Quantitative Genetics of Sperm Precedence in hsophila melumgt"

Transcription

1 Copyright by the Genetics Society of America Quantitative Genetics of Sperm Precedence in hsophila melumgt" Kimberly A. Hughes' Committee on Evolutionaq Biology, University of Chicago, Chicago, Illinois Manuscript received May 20, 1996 Accepted for publication October 4, 1996 ABSTRACT To assess the genetic basis of sperm competition under conditions in which it occurs, I estimated additive, dominance, homozygous and environmental variance components, the effects of inbreeding, and the weighted average dominance of segregating alleles for two measures of sperm precedence in a large, outbred laboratory population. Both first and second male precedence show significant decline on inbreeding. Second male precedence demonstrates significant dominance variance homozygous and genetic variance, but the additive variance is low and not significantly different from zero. first For male precedence, the variance among homozygous lines is again significant, and dominance variance is larger than the additive variance, but is not statistically significant. In contrast, malematingsuccessand other fitness components in Drosophila generally exhibit significant additive variance and little or no dominance variance. Other recent experiments have shown significant genotypic variation for sperm precedence and have associated it with allelic variants of accessory-gland proteins. The contrast between sperm precedence and other male fitness traits in the structure of quantitative genetic variation suggests that different mechanisms maybe responsible for the maintenance of variation in these traits. The pattern of genetic variation and inbreeding decline shown in this experiment suggests that one or a few genes with major effects on sperm precedence may be segregating in this population. HE existence and relevance of sperm competition T in Drosophila mlanogaster has been a contentious issue in evolutionary genetics. Some of the available evidence suggests that female D. mlanogaster remate only when they have depleted their reserves of stored sperm (MANNING 1962; GROMKO and F'YLE 1978; GROMKO and MARKOW 1993). Under these conditions, competition between gametes from different males will have little effect on male reproductive success. Other results suggest that females may readily remate before sperm stores are depleted, at least under some conditions (GROMKO et al. 1984; BELLEN and KRIGER 1987; HARSHMAN et al. 1988; CLARK et al. 1995). If so, relative fertilization success is an important aspect of male fitness and should be subject to strong directional selection. Mechanistic causes of differences in this trait could involve behavioral, physiological, or morphological differences between males. The differing results reported in the above experiments have been related to differences in experimental design (see DISCUSSION). In this paper, the term sperm pecehce will be used to describe relative fertilization success of different males that have mated with the same female. The tendency for females to mate with a second male if she has recently mated with another is only one prerequisite for the evolution of mechanisms to promote sperm precedence. Another necessary condition is the availability of heritable genetic variation for traits that affect the ' A-esent address: Kimberly A. Hughes, Department of Life Science, Arizona State University, West Campus, P.O. Box 37100, Phoenix, AZ hughes@asuvm.inre.asu.edu probability of sperm use when sperm from multiple males is available.several studies havesuggested that genotypic variation for sperm precedence exists both within and between populations. PROUT and BUND- GMRD (1977) demonstrated that different laboratory strains of D. mlanogaster exhibit different degrees of spermcompetitive ability. Similarly, GILBERT and RICH- MOND (1981) found that different marker strains differed in their ability to sire offspring when mated to nonvirgin females. CLARK et al. (1995) measured significant variation associated with homozygosity for different chromosomes extracted from natural populations. They also showed that this variation was associated with SSCP variation at four genes coding for accessory gland pre teins, which are found in semen and are transferred to females during copulation. Together, these three studies provide compelling evidence that genetic variation affects sperm precedence. However, for reasons of experimental design, the amount of heritable variation in sperm precedence in the populations of D. mlanogaster used in these studies was not estimable. While not a direct estimate of heritable variation for this trait, the experiment of SERVICE and FALES (1993) is at least suggestive that some variation for sperm-competitive ability is heritable. In this experiment, lines selected for different life-history patterns showed significant differences in s p a defense, where sperm defense is the proportion of offspring sired by the first mate of a female, when she has had two different mates. Sperm offense is the term the authors apply to the proportion of offspring sired by the second mate. This charac- Genetics (January, 1997)

2 140 K. A. Hughes ter did not differ significantly between strains. Genetic divergence in sperm defense could have resulted from direct selection on some aspect of sperm precedence, or through an indirect response to selection on a genetically correlated trait. Since genetic correlations are very sensitive to changes in gene frequency (BOHREN et al. 1966; FALCONER 1989, p. 319), they could arise during the course of artificial selection, even if little additive genetic variation was present for sperm defense in the original population. Thus, a correlated response to artificial selection applied to other traits does not conclusively demonstrate the existence of heritable variation for sperm precedence in the ancestral population. Several questions about the evolutionary significance of sperm precedence can be investigated with a formal quantitative-genetic analysis. Some of the following issues are difficult or impossible to address using other empirical approaches. (1) Partitioning genetic variation. The extent of the genetic variation for sperm competition can be measured and can be partitioned into components associated with heritable and with nonheritable variation. The existence of significant genetic variation for sperm precedence would corroborate the studies cited above, while the detection of significant additive variation would provide a direct demonstration of heritable intrapopulation variation in D. melanogaster. (2) Maintenance of genetic variation. Unlike previous experiments, a quantitative genetic analysis provides a test of hypotheses for the maintenance of genetic variation for sperm precedence. Theory predicts that alleles maintained by pure balancing selection will contribute to the nonheritable (nonadditive) portion of the genetic variation, but not to the heritable variation (& DANE 1949). Alternately, if genetic variation for sperm precedence is maintained primarily by mutation-selection balance, much of the genetic variation should exist as additive variation. Comparisons between the genetic components of variation therefore provide a test of the hypothesis that genetic variation is maintained by balancing selection (CHARLESWORTH 1987). (3) Arejirst and second mab effts due to the same mechanism? Sperm precedence is usually measured as two different characters: performance as a first male (a virgin female s first mate) and performance as a second male (the male mated to a nonvirgin female). We do not know the biological mechanisms involved in sperm precedence in this species, so the mechanisms controlling performance as a first male might be similar to or very different from those controlling performance as a second male. Genetic variation at loci affecting these mechanisms will contribute to the genetic correlation between the two traits. If similar mechanisms are responsible for both first- and second-male performance, then one expects a large positive or negative value for the genetic correlation between the traits. If the mechanisms controlling performance as first or sec- ond males are different, then large correlations are not expected. (4) Is spermprecedencegenetically similar toothermale jitness components? One can make inferences about the genetic