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In animal populations, sib mating is often the primary source of inbreeding depression (ID). We used recently wild-caught Drosophila melanogaster to test whether such ID is amplified by environmental stress and, in males, by sexual selection. We also investigated whether increased ID because of stress (increased larval competition) persisted beyond the stressed stage and whether the effects of stress and sexual selection interacted. Sib mating resulted in substantial cumulative fitness losses (egg to adult reproduction) of 50% (benign) and 73% (stressed). Stress increased ID during the larval period (23% vs. 63%), but not during post-stress reproductive stages (36% vs. 31%), indicating larval stress may have purged some adult genetic load (although ID was uncorrelated across stages). Sexual selection exacerbated inbreeding depression, with inbred male offspring suffering a higher reproductive cost than females, independent of stress (57% vs. 14% benign, 49% vs. 11% stress).
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Inbreeding, or the mating of relatives, and the associated cost to fitness known as inbreeding depression have long been a focus in evolutionary biology, ecology and conservation biology (Charlesworth & Charlesworth, 1987; Ralls et al., 1988; Keller & Waller, 2002; Kristensen & Sorensen, 2005). Inbreeding depression is a common phenomenon in nature and has been documented in a wide range of taxa (Crnokrak & Roff, 1999; Keller & Waller, 2002). As a result, inbreeding is recognized as a potent force influencing the persistence of natural populations (Keller & Waller, 2002; Spielman et al., 2004; Frankham, 2008) as well as shaping the evolution of life history, morphology, physiology and behaviour (Charpentier et al., 2007). However, we have a poor understanding of the factors that contribute to the considerable variation in the severity of inbreeding depression that has been observed across taxa, populations and even life-history stages (Hedrick & Kalinowski, 2000; Keller & Waller, 2002; Pemberton, 2008). Currently, sexual selection and environmental stress have become the focus of much investigation owing to the potential role they play in determining the magnitude of inbreeding depression. It has been hypothesized that sexual selection can reduce inbreeding depression by increasing selection against deleterious alleles (Radwan et al., 2004; Jarzebowska & Radwan, 2009), whereas environmental stress often amplifies the negative effects of inbreeding on fitness (Armbruster & Reed, 2005). In general, both sexual selection and environmental stress are predicted to increase the effectiveness with which deleterious alleles are purged from a population (Whitlock & Bourguet, 2000; Kristensen et al., 2003; Swindell & Bouzat., 2006; Whitlock & Agrawal, 2009). However, it is unknown how these two forces interact to shape the genetic architecture and expression of inbreeding depression in natural populations.
Recent interest in the role of sexual selection in population persistence has centred around the potential for sexual selection to purge mutational load, thereby preventing mutational meltdown and reducing rates of extinction in small populations (Whitlock, 2000; Sharp & Agrawal, 2008; Jarzebowska & Radwan, 2009). Although there is some evidence that sexual selection can effectively remove deleterious mutations from populations (Whitlock & Agrawal, 2009), much less is known about the effects of sexual selection on the severity of inbreeding depression. Sexual selection theory predicts that females who choose superior males increase the chances of survival of their offspring by selection of beneficial alleles and implies that males expressing deleterious alleles are less likely to find mates (Williams, 1966; Whitlock & Bourguet, 2000). Being inbred may therefore be more costly for males than for females. Several non-Drosophila studies provide evidence that aspects of sexual selection such as male–male competition and female choice exacerbate inbreeding depression for components of male mating success and for lifetime reproductive success (Potts et al., 1994; Pray et al., 1994; Meagher et al., 2000; Slate et al., 2000; Höglund et al., 2002). One study suggests that in Drosophila, the pattern may be similar (Miller & Hedrick, 1993). Inbreeding depression was significantly greater in males when sexual selection was included as a component of male fitness (as measured by competitive male mating success). Miller & Hedrick (1993) reported substantially higher levels of inbreeding depression for competitive male mating ability (72.5%) than for female fecundity (1.5%, nonsignificant). This bias was not found when sexual selection was not incorporated as a component of male reproductive fitness (Robinson et al., 2009).
