Behavioral Ecology Advance Access originally published online on June 22, 2005
Behavioral Ecology 2005 16(5):889-897; doi:10.1093/beheco/ari066
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Relative influence of male and female genital morphology on paternity in the dung beetle Onthophagus taurus
Evolutionary Biology Research Group, School of Animal Biology (M092), University of Western Australia, Nedlands, WA 6009, Australia
Address correspondence to C.M. House. E-mail: clarissa.house{at}manchester.ac.uk.
Received 23 June 2004; revised 16 May 2005; accepted 23 May 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Studies of fertilization success have demonstrated that male effects are often a strong and important source of variation in P2 (the proportion of offspring that are fertilized by the second male to mate). More recently there has been emphasis on female processes that occur during and after copulation that might bias the outcome of male-male interactions. Here we used the sterile male technique to evaluate whether female genital morphology influences the repeatability of P2 when the same pair of male dung beetles, Onthophagus taurus, copulated with a series of full-sib females or unrelated females that were all unrelated to the male pair. Repeatability estimates of measures of female genital morphology showed that full-sib females varied less in their genital morphology than did unrelated females. Therefore, if female genital traits are an important source of variation in male fertilization success, P2 should be more repeatable across full-sib than unrelated females. Contrary to this prediction, we show that the repeatability of P2 did not differ between female groups. Moreover, specific dimensions of the female genitalia (sclerotized vagina and bursa) did not contribute significantly to variance in P2. In contrast, male effects had consistent and repeatable influences on paternity across females. These were partly explained by variation in the morphology of male genital sclerites.
Key words: female genitalia, Onthophagus taurus, repeatability, sperm competition.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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For many species there is considerable variation in paternity when two males mate with the same female within a single reproductive cycle (see Simmons, 2001
). There is extensive theoretical and empirical evidence to suggest that an important source of variation in paternity arises from sperm competition (Parker, 1970). Sperm competition can favor morphological (Waage, 1979), behavioral (Alcock, 1994), and physiological adaptations (Chapman et al., 1995) in males that ensure that a given male's ejaculate dominates within the competitive arena of the female genital tract (Birkhead and Møller, 1992, 1998; Simmons, 2001; Smith, 1984).
When females receive sperm from more than one male the opportunity arises for them to influence the outcome of sperm competition. Thornhill (1983, 1984) defined female processes that occur during and after copulation, which act to bias paternity towards a particular male, as cryptic female choice. Eberhard (1996) identified 20 mechanisms by which female behavior, physiology, and morphology could explain variance in fertilization success. These mechanisms can be summarized into two broad categories: female influences over (1) the degree of insemination and (2) the transfer and utilization of sperm after an ejaculate is deposited within the female genital tract. Females may limit sperm transfer by terminating copulation prematurely (Thornhill, 1976), removing the male's spermatophore (Sakaluk and Eggert, 1996; Simmons, 1986), or consuming the copulating male (Elgar et al., 2000). Variance in fertilization success postcopula may occur if the morphology of the female genital tract, muscular contractions, and/or mucosal secretions cause sperm to be differentially stored and utilized (Bloch Qazi et al., 1998; Cordoba-Aguilar, 1999; Edvardsson and Arnqvist, 2000; Hosken et al., 1999; Neubaum and Wolfner, 1999; Rodriguez, 1994, 1995; Simmons and Achmann, 2000).
The primary objective of this study was to determine whether variation in the genital morphology of the female dung beetle, Onthophagus taurus, explains some of the variation in P2 (the proportion of offspring sired by the last male to mate with a doubly mated female). Comparative studies in a range of animal taxa show that female genital morphology is simpler than male genital morphology (Eberhard, 1996). Nonetheless, it is argued that the female genital tract provides the arena where male sperm compete for fertilizations and thereby imposes selection on both male and female genital traits (Eberhard, 1996; Parker, 1970; Telford and Jennions, 1998). Phylogenetic analysis of genital morphology in 36 species of carabid beetle (Liebherr, 1992), 113 species of Drosophila (Pitnick et al., 1999), and 13 species of diopsid stalk-eyed flies (Presgraves et al., 1999) strongly supports this prediction. In each instance, female and male genitalia were found to coevolve, suggesting that a change in the genital morphology of one sex causes a change in the genital traits of the opposite sex. The findings of within-species studies that have estimated the influence of female genitalia on the variance in paternity are, however, less conclusive. In just two insect species, the red flour beetle (Tribolium castaneum, Fedina and Lewis, 2004) and the yellow dung fly (Scatophaga stercoraria, Parker et al., 1999), the dimensions of the spermatheca affected the rate of sperm displacement and subsequently P2. It is not known if other aspects of the female genital tract can influence paternity. Thus, further detailed studies are required to determine the generality of the influence of female genital morphology on variance in paternity.