basis of variation in sperm precedence by comparing the estimates of genetic variance to similar estimates for male fitness components. The focus of much of the controversy concerning sperm precedence in D. melanogaster is whether this trait is an important fitness component for male flies. Variance component estimates for male mating success, longevity, and fecundity are available for the same population of flies (HUGHES 1995a,b). That these traits are major fitness components is not disputed. They can therefore provide a benchmark for comparison to sperm precedence. Also, variance component estimates for these other traits have provided an opportunity to test hypotheses for the maintenance of variation (HUGHES 1995a,b; CHARLES- WORTH and HUGHES 1997). Similar estimates for sperm precedence will allow comparisons of the forces maintaining variation in different traits. In many previous experiments on the genetics of sperm precedence, it has not been possible to control for confounding effects of differential male mating propensity or differential larval survival. For example, in the assays conducted by PROUT and BUNDGAARD (1977) and CLARK et al. (1995), apparent differences between genotypes in sperm precedence might be attributable to difference in the number of copulations achieved by males of the different genotypes. Differential survival of larvae sired by males of different genotypes could also lead to apparent differences in sperm precedence. In thexperiments described below, independent assays of male mating propensity provide a means of determining if genotypic differences in mating ability have been confounded with differences in sperm precedence. Independent assays of male productivity (which includes larval survival and larval competitive ability) provide a test of the hypothesis that apparent differences in sperm precedence are due to differences in larval performance. MATERIALS AND METHODS Experimental populations: I estimated additive, dominance, and environmental variance components for two measures of sperm precedence. I assayed the progeny of 1640 D. melanogaster females, which had been mated to males representing 192 different genotypes (156 heterozygous genotypes and 36 homozygous genotypes). In all, 126,424 progeny were counted. Because I was interested in the standing genetic variation in a stable population and wished to avoid the confounding effects of novel environments, I used flies derived from a large, outbred laboratory population. This population has become adapted to laboratory conditions over many generations without substantial loss of genetic variation. The experimental flies were obtained from the Npopulation, maintained in the laboratory of Dr. BRIAN CHARLESWORTH. This population was originally derived in 1977 from 21 (inversion-free) isofemale lines from a stock collected by Dr. P. T. IVES in 1975 in Amherst,

3 Sperm Precedence in Drosophila 141 Block 1... Block 5 D A M S chromosome *a Lines 1 0 x x x x 2 0 x x x x 3 0 x x x x 4 o x x x x 5 x x x x o 6 x x x x x x x x o x x x x x x x x o x x x x 0 x x x x 0 x x x x 0 FIGURE 1.-North Carolina I1 breeding design. Series of crosses for a modified NCII breeding program. Each x represents a cross between females of the line represented by the column number and males of the line represented by the row numbers; o is used to represent crosses that result in flies homozygous for chromosome IZZ. Every cross is of the form TM6/+, X TM6/+,. The wild-type males produced from each cross will have third-chromosome genotype +r. These genotypes are constructed by crossing males from lines represented by column numbers to females from lines represented by row numbers. Each o represents a cross between females and males from the same line. The wild-type males produced from these crosses will have third-chromosome genotype MA. It has been maintained at large population size since that time, and repeated quantitative genetic analyses of sterno-pleural bristle number indicates there has been little erosion of genetic variability over time (B. CHARLESWORTH, unpublished data). Flies have been cultured in bottles since the establishment of the laboratory population. Female and male flies are thus confined together in a limited space for extended periods of time. Culture conditions for the Npopulation have therefore resembled the continuous confinement experimental paradigm described in the Introduction. Details of the maintenance of these lines are given in CHARLESWORTH and CHARLESWORTH (1985). Females and control males bearing the scarlet eye-color mutation were obtained as follows. By means of eight generations of backcrosses, the scarlet (st) mutation was introduced onto the genetic background of the Nstock to eliminate any effects on male performance of the st/st genetic background. Outbred st/st males from this population have near-normal mating ability (HUGHES 1995a). Breeding design and genetic analysis: A quantitative genetic breeding design known as the North Carolina Design I1 (COMSTOCK and ROBINSON 1952) was used to generate the experimental organisms (Figure 1). A third-chromosome balanced-lethal system, TM6/Sb, was placed on an N genetic background to produce a stock that is +/ +, +/ +, TM&/Sb for the X, second, and third chromosomes, respectively (+ represents a wild-type chromosome derived from the Npopulation). The TM6 chromosome contains multiple, overlap- ping inversions that function as an effective suppresser of crossing-over for the third chromosome (LINDSLEY and ZIMM 1992). The TM6 chromosome also contains the dominant marker, Dichaete (D), allowing the identification of individuals bearing the chromosome and visible recessive mutations that allow detection of crossover events. The resulting balanced- lethal system was then used to produce lines of flies carrying isogenic third chromosomes, balanced over the TM6 chrome some. Different lines were intercrossed in the breeding design (COMSTOCK and ROBINSON 1952) to generate the experimental material. Eight different chromosomal lines were intercrossed in each of five independent blocks of the experiment. A total of 40 independent third chromosomes were therefore sampled from the Npopulation. Hybrid dysgenesis would not have been induced in the progeny of these crosses, because both the Nstock and the balanced-lethal stock derived from it are of the I/Pcytotype. A detailed description of the crosses used to produce the balancer stock, the st/st stock, and the experimental males is given in HUGHES (1995a). The NCII breeding design with reciprocals and homozygous crosses allowed the computation of six components of genetic variance: environmental, additive, dominance, maternalextranuclear, paternal-extranuclear, and variance due to interactions involving the extranuclear effects (COCKERHAM and WEIR 1977). These variance components will be referred to as V, V, V, VM, V, and V, respectively. Variance components were calculated by using the restricted maximum likelihood (REML) procedure in the Quercus package (SHAW and SHAW 1994) and by standard ANOVA methods [Henderson s synthesis method (SEARLE 1971)l. Because of the family structure of the data, variance partitioning was used to calculate genotypic correlations between traits. Genotypic components of covariance were calculated as COV,[x, y] = 1/2(VG[x + y] - VG[4 - VG[y]), where COVG[x,y] is the genetic covariance of two traits, x and y, and VG[x] and V&] are the respective variances. Correlation coefficients (r) were then computed from the standard formula rg = COV~, ~I/~ (V~[XI*V~[~I). Because measurements of sperm precedence were made for genotypes either heterozygous or homozygous with respect to the entire third chromosome [-40% of the D. melanoguster genome (CHARLESWORTH et al. 1992)], I was able to calculate the inbreeding decline associated with these characters. The inbreeding decline was calculated for each block of the experiment as (wo- wi)/wo, where w,, is the mean of the trait for all the heterozygous lines within a block, and w, is the mean for all homozygous lines within a block (JOHNSTON and SCHOEN 1994; HUGHES 1995b). The estimates for each block were then averaged to give the overall estimate of inbreeding decline for the trait. Measurement of sperm precedence: If a female copulates withtwo or more males, sperm from different males may compete for fertilization opportunities. The degree to which a male s sperm can fertilize available eggs, at the expense of another male s sperm, is here termed sperm precedence. The term sperm displacement has also been used to describe very similar measures of male reproductive performance (CLARK et al. 1995). Males from two replicates of each reciprocal cross were assayed for (1) first male precedence, the ability of a male to sire offspring when a female subsequently mates with a different male and (2) second mab precedence, the ability of a male to sire offspring when a female has previously mated with a different male. The alternative male in both cases was homozygous for the scarlet eyecolor mutation, as described above. Virgin females used in the experiments were also drawn from the st/st stock with N genetic background. All genotypes in a single block were tested at the same time to control for any time effect. Different blocks were set up at different times over the course of 18 months. All flies used in the experiments were 5-7 days old at the time of their first mating, and there were no systematic differences in the ages of either control or experimental flies used in assays of different genotypes within a block. For assays of first male precedence, virgin st/st females were

