Recurrent gene co-amplification on Drosophila X and Y chromosomes
[['Selfish genetic elements whose evolutionary trajectories are in conflict with those of their host were first described almost 100 years ago [1] .', 'However , only in recent decades has it become apparent that the antagonistic coevolution resulting from genetic conflict has shaped genome content and structure across the tree of life , from bacteria to plants and animals [2] .', 'Antagonistic coevolution can occur between organisms , as in the evolutionary “arms race” experienced between pathogens and their hosts , or within genomes among genetic elements with different inheritance patterns ( such as mobile elements , or X and Y chromosomes [3] ) .', 'For instance , selfish genetic elements can manipulate meiosis ( or gametogenesis ) so that they are transmitted to more than 50% of offspring ( so called “segregation distorters” or “meiotic drivers” ) [4] .', 'These processes can leave behind a variety of distinct genetic signatures .', 'For example , genes involved in pathogen virulence and host resistance consistently show elevated levels of amino acid substitutions ( i . e . dN/dS ) whereas genes involved in intra-genomic conflict , such as segregation distorters and their suppressors , often have high rates of lineage-specific duplications and gene amplifications [5 , 6] .', 'A well-studied cryptic sex chromosome drive system in mouse involves the convergent acquisition and amplification of the same gene families ( Slx/Sly ) on both the X and Y chromosome , and careful experimentation has shown that the co-amplified genes are in a co-evolutionary battle over sex chromosome transmission , whereby the X-and Y-linked copies of a gene family directly compete with each other [7 , 8] .', 'Sly knockdowns show female-biased sex ratios , while Slx deficiency causes a sex ratio distortion towards males .', 'A similar mechanisms of cryptic segregation distortion has been implicated in the Stellate/ Suppressor of Stellate ( Ste/Su ( Ste ) ) system in D . melanogaster , where the expression of the X-linked gene Ste leads to the production of defective sperm , and Su ( Ste ) , which is a multi-gene copy of Ste that moved to the Y-chromosome , silences Ste [9 , 10] .', 'Although rapid rates of amino acid substitution , gene duplication , and gene amplification are all characteristics of evolutionary conflict , these processes are also associated with strong selection in the absence of conflict , or in some cases , even neutral evolution .', 'This makes identification of conflict from genomic data alone difficult [11] .', 'For example , recent studies have shown that the Y chromosomes of many organisms contain testes-specific genes that have amplified in copy number [12–14] .', 'Some of these Y-linked gene families , such as those in mice , have been shown to be involved in sex chromosome drive , whereas for other gene families , the extra copies may either act to increase gene dosage or prevent degeneration by providing a substrate for non-allelic gene conversion [15] .', 'Alternatively , the extra copies may be neutral or even slightly deleterious , yet they remain on the Y due to the reduced efficiency of selection on this non-recombining chromosome [16] .', 'One key signature that appears to be unique to Y-amplified genes involved in sex chromosome drive is that their X-linked homologs have duplicated as well .', 'This pattern is consistent with antagonistic co-evolution resulting from repeated bouts of sex ratio distortion and suppression ( see Discussion ) .', 'Here , we use bioinformatics and functional genomic analyses to assess the prevalence of sex chromosome gene amplification across Drosophila species .', 'Consistent with a role for Y-amplified genes unrelated to genetic conflict ( see Discussion ) , we find hundreds of genes that appear to be present in multiple copies on the Y chromosomes of many Drosophila species .', 'However , we also find a second category of Y-amplified genes whose X homolog has been duplicated as well .', 'We show that species with young sex chromosomes have repeatedly evolved genes that have co-amplified on the X and the Y and show functions and expression patterns that are consistent with genetic conflict .', 'We explore a variety of evolutionary scenarios that could give rise to this pattern of X-Y co-amplification based on detailed investigation of the S-Lap and GAPsec genes that have been independently co-amplified on the X and Y chromosomes of multiple species in the obscura group .', 'We find that gene expression levels and