Different Forces Drive the Evolution of the Acp26Aa and Acp26Ab Accessory Gland Genes in the Drosophila melanogaster Species Complex

Corresponding author: Montserrat Aguadé, Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071 Barcelona, Spain. E-mail address: aguade@porthos.bio.ub.es.

Communicating editor: A. G. CLARK

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Acp26Aa and Acp26Ab genes that code for male accessory gland proteins are tandemly arranged in the species of the Drosophila melanogaster complex. An ~1.6-kb region encompassing both genes has been sequenced in 10, 24, and 18 lines from Spain, Ivory Coast, and Malawi, respectively; the previously studied 10 lines from North Carolina have also been included in the analyses. A total of 110 nucleotide and 4 length polymorphisms were detected. Silent variation for the whole Acp26A region was slightly higher in African than in non-African populations, while for both genes nonsynonymous variation was similar in all populations studied. Based on F_st estimates no major genetic differentiation was detected between East and West Africa, while in general non-African populations were strongly differentiated from both African populations. Comparison of polymorphism and divergence at synonymous and nonsynonymous sites revealed that directional selection acting on amino acid replacement changes has driven the evolution of the Acp26Aa protein in the last 2.5 myr.

IN sexually reproducing organisms both external and internal fertilization require the interaction between proteins synthesized exclusively in the male or the female reproductive organs. In the case of external fertilization there is a relatively reduced number of gamete surface proteins that mediate species-specific gamete recognition (PALUMBI 1994 ). The situation is more complex in organisms with internal fertilization as, in addition to fertilization, mating often triggers in the female a series of physiological and behavioral responses; also, in the case of multiple matings there may be sperm competition mediated by male-specific proteins. In both cases there is an ample field for natural selection to shape variation within and between species in the genes coding for these proteins.

The study of nucleotide variation in the genes coding for sperm lysin in abalone (LEE and VACQUIER 1995 ) and sperm bindin in sea urchins (METZ and PALUMBI 1996 ) has revealed the action of positive Darwinian selection in shaping their protein evolution. In the case of sperm lysin the ratio between nonsynonymous and synonymous divergence was significantly higher than one, but no polymorphism was detected in the small sample of individuals analyzed from a particular species. On the other hand, an excess of nonsynonymous substitutions between species was detected in a 40-codon domain region of sperm bindin; there was also a high level of nonsynonymous polymorphism in that same region in at least one of the species analyzed.

In Drosophila the seminal fluid contains many proteins that are synthesized in the male reproductive tissues, primarily in the accessory glands [male Accessory gland proteins (Acps)], which are transferred to the female during copulation. Some of these proteins, like Acp36DE, remain in the female's genital tract and seem to participate in sperm storage (BERTRAM et al. 1996 ; WOLFNER 1997 ). Other Acps (e.g., Acp70A, Acp26Aa) move to the hemolymph and thereafter they interact with still unidentified female receptors and induce behavioral and physiological changes in the female. It has been also shown that variation in some of these genes can account for differences in sperm competition (CLARK et al. 1995 ). In that study one of the regions that revealed a positive correlation between nucleotide variation and the ability to defend sperm displacement was the Acp26A region, which contains the Acp26Aa and Acp26Ab genes arranged in tandem (MONSMA and WOLFNER 1988 ). However, in a later experiment using males with a null mutation of Acp26Aa that produced no Acp26Aa protein (HERNDORN and WOLFNER 1995 ), the absence of Acp26Aa in these males had no effect on sperm competition. One of the well-established functions of Acp26Aa in Drosophila melanogaster is to stimulate egg laying for a period of 24 hr after mating (HERNDORN and WOLFNER 1995 ). Given that the sex-peptide or Acp70A also induces egg laying but for a longer period of up to 7 days (KUBLI 1996 ), it has been proposed that Acp26Aa would increase egg deposition as opposed to Acp70A, which would stimulate the production of eggs by the ovary (KUBLI 1996 ; WOLFNER 1997 ). On the other hand, little is known about the function of the Acp26Ab gene product.

