Journal of Heredity 2004:95(2):114-118
© 2004 The American Genetic Association
Molecular Evolution of X-linked Accessory Gland Proteins in Drosophila pseudoobscura
From the Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803. We thank S. Dixon, P. Michalak, and D. Ortíz-Barrientos for constructive comments on the manuscript and R. Staten for technical assistance. This work was funded by a Sigma Xi grant-in-aid of research to B.A.C., as well as National Science Foundation grants 9980797, 0211007, and 0314552, and Louisiana Board of Regents Governor's Biotechnology Initiative grant 005 to M.A.F.N. National Institutes of Health grant P20 RR16456 from the Biomedical Research Infrastructure Network Program of the National Center for Research Resources provided support for L.S.S as an undergraduate fellow.
Address correspondence to M. A. F. Noor at the address above, or e-mail: mnoor{at}lsu.edu.
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In Drosophila melanogaster and Drosophila simulans, positive Darwinian selection drives high rates of evolution of male reproductive genes, and accessory gland proteins (Acps) in particular. Here, we tested whether 13 X-linked male-specific genes, 4 Acps and 9 non-Acps, are under selective forces in the Drosophila pseudoobscura species group, much as those in the D. melanogaster group. We observed a statistically significant correlation in relative rates of nonsynonymous evolution between the two species groups tested. One Acp examined had a higher rate of nonsynonymous substitution than predicted by a neutral model in both species groups, suggesting its divergence was driven by positive Darwinian selection. To further test for the signature of selection, we examined polymorphism of three Acps within D. pseudoobscura. From this test, no Acp individually bore the signature of positive selection, but the 3 Acps together possessed an excess of nonsynonymous differences between species, relative to polymorphism within species. We conclude that faster evolution of Acps in the D. pseudoobscura group appears to be driven by positive selection, as previously suggested in the D. melanogaster group.
In Drosophila species, the male accessory gland secretes seminal fluids transferred to the female during copulation. The seminal fluid of Drosophila melanogaster contains at least 83 unique accessory gland proteins, or Acps (Chen et al. 1988; Swanson et al. 2001a). These Acps stimulate a series of behavioral and physiological changes in recipient females. For example, Acps are responsible for stimulating egg production and ovulation (Heifetz et al. 2000), increasing egg-laying rate during oogenesis (Heifetz et al. 2001), decreasing frequency of remating (Neubaum and Wolfner 1999), and reducing the female's lifespan (Fowler and Partridge, 1989). Acps may also be important for efficient sperm storage in females (Tram and Wolfner 1999), and may thereby mediate sperm displacement (Clark et al. 1995). Because many such adaptations are advantageous for males but disadvantageous for females, the potential for "sexual conflict" and rapid evolution exists (see reviews in Pizzari and Snook 2003; Swanson 2003). Hence, the functionally diverse and important roles of Acps suggest Acp genes could experience accelerated rates of evolution by means of positive Darwinian selection.
Several studies have tested for the signature of positive selection on Acps and other male-specific genes in the D. melanogaster group (see reviews in Singh and Kulathinal 2000; Swanson and Vacquier 2002; Wolfner 2002). Patterns of variation in genes under positive selection deviate from a neutral model of evolution by possessing an increased number of nonsynonymous nucleotide differences between species. Thus, directional selection can be identified by comparing the ratio of nonsynonymous to synonymous polymorphisms within species to the ratio of nonsynonymous and synonymous fixed differences between species (McDonald and Kreitman 1991). Positive selection can also be identified by determining the nonsynonymous and synonymous nucleotide substitution rates (dN/dS) for coding sequences between two closely related species. Genes with higher dN/dS relative to other coding sequences are experiencing faster amino acid evolution, and a ratio greater than unity is considered a signature of strong positive selection. Consistent with these two predictions of positive selection, Acps evolve faster than non-Acp genes, and there is a significantly higher fraction of fixed nonsynonymous substitutions than predicted by a neutral model of evolution in the D. melanogaster group (e.g., Begun et al. 2000; Swanson et al. 2001a).
