Effects of fine‐scale genetic structure on male mating success in gynodioecious Beta vulgaris ssp. maritima

Plant mating systems are known to influence population genetic structure because pollen and seed dispersal are often spatially restricted. However, the reciprocal outcomes of population structure on the dynamics of polymorphic mating systems have received little attention. In gynodioecious sea beet (Beta vulgaris ssp. maritima), three sexual types co‐occur: females carrying a cytoplasmic male sterility (CMS) gene, hermaphrodites carrying a non‐CMS cytoplasm and restored hermaphrodites that carry CMS genes and nuclear restorer alleles. This study investigated the effects of fine‐scale genetic structure on male reproductive success of the two hermaphroditic forms. Our study population was strongly structured and characterized by contrasting local sex‐ratios. Pollen flow was constrained over short distances and depended on local plant density. Interestingly, restored hermaphrodites sired significantly more seedlings than non‐CMS hermaphrodites, despite the previous observation that the former produce pollen of lower quality than the latter. This result was explained by the higher frequency of females in the local vicinity of restored (CMS) hermaphrodites as compared to non‐CMS hermaphrodites. Population structure thus strongly influences individual fitness and may locally counteract the expected effects of selection, suggesting that understanding fine scale population processes is central to predicting the evolution of gender polymorphism in angiosperms.     

Paternal leakage sustains the cytoplasmic polymorphism underlying gynodioecy but remains invasible by nuclear restorers

Cytoplasmic male sterility (CMS) in plants often results in gynodioecious populations, composed of hermaphrodites and male-sterile females. All models of gynodioecy assume maternal inheritance of the cytoplasmic alleles and postulate a variety of negatively frequency-dependent mechanisms to maintain the cytoplasmic polymorphisms observed in many natural populations. However, in some plant species, mitochondria are transmitted at least occasionally by pollen, a process called paternal leakage. We show that even a small amount of paternal leakage is sufficient to sustain a permanent, stable cytoplasmic polymorphism. Because only hermaphrodites provide pollen in gynodioecious species, the effects of paternal leakage are biased and occur more often from the non-CMS male-fertile haplotype to the CMS male-sterile haplotype. We also show that a nuclear restorer disrupts the polymorphic cytoplasmic equilibrium, leading to fixation of both the CMS allele and the restorer. Although a dominant nuclear restorer fixes, it fixes much more slowly than in the standard CMS models. Although a stable cytonuclear polymorphism is possible with "matching alleles" nuclear restoration, oscillations to low frequencies present a risk of loss by drift. Paternal leakage enhances the stability of joint cytonuclear polymorphism by reducing the chance that a CMS allele is lost by drift.     

Relative fitness of females and hermaphrodites in a natural gynodioecious population of wild radish,Raphanus sativus L. (Brassicaceae): comparison based on molecular genotyping

In many gynodioecious species, sex determination involves both cytoplasmic male‐sterility (CMS) genes and nuclear genes that restore male function. Differences in fitness among genotypes affect the dynamics of those genes, and thus that of gynodioecy. We used a molecular marker to discriminate between hermaphrodites with and without a CMS gene in gynodioecious Raphanus sativus. We compared fitness through female function among the three genotypes: females, hermaphrodites with the CMS gene and those without it. Although there was no significant difference among the genotypes in seed size, hermaphrodites without the CMS gene produced significantly more seeds, and seeds with a higher germination rate than the other genotypes, suggesting no fitness advantage for females and no benefit to bearing the CMS gene. Despite the lack of fitness advantage for females in the parameter values we estimated, a theoretical model of gynodioecy shows it can be maintained if restorer genes impose a cost paid in pollen production. In addition, we found that females invest more resources into female reproduction than hermaphrodites when they become larger. If environmental conditions enable females to grow larger this would facilitate the dynamics of CMS genes.     

