Molecular Population Genetics of Male Accessory Gland Proteins in Drosophila

Corresponding author: David J. Begun, Section of Evolution and Ecology, University of California, Davis, CA 95616., djbegun{at}ucdavis.edu (E-mail)

Communicating editor: W. STEPHAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila seminal proteins have an unusually high rate of molecular sequence evolution, suggesting either a high rate of neutral substitution or rapid adaptive evolution. To further quantify patterns of polymorphism and divergence in genes encoding seminal proteins, also called accessory gland proteins (Acp's), we conducted a sequencing survey of 10 Acp genes in samples of Drosophila melanogaster and D. simulans (Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB). Mean heterozygosity at replacement sites in D. simulans was 0.0074 for Acp genes and 0.0013 for a set of 19 non-Acp genes, and mean melanogaster-simulans divergence at replacement sites was 0.0497 for Acp genes and 0.0107 at non-Acp genes. The elevated divergence of Acp genes is thus accompanied by elevated within-species polymorphism. In addition to the already-reported departures of Acp26A, Acp29AB, and Acp70A from neutrality, our data reject neutrality at Acp29AB and Acp36DE in the direction of excess replacements in interspecific comparisons.

MOLECULAR population genetic analysis of particular classes of proteins or genetic pathways may eventually allow us to make general inferences about the connection between the functional and evolutionary properties of genes. Sexual phenotypes involved in male-male and male-female interactions in Drosophila have attracted much recent attention (CHAPMAN et al. 1995 ; RICE 1996 ; CLARK et al. 1999 ; HOLLAND and RICE 1999 ). A large proportion of wild-caught Drosophila melanogaster females carry sperm from two or more males in their reproductive tract (COBBS 1977 ; GRIFFITHS et al. 1982 ; HARSHMAN and CLARK 1998 ; IMHOF et al. 1998 ). The presence of sperm from two or more males in the female reproductive tract promotes "competition" between males for access to fertilizations. As is true for most insects (SMITH 1984 ; SIMMONS and SIVA-JOTHY 1998 ), if two Drosophila melanogaster males mate in succession with a single female, the sperm from the second male fertilizes the majority of eggs (GROMKO and PYLE 1978 ; GROMKO et al. 1984 ). Sperm competition is a complex phenomenon, the outcome of which may be affected by a large number of different gene products and contexts (SIMMONS and SIVA-JOTHY 1998 ). For example, Drosophila seminal fluid (as opposed to sperm alone) has been shown to play a role in sperm displacement (HARSHMAN and PROUT 1994 ). In addition to its role in sperm competition, seminal fluid stimulates oviposition and reduces female receptivity to future matings (RICHMOND et al. 1980 ; CHEN 1984 , CHEN 1996 ). There is abundant genetic variation affecting both male and female components of sperm use in Drosophila (CLARK et al. 1995 , CLARK et al. 1999 ; RICE 1996 ; CLARK and BEGUN 1998 ). Given our intuition that the outcome of sperm competition should be highly correlated with male fitness, the large amount of genetic variation affecting the trait is somewhat surprising.

Accessory gland proteins are an important component of Drosophila seminal fluid. Two-dimension gel electrophoresis indicates that there are large numbers of seminal proteins (THOMAS and SINGH 1992 ), the functions of most of which are unknown. Protein electrophoretic analysis of accessory gland proteins showed that on average they are more polymorphic than, and evolve faster than, the other proteins examined (COULTHART and SINGH 1988 ; THOMAS and SINGH 1992 ; CIVETTA and SINGH 1995 ). However, those data provide no means of distinguishing whether rapid evolution is best explained as a result of natural selection or rather as a result of reduced functional constraints. Molecular population genetic analysis of accessory gland protein encoding genes Acp26Aa and Acp26Ab suggested that natural selection has caused amino acid evolution of the former, but not the latter (AGUADE 1997 , AGUADE 1998 ; TSAUR and WU 1998 ). Analysis of Acp29AB (AGUADE 1999 ) also provided weak evidence for adaptive protein evolution in D. melanogaster and D. simulans, yet analysis of Acp70A (CIRERA and AGUADE 1997 ) provided no support for the notion that selection has caused interspecific divergence in this gene. Therefore, despite the broad appeal of the idea that amino acid evolution of seminal fluid proteins is driven primarily by selection, it is supported by little data. Here we present an analysis of variation in 13 accessory gland proteins in D. melanogaster and D. simulans. These data allow us to test the general hypothesis that interspecific divergence in this class of proteins is primarily driven by natural selection.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

