The Cost of Keeping Eggs Fresh: Quantitative Genetic Variation in Females that Mate Late Relative to Sexual Maturation

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1 vol. 169, no. 3 the american naturalist march 2007 The Cost of Keeping Eggs Fresh: Quantitative Genetic Variation in Females that Mate Late Relative to Sexual Maturation Patricia J. Moore, 1,* W. Edwin Harris, 2, and Allen J. Moore 1, 1. School of Biosciences, University of Exeter, Cornwall Campus, Penryn, Cornwall TR10 9EZ, United Kingdom; 2. Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom Submitted June 12, 2006; Accepted September 26, 2006; Electronically published January 22, 2007 abstract: In many species, females abandon mate choice to ensure that eggs are fertilized before they are lost. But why do females not just maintain oocytes longer if there is a benefit to mate choice? We conducted a quantitative genetic study in the cockroach Nauphoeta cinerea to test whether genetic constraints prevent the evolution of oocyte maintenance or selection against oocyte loss is weak when females mate late relative to sexual maturity. We found standing genetic variation within the population and no evidence for genetic constraints. Levels of genetic variation are of the magnitude found for life-history traits in general, suggesting that this trait has been exposed to selection. We unexpectedly found two categories of females: those that delay reproduction and those that reproduce at a normal time when mating late, which could indicate alternative strategies. However, frequency-dependent selection does not maintain this variation as females that delay always reproduce less well. Given these findings, we suggest that there may be advantages to egg degradation. The evolution of maintenance of fertilizable oocytes over time would then be constrained by the need to maintain the mechanism by which females control the distribution of resources between current and future reproductive events. Keywords: delayed reproduction, genetic constraints, life history, Nauphoeta cinerea, oocyte maintenance, threshold character. A core assumption of life-history theory is that reproduction is costly (Roff 1992; Stearns 1992). Organisms face * Corresponding author; p.j.moore@exeter.ac.uk. ed.harris@manchester.ac.uk. a.j.moore@exeter.ac.uk. Am. Nat Vol. 169, pp by The University of Chicago /2007/ $ All rights reserved. trade-offs in which different physiological processes compete for limited resources (Gustafsson et al. 1994), and the cost of reproduction is partly determined by the trade-off in resource allocation between gametes and somatic cells. Some resource allocation decisions are developmental, with allocation to germ or soma occurring before an individual reaches adulthood (Jervis et al. 2005). There also can be phenotypic plasticity in adult allocation, and individuals can respond to changes in environmental conditions (Kaitala et al. 1997). Females can delay reproduction and oocyte maturation under suboptimal conditions and wait for more favorable conditions (e.g., Byers et al. 2005). Nonetheless, given that producing oocytes comes at the expense of other physiological functions, once oocytes have been produced, females need to use them rather than risk losing that investment. In addition to decisions regarding allocation of resources, reproduction requires coordination between reproductive physiology and social conditions. Both highquality oocytes and the sperm to fertilize them are required for reproduction. Thus, females need to coordinate sexual maturation and investment in oocytes with availability of acceptable mates (Tsitrone et al. 2003a, 2003b). In species where females mate nonrandomly, females may be required to delay mating if high-quality mates are not encountered when females reach sexual maturity (Andersson 1994). Exercising female mate choice poses a potential cost, however, because eggs ready for fertilization might deteriorate while the females are searching for acceptable males (Jennions and Petrie 1997). If there is a cost to a mistiming of sexual maturation and mating and timing of maturation is canalized, females are predicted to respond to the risk of lost fertility by reducing their choosiness (Moore and Moore 2001). In unmated females with mature reproductive systems (or mature eggs), behavioral responses to avoid a delay in availability of sperm are observed. Thus, females have been shown to become less choosy when gravid in midwife toads (Lea et al. 2000) and with age in crickets, guppies, and cockroaches (Gray 1999; Kodric-

2 312 The American Naturalist Brown and Nicoletto 2001; Moore and Moore 2001). When faced with the loss of expensive oocytes, any male may be better than no male at all. Oocytes, once produced, degenerate with time (Fissore et al. 2002). Thus, females are under time pressure to fertilize oocytes once they are mature. But why must oocytes be lost over time? Why are females unable to maintain mature oocytes? Mature sperm can be stored for long periods of time in both the male and female reproductive tract (Birkhead and Møller 1993; Simmons 2001), so it is reasonable to suppose that similar storage functions could evolve for oocytes. If mate choice is adaptive, why abandon this behavioral strategy rather than evolving a mechanism to preserve eggs longer? We investigated the question as to why mature eggs are not maintained in the cockroach Nauphoeta cinerea. This hemimetabolous insect is ovoviviparous; although no nutrients are passed to developing offspring, females brood eggs internally and give live birth (Roth and Willis 1954). Thus, there is a relatively large maternal investment in N. cinerea. In contrast, males make only genetic contributions to their offspring (Moore and Moore 1988; Moore 