Disentangling the causes of heterogeneity in male fecundity in gynodioecious Beta vulgaris ssp. maritima

Variation among individuals in reproductive success is advocated as a major process driving evolution of sexual polymorphisms in plants, such as gynodioecy where females and hermaphrodites coexist. In gynodioecious Beta vulgaris ssp. maritima, sex determination involves cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. Both restored CMS and non-CMS hermaphrodites co-occur. Genotype-specific differences in male fitness are theoretically expected to explain the maintenance of cytonuclear polymorphism. Using genotypic information on seedlings and flowering plants within two metapopulations, we investigated whether male fecundity was influenced by ecological, phenotypic and genetic factors, while taking into account the shape and scale of pollen dispersal. Along with spatially restricted pollen flow, we showed that male fecundity was affected by flowering synchrony, investment in reproduction, pollen production and cytoplasmic identity of potential fathers. Siring success of non-CMS hermaphrodites was higher than that of restored CMS hermaphrodites. However, the magnitude of the difference in fecundity depended on the likelihood of carrying restorer alleles for non-CMS hermaphrodites. Our results suggest the occurrence of a cost of silent restorers, a condition supported by scarce empirical evidence, but theoretically required to maintain a stable sexual polymorphism in gynodioecious species.     

Sex‐specific fitness variation in gynodioecious Beta vulgaris ssp. maritima: do empirical observations fit theoretical predictions?

In gynodioecious species, in which hermaphroditic and female plants co-occur, the maintenance of sexual polymorphism relies on the genetic determination of sex and on the relative fitness of the different phenotypes. Flower production, components of male fitness (pollen quantity and pollen quality) and female fitness (fruit and seed set) were measured in gynodioecious Beta vulgaris spp. maritima, in which sex is determined by interactions between cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. The results suggested that (i) female had a marginal advantage over hermaphrodites in terms of flower production only, (ii) restored CMS hermaphrodites (carrying both CMS genes and nuclear restorers) suffered a slight decrease in fruit production compared to non-CMS hermaphrodites and (iii) restored CMS hermaphrodites were poor pollen producers compared to non-CMS hermaphrodites, probably as a consequence of complex determination of restoration. These observations potentially have important consequences for the conditions of maintenance of sexual polymorphism in B. vulgaris and are discussed in the light of existing theory on evolutionary dynamics of gynodioecy.     

A cost of restoration of male fertility in a gynodioecious species,Lobelia siphilitica

Abstract.— Models allowing the coexistence of females and hermaphrodites in gynodioecious populations assume a simple genetic system of sex determination, a seed fitness advantage of females (compensation), and a negative pleiotropic effect of nuclear sex-determining genes on fitness (cost of restoration). In Lobelia siphilitica , sex is determined by both mitochondrial genes causing cytoplasmic male sterility (CMS) and nuclear genes that restore fertility when present with specific CMS haplotypes (nuclear restorers). I tested for a cost of restoration in L. siphilitica by measuring restored hermaphrodites for five fitness components and estimating the number of nuclear restorers by crosses with females carrying CMS1 and CMS2. A cost of restoration appears as a significant negative coefficient (B) in the regression model explaining fitness. I found that hermaphrodites carrying more nuclear restorer genes for CMS2 (or restorer genes of greater effect) have lower pollen viability (B =– 1.08, P = 0.001). This pollen viability cost of restoration in L. siphilitica supports the theoretical prediction that negative pleiotropic effects of restorers will exist in populations of gynodioecious species containing females. The existence of such a cost supports the view that gynodioecy can be a stable breeding system in nature.     

Fine scale spatial structuring of sex and mitochondria in Silene vulgaris

Fine scale spatial structure (FSSS) of cytoplasmic genes in plants is thought to be generated via founder events and can be amplified when seeds germinate close to their mother. In gynodioecious species these processes are expected to generate FSSS in sex ratio because maternally inherited cytoplasmic male sterility genes partially influence sex expression. Here we document a striking example of FSSS in both mitochondrial genetic markers and sex in roadside populations of Silene vulgaris. We show that in one population FSSS of sexes influences relative fruit production of females compared to hermaphrodites. Furthermore, FSSS in sex ratio is expected to persist into future generations because offspring sex ratios from females are female-biased whereas offspring sex ratios from hermaphrodites are hermaphrodite-biased. Earlier studies indicated that pollen limitation is the most likely mechanism underlying negative frequency dependent fitness of females. Our results support the theoretical predictions that FSSS in sex ratio can reduce female fitness by decreasing the frequency at which females experience hermaphrodites. We argue that the influence of FSSS on female fitness is complementary to the influence of larger scale population structure on female fitness, and that population structure at both scales will act to decrease female frequencies in gynodioecious species. Better comprehension of the spatial structure of genders and genes controlling sex expression at a local scale is required for future progress toward understanding sex ratio evolution in gynodioecious plants.     

