Founder effects and sex ratio in the gynodioecious Thymus vulgaris L.

Thymus vulgaris is a gynodioecious species (in which females and hermaphrodites coexist) with a highly variable frequency of females among natural populations (5–95%) and a high average female frequency (60%). Sex determination involves both cytoplasmic genes responsible for male sterility, i.e. the female phenotype, and specific nuclear factors responsible for the restoration of male fertility, and thus a hermaphrodite phenotype. In this study, molecular markers of the mitochondrial genome have been used to quantify the cytoplasmic diversity in 11 clumps of individuals observed in four recently founded populations. The very low diversity within patches in conjunction with the strong diversity among patches strongly suggests that clumps of individuals are the result of single matrilinear families. In clumps that contain mainly females, all the analysed females showed the same cytoplasmic pattern. This pattern differed from that shown by neighbouring hermaphrodites, indicating that the determination of sex is locally cytoplasmic. A comparison of genetic diversity before and after fire in one population showed that disturbances may cause a reduction in genetic diversity and a concurrent induction of local cytoplasmic determination of sex. Such cytoplasmic determination of sex in colonizing populations, together with the greater seed set of females, may largely improve the colonizing ability of the 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.     

Inbreeding effects on progeny sex ratio and gender variation in the gynodioecious Silene vulgaris (Caryophyllaceae)

In gynodioecious species, sex expression is generally determined through cytoplasmic male sterility genes interacting with nuclear restorers of the male function. With dominant restorers, there may be an excess of females in the progeny of self-fertilized compared with cross-fertilized hermaphrodites. Moreover, the effect of inbreeding on late stages of the life cycle remains poorly explored. Here, we used hermaphrodites of the gynodioecious Silene vulgaris originating from three populations located in different valleys in the Alps to investigate the effects of two generations of self- and cross-fertilization on sex ratio and gender variation. We detected an increase in females in the progeny of selfed compared with outcrossed hermaphrodites and inbreeding depression for female and male fertility. Male fertility correlated positively with sex ratio differences between outbred and inbred progeny, suggesting that dominant restorers are likely to influence male fertility qualitatively and quantitatively in S. vulgaris. We argue that the excess of females in the progeny of selfed compared with outcrossed hermaphrodites and inbreeding depression for gamete production may contribute to the maintenance of females in gynodioecious populations of S. vulgaris because purging of the genetic load is less likely to occur.     

Genetics and adaptation in structured populations: sex ratio evolution in Silene vulgaris

Theoretical models suggest that population structure can interact with frequency dependent selection to affect fitness in such a way that adaptation is dependent not only on the genotype of an individual and the genotypes with which it co-occurs within populations (demes), but also the distribution of genotypes among populations. A canonical example is the evolution of altruistic behavior, where the costs and benefits of cooperation depend on the local frequency of other altruists, and can vary from one population to another. Here we review research on sex ratio evolution that we have conducted over the past several years on the gynodioecious herb Silene vulgaris in which we combine studies of negative frequency dependent fitness on female phenotypes with studies of the population structure of cytoplasmic genes affecting sex expression. This is presented as a contrast to a hypothetical example of selection on similar genotypes and phenotypes, but in the absence of population structure. Sex ratio evolution in Silene vulgaris provides one of the clearest examples of how selection occurs at multiple levels and how population structure, per se, can influence adaptive evolution.     

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.     

Maternal sex and mate relatedness affect offspring quality in the gynodioecious Silene acaulis

In gynodioecious species, females sacrifice fitness by not producing pollen, and hence must have a fitness advantage over hermaphrodites. Because females are obligately outcrossed, they may derive a fitness advantage by avoiding selfing and inbreeding depression. However, both sexes are capable of biparental inbreeding, and there are currently few estimates of the independent effects of maternal sex and multiple levels of inbreeding on female advantage. To test these hypotheses, females and hermaphrodites from six Alaskan populations of Silene acaulis were crossed with pollen from self (hermaphrodites only), a sibling, a random plant within the same population, and a plant from a different population. Germination, survivorship and early growth revealed inbreeding depression for selfs and higher germination but reduced growth in sib-crosses, relative to outcrosses. Independent of mate relatedness, females germinated more seeds that grew faster than offspring from hermaphrodites. This indicates that inbreeding depression as well as maternal sex can influence breeding system evolution. The effect of maternal sex may be explained by higher performance of female genotypes and a greater abundance of female genotypes among the offspring of female mothers.     

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.     

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.     

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.     

