Comparative genomics of the closely related fungal genera Cryptococcus and Kwoniella reveals karyotype dynamics and suggests evolutionary mechanisms of pathogenesis.

In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

Comparative genomics of Cryptococcus and Kwoniella reveals pathogenesis evolution and contrasting karyotype dynamics via intercentromeric recombination or chromosome fusion.

A large-scale comparative genomic analysis was conducted for the global human fungal pathogens within the Cryptococcus genus, compared to non-pathogenic Cryptococcus species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species of both genera, resulting in a dataset encompassing virtually all of their known diversity. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at pre-adaptive pathogenic potential, our analysis found evidence in pathogenic Cryptococcus species of specific examples of gene gain (via horizontal gene transfer) and gene loss, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the two genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5 or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes, underwent chromosome reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Taken together, our findings advance our understanding of genomic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

Frequent transitions in mating-type locus chromosomal organization in Malassezia and early steps in sexual reproduction.

Fungi in the basidiomycete genus Malassezia are the most prevalent eukaryotic microbes resident on the skin of human and other warm-blooded animals and have been implicated in skin diseases and systemic disorders. Analysis of Malassezia genomes revealed that key adaptations to the skin microenvironment have a direct genomic basis, and the identification of mating/meiotic genes suggests a capacity to reproduce sexually, even though no sexual cycle has yet been observed. In contrast to other bipolar or tetrapolar basidiomycetes that have either two linked mating-type-determining (MAT) loci or two MAT loci on separate chromosomes, in Malassezia species studied thus far the two MAT loci are arranged in a pseudobipolar configuration (linked on the same chromosome but capable of recombining). By generating additional chromosome-level genome assemblies, and an improved Malassezia phylogeny, we infer that the pseudobipolar arrangement was the ancestral state of this group and revealed six independent transitions to tetrapolarity, seemingly driven by centromere fission or translocations in centromere-flanking regions. Additionally, in an approach to uncover a sexual cycle, Malassezia furfur strains were engineered to express different MAT alleles in the same cell. The resulting strains produce hyphae reminiscent of early steps in sexual development and display upregulation of genes associated with sexual development as well as others encoding lipases and a protease potentially relevant for pathogenesis of the fungus. Our study reveals a previously unseen genomic relocation of mating-type loci in fungi and provides insight toward the identification of a sexual cycle in Malassezia, with possible implications for pathogenicity.

Frequent transitions in mating-type locus chromosomal organization in Malassezia and early steps in sexual reproduction.

Fungi in the basidiomycete genus Malassezia are the most prevalent eukaryotic microbes resident on the skin of human and other warm-blooded animals and have been implicated in skin diseases and systemic disorders. Analysis of Malassezia genomes revealed that key adaptations to the skin microenvironment have a direct genomic basis, and the identification of mating/meiotic genes suggests a capacity to reproduce sexually, even though no sexual cycle has yet been observed. In contrast to other bipolar or tetrapolar basidiomycetes that have either two linked mating-type-determining ( MAT ) loci or two MAT loci on separate chromosomes, in Malassezia species studied thus far the two MAT loci are arranged in a pseudobipolar configuration (linked on the same chromosome but capable of recombining). By incorporating newly generated chromosome-level genome assemblies, and an improved Malassezia phylogeny, we infer that the pseudobipolar arrangement was the ancestral state of this group and revealed six independent transitions to tetrapolarity, seemingly driven by centromere fission or translocations in centromere- flanking regions. Additionally, in an approach to uncover a sexual cycle, Malassezia furfur strains were engineered to express different MAT alleles in the same cell. The resulting strains produce hyphae reminiscent of early steps in sexual development and display upregulation of genes associated with sexual development as well as others encoding lipases and a protease potentially relevant for pathogenesis of the fungus. Our study reveals a previously unseen genomic relocation of mating-type loci in fungi and provides insight towards the discovery of a sexual cycle in Malassezia , with possible implications for pathogenicity. Significance Statement: Malassezia , the dominant fungal group of the mammalian skin microbiome, is associated with numerous skin disorders. Sexual development and yeast-to-hyphae transitions, governed by genes at two mating-type ( MAT ) loci, are thought to be important for fungal pathogenicity. However, Malassezia sexual reproduction has never been observed. Here, we used chromosome-level assemblies and comparative genomics to uncover unforeseen transitions in MAT loci organization within Malassezia , possibly related with fragility of centromeric-associated regions. Additionally, by expressing different MAT alleles in the same cell, we show that Malassezia can undergo hyphal development and this phenotype is associated with increased expression of key mating genes along with other genes known to be virulence factors, providing a possible connection between hyphal development, sexual reproduction, and pathogenicity.

