Evidence of the accumulation of allele-specific non-synonymous substitutions in the young region of recombination suppression within the mating-type chromosomes of Neurospora tetrasperma

Currently, little is known about the origin and early evolution of sex chromosomes. This is largely due to the fact that ancient non-recombining sex chromosomes are highly degenerated, and thus provide little information about the early genomic events in their evolution. The Neurospora tetrasperma mating-type (mat) chromosomes contain a young (<6 Mya) and large region (>6.6 Mb) of suppressed recombination, thereby providing a model system to study early stages of sex chromosome evolution. Here, we examined alleles of 207 genes located on the N. tetrasperma mat a and mat A chromosomes to test for signs of genomic alterations at the protein level in the young region of recombination suppression. We report that the N. tetrasperma mat a and mat A chromosomes have each independently accumulated allele-specific non-synonymous codon substitutions in a time-dependent, and gene-specific manner in the recombinationally suppressed region. In addition, examination of the ratio (ω) of non-synonymous substitutions (dN) to synonymous substitutions (dS) using maximum likelihood analyses, indicates that such changes are associated with relaxed purifying selection, a finding consistent with genomic degeneration. We also reveal that sex specific biases in mutation rates or selection pressures are not necessary for genomic alterations in sex chromosomes, and that recombination suppression in itself is sufficient to explain these results. The present findings extend our current understanding of genomic events associated within the young region of recombination suppression in these fungal sex-regulating 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.     

Chaos of Rearrangements in the Mating-Type Chromosomes of the Anther-Smut Fungus Microbotryum lychnidis-dioicae

Sex chromosomes in plants and animals and fungal mating-type chromosomes often show exceptional genome features, with extensive suppression of homologous recombination and cytological differentiation between members of the diploid chromosome pair. Despite strong interest in the genetics of these chromosomes, their large regions of suppressed recombination often are enriched in transposable elements and therefore can be challenging to assemble. Here we show that the latest improvements of the PacBio sequencing yield assembly of the whole genome of the anther-smut fungus, Microbotryum lychnidis-dioicae (the pathogenic fungus causing anther-smut disease of Silene latifolia), into finished chromosomes or chromosome arms, even for the repeat-rich mating-type chromosomes and centromeres. Suppressed recombination of the mating-type chromosomes is revealed to span nearly 90% of their lengths, with extreme levels of rearrangements, transposable element accumulation, and differentiation between the two mating types. We observed no correlation between allelic divergence and physical position in the nonrecombining regions of the mating-type chromosomes. This may result from gene conversion or from rearrangements of ancient evolutionary strata, i.e., successive steps of suppressed recombination. Centromeres were found to be composed mainly of copia-like transposable elements and to possess specific minisatellite repeats identical between the different chromosomes. We also identified subtelomeric motifs. In addition, extensive signs of degeneration were detected in the nonrecombining regions in the form of transposable element accumulation and of hundreds of gene losses on each mating-type chromosome. Furthermore, our study highlights the potential of the latest breakthrough PacBio chemistry to resolve complex genome architectures.     

Degeneration in codon usage within the region of suppressed recombination in the mating-type chromosomes of Neurospora tetrasperma

The origin and early evolution of sex chromosomes are currently poorly understood. The Neurospora tetrasperma mating-type (mat) chromosomes have recently emerged as a model system for the study of early sex chromosome evolution, since they contain a young (<6 million years ago [Mya]), large (>6.6-Mb) region of suppressed recombination. Here we examined preferred-codon usage in 290 genes (121,831 codon positions) in order to test for early signs of genomic degeneration in N. tetrasperma mat chromosomes. We report several key findings about codon usage in the region of recombination suppression, including the following: (i) this region has been subjected to marked and largely independent degeneration among gene alleles; (ii) the level of degeneration is magnified over longer periods of recombination suppression; and (iii) both mat a and mat A chromosomes have been subjected to deterioration. The frequency of shifts from preferred codons to nonpreferred codons is greater for shorter genes than for longer genes, suggesting that short genes play an especially significant role in early sex chromosome evolution. Furthermore, we show that these degenerative changes in codon usage are best explained by altered selection efficiency in the recombinationally suppressed region. These findings demonstrate that the fungus N. tetrasperma provides an effective system for the study of degenerative genomic changes in young regions of recombination suppression in sex-regulating chromosomes.     

