SUMMARY Understanding how nature builds new traits is a fundamental goal of evolutionary genetics. Unbiased experimental dissection of trait variation from the wild has to date used linkage or association mapping, which are suitable only for crosses between compatible individuals of a given species. In the first funding period of this methods-development R01, PI Brem developed RH-seq, an approach for the unbiased mapping of natural trait variation that can be applied to reproductively isolated species. Our RH-seq projects in invertebrate test cases have put the complex genetics of ancient traits within reach for the first time in the experimental literature. We now want to advance strategies that investigate deeper themes in complex genetics between species—namely whether evolution uses concerted molecular mechanisms across the loci underlying a polygenic adaptation, and how these loci work together to drive phenotype. To test-drive these approaches, in our first Aim we will use an ecologically relevant model system, a thermotolerance divergence between yeast species that last shared an ancestor five million years ago. In our second Aim, we will port our ideas and tools for interspecies genetics to mouse primary cells. The latter will use as a testbed a cell-autonomous, pro- inflammatory aging program called cellular senescence, which we have found to diverge between between sister species of mice. We will develop RH-seq for unbiased genetic mapping of senescence traits, and we will pursue epistatic and molecular mechanisms of the underlying loci as a parallel to our yeast model. Together, our yeast and mouse projects will advance methods for the analysis of polygenic traits as they differ between species, and accelerate the dissection of such ancient characters in systems across Eukarya.