small RNA production from these co-amplified genes are most consistent with a cryptic sex ratio drive system , however additional experiments are necessary to test these claims .', 'We develop a model for how such a system could have evolved and present evidence suggesting that the same genes appear to have become involved in a meiotic conflict independently among multiple species of this group .'], ['To analyze gene content evolution and identify amplified X- and Y-linked genes , we sequenced both male and female genomic DNA in 26 Drosophila species from across the Drosophila phylogeny ( S1 Table ) .', 'Roughly half of the species considered ( 11 out of 26 ) harbor the typical sex chromosome complement of Drosophila ( that is , a single pair of ancient sex chromosomes , shared by all members of Drosophila ) .', 'In addition to the ancestral pair of sex chromosomes , the other 15 species have a younger pair of “neo-sex” chromosomes , which formed when an autosome became fused to one or both of the ancient X and Y chromosomes ( S1 Fig ) .', 'These younger “neo-sex” chromosomes are at various stages of evolving the typical properties of ancestral sex chromosomes , with neo-Y chromosomes losing their original genes and acquiring a genetically inert heterochromatic appearance , and neo-X chromosomes acquiring their unique gene content and sex-specific expression patterns [16 , 17] .', 'We identified putative Y-amplified genes based on male and female gene coverage without relying on a genome assembly ( S2 Fig , see Methods ) and validated our approach using a high-quality genome assembly from D . miranda ( S3 Fig ) .', 'Using this approach , we identify ( depending on our cutoffs ) 100s of genes that have multiple copies on the Y across the 26 species investigated ( S2 and S3 Tables , S4 Fig ) .', 'Genes might amplify on the Y for a variety of reasons , but co-amplification of testis genes may be a defining feature of genes evolving under genetic conflict ( see Discussion ) .', 'Here we define co-amplified genes as being amplified on the Y , based on male and female gene coverage , and as having at least two copies on the X chromosome in our genome assemblies .', 'Among these multi-copy gene families on the Y , we found 35 amplified Y-linked genes with co-amplified X homologs in 10 species ( Fig 1 , S4 Table ) .', 'We infer that the copy number of these co-amplified X/Y genes ranges from 8 copies on the Y up to 297 Y-linked copies ( for an uncharacterized testis gene in D . melanogaster that amplified on the Y of D . robusta ) , with a mean copy number of 58 ( S5 Table ) .', 'We detect between 2–4 X-linked copies in our assemblies for these co-amplified X/Y genes ( S5 Table ) .', 'The number of assembled X copies is likely an underestimate since recent gene duplicates are typically collapsed in assemblies derived from short read sequencing data , but investigations of high-quality genome assemblies derived from long-read technologies confirm that co-amplified genes have considerably fewer copies on the X than the Y chromosome ( S6 Table; see also Discussion ) .', 'We next sought to investigate the putative functions of these co-amplified genes .', 'We found that many are expressed in reproductive tissues in D . melanogaster ( S4 Table ) .', 'Of the candidate genes that we find , 76% are expressed in the testes in D . melanogaster ( versus 56% genome-wide , FlyAtlas data ) which is significantly more than expected by chance ( one-sided Fisher’s Exact Test P = 0 . 011 ) .', 'Several of the genes have meiosis-related functions ( Fig 1 , S4 Table ) .', 'For example , we identify genes that are associated with spindle assembly involved in male meiosis ( fest ) , chromosome segregation ( mars ) , or male meiosis cytokinesis ( scra ) , amongst others ( Fig 1 ) .', 'Indeed , GO enrichment analysis reveals the following terms to be enriched among co-amplified X/Y genes: sperm chromatin condensation , spindle organization , cell cycle process , and mitotic spindle organization ( S5A Fig , S7 Table; note that the nominal P-values are significant but not after correcting for multiple hypothesis testing ) .', 'Genes only amplified on the Y chromosome , on the other hand , show GO enrichment for different categories of metabolic processes , translation , and biosynthetic processes ( S5B Fig , S7 Table ) .', 'Amplified Y genes were detected in each species investigated ( S2 Table ) .', 'Interestingly , however , co-amplification of genes is much more common in species with recently added neo-sex chromosomes: of the 10 species where we