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.     

Multiple CMS-restorer gene polymorphism in gynodioecious Plantago coronopus

The mode of inheritance of the male sterility trait is crucial for understanding the evolutionary dynamics of the sexual system gynodioecy, which is the co-occurrence of female and hermaphrodite plants in natural populations. Both cytoplasmic (CMS) and nuclear (restorer) genes are known to be involved. Theoretical models usually assume a limited number of CMS genes with each a single restorer gene, while reality is more complex. In this study, it is shown that in the gynodioecious species Plantago coronopus two new CMS-restorer polymorphisms exist in addition to the two that were already known, which means four CMS-restorer systems at the species level. Furthermore, three CMS types were shown to co-occur within a single population. All new CMS types showed a multilocus system for male fertility restoration, in which both recessive and dominant restorer alleles occur. Our finding of more than two co-occurring CMS-restorer systems each with multiple restorer genes raises the question how this complex of male sterility systems is maintained in natural populations.     

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.     

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.     

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.     

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.     

Consequences of prairie fragmentation on the progeny sex ratio of a gynodioecious species, Lobelia spicata (Campanulaceae)

Habitat fragmentation of prairie ecosystems has resulted in increased isolation and decreased size of plant populations. In large populations, frequency-dependent selection is expected to maintain genetic diversity of sex determining factors associated with gynodioecy, that is, nuclear restorer genes that reverse cytoplasmic male sterility (nucleocytoplasmic gynodioecy). However, genetic drift will have a greater influence on small isolated populations that result from habitat fragmentation. The genetic model for nucleocytoplasmic gynodioecy implies that the proportion of female progeny produced by hermaphroditic and female plants will show more extreme differences in populations with reduced allelic diversity, and that restoration of male function will increase with inbreeding. We investigated potential impacts of effects resulting from reduced population sizes by comparison of progeny sex ratios produced by female and hermaphroditic plants in small and large populations of the gynodioecious prairie species, Lobelia spicata. A four-way contingency analysis of the impact of population size, population sex ratio, and maternal gender on progeny sex ratios showed that progeny sex ratios of hermaphroditic plants were strongly influenced by population size, whereas progeny sex ratios of female plants were strongly influenced by population sex ratio. Further, analysis of variation in progeny-type distribution indicated decreased restoration and increased loss of male function in smaller and isolated populations. These results are consistent with reduced allelic diversity or low allelic frequency at restorer loci in small and isolated populations. The consequent decrease in male function has the potential to impede seed production in these fragmented prairies.     

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.     

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.     

Modelling the maintenance of male-fertile cytoplasm in a gynodioecious population

Gynodioecy is the co-occurrence of females and hermaphrodites in populations. It is usually due to the combined action of cytoplasmic male sterility (CMS) genes and nuclear genes that restore male fertility. According to previous theoretical studies, it is very difficult to explain the maintenance of gynodioecy with CMS and male-fertile cytotypes, although it has been observed in some species. However, only very specific situations have been investigated so far. We present a model to investigate the conditions that promote the maintenance of this breeding system in the case of an outcrossed species when CMS and male-fertile (non-CMS) cytotypes are present in an infinite panmictic population. We show that the type of cost of restoration strongly affects the conditions for stable maintenance of gynodioecy. Stable nuclear-cytoplasmic gynodioecy requires a female advantage, which is a classical condition for gynodioecy, but also a cost of CMS for female fitness, which had been rarely investigated. A cost of restoration is also needed, which could affect either pollen or seeds. Finally, we found that gynodioecy was attainable for a large set of parameter values, including low differences in fitness among genotypes and phenotypes. Our theoretical predictions are compared with previous theoretical work and with results of empirical studies on various gynodioecious species.     

Genetic determination of male sterility in gynodioecious Silene nutans

Gynodioecy, the coexistence of female and hermaphrodite plants within a species, is often under nuclear–cytoplasmic sex determination, involving cytoplasmic male sterility (CMS) genes and nuclear restorers. A good knowledge of CMS and restorer polymorphism is essential for understanding the evolution and maintenance of gynodioecy, but reciprocal crossing studies remain scarce. Although mitochondrial diversity has been studied in a few gynodioecious species, the relationship between mitotype diversity and CMS status is poorly known. From a French sample of Silene nutans, a gynodioecious species whose sex determination remains unknown, we chose the four most divergent mitotypes that we had sampled at the cytochrome b gene and tested by reciprocal crosses whether they carry distinct CMS genes. We show that gynodioecy in S. nutans is under nuclear–cytoplasmic control, with at least two different CMSs and up to four restorers with epistatic interactions. Female occurrence and frequency were highly dependent on the mitotype, suggesting that the level of restoration varies greatly among CMSs. Two of the mitotypes, which have broad geographic distributions, represent different CMSs and are very unequally restored. We discuss the dynamics of gynodioecy at the large-scale meta-population level.     

SEX RATIO VARIATION AMONG GYNODIOECIOUS POPULATIONS OF SEA BEET: CAN IT BE EXPLAINED BY NEGATIVE FREQUENCY‐DEPENDENT SELECTION?