RECENT GENE‐CAPTURE ON THE UV SEX CHROMOSOMES OF THE MOSS CERATODON PURPUREUS

Sex chromosomes evolve from ordinary autosomes through the expansion and subsequent degeneration of a region of suppressed recombination that is inherited through one sex. Here we investigate the relative timing of these processes in the UV sex chromosomes of the moss Ceratodon purpureus using molecular population genetic analyses of eight newly discovered sex‐linked loci. In this system, recombination is suppressed on both the female‐transmitted (U) sex chromosome and the male‐transmitted (V) chromosome. Genes on both chromosomes therefore should show the deleterious effects of suppressed recombination and sex‐limited transmission, while purifying selection should maintain homologs of genes essential for both sexes on both sex chromosomes. Based on analyses of eight sex‐linked loci, we show that the nonrecombining portions of the U and V chromosomes expanded in at least two events (～0.6–1.3 MYA and ～2.8–3.5 MYA), after the divergence of C. purpureus from its dioecious sister species, Trichodon cylindricus and Cheilothela chloropus. Both U‐ and V‐linked copies showed reduced nucleotide diversity and limited population structure, compared to autosomal loci, suggesting that the sex chromosomes experienced more recent selective sweeps that the autosomes. Collectively these results highlight the dynamic nature of gene composition and molecular evolution on nonrecombining portions of the U and V sex chromosomes.     

Fast Accumulation of Nonsynonymous Mutations on the Female-Specific W Chromosome in Birds

Following cessation of recombination during sex chromosome evolution, the nonrecombining sex chromosome is affected by a number of degenerative forces, possibly resulting in the fixation of deleterious mutations. This might take place because of weak selection against recessive or partly recessive deleterious mutations due to permanent heterozygosity of nonrecombining chromosomes. Furthermore, population genetic processes, such as selective sweeps, background selection, and Muller’s ratchet, result in a reduction in N_e, which increase the likelihood of fixation of deleterious mutations. Theory thus predicts that nonrecombining genes should show increased levels of nonsynonymous (d_N) to synonymous substitutions (d_S). We tested this in an avian system by estimating the ratio between d_N and d_S in six gametologous gene pairs located on the Z chromosome and the nonrecombining, female-specific W chromosome. In comparisons, we found a significantly higher d_N/d_S ratio for the W-linked than the Z-linked copy in three of the investigated genes. In a concatenated alignment of all six genes, the d_N/d_S ratio was six times higher for W-linked than Z-linked genes. By using human and mouse as outgroup in maximum likelihood analyses, W-linked genes were found to evolve differently compared with their Z-linked gametologues and outgroup sequences. This seems not to be a consequence of functional diversification because d_N/d_S ratios between gametologous gene copies were consistently low. We conclude that deleterious mutations are accumulating at a high rate on the avian W chromosome, probably as a result of the lack of recombination in this female-specific chromosome. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Deborah Charlesworth]     

Positive and negative selection on mammalian Y chromosomes

Y chromosomes are genetically degenerate in most organisms studied. The loss of genes from Y chromosomes is thought to be due to the inefficiency of purifying selection in nonrecombining regions, which leads to the accumulation of deleterious mutations via the processes of hitchhiking, background selection, and Muller's ratchet. As the severity of these processes depends on the number of functional genes linked together on the nonrecombining Y, it is not clear whether these processes are still at work on the old, gene-poor mammalian Y chromosomes. If purifying selection is indeed less efficient in the Y-linked, compared to the X-linked genes, deleterious nonsynonymous substitutions are expected to accumulate faster on the Y chromosome. However, positive selection on Y-linked genes could also increase the rate of amino acid-changing substitutions. Thus, the previous reports of an elevated nonsynonymous substitution rate in Y-linked genes are still open to interpretation. Here, we report evidence for positive selection in two out of three studied mammalian Y-linked genes, suggesting that adaptive Darwinian evolution may be common on mammalian Y chromosomes. Taking positive selection into account, we demonstrate that purifying selection is less efficient in mammalian Y-linked genes compared to their X-linked homologues, suggesting that these genes continue to degenerate.     

