Has recombination changed during the recent evolution of the guppy Y chromosome?

Genome sequencing and genetic mapping of molecular markers have demonstrated nearly complete Y-linkage across much of the guppy (Poecilia reticulata) XY chromosome pair. Predominant Y-linkage of factors controlling visible male-specific coloration traits also suggested that these polymorphisms are sexually antagonistic (SA). However, occasional exchanges with the X are detected, and recombination patterns also appear to differ between natural guppy populations, suggesting ongoing evolution of recombination suppression under selection created by partially sex-linked SA polymorphisms. We used molecular markers to directly estimate genetic maps in sires from 4 guppy populations. The maps are very similar, suggesting that their crossover patterns have not recently changed. Our maps are consistent with population genomic results showing that variants within the terminal 5 Mb of the 26.5 Mb sex chromosome, chromosome 12, are most clearly associated with the maleness factor, albeit incompletely. We also confirmed occasional crossovers proximal to the male-determining region, defining a second, rarely recombining, pseudo-autosomal region, PAR2. This fish species may therefore have no completely male-specific region (MSY) more extensive than the male-determining factor. The positions of the few crossover events suggest a location for the male-determining factor within a physically small repetitive region. A sex-reversed XX male had few crossovers in PAR2, suggesting that this region's low crossover rate depends on the phenotypic, not the genetic, sex. Thus, rare individuals whose phenotypic and genetic sexes differ, and/or occasional PAR2 crossovers in males can explain the failure to detect fully Y-linked variants.

How did the guppy Y chromosome evolve?

The sex chromosome pairs of many species do not undergo genetic recombination, unlike the autosomes. It has been proposed that the suppressed recombination results from natural selection favouring close linkage between sex-determining genes and mutations on this chromosome with advantages in one sex, but disadvantages in the other (these are called sexually antagonistic mutations). No example of such selection leading to suppressed recombination has been described, but populations of the guppy display sexually antagonistic mutations (affecting male coloration), and would be expected to evolve suppressed recombination. In extant close relatives of the guppy, the Y chromosomes have suppressed recombination, and have lost all the genes present on the X (this is called genetic degeneration). However, the guppy Y occasionally recombines with its X, despite carrying sexually antagonistic mutations. We describe evidence that a new Y evolved recently in the guppy, from an X chromosome like that in these relatives, replacing the old, degenerated Y, and explaining why the guppy pair still recombine. The male coloration factors probably arose after the new Y evolved, and have already evolved expression that is confined to males, a different way to avoid the conflict between the sexes.

PromethION Sequencing and Assembly of the Genome of Micropoecilia picta, a Fish with a Highly Degenerated Y Chromosome.

We here describe sequencing and assembly of both the autosomes and the sex chromosome in Micropoecilia picta, the closest related species to the guppy, Poecilia reticulata. Poecilia (Micropoecilia) picta is a close outgroup for studying the guppy, an important organism for studies in evolutionary ecology and in sex chromosome evolution. The guppy XY pair (LG12) has long been studied as a test case for the importance of sexually antagonistic variants in selection for suppressed recombination between Y and X chromosomes. The guppy Y chromosome is not degenerated, but appears to carry functional copies of all genes that are present on its X counterpart. The X chromosomes of M. picta (and its relative Micropoecilia parae) are homologous to the guppy XY pair, but their Y chromosomes are highly degenerated, and no genes can be identified in the fully Y-linked region. A complete genome sequence of a M. picta male may therefore contribute to understanding how the guppy Y evolved. These fish species' genomes are estimated to be about 750 Mb, with high densities of repetitive sequences, suggesting that long-read sequencing is needed. We evaluated several assembly approaches, and used our results to investigate the extent of Y chromosome degeneration in this species.

Meiosis and beyond - understanding the mechanistic and evolutionary processes shaping the germline genome.

The separation of germ cell populations from the soma is part of the evolutionary transition to multicellularity. Only genetic information present in the germ cells will be inherited by future generations, and any molecular processes affecting the germline genome are therefore likely to be passed on. Despite its prevalence across taxonomic kingdoms, we are only starting to understand details of the underlying micro-evolutionary processes occurring at the germline genome level. These include segregation, recombination, mutation and selection and can occur at any stage during germline differentiation and mitotic germline proliferation to meiosis and post-meiotic gamete maturation. Selection acting on germ cells at any stage from the diploid germ cell to the haploid gametes may cause significant deviations from Mendelian inheritance and may be more widespread than previously assumed. The mechanisms that affect and potentially alter the genomic sequence and allele frequencies in the germline are pivotal to our understanding of heritability. With the rise of new sequencing technologies, we are now able to address some of these unanswered questions. In this review, we comment on the most recent developments in this field and identify current gaps in our knowledge.

