EXPERIMENTAL EVIDENCE FOR FREQUENCY DEPENDENT SELF-FERTILIZATION IN THE GYNODIOECIOUS PLANT, SILENE VULGARIS

After over a half century of empirical and theoretical research regarding the evolution and maintenance of gynodioecy in plants, unexplored factors influencing the relative fitnesses of females and hermaphrodites remain. Theoretical studies suggest that hermaphrodite self-fertilization (selfing) rate influences the maintenance of gynodioecy and we hypothesized that population sex ratio may influence hermaphrodite selfing rate. An experimental test for frequency-dependent self-fertilization was conducted using replicated populations constructed with different sex ratios of the gynodioecious plant Silene vulgaris . We found that hermaphrodite selfing increased with decreased hermaphrodite frequency, whereas evidence for increased inbreeding depression was equivocal. We argue that incorporation of context dependent inbreeding into future models of the evolution of gynodioecy is likely to yield novel insights into sex ratio evolution.     

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.     

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.     

Recent advances in the study of gynodioecy: the interface of theory and empiricism

Background

In this review we report on recent literature concerned with studies of gynodioecy, or the co-occurrence of female and hermaphrodite individuals in natural plant populations. Rather than review this literature in its entirety, our focus is on the interplay between theoretical and empirical approaches to the study of gynodioecy.

Scope

Five areas of active inquiry are considered. These are the cost of restoration, the influence of population structure on spatial sex-ratio variation, the influence of inbreeding on sex expression, the signature of cyto-nuclear coevolution on the mitochondrial genome, and the consequences of mitochondrial paternal leakage.

Conclusions

Recent advances in the study of gynodioecy have been made by considering both the ecology of female:hermaphrodite fitness differences and the genetics of sex expression. Indeed theory has guided empiricism and empiricism has guided theory. Future advances will require that some of the methods currently available only for model organisms be applied to a wider range of species.Key words: Breeding system, gynodioecy, cytoplasmic male sterility, restoration, sex ratio, inbreeding, population structure, genetic conflict     

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.     

Can females benefit from selfing avoidance? Genetic associations and the evolution of plant gender

Evolutionary stability of dioecy and nuclear gynodioecy in higher plants requires that females produce over twice as many successful seeds as hermaphrodites. This fitness differential is widely thought to derive primarily from the advantages of outcrossing caused by high selfing rates and inbreeding depression in the hermaphrodite. This study hypothesized that (i) extraordinarily high deleterious mutation rates are necessary to double female seed success due to outcrossing, and (ii) the large difference in outcrossing rates between sex morphs causes differential purging of these mutations, resulting in additional genetic selection on male sterility. Using genetically explicit models, I showed that the phenotypic outcrossing advantage requires at least one new highly recessive deleterious mutation per genome per generation, regardless of selection coefficient. However, under this mutational regime, differential purging created strong genetic selection against recessive male sterility that overwhelmed the phenotypic selection in favour of outcrossing. In very small populations and for dominant male sterility, this genetic selection was weaker or absent. This first genetically explicit study of the outcrossing advantage of unisexual females may shed new light on both the genetic and selective conditions for the evolution of gynodioecy and dioecy.     

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.     

Sex-biased seed predation and the maintenance of females in a gynodioecious plant

We investigated genetic and ecological factors contributing to the maintenance of females in populations of the gynodioecious plant, Sidalcea hendersonii (Malvaceae). Our crossing experiments indicated that male sterility is controlled by a dominant nuclear allele. Nuclear determination of sex theoretically requires much higher female fitness to account for the high frequency of female plants often observed during surveys of S. hendersonii populations. Females were, in fact, shown to have higher fitness in an experimental population, producing more viable seed and surviving offspring than hermaphrodite plants. In natural populations, however, differences in viable seed production were not evident unless seed predation was considered. Sex-biased seed predation appears to play an important role in the maintenance of gynodioecy in S. hendersonii. In populations with high female frequencies, weevil larvae destroyed significantly more seeds from hermaphrodite plants than females, substantially reducing their seed production. To our knowledge, this study provides the first evidence that sex-biased predation may be responsible for high female frequencies in natural populations of a gynodioecious species.     

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.     

Pollination success across an elevation and sex ratio gradient in gynodioecious Daphne laureola

Gynodioecy is a dimorphic breeding system in which hermaphrodite and female individuals coexist in populations. Sex ratio and gender-relative lifetime seed production determine the stability of gynodioecy, and both genetic and ecological factors may influence these parameters. I analyzed the consequences of variation in population sex ratio and site elevation for the relative pollination success of female and hermaphrodite individuals of Daphne laureola in southern Spain, where previous studies failed to detect female fecundity advantages at two mid-elevation sites. Pollination success, estimated as stigmatic pollen loads, number of pollen tubes per style, and percentage of fertilized flowers, was higher for hermaphrodites than females in populations with 20-56% females. Furthermore, female quantitative disadvantage in pollination success increased with elevation, suggesting that the higher availability of pollen due to the increased proportion of hermaphrodites could not mitigate the negative effect that other factors associated with elevation apparently had on pollination. Supplemental hand pollinations showed that female seed production was pollen limited in populations with a proportion of females >50%, although both pollination success and natural fruit set of females in these sites were the highest recorded.     

