Population genetics of the Y chromosome of Drosophila melanogaster: rDNA variation and phenotypic correlates

One means of examining the evolutionary significance of molecular variation on the Y chromosome is to identify phenotypes specifically affected by Y-linked genes, and to quantify the phenotypic variation and its correlation to the molecular variation. The functional importance of the Y-linked array of rRNA genes is demonstrated by the ability of Y chromosome to rescue X-linked bobbed lethal alleles, whose lethality is seen in homozygous females. Because low numbers of X-linked rDNA gene copies result in increased developmental time and shortened bristles, and because there is considerable natural variation in Y-linked copy number, a careful examination of Y-linked variation in these two traits may uncover a mode of selection acting on the multigene family. In this study, 36 Y-chromosome replacement lines were tested to detect subtle variation in bristle phenotypes and developmental rates. Correlations among these traits, rDNA gene copy number, and intergenic sequence length were quantified. The absence of significant correlations between phenotypic characters and rDNA copy number of intergenic sequence length suggests that the extant molecular variation in Y-linked rDNA can have at most very small selective effects.     

Natural variation of the Y chromosome suppresses sex ratio distortion and modulates testis-specific gene expression in Drosophila simulans

X-linked sex-ratio distorters that disrupt spermatogenesis can cause a deficiency in functional Y-bearing sperm and a female-biased sex ratio. Y-linked modifiers that restore a normal sex ratio might be abundant and favored when a X-linked distorter is present. Here we investigated natural variation of Y-linked suppressors of sex-ratio in the Winters systems and the ability of these chromosomes to modulate gene expression in Drosophila simulans. Seventy-eight Y chromosomes of worldwide origin were assayed for their resistance to the X-linked sex-ratio distorter gene Dox. Y chromosome diversity caused males to sire ∼63% to ∼98% female progeny. Genome-wide gene expression analysis revealed hundreds of genes differentially expressed between isogenic males with sensitive (high sex ratio) and resistant (low sex ratio) Y chromosomes from the same population. Although the expression of about 75% of all testis-specific genes remained unchanged across Y chromosomes, a subset of post-meiotic genes was upregulated by resistant Y chromosomes. Conversely, a set of accessory gland-specific genes and mitochondrial genes were downregulated in males with resistant Y chromosomes. The D. simulans Y chromosome also modulated gene expression in XXY females in which the Y-linked protein-coding genes are not transcribed. The data suggest that the Y chromosome might exert its regulatory functions through epigenetic mechanisms that do not require the expression of protein-coding genes. The gene network that modulates sex ratio distortion by the Y chromosome is poorly understood, other than that it might include interactions with mitochondria and enriched for genes expressed in post-meiotic stages of spermatogenesis.     

Indirect evidence from DNA sequence diversity for genetic degeneration of the Y-chromosome in dioecious species of the plant Silene: the SlY4/SlX4 and DD44-X/DD44-Y gene pairs

The action of natural selection is expected to reduce the effective population size of a nonrecombining chromosome, and this is thought to be the chief factor leading to genetic degeneration of Y-chromosomes, which cease recombining during their evolution from ordinary chromosomes. Low effective population size of Y chromosomes can be tested by studying DNA sequence diversity of Y-linked genes. In the dioecious plant, Silene latifolia, which has sex chromosomes, one comparison (SlX1 vs. SlY1) indeed finds lower Y diversity compared with the homologous X-linked gene, and one Y-linked gene with no X-linked homologue has lower species-wide diversity than a homologous autosomal copy (SlAp3Y vs. SlAp3A). To test whether this is a general pattern for Y-linked genes, we studied two further recently described X and Y homologous gene pairs in samples from several populations of S. latifolia and S. dioica. Diversity is reduced for both Y-linked genes, compared with their X-linked homologues. Our new data are analysed to show that the low Y effective size cannot be explained by different levels of gene flow for the X vs. the Y chromosomes, either between populations or between these closely related species. Thus, all four Y-linked genes that have now been studied in these plants (the two studied here, and two previously studied genes, have low diversity). This supports other evidence for an ongoing degeneration process in these species.     