4 142 K. A. H Iughes first mated to males of each of the wild-type genotypes represented in Figure 1. One virgin female was placed in a standard culture vial with two virgin males of the appropriate genotype. The flies were kept together for 2 hr, then the males were discarded. This time period was chosen because, in similar experiments, it has been shown that almost all females mate within 2 hr in this kind of mating trial, but extremely few mate more than once (PROUT and BUNDGAARD 1977; HUGHES 1995a). Three days later (day three), females were transferred to fresh vials, along with two virgin st/st males. These flies were kept together for 24 hr, and then females were transferred to freshlaying vials and the males were discarded (day four). Femaleswere transferred again on dayseven, and the first layingvialswere retained. On day10,all females werediscarded, and the second layingvialswere retained. Progeny from first laying vials were scored on days20 and 21 (there were too many progeny to be scored in a single day). Progeny from second laying vials were scored on days 23 and 24. Progeny expressing the scarlet phenotype were scored as having been sired by the st/st male; progeny expressing wild-type eye color were scored as having been sired by the experimental male. Assays of second male precedence were carried out in an analogous manner, except that the first matings (of st/st females to st/st males) took place en masse in culture bottles. Sperm precedence was calculated as a/( b + l), where a is the number of progeny sired by the second male, and b is the number of progeny sired by the first male, and a and b are calculated from the totals from both laying vials. This estimator of relative success is approximately unbiased (HALDANE 1955). Both first and second male precedence were calculated in this way, so that the two traits would be measured on the same scale (most matings showed strong second male precedence, whether the second male was a st/st control or a wildtype experimental male). This method yields a measure of first male precedence that declines with increasing male fitness, so some genetic parameters were rescaled so that their values could be interpreted in the usual manner (so that positive inbreeding decline indicates that homozygotes are less fit than heterozygotes, for example). All data were standardized by the mean value for the trait in the appropriate block to eliminate differences in mean values between blocks and to provide estimates of standardized variance components (HOULE 1992). Cube-root transformations of the raw data were used for significance testing, since this transformation improved the fit of both data and residuals to a normal distribution, in conformance with assumptions of the analyses. Measurement of other male reproductive traih Genotypic differences in male mating propensity and in male productivity were assayed as described in HUGHES (1995a,b). Briefly, mating propensity was assayed by letting males of each genotype compete with males of the st/st genotype for matings with st/st virgin females. These measures of mating success were used to test the hypothesis that apparent differences in sperm precedence were actually due to differences in mating propensity (and concomitant differences in the number of copulations achieved by males of different genotypes). Male productivity was measured by allowing scarlet females that had mated with a male of an experimental genotype to lay eggs in a vial along with a scarlet female that had mated with a scarlet male. Offspring of the experimental males (having genotype +/st) are therefore raised in an environment in which they must compete with larvae with the st/st genotype. This is the same sort of competitive environment in which larvae produced in the sperm precedence trials are reared. Apparent differences among experimental genotypes in sperm precedence could result from differences in the ability of their offspring to survive in this competitive environment. The assays of male productivity were therefore used to test this possibility. It should be noted that these measures of male productivity also include any differences in the number of zygotes actually sired by males of different genotypes. Such differences could be functionally related to real differences in sperm precedence. Removal of these effects may therefore remove some differences actually attributable to sperm precedence. In this experiment there is no way to distinguish between these two possibilities. Statistical analysis REML methods were used to test the significance of maternal, paternal, and higherarder variance components. Significance was evaluated by comparing the likelihood values for a model including the variance component of interest to a reduced model that does not include that variance component (SHAW 1987). The test statistic comparing the two likelihoods is then given by twice the difference between the log likelihoods. Significance is evaluated by comparing the difference in log likelihoods to a chi-square distribution with degrees of freedom equal to the difference in the number of parameters included in the two models. The computation of analogous sums of squares by the ANOVA Method (this is the classical factorial ANOVA, modified for unbalanced data, also called Henderson s Method I), combined with variance component estimation by synthesis (HART- LEY 1967), produces unbiased estimates for random models (SEARLE 1971). Because REML estimates have more stringent distributional requirements, Henderson s method wasused to calculate additive, dominance, and environmental components of variance of variance. Significance for these variances was evaluated by standard Rests. The significance of genetic correlations and inbreeding decline was tested by computing t-tests, which compare the mean estimate over the five blocks with the empirical standard error of the five independent block estimates. Tests on inbreeding decline were conducted on differences in performance between inbred and outbred lines, rather than on inbreeding decline itself, to avoid conducting significance tests on ratios. Data analysis was carried out in two different ways. For one set of analyses, only females that had produced at least one offspring from each mating were included (restricted data set). This insures that only females that had actually mated with both males were included in the data set. In the other set of analyses, it was assumed that all females had mated with the first males, and all females that produced at least one offspring from their second mating were included in the analysis (complete data set). This assumption guarantees that matings exhibiting 100% second male precedence are included (these matings would be excluded from the restricted data set). This is a reasonable assumption since, in independent assays, 96% of similar-agedvirginfemales mated and produced offspring under the same mating conditions (K. A. HUGHES, unpublished data). Both analyses were conducted to examine whether either assumption biased the results. Results of the two typesofanalyseswere entirely consistent, indicating that little or no bias results from making this assumption. Unless otherwise specified, reported results refer to the restricted data set. Unless otherwise indicated, analyses were conducted after the removal of very low scoring chromosomal homozygotes (those for which line means were <50% of the mean for heterozygous lines within a block). Because such chromosomes may contain severely detrimental alleles that have different genetic properties from high-fitness chromosomes, heterozygous crosses involving these chromosomes were also excluded from the analyses, except where indicated. RESULTS Untransformed variates for first and second male precedence are shown in Figure 2 (homozygous lines) and

