Fungi are enigmatic organisms that flourish in soil, on decaying plants, or during infection of animals or plants. Growing in myriad forms, from single-celled yeast to multicellular molds and mushrooms, fungi have also evolved a variety of strategies to reproduce. Normally, fungi reproduce in one of two ways: either they reproduce asexually, with one individual producing a new individual identical to itself, or they reproduce sexually, with two individuals of different ‘mating types’ contributing to produce a new individual. However, individuals of some species exhibit ‘homothallism’ or self-fertility: these individuals can produce reproductive cells that are universally compatible, and therefore can reproduce sexually with themselves or with any other cell in the population.

Homothallism has evolved multiple times throughout the fungal kingdom, suggesting it confers advantage when population numbers are low or mates are hard to find. Yet some homothallic fungi been overlooked compared to heterothallic species, whose mating types have been well characterised. Understanding the genetic basis of homothallism and how it evolved in different species can provide insights into pathogenic species that cause fungal disease.

With that in mind, Passer, Clancey et al. explored the genetic basis of homothallism in Cryptococcus depauperatus, a close relative of C. neoformans, a species that causes fungal infections in humans. A combination of genetic sequencing techniques and experiments were applied to analyse, compare, and manipulate C. depauperatus’ genome to see how this species evolved self-fertility.

Passer, Clancey et al. showed that C. depauperatus evolved the ability to reproduce sexually by itself via a unique evolutionary pathway. The result is a form of homothallism never reported in fungi before. C. depauperatus lost some of the genes that control mating in other species of fungi, and acquired genes from the opposing mating types of a heterothallic ancestor to become self-fertile.

Passer, Clancey et al. also found that, unlike other Cryptococcus species that switch between asexual and sexual reproduction, C. depauperatus grows only as long, branching filaments called hyphae, a sexual form. The species reproduces sexually with itself throughout its life cycle and is unable to produce a yeast (asexual) form, in contrast to other closely related species.

This work offers new insights into how different modes of sexual reproduction have evolved in fungi. It also provides another interesting case of how genome plasticity and evolutionary pressures can produce similar outcomes, homothallism, via different evolutionary paths. Lastly, assembling the complete genome of C. depauperatus will foster comparative studies between pathogenic and non-pathogenic Cryptococcus species.

Sexual reproduction, generally defined as the production of viable and fertile offspring by combining genetic information from mating partners of two different types, is a process conserved across the eukaryotic tree of life (Goodenough and Heitman, 2014; Heitman, 2015). Sexual reproduction has many benefits, such as generating novel genetic combinations and removing deleterious mutations (Agrawal and Whitlock, 2012), but it also has many costs, in that it is energetically expensive, time-consuming, and in some species two parents are required to produce one progeny. Therefore, some organisms, such as fungi, balance sexual reproduction with asexual mitotic cycles in order to populate an environmental niche (a strategy known as facultative sexual reproduction) (Williams, 1975; Maynard-Smith, 1978).

In fungi, sexual reproduction usually involves the fusion of haploid partners of opposite mating type, a situation known as heterothallism. The mechanisms of mating compatibility under heterothallism are quite diverse across fungal taxa and frequently involve genomic structures ranging from a mating-type (MAT) locus region containing only one or two genes (e.g., the Mucoromycota and Ascomycota phyla) (Bennett and Turgeon, 2016; Lee and Idnurm, 2017), to highly complex regions containing several genes at one (bipolar) or two (tetrapolar) MAT loci (e.g., most Basidiomycota) (Coelho et al., 2017), and even to mating-type chromosomes that exhibit large non-recombining regions resembling sex chromosomes of plants and animals (e.g., Microbotryum spp. and Neurospora tetrasperma) (Menkis et al., 2008; Branco et al., 2017; Sun et al., 2017; Branco et al., 2018). In most of these systems, and similar to the presence of separate sexes in different individuals in animals and plants, the probability of encountering a suitable mating partner when only two mating types exist in a population at equilibrium cannot exceed 50%, which may pose a substantial fitness reduction in environments where population densities are very low and compatible mating partners are scarce (Hoekstra, 1987). As a possible evolutionary response to such selective pressures, many fungal species evolved the ability to reproduce sexually without the need for another individual, a state known as homothallism (Ni et al., 2011; Wilson et al., 2015), analogous to the evolution of hermaphroditism or parthenogenesis in plants and animals (Jarne and Charlesworth, 1993; Neaves and Baumann, 2011; Busch and Delph, 2012). In such fungi, haploid cells are universally compatible for mating, and a single isolate can undergo sexual reproduction alone (Ni et al., 2011; Wilson et al., 2015). This is usually achieved either by combining the genes of opposite mating types within a single genome or by undergoing mating-type switching (Ni et al., 2011; Gioti et al., 2012; Fu et al., 2015; Wilson et al., 2015; David-Palma et al., 2016; Krassowski et al., 2019; Cabrita et al., 2021).