Inbreeding depression is also widely recognized as being negatively affected by environmental stress. However, the magnitude of inbreeding depression reported under stressful conditions is highly variable (see Armbruster & Reed, 2005). As a result, several authors have argued that the current body of research demonstrates a lack of consistent or predictable effects of environmental stress on the expression of inbreeding depression (Keller & Waller, 2002; Armbruster & Reed, 2005; Waller et al., 2008). Furthermore, most of this research has focused on overall survival or single fitness components, which has left another important question unanswered: what is the effect of early stress on inbreeding depression in later life-history stages? It is unclear whether stress has a long-lasting effect on the physiological functioning of an organism, exacerbating the effects of inbreeding on fitness even after the source of stress is removed. Two alternative hypotheses predict different changes in the level of inbreeding depression following exposure to stress during development. Environmental stress could amplify inbreeding depression in later life-history stages because of greater vulnerability of inbred individuals to long-lasting phenotypic effects of stress. Alternatively, stress may purge genetic load during development, thus reducing levels of inbreeding depression in later life-history stages. This can occur if stress increases selection against deleterious mutations that affect fitness at multiple life-history stages (Haldane, 1957). This hypothesis predicts that stress will amplify inbreeding depression during the stage at which individuals are exposed (e.g. larval survival) but will have either no effect or reduce inbreeding depression for later performance (e.g. reproduction), as observed by Montalvo (1994) in the blue columbine (Aquilegia caerulea). These alternatives are not mutually exclusive, but it is important to understand their relative importance.
In D. melanogaster, given that females exhibit strong sperm precedence (see review in Manier et al., 2010) and lay multiple eggs on a single fruit (Nunney, 1990), full-sib mating is expected to occur in the wild among offspring from a single fruit. However, despite the extensive use of D. melanogaster as a model for the effects of inbreeding on fitness, an accurate measure of the cost of sib mating in wild populations is lacking. The vast majority of studies have used populations maintained under laboratory conditions for well over 20 generations (Tantawy, 1957; Sharp, 1984; Mackay, 1985; Miller & Hedrick, 1993; Miller et al., 1993; Garcia et al., 1994; Hughes, 1995, 1997; Fowler & Whitlock, 2002; Hughes et al., 2002). The few exceptions are those studies that have measured the fitness effects of individuals made homozygous for various chromosomes extracted from males taken directly from wild populations. Laboratory-adapted populations may have a different genetic architecture relative to natural populations, for example because of bottleneck events occurring during the establishment or maintenance of a laboratory population. This may result in differences in the levels of inbreeding depression. Concerns have been raised regarding potential underestimation of the levels of inbreeding depression found in nature (Sheilds, 1993; Hedrick & Kalinowski, 2000; Joron & Brakefield, 2003) when laboratory populations are used. To investigate the effect of inbreeding in flies with a natural genetic architecture, the cost of sib mating needs to be measured from recently wild-caught populations.
The aim of this study was to evaluate how inbreeding depression is influenced by both sexual selection and environmental stress using two wild-caught populations of D. melanogaster. Specifically this study addresses three main questions: (i) How large is the fitness cost of sib mating when developmental and reproductive costs are included? (ii) Are reproductive costs in the inbred offspring greater in males than in females? (iii) Given a stressful environment during larval development, does inbreeding depression increase for larval survival and/or adult reproduction? The cost of one generation of full-sib mating was measured in two populations of D. melanogaster collected from Northern California after only one generation of controlled outcrossing in the laboratory (to avoid any unintentional purging of deleterious recessive alleles). We measured egg hatchability, larval-to-adult survival, female fecundity and male mating success of inbred offspring and evaluated the role of stress on inbreeding depression by comparing the effect of rearing larvae under the conditions of low and high food competition. In addition, we evaluated the effect of the age of females on inbreeding depression for female fecundity by measuring the number of offspring produced separately during the early (days 1–8) and late (days 8–16) stages in the female’s life. Previous work in Drosophila has shown that inbreeding depression increases with age (Hughes, 1995; Hughes et al., 2002).
