Ecological divergence of sympatricSaccharomycesspecies across wild and fermentative environments in the neotropics

The common yeastSaccharomyces cerevisiaeis widely associated with anthropogenic habitats and dominates agave fermentations in Mexico, despite the absence of starter cultures in artisanal production. Yet, the origins and dispersal of the microorganisms involved in this fermentation system—including wildS. cerevisiaeand its sister speciesS. paradoxus—remain poorly understood. Here, we analyzed the distribution of these two species across three regions in Mexico by collecting a total of 861 samples from 15 traditional distilleries and 25 nearby natural sites, including tree bark, insects, fermentation tanks, and distillery surfaces. Among 4,006 isolates identified by MALDI-TOF biotyping,S. cerevisiaeandS. paradoxuswere recovered at overall rates of 21% and 3.4%, respectively, with the former more frequently found in distilleries and the latter in natural habitats. Notably,Saccharomycesisolates were not exclusively associated with oak trees, as strains were also recovered from leguminous, pine, and Anacardiaceae trees, suggesting that the oak specialization observed in temperate regions may not hold in tropical environments. Genome sequencing of 100Saccharomycesisolates and SNP-based analyses showed thatS. cerevisiaestrains clustered by geography, not by substrate, and were largely confined to two Mexican Agave clades, with minor representation from the North American Oak, Pacific West Coast, and Wine clades. Strains isolated from agave fermentations belonged to the same populations as those from plant substrates, with insects, especially Drosophilidae, likely mediating their dispersal. Meanwhile,S. paradoxusisolates grouped into the SpA (Eurasian) and SpB (North American) lineages, with three novel SpB subclades identified. Intriguingly, one of these, the SpB_MxAgave population, was exclusively associated with agave fermentations, providing the first evidence of a persistent association ofS. paradoxuswith an anthropogenic habitat. Our study sheds light on the ecological dynamics and diversity ofS. cerevisiaeandS. paradoxuspopulations in the neotropics.