Ecological and genetic factors contributing to the low frequency of male sterility in Chamaecrista Fasciculata (Fabaceae)

Bumble bee pollinated Chamaecrista fasciculata provides pollen as the sole reward to its pollinators. Male sterility, expressed as an absence or nearly complete absence of pollen production, occurs in low frequency in populations of C. fasciculata. Here we describe experiments, using C. fasciculata, to examine frequently cited determinants of the spread and maintenance of male sterility: compensation and the genetic basis of male sterility. In addition, we examine the role the pollination system plays in determining the reproductive success of the male steriles. Seventeen populations in Maryland, Illinois, and Kansas were surveyed and found to range from 0 to 6% male sterility per population. An artificial population of male-sterile simulants and hermaphrodites was created to examine how the local frequency of nonrewarding male steriles might affect male-sterile female reproductive success. Male steriles performed equally poorly, with respect to seed production, whether surrounded by other male-sterile simulants or hermaphrodites. Compensation was examined by comparison of male steriles and hermaphrodites with respect to several reproductive and nonreproductive characters. Male steriles outperformed hermaphrodites in terms of nonreproductive biomass, but performed equally in terms of ovule number and produced many fewer flowers. The genetic basis of male sterility was examined by performing both intra- and interpopulational crosses of male steriles to hermaphrodites and indicate that male sterility is not purely cytoplasmic. The low frequency of male sterility in C. fasciculata populations may reflect reduced female reproductive success because of pollinator avoidance, lack of reproductive compensation, and a mode of inheritance that is not purely cytoplasmic.     

How much better are females? The occurrence of female advantage,its proximal causes and its variation within and among gynodioecious species

Background

Gynodioecy is a reproductive system of interest for evolutionary biologists, as it poses the question of how females can be maintained while competing with hermaphrodites that possess both male and female functions. One necessary condition for the maintenance of this polymorphism is the occurrence of a female advantage, i.e. a better seed production or quality by females compared with hermaphrodites. Theoretically, its magnitude can be low when sterility mutations are cytoplasmic, while a 2-fold advantage is needed in the case of nuclear sterility. Such a difference is often thought to be due to reduced inbreeding depression in obligatory outcrossed females. Finally, variation in sex ratio and female advantage occur among populations of some gynodioecious species, though the prevalence of such variation is unknown.

Scope

By reviewing and analysing the data published on 48 gynodioecious species, we examined three important issues about female advantage. (1) Are reduced selfing and inbreeding depression likely to be the major cause of female advantage? (2) What is the magnitude of female advantage and does it fit theoretical predictions? (3) Does the occurrence or the magnitude of female advantage vary among populations within species and why?

Conclusions

It was found that a female advantage occurred in 40 species, with a magnitude comprised between 1 and 2 in the majority of cases. In many species, reduced selfing may not be a necessary cause of this advantage. Finally, female advantage varied among populations in some species, but both positive and negative correlations were found with female frequency. The role of reduced selfing in females for the evolution of gynodioecy, as well as the various processes that affect sex ratios and female advantage in populations are discussed.     

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.     

Exploring the genetic basis and proximate causes of female fertility advantage in gynodioecious Thymus vulgaris

In many gynodioecous species, females produce more viable seeds than hermaphrodites. Knowledge of the relative contribution of inbreeding depression in hermaphrodites and maternal sex effects to the female fertility advantage and the genetic basis of variation in female fertility advantage is central to our understanding of the evolution of gender specialization. In this study we examine the relative contribution of inbreeding and maternal sex to the female fertility advantage in gynodioecious Thymus vulgaris and quantify whether there is genetically based variation in female fertility advantage for plants from four populations. Following controlled self and outcross (sib, within-population, and between-population) pollination, females had a more than twofold fertility advantage (based on the number of germinating seeds per fruit), regardless of the population of origin and the type of pollination. Inbreeding depression on viable seed production by hermaphrodites occurred in two populations, where inbreeding had been previously detected. Biparental inbreeding depression on viable seed production occurred in three of four populations for females, but in only one population for hermaphrodites. Whereas the maternal sex effect may consistently enhance female fertility advantage, inbreeding effects may be limited to particular population contexts where inbreeding may occur. A significant family x maternal sex interaction effect on viable seed production was observed, illustrating that the extent of female fertility advantage varies significantly among families. This result is due to greater variation in hermaphrodite (relative to female) seed fertility between families. Despite this genetic variation in female fertility advantage and the highly female biased sex ratios in populations of T. vulgaris, gynodioecy is a stable polymorphism, suggesting that strong genetic and/or ecological constraints influence the stability of this polymorphism.     

