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.     

Arms races with mitochondrial genome soft sweeps in a gynodioecious plant,Plantago lanceolata

Biological situations involving conflict can create arms race situations with repeated fixations of different functional variants, producing selective sweeps and lowering neutral diversity in genome regions linked to the functional locus. However, they can sometimes lead to balancing selection, potentially creating long coalescent times for sites with functionally different variants, and, if recombination occurs rarely, for extended haplotypes carrying such variants. We tested between these possibilities in a gynodioecious plant, Plantago lanceolata, in which cytoplasmic male‐sterility factors conflict with nuclear restorers of male fertility. We find low mitochondrial diversity, which does not support very long‐term coexistence of highly diverged mitochondrial haplotypes. Interestingly, however, we found a derived haplotype that is associated with male fertility in a restricted geographic region, and that has fixed differences from the ancestral sequence in several genes, suggesting that it did not arise very recently. Taken together, the results suggest arms race events that involved “soft" selective sweeps involving a moderately old‐established haplotype, consistent with the frequency fluctuations predicted by theoretical models of gynodioecy.     

Dioecy Is Associated with High Genetic Diversity and Adaptation Rates in the Plant Genus Silene

About 15,000 angiosperm species (∼6%) have separate sexes, a phenomenon known as dioecy. Why dioecious taxa are so rare is still an open question. Early work reported lower species richness in dioecious compared with nondioecious sister clades, raising the hypothesis that dioecy may be an evolutionary dead-end. This hypothesis has been recently challenged by macroevolutionary analyses that detected no or even positive effect of dioecy on diversification. However, the possible genetic consequences of dioecy at the population level, which could drive the long-term fate of dioecious lineages, have not been tested so far. Here, we used a population genomics approach in the Silene genus to look for possible effects of dioecy, especially for potential evidence of evolutionary handicaps of dioecy underlying the dead-end hypothesis. We collected individual-based RNA-seq data from several populations in 13 closely related species with different sexual systems: seven dioecious, three hermaphroditic, and three gynodioecious species. We show that dioecy is associated with increased genetic diversity, as well as higher selection efficacy both against deleterious mutations and for beneficial mutations. The results hold after controlling for phylogenetic inertia, differences in species census population sizes and geographic ranges. We conclude that dioecious Silene species neither show signs of increased mutational load nor genetic evidence for extinction risk. We discuss these observations in the light of the possible demographic differences between dioecious and self-compatible hermaphroditic species and how this could be related to alternatives to the dead-end hypothesis to explain the rarity of dioecy.     

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.     

An angiosperm-wide analysis of the gynodioecy–dioecy pathway

Background and Aims

About 6 % of an estimated total of 240 000 species of angiosperms are dioecious. The main precursors of this sexual system are thought to be monoecy and gynodioecy. A previous angiosperm-wide study revealed that many dioecious species have evolved through the monoecy pathway; some case studies and a large body of theoretical research also provide evidence in support of the gynodioecy pathway. If plants have evolved through the gynodioecy pathway, gynodioecious and dioecious species should co-occur in the same genera. However, to date, no large-scale analysis has been conducted to determine the prevalence of the gynodioecy pathway in angiosperms. In this study, this gap in knowledge was addressed by performing an angiosperm-wide survey in order to test for co-occurrence as evidence of the gynodioecy pathway.

Methods

Data from different sources were compiled to obtain (to our knowledge) the largest dataset on gynodioecy available, with 275 genera that include at least one gynodioecious species. This dataset was combined with a dioecy dataset from the literature, and a study was made of how often dioecious and gynodioecious species could be found in the same genera using a contingency table framework.

Key Results

It was found that, overall, angiosperm genera with both gynodioecious and dioecious species occur more frequently than expected, in agreement with the gynodioecy pathway. Importantly, this trend holds when studying different classes separately (or sub-classes, orders and families), suggesting that the gynodioecy pathway is not restricted to a few taxa but may instead be widespread in angiosperms.

