Mating-type Chromosomes Research Articles

High-Quality Genome Assemblies of 4 Members of the Podospora anserina Species Complex.

The filamentous fungus Podospora anserina is a model organism used extensively in the study of molecular biology, senescence, prion biology, meiotic drive, mating-type chromosome evolution, and plant biomass degradation. It has recently been established that P. anserina is a member of a complex of 7 closely related species. In addition to P. anserina, high-quality genomic resources are available for 2 of these taxa. Here, we provide chromosome-level annotated assemblies of the 4 remaining species of the complex, as well as a comprehensive data set of annotated assemblies from a total of 28 Podospora genomes. We find that all 7 species have genomes of around 35 Mb arranged in 7 chromosomes that are mostly collinear and less than 2% divergent from each other at genic regions. We further attempt to resolve their phylogenetic relationships, finding significant levels of phylogenetic conflict as expected from a rapid and recent diversification.

Intratetrad mating as the driving force behind the formation of sex chromosomes in fungi

In some fungi-ascomycetes and basidiomycetes, the 4 haploid spores formed as a result of meiosis can fuse in pairs, forming a dikaryon or diploid. The consequence of such intratetrad mating is the preservation of heterozygosity of genes linked to the mating-type (MAT) locus. If the MAT is linked to the centromere physically or genetically (as a result of the suppression of recombination), the centromere regions of all chromosomes are preserved in a heterozygous state. Suppression of recombination in the MAT chromosome contributes to the accumulation of lethal mutations and chromosomal rearrangements in it. Two MAT chromosomes cease to be homologous and become analogues of the sex chromosomes of animals and plants.

Dynamics of transposable element accumulation in the non-recombining regions of mating-type chromosomes in anther-smut fungi

In the absence of recombination, the number of transposable elements (TEs) increases due to less efficient selection, but the dynamics of such TE accumulations are not well characterized. Leveraging a dataset of 21 independent events of recombination cessation of different ages in mating-type chromosomes of Microbotryum fungi, we show that TEs rapidly accumulated in regions lacking recombination, but that TE content reached a plateau at ca. 50% of occupied base pairs by 1.5 million years following recombination suppression. The same TE superfamilies have expanded in independently evolved non-recombining regions, in particular rolling-circle replication elements (Helitrons). Long-terminal repeat (LTR) retrotransposons of the Copia and Ty3 superfamilies also expanded, through transposition bursts (distinguished from gene conversion based on LTR divergence), with both non-recombining regions and autosomes affected, suggesting that non-recombining regions constitute TE reservoirs. This study improves our knowledge of genome evolution by showing that TEs can accumulate through bursts, following non-linear decelerating dynamics.

Sheltering of deleterious mutations explains the stepwise extension of recombination suppression on sex chromosomes and other supergenes.

Many organisms have sex chromosomes with large nonrecombining regions that have expanded stepwise, generating "evolutionary strata" of differentiation. The reasons for this remain poorly understood, but the principal hypotheses proposed to date are based on antagonistic selection due to differences between sexes. However, it has proved difficult to obtain empirical evidence of a role for sexually antagonistic selection in extending recombination suppression, and antagonistic selection has been shown to be unlikely to account for the evolutionary strata observed on fungal mating-type chromosomes. We show here, by mathematical modeling and stochastic simulation, that recombination suppression on sex chromosomes and around supergenes can expand under a wide range of parameter values simply because it shelters recessive deleterious mutations, which are ubiquitous in genomes. Permanently heterozygous alleles, such as the male-determining allele in XY systems, protect linked chromosomal inversions against the expression of their recessive mutation load, leading to the successive accumulation of inversions around these alleles without antagonistic selection. Similar results were obtained with models assuming recombination-suppressing mechanisms other than chromosomal inversions and for supergenes other than sex chromosomes, including those without XY-like asymmetry, such as fungal mating-type chromosomes. However, inversions capturing a permanently heterozygous allele were found to be less likely to spread when the mutation load segregating in populations was lower (e.g., under large effective population sizes or low mutation rates). This may explain why sex chromosomes remain homomorphic in some organisms but are highly divergent in others. Here, we model a simple and testable hypothesis explaining the stepwise extensions of recombination suppression on sex chromosomes, mating-type chromosomes, and supergenes in general.

Tempo of Degeneration Across Independently Evolved Nonrecombining Regions.