5 1 10 2o Sperm Precedence in Drosophila First Male Precedence (Defense Second P1) Male Precedence (Offense P2) FIGURE 2.-Histograms of line means for chromosomal heterozygotes. Variates are untransformed and depicted on a scale that shows the proportion of offspring sired by each genotype in the experimental design, when that genotype is used as the first or second male. This scale corresponds to the traits P1 and P2, or sperm offense and sperm defense, respectively, used by CLARK et nl. (1995). Figure 3 (heterozygous lines). The scale chosen for the illustration is the proportion of offspring sired by each genotype in the experimental design, when that genotype is used as the first or second male to mate with a female. This scale corresponds to the traits P1 and P2, or sperm offense and sperm defense, respectively, used by CLARK et al. (1995). This scale was chosen for illustration for ease of comparison with previous studies and for ease of interpretation. REML analyses of all six variance components were first computed for each trait. Neither VI,, V,, nor V, were significantly different from zero for first or second male precedence. This indicates that there were no significant maternal, paternal, or higher-order-interaction effects on these traits. Variance components were then recalculated as described above, including only V,, v,, and V, in the models. This formulation of the statistical model is equivalent to the biological assumption that only additive effects of alleles and dominance interactions between alleles contribute to the genetic variance. First male precedence: For the restricted data set, this trait does not demonstrate significant additive or nonadditive genetic variation. The variance components, calculated for several different scenarios, are given in Table 1. Variance components for the restricted data set, when severely deleterious homozygous lines are removed, correspond to the following genetic coefficients of variation: CV,, 0%; CV,,, 19%; and CV,, 94%. These and subsequent variance components and standard deviations were computed on untransformed data, but all statistical tests were conducted on transformed data. Results for the complete data set are similar when severely deleterious homozygous lines are excluded (Table 1). Genetic coefficients of variation are as follows: Cy,, 0%; CV,,, 12%; and CV,, 88%. Again, neither of the genetic variance components are significantly different from 0. The estimates are not much affected by inclusion of heterozygous crosses involving chromosomes that perform very poorly as homozygotes, and genetic variance components remain nonsignificant (Table 1). Second male precedence: This trait demonstrates significant dominance, but no significant additive genetic variation, and this pattern exists whether the analysis is conducted on the restricted or the complete data set. It also persists whether or not poorly performing homozygous lines are included (Table 1). After deleting extremely low performing homozygous lines, the estimates of genetic coefficients of variation are as follows: Cy,, 0%; CV,, 21%; and C&, 88%. Results for the complete data set are very similar: CV,, 0%; CV,,, 30%; and CV,, 91%. It appears that substantial genetic variance exists for this character, but little or none of this variation is heritable. As for first male precedence, the variance component estimates are not substantially affected by the inclusion of chromosomes that perform very poorly as homozygotes. FIGLTRE 3.-Histograms of line means for chrome soma1 homozygotes. Variates are depicted as in Figure 2. First Male Precedence (Defense Second P1) Male Precedence (Offense P2)

6 144 K. A. Hughes TABLE 1 Genetic and environmental variance for sperm precedence Trait V* VD VF VE First male precedence (F32,3>0 = 1.1, P= 0.3) (F3y,29> 1.0, P = 0.5) (F = 1.0, P = 0.4) Second male precedence (F33,346 = 1.5, P = 0.05) (F = 1.6, P = 0.03) (F45,503 = 1.4, P = 0.05) The top number for each trait represents the variance component estimate for the restricted data set. The middle number is the estimate from the complete data set. The bottom number is the estimate from the complete data set, including severely deleterious homozygous lines. All values are times lo-. For genetic variance components with positive estimates, Fvalues and Pvalues associated from the analysis of transformed variates are shown. Genetic variance when differences in mating ability and larval survival are removed Variance component estimates for both first and second male precedence are very similar after the effects of male mating success and productivity are removed (Table 2). The significance of V, for second male precedence becomes only marginal, but the estimates for VA, V,, and V, are practically unchanged. VA remains nonsignificant for both traits, although V, for second male precence, corrected for differences in male mating success, becomes marginally significant at P = The estimates of V, remain considerably larger than the estimates of VA for all traits. Differences in male mating propensity and in productivity therefore do not appear to have contributed much to the observed genetic variation for second male precedence. Regressions of sperm precedence traits on male mating success and productivity do not approach significance (data not shown), except in one case. The regression of first male precedence on male productivity is significant at P = This suggests that some of the genetic variance in sperm precedence may be due to variation in larval viability. However, this is believed to be unlikely, as outlined in the DISCUSSION. Geneticvariance of homozygouslines:both traits exhibit significant homozygous genetic variance (V,). Lines homozygous for the entire third chromosome yield very similar estimates of Vc for both sperm precedence traits: for first male precedence V, = and CV, = 52.5% (t = 2.58, P = 0.03), while for second male precedence V, = 0.29 and CV, = 54.1 % (t = 3.24, P = 0.02). Both estimates are also significant by nonparametric signed-rank tests (Z = 7.50, P = 0.03 for both estimates). For the complete data set, V, estimates for both traits are also significant and very similar (first male precedence: V, = 0.09, CV, = 29.5%, t = 2.49, P = 0.03; second male precedence: V, = 0.09, CV, = 29.8%, t = 6.60, P = 0.002). Environmental and sampling variance: Estimates of the environmental Variation for both first and second male precedence are much larger than corresponding estimates of genetic variation (Table 1). However, only a portion of this environmental variance is truly environmentally caused variation in the character. A large part of the variation is sampling variance, due to the binomial sampling process of competition between sperm from two different males. It is therefore worthwhile to ask how large a component of the environmental variation is due to sampling variance. The approximate sampling variance of the estimator of sperm precedence isgivenby &DANE (1955) as v(l + v) n, where v is the ratio of numbers of off- TABLE 2 Genetic and environmental variance for sperm precedence after removing effects of mating success and male productivity Residuals v, VA v, VF First male precedence on: Mating success (F3>,136 = 1.1, P= 0.3) Productivity (F3>,126 = 1.4, P= 0.1) Second male precedence on: Mating success (F26,IOj = 1.3, P= 0.1) Productivity (F27,//, = 1.4, P= ) 68.1 Values for each trait represent the variance component estimate for the restricted data set. Effects of male mating success and productivity were removed by linear regression analysis. All values are times lo-. For genetic variance components with positive estimates, F values and P values associated from the analysis of transformed variates are shown.