This study is devoted to assess sex ratio variation among 33 populations of the gynodioecious Beta vulgaris ssp. maritima in Brittany (France) and to explore the causes of this variation. We showed that three different CMS (cytoplasmic male sterility) cytotypes occurred in populations, but strongly differed for their frequencies and the frequency of their associated nuclear restorer alleles (which counteract the effect of CMS and restore male fertility). No correlation was found between CMS and restorer frequencies within populations, which has been previously interpreted as a result of stochasticity. However, neutral genetic variation did not indicate recent population bottlenecks in studied populations. Moreover, no significant correlation was found between female frequency or variance and current population size. Consequently, stochastic processes could not be the major cause of sex ratio variation. Alternatively, empirical estimations of the variation of females, CMS genes and nuclear restorer allele's frequencies were compared to theoretical predictions based on a frequency‐dependent selection model of gynodioecy. In particular, we showed that an absence of correlation between CMS and restorer frequencies could also occur without stochasticity. The large variation of sex ratio in Beta vulgaris could thus be explained by frequency‐dependent selection acting on CMS genes and restorer alleles.     

Disentangling the effects of mating systems and mutation rates on cytoplamic diversity in gynodioecious Silene nutans and dioecious Silene otites

Many flowering plant species exhibit a variety of distinct sexual morphs, the two most common cases being the co-occurrence of females and males (dioecy) or the co-occurrence of hermaphrodites and females (gynodioecy). In this study, we compared DNA sequence variability of the three genomes (nuclear, mitochondrial and chloroplastic) of a gynodioecious species, Silene nutans, with that of a closely related dioecious species, Silene otites. In the light of theoretical models, we expect cytoplasmic diversity to differ between the two species due to the selective dynamics that acts on cytoplasmic genomes in gynodioecious species: under an epidemic scenario, the gynodioecious species is expected to exhibit lower cytoplasmic diversity than the dioecious species, while the opposite is expected in the case of balancing selection maintaining sterility cytoplasms in the gynodioecious species. We found no difference between the species for nuclear gene diversity, but, for the cytoplasmic loci, the gynodioecious S. nutans had more haplotypes, and higher nucleotide diversity, than the dioecious relative, S. otites, even though the latter has a relatively high rate of mitochondrial synonymous substitutions, and therefore presumably a higher mutation rate. Therefore, as the mitochondrial mutation rate cannot account for the higher cytoplasmic diversity found in S. nutans, our findings support the hypothesis that gynodioecy in S. nutans has been maintained by balancing selection rather than by epidemic-like dynamics.     

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.     

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.     

The Effect of Breeding System on Polymorphism in Mitochondrial Genes of Silene

Gynodioecy is a breeding system characterized by the co-occurrence of hermaphrodite and female individuals, generally as the result of nuclear–cytoplasmic interactions. The question remains whether the genetic factors controlling gynodioecy are maintained in species over long evolutionary timescales by balancing selection or are continually arising and being replaced in epidemic sweeps. If balancing selection maintains these factors, then neutral cytoplasmic diversity should be greater in gynodioecious than hermaphroditic species. In contrast, epidemic sweeps of factors controlling gynodioecy should decrease cytoplasmic diversity in gynodioecious relative to hermaphroditic species. We took a comparative approach in which we sequenced two mitochondrial genes, cytochrome b (cob) and cytochrome oxidase (cox1), for multiple populations of several hermaphroditic, gynodioecious, and dioecious species in the genus Silene. Breeding system was predictive of polymorphism. Gynodioecious species harbor many old haplotypes while hermaphroditic and dioecious species have little to no nucleotide diversity. The genealogical structure of neither gene departed from neutral expectations. Taken together, our results suggest that balancing selection acts on cytoplasmic male-sterility factors in several gynodioecious species in the genus.     

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.     

Effective population sizes for cytoplasmic and nuclear genes in a gynodioecious species. The role of the sex determination system

Equations are derived for the effective sizes of gynodioecious populations with respect to both nuclear and cytoplasmic genes (N(ec) and N(en), respectively). Compared to hermaphroditism, gynodioecy generally reduces effective population sizes for both kinds of loci to an extent depending on the frequency of females, the sex determination system, and the selfing rate of hermaphrodites. This reduction is due to fitness differences between the sexes and is highly influenced by the mode of inheritance of this fitness. In absence of selfing, nuclear gynodioecy results in a reduction of N(ec) that depends strongly on the dominance of male sterility alleles, while N(en) remains equal to the census number (N). With cytonuclear gynodioecy, both cytoplasmic and nuclear effective sizes are reduced, and at the extreme, dioecy results in the minimum N(ec) values and either minimum or maximum N(en) values (for low or high frequency of females, respectively). When selfing occurs, gynodioecy either increases or decreases N(en) as compared to hermaphroditism with the same selfing rate of hermaphrodites. Unexpectedly, N(ec) also varies with the selfing rate. Thus the genetic sex-determination system appears as a major factor for the nuclear and cytoplasmic genetic diversities of gynodioecious species.