Recombination is beneficial over the long term, allowing more effective selection. Despite long-term advantages of recombination, local recombination suppression can evolve and lead to genomic degeneration, in particular on sex chromosomes. Here, we investigated the tempo of degeneration in nonrecombining regions, that is, the function curve for the accumulation of deleterious mutations over time, leveraging on 22 independent events of recombination suppression identified on mating-type chromosomes of anther-smut fungi, including newly identified ones. Using previously available and newly generated high-quality genome assemblies of alternative mating types of 13 Microbotryum species, we estimated degeneration levels in terms of accumulation of nonoptimal codons and nonsynonymous substitutions in nonrecombining regions. We found a reduced frequency of optimal codons in the nonrecombining regions compared with autosomes, that was not due to less frequent GC-biased gene conversion or lower ancestral expression levels compared with recombining regions. The frequency of optimal codons rapidly decreased following recombination suppression and reached an asymptote after ca. 3 Ma. The strength of purifying selection remained virtually constant at dN/dS = 0.55, that is, at an intermediate level between purifying selection and neutral evolution. Accordingly, nonsynonymous differences between mating-type chromosomes increased linearly with stratum age, at a rate of 0.015 per My. We thus develop a method for disentangling effects of reduced selection efficacy from GC-biased gene conversion in the evolution of codon usage and we quantify the tempo of degeneration in nonrecombining regions, which is important for our knowledge on genomic evolution and on the maintenance of regions without recombination.

Onset and stepwise extensions of recombination suppression are common in mating-type chromosomes of Microbotryum anther-smut fungi.

Sex chromosomes and mating-type chromosomes can display large genomic regions without recombination. Recombination suppression often extended stepwise with time away from the sex- or mating-type-determining genes, generating evolutionary strata of differentiation between alternative sex or mating-type chromosomes. In anther-smut fungi of the Microbotryum genus, recombination suppression evolved repeatedly, linking the two mating-type loci and extended multiple times in regions distal to the mating-type genes. Here, we obtained high-quality genome assemblies of alternative mating types for four Microbotryum fungi. We found an additional event of independent chromosomal rearrangements bringing the two mating-type loci on the same chromosome followed by recombination suppression linking them. We also found, in a new clade analysed here, that recombination suppression between the two mating-type loci occurred in several steps, with first an ancestral recombination suppression between one of the mating-type locus and its centromere; later, completion of recombination suppression up to the second mating-type locus occurred independently in three species. The estimated dates of recombination suppression between the mating-type loci ranged from 0.15 to 3.58million years ago. In total, this makes at least nine independent events of linkage between the mating-type loci across the Microbotryum genus. Several mating-type locus linkage events occurred through the same types of chromosomal rearrangements, where similar chromosome fissions at centromeres represent convergence in the genomic changes leading to the phenotypic convergence. These findings further highlight Microbotryum fungi as excellent models to study the evolution of recombination suppression.

Evolutionary Genomics of Sex-Related Chromosomes at the Base of the Green Lineage.

Although sex is now accepted as a ubiquitous and ancestral feature of eukaryotes, direct observation of sex is still lacking in most unicellular eukaryotic lineages. Evidence of sex is frequently indirect and inferred from the identification of genes involved in meiosis from whole genome data and/or the detection of recombination signatures from genetic diversity in natural populations. In haploid unicellular eukaryotes, sex-related chromosomes are named mating-type (MTs) chromosomes and generally carry large genomic regions where recombination is suppressed. These regions have been characterized in Fungi and Chlorophyta and determine gamete compatibility and fusion. Two candidate MT+ and MT− alleles, spanning 450–650 kb, have recently been described in Ostreococcus tauri, a marine phytoplanktonic alga from the Mamiellophyceae class, an early diverging branch in the green lineage. Here, we investigate the architecture and evolution of these candidate MT+ and MT− alleles. We analyzed the phylogenetic profile and GC content of MT gene families in eight different genomes whose divergence has been previously estimated at up to 640 Myr, and found evidence that the divergence of the two MT alleles predates speciation in the Ostreococcus genus. Phylogenetic profiles of MT trans-specific polymorphisms in gametologs disclosed candidate MTs in two additional species, and possibly a third. These Mamiellales MT candidates are likely to be the oldest mating-type loci described to date, which makes them fascinating models to investigate the evolutionary mechanisms of haploid sex determination in eukaryotes.

Mating-Type Locus Organization and Mating-Type Chromosome Differentiation in the Bipolar Edible Button Mushroom Agaricus bisporus.

