Sex in Drosophila mauritiana: A Very High Level of Amino Acid Polymorphism in a Male Reproductive Protein Gene, Acp26Aa

Shun-Chern Tsaur1,, Chau-Ti Ting2, and Chung-I WuGo,

Department of Ecology and Evolution, University of Chicago


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Many genes pertaining to male reproductive functions have been shown to evolve rapidly between species, and evidence increasingly suggest the influence of positive Darwinian selection. The accessory gland protein gene (Acp26Aa) of Drosophila is one such example. In order to understand the mechanism of selection, it is often helpful to examine the pattern of polymorphism. We report here that the level of amino acid polymorphism in the N-terminal quarter of Acp26Aa is high in Drosophila melanogaster and is unprecedented in its sibling species Drosophila mauritiana. We postulate that (1) this N-terminal segment may play a role in sperm competition, and (2) D. mauritiana may have been under much more intense sexual selection than other species. Both postulates have important ramifications and deserve to be tested rigorously.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Many studies in the last decade have attempted to detect sexual selection at the molecular level (Lee, Ota, and Vacquier 1995Citation ; Metz and Palumbi 1996Citation ; Sutton and Wilkinson 1997Citation ; Aguade 1998, 1999Citation ; Coulthart and Singh 1988Citation ; Nurminsky et al. 1998Citation ; Ting et al. 1998Citation ; Tsaur, Ting, and Wu 1998Citation ; Ting, Tsaur, and Wu 2000Citation ; Wyckoff, Wang, and Wu 2000Citation ). These studies have suggested that genes of male reproduction may often evolve rapidly between species, presumably driven by positive selection. The mechanism(s) of positive selection can be revealed in some detail by studying the pattern of polymorphism within species. For example, if an advantageous mutation has recently swept through the population, the level of polymorphism in the vicinity may be reduced due to genetic hitchhiking (Maynard Smith and Haigh 1974Citation ; Stephan, Wiehe, and Lenz 1992Citation ; Fay and Wu 2000Citation ). Some studies cited above do indeed show a reduced level of polymorphism (Nurminsky et al. 1998Citation ; Ting, Tsaur, and Wu 2000Citation ). On the other hand, if the mechanism of selection is complex, one may sometimes observe a high level of amino acid polymorphism. This may happen, for example, if selection is frequency-dependent due to competition among males in fertilization (Eberhard 1985Citation ; Wu, Johnson, and Palopoli 1996Citation ) or interaction between sexes (Rice 1996Citation ). The bindin gene in sea urchins appears to represent such a case (Metz and Palumbi 1996Citation ).

The product of the Acp26Aa (accessory gland protein) gene is one of a group of specialized proteins transferred to the Drosophila female in the male's ejaculate (Monsma, Harada, and Wolfner 1990Citation ). Population genetic analyses (Aguade, Miyashita, and Langley 1992Citation ; Aguade 1998Citation ; Tsaur, Ting, and Wu 1998Citation ) and long-term evolutionary comparisons (Tsaur and Wu 1997Citation ) have indicated strong positive selection operating on this gene. The gene product stimulates egg laying (Herndon and Wolfner 1995Citation ) and may be involved in sperm competition (Clark et al. 1995Citation ), although a convincing proof is still lacking (Herndon and Wolfner 1995Citation ). To shed some light on this issue, we compare levels of polymorphism in different parts of proteins in two species.

For males, the dilemma of using ejaculatory proteins to assist in sperm competition could give rise to the difficulty in distinguishing self from foes. While there may be ways to discriminate, a window of opportunity does exist in Drosophila which competing males may effectively exploit. For example, Drosophila melanogaster males do not ejaculate sperm until they are at least 5 min into copulation (Hall et al. 1980Citation ); thus, some ejaculatory proteins' actions could precede sperm entry. In the case of Acp26Aa, the N-terminus presented in figure 1 will have been cleaved and degraded by the time sperm from the copulating males begin to enter (Park and Wolfner 1995Citation ). The cleavage is known to be mediated by the ejaculate itself but would occur only in the female's reproductive tract (Park and Wolfner 1995Citation ). If this portion of peptide functions in reducing the competitiveness of the stored sperm, the pattern of polymorphism and divergence in this short stretch of protein may reveal a signature of positive selection that is different from that of the rest of the protein.



