The effects of inbreeding, genetic dissimilarity and phenotype on male reproductive success in a dioecious plant

Pollen fate can strongly affect the genetic structure of populations with restricted gene flow and significant inbreeding risk. We established an experimental population of inbred and outbred Silene latifolia plants to evaluate the effects of (i) inbreeding depression, (ii) phenotypic variation and (iii) relatedness between mates on male fitness under natural pollination. Paternity analysis revealed that outbred males sired significantly more offspring than inbred males. Independently of the effects of inbreeding, male fitness depended on several male traits, including a sexually dimorphic (flower number) and a gametophytic trait (in vitro pollen germination rate). In addition, full-sib matings were less frequent than randomly expected. Thus, inbreeding, phenotype and genetic dissimilarity simultaneously affect male fitness in this animal-pollinated plant. While inbreeding depression might threaten population persistence, the deficiency of effective matings between sibs and the higher fitness of outbred males will reduce its occurrence and counter genetic erosion.     

Seed paternity in flowering plants: an evolutionary perspective

The ultimate importance of paternal contributions to fitness and of post-pollination selection in flowering plants have remained elusive, largely because of the technical difficulty of assigning paternity. I review empirical studies that use heritable markers to investigate per-fruit seed paternity in natural populations and after experimental multiple-donor pollination. Thirty-one studies covering 23 species from 16 plant families document that in natural populations seeds from a single fruit are often fathered by multiple pollen donors (5 species from 5 families), that donors can differ significantly in seed-siring success (8 species from 6 families), that variation in pollen tube growth rates can be heritable (n = 1 out of 4 studies), that donor and recipient genotypes can simultaneously affect paternity (n = 2), and that temporal order of pollen deposition (n = 1) and environmental effects(n = 2) affect the outcome of pollen competition. These studies also investigate the role of post-pollination selection in the avoidance of inbreeding and for species boundaries. Most studies of male reproductive success in plants to date base on isozyme electrophoresis. The availability in the last decade of highly polymorphic molecular markers such as microsatellite DNA has been expected to open new possibilities to investigate competition and selection during the gametophytic phase. Yet, to date, there is still need for greater data wealth on seed paternity to test theories of sex allocation and to gain deeper understanding of floral trait evolution and of the evolutionary consequences of post-pollination selection in flowering plants.     

Effects of inbreeding and pollen donor provenance and diversity on offspring performance under environmental stress in the rare plant Cochlearia bavarica

Habitat degradation and loss can reduce size and genetic variability of natural populations, increasing individual homozygosity and average relatedness between individuals. While the resulting inbreeding depression may be reduced by natural selection under prevailing environmental conditions, it may increase again under environmental stress. To investigate the effect of environmental stress on offspring performance and the expression of inbreeding depression, we hand-pollinated maternal plants in small (< 100, n=5) and large populations (> 400 flowering plants, n=5) of the rare plant Cochlearia bavarica (Brassicaceae) and raised the offspring under experimentally manipulated water and light regimes (normal or reduced supply). In addition to considering natural variation in inbreeding levels due to population size, we manipulated pollen donor provenance and diversity. Maternal plants were pollinated with nine donors from a different population or with one or nine donors from the same population. One further inflorescence of each maternal plant was exposed to free pollination. Offspring growth and survival were monitored over 300 days. Offspring performance varied significantly among populations and maternal plants. Environmental stress interacted significantly with these factors. However, there was no general indication that offspring from small populations were more negatively affected. In seven out of 10 populations, offspring derived from between-population pollination performed better than offspring derived from within-population pollination. Also, in five out of 10 populations, average offspring size was higher after within-population pollination with nine than after pollination with one pollen donor. These results suggest low genetic diversity within C. bavarica populations, both smaller and larger ones. Interactions between environmental stress and pollination treatment indicated that using pollen donors from outside a population or increasing the number of pollen donors can reduce inbreeding depression, but that this beneficial effect is impaired under stressful conditions.     