found co-amplified genes , nine harbor neo-sex chromosomes ( Fig 1 ) , and in the vast majority of cases the amplified genes were ancestrally present on the chromosome that formed the neo-sex chromosomes ( Fig 1 ) .', 'We decided to more carefully characterize two co-amplified genes in D . pseudoobscura , a species with a high quality PacBio-based genome assembly .', 'D . pseudoobscura currently lacks an assembled Y chromosome , but we inferred Y-linkage of contigs based on male and female read coverage using Illumina data ( see Methods ) .', 'We identified two adjacent genes that exist in multiple copies on the X and Y chromosome of D . pseudoobscura: S-Lap1 ( Dpse\\GA19547 ) and GAPsec ( Dpse\\GA28668 ) .', 'S-Lap1 is a member of a leucyl aminopeptidase gene family that encodes the major protein constituents of Drosophila sperm [18] , while GAPsec is a GTPase activating protein .', 'This situation is reminiscent of the Segregation distorter meiotic drive system in D . melanogaster , where the distorter is a truncated tandem duplication of RanGAP , which is also a GTPase activator [19] .', 'Both S-Lap1 and GAPsec show partial tandem duplications on the X ( Fig 2A ) , and we detect roughly 100 ( partial and full-length ) copies of both S-Lap1 and GAPsec on the Y chromosome ( the Y-linked contigs contain 127 copies of S-Lap1 and 91 copies of GAPsec; Figs 2B and 3 ) .', 'S-Lap1 and S-Lap2 are present in all Drosophila species investigated ( Fig 2A ) , and probably originated in an ancestor of Drosophila; phylogenetic clustering of S-Lap1 and S-Lap2 in certain species groups ( Fig 2C ) probably resulted from gene conversion homogenizing gene duplicates within a clade [20] .', 'In most species of the Drosophila clade , S-Lap1 and S-Lap2 are similar in size; in D . pseudoobscura and its sister D . persimilis , however , S-Lap2 has acquired a large deletion , removing more than half of the 3’ end of the gene ( Fig 2A and 2C; S6A Fig ) .', 'The partial duplication of GAPsec , on the other hand , is only found in D . pseudoobscura and its close relative D . persimilis ( Fig 2A and 2C; S6B Fig ) .', 'S-Lap1 and GAPsec probably dispersed onto the Y chromosome simultaneously , as there are multiple locations on the Y that preserve their X orientation ( Fig 2B ) ; note however , that the amplified copies on the Y do not include the tandemly duplicated copies .', 'For both S-Lap1 and GAPsec , the X- and Y-linked copies are highly expressed in testis of D . pseudoobscura ( S4 and S8 Tables , Fig 4A ) .', 'We used stranded RNA-seq and small RNA profiles from wildtype D . pseudoobscura testes , to obtain insights into the evolutionary mechanism responsible for the co-amplification of S-Lap1 and GAPsec .', 'Interestingly , we detect both sense and antisense transcripts and small RNAs derived from S-Lap1 ( Fig 4A , S8 Table; see S7 Fig for the size distribution of small RNAs mapping and S8 Fig for cross-mapping of RNA-seq and small RNA reads between different gene copies ) .', 'In particular , stranded RNA-seq data reveal that the X-linked copy of S-Lap1 duplicate produces both sense and anti-sense transcripts , resulting in the production of small RNAs ( see Fig 4A , S8 Table ) .', 'Close inspection of this genomic region in D . pseudoobscura shows that the duplicated GAPsec gene is directly adjacent to where the S-Lap1 duplicate antisense transcript begins ( Fig 4A ) .', 'Intriguingly , this segment scores highly as a potential promoter sequence when using the Berkeley Drosophila Genome Project ( BDGP ) neural network promoter prediction algorithm [21] ( score = 0 . 89 , highest possible score = 1 ) .', 'Thus , this suggests that the partial duplication of GAPsec provided a promoter-like sequence in D . pseudoobscura for antisense transcription of S-Lap1 duplicate .', 'Note that this putative promoter sequence is not part of the Y copies of S-Lap1 ( which lack the GAPsec duplicate ) , and we detect virtually no antisense transcripts that originate from the Y-amplified copies of S-Lap1 ( S8 Table ) .', 'CAGE-seq data support that the GAPsec duplicate generated a new TSS resulting in antisense transcription of S-Lap1 duplicate ( Fig 4A ) .', 'How unusual is antisense RNA expression , and the production of small RNAs for testis genes ?', 'To see if these features of S-Lap1 are commonly observed for other genes in D . pseudoobscura testis , we used our RNA-seq and small RNA data to identify additional genes that are expressed in testis ( rpkm> = 2 ) , show antisense expression ( at least 75% of sense expression ) , and the production of small