PROJECT SUMMARY Populations must be able to adapt in environmental conditions that may change either suddenly (within one generation) or gradually (over multiple generations). Theoretical models predict that these differences in the rate of environmental change will fundamentally influence the number and effect sizes of mutations that fix, but few studies have mechanistically examined the genetics of adaptation in environments that become more stressful over time. Moreover, the theoretical models do not always account for well-known phenomena that introduce evolutionary constraints, such as genotype by environment (GxE) interactions and pleiotropy. The goal of this research is to compare patterns of genome evolution and effects of mutations in RNA viruses under sudden or gradual environmental change, and to use these data to evaluate theoretical models of adaptation in environments that change at different rates. The project uses temperature-resistant populations of the model bacteriophage ɸ6 Cystovirus that were previously generated through an evolution experiment in which viral populations were exposed to a heat shock temperature that was increased either gradually (Gradual populations) or suddenly (Sudden populations). Here, we propose to use these populations to examine patterns of mutation fixation and to characterize the role of GxE interactions and pleiotropy in environments that change at different rates. Specifically, we will 1) evaluate the number of mutations, their times to fixation, and haplotype diversity of Sudden and Gradual populations; and 2) measure the effects of sequential mutations from select lineages on both viral thermostability and growth rate, and correlate those effects with the rate of environmental change experienced by the lineage. Our study will address the central question in evolutionary biology of how changes to the strength and tempo of selection influence adaptation, and will illuminate the mechanistic underpinnings of adaptation in different rates of environmental change. Establishing the selective pressures and constraints at play in changing environments will give us tools to predict or control viral evolution.