1994). This asymmetry in parental investment is expected to result in sexual selection (Trivers 1972), and adaptive female mate choice has been documented in N. cinerea (reviewed in Moore 1990; Moore and Pizzari 2005). However, females become less choosy as fertility degrades (Moore and Moore 2001). In insects, reproductive behavior is controlled through interactions between the brain, the corpora allata, and the ovaries and is mediated by juvenile hormone (Roth 1964a, 1964b; Buschor et al. 1984). In N. cinerea there is a period of sexual maturation following eclosion to the adult stage. Newly emerged and young adult females are not receptive to male courtship and will not mate, and only low levels of juvenile hormone are released leading to gradual oocyte maturation and yolk deposition. Once sexually mature, females respond to male courtship and will copulate. Reproduction in N. cinerea consists of cycles of oocyte maturation, pregnancy, and parturition, a cycle that normally lasts about 45 days (Moore 1994). Females that mate past the time when they first become sexually receptive have reduced fertility (Moore and Moore 2001). Both the number of clutches and the size of clutches are reduced. The reduction in the number of offspring produced when females mate late relative to sexual maturation is due to a reduction in the number of oocytes fertilized and not to embryo death in utero (Moore and Harris 2003). We have shown that the loss of fertility as females delay mating is correlated to a loss of oocytes via apoptosis (Moore and Sharma 2005). However, apoptosis not only removes mature follicles ready for fertilization but also removes immature oocytes that will be utilized in future reproductive cycles. Thus, the time at which a female mates, relative to sexual maturation, has long-term consequences for lifetime reproductive success. During the course of our experiments documenting the cost of mating late relative to sexual maturation (Moore and Moore 2001), we observed substantial variation in the reproductive potential of females that mate late. Some females are able to cope well with a delay in mating relative to sexual maturity, while others perform very poorly. Given this variation in the ability of females to utilize oocytes over time, we investigated the potential for oocytes to be maintained over time. Two main explanations for a lack of evolution are genetic constraints and a lack of selection. A genetic constraint on evolution will arise even in the presence of selection if oocytes are not maintained, because there is no additive genetic variance underlying the observed differences in fertility following sexual maturation (no evolvability). There also may be genetic constraints because of trade-offs arising from pleiotropy or linkage disequilibrium among life-history traits that constrain the evolutionary response to maintaining oocytes (Merilä and Sheldon 1999). This would be reflected in a pattern of strong genetic correlations or covariances (Arnold 1992). Alternatively, genetic variation may be hidden to selection if females never experience the environment where loss of oocytes is expressed, and loss of oocytes is genetically robust (i.e., genetic variation has relatively small phenotypic effects) under typical environmental conditions (de Visser et al. 2003; Hermisson and Wagner 2004). This would be indicated by higher than normal genetic variation under the unusual environment (Hoffmann and Merilä 1999; de Visser et al. 2003; Hermisson and Wagner 2004). We therefore sought to examine how these alternatives might affect the N. cinerea system by undertaking a quantitative genetic study designed to examine the levels of genetic variation in fertility-related traits in females that mate late relative to sexual maturation. In the course of the experiment, however, we uncovered unexpected but obvious differences among females. While the design of the experiment, mating females at 18 days after adult eclosion, is the same as in our other studies in this area (Moore and Moore 2001; Moore and Harris 2003; Moore and Sharma 2005), the large sample sizes required by the half-sib breeding design allowed us to detect a previously undocumented pattern in female response to this treatment. It became obvious that there was a bimodal distribution of the time to parturition of the first clutch of offspring, suggesting two categories of females. We have never seen this pattern in females that mate at sexual maturity, where time to parturition of the first clutch is unimodal and normally distributed. This presented a third possibility: for many genetically determined dichotomous (threshold) traits, a

3 The Cost of Keeping Eggs Fresh 313 trade-off of fitness associated with different phenotypes maintains variation (Roff 1996; Hazel et al. 2004). Thus, following our analysis of genetic variation, we looked for differences in components of fitness between females with early or delayed parturition of the first clutch as well as the genetic basis for the expression of this threshold trait. Methods Animal Husbandry Animals used to generate our quantitative genetic breeding design were collected from eight outbred, mixed-sex, massrearing colonies housed in standard rearing conditions with a 12L : 12D photoperiod at 27 C and ad lib. access to rodent chow and water. These colonies contained several thousand individuals, and there was extensive enforced gene flow among the colonies. The colonies have been in culture for more than 50 years. This represents approximately 150 generations, so the colonies were likely to be at genetic equilibrium for our experiment. These cockroaches breed continuously throughout the year and have no period of reproductive diapause. Late-instar nymphs were separated by sex