RNAs ( rpkm> = 100 ) .', 'In addition to the S-Lap1 gene , this screen revealed 6 additional genes that produced antisense transcripts and small RNAs in the testes of D . pseudoobscura ( S9 Fig , S9 Table ) .', 'Interestingly , all of the identified genes are members of gene families ( i . e . we detect at least 2 gene copies in the D . pseudoobscura assembly ) , and for 5 out of 6 genes , at least one copy is located on one of the sex chromosomes , and another copy is found on the other sex chromosome , or an autosome .', 'In most Drosophila species , S-Lap1 and GAPsec are located on an autosome ( chromosome 3L in D . melanogaster ) .', 'In the D . pseudoobscura and affinis group , however , this chromosome arm fused with the sex chromosomes about 15MY ago , causing S-Lap1 and GAPsec to become sex-linked .', 'Intriguingly , patterns of molecular evolution at S-Lap1 and GAPsec suggest that they may have independently co-amplified in several members of the pseudoobscura species group .', 'We used high-quality PacBio genome assemblies for two additional members of that species group [22] , D . miranda , which diverged form D . pseudoobscura about 2–4 MY ago , and D . athabasca , which diverged 10–15 MY ago [23] .', 'While our Illumina sequencing-based approach failed to detect co-amplified X and Y genes in these species ( they have similar M/F coverage ) , examination of the assembled PacBio genomes revealed that both gene pairs independently amplified on the sex chromosomes of both D . miranda and D . athabasca ( see Fig 5 ) .', 'We identify tandem duplications of the entire genomic region containing a total of 11 copies of S-Lap1 and 6 copies of GAPsec on chromosome XR in D . miranda , and these two genes have amplified 5 and 4 times , respectively , on the neo-Y chromosome of D . miranda ( Fig 5A ) .', 'Both the nature of the duplication event and patterns of sequence evolution suggest that co-amplification of S-Lap1 and GAPsec occurred independently in D . miranda .', 'Here , the XR copies arose from individual duplications of these two genes followed by three tandem duplications of the entire genomic region encompassing S-Lap1 and GAPsec , producing a total of 11 copies of S-Lap1 and 6 copies of GAPsec .', 'All six of the X-linked copies of GAPsec are highly similar to each other ( >99% identical ) , and more similar to their Y-linked paralogs than they are to D . pseudoobscura ( Fig 5C ) .', 'Also , S-Lap1 and GAPsec appear to have moved only to a single location on the neo-Y of D . miranda , instead of being dispersed all across the Y , as in D . pseudoobscura ( Fig 5A ) .', 'Patterns of gene expression and short RNA production in D . miranda mimic that of D . pseudoobscura , with SLap-1 ( and GAPsec ) transcripts being produced from both strands , and small RNAs are generated across that genomic region ( Fig 4B ) .', 'The mechanism of antisense production appears to differ from D . pseudoobscura ( Fig 4 ) .', 'In particular , transcriptional read-through at both S-Lap1 and GAPsec appear to generate anti-sense transcripts of both genes .', 'Close inspection of this genomic region in D . miranda reveals sequence differences between the X and Y copies that may account for antisense production at X-linked gene copies .', 'In particular , we detect a polyadenylation signal ( AATAAA ) for GAPsec that is present in most ( 3 of the 4 ) Y copies , and in the homologous copy on the X in D . pseudoobscura , but which is missing in the D . miranda X-linked copies of GAPsec .', 'This mutational event could account for the creation of read-through transcripts on the X of D . miranda , leading to production of antisense transcripts for S-Lap1 and initiation of RNAi , analogous to the model proposed for D . pseudoobscura .', 'Likewise , we infer independent amplifications of S-Lap1 and GAPsec on the X chromosome of D . athabasca , another member of the pseudoobscura group for which we have generated a high-quality female genome assembly ( Fig 5B ) .', 'We detect 6 copies of GAPsec and 5 copies of S-Lap1 on the X chromosome of D . athabasca , and genomic coverage analysis suggests a similar number of copies on the Y chromosome ( i . e . males show similar coverage of S-Lap1 and GAPsec as females , suggesting similar copy numbers on the X and Y ) .', 'This suggests that S-Lap1 and GAPsec are independently involved in co-amplification in species where this locus has become sex-linked .'], ['Genes may amplify on the Y chromosome for a variety of reasons , and our current data do not allow us to evaluate their relative importance .', 