Over time, species evolve substantial phenotype differences. Yet, genetic analysis of these traits is limited by reproductive barriers to those phenotypes that distinguish closely related species. Here, we conduct a genome-wide non-complementation screen to identify genes that contribute to a major difference in thermal growth profile between two Saccharomyces species. S. cerevisiae is capable of growing at temperatures exceeding 40C, whereas S. uvarum cannot grow above 33C but outperforms S. cerevisiae at 4C. The screen revealed only a single nuclear-encoded gene with a modest contribution to heat tolerance, but a large effect of the species' mitochondrial DNA (mitotype). Furthermore, we found that, while the S. cerevisiae mitotype confers heat tolerance, the S. uvarum mitotype confers cold tolerance. Recombinant mitotypes indicate multiple genes contribute to thermal divergence. Mitochondrial allele replacements showed that divergence in the coding sequence of COX1 has a moderate effect on both heat and cold tolerance, but it does not explain the entire difference between the two mitochondrial genomes. Our results highlight a polygenic architecture for interspecific phenotypic divergence and point to the mitochondrial genome as an evolutionary hotspot for not only reproductive incompatibilities, but also thermal divergence in yeast.

24S. eubayanus, the wild, cold-tolerant parent of hybrid lager-brewing yeasts, has a 25 complex and understudied natural history. The exploration of this diversity can be used both to 26 develop new brewing applications and to enlighten our understanding of the dynamics of yeast 27 evolution in the wild. Here, we integrate whole genome sequence and phenotypic data of 200 S. 28 eubayanus strains, the largest collection to date. S. eubayanus has a multilayered population 29 structure, consisting of two major populations that are further structured into six subpopulations. 30Four of these subpopulations are found exclusively in the Patagonian region of South America; 31 one is found predominantly in Patagonia and sparsely in Oceania and North America; and one is 32 specific to the Holarctic ecozone. S. eubayanus is most abundant and genetically diverse in 33 Patagonia, where some locations harbor more genetic diversity than is found outside of South 34America. All but one subpopulation shows isolation-by-distance, and gene flow between 35 subpopulations is low. However, there are strong signals of ancient and recent outcrossing, 36 including two admixed lineages, one that is sympatric with and one that is mostly isolated from 37 its parental populations. Despite S. eubayanus' extensive genetic diversity, it has relatively little 38 phenotypic diversity, and all subpopulations performed similarly under most conditions tested. 39Using our extensive biogeographical data, we constructed a robust model that predicted all 40 known and a handful of additional regions of the globe that are climatically suitable for S. 41 eubayanus, including Europe. We conclude that this industrially relevant species has rich wild 42 diversity with many factors contributing to its complex distribution and biology. 43 44 45 46