In heterothallic basidiomycete fungi, sexual compatibility is restricted by mating types, typically controlled by two loci: PR, encoding pheromone precursors and pheromone receptors, and HD, encoding two types of homeodomain transcription factors. We analysed the single mating-type locus of the commercial button mushroom variety, Agaricus bisporus var. bisporus, and of the related variety burnettii. We identified the location of the mating-type locus using genetic map and genome information, corresponding to the HD locus, the PR locus having lost its mating-type role. We found the mip1 and β-fg genes flanking the HD genes as in several Agaricomycetes, two copies of the β-fg gene, an additional HD2 copy in the reference genome of A. bisporus var. bisporus and an additional HD1 copy in the reference genome of A. bisporus var. burnettii. We detected a 140 kb-long inversion between mating types in an A. bisporus var. burnettii heterokaryon, trapping the HD genes, the mip1 gene and fragments of additional genes. The two varieties had islands of transposable elements at the mating-type locus, spanning 35 kb in the A. bisporus var. burnettii reference genome. Linkage analyses showed a region with low recombination in the mating-type locus region in the A. bisporus var. burnettii variety. We found high differentiation between β-fg alleles in both varieties, indicating an ancient event of recombination suppression, followed more recently by a suppression of recombination at the mip1 gene through the inversion in A. bisporus var. burnettii and a suppression of recombination across whole chromosomes in A. bisporus var. bisporus, constituting stepwise recombination suppression as in many other mating-type chromosomes and sex chromosomes.

Size Variation of the Nonrecombining Region on the Mating-Type Chromosomes in the Fungal Podospora anserina Species Complex.

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.

Differential Gene Expression between Fungal Mating Types Is Associated with Sequence Degeneration.

Degenerative mutations in non-recombining regions, such as in sex chromosomes, may lead to differential expression between alleles if mutations occur stochastically in one or the other allele. Reduced allelic expression due to degeneration has indeed been suggested to occur in various sex-chromosome systems. However, whether an association occurs between specific signatures of degeneration and differential expression between alleles has not been extensively tested, and sexual antagonism can also cause differential expression on sex chromosomes. The anther-smut fungus Microbotryum lychnidis-dioicae is ideal for testing associations between specific degenerative signatures and differential expression because 1) there are multiple evolutionary strata on the mating-type chromosomes, reflecting successive recombination suppression linked to mating-type loci; 2) separate haploid cultures of opposite mating types help identify differential expression between alleles; and 3) there is no sexual antagonism as a confounding factor accounting for differential expression. We found that differentially expressed genes were enriched in the four oldest evolutionary strata compared with other genomic compartments, and that, within compartments, several signatures of sequence degeneration were greater for differentially expressed than non-differentially expressed genes. Two particular degenerative signatures were significantly associated with lower expression levels within differentially expressed allele pairs: upstream insertion of transposable elements and mutations truncating the protein length. Other degenerative mutations associated with differential expression included nonsynonymous substitutions and altered intron or GC content. The association between differential expression and allele degeneration is relevant for a broad range of taxa where mating compatibility or sex is determined by genes located in large regions where recombination is suppressed.

Higher Gene Flow in Sex-Related Chromosomes than in Autosomes during Fungal Divergence.

Nonrecombining sex chromosomes are widely found to be more differentiated than autosomes among closely related species, due to smaller effective population size and/or to a disproportionally large-X effect in reproductive isolation. Although fungal mating-type chromosomes can also display large nonrecombining regions, their levels of differentiation compared with autosomes have been little studied. Anther-smut fungi from the Microbotryum genus are castrating pathogens of Caryophyllaceae plants with largely nonrecombining mating-type chromosomes. Using whole genome sequences of 40 fungal strains, we quantified genetic differentiation among strains isolated from the geographically overlapping North American species and subspecies of Silene virginica and S. caroliniana. We inferred that gene flow likely occurred at the early stages of divergence and then completely stopped. We identified large autosomal genomic regions with chromosomal inversions, with higher genetic divergence than the rest of the genomes and highly enriched in selective sweeps, supporting a role of rearrangements in preventing gene flow in genomic regions involved in ecological divergence. Unexpectedly, the nonrecombining mating-type chromosomes showed lower divergence than autosomes due to higher gene flow, which may be promoted by adaptive introgressions of less degenerated mating-type chromosomes. The fact that both mating-type chromosomes are always heterozygous and nonrecombining may explain such patterns that oppose to those found for XY or ZW sex chromosomes. The specific features of mating-type chromosomes may also apply to the UV sex chromosomes determining sexes at the haploid stage in algae and bryophytes and may help test general hypotheses on the evolutionary specificities of sex-related chromosomes.