TESTING MODELS OF SEX RATIO EVOLUTION IN A GYNODIOECIOUS PLANT: FEMALE FREQUENCY COVARIES WITH THE COST OF MALE FERTILITY RESTORATION

In many gynodioecious species, cytoplasmic male sterility genes (CMS) and nuclear male fertility restorers (Rf) jointly determine whether a plant is female or hermaphrodite. Equilibrium models of cytonuclear gynodioecy, which describe the effect of natural selection within populations on the sex ratio, predict that the frequency of females in a population will primarily depend on the cost of male fertility restoration, a negative pleiotropic effect of Rf alleles on hermaphrodite fitness. Specifically, when the cost of restoration is higher, the frequency of females at equilibrium is predicted to be higher. To test this prediction, we estimated variation in the cost of restoration across 26 populations of Lobelia siphilitica, a species in which Rf alleles can have negative pleiotropic effects on pollen viability. We found that L. siphilitica populations with many females were more likely to contain hermaphrodites with low pollen viability. This is consistent with the prediction that the cost of restoration is a key determinant of variation in female frequency. Our results suggest that equilibrium models can explain variation in sex ratio among natural populations of gynodioecious species.     

Consequences of inbreeding for offspring fitness and gender in Silene vulgaris,a gynodioecious plant

Abstract In gynodioecious plants, hermaphrodite and female plants co‐occur in the same population. In these systems gender typically depends on whether a maternally inherited cytoplasmic male sterility factor (CMS) is counteracted by nuclear restorer alleles. These restorer alleles are often genetically dominant. Although plants of the female morph are obligatorily outcrossing, hermaphrodites may self. This selfing increases homozygosity and may thus have two effects: (1) it may decrease fitness (i.e. result in inbreeding depression) and (ii) it may increase homozygosity of the nuclear restorer alleles and therefore increase the production of females. This, in turn, enhances outcrossing in the following generation. In order to test the latter hypothesis, experimental crosses were conducted using individuals derived from four natural populations of Silene vulgaris, a gynodioecious plant. Treatments included self‐fertilization of hermaphrodites, outcrossing of hermaphrodites and females using pollen derived from the same source population as the pollen recipients, and outcrossing hermaphrodites and females using pollen derived from different source populations. Offspring were scored for seed germination, survivorship to flowering and gender. The products of self‐fertilization had reduced survivorship at both life stages when compared with the offspring of outcrossed hermaphrodites or females. In one population the fitness of offspring produced by within‐population outcrossing of females was significantly less than the fitness of offspring produced by crossing females with hermaphrodites from other populations. Self‐fertilization of hermaphrodites produced a smaller proportion of hermaphroditic offspring than did outcrossing hermaphrodites. Outcrossing females within populations produced a smaller proportion of hermaphrodite offspring than did crossing females with hermaphrodites from other populations. These results are consistent with a cytonuclear system of sex determination with dominant nuclear restorers, and are discussed with regard to how the mating system and the genetics of sex determination interact to influence the evolution of inbreeding depression.     

Modeling gynodioecy: novel scenarios for maintaining polymorphism

Nuclear-cytoplasmic gynodioecy is a breeding system of plants in which females and hermaphrodites co-occur in populations, and gender is jointly determined by cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. Persistent polymorphism at both CMS and nuclear-restorer loci is necessary to maintain this breeding system. Theoretical models have explained how nuclear-cytoplasmic gynodioecy can be stable for certain assumptions. However, recent advances in our understanding of the genetics, population biology, and molecular mechanisms of sex determination in nuclear-cytoplasmic gynodioecious species suggest the utility of new models with different underlying assumptions. In this article, we examine different negative pleiotropic fitness effects of nuclear restorers (costs of restoration) using genetic and population assumptions based on recent literature. Specifically, we model populations with two CMS types and separate nuclear restorer loci for each CMS type. Under these assumptions, both overdominance for fitness and frequency-dependent selection at nuclear-restorer loci can support nuclear-cytoplasmic gynodioecy. Costs of restoration can be either dependent or independent of the cytoplasmic background. Seed fitness costs are more vulnerable to fixation of CMS types than pollen costs. Survivorship costs are effective at maintaining polymorphism even when total reproductive effects are low. Overall, our models display differences in the stability of nuclear-cytoplasmic gynodioecy and predicted population sex ratios that should be informative to researchers studying gynodioecy in the wild.     

