Molecular Evolution and Population Genetics of Duplicated Accessory Gland Protein Genes in Drosophila
http://www.100md.com
分子生物学进展 2004年第9期
* Section of Integrative Biology, University of Texas-Austin
Section of Evolution and Ecology, University of California-Davis
E-mail: aholloway@mail.utexas.edu.
Abstract
To investigate the potential importance of gene duplication in D. melanogaster accessory gland protein (Acp) gene evolution we carried out a computational analysis comparing annotated D. melanogaster Acp genes to the entire D. melanogaster genome. We found that two known Acp genes are actually members of small multigene families. Polymorphism and divergence data from these duplicated genes suggest that in at least four cases, protein divergence between D. melanogaster and D. simulans is a result of directional selection. One putative Acp revealed by our computational analysis shows evidence of a recent selective sweep in a non-African population (but not in an African population). These data support the idea that selection on reproduction-related genes may drive divergence of populations within species, and strengthen the conclusion that Acps may often be under directional selection in Drosophila.
Key Words: accessory gland protein ? Drosophila ? gene duplication ? gene expression ? molecular evolution ? selection
At least three classes of models have been proposed to explain the evolutionary processes for the retention and subsequent divergence of gene duplicates. Lynch and Force (2000) suggest that ancestral genes with multiple functions in different tissues or developmental stages may have high rates of retention of duplicates under mutation-selection balance. In this model, degenerative mutations result in subfunctionalization, which favors retention and subsequent evolution of duplicates. A second class of models invokes fixation of duplications by genetic drift (e.g., Lynch and Conery 2003; Walsh 2003). Finally, a third class of models relies on new, beneficial mutations driving adaptive divergence (and thus retention) of duplicates (Hughes 1994). One would expect new duplicates from classes of proteins under chronic directional selection to have unusually high fixation probabilities because a higher proportion of new mutations may be beneficial in such genes. For example, if reproduction-related proteins experience directional selection more frequently than other proteins (Civetta and Singh 1998; Nurminsky et al. 1998; Swanson and Vacquier 2002; Ranz et al. 2003), then perhaps a large number of duplicate reproduction-related genes spread through populations and diverge under directional selection.
We investigated duplication and divergence in reproduction-related accessory gland proteins genes (Acps) in Drosophila. Acps are male-specific seminal fluid proteins that affect multiple aspects of female physiology and behavior (for review see Wolfner 1997). We carried out Blast comparisons of the 13 annotated Acps (see Methods) to the D. melanogaster reference sequence (Flybase Consortium 2003). These Blast analyses suggested that two genes, Acp29AB and Acp53Ea, are members of small multigene families.
E-values returned from the tBlastN search (default parameters) with Acp29AB as the query sequence were 1.5 x 10–47 and 2.6 x 10–35 for Lectin29Ca and Lectin30A, respectively. Intraspecific paralogous protein divergence was, on average, 31% between Acp29AB and Lectin29Ca, 35.5% between Acp29AB and Lectin30A, and 38% between Lectin29Ca and Lectin30A. Lectin29Ca is 356 bases distal to Acp29AB and Lectin30A is approximately 1 Mbase distal to these tandem duplicates. Acp29AB is 234 amino acids, while Lectin29Ca and Lectin30A are 236 and 223 amino acids long, respectively. Each gene is composed of a single exon. Our analysis of Lectin30A and comparison to its paralogs suggested that the 5' end was incorrectly annotated. We confirmed this hypothesis by RACE, and we used our annotation in all analyses. The three members of the Acp29AB family are predicted to be lectin galactose binding proteins (Theopold et al. 1999) and to have a signal sequence (SignalP v2.0, Nielsen et al. 1997). The tBlastN search returned several other more distantly related putative Acp29AB paralogs, primarily lectins (Lectin21Cb, Lectin24Db, Lectin22C, Lectin 21Ca, Lectin24A, Lectin28C, and CG15818). However, we will not present data from these genes in this report.
