Abstract
From yeasts to humans, introgressive hybridization significantly influences the evolutionary history of living organisms by introducing new genetic diversity. Strains of Saccharomyces cerevisiae worldwide exhibit introgressions from the sister species S. paradoxus, despite the average sequence identity between these species being lower than 90%. While S. cerevisiae isolates from the Neotropics are known for their high levels of introgression, the hybridization events originating them remain unclear. Here, we sequence 216 S. cerevisiae isolates from open, spontaneous agave fermentation across Mexico. The genomes of these strains reveal considerable genetic diversity and population structure linked to geographic distribution, which had been overlooked due to undersampling of this megadiverse region. These strains, along with those from French Guiana, Ecuador, and Brazil, form a broader Neotropical phylogenetic cluster that is notably enriched in introgressions. Surprisingly, their origins and the observed conservation patterns of these introgressions indicate multiple hybridization events, suggesting flexible species barriers in this region. Our findings underscore concurrent evolutionary processes-geographical stratification and multiple introgressions-that shape the genomes of a diverse lineage of S. cerevisiae. Neotropical yeasts thus provide a natural laboratory for exploring the mechanisms and adaptive significance of introgressive hybridization in eukaryotic genome evolution.