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Mol Biol Evol. 2008 October; 25(10): 2157–2166. Published online 2008 July 24. doi: 10.1093/molbev/msn162. | PMCID: PMC2727386 |
Copyright © The Author 2008. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org Recent Origins of Sperm Genes in Drosophila Steve Dorus,  Zoë N. Freeman, Elizabeth R. Parker, Benjamin D. Heath, and Timothy L. Karr Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom Accepted July 18, 2008. Newly created genes often acquire testis-specific or enhanced expression but neither the mechanisms responsible for this specificity nor the functional consequences of these evolutionary processes are well understood. Genomic analyses of the Drosophila melanogaster sperm proteome has identified 2 recently evolved gene families on the melanogaster lineage and 4 genes created by retrotransposition during the evolution of the melanogaster group that encode novel sperm components. The expanded Mst35B (protamine) and tektin gene families are the result of tandem duplication events with all family members displaying testis-specific expression. The Mst35B family encodes rapidly evolving protamines that display a robust signature of positive selection within the DNA-binding high-mobility group box consistent with functional diversification in genome repackaging during sperm nuclear remodeling. The Mst35B paralogs also reside in a significant regional cluster of testis-overexpressed genes. Tektins, known components of the axoneme, are encoded by 3 nearly identical X-linked genes, a finding consistent with very recent gene family expansion. In addition to localized duplication events, the evolution of the sperm proteome has also been driven by recent retrotransposition events resulting in Cdlc2, CG13340, Vha36, and CG4706. Cdlc2, CG13340, and Vha36 all display high levels of overexpression in the testis, and Cdlc2 and CG13340 reside within testis-overexpressed gene clusters. Thus, gene creation is a dynamic force in the evolution of sperm composition and possibly function, which further suggests that acquisition of molecular functionality in sperm may be an influential pathway in the fixation of new genes. Keywords: sperm, gene duplication, retrotransposition, testis, protamines, proteomics The fundamental role of gene duplication in the evolution of functional novelty and biological diversity has long been recognized and is believed to be important to the evolution of species-specific traits ( Ohno 1970). One primary mechanism of gene duplication involves segmental duplication of chromosomal regions resulting in tandem copies of genes that initially share features such as intron/exon boundaries and potentially cis-acting regulatory regions ( Samonte and Eichler 2002). Alternatively, genes can be duplicated through retrotransposition, a process involving the insertion of a retrotranscribed mRNA molecular within a different genomic region ( Long et al. 2003). This process results in a new gene lacking introns that is generally decoupled from cis-regulatory regions associated with the parental loci ( Brosius 1999; Long et al. 2003). Although the possible fates of newly duplicated genes have been explored from an evolutionary perspective ( Force et al. 1999; Lynch and Conery 2000; Lynch and Force 2000) and, to a much lesser extent, through genetic ( Loppin et al. 2005) and functional studies ( Brown et al. 1998; Maston and Ruvolo 2002; Kalamegham et al. 2007), questions remain concerning the early evolution of new genes. Of particular interest are the mechanisms by which new genes acquire spatiotemporal expression patterns and how this influences the acquisition of their ultimate cellular function. For example, recent studies have demonstrated that a preponderance of new retrogenes acquire testis-specific expression both in Drosophila ( Betran et al. 2002) and primates ( Dorus et al. 2003; Emerson et al. 2004, Marques et al. 2005; Vinckenbosch et al. 2006). Despite being a widespread evolutionary process, little is known about the specific ramifications of new gene creation on testes function, spermatogenesis, or sperm composition. There are, however, 3 notable exceptions to this. The first involves the evolutionary history of K81, a strict paternal effect gene created through retrotransposition prior to the divergence of the melanogaster subgroup ( Loppin et al. 2005). As a paternal effect lethal mutation, K81 flies are viable, produce motile sperm, and have no adult phenotype. Instead, the phenotype is manifest during fertilization following sperm entrance into the egg ( Yasuda et al. 1995). In wild-type eggs fertilized by sperm from K81 homozygous males, paternal chromosomes systematically fail to properly separate sister chromatids during the first zygotic division leading to lethality early in embryogenesis ( Loppin et al. 2005). This unique phenotype could result from a defect of sperm chromatin remodeling or paternal DNA replication during male pronuclear formation. Interestingly, the