POPULATION SEX STRUCTURE AND REPRODUCTIVE FITNESS IN GYNODIOECIOUS SIDALCEA MALVIFLORA MALVIFLORA (MALVACEAE)

The spatial distribution of females and hermaphrodites within gynodioecious populations is expected to exert considerable selective pressure on gender fitness through pollen limitation of seed set. If pollen flow is predominantly local, seed set in individual plants may be sensitive to the proximity of pollen donors; pollen limitation of seed set may occur if hermaphrodites are locally rare. Under such circumstances, female fitness will be negatively frequency dependent and hermaphrodite fitness will be positively frequency dependent. Given local seed dispersal, a nonrandom clumped distribution of the genders is expected in gynodioecious populations due to the heritability of gender in gynodioecious species. If gender fitness is frequency dependent, such structure should favor hermaphrodites and select against females. To test this hypothesis, I quantified the distribution of the genders in terms of nearest neighbors and neighborhood sex ratio in two populations of gynodioecious Sidalcea malviflora malviflora. I then measured the effect of neighborhood sex ratio on open-pollinated seed set and pollen limitation in both manipulated and unmanipulated neighborhoods. Results indicate that the genders have a patchy distribution and that both genders are pollen limited and show an increase in seed set with an increase in neighborhood hermaphrodite frequency. The observed population sex structure favors hermaphrodites and disadvantages females. These results highlight the importance that population-level traits can have in determining individual fitness and the evolution of sex ratios in gynodioecious species.     

HOW SELFING,INBREEDING DEPRESSION,AND POLLEN LIMITATION IMPACT NUCLEAR‐CYTOPLASMIC GYNODIOECY: A MODEL

Gynodioecy, the co‐occurrence of females and hermaphrodites, is often due to conflicting interactions between cytoplasmic male sterility genes and nuclear restorers. Although gynodioecy often occurs in self‐compatible species, the effect of self‐pollination, inbreeding depression, and pollen limitation acting differently on females and hermaphrodites remains poorly known in the case of nuclear‐cytoplasmic gynodioecy (NCG). In this study, we model NCG in an infinite population and we study the effect of selfing rate, inbreeding depression, and pollen limitation on the maintenance of gynodioecy and on sex ratios at equilibrium. We found that selfing and inbreeding depression have a strong impact, which depends on whether restorer cost acts on male or female fitness. When cost affects male fitness, the strength of cost has no effect, whereas selfing and inbreeding depression only impact gynodioecy by modifying the value of the female advantage. When cost affects female fitness, selfing facilitates NCG and reduces the role of strength of the cost, even when no inbreeding depression occurs, whereas inbreeding depression globally restricts the maintenance of the polymorphism. Finally, we found that pollen limitation could additionally strongly modify the dynamic of gynodioecy. We discuss our findings in the light of empirical data available in gynodioecious species.     

When is it worth being a self-compatible hermaphrodite? Context-dependent effects of self-pollination on female advantage in gynodioecious Silene nutans

In gynodioecious plant species with nuclear‐cytoplasmic sex determination, females and hermaphrodites plants can coexist whenever female have higher seed fitness than hermaphrodites. Although the effect of self fertilization on seed fitness in hermaphrodites has been considered theoretically, this effect is far from intuitive, because it can either increase the relative seed fitness of the females (if it leads hermaphrodites to produce inbred, low quality offspring) or decrease it (if it provides reproductive assurance to hermaphrodites). Hence, empirical investigation is needed to document whether relative seed fitness varies with whether pollen is or is not limiting to seed production. In the current study, we measured fruit set and seed production in both females and hermaphrodites and the selfing rate in hermaphrodites in two experimental patches that differed in sex ratios in the gynodioecious plant Silene nutans. We found an impact of plant gender, patch, and their interaction, with females suffering from stronger pollen limitation when locally frequent. In the most pollen‐limited situation, the selfing rate of hermaphrodites increased and provided hermaphrodites with a type of reproductive assurance that is not available to females. By integrating both the beneficial (reproductive assurance) and costly effects (through inbreeding depression) of self‐pollination, we showed that whether females did or did not exhibit higher seed fitness depended on the degree of pollen limitation on seed production.     