Surprising Differences in the Variability of Y Chromosomes in African and Cosmopolitan Populations of Drosophila melanogaster

The nonrecombining Drosophila melanogaster Y chromosome is heterochromatic and has few genes. Despite these limitations, there remains ample opportunity for natural selection to act on the genes that are vital for male fertility and on Y factors that modulate gene expression elsewhere in the genome. Y chromosomes of many organisms have low levels of nucleotide variability, but a formal survey of D. melanogaster Y chromosome variation had yet to be performed. Here we surveyed Y-linked variation in six populations of D. melanogaster spread across the globe. We find surprisingly low levels of variability in African relative to Cosmopolitan (i.e., non-African) populations. While the low levels of Cosmopolitan Y chromosome polymorphism can be explained by the demographic histories of these populations, the staggeringly low polymorphism of African Y chromosomes cannot be explained by demographic history. An explanation that is entirely consistent with the data is that the Y chromosomes of Zimbabwe and Uganda populations have experienced recent selective sweeps. Interestingly, the Zimbabwe and Uganda Y chromosomes differ: in Zimbabwe, a European Y chromosome appears to have swept through the population.     

The Y chromosome that lost the male-determining function behaves as an X chromosome in the medaka fish, Oryzias latipes

The medaka, Oryzias latipes, has an XX/XY sex-determination system, and a Y-linked DM-domain gene, DMY, is the sex-determining gene in this species. Since DMY appears to have arisen from a duplicated copy of the autosomal DMRT1 gene approximately 10 million years ago, the medaka Y chromosome is considered to be one of the youngest male-determining chromosomes in vertebrates. In the screening process of sex-reversal mutants from wild populations, we found a population that contained a number of XY females. PCR, direct sequencing, and RT-PCR analyses revealed two different null DMY mutations in this population. One mutation caused loss of expression during the sex-determining period, while the other comprised a large deletion in putative functional domains. YY females with the mutant-type DMY genes on their Y chromosomes were fully fertile, indicating that the X and Y chromosomes were functionally the same except for the male-determining function. In addition, we investigated the frequencies of the sex chromosome types in this population over four successive generations. The Y chromosomes bearing the mutant-type DMY genes were detected every year with no significant differences in their frequencies. These results demonstrate that aberrant Y chromosomes behaving as X chromosomes have been maintained in this population.     

Molecular evolution of a Y chromosome to autosome gene duplication in Drosophila

In contrast to the rest of the genome, the Y chromosome is restricted to males and lacks recombination. As a result, Y chromosomes are unable to respond efficiently to selection, and newly formed Y chromosomes degenerate until few genes remain. The rapid loss of genes from newly formed Y chromosomes has been well studied, but gene loss from highly degenerate Y chromosomes has only recently received attention. Here, we identify and characterize a Y to autosome duplication of the male fertility gene kl-5 that occurred during the evolution of the testacea group species of Drosophila. The duplication was likely DNA based, as other Y-linked genes remain on the Y chromosome, the locations of introns are conserved, and expression analyses suggest that regulatory elements remain linked. Genetic mapping reveals that the autosomal copy of kl-5 resides on the dot chromosome, a tiny autosome with strongly suppressed recombination. Molecular evolutionary analyses show that autosomal copies of kl-5 have reduced polymorphism and little recombination. Importantly, the rate of protein evolution of kl-5 has increased significantly in lineages where it is on the dot versus Y linked. Further analyses suggest this pattern is a consequence of relaxed purifying selection, rather than adaptive evolution. Thus, although the initial fixation of the kl-5 duplication may have been advantageous, slightly deleterious mutations have accumulated in the dot-linked copies of kl-5 faster than in the Y-linked copies. Because the dot chromosome contains seven times more genes than the Y and is exposed to selection in both males and females, these results suggest that the dot suffers the deleterious effects of genetic linkage to more selective targets compared with the Y chromosome. Thus, a highly degenerate Y chromosome may not be the worst environment in the genome, as is generally thought, but may in fact be protected from the accumulation of deleterious mutations relative to other nonrecombining regions that contain more genes.     