Sperm competition studies in O. taurus indicate that sperm mix randomly in the female genital tract and compete for fertilizations under the principle of a fair raffle (Simmons et al., 2004; Tomkins and Simmons, 2000). After two males copulate with a female there is considerable variation in P2, some of which is explained by differences in males and their genital morphology (House and Simmons, 2003). There is no effect of male size or copula duration on P2, as may be expected if females do not prefer males of a certain body size or prolonged copula stimulation (House and Simmons, 2003; Tomkins and Simmons, 2000). However, it is yet to be ascertained whether the female genital tract influences fertilization success in this species.
Previously, we have shown that male effects are likely to be a major source of variation in P2 in O. taurus (House and Simmons, 2003). In this study, we mate the same pair of males to both full-sib females and genetically unrelated females to partition male and female effects and to examine the relationship between female genital morphology and paternity. More specifically, our study was designed to test three hypotheses. First, we examine whether measures of female genital morphology are repeatable within families. If so, we predict that the variation in female genital morphology would be greater between families than between sisters from the same family. Repeatability estimates provide an upper estimate of trait heritability, and this is suggestive of underlying genetic variation (Falconer and Mackay, 1996). Second, we determine whether repeatability estimates of P2 are higher when the same pair of males is mated to a series of full-sib females that are unrelated to the male pair, compared with a series of unrelated females. If sisters vary less in their genital morphology than unrelated females and female genital traits are an important source of variation in fertilization success, P2 should be more repeatable when competing males mate with sisters. Finally, we examine whether specific measures of female genital morphology influence the variation in paternity. A significant relationship between female genitalia and P2 would indicate that variation in female genital traits imposes selection on male genital morphology.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Beetle collection
Approximately 300 field-mated female dung beetles were collected from fresh cattle dung from a pasture in Denmark, southwest Western Australia. Females were placed in individual breeding chambers (25 x 6 cm PVC piping) that were three-quarters filled with moist sand and topped with 250 ml of cow dung and left to construct brood masses for 10 days. A brood mass is a mass of dung that provides the nutritional resources for the growth and development of a single offspring (Hunt and Simmons, 1997
). Brood masses were sieved from the sand after 10 days and incubated at 27°C for 3 weeks. Emerging offspring were maintained in single-sex cultures with unlimited access to fresh cow dung for 2 weeks. These stock beetles were used to generate animals for our experimental design.
Experimental animals
To obtain a random sample of males for our experiment, 150 virgin males and 150 virgin females were randomly selected and maintained in a mixed-sex population for 1 week to ensure that all females were reproductively mature and had mated. Mated females were then placed in individual breeding chambers for 10 days. Chambers were then sieved, and brood masses were incubated at 27°C for 3 weeks. Offspring were sexed at emergence and maintained in single-sex cultures. The resulting virgin male offspring constitute our experimental males. To ensure that these males did not mate with their sisters, all female offspring were discarded.
Concurrently, experimental females were obtained by randomly pairing 150 virgin males and 150 virgin females and maintaining each pair in an individual container (5 x 7 x 7 cm) for 1 week to mate. Mated females where placed in individual breeding chambers for 10 days to obtain brood masses. Chambers were then sieved, and the brood masses were incubated at 27°C for 3 weeks. Offspring were sexed at emergence. Virgin experimental females were maintained in individual containers (5 x 7 x 7 cm) in their full-sib family groups and their brothers were discarded.
O. taurus are sexually immature at emergence and require 12–14 days of constant feeding before their gonads mature (Hunt and Simmons, 1997, 1998). Thus, to ensure they were sexually mature, all experimental animals were provided with unlimited access to fresh cow dung for 2 weeks before the start of the experiment. At the commencement of the experiment, male courtship and female receptivity to courtship confirmed that the experimental beetles were sexually mature.