Nucleotide variation in the Acp26A region has been surveyed in a North American population of D. melanogaster and also in one allele of the sibling species D. simulans, D. mauritiana, and D. sechellia (AGUADE et al. 1992 ). For the Acp26Aa gene this study revealed a high degree of amino acid replacement variation both within and between species. In fact, estimates of polymorphism and divergence at nonsynonymous sites were rather high, and of the same order as the corresponding estimates (polymorphism and divergence) at synonymous sites. Variation was not randomly distributed along the coding region, but there was an excess of both nonsynonymous and synonymous divergence in the 3' half of the gene. The second exon of the Acp26Aa gene was later compared between D. melanogaster and D. simulans, and the more distantly related species of the melanogaster group, D. yakuba and D. teissieri (TSAUR and WU 1997 ). In the different comparisons the estimates of K_a were generally higher than the corresponding estimates of K_s, pointing again to positive selection as a major force in the evolution of this gene. On the other hand, the Acp26Ab gene did not show the elevated nonsynonymous variation observed in the Acp26Aa gene.

However, variation at these two genes has been studied only in a sample from North America. To obtain a better insight into the role played by selection in the evolution of these genes, nucleotide variation at the Acp26Aa and Acp26Ab genes has been surveyed in three other populations: a European population (Montblanc, Spain) as a second representative of non-African populations, and two populations from equatorial Africa (Ivory Coast and Malawi in West and East Africa, respectively).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks:
Ten isofemale lines collected in 1993 in Montblanc (Spain) were isogenized for the second chromosome by the corresponding series of crosses with a balancer stock. Twenty-four lines from Lamto (Ivory Coast) and 18 lines from Malawi, kindly provided by M. Veuille and V. Bénassi (see BENASSI et al. 1993 ; BENASSI and VEUILLE 1995 ), were used in this study. African lines were either isogenic for the second chromosome or maintained as heterozygotes over a balancer chromosome. The African isogenic lines were used directly. On the other hand, one male from each of the African lines maintained as heterozygotes was crossed with females carrying the deficiency Df(2L)Gdh-A,dpL, which covers cytological positions 25D7 to 26A8-9; individuals heterozygous for the deficiency and, therefore, hemizygous for the wild Acp26A locus were selected from the progeny. Ten lines from North Carolina, previously studied in AGUADE et al. 1992 , were also included in the analyses.

DNA extraction, PCR amplification, and sequencing:
DNA was extracted from 10 adult flies by a modification of protocol 48 in ASHBURNER 1989 . For each of the lines an ~1.7-kb region encompassing both the Acp26Aa and Acp26Ab transcriptional units was PCR-amplified using primers 23 nucleotides long (AGUADE et al. 1992 ). The conditions for amplification were 94° for denaturing, 45° for annealing, and 65° for extension over 30 cycles. Sequencing primers were spaced on average 250 nucleotides. PCR products were made single-stranded with the lambda exonuclease procedure (HIGUCHI and OCHMAN 1989 ) and sequenced using Sequenase (United States Biochemical, Cleveland, OH). Alternatively, they were purified with Qiaquick columns (QIAGEN Inc., Chatsworth, CA) and sequenced using the Dye Terminator chemistry (Perkin Elmer, Norwalk, CT) according to the manufacturer's directions with slight modifications; the products were separated with an ABI 377 automated DNA sequencer (Perkin Elmer). All lines were sequenced on both strands. The sequences of D. simulans, D. mauritiana, and D. sechellia used in the analyses are those reported in AGUADE et al. 1992 . The sequences newly reported in this article have been deposited in the EMBL sequence database library under accession numbers AJ231350–AJ231401.

Sequence analysis:
Sequences were edited for further analyses using the MacClade version 3.0.6 program (MADDISON and MADDISON 1992 ). The DnaSP version 2.80 program (ROZAS and ROZAS 1997 ) was used for most intraspecific and some interspecific analyses.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nucleotide variation:
Figure 1 and Table 1 give a summary of nucleotide sequence variation in the four populations surveyed. A total of 110 nucleotide and 4 length polymorphisms were detected in the 1614-nucleotide region studied (excluding alignment gaps). Five nucleotide polymorphisms segregated for three variants resulting in a total of 115 mutations segregating in the sampled populations. Only 1 length polymorphism (an 8-bp deletion from nucleotide 1575 to 1582) segregated in three populations (two African and one non-African), while the other three deletions were singletons. Nucleotide polymorphism at position 1578 was not considered in further analyses as some of the lines segregated for a deletion spanning this position.

