Our understanding of the role of positive selection in the evolution of genes with male-biased expression can be hindered by two observations. First, male-biased genes tend to be overrepresented among lineage-specific genes. Second, novel genes are prone to experience bursts of adaptive evolution shortly after their formation. A thorough study of the forces acting on male-biased genes therefore would benefit from phylogenywide analyses that could distinguish evolutionary trends associated with gene formation and later events, while at the same time tackling the interesting question of whether adaptive evolution is indeed idiosyncratic. Here we investigate the roughex (rux) gene, a dose-dependent regulator of Drosophila spermatogenesis with a C-terminal domain responsible for nuclear localization that shows a distinct amino acid sequence in the melanogaster subgroup. We collected polymorphism and divergence data in eight populations of six Drosophila species, for a total of 99 rux sequences, to study rates and patterns of evolution at this male-biased gene. Our results from two phylogeny-based methods (PAML and HyPhy) as well as from population genetics analyses (McDonald–Kreitman-based tests) indicate that amino acid replacements have contributed disproportionately to divergence, consistent with adaptive evolution at the Rux protein. Analyses based on extant variation show also the signature of recent selective sweeps in several of the populations surveyed. Most important, we detect the significant and consistent signature of positive selection in several independent Drosophila lineages, which evidences recurrent and concurrent events of adaptive evolution after rux formation.

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GENOMEWIDE analyses of gene expression in a variety of organisms including Drosophila have revealed differences in messenger RNA abundances between the two sexes for a substantial fraction of genes (JIN et al. 2001; ARBEITMAN et al. 2002; PARISI et al. 2003; RANZ et al. 2003; GIBSON et al. 2004; REINKE et al. 2004; RINN et al. 2004). On the other hand, several molecular evolutionary studies have now described that genes with sex-biased expression show fast rates of protein evolution (NUZHDIN et al. 2004; ZHANG et al. 2004; ZHANG and PARSCH 2005). This observation is consistent with earlier findings showing that reproductive proteins evolve more quickly than proteins with non-sex-related functions, granted that much of the difference in expression profiles between sexes occurs in the germ line (THOMAS and SINGH 1992; CIVETTA and SINGH 1995; VACQUIER 1998; BEGUN et al. 2000; WYCKOFF et al. 2000; BIRKHEAD and PIZZARI 2002; SWANSON and VACQUIER 2002; CASTILLO-DAVIS et al. 2004). Additionally, there are several examples of genes where these high rates of amino acid replacements result from adaptive protein evolution likely triggered by sexual selection and/or sexual conflict (AGUADÉ et al. 1992; CIVETTA and SINGH 1998; AGUADÉ 1999; BIRKHEAD and PIZZARI 2002; SWANSON and VACQUIER 2002; BEGUN and LINDFORS 2005; ELLEGREN and PARSCH 2007). Although there is increasing evidence for adaptive evolution in female reproductive genes (SWANSON et al. 2001, 2003; TURNER and HOEKSTRA 2006; KELLEHER et al. 2007; LAWNICZAK and BEGUN 2007), most reported cases pertain to the male class (TORGERSON et al. 2002; SWANSON et al. 2003; GOOD and NACHMAN 2005; NIELSEN et al. 2005; CIVETTA et al. 2006; PROSCHEL et al. 2006; BAUER DUMONT et al. 2007).

Our understanding of how natural selection works on male-biased genes in Drosophila can be confounded, however, by two features. First, male-biased genes have higher effective birth and extinction rates, and this appears to be widespread among several Drosophila genomes (BEGUN and LINDFORS 2005; WAGSTAFF and BEGUN 2005a,b; BEGUN et al. 2006, 2007; LEVINE et al. 2006; HAERTY et al. 2007; ZHANG et al. 2007). In agreement with this idea, a large fraction of novel genes show sex-specific expression, and testis-specific expression in particular (LEVINE et al. 2006). Second, novel genes, evolved through gene duplication (LONG et al. 2003) or by "de novo" recruitment of noncoding DNA (LEVINE et al. 2006), are prone to bursts of adaptive evolution shortly after their birth (LONG and LANGLEY 1993; BETRAN and LONG 2003; JONES and BEGUN 2005; CHEN et al. 2007). Thus, a thorough study of selective trends acting on male-biased genes would benefit from phylogenywide analyses that could take into account evolutionary trends occurring shortly after gene formation and later events.

