Slow inbred lines of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> express as much inbreeding depression as fast inbred lines under semi-natural conditions

Selection may reduce the deleterious consequences of inbreeding. This may be due to purging of recessive deleterious alleles or balancing selection favouring heterozygote offspring. Such selection is expected to be more efficient at slower compared to at faster rates of inbreeding. In this study we tested the impact of inbreeding and the rate of inbreeding on fitness related traits (egg productivity, egg-to-adult viability, developmental time and behaviour) under cold and benign semi-natural thermal conditions using Drosophila melanogaster as a model organism. We used non-inbred control and slow and fast inbred lines (both with an expected inbreeding level of 0.25). The results show that contrary to expectations the slow inbred lines do not maintain higher average fitness than the fast inbred lines. Furthermore, we found that stressful environmental conditions increased the level of inbreeding depression but the impact of inbreeding rate on the level of inbreeding depression was not affected by the environmental conditions. The results do not support the hypothesis that inbreeding depression is less severe with slow compared to fast rates of inbreeding and illustrate that although selection may be more efficient with slower rates of inbreeding this does not necessary lead to less inbreeding depression.     

Trait specific consequences of fast and slow inbreeding: lessons from captive populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The increased homozygosity due to inbreeding leads to expression of deleterious recessive alleles, which may cause inbreeding depression in small populations. The severity of inbreeding depression has been suggested to depend on the rate of inbreeding, with slower inbreeding being more effective in purging deleterious alleles of smaller effect. The effectiveness of purging is however dependent on various factors such as the effect of the deleterious, recessive alleles, the genetic background of inbreeding depression and the environment in which purging occurs. Investigations have shown inconclusive results as to whether purging efficiently diminish inbreeding depression. Here we used an ecologically relevant inbreeding coefficient (f ≈ 0.25) and generated ten slow and ten fast inbred lines of Drosophila melanogaster by keeping the effective population size constant at respectively 32 and 2 for 19 or 2 generations. These inbred lines were contrasted to non-inbred control lines. We investigated the effect of inbreeding and inbreeding rate in traits associated with fitness including heat, cold and desiccation stress resistance, egg-to-adult viability, development time, productivity, metabolic rate and wet weight under laboratory conditions. The results showed highly trait specific consequences of inbreeding and generally no support for the hypothesis that slow inbreeding is less deleterious than fast inbreeding. Egg-to-adult viability and development time were investigated under both benign and heat stress conditions. Reduced viability and increased developmental time were observed at stressful temperatures and inbreeding depression was on average more severe at stressful compared to benign temperatures.     

Inbreeding reveals stronger net selection on Drosophila melanogaster males: implications for mutation load and the fitness of sexual females

Stronger selection on males has the potential to lower the deleterious mutation load of females, reducing the cost of sex. However, few studies have directly quantified the strength of selection for both sexes. As the magnitude of inbreeding depression (ID) is related to the strength of selection, we measured the cost of inbreeding for both males and females in a laboratory population of Drosophila melanogaster. Using a novel technique for inbreeding, we found significant ID for both juvenile viability and adult fitness in both sexes. The genetic variation responsible for this depression in fitness appeared to be recessive for adult fitness (h=0.11) and partially additive for juvenile viability (h=0.29). ID was identical across the sexes in terms of juvenile viability but was significantly more deleterious for males than females as adults, even though female X-chromosome homogamety should predispose them to a higher inbreeding load. We estimated the strength of selection on adult males to be 1.24 greater than on adult females, and this appears to be a consequence of selection arising from competition for mates. Combined with the generally positive intersexual genetic correlation for inbred lines, our results suggest that the mutation load of sexual females could be meaningfully reduced by stronger selection acting on males.     

No evidence for optimal fitness at intermediate levels of inbreeding in Drosophila melanogaster

Optimal outbreeding theory predicts fitness benefits to intermediate levels of inbreeding. In the present study, we test for linear (consistent with inbreeding depression) and nonlinear (consistent with optimal outbreeding) effects of inbreeding on reproductive fitness in male and female Drosophila melanogaster . We found linear declines in fitness associated with increased inbreeding for egg-to-adult viability, but not the number of eggs laid or sperm competitive ability. Egg-to-adult viability was also lower in the progeny of inbred males and females mated to unrelated individuals. However, there was no evidence for optimal fitness at intermediate levels of inbreeding for any trait. The present study highlights the importance of considering biologically realistic levels of inbreeding and cross-generational effects when investigating the costs and benefits of mating with relatives.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 501–510.     

