Genome-scale characterization of wild yeasts reveals cryptic diversity and population structure across three genera

Environmental surveys of wild yeasts typically rely on ribosomal barcodes, which cannot resolve cryptic species, interspecific gene flow, mixed cultures, or population structure. To determine what genome-scale characterization adds, we sequenced a representative panel of wild yeasts spanning the genera Saccharomyces , Schizosaccharomyces , and Lachancea using Oxford Nanopore long-read whole-genome sequencing and placed each isolate within published reference datasets. Whole-genome analyses revealed biologically important features that barcoding alone could not detect. A shagbark-hickory isolate resolved as a genuine two-species co-culture. An oak-bark isolate proved to be Schizosaccharomyces versatilis , a recently reinstated species represented by very few known strains, and its analysis demonstrated that standard assembly-quality benchmarks can be misleading for deep-branching taxa. Three Lachancea thermotolerans isolates formed a distinct, previously unsampled population within the wild tree-associated lineage, extending its known geographic range. In contrast, an apparent signal of Saccharomyces eubayanus introgression in two beer-associated S. cerevisiae isolates disappeared after analysis with matched negative controls and de novo assemblies, showing that it reflected mapping artifacts rather than genuine ancestry. Together, these results demonstrate that inexpensive long-read whole-genome sequencing transforms wild-yeast bioprospecting from species identification into a genome-scale framework for resolving cryptic diversity, population structure, and mixed cultures while providing stronger support – and stronger limits – for evolutionary inference.