Adaptation of to Long-Term Serial Passage in Complex Medium: Evidence of Parallel Evolution.

Experimental evolution of bacterial populations in the laboratory has led to identification of several themes, including parallel evolution of populations adapting to carbon starvation, heat stress, and pH stress. However, most of these experiments study growth in defined and/or constant environments. We hypothesized that while there would likely continue to be parallelism in more complex and changing environments, there would also be more variation in what types of mutations would benefit the cells. In order to test our hypothesis, we serially passaged in a complex medium (Luria-Bertani broth) throughout the five phases of bacterial growth. This passaging scheme allowed cells to experience a wide variety of stresses, including nutrient limitation, oxidative stress, and pH variation, and therefore allowed them to adapt to several conditions. After every ~30 generations of growth, for a total of ~300 generations, we compared both the growth phenotypes and genotypes of aged populations to the parent population. After as few as 30 generations, populations exhibit changes in growth phenotype and accumulate potentially adaptive mutations. There were many genes with mutant alleles in different populations, indicating potential parallel evolution. We examined 8 of these alleles by constructing the point mutations in the parental genetic background and competed those cells with the parent population; five of these alleles were found to be adaptive. The variety and swiftness of adaptive mutations arising in the populations indicate that the cells are adapting to a complex set of stresses, while the parallel nature of several of the mutations indicates that this behavior may be generalized to bacterial evolution. With a growing body of work directed toward understanding the mechanisms of evolution using experimental systems, it is crucial to decipher what effects the experimental setup has on the outcome. If the goal of experimental laboratory evolution is to elucidate underlying evolutionary mechanisms and trends, these must be demonstrated in a variety of systems and environments. Here, we perform experimental evolution in a complex medium allowing the cells to transition through all five phases of growth, including death phase and long-term stationary phase. We show that the swiftness of selection and the specific targets of adaptive evolution are different in this system compared to others. We also observe parallel evolution where different mutations in the same genes are under positive natural selection. Together, these data show that while some outcomes of microbial evolution experiments may be generalizable, many outcomes will be environment or system specific.