Obligate sexual reproduction of a homothallic fungus closely related to the Cryptococcus pathogenic species complex.

.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.

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.

Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans.

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications.

Multiple Pathways to Homothallism in Closely Related Yeast Lineages in the Basidiomycota.

Sexual reproduction in fungi relies on proteins with well-known functions encoded by the mating type (MAT) loci. In the Basidiomycota, MAT loci are often bipartite, with the P/R locus encoding pheromone precursors and pheromone receptors and the HD locus encoding heterodimerizing homeodomain transcription factors (Hd1/Hd2). The interplay between different alleles of these genes within a single species usually generates at least two compatible mating types. However, a minority of species are homothallic, reproducing sexually without an obligate need for a compatible partner. Here, we examine the organization and function of the MAT loci of Cystofilobasidium capitatum, a species in the order Cystofilobasidiales, which is unusually rich in homothallic species. We determined MAT gene content and organization in C. capitatum and found that it resembles a mating type of the closely related heterothallic species Cystofilobasidium ferigula To explain the homothallic sexual reproduction observed in C. capitatum, we examined HD protein interactions in the two Cystofilobasidium species and determined C. capitatum MAT gene expression both in a natural setting and upon heterologous expression in Phaffia rhodozyma, a homothallic species belonging to a clade sister to that of Cystofilobasidium. We conclude that the molecular basis for homothallism in C. capitatum appears to be distinct from that previously established for P. rhodozyma Unlike in the latter species, homothallism in C. capitatum may involve constitutive activation or dispensability of the pheromone receptor and the functional replacement of the usual Hd1/Hd2 heterodimer by an Hd2 homodimer. Overall, our results suggest that homothallism evolved multiple times within the Cystofilobasidiales.IMPORTANCE Sexual reproduction is important for the biology of eukaryotes because it strongly impacts the dynamics of genetic variation. In fungi, although sexual reproduction is usually associated with the fusion between cells belonging to different individuals (heterothallism), sometimes a single individual is capable of completing the sexual cycle alone (homothallism). Homothallic species are unusually common in a fungal lineage named Cystofilobasidiales. Here, we studied the genetic bases of homothallism in one species in this lineage, Cystofilobasidium capitatum, and found it to be different in several aspects from those of another homothallic species, Phaffia rhodozyma, belonging to the genus most closely related to Cystofilobasidium Our results strongly suggest that homothallism evolved independently in Phaffia and Cystofilobasidium, lending support to the idea that transitions between heterothallism and homothallism are not as infrequent as previously thought. Our work also helps to establish the Cystofilobasidiales as a model lineage in which to study these transitions.

Factors enforcing the species boundary between the human pathogens Cryptococcus neoformans and Cryptococcus deneoformans.

Hybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species.

The Untapped Australasian Diversity of Astaxanthin-Producing Yeasts with Biotechnological Potential-Phaffia australis sp. nov. and Phaffia tasmanica sp. nov.

Phaffia is an orange-colored basidiomycetous yeast genus of the order Cystofilobasidiales that contains a single species, P. rhodozyma. This species is the only fungus known to produce the economically relevant carotenoid astaxanthin. Although Phaffia was originally found in the Northern hemisphere, its diversity in the southern part of the globe has been shown to be much greater. Here we analyze the genomes of two Australasian lineages that are markedly distinct from P. rhodozyma. The two divergent lineages were investigated within a comprehensive phylogenomic study of representatives of the Cystofilobasidiales that supported the recognition of two novel Phaffia species, for which we propose the names of P. australis sp. nov. and P. tasmanica sp. nov. Comparative genomics and other analyses confirmed that the two new species have the typical Phaffia hallmark-the six genes necessary for the biosynthesis of astaxanthin could be retrieved from the draft genome sequences, and this carotenoid was detected in culture extracts. In addition, the organization of the mating-type (MAT) loci is similar to that of P. rhodozyma, with synteny throughout most regions. Moreover, cases of trans-specific polymorphism involving pheromone receptor genes and pheromone precursor proteins in the three Phaffia species, together with their shared homothallism, provide additional support for their classification in a single genus.