E-values returned from the tBlastN search with Acp53Ea as the query sequence were 2.1 x 10–5 for CG8626 and 9.4 x 10–4 for CG15616. CG8626 and CG15616 will hereafter be referred to as Acp53C14a and Acp53C14b, respectively, based on putative function, genomic location, and gene structure. Another more highly diverged putative duplicate identified by B. Wagstaff (personal communication) did not appear in our Blast results. However, this gene (Acp53C14c) appears to be another tandem duplicate and shows male-limited expression (B. Wagstaff, personal communication). Intraspecific paralogous protein divergence was 48.5% between Acp53Ea and Acp53C14a, 42.5% between Acp53Ea and Acp53C14b, and 45% between Acp53C14a and Acp53C14b. The divergence of Acp53C14c from other putative Acp53Ea duplicates was >65%. These genes are tandem duplications, with Acp53C14a located 423 bp proximal to Acp53C14b, Acp53Ea 487 bases distal to Acp53C14b, and Acp53C14c 519 bp distal to Acp53Ea. Acp53Ea, Acp53C14a, Acp53C14b, and Acp53C14c are predicted to be 120, 121, 132, and 124 amino acids long, respectively. Each is composed of two exons with a 50–60 nt intron roughly 40 bases from the initiation codon. All genes are predicted to be peptide hormones and to have a signal sequence (SignalP v2.0, Nielsen et al. 1997).
The high levels of silent and replacement divergence among putative paralogs suggest that the duplication events predate the split of D. yakuba from the D. melanogaster/D. simulans lineage. Nevertheless, the conserved gene structures, expression patterns, presence of predicted signal peptides, and, for most cases, tandem organization all indicate that we have correctly identified paralogous genes.
Acp29AB and Acp53Ea are expressed only in male accessory glands (Wolfner et al. 1997). Our RT-PCR experiments showed that the only detectable expression of Lectin29Ca, Acp53C14a, and Acp53C14b is in accessory glands (fig. 1). We were unable to detect an RT-PCR product from Lectin30A. However, given that our RACE products were derived from male cDNA, we are certain the gene is expressed in males (perhaps at low levels). The fact that the Acp duplicates identified here share accessory-gland enriched expression further supports the inference of paralogy and suggests that subfunctionalization, at least with respect to gene expression (sensu Lynch and Force 2000), cannot explain fixation of Acp duplicates. Levels of protein polymorphism and divergence for these Acp genes and putative duplicates (table 1) were higher than those typically seen in D. simulans and D. melanogaster genes, as was the case for previous surveys of Acp variation (Begun et al. 2000; Swanson et al. 2001). There was, however, a major exception in the Acp29AB family.
FIG. 1. RT-PCR analysis of tissue-specific expression of putative duplicates. (A) Lectin29Ca, (B) Acp53C14a, and (C) Acp53C14b. Lane assignments for each gel: (1) 1 kB ladder, (2) whole females, (3) males without reproductive tracts, (4) testes, (5) accessory glands, and (6) negative control
Table 1 Silent and Replacement Site Heterozygosity and Divergence for Acp29AB and Acp53Ea Gene Families in D. melanogaster and D. simulans.
Lectin29Ca had no silent polymorphisms and only a single replacement polymorphism in our U.S. D. melanogaster sample (table 2). This is highly unusual given the relatively high levels of variation in D. melanogaster generally and in Acp genes specifically. Low levels of heterozygosity are even more surprising given high levels of silent and replacement divergence at this gene. We used the HKA test (Hudson, Kreitman, and Aguade 1987) to compare polymorphism and divergence data from Lectin29Ca and vermilion (we chose vermilion because of the availability of molecular population genetic data for both U.S. and African samples and because there is no evidence that vermilion has been subject to directional or balancing selection [Begun and Aquadro 1995]). Lectin29Ca versus the coding region of vermilion for the U.S. sample showed a highly significant departure from neutrality (P = 0.0022), consistent with a hitchhiking event reducing variation in Lectin29Ca. In contrast, the African sample showed considerably higher levels of polymorphism but similar levels of divergence (table 2). The HKA test of African variation at Lectin29Ca and vermilion showed no deviation from the neutral model (P = 0.7875). Given that the North American populations are thought to be recently derived from ancestral African populations (David and Capy 1988), the data support the idea that a selective sweep at Lectin29Ca occurred in the very recent past. Note that Acp29AB is only 356 bp upstream of Lectin29Ca, yet it shows normal levels of heterozygosity in the California sample. This suggests that the window of reduced heterozygosity associated with Lectin29Ca may be quite small, though additional population genetic data from the other flanking region would be necessary to determine if this is indeed the case. It seems notable that of three solid cases of individual genes with evidence for recent selection in non-African D. melanogaster samples (desat, Takahashi et al. 2001; Acp36DE, Begun et al. 2000; Lectin29Ca, this report), two are Acps. Such observations support the notion that selection on sexual traits can cause rapid divergence of Drosophila populations (Knowles and Markow 2001; Miller, Starmer, and Pitnick 2003; Pitnick et al. 2003). Additional work will be required to precisely determine the extent of the "swept" region of Lectin29Ca in non-African populations and to identify putative mutations that might be targets of selection.