Genetic variation among females affects paternity in a dioecious plant

Flowering plants rely on vectors for pollen transfer, and cannot choose their mates. Although recipient plants are unable to choose which pollen they receive, post‐pollination selection (acting pre‐ or post‐zygotically) may modify the outcome of pollination. Here we show that genetic variation among pollen recipients can predict the outcome of pollen competition (seed paternity) in the dioecious white campion. To investigate whether genetic variation among pollen recipients affects paternity, we applied the same pollen mixture from two males to three females, two of which full sisters and the third one chosen at random (unrelated). To control for maternal environmental effects, the plants used for these crosses were greenhouse‐reared F₁. We replicated this in two populations, for a total of 51 crosses, and genotyped a total of 772 offspring to assign paternity. If genetic variation affects paternity, we expected greater similarity of paternity success of the focal male with the sisters, compared to the unrelated female. Paternity of the focal male was significantly more repeatable over sisters, compared to repeatability over the mean of sisters and the unrelated females. When populations were analyzed separately, this was significant in one of the two populations. Paternity was not significantly correlated with stigma size. This provides evidence that in at least one population, genetic variation among individual plants influences the donors’ paternity success, as assessed through genetic analysis of the seedling. Since due to gravity‐dispersed seeds natural patches may often consist of related plants, the observed effect may contribute to variation in male reproductive success.     

Influence of pollen limitation and inbreeding depression in the maintenance of incomplete dichogamy in Salvia elegans

The widespread presence of incomplete dichogamy (i.e., partial separation in time between male and female phases) in flowering plants is a long‐standing question in floral evolution. In this study, we proposed four scenarios in which depending on the particular combination of pollen limitation and inbreeding depression, the presence of complete dichogamy, incomplete dichogamy, or adichogamy may be favored. Moreover, we evaluated the role of pollen limitation and inbreeding depression in a natural population of Salvia elegans to test the validity of our predicted scenarios. Our results indicate that S. elegans is partially protandrous as pollen viability and stigma receptivity overlap in the last days of life of the flower. Furthermore, through pollination treatments, we found no evidence of pollen limitation or inbreeding depression in any of the evaluated fitness components. As expected by one of the proposed scenarios, incomplete dichogamy seems to be favored in plants with absence of inbreeding depression and pollen limitation as a way to diminish interference between male and female functions.     

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.     

INBREEDING DEPRESSION IN ANDRODIOECIOUS POPULATIONS OF DATISCA GLOMERATA (DATISCACEAE)

Several workers have suggested that the rarity of androdioecy (the presence of males and hermaphrodites in a breeding population) in nature is due to the large fitness gain required by male plants in order to be maintained by selection. As part of an ongoing investigation of this hypothesis, we tested the effects of selfing on fitness in functionally androdioecious populations of Datisca glomerata. We compared progeny from self-fertilizations, cross-fertilizations with pollen from male plants only, cross-fertilizations with pollen from hermaphrodite plants only, and open-pollinated flowers for several measures of progeny fitness including seed weight, germination rate, and seedling weight. Significant inbreeding depression was observed for androdioecious populations of D. glomerata for both seed and seedling weights. However, no significant differences were observed across treatments for seed germination percentages. The observation of significant levels of inbreeding depression in this study, combined with prior evidence of threefold greater pollen production by males, may at least partially account for the large fitness increase required by males to be maintained by selection.     