To determine whether these conclusions apply to other Drosophila species groups with different effective population sizes and genetic backgrounds, and perhaps with unique selective constraints, we conducted tests for positive Darwinian selection and relatively faster evolution of four X-linked Acps in the Drosophila pseudoobscura species group. D. pseudoobscura diverged from D. melanogaster approximately 21 to 46 million years ago (Beckenbach et al. 1993), and, within each group respectively, Drosophila miranda and Drosophila simulans diverged approximately 2 to 3 million years ago (Lemeunier et al. 1986; Wang et al. 1997; see Figure 1). This system provides a means of conducting a paired comparison of positive selection and evolutionary rates for homologous genes in both species groups. First, we evaluate if relative rates of nonsynonymous substitution (dN/dS) for male-specific genes, including Acps, are comparable in the D. pseudoobscura and D. melanogaster groups. Second, we test for evidence of directional selection on D. pseudoobscura Acps. We predict the X-linked Acp genes in D. pseudoobscura will also be under positive Darwinian selection and evolve faster than X-linked non-Acp genes, as with the trends identified in the D. melanogaster group.
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Drosophila Stocks
D. pseudoobscura stocks used in the present study were collected from James Reserve, California (one strain), Mather, California (one strain), Mesa Verde, Colorado (one strain), Flagstaff, Arizona (one strain and seven wild-caught males), and Zapotitlan, Mexico (one strain). A stock of D. miranda collected from Mather, California, was used as an outgroup for divergence data analyses.
DNA Isolation, PCR Amplification, and Sequencing
Genomic DNA was isolated from adult males of D. pseudoobscura and D. miranda with the single fly squish protocol (Gloor and Engels 1992). Three X-linked Acps (CG2206, CG13309, and CG16707) were amplified by means of Polymerase Chain Reactions (PCRs), using DNA from each of the three species in 50 µl volumes. Primers for PCR amplification were designed from the D. pseudoobscura genome sequence available at the Baylor College of Medicine Human Genome Sequencing Center's Drosophila Genome project. Sizes of PCR products were confirmed by electrophoresis on 1% agarose gels and purified for sequencing with a Qiaquick PCR Gel Extraction Kit (Qiagen). Purified PCR product for each Acp was sequenced in both directions with ABI Big Dye version 3 Terminators on an ABI 3700 DNA sequencer (Perkin-Elmer). Sequences were submitted to the GenBank database under accession numbers AY452754–AY452764 and AY454152.
Data Analyses
DNA sequences were aligned with use of ClustalW in BioEdit 5.0.9 (Thompson et al. 1994). Open reading frames and introns were identified for each Acp by comparison of D. melanogaster ORFs and, in the case of CG16707, from reverse transcription–PCR sequences from D. pseudoobscura. Maximum likelihood estimates of nonsynonymous and synonymous nucleotide substitution rates (dN/dS) were estimated with PAML (Yang and Nielsen 2000). The dN/dS values of partial coding sequences for four Acp and nine non-Acp X-linked genes in D. pseudoobscura–D. miranda were compared to dN/dS values of the homologous genes in D. melanogaster–D. simulans from Counterman et al. (in press; see Table 1). These genes were identified from an expressed sequence tag (EST) library made from D. simulans male accessory glands, and all of these genes, including non-Acps, are male-specific in their expression (Swanson et al. 2001). Regression analyses were used to test for similarity in dN/dS values between the D. pseudoobscura–D. miranda and D. melanogaster–D. simulans groups. A nonparametric Mann–Whitney U test comparing dN/dS of Acp and non-Acp genes was used to determine if Acps was evolving faster than non-Acps in the D. pseudoobscura group. The values for CG4111 were excluded from both of these analyses, because no synonymous nucleotide differences separated D. pseudoobscura and D. miranda. Further, to identify if Acps was experiencing positive selection in D. pseudoobscura, we calculated Fu and Li's D* (Fu and Li 1993) and Tajima's D (Tajima 1989) with SITES (Hey and Wakeley 1997), and performed a McDonald–Kreitman test (McDonald and Kreitman 1991) with DnaSP (Rozas and Rozas 1999), using D. miranda as an outgroup. Also using DnaSP, we examined codon usage bias by calculating the effective number of codons (ENC) and codon bias index (CBI) for D. pseudoobscura and D. miranda.
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dN/dS Correlation Between Species Groups
We detected a significant correlation of dN/dS values across these genes between the D. pseudoobscura–D. miranda and D. melanogaster–D. simulans groups (r2 =.72; P =.0005), largely driven by one Acp gene that is fast-evolving in both species groups (see Figure 2). The nonparametric Spearman regression analysis also indicated a marginally significant positive relationship (r2 =.32; P =.056). Exclusion of the fast-evolving Acp gene makes these correlations nonsignificant but still positive.
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Faster Acp Versus Non-Acp Genes in D. pseudoobscura
Estimates of dN/dS between D. pseudoobscura and D. miranda were significantly higher for the three Acp genes tested than for the nine male-specific non-Acp genes (Mann–Whitney U, P =.0199; t test P =.0157). Only two of the non-Acp genes had dN/dS estimates higher than the Acp gene with the lowest dN/dS. This result is conservative, because the excluded Acp (CG4111) bore no synonymous differences but one nonsynonymous difference between D. pseudoobscura and D. miranda.
