Genomic changes following the reversal of a Y chromosome to an autosome in Drosophila pseudoobscura

Robertsonian translocations resulting in fusions between sex chromosomes and autosomes shape karyotype evolution by creating new sex chromosomes from autosomes. These translocations can also reverse sex chromosomes back into autosomes, which is especially intriguing given the dramatic differences between autosomes and sex chromosomes. To study the genomic events following a Y chromosome reversal, we investigated an autosome‐Y translocation in Drosophila pseudoobscura. The ancestral Y chromosome fused to a small autosome (the dot chromosome) approximately 10–15 Mya. We used single molecule real‐time sequencing reads to assemble the D. pseudoobscura dot chromosome, including this Y‐to‐dot translocation. We find that the intervening sequence between the ancestral Y and the rest of the dot chromosome is only ～78 Kb and is not repeat‐dense, suggesting that the centromere now falls outside, rather than between, the fused chromosomes. The Y‐to‐dot region is 100 times smaller than the D. melanogaster Y chromosome, owing to changes in repeat landscape. However, we do not find a consistent reduction in intron sizes across the Y‐to‐dot region. Instead, deletions in intergenic regions and possibly a small ancestral Y chromosome size may explain the compact size of the Y‐to‐dot translocation.     

Biased distribution of repetitive elements: a landmark for neo-Y chromosome evolution in Drosophila miranda

It is generally assumed that the sex chromosomes developed from a pair of homologs. Over evolution, the proto-Y chromosome, with a very short differential segment, matured in its final stage into a heterochromatic and, for the most part, genetically eroded Y chromosome. The constraints on the evolution of the proto-Y chromosome have been speculated upon since the sex chromosomes were discovered. Several models have been suggested. Drosophila miranda has proved to be a unique and potent model system to study Y-chromosome evolution. We use selected test genes distributed along the neo-Y chromosome as entry gates to analyze the molecular mechanisms involved in the process of Y-chromosome evolution. Here, we report our findings on the Krüppel gene (Kr), which is located distally on the neo-sex chromosome pair.     

Reduced levels of microsatellite variability on the neo-Y chromosome of Drosophila miranda

BACKGROUND: In many species, sex is determined by a system involving X and Y chromosomes, the latter having lost much of their genetic activity. Sex chromosomes have evolved independently many times, and several different mechanisms responsible for the degeneration of the Y chromosome have been proposed. Here, we have taken advantage of the secondary sex chromosome pair in Drosophila miranda to test for the effects of evolutionary forces involved in the early stages of Y-chromosome degeneration. Because of a fusion of one of the autosomes to the Y chromosome, a neo-Y chromosome and a neo-X chromosome have been formed, resulting in the transmission of formerly autosomal genes in association with the sex chromosomes. RESULTS: We found a 25-fold lower level of variation at microsatellites located on the neo-Y chromosome compared with homologous loci on the neo-X chromosome, or with autosomal and X-linked microsatellites. Sequence analyses of the region flanking the microsatellites suggested that the neo-sex chromosomes originated about 1 million years ago. CONCLUSIONS: Variability of the neo-Y chromosome of D. miranda is substantially reduced below expectations at mutation-drift equilibrium. Such a reduction is predicted by theories of the degeneration of the Y chromosome. Another possibility is that there is little or no mutation at microsatellite loci on a non-recombining chromosome such as the neo-Y, but this seems inconsistent with other data.     

Dosage compensation of a retina-specific gene in Drosophila miranda

The X1R chromosome of Drosophila miranda and the 3L autosome of Drosophila melanogaster are thought to have originated from the ancestral D chromosomal element and therefore may contain the same set of genes. It is expected that these genes will be dosage compensated in D. miranda because of their X linkage. To test these possibilities and to study evolution of the dosage compensation mechanism, we used the 3L-linked autosomal head-specific gene 507ml of D. melanogaster to isolate the homologous gene (507 mr) from a D. miranda genomic library. In situ hybridization showed that gene 507 is located at the 12A region of the X1R chromosome of D. miranda, indicating that the chromosomal homology deduced by cytogenetic means is correct. Restriction analysis and cross-specific DNA and RNA blot hybridization revealed the presence of extensive restriction pattern polymorphism and lack of sequence similarity in some areas of the 507 mr and 507 ml DNA, including the 3 portion of the transcribed region. However, the 5 portion of the transcribed region and the DNA sequences, located approximately 0.8 kb upstream and 3 kb downstream from the 507 ml gene showed a high degreee of similarity with the DNA sequences of comparable regions of the 507 mr gene. In both species gene 507 codes for a highly abundant 1.8 kb RNA which is expressed in the retina of the compound eye. Although in D. miranda the males have one and the females have two copies of the 507 gene, the steady-state levels of the 507 mRNA in both sexes were found to be similar, indicating that gene 507 is dosage compensated in D. miranda. Thus, along with the disparate rates of evolution in different areas of the DNA associated with gene 507, in D. miranda this gene has come under the regulation of the X chromosomal dosage compensation mechanism.by M.L. Pardue     