View larger version (79K): In this window In a new window Download PPT slide   Figure 1. Nucleotide and length polymorphisms in the Acp26Aa and Acp26Ab transcription units in four natural populations of D. melanogaster. The polyadenylation signal of the Acp26Aa gene (MONSMA and WOLFNER 1988 ) was considered for simplicity the limit between transcription units Acp26Aa and Acp26Ab. Polymorphisms were numbered as in AGUADE et al. 1992 . Lines from North Carolina (NC), Montblanc (Mo), Lamto (La), and Malawi (Ma) are presented sequentially. Thick-bordered boxes above the sequence information represent the two exons of Acp26Aa and the second exon of Acp26Ab, while thin-bordered boxes represent the single intron of each gene. Dots represent the same nucleotide as in the first sequence. n, Nonsynonymous; *, singletons as referred to the complete data set; d, deletion. The extent of deletions is indicated by the location of their first and last nucleotide. Nucleotide polymorphisms included in a region for which a deletion is segregating are included in square brackets.

Unlike the samples from Montblanc and North Carolina, the African samples segregated for the In(2L)t inversion. The region studied is located on band 26A and therefore is included in the region affected by the inversion, which extends between bands 22D3-E1 and 34A8-9. Only 1 of the 18 lines studied from Malawi presented the inversion, while 9 of the 24 lines studied from the Ivory Coast did (M. VEUILLE, personal communication). The 2 sets of lines of this latter population were considered together for further analyses as they were not genetically differentiated for the Acp26A region when using the permutation test proposed by HUDSON et al. 1992A and K_s^* as the test statistic.

Nucleotide variation for the whole region studied was estimated separately for each population (Table 1). The level of both total and silent polymorphism was slightly higher for the two African populations, especially when compared to the North American sample. The level of synonymous variation in the exons was higher than that of silent variation in noncoding regions. The region between the two genes, which includes regulatory sequences of both genes, showed the lowest level of silent polymorphism.

As in the original study of this region (AGUADE et al. 1992 ), the level of nonsynonymous variation of both the Acp26Aa and Acp26Ab genes was rather high as compared to other regions of the genome; the ratio of nonsynomymous to synonymous polymorphism was higher for the Acp26Aa gene. Figure 2 shows the amino acid variation of the two proteins in the populations sampled. The mean number of amino acid differences between lines varied between 4 and 5 for Acp26Aa and was approximately equal to 1 for Acp26Ab. Only the sample from Lamto presented one not-in-frame 3-bp deletion in exon 2 of the Acp26Aa gene that resulted in one amino acid change in addition to one amino acid deletion. Polymorphisms at adjacent residues 24 and 25 involved the same 2 amino acids (Gln/Lys), and also those at nearby residues 76 and 79 (Asp/Asn). Residue 90 of the Acp26Ab protein segregated for 2 amino acid variants (Ala/Glu) at intermediate frequencies in the African populations, while it was monomorphic for Ala in the non-African samples. No amino acid polymorphism was detected in any of the five putative glycosylation sites (MONSMA and WOLFNER 1988 ) and in any of the three peptidase sites (PARK and WOLFNER 1995 ) of Acp26Aa; also the 11-amino-acid residues conserved relative to the Aplysia Egg Laying Hormone (ELH; MONSMA and WOLFNER 1988 ) were monomorphic in all the samples studied.

View larger version (36K): In this window In a new window Download PPT slide   Figure 2. Amino acid replacement polymorphism in Acp26Aa and Acp26Ab in four natural populations of D. melanogaster. Numbers indicate the amino acid residue in the D. melanogaster protein. Dots indicate same amino acid as in the first sequence. *, Singletons as referred to the complete data set; d, deletion; C, conservative; NC, nonconservative without charge change; R, nonconservative with charge change.

Genetic differentiation between populations:
Table 2 summarizes the analysis of population differentiation. The F_st parameter, which measures the proportion of nucleotide diversity attributable to variation between populations, was estimated from the average number of differences between alleles according to HUDSON et al. 1992B . Population differentiation was assessed by the permutation test (HUDSON et al. 1992A ) using K_s^* as the test statistic. The analysis was performed for the whole region studied considering either all variation or only silent variation, and also separately for the Acp26Aa and Acp26Ab transcription units. In all cases the comparison between the two African populations presented the lowest F_st estimates. These populations did not show any significant differentiation for the Acp26Ab transcription unit, while for the Acp26Aa transcription unit, and for the whole region, they were either marginally (0.05 < P < 0.10) or slightly (P = 0.04) differentiated. The non-African populations showed a significant differentiation in all cases from the Malawi population and in most cases from the Ivory Coast population.