Table 2 Silent and Replacement Variation for Acp29AB and Acp53Ea Gene Families in D. melanogaster and D. simulans.
Numbers of polymorphic and fixed, silent, and replacement mutations were compared to predictions of the neutral model (McDonald and Kreitman 1991). Of the six genes in our study, four (Acp29AB, Lectin30A, Acp53C14b, and Acp53C14c) reject the null hypothesis in a direction consistent with adaptive protein evolution (table 2). Moreover, our population genetic data support the notion that all three members of the Acp29AB family have been influenced by recent directional selection. Overall, our results put on firmer ground the conclusion that adaptive protein evolution is a major cause of divergence of Acp proteins in D. melanogaster and D. simulans.
Methods
Amino acid sequences of 13 annotated Acps from D. melanogaster (Acp26Aa, Acp26Ab, Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp70A, Acp76A, Acp95EF, and Acp 98AB) were compared to the D. melanogaster reference sequence (Genome Release 3.0, Flybase Consortium 2003) by tBlastN searches using default parameters. Sequences were aligned by manual curation and molecular population genetics of putative duplicates with <50% nucleotide divergence from a known Acp were investigated. Nucleotide divergence (or uncorrected pairwise-distance) was calculated as a measure of the number of mismatched nucleotides/total number of nucleotides at 1st and 2nd codon positions. Duplicate genes are and considered putative Acps but are referred to as Acps for convenience.
Candidate Acp duplicates were subjected to RT-PCR to determine whether their expression was restricted to accessory glands. mRNA was extracted from four tissues of D. melanogaster: male accessory glands, testes, male carcasses, and whole females. First strand synthesis was carried out using an oligo-dT primer and Superscript Reverse Transcriptase (Invitrogen, San Diego, Calif.). RT-PCR was carried out using gene-specific primers on RNA/DNA heteroduplex isolated from each tissue.
D. simulans population genetic data are from inbred lines established from flies collected at the Wolfskill Orchard in Winters, Calif. (Begun and Whitley 2000). D. melanogaster population data are from isochromosomal lines derived from the Wolfskill Orchard or from isofemale lines from Malawi, Africa. D. yakuba sequences are from an isofemale line. Some D. simulans Acp29AB sequences are from Begun et al. (2000). In most cases DNA sequencing was carried out directly on PCR products. For cases in which inbred lines were not available, PCR products were cloned prior to sequencing.
Sequences were assembled using SeqMan (DNAstar, Inc., Madison, WI) and manually curated in MacClade 4.0 (Maddison and Maddison 2000). Alignments are available upon request from the authors. Summary statistics and tests of the neutral equilibrium model were carried out using DnaSP version 3.53 (Rozas and Rozas 1999). SignalP version 2.0 was used to predict presence/absence of signal peptides characteristic of Acps and other secreted proteins (Nielsen et al. 1997). Sequences were submitted to GenBank under accession numbers AY635196-AY635290.
Acknowledgements
We thank A. Kern and S. Joseph for useful comments on the manuscript. Grants from the University of Texas Graduate Program in Evolution, Ecology, and Behavior to A.K.H. and the NIH (GM55298) and NSF (DEB-0327049) to D.J.B supported this work.
Literature Cited
Begun, D. J. 2002. Protein variation in Drosophila simulans, and comparison of genes from centromeric versus noncentromeric regions of chromosome 3. Mol. Biol. Evol. 19:201-203.
Begun, D. J., and C. F. Aquadro. 1995. Molecular variation at the vermilion locus in geographically diverse populations of Drosophila melanogaster and D. simulans. Genetics 140:1019-1032.
Begun, D. J., and P. Whitley. 2000. Reduced X-linked nucleotide polymorphism in Drosophila simulans. Proc. Natl. Acad. Sci. USA 97:5960-5965.
Begun, D. J., P. Whitley, B. L. Todd, H. M. Waldrip-Dail, and A. G. Clark. 2000. Molecular population genetics of male accessory gland proteins in Drosophila. Genetics 156:1879-1888.