K81 gene product is not an integral sperm protein but is expressed in primary spermatocytes where it presumably regulates aspects of spermatogenesis required for proper sperm function in the egg. The second is the well-studied case of Sdic, a newly created gene encoding a protein present in the sperm tail ( Nurminsky et al. 1998). Sdic is an unusual case of an X-linked chimeric gene specific to Drosophila melanogaster that was created through the duplication of annexinX ( AnnX) and subsequent fusion with Cdic, a cytoplasmic dynein. This chimeric gene has also undergone a series of tandem gene duplication events resulting in a 4 gene cluster ( Ponce and Hartl 2006). Although the precise function of sdic in sperm is not known, further analysis of this novel gene and other new testis-enriched genes should provide important insights into the processes involved in the acquisition of testes expression and integration of novel proteins as components of mature sperm. The third example is the Drosophila gene, mojoless ( mjl), which was created approximately 50 MYA through retrotransposition ( Kalamegham et al. 2007). Evolutionary analysis indicate that this gene represents a retrotransposed copy of shaggy ( sgg), a glycogen synthase kinase-3 encoding gene. By unknown mechanisms, mjl acquired male germ line expression and testis function as RNA interference (RNAi) resulted in male infertility. Interestingly, mjl partially rescues the sgg mutant phenotype indicating that it maintains some ancestral biochemical function despite its newly acquired role in male fertility. Genomic analyses of the D. melanogaster sperm proteome (DmSP) ( Dorus et al. 2006) have allowed us to evaluate the importance of gene creation in the evolution of Drosophila sperm. These analyses determined that the Mst35B ( protamine) and tektin gene families have been recently expanded through tandem gene duplication during D. melanogaster evolution. Among these gene families, positive selection driving the functional divergence of the Mst35B gene family was also observed. Remarkably, the X-linked tektin gene family is comprised of 3 closely related paralogs, with 2 of the loci sharing complete nucleotide identity. This finding is indicative of an extremely recent set of duplication events during D. melanogaster evolution. A survey of retrotransposed genes also identified 4 newly created genes, CG13340, Vha36, CG4706, and Cdlc2, which encode integral sperm components. Finally, Mst35B genes, the tektins, and Cdlc2 are demonstrated to have testis-specific expression patterns. Interestingly, the Mst35B genes, Cdlc2 and CG13340 also localize to genomic regions with significant clustering of testis-overexpressed genes. The use of whole-sperm proteomics provides an alternative experimental approach for the study of new gene creation as it focuses specifically on proteins that are developmentally incorporated as integral sperm components. This also eliminates a reliance on gene expression in the assessment of putative functionality of novel genes. Our analysis of the DmSP thus provides a new perspective on the dynamic influence of gene creation in spermatogenesis, and specifically on sperm evolution, and suggests that the acquisition of sperm functionality may be a common evolutionary conduit for the fixation and functional evolution of new genes. Bioinformatic Identification of Novel Sperm Genes in D. melanogaster Nucleotide coding sequences were downloaded from FlyBase ( http://flybase.bio.indiana.edu/) for all sperm components characterized using whole-sperm mass spectrometry (MS) ( Dorus et al. 2006). Local batch BlastN was used to compare these sequences against all coding gene sequences in the D. melanogaster genome (R5.7). Any comparison with significant nucleotide identity (>70% identity with >50% coding sequence coverage) was downloaded for further analysis. “In-frame,” pairwise ClustalX alignments of the nucleotide sequences were conducted for these genes, and pairwise dS estimates were calculated using the method of Nei and Gojobori (1986). Candidate gene duplication events specific to D. melanogaster were tentatively identified by choosing paralogs with dS values lower than 0.175, a value slightly greater than the average synonymous divergence between D. melanogaster and Drosophila simulans orthologs ( Dorus et al. 2006). Genes identified by these criteria were subject to further comparative genomic analyses (see below). A complete list of pairwise comparisons with dS less than 0.175 is provided in supplementary table 1 ( Supplementary Material online). Comparative Genomic and Evolutionary Analyses The evolutionary history of putative duplication events was inferred from evolutionary divergence estimates complemented by comparisons of syntentic genomic regions in the melanogaster group and Drosophila pseudoobscura ( Clark et al. 2007). Duplication events determined to be specific to the D. melanogaster lineage were subject to additional analyses. Available orthologous Drosophila coding sequences