AbstractThe subseafloor is a vast global habitat that supports microorganisms that have a global scale impact on geochemical cycles. Much of the subseafloor contains endemic microbial populations that consist of small populations under growth-limited conditions. For small population sizes, the impacts of stochastic evolutionary events can have large impacts on intraspecific population dynamics and allele frequencies. These conditions are fundamentally different than those experienced by most microorganisms in surface environments, and it is unknown how small population sizes and growth-limiting conditions influence evolution and population structure. Using a two-year, high-resolution environmental time-series, we examine the dynamics of 10 microbial populations from cold, oxic crustal fluids collected from the subseafloor site North Pond, located near the mid-Atlantic ridge. The 10 microbial populations were divided into groups with distinct patterns of population dynamics based on abundance, nucleotide diversity, and changes in allele frequency. Results reveal rapid allele frequency shifts linked to different types of population interactions, including sweeps, dispersal, and clonal expansion. Dispersal plays an important role in structuring the most abundant populations in the crustal fluids. Microbial populations in the subseafloor of North Pond are highly dynamic and evolution is governed largely by the stochastic forces of dispersal and drift.ImportanceThe cold, oxic subseafloor is an understudied habitat that is difficult to access, yet important to global biogeochemical cycles and starkly different compared microbial habitats on the surface of the Earth. Our understanding of microbial evolution and population dynamics has been largely molded by studies of microbes living in surface habitats that can host 10-1,000 times more microbial biomass than has been observed in the subsurface. This study provides an opportunity to observe evolution in action within a low biomass, growth-limited environment and reveals that while microbial populations in the subseafloor can be influenced by changes in selection pressure and small-scale gene sweeps, the stochastic forces of genetic drift and dispersal have an important impact on the evolution of microbial populations. Much of the microbial life on the planet exists under growth-limited conditions and the subseafloor provides a natural laboratory to explore these fundamental biological questions.