Environmental dependence of inbreeding depression and purging in Drosophila melanogaster

Elimination or reduction of inbreeding depression by natural selection at the contributing loci (purging) has been hypothesized to effectively mitigate the negative effects of inbreeding in small isolated populations. This may, however, only be valid when the environmental conditions are relatively constant. We tested this assumption using Drosophila melanogaster as a model organism. By means of chromosome balancers, chromosomes were sampled from a wild population and their viability was estimated in both homozygous and heterozygous conditions in a favourable environment. Around 50% of the chromosomes were found to carry a lethal or sublethal mutation, which upon inbreeding would cause a considerable amount of inbreeding depression. These detrimentals were artificially purged by selecting only chromosomes that in homozygous condition had a viability comparable to that of the heterozygotes (quasi-normals), thereby removing most deleterious recessive alleles. Next, these quasi-normals were tested both for egg-to-adult viability and for total fitness under different environmental stress conditions: high-temperature stress, DDT stress, ethanol stress, and crowding. Under these altered stressful conditions, particularly for high temperature and DDT, novel recessive deleterious effects were expressed that were not apparent under control conditions. Some of these chromosomes were even found to carry lethal or near-lethal mutations under stress. Compared with heterozygotes, homozygotes showed on average 25% additional reduction in total fitness. Our results show that, except for mutations that affect fitness under all environmental conditions, inbreeding depression may be due to different loci in different environments. Hence purging of deleterious recessive alleles can be effective only for the particular environment in which the purging occurred, because additional load will become expressed under changing environmental conditions. These results not only indicate that inbreeding depression is environment dependent, but also that inbreeding depression may become more severe under changing stressful conditions. These observations have significant consequences for conservation biology.     

Inbreeding depression across a nutritional stress continuum

Many natural populations experience inbreeding and genetic drift as a consequence of nonrandom mating or low population size. Furthermore, they face environmental challenges that may interact synergistically with deleterious consequences of increased homozygosity and further decrease fitness. Most studies on inbreeding–environment (I-E) interactions use one or two stress levels, whereby the resolution of the possible stress and inbreeding depression interaction is low. Here we produced Drosophila melanogaster replicate populations, maintained at three different population sizes (10, 50 and a control size of 500) for 25 generations. A nutritional stress gradient was imposed on the replicate populations by exposing them to 11 different concentrations of yeast in the developmental medium. We assessed the consequences of nutritional stress by scoring egg-to-adult viability and body mass of emerged flies. We found: (1) unequivocal evidence for I-E interactions in egg-to-adult viability and to a lesser extent in dry body mass, with inbreeding depression being more severe under higher levels of nutritional stress; (2) a steeper increase in inbreeding depression for replicate populations of size 10 with increasing nutritional stress than for replicate populations of size 50; (3) a nonlinear norm of reaction between inbreeding depression and nutritional stress; and (4) a faster increase in number of lethal equivalents in replicate populations of size 10 compared with replicate populations of size 50 with increasing nutritional stress levels. Our data provide novel and strong evidence that deleterious fitness consequences of I-E interactions are more pronounced at higher nutritional stress and at higher inbreeding levels.     

Strong inbreeding depression in male mating behaviour in a poeciliid fish

The magnitude of inbreeding depression is often larger in traits closely related to fitness, such as survival and fecundity, compared to morphological traits. Reproductive behaviour is also closely associated with fitness, and therefore expected to show strong inbreeding depression. Despite this, little is known about how reproductive behaviour is affected by inbreeding. Here we show that one generation of full‐sib mating results in a decrease in male reproductive performance in the least killifish (Heterandria formosa). Inbred males performed less gonopodial thrusts and thrust attempts than outbred males (δ = 0.38). We show that this behaviour is closely linked with fitness as gonopodial performance correlates with paternity success. Other traits that show inbreeding depression are offspring viability (δ = 0.06) and maturation time of males (δ = 0.19) and females (δ = 0.14). Outbred matings produced a female biased sex ratio whereas inbred matings produced an even sex ratio.     