were aligned in-frame using ClustalX. Phylogenies were determined by exhaustive parsimony analysis as implemented by PAUP ( Swofford 2003), and sequence analysis and ancestral node reconstruction were conducted using maximum likelihood methods implemented by the codeml program in the PAMLv3.14 package ( Yang 1996). Branch-specific ω values were obtained using the free-ratio model and branch-specific rate acceleration assessed using 2-ratio models ( Yang 1998). Pairwise dN and dS values were estimated using the method of Nei and Gojobori (1986). Model A of codeml was used to detect positive selection on codons along specific foreground branches ( Zhang et al. 2005). To test the significance of the results from Model A, we used the more stringent comparison of Model A to itself with ω constrained to 1.0 (Test 2; Model A fix) and also the more conservative χ 2 to calculate P ( Zhang et al. 2005). Parameter estimates and log-likelihood values under different models are provided in supplementary table 2 ( Supplementary Material online). Likelihood ratio statistics (2Δ ![[ell]](/corehtml/pmc/pmcents/x2113.gif) ) for alternative hypothesis testing and positively selected sites by Bayes empirical methods are provided in supplementary table 3 ( Supplementary Material online). Sliding-window analyses were conducted using the Swaap v. 1.0.2 software package with a window size of 60 bp and a sliding increment of 3 bp ( Pride and Blaser 2002). Reverse Transcriptase–Polymerase Chain Reaction Ten male carcasses (minus gonads) and testes from 25 male equivalents were dissected, washed (3×) in phosphate-buffered saline, as described by Snook and Markow (2002), and flash frozen in liquid nitrogen. Total RNA was then isolated using manufacturer's protocols (Ambion RNAqueous Kit), and RNA was resuspended in TE and quantified. Reverse transcription was carried out by standard protocols (Promega Improm-II Reverse Transcription System) using 75-ng input RNA (final concentration 3.75 ng/μl) from whole males (minus gonads) and testis. Gene-specific polymerase chain reaction (PCR) primers were designed for all newly created genes with the exception of the tektin gene family members (primer sequences are provided in supplementary table 4, Supplementary Material online). Tektin gene family mRNA sequences are nearly identical (>99%) at the nucleotide level thus precluding gene-specific PCR amplification. PCR was conducted using equal quantities of complementary DNA from the whole fly (minus gonads) and testis, and equal volumes of each PCR were loaded onto the same 1% agarose gel. Optical density of the relevant bands were quantified using AIDA software (RayTest, Pforzheim, Germany). Due to the extremely high level of nucleotide identity between tektin paralogs, a detailed inspection of expressed sequence tag (EST) sequences was required to assess gene-specific expression patterns. We, therefore, examined the D. melanogaster EST database ( http://www.ncbi.nlm.nih.gov/) using BlastN and manually determined the gene from which each EST originated when possible. Surveys of expression data were also conducted from previous microarray studies ( Parisi et al. 2004; Chintapalli et al. 2007) to confirm testis overexpression compared with the whole fly and to analyze the expression profiles of neighboring genes in regions containing newly created sperm genes (see below). Genomic Analyses of Gene Clustering Based on Testis Overexpression Drosophila melanogaster microarray gene expression data were obtained from Chintapalli et al. (2007) ( http://www.flyatlas.org/). An average was taken for the expression quantification data for all genes assayed in duplicate within this data set. The identification of testis-overexpressed gene clusters was performed using 2 alternative approaches based upon a stringent adjacent gene model ( Li et al. 2005). The first approach, based on our empirical observations (see and ), determined the genome-wide probability of finding 6 genes with >5.0-fold enrichment in the testis (compared with the whole fly) in a group of 7 adjacent genes. The second approach calculated the probability that the average fold enhancement of testis expression (compared with the whole fly) for 7 adjacent genes was equal to or greater than that observed for the genomic regions containing Mst35B (10.62×) and Cdlc2 (9.21×). The probability of retrotransposed genes relocating next to or within previously existing testis-overexpressed gene clusters was calculated assuming a stochastic model of gene insertion. Determination of all possible insertion locations was based on the total number of genes in the melanogaster genome (R5.7) and significant testis-overexpressed gene clusters were defined as any adjacent set of 7 genes that include 6 or more genes with >5.0-fold expressional enrichment in the testis. Mst35B Protamine Gene Duplication in D. melanogaster MS of purified sperm identified a tryptic peptide encoded by Mst35Ba and Mst35Bb, genes encoding the integral sperm proteins, dProtamineA