7 Drosophila in Precedence Sperm 145 zygotes TABLE 3 Genetic correlations between first and second sperm precedence components Homozygotes r X2 P r X2 P spring sired by the second male to numbers sired by the first, and n is the sample size. For the restricted data set, the mean values of v for first and second male precedence are 28 and 24, respectively, with corresponding sample sizes (harmonic mean of individuals per trial) of 68 and 53, respectively. HALDANE'S formula then indicates that the corresponding expected sampling variances are 346 and 283. The variance components reported in Table 1 were calculated on standardized estimates, so we also need to know the mean values over all vials of first and second male precedence: 16.5 and 18.0, respectively. Using these estimates, and the relation v[y] = v[x] (dy/dx)', the expected sampling variance of the standardized variates can be approximated as 1.27 for first male precedence, and 0.88 for second male precedence. Comparing these values to those in the third column of Table 1, it can be seen that the expected sampling variance can account for all or nearly all of the environmental variance for both traits. There is thus no compelling evidence for variation in sperm precedence caused by variation in the environment. Correlations between first and second male precedence: Genotypic correlations between first and second male precedence can be caused by pleiotropic effects of alleles on the two traits, or by linkage between alleles at different loci. CLARK et al. (1995) report a small and nonsignificant correlation between sperm offense and sperm defense in homozygous line means. I therefore calculated genotypic correlations for comparison. Since the complete data set yielded significant estimates of genetic variance components for both traits, correlations were calculated from these data. Genotypic covariances were calculated on homozygous and heterozygous lines separately. In the analysisof homozygous lines, severely deleterious lines were excluded. The estimate of the genetic correlation for heterozygous lines was large and positive (Table 3), but was not highly significant (P = 0.12). This high correlation was not evident among the homozygous lines. For homozygous lines, the correlation between line means was also calculated and found to be very close to the estimate obtained by REML variance-component estimation (ylm = 0.33). A similar calculation for heterozygous lines is clearly inappropriate, as these lines are not independent. The result for homozygous lines is consistent with the small, nonsignificant correlation reported by CLARK et al. (1995). The high correlation among heterozygous lines is somewhat unexpected, given the earlier results. Implications of the large positive correlation, if real, are described in the DISCUSSION. Genotypic correlations between sperm precedence and other male fitness components: Genotypic correlations between sperm precedence and other male fitness traits were also calculated (Table 4). Only one of the four correlations is significant, that between first male precedence and male productivity. Although large (rg = 0.648), this correlation is only marginally significant after corrections for multiple tests are made (P < 0.06). Nevertheless, like the analyses of residuals, this result is suggestive that the traits measured in the assays of male productivity and sperm precedence are not biologically independent. Genotypic correlations between sperm precedence and female remating: Since rematings were not directly observed, there is a possibility that the results have been affected by multiple female remating. Unless there are systematic differences between male genotypes in the likelihood that they will induce females to multiply remate, variation due to multiple remating will contribute to the environmental, but not to the genetic components of variation. Multiple remating should not, therefore, affect conclusions based on comparisons of additive, dominance, and homozygous genetic variance, and the inbred load. However,systematic differences between male genotypes might affect these conclusions. To test for differences between male genotypes, I have investigated the properties of another trait, the probability that females mated to a given male genotype re- mated at least once (CLARK et al. 1995). The results indicate that there is no significant variation among either first-or second-male genotypes in the probability of female remating (results not shown). There is a marginally significant positive correlation between first male precedence (sperm defense) and the probability that a female remates in her second mating (r = 0.19, P = 0.09) for heterozygous line means, but not for homozygous lines (r = 0.02, P = 0.94). There is no significant correlation between probability of remating and second male precedence (sperm offense; results not shown). Inbreeding decline: The inbreeding decline for second male precedence is large and significant (Table 5). Males homozygous for the third chromosome sire only 67% as many offspring as heterozygotes when they mate with a female that is already inseminated. The inbreeding decline associated with this character (0.33) is very similar to that measured for the mating ability of young flies in the same population (0.35) (HUGHES 1995b). The value is somewhat smaller than that for mating ability in older males (0.5'7). A value of 0.33 for the inbreeding decline corresponds to an estimate of 0.40 for the inbred genetic load (B) of the third chromo-

8 146 K A. Hughes TABLE 4 Correlations between sperm precedence and other male fitness components Correlations with male mating success Correlations with male productivity Sperm Homozygotes Heterozygotes Homozygotes Heterozygotes precedence trait r S E t P r SE t P r S E t P r S E t P First male precedence 0.30 (0.13) (0.14) * (0.11) (0.08) Second male precedence 0.16 (0.33) (0.13) (0.35) (0.11) Correlations between sperm-precedence traits and other components of male reproductive fitness, calculated by standard ANOVA methods. Reported values for r are the means over the five blocks. Numbers in parentheses are empirical SE from the five independent estimates. Values of t and corresponding significance levels are also calculated from the five independent estimates. *P < The correlation between first male precedence and productivity is not significant after correction for multiple tests. some, where B is expressed as the difference between heterozygotes and homozygotes in log mean values (GREENBERG and CROW 1960; SIMMONS and CROW 1977; CHARLESWORTH and CHARLESWORTH 1987; MUKAI 1988; CHARLESWORTH and HUGHES 1996). The inbreeding decline for first male precedence (0.25) is similar in magnitude to that for second male precedence (Table 5). This value