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.     

Sex‐specific fitness variation in gynodioecious Beta vulgaris ssp. maritima: do empirical observations fit theoretical predictions?

In gynodioecious species, in which hermaphroditic and female plants co-occur, the maintenance of sexual polymorphism relies on the genetic determination of sex and on the relative fitness of the different phenotypes. Flower production, components of male fitness (pollen quantity and pollen quality) and female fitness (fruit and seed set) were measured in gynodioecious Beta vulgaris spp. maritima, in which sex is determined by interactions between cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. The results suggested that (i) female had a marginal advantage over hermaphrodites in terms of flower production only, (ii) restored CMS hermaphrodites (carrying both CMS genes and nuclear restorers) suffered a slight decrease in fruit production compared to non-CMS hermaphrodites and (iii) restored CMS hermaphrodites were poor pollen producers compared to non-CMS hermaphrodites, probably as a consequence of complex determination of restoration. These observations potentially have important consequences for the conditions of maintenance of sexual polymorphism in B. vulgaris and are discussed in the light of existing theory on evolutionary dynamics of gynodioecy.     

Disentangling the causes of heterogeneity in male fecundity in gynodioecious Beta vulgaris ssp. maritima

Variation among individuals in reproductive success is advocated as a major process driving evolution of sexual polymorphisms in plants, such as gynodioecy where females and hermaphrodites coexist. In gynodioecious Beta vulgaris ssp. maritima, sex determination involves cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. Both restored CMS and non-CMS hermaphrodites co-occur. Genotype-specific differences in male fitness are theoretically expected to explain the maintenance of cytonuclear polymorphism. Using genotypic information on seedlings and flowering plants within two metapopulations, we investigated whether male fecundity was influenced by ecological, phenotypic and genetic factors, while taking into account the shape and scale of pollen dispersal. Along with spatially restricted pollen flow, we showed that male fecundity was affected by flowering synchrony, investment in reproduction, pollen production and cytoplasmic identity of potential fathers. Siring success of non-CMS hermaphrodites was higher than that of restored CMS hermaphrodites. However, the magnitude of the difference in fecundity depended on the likelihood of carrying restorer alleles for non-CMS hermaphrodites. Our results suggest the occurrence of a cost of silent restorers, a condition supported by scarce empirical evidence, but theoretically required to maintain a stable sexual polymorphism in gynodioecious species.     