and dProtamineB ( table 1). Both of these protamines have also been previously demonstrated to localize to sperm ( Raja and Renkawitz-Pohl 2005). Mst35Ba and Mst35Bb represent a gene duplication specific to D. melanogaster as levels of synonymous divergence ( dS) were significantly lower between Mst35B paralogs than orthologous comparisons between D. melanogaster and the single copy present in D. simulans. In concert with this result, divergence at these loci was also significantly lower than the average dS between D. melanogaster and D. simulans ( supplementary table 5, Supplementary Material online). Comparative genomic analysis within the melanogaster subgroup identified a single orthologous Mst35B gene with syntenic correspondence. Finally, close genomic proximity of Mst35Ba and Mst35Bb (449 bp) and the conservation of gene structure (intron/exon boundaries) between these paralogs suggest that they were created through a tandem duplication event (). | Table 1MS Identification and Testis Enrichment of Novel Sperm Genes in Drosophila melanogaster |
Mst35B Paralogs Are Testis-Specific and Reside in a Testis-Overexpressed Gene Cluster As expected, given their presumed function in genome repackaging during spermatogenesis, both Mst35B paralogs are significantly overexpressed in the testis in comparison to the whole fly (minus gonads) as determined by gene-specific reverse transcriptase–polymerase chain reaction (RT-PCR; ). This finding is consistent with 2 previous microarray studies ( Parisi et al. 2004; Chintapalli et al. 2007) and our own analysis of D. melanogaster EST databases ( table 1). The Mst35B cis-acting promoters have not been characterized, so it cannot be determined if promoter regions have been duplicated along with the protein-coding exons. However, Mst35B loci are embedded within an ~70-kb region containing 5 additional genes overexpressed in the testis (). Two analyses of the genome-wide clustering of testis-overexpressed genes confirm significant clustering within this region. First, an analysis of genome regions containing at least 6 out of 7 genes with >5-fold enrichment in the testis demonstrates that they are exceedingly rare with a frequency of 0.00093 within the genome. Second, the 10.62-fold average testis enrichment in this gene cluster is extremely high and also found at an extremely low frequency in the remainder of the genome (0.0013). Despite the concentration of testis-overexpressed genes in this genomic region, only the Mst35B paralogs encode empirically identified sperm proteins ( Dorus et al. 2006). Positive Selection on Mst35B during D. melanogaster Evolution Maximum likelihood analyses provided evidence of positive selection in the recent evolutionary history of these paralogs. First, a significant evolutionary rate increase was observed for the Mst35Ba lineage following the duplication event ( P = 0.012) in comparison to the remainder of the phylogeny (; supplementary tables 2 and 3, Supplementary Material online). Although branch-specific maximum likelihood analyses did not identify the Mst35Ba lineage (ω = 2.9) as significantly greater than 1.0, branch-site models (Model A) provided significant evidence for the influence of positive selection on a class of sites on this lineage ( P= 0.029). Additionally, a significant asymmetry in evolutionary rates is observed between the Mst35Ba and Mst35Bb lineages (). Although asymmetry is common ( Conant and Wagner 2003; Fares et al. 2006), significant rate asymmetry is less likely among local “DNA-based” duplicates ( Cusack and Wolfe 2007). Protamines contain a DNA-binding high-mobility group (HMG) box domain that has been implicated in both gene regulation ( Travers 2002) and chromatin remodeling ( Ragab et al. 2005). A sliding-window analysis of the Mst35B paralogs distinctly identified the HMG box domain as the rapidly evolving region within the gene (). Consistent with this observation, an ω > 1.0 was observed on the MstBa lineage for this domain () that contained 6 sites in the ω 2 (ω > 1.0) category using branch-site maximum likelihood analysis (). Finally, the concentration of these codon sites within the 44 amino acid HMG box domain is significantly higher than the 3 sites identified in the remainder of the gene ( P= 0.032; 2-tailed Fisher's exact test). This finding is suggestive of functional diversification of the HMG box and consistent with the rapid evolution of DNA-/RNA-binding sperm proteins in Drosophila ( Dorus et al. 2006) and protamines in other taxa ( Wyckoff et al. 2000). Extremely Recent Expansion of an X-linked Sperm Structural Gene Family Our previous analysis of the DmSP also identified 11 unique protein fragments specific to the proteins encoded by the X-linked tektin gene family, which includes CG17450 and its paralogs, CG32820 and CG32819 ( table 1). Due to the very high level of amino acid identity, MS was unable to determine if all, or only a subset, of the tektins are present in mature sperm. However, tektins are known sperm axonemal