and housed under standard rearing conditions in 30 # 18 # 12-cm plastic boxes. Newly emerged virgin adults were collected daily as tenerals from the single-sex colonies for use as sires and dams in a halfsib breeding design. Sexual maturity (and therefore receptivity to mating) at 27 C is not reached until 4 to 7 days of age for females (Roth 1964a). Males begin to be able to produce spermataphores when as young as 2 days of age and are fully competent at 6 7 days of age (Roth 1964a) which parallels the development of their attractiveness to females and pheromone development (Moore et al. 1995). Breeding Design We used a standard paternal half-sib breeding design (dams nested within sires; Lynch and Walsh 1998) to estimate additive and nonadditive genetic plus common environment contributions to variation in reproduction in females where mating is delayed relative to sexual maturation. Fifty virgin adult males (9 12 days after adult eclosion) were mated to four unrelated virgin adult females (6 9 days after adult eclosion) sequentially over 3 days. Immediately after mating, the females were placed in individual 11 # 11 # 3-cm plastic boxes, provided with ad lib. rodent chow and water, and housed in incubators in standard rearing conditions. Females were checked daily for parturition of their first clutch. Following birth of the clutch, offspring were counted. To control for density effects, 20 offspring from each clutch were placed into a 17 # 12 # 5-cm plastic box, provided with ad lib. rodent chow and water, and housed under standard rearing conditions until adults began to emerge. Clutches were checked daily for newly emerged adults. Four adult females were collected from each clutch for analysis of fecundity when forced to mate late relative to development of sexual maturity. At 18 days after adult eclosion, each adult daughter was mated to a 10-day-old virgin male, isolated from the mass colonies as above. Following mating, we placed these females into 11 # 11 # 3-cm plastic boxes with ad lib. food and water. All females were housed in individual boxes in a walk-in incubator at 27 C. We checked females daily for the presence of newborn offspring. For each clutch of offspring produced, we recorded the date and number of offspring born. We removed all of the offspring, and the female was returned to her individual box until birth of the next clutch, when this procedure was repeated. Observations continued until the death of the female. Life-History Traits We analyzed two life-history traits, longevity and fecundity, in the daughters produced from our breeding design. Because we measured all of the clutches produced by females, we also had information on the embryonic development time of the offspring of these daughters, providing a measure of offspring quality for different females. We did not measure female body size as a variable because in previous experiments we never found an influence of body size on fecundity (R. F. Preziosi, P. J. Moore, and A. J. Moore, unpublished data). Fecundity. We examined the fecundity of females when mating is delayed relative to sexual maturation by analyzing the number of offspring produced in each of the first three clutches as a series of separate traits (first clutch, second clutch, etc.). We also scored the total numbers of clutches a female produced over her lifetime and the total number of offspring produced in all clutches combined. Life span. We measured the total life span of females that delayed mating relative to sexual maturation from adult eclosion to death. Offspring development. The development time of the first clutch was measured as the number of days from mating to parturition of the first clutch. The development time of subsequent clutches was measured as the number of days between parturition of each clutch. Statistical Analyses Data from all characters we measured were normally distributed. It was not possible to transform time to first parturition to normality (see Results ). While a few fe-

4 314 The American Naturalist males in the study produced as many as nine clutches over their lifetime, clutch size is reported only for clutches 1 3 (for genetic analyses) and 1 6 (for phenotypic analyses of fitness differences) because the number of females producing clutches declined rapidly, precluding meaningful analyses. The total number of offspring produced, however, includes those from all clutches. Analysis of genetic variation. To analyze genetic variation in fecundity-related traits, we fitted a standard linear mixed-effects ANOVA using restricted maximum likelihood (REML) with dam nested in sire to estimate sire and dam variance and covariance components using the varcomp function in S-PLUS, version 6.2 (Pinheiro and Bates 2000). Statistical significance of additive and dam variances was determined by F-tests with Satterthwaitecorrected degrees of freedom (Lynch and Walsh 1998). We calculated narrow-sense and broad-sense heritabilities from the sire and dam variances estimated by REML. We used weighted family means to estimate the standard error of heritability estimates (Lynch and Walsh 1998). We calculated coefficients of variation for the traits we measured to provide standardized estimates of phenotypic, additive genetic, and residual variation to facilitate direct comparison of traits with different scales of measure (Houle 1992). We estimated additive genetic correlations among all characters using covariances from a REML analysis. Standard error for genetic correlations was calculated in S-PLUS using the jackknife method applied to pseudovalues (Potvin and Roff 1993; Roff and Preziosi 1994; Roff 2006). Analysis of fitness trade-offs. Initial examination of the distribution of development times of the first clutch of offspring led