Multiple sex chromosomes in Drosophila miranda: A system to study the degeneration of a chromosome

Drosophila miranda possesses an intriguing sex chromosome constitution. While female metaphase plates have 10 chromosomes (diploid set), in males only 9 chromosomes can be identified. The missing homologue has been translocated to the Y, forming a neo-Y chromosome which is polytenized in the salivary gland cells. This report presents a detailed characterization of DNA, isolated from D. miranda flies. In situ hybridizations, using cRNA transcribed from unfractionated D. miranda DNA, reveal hybridization to the neo-Y with label distributed over the entire chromosome. The original partner of the translocated chromosome, X², is essentially unlabelled. These results suggest that repetitive DNA sequences invade the translocated chromosome. This result is discussed with reference to the hypothesis of degeneration of the Y chromosome, formulated by Muller (1918, 1932a).     

Is the Y chromosome of Drosophila an evolved supernumerary chromosome?

The Y chromosomes of most Drosophila species are necessary for male fertility but they are not involved in sex determination. They have many puzzling properties that resemble the effects caused by B chromosomes. Classical genetic and molecular studies reveal substantial affinities between Y and B chromosomes and suggest that the Y chromosomes of Drosophila are not degenerated homologues of the X chromosomes, but rather that their Y chromosomes evolved as specialized supernumeraries similar to classical B chromosomes.     

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.     

Origin and evolution of the Drosophila Y chromosome

Three recent findings are making a deep impact on our understanding of the Drosophila Y. First, the sequencing of the Drosophila genome and the development of proper computational methods increased the number of known single-copy Y-linked genes from 1 to 16, and revealed a chromosome packed with genes acquired from the autosomes. Second, this, coupled with the finding that B-chromosomes are able to show very regular segregation from the X chromosome, reinforce the hypothesis that the Drosophila Y is a specialized B-chromosome, instead of a degenerated homologue of the X. Third and finally, Y chromosomes seem to have a strong effect on male fitness.     

Genome analysis: More Drosophila Y chromosome genes

The Drosophila melanogaster Y chromosome has long been known to contain few functional genes other than several required for male fertility. The D. melanogaster genome sequence has now allowed characterization of two more male fertility genes, shedding light on the function and evolution of Y chromosomes.     

A selective sweep associated with a recent gene transposition in Drosophila miranda

In Drosophila miranda, a chromosome fusion between the Y chromosome and the autosome corresponding to Muller's element C has created a new sex chromosome system. The chromosome attached to the ancestral Y chromosome is transmitted paternally and hence is not exposed to crossing over. This chromosome, conventionally called the neo-Y, and the homologous neo-X chromosome display many properties of evolving sex chromosomes. We report here the transposition of the exuperantia1 (exu1) locus from a neo-sex chromosome to the ancestral X chromosome of D. miranda. Exu1 is known to have several critical developmental functions, including a male-specific role in spermatogenesis. The ancestral location of exu1 is conserved in the sibling species of D. miranda, as well as in a more distantly related species. The transposition of exu1 can be interpreted as an adaptive fixation, driven by a selective advantage conferred by its effect on dosage compensation. This explanation is supported by the pattern of within-species sequence variation at exu1 and the nearby exu2 locus. The implications of this phenomenon for genome evolution are discussed.     

Analysis of Y chromosome nucleolar organizer mutants in Drosophila melanogaster

Five Y chromosome nucleolar organizer (Y-NO) mutants were analyzed with respect to their rRNA gene numbers, phenotypes and additivity tests with other NO mutants. Four of these are indicative of a class of mutants in which most of the rRNA genes are transcribing functional rRNA. The other mutant has 80 genes, however, lethality and additivity tests suggests that many if not all of these rRNA genes are non-functional. The basis for the observed suppression of rRNA genes of the Y-NO region is discussed.     

Telomere repeats within the neo-Y-chromosome of Drosophila miranda

The clone Dmir1098, isolated from a genomic lambda library of Drosophila miranda labels exclusively the tips of the giant chromosomes in the highly polytenized nuclei of the female larval salivary glands. However, the in situ hybridizations to male metaphase plates, using the same probe, reveal a massive labeling block within the neo-Y-chromosome in addition to the labeling blocks at both chromosome ends. From the comparison with the Y chromosome labeling pattern of D. pseudoobscura, a sibling species to D. miranda, an end-to-end fusion mechanism involving the telomere repeats would be the most straightforward explanation for the karyotype change in D. miranda.     