microtubule–binding proteins ( Cao et al. 2006) and given their testis-specific expression (see below), it is reasonable to assume that all 3 loci encode sperm structural proteins. CG17450, CG32820, and CG32819 were identified as recent duplicates in D. melanogaster based on the extremely low levels of synonymous divergence between them ( supplementary table 5, Supplementary Material online). Remarkably, no nucleotide divergence (either synonymous or nonsynonymous) exists between CG32819 and CG32820, a finding suggestive of a very recent evolutionary origin. Comparative genomic analyses also confirmed the recent timing of these duplication events: single regions with syntenic correspondence to D. melanogaster CG17450 were identified throughout the melanogaster subgroup, whereas CG32820 and CG32819 are unique to D. melanogaster. Colocalization and conservation of itron/exon boundaries strongly suggest that expansion of the gene cluster occurred through local tandem duplications. The most parsimonious explanation is that duplication of the ancestral region (also including the ancestor of CG33502) created a second region containing either CG32820/ CG32857 or CG32819/ CG33500 followed by another more recent 2 gene duplication event, resulting in the final gene pair ( supplementary fig. 1, Supplementary Material online). Complete nucleotide identity exists between the ~6-kb region containing genes CG32820/CG32857 and CG32819/ CG33500. RT-PCR assays of the tektin loci demonstrated specific expression in the testis (). Nucleotide identity among these loci precludes the design of gene-specific primers. However, at least 2 of the 3 loci are expressed and have enhanced expression in the testis based on detailed analysis of ESTs ( table 1). Unlike the Mst35B gene family, no evidence exists for clustering of testis-overexpressed loci in genomic regions around these tektins ( supplementary fig. 1, Supplementary Material online). Maximum likelihood analysis of the tektin gene family failed to identify the evidence of accelerated evolution or the signature of positive selection (). In fact, dN/ dS ratios are significantly below 1.0 on all lineages of the phylogeny, and only a modest increase is observed on the lineages following gene duplication events. Sperm Genes Created through Retrotransposition The DmSP contains 4 previously identified retrogenes created during the evolution of the melanogaster group ( Betran et al. 2002) ( table 2). However, only 2 of the parental loci ( CG32063 and ctp) were also found in the DmSP. The other 2 retroposed DmSP genes arose from parental loci not highly expressed in the testis and therefore acquired testis expression and sperm localization by other mechanisms following their creation. Although based on a limited number of events, the status of parental loci as a sperm gene does not appear to be a prerequisite for the future acquisition of sperm function by the retrogene. However, retrotransposition of sperm genes consistently results in the creation of novel sperm retrogenes. It is also noteworthy that 2 of these events involved the retrotransposition of X-linked genes to create autosomal sperm loci ( table 2). Analysis of a complete suite of retroposition events in Drosophila will be needed to fully characterize the dynamics of retrogene inclusion in the DmSP. | Table 2Recent Retrotransposition Events Resulting in the Creation of Novel Sperm Components |
Retrotransposition into Testis-Overexpressed Gene Clusters Given that retrotransposition events do not usually transfer cis-acting regulatory sequences, we sought to determine if the establishment of testis-specific expression may be related to expressional characteristics of the genomic region within which the new gene has been relocated. Our analysis identified one retrogene, Cdlc2, which resides in a ~15-kb genomic region with expressional characteristics consistent with this possibility. Cdlc2 is testis-specific by RT-PCR () and retrotransposed into a significant cluster of testis-overexpressed genes (9.21-fold average expressional enrichment in the testis, ). The parental gene of Cdlc2, ctp, is present in the DmSP but not overexpressed in the testis. Clusters of testis-overexpressed genes with common properties of the Cdlc2 region are exceedingly rare in the genome, comprising less than 0.01% of all possible adjacent groups of 7 genes. The probability of a retrogene inserting within such a cluster is also exceedingly small (0.7%) further suggesting a role for regional expression regulation in the establishment of testis-specific expression. It is also noteworthy that the testis-overexpressed retrogene, CG13340, has also been previously demonstrated, using a different analytical method, to reside in a much larger genomic region (~234 kb) enriched for testis-expressed genes ( Parisi et al. 2004). Gene duplication, and the subsequent evolution of genetic novelty, is now recognized as a critical process in evolution ( Ohno 1970; Long et al. 2003). Although understood as a widespread