Experimental protocol
Sterile male technique
We used the sterile male technique to measure the fertilization success of a given experimental male in each mating pair (Boorman and Parker, 1976). This technique has been used extensively in studies of paternity in O. taurus (see Hunt and Simmons, 2001, 2002; Tomkins and Simmons, 2000). In this study, experimental virgin males were sterilized by exposure to 10 krad of gamma radiation from a 60Co source at a dose rate of 1 krad min–1 under nitrogen gas anesthesia. Irradiated males show no change in courtship or mating behavior for 7 days postirradiation, but eggs that are fertilized by irradiated sperm fail to hatch due to lethal mutations (Hunt and Simmons, 2002). Thus, eggs that are fertilized by the irradiated male are reliably identified when a normal (N) and irradiated (R) male copulate with the same female (Simmons, 2001). Copulations involving irradiated males were commenced directly after the effect of the nitrogen anesthetic wore off because irradiation does not influence the time taken for males to initiate courtship or the probability of gaining a mating (Hunt and Simmons, 2002). Between copulations, normal and irradiated males were placed in separate containers but were maintained under identical culture conditions.
Mating design
Each experimental replicate consisted of two unrelated virgin males and six virgin females unrelated to the male pair (three full-sib females and three unrelated females) (Figure 1). The males in each replicate were randomly paired, and one male in the pair was irradiated so that each of the six females mated with both a normal (N) and an irradiated (R) male. The irradiation sequence (RN or NR) was randomized across replicates. Control double matings were also performed (NN or RR). This provided an estimation of natural levels of infertility in females that mated to normal males (n = 8) and residual fertility in females that mated to irradiated males (n = 8).
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Within each replicate, the same pair of unrelated experimental males copulated with three full-sib females and three unrelated females. To assign experimental females to each male pair, each full-sib family was given a number between 1 and 150. Each male pair was then randomly assigned a full-sib family using a random number table, and three females from this family served as the related females. Individual females from the remaining families were then randomly assigned to male pairs. Three females from three different families represented the unrelated female group. If a family was represented more than once in the unrelated group for a given replicate, a new female was reassigned at random. This protocol ensured that unrelated females were not genetically related to each other, to the females in the full-sib group, or to the two males with which they mated.
For each replicate, the mating role (first or second male) for each male of the pair was the same for all six females. Each female copulated with both males before the pair was mated to the next female. Thus, any effect of copula number on ejaculate volume should be minimal, as females received ejaculates from males that had completed an equal number of prior copulations. The order that males were mated to full-sib and unrelated females was randomized across replicates (Figure 1).
All copulations were performed in a constant temperature room set at 27°C and were observed under red lighting in transparent chambers (1 x 5 cm) that had a 1-cm-thick plaster-of-Paris base that had been moistened with water and dung. A single male was introduced into a chamber, followed by a single female. At the completion of copulation, the female's first mate was removed and the second male introduced. After copulation, both males were placed in individual plastic boxes (5 x 7 x 7 cm) that were half-filled with moist sand and supplied with 10 ml of cow dung. After a minimum period of 1 h, rested males were reintroduced into the mating chambers and a new female was introduced. The process was repeated until all females in a replicate were twice mated. Females that would not mate twice on the same day were housed in a manner that was identical to that of the "resting" males. Recalcitrant females were presented with opportunities to mate on successive days. The interval between successive matings is not a significant determinant of fertilization success in this species (House and Simmons, 2003; Tomkins and Simmons, 2000).
Twice-mated experimental females were placed in individual breeding chambers that were three-quarters filled with sand and 250 ml of cow dung. Chambers were maintained for 10 days and then sieved and the female and her brood masses collected. After 10 days, females produced between 6–28 brood masses (mean ± SE: 14.4 ± 4.5). Both the experimental males and females were placed in 1.5-ml Eppendorf vials, frozen, and stored in 70% ethanol for morphometric analysis. Brood masses were opened and recorded as fertile if they contained larvae or sterile if they contained a rotting egg in order to assign paternity.
Morphometrics
The primary vagina of female O. taurus has a single sclerotized portion that supports the bursa. The infundibulum is positioned at right angles to the sclerotized portion of the vagina and runs into the spermathecal duct that attaches to the kidney-shaped spermatheca (Figure 2). The genitalia of male O. taurus have an inflatable endophallus, which has several chitinous sclerites (see House and Simmons, 2003, for a view of the sclerites captured in situ). Tissue connected to the female and male genitalia was macerated in 10% KOH (30 min) and cleared in 50% aqueous lactic acid solution (30 min) before being mounted on slides with Eukitt fixative. A circular, 13-mm-diameter cover slip was placed on top of the female genitalia to prevent the bursa from being crushed.