'In particular , multi-copy genes may simply arise on the Y at a higher rate , since the high repeat content on the Y facilitates structural re-arrangements that can promote gene family expansion [25] .', 'Additionally , the efficacy of natural selection is reduced on the non-recombining Y , and Y chromosomes across diverse taxa accumulate functionless and deleterious repetitive DNA [16] .', 'Amplified Y genes thus may either provide no benefit for their carriers , or could in fact be slightly deleterious , yet natural selection is unable to remove them [26] .', 'Heterochromatin formation on the Y may further dampen any functional consequences of gene family expansion , and multi-copy Y genes may simply be more tolerated on the silenced Y . Finally , some multi-copy Y genes may actually contribute to male fitness and fertility [12–14 , 27] .', 'Gene family expansion on the Y chromosome may help to compensate for reduced gene dose on the heterochromatic and transcriptionally repressed Y chromosome [16 , 17] .', 'Y chromosomes are transmitted from father to son , and are thus an ideal genomic location for genes that specifically enhance male fitness [28] .', 'Y chromosomes of several species , including mammals and Drosophila , have been shown to contain multi-copy gene families that are expressed in testis and contribute to male fertility [12–14] .', 'Our analysis shows that multi-copy Y genes are common across flies , and it will be of great interest to identify the diverse evolutionary processes driving their amplification .', 'Co-amplification of X/Y genes , on the other hand , is more difficult to explain under scenarios that do not involve genetic conflict .', 'Several factors that may explain accumulation of genes on the Y do not apply to the X: The repeat content of X chromosomes is comparable to that of autosomes [22]; natural selection efficiently purges deleterious mutations from the recombining X; and transcription of the X chromosome in Drosophila males is increased in somatic tissues , rather than reduced [29] .', 'In addition , co-amplified X and Y genes are enriched for meiosis functions ( see also [24] ) , and the X-linked copies of co-amplified genes are highly expressed in testis [24] .', 'Functions in chromatin formation and chromosome segregation might be expected for selfish genes that are trying to interfere with proper condensation of the heterochromatic Y chromosome , or with fair segregation of homologous chromosomes .', 'Testis expression of co-amplified X-linked genes is unusual , as testis-expressed genes are underrepresented on the X chromosome of Drosophila ( [30] , but also see [31] ) , but can be understood under intragenomic conflict models [32–36] .', 'Most importantly , production of double-stranded RNA and triggering of the RNAi pathway is inconsistent with gene amplification boosting gene product , but instead has the opposite effect and results in transcriptional down-regulation of co-amplified X/Y genes .', 'Could other evolutionary forces or properties of sex chromosomes account for co-amplification of sex-linked genes ?', 'In several species , X chromosomes are down-regulated during spermatogenesis .', 'While there has been considerable debate about the exact mechanisms of male germline X inactivation in Drosophila , testis genes appear transcriptionally repressed during spermatogenesis on old X chromosomes in Drosophila [37–39] .', 'Co-amplification of testis genes could compensate for reduced expression of inactivated sex-linked genes and may thus be an adaptation to counter silencing of sex-linked genes in spermatogenesis .', 'However , this model would not explain why co-amplified genes are frequent targets by endo-siRNA [24] , which instead indicates a conflict between the X- and Y-linked copies , and not coordinated selection for their up-regulation .', 'Also , many copies of co-amplified genes are truncated ( as we observe for S-Lap or GAPsec , but also for others; see S6 Table ) , suggesting that the duplicated copies do not have the same function as their parent copies .', 'Gene amplification to counter male germline X inactivation would also predict that ( co ) -amplified genes are more abundant on older sex chromosomes , where inactivation of the X in spermatogenesis is complete .', 'In contrast , we find that co-amplified genes are more common in species with young neo-sex chromosomes ( Fig 1 ) .', 'In particular , the young neo-X chromosome of D . miranda shows no signs of reduced expression in testis [40] , yet we detect the largest numbers of co-amplified genes in