Civetta, A., and R. S. Singh. 1998. Sex-related genes, directional sexual selection, and speciation. Mol. Biol. Evol. 15:901-909.
David, J., and P. Capy. 1988. Genetic variation of Drosophila melanogaster natural populations. Trends Genet. 4:106-111.
Flybase Consortium. 2003. The FlyBase database of the Drosophila genome projects and community literature. Nucleic Acids Res. 31:172-175 (www.flybase.org.).
Hudson, R. R., M. Kreitman, and M. Aguade. 1987. A test of neutral molecular evolution based on nucleotide data. Genetics 116:153-159.
Hughes, A. L. 1994. The evolution of functionally novel proteins after gene duplication. Proc. R. Soc. Lond. B Biol. Sci. 256:119-124.
Knowles, L. L., and T. A. Markow. 2001. Sexually antagonistic coevolution of a postmating-prezygotic reproductive character in desert Drosophila. Proc. Natl. Acad. Sci. USA 98:8692-8696.
Lynch, M., and J. S. Conery. 2003. The origins of genome complexity. Science 302:1401-1404.
Lynch, M., and A. Force. 2000. The probability of duplicate gene preservation by subfunctionalization. Genetics 154:459-473.
Maddison, D. R., and W. P. Maddison. 2000. MacClade 4: analysis of phylogeny and character evolution. Sinauer Associates, Sunderland, Mass.
McDonald, J. H., and M. Kreitman. 1991. Adaptive protein evolution at the Adh locus in Drosophila. Nature 351:652-654.
Miller, G. T., W. T. Starmer, and S. Pitnick. 2003. Quantitative genetic analysis of among-population variation in sperm and female sperm-storage organ length in Drosophila mojavensis. Genet. Res. 81:213-220.
Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1-6.
Nurminsky, D. I., M. V. Nurminskaya, D. De Aguiar, and D. L. Hartl. 1998. Selective sweep of a newly evolved sperm-specific gene in Drosophila. Nature 396:572-575.
Pitnick, S., G. T. Miller, K. Schneider, and T. A. Markow. 2003. Ejaculate-female coevolution in Drosophila mojavensis. Proc. R. Soc. Lond. B Biol. Sci. 270:1507-1512.
Ranz, J. M., C. I. Castillo-Davis, C. D. Meiklejohn, and D. L. Hartl. 2003. Sex-dependent gene expression and evolution of the Drosophila transcriptome. Science 300:1742-1745.
Rozas, J., and R. Rozas. 1999. DnaSP 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174-175.
Swanson, W. J., A. G. Clark, H. M. Waldrip-Dail, M. F. Wolfner, and C. F. Aquadro. 2001. Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila. Proc. Natl. Acad. Sci. USA 98:7375-7379.
Swanson, W. J., and V. D. Vacquier. 2002. The rapid evolution of reproductive proteins. Nat. Rev. Genet. 3:137-144.
Takahashi, A., S.-C. Tsaur, J. A. Coyne, and C.-I. Wu. 2001. The nucleotide changes governing cuticular hydrocarbon variation and their evolution in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 98:3920-3925.
Theopold, U., M. Rissler, M. Fabbri, O. Schmidt, and S. Natori. 1999. Insect glycobiology: a lectin multigene family in Drosophila melanogaster. Biochem. Biophys. Res. Comm. 261:923-927.
Walsh, J. B. 2003. Population-genetic models of the fates of duplicate genes. Genetica 118:279-294.
Wolfner, M. F. 1997. Tokens of love: functions and regulation of Drosophila male accessory gland products. Insect Biochem. Mol. Biol. 27:179-192.
Wolfner, M. F., H. A. Harada, M. J. Bertram, T. J. Stelick, K. W. Kraus, J. M. Kalb, Y. O. Lung, D. M. Neubaum, M. Park, and U. Tram. 1997. New genes for male accessory gland proteins in Drosophila melanogaster. Insect Biochem. Mol. Biol. 27:825-834.(Alisha K. Holloway* and D)
Section of Evolution and Ecology, University of California-Davis
E-mail: aholloway@mail.utexas.edu.
