Severe outbreeding and inbreeding depression maintain mating system differentiation in Epipactis (Orchidaceae)

In hermaphroditic plants, theory for mating system evolution predicts that populations will evolve to either complete autonomous selfing (AS) or complete outcrossing, depending on the balance between automatic selection favouring self‐fertilization and costs resulting from inbreeding depression (ID). Theory also predicts that selection for selfing can occur rapidly and is driven by purging of genetic load and the loss of ID. Therefore, selfing species are predicted to have low levels of ID or even to suffer from outbreeding depression (OD), whereas predominantly outcrossing species are expected to have high levels of ID. To test these predictions, we related the capacity of AS to the magnitude of early‐acting inbreeding or OD in both allogamous and autogamous species of the orchid genus Epipactis. For each species, the level of AS was assessed under controlled greenhouse conditions, whereas hand‐pollinations were performed to quantify early costs of inbreeding or OD acting at the level of fruit and seed production. In the autogamous species, the capacity of AS was high (> 0.72), whereas in the allogamous species AS was virtually absent (< 0.10). Consistent with our hypothesis, allogamous Epipactis species had significantly higher total ID (average: 0.46) than autogamous species, which showed severe costs of OD (average: ?0.45). Overall, our findings indicate that strong early‐acting ID represents an important mechanism that contributes to allogamy in Epipactis, whereas OD may maintain selfing in species that have evolved to complete selfing.     

Inbreeding depression is high in a self‐incompatible perennial herb population but absent in a self‐compatible population showing mixed mating

High inbreeding depression is thought to be one of the major factors preventing evolutionary transitions in hermaphroditic plants from self‐incompatibility (SI) and outcrossing toward self‐compatibility (SC) and selfing. However, when selfing does evolve, inbreeding depression can be quickly purged, allowing the evolution of complete self‐fertilization. In contrast, populations that show intermediate selfing rates (a mixed‐mating system) typically show levels of inbreeding depression similar to those in outcrossing species, suggesting that selection against inbreeding might be responsible for preventing the transition toward complete self‐fertilization. By implication, crosses among populations should reveal patterns of heterosis for mixed‐mating populations that are similar to those expected for outcrossing populations. Using hand‐pollination crosses, we compared levels of inbreeding depression and heterosis between populations of Linaria cavanillesii (Plantaginaceae), a perennial herb showing contrasting mating systems. The SI population showed high inbreeding depression, whereas the SC population displaying mixed mating showed no inbreeding depression. In contrast, we found that heterosis based on between‐population crosses was similar for SI and SC populations. Our results are consistent with the rapid purging of inbreeding depression in the derived SC population, despite the persistence of mixed mating. However, the maintenance of outcrossing after a transition to SC is inconsistent with the prediction that populations that have purged their inbreeding depression should evolve toward complete selfing, suggesting that the transition to SC in L. cavanillesii has been recent. SC in L. cavanillesii thus exemplifies a situation in which the mating system is likely not at an equilibrium with inbreeding depression.     

Little to no inbreeding depression in a tapeworm with mixed mating

Meta‐studies on hermaphrodites have found a negative relationship between primary selfing rates and levels of inbreeding depression (ID) and, thus, generally support purging in inbred systems. However, in plants, high among‐taxa variance in ID results in no difference in the mean ID between outcrossing and mixed‐mating taxa. Selective interference likely explains high ID among mixed‐mating taxa, whereas low levels of ID among mixed‐mating taxa are not as stressed. Among animal hermaphrodites, primarily molluscs, there are little data on mixed‐mating systems. To fill a taxonomic and mating system gap, we tested for ID in a mixed‐mating tapeworm, Oochoristica javaensis. We provide a direct estimate of ID across infection of an intermediate host by comparing selfing rates at two life history stages. We found little to no evidence for ID, and the level of ID falls in line with what is reported for highly selfing species even though O. javaensis has mixed mating. We discuss this result within the context of kin mating in O. javaensis. Our results emphasize that primary selfing rates alone may be insufficient to classify the inbreeding history in all species when testing for a relationship to ID. Mixed‐mating taxa, and possibly some outcrossing taxa, may exhibit low levels of ID if biparental inbreeding is also driving purging. We advocate that ID studies report estimates of inbreeding history (e.g. F_IS or identity disequilibrium) from nature‐derived adult samples to provide context rather than relying on primary selfing rates alone.     