Improved Reference Genome Uncovers Novel Sex-Linked Regions in the Guppy (Poecilia reticulata).

Theory predicts that the sexes can achieve greater fitness if loci with sexually antagonistic polymorphisms become linked to the sex determining loci, and this can favor the spread of reduced recombination around sex determining regions. Given that sex-linked regions are frequently repetitive and highly heterozygous, few complete Y chromosome assemblies are available to test these ideas. The guppy system (Poecilia reticulata) has long been invoked as an example of sex chromosome formation resulting from sexual conflict. Early genetics studies revealed that male color patterning genes are mostly but not entirely Y-linked, and that X-linkage may be most common in low-predation populations. More recent population genomic studies of guppies have reached varying conclusions about the size and placement of the Y-linked region. However, this previous work used a reference genome assembled from short-read sequences from a female guppy. Here, we present a new guppy reference genome assembly from a male, using long-read PacBio single-molecule real-time sequencing and chromosome contact information. Our new assembly sequences across repeat- and GC-rich regions and thus closes gaps and corrects mis-assemblies found in the short-read female-derived guppy genome. Using this improved reference genome, we then employed broad population sampling to detect sex differences across the genome. We identified two small regions that showed consistent male-specific signals. Moreover, our results help reconcile the contradictory conclusions put forth by past population genomic studies of the guppy sex chromosome. Our results are consistent with a small Y-specific region and rare recombination in male guppies.

Locating the Sex Determining Region of Linkage Group 12 of Guppy (Poecilia reticulata).

Despite over 100 years of study, the location of the fully sex-linked region of the guppy (Poecilia reticulata) carrying the male-determining locus, and the regions where the XY pair recombine, remain unclear. Previous population genomics studies to determine these regions used small samples from recently bottlenecked captive populations, which increase the false positive rate of associations between individuals' sexes and SNPs. Using new data from multiple natural populations, we show that a recently proposed candidate for this species' male-determining gene is probably not completely sex-linked, leaving the maleness factor still unidentified. Variants in the chromosome 12 region carrying the candidate gene sometimes show linkage disequilibrium with the sex-determining factor, but no consistently male-specific variant has yet been found. Our genetic mapping with molecular markers spread across chromosome 12 confirms that this is the guppy XY pair. We describe two families with recombinants between the X and Y chromosomes, which confirm that the male-determining locus is in the region identified by all previous studies, near the terminal pseudo-autosomal region (PAR), which crosses over at a very high rate in males. We correct the PAR marker order, and assign two unplaced scaffolds to the PAR. We also detect a duplication, with one copy in the male-determining region, explaining signals of sex linkage in a more proximal region.

Using GC Content to Compare Recombination Patterns on the Sex Chromosomes and Autosomes of the Guppy, Poecilia reticulata, and Its Close Outgroup Species.

Genetic and physical mapping of the guppy (Poecilia reticulata) have shown that recombination patterns differ greatly between males and females. Crossover events occur evenly across the chromosomes in females, but in male meiosis they are restricted to the tip furthest from the centromere of each chromosome, creating very high recombination rates per megabase, as in pseudoautosomal regions of mammalian sex chromosomes. We used GC content to indirectly infer recombination patterns on guppy chromosomes, based on evidence that recombination is associated with GC-biased gene conversion, so that genome regions with high recombination rates should be detectable by high GC content. We used intron sequences and third positions of codons to make comparisons between sequences that are matched, as far as possible, and are all probably under weak selection. Almost all guppy chromosomes, including the sex chromosome (LG12), have very high GC values near their assembly ends, suggesting high recombination rates due to strong crossover localization in male meiosis. Our test does not suggest that the guppy XY pair has stronger crossover localization than the autosomes, or than the homologous chromosome in the close relative, the platyfish (Xiphophorus maculatus). We therefore conclude that the guppy XY pair has not recently undergone an evolutionary change to a different recombination pattern, or reduced its crossover rate, but that the guppy evolved Y-linkage due to acquiring a male-determining factor that also conferred the male crossover pattern. We also identify the centromere ends of guppy chromosomes, which were not determined in the genome assembly.

