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.     

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.     

The spatial structure of sexual and cytonuclear polymorphism in the gynodioecious Beta vulgaris ssp. maritima: I/ at a local scale

We have analyzed the spatial distribution of the sex phenotypes and of mitochondrial, chloroplast, and nuclear markers within two gynodioecious populations of Beta vulgaris ssp. maritima. Within both populations, sexual phenotype variation is controlled mainly by the cytoplasmic genotype, although in one study population a joint polymorphism of cytonuclear factors is clearly involved. In spite of contrasts in the ecology (mainly due to different habitats), a clear common feature in both populations is the highly patchy distribution of cytoplasmic haplotypes, contrasting with the wide distribution of nuclear diversity. This high contrast between cytoplasmic vs. nuclear spatial structure may have important consequences for the maintenance of gynodioecy. It provides opportunities for differential selection since nuclear restorer alleles are expected to be selected for in the presence of their specific cytoplasmic male sterile (CMS) type, but to be neutral (or selected against if there is a cost of restoration) in the absence of their CMS type. Selective processes in such a cytonuclear landscape may explain the polymorphism we observed at restorer loci for two CMS types.     

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.     

Local population structure and sex ratio: evolution in gynodioecious plants

Although the influence of population structure on evolution has been explored previously in a variety of theoretical studies, there are few examples of specific traits whose fitness is likely to be modified by the local structure. Here we focus on a specific trait, sex expression in gynodioecious plants, and derive a model in which the fitness of females and hermaphrodites is a function of the local sex ratio. By using the concept d genes. As a consequence, when local demes vary in sex ratio, a polymorphism for a cytoplasmic male sterility (CMS) allele can be maintained in the absence of nuclear alleles that restore male function. When of subjective frequencies, it is shown that among-deme variance in the local sex ratio reduces the average fitness of females when pollen availability limits fertility. In contrast, sex ratio variance increases the fitness of hermaphrodites from the perspective of maternally inherited genes and lessens the negative impact of pollen limitation on hermaphrodite fitness when it is measured from the perspective of biparentally inheriterestorer alleles are introduced into the model, polymorphism cannot be maintained simultaneously at both the cytoplasmic and nuclear loci. In that case, the CMS allele spreads to fixation, and the equilibrium frequency of females is an inverse function of the equilibrium frequency of the restorer allele, which increases with increased structure. The results exemplify how population structure can greatly alter the fitness and evolution of a frequency-dependent trait.     

Offspring sex ratio under inbreeding and outbreeding in a gynodioecious plant

Silene vulgaris is a gynodioecious plant native to Eurasia and now found throughout much of North America. Using hermaphrodite plants from three geographic regions (Stamford, NY; Broadway,VA; and Giles Co., VA) and four local populations within each region, we employed a hierarchical crossing design to explore the geographic structure of sex determining genes. Sex determination in this species is cytonuclear involving multiple cytoplasmic male sterility and nuclear restorer loci. Due to dominance effects within nuclear restorer loci, self-fertilization of hermaphrodites heterozygous at restorer loci should produce some homozygous recessive female offspring. Female offspring may also result from outcrossing among related individuals. At greater geographic and genetic distances, mismatches between cytoplasmic and nuclear sex determining genes should also produce high frequencies of female offspring if coevolution between cytoplasmic and nuclear sex determining alleles occurs independently among widely separated populations. We found evidence of dominance effects among nuclear restorer loci but no evidence of nuclear-cytoplasmic mismatches at the regional level. Of 63 maternal lines, 55 produced at least one female offspring when self-fertilized. Outcrossing within populations produced significantly fewer female offspring than self-fertilization. Outcrossing among regions produced the lowest proportion of female offspring, significantly fewer than outcrossing among populations within regions. Regions responded differently to among-region outcrossing with pollen donors from the two Virginia regions producing far fewer female offspring with New York dams than crosses among New York populations. These results indicate that nuclear restoration is complex, involving multiple loci with epistatic interactions and that most hermaphrodites in nature are heterozygous at one or more restorer locus. Further, regional differences in restorer frequencies indicate significant genetic structure for sex determining genes at large geographic scales, perhaps reflecting invasion history.     

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.     

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.     