Homothallism in fungi evolved multiple times independently (Billiard et al., 2012; Gioti et al., 2012; Wilson et al., 2015; Hanson and Wolfe, 2017; Krassowski et al., 2019; Sun et al., 2019b; Cabrita et al., 2021), indicating that it provides a selective advantage under certain conditions, for instance, (i) to allow reproductive assurance, which may be a substantial benefit in patchy habitats (Murtagh et al., 2000; Nieuwenhuis and Immler, 2016; Nieuwenhuis et al., 2018), (ii) to increase compatibility to promote outcrossing (Heitman, 2015), or (iii) even to reduce outbreeding depression by avoiding breaking up locally co-adapted gene complexes (Epinat and Lenormand, 2009), which has been hypothesized to be advantageous for pathogenic fungi (Alby et al., 2009; Heitman, 2010; Hauser, 2021).

Cryptococcus is a fungal genus within the Basidiomycota that comprises both pathogenic and closely related non-pathogenic saprobic species. The non-pathogenic species currently include Cryptococcus wingfieldii, Cryptococcus amylolentus, Cryptococcus floricola, Cryptococcus depauperatus, and Cryptococcus luteus (Liu et al., 2015; Passer et al., 2019). The pathogenic clade, which is responsible for over 200,000 human infections annually (Rajasingham et al., 2017), currently has seven recognized species distributed into three subgroups: Cryptococcus neoformans, Cryptococcus deneoformans, and the Cryptococcus gattii species complex (Hagen et al., 2015). A new lineage within the C. gattii species complex has recently been isolated from middens, midden soil, or tree holes associated with the Southern tree hyrax (Dendrohyrax arboreus) in Zambia and termed C. gattii VGV; no human infections have thus far been attributed to this novel lineage (Farrer et al., 2019).

The heterothallic reproductive cycle of C. neoformans and C. gattii (also designated as bisexual mating or opposite-sex mating) has been known since the 1970s and readily occurs under laboratory conditions (Kwon-Chung, 1975; Kwon-Chung, 1976a; Kwon-Chung, 1976b). All of the species in the pathogenic Cryptococcus species complex have a bipolar mating system, in which the α and a mating types are determined by a single, unusually large (∼120kb in size), MAT locus that encompasses more than 20 genes (Lengeler et al., 2002; Fraser et al., 2004; Loftus et al., 2005). Among these genes are those encoding the mating-type-specific pheromones (MFα or MFa) and G protein-coupled receptors (GPCR) (Ste3α or Ste3a) that initiate recognition of compatible mating partners, and the homeodomain transcription factors (HD1/Sxi1α or HD2/Sxi2a), which establish cell-type identity and orchestrate progression through the sexual cycle (Hull et al., 2005; Sun et al., 2019a). In addition, the MAT locus contains essential genes (Fraser et al., 2004; Ianiri et al., 2020) and genes that contribute to virulence (Sun et al., 2019a). Importantly, comparative genomic studies with the closely related species C. amylolentus uncovered that the single MAT locus in the pathogenic Cryptococcus species is the result of a fusion of ancestrally unlinked pheromone/receptor (P/R) and homeodomain (HD) loci, possibly initiated through ectopic inter-centromeric recombination (Sun et al., 2017).