Selection, recombination and demographic history in Drosophila miranda

Selection, recombination, and the demographic history of a species can all have profound effects on genomewide patterns of variability. To assess the impact of these forces in the genome of Drosophila miranda, we examine polymorphism and divergence patterns at 62 loci scattered across the genome. In accordance with recent findings in D. melanogaster, we find that noncoding DNA generally evolves more slowly than synonymous sites, that the distribution of polymorphism frequencies in noncoding DNA is significantly skewed toward rare variants relative to synonymous sites, and that long introns evolve significantly slower than short introns or synonymous sites. These observations suggest that most noncoding DNA is functionally constrained and evolving under purifying selection. However, in contrast to findings in the D. melanogaster species group, we find little evidence of adaptive evolution acting on either coding or noncoding sequences in D. miranda. Levels of linkage disequilibrium (LD) in D. miranda are comparable to those observed in D. melanogaster, but vary considerably among chromosomes. These patterns suggest a significantly lower rate of recombination on autosomes, possibly due to the presence of polymorphic autosomal inversions and/or differences in chromosome sizes. All chromosomes show significant departures from the standard neutral model, including too much heterogeneity in synonymous site polymorphism relative to divergence among loci and a general excess of rare synonymous polymorphisms. These departures from neutral equilibrium expectations are discussed in the context of nonequilibrium models of demography and selection.     

The Y chromosome of Drosophila melanogaster exhibits chromosome-wide imprinting

Genomic imprinting is well known as a regulatory property of a few specific chromosomal regions and leads to differential behavior of maternally and paternally inherited alleles. We surveyed the activity of two reporter genes in 23 independent P-element insertions on the heterochromatic Y chromosome of Drosophila melanogaster and found that all but one location showed differential expression of one or both genes according to the parental source of the chromosome. In contrast, genes inserted in autosomal heterochromatin generally did not show imprint-regulated expression. The imprints were established on Y-linked transgenes inserted into many different sequences and locations. We conclude that genomic imprinting affecting gene expression is a general property of the Drosophila Y chromosome and distinguishes the Y from the autosomal complement.     

Accumulation of Spock and Worf,two novel non-LTR retrotransposons,on the neo-Y chromosome of Drosophila miranda

Transposable elements constitute a major fraction of eukaryotic genomes. Here, I characterize two novel non-LTR retrotransposons, cloned from the neo-Y chromosome of Drosophila miranda. Worf is 4.1 kb in size and shows homology to the T1-2 non-LTR transposon characterized in Anopheles. Spock is 4.9 kb in size and shows similarity to the Doc element of D. melanogaster. Southern blot analysis of both elements yielded stronger signals for male DNA. In situ hybridization to polytene chromosomes revealed that both elements are accumulating on the neo-Y chromosome of D. miranda. PCR analysis was conducted to investigate the frequency of spock and worf and of the previously identified transposons, TRIM and TRAM, at individual chromosomal sites among 12 strains of D. miranda. Contrary to the observation that element frequencies are usually kept low at individual sites in Drosophila, the four transposons investigated are fixed at their genomic locations on the neo-Y chromosome. These results support the hypothesis that transposons accumulate in nonrecombining regions and may be one cause of the heteromorphism of sex chromosomes.     

Frequency of telomere repeat units in the Drosophila miranda genome

Cloned DNA fragments of Drosophila miranda which label all chromosome ends show a basic tandem repeat unit of 4.4 kb. The D. miranda telomere specific tandem repeats do not cross-hybridize with genomic D. melanogaster DNA which itself contains telomere repeat units of 3 kb. For a more detailed analysis of the functional criteria of telomere specific sequences we determined the repetition frequency of the tandem repeat units. As a low estimate we found a repetition frequency of 20 for female D. miranda DNA. This is on average equivalent to 2 telomere repeat units per chromosome end in the female D. miranda karyotype. However, a variable number of tandem repeat units per chromosome end would describe more closely the obtained differences in the labeling intensity between the individual chromosomes (X¹L-5). For the D. miranda male DNA we determined a repetition frequency of 90. The frequency difference of 70 copies between male and female DNA must be due to the Y-chromosome.     

A survey of chromosomal and nucleotide sequence variation in Drosophila miranda

There have recently been several studies of the evolution of Y chromosome degeneration and dosage compensation using the neo-sex chromosomes of Drosophila miranda as a model system. To understand these evolutionary processes more fully, it is necessary to document the general pattern of genetic variation in this species. Here we report a survey of chromosomal variation, as well as polymorphism and divergence data, for 12 nuclear genes of D. miranda. These genes exhibit varying levels of DNA sequence polymorphism. Compared to its well-studied sibling species D. pseudoobscura, D. miranda has much less nucleotide sequence variation, and the effective population size of this species is inferred to be several-fold lower. Nevertheless, it harbors a few inversion polymorphisms, one of which involves the neo-X chromosome. There is no convincing evidence for a recent population expansion in D. miranda, in contrast to D. pseudoobscura. The pattern of population subdivision previously observed for the X-linked gene period is not seen for the other loci, suggesting that there is no general population subdivision in D. miranda. However, data on an additional region of period confirm population subdivision for this gene, suggesting that local selection is operating at or near period to promote differentiation between populations.