Virulence-associated variants in <i>Cryptococcus neoformans</i> sequence type 93 are less likely to be associated with population structure compared to independent rare mutations

<i>Cryptococcus neoformans</i> is a pathogenic yeast that is the causative agent of cryptococcal meningitis. While it is well known that the genotype of <i>C. neoformans</i> impacts patient outcomes, the reason for this association has not been well elucidated. In this study, we examined the relationship between two subpopulations in the sequence type 93 clade of <i>C. neoformans</i>: ST93A and ST93B. We found extensive linkage disequilibrium (LD) among the single nucleotide polymorphisms (SNPs) that differentiate ST93A from ST93B. We also found differences in the extent of linkage among SNPs within each subpopulation; LD was more extensive within ST93B than ST93A. SNPs associated with virulence were in long-range linkage disequilibrium with less frequency than recurrent SNPs not associated with virulence. We investigated the karyotype of ST93A and ST93B using contour-clamped gel electrophoresis and long-read sequencing and found that the extensive long-range linkage was not due to chromosomal rearrangements. Overall, we found that the two subpopulations in ST93 are driven by SNPs in LD. We additionally found that recurrent SNPs associated with virulence were less frequently evolutionarily linked and were two times more likely to be independent, congruent mutations rather than tied to phylogeny.IMPORTANCE<i>Cryptococcus neoformans</i> is an important pathogen that is widely distributed and ubiquitous in the environment. The majority of the human population has a latent, controlled infection suggesting that <i>C. neoformans</i> is uniquely adapted to cause infection. In spite of this, the reason <i>C. neoformans</i> is a pathogen remains unknown; interestingly, most environmental isolates are avirulent but are genetically very similar to disease-causing virulent isolates. Recent evidence from genome-wide association studies shows that small mutations in key virulence-associated genes are associated with the virulence of specific isolates. The data presented here provide an evolutionary framework for those small mutations. The mutations that impact disease are not being collected over long-term evolution. The mutations may instead occur independently during infection. Identifying these genes that are more likely to be mutated during infection will be fundamental for understanding <i>C. neoformans</i> virulence.