Over the past few decades, experimental procedures have been developed and refined for the study of microbial evolution under controlled laboratory conditions. Whether such information from test-tube studies pertains to the evolution of microorganisms and their hosts in nature is uncertain owing to the challenges associated with tracking genotypes and populations in the wild. One promising and recent development involves the insertion of barcodes into the genomes of microorganisms, which can then be retrieved through quantitative sequencing technologies. GEMS postdoc John McMullen has developed a population genomics pipeline for molecular barcoding of closely related strains with the goal of quantifying fitness costs associated with the varying lifestyles of microbes. McMullen, together with Jay Lennon, Cari Vanderpool, and Hunter Cobbley is using this strategy to study the evolutionary ecology of Rhizobium leguminosarum, both during its interactions with clover hosts and in the soil environment. In particular, they are focusing on bottom-up and top-down pressures of nitrogen addition and phage parasitism, respectively. The project goal is to quantitatively track genotypes (i.e., phage defense systems) in nested symbionts to understand trade-offs and evolutionary constraints on mutualisms involving plants, microbes, and mobile genetic elements. The proposed work addresses long-standing limitations that preclude a full understanding of the legume-rhizobium symbiosis, including challenges associated with quantifying fitness and evolutionary trajectories of bacteria in soil and plant life-stages in the face of disruptive effects. Molecular-based mark-recapture approaches will synergize with other GEMS-related research that seek to quantitatively understand the evolution of microbial populations (e.g., Pseudomonas, Streptomycetes, Bombella) embedded in complex communities.