The Paradox of Clonality and the Evolution of Self-Incompatibility

In the January issue of New Phytologist Vallejo-Marín and O''Brien¹ documented that in the genus Solanum (Solanaceae) clonality and self-incompatibility, a common genetic mechanism enforcing cross-fertilization, co-occur more often than expected by chance. Using a phylogenetic approach the authors showed that the statistical association between clonality and self-incompatibility persists even after taking into account phylogenetic relationships among species, uncertainty in the phylogenetic reconstruction, and associations between clonality and life history (annual/perennial). Vallejo-Marín and O''Brien¹ suggest that clonality and self-incompatibility tend to co-occur because clonality, by allowing the persistence and propagation of a genotype in environments with limited pollinator or mate availability, reduces the selective pressure favoring the breakdown of self-incompatibility. In addition to promoting the maintenance of self-incompatibility, when clonality results in the spatial aggregation of genetically identical individuals, clonality may promote its breakdown by restricting pollen transfer between different genotypes. Here I call attention to these contradictory predictions of the effects of clonality on the evolution of self-incompatibility, and suggest that the outcome of this paradox depend on both the extent to which clonal propagation compensates for limited seed production, and on the extent to which clonality reduces pollen transfer between genotypes.Key Words: asexual reproduction, clonality, mating system, pollen limitation, reproductive assurance, reproductive compensation, SolanumFlowering plants display a variety of mechanisms preventing self-fertilization, among which self-incompatibility is one of the best studied.^2,3 In the genus Solanum (Solanaceae) ancestrally present self-incompatibility has broken down multiple independent times through out the evolutionary history of this group to give rise to self-compatible lineages.^4,5 The breakdown of outcrossing mechanisms, including self-incompatibility, is one of the most common and best studied evolutionary transitions in flowering plants.⁶Self-incompatibility is a genetic mechanism by which the maternal plant can recognize and reject pollen grains expressing alleles in common with the maternal genotype.⁷ The reproductive advantages of self-incompatibility can be understood as a balance between two forces. On one hand the rejection of pollen grains is a mechanism for the preferential support of outbred progeny which, generally, is of higher genetic quality. On the other hand, if pollen or pollinator availability is low, pollen grains that are rejected by the self-incompatibility mechanism may not be substituted, and thus some ovules would go unfertilized. The evolutionary maintenance of self-incompatibility depends on the relative benefit of producing higher quality offspring and the relative costs incurred by potential reduction in offspring number⁸Plant clonality can affect the relative benefits and costs of self-incompatibility through its effects on the persistence and spatial distribution of genotypes. For instance, Vallejo-Marín and O''Brien¹ suggest that clonality provides reproductive assurance in colonizing taxa by allowing genotypes to persist and propagate even in the absence of conditions conducive to seed production. The reproductive assurance conferred by clonality would then relief the evolutionary costs of self-incompatibility incurred through reduced seed number, favoring the maintenance of self-incompatibility (Fig. 1).^1,9,10 In contrast, clonality may increase the evolutionary costs of self-incompatibility, by restricting pollen transfer between genotypes.^11,12 In some forms of clonal growth, dispersal of asexual propagules is very localized, resulting in an aggregation or clumping of genotypes. If spatial clumping restricts pollen flow between distinct genetic individuals, plants may not receive enough compatible pollen to fertilize all ovules (incomplete reproductive compensation) thus resulting in reduced seed set, which may favor the breakdown of self-incompatibility (Fig. 1).^8,12,13 It is important to note that both the maintenance and the breakdown effects mentioned above are expected to occur in the same ecological conditions, namely when the availability of pollen, pollinators, or compatible mates limit seed set (collectively known as pollen limitation).Open in a separate windowFigure 1Potential consequences of clonality for the evolutionary maintenance of self-incompatibility (SI). Full reproductive compensation occurs when pollen grains rejected by the SI system can be replaced with other compatible pollen grains. Under full reproductive compensation, seed set is independent of the level of SI expression.⁸ When pollen receipt is limited, rejected pollen grains cannot be always substituted (incomplete reproductive compensation) and consequently seed set is negatively related to the level of SI expression.⁸ Notice that under similar ecological conditions, e.g., when pollen receipt is limited (outside arrows), clonality may both favor the maintenance of SI through reproductive assurance, and facilitate its breakdown through increasing within-genotype pollen transfer and reducing seed set.A contrasting ecological scenario, may in turn favor the maintenance of self-incompatibility in clonal taxa. When pollen is abundant, and pollinators transport pollen through larger distances, enough pollen might still be received to fertilize all ovules even after accounting for pollen rejected by the self-incompatibility system (full reproductive compensation; Fig. 1). In this case, seed number will not be affected by the expression of self-incompatibility, but seed quality may be increased through the rejection of inbred pollen.¹⁴ In other words, when pollen receipt does not limit seed number, the presence of clonality is expected to favor self-incompatibility as a mechanism to successfully screen inbred pollen originated from different individuals of the same genotype or clone.To summarize, under the same ecological conditions (i.e., pollen limitation) the co-occurrence of clonality and self-incompatibility may have contrasting effects (Fig. 1). On one hand clonality favors the maintenance of self-incompatibility by providing reproductive assurance. On the other hand clonality may favor the breakdown of self-incompatibility, because rejected pollen cannot be compensated for, ensuing in a reduction in seed number. The resolution of the paradox of clonality for the evolution of self-incompatibility is likely to depend on the degree to which clonal propagation compensates for limited reproduction through seeds (e.g., during population establishment), as well as on the extent to which clonality reduces pollen flow between established genotypes, which in turn is affected by characteristics such as clonal architecture, plant density, and pollinator type and availability.^12,15,16Vallejo-Marín and O''Brien''s data¹ suggest that in the colonizing genus Solanum, which is expected to experience pollen limiting conditions, clonality reduces the costs of self-incompatibility through reproductive assurance (maintenance effect) more than it increases the costs of self-incompatibility due to pollen flow within spatially clumped genotypes (breakdown effect). Future comparative studies of the association between self-incompatibility and clonality in other groups could help us determine the general conditions in which the maintenance effect outweighs the breakdown effect.     