Under the proper environmental conditions (e.g., V8 media in dark, dry conditions), Cryptococcus cells secrete pheromones unique to the mating type of the cell (a cells produce the MFa pheromone, and α cells produce the MFα pheromone) (Davidson et al., 2000; McClelland et al., 2002). These pheromones bind to the Ste3α and Ste3a receptors, respectively, which signal through the Ste20 protein to a mitogen-activated protein kinase (MAPK) signaling cascade that includes the Ste11, Ste7, Cpk1, and Ste50 proteins (Sun et al., 2019a; Zhao et al., 2019). The final target of this signaling cascade is the transcription factor Mat2 (Lin et al., 2010; Feretzaki and Heitman, 2013), which directly or indirectly activates genes involved in mating and the yeast-to-hyphal morphological transition, initiated by the MATα parent that extends a conjugation tube towards the enlarged MATa mating partner (Zhao et al., 2019; Sun et al., 2020). The two mating partners fuse to form a dikaryotic zygote from which a hyphal filament protrudes and extends to form a dikaryotic hypha. During hyphal growth, fused clamp connections form across the septa to ensure that each hyphal compartment maintains two unfused, paired parental nuclei (Kwon-Chung, 1976b; Lin, 2009). Following the extension of the hyphal filament, the tip differentiates into a basidium, in which karyogamy and meiosis occur followed by repeated mitotic divisions to produce four chains of spores. Eventually, the spores are released from the basidia, disseminate (acting as infectious propagules) (Reedy et al., 2007; Velagapudi et al., 2009), and grow as yeast cells until encountering mating stimuli again (Kwon-Chung, 1975; Kwon-Chung, 1976b; Sun et al., 2019c; Zhao et al., 2019; Sun et al., 2020).

C. deneoformans and C. gattii can also participate in an unusual form of homothallism, termed unisexual reproduction, during which haploid cells of a single mating type undergo ploidy changes and meiosis to produce genetically identical progeny (Lin et al., 2005; Lin et al., 2010; Ni et al., 2011; Feretzaki and Heitman, 2013; Ni et al., 2013; Fu et al., 2015; Wilson et al., 2021). The features of unisexual reproduction are similar to those observed during opposite-sex mating, and there are two ways in which unisexual reproduction is initiated: (i) two cells of the same mating type can fuse, produce hyphae with unfused clamp connections (termed a monokaryon), form basidia, and basidiospores, or (ii) a single cell can undergo endoreplication forming a diploid cell that then produces a similar hyphal filament and completes the sexual cycle (Lin et al., 2005; Zhao et al., 2019).

Interestingly, many of the species that are closely related to members of the pathogenic Cryptococcus species, such as C. amylolentus, C. depauperatus, and C. luteus, are not known to cause disease in plants or animals (Findley et al., 2009; Rodriguez-Carres et al., 2010) and are instead regarded as saprobes or mycoparasites (i.e., parasites of other fungi) (Sivakumaran et al., 2003; Begerow et al., 2017). C. depauperatus, in particular, was first identified by Petch, 1931 on scale insects and originally described as the type specimen of Aspergillus depauperatus. Although initially considered as an insect-associated fungus, C. depauperatus was later reassessed as a possible mycoparasite of the entomopathogenic fungus Akanthomyces lecanii considering that (i) specimens from which C. depauperatus had been isolated or identified from also contained A. lecanii (Petch, 1931; Malloch et al., 1978; Samson et al., 1983; Kubátová, 1992), and (ii) the fact that C. depauperatus, as with other mycoparasitic basidiomycetes, can produce haustorial branches (Ginns and Malloch, 2003). There are only two strains of C. depauperatus available: CBS7841, which was isolated from a dead spider in Canada (Malloch et al., 1978), and CBS7855, which was isolated from a dead caterpillar in the Czech Republic (Kubátová, 1992). Interestingly, C. depauperatus, possibly along with C. luteus, are the only naturally occurring Cryptococcus species with no known yeast phase (Malloch et al., 1978; Kwon-Chung et al., 1995; Roberts, 1997; Ginns and Bernicchia, 2000). Indeed, in contrast to the dimorphic growth of C. neoformans and C. gattii, which are usually yeasts in the asexual stage and produce hyphae, basidia, and basidiospores during the sexual stage, C. depauperatus grows by continuously producing hyphae, basidia, and basidiospores under typical laboratory conditions (Kwon-Chung et al., 1995) with no budding yeast cells observed during its entire life cycle. This species also displays a slow growth phenotype compared to other Cryptococcus species (Findley et al., 2009), possibly as a consequence of being continuously engaged in an energetically costly sexual cycle. Early data from fluorescent-activated cell sorting (FACS) indicates the spores are haploid, and random amplified polymorphic DNA (RAPD) analyses found the two strains to be genetically distinct (Rodriguez-Carres et al., 2010). Given the geographical range of the two isolates, they may represent different populations or even isolates of two closely related but distinct species. However, given the small sample size (n = 2), it is difficult to know if these differences are strain- or species-specific.