Here we analyze the molecular evolution of the roughex (rux) gene, a dose-dependent regulator of spermatogenesis (GONCZY et al. 1994), in several independent lineages of Drosophila. rux is indeed a male-biased gene, with transcript levels in males significantly higher than in females (PARISI et al. 2003; RANZ et al. 2003; RIFKIN et al. 2003; GIBSON et al. 2004), and earlier evolutionary analyses of the Rux protein by AVEDISOV et al. (2001) reported rapid rates of amino acid replacements attributed to low overall selective constraints. Interestingly, our analysis of the recently available 12 Drosophila genomes (CLARK et al. 2007) reveals that the Rux domain responsible for proper intracellular (nuclear) localization is present only in the melanogaster subgroup. This suggests that the rux function (sensu Drosophila melanogaster) likely evolved 8–15 million years ago (LACHAISE et al. 1988). In all, rux is an ideal candidate for investigating whether adaptive evolution in male-biased proteins occurs repeatedly in independent lineages or if, in contrast, adaptation is idiosyncratic in nature (LEVINE and BEGUN 2007).

To obtain a comprehensive picture of the evolutionary trends in rux we analyzed polymorphism data in eight populations from six species of the melanogaster subgroup (for a total of 99 chromosomes). Our study reveals the signature of recurrent events of adaptive evolution, with a significant excess of amino acid replacements fixed between species. More interestingly, positive (Darwinian) selection in the Rux protein has occurred independently in several lineages and these concurrent events are detected as bursts of amino acid replacements after rux formation as well as patterns of polymorphism consistent with more contemporary selective sweeps in different species.

Drosophila stocks and population surveys:

To obtain polymorphism and divergence data we surveyed eight populations in six Drosophila species: one population each of D. santomea, D. melanogaster, D. mauritiana, and D. sechellia and two populations each (one island and one mainland) of D. simulans and D. yakuba. The Tucson Drosophila Stock Center made available D. simulans flies from the Democratic Republic of Congo, and we used also ethanol-preserved males of D. simulans collected in the island of São Tomé. T. F. Mackay kindly provided isofemale lines of D. yakuba from Abidjan, Ivory Coast, and the "island" populations of D. yakuba and D. santomea were collected in São Tomé (LLOPART et al. 2005). Highly inbred lines of D. melanogaster from Raleigh, North Carolina, were kindly supplied by T. F. Mackay and are described in FRY et al. (1998). D. mauritiana and D. sechellia lines were generously provided by J. A. Coyne. One additional stock of D. mauritiana (Tucson Drosophila Stock Center no. 14021-0241.07) was also used to investigate the exon–intron structure of the rux gene.

DNA preparation and sequencing:

We extracted genomic DNA from single males using the Puregene DNA isolation kit for paraffin-embedded tissue (Gentra Systems, Minneapolis) with minor modifications. PCR reactions were performed using primers designed to either D. melanogaster or D. yakuba sequences from GenBank. To control for possible demography that may mimic the fingerprints of selection in island populations we used multilocus data from LLOPART et al. (2005) in the cases of D. yakuba and D. santomea, and for the population of D. simulans collected in São Tomé we amplified the putatively neutral gene Xdh. After cleanup of the PCR fragments using the Wizard MagneSil system (Promega, Madison, WI) both strands were sequenced directly on a 3730 DNA analyzer using the Big Dye 3.1 chemistry (Applied Biosystems, Foster City, CA). To edit the sequences and assemble the contigs we used the software Sequencher 4.1 (Gene Codes, Ann Arbor, MI). Haplotypes in the Xdh region were determined experimentally by performing cycle-sequencing reactions using allele-specific primers, whereas rux haplotypes were inferred directly because it is an X-linked gene. Alignment of the amino acid sequences was obtained using ClustalX (THOMPSON et al. 1997). We surveyed exon 2 (1000 bp) of the rux gene. One additional region encompassing exon 1 (22 bp), intron 1 (100 bp), and 5'-noncoding sequence (600 bp) was also investigated in the D. melanogaster, D. simulans, D. mauritiana, and D. sechellia populations. In the 5'-noncoding region of D. sechellia we discovered the remnants of an unknown non-LTR retrotransposon (sequence AF237761 in GenBank), which was excluded from the study. We restricted our analyses to the portion of the 5'-noncoding sequence that could be aligned among all species. The D. teissieri, D. erecta, and D. orena rux sequences used here were published elsewhere (AVEDISOV et al. 2001), and we also used previously reported sequences of the rux gene in the D. yakuba and D. santomea populations from São Tomé (LLOPART et al. 2005).

To investigate the exon–intron structure of the rux gene in D. mauritiana we extracted total RNA from 1-day-old adult females and males using the RNAeasy kit (QIAGEN, Valencia, CA), and first-strand cDNA was obtained using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). RT–PCR reactions were performed using primers designed to two exons. Cleanup of PCR products and sequencing were carried out as described above. Sequences newly reported in this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. EU476916–EU477049.