Preventing inbreeding depression in Anacamptis coriophora (Orchidaceae) as a model of food-rewarding orchid

Pollination of deceptive orchids has enabled scientists to understand how these species avoid inbreeding depression by reducing the number of pollinator visits per inflorescence. In rewarding species, which receive a higher rate of visits per plant, geitonogamy is usually higher and therefore the risk of inbreeding increases. In this study, we assess the breeding system of the rewarding orchid A. coriophora, and the spatio-temporal changes in its fitness as well as variation in nectar content after pollination. We found that the species partially selects allogamous pollen if pollinia from the same stalk and other plants arrive to the stigma. Furthermore, when self-pollination occurs, despite successful fructification, seed viability is significantly lower than that of cross-pollinated plants. A. coriophora exhibits spatio-temporal variation in fitness that does not correlate with any plant feature. Moreover, nectar volume is reduced after pollination, but the sugar concentration is maintained. This study emphasizes how essential the pre-zygotic and post-zygotic reproductive barriers are for rewarding orchids to avoid inbreeding depression.

     

Postcopulatory inbreeding avoidance in guppies

In many species, the negative fitness effects of inbreeding have facilitated the evolution of a wide range of inbreeding avoidance mechanisms. Although avoidance mechanisms operating prior to mating are well documented, evidence for postcopulatory mechanisms of inbreeding avoidance remain scarce. Here, we examine the potential for paternity biases to favour unrelated males when their sperm compete for fertilizations though postcopulatory inbreeding avoidance mechanisms in the guppy, Poecilia reticulata. To test this possibility, we used a series of artificial inseminations to deliver an equal number of sperm from a related (either full sibling or half sibling) and unrelated male to a female while statistically controlling for differences in sperm quality between rival ejaculates. In this way, we were able to focus exclusively on postcopulatory mechanisms of inbreeding avoidance and account for differences in sperm competitiveness between rival males. Under these carefully controlled conditions, we report a significant bias in paternity towards unrelated males, although this effect was only apparent when the related male was a full sibling. We also show that sperm competition generally favours males with highly viable sperm and thus that some variance in sperm competitiveness can be attributed to difference in sperm quality. Our findings for postcopulatory inbreeding avoidance are consistent with prior work on guppies, revealing that sperm competition success declines linearly with the level of relatedness, but also that such effects are only apparent at relatedness levels of full siblings or higher. These findings reveal that postcopulatory processes alone can facilitate inbreeding avoidance.     

Resolving the conundrum of inbreeding depression but no inbreeding avoidance: Estimating sex‐specific selection on inbreeding by song sparrows (Melospiza melodia)

Inbreeding avoidance among interacting females and males is not always observed despite inbreeding depression in offspring fitness, creating an apparent “inbreeding paradox.” This paradox could be resolved if selection against inbreeding was in fact weak, despite inbreeding depression. However, the net magnitude and direction of selection on the degree to which females and males inbreed by pairing with relatives has not been explicitly estimated. We used long‐term pedigree data to estimate phenotypic selection gradients on the degree of inbreeding that female and male song sparrows (Melospiza melodia) expressed by forming socially persistent breeding pairs with relatives. Fitness was measured as the total numbers of offspring and grand offspring contributed to the population, and as corresponding expected numbers of identical‐by‐descent allele copies, thereby accounting for variation in offspring survival, reproduction, and relatedness associated with variation in parental inbreeding. Estimated selection gradients on the degree to which individuals paired with relatives were weakly positive in females, but negative in males that formed at least one socially persistent pairing. However, males that paired had higher mean fitness than males that remained socially unpaired. These analyses suggest that net selection against inbreeding may be weak in both sexes despite strong inbreeding depression, thereby resolving the “inbreeding paradox.”     

The realized effect of postpollination sexual selection in a natural plant population

The ultimate importance of postpollination sexual selection has remained elusive, largely because of the difficulty of assigning paternity in the field. Here I use a powerful new molecular marker (AFLP) for paternity analysis in a natural population of the outcrossing angiosperm Persoonia mollis (Proteaceae) to assess male reproductive success following equal pollination of 15 pollen donors on each of 6310 pistils. These results were contrasted with male reproductive success of these same plants following natural mating. Following equal pollination, there was a significant departure from equal siring success, indicating a potential for postpollination sexual selection. The most successful pollen donor sired more than twice the expected number of seeds, and this was largely consistent across recipient plants. However, siring success following natural mating was significantly different from siring success following artificial pollination and showed that the reproductive gains to be made from superior pollen performance did not translate into increased reproductive success following natural mating. As the ecological context for post-pollination sexual selection is strong in P. mollis, I suggest that pollen competition may ultimately have only a weak effect on non-random male mating success under natural conditions because the realized opportunities for pollen competition within pistils are limited.     