Linkage disequilibrium:
In light of the results of genetic differentiation between populations, linkage disequilibrium or pairwise gametic association was estimated separately for each population and also for the pooled African sample. Table 3 shows the total number of pairwise comparisons per population, the number of comparisons that presented a significant association (using the ² test and without considering the Bonferroni correction for multiple comparisons), and the proportion of significant comparisons. In all cases this proportion was higher in the non-African than in the African samples. Also in the African samples the proportion was in all cases higher for the Acp26Ab transcription unit. For the two larger samples from Africa and for the pooled African sample, Table 3 also shows the number of significant comparisons when the Bonferroni correction was applied. In the pooled African sample, 8 of the 15 significant associations in the Acp26Ab transcription unit were in the trailer between polymorphisms 1490 and 1572; in this smaller region this represented 22% of significant associations (8 out of 36 comparisons). The trailer presented in fact three major haplotypes (represented by lines La32, La36, and La108) in the African samples. These major haplotypes were also present in the non-African samples, although only two segregated in each population. However, even in this small region (between sites 1490 and 1572) five recombination events could be inferred in the history of the African pooled sample using the four-gamete test (HUDSON and KAPLAN 1985 ), while none was in the non-African samples.

Tests of neutrality:
Tests of neutrality based on polymorphism data were applied to the whole region and also to the two transcription units separately. In the samples studied neither the Tajima test (TAJIMA 1989 ) nor the Fu and Li test (FU and LI 1993 ) detected any departure from neutral expectations.

Tests of neutrality based on polymorphism and divergence data were also applied. The direct relationship between levels of polymorphism and divergence in a given region, predicted by the neutral theory of molecular evolution, constitutes the common null hypothesis of these tests. Table 4 summarizes the results of the McDonald and Kreitman (MK) test (MCDONALD and KREITMAN 1991 ). This test, which compares nonsynonymous and synonymous changes in a coding region, was applied separately to the Acp26Aa and Acp26Ab genes. In the Acp26Aa gene there was in all cases an excess of nonsynonymous differences fixed between species; this excess was significant in comparisons involving the Montblanc, Lamto, and Malawi populations, and only marginally significant in the North Carolina comparison. On the other hand, the MK test did not detect any departure from neutral expectations in the much shorter Acp26Ab coding region. The Hudson, Kreitman, and Aguadé (HKA) test (HUDSON et al. 1987 ) was applied both for all changes and for silent changes (analysis not shown); in this test the regions compared were the two transcription units included in the fragment studied (Acp26Aa and Acp26Ab). When all changes were considered, most of the comparisons revealed a significant departure from the neutral expectations, while no departure was detected when only silent variation was considered. The difference in the results of these two applications of the HKA test is consistent with the excess of nonsynonymous divergence in the Acp26Aa gene already detected using the MK test.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Pattern of nucleotide variation:
In the few studies that compare nucleotide variation between East African and non-African populations, the estimated nucleotide variation (measured as or ) was much higher in the African populations, while the level of linkage disequilibrium was higher in the non-African samples (BEGUN and AQUADRO 1993 , BEGUN and AQUADRO 1995 ). The analysis of silent (and presumably neutral) variation within the Adh^S allele in samples from both East and West Africa and in non-African samples revealed a major differentiation of non-African and West African populations relative to East African samples (BENASSI and VEUILLE 1995 ).

In this study variation both at the Acp26Aa and Acp26Ab genes was surveyed in two non-African samples and in a subsample of the same two African populations studied by BENASSI and VEUILLE 1995 . As in previous studies the level of variation was higher in African than in non-African populations, although the differences were not very conspicuous (Table 1). In addition, both genes presented similar levels of nonsynonymous variation in African and non-African populations. As in previous nuclear DNA studies comparing African and non-African populations, polymorphisms in the North American and European samples were a subset of those present in the two African populations. However, in contrast to previous studies (BEGUN and AQUADRO 1993 , BEGUN and AQUADRO 1995 ) most of the nonrare polymorphisms segregating in the African samples were also segregating in the non-African samples. Also the two African samples shared most nonunique polymorphisms.

Unlike the study of the Adh region (BENASSI and VEUILLE 1995 ), no major differentiation for the Acp26A region was detected between West and East Africa. F_st estimates were rather low both for the Acp26Aa (0.02–0.03) and Acp26Ab (0.01) transcription units. In the case of the Acp26Aa region the two populations were marginally or slightly differentiated, while in the case of the Acp26Ab region they did not show any differentiation. In contrast to silent variation within Adh^S, variation at the Acp26Aa and Acp26Ab regions might be governed by selection (see below). Also non-African populations showed in general a strong differentiation from both East and West Africa; this differentiation could be, as for other genes, both a result of drift during the out-of-Africa expansion and of selection acting differentially during that process.