Conclusions

This work complements that previously carried out on the monoecy pathway and suggests that gynodioecy is also a common pathway in angiosperms. The results also identify angiosperm families where some (or all) dioecious species may have evolved from gynodioecious precursors. These families could be the targets of future small-scale studies on transitions to dioecy taking phylogeny explicitly into account.     

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.     

Emergence of gynodioecy in wild beet (Beta vulgaris ssp. maritima L.): a genealogical approach using chloroplastic nucleotide sequences

Gynodioecy is a breeding system where both hermaphroditic and female individuals coexist within plant populations. This dimorphism is the result of a genomic interaction between maternally inherited cytoplasmic male sterility (CMS) genes and bi-parentally inherited nuclear male fertility restorers. As opposed to other gynodioecious species, where every cytoplasm seems to be associated with male sterility, wild beet Beta vulgaris ssp. maritima exhibits a minority of sterilizing cytoplasms among numerous non-sterilizing ones. Many studies on population genetics have explored the molecular diversity of different CMS cytoplasms, but questions remain concerning their evolutionary dynamics. In this paper we report one of the first investigations on phylogenetic relationships between CMS and non-CMS lineages. We investigated the phylogenetic relationships between 35 individuals exhibiting different mitochondrial haplotypes. Relying on the high linkage disequilibrium between chloroplastic and mitochondrial genomes, we chose to analyse the nucleotide sequence diversity of three chloroplastic fragments (trnK intron, trnD-trnT and trnL-trnF intergenic spacers). Nucleotide diversity appeared to be low, suggesting a recent bottleneck during the evolutionary history of B. vulgaris ssp. maritima. Statistical parsimony analyses revealed a star-like genealogy and showed that sterilizing haplotypes all belong to different lineages derived from an ancestral non-sterilizing cytoplasm. These results suggest a rapid evolution of male sterility in this taxon. The emergence of gynodioecy in wild beet is confronted with theoretical expectations, describing either gynodioecy dynamics as the maintenance of CMS factors through balancing selection or as a constant turnover of new CMSs.     

The evolution of reproductive systems and sex-determining mechanisms within rumex (polygonaceae) inferred from nuclear and chloroplastidial sequence data

The genus Rumex includes hermaphroditic, polygamous, gynodioecious, monoecious, and dioecious species, with the dioecious species being represented by different sex-determining mechanisms and sex-chromosome systems. Therefore, this genus represents an exceptional case study to test several hypotheses concerning the evolution of both mating systems and the genetic control of sex determination in plants. Here, we compare nuclear intergenic transcribed spacers and chloroplast intergenic sequences of 31 species of Rumex. Our phylogenetic analysis supports a systematic classification of the genus, which differs from that currently accepted. In contrast to the current view, this new phylogeny suggests a common origin for all Eurasian and American dioecious species of Rumex, with gynodioecy as an intermediate state on the way to dioecy. Our results support the contention that sex determination based on the balance between the number of X chromosomes and the number of autosomes (X/A balance) has evolved secondarily from male-determining Y mechanisms and that multiple sex-chromosome systems, XX/XY1Y2, were derived twice from an XX/XY system. The resulting phylogeny is consistent with a classification of Rumex species according to their basic chromosome number, implying that the evolution of Rumex species might have followed a process of chromosomal reduction from x = 10 toward x = 7 through intermediate stages (x = 9 and x = 8).     

THE ROLE OF ANDRODIOECY AND GYNODIOECY IN MEDIATING EVOLUTIONARY TRANSITIONS BETWEEN DIOECY AND HERMAPHRODITISM IN THE ANIMALIA