Anther-stigma separation is associated with inbreeding depression in Datura stramonium,a predominantly self-fertilizing annual

Abstract.— Genetically based variation in outcrossing rate generates lineages within populations that differ in their history of inbreeding. According to some models, mating-system modifiers in such populations will demonstrate both linkage and identity disequilibrium with fitness loci, resulting in lineage-specific inbreeding depression. Other models assert that differences among families in levels of inbreeding depression are mainly attributable to random accumulation of genetic load, unrelated to variation at mating-system loci. We measured female reproductive success of selfed and outcrossed progeny from naturally occurring lineages of Datura stramonium , a predominantly self-fertilizing annual weed that has heritable variation in stigma-anther separation, a trait that influences selfing rates. Progeny from inbred lineages (as identified by high degree of anther-stigma overlap) showed equal levels of seed production, regardless of cross type. Progeny from mixed lineages (as identified by relatively high separation between anthers and stigma) showed moderate levels of inbreeding depression. We found a significant correlation between anther-stigma separation and relative fitness of selfed and outcrossed progeny, suggesting that family-level inbreeding depression may be related to differences among lineages in inbreeding history in this population. Negative inbreeding depression in putatively inbred lineages may be due in part to additive effects or to epistatic interactions among loci.     

Family level inbreeding depression and the evolution of plant mating systems

Variation in the magnitude of inbreeding depression (ID) among families may have important consequences for mating system evolution. Experimental studies have shown that such variation is a common feature of natural plant populations. Unfortunately, the genetic and evolutionary significance of family level estimates remains obscure. Almost any kind of genetic variation will generate differences in ID among families, and as a consequence, a non-zero variance in family level ID is not sufficient to distinguish genetic architectures with wholly different implications for mating system evolution. Quantitative genetic methods provide a means to extract more information from ID experiments. Estimates of quantitative genetic variance components directly inform questions about the genetic basis of ID and should ultimately allow tests of alternative theories of mating system evolution.     

Reduced inbreeding depression due to historical inbreeding in Drosophila melanogaster: evidence for purging

An important issue in conservation biology and the study of evolution is the extent to which inbreeding depression can be reduced or reversed by natural selection. If the deleterious recessive alleles causing inbreeding depression can be 'purged' by natural selection, outbred populations that have a history of inbreeding are expected to be less susceptible to inbreeding depression. This expectation, however, has not been realized in previous laboratory experiments. In the present study, we used Drosophila melanogaster as a model system to test for an association between inbreeding history and inbreeding depression. We created six 'purged' populations from experimental lineages that had been maintained at a population size of 10 male-female pairs for 19 generations. We then measured the inbreeding depression that resulted from one generation of full-sib mating in the purged populations and in the original base population. The magnitude of inbreeding depression in the purged populations was approximately one-third of that observed in the original base population. In contrast to previous laboratory experiments, therefore, we found that inbreeding depression was reduced in populations that have a history of inbreeding. The large purging effects observed in this study may be attributable to the rate of historical inbreeding examined, which was slower than that considered in previous experiments.     

Effect of recurrent selfing on inbreeding depression and mating system evolution in an autopolyploid plant

Genome duplication resulting in polyploidy can have significant consequences for the evolution of mating systems. Most theory predicts that self‐fertilization will be selectively favored in polyploids; however, many autopolyploids are outcrossing or mixed‐mating. Here, we examine the hypothesis that the evolution of selfing is restricted in autopolyploids because the genetic cost of selfing (i.e., inbreeding depression) increases monotonically with successive generations of inbreeding. Using the herbaceous, autotetraploid plant Chamerion angustifolium, we generated populations with different inbreeding coefficients (F= 0, 0.17 and 0.36) through three consecutive generations of selfing and compared their magnitudes of inbreeding depression in a common environment. Mating system estimates for four natural populations confirmed that tetraploid selfing rates (s_m= 0.25, SE = 0.02) are similar to those of diploids (s_m= 0.12, SE = 0.12; F_1,2= 1.34, P= 0.37) indicating that both cytotypes are predominantly outcrossing. Compared to an outbred control line, mean inbreeding depression for seed production, survival, and height (vegetative and total) in the inbred line differed among generations (inbreeding coefficients). Across all stages, inbreeding depression (relative to control) was positively related to generation (inbreeding coefficient). Although the initial costs of inbreeding in extant and newly synthesized polyploids may be low compared to diploids, the monotonic increase in inbreeding depression with repeated inbreeding may limit the extent to which selfing variants are favored.     