D. simulans data for nine Acp genes (Acp28AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB) are from 10–14 highly inbred lines made from females captured at the Wolfskill Orchard in Winter, California. D. melanogaster data from the United States for these same nine genes are from 10–12 isogenic stocks derived from females also captured at Wolfskill. D. melanogaster data from Africa for these genes were from six to eight homozygous chromosomes isolated from a Zimbabwe sample or from single Zimbabwe chromosomes placed over appropriate deficiencies. Populations of D. melanogaster from Zimbabwe are often different from those in other locations and may represent populations that are closer to equilibrium (BEGUN and AQUADRO 1993 ). DNA sequences were obtained directly from PCR products on ABI (Foster City, CA) 377 and Beckman (Fullerton, CA) CEQ-2000 automated sequencers. Data from Acp26Aa and Acp26Ab were from AGUADÉ et al. (1994) and AGUADE 1998 . Data for Acp70A were from CIRERA and AGUADE 1997 . Sequences were analyzed using DnaSP (ROZAS and ROZAS 1999 ), SITES (HEY and WAKELEY 1997 ), and the molecular evolutionary analysis package (E. MORIYAMA, unpublished data). Sequences can be found under GenBank accession nos. AY010527, AY010711.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Summaries of polymorphism and divergence in Acp genes are presented in Table 1 Table 2 Table 3 and Fig 1 and Fig 2. Most Acp genes are short, with the consequence that for many genes there are too few segregating sites or interspecific differences to test evolutionary models or estimate model parameters with much confidence. Consequently, the most powerful analyses of these data ask questions about polymorphism and divergence across the whole set of genes. We present these analyses first. We then note interesting observations at individual genes.

View larger version (22K): In this window In a new window Download PPT slide   Figure 1. Proportion of sites polymorphic [polymorphic/(polymorphic + fixed)] in the Acp36DE genes of D. melanogaster samples from Zimbabwe (top) and the United States (bottom), as determined from the runs test of MCDONALD 1996 .

View larger version (9K): In this window In a new window Download PPT slide   Figure 2. Organization of the 3' end of the Acp98AB gene in D. melanogaster and D. simulans.

Polymorphism and divergence in Acp genes:
There are 9 Acp loci (Acp28AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, and Acp98AB) for which we have polymorphism data from D. simulans and D. melanogaster (Table 2). The mean replacement (WATTERSON 1975 ) for these 9 D. simulans Acp genes (weighted by length of surveyed region) is 0.0074, compared to a mean replacement of 0.0013 for 19 D. simulans genes scattered across chromosome arm 3R (BEGUN and WHITLEY 2000 ). None of these Acp genes or other genes on 3R are in regions of very low recombination in D. simulans, so levels of heterozygosity between Acp genes and other genes can be compared without accounting for effects of differences in recombination rate between classes of proteins. Mean replacement divergence for the 9 Acp genes is 0.0497, while the mean replacement divergence for 19 genes on 3R is 0.0107. Thus, replacement heterozygosity is about five to six times greater for Acp's than for 19 genes on 3R (BEGUN and WHITLEY 2000 ), while the replacement divergence is about four to five times greater than for the 19 genes on 3R. Mean (weighted by length) silent heterozygosity for the nine D. simulans Acp genes is 0.028. This is close to the average silent heterozygosity, 0.035, for the 19 D. simulans genes surveyed on 3R. The average silent divergence for these 9 Acp genes is 0.117, also very similar to previous results from other genes compared in these two species (BAUER and AQUADRO 1997 ; BEGUN and WHITLEY 2000 ).

