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.     

Microsatellites reveal substantial among-population genetic differentiation and strong inbreeding in the relict fern Dryopteris aemula

Background and Aims

A previous study detected no allozyme diversity in Iberian populations of the buckler-fern Dryopteris aemula. The use of a more sensitive marker, such as microsatellites, was thus needed to reveal the genetic diversity, breeding system and spatial genetic structure of this species in natural populations.

Methods

Eight microsatellite loci for D. aemula were developed and their cross-amplification with other ferns was tested. Five polymorphic loci were used to characterize the amount and distribution of genetic diversity of D. aemula in three populations from the Iberian Peninsula and one population from the Azores.

Key Results

Most microsatellite markers developed were transferable to taxa close to D. aemula. Overall genetic variation was low (H_T = 0·447), but was higher in the Azorean population than in the Iberian populations of this species. Among-population genetic differentiation was high (F_ST = 0·520). All loci strongly departed from Hardy–Weinberg equilibrium. In the population where genetic structure was studied, no spatial autocorrelation was found in any distance class.

Conclusions

The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy–Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa.     

The role of inbreeding and outbreeding in herbivore resistance and tolerance in Vincetoxicum hirundinaria

Background and Aims

Inbreeding via self-fertilization may have negative effects on plant fitness (i.e. inbreeding depression). Outbreeding, or cross-fertilization between genetically dissimilar parental plants, may also disrupt local adaptation or allelic co-adaptation in the offspring and again lead to reduced plant fitness (i.e. outbreeding depression). Inbreeding and outbreeding may also increase plant vulnerability to natural enemies by altering plant quality or defence. The effects of inbreeding and outbreeding on plant size and response to herbivory in the perennial herb, Vincetoxicum hirundinaria, were investigated.

Methods

Greenhouse experiments were conducted using inbred and outbred (within- and between-population) offspring of 20 maternal plants from four different populations, quantifying plant germination, size, resistance against the specialist folivore, Abrostola asclepiadis, and tolerance of simulated defoliation.

Key Results

Selfed plants were smaller and more susceptible to damage by A. asclepiadis than outcrossed plants. However, herbivore biomass on selfed and outcrossed plants did not differ. The effects of inbreeding on plant performance and resistance did not differ among plant populations or families, and no inbreeding depression at all was found in tolerance of defoliation. Between-population outcrossing had no effect on plant performance or resistance against A. asclepiadis, indicating a lack of outbreeding depression.

Conclusions

Since inbreeding depression negatively affects plant size and herbivore resistance, inbreeding may modify the evolution of the interaction between V. hirundinaria and its specialist folivore. The results further suggest that herbivory may contribute to the maintenance of a mixed mating system of the host plants by selecting for outcrossing and reduced susceptibility to herbivore attack, and thus add to the growing body of evidence on the effects of inbreeding on the mating system evolution of the host plants and the dynamics of plant–herbivore interactions.     

Disease-mediated inbreeding depression in a large,open population of cooperative crows

Disease-mediated inbreeding depression is a potential cost of living in groups with kin, but its general magnitude in wild populations is unclear. We examined the relationships between inbreeding, survival and disease for 312 offspring, produced by 35 parental pairs, in a large, open population of cooperatively breeding American crows (Corvus brachyrhynchos). Genetic analyses of parentage, parental relatedness coefficients and pedigree information suggested that 23 per cent of parental dyads were first- or second-order kin. Heterozygosity–heterozygosity correlations suggested that a microsatellite-based index of individual heterozygosity predicted individual genome-wide heterozygosity in this population. After excluding birds that died traumatically, survival probability was lower for relatively inbred birds during the 2–50 months after banding: the hazard rate for the most inbred birds was 170 per cent higher than that for the least inbred birds across the range of inbreeding index values. Birds that died with disease symptoms had higher inbreeding indices than birds with other fates. Our results suggest that avoidance of close inbreeding and the absence of inbreeding depression in large, open populations should not be assumed in taxa with kin-based social systems, and that microsatellite-based indices of individual heterozygosity can be an appropriate tool for examining the inbreeding depression in populations where incest and close inbreeding occur.     