Considering the striking resemblance of its growth with the sexual life cycle of other Cryptococcus species, the hypothesis was put forth that C. depauperatus is homothallic and only the sexual developmental program is active at any given time during the life cycle of this species (Malloch et al., 1978; Kwon-Chung et al., 1995; Rodriguez-Carres et al., 2010). However, to date, a thorough characterization of the C. depauperatus genomes has not been completed, leaving unanswered questions. Here, we carried out an in-depth genomic, genetic, and phenotypic study of C. depauperatus. Newly generated chromosome-level genome assemblies of CBS7841 and CB7855 revealed 98% genome-wide shared identity and a uniform pattern of divergence across the genome. We found a putative MAT locus containing genes phylogenetically aligned with MATa alleles of other species, as well as a few unlinked MATα gene alleles, including the MFα gene, but no homologs of the mating-type determinants SXI1 and SXI2. This led to the hypothesis that compatible pheromone and pheromone-receptor genes could be the key components underlying the homothallic mating behavior of C. depauperatus. Agrobacterium-mediated transformation was developed to delete genes by homologous recombination. Deletion of key mating and meiosis genes (MFα, STE3, and DMC1) showed severe defects in basidia and/or spore production, but not in hyphal growth. Furthermore, with the first genetic mutants isolated in this species, we identified recombinant meiotic progeny generated from intra-strain genetic crosses. These data support the hypothesis that C. depauperatus is an obligately sexual, homothallic fungal species.

Genetically controlled self-incompatibility systems are thought to have evolved to prevent inbreeding and promote outcrossing, but seeking a compatible mate is not always an easy task. This hurdle is particularly relevant for species with low population densities or that have spatially structured populations where dispersal between different patches is limited (Murtagh et al., 2000; Busch and Delph, 2012). Such a scenario of high-cost to mate finding has been proposed as an explanation for the emergence of reproductive mechanisms that overcome the low encounter rate, including hermaphroditism in many animals and plants, and homothallism in fungi, both systems allowing for reproductive assurance and the persistence of populations in a specific environment where mates are scarce or unavailable.

In this study, we present four lines of evidence that C. depauperatus is continuously undergoing sexual reproduction. The first evidence is the significantly increased expression of key mating and meiosis genes in conditions that promote sporulation. Second, the isolation of double-drug-resistant progeny from co-cultures of can1 and fur1 single-drug-resistant mutants showed exchange of genetic material occurred after co-incubation of marked parental strains. Third, we identified that this exchange of genetic material most likely involves mating, cell–cell fusion, karyogamy, and meiosis as demonstrated by the fact that sporulation, and thus the production of recombinant progeny, was severely impaired upon deletion of key components required for the pheromone signaling cascade (STE3) or completely abolished when deleting a meiotic-specific recombinase (DMC1). Fourth, we detected evidence of recombination on Chr 3 in CBS7841 upon analysis of the segregation patterns of UV-induced single-nucleotide variants. Taken together, these findings provide robust evidence of meiotic sexual reproduction, and we propose that C. depauperatus represents the first obligately sexual homothallic species to be identified in the Cryptococcus genus.

Although undergoing a meiotic cycle, our genomic analyses also revealed that two meiotic genes, MSH4 and MSH5, were lost in C. depauperatus while being retained in other Cryptococcus species. In S. cerevisiae, Msh4 and Msh5, along with other factors, repair most (~80%) of the double-strand breaks (DSBs) initiated by Spo11 as crossovers that show interference (a.k.a. class I crossovers) (de los de los Santos et al., 2003; Argueso et al., 2004). Thus, the loss of MSH4 and MSH5 in C. depauperatus, possibly along with loss of class I crossover formation and crossover interference, indicates that the crossover homeostasis machinery might not be completely intact in this species. If confirmed, such scenario may be analogous to the loss of Msh4 and Msh5 in the fission yeast Schizosaccharomyces pombe, along with class I crossover formation and crossover interference losses (Hollingsworth and Brill, 2004; Malik et al., 2007), or parallel the loss of class I crossovers via loss-of-function mutations in Msh4 and/or Msh5 in model species as diverse as S. cerevisiae, Caenorhabditis elegans, and Arabidopsis thaliana, where such events result in fewer crossovers, and those that remain are interference-independent (i.e., produced by a pathway involving instead the Mus81-Mms4/Eme1 protein complex) (Zalevsky et al., 1999; Kelly et al., 2000; Higgins et al., 2004; Hollingsworth and Brill, 2004).