HGT in the human and skin commensal Malassezia: A bacterially derived flavohemoglobin is required for NO resistance and host interaction.

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn's disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in Malassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.

Centromere scission drives chromosome shuffling and reproductive isolation.

A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promoting speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans The resulting DSBs were repaired in a complex manner, leading to the formation of multiple interchromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced ∼3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages.

Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly.

Loss of centromere function drives karyotype evolution in closely related Malassezia species.

Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species. Malassezia species show variable karyotypes ranging between six and nine chromosomes. Here, we experimentally identified all eight centromeres in M. sympodialis as 3-5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur, which has seven chromosomes, and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with nine chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome-chromosome fusion. We propose that AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation.

Millions of yeast, bacteria and other microbes live in or on the human body. A type of yeast known as Malassezia is one of the most abundantmicrobes living on our skin. Generally, Malassezia do not cause symptoms in humans but are associated with dandruff, dermatitis and other skin conditions in susceptible individuals. They have also been found in the human gut, where they exacerbate Crohn's disease and pancreatic cancer. There are 18 closely related species of Malassezia and all have an unusually small amount of genetic material compared with other types of yeast. In yeast, like in humans, the genetic material is divided among several chromosomes. The number of chromosomes in different Malassezia species varies between six and nine. A region of each chromosome known as the centromere is responsible for ensuring that the equal numbers of chromosomes are passed on to their offspring. This means that any defects in centromeres can lead to the daughter yeast cells inheriting unequal numbers of chromosomes. Changes in chromosome number can drive the evolution of new species, but it remains unclear if and how centromere loss may have contributed to the evolution of Malassezia species. Sankaranarayanan et al. have now used biochemical, molecular genetic, and comparative genomic approaches to study the chromosomes of Malassezia species. The experiments revealed that nine Malassezia species had centromeres that shared common features such as being rich in adenine and thymine nucleotides, two of the building blocks of DNA. Sankaranarayanan et al. propose that these adenines and thymines make the centromeres more fragile leading to occasional breaks. This may have contributed to the loss of centromeres in some Malassezia cells and helped new species to evolve with fewer chromosomes. A better understanding of how Malassezia organize their genetic material should enable in-depth studies of how these yeasts interact with their human hosts and how they contribute to skin disease, cancer, Crohn's disease and other health conditions. More broadly, these findings may help scientists to better understand how changes in chromosomes cause new species to evolve.

Genetic and genomic evolution of sexual reproduction: echoes from LECA to the fungal kingdom.

Sexual reproduction is vastly diverse and yet highly conserved across the eukaryotic domain. This ubiquity suggests that the last eukaryotic common ancestor (LECA) was sexual. It is hypothesized that several critical processes in sexual reproduction, including cell fusion and meiosis, were acquired during the evolution from the first eukaryotic common ancestor (FECA) to the sexual LECA. However, it is challenging to delineate the exact origin and evolution of sexual reproduction given that both FECA and LECA are extinct. Studies of diverse eukaryotes have helped to shed light on this sexual evolutionary trajectory, revealing that a primordial sexual ploidy cycle likely involved endoreplication followed by concerted chromosome loss and that cell-cell fusion, meiosis, and sex determination later arose to shape modern sexual reproduction. Despite the general conservation of sexual reproduction processes throughout eukaryotes, modern sexual cycles are immensely diverse and complex. This diversity and complexity has become readily apparent in the fungal kingdom with the recent rapid expansion of whole-genome sequencing. This abundance of data, the variety of genetic tools available to manipulate and characterize fungi, and the thorough characterization of many fungal sexual cycles make the fungal kingdom an excellent forum, in which to study the conservation and diversification of sexual reproduction.

The Evolution of Sexual Reproduction and the Mating-Type Locus: Links to Pathogenesis of Cryptococcus Human Pathogenic Fungi.