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Fig. 1.—Amino acid sequences of the 56 residues of the N-segments from 23 Drosophila mauritiana alleles (mau), and one each from Drosophila sechellia (sec), Drosophila simulans (sim), and Drosophila melanogaster (mel), are shown. Dashes denote deletions. The segment ends with a conserved pair of lysines (KK) which represent the putative cleavage site according to Park and Wolfner (1995). The segment shown is cleaved and degraded in the female's reproductive tract shortly after ejaculation (see Materials and Methods)

 
A second aspect of sexual selection driving rapid molecular evolution is the possibility of a lineage-specific effect that is correlated with sexual behavior. In other words, species that are under stronger pressure of sexual selection (being more promiscuous, for example) should bear the signature over many genes of male reproduction. There is a hint of that in higher primates (Wyckoff, Wang, and Wu 2000Citation ), but the pattern has not been documented in Drosophila. Among the sibling species of Drosophila simulans, Drosophila mauritiana is most noteworthy in this regard. The degree of hybrid male sterility is much greater between D. mauritiana and D. simulans than that between Drosophila sechellia and D. simulans (Wu and Davis 1993Citation ; Cabot et al. 1994Citation ; Perez and Wu 1995Citation ; Hollocher and Wu 1996Citation ; Palopoli, Davis, and Wu 1996Citation ), even though the former pairing is phylogenetically more closely related (Caccone et al. 1996Citation ; Harr et al. 1998Citation ; Ting, Tsaur, and Wu 2000Citation ). Because hybrid male sterility is a reflection of the divergence in the spermatogenic programs, genes involved in spermatogenesis in D. mauritiana must have been evolving most rapidly since speciation. At the molecular level, a much elevated rate of amino acid substitution along the D. mauritiana lineage has been reported for the Odysseus gene of hybrid male sterility (Ting et al. 1998Citation ). Is the acceleration in the rate of evolution observable among other male reproductive genes in the lineage of D. mauritiana? We shall compare the divergence and polymorphism of Acp26Aa in this group of Drosophila.


    Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We sequenced 22 D. mauritiana isofemale lines (18 from the National Institute of Genetics, Mishima, Japan, and 4 from the National Drosophila Species Resource Center, Bowling Green, Ohio) and included the one from Aguade, Miyashita, and Langley (1992)Citation in our analysis. We compared these sequences with the 49 D. melanogaster alleles from previous analyses (Aguade, Miyashita, and Langley 1992Citation ; Tsaur, Ting, and Wu 1998Citation ). The DNA sequences reported in this paper have been deposited in the GenBank database under the accession numbers AF302208AF302229.

Acp26Aa has a small exon 1 (with 11 codons) and a larger exon 2 (with 250–260 codons). As in Tsaur, Ting, and Wu (1998)Citation , we obtained only the sequences of the larger exon 2 of Acp26Aa, because exon 1 likely codes for the signal peptide and has no bearing on the secreted protein (Monsma and Wolfner 1988Citation ). A small portion of exon 2 (up to six amino acids) may also be included in the signal peptide, although the inference was made only for D. melanogaster. We therefore included the entire exon 2 of D. mauritiana in our analysis. Figure 1 corresponds to nucleotide positions 118–285 of Aguade, Miyashita, and Langley (1992)Citation . Primers, PCR conditions, sequencing, and analysis followed the descriptions of Tsaur, Ting, and Wu (1998)Citation . Synonymous and nonsynonymous nucleotide differences were computed using the MEGA program package, version 1.01.


    Results
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
As stated in the Introduction, we shall contrast the cleaved N-terminus of ACP26Aa with the rest of the protein. Figure 1 displays the 56 amino acids of the cleaved N-terminal end (see Materials and Methods for details), referred to as the "N-segment." The remaining 199 amino acids near the C-terminus will be referred to as the "C-segment." Even a cursory inspection leaves an impression of extreme polymorphism in the N-segment of D. mauritiana. There are also two deletions (6 and 11 codons, respectively) and one insertion (1 codon), with none being rare (13, 6, and 3 occurrences out of 23, respectively). For an endemic species like D. mauritiana, which is generally not highly polymorphic (Hilton, Kliman, and Hey 1994Citation ; Caccone et al. 1996Citation ; Ting, Tsaur, and Wu 2000Citation ), such a high level of polymorphism is unprecedented. The C-segment is much less polymorphic than the N-segment but is still more so than other regions surveyed, perhaps as a result of tight linkage with the N-segment. Sequences of the C-segment can be found in the GenBank deposition. (For the D. melanogaster sequences, please refer to Aguade, Miyashita and Langley [1992]Citation and Tsaur, Ting, and Wu [1998]Citation .)