Dioecy (gonochorism) is dominant within the Animalia, although a recent review suggests hermaphroditism is also common. Evolutionary transitions from dioecy to hermaphroditism (or vice versa) have occurred frequently in animals, but few studies suggest the advantage of such transitions. In particular, few studies assess how hermaphroditism evolves from dioecy or whether androdioecy or gynodioecy should be an “intermediate” stage, as noted in plants. Herein, these transitions are assessed by documenting the numbers of androdioecious and gynodioecious animals and inferring their ancestral reproductive mode. Both systems are rare, but androdioecy was an order of magnitude more common than gynodioecy. Transitions from dioecious ancestors were commonly to androdioecy rather than gynodioecy. Hermaphrodites evolving from sexually dimorphic dioecious ancestors appear to be constrained to those with female‐biased sex allocation; such hermaphrodites replace females to coexist with males. Hermaphrodites evolving from sexually monomorphic dioecious ancestors were not similarly constrained. Species transitioning from hermaphroditic ancestors were more commonly androdioecious than gynodioecious, contrasting with similar transitions in plants. In animals, such transitions were associated with size specialization between the sexes, whereas in plants these transitions were to avoid inbreeding depression. Further research should frame these reproductive transitions in a theoretical context, similar to botanical studies.     

Mathematical Study of the Evolution of Gynodioecy with Cytoplasmic Inheritance under the Effect of a Nuclear Restorer Gene

A study is described of the influence of the introduction of a dominant nuclear restorer gene into a cytoplasmic gynodioecious plant population. This study includes the consideration of separate effects on the relative female fertility of nuclear, cytoplasmic and sex (phenotypic) factors. Under these assumptions, the introduction of a dominant nuclear restorer gene into a cytoplasmic gynodioecious population can lead to several different situations: persistence of cytoplasmic gynodioecy, appearance of a nuclear-cytoplasmic gynodioecy, appearance of a nuclear gynodioecy or complete restoration of male fertility. The development of stable nuclear-cytoplasmic gynodioecy in a mathematical model is new and is possible because of the consideration of the separate relative female fertilities. The possibility of a transformation of cytoplasmic gynodioecy into a nuclear one has never been obtained before. It could constitute a route for the appearance of this latter kind of gynodioecy in plant populations. Finally, the possibilities of evolution of gynodioecy from one kind to the other, and towards dioecy, are discussed, as are some theoretical schemes that seem to correspond to observed actual situations.     

When gametophytic self-incompatibility meets gynodioecy.

The occurrence of gynodioecy among angiosperms appears to be associated with self-compatibility. We use individual-based simulations to investigate the conditions for breakdown of a gametophytic self-incompatibility system in gynodioecious populations and make a comparison with hermaphroditic populations where the conditions are well known. We study three types of mutations causing self-compatibility. We track the fate of these mutations in both gynodioecious and hermaphroditic populations, where we vary the number of S-alleles, inbreeding depression and selfing rate. We find that the conditions for breakdown are less stringent if the population is gynodioecious and that the breakdown of self-incompatibility tends to promote stability of gynodioecious populations since it results in a higher frequency of females. We also find that fecundity selection has a large effect on the probability of breakdown of self-incompatibility, in particular if caused by a mutation destroying the female function of the S-locus.     

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.     

Reduced recombination in gynodioecious populations of a facultative apomictic orchid

Many plants combine sexual reproduction with some form of asexual reproduction to different degrees, and lower genetic diversity is expected with asexuality. Moreover, the ratios of sexual morphs in species with gender dimorphism are expected to vary in proportion to the reproductive success of the sexual process. Hence, sex ratios can directly influence the genetic structure and diversity of a population. We investigated genotypic diversity in 23 populations of a facultative, apomictic gynodioecious orchid, Satyrium ciliatum, to examine the effect on genotypic diversity of variation in the frequency of females and in the amount of sexual reproduction. The study involved one pure female, seven gynodioecious (both females and hermaphrodites present) and 15 hermaphroditic populations. Pollinia receipt was higher in hermaphroditic than in gynodioecious populations. Analyses of variation in ISSRs demonstrated that genotypic diversity was high in all populations and was not significantly different between hermaphroditic and gynodioecious populations. We used character compatibility analysis to determine the extent to which recombination by sexual reproduction contributed to genotypic diversity. The results indicate that the contribution of recombination to genotypic diversity is higher in hermaphroditic than in gynodioecious populations, consistent with the finding that hermaphroditic populations received higher amounts of pollinia. Our finding of reduced recombination in gynodioecious populations suggests that maintenance of sex in hermaphrodites plays an important role in generating genotypic diversity in this apomictic orchid.     