We have polymorphism data from African D. melanogaster samples for these nine Acp genes; the mean replacement heterozygosity is 0.0025, compared to 0.0074 for D. simulans. The approximately threefold difference in heterozygosity is roughly similar to that previously observed between these two species (e.g., MORIYAMA and POWELL 1996 ). There are five Acp genes (29AB, 32CD, 33A, 36DE, and 53Ea) for which we have polymorphism from both African and non-African samples of D. melanogaster, as well as for D. simulans. The mean replacement heterozygosity is essentially the same in the African and non-African D. melanogaster samples for these five loci (0.0027 and 0.0026, respectively). Silent site heterozygosity is also very similar for these five genes in the African and non-African samples (Table 3) and for a slightly larger sample of seven Acp genes for which we have survey data for African and non-African samples (silent for the two samples are 0.018 and 0.014, respectively). Possible exceptions to this generalization can be found in the case of Acp36DE (for which the African sample is about twice as variable as the non-African sample) and Acp95EF (for which the non-African sample is completely lacking variation, while the African sample has more "typical" levels of silent heterozygosity).

Comparison of numbers of silent and replacement polymorphisms in D. melanogaster and D. simulans suggests that the ratio of replacement to silent polymorphism for autosomal genes is different in the two species (BEGUN 1996 ; MORIYAMA and POWELL 1996 ). For nine Acp genes (all Acp's are autosomal; WOLFNER 1997 ) for which we have population samples for D. simulans and African D. melanogaster samples, the numbers of silent and replacement polymorphisms in D. simulans are 76 and 81, respectively; the numbers of silent and replacement polymorphisms in African D. melanogaster are 42 and 21, respectively. The 2 x 2 contingency table is significantly heterogeneous by a G-test (P = 0.013). This heterogeneity supports the earlier analysis of data from non-Acp genes (BEGUN 1996 ; MORIYAMA and POWELL 1996 ). Importantly, however, data from Acp's is opposite in direction from the data from other loci. As previously noted (AQUADRO et al. 1988 ; BEGUN 1996 ), the general pattern is one of proportionally more replacement polymorphism in D. melanogaster than in D. simulans (relative to silent polymorphism). For Acp loci, it is D. simulans rather than D. melanogaster that appears to have proportionally more replacement polymorphism.

Joint analysis of polymorphism and divergence at silent and replacement sites can provide more powerful tests of evolutionary models than can analysis of polymorphism alone (e.g., HUDSON et al. 1987 ). The numbers of silent and replacement polymorphisms in the two species can be compared to the numbers of silent and replacement "fixed" differences between species at the Acp genes (MCDONALD and KREITMAN 1991 ). The null hypothesis under neutrality is that the ratio of silent to replacement polymorphism will be similar to the ratio of silent to replacement divergence. We include data from all the genes in Table 2 for our analysis of silent polymorphism and fixations. Where possible, we use polymorphism data from African D. melanogaster samples (Table 1). The total numbers of polymorphisms and fixed differences are shown in Table 3. The contingency table reflects significant heterogeneity (G = 17.5; P < 0.001). This is not surprising given the significant deviations from homogeneity observed at Acp26Aa and Acp36DE individually. If we remove the data from these two genes the resulting 2 x 2 contingency table (Table 3) is not significantly heterogeneous. By this criterion there is no compelling evidence that directional selection is the predominant cause of amino acid substitutions in these genes (although the strength of such a conclusion is compromised by the reduction in statistical power associated with removing large numbers of sites from Acp26Aa and Acp36DE from the analysis).

