Genomic rearrangements at the MAT locus suggest reproductive shifts in Rhodotorula yeasts

Rhodotorula, a red yeast genus inhabiting diverse environmental and clinical niches, is a promising microbial source for carotenoid and lipid production. Despite the coexistence of sexual and asexual species, the evolutionary mechanisms underlying their reproductive diversity remain poorly understood. Here, we present a global genomic survey of 249 Rhodotorula strains using Oxford Nanopore, PacBio, and Illumina sequencing. Phylogenomic analysis resolved a robust species tree, delineating three major clades with substantial cryptic species diversity. Chromosome-level assemblies revealed a constrained tetrapolar mating system characterized by unlinked pheromone/receptor (P/R) and homeodomain (HD) mating-type (MAT) loci but displaying infrequent recombination. This genomic architecture is widely conserved, even in species lacking observed sexual cycles, suggesting widespread sexual potential. The P/R locus functions as a supergene, exhibiting conserved gene content and order within mating types but variable configurations between compatible types. Clade-specific biases in mating-type allele distributions indicates asymmetric evolutionary pressures. Structural rearrangements and trans-specific polymorphism of mating-specific allele at the P/R locus, may drive sequence divergence, potentially generating novel mating compatibility within the same mating type. The intermediate P/R genomic states retaining pheromone genes from the opposite mating type may preserve residual functions or facilitate transitions in mating-type architecture. Together with the presence of homozygous hybrid strains, these genomic signatures suggest a potential shift toward same-sex-like mating. Our findings elucidate how mating-type architecture and allele dynamics underpin the evolution of reproductive strategies in fungi and suggest that innovations in MAT system may be a key evolutionary lever in fungal adaptation across environments. Significance StatementRhodotorula yeasts are a widespread genus and occupy a unique intersection of biology serving both as emerging fungal pathogens and industrial workhorses producing carotenoids and lipids. Yet, despite their growing importance, the genetic mechanisms shaping their reproductive strategies remain elusive. By leveraging a global dataset of 249 genomes, we uncover the hidden architecture of sexual identity in Rhodotorula: a conserved but flexible MAT locus structure, varying degrees of recombination suppression, and lineage-specific mating-type imbalances. Our discovery of same-sex-like mating signatures and widespread interspecies hybrids reveals a dynamic evolutionary landscape, where sexual plasticity fuels adaptation, speciation, and possibly pathogenesis. These insights redefine our understanding of sexual evolution in basidiomycetous yeasts and open new avenues for biotechnology and medical mycology.