Interpopulation variation in mating system and late-stage inbreeding depression in Magnolia stellata

Inbreeding has the potential to cause evolutionary changes in populations, although these changes are likely to drive populations to extinction through inbreeding depression and reductions in genetic diversity. We investigated the mating system and late-stage inbreeding depression (δ) in 10 populations of Magnolia stellata using nine microsatellite markers and evaluated the effects of population size and the degree of population isolation through inbreeding and inbreeding depression on the persistence of populations. The outcrossing rates were very similar (~0.7) among populations, but the correlations of paternity, fractions of biparental inbreeding and inbreeding coefficients at the seed stage ( F _S) varied among populations, suggesting that the level of outcrossing was similar among populations, while the quality of it was not. A significant negative correlation was detected between F _S and population size. The average value of δ was 0.709, and the values in six of the 10 populations were significant. The values of δ differed among populations, although clear relationships with population size and the degree of population isolation were not detected. However, in one population, which was very small and located in the edge of the species' range, we obtained a very low value of δ (–0.096), which may be indicative of purging or the fixation of deleterious alleles. Existing M. stellata populations that are small (and thus might be expected to have higher frequencies of inbreeding) and have large values of δ may be in danger of declining, even if the populations are located within the central region of the species' range.     

Variation of selfing rate and inbreeding depression among individuals and across generations within an admixed Cedrus population

We investigated the variation and short-term evolution of the selfing rate and inbreeding depression (ID) across three generations within a cedar forest that was established from admixture ca 1860. The mean selfing rate was 9.5%, ranging from 0 to 48% among 20 seed trees (estimated from paternally inherited chloroplast DNA). We computed the probability of selfing for each seed and we investigated ID by comparing selfed and outcrossed seeds within progenies, thus avoiding maternal effects. In all progenies, the germination rate was high (88–100%) and seedling mortality was low (0–12%). The germination dynamics differed significantly between selfed and outcrossed seeds within progenies in the founder gene pool but not in the following generations. This transient effect of selfing could be attributed to epistatic interactions in the original admixture. Regarding the seedling growth traits, the ID was low but significant: 8 and 6% for height and diameter growth, respectively. These rates did not vary among generations, suggesting minor gene effects. At this early stage, outcrossed seedlings outcompeted their selfed relatives, but not necessarily other selfed seedlings from other progenies. Thus, purging these slightly deleterious genes may only occur through within-family selection. Processes that maintain a high level of genetic diversity for fitness-related traits among progenies also reduce the efficiency of purging this part of the genetic load.     

Inbreeding and outbreeding depression in Caenorhabditis nematodes

The nematode Caenorhabditis elegans reproduces primarily by self-fertilization of hermaphrodites, yet males are present at low frequencies in natural populations (androdioecy). The ancestral state of C. elegans was probably gonochorism (separate males and females), as in its relative C. remanei. Males may be maintained in C. elegans because outcrossed individuals escape inbreeding depression. The level of inbreeding depression is, however, expected to be low in such a highly selfing species, compared with an outcrosser like C. remanei. To investigate these issues, we measured life-history traits in the progeny of inbred versus outcrossed C. elegans and C. remanei individuals derived from recently isolated natural populations. In addition, we maintained inbred lines of C. remanei through 13 generations of full-sibling mating. Highly inbred C. remanei showed dramatic reductions in brood size and relative fitness compared to outcrossed individuals, with evidence of both direct genetic and maternal-effect inbreeding depression. This decline in fitness accumulated over time, causing extinction of nearly 90% of inbred lines, with no evidence of purging of deleterious mutations from the remaining lines. In contrast, pure strains of C. elegans performed better than crosses between strains, indicating outbreeding depression. The results are discussed in relation to the evolution of androdioecy and the effect of mating system on the level of inbreeding depression.     