Heterosis is common and inbreeding depression absent in natural populations of Arabidopsis thaliana

The importance of genetic drift in shaping patterns of adaptive genetic variation in nature is poorly known. Genetic drift should drive partially recessive deleterious mutations to high frequency, and inter‐population crosses may therefore exhibit heterosis (increased fitness relative to intra‐population crosses). Low genetic diversity and greater genetic distance between populations should increase the magnitude of heterosis. Moreover, drift and selection should remove strongly deleterious recessive alleles from individual populations, resulting in reduced inbreeding depression. To estimate heterosis, we crossed 90 independent line pairs of Arabidopsis thaliana from 15 pairs of natural populations sampled across Fennoscandia and crossed an additional 41 line pairs from a subset of four of these populations to estimate inbreeding depression. We measured lifetime fitness of crosses relative to parents in a large outdoor common garden (8,448 plants in total) in central Sweden. To examine the effects of genetic diversity and genetic distance on heterosis, we genotyped parental lines for 869 SNPs. Overall, genetic variation within populations was low (median expected heterozygosity = 0.02), and genetic differentiation was high (median F_ST = 0.82). Crosses between 10 of 15 population pairs exhibited significant heterosis, with magnitudes of heterosis as high as 117%. We found no significant inbreeding depression, suggesting that the observed heterosis is due to fixation of mildly deleterious alleles within populations. Widespread and substantial heterosis indicates an important role for drift in shaping genetic variation, but there was no significant relationship between fitness of crosses relative to parents and genetic diversity or genetic distance between populations.     

Between a rock and a hard place: evaluating the relative risks of inbreeding and outbreeding for conservation and management

As populations become increasingly fragmented, managers are often faced with the dilemma that intentional hybridization might save a population from inbreeding depression but it might also induce outbreeding depression. While empirical evidence for inbreeding depression is vastly greater than that for outbreeding depression, the available data suggest that risks of outbreeding, particularly in the second generation, are on par with the risks of inbreeding. Predicting the relative risks in any particular situation is complicated by variation among taxa, characters being measured, level of divergence between hybridizing populations, mating history, environmental conditions and the potential for inbreeding and outbreeding effects to be occurring simultaneously. Further work on consequences of interpopulation hybridization is sorely needed with particular emphasis on the taxonomic scope, the duration of fitness problems and the joint effects of inbreeding and outbreeding. Meanwhile, managers can minimize the risks of both inbreeding and outbreeding by using intentional hybridization only for populations clearly suffering from inbreeding depression, maximizing the genetic and adaptive similarity between populations, and testing the effects of hybridization for at least two generations whenever possible.     

Mating structure and inbreeding and outbreeding depression in the rare plant Gentianella germanica (Gentianaceae)

Isolation and small size of populations as a result of habitat destruction and fragmentation may negatively affect plant fitness through pollinator limitation and increased levels of inbreeding. To increase genetic variation in small populations of rare plants artificial gene flow has been suggested as a management tool. We investigated whether pollinator limitation and inbreeding depression could reduce fitness in Gentianella germanica, an endangered biennial of increasingly fragmented calcareous grasslands in Central Europe. We experimentally excluded pollinators and generated progenies by hand-pollinating flowers with pollen from different distances. G. germanica was highly selfing. Pollinator exclusion strongly reduced seed set, indicating that pollinator limitation could potentially reduce plant fitness. Germination rate as well as number of leaves and rosette size of progeny from 10-m crosses was higher than that of progeny from open pollinations, self-, 1-m, and interpopulation crosses. After 6 mo of growth differences in the number of surviving plants persisted, whereas differences in plant size did not. The results suggest that inbreeding depression may reduce plant performance in G. germanica. Outbreeding depression in the performance of progeny from interpopulation crosses indicates that caution is necessary in using artificial interpopulation gene flow as a management tool.     

Evidence for an epigenetic role in inbreeding depression

Inbreeding depression (i.e. negative fitness effects of inbreeding) is central in evolutionary biology, affecting numerous aspects of population dynamics and demography, such as the evolution of mating systems, dispersal behaviour and the genetics of quantitative traits. Inbreeding depression is commonly observed in animals and plants. Here, we demonstrate that, in addition to genetic processes, epigenetic processes may play an important role in causing inbreeding effects. We compared epigenetic markers of outbred and inbred offspring of the perennial plant Scabiosa columbaria and found that inbreeding increases DNA methylation. Moreover, we found that inbreeding depression disappears when epigenetic variation is modified by treatment with a demethylation agent, linking inbreeding depression firmly to epigenetic variation. Our results suggest an as yet unknown mechanism for inbreeding effects and demonstrate the importance of evaluating the role of epigenetic processes in inbreeding depression.     