is significant by the Wilcoxon test, and is marginally significant by the t-test. Inbreeding decline calculated on the complete data set is consistent with that from the restricted data set. For second male precedence, inbreeding decline is somewhat higher than that from the restricted data, 0.49 ( t = 2.06, one-tailed P = 0.05). For first male precedence, the mean estimate is the same as that for the restricted data set, but is now significant by a t-test: 0.25 (t = 2.52, one-tailed P = 0.03). Minimumnumber of loci affectingspermprecedence: Given the above estimates of B and V,, it is possible to place a lower bound on the number of loci affecting a quantitative trait and an upper bound on the mean homozygous effect of segregating loci. These relationships were first derived by CHARLESWORTH (1969) and MUKAI et al. (1974) for the special case of pure balancing selection, and have recently been generalized to cases in which variation is maintained by mutation-selection balance or by antagonistic pleiotropy coupled with marginal overdominance (CHARLES WORTH and HUGHES 1997). As shown in these papers, if Bi is the inbred load associated with locus z, m is the number of loci affecting a trait, and the total inbred load and dominance variance are expressed as B = &Bj = mb and V, = XBT = m(b2 + VB), respectively, then the following inequalities hold: m 2 B2/VD and B I V,/ B. Applying these inequalities to the values in Tables 1 and 5 give m > and B < for first male precedence. For second male precedence, m and - B These values for m are very small, and the TABLE 5 Inbreeding decline in first and second male precedence, block means male First precedence Second male precedence Inbreeding Homozygous Heterozygous decline Heterozygous Homozygous decline Block lines (w,) lines (w,) (1- [wo/wil) lines (wo) lines (w,) (1 - [Wi/W,I) Means: w, = 16.4 w, = = 0.25 i& = 16.6 w, = = 0.33 Load: B = 0.29 B = 0.40 Tests: t = 1.67 (P = 0.08); 2 = 7.50 (P = 0.03) t = 2.49 (P = 0.03); Z = 7.50 (P = 0.03) Entries in the table are the means for raw data values from the restricted data set. Significance was calculated on transformed data. t and 2 are, respectively, parametric and nonparametric test statistics for the difference in means between heterozygous and homozygous lines. Both tessts are one-tailed. Both first and second male precedence are reported as u/(b + l), where a is the number of progeny sired by the second male, and b is the number of progeny sired by the first male. Inbreedingdecline values have been scaled so that they are positive when homozygous lines are inferior to heterozygous lines: B is calculated as In[ w,] - In[ wo] for first male precedence and ln[w,] - In[w,] for second male precedence. Inbre

9 Precedence Sperm in Drosophila 147 TABLE 6 Bounds on m and for life-history traits in D. melanogaster m2r Character Egg-to-adult viability Egg-to-adult viability Virgin male longevity Sperm precedence of first-mated male Sperm precedence of second-mated male Source SIMMONS and CROW(1977), Table 1, second chromosome MUKAI(1988), natural populations, second chromosome HUGHES(1995a,b), Nlaboratory population, third chromosome Present analysis Present analysis Modified from CHARI.ESWORTH and HUGHES(1997). is the maximum average inbred load per segregating ~ocus, andm is the minimum number of segregating alleles per locus, with respect to the third chromosome. values of R are very large, compared to estimates of these parameters for other male life-history traits measured for the same genotypes (Table 6, modified from CHARLESWORTH and HUGHES1997). These results are therefore consistent with the interpretation that sperm precedence is influenced by relatively few polymorphic loci of relatively large homozygous effects. Two simple statistical tests can be used to addressthis hypothesis. Both are based on the idea that sibships in which a major gene is segregating will demonstrate higher phenotypic variance than other sibships in the same experiment (FAIN1978; MITCHELL-OLDS and BERGELSON 1990). For chromosomal heterozygotes, Levene s test indicates significant heterogeneity of variance among sibships, as expected when a major gene is segregating (first male precedence: F5x,r99 = 1.76, P < 0.01; second male precedence: F5x,214 = 1.91, P < 0.001). Quadratic regressions of sibship variance on sibship mean show maximal variance at intermediatevalues, as expected on the hypothesis of a major gene, but the quadratic terms are not significant (first male precedence: t = 1.37, P < 0.2; second male precedence: t = 1.50, P < 0.15). Histograms of untransformedline means show that, for secondmale precedence, chromosomal homozygotes cluster in the tails of the distribution relative to chromosomal heterozygotes (Figure 4), but this pattern is not as striking for first male precedence. Although not conclusive, these results suggest that at least one gene of major effect is segregating in this population. Average dominance of genes affecting sperm precedence: MUKAI et al. (1972) showed thatthe average dominance of genes affecting a life-history trait can be estimated by the regression of the trait value in heterozygotes on thesum of the trait in the two corresponding homozygous lines. Let h be the dominance coefficient of a diallelic locus, such that the fitnesses of the three genotypes are 1, 1-hs, and 1-s, respectively. This regression then yields an estimate of the weighted average of h over all loci, where the weights are proportional to the homozygous genetic variance of each locus. Under a model of overdominance, A should clearly be negative. The estimates of A for first and second male precedenceare 0.01 (SE = 0.15) and (SE = 0.42), respectively. These values are low, compared to similar values estimated for othermale life-history traits (HUGHES 1995b), and suggest that loci with deleterious effects on sperm precedence are largelyrecessive in their effects.however, these values for A, like most other published values, are associated with very large standard errors and are not significantly different from values representative either of alleles with additive effects or of alleles with overdominant effects. x, DISCUSSION Genetic variation for sperm precedence: The N p o p ulation used in this experiment demonstrates significant V,, for one measures of sperm precedence, and significant inbreeding depression and significant variance amonghomozygous lines for both traits. A recent experiment ona different populationof D. melanogmter measured significant variation among lines isogenic for second and third chromosomes and found significant O Precedence First Precedence Male Second Male O , FIGURE4.-Histograms of line means for untransformed, standardized variates. lines homozygous for the third chromosome; B H B, chromosomal heterozygotes.