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Morphological variation in the female genital traits was examined using linear measurements. The area and length of the outline of the bursa; the width and length of the outline of the sclerotized vagina; and the width, area, and length of the outline of the spermatheca were measured (Figure 2). Variation in four of the males' genital sclerites was also measured. The length of the outline and the distance between and the area bound by distinctive landmarks on four of the male sclerites were recorded (see House and Simmons, 2003
, for schematic diagrams of the sclerites). All measurements were extracted using the measurement explorer function of the Optimas Image Analysis package. Female body size (pronotum width) was measured using the eyepiece graticule in a binocular microscope. Pronotum width is a standard measure of body size in Onthophagus (Emlen, 1997). Repeated measures of each female genital trait were taken from a cohort of 19 beetles. Variation between subjects was significantly greater than within subjects for one measure of the bursa, two measures of the sclerotized vagina, and two measures of the spermatheca (range of repeatability estimates 0.9–0.94; F1,18 = 9.61–18.06, all p = .0001). Measures of the area of the bursa and spermatheca were not repeatable, so these measures were not retained in the final analysis. Repeated measures of the male genital sclerites were taken from a cohort of 20 beetles. Variation between subjects was significantly greater than within subjects for the measures of all five of the sclerites (range of repeatability estimates 0.73–0.97; F1,19 = 3.71–34.69, p = .0014–.0001).
Statistical analysis
The statistical package JMP was used for descriptive analyses of the properties of female and male genital morphology. To describe the variation in female and male genitalia we performed principal component analysis (PCA) on correlations. This approach has the advantage of conserving degrees of freedom and reduced the risk of type II error in our subsequent analyses (Quinn and Keough, 2002). The PCA of female genital morphology included the length of the bursa outline and the width and length of the outline of the sclerotized vagina. Measures of the spermatheca were not included as PCA does not accommodate missing values for any of the original variables and the spermatheca was not always found during dissection. For the PCA of male genital morphology a separate analysis was performed for the different measures of a single sclerite. This approach aided our interpretation of pattern of principal components (PCs) and was appropriate, given that there was a minimum of two variables (i.e., measurements) for each sclerite (Bryant and Yarnold, 2003).
The statistical package SPSS v. 11 was used to estimate the variance in P2 when the same pair of males within a replicate mated to related or unrelated females. Theoretically our design was fully balanced, with a total of 60 replicate male pairs. In 30 of these replicates, the first male was normal and the second male was irradiated (NR), while in the other 30 replicates the first male was irradiated and the second male was normal (RN). Within each irradiation sequence, 15 replicate male pairs copulated with full-sib females first and then unrelated females (Figure 1). The remaining replicate male pairs copulated with unrelated females and then full-sib females (Figure 1). Our experimental design became unbalanced, however, as some male pairs did not complete their allocated number of copulations and some doubly mated females produced too few broods. P2 values that are based on few total offspring may be unreliable. Therefore, in our estimates of variance in P2, we excluded females that produced fewer than 10 offspring (n = 27) and used restricted maximum likelihood to estimate variance components. Repeatability (R) estimates were calculated as 
W/(W + E), where W is the variation in P2 between replicate male pairs, and E represents the differences in P2 among females that copulated with the same males in a pair (Becker, 1984). The standard error (SE) of R was calculated according to the method outlined by Becker (1984) for an unbalanced design. All data are presented as means ± SE.
To determine whether measures of female or male genital morphology explained variation in P2, we used generalized linear models (GLMs) with binomial errors and logit link functions. These analyses were conducted in S-Plus 6.4 using the Mass library of Venables and Ripley (2002) and the glm function. The response variable was equal to the total number of offspring sired by the second male to mate divided by the total number of offspring produced by the female. GLMs give low weight to estimates with small binomial denominators; so all females that produced offspring were included. Overdispersion in the data was corrected by testing the fit of the model using the F statistic rather than chi square (Crawley, 2002). The sequential deletion method outlined in Crawley (2002) was used to derive our minimal model.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Female genital morphology
The phenotypic correlation between the width and length of the outline of the sclerotized portion of the vagina was positive and statistically significant. Similarly, the phenotypic correlations between the length of the outline of the bursa and the width and length of the outline of the sclerotized portion of the vagina were positive and statistically significant (Table 1). For the measures of female genital morphology other than the spermatheca, most of the variation was explained by PC1 (Table 2). The remaining PCs had eigenvalues that were less than one and were not included in our further analyses (Bryant and Yarnold, 2003
). Female PC1 was used as our measure of genital tract morphology for our analyses of paternity. The width and length of the outline of the spermatheca were significantly correlated with body size (Table 1). Because some of the females in our paternity experiments had missing values for measurements of the spermatheca, we use female body size in our analyses.