Table 1 summarizes the polymorphisms and divergence of Acp26Aa within and between D. mauritiana and D. melanogaster. Insertions/deletions and nonsynonymous (R, for amino acid replacement) and synonymous (S) nucleotide changes are separately tallied. The C-segment shows a significantly larger R : S ratio in the between-species comparison (59:17) than in the polymorphism data (11:25 and 7:10, respectively) by the McDonald and Kreitman (1991)Citation test. This difference is usually interpreted to mean that directional selection is driving amino acid replacements, which would enhance the level of divergence much more than that of polymorphism. This conclusion has been drawn previously using the entire Acp26Aa gene (Aguade 1998Citation ; Tsaur, Ting, and Wu 1998Citation ). Interestingly, the difference is not observable in the N segment, although it is not because of a low value of R between species. In fact, the R : S ratio is larger, albeit insignificantly, in the N-segment (11:1) than in the C-segment (59:17) in the divergence data. In other words, the R : S ratios are high in the N-segment in both the within-species and the between-species data. That the N-segment shows an excess of replacement polymorphism can also be seen by comparing the and C-segments. For D. mauritiana, the R : S ratios are 20:2 and 11:25, respectively (P < 0.001 by Fisher's exact test), while they are 13:6 and 7:10 for D. melanogaster (P < 0.05). The same conclusion can be reached by the HKA test (Hudson, Kreitman, and Aguade 1987Citation ) with respect to amino acid replacements.


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Table 1 Numbers of Divergent and Polymorphic Sites in the N-Segment (56 residues) and the C-Segment (199 residues)

 
Is the high level of amino acid polymorphism in the N-segment attributable to some form of polymorphism-enhancing selection? For D. mauritiana, the average of the pairwise Ka (number of nonsynonymous changes per nonsynonymous site) values (0.0367 ± 0.0013) is significantly higher than that of Ks (number of synonymous changes per synonymous site)(0.0055 ± 0.0011) in the N-segment. While the latter is well within the range of within-species variation among six other genes (0.0003–0.012, with a mean of <0.005; Hilton, Kliman, and Hey 1994Citation ; Ting, Tsaur, and Wu 2000Citation ), the average Ka is significantly higher than the average level of silent variation in this species. This difference in replacement and silent polymorphisms does not appear to be solely accountable by the relaxation of negative selection, at least in D. mauritiana.

We also note that all D. mauritiana sequences evolved faster than those of D. sechellia and D. simulans in the N-segment when D. melanogaster sequences were used as the outgroup. For instance, five of the six replacement differences between D. simulans and D. mauritiana occurred in the lineage to D. mauritiana. In general, the patterns of DNA evolution in the Acp26Aa sequences are exaggerated in D. mauritiana in comparison with D. melanogaster.

The above analysis on the comparisons between synonymous and nonsynonymous changes is a direct assessment of positive selection. Previously, we noticed that the number of high-frequency variant sites in Acp26Aa of D. melanogaster was in excess of the neutral expectation (Tsaur, Ting, and Wu 1998Citation ), which has recently been shown to be the unique signature of hitchhiking (Fay and Wu 2000Citation ). In D. mauritiana, we can also visualize many high-frequency variants in the N-segment, which is further affected by an insertion and two deletions of amino acids, all at intermediate frequencies (26%–57%). None of the statistics (Tajima 1989Citation ; Fu and Li 1993Citation ; Fay and Wu 2000Citation ), however, could conclude a significant departure from neutrality in this frequency spectrum. While a small DNA region like the N-segment may often fail to yield the required statistical resolution, the H statistic of Fay and Wu (2000)Citation can sometimes reject the neutral model in favor of genetic hitchhiking with as few as one segregating site. We suggest that because these tests were designed to examine the impact of selection on linked neutral variations but the N-segment is likely to be under selection at a number of sites simultaneously, the tests may not be directly applicable. The occurrences of multiple mutants at many of the same sites also violate the assumptions of the infinite-sites model, which is the basis of many statistical tests.