Cryptococcus species utilize a variety of sexual reproduction mechanisms, which generate genetic diversity, purge deleterious mutations, and contribute to their ability to occupy myriad environmental niches and exhibit a range of pathogenic potential. The bisexual and unisexual cycles of pathogenic Cryptococcus species are stimulated by properties associated with their environmental niches and proceed through well-characterized signaling pathways and corresponding morphological changes. Genes governing mating are encoded by the mating-type (MAT) loci and influence pathogenesis, population dynamics, and lineage divergence in Cryptococcus. MAT has undergone significant evolutionary changes within the Cryptococcus genus, including transition from the ancestral tetrapolar state in nonpathogenic species to a bipolar mating system in pathogenic species, as well as several internal reconfigurations. Owing to the variety of established sexual reproduction mechanisms and the robust characterization of the evolution of mating and MAT in this genus, Cryptococcus species provide key insights into the evolution of sexual reproduction.

Convergent evolution of linked mating-type loci in basidiomycete fungi.

Sexual development is a key evolutionary innovation of eukaryotes. In many species, mating involves interaction between compatible mating partners that can undergo cell and nuclear fusion and subsequent steps of development including meiosis. Mating compatibility in fungi is governed by the mating type (MAT) loci. In basidiomycetes, the ancestral state is hypothesized to be tetrapolar, with two genetically unlinked MAT loci containing homeodomain transcription factor genes (HD locus) and pheromone and pheromone receptor genes (P/R locus), respectively. Alleles at both loci must differ between mating partners for completion of sexual development. However, there are also basidiomycetes with bipolar mating systems, which can arise through genomic linkage of the HD and P/R loci. In the order Tremellales, bipolarity is found only in the pathogenic Cryptococcus species. Here, we describe the analysis of MAT loci from 24 species of the Trichosporonales, a sister order to the Tremellales. In all of the species analyzed, the MAT loci are fused and a single HD gene is present in each mating type, similar to the organization in the pathogenic Cryptococci. However, the HD and P/R allele combinations in the Trichosporonales are different from those in the pathogenic Cryptococci. This and the existence of tetrapolar species in the Tremellales suggest that fusion of the HD and P/R loci occurred independently in the Trichosporonales and pathogenic Cryptococci, supporting the hypothesis of convergent evolution towards fused MAT regions, similar to previous findings in other fungal groups. Unlike the fused MAT loci in several other basidiomycete lineages though, the gene content and gene order within the fused MAT loci are highly conserved in the Trichosporonales, and there is no apparent suppression of recombination extending from the MAT loci to adjacent chromosomal regions, suggesting different mechanisms for the evolution of physically linked MAT loci in these groups.

Mating-System Evolution: All Roads Lead to Selfing.

Fungi reproduce via both heterothallic outcrossing and homothallic selfing modes, and transitions between the two are common throughout the tree of life. A new study reports that the transition from heterothallism to homothallism is common and has repeatedly punctuated the evolutionary trajectory across a major lineage of the fungal kingdom.

Genetic and Genomic Analyses Reveal Boundaries between Species Closely Related to Cryptococcus Pathogens.

Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes >200,000 new human infections annually. Elucidation of how these species evolved into important human-pathogenic species remains challenging and can be advanced by studying the most closely related nonpathogenic species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii However, these species have only four known isolates, and available data were insufficient to determine species boundaries within this group. By analyzing full-length chromosome assemblies, we reappraised the phylogenetic relationships of the four available strains, confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. The genomes of the three species are ∼6% divergent and exhibit significant chromosomal rearrangements, including inversions and a reciprocal translocation that involved intercentromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can still undergo sexual reproduction with C. amylolentus However, most of the resulting spores were inviable or sterile or showed reduced recombination during meiosis, indicating that intrinsic postzygotic barriers had been established. Our study and genomic data will foster additional studies addressing fungal speciation and transitions between nonpathogenic and pathogenic Cryptococcus lineages.IMPORTANCE The evolutionary drivers of speciation are critical to our understanding of how new pathogens arise from nonpathogenic lineages and adapt to new environments. Here we focus on the Cryptococcus amylolentus species complex, a nonpathogenic fungal lineage closely related to the human-pathogenic Cryptococcus neoformans/Cryptococcus gattii complex. Using genetic and genomic analyses, we reexamined the species boundaries of four available isolates within the C. amylolentus complex and revealed three genetically isolated species. Their genomes are ∼6% divergent and exhibit chromosome rearrangements, including translocations and small-scale inversions. Although two of the species (C. amylolentus and newly described C. floricola) were still able to interbreed, the resulting hybrid progeny were usually inviable or sterile, indicating that barriers to reproduction had already been established. These results advance our understanding of speciation in fungi and highlight the power of genomics in assisting our ability to correctly identify and discriminate fungal species.