizobia in root nodules, but the processes by which rhizobia evolve to provide benefits to novel hosts remain poorly understood. Using cycles of in planta and in vitro evolution, we experimentally simulated lifestyles where rhizobia repeatedly interact with novel plant genotypes with which they initially provide negligible benefits. Using a full-factorial replicated design, we independently evolved two rhizobia strains in associations with each of two Lotus japonicus genotypes that vary in regulation of nodule formation. We evaluated phenotypic evolution of rhizobia by quantifying fitness, growth effects and histological features on hosts, and molecular evolution via genome resequencing. Rhizobia evolved enhanced host benefits and caused changes in nodule development in one of the four host-symbiont combinations, that appeared to be driven by reduced costs during symbiosis, rather than increased nitrogen fixation. Descendant populations included genetic changes that could alter rhizobial infection or proliferation in host tissues, but lack of evidence for fixation of these mutations weakens the results. Evolution of enhanced rhizobial benefits occurred only in a subset of experiments, suggesting a role for host-symbiont genotype interactions in mediating the evolution of enhanced benefits from symbionts.