Abstract
To investigate the potential importance of gene duplication in D. melanogaster accessory gland protein (Acp) gene evolution we carried out a computational analysis comparing annotated D. melanogaster Acp genes to the entire D. melanogaster genome. We found that two known Acp genes are actually members of small multigene families. Polymorphism and divergence data from these duplicated genes suggest that in at least four cases, protein divergence between D. melanogaster and D. simulans is a result of directional selection. One putative Acp revealed by our computational analysis shows evidence of a recent selective sweep in a non-African population (but not in an African population). These data support the idea that selection on reproduction-related genes may drive divergence of populations within species, and strengthen the conclusion that Acps may often be under directional selection in Drosophila.
Key Words: accessory gland protein ? Drosophila ? gene duplication ? gene expression ? molecular evolution ? selection
At least three classes of models have been proposed to explain the evolutionary processes for the retention and subsequent divergence of gene duplicates. Lynch and Force (2000) suggest that ancestral genes with multiple functions in different tissues or developmental stages may have high rates of retention of duplicates under mutation-selection balance. In this model, degenerative mutations result in subfunctionalization, which favors retention and subsequent evolution of duplicates. A second class of models invokes fixation of duplications by genetic drift (e.g., Lynch and Conery 2003; Walsh 2003). Finally, a third class of models relies on new, beneficial mutations driving adaptive divergence (and thus retention) of duplicates (Hughes 1994). One would expect new duplicates from classes of proteins under chronic directional selection to have unusually high fixation probabilities because a higher proportion of new mutations may be beneficial in such genes. For example, if reproduction-related proteins experience directional selection more frequently than other proteins (Civetta and Singh 1998; Nurminsky et al. 1998; Swanson and Vacquier 2002; Ranz et al. 2003), then perhaps a large number of duplicate reproduction-related genes spread through populations and diverge under directional selection.
We investigated duplication and divergence in reproduction-related accessory gland proteins genes (Acps) in Drosophila. Acps are male-specific seminal fluid proteins that affect multiple aspects of female physiology and behavior (for review see Wolfner 1997). We carried out Blast comparisons of the 13 annotated Acps (see Methods) to the D. melanogaster reference sequence (Flybase Consortium 2003). These Blast analyses suggested that two genes, Acp29AB and Acp53Ea, are members of small multigene families.
E-values returned from the tBlastN search (default parameters) with Acp29AB as the query sequence were 1.5 x 10–47 and 2.6 x 10–35 for Lectin29Ca and Lectin30A, respectively. Intraspecific paralogous protein divergence was, on average, 31% between Acp29AB and Lectin29Ca, 35.5% between Acp29AB and Lectin30A, and 38% between Lectin29Ca and Lectin30A. Lectin29Ca is 356 bases distal to Acp29AB and Lectin30A is approximately 1 Mbase distal to these tandem duplicates. Acp29AB is 234 amino acids, while Lectin29Ca and Lectin30A are 236 and 223 amino acids long, respectively. Each gene is composed of a single exon. Our analysis of Lectin30A and comparison to its paralogs suggested that the 5' end was incorrectly annotated. We confirmed this hypothesis by RACE, and we used our annotation in all analyses. The three members of the Acp29AB family are predicted to be lectin galactose binding proteins (Theopold et al. 1999) and to have a signal sequence (SignalP v2.0, Nielsen et al. 1997). The tBlastN search returned several other more distantly related putative Acp29AB paralogs, primarily lectins (Lectin21Cb, Lectin24Db, Lectin22C, Lectin 21Ca, Lectin24A, Lectin28C, and CG15818). However, we will not present data from these genes in this report.
E-values returned from the tBlastN search with Acp53Ea as the query sequence were 2.1 x 10–5 for CG8626 and 9.4 x 10–4 for CG15616. CG8626 and CG15616 will hereafter be referred to as Acp53C14a and Acp53C14b, respectively, based on putative function, genomic location, and gene structure. Another more highly diverged putative duplicate identified by B. Wagstaff (personal communication) did not appear in our Blast results. However, this gene (Acp53C14c) appears to be another tandem duplicate and shows male-limited expression (B. Wagstaff, personal communication). Intraspecific paralogous protein divergence was 48.5% between Acp53Ea and Acp53C14a, 42.5% between Acp53Ea and Acp53C14b, and 45% between Acp53C14a and Acp53C14b. The divergence of Acp53C14c from other putative Acp53Ea duplicates was >65%. These genes are tandem duplications, with Acp53C14a located 423 bp proximal to Acp53C14b, Acp53Ea 487 bases distal to Acp53C14b, and Acp53C14c 519 bp distal to Acp53Ea. Acp53Ea, Acp53C14a, Acp53C14b, and Acp53C14c are predicted to be 120, 121, 132, and 124 amino acids long, respectively. Each is composed of two exons with a 50–60 nt intron roughly 40 bases from the initiation codon. All genes are predicted to be peptide hormones and to have a signal sequence (SignalP v2.0, Nielsen et al. 1997).
