Editor's evaluation: Obligate sexual reproduction of a homothallic fungus closely related to the Cryptococcus pathogenic species complex

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Sexual reproduction is a ubiquitous, ancient eukaryotic trait. While most sexual organisms have to find a mating partner, species as diverse as animals, plants, and fungi have evolved the ability to reproduce sexually without requiring another individual. Here, we uncovered the mechanism of self-compatibility (homothallism) in Cryptococcus depauperatus, a fungal species closely related to the human fungal pathogens Cryptococcus neoformans and Cryptococcus gattii. In contrast to C. neoformans or C. gattii, which grow as a yeast asexually, and produce hyphae, basidia, and infectious spores during sexual reproduction, C. depauperatus grows exclusively as hyphae decorated with basidia and abundant spores, thus continuously engaged in sexual reproduction. Through comparative genomics and analyses of mutants defective in key mating/meiosis genes, we demonstrate the C. depauperatus sexual cycle involves meiosis and that self-compatibility is orchestrated by an unlinked mating receptor (Ste3a) and pheromone ligand (MFα) pair derived from opposite mating types of a heterothallic (self-sterile) ancestor. We identified a putative mating-type (MAT) determining region containing genes phylogenetically aligned with MATa alleles of other species, and a few MATα gene alleles scattered throughout the genome, but no homologs of the mating-type homeodomain genes SXI1 (HD1) and SXI2 (HD2). Comparative analyses suggest a dramatic remodeling of the MAT locus possibly owing to reduced selective constraints to maintain mating-type genes in tight linkage, associated with a transition to self-fertility. Our findings support C. depauperatus as an obligately sexual, homothallic fungus and provide insight into repeated transitions between sexual reproduction modes that have occurred throughout the fungal kingdom. Editor's evaluation There are various ways in which self-fertility has arisen in the fungal kingdom. This study describes a novel form of self-fertility that evolved in a species closely related to the Cryptococcus species causing serious human lung and brain infections, in which sexual development is achieved by self-signaling of a cognate pheromone and pheromone-receptor pair. Through a combination of high-quality genomic analysis and experimental gene expression and manipulation work, the study significantly adds to our understanding of the evolution and flexibility of fungal breeding systems. https://doi.org/10.7554/eLife.79114.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest 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. Introduction 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. Results CBS7841 and CBS7855 genomes are overall syntenic and present homogeneous genome-wide divergence To establish the basis of the C. depauperatus sexual cycle and explore the genomic variation between the two available isolates, we sequenced, assembled, and annotated the genomes of CBS7841 and CBS7855 with both Oxford Nanopore and Illumina reads. The resulting assemblies are approximately 16.28 Mb (CBS7841) and 16.33 Mb (CBS7855) in size and comprised eight contigs with telomeric repeats TAA(C)4,5 at both ends, corresponding to eight chromosomes (Figure 1, Figure 1—figure supplement 1). Each contig contains a large, open reading frame (ORF)-free region, rich in long terminal repeat (LTR) retrotransposons, which have been shown to be coincident with centromeres in other Cryptococcus species (Janbon et al., 2014; Sun et al., 2017; Yadav et al., 2018; Schotanus and Heitman, 2020) and are predicted to constitute functional centromeres in C. depauperatus (Figure 1A). Figure 1 with 5 supplements see all Download asset asset between the two C. depauperatus strains and of C. depauperatus. the genome assemblies of C. depauperatus CBS7841 and The two assemblies are overall for to see Figure 1—figure supplement for genomic features are in different for each as shown in the C. depauperatus as a species to the human pathogenic Cryptococcus The tree was by a on a data of protein of genes shared across strains of seven Cryptococcus species and an of the = A tree by was with the Figure 1—figure supplement The of each was by of the and in the and probability in the are given in of each of the three of are the gene factor the factor and support for the and for sexual reproductive structures with spore of C. neoformans C. depauperatus CBS7841 and C. amylolentus = and