us to divide the females into two populations based on the number of days between mating and parturition of the first clutch (see Results ; fig. 1). In Nauphoeta cinerea, over 90% of females mated concurrently to development of sexual maturity produce their first clutch within 34 to 55 days after mating (Roth 1964b; Moore 1994; P. Moore, unpublished data). This includes a 7-day period of oocyte maturation and a day period of embryonic development. After the first clutch is produced, subsequent clutches are born about 50 days apart (Roth 1964b). Therefore, in this study, normal reproductive timing was defined as between 40 and 60 days postmating, and delayed reproductive timing was defined as parturition of the first clutch 60 days or more postmating. We used ANOVA or t-tests (Sokal and Rolf 1995) to examine differences in both developmental timing and fecundity traits between categories of females (normal or delayed developmental timing). The strength of pairwise relationships between variables was measured using Pearson product-moment correlations. The hypothesis that Figure 1: Frequency distribution of development of first clutch of offspring for females that mate late relative to sexual maturation. Females fell into one of two categories, based on the development time of their first clutch of offspring. One group of females produced a first clutch with normal timing, within 60 days of mating (hatched bars). The other group of females produced a first clutch with delayed timing, taking more than 60 days to parturition (solid bars). there was no difference between the survival curves for the two groups of females was tested using the log rank test. All means are reported with standard errors. Analyses were performed with JMP Professional, version 5.0.1a. Heritability of the threshold trait. In order to analyze whether there was an inherited component to the membership in the group of females that had normal timing of parturition of the first clutch of offspring or the group that had delayed timing, we analyzed the heritability of liability for delayed production of offspring. This method assumes that there is an underlying continuous trait, influenced by multiple additive genetic effects, and that, although there is a normal distribution of the underlying character, individuals that are above or below a threshold express a different trait. Each female was categorized as either normal (0) or delayed (1), based on the developmental timing of her first clutch of offspring (see below). We then used the method of Reich et al. (1972) to measure the heritability and associated standard error for the threshold of producing a delayed clutch, assuming that all affected (delayed) individuals were probands and applying the formula from Hill and Smith (Elston et al. 1977). Calculations of heritability on the underlying scale (Roff 1997), using REML ANOVA analysis on the half-sib data, with normal and delayed scored as 0 or 1 and the mean proportion of delayed reproduction (p) associated with sire classes, are also presented.

5 The Cost of Keeping Eggs Fresh 315 Table 1: ANOVA tables of nested half-sib/full-sib analyses for each trait Trait/source df MS F P Total no. offspring: Sire 41 5, Dam within sire 102 3, Error 322 2, No. clutches: Sire Dam within sire Error No. offspring, first clutch: Sire Dam within sire !.001 Error No. offspring, second clutch: Sire Dam within sire Error No. offspring, third clutch: Sire Dam within sire Error Life span: Sire 41 16, Dam within sire , Error ,432.8 Note: Significance was estimated using Satterthwaite-corrected degrees of freedom (Lynch and Walsh 1998). Results Genetic Variation We examined the patterns of genetic variation for fecundity traits in females that were prevented from mating until 18 days after adult eclosion. There was significant additive genetic (sire) variance for total number of offspring, number of clutches, number of offspring in the second clutch, and number of offspring in the third clutch (table 1). There were significant dam variances for total number of offspring, number of offspring in the first clutch, and number of offspring in the third clutch (table 1). The coefficients of additive genetic variation for all but life span suggest that there is scope for an evolutionary response to selection in fecundity-related traits (table 2). The narrow-sense heritability for the number of offspring in the first clutch was low, whereas other clutch sizes showed higher heritabilities (table 2). This reflected a higher phenotypic variance rather than lower additive genetic variance in the first clutch. The coefficients of additive genetic variance were of similar magnitude for the number of offspring in all three clutches. There was no measurable heritability for life span, Table 2: Description of phenotypic and genetic variation in life-history traits of females that mate late relative to sexual maturation Trait Mean V P V A V G h 2 (SE) H 2 (SE) CV A CV R Total no. offspring , (.15).20 (.14) No. clutches over a lifetime (.15).20 (.14) No. offspring, first clutch (.15).25 (.12) No. offspring, second clutch (.12).09 (.13) No. offspring, third clutch (.16).25 (.16) Life span (days) , , (.12).11 (.13) Note: All traits were analyzed using 42 sires, 145 dams, and 502 offspring. Additive genetic variance (V A ) was calculated as 4 # sire variance; genetic variance (V G ) was calculated as 2 # sire dam variance (Lynch and Walsh 1998). Significance of additive and dam variances is provided in table 1. V P is phenotypic variance; h 2 is the narrow-sense heritability; H 2 is the broad-sense heritability; CV A is the additive genetic coefficient of variation; CV R is the residual coefficient of variation (both CV A and CV R were calculated according to Houle 1992).