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.     

Functional characterization of gynodioecy in Fragaria vesca ssp. bracteata (Rosaceae)

Background and Aims

Gynodioecy is a phylogenetically widespread and important sexual system where females coexist with hermaphrodites. Because dioecy can arise from gynodioecy, characterization of gynodioecy in close relatives of dioecious and sub-dioecious species can provide insight into this transition. Thus, we sought to determine whether Fragaria vesca ssp. bracteata, a close relative to F. chiloensis and F. virginiana, exhibits the functional and population genetic hallmarks of a gynodioecious species.

Methods

We compared reproductive allocation of females and hermaphrodites grown in the greenhouse and estimated genetic diversity (allelic diversity, heterozygosity) and inbreeding coefficients for field-collected adults of both sexes using simple sequence repeat (SSR) markers. We estimated mating system and early seed fitness from open-pollinated families of both sex morphs.

Key Results

Under greenhouse conditions, females and hermaphrodites allocated similarly to all reproductive traits except flower number, and, as a consequence, females produced 30 % fewer seeds per plant than hermaphrodites. Under natural conditions, hermaphrodites produce seeds by self-fertilization approx. 75 % of the time, and females produced outcrossed seeds with very little biparental inbreeding. Consistent with inbreeding depression, seeds from open-pollinated hermaphrodites were less likely to germinate than those from females, and family-level estimates of hermaphrodite selfing rates were negatively correlated with germination success and speed. Furthermore, estimates of inbreeding depression based on genetic markers and population genetic theory indicate that inbreeding depression in the field could be high.

Conclusions

The joint consideration of allocation and mating system suggests that compensation may be sufficient to maintain females given the current understanding of sex determination. Fragaria vesca ssp. bracteata exhibited similar sex morph-dependent patterns of mating system and genetic diversity, but less reproductive trait dimorphism, than its sub-dioecious and dioecious congeners.     

Evidence of restoration cost in the annual gynodioecious Phacelia dubia

A negative pleiotropic effect on fitness of nuclear sex‐determining genes (cost of restoration) could explain nuclear–cytoplasmic gynodioecy but rarely has been demonstrated empirically. In a gynodioecious Phacelia dubia population, maternal lineages produce only hermaphroditic progenies irrespective of the pollen parent (N) or can segregate females (S). Natural progenies of N maternal plants had lower seed viability than that of S. Full‐sib progenies of unrelated hermaphrodites from all possible matings between N and S lineages had similar pollen filling but differed in sporophyte performance, mainly at seed germination stage. A discrete multivariate analysis reveals that the performance of N_♀ × S_♂ progeny at early stages of development was significantly lower than that of the other three types of mating in agreement with the silent‐cost‐of‐restoration hypothesis, affecting the sporophyte. The restoration cost and male sterility appear to be dominant and consequence of nuclear–cytoplasmic incompatibilities that may maintain nuclear–cytoplasmic polymorphism by frequency‐dependent selection.     

FREQUENCY-DEPENDENT FITNESS IN SILENE VULGARIS,A GYNODIOECIOUS PLANT

In gynodioecious plants the selective processes that determine the relative number of female and hermaphroditic individuals are often frequency dependent. Frequency-dependent fitness can occur in the two sexes through a variety of mechanisms, especially given pollen limitation and inbreeding depression when hermaphrodites are rare. Frequency dependence in several components of the fitness of female and hermaphroditic Silene vulgaris was tested in experiments in which the relative numbers of the two sexes was varied among 12 artificial populations. In females, the proportion of flowers that set fruit covaried positively among populations with the frequency of hermaphrodites in two separate experiments, whereas the number of flowers/plant covaried negatively in one case. In hermaphrodites, the number of seeds/fruit covaried positively with the frequency of hermaphrodites, whereas the fitness of hermaphrodites estimated through pollen transfer covaried negatively. The results are discussed as they relate to the selective maintenance of gynodioecy in S. vulgaris and in light of a recent model of the effect of population structure on selection in gynodioecious systems.     