Interactive effects of environmental stress and inbreeding on reproductive traits in a wild bird population

1. Conservation biologists are concerned about the interactive effects of environmental stress and inbreeding because such interactions could affect the dynamics and extinction risk of small and isolated populations, but few studies have tested for these interactions in nature. 2. We used data from the long-term population study of song sparrows Melospiza melodia on Mandarte Island to examine the joint effects of inbreeding and environmental stress on four fitness traits that are known to be affected by the inbreeding level of adult birds: hatching success, laying date, male mating success and fledgling survival. 3. We found that inbreeding depression interacted with environmental stress to reduce hatching success in the nests of inbred females during periods of rain. 4. For laying date, we found equivocal support for an interaction between parental inbreeding and environmental stress. In this case, however, inbred females experienced less inbreeding depression in more stressful, cooler years. 5. For two other traits, we found no evidence that the strength of inbreeding depression varied with environmental stress. First, mated males fathered fewer nests per season if inbred or if the ratio of males to females in the population was high, but inbreeding depression did not depend on sex ratio. Second, fledglings survived poorly during rainy periods and if their father was inbred, but the effects of paternal inbreeding and rain did not interact. 6. Thus, even for a single species, interactions between the inbreeding level and environmental stress may not occur in all traits affected by inbreeding depression, and interactions that do occur will not always act synergistically to further decrease fitness.     

Detecting purging of inbreeding depression by a slow rate of inbreeding for various traits: the impact of environmental and experimental conditions

Inbreeding depression (ID) has since long been recognized as a significant factor in evolutionary biology. It is mainly the consequence of (partially) recessive deleterious mutations maintained by mutation-selection balance in large random mating populations. When population size is reduced, recessive alleles are increasingly found in homozygous condition due to drift and inbreeding and become more prone to selection. Particularly at slow rates of drift and inbreeding, selection will be more effective in purging such alleles, thereby reducing the amount of ID. Here we test assumptions of the efficiency of purging in relation to the inbreeding rate and the experimental conditions for four traits in D. melanogaster. We investigated the magnitude of ID for lines that were inbred to a similar level, F ≈ 0.50, reached either by three generations of full-sib mating (fast inbreeding), or by 12 consecutive generations with a small population size (slow inbreeding). This was done on two different food media. We observed significant ID for egg-to-adult viability and heat shock mortality, but only for egg-to-adult viability a significant part of the expressed inbreeding depression was effectively purged under slow inbreeding. For other traits like developmental time and starvation resistance, however, adaptation to the experimental and environmental conditions during inbreeding might affect the likelihood of purging to occur or being detected. We discuss factors that can affect the efficiency of purging and why empirical evidence for purging may be ambiguous.Subject terms: Evolutionary genetics, Inbreeding     

Inbreeding-environment interactions for fitness: complex relationships between inbreeding depression and temperature stress in a seed-feeding beetle

It is commonly argued that inbred individuals should be more sensitive to environmental stress than are outbred individuals, presumably because stress increases the expression of deleterious recessive alleles. However, the degree to which inbreeding depression is dependent on environmental conditions is not clear. We use two populations of the seed-feeding beetle, Callosobruchus maculatus, to test the hypotheses that (a) inbreeding depression varies among rearing temperatures, (b) inbreeding depression is greatest at the more stressful rearing temperatures, (c) the degree to which high or low temperature is stressful for larval development varies with inbreeding level, and (d) inbreeding depression is positively correlated between different environments. Inbreeding depression (δ) on larval development varied among temperatures (i.e., there was a significant inbreeding-environment interaction). Positive correlations for degree of inbreeding depression were consistently found between all pairs of temperatures, suggesting that at least some loci affected inbreeding depression across all temperatures examined. Despite variation in inbreeding depression among temperatures, inbreeding depression did not increase consistently with our proxy for developmental stress. However, inbreeding changed which environments are benign versus stressful for beetles; although 20°C was not a stressful rearing temperature for outbred beetles, it became the most stressful environment for inbred larvae. The finding that inbreeding-environment interactions can cause normally benign environments to become stressful for inbred populations has important consequences for many areas of evolutionary genetics, artificial breeding (for conservation or food production), and conservation of natural populations.     

