Roberta Bergero

Evolutionary Strata on the X Chromosomes of the Dioecious Plant Silene latifolia: Evidence From New Sex-Linked Genes

Despite its recent evolutionary origin, the sex chromosome system of the plant Silene latifolia shows signs of progressive suppression of recombination having created evolutionary strata of different X–Y divergence on sex chromosomes. However, even after 8 years of effort, this result is based on analyses of five sex-linked gene sequences, and the maximum divergence (and thus the age of this plants sex chromosome system) has remained uncertain. More genes are therefore needed. Here, by segregation analysis of intron size variants (ISVS) and single nucleotide polymorphisms (SNPs), we identify three new Y-linked genes, one being duplicated on the Y chromosome, and test for evolutionary strata. All the new genes have homologs on the X and Y chromosomes. Synonymous divergence estimated between the X and Y homolog pairs is within the range of those already reported. Genetic mapping of the new X-linked loci shows that the map is the same in all three families that have been studied so far and that X–Y divergence increases with genetic distance from the pseudoautosomal region. We can now conclude that the divergence value is saturated, confirming the cessation of X–Y recombination in the evolution of the sex chromosomes at ∼10–20 MYA.

Evidence for Degeneration of the Y Chromosome in the Dioecious Plant Silene latifolia

The human Y--probably because of its nonrecombining nature--has lost 97% of its genes since X and Y chromosomes started to diverge [1, 2]. There are clear signs of degeneration in the Drosophila miranda neoY chromosome (an autosome fused to the Y chromosome), with neoY genes showing faster protein evolution [3-6], accumulation of unpreferred codons [6], more insertions of transposable elements [5, 7], and lower levels of expression [8] than neoX genes. In the many other taxa with sex chromosomes, Y degeneration has hardly been studied. In plants, many genes are expressed in pollen [9], and strong pollen selection may oppose the degeneration of plant Y chromosomes [10]. Silene latifolia is a dioecious plant with young heteromorphic sex chromosomes [11, 12]. Here we test whether the S. latifolia Y chromosome is undergoing genetic degeneration by analyzing seven sex-linked genes. S. latifolia Y-linked genes tend to evolve faster at the protein level than their X-linked homologs, and they have lower expression levels. Several Y gene introns have increased in length, with evidence for transposable-element accumulation. We detect signs of degeneration in most of the Y-linked gene sequences analyzed, similar to those of animal Y-linked and neo-Y chromosome genes.

Preservation of the Y Transcriptome in a 10-Million-Year-Old Plant Sex Chromosome System

Classical genetic studies discovered loss of genes from the ancient sex chromosome systems of several animals (genetic degeneration), and complete genome sequencing confirms that the heterogametic sex is hemizygous for most sex-linked genes. Genetic degeneration is thought to result from the absence of recombination between the sex chromosome pair (reviewed by [1]) and is very rapid after sex chromosome-autosome fusions in Drosophila [2-4]. Plant sex chromosome systems allow study of the time course of degeneration, because they evolved from a state wholly without sex chromosomes (rather than after a large genome region fused to a preexisting sex chromosome), and, in several taxa, recombination stopped very recently. However, despite increasing genetic and physical mapping of plant nonrecombining sex-determining regions [5-8], it remains very difficult to discover sex-linked genes, and it is unclear whether Y-linked genes are losing full function. We therefore developed a high-throughput method using RNA-Seq to identify sex linkage in Silene latifolia. Recombination suppression between this plants XY sex chromosome pair started only about 10 million years ago [9]. Our approach identifies several hundred new sex-linked genes, and we show that this young Y chromosome retains many genes, yet these already have slightly reduced gene expression and are accumulating changes likely to reduce protein functions.

Defining Regions and Rearrangements of the Silene latifolia Y Chromosome

We combine data from published marker genotyping of three sets of S. latifolia Y chromosome deletion mutants with changed sex phenotypes and add genotypes for several new genic markers to refine the deletion map of the Y chromosome and compare it with the X chromosome genetic map. We conclude that the Y chromosome of this species has been derived through multiple rearrangements of the ancestral gene arrangement and that none of the rearrangements so far detected was involved in stopping X–Y recombination. Different Y genotypes may also differ in their gene content and possibly arrangements, suggesting that mapping the Y-linked sex-determining genes will be difficult, even if many further genic markers are obtained. Even in determining the map of Y chromosome markers to discover all the rearrangements, physical mapping by FISH or other experiments will be essential. Future deletion mapping work should ensure that markers are studied in the parents of deletion mutants and should probably include additional deletions that were not ascertained by causing mutant sex phenotypes.

