Genomic plasticity of the mating-type loci underlies reproductive strategy transitions in Rhodotorula yeasts

Transitions from canonical outcrossing to rare, cryptic, or noncanonical reproduction are widespread in fungi, but how mating-type (MAT) systems are reorganized at the genomic and population levels during these shifts remains unclear. The coexistence of sexually reproducing species and putatively asexual lineages makes Rhodotorula a natural comparative system for addressing this question. We analyzed 249 globally sampled strains using a combination of long-read and short-read sequencing. Phylogenomics resolved three major clades and several closely related species complexes. Chromosome-scale assemblies confirmed physical separation of the pheromone/receptor (P/R) and homeodomain (HD) loci, whereas non-random P/R-HD associations revealed constrained tetrapolar inheritance. The P/R locus showed conserved synteny within mating types but extensive structural divergence between A1 and A2 alleles, whereas the HD locus remained compact and conserved. Mating-type distributions varied across clades, with both A1 and A2 present in Clade C, lineage-structured partitioning in Clade B, and pronounced A2 dominance in Clade A. Genome-wide linkage disequilibrium (LD) decay in putatively asexual R. mucilaginosa, although slower than in sexual R. diobovata, was consistent with historical recombination in a species without an observed sexual cycle. Clade A further exhibited deep trans-specific polymorphism of STE3.A2 and relaxed purifying selection, mosaic A2 P/R configurations retaining RHA.A1, A1/A2 and A2/A2 hybrid genomes, and A2-associated gene content beyond MAT loci. In conclusion, our findings underscore the dynamic nature of MAT-locus remodeling in Rhodotorula and establish this genus as a valuable model for investigating transitions from heterothallism toward reproductive strategies less dependent on opposite-type partners.