this species ( Fig 1 ) .', 'Co-amplification of X and Y-linked genes could also allow meiotic pairing between diverging sex chromosomes .', 'In particular , the ribosomal RNA gene cluster is present on both the X and the Y in D . melanogaster and functions as an X-Y pairing site during male meiosis [41] .', 'This model , however , would not explain meiosis-specific function and testis-expression of co-amplified genes , or their targeting by endo-siRNA .', 'Also , acquiring homologous pairing sites should also be more important on more divergent , heteromorphic sex chromosomes , counter to our finding of co-amplified genes being more common on young neo-sex chromosomes .', 'Co-amplification of genes on young sex chromosomes with meiosis-related functions , expression in testis , and targeting by endo-siRNAs can all be understood under a model of RNAi mediated cryptic sex chromosome drive .', 'How would co-amplification of meiosis-related genes on the X and Y cause meiotic drive and its suppression ?', 'If amplified Y genes are involved in a battle with the X over fair transmission , changes in gene copy number may tip the balance over inclusion into functional sperm , and could result in repeated co-amplification of distorters and suppressors on the sex chromosomes ( Fig 6A ) .', 'In particular , an X-linked gene involved in chromosome segregation may evolve a duplicate that acquires the ability to incapacitate Y-bearing sperm ( Fig 6A ) .', 'Invasion of this sex-ratio distorter skews the population sex ratio and creates a selective advantage to evolve a Y-linked suppressor that is resistant to the distorter .', 'Suppression may be achieved at the molecular level by increased copy number of the wildtype function or by inactivation of X-linked drivers using RNAi [33 , 34 , 42] .', 'If both driver and suppressor are dosage sensitive , they would undergo iterated cycles of expansion , resulting in rapid co-amplification of both driver and suppressor on the X and Y chromosome [43] .', 'Such a model is consistent with what we observe for the S-Lap and GAPsec genes in D . pseudoobscura ( Fig 6B ) .', 'S-Lap1 is the most abundant sperm protein in D . melanogaster [18] but its function is poorly characterized .', 'If this protein is crucial for generating Y-bearing sperm , depletion of S-Lap1 during spermatogenesis would result in drive .', 'S-Lap1 was duplicated in an ancestor of D . pseudoobscura , and a partial duplication of GAPsec ( and truncation of S-Lap1-duplicate ) created a TSS for anti-sense transcription of S-Lap1 duplicate .', 'Anti-sense production of S-Lap1-duplicate transcript may trigger siRNA production and silencing of S-Lap1 , which could result in elimination of Y-bearing sperm .', 'Acquisition of multiple copies of S-Lap1 on the Y chromosome could restore S-Lap1 function , and create a cryptic drive system in D . pseudoobscura ( Fig 6B ) .', 'It is of course also possible that the Y-linked copies of S-Lap1 interfere with the production of X-linked sperm ( i . e . that the Y chromosome is the driver ) , and S-Lap1-duplicate on the X silences Y-copies through production of antisense RNA and RNAi .', 'A similar model of cryptic drive could also explain patterns of molecular evolution and gene expression at S-Lap and GAPsec in D . miranda , where read-through transcription generates anti-sense transcripts that trigger RNAi .', 'Detailed molecular testing will be necessary to characterize the wildtype function of S-Lap1 , and the cellular basis of the putative drive phenotype and its suppression .', 'Below , we discuss how several aspects of the co-amplified genes that we have identified would make sense under a model of sex chromosome drive .', 'Most of the species where we identify co-amplified X/Y genes harbor neo-sex chromosomes .', 'Under a drive model , this would make sense if sex ratio distorters have repeatedly evolved to exploit genomic vulnerabilities associated with the formation of new sex chromosomes .', 'Different features of young vs . old sex chromosomes create different susceptibilities to sex chromosome drive .', 'Old Y chromosomes are typically highly repetitive and heterochromatic , a feature that may easily be exploited by a driver on the X . Also , old sex chromosomes show much higher levels of sequence divergence , which makes identification and targeting of the homolog by a driver easier .', 'Yet , young Y chromosomes typically contain many more genes that can evolve to cheat meiosis , thereby increasing the chances of a Y-linked driver .', 'Finally , young X chromosomes may not yet be