The evolution and spread of microbial communities has profoundly impacted our planet’s biogeochemical cycles and biosphere, yet we lack a fundamental understanding of the processes that drive microbial evolution. This is particularly the case for the deep biosphere, where it is likely that microbial populations from different habitats in the subsurface are subject to vastly different evolutionary forces. For example, in low-energy environments with limited fluid flux, genetic drift may be a significant evolutionary driver. In contrast, hydrothermal systems have extensive fluid flux and are dominated by physiochemical gradients, and therefore selection pressure from environmental conditions may drive diversification. We do not yet know the relative extent to which natural selection or genetic drift drive evolution in the subsurface, nor have we identified the strongest selection pressures in different subsurface environments. By comparing multiple genomes of the same lineage, it will be possible to assess the size of the pangenome, estimate the nature and rates of gene gain or loss, and evaluate the relative influence of genetic drift and selection on the evolutionary trajectory of a given microbial population. To address these evolutionary questions, we propose to sequence five single cell amplified genomes (SAGs) from the Methanothermococcales that have been sorted from a single sample at the Von Damm vent field on the Mid Cayman Rise. PIs Rika Anderson and Julie A. Huber.