The high levels of silent and replacement divergence among putative paralogs suggest that the duplication events predate the split of D. yakuba from the D. melanogaster/D. simulans lineage. Nevertheless, the conserved gene structures, expression patterns, presence of predicted signal peptides, and, for most cases, tandem organization all indicate that we have correctly identified paralogous genes.
Acp29AB and Acp53Ea are expressed only in male accessory glands (Wolfner et al. 1997). Our RT-PCR experiments showed that the only detectable expression of Lectin29Ca, Acp53C14a, and Acp53C14b is in accessory glands (fig. 1). We were unable to detect an RT-PCR product from Lectin30A. However, given that our RACE products were derived from male cDNA, we are certain the gene is expressed in males (perhaps at low levels). The fact that the Acp duplicates identified here share accessory-gland enriched expression further supports the inference of paralogy and suggests that subfunctionalization, at least with respect to gene expression (sensu Lynch and Force 2000), cannot explain fixation of Acp duplicates. Levels of protein polymorphism and divergence for these Acp genes and putative duplicates (table 1) were higher than those typically seen in D. simulans and D. melanogaster genes, as was the case for previous surveys of Acp variation (Begun et al. 2000; Swanson et al. 2001). There was, however, a major exception in the Acp29AB family.
FIG. 1. RT-PCR analysis of tissue-specific expression of putative duplicates. (A) Lectin29Ca, (B) Acp53C14a, and (C) Acp53C14b. Lane assignments for each gel: (1) 1 kB ladder, (2) whole females, (3) males without reproductive tracts, (4) testes, (5) accessory glands, and (6) negative control
Table 1 Silent and Replacement Site Heterozygosity and Divergence for Acp29AB and Acp53Ea Gene Families in D. melanogaster and D. simulans.
Lectin29Ca had no silent polymorphisms and only a single replacement polymorphism in our U.S. D. melanogaster sample (table 2). This is highly unusual given the relatively high levels of variation in D. melanogaster generally and in Acp genes specifically. Low levels of heterozygosity are even more surprising given high levels of silent and replacement divergence at this gene. We used the HKA test (Hudson, Kreitman, and Aguade 1987) to compare polymorphism and divergence data from Lectin29Ca and vermilion (we chose vermilion because of the availability of molecular population genetic data for both U.S. and African samples and because there is no evidence that vermilion has been subject to directional or balancing selection [Begun and Aquadro 1995]). Lectin29Ca versus the coding region of vermilion for the U.S. sample showed a highly significant departure from neutrality (P = 0.0022), consistent with a hitchhiking event reducing variation in Lectin29Ca. In contrast, the African sample showed considerably higher levels of polymorphism but similar levels of divergence (table 2). The HKA test of African variation at Lectin29Ca and vermilion showed no deviation from the neutral model (P = 0.7875). Given that the North American populations are thought to be recently derived from ancestral African populations (David and Capy 1988), the data support the idea that a selective sweep at Lectin29Ca occurred in the very recent past. Note that Acp29AB is only 356 bp upstream of Lectin29Ca, yet it shows normal levels of heterozygosity in the California sample. This suggests that the window of reduced heterozygosity associated with Lectin29Ca may be quite small, though additional population genetic data from the other flanking region would be necessary to determine if this is indeed the case. It seems notable that of three solid cases of individual genes with evidence for recent selection in non-African D. melanogaster samples (desat, Takahashi et al. 2001; Acp36DE, Begun et al. 2000; Lectin29Ca, this report), two are Acps. Such observations support the notion that selection on sexual traits can cause rapid divergence of Drosophila populations (Knowles and Markow 2001; Miller, Starmer, and Pitnick 2003; Pitnick et al. 2003). Additional work will be required to precisely determine the extent of the "swept" region of Lectin29Ca in non-African populations and to identify putative mutations that might be targets of selection.
Table 2 Silent and Replacement Variation for Acp29AB and Acp53Ea Gene Families in D. melanogaster and D. simulans.