6 316 The American Naturalist Table 3: Phenotypic and additive genetic correlations for life-history traits of females that mate late relative to sexual maturation Total no. offspring No. clutches First clutch No. offspring Second clutch Third clutch Normal or delayed reproduction Life span Total no. offspring.88 (!.001).54 (!.001).60 (!.001).69 (!.001).29 (!.001).28 (!.001) No. clutches over a lifetime.93 (.02).35 (!.001).30 (!.001).31 (!.001).17 (!.001).51 (!.001) No. offspring, first clutch.16 (.53).17 (.37).29 (!.001).16 (!.001).05 (.337).07 (.151) No. offspring, second clutch.28 (.84).43 (.46).31 (.22).55 (!.001).24 (!.001).07 (.195) No. offspring, third clutch.93 (.03).67 (1.07).14 (.10).11 (.75).40 (!.001).09 (.105) Normal or delayed reproduction 1.26 (.10).86 (.13).18 (.79).29 (.19) 1.01 (.38).09 (.090) Note: Phenotypic correlations are presented on the upper diagonal, with significance in parentheses. Genetic correlations are presented on the lower diagonal, with SE in parentheses. Each r G value is based on restricted maximum likelihood analysis, with SE obtained by jackknife. No genetic correlations could be calculated between fecundity traits and life span, given the lack of genetic variance for life span. and the coefficient of additive genetic variance for life span was relatively low (table 1); we therefore did not consider the genetic aspects of life span further as a potential constraint on evolution of life histories in females that mate relatively late. Broad-sense and narrow-sense heritabilities were similar for all traits (table 2), indicating that there were few common environment or nonadditive effects. There were no obvious trade-offs among fitness components. Phenotypic correlations show expected relationships between fecundity-related traits (table 3). There were significant positive phenotypic correlations between all of these traits except life span and individual clutch sizes, which were not significantly different from zero. Very high correlations reflected the ovoviviparous reproduction of Nauphoeta cinerea females and the necessary relationship between number of clutches and overall fecundity. The phenotypic correlations between total number of offspring and number of offspring in each clutch also were moderately high. However, there was a weaker (although still strong) relationship between the numbers of offspring in each clutch. There were also significant phenotypic correlations between numbers of offspring in individual clutches and total number of clutches produced over a life span. There was no evidence of any genetic constraints due to high negative genetic correlations among fecundity traits (table 3). Two pairs of traits were virtually genetically identical: number of clutches over a lifetime and total number of offspring, and number of offspring in the third clutch and total number of offspring. There was no overwhelming or obvious pattern of covariation. There was a mix of positive and negative genetic correlations, although the positive correlations were all higher than the negative correlations. While there was a positive genetic correlation between the number of offspring in the first and second clutch, most genetic correlations with the size of the first clutch were negative and small. Trade-Offs among Alternative Responses to Mating Late Relative to Sexual Maturation Mating late relative to development of sexual maturity had an obvious effect on schedule of reproduction for some females, with a bimodal distribution for timing of parturition of their first clutch of offspring (fig. 1). Among the daughters mated at 18 days after adult eclosion, 50.7% produced a first clutch within 60 days from mating and thus had normal reproductive timing, based on our previous experience mating females at 7 days after adult eclosion. The mean time to parturition of the first clutch within this category was days. Parturition occurred between 33 and 60 days after mating. Among the offspring mated at 18 days after adult eclosion, 42.1% had a delayed first reproductive event, with the parturition of the first clutch more than 60 days after mating. The mean development time of this category was days, with the range between 61 and 154 days. This effect was seen only in the first clutch, as development times of offspring born after the first reproductive event were not significantly different between females with normal timing of the first reproductive event and females with delayed timing of the first reproductive event (table 4). The category into which a female fell had significant fitness consequences, but there were no trade-offs between the groups. Females that produced a first clutch with normal timing had more offspring over their lifetime than females that had a delayed first parturition (fig. 2A). Females with normal first parturition had an average of