Male attractiveness, fertility and susceptibility to oxidative stress are influenced by inbreeding in Drosophila simulans

Inbreeding frequently leads to inbreeding depression, a reduction in the trait values of inbred individuals. Inbreeding depression has been documented in sexually selected characters in several taxa, and while there is correlational evidence that male fertility is especially susceptible to inbreeding depression, there have been few direct experimental examinations of this. Here, we assessed inbreeding depression in male fertility and a range of other male fitness correlates in Drosophila simulans. We found that male fertility and attractiveness were especially susceptible to inbreeding depression. Additionally, levels of testicular oxidative stress were significantly elevated in inbred males, although sperm viability did not differ between inbred and outbred males. Copulation duration, induction of oviposition, and the proportion of eggs hatching did not differ for females mated to inbred or outbred males. Nevertheless, our results clearly show that key male fitness components are impaired by inbreeding and provide evidence that aspects of male fertility are especially susceptible to inbreeding depression.     

The variance in inbreeding depression and the recovery of fitness in bottlenecked populations

Theoretical analyses of inbreeding suggest that following an increased degree of inbreeding there may be a temporary recovery of fitness, because of selection either within or among inbred lineages. This is possible because selection can act more efficiently to remove deleterious alleles given the greater homozygosity of such populations. If common, recovery of fitness following inbreeding may be important for understanding some evolutionary processes and for management strategies of remnant populations, yet empirical evidence for such recovery in animals is scant. Here we describe the effects of single-pair population bottlenecks on a measure of fitness in Drosophila melanogaster. We compared a large number of families from each of 52 inbred lines with many families from the outbred population from which the inbred lineages were derived. Measures were made at the third and the 20th generations after the bottleneck. In both generations there was, on average, substantial inbreeding depression together with a highly significant variance among the inbred lines in the amount of fitness reduction. The average fitness of inbred lines was correlated across generations. Our data provide evidence for the possibility of recovery of fitness at two levels, because (i) the average fitness reduction in the F20 generation was significantly less than in the F3 generation, which implies that selection within lines has occurred, and (ii) the large variance in inbreeding depression among inbred lines implies that selection among them is possible. The high variance in inbreeding depression among replicate lines implies that modes of evolution which require a low level of inbreeding depression can function at least in a fraction of inbred populations within a species and that results from studies with low levels of replication should be treated with caution.     

A genetic perspective on mating systems and sex ratios of parasitoid wasps

Parasitoid sex ratios are influenced by mating systems, whether complete inbreeding, partial inbreeding, complete inbreeding avoidance, or production of all-male broods by unmated females. Population genetic theory demonstrates that inbreeding is possible in haplodiploids because the purging of deleterious and lethal mutations through haploid males reduces inbreeding depression. However, this purging does not act quickly for deleterious mutations or female-limited traits (e.g., fecundity, host searching, sex ratio). The relationship between sex ratio, inbreeding, and inbreeding depression has not been explored in depth in parasitoids. The gregarious egg parasitoid, Trichogramma pretiosum Riley, collected from Riverside, CA (USA) produced a female-biased sex ratio of 0.24 (proportion of males). Six generations of sibling mating in the laboratory uncovered considerable inbreeding depression (∼ 20%) in fecundity and sex ratio. A population genetic study (based upon allozymes) showed the population was inbred (F _it = 0.246), which corresponds to 56.6% sib-mating. However, average relatedness among females emerging from the same host egg was only 0.646, which is less than expected (0.75) if ovipositing females mate randomly. This lower relatedness could arise from inbreeding avoidance, multiple mating by females, or superparasitism. A review of the literature in general shows relatively low inbreeding depression in haplodiploid species, but indicates that inbreeding depression can be as high as that found in Drosophila. Finally, mating systems and inbreeding depression are thought to evolve in concert (in plants), but similar dynamic models of the joint evolution of sex ratio, mating systems, and inbreeding depression have not been developed for parasitoid wasps. Received: November 13, 1998 /Accepted: January 8, 1999     

Genome‐wide regulatory deterioration impedes adaptive responses to stress in inbred populations of Drosophila melanogaster*

Inbreeding depression is often intensified under environmental stress (i.e., inbreeding–stress interaction). Although the fitness consequences of this phenomenon are well‐described, underlying mechanisms such as an increased expression of deleterious alleles under stress, or a lower capacity for adaptive responses to stress with inbreeding, have rarely been investigated. We investigated a fitness component (egg‐to‐adult viability) and gene‐expression patterns using RNA‐seq analyses in noninbred control lines and in inbred lines of Drosophila melanogaster exposed to benign temperature or heat stress. We find little support for an increase in the cumulative expression of deleterious alleles under stress. Instead, inbred individuals had a reduced ability to induce an adaptive gene regulatory stress response compared to controls. The decrease in egg‐to‐adult viability due to stress was most pronounced in the lines with the largest deviation in the adaptive stress response (R² = 0.48). Thus, we find strong evidence for a lower capacity of inbred individuals to respond by gene regulation to stress and that this is the main driver of inbreeding‐stress interactions. In comparison, the altered gene expression due to inbreeding at benign temperature showed no correlation with fitness and was pronounced in genomic regions experiencing the highest increase in homozygosity.     