Role of parasite transmission in promoting inbreeding: II. Pedigree reconstruction reveals sib‐transmission and consequent kin‐mating

Even though parasitic flatworms are one of the most species‐rich groups of hermaphroditic organisms, we know virtually nothing of their mating systems (selfing or kin‐mating rates) in nature. Hence, we lack an understanding of the role of inbreeding in parasite evolution. The natural mating systems of parasitic flatworms have remained elusive due to the inherent difficulty in generating progeny‐array data in many parasite systems. New developments in pedigree reconstruction allow direct inference of realized selfing rates in nature by simply using a sample of genotyped individuals. We built upon this advancement by utilizing the closed mating systems, that is, individual hosts, of endoparasites. In particular, we created a novel means to use pedigree reconstruction data to estimate potential kin‐mating rates. With data from natural populations of a tapeworm, we demonstrated how our newly developed methods can be used to test for cosibling transmission and inbreeding depression. We then showed how independent estimates of the two mating system components, selfing and kin‐mating rates, account for the observed levels of inbreeding in the populations. Thus, our results suggest that these natural parasite populations are in inbreeding equilibrium. Pedigree reconstruction analyses along with the new companion methods we developed will be broadly applicable across a myriad of parasite species. As such, we foresee that a new frontier will emerge wherein the diverse life histories of flatworm parasites could be utilized in comparative evolutionary studies to broadly address ecological factors or life history traits that drive mating systems and hence inbreeding in natural populations.     

The effects of age and environment on the expression of inbreeding depression in Eucalyptus globulus

Inbreeding adversely affects fitness traits in many plant and animal species, and the magnitude, stability and genetic basis of inbreeding depression (ID) will have short- and long-term evolutionary consequences. The effects of four degrees of inbreeding (selfing, f=50%; full- and half-sib matings, f=25 and 12.5%; and unrelated outcrosses, f=0%) on survival and growth of an island population of Eucalyptus globulus were studied at two sites for over 14 years. For selfs, ID in survival increased over time, reaching a maximum of 49% by age 14 years. However, their inbreeding depression for stem diameter remained relatively stable with age, and ranged from 28 to 36% across years and sites. ID for survival was markedly greater on the more productive site, possibly due to greater and earlier onset of inter-tree competition, but was similar on both sites for the diameter of survivors. The deleterious trait response to increasing inbreeding coefficients was linear for survival and diameter. Non-significant quadratic effects suggested that epistasis did not contribute considerably to the observed ID at the population level. Among- and within-family coefficients of variation for diameter increased with inbreeding degree, and the variance among the outcrossed families was significant only on the more productive site. The performance of self-families for diameter was highly stable between sites. This suggests that, for species with mixed mating systems, environmentally stable inbreeding effects in open-pollinated progenies may tend to mask the additive genotype-by-environment interaction for fitness traits and the adaptive response to the environment.     

An explicit model for the inbreeding load in the evolutionary analysis of selfing

I present analytical predictions for the equilibrium inbreeding load expected in a population under mutation, selection, and a regular mating system for any population size and for any magnitude and recessivity of the deleterious effects. Using this prediction, I deduce the relative fitness of mutant alleles with small effect on selfing to explore the situations where selfing or outcrossing are expected to evolve. The results obtained are in agreement with previous literature, showing that natural selection is expected to lead to stable equilibria where populations show either complete outcrossing or complete selfing, and that selfing is promoted by large deleterious mutation rates. I find that the evolution of selfing is favored by a large recessivity of deleterious effects, while the magnitude of homozygous deleterious effects only becomes relevant in relatively small populations. This result contradicts the standard assumption that purging in large populations will only promote selfing when homozygous deleterious effects are large, and implies that previously published results obtained assuming lethal mutations in large populations can be extrapolated to nonlethal alleles of similar recessivity. This conclusion and the general approach used in this analysis can be useful in the study of the evolution of mating systems.     