7 The Cost of Keeping Eggs Fresh 317 Table 4: Mean development time ( 1 SE) of offspring in females with normal or delayed production of the first clutch with test of differences between means Clutch no., timing of first clutch Mean days to parturition Sample size t statistic from t-test Probability Normal Delayed Normal Delayed Normal Delayed Normal Delayed Normal Delayed Note: Development time of clutches is measured as the number of days between parturition of the clutches offspring over a lifetime, whereas females with delayed first parturition had an average of offspring ( F p 36.01, df p 1, 405, P!.0001). There was no difference in the number of offspring in the first clutch between females with normal first parturition and those with delayed first parturition (table 5). Instead, differences in lifetime reproductive success reflect decreased numbers of offspring in subsequent clutches (fig. 2B; table 5) and number of clutches produced. The pattern of fertility is similar in the two categories, with a rise in offspring numbers between the first and second clutch and then a reduction in clutch size over time. However, the increase in clutch size between the first and second clutch is smaller in the females that had a delayed first clutch compared with females with normal timing of the first clutch, and clutch size drops off more rapidly between the second and third clutch. Females with normal timing of first parturition also had more clutches over their lifetime (F p 11.89, df p 1, 405, P p.001). The mean number of clutches for females with normal reproductive timing of the first clutch was , and for females with delayed reproductive timing, the mean number of clutches was In females that had normal timing of first parturition, there was no significant correlation between the number of offspring in the first clutch and the number of clutches produced over a lifetime ( r p 0.03, P p.657, N p 236). However, in females that had a delayed first parturition, there was a significant positive correlation between the number of offspring in the first clutch and the number of clutches produced over a lifetime ( r p 0.28, P p.0001, N p 196). There was no trade-off between decreased reproduction and survival between the two categories, as decreased reproductive output did not result in longer life span in females that delayed production of their first clutch (fig. 3). There was no significant difference in the survival curves between females that had normal timing of first parturition and those that had delayed first parturition 2 ( x p 1.529, P p.216). There was no difference in survivorship between the two classes of females, even when we censored the data at 300 or 400 days (data not shown). Females that had a normal first parturition lived an average of days, and females that had a delayed first par- turition lived an average of days. Given the length of time between clutches, this difference would not translate into more offspring. Moreover, the pattern of correlation between life span and clutch size was the same for both categories. We found negative correlations between the average numbers of offspring per clutch and life span for both females with normal first parturition ( r p 0.28, P!.001, N p 226) and those with delayed first parturition ( r p 0.20, P p.006, N p 192). Is Membership Random in the Two Categories of Reproductive Timing? Given the fitness costs of delaying the production of the first clutch, why do all females not produce offspring normally? As the distribution of timing of first parturition suggests two discrete categories of females, we analyzed our timing of first parturition as a threshold trait. We found a clear genetic influence on category membership. The heritability for the threshold for delayed production

8 318 The American Naturalist Discussion Figure 2: Mean number of offspring ( SE) born to females that produce a first clutch within normal timing, and females that delay production of a first clutch. A, Females that produced a first clutch with normal developmental time (dashed line) accumulated a greater mean number of offspring over their lifetime compared with females that had delayed parturition of their first clutch (solid line). B, While there was no difference in numbers of offspring in the first clutch between females with normal parturition (hatched bars) and delayed parturition (solid bars) of the first clutch, there were significantly fewer offspring in the subsequent clutches of offspring in the females that had delayed parturition. Bars represent standard errors. of a first clutch within the first reproductive cycle was 0.80 ( SE p 0.22). Heritability on the underlying scale was 0.72 (0.16). Genetic correlations between category of reproduction and overall reproduction supports the phenotypic analyses (table 3). Total number of offspring and number of clutches have a high (approximately 1) negative genetic correlation with category (normal, scored 0, or delayed, scored 1). However, the genetic correlations between reproduction in first or second clutches and category are much smaller. Thus, the genes that influence membership in a category are the same or linked with total fecundity and number of clutches, as they must be, but less strongly linked to genes influencing fecundity in individual clutches. There is no evidence that females that delay mating are genetically predisposed to producing larger clutches; in fact, the opposite appears to be true. Females are expected to have evolved adaptations that allow them to minimize the costs that might arise when, for instance, they have fertilizable eggs that will degenerate over time