transcriptionally inactive during spermatogenesis and thus express more drivers .', 'In many species , including Drosophila , expression from the X chromosome is reduced during spermatogenesis [37] .', 'Low gene number and high repeat content makes Y chromosomes especially vulnerable to meiotic drive , and silencing of the X during spermatogenesis may have evolved as a genome defense against driving X’s [32] .', 'Suppression of transcription during spermatogenesis may not have fully evolved on young X chromosomes , allowing the expression of more X-linked drivers .', 'This may account for the prevalence of co-amplified X/Y genes in species with recently formed neo-sex chromosomes .', 'The RNAi pathway could be utilized in different ways to either create a meiotic driver , or to suppress it .', 'For example , a gene on the X ( or it’s duplicate ) may gain a novel function that disrupts segregation of the Y chromosome .', 'The homologous Y gene ( or duplicates of it ) may then silence the driving X by producing anti-sense transcripts that generate dsRNAs and launch the RNAi response , to silence the X-linked driver .', 'This scenario resembles the Winters sex ratio system , even though the suppressors of X-linked drive are autosomal and not Y-linked [42] .', 'The RNAi machinery can also be hijacked to create a driving X . In particular , if an X-linked gene is required for producing Y-bearing sperm , an X chromosome that silences this gene could evolve a drive phenotype .', 'It could do so by antisense RNA production of this X-linked gene ( or duplicates of it ) , thereby triggering the RNAi response to inactivate the gene .', 'The organism could restore the wildtype function of this gene by increasing its dose through its amplification on the Y , and even non-functional copies may act as a decoy to soak up endo-siRNA that are targeting this locus .', 'This pathway may underlie the putative GAPsec /S-Lap1 drive system in D . pseudoobscura , where the X-linked duplicates of S-Lap1 produce the vast majority of antisense transcripts ( roughly 95% , see S8 Table ) , while the Y-linked S-Lap1 copies predominantly generate sense RNA ( >99 . 9% ) .', 'Most of the small RNAs are produced from S-Lap1 duplicate ( the putative driver ) and the Y-linked copies of S-Lap1 ( about 96% in total ) , consistent with the idea that amplification of this spermatogenesis gene allows restoration of wildtype function , possibly by acting as a decoy to dilute RNAi induced silencing triggered by antisense transcripts of S-Lap1 duplicate .', 'Under either scenario , if both the driver and suppressor are dosage-sensitive , this can lead to the repeated invasion of driving and suppressing chromosomes through co-amplification of genes on the X and Y chromosome .', 'In order to trigger the RNAi response , the production of dsRNA is required .', 'This can be achieved in multiple ways .', 'In the D . simulans Winters system , the two suppressor genes both encode related long inverted repeats that can form hairpin RNAs ( hpRNAs ) , which are then processed by the RNAi machinery to generate siRNAs that repress the paralogous distorters [42] .', 'Alternatively , the production of dsRNA can occur through anti-sense transcription of the target genes , and this mechanism is creating siRNAs in the putative drive involving GAPsec and S-Lap1 , both in D . pseudoobscura and D . miranda .', 'Our data further support growing evidence that the production of antisense transcripts , hairpin RNAs and small RNAs may underlie some silenced meiotic drive systems [24 , 42] .', 'RNA interference ( RNAi ) related pathways provide defense against viruses and transposable elements , and have been implicated in the suppression of meiotic drive elements [42] .', 'Intriguingly , genes in these pathways often evolve rapidly , and show frequent gene duplication and loss over long evolutionary time periods .', 'Argonaute 2 ( Ago2 ) , for example , is one of the key RNAi genes in insects , and has repeatedly formed new testis-specific duplicates in the recent history of the Drosophila obscura group [44] .', 'Analysis of additional RNAi-pathway genes confirms that they undergo frequent independent duplications and that their history has been particularly labile within the Drosophila obscura group [45] .', 'Our finding suggests that the presence of young sex chromosomes in this species group makes them especially vulnerable to the invasion of meiotic drive elements , and may thus drive the rapid evolution of RNAi genes in this clade .', 'It will be of interest to study the dynamics of RNAi genes in other species groups that have gained novel sex chromosomes , to see if diversification of RNAi genes is correlated with the emergence of new sex chromosomes .', 'It is important to note that RNAi is only one means by which co-amplified genes could compete with each other to create and suppress sex chromosome drive .', 'In particular , drive and silencing for the well-studied , co-amplified Slx/Sly genes in mice involves competition of SLX and SLY at the protein level for entry to the nucleus and for nuclear binding sites [7 , 8][46] .', 'Also , not all drive systems lead to ge