Segregation of male‐sterility alleles across a species boundary

Hybrid zones may serve as bridges permitting gene flow between species, including alleles influencing the evolution of breeding systems. Using greenhouse crosses, we assessed the likelihood that a hybrid zone could serve as a conduit for transfer of nuclear male‐sterility alleles between a gynodioecious species and a hermaphroditic species with very rare females in some populations. Segregation patterns in progeny of crosses between rare females of hermaphroditic Schiedea menziesii and hermaphroditic plants of gynodioecious Schiedea salicaria heterozygous at the male‐sterility locus, and between female S. salicaria and hermaphroditic plants from the hybrid zone, were used to determine whether male‐sterility was controlled at the same locus in the parental species and the hybrid zone. Segregations of females and hermaphrodites in approximately equal ratios from many of the crosses indicate that the same nuclear male‐sterility allele occurs in the parent species and the hybrid zone. These rare male‐sterility alleles in S. menziesii may result from gene flow from S. salicaria through the hybrid zone, presumably facilitated by wind pollination in S. salicaria. Alternatively, rare male‐sterility alleles might result from a reversal from gynodioecy to hermaphroditism in S. menziesii, or possibly de novo evolution of male sterility. Phylogenetic analysis indicates that some species of Schiedea have probably evolved separate sexes independently, but not in the lineage containing S. salicaria and S. menziesii. High levels of selfing and expression of strong inbreeding depression in S. menziesii, which together should favour females in populations, argue against a reversal from gynodioecy to hermaphroditism in S. menziesii.     

MK17, a specific marker closely linked to the gynoecium suppression region on the Y chromosome in Silene latifolia

The aim of this work was to isolate new DNA markers linked to the Silene latifolia Y chromosome. To do this we created a chromosome-specific plasmid library after DOP-PCR amplification of laser-microdissected Y-chromosomes. The library screening led to the isolation of several clones yielding mostly to exclusive male specific hybridization signals. Subsequent PCR confirmed the Y-unique linkage for one of the sequences. This DNA sequence called MK17 has no homology to any known DNA sequence and it is not expressed. Based on PCR and Southern analyses, MK17 is present only in dioecious species of the Elisanthe section of the genus Silene (S. latifolia, S. dioica, and S. diclinis) and it is absent in related gynodioecious and hermaphroditic species. The mapping analysis using a panel of deletion mutants showed that MK17 is closely linked to the region controlling suppression of gynoecium development. Hence MK17 represents a valuable marker to isolate genes controlling the gynoecium development suppression on the Y chromosome of S. latifolia.     

Mating system dynamics of Ocotea tenera (Lauraceae), a gynodioecious tropical tree

Progeny arrays of Ocotea tenera (Lauraceae), a gynodioecious tree endemic to Costa Rica, were electrophoretically surveyed for allozyme variation to estimate the outcrossing rate in the overall population and to test for differences in outcrossing rates between hermaphroditic and female trees. Multilocus outcrossing rate estimates across 3 yr indicated O. tenera predominantly outcrosses. However, significant heterogeneity in single-locus outcrossing rates was found among loci. Two loci (Fe1, Fe2) gave high outcrossing estimates, and a third locus (Gdh) gave much lower outcrossing estimates. Heterogeneity in Gdh pollen allele frequencies, consanguineous matings, and selection against homozygous zygote genotypes at the Fe1 and Fe2 loci are factors contributing to the discrepancy in outcrossing rate estimates among loci. There were no differences in the mating systems of hermaphroditic and female trees, which suggests that factors beyond prevention of self-fertilization may have also promoted the evolution of gynodioecy in O. tenera.