Extensive Divergence Between Mating-Type Chromosomes of the Anther-Smut Fungus

Genomic regions that determine mating compatibility are subject to distinct evolutionary forces that can lead to a cessation of meiotic recombination and the accumulation of structural changes between members of the homologous chromosome pair. The relatively recent discovery of dimorphic mating-type chromosomes in fungi can aid the understanding of sex chromosome evolution that is common to dioecious plants and animals. For the anther-smut fungus, Microbotryum lychnidis-dioicae (= M. violaceum isolated from Silene latifolia), the extent of recombination cessation on the dimorphic mating-type chromosomes has been conflictingly reported. Comparison of restriction digest optical maps for the two mating-type chromosomes shows that divergence extends over 90% of the chromosome lengths, flanked at either end by two pseudoautosomal regions. Evidence to support the expansion of recombination cessation in stages from the mating-type locus toward the pseudoautosomal regions was not found, but evidence of such expansion could be obscured by ongoing processes that affect genome structure. This study encourages the comparison of forces that may drive large-scale recombination suppression in fungi and other eukaryotes characterized by dimorphic chromosome pairs associated with sexual life cycles.     

Detecting positive selection within genomes: the problem of biased gene conversion

The identification of loci influenced by positive selection is a major goal of evolutionary genetics. A popular approach is to perform scans of alignments on a genome-wide scale in order to find regions evolving at accelerated rates on a particular branch of a phylogenetic tree. However, positive selection is not the only process that can lead to accelerated evolution. Notably, GC-biased gene conversion (gBGC) is a recombination-associated process that results in the biased fixation of G and C nucleotides. This process can potentially generate bursts of nucleotide substitutions within hotspots of meiotic recombination. Here, we analyse the results of a scan for positive selection on genes on branches across the primate phylogeny. We show that genes identified as targets of positive selection have a significant tendency to exhibit the genomic signature of gBGC. Using a maximum-likelihood framework, we estimate that more than 20 per cent of cases of significantly elevated non-synonymous to synonymous substitution rates ratio (d_N/d_S), particularly in shorter branches, could be due to gBGC. We demonstrate that in some cases, gBGC can lead to very high d_N/d_S (more than 2). Our results indicate that gBGC significantly affects the evolution of coding sequences in primates, often leading to patterns of evolution that can be mistaken for positive selection.     

The Trouble with Sliding Windows and the Selective Pressure in BRCA1

Sliding-window analysis has widely been used to uncover synonymous (silent, d_S) and nonsynonymous (replacement, d_N) rate variation along the protein sequence and to detect regions of a protein under selective constraint (indicated by d_N<d_S) or positive selection (indicated by d_N>d_S). The approach compares two or more protein-coding genes and plots estimates dˆ _S and dˆ _N from each sliding window along the sequence. Here we demonstrate that the approach produces artifactual trends of synonymous and nonsynonymous rate variation, with greater variation in dˆ _S than in dˆ _N. Such trends are generated even if the true d_S and d_N are constant along the whole protein and different codons are evolving independently. Many published tests of negative and positive selection using sliding windows that we have examined appear to be invalid because they fail to correct for multiple testing. Instead, likelihood ratio tests provide a more rigorous framework for detecting signals of natural selection affecting protein evolution. We demonstrate that a previous finding that a particular region of the BRCA1 gene experienced a synonymous rate reduction driven by purifying selection is likely an artifact of the sliding window analysis. We evaluate various sliding-window analyses in molecular evolution, population genetics, and comparative genomics, and argue that the approach is not generally valid if it is not known a priori that a trend exists and if no correction for multiple testing is applied.     