Nonsynonymous variation at the Acp26Aa gene is governed by directional selection:
When the whole gene was considered, K_a and K_s estimates between D. melanogaster and the closely related species D. simulans, D. mauritiana, and D. sechellia were rather similar, and no significant excess of replacement changes could be detected (AGUADE et al. 1992 ). However, when the region corresponding to the second exon of the gene was compared between both D. melanogaster and D. simulans, and the more distantly related species D. yakuba and D. teissieri (TSAUR and WU 1997 ), the average K_a/K_s ratio for the four comparisons was ~1.5; only the K_a/K_s ratio for the branch separating both species pairs was significantly >1, which constituted unambigous evidence that at least in the past the evolution of Acp26Aa had been driven by positive selection.

In this study another three populations of D. melanogaster were sampled. Application of the MK test to the different populations surveyed (and to the complete D. melanogaster sample) revealed a significant excess of nonsynonymous fixed differences between D. melanogaster and each of the three sibling species (Table 4). This excess of nonsynonymous divergence was also revealed by the results of the HKA tests performed comparing all variation (most tests significant) and only silent variation (all tests nonsignificant) between the Acp26Aa and Acp26Ab regions (analysis not shown). We can now assert that directional selection not only drove the evolution of Acp26Aa in the distant past (between the D. melanogaster-D. simulans split, 2.5 mya, and the D. yakuba-D. teissieri split, 6 mya) but also in the last 2.5 my.

Acp26Aa is processed in the female genital tract just after mating; the protein is cleaved three times after residues 48, 68, and 115 or 117, generating four peptides (PARK and WOLFNER 1995 ). Only the processed protein is active, and secretions from the male accessory gland main cells are needed for the processing. As one of the known functions of Acp26Aa is the stimulation of egg laying in the mated female, the last peptide would be the candidate for that function as it contains the region of similarity to the ELH in the four species of the melanogaster complex. To test whether positive selection has driven the evolution of the different parts of the protein, the Acp26Aa coding region was first divided in two fragments that included the first three peptides and the last peptide, respectively. Application of the MK test revealed an excess of fixed nonsynonymous differences in both fragments, although significance was higher in the second fragment (Table 5). To further explore the distribution of fixed and polymorphic changes, both fragments were subdivided, despite the fact that subdivision could lower the statistical power of the MK test; the first fragment was divided in two, corresponding to the regions coding for the first two peptides and for the third peptide, and the second was in two equally sized fragments (Table 5). Although the contribution of the different peptides to the function(s) of the Acp26Aa protein is not known, this analysis indicates that directional selection is governing at least the evolution of the third and fourth peptides in the melanogaster species complex; it also indicates that selection might be acting differentially along the fourth peptide.

Directional selection is, however, expected to cause a reduction of polymorphism in the region closely linked to the favored variant. Although all D. melanogaster populations harbored an important amount of nonsynonymous and synonymous polymorphisms at the Acp26Aa region, some regions showed a very low level of polymorphism without the corresponding reduction in the levels of fixed differences. In fact, in the sample from North Carolina the region extending from polymorphic site 417 to the end of the Acp26Aa transcription unit (Figure 1) presented 5 singletons out of 6 polymorphisms; also in the sample from Malawi the region spanning polymorphic sites 270 to 693 presented 10 singletons out of 11 polymorphisms. Nevertheless, it could be argued that the sample from North Carolina is not at equilibrium, and that the observed distribution of polymorphism is still reflecting its rather recent origin; this argument would generally not be made for the East African sample. Also the extent of the fragment with reduced polymorphism in Malawi (~400 bp) would be consistent with the region expected to be affected by a selective sweep in a region of high recombination like the Acp26Aa region. On the other hand, as the coalescence time of any given sample of sequences is 4N generations, selective sweeps that occurred before that time would have no effect on the observed polymorphism. Under the assumption of mutation-drift equilibrium this time can be estimated from the ratio between (4Nµ) and divergence (2tµ). Using restriction map polymorphism in D. melanogaster, and divergence between D. melanogaster and D. simulans, this time was estimated to represent 25% of the time since the split of the melanogaster and simulans lineages (EANES et al. 1996 ). Therefore, only selective sweeps occurring in the last ~600,000 years would have any effect on extant polymorphism in D. melanogaster.