Pollen competition reduces inbreeding depression in Collinsia heterophylla (Plantaginaceae)

We tested two predictions of the hypothesis that competition between self-pollen may mitigate negative genetic effects of inbreeding in plants: (1) intense competition among self-pollen increases offspring fitness; and (2) pollen competition reduces the measured strength of inbreeding depression. We used Collinsia heterophylla (Plantaginaceae), an annual with a mixed mating system, to perform controlled crosses in which we varied both the size of the pollen load and the source of pollen (self vs. outcross). Fitness of selfed offspring was higher in the high pollen-load treatment. Our second prediction was also upheld: inbreeding depression was, on average, lower when large pollen loads were applied (11%) relative to the low pollen-load treatment (28%). The reduction was significant for two fitness components relatively late in the life-cycle: number of surviving seedlings and pollen-tube growth rate in vitro. These findings suggest that intermittent inbreeding, which leads to self-fertilization in plants with genetic loads, may select for traits that enhance pollen competition.     

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.     

Pollen limitation and inbreeding depression in an 'old rare' bumblebee-pollinated grassland herb

Habitat fragmentation and reduction of population size have been found to negatively affect plant reproduction in 'new rare' species that were formerly common. This has been attributed primarily to effects of increased inbreeding but also to pollen limitation. In contrast, little is known about the reproduction of 'old rare' species that are naturally restricted to small and isolated habitats and thus may have developed strategies to cope with long-term isolation and small population size. Here we study the effects of pollen source and quantity on reproduction of the 'old rare' bumblebee-pollinated herb, Astragalus exscapus. In two populations of this species, we tested for pollen autodeposition, inbreeding depression and outbreeding depression. Caged plants were left unpollinated or were pollinated with pollen from the same plant, from the same population or from a distant population (50 km). Additionally, we tested for pollen limitation by pollen supplementation in four populations of different size and density. In the absence of pollinators, plants did not produce seed whereas self-pollinated plants did. This indicates a self-compatible breeding system dependent on insect pollination. Both self-pollination and, in one of the two populations, cross-pollination with pollen from plants from the distant population resulted in a lower number of seeds per flower than cross-pollination with pollen from plants from the resident population, indicating inbreeding and outbreeding depression. Pollen addition enhanced fruit set and number of seeds per flower in three of the four populations, indicating pollen limitation. The degree of pollen limitation was lowest in the smallest but densest population. Our results suggest that, similar to 'new rare' plant species, also 'old rare' species may be at risk of inbreeding depression and pollen limitation.     

Inbreeding depression in male gametic performance

One key objective in evolutionary ecology is to understand the magnitude of inbreeding depression expressed across sex‐specific components of fitness. One major component of male fitness is fertilization success, which depends on male gametic performance (sperm and pollen performance in animals and plants, respectively). Inbreeding depression in male gametic performance could create sex‐specific inbreeding depression in fitness, increase the benefit of inbreeding avoidance and reduce the efficacy of artificial insemination and pollination. However, there has been no assessment of the degree to which inbreeding generally depresses male gametic performance and hence post‐copulatory or post‐pollination fertilization success. Because inbreeding depression is understood to be a property of diploid entities, it is not clear what degree of inbreeding depression in haploid gametic performance should be expected. Here, we first summarize how inbreeding depression in male gametic performance could potentially arise through gene expression in associated diploid cells and/or reduced genetic diversity among haploid gametes. We then review published studies that estimate the magnitude of inbreeding depression in traits measuring components of sperm or pollen quantity, quality and competitiveness. Across 51 published studies covering 183 study traits, the grand mean inbreeding load was approximately one haploid lethal equivalent, suggesting that inbreeding depresses male gametic performance across diverse systems and traits. However, there was an almost complete lack of explicit estimates from wild populations. Future studies should quantify inbreeding depression in systematic sets of gametic traits under naturally competitive and noncompetitive conditions and quantify the degree to which gamete phenotypes and performance reflect haploid vs. diploid gene expression.     