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Repeatability of female genital morphology
On average, families of full-sib females share half of their genes in common and were reared in a common environment (Falconer and Mackay, 1996
). Therefore, measures of morphology across full-sib females are predicted to be repeatable. Repeated-measures ANOVA indicated that variation in the pronotum width and the length of the outline of the bursa and sclerotized portion of the vagina were significantly greater between families than within families (Table 3). The width of the sclerotized vagina and the width and length of the outline of the spermatheca were nonrepeatable within families (Table 3).
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Estimation of male fertilization success
Females (n = 8) that copulated with two irradiated males (RR) produced a total of 127 brood masses that each contained a single undeveloped egg (z). Females (n = 8) that copulated with two normal males (NN) produced a total of 150 brood masses, of which a proportion of 0.93 ± 0.08 contained a viable egg (p). Estimates of fertilization success were calculated using the methodology outlined in Cook et al. (1997)
. The proportion of eggs sired by a normal male (PN) was calculated as PN = (x – z)/(p – z), where x is the proportion of eggs that hatch from a double mating (NR or RN), p is the proportion hatching after a NN mating, and z is the proportion hatching after a RR mating. In cases when the irradiation sequence was RN, P2 = PN, and when the sequence was reversed, P2 = 1 – PN. Values of P2 greater than 1 or less than 0 can occur when estimates of x are higher in RN experimental males than NN control males or lower in NR experimental males than in RR control males. P2 values were therefore corrected using the formula given by Cook et al. (1997) so that the data fell within the range 0–1. When the irradiation sequence was NR, the mean P2 was 0.40 ± 0.04 when 15 replicate pairs of males copulated with full-sib females and 0.31 ± 0.05 when 11 replicate pairs of males copulated with unrelated females. In contrast, when the irradiation sequence was RN, the mean P2 was 0.84 ± 0.02 when 16 replicate pairs of males copulated with full-sib females and 0.81 ± 0.03 when 16 replicate pairs of males copulated with unrelated females (Figure 3).
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Variance and repeatability of P2
The pronotum width and the length of the outline of the bursa and sclerotized vagina were repeatable within families of full-sib females (Table 3; see above). If the size of these female traits affect P2, related females should generate consistent P2 values due to the similarity of their body size, which is correlated with spermatheca size and some dimensions of their genital tract. Thus, we predict that the repeatability of P2 will be higher when males copulate with a group of related females and lower when males copulate with unrelated females.
The P2 values were arcsine square-root transformed, and the data were split into two groups; (1) the pairs of males that were mated to females that were full-sibs of one another (replicate male pairs, n = 31, total number of females N = 74) and (2) the pairs of males that were mated to females that were unrelated to one another (replicate male pairs, n = 27, total number of females N = 70). Within these groups, the components of variance in P2 due to the irradiation sequence, "male pair," and error were estimated (Table 4). The differences between the variance components across the two groups were tested according to Zar (1984: p. 122). The F ratios for irradiation sequence (F26,30 = 1.01), male pair (F26,30 = 1.86), and error (F26,30 = 1.20) variance were smaller than the critical value of 2.11. Therefore, repeatability in P2 did not differ between male pairs mated with full-sib females and male pairs mated with unrelated females (Table 4). Finally, we estimated the overall repeatability of P2 for replicate male pairs (n = 45) by pooling the data across the irradiation sequence, full-sib sisters, and unrelated females. This yielded a repeatability estimate of 0.49 ± 0.08 for P2. Thus, when two males copulated with the same set of females, fertilization success was highly repeatable with males consistently fertilizing a similar proportion of each female's eggs. However, between replicates there was considerable variation in the proportion of eggs sired by the second male to copulate.