Heterosis and inbreeding depression in bottlenecked populations: a test in the hermaphroditic freshwater snail Lymnaea stagnalis

Small population size is expected to induce heterosis, due to the random fixation and accumulation of mildly deleterious mutations, whereas within‐population inbreeding depression should decrease due to increased homozygosity. Population bottlenecks, although less effective, may have similar consequences. We tested this hypothesis in the self‐fertile freshwater snail Lymnaea stagnalis, by subjecting experimental populations to a single bottleneck of varied magnitude. Although patterns were not strong, heterosis was significant in the most severely bottlenecked populations, under stressful conditions. This was mainly due to hatching rate, suggesting that early acting and highly deleterious alleles were involved. Although L. stagnalis is a preferential outcrosser, inbreeding depression was very low and showed no clear relationship with bottleneck size. In the less reduced populations, inbreeding depression for hatching success increased under high inbreeding. This may be consistent with the occurence of synergistic epistasis between fitness loci, which may contribute to favour outcrossing in L. stagnalis.     

The decline in fitness with inbreeding: evidence for negative dominance‐by‐dominance epistasis in Drosophila melanogaster

Genetic interactions can play an important role in the evolution of reproductive strategies. In particular, negative dominance‐by‐dominance epistasis for fitness can theoretically favour sex and recombination. This form of epistasis can be detected statistically because it generates nonlinearity in the relationship between fitness and inbreeding coefficient. Measures of fitness in progressively inbred lines tend to show limited evidence for epistasis. However, tests of this kind can be biased against detecting an accelerating decline due to line losses at higher inbreeding levels. We tested for dominance‐by‐dominance epistasis in Drosophila melanogaster by examining viability at five inbreeding levels that were generated simultaneously, avoiding the bias against detecting nonlinearity that has affected previous studies. We find an accelerating rate of fitness decline with inbreeding, indicating that dominance‐by‐dominance epistasis is negative on average, which should favour sex and recombination.     

Inbreeding avoidance and the evolution of gender dimorphism in Wurmbea biglandulosa (Colchicaceae)

How females establish in populations of cosexuals is central to understanding the evolution of gender dimorphism in angiosperms. Inbreeding avoidance hypotheses propose that females can establish and be maintained if cosexual fitness is reduced because they self-fertilize, and their progeny express inbreeding depression. Here we assess the role of inbreeding avoidance in maintaining sexual system variation in Wurmbea biglandulosa. We estimated costs of self-pollination, mating patterns, and inbreeding depression in gender monomorphic (cosexuals only) and dimorphic (males and females) populations. Costs of selfing, estimated from seed set of experimentally self- and cross-pollinated flowers, were severe in both males and cosexuals (inbreeding depression, sigma = 0.86). In a field experiment, intact males that could self produced fewer seeds than both emasculated males and females, whereas seed set of intact and emasculated cosexuals did not differ. Thus, pollinator-mediated selfing reduces fitness of males but not cosexuals under natural conditions. Outcrossing rates of males revealed substantial selfing (t = 0.68), whereas females and cosexuals were outcrossed (0.92 and 0.97). For males, progeny inbreeding coefficients exceeded parental coefficients (0.220 vs. 0.009), whereas for females and cosexuals these coefficients did not differ and approached zero. Differences in coefficients between males and their progeny indicate that selfed progeny express severe inbreeding depression (sigma = 0.93). Combined with inbreeding depression for seed set, cumulative sigma = 0.99, indicating that most or all selfed zygotes fail to reach reproductive maturity. We propose that present sexual system variation in W. biglandulosa is maintained by high inbreeding depression coupled with differences in selfing rates among monomorphic and dimorphic populations.     

Variation in self-fertility,inbreeding depression and levels of inbreeding in four Cyclamen species

The ability to self in the absence of pollinators, i.e. reproductive assurance, and the detrimental consequences of inbreeding, i.e. inbreeding depression, are central factors influencing plant mating system evolution. The purpose of this study was to quantify whether self-fertility and inbreeding depression are related to levels of inbreeding in four Cyclamen species, namely C. balearicum (mean F_is = 0.930), C. creticum (mean F_is = 0.748), C. repandum (mean F_is = 0.658) and C. hederifolium (mean F_is = 0.329). C. balearicum showed a markedly greater capacity to autonomously self-fertilize than the three other species, which may have favoured inbreeding in this species. Levels of inbreeding depression were highest in C. creticum and C. hederifolium at the fruit maturation (δ = 0.18 and 0.20, respectively) and seed number (δ = 0.32 and 0.30, respectively) stages, and for C. repandum at the seed weight stage (δ = 0.23). Although C. balearicum showed inbreeding depression on seed germination (δ = 0.45), this may be an artefact of the generally low levels of seed germination in the experiment. Overall, we observed only limited evidence for the predicted negative relation between inbreeding coefficients and levels of inbreeding since C. creticum had high levels of inbreeding and inbreeding depression. Other factors may thus influence the relationship between inbreeding and inbreeding depression in these species.     