Understanding Adaptation, Coevolution, Host Specialization, and Mating System in Castrating Anther-Smut Fungi by Combining Population and Comparative Genomics

Anther-smut fungi provide a powerful system to study host-pathogen specialization and coevolution, with hundreds of Microbotryum species specialized on diverse Caryophyllaceae plants, castrating their hosts through manipulation of the hosts' reproductive organs to facilitate disease transmission. Microbotryum fungi have exceptional genomic characteristics, including dimorphic mating-type chromosomes, that make this genus anexcellent model for studying the evolution of mating systems and their influence on population genetics structure and adaptive potential. Important insights into adaptation, coevolution, host specialization, and mating system evolution have been gained using anther-smut fungi, with new insights made possible by the recent advent of genomic approaches. We illustrate with Microbotryum case studies how using a combination of comparative genomics, population genomics, and transcriptomics approaches enables the integration of different evolutionary perspectives across different timescales. We also highlight current challenges and suggest future studies that will contribute to advancing our understanding of the mechanisms underlying adaptive processes in populations of fungal pathogens.

Little Evidence of Antagonistic Selection in the Evolutionary Strata of Fungal Mating-Type Chromosomes (Microbotryum lychnidis-dioicae).

Recombination suppression on sex chromosomes often extends in a stepwise manner, generating evolutionary strata of differentiation between sex chromosomes. Sexual antagonism is a widely accepted explanation for evolutionary strata, postulating that sets of genes beneficial in only one sex are successively linked to the sex-determining locus. The anther-smut fungus Microbotryum lychnidis-dioicae has mating-type chromosomes with evolutionary strata, only some of which link mating-type genes. Male and female roles are non-existent in this fungus, but mating-type antagonistic selection can also generate evolutionary strata, although the life cycle of the fungus suggests it should be restricted to few traits. Here, we tested the hypothesis that mating-type antagonism may have triggered recombination suppression beyond mating-type genes in M. lychnidis-dioicae by searching for footprints of antagonistic selection in evolutionary strata not linking mating-type loci. We found that these evolutionary strata (i) were not enriched in genes upregulated in the haploid phase, where cells are of alternative mating types, (ii) carried no gene differentially expressed between mating types, and (iii) carried no genes displaying footprints of specialization in terms of protein sequences (dN/dS) between mating types after recommended filtering. Without filtering, eleven genes showed signs of positive selection in the strata not linking mating-type genes, which constituted an enrichment compared to autosomes, but their functions were not obviously involved in antagonistic selection. Thus, we found no strong evidence that antagonistic selection has contributed to extending recombination suppression beyond mating-type genes. Alternative hypotheses should therefore be explored to improve our understanding of the sex-related chromosome evolution.

Convergent recombination cessation between mating-type genes and centromeres in selfing anther-smut fungi.

The degree of selfing has major impacts on adaptability and is often controlled by molecular mechanisms determining mating compatibility. Changes in compatibility systems are therefore important evolutionary events, but their underlying genomic mechanisms are often poorly understood. Fungi display frequent shifts in compatibility systems, and their small genomes facilitate elucidation of the mechanisms involved. In particular, linkage between the pre- and postmating compatibility loci has evolved repeatedly, increasing the odds of gamete compatibility under selfing. Here, we studied the mating-type chromosomes of two anther-smut fungi with unlinked mating-type loci despite a self-fertilization mating system. Segregation analyses and comparisons of high-quality genome assemblies revealed that these two species displayed linkage between mating-type loci and their respective centromeres. This arrangement renders the same improved odds of gamete compatibility as direct linkage of the two mating-type loci under the automictic mating (intratetrad selfing) of anther-smut fungi. Recombination cessation was found associated with a large inversion in only one of the four linkage events. The lack of trans-specific polymorphism at genes located in nonrecombining regions and linkage date estimates indicated that the events of recombination cessation occurred independently in the two sister species. Our study shows that natural selection can repeatedly lead to similar genomic patterns and phenotypes, and that different evolutionary paths can lead to distinct yet equally beneficial responses to selection. Our study further highlights that automixis and gene linkage to centromeres have important genetic and evolutionary consequences, while being poorly recognized despite being present in a broad range of taxa.