10 148 K. A. Hughes associations between sperm precedence and allelic variants of accessory-gland proteins (CLARK et al. 1995). A third study has recently demonstrated divergence for sperm offense, but not sperm defense, in lines selected for early and late life performance (SERVICE and FALES 1991). Together, these three experiments strongly suggest that Drosophila populations possess intrapopulation genetic Variation for physiological and/or behavioral traits that influence male fitness through their effects on sperm precedence. The results presented here indicate that the genetic variance for sperm precedence in the Npopulation is mainly nonadditive. This is a somewhat surprising result, since other fitness components in Drosophila generally exhibit additive genetic variance, and little nonadditive variation (ROSE and CHARLESWORTH 1981; MUM 1985; HUGHES 1995a). For fitness components, large amounts of dominance variance relative to additive variance are expected when allele frequencies are intermediate and alleles affecting a trait are overdominant (HALDANE 1949). High ratios of V, to V, can also occur when variation is due to low-frequency completely recessive alleles (FALCONER 1989, p. 131), or when variation is due to mutation-selection balance, but selection on the trait isvery weak, as occurs for lifehistory traits expressed late in life ( CHARLESWORTH and HUGHES 1996). Contrary to the last scenario, molecular evidence suggests that loci presumed to affect male fertilization success are to be under strong selection. AGUADE et al. (1992) found that accessory-gland proteins at two different loci experience unusually high substitution rates. One of these loci (Acp26Aa, on the second chromosome) was associated with variation in sperm competitive ability in the study of CLARK et al. (1995). A survey by COULTHART and SINCH (1988) also found that accessory-gland proteins are more polymorphic than testesspecific proteins in both D. melanogasterand D. simulans. Esterase-6 (a thirdchromosome gene for which effects on both sperm precedence and on female mating have been reported) has also been reported to demonstrate high levels of molecular diversity (OAKESHOTT et al. 1989). If loci such as these have measurable effects on male reproductive success, then selection operating on gamete competition could indeed be very strong. Strong directional selection could lead to rapid divergence and account for low levels of additive genetic variance within populations. However, if this is the only form of selection operating on accessory-gland proteins and esterase- 6, then the high levels of polymorphism within populations is something of a paradox. Overdominant selection, frequency-dependent selection, or some other form of balancing selection, would need to be invoked to explain the high allelic diversity within populations. Two single-locus models of the population genetics of sperm displacement have described conditions in which selection can lead to stable polymorphism. PROUT and BUNDGAARD (1977) found that, for a diallic locus, either overdominance or nontransitivity of alleles with respect to sperm precedence can lead to polymorphism. In addition, PROUT and CLARK (1996) have recently shown that pleiotropic effects on male mating success and fecundity can increase the opportunity for stable polymorphism for alleles affecting sperm precedence. The maintenance of polymorphism in these models was not necessarily dependent on induced overdominance, since certain combinations of parameters lead to frequency-dependent fitnesses of alternative genotypes, and to an advantage for the rare allele. Female preference, male-male competition, and the maintenance of variation in sperm precedence: As noted in the Introduction, NEWPORT and GROMKO (1984) have argued that the design of laboratory experiments involving female remating is critical to the interpretation of sperm-precedence results. They note that experiments in which females readily remate are typically those in which the females were continuously confined with males for extended periods. When females are exposed to males for only a brief period each day, they tend not to remate until sperm stores are depleted. Because mating females collected from a natural population tended to have smaller reserves of stored sperm than nonmating females ( GROMKO and MARKOW 1993), these authors argue that the brief encounter protocol is more reflective of the conditions under which sperm precedence has evolved in natural populations. This would suggest that interejaculate competition has not been under strong selection in nature, since ejaculates from different males are not normally present in the same female. It would also suggest that female behavior, rather than direct competition between ejaculates from different males, may be the most important determinant of male fertilization success. It is worth noting that intersexual and intrasexual selection may not be mutually exclusive hypotheses in this case. SER- VICE and VOSSBRINK (1996), and CLARK et al. (1995) described evidence that males of different genotypes may induce variation in female remating behavior that contributes to observed variation in sperm precedence traits. As noted above, molecular evidence suggests that accessory-gland loci have both unusually high substitution rates and unusually high levels of polymorphism between populations (see below). High substitution rates are potentially consistent with either strong interejaculate competition, or strong selection due to female remating preferences. It is more difficult to make a case for the maintenance of high levels of polymorphism via female preference, but it is possible that some form of frequency-dependent mechanism could exist. Another intriguing possibility is that variation is maintained by antagonistic selection acting on accessory gland proteins with pleiotropic effects on male fertilization SUC-

11 Drosophila in Precedence Sperm 149 cess and female life-history traits. This third hypothesis is consistent with two recent studies. An experimental study by RICE (1996) has shown that relaxing femalespecific selection led to the rapid evolution of some property of the male ejaculate that decreased the longevity of females that were exposed to it. The theoretical treatment of PROUT and CLARK (1996), described above, suggests that pleiotropy of this kind can greatly expand the opportunity for polymorphism. In the Npopulation used in the present experiment, continuous confinement of females and males together in culture bottles has been the norm for hundreds of generations. A continuous confinement protocol was therefore justified in the partitioning of genetic variance for sperm competition. However, if continuous confinement decreases the ability of females to exert a preference, these culture conditions may have increased the importance of intrasexual (among-male) competition relative to intersexual selection. It is therefore quite interesting that under these conditions, the maintenance of high levels of genetic variance is still apparent. This result does not rule out a role for female preference, but does suggest that variation is maintained even under conditions that may decrease the strength of female preference compared to that present in natural populations. Comparison of this population to others maintained under different culture conditions could provide insight into the role of female-mediated selection. Arethere genes of major effect? One interesting possibility is that only a few polymorphic loci, subject to one of the forms of balancing selection described above, affect sperm precedence. A test of this possibility is provided by calculating m, the minimum number of loci affecting the traits, and E, the maximum inbred load associated with these loci. If variation is maintained by mutation-selection balance, we expect the