Positive Selection on Acp Genes in D. pseudoobscura
Genes under positive selection are commonly identified by a dN/dS greater than 1, which suggests there has been a relative accumulation of nonsynonymous substitutions, a dearth of synonymous substitutions relative to a neutral expectation, or both. Under this conservative test of positive selection, one Acp studied here, CG16707, exhibited a dN/dS value greater than 1 in both Drosophila species groups.
Positive selection was also detected when polymorphism and divergence data were examined for the pooled Acps. We obtained polymorphism and divergence data from D. pseudoobscura and D. miranda for CG13309, CG2206, and CG16707 (Table 2). The neutral model predicts the ratio of nonsynonymous to synonymous polymorphisms will be similar to the ratio of nonsynonymous to synonymous fixed differences between the species (McDonald and Kreitman 1991). Using this test, our analyses did not detect the signature of positive selection on any individual Acp genes (Table 1). However, positive selection was detected when the Acps were pooled (as done by Begun et al. 2000), indicated by a significant excess of fixed nonsynonymous substitutions (P =.003). Tajima's D and Fu and Li's D* tests for neutrality provided no evidence of directional selection on the Acp genes individually (data not shown).
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To evaluate the strength of selection at synonymous sites, we tested for significant differences in codon usage bias. Using ENC and CBI, we detected no significant difference in codon usage bias between Acps and non-Acps with either D. pseudoobscura (Mann–Whitney U, P =.637, P =.518) or D. miranda (Mann–Whitney U, P =.814, P =.4504).
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Among the genes tested, positive Darwinian selection appears to be driving faster evolution of Acps as compared to male-specific non-Acp genes in D. pseudoobscura. We also observed a trend for similarity in relative rates of nonsynonymous substitution between the D. pseudoobscura and D. melanogaster species groups, suggesting these genes are under generally similar selective constraints, despite millions of years of evolutionary divergence. Understanding the evolutionary forces affecting Acp divergence may provide insight into the functional importance of male-female protein interactions during the evolution of reproductive isolation.
Faster Acp evolution, compared to evolution of non-Acp genes, suggests directional selection is promoting divergence of Acps, possibly because of their functional importance in reproduction. High divergence of proteins whose function entails interacting with other proteins may lead to functional incompatibilities. Genes with accelerated rates of evolution increase the frequency with which incompatibilities evolve between closely related organisms. Acps in Drosophila evolve fast, increasing the rate at which reproductive incompatibilities may arise, and are therefore good candidates for genes involved in speciation.
The correlation in dN/dS we observed between these species groups was statistically significant, albeit less than unity largely because of one fast-evolving Acp in the two groups. This lack of correlative unity suggests that these genes may experience some unique selective constraints in each group, that selection on these genes is episodic, or both. The primary cause of differing selective constraints is a change in function of the gene from one group to another. Acp functions can change by loss of function or evolution of novel functions. However, substitutions resulting in loss of function would have caused dN/dS to approach 1, and only one gene studied possessed a dN/dS near 1. Furthermore, reverse transcription–PCR confirmed that the most divergent Acp tested (CG16707) is transcribed in both D. pseudoobscura and D. miranda (Stevison LS, unpublished data). Development of novel functions, including subfunctionalization (Force et al. 1999), is a more likely explanation than loss of function for the difference in selective constraints between these groups. Relaxed selective constraints on reproductive proteins in females, which interact with Acps, could result in the evolution of divergent novel Acp functions through random mutation. Evidence of fast evolution and positive selection of female reproductive proteins suggests antagonistic evolution of the sexes may be one of the forces driving the divergence of Acps (Swanson et al. 2001b; Swanson 2003). Such antagonistic coevolution would also likely be episodic in nature.
Although specific functions of Acps are not yet known, recent studies demonstrate the power of identifying reproductive gene functions when addressing reproductive isolation and speciation (Chapman et al. 2003; Liu and Kubli 2003; Swanson 2003). We conclude that the rapid rates of Acp evolution in D. melanogaster group apply in the D. pseudoobscura group, as well, and perhaps more broadly. It appears likely that some sort of sexual selection is driving the evolution of Acps and some other male-specific genes associated with reproduction.
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Corresponding Editor: R. C. Woodruff
* These authors contributed equally. 
Received August 29, 2003
Accepted November 25, 2003
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