A comparison of male and female responses to inbreeding in Cucurbita pepo subsp. texana (Cucurbitaceae)

Accurate estimates of inbreeding depression are necessary in order to predict the evolutionary dynamics of a population, but many studies estimate inbreeding depression based solely on components of female function such as fruit set, seed set, and seed quality. Because total fitness is achieved through both male and female functions in hermaphroditic plants, estimates of both male and female fitness are needed to estimate accurately the magnitude of inbreeding depression. Seedlings of a wild gourd, Cucurbita pepo subsp. texana, with coefficients of inbreeding of 0 and 0.75 were planted in an experimental garden, and several components of male and female fitness were measured over the course of the growing season. Fitness in inbred plants was confounded by both maternal and genetic inbreeding effects. Inbred individuals produced significantly fewer fruits than outcrossed individuals, and percentage germination of seeds from inbred individuals was significantly lower than seeds from outcrossed individuals. Inbred plants also produced significantly fewer staminate flowers and marginally fewer and smaller pollen grains per flower. Pollen from inbred plants also grew significantly more slowly in vitro than pollen from outcrossed plants. Multiplicative estimates of inbreeding depression revealed inbreeding depression for both male and female functions in wild gourd, but inbreeding depression through female function was stronger than inbreeding depression through male function.     

Heat stress but not inbreeding affects offensive sperm competitiveness in Callosobruchus maculatus

Environmental and genetic stress have well‐known detrimental effects on ejaculate quality, but their concomitant effect on male fitness remains poorly understood. We used competitive fertilization assays to expose the effects of stress on offensive sperm competitive ability in the beetle Callosobruchus maculatus, a species where ejaculates make up more than 5% of male body mass. To examine the effects of environmental and genetic stress, males derived from outcrosses or sib matings were heat shocked at 50°C for 50 min during the pupal stage, while their siblings were maintained at a standard rearing temperature of 28°C. Heat‐shocked males achieved only half the offensive paternity success of their siblings. While this population exhibited inbreeding depression in body size, sperm competitiveness was unaffected by inbreeding, nor did the effect of heat shock stress on sperm competitiveness depend on inbreeding status. In contrast, pupal emergence success was increased by 34% among heat‐stressed individuals, regardless of their inbreeding status. Heat‐shocked males' ejaculate size was 19% reduced, but they exhibited 25% increased mating duration in single mating trials. Our results highlight both the importance of stress in postcopulatory sexual selection, and the variability among stressors in affecting male fitness.     

Elevation-related variation in the population characteristics of distylous Primula nivalis affects female fitness and inbreeding depression

The population characteristics of distylous species are highly sensitive to stochastic natural selection pressure.Therefore,populations growing under different environmental conditions may vary in floral morph ratios,potentially affecting female fitness and leading to inbreeding depression.However,the variation in offspring quality among populations as a result of inbreeding depression is poorly understood in distylous species.This study investigates variations in plant density,seed mass,seed viabilityfemale fitness,and post-dispersal inbreeding depression in both sexual morphs(long-styled and shortstyled plants)of the distylous Primula nivalis that were subjected to different pollination treatments along an elevational gradient from 1657 to 2704 m a.s.l.Population characteristics(morph plant density and ratio)and fruit set were significantly affected by sexual morph and elevation.Plant density and fruitset frequencies were lower for short-styled than for long-styled plants at 2704 m a.s.l.The seeds from the cross-pollinated flowers of both morphs were higher in quality than those of self-pollinated flowers.The female fitness of seeds from cross-pollinated flowers of both morphs was higher than that of seeds from open-pollinated and self-pollinated flowers.The female fitness of seeds from long-styled flowers was higher than that of seeds from short-styled flowers at all elevations.Inbreeding depression increased with elevation among plants with short-styled flowers but not among those with long-styled flowers.Variation in the elevation-dependent mating system might influence female fitness and affect inbreeding depression in both floral morphs.In conclusion,the low quality of seeds from short-styled flowers at high elevations might decrease short-styled flower frequency,affecting population characteristics.     