Arms races with mitochondrial genome soft sweeps in a gynodioecious plant, Plantago lanceolata.

Biological situations involving conflict can create arms race situations with repeated fixations of different functional variants, producing selective sweeps and lowering neutral diversity in genome regions linked to the functional locus. However, they can sometimes lead to balancing selection, potentially creating long coalescent times for sites with functionally different variants, and, if recombination occurs rarely, for extended haplotypes carrying such variants. We tested between these possibilities in a gynodioecious plant, Plantago lanceolata, in which cytoplasmic male-sterility factors conflict with nuclear restorers of male fertility. We find low mitochondrial diversity, which does not support very long-term coexistence of highly diverged mitochondrial haplotypes. Interestingly, however, we found a derived haplotype that is associated with male fertility in a restricted geographic region, and that has fixed differences from the ancestral sequence in several genes, suggesting that it did not arise very recently. Taken together, the results suggest arms race events that involved "soft" selective sweeps involving a moderately old-established haplotype, consistent with the frequency fluctuations predicted by theoretical models of gynodioecy.

Exaggerated heterochiasmy in a fish with sex-linked male coloration polymorphisms.

It is often stated that polymorphisms for mutations affecting fitness of males and females in opposite directions [sexually antagonistic (SA) polymorphisms] are the main selective force for the evolution of recombination suppression between sex chromosomes. However, empirical evidence to discriminate between different hypotheses is difficult to obtain. We report genetic mapping results in laboratory-raised families of the guppy (Poecilia reticulata), a sexually dimorphic fish with SA polymorphisms for male coloration genes, mostly on the sex chromosomes. Comparison of the genetic and physical maps shows that crossovers are distributed very differently in the two sexes (heterochiasmy); in male meiosis, they are restricted to the termini of all four chromosomes studied, including chromosome 12, which carries the sex-determining locus. Genome resequencing of male and female guppies from a population also indicates sex linkage of variants across almost the entire chromosome 12. More than 90% of the chromosome carrying the male-determining locus is therefore transmitted largely through the male lineage. A lack of heterochiasmy in a related fish species suggests that it originated recently in the lineage leading to the guppy. Our findings do not support the hypothesis that suppressed recombination evolved in response to the presence of SA polymorphisms. Instead, a low frequency of recombination on a chromosome that carries a male-determining locus and has not undergone genetic degeneration has probably facilitated the establishment of male-beneficial coloration polymorphisms.

Evolution of sex determination and heterogamety changes in section Otites of the genus Silene.

Switches in heterogamety are known to occur in both animals and plants. Although plant sex determination systems probably often evolved more recently than those in several well-studied animals, including mammals, and have had less time for switches to occur, we previously detected a switch in heterogamety in the plant genus Silene: section Otites has both female and male heterogamety, whereas S. latifolia and its close relatives, in a different section of the genus, Melandrium (subgenus Behenantha), all have male heterogamety. Here we analyse the evolution of sex chromosomes in section Otites, which is estimated to have evolved only about 0.55 MYA. Our study confirms female heterogamety in S. otites and newly reveals female heterogamety in S. borysthenica. Sequence analyses and genetic mapping show that the sex-linked regions of these two species are the same, but the region in S. colpophylla, a close relative with male heterogamety, is different. The sex chromosome pairs of S. colpophylla and S. otites each correspond to an autosome of the other species, and both differ from the XY pair in S. latifolia. Silene section Otites species are suitable for detailed studies of the events involved in such changes, and our phylogenetic analysis suggests a possible change from female to male heterogamety within this section. Our analyses suggest a possibility that has so far not been considered, change in heterogamety through hybridization, in which a male-determining chromosome from one species is introgressed into another one, and over-rides its previous sex-determining system.

A new physical mapping approach refines the sex-determining gene positions on the Silene latifolia Y-chromosome.

Sex chromosomes are particularly interesting regions of the genome for both molecular genetics and evolutionary studies; yet, for most species, we lack basic information, such as the gene order along the chromosome. Because they lack recombination, Y-linked genes cannot be mapped genetically, leaving physical mapping as the only option for establishing the extent of synteny and homology with the X chromosome. Here, we developed a novel and general method for deletion mapping of non-recombining regions by solving "the travelling salesman problem", and evaluate its accuracy using simulated datasets. Unlike the existing radiation hybrid approach, this method allows us to combine deletion mutants from different experiments and sources. We applied our method to a set of newly generated deletion mutants in the dioecious plant Silene latifolia and refined the locations of the sex-determining loci on its Y chromosome map.