Epistatic and cytonuclear interactions govern outbreeding depression in the autotetraploid Campanulastrum americanum

The consequences of combining divergent genomes among populations of a diploid species often involve F1 hybrid vigor followed by hybrid breakdown in later recombinant generations. As many as 70% of plant species are thought to have polyploid origins; yet little is known about the genetic architecture of divergence in polyploids and how it may differ from diploid species. We investigated the genetic architecture of population divergence using controlled crosses among five populations of the autotetraploid herb, Campanulastrum americanum. Plants were reciprocally hybridized to produce F1, F2, and F1-backcross generations that were grown with parental types in a greenhouse and measured for performance. In contrast to diploid expectations, most F1 hybrids lacked heterosis and instead showed strong outbreeding depression for early life traits. Recombinant hybrid generations often showed a recovery of performance to levels approximating, or at times even exceeding, the parental values. This pattern was also evident for an index of cumulative fitness. Analyses of line means indicated nonadditive gene action, especially forms of digenic epistasis, often influenced hybrid performance. However, standard diploid genetic models were not adequate for describing the underlying genetic architecture in a number of cases. Differences between reciprocal hybrids indicated that cytoplasmic and/or cytonuclear interactions also contributed to divergence. An enhanced role of epistasis in population differentiation may be the norm in polyploids, which have more gene copies. This study, the first of its kind on a natural autotetraploid, suggests that gene duplication may cause polyploid populations to diverge in a fundamentally different way than diploids.     

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.     

Dispersal as a means of inbreeding avoidance in a wild bird population

The long-term study of animal populations facilitates detailed analysis of processes otherwise difficult to measure, and whose significance may appear only when a large sample size from many years is available for analysis. For example, inbreeding is a rare event in most natural populations, and therefore many years of data are needed to estimate its effect on fitness. A key behaviour hypothesized to play an important role in avoiding inbreeding is natal dispersal. However, the functional significance of natal dispersal with respect to inbreeding has been much debated but subject to very few empirical tests. We analysed 44 years of data from a wild great tit Parus major population involving over 5000 natal dispersal events within Wytham Woods, UK. Individuals breeding with a relative dispersed over several-fold shorter distances than those outbreeding; within the class of inbreeding birds, increased inbreeding was associated with reduced dispersal distance, for both males and females. This led to a 3.4-fold increase (2.3-5, 95% CI) in the likelihood of close (f=0.25) inbreeding relative to the population average when individuals dispersed less than 200m. In the light of our results, and published evidence showing little support for active inbreeding avoidance in vertebrates, we suggest that dispersal should be considered as a mechanism of prime importance for inbreeding avoidance in wild populations.     

The estimation of population differentiation with microsatellite markers

Microsatellite markers are routinely used to investigate the genetic structuring of natural populations. The knowledge of how genetic variation is partitioned among populations may have important implications not only in evolutionary biology and ecology, but also in conservation biology. Hence, reliable estimates of population differentiation are crucial to understand the connectivity among populations and represent important tools to develop conservation strategies. The estimation of differentiation is c from Wright's FST and/or Slatkin's RST, an FST -analogue assuming a stepwise mutation model. Both these statistics have their drawbacks. Furthermore, there is no clear consensus over their relative accuracy. In this review, we first discuss the consequences of different temporal and spatial sampling strategies on differentiation estimation. Then, we move to statistical problems directly associated with the estimation of population structuring itself, with particular emphasis on the effects of high mutation rates and mutation patterns of microsatellite loci. Finally, we discuss the biological interpretation of population structuring estimates.     

Heterosis and outbreeding depression in descendants of natural immigrants to an inbred population of song sparrows (Melospiza melodia)

