25Saccharomyces eubayanus represents missing cryotolerant ancestor of lager yeast hybrid 26 and can be found in Patagonia in association with Nothofagus forests. The limited number 27 of isolates and associated genomes available has prevented to resolve the S. eubayanus 28 origin and evolution. Here, we present a sampling effort at an unprecedented scale and 29 report the isolation of 160 strains from ten sampling sites along 2,000 km distance in South 30America. We sequenced the genome of 82 strains and, together with other 25 available 31 genomes, performed comprehensive phylogenetic analysis. Our results revealed the 32 presence of three main Patagonia-B lineages together with dozens of admixed strains 33 distributed in three mosaic clusters. The PB-1 lineage isolated from Tierra del Fuego 34 exhibited the highest genetic diversity, lowest LD blocks and highest Fis values compared 35 to the other lineages, suggesting a successful adaptation to cold temperatures in extreme 36 environments and greater inbreeding rates in Tierra del Fuego. Differences between 37 lineages and strains were found in terms of aneuploidy and pangenome content, evidencing 38 a lateral gene transfer event in PB-2 strains from an unknown donor species. Overall, the 39 Patagonian lineages, particularly southern populations, showed a greater global genetic 40 diversity compared to Holarctic and Chinese lineages, supporting the scenario of a S. 41 eubayanus colonization from Patagonia and then spread towards northern and western 42 regions, including the Holarctic (North America and China) and New Zealand. Interestingly, 43 fermentative capacity and maltose consumption resulted negatively correlated with latitude, 44 indicating a better fermentative performance in norther populations. Our genome analysis 45 together with previous reports in the sister species S. uvarum strongly suggests that the S.
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