Inference about the Rux nuclear domain:

Earlier studies using Southern blot analysis and PCR with degenerated primers failed to detect rux homologous sequences in several species outside the melanogaster subgroup (AVEDISOV et al. 2001). To explore the possibility that rux may be a novel gene emerged in the melanogaster subgroup, we searched all available Drosophila genomes using both BLASTP and TBLASTN programs, using default parameters (http://flybase.net/blast/). While BLASTP is used for finding similar amino acid sequences in annotated protein databases, TBLASTN allows you to compare a protein sequence to the six-frame translations of a nucleotide database. Our searches were performed using the complete amino acid sequence, the C-terminal domain (amino acids 188–335 in AVEDISOV et al. 2001), and the non-C portion of the D. melanogaster Rux (supplemental Figure S1).

Inference about selection:

To detect the effects of positive selection using divergence data (one sequence per species analyzed) we first applied the maximum-likelihood method (NIELSEN and YANG 1998; YANG et al. 2000) implemented in the program codeml of PAML (version 3.15) (YANG 1997). This method is based on the comparison of several site-specific models with different assumptions regarding the ratio of nonsynonymous to synonymous substitution rates ( = d_N/d_S) used as an estimate of the selective pressure across sites. We compared models M1 (nearly neutral) vs. M2a (positive selection) and M7 (beta) vs. M8 (beta and ), using equilibrium codon frequencies calculated from the average nucleotide frequencies at third codon positions (CodonFreq = 2). Model M1 assumes two classes of sites, conserved sites with estimated from the data ( < 1) and neutral sites ( = 1), while M2a considers an extra third class of sites under positive selection ( > 1) (WONG et al. 2004; YANG et al. 2005). In M7 and M8 varies across sites following a beta distribution, with M8 having the additional discrete class of sites with > 1.

We identified positively selected sites at which nonsynonymous substitutions occur at a higher rate than synonymous ones with the Bayes Empirical Bayes (BEB) calculation of posterior probabilities for site classes (YANG et al. 2005), implemented in codeml (PAML, models M2a and M8). To account for potential heterogeneity across sites in d_S we also applied two recently developed methods (KOSAKOVSKY POND and FROST 2005) executed by the software HyPhy (POND and FROST 2005) and available in the web-based interface Datamonkey (http://www.datamonkey.org/; POND and FROST 2005). Both methods are based on maximum-likelihood estimates, but one assumes that substitution rates across sites can vary according to a gamma distribution and infers the rate at which individual sites evolve (random-effects models, REL) while the other estimates the ratio of nonsynonymous to synonymous substitutions on a site-by-site basis (fixed-effects models, FEL).

McDonald and Kreitman (MK) tests (MCDONALD and KREITMAN 1991) as well as basic calculations of polymorphism statistics were performed using the program DnaSP 4.10 (ROZAS et al. 2003). To estimate the selection coefficients () associated with nonlethal amino acid replacement mutations we used the Markov chain Monte Carlo (MCMC) method implemented in the web-based program MKPRF (http://cbsuapps.tc.cornell.edu/mkprf.aspx) (BUSTAMANTE et al. 2002, 2003; BARRIER et al. 2003). Posterior means and probabilities of 0 were estimated after 10,000 iterations (1000 burn-in iterations).

To contrast whether polymorphism patterns were compatible with a standard neutral model we applied the DH test (ZENG et al. 2006), which is based on the combined study of D (TAJIMA 1989b) and H (FAY and WU 2000) statistics. The H statistic was calculated for D. yakuba and D. santomea using D. teissieri as outgroup and for D. simulans, D. mauritiana, and D. sechellia using D. melanogaster as outgroup, and for the D. melanogaster polymorphisms we used the D. simulans sequence. Statistical significance of the DH test was assessed using neutral coalescent simulations produced by the program ms (http://home.uchicago.edu/rhudson1/; HUDSON 2002). Probabilities were estimated by generating 10,000 replicate samples with the same number of mutations as that observed in the data. Simulations were performed under the assumption of no recombination and with likely levels of intragenic recombination estimated using the maximum-likelihood method implemented in the program MaxHap (http://home.uchicago.edu/rhudson1/; HUDSON 2001).

To determine whether polymorphism patterns at the rux gene could be explained by demographic events in island populations, we simulated different population dynamics using the program ms (http://home.uchicago.edu/rhudson1/; HUDSON 2002). We modeled an ancestral panmictic population that undergoes a size reduction (i.e., island colonization), experiences reduced constant size for some time period, and finally expands by growing exponentially. The population growth is specified by the equation N₂ = N₁exp^–t, where N₂ is the current population size, N₁ is the size prior to the expansion, is the growth parameter, and t is the time at which the growth began. Simulations to evaluate DH values in the D. yakuba and D. simulans island populations consider variables to be both the time at which the size reduction occurred and the period of constant reduced size. In contrast, the modeling of D. santomea assumes that the island colonization occurred 0.45 million years ago, an estimate of the time of the D. yakuba–D. santomea split (LLOPART et al. 2002). A total of 10,000 replicates were used to estimate P-values.