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.

Sequencing and comparison of the mitochondrial COI gene from isolates of Arbuscular Mycorrhizal Fungi belonging to Gigasporaceae and Glomeraceae families.

Arbuscular Mycorrhizal Fungi (AMF) are well known for their ecological importance and their positive influence on plants. The genetics and phylogeny of this group of fungi have long been debated. Nuclear markers are the main tools used for phylogenetic analyses, but they have sometimes proved difficult to use because of their extreme variability. Therefore, the attention of researchers has been moving towards other genomic markers, in particular those from the mitochondrial DNA. In this study, 46 sequences of different AMF isolates belonging to two main clades Gigasporaceae and Glomeraceae have been obtained from the mitochondrial gene coding for the Cytochrome c Oxidase I (COI), representing the largest dataset to date of AMF COI sequences. A very low level of divergence was recorded in the COI sequences from the Gigasporaceae, which could reflect either a slow rate of evolution or a more recent evolutionary divergence of this group. On the other hand, the COI sequence divergence between Gigasporaceae and Glomeraceae was high, with synonymous divergence reaching saturated levels. This work also showed the difficulty in developing valuable mitochondrial markers able to effectively distinguish all Glomeromycota species, especially those belonging to Gigasporaceae, yet it represents a first step towards the development of a full mtDNA-based dataset which can be used for further phylogenetic investigations of this fungal phylum.

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.

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.

SlWUS1; an X-linked gene having no homologous Y-linked copy in Silene latifolia.

The dioecious plant Silene latifolia has heteromorphic sex chromosomes, and comparison of the positions of sex-linked genes indicates that at least three large inversions have occurred during the evolution of the Y chromosome. In this article, we describe the isolation of a new sex-linked gene from S. latifolia, which provides new information on the evolution of this plant's young sex chromosomes. By using reverse-transcription polymerase chain reaction degenerate primers based on the Arabidopsis thaliana sequence of WUSCHEL, a flower-development gene, we found two copies in S. latifolia, which we named SlWUS1 and SlWUS2. Southern blot and genetic segregation analysis showed that SlWUS1 is located on the X chromosome and SlWUS2 is autosomal. No Y-linked copy of SlWUS1 was found by either Southern blot analysis under low-stringency conditions or polymerase chain reaction with degenerate primers, so we conclude that SlWUS1 probably has no Y-linked homolog. It is unknown whether the Y chromosome lost the SlWUS1 copy by degeneration of this individual gene or whether deletion of a larger genome region was involved. Several tests lead us to conclude that dosage compensation has not evolved for this sex-linked gene. We mapped the ortholog in the nondioecious relative S. vulgaris (SvWUS1), to compare the location in a species that has no history of having sex chromosomes. SvWUS1 maps to the same linkage group as other fully X-linked genes, indicating that it was not added to the X, but was lost from the Y. Its location differs in the maps from the two species, raising the possibility that the X chromosome, as well as the Y, may have been rearranged.

Does local adaptation cause high population differentiation of Silene latifolia Y chromosomes?

Natural selection can reduce the effective population size of the nonrecombining Y chromosome, whereas local adaptation of Y-linked genes can increase the population divergence and overall intra-species polymorphism of Y-linked sequences. The plant Silene latifolia evolved a Y chromosome relatively recently, and most known X-linked genes have functional Y homologues, making the species interesting for comparisons of X- and Y-linked diversity and subdivision. Y-linked genes show higher population differentiation, compared to X-linked genes, and this might be maintained by local adaptation in Y-linked genes (or low sequence diversity). Here we attempt to test between these causes by investigating DNA polymorphism and population differentiation using a larger set of Y-linked and X-linked S. latifolia genes (than used previously), and show that net sequence divergence for Y-linked sequences (measured by D(a) , also known as δ) is low, and not consistently higher than X-linked genes. This does not support local adaptation, instead, the higher values of differentiation measures for the Y-linked genes probably result largely from reduced total variation on the Y chromosome, which in turn reflect deterministic processes lowering effective population sizes of evolving Y-chromosomes.