Despite this statistical inference regarding amino acid evolution pooled across loci, we should not be too quick to conclude that polymorphism and divergence data provide no support for the notion that directional selection plays a role in Acp protein evolution. Under the neutral model one expects the ratio of replacement to silent fixations to equal the ratio of replacement to silent polymorphisms. However, data sets from genes evolving under the neutral model should show minor deviations from this expectation because of sampling and stochastic variances. This suggests that for a sample of several loci, a null hypothesis under the neutral model is that half the loci are expected to show a greater ratio of replacement to silent fixations than the ratio of replacement to silent polymorphisms, while half are expected to show a smaller ratio of replacement to silent fixations compared to the ratio of replacement to silent polymorphisms.

The data from Acp's appear to be inconsistent with this prediction. Note that there is a consistent pattern across genes of greater ratios of replacement to silent fixations compared to ratios of replacement to silent polymorphisms. For the seven Acp genes for which there are at least five total polymorphisms and at least five total fixations, all seven show proportionally more replacement fixations than replacement polymorphisms. If, under the neutral model, a locus has a 50% probability of a greater ratio of replacement to silent divergence than replacement to silent polymorphism, our observation of seven loci with proportionally more replacement fixations than replacement polymorphisms is highly unlikely (binomial probability, P = 0.008). How do the Acp's compare to other genes sequenced in these species? The data set from BEGUN and WHITLEY 2000 may be most appropriate, as there is no bias in the genes sampled toward those thought to be under natural selection on the basis of other types of data (e.g., allozyme data). Among 35 genes that satisfy the criterion of at least five total polymorphisms and five total fixations, 25 show a greater ratio of replacement to silent fixations compared to the ratio of replacement to silent polymorphism; only 10 show a smaller ratio of replacement to silent fixations compared to the ratio of replacement to silent polymorphism. Comparison of the ratios in the two sets of loci (7:0 to 25:10) is not significant by Fisher's exact test. This comparison is somewhat problematic, however, in that the sample of 35 genes from BEGUN and WHITLEY 2000 includes no polymorphism data from this species. Unfortunately, only five Acp's satisfy our criterion of at least five total polymorphisms and at least five total fixations when polymorphism data from D. melanogaster are removed. Comparison of replacement to silent fixations vs. replacement to silent polymorphisms for this subset of the Acp's vs. other loci yields 3:2 to 25:10, which is not significant. However, given that only five genes are analyzed one cannot draw a strong conclusion. As noted earlier, the probability of observing 7 of 7 loci with greater ratios of replacement to silent fixations than replacement to silent polymorphisms is unlikely under the neutral model. However, our "random" sample of 35 loci shows 25 with a greater ratio of replacement to silent fixations than of replacement to silent polymorphism; this too is unlikely under the neutral model (P = 0.01). We should not conclude from this, however, that adaptive amino acid fixations are the cause of the pattern. The data are confounded by the pooling of substitutions that occurred along two lineages (D. melanogaster and D. simulans) with polymorphism data. AKASHI 1996 showed that relative to silent substitution rates, the amino acid substitution rate is higher in the melanogaster lineage than in the simulans lineage. The extent to which this pattern results from directional selection rather than genetic drift remains unclear.

In general, then, despite the large amount of data from Acp's, we are still unable to make strong statements regarding the importance of directional selection in the evolution of these genes. The addition of data from outgroup species for Acp's and other loci will help address this uncertainty.

As we might expect given the results of AKASHI 1996 , average codon bias (WRIGHT 1990 ; Table 4) in D. simulans Acp genes is greater than the average codon bias in D. melanogaster Acp genes [effective number of codons (ENC) = 50.6 and 53.3, respectively]. The absolute levels of codon bias in Acp genes are low compared to the average degree of codon bias in Drosophila genes (POWELL and MORIYAMA 1997 ; MCVEAN and VIEIRA 1999 ). The accessory gland protein-coding genes were isolated in such a way that favors overrepresentation of highly expressed genes (DIBENEDETTO et al. 1990 ; BERTRAM et al. 1996 ; WOLFNER et al. 1997 ). Furthermore, accessory gland proteins are fairly short. Both factors should favor higher, rather than lower degrees of codon bias (reviewed in POWELL and MORIYAMA 1997 ). One possible explanation for the relatively low levels of codon bias in Acp's is the notion that rapid rates of protein evolution (as we see in Acp's) should be correlated with lower levels of codon bias (e.g., AKASHI 1994 ). An unusual aspect of codon bias of Acp genes is that Acp63F and Acp95EF have quite different ENC values in the two species (with D. melanogaster showing much less bias than D. simulans). Data from outgroup species for these genes would be required to understand how evolution of silent sites has proceeded differently along the two lineages.