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In the above analysis P2 values were arcsine square-root transformed, but despite the transformation the data remained significantly different from a normal distribution. However, more importantly for parametric analysis, the residuals from the analysis were normally distributed for all treatment combinations (NR: full-sib females, K = 0.12, n = 34, p = .20; NR: unrelated females, K = 0.11, n = 29, p = .20; RN: full-sib females, K = 0.08, n = 40, p = .20. RN: unrelated females, K = 0.11, n = 41, p = .20) (Grafen and Hails, 2002
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Female morphology and variation in P2
We used a GLM to determine whether specific measures of female genital morphology explained some of the variation in P2. The full model included the fixed factor irradiation sequence (NR or RN). As replicate male pairs were observed under a given irradiation sequence (NR, n = 25 or RN, n = 26), they were nested within the irradiation sequence. PC1 for female genital morphology, copulation number (which refers to the sequence in which the same pair of males copulated with the females in a replicate, and female body size, which was significantly correlated with the dimensions of the spermatheca) were entered as covariates (n = 163). The full model showed that there were negative trends between P2 and copulation sequence and female body size, whereas the trend between P2 and female PC1 was positive (Table 5). The full model was compared to reduced models that sequentially excluded the least significant variables. Log-likelihood ratio (LLR) tests were used to determine whether their inclusion increased the fit of the model to our data. Comparison of the models showed that copulation sequence, female PC1, and female body size failed to explain a significant amount of the variance in P2 (copulation sequence, LLR = –1.02, df = 1, p = .59; PC1, LLR = –5.08, df = 1, p = .23; female body size, LLR = –4.25, df = 1, p = .27), whereas replicate male pairs (LLR = –373.61, df = 50, p < .001) and the irradiation sequence (LLR = 352.39, df = 1, p < .0001) explained a significant amount of the variance in P2. Thus, there is no evidence that any of the female morphological traits that we measured biased sperm usage when females copulate with two males. Moreover, the copulation sequence did not explain a significant proportion of the variance in P2 as might be expected if one male in the pair became sperm depleted or if there were a progressive reduction in the viability of the sperm from irradiated males.
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Male morphology and variance in P2
For measures of the male genital sclerite 1, 2, 4, and 5, most of the variation was explained by PC1 (Table 2). The remaining PCs had eigenvalues that were less than 1 and were not included in our further analyses (Bryant and Yarnold, 2003
). As the male pair was a significant source of variance in P2 (
17%, Table 4), we used a GLM to determine whether male PC1 for sclerite 1, 2, 4, and 5 explained any of this variation.
The GLM included the fixed factor irradiation sequence and covariates; the PCs that characterized the variation in sclerite 4 and 5 of the first male and sclerite 1 and 2 of the second male. These PCs were selected as we have previously shown that they significantly influence P2 in this species (House and Simmons, 2003). The competing males' average fertilization success was used as the response variable. The full model showed that the parameter estimates were negative for PC1 of sclerite 4 of the first male and PC1 of sclerite 1 of the second male. In contrast, the parameter estimates were positive for PC1 of sclerite 5 of the first male and PC1 of sclerite 2 of the second male (Table 6). For each of the sclerites, a negative PC score corresponds with smaller sclerite dimensions, and a positive PC score corresponds with larger sclerite dimensions. We sequentially excluded terms from the model to determine whether their inclusion increased the fit of the model to the data. Comparison of the reduced models showed that PC1 of sclerite 5 and PC1 of sclerite 1 did not explain a significant amount of the variance in P2 (PC1 of sclerite 5, LLR = –0.32, df = 1, p = .78; PC1 of sclerite 1, LLR = –11.69, df = 1, p = .09), whereas PC1 of sclerite 4 (LLR = –15.65, df = 1, p = .05), PC1 of sclerite 2 (LLR = –17.27, df = 1, p = .04), and the irradiation sequence (LLR = –151.86, df = 1, p < .0001) explained a significant amount of the variance in P2. Thus, P2 was significantly decreased when the first male's fourth sclerite was small, and P2 was significantly increased when the second male's second sclerite was enlarged.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Our study does not provide support for the prediction that female genital morphology influences a male's fertilization success. Female body size, which was correlated with the size of the female's spermatheca, and the dimensions of the bursa and the sclerotized portion of the vagina were repeatable among families of full-sib females. However, reducing the morphological variation among females that mated to the same male pair did not increase the repeatability of a male's P2. Instead, the variation among males was important, with a given pair of males generating similar P2 values whether or not their series of partners were related or unrelated to each other.