Protein divergence in Rux:

Rates of evolution at nonsynonymous (d_N) and synonymous (d_S) sites in the rux gene were first estimated by maximum likelihood using PAML. Consistent with an overall pattern of functional constraints on protein evolution in the nine species of the melanogaster subgroup, the average d_N/d_S ratio () across sites and lineages is <1 ( = 0.47; Table 1). Likelihood-ratio tests comparing models that assume no positive selection (M1 and M7) to models that allow for a fraction of sites evolving under positive selection (M2a and M8) show, however, that the observed pattern of divergence at rux is best explained by the inclusion of a class of sites under positive selective pressure (P 0.0001). These comparisons suggest also that 5–7% of codons are evolving by Darwinian selection with estimates of 4. The BEB method identifies 17 codon sites as positively selected, with 3 sites having a very high (P > 0.95) posterior probability (Table 1).

View this table: In this window In a new window   TABLE 1 Results of positive selection in the rux gene from PAML and HyPhy

 
We also investigated the signature of positive selection based on the d_N/d_S ratio by applying the FEL and REL methods (implemented in the HyPhy package), which incorporate not only nonsynonymous but also synonymous rate variation among codon sites explicitly (KOSAKOVSKY POND and FROST 2005; POND and FROST 2005). Results from FEL and REL show good agreement with those obtained using PAML (Table 1). In the nine species of the melanogaster subgroup the REL method identifies seven sites with very strong evidence for positive selection (posterior probabilities >0.95) and the FEL approach detects three positively selected sites (P < 0.05). Notably, several codon sites are identified by all three methods (BEB, FEL, and REL) as positively selected.

To investigate whether the results suggesting positive selection based on divergence data are due to the effect of a single lineage we analyzed separately the two main clades of the melanogaster subgroup. The first clade (D. yakuba–D. erecta) includes D. yakuba, D. santomea, D. teissieri, D. orena, and D. erecta, while the second clade (D. simulans–D. melanogaster) includes D. simulans, D. mauritiana, D. sechellia, and D. melanogaster (Figure 1). In both clades an extra class of nonsynonymous sites with significantly >1 is required to best explain the data (Table 1). The BEB method identifies 33 and 15 codon sites positively selected in the D. yakuba–D. erecta and the D. simulans–D. melanogaster clades, respectively. The methods implemented in HyPhy (FEL and REL) also detect codon sites with > 1 in both clades.

View larger version (14K): In this window In a new window Download PPT slide   FIGURE 1.— Phylogenetic relationship among the nine species of the D. melanogaster subgroup inferred from rux gene genealogy. The proposed phylogeny is in complete agreement with that obtained from the 12 Drosophila Genomes Project (CLARK et al. 2007).

 
Remarkably, the distribution of codon sites under positive selection across the gene differs between the two clades of the melanogaster subgroup (Figure 2). In the D. yakuba–D. erecta clade PAML identifies sites possibly under positive selection to be evenly distributed along the protein sequence, with sites with the highest posterior probabilities located in the N-terminal two-thirds of the protein (amino acids 1–217 in AVEDISOV et al. 2001). In contrast, in the D. simulans–D. melanogaster clade the signature of positive selection is detected only in the C-terminal domain (amino acids 188–335 in AVEDISOV et al. 2001) of the protein. An equivalent distribution of sites with significantly greater than one is obtained by FEL and REL methods, with positive selection in the D. simulans–D. melanogaster clade detected only in the C-terminal third of the protein. Thus, results of both PAML and HyPhy show evidence for adaptive evolution in the melanogaster subgroup as well as in the two main clades within the subgroup with spatial differences across the Rux protein. (supplemental Table S1 lists all codon sites identified as positively selected using PAML and HyPhy methods in the different taxa.)

View larger version (15K): In this window In a new window Download PPT slide   FIGURE 2.— Distribution of positively selected sites along exon 2 of the rux gene identified by PAML (BEB) (diamonds, posterior probabilities >0.50), FEL (circles, posterior probabilities >0.50), and REL (triangles, P < 0.20) in species of (A) the melanogaster subgroup, (B) the D. yakuba–D. erecta clade, and (C) the D. simulans–D. melanogaster clade.