process, the specific molecular impact of gene duplication on the evolution of protein function is still being elucidated. Furthermore, the complexity of whole-cell systems makes it technically difficult to integrate the role of gene duplication and neofunctionalization into a framework of cellular function and evolution. We have approached this general problem by first identifying the principle protein components of whole D. melanogaster sperm, a cell type of central importance to sexual reproduction and Darwinian fitness ( Dorus et al. 2006). Sperm proteome characterization forms a powerful basis for the study of gene creation and function as it allows for the conclusive identification of new genes encoding novel sperm components during spermatogenesis. Our previous evolutionary analysis of the DmSP did not identify widespread rapid evolution of genes encoding sperm components ( Dorus et al. 2006). The absence of rapid evolution across the DmSP is perhaps not surprising as many integral sperm proteins are involved in essential and oftentimes ubiquitous functions, such as metabolism and energetics. This finding highlights the central role of sperm in sexual reproduction and likely reflects the ancient evolutionary history of sperm ( Baccetti 1982, 1986). In this study, we have utilized an alternative approach to specifically determine if gene creation is a mechanism driving the evolution of sperm form and function and demonstrate that Drosophila sperm has, in fact, been impacted by the recent origination and diversification of novel sperm genes. Proteomic and comparative genomic approaches used in this study revealed the expansion of the Mst35B and tektin gene families during the evolution of D. melanogaster. In both cases, gene family expansion occurred through tandem duplication events and resulted in the creation of testis-specific sperm genes. Despite these similarities, the selective forces influencing the evolutionary histories of these expanded gene families are quite distinct. In the case of Mst35B, rapid evolution and a significant signature of positive selection was observed that is highly concentrated within the DNA-binding HMG box domain. This finding is consistent with the evolution of the DmSP where DNA-/RNA-binding proteins were found to evolve at an accelerated rate similar to that of male accessory gland genes ( Dorus et al. 2006). It is true, however, that relaxation of functional constraint could also contribute to the observed evolutionary acceleration of these new genes. HMG box domains are found in a variety of critical proteins essential for sex determination or sex differentiation, such as the testis-determining SRY and SOX9 genes in mammals ( Harley et al. 2003) and the mating-type ( MAT) locus of yeast ( Butler et al. 2004). Protamines have also been shown to be influenced by positive selection in primates ( Wyckoff et al. 2000), but the underlying selective forces are unclear ( Rooney and Zhang 1999). It is also known that protamine mutants are not haploinsufficient in Drosophila, but detailed complementation analyses of Mst35Ba and Mst35Bb mutants have yet to be conducted ( Raja and Renkawitz-Pohl 2005). Such analyses will be crucial in determining to what extent the rapid evolution of the HMG box domain has resulted in neofunctionalization that may be relevant to the process of genome condensation and packaging during spermatogenesis. The tektin gene family, on the other hand, has originated much more recently and appears to be under strong selective constraint. This gene family encodes tektin-like cytoskeletal proteins that interact directly with microtubules in the sperm axoneme ( Steffen and Linck 1988), and their more constrained evolutionary characteristics are consistent with the conservative evolution of sperm structural components ( Dorus et al. 2006). Selective pressures, associated with expression level requirements during spermatogenesis, may have driven the rapid and recent increase in copy number. We note that 2 of the tektin genes, CG32819 and CG32820, are identical at the nucleotide level suggesting an extremely recent duplication event. Our comparative analysis of all D. melanogaster genes reveals that the incidence of genes sharing identical exon and intron sequences is rare (<0.3% of all genes) and that this gene pair may therefore be among the youngest genes in the genome. Population genetic screens will be essential to determine if the most recent duplication event is fixed within worldwide melanogaster populations or whether it represents a copy number polymorphism. Although evident from comparative genomic analyses that this duplication event occurred on the melanogaster lineage, it could be posited that gene conversion is responsible for complete sequence homogenization between tektin gene copies. Explicit tests for gene conversion are confounded by the fact that the gene cluster itself originated through unequal crossover events. However, we do not favor gene conversion as an explanation for several