Maternal sex effects and inbreeding depression under varied environmental conditions in gynodioecious Fragaria vesca subsp. bracteata

Background and Aims

Gynodioecy (coexistence of females and hermaphrodites) is a sexual system that occurs in numerous flowering plant lineages. Thus, understanding the features that affect its maintenance has wide importance. Models predict that females must have a seed fitness advantage over hermaphrodites, and this may be achieved via seed quality or quantity. Females in a population of Fragaria vesca subsp. bracteata, a long-lived gynodioecious perennial, do not demonstrate a seed quantity advantage, so this study explored whether females produced better quality seed via maternal sex effects or avoidance of inbreeding depression (IBD).

Methods

Families of selfed and outcrossed seed were created using hermaphrodite mothers and families of outcrossed seed were created using female mothers. The effects of these pollination treatments were assessed under benign conditions early in life and under varied conditions later in life. To test for an effect of maternal sex, fitness components and traits associated with acclimation to variable environments of progeny of outbred hermaphrodites and females were compared. To test for expression of IBD, fitness parameters between inbred and outbred progeny of hermaphrodites were compared.

Key Results

Offspring of females were more likely to germinate in benign conditions and survive in harsh resource environments than outbred progeny of hermaphrodites. IBD was low across most life stages, and both the effect of maternal sex on progeny quality and the expression of IBD depended on both maternal family and resource condition of the progeny.

Conclusions

The effect of maternal sex and IBD on progeny quality depended on resource conditions, maternal lineage and progeny life stage. In conjunction with known lack of differences in seed quantity, the quality advantages and IBD observed here are still unlikely to be sufficient for maintenance of gynodioecy under nuclear inheritance of male sterility.     

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.     

The identification of allergen proteins in sugar beet (Beta vulgaris) pollen causing occupational allergy in greenhouses

Background

During production of sugar beet (Beta vulgaris) seeds in greenhouses, workers frequently develop allergic symptoms. The aim of this study was to identify and characterize possible allergens in sugar beet pollen.

Methods

Sera from individuals at a local sugar beet seed producing company, having positive SPT and specific IgE to sugar beet pollen extract, were used for immunoblotting. Proteins in sugar beet pollen extracts were separated by 1- and 2-dimensional electrophoresis, and IgE-reactive proteins analyzed by liquid chromatography tandem mass spectrometry.

Results

A 14 kDa protein was identified as an allergen, since IgE-binding was inhibited by the well-characterized allergen Che a 2, profilin, from the related species Chenopodium album. The presence of 17 kDa and 14 kDa protein homologues to both the allergens Che a 1 and Che a 2 were detected in an extract from sugar beet pollen, and partial amino acid sequences were determined, using inclusion lists for tandem mass spectrometry based on homologous sequences.

Conclusion

Two occupational allergens were identified in sugar beet pollen showing sequence similarity with Chenopodium allergens. Sequence data were obtained by mass spectrometry (70 and 25%, respectively for Beta v 1 and Beta v 2), and can be used for cloning and recombinant expression of the allergens. As for treatment of Chenopodium pollinosis, immunotherapy with sugar beet pollen extracts may be feasible.     

