Summary: Genomic regions determining sexual compatibility often display recombination suppression, as occurs in sex chromosomes, plant self‐incompatibility loci and fungal mating‐type loci. Regions lacking recombination can extend beyond the genes determining sexes or mating types, by several successive steps of recombination suppression. Here we review the evidence for recombination suppression around mating‐type loci in fungi, sometimes encompassing vast regions of the mating‐type chromosomes. The suppression of recombination at mating‐type loci in fungi has long been recognized and maintains the multiallelic combinations required for correct compatibility determination. We review more recent evidence for expansions of recombination suppression beyond mating‐type genes in fungi (‘evolutionary strata’), which have been little studied and may be more pervasive than commonly thought. We discuss testable hypotheses for the ultimate (evolutionary) and proximate (mechanistic) causes for such expansions of recombination suppression, including (1) antagonistic selection, (2) association of additional functions to mating‐type, such as uniparental mitochondria inheritance, (3) accumulation in the margin of nonrecombining regions of various factors, including deleterious mutations or transposable elements resulting from relaxed selection, or neutral rearrangements resulting from genetic drift. The study of recombination suppression in fungi could thus contribute to our understanding of recombination suppression expansion across a broader range of organisms.

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.

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 3.58 million 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.

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 3.58 million 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.

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 non-recombining regions, i.e., 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 non-optimal codons and non-synonymous substitutions in non-recombining regions. We found a reduced frequency of optimal codons in the non-recombining regions compared to autosomes, that was not due to less frequent GC-biased gene conversion or lower ancestral expression levels compared to recombining regions. The frequency of optimal codons rapidly decreased following recombination suppression and reached an asymptote after ca 3 Mya. The strength of purifying selection remained virtually constant at d _N /d _S = 0.55, i.e . at an intermediate level between purifying selection and neutral evolution. Accordingly, non-synonymous 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 non-recombining regions, which is important for our knowledge on genomic evolution and on the maintenance of regions without recombination.

Sex chromosome evolution is traditionally viewed as a unidirectional process of recombination suppression followed by progressive Y degeneration, yet many vertebrates deviate from this canonical trajectory. Frogs represent a striking exception: they retain homomorphic sex chromosomes, exhibit extreme heterochiasmy, and undergo X-Y recombination in sex-reversed females, providing a powerful system to explore alternative evolutionary pathways. Here, we investigate a Swiss Alpine population of the European common frog ( Rana temporaria ) with homomorphic XY chromosomes and three coexisting male genotypes, using whole genome sequencing of pooled samples carrying distinct Y haplotypes, whole genome sequencing of doubled haploid YY individuals, and transcriptomic (RNA-seq) data. We uncover extreme heterogeneity in X–Y divergence: two fully differentiated Y haplotypes span nearly 90% of the sex chromosome (∼618–625 Mb), forming the largest non-recombining region (large NRR) described in a vertebrate to date, whereas a semi-differentiated Y harbors only a NRR of 4.64 Mb (small NRR), and XX males display no detectable X-Y divergence. Despite extensive recombination suppression, Y degeneration is minimal even within the large NRR: we detect elevated transposable element insertions but no gene loss, Y-linked gene copy decay, faster-X effects, or enrichment of sex-biased genes. All Y haplotypes share a small NRR encompassing the candidate master sex-determining gene Dmrt1 , defining a common sex-determining locus. Our data support a dynamic model in which extreme heterochiasmy and recurrent X–Y recombination via sex reversal repeatedly reset X-Y divergence, generating and potentially maintaining multiple Y haplotypes with distinct evolutionary histories. The long-term coexistence of these haplotypes across the species range is compatible with a dynamic equilibrium between drift-driven loss and mutation- and recombination-driven renewal of Y chromosome variation. Together, our findings reveal a non-canonical, reversible pathway of sex chromosome evolution shaped by sex-specific recombination patterns and sex reversal, challenging the universality of classical models and highlighting the value of non-model vertebrates for understanding sex chromosome diversity.