The Acp26Aa and Acp26Ab genes have different evolutionary histories as expected in a high recombination region like the Acp26A region. The function of the Acp26Ab protein after its transfer to the female is not known; there is no evidence of selection either driving its evolution or maintaining the different protein variants. Unlike the Acp26Aa gene, the level of nonsynonymous variation in the Acp26Ab coding region is, both within and between species, roughly an order of magnitude lower than that of synonymous variation (AGUADE et al. 1992 ), indicating a certain degree of functional constraint at the protein level.

	ACKNOWLEDGMENTS

I thank Michel Veuille and Véronique Bénassi for the African lines and for sharing unpublished information on the chromosomal polymorphism of these lines. I also thank the Umeå Stock Center for the Df(2L)Gdh-A,dpL/SM1 line, Serveis Científico-Tècnics from Universitat de Barcelona for automated sequencing facilities, and Julio Rozas, Carmen Segarra, and Griselda Ribó for comments on the manuscript. Special thanks are given to David Salguero for his excellent technical assistance. This work was supported by grants PB94-0923 from Dirección General de Investigación Científica y Técnica, Spain, and 1995SGR-577 from Comissió Interdepartamental de Recerca i Tecnologia, Generalitat de Catalunya.

Manuscript received February 4, 1998; Accepted for publication June 16, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AGUADÉ, M., N. MIYASHITA, and C. H. LANGLEY, 1992  Polymorphism and divergence in the Mst26A male accessory gland gene region in Drosophila. Genetics 132:755-770[Abstract].

ASHBURNER, M., 1989 Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

BEGUN, D. J. and C. F. AQUADRO, 1993  African and North American populations of Drosophila melanogaster are very different at the DNA level. Nature 356:519-520.

BEGUN, D. J. and C. F. AQUADRO, 1995  Molecular variation at the vermillion locus in geographically diverse populations of Drosophila melanogaster and D. simulans.. Genetics 140:1019-1032[Abstract].

BÉNASSI, V. and M. VEUILLE, 1995  Comparative population structuring of molecular and allozyme variation of Drosophila melanogaster Adh between Europe, West Africa and East Africa. Genet. Res. 65:95-103[Medline].

BÉNASSI, V., S. AULARD, S. MAZEAU, and M. VEUILLE, 1993  Molecular variation of Adh and P6 genes in African populations of Drosophila melanogaster and its relation to chromosomal inversions. Genetics 134:789-799[Abstract].

BERTRAM, M. J., D. M. NEUBAUM, and M. F. WOLFNER, 1996  Localization of the Drosophila accessory gland protein Acp36DE in the mated female suggests a role in sperm storage. Inst. Biochem. Mol. Biol. 26:971-980.

CLARK, A. G., M. AGUADÉ, T. PROUT, L. HARSHMAN, and C. H. LANGLEY, 1995  Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster.. Genetics 139:189-201[Abstract].

EANES, W. F., M. KIRCHNER, J. YOON, C. H. BIERMANN, and I.-N. WANG et al., 1996  Historical selection, amino acid polymorphism and lineage-specific divergence at the G6pd locus in Drosophila melanogaster and D. simulans.. Genetics 144:1027-1041[Abstract].

FU, Y. X. and W-S. LI, 1993  Statistical tests of neutrality of mutations. Genetics 133:693-709[Abstract].

HERNDORN, L. A. and M. F. WOLFNER, 1995  A Drosophila seminal fluid protein, Acp26Aa, stimulates egg-laying in females for one day after mating. Proc. Natl. Acad. Sci. USA 92:10114-10118[Abstract/Free Full Text].

HIGUCHI, R. G. and H. OCHMAN, 1989  Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Res. 17:5865[Free Full Text].

HUDSON, R. R. and N. L. KAPLAN, 1985  Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111:147-164[Abstract/Free Full Text].

HUDSON, R. R., M. KREITMAN, and M. AGUADÉ, 1987  A test of neutral molecular evolution based on nucleotide data. Genetics 116:153-159[Abstract/Free Full Text].

HUDSON, R. R., D. D. BOOS, and N. L. KAPLAN, 1992a  A statistical test for detecting geographic subdivision. Mol. Biol. Evol. 9:138-151[Abstract].

HUDSON, R. R., M. SLATKIN, and W. P. MADDISON, 1992b  Estimation of levels of gene flow from DNA sequence data. Genetics 132:583-589[Abstract].

KUBLI, E., 1996  The Drosophila sex-peptide: a peptide pheromone involved in reproduction. Adv. Dev. Biochem. 4:99-128.