Isolation and characterization of Y chromosome sequences from the African malaria mosquito Anopheles gambiae

The karyotype of the African malaria mosquito Anopheles gambiae contains two pairs of autosomes and a pair of sex chromosomes. The Y chromosome, constituting approximately 10% of the genome, remains virtually unexplored, despite the recent completion of the A. gambiae genome project. Here we report the identification and characterization of Y chromosome sequences of total length approaching 150 kb. We developed 11 Y-specific PCR markers that consistently yielded male-specific products in specimens from both laboratory colony and natural populations. The markers are characterized by low sequence polymorphism in samples collected across Africa and by presence in more than one copy on the Y. Screening of the A. gambiae BAC library using these markers allowed detection of 90 Y-linked BAC clones. Analysis of the BAC sequences and other Y-derived fragments showed massive accumulation of a few transposable elements. Nevertheless, more complex sequences are apparently present on the Y; these include portions of an approximately 48-kb-long unmapped AAAB01008227 scaffold from the whole genome shotgun assembly. Anopheles Y appears not to harbor any of the genes identified in Drosophila Y. However, experiments suggest that one of the ORFs from the AAAB01008227 scaffold represents a fragment of a gene with male-specific expression.     

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.     

Evolution of DNA in Heterochromatin: the Drosophila Melanogaster Sibling Species Subgroup as a Resource

The Drosophila melanogasterspecies subgroup is a closely-knit collection of eight sibling species whose relationships are well defined. These species are too close for most evolutionary studies of euchromatic genes but are ideal to investigate the major changes that occur to DNA in heterochromatin over short periods during evolution. For example, it is not known whether the locations of genes in heterochromatin are conserved over this time. The 18S and 28S ribosomal RNA genes can be considered as genuine heterochromatic genes. In D. melanogasterthe rRNA genes are located at two sites, one each on the X and Y chromosome. In the other seven sibling species, rRNA genes are also located on the sex chromosomes but the positions often vary significantly, particularly on the Y. Furthermore, rDNA has been lost from the Y chromosome of both D. simulansand D. sechellia, presumably after separation of the line leading to present-day D. mauritiana.We conclude that changes to chromosomal position and copy number of rDNA arrays occur over much shorter evolutionary timespans than previously thought. In these respects the rDNA behaves more like the tandemly repeated satellite DNAs than euchromatic genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structure,molecular evolution and maintenance of copy number of extended repeated structures in the X-heterochromatin of Drosophila melanogaster

The 60 kb repeats located in the distal heterochromatin of the X chromosome of Drosophila melanogaster were cloned in overlapping cosmids. These regions, designated as SCLRs, comprised the following types of repeated elements Stellate genes, which are known to be involved in spermatogenesis; copia-like retrotransposons; LINE elements, including amplified Type rDNA insertions; and rDNA fragments. The following steps in SCLR formation were hypothesized: insertion of mobile elements into the rDNA and Stellate gene clusters: internal tandem duplication events; recombination between the rDNA cluster and Stellate tandem repeat; and amplification of the whole SCLR structure. There are about nine SCLR copies per haploid genome, but there is approximately a twofold variation in copy number between fly stocks. The SCLR copy number differences between closely related stocks are suggested to be the result of unequal sister chromatid exchange (USCE). The restricted variation in SCLR copy number between unrelated stocks and the absence of chromosomes free of SCLRs suggests that natural selection is active in copy number maintenance.     