Patterns at individual genes:
Acp36DE: We observed 24 silent and 25 replacement polymorphisms in our sample of 6 D. simulans alleles, and we observed 54 silent and 23 replacement polymorphisms in our sample of 16 D. melanogaster alleles (Table 1). Levels of silent polymorphism as measured by and are about the same in the D. simulans sample and the Zimbabwe D. melanogaster sample. As has been observed in several genes (e.g., BEGUN and AQUADRO 1993 ), the amount of silent variation is less in U.S. samples than in Zimbabwe samples of D. melanogaster. Comparison of replacement and silent polymorphism in the three samples reveals that the ratio of replacement to silent polymorphism is the same in the D. simulans sample and the U.S. D. melanogaster sample. However, the ratio of replacement to silent polymorphism is lower in the Zimbabwe D. melanogaster sample. In fact, although the U.S. sample is about half as polymorphic as the Zimbabwe sample for silent variation, the two samples are about equally variable for replacement variation.

Tests of homogeneity of the silent and replacement polymorphic and fixed variants (MCDONALD and KREITMAN 1991 ) show that silent and replacement polymorphism and divergence of the D. simulans and Zimbabwe D. melanogaster samples are significantly heterogeneous (P = 0.03). Under the assumption that amino acid mutations are likely to be under stronger selection than are silent mutations, the data support the idea that there is an excess of amino acid fixations at Acp36DE. However, the polymorphism and divergence from the D. simulans and U.S. D. melanogaster sample (Table 1) are not significantly heterogeneous (P = 0.51). In other words, the results of the two tests are indicative of significant differences in the configuration of silent and replacement variation in the Zimbabwe and U.S. D. melanogaster samples. A sliding window analysis of for silent sites in the two D. melanogaster samples (Fig 1) shows that the distribution of variation in the two samples is roughly similar, the only exception being a region around nucleotides 750–1500, where there is a large drop in the amount of variation in the U.S. sample. We performed the "runs" tests of MCDONALD 1996 on the two D. melanogaster samples. There was a highly significant deviation from the null hypothesis in the U.S. sample, but no deviation in the Zimbabwe sample (Table 1). Inspection of the silent and replacement variation in the U.S. and Zimbabwe D. melanogaster samples over this region shows that there are eight silent and two replacement polymorphisms in Zimbabwe and zero silent and six replacement polymorphisms in the United States. These ratios of silent to replacement polymorphisms are significantly different by Fisher's exact test (P = 0.009), although the P value cannot be taken literally because the test was performed on a subset of the data that appeared "unusual." Nevertheless, the results suggest different types of deviations from the null hypothesis in the Zimbabwe and U.S. D. melanogaster samples. The comparison of Zimbabwe D. melanogaster and D. simulans provides evidence of amino acid differences between species caused by natural selection. The U.S. D. melanogaster sample provides evidence for a very recent perturbation of the polymorphism by natural selection. Specifically, a small region within the Acp36DE protein in the U.S. D. melanogaster sample appears to be depauperate of silent variation and to have "extra" amino acid variation. We cannot rule out the possibility that selection on silent sites contributes to rejection of the null model; however, the fact that selection acting on codon bias at Acp36DE is weak supports the idea that selection on amino acids is a likely cause of rejection of the neutral model.