Effects of low-efficiency pollinators on plant fitness and floral trait evolution in <Emphasis Type="Italic">Campanula americana</Emphasis> (Campanulaceae)

Floral visitors vary in their pollination efficiency and their preferences for floral traits. If low-efficiency pollinators decrease the amount of pollen available to higher efficiency visitors, then low-efficiency visitors may actually have negative fitness consequences for the plants that they visit. We used experimental arrays in two populations to determine the floral preferences and the fitness effects of low-efficiency (or ugly) pollinators on Campanula americana. These ugly pollinators (halictid bees) preferentially visited flowers with pollen over flowers that had had their pollen removed. C. americana pollen color varies quantitatively from light tan to dark purple, and we found that natural variation in pollen color influenced the magnitude of halictid preferences for flowers with pollen. In general, preferences for flowers with pollen were stronger when the ugly pollinators foraged in arrays of flowers with tan-colored pollen than in arrays with purple-colored pollen. When plants received few visits by efficient Bombus pollinators, visits by ugly pollinators significantly decreased siring success relative to plants where visits by ugly pollinators were prevented. In contrast, ugly pollinators did not influence siring success when higher efficiency pollinators were more abundant. Thus, the relationship between low-efficiency pollinators and the plants that they visit varies from commensalistic to antagonistic depending on the presence of other pollinators in the community. Our findings suggest that the negative fitness effects and floral preferences of low-efficiency or ugly pollinators may contribute to the maintenance of a pollen color polymorphism in C. americana.     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

Differential costs of reproduction in females and hermaphrodites in a gynodioecious plant

Background and Aims

Plants exhibit a variety of reproductive systems where unisexual (females or males) morphs coexist with hermaphrodites. The maintenance of dimorphic and polymorphic reproductive systems may be problematic. For example, to coexist with hermaphrodites the females of gynodioecious species have to compensate for the lack of male function. In our study species, Geranium sylvaticum, a perennial gynodioecious herb, the relative seed fitness advantage of females varies significantly between years within populations as well as among populations. Differences in reproductive investment between females and hermaphrodites may lead to differences in future survival, growth and reproductive success, i.e. to differential costs of reproduction. Since females of this species produce more seeds, higher costs of reproduction in females than in hermaphrodites were expected. Due to the higher costs of reproduction, the yearly variation in reproductive output of females might be more pronounced than that of hermaphrodites.

Methods

Using supplemental hand-pollination of females and hermaphrodites of G. sylvaticum we examined if increased reproductive output leads to differential costs of reproduction in terms of survival, probability of flowering, and seed production in the following year.

Key Results

Experimentally increased reproductive output had differential effects on the reproduction of females and hermaphrodites. In hermaphrodites, the probability of flowering decreased significantly in the following year, whereas in females the costs were expressed in terms of decreased future seed production.

Conclusions

When combining the probability of flowering and seed production per plant to estimate the multiplicative change in fitness, female plants showed a 56 % and hermaphrodites showed a 39 % decrease in fitness due to experimentally increased reproduction. Therefore, in total, female plants seem to be more sensitive to the cost of reproduction in terms of seed fitness than hermaphrodites.     

THE EFFECT OF POLLEN VERSUS SEED FLOW ON THE MAINTENANCE OF NUCLEAR‐CYTOPLASMIC GYNODIOECY

Gynodioecy, where females co‐occur with hermaphrodites, is a relatively common sexual system in plants that is often the result of a genetic conflict between maternally inherited male sterility genes in the mitochondrial genome and the biparentally inherited male fertility restorer genes in the nucleus. Previous models have shown that nuclear‐cytoplasmic gynodioecy can be maintained under certain conditions by negative frequency‐dependent selection, but the effect of other evolutionary processes such as genetic drift and population subdivision is only partially understood. Here, we investigate the joint effects of frequency‐dependent selection, drift, and migration through either pollen or seeds on the maintenance of nuclear‐cytoplasmic gynodioecy in a subdivided population. We find that the combination of drift and selection causes the loss of gynodioecy under scenarios that would maintain it under the influence of selection alone, and that both seed and, more surprisingly, pollen flow can maintain the polymorphism. In particular, although pollen flow could not avoid the loss of cytoplasmic polymorphism within demes, it allowed the maintenance of nuclear‐cytoplasmic polymorphism at the metapopulation level.     