 

Rux polymorphism:

We surveyed patterns of nucleotide polymorphism at the rux gene in six species, with a total of 99 chromosomes from eight populations. Table 2 lists summary statistics of sequence polymorphism. In agreement with previous reports, D. sechellia shows extremely reduced levels of polymorphism, a pattern that is suggestive of a severely reduced effective population size (KLIMAN et al. 2000). Another overall feature that stands out from our survey is the high number of nonsynonymous polymorphisms observed in the rux gene of D. mauritiana (18 replacement and 11 synonymous polymorphisms in a sample of 6 chromosomes). Furthermore, all these polymorphisms are located in the C-terminal half of the protein and most (10) are clustered in an 80-bp-long region of exon 2. This region corresponds to a six-amino-acid repeat unit that has expanded in D. simulans, D. mauritiana, and D. sechellia but is not present in either D. melanogaster or the D. yakuba–D. erecta clade. To investigate the possibility that the abundance of replacement polymorphisms is due to a change in the exon–intron structure of the rux gene in D. mauritiana we obtained the sequence of the cDNA. Sequence analyses indicate that the exon–intron boundaries are conserved between D. mauritiana and D. melanogaster and that the hypervariable 80-bp-long region is present in the D. mauritiana cDNA (supplemental Figure S2). Thus the region where most amino acid replacement polymorphisms cluster does not represent a new intron evolved in D. mauritiana, but a portion of the coding sequence with severely reduced selective constraints in this species.

View this table: In this window In a new window   TABLE 2 Polymorphism in the rux gene

 

Inference about selection using patterns of divergence and polymorphism:

To determine whether patterns of variation in the rux gene are consistent with a neutral model we applied the MK test that compares patterns of divergence and polymorphism (MCDONALD and KREITMAN 1991). This test contrasts the ratio of polymorphic to fixed differences at amino acid replacement and synonymous or silent sites and is based on the prediction of the neutral theory that divergence between species correlates with variation within species (KIMURA 1983). We used two different approaches to assess the statistical significance of the difference in the ratios: the 2 x 2 goodness-of-fit test proposed originally (MCDONALD and KREITMAN 1991) and a maximum-likelihood method based on the Poisson random field framework (MKPRF) (SAWYER and HARTL 1992; BUSTAMANTE et al. 2003). This latter approach also allows us to estimate directly the direction and strength of selection (; = 2N_es) acting on amino acid replacement mutations in the rux gene. The results of the MK and MKPRF tests as well as estimates of on nonlethal amino acid replacements are shown in Table 3.

View this table: In this window In a new window   TABLE 3 Adaptive evolution in the Rux protein

 
The comparison between D. yakuba and D. santomea indicates that amino acid replacements have contributed disproportionately to divergence between these sibling species (G = 12.49, P = 0.0004), consistent with adaptive evolution in the Rux protein. MK-based tests between very closely related species, however, should be interpreted carefully because some of the observed fixed differences may be due to ancestral polymorphism (CHARLESWORTH et al. 2005). To minimize these confounding effects we performed MK tests using the more distant D. teissieri and also detected the signature of adaptive evolution in both D. teissieri–D. yakuba (G = 5.52, P = 0.019) and D. teissieri–D. santomea (G = 8.30, P = 0.004) comparisons. Congruently, estimates of are positive (+2.56 and +6.39) and significantly >0 (P = 0.0098 and P = 0.0008) for the D. teissieri–D. yakuba and D. teissieri–D. santomea comparisons, respectively. In the D. simulans–D. melanogaster clade there is also a minor but significant excess of amino acid replacements fixed between these species and the estimate of is also >0 ( = +2.24, P = 0.011). When we remove singletons to reduce the confounding effects of weakly deleterious mutations that will not contribute to long-term evolutionary trends, the excess of amino acid replacements between species remains significant in both comparisons (G = 8.01, P = 0.0045 for D. yakuba–D. santomea and G = 6.51, P = 0.012 for D. simulans–D. melanogaster). We find no evidence for recent selection at the Rux protein in the D. simulans lineage after the split from its closer relatives D. mauritiana and D. sechellia.

Finally, we performed lineage-specific MK tests to determine whether the excess of adaptive nonsynonymous substitutions is associated with a single lineage or results from independent events in different lineages. Using parsimonious criteria we assigned fixed differences to either the D. yakuba or the D. santomea lineages, using the D. teissieri sequence as the outgroup, and D. erecta was used to polarize fixed differences in the lineage leading to the ancestor of D. yakuba and D. santomea after the split from D. teissieri. (The D. orena sequence was not used as the outgroup because it shows extreme GC bias at some loci; see TAKANO-SHIMIZU 1999.) In the comparison between D. melanogaster and D. simulans, the D. yakuba sequence was used as the outgroup. Lineage-specific MK tests indicate a rapid accumulation of amino acid replacements in three lineages of the melanogaster subgroup (D. yakuba, D. santomea, and D. melanogaster lineages), as well as in the lineage leading to the D. yakuba and D. santomea pair after the split from D. teissieri (Figure 3). While the high number of amino acid replacements in the comparison D. yakuba–D. santomea is due to an excess of fixations in each lineage after the split, in the D. melanogaster–D. simulans comparison only the D. melanogaster lineage shows evidence of adaptive evolution (Figure 3). Following SMITH and EYRE-WALKER (2002) we estimated the number of adaptive amino acid substitutions in the D. yakuba, D. santomea, and D. melanogaster lineages to be 4.84, 5.50, and 12.92, respectively.