Acp29AB: A test of the contingency table of polymorphic/fixed and silent/replacement variation at Acp29AB results is a significant rejection of homogeneity (Table 1). A previous analysis of Acp29AB yielded similar results (AGUADE 1999 ). As was the case for Acp36DE, the configuration of the contingency table is most readily interpreted in terms of an excess of amino acid fixations between species. Our estimate of silent heterozygosity for a U.S. sample of D. melanogaster was similar to that reported by AGUADE 1998 for a French sample (0.024 vs. 0.023). AGUADE 1999 reported very similar levels of silent heterozygosity in French and African (Lamto and Malawi) samples. Our African sample from Zimbabwe seems to be different from the African samples used by AGUADE 1999 , as our silent heterozygosity is only about one-third that of samples from other locations. Comparison of the Zimbabwe D. melanogaster to the African data from AGUADE 1999 confirms that the two samples are in fact significantly different (K_st = 0.07, P < 0.001 by a permutation test of HUDSON et al. 1992 ). The Zimbabwe D. melanogaster sample was polymorphic at four amino acid residues; these four residues were also polymorphic in the D. melanogaster sample analyzed by AGUADE 1999 . AGUADE 1999 reported two polymorphic residues that were monomorphic in our sample; 105 (present twice in AGUADE 1999 ) and 215 (present once in AGUADE 1999 ).

Acp32CD: Acp32CD appears to be an unusual Acp gene in that it is evolving slowly at both silent and replacement sites. Acp32CD also harbors relatively little polymorphism. This is especially true of silent sites. No silent polymorphisms were observed in D. melanogaster and only two silent polymorphisms were seen in D. simulans. Replacement polymorphism was also low in both species, although not atypically low relative to levels of replacement polymorphism at other Acp genes (Table 2). Under the neutral model these observations would be interpreted as a result of unusually high functional constraints in this gene compared to functional constraints on other Acp's (KIMURA 1983 ). An alternative viewpoint (GILLESPIE 1991 ) is that directional selection impinges on this locus to a lesser degree than it does at other Acp loci. As a consequence of low polymorphism and low divergence in a short gene region of just 415 bp, the contingency table of polymorphisms and fixed differences is not significantly heterogeneous. It should be noted, however, that with a contingency table containing only two fixed differences (one silent and one replacement), it is impossible to reject the null hypothesis.

Acp62F: Acp62F shows an unusual pattern of polymorphism and divergence not previously observed in samples of genes from D. simulans and D. melanogaster. The silent heterozygosity in D. simulans (0.092) is very high compared to the average silent heterozygosity of autosomal genes (BEGUN and WHITLEY 2000 ) in this species (0.035), although the 95% confidence interval (C.I.; KREITMAN and HUDSON 1991 ) for D. simulans Acp62F (0.020–0.202) does overlap the average silent for autosomal loci in D. simulans (BEGUN and WHITLEY 2000 ). Perhaps more surprising than the high heterozygosity in D. simulans is the fact that two of the D. simulans alleles, DS6C and DS7C, cluster with D. melanogaster alleles rather than other D. simulans alleles in both neighbor-joining and maximum-likelihood trees (data not shown), although sequence data from an outgroup species will be required to root such a tree. Balancing selection is one explanation for high levels of polymorphism of the D. simulans Acp62F. However, a Hudson-Kreitman-Aguadé (HKA) test (HUDSON et al. 1987 ) of Acp62F vs. AATS-GluPro (a "typical" D. simulans autosomal locus in terms of polymorphism and divergence; BEGUN and WHITLEY 2000 ) provides no evidence for deviations from the neutral model (² = 1.10, P = 0.29).

Acp98AB: Acp98AB is unusual in that a mutation in a termination codon has created a "fixed" length difference between species in the protein. The final base of the TGA termination codon in D. melanogaster is a C in D. simulans, with the result that the codon homologous to the D. melanogaster stop codon codes for cysteine in D. simulans (Fig 2). The two species have the same sequence for the next 12 bases; the last triplet among these 12 bases codes for a termination codon in D. simulans. This unusual mutation at a stop codon was independently confirmed with a PCR-restriction fragment length polymorphism analysis of the mutation. Acp98AB is also unusual in the patterns of polymorphisms and divergence in the amino acid residues that are shared by both species. There are no polymorphisms of any type within either species and no differences between species at silent sites. However, there are eight amino acid differences between species. This pattern is suggestive of adaptive protein evolution. However, the small size of the locus (160 bp) diminishes our ability to carry out hypothesis tests of variation at this gene.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