lower bound on m to be large, and the upper bound on to be small, since the load contributed by partially recessive alleles is between one and two times the mutation rate (CROW and KIMURA 1970). If variation is maintained by balancing selection acting on a few loci, then the converse will be true. The small values of m and large values of calculated for sperm precedence (Table 6) lend support to the balancing selection hypothesis. This conclusion must be treated with caution, since the available statistical tests are somewhat unsophisticated. Only two of four tests based on variances within sibships were significant, but these tests are known to be of low power for moderate sibship sizes (FAIN 1978). It should be noted that the parameters m and E are not related to the effective number of loci parameter described by WRIGHT (1968) and by LANDE (1981), and that the method provides a fairly assumption-free way of detecting variability maintained by selection. However, the presence of epistatic variance can potentially inflate the estimate of V, and lead to an underestimate of m. TACHIDA and COCKERHAM (1988) have argued that there is not much support for the notion that epistasis contributes substantially to the genetic variance in fitness-related traits in D. melanogaster. But their inference is based in part on the observation of generally low levels of V, compared to V, for life-history traits, which does not seem to hold for sperm precedence. No other estimates are currently available for the number of segregating alleles affecting sperm precedence. One candidate locus for the third chromosome is esterase6 (GILBERT and RICHMOND 1981). However, CLARK et al. (1995) found no significant association between alleles of esterase-6 and variation in sperm precedence in chromosomal homozygotes. Effects of mating success and larval viability: A potential concern in these experiments is that the estimated variance components do not reflect genetic variation for some quality of the male ejaculate, but are influenced by variation for some other aspect of male reproductive performance. For example, genetic variation in mating success could cause differences between genotypes in the number of successful matings that first or second males achieve. Differences in sperm precedence would then reflect differences in total quantity of sperm transferred, due only to differences in total number of copulations. Several lines of evidence argue against this interpretation. First, there are no significant correlations between male mating success and the two measures of spermprecedence traits measured. Second, the pattern of genetic variation seen here isvery different from that observed for male mating success in the same population of flies (HUGHES 1995a). In that study, mating success demonstrated significant additive genetic variation and no significant dominance variance. Third, the methods were chosen to minimize the probability of more than one copulation by both the first and second mates (see MATERIALS AND METHODS). Finally, when the effectsof differences in male mating success are removed by taking the residuals of the regression of sperm precedence on mating success, the results are qualitatively the same. Differences in larval viability could also cause apparent differences in sperm precedence. The lackof a genetic correlation between second male precedence and male productivity (which includes larval viability) argues against this interpretation. However, there is a marginally significant positive genetic correlation between productivity and first male precedence, and a significant regression coefficient offirst male precedence on male productivity. However, there is little sup port for the notion that larval characteristics have lead to inflation of the estimate of V, for sperm precedence characters, because the larvae produced in the spermprecedence trials were not genetically identical to the flies used in the NCII breeding design. This is because the dams of the larvae were all from the st/st marker

12 150 K. A. Hughes stock. To assay sperm precedence and male productivity, the male flies produced in the breeding design (to which the variance components are applicable) were mated to randomly chosen females bearing the st mutation on an outbred Ngenetic background. The larvae produced from these matings are related only through their sires. Therefore only an additive component of genetic variation in larval viability could contribute to genetic variation in the sperm precedence traits. This could affect the estimates of V,, but wouldhave no effect on the VD estimates. Nonadditive variation in larval fitness would only contribute to VD if there were nonadditive interactions involving alleles closely linked to the st mutation itself, since this is the only part of the genome that is uniform among the different heterozygous larval genotypes. It therefore appears unlikely that the nonadditive variation that was detected is due to differences in larval fitness components. If this variation is not attributable to larval performance or to male mating success, then it must be due to genetic differences in male fertilization success. Mechanisms for sperm precedence: If, as seems likely, the relevant genetic variation is due to male fertility, then one potential mechanism yields a testable prediction about the observed variance components. If genetic variation in sperm precedence is caused simply by differences in the number of viable sperm transferred during copulation, these differences should lead to a high genetic correlation between first- and second-male precedence. The low correlation reported here and in CLARK et al. (1995) suggests that, for chromosomal homozygotes, differences among males that are the last mates of females do not have the same causes as do differences among males that are the jirst mates of females. However, the correlation in heterozygous lines approaches +l. The correlation is not highly significant, especially if corrections for multiple tests are ap plied. Indeed, genetic correlations are notoriously difficult to measure accurately. The high correlation reported here is also potentially inflated by an artifact of estimation. Because the genetic covariance is estimated only for those replicates that have nonmissing values for both traits, the genetic variances that are used to calculate the correlation are based only on this subset of the data. For this subset, the genetic variance for first male precedence is very close to zero. This term appears in the denominator, so the correlation is very sensitive to inaccuracies in the estimation of this variance component. Nevertheless, the genetic correlation between these two traits isof considerable interest, and more estimates are needed to resolve the discrepancy between available estimates. Mechanistic explanations for genetic variation in sperm precedence remain obscure. One recent experiment has shown that seminal fluid from second mates reduces the number of progeny sired by the first male, even when the second males are sterile (IIARSHW and PROUT 1994). This suggests that second-male seminal fluid interferes with the function of stored sperm from the first male. This mechanism is consistent with the low genetic correlation reported in CLARK et al. (1995) and with the significant genetic variation in second male precedence reported here. Further experiments will be needed to illuminate mechanisms causing a first male effect, or causing a correlation between first and second male effects. I thank B. CHARLESWORTH for suggestions on the design and analysis of these experiments, and for comments on an early draft of the manuscript. The comments of A. CLARK, M. GROMKO, and T. PROUT were extremely helpful and greatly improved the final product. I also thank R. SHAW and F. 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