Department of Ecology and Evolution, University of Chicago
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Abstract |
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Introduction |
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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 1990
). Population genetic analyses (Aguade, Miyashita, and Langley 1992
; Aguade 1998
; Tsaur, Ting, and Wu 1998
) and long-term evolutionary comparisons (Tsaur and Wu 1997
) have indicated strong positive selection operating on this gene. The gene product stimulates egg laying (Herndon and Wolfner 1995
) and may be involved in sperm competition (Clark et al. 1995
), although a convincing proof is still lacking (Herndon and Wolfner 1995
). 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. 1980
); 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 1995
). The cleavage is known to be mediated by the ejaculate itself but would occur only in the female's reproductive tract (Park and Wolfner 1995
). 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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Materials and Methods |
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Acp26Aa has a small exon 1 (with 11 codons) and a larger exon 2 (with 250260 codons). As in Tsaur, Ting, and Wu (1998)
, 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 1988
). 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 118285 of Aguade, Miyashita, and Langley (1992)
. Primers, PCR conditions, sequencing, and analysis followed the descriptions of Tsaur, Ting, and Wu (1998)
. Synonymous and nonsynonymous nucleotide differences were computed using the MEGA program package, version 1.01.
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Results |
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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)
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 1998
; Tsaur, Ting, and Wu 1998
). 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 1987
) with respect to amino acid replacements.
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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 1998
), which has recently been shown to be the unique signature of hitchhiking (Fay and Wu 2000
). 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 1989
; Fu and Li 1993
; Fay and Wu 2000
), 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)
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 1987
). 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.
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Discussion |
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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)
, 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 1995
).
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 1996
). Such a phylogenetic relationship is not supported by molecular studies (Caccone et al. 1996
; Harr et al. 1998
). In fact, recent analyses strongly support a closest kinship between D. simulans and D. mauritiana (Harr et al. 1998
; Ting, Tsaur, and Wu 2000
). As in the OdsH gene (Ting et al. 1998
), 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 1998
). For example, the chimpanzee and the bonobo may experience a greater pressure of sperm competition than the gorilla (De Waal and Lanting 1997
), and the evolution of the protamine gene complex indeed suggests such a trend (Wyckoff, Wang, and Wu 2000
). 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.
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Acknowledgements |
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Footnotes |
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1 Present address: Institute of Zoology, Academia Sinica, Taipei, Taiwan, Republic of China.
2 Present address: Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
3 Keywords: Acp26Aa,
Drosophila mauritiana,
sexual selection
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
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