Variation in pollen production and pollen viability in natural populations of gynodioecious Beta vulgaris ssp. maritima: evidence for a cost of restoration of male function?

Gynodioecious species are defined by the co-occurrence of two clearly separated categories of plants: females and hermaphrodites. The hermaphroditic category may, however, not be homogeneous, as male fitness may vary among hermaphrodites as a result of many biological factors. In this study, we analysed estimates of pollen quantity and viability in the gynodioecious Beta vulgaris ssp. maritima, comparing hermaphrodites bearing a male-fertile cytotype and hermaphrodites bearing cytoplasmic male sterility (CMS) genes, which are counteracted by nuclear restoration factors. We show that: (i) pollen quantity continuously varies among restored hermaphrodites, suggesting a complex genetic determination of nuclear restoration; (ii) pollen viability was lower in restored (CMS) hermaphrodites than in non-CMS hermaphrodites, probably because of incomplete restoration in some of these plants; and (iii) pollen quantity and viability also varied among hermaphrodites with male-fertile cytotypes, possibly a result of a silent cost of restoration. Finally, we discuss the consequences of these results for pollen flow and the dynamics of gynodioecy.     

On the gynodioecious polymorphism in Saxifraga granulata L. (Saxifragaceae)

Sexual and vegetative fitness components in hermaphrodite and female plants of the self-compatible, perennial herb Saxifraga granulata are compared using material derived from a gynodioecious population in northern England.
Females produced only 57% as many seeds as hermaphrodites, but their ovule offspring were 1.28 times as fit as those of hermaphrodites, and females were more vegetatively vigorous. The advantages to females in ovule offspring quality and in vegetative reproduction counteract their disadvantages in pollen and seed production and therefore probably play a role in the maintenance of the gynodioecious polymorphism. Pollination ecology, resource reallocation and inbreeding depression all appear to contribute to the observed sex differences in fitness.     

Reproductive assurance drives transitions to self-fertilization in experimental <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background

Evolutionary transitions from outcrossing between individuals to selfing are partly responsible for the great diversity of animal and plant reproduction systems. The hypothesis of `reproductive assurance’ suggests that transitions to selfing occur because selfers that are able to reproduce on their own ensure the persistence of populations in environments where mates or pollination agents are unavailable. Here we test this hypothesis by performing experimental evolution in Caenorhabditis elegans.

Results

We show that self-compatible hermaphrodites provide reproductive assurance to a male-female population facing a novel environment where outcrossing is limiting. Invasions of hermaphrodites in male-female populations, and subsequent experimental evolution in the novel environment, led to successful transitions to selfing and adaptation. Adaptation was not due to the loss of males during transitions, as shown by evolution experiments in exclusively hermaphroditic populations and in male-hermaphrodite populations. Instead, adaptation was due to the displacement of females by hermaphrodites. Genotyping of single-nucleotide polymorphisms further indicated that the observed evolution of selfing rates was not due to selection of standing genetic diversity. Finally, numerical modelling and evolution experiments in male-female populations demonstrate that the improvement of male fitness components may diminish the opportunity for reproductive assurance.

Conclusions

Our findings support the hypothesis that reproductive assurance can drive the transition from outcrossing to selfing, and further suggest that the success of transitions to selfing hinges on adaptation of obligate outcrossing populations to the environment where outcrossing was once a limiting factor.