Multiple convergent supergene evolution events in mating-type chromosomes

Convergent adaptation provides unique insights into the predictability of evolution and ultimately into processes of biological diversification. Supergenes (beneficial gene linkage) are striking examples of adaptation, but little is known about their prevalence or evolution. A recent study on anther-smut fungi documented supergene formation by rearrangements linking two key mating-type loci, controlling pre- and post-mating compatibility. Here further high-quality genome assemblies reveal four additional independent cases of chromosomal rearrangements leading to regions of suppressed recombination linking these mating-type loci in closely related species. Such convergent transitions in genomic architecture of mating-type determination indicate strong selection favoring linkage of mating-type loci into cosegregating supergenes. We find independent evolutionary strata (stepwise recombination suppression) in several species, with extensive rearrangements, gene losses, and transposable element accumulation. We thus show remarkable convergence in mating-type chromosome evolution, recurrent supergene formation, and repeated evolution of similar phenotypes through different genomic changes.

Self-fertility in Chromocrea spinulosa is a consequence of direct repeat-mediated loss of MAT1-2, subsequent imbalance of nuclei differing in mating type, and recognition between unlike nuclei in a common cytoplasm.

The filamentous fungus Chromocrea spinulosa (Trichoderma spinulosum) exhibits both self-fertile (homothallic) and self-sterile (heterothallic) sexual reproductive behavior. Self-fertile strains produce progeny cohorts that are 50% homothallic, 50% heterothallic. Heterothallic progeny can mate only with homothallic strains, and progeny also segregate 50% homothallic, 50% heterothallic. Sequencing of the mating type (MAT) region of homothallic and heterothallic strains revealed that both carry an intact MAT1-1 locus with three MAT1-1 genes (MAT1-1-1, MAT1-1-2, MAT1-1-3), as previously described for the Sordariomycete group of filamentous fungi. Homothallic strains, however, have a second version of MAT with the MAT1-2 locus genetically linked to MAT1-1. In this version, the MAT1-1-1 open reading frame is split into a large and small fragment and the truncated ends are bordered by 115bp direct repeats (DR). The MAT1-2-1 gene and additional sequences are inserted between the repeats. To understand the mechanism whereby C. spinulosa can exhibit both homothallic and heterothallic behavior, we utilized molecular manipulation to delete one of the DRs from a homothallic strain and insert MAT1-2 into a heterothallic strain. Mating assays indicated that: i) the DRs are key to homothallic behavior, ii) looping out of MAT1-2-1 via intra-molecular homologous recombination between the DRs in self-fertile strains results in two nuclear types in an individual (one carrying both MAT1-1 and MAT1-2 and one carrying MAT1-1 only), iii) self-fertility is achieved by inter-nuclear recognition between these two nuclear types before meiosis, iv) the two types of nuclei are in unequal proportion, v) having both an intact MAT1-1-1 and MAT1-2-1 gene in a single nucleus is not sufficient for self-fertility, and vi) the large truncated MAT1-1-1 fragment is expressed. Comparisons with MAT regions of Trichoderma reesei and Trichoderma virens suggest that several crossovers between misaligned parental MAT chromosomes may have led to the MAT architecture of homothallic C. spinulosa.

Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism

Sex chromosomes can display successive steps of recombination suppression known as "evolutionary strata," which are thought to result from the successive linkage of sexually antagonistic genes to sex-determining genes. However, there is little evidence to support this explanation. Here we investigate whether evolutionary strata can evolve without sexual antagonism using fungi that display suppressed recombination extending beyond loci determining mating compatibility despite lack of male/female roles associated with their mating types. By comparing full-length chromosome assemblies from five anther-smut fungi with or without recombination suppression in their mating-type chromosomes, we inferred the ancestral gene order and derived chromosomal arrangements in this group. This approach shed light on the chromosomal fusion underlying the linkage of mating-type loci in fungi and provided evidence for multiple clearly resolved evolutionary strata over a range of ages (0.9-2.1 million years) in mating-type chromosomes. Several evolutionary strata did not include genes involved in mating-type determination. The existence of strata devoid of mating-type genes, despite the lack of sexual antagonism, calls for a unified theory of sex-related chromosome evolution, incorporating, for example, the influence of partially linked deleterious mutations and the maintenance of neutral rearrangement polymorphism due to balancing selection on sexes and mating types.

Introgression maintains the genetic integrity of the mating-type determining chromosome of the fungus Neurospora tetrasperma.