but no sperm with which to fertilize them. Evolution requires both selection and the presence of heritable variation in the selected trait (Lynch and Walsh 1998). In this study, we examined the evolutionary potential of maintaining fertility after sexual maturation by examining the pattern of genetic variation underlying fecundity of females that mate late relative to sexual maturation in Nauphoeta cinerea. We found that fecundity has the potential to evolve because the necessary standing genetic variation exists within the population. There were no genetic trade-offs among the life-history traits we measured. However, there was also no evidence that fecundity following mating late relative to sexual maturation has been protected from selection, because the pattern of additive genetic variation is similar to other life-history traits (Mousseau and Roff 1987; Merilä and Sheldon 1999). Unexpectedly, we found that females had one of two separate responses to mating late relative to sexual reproduction ability, and membership in these categories had a strong genetic component. Again, however, there were no tradeoffs in fitness between these categories, providing necessary frequency-dependent advantages to maintain a threshold trait. Thus, we need to ask why females have not evolved the ability to maintain fertility over time. Genetic Variation in the Ability to Cope with Mating Late Relative to Sexual Maturation One potential explanation to the question of why the ability to maintain high-quality oocytes following maturation has not evolved are genetic constraints arising from the lack of genetic variation in the control of the onset of apoptosis following maturation or constraints imposed by genetic correlations between fitness traits. Our results allow us to reject this hypothesis. There was a significant contribution of additive genetic variance to both the number of clutches produced and the number of total offspring. Indeed, the genetic correlation between these two traits indicates that, genetically, they are essentially the same trait. In addition, negative genetic correlations between size of the first clutch and other fitness traits were of small size. Genetic correlations between type of reproduction (delayed or normal) and individual clutch size suggest no trade-off between clutch size and delaying reproduction. Thus, there is additive genetic variance in fertility and no obvious genetic trade-offs when females are mated at 18 days after adult eclosion. Thus, fertility following mating late has the genetic potential to evolve.

9 The Cost of Keeping Eggs Fresh 319 Table 5: Mean number of offspring ( 1 SE) born to females with normal or delayed production of the first clutch with test of differences between means Clutch no., timing of first clutch Mean no. offspring Sample size t statistic from t-test Probability Normal Delayed !.001 Normal Delayed !.001 Normal Delayed !.001 Normal Delayed !.001 Normal Delayed !.002 Normal Delayed Our study measures only a small number of life-history traits, and therefore it is possible that there are genetic constraints we did not measure. For instance, we did not measure body size in our offspring because body size has never been a significant variable in our previous studies. However, the patterns we see are similar to those in other studies of the genetics of life history (Mousseau and Roff 1987; Merilä and Sheldon 1999, 2000). Empirically, there are fewer genetic constraints on life-history evolution than might be predicted by theory (Sheldon et al. 2003; Charmantier et al. 2006). Our study fits well within this general finding. Given the severe reduction in lifetime reproductive success in females that coped poorly with mating late, we might expect strong selection to eliminate variation in response to postponed mating from the population. One possibility is that females cannot maintain mature oocytes because although the genetic variation available for adaptation is available in the population, it is protected from selection (Merilä and Sheldon 1999). Under rare unfavorable conditions, selection on alleles resulting in decreased fitness will be much less effective (Hoffmann and Merilä 1999). This could occur for N. cinerea because the variation in fecundity that we measured would be expressed only in rare social environments where males are infrequently encountered, because the behavioral adaptations lead to females accepting less preferable mates in order to acquire sperm. We cannot measure selection directly, but we can look for signatures of selection in our genetic analysis. If it were the case that fecundity following mating late has not experienced selection, we would expect to see higher levels of genetic variation as would be anticipated for protected traits. Under the hypothesis that selection can only occur under rare conditions, fecundity variation should be genetically robust, and we should see increased genetic variation under the novel environment (de Visser Figure 3: Survival curves for females that produced a first clutch with normal reproductive timing (gray line, N p 225) and females that had delayed parturition of their first clutch (black line, N p 170). There was no significant difference between the two curves.