     

Siring success and paternal effects in heterodichogamous Acer opalus

Background and Aims

Heterodichogamy (a dimorphic breeding system comprising protandrous and protogynous individuals) is a potential starting point in the evolution of dioecy from hermaphroditism. In the genus Acer, previous work suggests that dioecy evolved from heterodichogamy through an initial spread of unisexual males. Here, the question is asked as to whether the different morphs in Acer opalus, a species in which males co-exist with heterodichogamous hermaphrodites, differ in various components of male in fitness.

Methods

Several components of male fertility were analysed. Pollination rates in the male phase were recorded across one flowering period. Pollen viability was compared among morphs through hand pollinations both with pollen from a single sexual morph and also simulating a situation of pollen competition; in the latter experiment, paternity was assessed with microsatellite markers. It was also determined whether effects of genetic relatedness between pollen donors and recipients could influence the siring success. Finally, paternal effects occurring beyond the fertilization process were tested for by measuring the height reached by seedlings with different sires over three consecutive growing seasons.

Key Results

The males and protandrous morphs had higher pollination rates than the protogynous morph, and the seedlings they sired grew taller. No differences in male fertility were found between males and protandrous individuals. Departures from random mating due to effects of genetic relatedness among sires and pollen recipients were also ruled out.

Conclusions

Males and protandrous individuals are probably better sires than protogynous individuals, as shown by the higher pollination rates and the differential growth of the seedlings sired by these morphs. In contrast, the fertility of males was not higher than the male fertility of the protandrous morph. While the appearance of males in sexually specialized heterodichogamous populations is possible, even in the absence of a fitness advantage, it is not clear that males can be maintained at an evolutionary equilibrium with two classes of heterodichogamous hermaphrodites.Key words: Acer opalus, heterodichogamy, male fertility, microsatellites, paternal effects, pollen competition, pollination rates, genetic relatedness     

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.     

Differential gender selection on flower size in two Neotropical savanna congeneric species

Larger flowers greatly increase among-individual pollen exchange within populations. However, water costs associated to transpirational cooling also increase with increasing flower size. Overall, the interplay between pollen and resource limitation determines the intensity of selection on flower size and this process is mostly dependent on gender and ecological context. To examine how pollinators and water use affect flower size, we determined corolla transpiration, pollen limitation, and selection through male and female fitness in two Kielmeyera species from the Brazilian cerrado flowering at different seasons. Hand-pollination experiments suggested pollen limitation through female fitness in both species, but K. coriacea showed lower limitation levels than K. regalis. For male fitness, the percentage of pollen removal was 1.5-times higher in K. coriacea. Higher air temperature and water deficit during flowering season of K. coriacea resulted in 4-fold higher corolla transpiration rates compared to K. regalis. Selection on flower size through male function was positive and significantly higher than selection through female components in both species. We also detected stabilizing selection in K. coriacea and positive selection in K. regalis on flower size through seed number. Our results suggest that selection on flower size in K. coriacea was mainly limited by water, whereas in K. regalis it was more limited by pollen. We demonstrate that differences in pollen and abiotic resource limitation determine gender-specific selection on flower size.     