Patterns of Codon Usage Bias in Silene latifolia

Patterns of codon usage bias (CUB) convey useful information about the selection on synonymous codons induced by gene expression and contribute to an understanding of substitution patterns observed at synonymous sites. They can also be informative about the distinctive evolutionary properties of sex chromosomes such as genetic degeneration of the Y chromosome, dosage compensation, and hemizygosity of the X chromosome in males, which can affect the selection on codon usage. Here, we study CUB in Silene latifolia, a species of interest for studying the early stages of sex chromosome evolution. We have obtained a large expressed sequence tag data set containing more than 1,608 sequence fragments by 454 sequencing. Using three different methods, we conservatively define 21 preferred codons. Interestingly, the preferred codons in S. latifolia are almost identical to those in Arabidopsis thaliana, despite their long divergence time (we estimate average nonsynonymous site divergence to be 0.216, and synonymous sites are saturated). The agreement suggests that the nature of selection on codon usage has not changed significantly during the long evolutionary time separating the two species. As in many other organisms, the frequency of preferred codons is negatively correlated with protein length. For the 43 genes with both exon and intron sequences, we find a positive correlation between gene expression levels and GC content at third codon positions, but a strong negative correlation between expression and intron GC content, suggesting that the CUB we detect in S. latifolia is more likely to be due to natural selection than to mutational bias. Using polymorphism data, we detect evidence of ongoing natural selection on CUB, but we find little support for effects of biased gene conversion. An analysis of ten sex-linked genes reveals that the X chromosome has experienced significantly more unpreferred to preferred than preferred to unpreferred substitutions, suggesting that it may be evolving higher CUB. In contrast, numbers of substitutions between preferred and unpreferred codons are similar in both directions in the Y-linked genes, contrary to the expectation of genetic degeneration.

Expansion of the Pseudo-autosomal Region and Ongoing Recombination Suppression in the Silene latifolia Sex Chromosomes

There are two very interesting aspects to the evolution of sex chromosomes: what happens after recombination between these chromosome pairs stops and why suppressed recombination evolves. The former question has been intensively studied in a diversity of organisms, but the latter has been studied largely theoretically. To obtain empirical data, we used codominant genic markers in genetic mapping of the dioecious plant Silene latifolia, together with comparative mapping of S. latifolia sex-linked genes in S. vulgaris (a related hermaphrodite species without sex chromosomes). We mapped 29 S. latifolia fully sex-linked genes (including 21 newly discovered from transcriptome sequencing), plus 6 genes in a recombining pseudo-autosomal region (PAR) whose genetic map length is ∼25 cM in both male and female meiosis, suggesting that the PAR may contain many genes. Our comparative mapping shows that most fully sex-linked genes in S. latifolia are located on a single S. vulgaris linkage group and were probably inherited from a single autosome of an ancestor. However, unexpectedly, our maps suggest that the S. latifolia PAR region expanded through translocation events. Some genes in these regions still recombine in S. latifolia, but some genes from both addition events are now fully sex-linked. Recombination suppression is therefore still ongoing in S. latifolia, and multiple recombination suppression events have occurred in a timescale of few million years, much shorter than the timescale of formation of the most recent evolutionary strata of mammal and bird sex chromosomes.

Active Miniature Transposons From a Plant Genome and Its Nonrecombining Y Chromosome

Mechanisms involved in eroding fitness of evolving Y chromosomes have been the focus of much theoretical and empirical work. Evolving Y chromosomes are expected to accumulate transposable elements (TEs), but it is not known whether such accumulation contributes to their genetic degeneration. Among TEs, miniature inverted-repeat transposable elements are nonautonomous DNA transposons, often inserted in introns and untranslated regions of genes. Thus, if they invade Y-linked genes and selection against their insertion is ineffective, they could contribute to genetic degeneration of evolving Y chromosomes. Here, we examine the population dynamics of active MITEs in the young Y chromosomes of the plant Silene latifolia and compare their distribution with those in recombining genomic regions. To isolate active MITEs, we developed a straightforward approach on the basis of the assumption that recent transposon insertions or excisions create singleton or low-frequency size polymorphisms that can be detected in alleles from natural populations. Transposon display was then used to infer the distribution of MITE insertion frequencies. The overall frequency spectrum showed an excess of singleton and low-frequency insertions, which suggests that these elements are readily removed from recombining chromosomes. In contrast, insertions on the Y chromosomes were present at high frequencies. Their potential contribution to Y degeneration is discussed.

