Previous genomic work comparing the more beneficial rhizobium symbionts from ambient nitrogen environments to the less beneficial rhizobia from high nitrogen environments showed that genetic variation at the symbiosis gene region of the pSym symbiosis plasmid differentiated high and low-quality populations. The specific loci in this region are canonical symbiosis genes, including regulators and enzymes required for nitrogen fixation, but the effects of specific mutations on symbiosis phenotypes have not been determined, despite the ability to manipulate Rhizobium in the laboratory. In addition, the phylogenetic evidence to date suggests HGT of this region of the plasmid, across diverse chromosomal backgrounds, making this an even more interesting candidate genetic region for functional validation to link selection and HGT. GEMS trainee David Vereau Gorbitz, along with Chase Schwarz, Kristen Farley, Cari Vanderpool, and Katy Heath integrate molecular genetics with quantitative genetics and evolutionary ecology to address this current gap in knowledge. Combining additional analyses of sequenced genomes with exploration of transcriptional profiles and targeted genetic manipulations will allow us to address evolutionary responses of mutualisms at a mechanistic level, integrating molecular processes with ecological processes in this system for the first time.