Sex-ratio evolution in nuclear-cytoplasmic gynodioecy when restoration is a threshold trait

Gynodioecious plant species, which have populations consisting of female and hermaphrodite individuals, usually have complex sex determination involving cytoplasmic male sterility (CMS) alleles interacting with nuclear restorers of fertility. In response to recent evidence, we present a model of sex-ratio evolution in which restoration of male fertility is a threshold trait. We find that females are maintained at low frequencies for all biologically relevant parameter values. Furthermore, this model predicts periodically high female frequencies (>50%) under conditions of lower female seed fecundity advantages (compensation, x = 5%) and pleiotropic fitness effects associated with restorers of fertility (costs of restoration, y = 20%) than in other models. This model explains the maintenance of females in species that have previously experienced invasions of CMS alleles and the evolution of multiple restorers. Sensitivity of the model to small changes in cost and compensation values and to initial conditions may explain why populations of the same species vary widely for sex ratio.     

A field guide to models of sex-ratio evolution in gynodioecious species

Gynodioecious plant species, species in which individuals are females or hermaphrodites, are ideal systems for studying connections between genetics, ecology, and long‐term evolutionary changes because sex determination can be complex, involving cytoplasmic and/or nuclear genes, and sex ratio is often variable across landscapes. Field data are needed to evaluate the many theories concerning this breeding system. In order to facilitate the gathering of relevant data, this paper introduces the four types of gynodiocy (nuclear, nuclear‐cytoplasmic and stochastic gynodioecy plus subdioecy), describes example species and expected patterns, discusses the various forces that drive the evolution of female frequencies, and gives concrete advice on where to start collecting data for different systems. For species in which females are relatively rare, we recommend reciprocal crosses to determine if sex‐determination is nuclear or nuclear‐cytoplasmic along with a search for correlations between female frequencies and ecological factors. For species in which females are common and sex ratios are highly variable, we recommend looking at female offspring sex ratios to determine if females are primarily produced in ephemeral epidemics. In the course of this discussion, we argue that the majority of natural gynodioecious species will have complex sex determination in which multiple cytoplasmic male sterility (CMS) genes interact with multiple nuclear restorers of fertility. Sex‐ratio evolution in such species will be primarily influenced by fitness differences among hermaphrodites (costs of restoration) and less influenced by fitness differences between the sexes (compensation). Metapopulation dynamics alone may explain population sex ratios of species in which females are associated with marginal environments or hybrid zones; however, we feel that in most cases equilibrium forces within populations and metapopulation dynamics among populations each explain portions of the sex‐ratio pattern.     

Pollen limitation of female reproductive success at fine spatial scale in a gynodioecious and wind-pollinated species, Beta vulgaris ssp. maritima

In sexually polymorphic plants, the spatial distribution of sexes is usually not random. Local variation in phenotype frequencies is expected to affect individual fitness of the different phenotypes. For gynodioecious species, with co-occurrence of hermaphrodites and females, if sexual phenotypes are structured in space and pollen flow is spatially restricted, local pollen availability should vary among patches. Female fitness may thus be low when hermaphrodites are locally rare. To test this hypothesis, we analysed how the reproductive output of females varied among patches within two natural study sites of the gynodioecious wind-pollinated Beta vulgaris ssp. maritima. Plants growing in female-biased areas and experiencing pollen limitation were found to have low fruit and seed sets but did not reallocate resources towards better offspring. Our results show that fine-scale processes influence individual fitness and the evolution of sex ratio in sexually polymorphic plants.     

Variation in pollen production and pollen viability in natural populations of gynodioecious Beta vulgaris ssp. maritima: evidence for a cost of restoration of male function?