Homothallism in fungi comes in many distinct forms. The simplest configuration is a single genome that encodes compatible sets of mating-type genes, either fused or unlinked (primary homothallism). Another strategy involves mating-type switching, either bidirectional or unidirectional, which leads to a mixed population of cells of opposite mating types that can mate. A third and the most recent form of homothallism to be described is unisexual reproduction where cells of the same mating type can undergo sexual reproduction despite having genes of only one mating type (reviewed in Ni et al., 2011; Roach et al., 2014; Fu et al., 2015; Wilson et al., 2015; Hanson and Wolfe, 2017; Wilson et al., 2021). Some other fungal species, referred to as pseudo-homothallic, can complete the sexual cycle without the apparent need for cell–cell fusion, but this is achieved by packaging two opposite mating-type nuclei derived from the same meiosis into the same cell (Lin and Heitman, 2007).

Our study reveals that homothallism in C. depauperatus is independent of the homeodomain transcription factors Sxi1/HD1 and Sxi2/HD2 as these genes are completely absent in the genomes of the two strains. Rather, it seems to be orchestrated by the expression in the same genome of a single interacting mating receptor (Ste3a)/pheromone ligand (MFα) pair that signals through a similar mating pathway characteristic of heterothallic Cryptococcus species. This is similar to findings from the ascomycete fungi Sordaria macrospora, N. crassa, and Trichoderma reesei, where one compatible pheromone/pheromone-receptor pair in mating partners seems to be necessary and sufficient for sexual development (Mayrhofer et al., 2006; Kim et al., 2012; Seibel et al., 2012).

Interestingly, despite a protein sequence that differs by two amino acids, introduction of the C. depauperatus MFα pheromone into a C. neoformans MATa strain induced robust hyphal formation, and this self-filamentous phenotype was abolished by deletion of either STE6 (encoding the pheromone exporter) or the STE3a gene (encoding the α-pheromone receptor). Likewise, deletion of the MFα and STE3a genes in C. depauperatus resulted in severe defects in basidia maturation and sporulation, but the C. depauperatus mfαΔ mutant, while unable to produce the pheromone itself, could sense the pheromone secreted from the ste3Δ mutant in a co-culture and sporulation was partially restored. Together, this indicates that the mechanisms for pheromone export and sensing between the two species are largely conserved and reveals that pheromone/receptor recognition can transcend species boundaries.

Intriguingly, the pheromone/receptor autocrine system of C. depauperatus seems to control the later stage of sexual development (basidia maturation), rather than hyphal development as it does during C. neoformans and C. deneoformans heterothallic mating. Indeed, we showed that the absence of the mating pheromone or the pheromone receptor did not result in obvious impairment of self-filamentation, except for an interference in hyphal polarity resulting in tip splitting. This is similar to previously described pheromone and receptor gene deletion mutants in the filamentous ascomycetes S. macrospora and Podospora anserina, as well as deletion of both receptor genes in Aspergillus nidulans, which cause no changes in hyphal growth (Seo et al., 2004; Coppin et al., 2005; Mayrhofer et al., 2006). Conversely, basidia maturation was severely impaired in C. depauperatus ste3Δ, mfαΔ, and ste3Δ mfαΔ mutants. While the reasons underlying such observations are presently unclear, it is important to note that some of the upstream components of the mating cascade critical for the paracrine induction of hyphal formation and dikaryon maintenance during heterothallic reproduction in C. neoformans and C. deneoformans (including MFα, Ste3, and Ste6) are seemingly bypassed in monokaryotic hyphal development and sporulation during unisexual reproduction in C. deneoformans (Gyawali et al., 2017; Tian et al., 2018). A reasonable explanation as to why the intercellular regulation mediated by pheromone is not strictly required for unisexual reproduction in C. deneoformans stems from the fact that the unisexual process in most cases occurs independently of cell–cell fusion, with cells transitioning to a diploid state either through cell fusion-independent karyogamy (minor route) or endoreplication (major route) (Fu and Heitman, 2017), and C. depauperatus may have adopted similar mechanisms.