View larger version (12K): In this window In a new window Download PPT slide   FIGURE 3.— Results of lineage-specific MK tests in the rux locus [goodness-of-fit (G) values and probabilities (P)]. Branch lengths do not represent real genetic distances.

 

Inferences about selection based on the frequency of polymorphic mutations:

To study whether patterns of intraspecific variation in the rux gene are consistent with a neutral model or, conversely, are suggestive of recent adaptive events we applied the DH test (ZENG et al. 2006). This test is based on the combined detection of both low- and high-frequency polymorphisms summarized by Tajima's D (TAJIMA 1989b) and Fay and Wu's H (FAY and WU 2000) statistics, respectively. An excess of low-frequency polymorphisms is expected after the removal of variation associated with a selective sweep (MAYNARD SMITH and HAIGH 1974; KAPLAN et al. 1989) or the elimination of deleterious mutations in small populations (CHARLESWORTH et al. 1993). When recombination occurs, however, a selective sweep is also expected to generate a combination of both high- and low-frequency mutations segregating in the population (FAY and WU 2000), the simultaneous detection of which (i.e., the DH test) is an effective way to identify the signature of positive selection using within-species variation (ZENG et al. 2007). Table 4 summarizes the results of DH tests for the rux gene under the conservative assumption of no recombination and under likely values of intragenic recombination estimated from the data (TAJIMA 1989b; WALL 1999; HUDSON 2001; ZENG et al. 2006, 2007). In all, three populations of the D. yakuba–D. erecta clade (Ivory Coast and São Tomé populations of D. yakuba and the São Tomé population of D. santomea) as well as the D. simulans population from São Tomé show polymorphisms in the rux gene that are skewed significantly toward low and high frequency of derived mutations.

View this table: In this window In a new window   TABLE 4 Neutrality tests based on polymorphism data of the rux gene

 
Although our results from the DH tests are consistent with a recent selective sweep in the rux region, demographic events, such as a recent population bottleneck or an expansion, can also skew the frequency spectrum, mimicking the effects of positive selection (TAJIMA 1989a). This possibility is particularly likely for D. santomea and the São Tomé population of D. yakuba, where there is evidence for nonequilibrium based on multilocus analyses (LLOPART et al. 2005; BACHTROG et al. 2006). Despite the fact that many of these nonselective scenarios are expected to have a smaller influence on H and DH than on other population statistics (PRZEWORSKI 2002; ZENG et al. 2006, 2007), we investigated whether demography alone could explain DH estimates as well as the number of polymorphisms observed at the rux locus in island populations. Using a number of combinations of changes in population sizes, we conducted coalescent simulations where a population undergoes an instantaneous reduction in size (i.e., invasion of an island) followed by a period of time for local adaptation and final expansion (see MATERIALS AND METHODS and supplemental Table S2 for a detailed description of the specific parameters used in the simulated demography). These simulations, however, are not intended to be exhaustive: we have inevitably missed more complex population dynamics. They serve nonetheless to assess how extreme cases of demography can possibly influence neutrality tests based on the frequency spectrum of polymorphisms in island populations. The results indicate that none of the simple demographic scenarios considered entirely explains the observed number of polymorphisms and the frequency spectrum in the rux locus (Table 5 and supplemental Table S2). While population dynamics that incorporate a very recent and brief population size reduction for D. yakuba and D. simulans are the most compatible with the number of polymorphisms observed today, they fail to produce significant DH tests. Likewise results in the D. santomea population show that none of the modeled demographic histories produces DHs that are equal or more extreme that those observed in the rux region.