We studied heterosis and outbreeding depression among immigrants and their descendants in a population of song sparrows on Mandarte Island, Canada. Using data spanning 19 generations, we compared survival, seasonal reproductive success, and lifetime reproductive success of immigrants, natives (birds with resident-hatched parents and grandparents), and their offspring (F1s, birds with an immigrant and a native parent, and F2s, birds with an immigrant grandparent and resident-hatched grandparent in each of their maternal and paternal lines). Lifetime reproductive success of immigrants was no worse than that of natives, but other measures of performance differed in several ways. Immigrant females laid later and showed a tendency to lay fewer clutches, but had relatively high success raising offspring per egg produced. The few immigrant males survived well but were less likely to breed than native males of the same age that were alive in the same year. Female F1s laid earlier than expected based on the average for immigrant and native females, and adult male F1s were more likely to breed than expected based on the average for immigrant and native males. The performance differences between immigrant and native females and between F1s and the average of immigrants and natives are consistent with the hypothesis that immigrants were disadvantaged by a lack of site experience and that immigrant offspring benefited from heterosis. However, we could not exclude the possibility that immigrants had a different strategy for optimizing reproductive success or that they experienced ecological compensation for life-history parameters. For example, the offspring of immigrants may have survived well because immigrants laid later and produced fewer clutches, thereby raising offspring during a period of milder climatic conditions. Although sample sizes were small, we found large performance differences between F1s and F2s, which suggested that either heterosis was associated with epistasis in F1s, that F2s experienced outbreeding depression, or that both phenomena occurred. These findings indicate that the performance of dispersers may be affected more by fine-scale genetic differentiation than previously assumed in this and comparable systems.     

A Bayesian method for the joint estimation of outcrossing rate and inbreeding depression

The population outcrossing rate (t) and adult inbreeding coefficient (F) are key parameters in mating system evolution. The magnitude of inbreeding depression as expressed in the field can be estimated given t and F via the method of Ritland (1990). For a given total sample size, the optimal design for the joint estimation of t and F requires sampling large numbers of families (100–400) with fewer offspring (1–4) per family. Unfortunately, the standard inference procedure (MLTR) yields significantly biased estimates for t and F when family sizes are small and maternal genotypes are unknown (a common occurrence when sampling natural populations). Here, we present a Bayesian method implemented in the program BORICE (Bayesian Outcrossing Rate and Inbreeding Coefficient Estimation) that effectively estimates t and F when family sizes are small and maternal genotype information is lacking. BORICE should enable wider use of the Ritland approach for field-based estimates of inbreeding depression. As proof of concept, we estimate t and F in a natural population of Mimulus guttatus. In addition, we describe how individual maternal inbreeding histories inferred by BORICE may prove useful in studies of inbreeding and its consequences.     

Optimal level of inbreeding in the common lizard

Mate choice with regard to genetic similarity has been rarely considered as a dynamic process. We examined this possibility in breeding populations of the common lizard (Lacerta vivipara) kept for several years in semi-natural conditions. We investigated whether they displayed a pattern of mate choice according to the genetic similarity and whether it was context-dependent. Mate choice depended on genetic similarity with the partner and also on age and condition. There was no systematic avoidance of inbreeding. Females of intermediate ages, more monogamous, did not mate with genetically similar partners, whereas younger and older females, more polyandrous, did but highest clutch proportions were associated with intermediate values of pair-relatedness. These results indicate dynamic mate choice, suggesting that individuals of different phenotypes select their partners in different ways according to their genetic similarity. We consider our results in the light of diverse and apparently contradictory theories concerning genetic compatibility, and particularly, optimal inbreeding and inclusive fitness.     

Mating system and early viability resistance to habitat fragmentation in a bird-pollinated eucalypt

Habitat fragmentation has been shown to disrupt ecosystem processes such as plant-pollinator mutualisms. Consequently, mating patterns in remnant tree populations are expected to shift towards increased inbreeding and reduced pollen diversity, with fitness consequences for future generations. However, mating patterns and phenotypic assessments of open-pollinated progeny have rarely been combined in a single study. Here, we collected seeds from 37 Eucalyptus incrassata trees from contrasting stand densities following recent clearance in a single South Australian population (intact woodland=12.6 trees ha⁻¹; isolated pasture=1.7 trees ha⁻¹; population area=10 km²). 649 progeny from these trees were genotyped at eight microsatellite loci. We estimated genetic diversity, spatial genetic structure, indirect contemporary pollen flow and mating patterns for adults older than the clearance events and open-pollinated progeny sired post-clearance. A proxy of early stage progeny viability was assessed in a common garden experiment. Density had no impact on mating patterns, adult and progeny genetic diversity or progeny growth, but was associated with increased mean pollen dispersal. Weak spatial genetic structure among adults suggests high historical gene flow. We observed preliminary evidence for inbreeding depression related to stress caused by fungal infection, but which was not associated with density. Higher observed heterozygosities in adults compared with progeny may relate to weak selection on progeny and lifetime-accumulated mortality of inbred adults. E. incrassata appears to be resistant to the negative mating pattern and fitness changes expected within fragmented landscapes. This pattern is likely explained by strong outcrossing and regular long-distance pollen flow.     