Species and Population Level Molecular Profiling Reveals Cryptic Recombination and Emergent Asymmetry in the Dimorphic Mating Locus of C. reinhardtii

Heteromorphic sex-determining regions or mating-type loci can contain large regions of non-recombining sequence where selection operates under different constraints than in freely recombining autosomal regions. Detailed studies of these non-recombining regions can provide insights into how genes are gained and lost, and how genetic isolation is maintained between mating haplotypes or sex chromosomes. The Chlamydomonas reinhardtii mating-type locus (MT) is a complex polygenic region characterized by sequence rearrangements and suppressed recombination between its two haplotypes, MT+ and MT−. We used new sequence information to redefine the genetic contents of MT and found repeated translocations from autosomes as well as sexually controlled expression patterns for several newly identified genes. We examined sequence diversity of MT genes from wild isolates of C. reinhardtii to investigate the impacts of recombination suppression. Our population data revealed two previously unreported types of genetic exchange in Chlamydomonas MT—gene conversion in the rearranged domains, and crossover exchanges in flanking domains—both of which contribute to maintenance of genetic homogeneity between haplotypes. To investigate the cause of blocked recombination in MT we assessed recombination rates in crosses where the parents were homozygous at MT. While normal recombination was restored in MT+×MT+ crosses, it was still suppressed in MT−×MT− crosses. These data revealed an underlying asymmetry in the two MT haplotypes and suggest that sequence rearrangements are insufficient to fully account for recombination suppression. Together our findings reveal new evolutionary dynamics for mating loci and have implications for the evolution of heteromorphic sex chromosomes and other non-recombining genomic regions.     

Integrating genomics,bioinformatics, and classical genetics to study the effects of recombination on genome evolution

This study presents compelling evidence that recombination significantly increases the silent GC content of a genome in a selectively neutral manner, resulting in a highly significant positive correlation between recombination and "GC3s" in the yeast Saccharomyces cerevisiae. Neither selection nor mutation can explain this relationship. A highly significant GC-biased mismatch repair system is documented for the first time in any member of the Kingdom Fungi. Much of the variation in the GC3s within yeast appears to result from GC-biased gene conversion. Evidence suggests that GC-biased mismatch repair exists in numerous organisms spanning six kingdoms. This transkingdom GC mismatch repair bias may have evolved in response to a ubiquitous AT mutational bias. A significant positive correlation between recombination and GC content is found in many of these same organisms, suggesting that the processes influencing the evolution of the yeast genome may be a general phenomenon. Nonrecombining regions of the genome and nonrecombining genomes would not be subject to this type of molecular drive. It is suggested that the low GC content characteristic of many nonrecombining genomes may be the result of three processes (1) a prevailing AT mutational bias, (2) random fixation of the most common types of mutation, and (3) the absence of the GC-biased gene conversion which, in recombining organisms, permits the reversal of the most common types of mutation. A model is proposed to explain the observation that introns, intergenic regions, and pseudogenes typically have lower GC content than the silent sites of corresponding open reading frames. This model is based on the observation that the greater the heterology between two sequences, the less likely it is that recombination will occur between them. According to this "Constraint" hypothesis, the formation and propagation of heteroduplex DNA is expected to occur, on average, more frequently within conserved coding and regulatory regions of the genome. In organisms possessing GC-biased mismatch repair, this would enhance the GC content of these regions through biased gene conversion. These findings have a number of important implications for the way we view genome evolution and suggest a new model for the evolution of sex.     

Massive changes in genome architecture accompany the transition to self-fertility in the filamentous fungus Neurospora tetrasperma

A large region of suppressed recombination surrounds the sex-determining locus of the self-fertile fungus Neurospora tetrasperma. This region encompasses nearly one-fifth of the N. tetrasperma genome and suppression of recombination is necessary for self-fertility. The similarity of the N. tetrasperma mating chromosome to plant and animal sex chromosomes and its recent origin (<5 MYA), combined with a long history of genetic and cytological research, make this fungus an ideal model for studying the evolutionary consequences of suppressed recombination. Here we compare genome sequences from two N. tetrasperma strains of opposite mating type to determine whether structural rearrangements are associated with the nonrecombining region and to examine the effect of suppressed recombination for the evolution of the genes within it. We find a series of three inversions encompassing the majority of the region of suppressed recombination and provide evidence for two different types of rearrangement mechanisms: the recently proposed mechanism of inversion via staggered single-strand breaks as well as ectopic recombination between transposable elements. In addition, we show that the N. tetrasperma mat a mating-type region appears to be accumulating deleterious substitutions at a faster rate than the other mating type (mat A) and thus may be in the early stages of degeneration.     