Gynodioecious species are defined by the co-occurrence of two clearly separated categories of plants: females and hermaphrodites. The hermaphroditic category may, however, not be homogeneous, as male fitness may vary among hermaphrodites as a result of many biological factors. In this study, we analysed estimates of pollen quantity and viability in the gynodioecious Beta vulgaris ssp. maritima, comparing hermaphrodites bearing a male-fertile cytotype and hermaphrodites bearing cytoplasmic male sterility (CMS) genes, which are counteracted by nuclear restoration factors. We show that: (i) pollen quantity continuously varies among restored hermaphrodites, suggesting a complex genetic determination of nuclear restoration; (ii) pollen viability was lower in restored (CMS) hermaphrodites than in non-CMS hermaphrodites, probably because of incomplete restoration in some of these plants; and (iii) pollen quantity and viability also varied among hermaphrodites with male-fertile cytotypes, possibly a result of a silent cost of restoration. Finally, we discuss the consequences of these results for pollen flow and the dynamics of gynodioecy.     

Consequences of genetic linkage for the maintenance of sexually antagonistic polymorphism in hermaphrodites

When selection differs between males and females, pleiotropic effects among genes expressed by both sexes can result in sexually antagonistic selection (SA), where beneficial alleles for one sex are deleterious for the other. For hermaphrodites, alleles with opposing fitness effects through each sex function represent analogous genetic constraints on fitness. Recent theory based on single‐locus models predicts that the maintenance of SA genetic variation should be greatly reduced in partially selfing populations. However, selfing also reduces the effective rate of recombination, which should facilitate selection on linked allelic combinations and expand opportunities for balancing selection in a multilocus context. Here, I develop a two‐locus model of SA selection for simultaneous hermaphrodites, and explore the joint influence of linkage, self‐fertilization, and dominance on the maintainance of SA polymorphism. I find that the effective reduction in recombination caused by selfing significantly expands the parameter space where SA polymorphism can be maintained relative to single‐locus models. In particular, linkage facilitates the invasion of male‐beneficial alleles, partially compensating for the “female‐bias” in the net direction of selection created by selfing. I discuss the implications of accounting for linkage among SA loci for the maintenance of SA genetic variation and mixed mating systems in hermaphrodites.     

The maintenance of nucleocytoplasmic polymorphism in a metapopulation: the case of gynodioecy

In gynodioecious species, gender is generally determined by epistatic interactions between cytoplasmic and nuclear loci. However, theoretical studies suggest that, for a joint polymorphism at both cytoplasmic and nuclear loci to be maintained in a panmictic population, selection must act differently on the various genotypes that determine the same gender. Here we show that, in a metapopulation with local extinction and restricted gene flow, nucleocytoplasmic polymorphism can be maintained without these differences. We use deterministic simulations. We assume that gene flow occurred only at recolonization. Founder effects create genetic variance between populations in the metapopulation, and local population growth is faster when the local frequency of females is high. Group selection phenomena are involved in the maintenance of the joint polymorphism in the metapopulation. The frequency of females in the metapopulation at equilibrium is higher than in a panmictic population with the same genetic system. However, these conclusions hold only if nuclear alleles restoring male fertility are dominant.     

An association between chloroplast DNA haplotypeand gender in a plant metapopulation

Theoretical models have shown that metapopulation structure can influence the evolution of the sex ratio in gynodioecious plants when the fitness of females and/or hermaphrodites varies among populations with the local sex ratio. Such frequency-dependent fitness has been demonstrated previously in natural populations of Silene vulgaris, a gynodioecious plant in which sex determination is cytonuclear. We investigated whether there was an association between chloroplast DNA (cpDNA) haplotype and sex expression, with the assumption that cpDNA haplotypes could be associated with specific male sterility variants likely found in the mitochondrial DNA, owing to co-transmission of the two cytoplasmic genomes. Two cpDNA haplotypes were studied in a collection of 111 individuals of known sex taken from 20 natural populations found in Virginia, USA. The set of haplotype 1 individuals consisted of 71.4% hermaphrodites, where as only 7.7% of the haplotype 2 individuals were hermaphroditic; a highly statistically significant difference. By extension of a previous model it was also shown that the differential clustering of these two haplotypes into local populations contributes to among-population sex ratio variation and has the potential to lower the fitness of haplotype 2 by greater than 50%, relative to that expected with no population structure. This revised version was published online in July 2006 with corrections to the Cover Date.