Pollen-tube growth rates in Collinsia heterophylla (Plantaginaceae): one-donor crosses reveal heritability but no effect on sporophytic-offspring fitness

Background and Aims

Evolutionary change in response to natural selection will occur only if a trait confers a selective advantage and there is heritable variation. Positive connections between pollen traits and fitness have been found, but few studies of heritability have been conducted, and they have yielded conflicting results. To understand better the evolutionary significance of pollen competition and its potential role in sexual selection, the heritability of pollen tube-growth rate and the relationship between this trait and sporophytic offspring fitness were investigated in Collinsia heterophylla.

Methods

Because the question being asked was if female function benefited from obtaining genetically superior fathers by enhancing pollen competition, one-donor (per flower) crosses were used in order to exclude confounding effects of post-fertilization competition/allocation caused by multiple paternity. Each recipient plant was crossed with an average of five pollen donors. Pollen-tube growth rate and sporophytic traits were measured in both generations.

Key Results

Pollen-tube growth rate in vitro differed among donors, and the differences were correlated with in vivo growth rate averaged over two to four maternal plants. Pollen-tube growth rate showed significant narrow-sense heritability and evolvability in a father–offspring regression. However, this pollen trait did not correlate significantly with sporophytic-offspring fitness.

Conclusions

These results suggest that pollen-tube growth rate can respond to selection via male function. The data presented here do not provide any support for the hypothesis that intense pollen competition enhances maternal plant fitness through increased paternity by higher-quality sporophytic fathers, although this advantage cannot be ruled out. These data are, however, consistent with the hypothesis that pollen competition is itself selectively advantageous, through both male and female function, by reducing the genetic load among successful gametophytic fathers (pollen), and reducing inbreeding depression associated with self–pollination in plants with mix-mating systems.Key words: Collinsia heterophylla, evolvability, female fitness, good genes, heritability, male fitness, mixed-mating system, Plantaginaceae, pollen competition, sexual selection     

Limiting inbreeding in disjunct and isolated populations of a woody shrub

Pollen movements and mating patterns are key features that influence population genetic structure. When gene flow is low, small populations are prone to increased genetic drift and inbreeding, but naturally disjunct species may have features that reduce inbreeding and contribute to their persistence despite genetic isolation. Using microsatellite loci, we investigated outcrossing levels, family mating parameters, pollen dispersal, and spatial genetic structure in three populations of Hakea oldfieldii, a fire‐sensitive shrub with naturally disjunct, isolated populations prone to reduction in size and extinction following fires. We mapped and genotyped a sample of 102 plants from a large population, and all plants from two smaller populations (28 and 20 individuals), and genotyped 158–210 progeny from each population. We found high outcrossing despite the possibility of geitonogamous pollination, small amounts of biparental inbreeding, a limited number of successful pollen parents within populations, and significant correlated paternity. The number of pollen parents for each seed parent was moderate. There was low but significant spatial genetic structure up to 10 m around plants, but the majority of successful pollen came from outside this area including substantial proportions from distant plants within populations. Seed production varied among seven populations investigated but was not correlated with census population size. We suggest there may be a mechanism to prevent self‐pollination in H. oldfieldii and that high outcrossing and pollen dispersal within populations would promote genetic diversity among the relatively small amount of seed stored in the canopy. These features of the mating system would contribute to the persistence of genetically isolated populations prone to fluctuations in size.