Avian Neo-Sex Chromosomes Reveal Dynamics of Recombination Suppression and W Degeneration

How the avian sex chromosomes first evolved from autosomes remains elusive as 100 million years (My) of divergence and degeneration obscure their evolutionary history. The Sylvioidea group of songbirds is interesting for understanding avian sex chromosome evolution because a chromosome fusion event ∼24 Ma formed “neo-sex chromosomes” consisting of an added (new) and an ancestral (old) part. Here, we report the complete female genome (ZW) of one Sylvioidea species, the great reed warbler (Acrocephalus arundinaceus). Our long-read assembly shows that the added region has been translocated to both Z and W, and whereas the added-Z has retained its gene order the added-W part has been heavily rearranged. Phylogenetic analyses show that recombination between the homologous added-Z and -W regions continued after the fusion event, and that recombination suppression across this region took several million years to be completed. Moreover, recombination suppression was initiated across multiple positions over the added-Z, which is not consistent with a simple linear progression starting from the fusion point. As expected following recombination suppression, the added-W show signs of degeneration including repeat accumulation and gene loss. Finally, we present evidence for nonrandom maintenance of slowly evolving and dosage-sensitive genes on both ancestral- and added-W, a process causing correlated evolution among orthologous genes across broad taxonomic groups, regardless of sex linkage.     

Inferring Adaptive Codon Preference to Understand Sources of Selection Shaping Codon Usage Bias

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify “adaptive codon preference,” a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated “preference” largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.     

The evolution of sex chromosomes in organisms with separate haploid sexes

The evolution of dimorphic sex chromosomes is driven largely by the evolution of reduced recombination and the subsequent accumulation of deleterious mutations. Although these processes are increasingly well understood in diploid organisms, the evolution of dimorphic sex chromosomes in haploid organisms (U/V) has been virtually unstudied theoretically. We analyze a model to investigate the evolution of linkage between fitness loci and the sex‐determining region in U/V species. In a second step, we test how prone nonrecombining regions are to degeneration due to accumulation of deleterious mutations. Our modeling predicts that the decay of recombination on the sex chromosomes and the addition of strata via fusions will be just as much a part of the evolution of haploid sex chromosomes as in diploid sex chromosome systems. Reduced recombination is broadly favored, as long as there is some fitness difference between haploid males and females. The degeneration of the sex‐determining region due to the accumulation of deleterious mutations is expected to be slower in haploid organisms because of the absence of masking. Nevertheless, balancing selection often drives greater differentiation between the U/V sex chromosomes than in X/Y and Z/W systems. We summarize empirical evidence for haploid sex chromosome evolution and discuss our predictions in light of these findings.     

Hotspots of Biased Nucleotide Substitutions in Human Genes

Genes that have experienced accelerated evolutionary rates on the human lineage during recent evolution are candidates for involvement in human-specific adaptations. To determine the forces that cause increased evolutionary rates in certain genes, we analyzed alignments of 10,238 human genes to their orthologues in chimpanzee and macaque. Using a likelihood ratio test, we identified protein-coding sequences with an accelerated rate of base substitutions along the human lineage. Exons evolving at a fast rate in humans have a significant tendency to contain clusters of AT-to-GC (weak-to-strong) biased substitutions. This pattern is also observed in noncoding sequence flanking rapidly evolving exons. Accelerated exons occur in regions with elevated male recombination rates and exhibit an excess of nonsynonymous substitutions relative to the genomic average. We next analyzed genes with significantly elevated ratios of nonsynonymous to synonymous rates of base substitution (d_N/d_S) along the human lineage, and those with an excess of amino acid replacement substitutions relative to human polymorphism. These genes also show evidence of clusters of weak-to-strong biased substitutions. These findings indicate that a recombination-associated process, such as biased gene conversion (BGC), is driving fixation of GC alleles in the human genome. This process can lead to accelerated evolution in coding sequences and excess amino acid replacement substitutions, thereby generating significant results for tests of positive selection.