The sterile male technique
In this study 63% of the variance in P2 was due to the irradiation sequence. The irradiation treatment decreased the fertilization capacity of irradiated sperm so that P2 was overestimated when the sequence was irradiated-normal (RN) and underestimated when the sequence was normal-irradiated (NR). This is often the case when the sterile technique is used to assign paternity (Simmons, 2001). The problem associated with the differential fertilization capacity was addressed by randomizing the irradiation sequence across the treatment combinations. Averaging across the NR and RN sequence the mean P2 was 0.62 ± 0.03, which is comparable to other studies in this species that have used the sterile male technique (0.58 ± 0.20, Tomkins and Simmons, 2000; 0.63 ± 0.03, House and Simmons, 2003), and the basic pattern of sperm mixing was revealed using molecular markers to assign paternity (Simmons et al., 2004).
Our study differs from those discussed above because the same pair of males copulated with multiple females (up to a maximum of six females). If the sperm of irradiated males declined in viability (Simmons, 2001), the number of copulations could have potentially increased the variance in P2 values when copulations occurred late in the sequence. However, there was no effect of copulation number on paternity, indicating that the sperm of irradiated males did not decline in fertilization capacity during the course of our experiment. Therefore, although we observed a large irradiation effect in our experiment, it is unlikely that this obscured any effects of female genital morphology on P2.
Repeatability of male effects on P2
Around 17% of the variance in P2 was explained by male effects, some of which was due to variation in male genital sclerites (Tables 4 and 6). The significant repeatability (R) of paternity suggests that there is a genetic basis for fertilization success because repeatability sets an upper limit to the heritability of a trait (Falconer and Mackay, 1996). Parker (1970) was the first to recognize that the female genital tract represents a unique environment within which sperm from different males compete to fertilize a female's ovum. Thus, from a quantitative genetic perspective, our finding is of interest as P2 was repeatable across a number of different female environments. The interaction between a male trait (i.e., fertilization success) and the environment (i.e., female genital tract) is an important consideration as trait expression is well known to vary in different environments (David et al., 2000; Jia and Greenfield, 1997; Jia et al., 2000; Rodriguez and Greenfield, 2003). Studies of Drosophila (Clark et al., 2000) and domestic fowl (Birkhead et al., 2004) have both found nontransitivity in paternity across different females, suggesting that incompatibilities between male and female genotypes may influence a male's fertilization success. In contrast, our results suggest that the best male genotypes, in terms of fertilization success, were the best in all the female environments that they encountered. Estimates of repeatability provide inflated estimates of genetic heritability of a trait because they include variance due to genes plus some environmental variance (Becker, 1984). Thus, a quantitative genetic study examining the additive genetic variance of fertilization success in homogenous (i.e., sisters) and heterogeneous (i.e., unrelated females) environments would greatly add to the findings of this study.
If the repeatability of P2 values is a product of consistent male influences, then by default, one or a number of the male traits that bias the outcome of fertilization must also have a genetic basis. Simmons and Kotiaho (2002) showed that testis size, ejaculate volume, and sperm length all have high heritabilities in O. taurus, which may contribute to the repeatability of P2. A causal relationship between sperm traits and fertilization success is yet to be confirmed in O. Taurus, but in other insect species there is evidence that male ejaculate traits can influence sperm competition (Hosken and Ward, 2001; Pitnick and Miller, 2000). In O. taurus, sperm competition is analogous to a raffle, thus the relative amounts of sperm contributed by competing males should be an important determinant of fertilization success (Tomkins and Simmons, 2000). In this study, P2 is highly variable between male pairs but repeatable within male pairs, as might be predicted if males transfer consistent ejaculate volumes and gain fertilizations in proportion to the number of sperm in their ejaculate.
In O. taurus, male genital traits are known to underlie their fertilization advantages or disadvantages. In a previous study, variation in the size and shape of the male genital sclerites explained a large proportion of the variation in paternity for this species (House and Simmons, 2003). Sclerites 4 and 5 influence the fertilization success of first males (i.e., served a defensive role), while sclerites 1 and 2 influenced the fertilization success of second males (i.e., served an offensive role). Consistent with our previous study, in the present study the second male was found to obtain a greater proportion of fertilizations when his second sclerite was large, whereas the fertilization success of the first male improved when his fourth sclerite was small. In contrast to our previous study, the effects of sclerites 5 and 1 were not significant although the trends were the same. This may reflect weaker selection on these genital traits or a lower statistical power in the present study. Interestingly, we have found significant but low levels of heritable variation in genital sclerite morphology (House and Simmons, in preparation), which will also contribute to the repeatability of P2 across male pairs.