LEE, Y. and V. D. VACQUIER, 1995  Positive selection is a general phenomenon in the evolution of abalone sperm lysin. Mol. Biol. Evol. 12:231-238[Abstract].

MADDISON, W. P., and D. R. MADDISON, 1992 MacClade: Analysis of Phylogeny and Character Evolution, Version 3.0. Sinauer Associates, Sunderland, MA.

MCDONALD, J. H. and M. KREITMAN, 1991  Adaptative protein evolution at the Adh locus in Drosophila. Nature 351:652-654[Medline].

METZ, E. C. and S. R. PALUMBI, 1996  Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin. Mol. Biol. Evol. 13:397-406[Abstract].

MONSMA, S. and M. F. WOLFNER, 1988  Structure and expression of a Drosophila male accessory gland gene whose products resemble a peptide pheromone precursor. Genes Dev. 2:1063-1073[Abstract/Free Full Text].

NEI, M., 1987 Molecular Evolutionary Genetics. Columbia University Press, New York.

PALUMBI, S. R., 1994  Genetic divergence, reproductive isolation, and marine speciation. Ann. Rev. Ecol. Syst. 25:547-572.

PARK, M. and M. F. WOLFNER, 1995  Male and female cooperate in the prohormone-like processing of a Drosophila melanogaster seminal fluid protein. Dev. Biol. 171:694-702[Medline].

ROZAS, J. and R. ROZAS, 1997  DnaSP version 2.0: a novel software package for extensive molecular population genetics analysis. Comput. Appl. Biosci. 13:307-311.

TAJIMA, F., 1989  Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585-595[Abstract/Free Full Text].

TSAUR, S.-H. and C.-I. WU, 1997  Positive selection and the molecular evolution of a gene of male reproduction, Acp26Aa of Drosophila. Mol. Biol. Evol. 14:544-549[Abstract].

WATTERSON, G. A., 1975  On the number of segregating sites in genetic models without recombination. Theor. Popul. Biol. 7:256-276[Medline].

WOLFNER, M. F., 1997  Tokens of love: functions and regulation of Drosophila male accessory gland products. Inst. Biochem. Mol. Biol. 27:179-192.

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				K. Ravi Ram and M. F. Wolfner Seminal influences: Drosophila Acps and the molecular interplay between males and females during reproduction Integr. Comp. Biol., September 1, 2007; 47(3): 427 - 445. [Abstract] [Full Text] [PDF]

				M. K. N. Lawniczak and D. J. Begun Molecular population genetics of female-expressed mating-induced serine proteases in Drosophila melanogaster Mol. Biol. Evol., September 1, 2007; 24(9): 1944 - 1951. [Abstract] [Full Text] [PDF]

				A. C. Fiumera, B. L. Dumont, and A. G. Clark Associations Between Sperm Competition and Natural Variation in Male Reproductive Genes on the Third Chromosome of Drosophila melanogaster Genetics, June 1, 2007; 176(2): 1245 - 1260. [Abstract] [Full Text] [PDF]

				A. Sanchez-Gracia and J. Rozas Unusual Pattern of Nucleotide Sequence Variation at the OS-E and OS-F Genomic Regions of Drosophila simulans Genetics, April 1, 2007; 175(4): 1923 - 1935. [Abstract] [Full Text] [PDF]

				A. Wong, S. N. Albright, and M. F. Wolfner Evidence for structural constraint on ovulin, a rapidly evolving Drosophila melanogaster seminal protein PNAS, December 5, 2006; 103(49): 18644 - 18649. [Abstract] [Full Text] [PDF]

				M. Proschel, Z. Zhang, and J. Parsch Widespread Adaptive Evolution of Drosophila Genes With Sex-Biased Expression Genetics, October 1, 2006; 174(2): 893 - 900. [Abstract] [Full Text] [PDF]

				J. A. Andres, L. S. Maroja, S. M. Bogdanowicz, W. J. Swanson, and R. G. Harrison Molecular Evolution of Seminal Proteins in Field Crickets Mol. Biol. Evol., August 1, 2006; 23(8): 1574 - 1584. [Abstract] [Full Text] [PDF]

				J. E. Pool, V. B. DuMont, J. L. Mueller, and C. F. Aquadro A Scan of Molecular Variation Leads to the Narrow Localization of a Selective Sweep Affecting Both Afrotropical and Cosmopolitan Populations of Drosophila melanogaster Genetics, February 1, 2006; 172(2): 1093 - 1105. [Abstract] [Full Text] [PDF]