Purging of inbreeding depression and fitness decline in bottlenecked populations of Drosophila melanogaster

Drastic reductions in population size, or bottlenecks, are thought to significantly erode genetic variability and reduce fitness. However, it has been suggested that a population can be purged of the genetic load responsible for reduced fitness when subjected to bottlenecks. To investigate this phenomenon, we put a number of Drosophila melanogaster isofemale lines known to differ in inbreeding depression through four ‘founder‐flush’ bottleneck cycles with flush sizes of 5 or 100 pairs and assayed for relative fitness (single‐pair productivity) after each cycle. Following the founder‐flush phase, the isofemale lines, with a large flush size and a history of inbreeding depression, recovered most of the fitness lost from early inbreeding, consistent with purging. The same isofemale lines, with a small flush size, did not regain fitness, consistent with the greater effect of genetic drift in these isofemale lines. On the other hand, the isofemale lines that did not show initial inbreeding depression declined in fitness after repeated bottlenecks, independent of the flush size. These results suggest that the nature of genetic variation in fitness may greatly influence the way in which populations respond to bottlenecks and that stochastic processes play an important role. Consequently, an attempt intentionally to purge a population of detrimental variation through inbreeding appears to be a risky strategy, particularly in the genetic management of endangered species.     

Deleterious mutation in related species of the plant genus Amsinckia with contrasting mating systems

Theory for the evolution of modifiers of the rate of mutation suggests that a lower rate of mutation may evolve after the breakdown of mechanisms that enforce outcrossing. Mutation accumulation (MA) experiments were conducted to compare deleterious mutation parameters in two closely related species of the plant genus Amsinckia, a group that exhibits wide variation in the mating system. One of the two species studied (A. douglasiana) is predominantly outcrossed in natural populations, where as the other species (A. gloriosa) is predominantly self-pollinated. Progeny assays of flower number per plant from generation 1 lines (control) and generation 11 lines (MA treatment) were conducted in both species. Dry weight measurements of progeny from the control and MA treatment in A. douglasiana also were made. Estimation of mutation parameters was conducted using maximum likelihood under the assumption of a gamma distribution of mutational effects. The two species exhibited similar rates and effects of deleterious mutation affecting flower number. Estimates of mutation rate for dry weight in A. douglasiana are close to those for flower number. Overall, the estimates of mutation parameters observed in these species are intermediate within the range reported for fitness components in other eukaryotes. The results are discussed within the context of evolutionary change in deleterious mutation accompanying mating system evolution and with respect to previous estimates of mutation parameters based on assays of inbreeding depression and the assumption of mutation-selection equilibrium.     

Relative fitness of two hermaphroditic mating types in the androdioecious clam shrimp,Eulimnadia texana

Androdioecy (populations of males and hermaphrodites) is a rare reproductive form, being described from only a handful of plants and animals. One of these is the shrimp Eulimnadia texana, which has populations comprised of three mating types: two hermaphroditic types (monogenics and amphigenics) and males. In a recent study, the amphigenic hermaphrodites were found to be in greater abundance than that predicted from a model of this mating system. Herein, we compare the relative fitness of offspring from amphigenic and monogenic siblings, attempting to understand the greater relative abundance of the former. Populations started with offspring from selfed monogenic hermaphrodites had a net reproductive rate (R) 87% that of offspring from their amphigenic siblings. Additionally, within populations of amphigenic offspring (which included males, monogenics and amphigenics), amphigenics survived longer than monogenics. These differences help to explain the increased relative abundance of amphigenics in natural populations, but amphigenics continue to be more abundant than expected.