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This study demonstrates a number of important features of the relationship between inbreeding and fitness under the conditions of sexual selection and environmental stress. Under standard laboratory conditions, sib mating resulted in a 50% cost to overall fitness in recently wild-caught D. melanogaster populations, corresponding to β = 2.77 lethal equivalents (Table 5). In addition, a striking difference was found in the level of inbreeding depression expressed in males and females (Fig. 1). Inbred males suffered an almost 2-fold higher cumulative loss in fitness than females, a result consistent with the study of Miller & Hedrick (1993). This difference between the sexes remained the same under high larval competition (Table 5), whereas the overall fitness cost to sib mating increased. When these cumulative effects are broken down, we found that egg hatchability was only slightly affected by inbreeding (< 2%), while even under benign conditions, relative larval survival dropped by about 20%. We also found that larval competitive stress amplified this larval inbreeding depression, but it did not increase inbreeding depression in the later life-history stages of adult reproduction. This result argues against the hypothesis that stress induces long-lasting negative phenotypic effects in inbred individuals but may be consistent with the possibility that stress, through purging some of the genetic load, can lower inbreeding depression at a later stage. All of these patterns were consistent across the two replicate populations. The only significant (but trivial) difference between them was a 2% level of inbreeding depression for egg hatchability in the Mayo population vs. 1% in Gala.
In this study, we emphasized the importance of testing wild-caught flies that retained their natural genetic architecture. However, we still tested the flies under laboratory conditions as recent advances in measuring inbreeding depression in wild populations using pedigree analysis (Pemberton, 2008) are not generally applicable to short-lived small animals such as Drosophila.
The Magnitude of inbreeding depression in recently wild-caught D. melanogaster
Table 6 summarizes literature on the effects of inbreeding on individual fitness components in D. melanogaster measured under standard benign laboratory conditions using fitness measures directly comparable to those measured in this study (see Simmons & Crow (1977) and Charlesworth & Charlesworth (1987) for reviews of estimates based on chromosomal homozygote populations). Only two studies (Miller & Hedrick, 1993; Robinson et al., 2009) measured inbreeding depression across the full spectrum of life-history stages in both sexes, and Robinson et al. (2009) excluded sexual selection in males. Our finding that mating between siblings caused a 50% reduction in overall cumulative fitness in recently caught populations of D. melanogaster under standard laboratory conditions (Fig. 1, Table 5) is consistent with results of Miller & Hedrick (1993). We found that rearing conditions that included larval competitive stress resulted in a larger 72% reduction in overall fitness.
Table 6. Summary of the effects of inbreeding on individual fitness components in Drosophila melanogaster measured under benign laboratory conditions. The number of haploid lethal equivalents (β) was calculated as described in the text (β = −[ln(winbred/woutbred)]/F), given a single level of inbreeding (F) or as the slope of the regression of ln(fitness) on F. For chromosome homozygotes, β was calculated according to Appendix 1. For fecundity measures, the age of adult females is the age range over which females laid eggs, and for mating success, it is the age at which males were tested. Hatchability and larval survival for both inbred (IB) and outbred (OB) individuals are included when the data were available. All values for this study are from the maternal analysis and include both the competitive index and per cent success (in parenthesis) for larval survival and male mating. Cumulative fitness for each sex is a multiplicative measure that was calculated across the same life-history stages for each study (see Methods).
