Diploids in the Cryptococcus neoformans Serotype A Population Homozygous for the α Mating Type Originate via Unisexual Mating

Abstract

The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the α mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual α population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (α-α) also occurs in nature as evidenced by the existence of natural diploid αADα hybrids that arose by fusion between two α cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, ∼500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the α MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct α cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens.

Analyses of Pediatric Isolates of Cryptococcus neoformans from South Africa

“…To the best of our knowledge, this is the first report of a clinical isolate with the AaAa genotype. Homozygous diploids of serotype A have been reported in other studies, and they most likely arose from samesex mating (35,36).…”

“…Ploidy was determined for each isolate by measuring cellular DNA content, using a modified protocol for flow cytometry (36,63). Control strains were the haploid H99 strain and our AD hybrid diploid strain RSA5272 (Table 1).…”

Multilocus Sequence Typing of Serially Collected Isolates of Cryptococcus from HIV-Infected Patients in South Africa

“…The ploidy of each isolate was determined by measurement of DNA content using a fluorescence-activated cell sorter (FACS) as described previously (22). Mating types were determine by PCR and confirmed by mating type assays on V8 medium as described previously (21).…”

“…The ploidy of C. neoformans has been reported to change during the course of infection (22,27). The six diploid isolates of molecular type VNI or VNII (all MAT␣/␣) were obtained from five patients: the incident and serial isolates of patient 9 had the same relatively common genotype of VNII, but the incident isolate (Table 2) (isolate number 17) was haploid, and the serial isolate (isolate number 18), which was collected 138 days later, was diploid, and its MIC to FLZ had increased from 4 to Ն64 g/ml.…”

The Evolution of Sex: a Perspective from the Fungal Kingdom

“…Notably, these isolates undergo limited hyphal growth and basidial production when grown in confrontation with a cells as a source of mating pheromone (which stimulates samesex mating), but no sporulation of these isolates has been observed as yet. Finally, a recent large-scale survey of ϳ500 C. neoformans serotype A isolates revealed that ϳ8% of the naturally occurring population of environmental and clinical isolates are diploid, and the majority of these are ␣AA␣ strains produced via same-sex mating, either by mating between genetically distinct individuals in the population or from selfmating (165). Taken together, these studies provide robust support that same-sex mating occurs both in the laboratory and in nature and occurs in all major lineages of Cryptococcus, including both serotype A and D strains and the sibling species C. gattii.…”