Under this scenario, we posit that the loss of the homeodomain genes SXI1 and SXI2 in C. depauperatus may have enabled additional changes in the regulation of the mating pathway and some genes have possibly taken on additional regulatory roles downstream of the mating pheromone cascade, including in basidia maturation, meiosis, and sporulation. Two possible candidate genes that could have gained additional regulatory roles include homologs of Mat2, an HMG transcription factor, and Znf2, a zinc finger transcription factor, which are central components governing opposite and unisexual mating in C. deneoformans (Lin et al., 2010). However, we did not observe an upregulation of these genes under sporulation conditions. Hence, homothallism in C. depauperatus does not completely bypass the requirements for heterothallic sexual reproduction in that it requires the interplay between a compatible pheromone/receptor pair of opposite mating types. These findings show an interesting parallel with studies in A. nidulans that have also revealed that homothallism in this species does not bypass the requirements for partner signaling, but instead requires activation of the heterothallic signaling pathway for sexual development in a single individual (Paoletti et al., 2007). Future studies should address whether other aspects of the mating pathway have been rewired in C. depauperatus due to the loss of the homeodomain transcription factors.

Our genomic and phylogenetic analysis revealed that the predicted MAT locus structure of C. depauperatus has been extensively remodeled compared to other Cryptococcus species. While C. depauperatus has retained genes necessary for sexual reproduction, some of the key genes, including the MFα gene encoding the mating pheromone, are found outside of the predicted MAT region. In heterothallic basidiomycete fungi, one or more pheromone genes are invariably linked to a pheromone receptor of the same mating specificity (Coelho et al., 2017) and maintained in this way due to suppression of recombination, which in some fungal lineages has been established hundreds of millions of years ago (Devier et al., 2009). We hypothesize that the MAT gene cohort in C. depauperatus has been dramatically rearranged due to reduced selective pressure to maintain these genes in tight linkage, likely associated with the development of self-compatibility. Additionally, all Cryptococcus species so far examined encode multiple identical copies of the MFα or MFa pheromone genes per mating type to increase the production of the mating pheromone per cell, and this is thought to enhance the functionality of the poorly diffusible small lipophilic peptides (Akada et al., 1989). In contrast, the presence of a single MFα gene copy in the C. depauperatus genome indicates that selection has acted to reduce the energetic burden of having extra gene copies without a direct benefit. Indeed, intercompatible MFα and Ste3a proteins are readily produced by the same cell, and thus, as our confrontation assays between mfαΔ and ste3Δ mutants suggested, long-distance communication is unlikely to be favored. Nevertheless, this system still allows admixture of genetic diversity via outcrossing.

Elucidating the genomic regions conferring sexual identity across a phylogeny can be a powerful approach to studying the mating-type loci structure and associated transitions between different modes of reproduction. The MAT locus identified on Chr 4 in both strains contains genes associated with C. amylolentus P/R and HD loci reminiscent of a past fusion event between the two regions in C. depauperatus. This event could have occurred in the common ancestor giving rise to C. depauperatus and the Cryptococcus pathogenic complex, or evolved instead independently in the two lineages. Our analyses cannot exclude the possibility as well that the two genomic regions in C. depauperatus fused together via chromosomal translocation only after homothallism was attained. Although there are several possible models to explain how the observed configuration of canonical MAT locus genes could have evolved from either a bipolar or tetrapolar mating system, we present two that we consider the most parsimonious given the present data and analyses (Figure 9).