|Life-History Stage||F||Type of Inbreeding||β||Source|
|Egg hatchability|| || ||%OB||%IB|| || |
|0.25||Full-sib mating||98.6||96.8||0.06||This study|
|0.25||Full-sib mating|| 96.0|| 90.0||0.26||Biémont, 1978|
|Egg-adult survival|| || ||%OB||%IB|| || |
|Egg-pupa survival||0.25–0.73||Full-sib mating||80.0||60.0||0.43||Garcia et al., 1994|
|Larval-adult survival||0.25||Full-sib mating||89.4||72.2||0.95* (0.85)||This study|
|0.25||Full-sib mating||85.6||66.7||1.00||Ehiobu et al., 1989|
|0.25–0.75||Full-sib mating||61.9||49.9||0.37||Tantawy, 1957|
|0.37||Chrom 2 homozygotes||NA||NA||0.30||Miller & Hedrick, 1993|
|Egg-adult survival||0.25–0.88||Full-sib mating||NA||NA||2.67*||Latter & Roberston, 1962|
|0.25||Full-sib mating||90.0||70.0||1.01||Biémont, 1978|
|0.25||Full-sib mating||80.0||72.0||0.42||Robinson et al., 2009|
|0.37||Chrom 2 homozygotes||NA||NA||0.82||Bijlsma et al., 1999|
|0.65||Chrom 3 homozygotes||NA||NA||0.89||Mackay, 1985|
|Adult female fitness|| || ||Age|| || || |
|Female fecundity||0.25||Full-sib mating||6–14 days|| ||0.00||This study|
|0.25|| ||15–22 days|| ||0.98|
|0.25||Full-sib mating||3–6 days|| ||1.10||Ehiobu et al., 1989|
|0.25||Full-sib mating||7–8 days|| ||0.57||Robinson et al., 2009|
|0.50||Full-sib mating||2–5 days|| ||1.12||Dahlgaard & Hoffmann, 2000|
|0.50||Full-sib mating||1–5 days|| ||0.30||Miller, 1994|
|0.55||Chrom 2 homozygotes||1–4 days|| ||0.00||Miller & Hedrick, 1993|
|0.98||Chrom 2 + 3 homozygotes||1–7 days|| ||0.34||Hughes et al., 2002|
| || ||8–14 days|| ||0.77|| |
| || ||15–21 days|| ||0.71|| |
|0.65||Chrom 3 homozygotes||Unknown|| ||1.25||Mackay, 1985|
|Adult male fitness|| || ||Age|| || || |
|Male mating success||0.25||Full-sib mating||14–18 days|| ||3.01* (1.22)||This study|
|0.25–0.98||Full-sib mating||4–7 days|| ||0.81*||Sharp, 1984|
|0.25||Full-sib mating||5 days|| ||5.39||Pendlebury & Kidwell, 1974|
|0.25||Full-sib mating||5 days|| ||2.25|| |
|0.37||Chrom 2 homozygotes||3 days|| ||1.02||Kosuda, 1983|
|0.37||Chrom 2 homozygotes||3 days|| ||3.49||Miller & Hedrick, 1993|
|0.37||Chrom 2 homozygotes||2 days|| ||1.86*||Brittnacher, 1981|
|0.44||Chrom 3 homozygotes||3 days|| ||0.43*||Hughes, 1995|
| ||Chrom 3 homozygotes||21 days|| ||1.14*|| |
|0.44||Chrom 3 homozygotes||3 days|| ||1.04*||Patridge et al., 1985|
|Cumulative fitness|| || ||Sex|| || || |
| ||0.25||Full-sib mating||Female|| ||1.60* (1.50)||This study|
| || ||Male|| ||4.25* (2.15)|| |
|0.25||Full-sib mating||Female|| ||0.91||Robinson et al., 2009|
| || ||Male|| ||0.92†|| |
|0.37||Chrom 2 homozygotes||Female|| ||0.27||Miller & Hedrick, 1993|
| || ||Male|| ||3.79‡|| |
|0.65||Chrom 3 homozygotes||Female|| ||2.13||Mackay, 1985|
The contribution of egg hatchability to cumulative inbreeding depression was statistically significant but very small (2%), similar to the 6% found by Biémont (1978). The very low inbreeding depression in egg hatchability suggests that a limited fraction of the offspring’s genes are expressed at this stage and/or that mutations in the genes involved in early development are generally not fully recessive, limiting the mutation selection build-up of deleterious alleles. In contrast, reductions in fitness under low and high larval competition, respectively, in larval-to-adult survival (22%, 63%), the fecundity of female offspring (15%, 11%) and the mating success of male offspring (51%, 48%) were all substantial. Overall, our estimates of lethal equivalents for larval-adult survival (0.95) and female fecundity (0, 6–14 days; 0.98, 15–22 days) are similar to those previously reported (Table 6). The finding that inbreeding depression for female fecundity increased with female age (Table 4) is in agreement with several studies demonstrating significant age effects in both females and males (Hughes, 1995; Hughes et al., 2002).