				B. J. Wagstaff and D. J. Begun Molecular Population Genetics of Accessory Gland Protein Genes and Testis-Expressed Genes in Drosophila mojavensis and D. arizonae Genetics, November 1, 2005; 171(3): 1083 - 1101. [Abstract] [Full Text] [PDF]

				T. M. Hambuch and J. Parsch Patterns of Synonymous Codon Usage in Drosophila melanogaster Genes With Sex-Biased Expression Genetics, August 1, 2005; 170(4): 1691 - 1700. [Abstract] [Full Text] [PDF]

				B. J. Wagstaff and D. J. Begun Comparative Genomics of Accessory Gland Protein Genes in Drosophila melanogaster and D. pseudoobscura Mol. Biol. Evol., April 1, 2005; 22(4): 818 - 832. [Abstract] [Full Text] [PDF]

				A. C. Fiumera, B. L. Dumont, and A. G. Clark Sperm Competitive Ability in Drosophila melanogaster Associated With Variation in Male Reproductive Proteins Genetics, January 1, 2005; 169(1): 243 - 257. [Abstract] [Full Text] [PDF]

				W. J. Swanson, A. Wong, M. F. Wolfner, and C. F. Aquadro Evolutionary Expressed Sequence Tag Analysis of Drosophila Female Reproductive Tracts Identifies Genes Subjected to Positive Selection Genetics, November 1, 2004; 168(3): 1457 - 1465. [Abstract] [Full Text] [PDF]

				Z. Zhang, T. M. Hambuch, and J. Parsch Molecular Evolution of Sex-Biased Genes in Drosophila Mol. Biol. Evol., November 1, 2004; 21(11): 2130 - 2139. [Abstract] [Full Text] [PDF]

				J. L. Mueller, D. R. Ripoll, C. F. Aquadro, and M. F. Wolfner Comparative structural modeling and inference of conserved protein classes in Drosophila seminal fluid PNAS, September 14, 2004; 101(37): 13542 - 13547. [Abstract] [Full Text] [PDF]

				E. Baudry, B. Viginier, and M. Veuille Non-African Populations of Drosophila melanogaster Have a Unique Origin Mol. Biol. Evol., August 1, 2004; 21(8): 1482 - 1491. [Abstract] [Full Text] [PDF]

				A. D. Kern, C. D. Jones, and D. J. Begun Molecular Population Genetics of Male Accessory Gland Proteins in the Drosophila simulans Complex Genetics, June 1, 2004; 167(2): 725 - 735. [Abstract] [Full Text] [PDF]

				G. Caracristi and C. Schlotterer Genetic Differentiation Between American and European Drosophila melanogaster Populations Could Be Attributed to Admixture of African Alleles Mol. Biol. Evol., May 1, 2003; 20(5): 792 - 799. [Abstract] [Full Text] [PDF]

				R. J. Kulathinal, L. Skwarek, R. A. Morton, and R. S. Singh Rapid Evolution of the Sex-Determining Gene, transformer: Structural Diversity and Rate Heterogeneity Among Sibling Species of Drosophila Mol. Biol. Evol., March 1, 2003; 20(3): 441 - 452. [Abstract] [Full Text] [PDF]

				S. Mousset, L. Brazier, M.-L. Cariou, F. Chartois, F. Depaulis, and M. Veuille Evidence of a High Rate of Selective Sweeps in African Drosophila melanogaster Genetics, February 1, 2003; 163(2): 599 - 609. [Abstract] [Full Text] [PDF]

				A. Civetta Positive Selection Within Sperm-Egg Adhesion Domains of Fertilin: An ADAM Gene with a Potential Role in Fertilization Mol. Biol. Evol., January 1, 2003; 20(1): 21 - 29. [Abstract] [Full Text] [PDF]

				M. Zurovcova and F. J. Ayala Polymorphism Patterns in Two Tightly Linked Developmental Genes, Idgf1 and Idgf3, of Drosophila melanogaster Genetics, September 1, 2002; 162(1): 177 - 188. [Abstract] [Full Text] [PDF]

				O. Lung, U. Tram, C. M. Finnerty, M. A. Eipper-Mains, J. M. Kalb, and M. F. Wolfner The Drosophila melanogaster Seminal Fluid Protein Acp62F Is a Protease Inhibitor That Is Toxic Upon Ectopic Expression Genetics, January 1, 2002; 160(1): 211 - 224. [Abstract] [Full Text] [PDF]