PROJECT SUMMARY The goal of the project is to understand the genetic basis and evolution of thermotolerance in the Saccharomyces species. Whereas much has been learned about the causes of phenotypic variation within species, reproductive barriers have limited genetic analysis to closely related, inter-fertile species. Consequently, we know little about the genetic basis of substantial phenotypic differences that arise over longer time periods and the importance of evolutionary hotspots and historical contingency. To overcome this reproductive barrier we will use hybrid, proteomic and transgenic analysis of yeast species. In Aim 1 we will map thermotolerance in interspecific hybrids using reciprocal chromosome loss and CRISPR induced mitotic recombination. This will enable us to measure the combined effects of many genes, but also identify thermotolerance genes and determine: whether they evolved through the cumulative effects of multiple changes, and whether their effects depend on changes in other genes. In Aim 2 we will investigate divergence in protein thermal stability and whether it results in temperature dependent fitness effects not detected by mapping. In Aim 3 we will examine the reuse of thermotolerance genes during thermal divergence along independent lineages. Doing so will determine whether thermal divergence requires certain genes or can be achieved in different ways. Together, these aims will push the limits of interspecific genetic analysis in order to provide insight into the acquisition of a complex and broadly relevant phenotype that has taken millions of years to evolve.