A Cryptic Cytoplasmic Male Sterility Unveils a Possible Gynodioecious Past for Arabidopsis thaliana

Gynodioecy, the coexistence of hermaphrodites and females (i.e. male-sterile plants) in natural plant populations, most often results from polymorphism at genetic loci involved in a particular interaction between the nuclear and cytoplasmic genetic compartments (cytonuclear epistasis): cytoplasmic male sterility (CMS). Although CMS clearly contributes to the coevolution of involved nuclear loci and cytoplasmic genomes in gynodioecious species, the occurrence of CMS genetic factors in the absence of sexual polymorphism (cryptic CMS) is not easily detected and rarely taken in consideration. We found cryptic CMS in the model plant Arabidopsis thaliana after crossing distantly related accessions, Sha and Mr-0. Male sterility resulted from an interaction between the Sha cytoplasm and two Mr-0 genomic regions located on chromosome 1 and chromosome 3. Additional accessions with either nuclear sterility maintainers or sterilizing cytoplasms were identified from crosses with either Sha or Mr-0. By comparing two very closely related cytoplasms with different male-sterility inducing abilities, we identified a novel mitochondrial ORF, named orf117Sha, that is most likely the sterilizing factor of the Sha cytoplasm. The presence of orf117Sha was investigated in worldwide natural accessions. It was found mainly associated with a single chlorotype in accessions belonging to a clade predominantly originating from Central Asia. More than one-third of accessions from this clade carried orf117Sha, indicating that the sterilizing-inducing cytoplasm had spread in this lineage. We also report the coexistence of the sterilizing cytoplasm with a non-sterilizing cytoplasm at a small, local scale in a natural population; in addition a correlation between cytotype and nuclear haplotype was detected in this population. Our results suggest that this CMS system induced sexual polymorphism in A. thaliana populations, at the time when the species was mainly outcrossing.     

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.     

NUCLEO-CYTOPLASMIC MALE STERILITY AND ALTERNATIVE ROUTES TO DIOECY

Population-genetic models of nucleo-cytoplasmic gynodioecy are shown to allow invasion of males and conversion to dioecy in a single cytotype. Pleiotropic effects of restorer alleles on fertility through male or female function can maintain a cytoplasmic polymorphism in a population that prevents evolution to dioecy regardless of the pollen fertility of males. However, a cytoplasmic polymorphism has little effect on, and may even reduce, the minimum pollen fertility required for the spread of males into an equilibrium gynodioecious population. Where the thresholds for dioecy are similar, the presence of males during a transient preequilibrium high frequency of females can accelerate evolution to dioecy by more than 50 times relative to nuclear male sterility. However, the appearance of a nonrestorable male-sterile cytotype generally eliminates males from both subdioecious and dioecious populations, converting them to purely cytoplasmic gynodioecy. These models contradict the previously suggested notion that nucleo-cytoplasmic gynodioecy represents a “stable” intermediate breeding system and instead show that such gynodioecy can generally evolve to subdioecy, and often to dioecy, as easily as nuclear gynodioecy.     

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.     

DO RECENT FINDINGS IN PLANT MITOCHONDRIAL MOLECULAR AND POPULATION GENETICS HAVE IMPLICATIONS FOR THE STUDY OF GYNODIOECY AND CYTONUCLEAR CONFLICT?

The coexistence of females and hermaphrodites in plant populations, or gynodioecy, is a puzzle recognized by Darwin. Correns identified cytoplasmic inheritance of one component of sex expression, now known as cytoplasmic male sterility (CMS). Lewis established cytonuclear inheritance of gynodioecy as an example of genetic conflict. Although biologists have since developed an understanding of the mechanisms allowing the joint maintenance of CMS and nuclear male fertility restorer genes, puzzles remain concerning the inheritance of sex expression and mechanisms governing the origination of CMS. Much of the theory of gynodioecy rests on the assumption of maternal inheritance of the mitochondrial genome. Here we review recent studies of the genetics of plant mitochondria, and their implications for the evolution and transmission of CMS. New studies of intragenomic recombination provide a plausible origin for the chimeric ORFs that characterize CMS. Moreover, evidence suggests that nonmaternal inheritance of mitochondria may be more common than once believed. These findings may have consequences for the maintenance of cytonuclear polymorphism, mitochondrial recombination, generation of gynomonoecious phenotypes, and interpretation of experimental crosses. Finally we point out that CMS can alter the nature of the cytonuclear conflict that may have originally selected for uniparental inheritance.     

The Dynamics of Gynodioecy in Plantago Lanceolata L. II. Mode of Action and Frequencies of Restorer Alleles

Male fertility in Plantago lanceolata is controlled by the interaction of cytoplasmic and nuclear genes. Different cytoplasmic male sterility (CMS) types can be either male sterile or hermaphrodite, depending on the presence of nuclear restorer alleles. In three CMS types of P. lanceolata (CMSI, CMSIIa, and CMSIIb) the number of loci involved in male fertility restoration was determined. In each CMS type, male fertility was restored by multiple genes with either dominant or recessive action and capable either of restoring male fertility independently or in interaction with each other (epistasis). Restorer allele frequencies for CMSI, CMSIIa and CMSIIb were determined by crossing hermaphrodites with ``standard' male steriles. Segregation of male steriles vs. non-male steriles was used to estimate overall restorer allele frequency. The frequency of restorer alleles was different for the CMS types: restorer alleles for CMSI were less frequent than for CMSIIa and CMSIIb. On the basis of the frequencies of male steriles and the CMS types an ``expected' restorer allele frequency could be calculated. The correlation between estimated and expected restorer allele frequency was significant.     