10 320 The American Naturalist et al. 2003; Hermisson and Wagner 2004). The pattern of genetic variation we see, however, allows us to reject the lack of selection hypothesis. The levels and pattern of genetic variation we see are similar to those seen for fecundity of females mated when sexually mature (e.g., lifetime 2 reproduction h p 0.19, number of clutches p 0.20, average clutch size p 0.11; Moore 1994) as well as of the level expected for life-history traits in general (Mousseau and Roff 1997; Merilä and Sheldon 1999). The relatively low (but nonzero) levels of additive genetic variation we found in fecundity suggest that the fitness cost of mating late relative to sexual maturity has resulted in selection. Fitness Performance following Mating Late Relative to Sexual Maturation The lifetime reproductive success of females that mate late relative to sexual maturation fell into two categories. One category of females produced a first clutch within a normal reproductive cycle and had a reproductive output similar to females that mate soon after reaching sexual maturity. The second category, however, had a delay in parturition of their first clutch. A bimodal response might indicate one group of females entering reproductive diapause. However, N. cinerea reproduce continually throughout the year and no diapause has ever been observed by us or reported in this species. The lifetime reproductive success of females in this second category was reduced both through missing a reproductive cycle and by having fewer offspring in the subsequent cycles. Thus, there was significant phenotypic variation among females in their ability to cope with the mistiming of mating relative to sexual maturation. There was significant heritable variation in the category that was expressed by a female. However, there is no evidence of fitness trade-offs maintaining alternative conditional strategies. The females who reproduced normally reproduced best, while females with delayed reproduction had severely compromised reproductive success. Females that start out with poor fecundity are unable to recover in successive reproductive cycles. Additionally, females that delayed reproduction did not accrue other types of benefits, such as longer life span. Thus, there is no evidence of frequency-dependent advantages that can lead to adaptive categorical traits (Roff 1996; Hazel et al. 2004). Might There Be a Cost of Maintaining Mature Oocytes? We have not discovered why oocytes are not maintained, but we can suggest a hypothesis. It may be that females have not evolved the ability to store mature oocytes simply because it is better that they do not. Females that have mature oocytes ready to fertilize under conditions where opportunities for reproduction are limited, such as when sperm are not available, face two choices. They could either maintain their oocytes while they wait for a better reproductive opportunity or resorb the nutrients invested in the current oocytes, recycling them into future reproduction. Interestingly, a similar bimodal response was seen in the seed beetle Callosobruchus maculatus when females were deprived of seeds on which to lay their eggs. One group of females dumped their eggs early while the other group retained their eggs until death (Messina and Slade 1999). Variation in oviposition rate in the absence of seeds is in part genetically based (Messina and Fry 2003). Thus, this may be a general response to a poor reproductive environment. The trade-off between current and future reproduction will depend on the ability of an individual to redirect resources invested in this reproductive event into future reproductive efforts (Tsitrone et al. 2003a, 2003b; Jervis et al. 2005). Animals have evolved a mechanism, oosorption, for recycling nutrients that are not oviposited, and we propose that this is a means by which resources are reallocated into survival when opportunities for reproduction are limited (Bell and Bohm 1975; Papaj 2000). Consider the category of females in our study that performed poorly and had reduced lifetime reproductive success when they mated late relative to sexual maturation. This might have reflected a more efficient or rapid switch to oosorption, which was highly costly in terms of lifetime fecundity. However, our experiment was conducted on females under good nutritional conditions. Perhaps females with loci leading to more efficient oosorption would be better able to cope with poor nutritional conditions or changes in food availability. Thus, the hidden genetic variation that was detrimental to fitness under these conditions would provide a benefit in another environmental context. We are exploring this hypothesis. Conclusion Given the ability of sperm to be stored in the male and female reproductive tracts, often for extreme lengths of time (Birkhead and Møller 1993; Simmons 2001), it is curious that oocytes seem so perishable. Even in species where females need to delay offspring development relative to mating, it is through delayed implantation of the embryo or delayed ovulation rather than maintenance of unfertilized, mature eggs (Weir and Rowlands 1973; Kaitala et al. 1997; Thom et al. 2004). The inability to store oocytes therefore requires an evolutionary explanation. We find no genetic constraint of the evolution of improved oocyte storage and no evidence of a lack of selection associated with the loss of current and future eggs. Females vary in how they respond to the mistiming of mating relative to

11 The Cost of Keeping Eggs Fresh 321 sexual maturation, falling into two genetically defined groups. There are no fitness trade-offs to belonging to one or the other group; those who delay reproduction always produce fewer offspring. This suggests that there must be some benefit to oosorption we have not measured. It is likely that this is dependent on nutritional environment. We therefore propose that although the genetic variance necessary for extending oocyte shelf life exists within the population and that there should be strong selection on increasing fecundity under certain social conditions, the evolution of this trait is likely to be constrained by complex trade-offs between resource allocation to current and future reproduction in response to changing nutritional environments. Acknowledgments We thank T. Montrose for expert technical assistance and R. Ward for help with setting up the experiment. E. Barrett, M. Edvardsson, J. Hunt, and T. Tregenza provided helpful comments. 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