Numbers of polymorphic and fixed, silent, and replacement mutations were compared to predictions of the neutral model (McDonald and Kreitman 1991). Of the six genes in our study, four (Acp29AB, Lectin30A, Acp53C14b, and Acp53C14c) reject the null hypothesis in a direction consistent with adaptive protein evolution (table 2). Moreover, our population genetic data support the notion that all three members of the Acp29AB family have been influenced by recent directional selection. Overall, our results put on firmer ground the conclusion that adaptive protein evolution is a major cause of divergence of Acp proteins in D. melanogaster and D. simulans.
Methods
Amino acid sequences of 13 annotated Acps from D. melanogaster (Acp26Aa, Acp26Ab, Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp70A, Acp76A, Acp95EF, and Acp 98AB) were compared to the D. melanogaster reference sequence (Genome Release 3.0, Flybase Consortium 2003) by tBlastN searches using default parameters. Sequences were aligned by manual curation and molecular population genetics of putative duplicates with <50% nucleotide divergence from a known Acp were investigated. Nucleotide divergence (or uncorrected pairwise-distance) was calculated as a measure of the number of mismatched nucleotides/total number of nucleotides at 1st and 2nd codon positions. Duplicate genes are and considered putative Acps but are referred to as Acps for convenience.
Candidate Acp duplicates were subjected to RT-PCR to determine whether their expression was restricted to accessory glands. mRNA was extracted from four tissues of D. melanogaster: male accessory glands, testes, male carcasses, and whole females. First strand synthesis was carried out using an oligo-dT primer and Superscript Reverse Transcriptase (Invitrogen, San Diego, Calif.). RT-PCR was carried out using gene-specific primers on RNA/DNA heteroduplex isolated from each tissue.
D. simulans population genetic data are from inbred lines established from flies collected at the Wolfskill Orchard in Winters, Calif. (Begun and Whitley 2000). D. melanogaster population data are from isochromosomal lines derived from the Wolfskill Orchard or from isofemale lines from Malawi, Africa. D. yakuba sequences are from an isofemale line. Some D. simulans Acp29AB sequences are from Begun et al. (2000). In most cases DNA sequencing was carried out directly on PCR products. For cases in which inbred lines were not available, PCR products were cloned prior to sequencing.
Sequences were assembled using SeqMan (DNAstar, Inc., Madison, WI) and manually curated in MacClade 4.0 (Maddison and Maddison 2000). Alignments are available upon request from the authors. Summary statistics and tests of the neutral equilibrium model were carried out using DnaSP version 3.53 (Rozas and Rozas 1999). SignalP version 2.0 was used to predict presence/absence of signal peptides characteristic of Acps and other secreted proteins (Nielsen et al. 1997). Sequences were submitted to GenBank under accession numbers AY635196-AY635290.
Acknowledgements
We thank A. Kern and S. Joseph for useful comments on the manuscript. Grants from the University of Texas Graduate Program in Evolution, Ecology, and Behavior to A.K.H. and the NIH (GM55298) and NSF (DEB-0327049) to D.J.B supported this work.
Literature Cited
Begun, D. J. 2002. Protein variation in Drosophila simulans, and comparison of genes from centromeric versus noncentromeric regions of chromosome 3. Mol. Biol. Evol. 19:201-203.
Begun, D. J., and C. F. Aquadro. 1995. Molecular variation at the vermilion locus in geographically diverse populations of Drosophila melanogaster and D. simulans. Genetics 140:1019-1032.
Begun, D. J., and P. Whitley. 2000. Reduced X-linked nucleotide polymorphism in Drosophila simulans. Proc. Natl. Acad. Sci. USA 97:5960-5965.
Begun, D. J., P. Whitley, B. L. Todd, H. M. Waldrip-Dail, and A. G. Clark. 2000. Molecular population genetics of male accessory gland proteins in Drosophila. Genetics 156:1879-1888.
Civetta, A., and R. S. Singh. 1998. Sex-related genes, directional sexual selection, and speciation. Mol. Biol. Evol. 15:901-909.
David, J., and P. Capy. 1988. Genetic variation of Drosophila melanogaster natural populations. Trends Genet. 4:106-111.
Flybase Consortium. 2003. The FlyBase database of the Drosophila genome projects and community literature. Nucleic Acids Res. 31:172-175 (www.flybase.org.).
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