There is abundant evidence that male sexual traits often evolve more quickly than other kinds of traits (e.g., EBERHARD 1985 ). Whether this pattern extends in a very general sense to genes required for male reproduction is an important issue of evolutionary genetics. A subset of the proteins of male reproduction in Drosophila are the accessory gland proteins. Although the functions of most Acp's are unknown, it is clear that seminal fluid of Drosophila affects female physiology (CHAPMAN et al. 1995 ; RICE 1996 ), behavior (CHEN et al. 1988 ), patterns of sperm storage by females (TRAM and WOLFNER 1999 ), and sperm competition between males (HARSHMAN and PROUT 1994 ). Accessory gland proteins are candidates for mediating at least some of these phenomena.

Evidence from protein gel electrophoresis has suggested that the accessory gland proteins tend to evolve more quickly and tend to be more polymorphic than most other proteins in Drosophila (COULTHART and SINGH 1988 ; THOMAS and SINGH 1992 ; CIVETTA and SINGH 1995 ). However, the nature of the electrophoretic data precluded accurate descriptions of the underlying genetic changes within and between species. The data presented here provide our first general picture of polymorphism and divergence of accessory gland proteins in two closely related Drosophila species.

Our data suggest that the patterns revealed by protein electrophoresis and by previous studies of DNA variation at Acp genes are in fact general properties of accessory gland proteins. As a group, Acp genes exhibit greater protein polymorphism and greater protein divergence than the "average" gene in D. simulans and D. melanogaster. The small size of the Acp genes reveals the limitations of molecular population genetics to test the null model of neutral variation within and between species. The three largest Acp genes, Acp26Aa, Acp29AB, and Acp36DE, each show patterns of polymorphism and divergence consistent with adaptive protein divergence. None of the remaining genes individually exhibit statistically significant deviations from the neutral model. A contingency table of polymorphic and fixed, silent, and replacement mutations of the Acp genes (omitting Acp26Aa, Acp29AB, and Acp36DE) does not reject the neutral model, even though there are large numbers of sites surveyed in the pooled data. The results provide no compelling evidence that a large fraction of amino acid substitutions in these proteins result from directional selection. This inference about replacement sites, however, does not speak strongly to the issue of whether most Acp loci have been targets of directional selection at relatively few amino acid positions. Statements regarding the ubiquity of directional selection across loci are even more difficult for this set of genes because many of the genes are quite small (leading to low statistical power).

One prediction from theoretical studies of genes affecting sperm displacement is that polymorphism in such genes can be maintained by balancing selection (PROUT and CLARK 1996 ). These theoretical results along with the empirical observations of abundant genetic variation in male components of sperm competition and the potential role of Acp's in such phenomena raise the issue of whether there is any evidence for balancing selection in the DNA data from these genes. One "signature" of balancing selection is excess polymorphism among sites that are tightly linked to a balanced polymorphism (HUDSON and KAPLAN 1988 ; KREITMAN and HUDSON 1991 ). The only potential candidate for balancing selection from the molecular data is the Acp62F gene in D. simulans, which shows an unusually high level of silent polymorphism and an unprecedented topology in which some D. simulans alleles show less divergence from D. melanogaster alleles than from other D. simulans alleles. However, given the small size of the regions surveyed for this article, additional data from flanking regions of these species as well as outgroup species will be required to determine if there is evidence for old, balanced polymorphisms in the Acp genes.

