The resource mutualism between legumes and rhizobia is an emerging model for how nested symbioses evolve. Nitrogen (N) fixation is a plasmid-encoded function in Rhizobium leguminosarum; thus, a greater understanding of the dynamics of plasmids in natural populations is critical for understanding how plasmid costs and benefits might shift under different selective environments and drive mutualism evolution. Previous work has shown that long-term N fertilization has resulted in fewer hosts, fewer symbiotic rhizobia, and lower-quality rhizobium partners for plant hosts. GEMS graduate student Sierra Bedwell, working with Katy Heath and Rachel Whitaker, hypothesizes that the abundance of plasmids (including pSym which carries the canonical symbiosis genes) might also change across environments and serve as a driver of mutualism decline. To test this, the team must consider strains in the soil in addition to nodule isolates, which are sampled in symbiosis with plants. The project aims to track plasmid dynamics in natural populations and widen the sample of Rhizobium strains from the soil towards better understanding the mechanisms of mutualism decline in nature.