reasons. First, complete sequence identity exists between the regions containing CG32819 and CG32820, whereas the region containing CG17450 is divergent throughout its length. It seems improbable that pervasive gene conversion occurs between 2 of the 3 neighboring loci without also homogenizing the third loci to an appreciable and detectable level. Second, complete nucleotide identity exists between 2 neighboring ~6-kb regions ( supplementary fig. 1, Supplementary Material online). Complete homogenization of such large regions is at odds with previous estimates of gene conversion tract length (~750 bp) in Drosophila ( Hilliker et al. 1994) and more recent estimates in experimental mammalian systems ( Ruksc et al. 2008). Lastly, population genetic studies indicate that gene conversion is less influential as a homogenizing force among X-linked paralogs than those located on autosomes ( Thornton and Long 2005). We therefore favor the interpretation that the tektin gene cluster is the result of very recent gene duplication events, the last of which may not have yet fixed in worldwide D. melanogaster populations. Our discovery of the tektin sperm gene cluster on the Drosophila X chromosome is somewhat unexpected for several reasons. First, it has been demonstrated that the Drosophila X chromosome contains a paucity of testis-overexpressed genes and testis-enriched gene clusters ( Boutanaev et al. 2002; Parisi et al. 2003, 2004). Second, there is also a paucity of integral sperm genes on the Drosophila X ( Dorus et al. 2006). Finally, it has been shown that genes with male-biased expression or functionality tend to move off of the X chromosome and onto the autosomes during evolution ( Betran et al. 2002; Dai et al. 2006; Shiao et al. 2007). To our knowledge, only one other example of a newly created, testis-specific X-linked gene cluster has been reported in D. melanogaster, the Sdic gene cluster ( Ponce and Hartl 2006). Therefore, the tektin and Sdic gene clusters represent an unusual and rare class of recently expanded X-linked sperm structural gene families. It has now been demonstrated in both primates and Drosophila that genes created through retrotransposition often acquire testis expression ( Betran et al. 2002; Dorus et al. 2003; Vinckenbosch et al. 2006; Bai et al. 2007; Shiao et al. 2007). Our results extend this observation by demonstrating that some retrotransposed genes in Drosophila are not only expressed during spermatogenesis but also encode protein components of sperm. Although the mechanisms responsible for the evolution of testis expression are not well understood, it has been argued that large, chromatin-mediated regulatory domains within the genome may promote testis expression, a hypothesis supported by the existence of testis-enriched clusters of genes ( Boutanaev et al. 2002; Parisi et al. 2004; Divina et al. 2005). Our identification of regions of significant clustering that contain new sperm genes also points to a possible link between regional gene regulation mechanisms, the establishment of testis expression, and subsequent functional acquisition. A more thorough analysis of the genomic distribution of testes-overexpressed genes will be necessary to determine the extent to which such a functional and mechanistic link exists. However, alternative mechanisms cannot be ruled out, including promoter sharing with neighboring genes ( Loppin et al. 2005), the direct inheritance of parental promoters ( Feral et al. 2001), the use of promoter elements from nearby transposable elements ( van de Lagemaat et al. 2003), and the acquisition of new untranslated regions from neighboring sequences ( Shiao et al. 2007). Little is currently known about the mechanisms underlying how, when or why proteins become incorporated into a functional sperm during evolution. It is clear from our previous study that integral sperm genes are nonrandomly distributed throughout the genome suggesting higher order regulatory properties may be involved. However, the retrogenes described in this study do not localize to any of the previously characterized DmSP gene clusters. Ultimately, a variety of complementary experimental approaches will be needed to elucidate the cellular and developmental mechanisms responsible for the inclusion of testis-expressed genes as novel sperm components. This study highlights the influence of new gene creation on the evolution of Drosophila sperm and provides strong evidence for the influence of positive selection on the functional divergence of the recently created Mst35B protamine paralogs. Analysis of newly retrotransposed sperm genes also suggests that localization in clusters of testis-overexpressed genes may be associated with the establishment of testis expression and acquisition of male-biased functions. The acquisition of functionality in sperm, and more generally in spermatogenesis, appears to be a common conduit in the fixation and early functional evolution new genes. 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