RESOURCE COMPENSATION AND THE EVOLUTION OF GYNODIOECY IN PHACELIA LINEARIS (HYDROPHYLLACEAE)

Gynodioecy is a dimorphic breeding system in which hermaphrodite and female individuals coexist in populations. Theoretical models have shown that if nuclear genes control sex expression, then gynodioecy can evolve only when females have large advantages in one or more fitness components. These female advantages must be large enough that females' expected lifetime production of viable seeds is more than twice that of hermaphrodites. Previous studies have found that cytoplasmic inheritance and/or a large offspring-vigor advantage of females (caused by hermaphrodite self-pollination and inbreeding depression of selfed seeds) account for this breeding system's evolution. This paper reports studies of gynodioecy in Phacelia linearis, an insect-pollinated annual plant in which gender inheritance appears to be nuclear. Twenty-six P. linearis populations surveyed in northern Utah, USA, contain a majority of perfect-flowered hermaphrodites, but most (22) also contain male-sterile individuals (females), at frequencies of up to 0.16. The hermaphrodite selfing rate is low (0.00–0.20 in four populations). Maternal gender does not consistently affect components of offspring vigor, such as seed size, germination rate, seedling survivorship, and vegetative size. Plants of the two genders do not differ in number of seeds per fruit or mean seed mass. Females produce significantly more fruits and seeds than hermaphrodites in natural populations. The ratio of the mean lifetime seed production of females to the mean lifetime seed production of hermaphrodites ranged from 1.31 to 2.52 in six natural populations. Females have greater shoot biomass than hermaphrodites and produce more seeds at any given shoot biomass than hermaphrodites, suggesting that their seed-production advantage arises from gender-specific patterns of resource allocation to growth and reproduction. The gender difference in plant size varies across environments and across genetic backgrounds. In this species nuclear gynodioecy appears to be evolutionarily stable mainly because of resource compensation by females, without a large outcrossing advantage of females.     

How geitonogamous selfing affects sex allocation in hermaphrodite plants

Does the mode of self-pollination affect the evolutionarily stable allocation to male vs. female function? We distinguish the following scenarios. (1) An ‘autogamous’ species, in which selfing occurs within the flower prior to opening. The pollen used in selfing is a constant fraction of all pollen grains produced. (2) A species with ‘abiotic pollination’, in which selfing occurs when pollen dispersed in one flower lands on the stigma of a nearby flower on the same plant (geitonogamy). The selfing rate increases with male allocation but a higher selfing rate does not mean a reduced export of pollen. (3) An ‘animal-pollinated’ species with geitonogamous selfing. Here the selfing rate also increases with male allocation, but pollen export to other plants in the population is a decelerating function of the number of simultaneously open flowers. In all three models selfing selects for increased female allocation. For model 3 this contradicts the general opinion that geitonogamous selfing does not affect evolutionarily stable allocations. In all models, the parent benefits more from a female-biased allocation than any other individual in the population. In addition, in models 2 and 3, greater male allocation results in more local mate competition. In model 3 and in model 2 with low levels of inbreeding depression, hermaphroditism is evolutionarily stable. In model 2 with high inbreeding depression, the population converges to a fitness minimum for the relative allocation to male function. In this case the fitness set is bowed inwards, corresponding with accelerating fitness gain curves. If the selfing rate increases with plant size, this is a sufficient condition for size-dependent sex allocation (more allocation towards seeds in large plants) to evolve. We discuss our results in relation to size-dependent sex allocation in plants and in relation to the evolution of dioecy.     

Male‐biased hermaphrodites in a gynodioecious shrub,Daphne jezoensis

Gynodioecy, a state where female and hermaphrodite plants coexist in populations, has been widely proposed an intermediate stage in the evolutionary pathway from hermaphroditism to dioecy. In the gynodioecy–dioecy pathway, hermaphrodites may gain most of their fitness through male function once females invade populations. To test this prediction, comprehensive studies on sex ratio variation across populations and reproductive characteristics of hermaphrodite and female phenotypes are necessary. This study examined the variation in sex ratio, sex expression, flower and fruit production and sexual dimorphism of morphological traits in a gynodioecious shrub, Daphne jezoensis, over multiple populations and years. Population sex ratio (hermaphrodite:female) was close to 1:1 or slightly hermaphrodite‐biased. Sex type of individual plants was largely fixed, but 15% of plants changed their sex during a 6‐year census. Hermaphrodite plants produced larger flowers and invested 2.5 times more resources in flower production than female plants, but they exhibited remarkably low fruit set (proportion of flowers setting fruits). Female plants produced six times more fruits than hermaphrodite plants. Low fruiting ability of hermaphrodite plants was retained even when hand‐pollination was performed. Fruit production of female plants was restricted by pollen limitation under natural conditions, irrespective of high potential fecundity, and this minimised the difference in resources allocated to reproduction between the sexes. Negative effects of previous flower and fruit production on current reproduction were not apparent in both sexes. This study suggests that gynodioecy in this species is functionally close to a dioecious mating system: smaller flower production with larger fruiting ability in female plants, and larger flower production with little fruiting ability in hermaphrodite plants.     