Another possibility is that rather than harboring old balanced polymorphisms, Acp genes may be subject to transient, yet strongly selected polymorphism (cf. GILLESPIE 1991 ). In such circumstances it is by no means clear that there is an expectation for excess polymorphism. The properties of samples of genes experiencing such selective histories are not well understood (but see GILLESPIE 1997 ). Acp36DE is particularly interesting with regard to models of the maintenance of variation. Haplotype variation at Acp36DE in U.S. samples of D. melanogaster was found to be correlated with sperm displacement phenotypes (CLARK et al. 1995 ). Subsequent evidence from direct experiments has confirmed a role in sperm storage for this protein (BERTRAM et al. 1996 ; NEUBAUM and WOLFNER 1999 ). Here we present evidence that natural selection has contributed to protein divergence between species. Our analysis also suggests that there has been a very recent episode of natural selection at this locus in U.S. populations of D. melanogaster. An important task for the future will be to determine the specific polymorphisms in Acp36DE having measurable effects on patterns of sperm use and to determine the history of these mutations. Such analyses will considerably improve our understanding of both the biology and the evolutionary dynamics of fitness variation associated with sperm use in Drosophila.

Even at this early stage, however, it is worth taking note of the fact that of the four Acp genes that appear to harbor alleles of large effect in D. melanogaster populations (CLARK et al. 1995 ), three of them, Acp26Aa, Acp29AB, and Acp36DE, also show evidence of adaptive protein evolution between species. Perhaps the appropriate model for Acp genes is one in which there is a succession of short-term balanced polymorphisms that give rise to fixations as the selective environment changes over time.

Perhaps the most interesting general observation regarding Acp protein variation in D. simulans and D. melanogaster is the observation of proportionally more replacement polymorphism in D. simulans. This pattern is in stark contrast to several observations in the species pair (AQUADRO et al. 1988 ; MORIYAMA and POWELL 1996 ). There are two sorts of explanations for such a pattern. First, under the neutral model we would expect such an observation if the neutral mutation rate for amino acids at Acp's were higher in D. simulans than in D. melanogaster. Alternatively, if balancing selection contributes to the amino acid polymorphism in these species, then one could explain the data by invoking stronger selection in D. simulans than in D. melanogaster. If this latter hypothesis were true, then one might predict that more of the Acp protein polymorphism would have measurable phenotypic effects in D. simulans than in D. melanogaster.

In a similar vein, one benefit of the data presented here is increased ability to identify polymorphisms or haplotypes that might have effects on patterns of sperm storage or sperm use. CLARK et al. 1995 demonstrated that there were associations between sperm displacement phenotypes in D. melanogaster and single-strand conformation polymorphism (SSCP) alleles at Acp genes 26Aa, 29AB, 36DE, and 53Ea. For example, they reported on phenotypic effects associated with six SSCP alleles at Acp53Ea in D. melanogaster (in a sample of 36 chromosomes). However, the SSCP data did not allow for identification of individual mutations that might be responsible for the phenotypic variation and did not even permit determination of the number of polymorphic sites in the sample. Our sequence analysis of Acp53Ea reveals only a single amino acid polymorphism in the non-African sample of D. melanogaster; this polymorphism is now an excellent candidate for further analysis of phenotypic effects. The continued function characterization of the Acp genes will be important for determining which phenotypes should be examined in such association studies. Given the small size of most Acp genes, direct experimental confirmation of intermediate frequency polymorphisms in Acp genes that contribute to variation in sperm storage and sperm use may be the most direct strategy for determining whether positive selection often acts on Acp polymorphisms in Drosophila.

Manuscript received January 3, 2000; Accepted for publication August 31, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AGUADÉ, M., 1997  Positive selection and the molecular evolution of a gene of male reproduction, Acp26Aa of Drosophila. Mol. Biol. Evol. 14:544-549[Abstract].

AGUADÉ, M., 1998  Different forces drive the evolution of the Acp26Aa and Acp26Ab accessory gland genes in the Drosophila melanogaster species complex. Genetics 150:1079-1089[Abstract/Free Full Text].

AGUADÉ, M., 1999  Positive selection drives the evolution of the Acp29AB accessory gland protein in Drosophila. Genetics 152:543-551[Abstract/Free Full Text].

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