['Introduction', 'Results', 'Discussion', 'Methods']
['Y chromosomes often contain amplified genes which can increase dosage of male fertility genes and counteract degeneration via gene conversion .', 'Here we identify genes with increased copy number on both X and Y chromosomes in various species of Drosophila , a pattern that has previously been associated with sex chromosome drive involving the Slx and Sly gene families in mice .', 'We show that recurrent X/Y co-amplification appears to be an important evolutionary force that has shaped gene content evolution of sex chromosomes in Drosophila .', 'We demonstrate that convergent acquisition and amplification of testis expressed gene families are common on Drosophila sex chromosomes , and especially on recently formed ones , and we carefully characterize one putative novel X/Y co-amplification system .', 'We find that co-amplification of the S-Lap1/GAPsec gene pair on both the X and the Y chromosome occurred independently several times in members of the D . obscura group , where this normally autosomal gene pair is sex-linked due to a sex chromosome—autosome fusion .', 'We explore several evolutionary scenarios that would explain this pattern of co-amplification .', 'Investigation of gene expression and short RNA profiles at the S-Lap1/GAPsec system suggest that , like Slx/Sly in mice , these genes may be remnants of a cryptic sex chromosome drive system , however additional transgenic experiments will be necessary to validate this model .', 'Regardless of whether sex chromosome drive is responsible for this co-amplification , our findings suggest that recurrent gene duplications between X and Y sex chromosomes could have a widespread effect on genomic and evolutionary patterns , including the epigenetic regulation of sex chromosomes , the distribution of sex-biased genes , and the evolution of hybrid sterility .']
['Sex chromosomes are hot spots for genetic conflict , and selfish genetic elements that increase their transmission are prone to originate on sex chromosomes .', 'Previous work has shown that genes that co-amplify on both the X and Y chromosome are involved in sex chromosome drive in mice .', 'Here , we use bioinformatic approaches to identify co-amplified genes in 26 Drosophila species , and we characterize a novel co-amplified gene family in D . pseudoobscura using functional genomic approaches .', 'Our comparative genomic analysis suggests that co-amplification of genes on sex chromosomes is rampant , and we detect co-amplified X/Y genes in dozens of fly species investigated , especially those with young sex chromosomes .', 'We find that co-amplified genes are often derived from well-characterized meiosis genes that are necessary for proper segregation , and are highly expressed in testis .', 'Finally , we show that co-amplified genes often produce antisense transcripts and are targeted by small RNAs .', 'Functional enrichment and expression patterns are consistent with a model where co-amplification of these genes is driven by intragenomic conflicts over transmission of the X and Y chromosome , and that RNAi mechanisms are utilized to launch evolutionary responses to counter sex ratio distortion .', 'This would imply a novel role for the RNAi pathway to defend the genome against selfish elements that try to manipulate fair transmission .']
['invertebrates', 'medicine and health sciences', 'animals', 'invertebrate genomics', 'animal models', 'drosophila melanogaster', 'model organisms', 'x-linked traits', 'experimental organism systems', 'y-linked traits', 'drosophila', 'research and analysis methods', 'genetic linkage', 'sex chromosomes', 'antisense rna', 'animal studies', 'chromosome biology', 'y chromosomes', 'insects', 'animal genomics', 'clinical genetics', 'arthropoda', 'biochemistry', 'rna', 'eukaryota', 'cell biology', 'nucleic acids', 'heredity', 'sex linkage', 'genetics', 'biology and life sciences', 'genomics', 'gene amplification', 'organisms', 'chromosomes']