View this table: In this window In a new window   TABLE 5 Neutrality tests based on polymorphism data of the rux gene under different simulated demographic scenarios in island populations

 
Despite the fact that none of the simulated demographic scenarios can fully explain the polymorphism patterns in the rux locus, we cannot formally rule out more complex neutral population dynamics (e.g., bottleneck followed by expansion combined with species introgression) having an effect on the frequency spectrum of polymorphisms in island populations. This appears to be the case in D. santomea for which the study of 29 loci showed 7 loci with more negative Tajima's D than rux (LLOPART et al. 2005). In contrast, none of the 29 loci analyzed in the island population of D. yakuba showed a more extreme D-value than that of rux, which strongly supports the notion of positive selection. As for the D. simulans population from São Tomé, there is no multilocus analysis yet to test mutation–drift equilibrium; meanwhile we collected polymorphism data for the putatively neutral locus Xdh. The analysis of the frequency spectrum at Xdh shows no significant departure from neutrality (_S = 0.064; D = 0.27, H = –12.35, P = 0.10 without recombination and P = 0.073 with recombination), which again would be consistent with positive selection on rux. Finally, we considered possible demographic effects for the D. yakuba population collected in Ivory Coast, the only mainland population for which there is a significant excess of low- and high-frequency derived polymorphisms. A study of 21 genes in this population of D. yakuba indicates a minor, if any, effect of demography on the frequency spectrum of rux (Ivory Coast population of D. yakuba mean D = –0.19; A. WILLIFORD, A. LLOPART and J. M. COMERON, unpublished data). This again supports the idea of recent positive selection acting on the rux gene in D. yakuba.

Here we combine several phylogeny-based methods with molecular population genetics analyses to show that variation between and within species at the rux gene in Drosophila is best explained by recurrent events of positive selection in independent lineages. In particular we detect a significant excess of amino acid replacements fixed between species that is observed when comparing to synonymous divergence (d_N/d_S approach using both PAML and HyPhy) as well as when contrasting levels of polymorphism and divergence for both replacement and silent mutations (MK and MKPRF approaches). This excess, we suggest, is the result of adaptive fixations of selectively advantageous amino acid replacements in the Rux protein and has occurred concurrently in different lineages.

Evidence for adaptive protein evolution in different lineages has been previously reported in genes responsible for reproductive isolation among Drosophila species, and in these cases positive selection is restricted to ancient events (BARBASH et al. 2004; PRESGRAVES and STEPHAN 2007). rux evolution is distinctive because our analyses of extant variation at the rux gene reveal also an excess of both high- and low-frequency polymorphisms (DH test) in four of eight populations surveyed, a signature of recent events of positive selection in recombining regions that cannot be explained by simple demographic scenarios alone. Although these departures in the frequency spectrum of polymorphisms are consistent with adaptive amino acid fixations, divergent gene expression cannot be ruled out as an additional target of selection (CHEN et al. 2007). Indeed male-biased genes are prone to show patterns of expression that are divergent between species (MEIKLEJOHN et al. 2003; MICHALAK and NOOR 2003; RANZ et al. 2003; NUZHDIN et al. 2004). In agreement with the possibility of selection at gene expression divergence, rux mRNA abundance differs significantly between males of D. melanogaster and D. simulans (PARISI et al. 2003; RANZ et al. 2003) and also between males of the very closely related D. yakuba and D. santomea (A. LLOPART, R. RATNAPPAN and J. M. COMERON, unpublished data).

A possible caveat to the use of the standard method to detect positive selection based on the d_N/d_S ratio (based on PAML software) is that a portion of synonymous mutations in Drosophila are not neutral (AKASHI 1995; AKASHI and SCHAEFFER 1997; KLIMAN 1999; MCVEAN and VIEIRA 2001; COMERON and GUTHRIE 2005; LLOPART et al. 2008). Therefore one could argue that in genes with strong selection on synonymous mutations (high codon bias) events of positive selection acting on amino acid replacements might be overestimated due to reduced rate of evolution at synonymous sites (reduced d_S and hence high d_N/d_S). Several lines of evidence suggest that our assessment of positive selection for rux based on maximum-likelihood estimates of d_N/d_S is not likely to be caused by selection on synonymous mutations. First, rux does not show a strong bias in synonymous codons, with 57% of D. melanogaster genes showing greater bias based on the frequency of preferred codons. Second, both PAML and HyPhy reveal that different clades exhibit different regions of the rux gene with codon sites under positive selection. Finally, it is worth noting that divergence estimates by PAML in Drosophila genes do not generate a negative relationship between d_S and the degree of codon bias (DUNN et al. 2001). This latter feature is likely a consequence of the methodology used by PAML to estimate the number of synonymous sites (BIERNE and EYRE-WALKER 2003) and, for our purposes, would also suggest that estimates of > 1 in the rux gene are not likely to be caused by reduced d_S associated with increased selection on synonymous sites.