P2 and female genital morphology
Theoretically, variation in the dimensions of the female bursa and spermatheca could influence the patterns of sperm storage and utilization (Walker, 1980). This would be most likely when the sperm-storage organs are of a fixed volume so that sperm from of a previous copulation are displaced from the spermatheca by the ejaculates of successive mates (Simmons, 2001). For example, in the flour beetle, T. castaneum, the spermatheca is full after two matings. After a third mating the numbers of sperm in the spermatheca remain constant but last male precedence increases, suggesting that sperm from previous copulations were displaced (Fedina and Lewis, 2004; Lewis and Jutkiewicz, 1998). The same is true for S. stercoraria, in which the storage capacity is reached after a single copulation (Parker and Simmons, 1991; Simmons, 2001).
In O. taurus, variation in the female genital traits that we have measured was not a significant source of variance in P2. During copulation the male ejaculates into the bursa. P2 tended to increase as the size of the vaginal sclerite and bursa increased, but variation in these structures explained less than 2% of the variance in P2. Sperm are transferred from the bursa to the spermatheca, the dimensions of which covary with body size. P2 decreased as female body size increases but again, variation in body size explained less than 2% of the overall variance. Thus, in contrast to T. castaneum and S. stercoraria, it is unlikely that the capacity of the sperm-storage organs of female O. taurus cause sperm to be displaced when the second male's sperm arrive. Rather, a male's share of fertilizations has been shown to be proportional to the relative number of copulations in which he has engaged (Hunt and Simmons, 2002) and the total number of males in competition (Simmons et al., 2004). The best explanation for this pattern is that the sperm-storage organs can accommodate sperm from many copulations, as occurs in a number of other insect species (Simmons, 1986; Siva-Jothy and Tsubaki, 1994).
Our failure to find a significant influence of female genital morphology on paternity does not mean that female influences are absent. If all females in the population prefer the same male traits, variance due to females will be indistinguishable from variance due to males (Pitnick and Brown, 2000). Thus, the effect of male genital morphology on paternity observed in this study could arise because of female preferences for males with particular genital configurations or because males use their genital sclerites in sperm competition with other males.
Postcopulatory cryptic female choice is difficult to distinguish from sperm competition (Simmons, 2001). However, whatever the mechanism underlying paternity bias in O. taurus, females that passively or actively select competitively superior sperm could potentially gain genetic benefits for their offspring in two ways. First, if P2 were a heritable trait, multiply mating females could improve the likelihood that their sons would produce competitively superior sperm (the so-called "sexy-sperm" process; Curtsinger, 1991; Harvey and May, 1989; Keller and Reeve, 1995; Sivinski, 1984). Alternatively, if a male's fertilization success were correlated with heritable variation in viability, females could improve the sperm competitiveness and overall fitness of their offspring (the so-called "good-sperm" process; Madsen et al., 1992; Parker, 1992; Yasui, 1997). In O. taurus, precopulatory courtship rate (Kotiaho et al., 2001) and sperm competition traits (Simmons and Kotiaho, 2002) are heritable and genetically correlated with condition. Thus, females that bias paternity in favor of males that signal their condition via courtship rate or the constituents of their seminal fluids could improve the genetic condition of their offspring.
In conclusion, in O. taurus, the dimensions of the female genital tract do not appear to influence a male's fertilization success. However, male effects were consistent as demonstrated by the high repeatability of P2 estimates. This may be due to intrinsic differences between males in traits that contribute to sperm competition success. In addition, female genital traits such as the sensory (Cordoba-Aguilar, 1999) or muscular (Neubaum and Wolfner, 1999) systems may still be found to play a role in influencing paternity.
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ACKNOWLEDGEMENTS |
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We thank Ernie Steiner for irradiating the beetles, Rob Brooks and Michael Jennions for statistical advice, and John Hunt for constructive criticisms of the manuscript. This research was funded by an Australian Postgraduate Award and a Foundation Bursary from the Australian Federation of University Women (WA) to C.M.H. and grants from the Australian Research Council to L.W.S.
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