Figure 9

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Both models envision an ancestral state with two cells of the opposite mating-type fusing to produce a self-filamentous diploid. If the ancestor of C. depauperatus had a bipolar mating configuration (Figure 9A), then the diploid cell could have undergone a round of meiosis, resulting in an F1 aneuploid 1n + 1 progeny containing the genome of the MATa parent plus the MAT chromosome of the MATα parent. This progeny would be self-filamentous and potentially able to undergo a sexual cycle. If the ancestor had instead a tetrapolar mating configuration (Figure 9B), two intra-MAT recombination steps are required to achieve homothallism. First, a recombination event at the HD locus would bring together compatible HD1 and HD2 alleles, whose gene products could heterodimerize and form an active transcription regulator. The haploid individuals carrying this compatible HD pair would shift from compatibility determined by two sequential checkpoints (P/R and HD) to compatibility determined by a single mechanism (P/R), representing the first step in a transition from heterothallism to homothallism. A complete transition to homothallism could then be achieved in a subsequent mating event by uniting in the same genome-compatible pheromones and receptors via recombination; the isolates resulting from such event would respond to their own pheromone. In both models, we posit that the selective pressure to maintain an intact MAT locus is likely relaxed once self-compatibility is established, allowing chromosomal rearrangements to distribute some of the MAT genes throughout the genome without a concomitant fitness disadvantage. The SXI1/HD1 and SXI2/HD2 genes are eventually lost once their function is bypassed or supplanted by other genes.

The varied mechanisms via which homothallism has evolved and is maintained in fungi suggest that it represents an evolutionarily significant mating strategy. Examples from other fungi provide additional insights as to how the homothallic lifestyle of C. depauperatus evolved. While the number of representative samples is presently limiting, increased sampling efforts over a broader range of niches, including surveys for mycoparasites, may prove a good strategy to uncover additional isolates of the same or from closely related species, as a recent study suggests (Guterres et al., 2021). A careful characterization of the ability to sexually reproduce will ultimately shed light on how this unique continuously active sexual development program emerged, making it a contrasting Cryptococcus species. Hence, C. depauperatus provides another vantage point and an opportunity to further understand both the evolution of sexual reproduction in the pathogenic and saprobic Cryptococcus lineages, and the frequent transitions between modes of sexual reproduction that have occurred throughout the fungal kingdom.

Sequencing reads and genome assemblies of C depauperatus CBS7841 and CBS7855 were submitted to GenBank under BioProjects PRJNA200572 and PRJNA200573, respectively. All other genomic data (RNA-seq and Illumina sequence of C depauperatus CBS7841 can1 mutants) are available under BioProject PRJNA803141. Source data files have been provided for Figures 1 to 7.

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   ID PRJNA200572. Cryptotoccus Sequencing.

   https://www.ncbi.nlm.nih.gov//bioproject/PRJNA200572
2. 1. Broad Institute

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   ID PRJNA200573. Cryptotoccus Sequencing.

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Author details

1. Andrew Ryan Passer

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Resources, Writing - preliminary draft as a thesis chapter, Writing – review and editing

   Contributed equally with

   Shelly Applen Clancey

   Competing interests

   No competing interests declared

2. Shelly Applen Clancey

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Contribution

   Conceptualization, Data curation, Investigation, Methodology, Resources, Validation, Writing – original draft, Writing – review and editing, Incorporating revisions from co-authors, Writing the final draft

   Contributed equally with

   Andrew Ryan Passer

   Competing interests

   No competing interests declared

3. Terrance Shea

   Broad Institute of MIT and Harvard, Cambridge, United States

   Contribution

   Formal analysis, Validation

   Competing interests

   No competing interests declared

4. Márcia David-Palma

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Anna Floyd Averette

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Contribution

   Technical support, Writing – review and editing

   Competing interests

   No competing interests declared

6. Teun Boekhout

   1. Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands
   2. Institute of Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, Amsterdam, Netherlands

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

7. Betina M Porcel

   Génomique Métabolique, CNRS, University Evry, Université Paris-Saclay, Evry, France

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

8. Minou Nowrousian

   Lehrstuhl für Molekulare und Zelluläre Botanik, Ruhr-Universität Bochum, Bochum, Germany

   Contribution

   Data curation, Funding acquisition, Investigation, Methodology, Resources, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0075-6695

9. Christina A Cuomo

   Broad Institute of MIT and Harvard, Cambridge, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5778-960X

10. Sheng Sun

    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

    Contribution

    Conceptualization, Formal analysis, Investigation, Methodology, Validation, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2895-1153

11. Joseph Heitman

    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

    Contribution

    Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    heitm001@duke.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6369-5995

12. Marco A Coelho

    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review and editing, Incorporating revisions from co-authors, Writing the final draft

    For correspondence

    marco.dias.coelho@duke.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-5716-0561

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