Gene Loss from a Plant Sex Chromosome System

Sex chromosomes have evolved independently in numerous animal and plant lineages. After recombination becomes suppressed between two homologous sex chromosomes, genes on the non-recombining Y chromosomes (and W chromosomes in ZW systems) undergo genetic degeneration, losing functions retained by their X- or Z-linked homologs, changing their expression, and becoming lost [1, 2]. Adaptive changes may also occur, both on the non-recombining Y chromosome, to shut down expression of maladapted genes [3], and on the X chromosome (or the Z in ZW systems), which may evolve dosage compensation to increase low expression or compensate for poor protein function in the heterogametic sex [2, 4, 5]. Although empirical approaches to studying genetic degeneration have been developed for model species [3, 6], the onset and dynamics of these changes are still poorly understood, particularly in de novo evolving sex chromosomes. Sex chromosomes of some plants evolved much more recently than those of mammals, birds, and Drosophila [7-9], making them suitable for studying the early stages of genetic degeneration in de novo evolving sex chromosomes. In plants, haploid selection should oppose gene loss from Y chromosomes, but recent work on sex chromosomes of two plant species has estimated that Y-linked transcripts are lacking for 10%-30% of X-linked genes [10-12]. Here, we provide evidence that, in Silene latifolia, this largely involved losses of Y-linked genes, and not suppressed expression of Y-linked alleles, or gene additions to the X chromosome. Our results also suggest that chromosome-wide dosage compensation does not occur in this plant.

Testing for the Footprint of Sexually Antagonistic Polymorphisms in the Pseudoautosomal Region of a Plant Sex Chromosome Pair

The existence of sexually antagonistic (SA) polymorphism is widely considered the most likely explanation for the evolution of suppressed recombination of sex chromosome pairs. This explanation is largely untested empirically, and no such polymorphisms have been identified, other than in fish, where no evidence directly implicates these genes in events causing loss of recombination. We tested for the presence of loci with SA polymorphism in the plant Silene latifolia, which is dioecious (with separate male and female individuals) and has a pair of highly heteromorphic sex chromosomes, with XY males. Suppressed recombination between much of the Y and X sex chromosomes evolved in several steps, and the results in Bergero et al. (2013) show that it is still ongoing in the recombining or pseudoautosomal, regions (PARs) of these chromosomes. We used molecular evolutionary approaches to test for the footprints of SA polymorphisms, based on sequence diversity levels in S. latifolia PAR genes identified by genetic mapping. Nucleotide diversity is high for at least four of six PAR genes identified, and our data suggest the existence of polymorphisms maintained by balancing selection in this genome region, since molecular evolutionary (HKA) tests exclude an elevated mutation rate, and other tests also suggest balancing selection. The presence of sexually antagonistic alleles at a locus or loci in the PAR is suggested by the very different X and Y chromosome allele frequencies for at least one PAR gene.

Slcyt, a Newly Identified Sex-Linked Gene, Has Recently Moved onto the X Chromosome in Silene latifolia (Caryophyllaceae)

The sex chromosomes of the plant species Silene latifolia (white campion) are very young (only 5-10 My old), and all 11 X-linked genes so far described have Y-linked homologues. Theory predicts that X chromosomes should accumulate a nonrandom set of genes. However, little is known about the importance of gene movements between the X and the autosomes in plants, or in any very young sex chromosome system. Here, we isolate from cDNA a new gene, Slcyt, on the S. latifolia X, which encodes a cytochrome B protein. We genetically mapped SlCyt and found that it is located approximately 1 cM from the pseudoautosomal region. Genes in this region of the X chromosome have low divergence values from their homologous Y-linked genes, indicating that the X only recently stopped recombining with the Y. Genetic mapping in Silene vulgaris suggests that Slcyt originally belonged to a different linkage group from that of the other S. latifolia X-linked genes. Silene latifolia has no Y-linked homologue of Slcyt, and also no autosomal paralogues seem to exist. Slcyt moved from an autosome to the X very recently, as the Cyt gene is also X linked in Silene dioica, the sister species to S. latifolia, but is probably autosomal in Silene diclinis, implying that a translocation to the X probably occurred after the split between S. diclinis and S. latifolia/S. dioica. Diversity at Slcyt is extremely low (pi(syn) = 0.16%), and we find an excess of high frequency-derived variants and a negative Tajimas D, suggesting that the translocation was driven by selection.