A haploid wild yeast resource for exploring the natural ecology of Saccharomyces cerevisiae.

Saccharomyces cerevisiae occurs predominantly in the diploid state in nature, limiting genetic analyses of wild populations. Here, we establish a haploid collection from 32 Taiwanese S. cerevisiae isolates through targeted HO disruption. This resource spans predomesticated Asian wild lineages and enables the investigation of reproductive isolation and ecological trait variation. Although all pairwise hybridizations formed zygotes, many yielded reduced spore viability, revealing strong postzygotic barriers. Genome analyses associated reduced hybrid fertility with lineage-specific structural variation, including elevated levels of intra-chromosomal inversions in H413-8/TW1 and inter-chromosomal rearrangements in PD35A/CHN-V, rather than sequence divergence alone. Phenotyping revealed ecological differentiation, with TW1 favoring cooler growth and a natural hybrid exhibiting heterosis with expanded thermotolerance. Most wild strains grew poorly on maltose, whereas anthropogenic strains displayed enhanced utilization linked to MAL + regulatory alleles and maltose-specific transporters. Together, this haploid collection links structural variation and metabolic divergence to ecological and reproductive differentiation in wild S. cerevisiae.