The effect of disease on the evolution of females and the genetic basis of sex in populations with cytoplasmic male sterility

The evolution of separate males and females is an important evolutionary transition that has occurred multiple times in flowering plants. While empirical studies have stressed the potential importance of natural enemies and organismal interactions in the evolution of separate sexes, there has been no treatment of natural enemies in the theoretical literature. We investigated the effects of disease on the evolution of females in gynodioecious populations composed of females and hermaphrodites, where sex is determined by the interaction of cytoplasmic male sterility (CMS) and nuclear restorer genes. When females are significantly more resistant than hermaphrodites, disease drives an increase in the frequency of females and sex determination becomes nuclear, creating the pre-conditions for the evolution of separate males and females. However, when females are only moderately more resistant, disease drives changes in the frequency of CMS and restorer alleles, but has little effect on the frequency of females. We discuss our results in the context of the evolution of mating systems and cyto-nuclear epistasis.     

Frequency-dependent pollinator discrimination acts against female plants in the gynodioecious Geranium maculatum

MethodsTo test the severity and consequences of this type of pollinator discrimination in Geranium maculatum, experimental populations with the range of sex ratios observed in nature were created, ranging from 13 % to 42 % females. Pollinators were observed in order to measure the strength of discrimination, and pollen deposition and seed production of both sexes were measured to determine the fitness consequences of this discrimination. Additionally a comparison was made across the sex ratios to determine whether discrimination was frequency-dependent.ConclusionsThe results suggest that pollinator discrimination negatively affects females'' relative fitness when they are rare. Thus, the initial spread of females in a population, the first step in the evolution of gynodioecy, may be made more difficult due to pollinator discrimination.     

Hybridization and regional sex ratios in Nemophila menziesii

I tested whether a region of high female frequencies in the gynodioecious plant, Nemophila menziesii, may be due to hybridization between regionally distributed populations with different corolla colours. I crossed plants in the greenhouse from populations with different corolla colours and found that hybrid crosses yielded higher frequencies of females than within-colour crosses. In the field, I found that populations with high female frequencies had intermediate mean corolla colours and higher variance in corolla colour, two traits suggesting hybridization. Nemophila menziesii has nuclear-cytoplasmic sex inheritance, thus if populations with different corolla colours are fixed for different male-sterile cytoplasms and matching nuclear restorer alleles, hybridization between populations with different corolla colour should yield high frequencies of females. Two populations that are all hermaphroditic in the field segregated females in hybrid crosses suggesting that field populations may contain sex ratio distorters but appear undistorted, a prediction of genomic conflict theory.     

Structural analysis of female and hermaphroditic flowers of a gynodioecious tree, Ocotea tenera (Lauraceae)

The evolution of gynodioecy from hermaphroditism involves modifications of floral structure such that male or female fitness is enhanced in hermaphrodites and females, respectively. We present an analysis of structural specialization of flowers of Ocotea tenera, in order to evaluate gender system evolution in this tropical tree species. Significant morphological and anatomical variation was found between high fruiting and low or nonfruiting trees. Female flowers were significantly smaller than hermaphroditic flowers, produced no viable pollen, and made relatively greater allocation to structures that increase female fitness. Hermaphroditic flowers were significantly larger than female flowers, produced copious quantities of pollen, and made relatively greater allocation to male structures. Analyses indicated that changes in allometries between whole-flower growth and growth of reproductive structures may have occurred, which enhance function of the flower and plant as a male or female. Efficiency of nutrient allocation for reproduction is argued to be a factor driving gender system evolution in Ocotea tenera.     

Determinants of sex allocation in a gynodioecious wild strawberry: implications for the evolution of dioecy and sexual dimorphism

One evolutionary pathway from plants with combined male and female functions (hermaphroditism) to those with separate sexes (dioecy) involves females coexisting with hermaphrodites (gynodioecy). The research presented here explores sex allocation in Fragaria virginiana (a gynodioecious wild strawberry), within the context of theory on the gynodioecy–dioecy transition. By growing clonally replicated plants in the greenhouse and surveying six populations in situ, I evaluated the effects of plant size, genotype, sexual identity, population of origin and female frequency on sex allocation. I found significant positive effects of plant size on most sex allocation traits studied. In addition to strong sex-specific allocation patterns, I found significant broad-sense heritabilities for all traits, suggesting that plants could respond to selection. Moreover, there was a negative genetic correlation between pollen production and fruit set per flower within hermaphrodites, lending support to a basic assumption of sex allocation theory. On the other hand, several sex allocation traits, namely pollen and ovules per flower in hermaphrodites, were positively genetically correlated, suggesting that they may act to constrain the evolution of sexual dimorphism. Populations differed in the frequency of females, and females were more prevalent on sites with lower soil moisture and where hermaphrodites were least likely to produce fruit, suggesting that females’ seed fitness relative to that of hermaphrodites may be strongly environment-dependent in this species.     

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.