Variation of female frequency and cytoplasmic male-sterility gene frequency among natural gynodioecious populations of wild radish (Raphanus sativus L.)

In gynodioecious plant populations, sex determination often involves both cytoplasmic male-sterility (CMS) genes and specific nuclear genes that restore male function. How gynodioecy is maintained under the joint dynamics of CMS and restorer genes remains controversial. Although many theoretical models deal with interactions between CMS genes and restorer genes with sexual phenotypes and predict changes in their frequencies, it is difficult to observe the frequencies because no molecular markers have been established for either CMS or restorer genes in well-studied gynodioecious plants. This is the first report of the frequency of a CMS gene determined using a molecular marker in natural populations of a gynodioecious plant. Using a set of CMS gene-specific polymerase chain reaction primers, we compared female and CMS gene frequencies in 18 natural populations of Raphanus sativus. Female frequency was relatively low, ranging from 0 to 0.21. In contrast, the CMS gene frequency was highly variable among populations, ranging from 0 to 1. Estimated restorer gene frequency seemed less variable than observed CMS gene frequency, probably due to higher gene flow than in the CMS gene. Genetic drift may play a role in maintaining high variability of the CMS gene, although other possibilities are not excluded.     

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.     

Evidence for effects of restorer genes on male and female reproductive functions of hermaphrodites in the gynodioecious species Thymus vulgaris L.

In Thymus vulgaris L., sex determination involves both the nuclear and the cytoplasmic genomes: the cytoplasm is responsible for male-sterility (the female phenotype) while specific nuclear genes may restore male fertility (the hermaphrodite phenotype). Previous observations have shown high variation among hermaphrodites for pollen and seed production. In order to investigate the origin of this variation, 12 female plants, four from each of three populations, were hand-pollinated with pollen from hermaphrodites from three different paternal populations. The sex-ratio (i.e. the frequency of hermaphrodites) produced and the reproductive functions of these offspring were measured. A strong positive correlation was observed between the sex-ratio within a family and both female and male reproductive functions of its hermaphrodites. No such correlation was found for females. This result suggests that restorer genes may be directly or indirectly involved both in sex determination and in the efficiency of resource allocation to reproductive functions. As a consequence, female advantage, i.e. the relative fecundity of females to hermaphrodites, is larger in families with low sex-ratio, and this might affect the evolution of this gynodioecious breeding system.     

Merging theory and mechanism in studies of gynodioecy

In gynodioecious species, females and hermaphrodites coexist and the genetics of sex determination is usually nuclear cytoplasmic. Maintaining nuclear-cytoplasmic gynodioecy requires polymorphism for the feminizing genes (contained in the mitochondria) and the genes that restore male fertility (contained in the nucleus). This complex polymorphism depends, in part, on there being negative pleiotropic effects (i.e. costs) of the nuclear restorer alleles. Here, we combine information from theoretical studies and studies on the molecular action of restorer alleles in crops to interpret the probable costs of such alleles, and suggest how various aspects of the theoretical models could be tested. In doing so, we highlight how crops can be used to address evolutionary questions about the maintenance of nuclear-cytoplasmic gynodioecy.     

Effects of CMS types and restorer alleles on plant performance in Plantago lanceolata L.: an indication for cost of restoration

In gynodioecious species, male steriles co-occur with hermaphrodites. Usually, the male sterile trait is maternally inherited, hence it is called Cytoplasmic Male Sterility (CMS). Nuclear loci restore male fertility in combination with their ‘own’ specific cytoplasmic types. In theory, two fitness components are important for the maintenance of this breeding system: a fitness advantage of the male steriles, and costs of restoration. The costs of restoration are alleged negative pleiotropic effects of restorer alleles. In this study the effects of different CMS types on plant performance and the cost of restoration were assessed in two experiments with Plantago lanceolata L. Biomass production differed significantly between the CMS types studied. In order to assess the costs of restoration, hermaphrodites with or without restorer alleles for a CMS type other than its own were compared. The studied restorer alleles caused a reduction in weight per seed, but the number of seeds produced was unaffected. The estimated cost of restoration measured as reduction of seed biomass was 13% for restorer alleles for CMSI. However, in the second experiment no pleiotropic effects of restorer alleles were detected, either because the assumptions for the experimental set-up were not valid or the costs of restoration may not always be expressed.