Trait‐specific consequences of inbreeding on adaptive phenotypic plasticity

Environmental changes may stress organisms and stimulate an adaptive phenotypic response. Effects of inbreeding often interact with the environment and can decrease fitness of inbred individuals exposed to stress more so than that of outbred individuals. Such an interaction may stem from a reduced ability of inbred individuals to respond plastically to environmental stress; however, this hypothesis has rarely been tested. In this study, we mimicked the genetic constitution of natural inbred populations by rearing replicate Drosophila melanogaster populations for 25 generations at a reduced population size (10 individuals). The replicate inbred populations, as well as control populations reared at a population size of 500, were exposed to a benign developmental temperature and two developmental temperatures at the lower and upper margins of their viable range. Flies developed at the three temperatures were assessed for traits known to vary across temperatures, namely abdominal pigmentation, wing size, and wing shape. We found no significant difference in phenotypic plasticity in pigmentation or in wing size between inbred and control populations, but a significantly higher plasticity in wing shape across temperatures in inbred compared to control populations. Given that the norms of reaction for the noninbred control populations are adaptive, we conclude that a reduced ability to induce an adaptive phenotypic response to temperature changes is not a general consequence of inbreeding and thus not a general explanation of inbreeding–environment interaction effects on fitness components.     

Seasonal stress drives predictable changes in inbreeding depression in field-tested captive populations of Drosophila melanogaster

Recent meta-analyses conducted across a broad range of taxa have demonstrated a strong linear relationship between the change in magnitude of inbreeding depression under stress and stress level, measured as fitness loss in outbred individuals. This suggests that a general underlying response may link stress and inbreeding depression. However, this relationship is based primarily on laboratory data, and it is unknown whether natural environments with multiple stressors and fluctuating stress levels alter how stress affects inbreeding depression. To test whether the same pattern persists in the field, we investigated the effect of seasonal variation on stress level and inbreeding depression in a 3-year field study measuring the productivity of captive populations of inbred and outbred Drosophila melanogaster. We found cold winter temperatures were most stressful and induced the greatest inbreeding depression. Furthermore, these data, collected under natural field conditions, conformed to the same predictive linear relationship seen in Drosophila laboratory studies, with inbreeding depression increasing by 0.17 lethal equivalents for every 10 per cent increase in stress level. Our results suggest that under natural conditions stress level is a primary determinant of the magnitude of inbreeding depression and should be considered when assessing extinction vulnerability in small populations.     

GENETIC VARIATION IN INBREEDING DEPRESSION IN THE RED FLOUR BEETLE TRIBOLIUM CASTANEUM

Inbreeding depression varies among species and among populations within a species. Few studies, however, have considered the extent to which inbreeding depression varies within a single population. We report on two experiments to provide evidence that inbreeding depression is genetically variable, such that within a single population some lineages suffer severe inbreeding depression, others suffer only mild inbreeding depression, and some lineages actually increase in phenotypic value at higher levels of inbreeding. We examine the effects of population structure on inbreeding depression for two traits in the first experiment (adult dry weight and female relative fitness), and for seven traits in the second experiment (female and male adult dry weight, female and male relative fitness, female and male developmental time, and egg-to-adult viability). In the first experiment, we collected data from 4 families within each of 38 lineages derived from a single ancestral stock population and maintained for four generations of full-sib mating. Both traits demonstrate significant inbreeding depression and provide evidence that even within a single lineage there is significant genetic variability in inbreeding depression. In the second experiment, we collected data from 5 replicates for each of 15 lineages derived from the same ancestral population used in the first experiment; these lineages were maintained for four generations of full-sib mating. We also collected data on outbred control beetles in each generation and incorporated these data into the analyses to account for environmental effects in an unbiased manner. All traits except female and male developmental time show significant inbreeding depression. All traits showing inbreeding depression are genetically variable in inbreeding depression, as is evident from a significant linear lineage-×-f component. For both experiments, the effect of population structure on inbreeding depression is further evident from the increasing amount of variation that can be explained by the models used to measure inbreeding depression when additional levels of population structure are included. Genetic variation in inbreeding depression has important implications for conservation biology and may be an important factor in mating-system evolution.     