Genome evolution is driven by a complex interplay of factors, including selection, recombination, and introgression. The regions determining sexual identity are particularly dynamic parts of eukaryotic genomes that are prone to molecular degeneration associated with suppressed recombination. In the fungus Neurospora tetrasperma, it has been proposed that this molecular degeneration is counteracted by the introgression of nondegenerated DNA from closely related species. In this study, we used comparative and population genomic analyses of 92 genomes from eight phylogenetically and reproductively isolated lineages of N. tetrasperma, and its three closest relatives, to investigate the factors shaping the evolutionary history of the genomes. We found that suppressed recombination extends across at least 6 Mbp (∼63%) of the mating-type (mat) chromosome in N. tetrasperma and is associated with decreased genetic diversity, which is likely the result primarily of selection at linked sites. Furthermore, analyses of molecular evolution revealed an increased mutational load in this region, relative to recombining regions. However, comparative genomic and phylogenetic analyses indicate that the mat chromosomes are temporarily regenerated via introgression from sister species; six of eight lineages show introgression into one of their mat chromosomes, with multiple Neurospora species acting as donors. The introgressed tracts have been fixed within lineages, suggesting that they confer an adaptive advantage in natural populations, and our analyses support the presence of selective sweeps in at least one lineage. Thus, these data strongly support the previously hypothesized role of introgression as a mechanism for the maintenance of mating-type determining chromosomal regions.

Deciphering evolutionary strata on plant sex chromosomes and fungal mating-type chromosomes through compositional segmentation

Sex chromosomes have evolved from a pair of homologous autosomes which differentiated into sex determination systems, such as XY or ZW system, as a consequence of successive recombination suppression between the gametologous chromosomes. Identifying the regions of recombination suppression, namely, the "evolutionary strata", is central to understanding the history and dynamics of sex chromosome evolution. Evolution of sex chromosomes as a consequence of serial recombination suppressions is well-studied for mammals and birds, but not for plants, although 48 dioecious plants have already been reported. Only two plants Silene latifolia and papaya have been studied until now for the presence of evolutionary strata on their X chromosomes, made possible by the sequencing of sex-linked genes on both the X and Y chromosomes, which is a requirement of all current methods that determine stratum structure based on the comparison of gametologous sex chromosomes. To circumvent this limitation and detect strata even if only the sequence of sex chromosome in the homogametic sex (i.e. X or Z chromosome) is available, we have developed an integrated segmentation and clustering method. In application to gene sequences on the papaya X chromosome and protein-coding sequences on the S. latifolia X chromosome, our method could decipher all known evolutionary strata, as reported by previous studies. Our method, after validating on known strata on the papaya and S. latifolia X chromosome, was applied to the chromosome 19 of Populus trichocarpa, an incipient sex chromosome, deciphering two, yet unknown, evolutionary strata. In addition, we applied this approach to the recently sequenced sex chromosome V of the brown alga Ectocarpus sp. that has a haploid sex determination system (UV system) recovering the sex determining and pseudoautosomal regions, and then to the mating-type chromosomes of an anther-smut fungus Microbotryum lychnidis-dioicae predicting five strata in the non-recombining region of both the chromosomes.

Breaking linkage between mating compatibility factors: Tetrapolarity in Microbotryum.

Linkage of genes determining separate self-incompatibility mechanisms is a general expectation of sexual eukaryotes that helps to resolve conflicts between reproductive assurance and recombination. However, in some organisms, multiple loci are required to be heterozygous in offspring while segregating independently in meiosis. This condition, termed "tetrapolarity" in basidiomycete fungi, originated in the ancestor to that phylum, and there have been multiple reports of subsequent transitions to "bipolarity" (i.e., linkage of separate mating factors). In the genus Microbotryum, we present the first report of the breaking of linkage between two haploid self-incompatibility factors and derivation of a tetrapolar breeding system. This breaking of linkage is associated with major alteration of genome structure, with the compatibility factors residing on separate mating-type chromosome pairs, reduced in size but retaining the structural dimorphism characteristic for regions of recombination suppression. The challenge to reproductive assurance from unlinked compatibility factors may be overcome by the automictic mating system in Microbotryum (i.e., mating among products of the same meiosis). As a curious outcome, this linkage transition and its effects upon outcrossing compatibility rates may reinforce automixis as a mating system. These observations contribute to understanding mating systems and linkage as fundamental principles of sexual life cycles, with potential impacts on conventional wisdom regarding mating-type evolution.