In the short N-segment of figure 1 , at least six recombination events can be detected, one between each pair of adjacent variant sites by the standard four-gametotype test (Hudson 1987Citation ). Adjacent variant sites are those where the mutant nucleotide occurs more than three times in the sample of 23 sequences. In some cases where only three haplotypes are present, for example, between residues 2 and 4 in figure 1 , recombination can still be inferred because the missing one is the ancestral type. The high level of recombination suggests either that the system is sufficiently old to allow many recombinations to occur among coexisting haplotypes or that recombinants themselves are favored, perhaps due to a mechanism that selects for rare alleles and enhances diversity. An exception may be that recombinants carrying the two large deletions are apparently selected against, as they are mutually exclusive among the haplotypes observed. In general, some forms of balancing or positive selection have to be invoked to fully account for the patterns of polymorphism and divergence in the N-segment of Acp26Aa.


    Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We have shown that a protein segment which could potentially be involved in sperm competition exhibits an extremely high level of amino acid polymorphism in D. mauritiana. Since the pattern in D. melanogaster deviates in the same direction but to a lesser extent, the same type of selective pressure may be operating in both species. It appears that the effect of selection on the N-segment of Acp26Aa is both the enhancement of polymorphism and the elevation of divergence. Moreover, as noted earlier, the high level of recombination may be suggestive of selection favoring diversity enhancement via recombination. The pattern is reminiscent of those of MHC (Hughes and Nei 1989Citation ) and bindin (Metz and Palumbi 1996Citation ).

If the N-segment can be shown to incapacitate stored sperm, a plausible model would be frequency-dependent selection. As in the "arms race" scenario, new amino acid mutations could be driven to an intermediate frequency when they are rare. It should be noted that the C-segment is also under strong positive selection, as previously shown by Aguade (1998)Citation , but does not exhibit a high level of polymorphism. The ACP26Aa protein, after the N-segment is cleaved, must have a separate function and experience a different selective pressure (see Herndon and Wolfner 1995Citation ).

Finally, we would like to address the possibility of a lineage effect in D. mauritiana. Previous suggestion of rapid evolution of the male reproductive system in this species, based on genetic data, can be negated if this species is a distant outgroup of D. simulans and D. sechellia (Palopoli, Davis, and Wu 1996Citation ). Such a phylogenetic relationship is not supported by molecular studies (Caccone et al. 1996Citation ; Harr et al. 1998Citation ). In fact, recent analyses strongly support a closest kinship between D. simulans and D. mauritiana (Harr et al. 1998Citation ; Ting, Tsaur, and Wu 2000Citation ). As in the OdsH gene (Ting et al. 1998Citation ), the accelerated rate of amino acid changes in Acp26Aa is more pronounced in D. mauritiana than in its sibling species. Lineage-dependence in sexual selection may be common among closely related species (Dixson 1998Citation ). For example, the chimpanzee and the bonobo may experience a greater pressure of sperm competition than the gorilla (De Waal and Lanting 1997Citation ), and the evolution of the protamine gene complex indeed suggests such a trend (Wyckoff, Wang, and Wu 2000Citation ). In this regard, D. mauritiana vis-à-vis its sibling species could be an ideal system for studying the genetic basis of diverging sexual systems which may have led to disparate pressure of sexual selection.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank Shigeo Hayashi for fly stocks and Steven Orzack, Ian Boussy, Justin Fay, and the two anonymous reviewers for helpful comments. This research was supported by NSF and NIH grants to C.-I.W.


    Footnotes
 
David Rand, Reviewing Editor

1 Present address: Institute of Zoology, Academia Sinica, Taipei, Taiwan, Republic of China. Back

2 Present address: Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China. Back

3 Keywords: Acp26Aa, Drosophila mauritiana, sexual selection Back

4 Address for correspondence and reprints: Chung-I. Wu, Department of Ecology and Evolution, University of Chicago, Chicago, Illinois 60637. E-mail: ciwu{at}uchicago.edu Back


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 Materials and Methods
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 Discussion
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Accepted for publication September 7, 2000.