Inbreeding depression is expected to vary among families, as they represent a random sampling of deleterious alleles in the population (Hedrick & Kalinowski, 2000; Haag et al., 2003). Interactions between inbreeding and families/lineages have been found in Drosophila (Bijlsma et al., 1999; Dahlgaard & Hoffmann, 2000; Reed et al., 2003), Daphnia (Haag et al., 2003), Peromyscus polionotus (Lacy et al., 1996) Tribolium castaneum (Pray & Goodnight, 1995), and plants (Dudash et al., 1997; Byers & Waller, 1999). In this study, highly significant interactions between inbreeding and family were observed for egg hatchability and female fecundity (Tables 1 and 2). However, this among-family variance is expected to decrease with an increase in the number of loci contributing to the trait (and hence potentially a source of deleterious alleles), a pattern found in this study for larval competition, both under low and high competition conditions, and male mating success (Table 2). This lack of a family effect for larval performance and male mating success may indicate the involvement of a large number of mildly deleterious alleles, which minimizes the sampling variance among the families.
Comparing among the traits, our results suggest that alleles having deleterious effects on the fitness of inbred larvae do not affect later life-history stages and/or that some deleterious alleles are purged at the larval stage so that they are not expressed at the later stages of female fecundity and male mating success. Under both low and high larval competition, the correlations between larval and adult traits were within the range 0–0.13. In addition, the nonsignificant positive correlation between male and female adult inbreeding depression under both benign and stressed larval conditions is consistent with the hypothesis that deleterious alleles show no consistent pattern in determining male mating success verses female fecundity.
Inbreeding depression is greater in males
Inbred male offspring showed a substantially greater loss of fitness than females, regardless of the female age (late female fecundity, β = 0.98; male mating success, β = 3.01, Table 6). Assuming no sex differences in egg-to-adult survival (Frankham & Wilcken, 2006), the cumulative relative fitness of inbred females was almost two-fold higher than inbred males (Table 5, Fig. 1), results similar to those of Miller & Hedrick (1993) (Table 6). In contrast, Robinson et al. (2009) did not incorporate sexual selection into the measure of adult male reproductive fitness and found no differences between the sexes in levels of inbreeding depression (both approximately 13%).
Marked sex differences in inbreeding depression have also been found in other animals. In house mice, Potts et al. (1994) found significant inbreeding depression for the acquisition of territories by males, whereas there was no detectable inbreeding depression for female fitness. In addition, the cost to sib mating in wild-caught mice has been shown to be almost four times greater for inbred males than for inbred females under semi-natural conditions (Meagher et al., 2000). Pray et al. (1994) also report that male red flour beetles (Tribolium castaneum) suffer greater costs of being inbred than females for proportion of offspring produced in a competitive social environment. Such results suggest that sexual selection via male–male competition and/or female choice may be responsible for amplifying the effects of inbreeding on male fitness. However, sexual dimorphism in inbreeding depression may result from competition for resources (food, water, territory) in general and therefore will not always be male biased. For example, wild female song sparrows exhibit greater inbreeding depression for lifetime reproductive success than males (Keller, 1998).
A finding that selection is greater against inbred males than inbred females can have important implications for the role of sexual selection in reducing genetic load in populations via female choice. Sexual selection is predicted to reduce the frequency of deleterious alleles in a population if males that carry a greater number of deleterious alleles are less likely to mate because of female choosiness (Whitlock & Bourguet, 2000), potentially reducing the risk of extinction in small populations (Whitlock, 2000). Recent empirical work in the bulb mite demonstrated that sexual selection can reduce both extinction rate and levels of inbreeding depression for small bottlenecked populations (Jarzebowska & Radwan, 2009), and a few other studies have demonstrated the effective removal of deleterious alleles from populations via sexual selection (Radwan, 2004; Radwan et al., 2004; Sharp & Agrawal, 2008; Hollis et al., 2009). However, our results illustrate a potential problem for the purging hypothesis, because it is assumed that deleterious alleles driven to a lower frequency by sexual selection result in an overall fitness benefit in females or in juveniles. We found no significant correlation between inbreeding depression in male reproduction and other traits. More empirical work is needed to determine whether sexual selection can in general alleviate mutational load in populations.
Inbreeding depression and environmental stress
Although we found that environmental stress (larval competition) increased inbreeding depression, we found no correlation between the benign and competitive environments in the inbreeding depression for larval survival (LCI r = −0.24) or in cumulative inbreeding depression (r = −0.09 female, r = −0.05 male). Several studies examining how purging of genetic load in different environments affects extinction rates suggest that environmental stress may increase the effectiveness of purging (Bijlsma et al., 2000; Swindell & Bouzat, 2006). However, the implications for population persistence are unclear if purging is environment specific and purging provides no fitness benefit in novel environments (Bijlsma et al., 1999; Leberg & Firmin, 2008).