MK-based methods to assess adaptive evolution on amino acid changes also assume neutrality of synonymous sites. Hence one could also contend that weak selection on synonymous sites would decrease the ratio of divergence to polymorphism for synonymous mutations and create the appearance of increased divergence for amino acid replacements under the MK framework. False positives, however, would appear only if replacement mutations were more neutral than synonymous mutations, an unlikely event based on the observation that both divergence and polymorphism levels are reduced at replacement sites (see also ANDOLFATTO 2005; SHAPIRO et al. 2007). More important, segregating mutations are also under stronger selective constraints when they are replacements than when they are synonymous based on an excess of low-frequency variants in the former class of mutations (SHAPIRO et al. 2007). Thus granted that weak selection on synonymous mutations might bias precise estimates of selection () based on the MKPRF framework, it is not likely that it could create the appearance of an excess of amino acid replacements between species in the absence of positive selection at the protein level. Another possible bias in the estimates of may arise from equilibrium departures at synonymous sites, perhaps associated with changes in effective population size (AKASHI 1996; MCVEAN and VIEIRA 2001; EYRE-WALKER 2002; COMERON and GUTHRIE 2005; KERN and BEGUN 2005; AKASHI et al. 2006). Although we cannot formally rule out this possible bias, we argue that, in the absence of adaptive evolution, it is unlikely that the bias would result in the detection of different positively selected sites in different clades.

Indeed, our results indicate also that amino acid replacements possibly subjected to positive selection are distributed in a different manner along the Rux protein in the two clades of the melanogaster subgroup. This is consistent with different portions of the protein having different selective histories in independent lineages. We observe that the N-terminal domain (amino acids 1–217 in AVEDISOV et al. 2001) evolves under positive selection at amino acid divergence solely in the D. yakuba–D. erecta clade. Our BLASTP and TBLASTN searches indicate that this domain is present in every genome of the 12 Drosophila species that have been sequenced (supplemental Figure S1). This domain is responsible for the physical interaction with the product of the gene CycA (AVEDISOV et al. 2000), and future studies will contrast whether its fast evolution is also accompanied by rapid coevolution of CycA, as seen in the interactors of the Drosophila hybrid inviability gene Nup96 (PRESGRAVES and STEPHAN 2007). For now, preliminary analyses based on d_N/d_S estimates in the melanogaster subgroup show no evidence of rapid protein evolution for CycA, with an overall < 0.1 and no increased probability for models that allow for positively selected sites (M1 vs. M2a and M7 vs. M8; P > 0.20 in both cases).

On the other hand, the C-terminal domain (amino acids 188–335 in AVEDISOV et al. 2001) shows evidence of positively selected sites in both clades of the melanogaster subgroup. Whole-genome cDNA microarray analyses show that some regions of the 3' end of the annotated rux gene are indeed transcribed in species outside the melanogaster subgroup (ZHANG et al. 2007; supplemental Figure S1). Nevertheless our BLASTP and TBLASTN searches indicate that this domain has no detectable amino acid similarity (supplemental Figure S1). This is consistent with the C-terminal domain being the result of extremely fast divergence rather than "de novo" formation. Functional analyses in D. melanogaster indicate, however, that the C-terminal domain is necessary for proper intracellular localization to the nucleus (AVEDISOV et al. 2000). Thus we suggest that this highly diverged domain, which comprises a third of the Rux protein (148 of 335 amino acids in D. melanogaster), may have promoted the evolution of a distinct function exclusive to the melanogaster subgroup. It is tempting to propose that rux, as a male-biased gene with mutants showing male, but not female, sterility, evolves under positive selection because of its role in spermatogenesis and that this is somehow achieved by postcopulatory sexual selection and/or sexual conflict (BIRKHEAD and PIZZARI 2002; SWANSON and VACQUIER 2002; ELLEGREN and PARSCH 2007). It is also possible, however, that adaptive evolution acting at the level of amino acid divergence may just be the result of its distinct protein domain. In all, adaptive evolution in the "ancestral" (i.e., N-terminal) domain of the Rux protein appears to be quite idiosyncratic (LEVINE and BEGUN 2007), while the C-terminal domain shows evidence of positive selection in independent Drosophila lineages within the melanogaster subgroup. This raises the interesting possibility that male-biased genes of either recent origin or ancient descent might be under different selective regimes.

We thank the editor and two anonymous reviewers for valuable comments and suggestions. We are also grateful to R. R. Hudson for making available ms and MaxHap programs. Our thanks also go to S. L. Kosakovsky Pond, S. Frost, and A. Poon for the Datamonkey webserver and to the Computational Biology Service Unit (Cornell University) for the MKPRF web server. Part of this work was carried out using computational resources of the Carver Center for Comparative Genomics (University of Iowa). This work was supported by the Roy J. Carver Charitable Trust grant 05-2258 and the National Science Foundation grant DEB-03-44209 to J.M.C. and by a Carver Scientific Research Initiative Grants Funding Initiative Award to A.L.

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. EU476916–EU477049.

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