Genetic Analysis of Stellate Elements of Drosophila Melanogaster

Repeated elements are remarkably important for male meiosis and spermiogenesis in Drosophila melanogaster. Pairing of the X and Y chromosomes is mediated by the ribosomal RNA genes of the Y chromosome and X chromosome heterochromatin, spermiogenesis depends on the fertility factors of the Y chromosome. Intriguingly, a peculiar genetic system of interaction between the Y-linked crystal locus and the X-linked Stellate elements seem to be also involved in male meiosis and spermiogenesis. Deletion of the crystal element of the Y, via an interaction with the Stellate elements of the X, causes meiotic abnormalities, gamete-genotype dependent failure of sperm development (meiotic drive), and deposition of protein crystals in spermatocytes. The current hypothesis is that the meiotic abnormalities observed in cry(-) males is due to an induced overexpression of the normally repressed Ste elements. An implication of this hypothesis is that the strength of the abnormalities would depend on the amount of the Ste copies. To test this point we have genetically and cytologically examined the relationship of Ste copy number and organization to meiotic behavior in cry(-) males. We found that heterochromatic as well as euchromatic Ste repeats are functional and that the abnormality in chromosome condensation and the frequency of nondisjunction are related to Ste copy number. Moreover, we found that meiosis is disrupted after synapsis and that cry-induced meiotic drive is probably not mediated by Ste.     

Evolution of the Ribosomal DNA Spacers of Drosophila melanogaster : Different Patterns of Variation on X and Y Chromosomes

Length variation of the ribosomal gene spacers of Drosophila melanogaster was studied. Analysis of 47 X chromosomal and 47 Y chromosomal linked rDNA arrays collected from five continents indicates that the arrays on the two chromosomes differ qualitatively. The Y-linked arrays from around the world share little or no similarity for either their overall length or the organization of their spacers. Most of the X-linked arrays do, however, share a major length spacer of 5.1 kb. In addition, those X-linked arrays that have a major 5.1-kb band have similar spacer organization as demonstrated by genomic DNA digestions with several restriction enzymes. These data strongly support the hypothesis that spacer length patterns on only X-linked genes are maintained primarily by natural selection.     

Highly efficient sex chromosome interchanges produced by I-CreI expression in Drosophila

The homing endonuclease I-CreI recognizes a site in the gene encoding the 23S rRNA of Chlamydomonas reinhardtii. A very similar sequence is present in the 28S rRNA genes that are located on the X and Y chromosomes of Drosophila melanogaster. In this work we show that I-CreI expression in Drosophila is capable of causing induced DNA damage and eliciting cell cycle arrest. Expression also caused recombination between the X and Y chromosomes in the heterochromatic regions where the rDNA is located, presumably as a result of a high frequency of double-strand breaks in these regions. Approximately 20% of the offspring of males expressing I-CreI showed exceptional inheritance of X- and Y-linked markers, consistent with chromosome exchange at rDNA loci. Cytogenetic analysis confirmed the structures of many of these products. Exchange between the X and Y chromosomes can be induced in males and females to produce derivative-altered Y chromosomes, attached-XY, and attached-X chromosomes. This method has advantages over the traditional use of X rays for generating X-Y interchanges because it is very frequent and it generates predictable products.     

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.     

Evolution of ribosomal RNA gene copy number on the sex chromosomes of Drosophila melanogaster

A diverse array of cellular and evolutionary forces--including unequal crossing-over, magnification, compensation, and natural selection--is at play modulating the number of copies of ribosomal RNA (rRNA) genes on the X and Y chromosomes of Drosophila. Accurate estimates of naturally occurring distributions of copy numbers on both the X and Y chromosomes are needed in order to explore the evolutionary end result of these forces. Estimates of relative copy numbers of the ribosomal DNA repeat, as well as of the type I and type II inserts, were obtained for a series of 96 X chromosomes and 144 Y chromosomes by using densitometric measurements of slot blots of genomic DNA from adult D. melanogaster bearing appropriate deficiencies that reveal chromosome-specific copy numbers. Estimates of copy number were put on an absolute scale with slot blots having serial dilutions both of the repeat and of genomic DNA from nonpolytene larval brain and imaginal discs. The distributions of rRNA copy number are decidedly skewed, with a long tail toward higher copy numbers. These distributions were fitted by a population genetic model that posits three different types of exchange events--sister-chromatid exchange, intrachromatid exchange, and interchromosomal crossing-over. In addition, the model incorporates natural selection, because experimental evidence shows that there is a minimum number of functional elements necessary for survival. Adequate fits of the model were found, indicating that either natural selection also eliminates chromosomes with high copy number or that the rate of intrachromatid exchange exceeds the rate of interchromosomal exchange.