Constant, cycling, hot and cold thermal environments: strong effects on mean viability but not on genetic estimates

It has frequently been suggested that trait heritabilities are environmentally sensitive, and there are genetic trade-offs between tolerating different environments such as hot and cold or constant and fluctuating temperatures. Future climate predictions suggest an increase in both temperatures and their fluctuations. How species will respond to these changes is uncertain, particularly as there is a lack of studies which compare genetic performances in constant vs. fluctuating environments. In this study, we used a nested full-sib/half-sib breeding design to examine how the genetic variances and heritabilities of egg-to-adult viability differ at high and low temperatures with and without daily fluctuations in temperatures using Drosophila melanogaster as a model organism. Although egg-to-adult viability was clearly sensitive to developmental temperatures, heritabilities were not particularly sensitive to developmental temperatures. Moreover, we found that egg-to-adult viabilities at different developmental temperatures were positively correlated, suggesting a common genetic background for egg-to-adult viability at different temperatures. Finding both a uniform genetic background coupled with rather low heritabilities insensitive to temperatures, our results suggest evolutionary responses are unlikely to be limited by temperature effects on genetic parameters or negative genetic correlations, but by the direct effects of stressful temperatures on egg-to-adult viability accompanied with low heritabilities.     

Effects of inbreeding on aversive learning in Drosophila

Inbreeding adversely affects life history traits as well as various other fitness‐related traits, but its effect on cognitive traits remains largely unexplored, despite their importance to fitness of many animals under natural conditions. We studied the effects of inbreeding on aversive learning (avoidance of an odour previously associated with mechanical shock) in multiple inbred lines of Drosophila melanogaster derived from a natural population through up to 12 generations of sib mating. Whereas the strongly inbred lines after 12 generations of inbreeding (0.75 < F < 0.93) consistently showed reduced egg‐to‐adult viability (on average by 28%), the reduction in learning performance varied among assays (average = 18% reduction), being most pronounced for intermediate conditioning intensity. Furthermore, moderately inbred lines (F = 0.38) showed no detectable decline in learning performance, but still had reduced egg‐to‐adult viability, which indicates that overall inbreeding effects on learning are mild. Learning performance varied among strongly inbred lines, indicating the presence of segregating variance for learning in the base population. However, the learning performance of some inbred lines matched that of outbred flies, supporting the dominance rather than the overdominance model of inbreeding depression for this trait. Across the inbred lines, learning performance was positively correlated with the egg‐to‐adult viability. This positive genetic correlation contradicts a trade‐off observed in previous selection experiments and suggests that much of the genetic variation for learning is owing to pleiotropic effects of genes affecting functions related to survival. These results suggest that genetic variation that affects learning specifically (rather than pleiotropically through general physiological condition) is either low or mostly due to alleles with additive (semi‐dominant) effects.     

Does inbreeding avoidance maintain gender dimorphism in Wurmbea dioica (Colchicaceae)?

The maintenance of females in gender dimorphic populations requires that they have a fitness advantage to compensate for their loss of male reproductive function. We assess whether inbreeding avoidance provides this advantage in two subdioecious Wurmbea dioica populations by estimating seed production, outcrossing rates and inbreeding depression. Fruiting males produced less than half as many seeds as females, owing to low outcrossing rates and early acting inbreeding depression. Inbreeding coefficients of fruiting males demonstrated that progeny were more inbred than their parents, implying that few selfed progeny reach maturity, as confirmed by inbreeding depression estimates that exceeded 0.85. In a glasshouse experiment, open-pollinated females exhibited a fitness advantage of 3.7 relative to fruiting males, but when we increased fruiting male outcrossing rate, female advantage was only 1.4. This reduced advantage is insufficient to maintain females if nuclear genes control sex. Thus, inbreeding avoidance could maintain females at high frequencies, although this is contingent upon high frequencies of fruiting males, which can be altered by environmentally determined gender plasticity.