A related phenomenon occurs when an environmental stress affects only one life-history stage of an organism. Does this stress amplify inbreeding depression across the entire life cycle of an organism or does it reduce inbreeding depression in later life-history stages because of genetic purging as a result of greater selection against individuals expressing deleterious recessive alleles (Armbruster & Reed, 2005; Waller et al., 2008)? We tested these hypotheses by measuring inbreeding depression during exposure to stress and after the stressor have been removed. We found that competitive stress only amplified inbreeding depression during the stage at which it was applied. Inbreeding depression affecting larval survival was substantially increased under conditions of competitive resource stress (Table 3, Fig. 1); however, female fecundity and male mating success, measured after the stress was applied, did not show increased inbreeding depression (Tables 2 and 3, Fig. 1). It is possible that the purging of individuals with the highest genetic load at the stressful stage (larval-to-adult development) could explain why inbreeding depression in the adult fitness stages was not increased (Table 3). However, the evidence for purging is weak as the among-family correlation linking inbreeding depression at the larval and reproductive stages is very close to zero under both benign and stressed conditions. This does not exclude the possibility of a purging effect, but for purging at an early stage to increase later fitness requires that some of the same deleterious alleles affected both stages, a scenario that would typically generate a positive correlation between inbreeding depression at the two stages under benign conditions.
Work in the blue columbine (Aquilegia caerulea) suggested that exposure to harsher conditions early in life (field vs. greenhouse germination) may lower inbreeding depression at later stages (Montalvo, 1994). This is presumably because of a reduction in selection against deleterious alleles under benign greenhouse conditions during the seedling period. Several other studies in plants show similar patterns of greater inbreeding depression for early life history (seed survival) than adult fitness (plant size) when seeds experienced stressful field conditions (Schoen, 1983; Kohn, 1988). Note that this role of purging cannot be detected in studies comparing lines or families that are genetically identical (e.g. when specific chromosomes are made homozygous).
Implications for inbreeding avoidance in D. melanogaster
In general, individuals that employ mechanisms to avoid mating with relatives have a selective advantage over those that do not, driving the evolution of mechanisms to avoid inbreeding (Pusey & Wolf, 1996; Panhuis & Nunney, 2007). This is because the genetic load of recessive deleterious alleles that cause inbreeding depression also creates conditions that favour genotypes that avoid inbreeding. Little is known about the levels of full-sib mating and the associated fitness costs in wild populations of Drosophila, the two factors that determine the strength of selective forces driving the evolution of avoidance mechanisms. In two cactophilic species of Drosophila and in D. melanogaster, it has been observed that females appear to reduce sperm use from related males, which may be beneficial by reducing inbreeding depression in their offspring (Markow, 1997; Panhuis & Nunney, 2007). Several other studies provide circumstantial evidence supporting the existence of post-mating, prefertilization inbreeding avoidance (PPIA) in D. melanogaster. For example, sperm competitive ability has been shown to decrease with the degree of relatedness between males and females (Clark et al., 1995, 1999; Clark & Begun, 1998; Mack et al., 2002). Our work clearly demonstrates massive fitness costs of mating with a sibling (Table 5, Fig. 1) in recently caught D. melanogaster especially if there is larval competition. Larval competition is found in nature (Nunney, 1990), and such large fitness costs provide a strong selective environment in which PPIA could evolve in this species. The level of sib mating is still unknown in wild populations of D. melanogaster; however, it appears that flies generally mate before dispersing from their natal site (unpublished data) so the frequency of such matings could be significant.
Finally, the way in which inbreeding depression is measured, using either a competitive index (LCI or MCI) versus using uncorrected per cent survival or mating success, is important in an ecological context. Estimating inbreeding depression using a competitive index is representative of situations in nature where multiple females lay eggs on a single fruit. Under these conditions, the inbred offspring of a female that has mated to a sibling are potentially competing against outbred offspring from other females. Alternatively, if only a single female lays eggs on a fruit, then raw per cent values would be an appropriate measure of fitness because inbred offspring are only competing with other inbred offspring. Inbreeding depression would be expected to be less under these conditions compared to conditions where multiple females lay eggs on a single fruit.