Among-individual variation in pollen limitation and inbreeding depression in a mixed-mating shrub

Background and Aims

Variation in inbreeding depression (δ) among individual plants is considered to play a central role in mating system evolution and population genetics. Moreover, such variation could be linked to individual susceptibility to pollen limitation (PL) because those individuals strongly affected by δ for seed production will require more outcross pollen for setting a given number of fruits or seeds. However, no study has tested explicitly for associations between PL and δ at the individual plant level. This study assesses the extent of among-individual variation in PL and δ, the consistency of δ across life stages, and the relationships between individual PL and δ in the mixed-mating shrub Myrtus communis.

Methods

Controlled hand-pollinations were performed in a natural M. communis population. Marked flowers were monitored until fruit production and a greenhouse experiment was conducted with the seeds produced.

Key Results

Compared with selfing, outcross-pollination enhanced seed number per fruit, germination rate and seedling growth, but did not enhance fruit-set. Only seed number per fruit was pollen limited and, thus, cumulative pollen limitation depended more on pollen quality (outcross pollen) than on quantity. The effects of δ varied considerably across life stages and individual plants. Cumulative δ was high across individuals (mean δ = 0·65), although there were no positive correlations between δ values at different life stages. Interestingly, maternal plants showing stronger δ for seed production were more pollen limited, but they were also less affected by δ for seedling growth because of a seed size/number trade-off.

Conclusions

Results show a general inconsistency in δ across life stages and individuals, suggesting that different deleterious loci are acting at different stages. The association between δ and PL at the individual level corroborates the idea that pollen limitation may be ‘genotype-dependent’ regardless of other factors.     

Microsatellite diversity and genetic structure of fragmented populations of the rare,fire-dependent shrub Grevillea macleayana

Recent habitat loss and fragmentation superimposed upon ancient patterns of population subdivision are likely to have produced low levels of neutral genetic diversity and marked genetic structure in many plant species. The genetic effects of habitat fragmentation may be most pronounced in species that form small populations, are fully self-compatible and have limited seed dispersal. However, long-lived seed banks, mobile pollinators and long adult lifespans may prevent or delay the accumulation of genetic effects. We studied a rare Australian shrub species, Grevillea macleayana (Proteaceae), that occurs in many small populations, is self-compatible and has restricted seed dispersal. However, it has a relatively long adult lifespan (c. 30 years), a long-lived seed bank that germinates after fire and is pollinated by birds that are numerous and highly mobile. These latter characteristics raise the possibility that populations in the past may have been effectively large and genetically homogeneous. Using six microsatellites, we found that G. macleayana may have relatively low within-population diversity (3.2-4.2 alleles/locus; Hexp = 0.420-0.530), significant population differentiation and moderate genetic structure (FST = 0.218) showing isolation by distance, consistent with historically low gene flow. The frequency distribution of allele sizes suggest that this geographical differentiation is being driven by mutation. We found a lack mutation-drift equilibrium in some populations that is indicative of population bottlenecks. Combined with evidence for large spatiotemporal variation of selfing rates, this suggests that fluctuating population sizes characterize the demography in this species, promoting genetic drift. We argue that natural patterns of pollen and seed dispersal, coupled with the patchy, fire-shaped distribution, may have restricted long-distance gene flow in the past.     

Efficient mitigation of founder effects during the establishment of a leading-edge oak population

Numerous plant species are shifting their range polewards in response to ongoing climate change. Range shifts typically involve the repeated establishment and growth of leading-edge populations well ahead of the main species range. How these populations recover from founder events and associated diversity loss remains poorly understood. To help fill this gap, we exhaustively investigated a newly established population of holm oak (Quercus ilex) growing more than 30 km ahead of the nearest larger stands. Pedigree reconstructions showed that plants belong to two non-overlapping generations and that the whole population originates from only two founder trees. The four first-generation trees that have reached maturity showed disparate mating patterns despite being full-sibs. Long-distance pollen immigration was notable despite the strong isolation of the stand: 6 per cent gene flow events in acorns collected on the trees (n = 255), and as much as 27 per cent among their established offspring (n = 33). Our results show that isolated leading-edge populations of wind-pollinated forest trees can rapidly restore their genetic diversity through the interacting effects of efficient long-distance pollen flow and purging of inbred individuals during recruitment. They imply that range expansions of these species are primarily constrained by initial propagule arrival rather than by subsequent gene flow.