identified and genes, respectively, for CBS7841 and These numbers are in the range the of genes predicted for other Cryptococcus species with complete genomes available from in C. to in C. et al., 2011; et al., 2014; et al., 2015; Sun et al., 2017; Passer et al., 2019; et al., 2021). of revealed a of of gene with over of the genes being present in both isolates (Figure 1—figure supplement 1). This indicates the of genes identified in C. depauperatus is not the result of gene but to genome size Indeed, the genomes of the two C. depauperatus isolates are the the described Cryptococcus species et al., 2011; et al., 2014; et al., 2015; Sun et al., 2017; Passer et al., 2019; et al., 2021). to the genome the to be overall conserved between the two C. depauperatus isolates, for large and three of which four are with at the Figure Figure 1—figure supplement 2), and the predicted regions that in between some of the homologous chromosomes (Figure 1—figure supplement by were with the contigs for CBS7841, but revealed a few for CBS7855, indicating there be in this (Figure 1—figure supplement The two isolates present divergence at the similar to the genetic divergence observed in species of the C. gattii complex (Farrer et al., 2015). A analysis a uniform pattern of divergence across the genome and no of between the two isolates as shown by the of genomic with divergence for the of which is found as a single on Figure 1—figure supplement this the two isolates are members of populations that have isolated since analyses C. depauperatus as to the human pathogenic Cryptococcus To establish evolutionary between C. depauperatus and other Cryptococcus we identified shared across strains Cryptococcus and as an (Figure both and a species where all of the support and were in the by the two (Figure Figure 1—figure supplement In both C. depauperatus as to the human pathogenic Cryptococcus of C. neoformans, C. deneoformans, and the C. gattii complex (Figure This with studies (Findley et al., 2009; Passer et al., that support for the C. amylolentus complex as the relative of the human pathogenic Cryptococcus clade, these studies To these we first the signal by the of genes or that are with a given in the species tree and for This analysis showed that the C. depauperatus and the human pathogenic Cryptococcus species had and compared to other branches in the of some of (Figure an of for this that over a of the for this we also the tree each of three for of each tree from the analysis (Figure 1—figure supplement there are three possible for how the taxa can be related as and in Figure 1—figure supplement In this the analysis more the again C. depauperatus with the human pathogenic Cryptococcus species, compared to the and Figure 1—figure supplement these analyses suggest that the by both and the for the C. depauperatus within the species tree on methods and taxa mating genes are found in and unlinked loci in the genome of C. depauperatus To the mechanisms of homothallism in C. depauperatus, we first the genomes MAT gene and of genes found within or to the MAT locus in C. neoformans, or the and loci in C. amylolentus the state of the identified out of genes in the genome of C. depauperatus (Figure data 1). of these genes (n = were to a region on in both CBS7841 and CBS7855, which we designated as the putative MAT locus (Figure Figure data 1). in this region with predicted key during mating include the pheromone receptor and the kinase The genes were found scattered throughout the genome, including and on and on 1, and a single mating pheromone gene on (Figure Figure data predicted to a with a of fungal mating pheromones et al., 2011; et al., 2017; Figure Among the genes in C. depauperatus, it was to the complete of homologs of the homeodomain transcription factors SXI1 (HD1) and SXI2 as these genes have in mating-type and of the sexual cycle in other in et al., 2017). To this we for proteins containing and This analysis genes in and each with a in the C. neoformans and A study in C. neoformans reported that these genes are transcription factors et al., 2015), and we they have similar in C. depauperatus, thus different from the homeodomain transcription factors and C. depauperatus has lost both the SXI1 and the SXI2 genes to our a unique case in the Figure with supplements see all Download asset asset The predicted MAT locus of C. depauperatus. the of between the C. amylolentus chromosomes containing the in and in MAT loci and the C. depauperatus CBS7841 within the MAT locus of C. neoformans whose

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