Sexually antagonistic polymorphism in simultaneous hermaphrodites

In hermaphrodites, pleiotropic genetic trade‐offs between female and male reproductive functions can lead to sexually antagonistic (SA) selection, where individual alleles have conflicting fitness effects on each sex function. Although an extensive theory of SA selection exists for dioecious species, these results have not been generalized to hermaphrodites. We develop population genetic models of SA selection in simultaneous hermaphrodites, and evaluate effects of dominance, selection on each sex function, self‐fertilization, and population size on the maintenance of polymorphism. Under obligate outcrossing, hermaphrodite model predictions converge exactly with those of dioecious populations. Self‐fertilization in hermaphrodites generates three points of divergence with dioecious theory. First, opportunities for stable polymorphism decline sharply and become less sensitive to dominance with increased selfing. Second, selfing introduces an asymmetry in the relative importance of selection through male versus female reproductive functions, expands the parameter space favorable for the evolutionary invasion of female‐beneficial alleles, and restricts invasion criteria for male‐beneficial alleles. Finally, contrary to models of unconditionally beneficial alleles, selfing decreases genetic hitchhiking effects of invading SA alleles, and should therefore decrease these population genetic signals of SA polymorphisms. We discuss implications of SA selection in hermaphrodites, including its potential role in the evolution of “selfing syndromes.”     

Reproductive characters in a gynodioecious species, Silene italica (Caryophyllaceae), with attention to the gynomonoecious phenotype

Gynodioecious populations (i.e. populations with female and hermaphrodite individuals) often contain a third phenotype with an intermediate sex expression. In Silene italica , this phenotype is characterized by a mixture of pistillate and perfect flowers and is thus gynomonoecious. To characterize sexual functions of these gynomonoecious individuals and their potential influence in the maintenance of gynodioecy, reproductive characters of the three sexual phenotypes were compared over 2 years in several families of S. italica produced by crossing. We found that gynomonoecious individuals were intermediate for flower production and female fertility characters, although they did not always significantly differ from female individuals. Perfect flowers of gynomonoecious and hermaphrodite plants were similar in size and pollen production. Gynomonoecious individuals were thus intermediate in female and in male functions. Family effects were found for most of the characters. The female advantage (i.e. the fertility of females compared to the female fertility of pollen producing plants) was not dramatically different when gynomonoecious plants were taken into account.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 583–591.     

The evolution of gynodioecy on a lattice

Gynodioecy is a breeding system in plants where populations consist of hermaphrodites and females. The females result from a genetic mutation which impairs pollen production in hermaphrodite plants. Most previous models for the evolution of gynodioecy do not take into account any spatial detail, which might be expected to play an important role in populations with short range interactions caused by poor or no locomotion.In this article we present a generalised mean-field analysis (which ignores any spatial detail), together with stochastic spatial simulations, to investigate the spatial effect on the evolution of gynodioecy. We show that, in a population of hermaphrodites where male sterility is caused by a dominant allele in a nuclear gene, mean-field calculations greatly underestimate the reproductive advantage females require to become viable under spatial constraints. This suggests that gynodioecy is less likely to evolve in plants with more localised pollination and seed setting. This may have implications for the evolution of dioecy, a breeding system in plants where the population consists of males and females, as gynodioecy is thought to be a route to dioecy. Our results also demonstrate that a lower frequency of females should be expected for gynodioecious populations when interactions are local. This is relevant when comparing the results of breeding experiments with observations of female frequency in the wild.     

Male sterility, protogyny, and pollen-pistil interference in Plantago maritima (Plantaginaceae), a wind-pollinated, self-incompatible perennial

Evolution and maintenance of male sterility in seed plants can be explained by the maternal inheritance of mitochondria, which encode the trait, and by adaptive functions that enhance female fecundity in male-sterile compared to hermaphrodite individuals. Protogyny and male sterility can independently decrease the negative effect of pollen-pistil interference in self-incompatible species. In Plantago maritima, which possesses both traits, protogyny increases seed set in hermaphrodite individuals. This is shown both by a significantly positive association between seed set and retarded dehiscence of the anthers and by a more than 50% reduction in seed set following self-pollination. Male sterility does not seem to increase seed set further, as female and hermaphrodite plants do not differ significantly in mean seed set per capsule. Bagging experiments demonstrate strong self-incompatibility in the study populations. Hence, in P. maritima male sterility seems neither to prevent selfing nor to reduce the effect of pollen-pistil interference. Females had significantly larger stigmas than hermaphrodites, but seed set varied negatively with stigma length among females, indicating that the evolution of unisexuality in P. maritima is not due to prefertilization sex allocation. I therefore conclude that the genetical system of nucleocytoplasmic determination of gender is the main cause for maintenance of male sterility in P. maritima.     

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.     

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.     

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.