The Origins of Specialization: Insights from Bacteria Held 25 Years in Captivity

Examples of ecological specialization abound in nature but the evolutionary and genetic causes of tradeoffs across environments are typically unknown. Natural selection itself may favor traits that improve fitness in one environment but reduce fitness elsewhere. Furthermore, an absence of selection on unused traits renders them susceptible to mutational erosion by genetic drift. Experimental evolution of microbial populations allows these potentially concurrent dynamics to be evaluated directly, rather than by historical inference. The 50,000 generation (and counting) Lenski Long-Term Evolution Experiment (LTEE), in which replicate E. coli populations have been passaged in a simple environment with only glucose for carbon and energy, has inspired multiple studies of their potential specialization. Earlier in this experiment, most changes were the side effects of selection, both broadening growth potential in some conditions and narrowing it in others, particularly in assays of diet breadth and thermotolerance. The fact that replicate populations experienced similar losses suggested they were becoming specialists because of tradeoffs imposed by selection. However a new study in this issue of PLOS Biology by Nicholas Leiby and Christopher Marx revisits these lines with powerful new growth assays and finds a surprising number of functional gains as well as losses, the latter of which were enriched in populations that had evolved higher mutation rates. Thus, these populations are steadily becoming glucose specialists by the relentless pressure of mutation accumulation, which has taken 25 years to detect. More surprising, the unpredictability of functional changes suggests that we still have much to learn about how the best-studied bacterium adapts to grow on the best-studied sugar.The wonder of biological diversity belies a puzzling subtext. Species are defined as much by their limits as their capabilities. Very few species in our common vernacular tolerate life in a wide range of environments, and those that do—the Norway rat, say—are not generally appealing. More often, we celebrate specialization to a particular condition: for example, orchid epiphytes growing tenuously in the cloud forest, only a subtle climate shift from extinction. Even grade school natural history teaches us that species are often unfit when living beyond their natural range.So it comes as a surprise that the causes of this rampant ecological specialization are poorly understood. “Use it or lose it,” but why? One common explanation is that natural selection tends to favor traits that simultaneously enhance fitness in one environment but compromise fitness elsewhere. This selective process is known as “antagonistic pleiotropy.” Another explanation is that a selective shadow falls upon unused traits, rendering them susceptible to mutational erosion by random genetic drift. This neutral process is known as “mutation accumulation” (Figure 1). These processes inevitably co-occur, and can be enhanced by the hybrid dynamic of genetic hitchhiking, in which neutral mutations affecting unused functions become linked to different mutations under positive selection. In most cases, the functional decay of a species can only be studied retrospectively, and distinguishing the roles of antagonistic pleiotropy and mutation accumulation is hampered by weak historical inference. Did selection, or an absence of selection, produce the blind cavefish [1]? There is little controversy that the sum of these dynamics can produce specialists, but their timing and relative importance is an open question.Open in a separate windowFigure 1Hypothetical dynamics of fitness in foreign environments by pleiotropy or mutation accumulation during long-term adaptation.Prolonged adaptation to one environment leads to decelerating fitness gains in the selective environment (solid black line), as beneficial mutations become limiting. Consequences of this adaptation for fitness in other environments may take different forms. No net change may occur if beneficial mutations generate no or inconsistent side effects (neutrality). However, the same mutations responsible for adaptation may also increase fitness in other environments (synergistic pleiotropy, dotted line), may decrease fitness in foreign environments at an equivalent rate if antagonistic effects correlate with selected effects (antagonistic pleiotropy, dotted line), or may decrease fitness at an increasing rate if subsequent mutations generate greater tradeoffs (antagonistic pleiotropy, dashed and dotted line). The uncertainty of the form of pleiotropic effects reflects a general lack of understanding of how mutations interact to affect fitness, particularly over the long term. Mutation accumulation (MA) in traits hidden from selection is expected to reduce fitness randomly but linearly on average, more slowly during evolution at a low mutation rate (MA, low U) or more rapidly at a high mutation rate (MA, high U). Evidence of all processes is now evident in this latest study of the evolution of diet breadth in the LTEE [20].The study of “evolution in action” using model experimental populations of rapidly reproducing organisms allows researchers to quantify both adaptation and any functional declines simultaneously. This approach is especially powerful when samples of evolving populations can be stored inanimate and studied at a later time under various conditions. Perhaps the best example of this approach is Richard Lenski''s Long-Term Evolution Experiment (LTEE), in which 12 populations of E. coli have been grown under simple conditions for more than 25 years and 50,000 generations [2],[3].When as a graduate student I wondered aloud whether the LTEE lines had become specialists, a colleague remarked: “Of course! You''ve selected for streamlined E. coli that have scuttled unused functions.” But with only a small amount of glucose as the sole carbon source available to the ancestor (the innovation by one population of using citrate for growth more than 30,000 generations in the future notwithstanding [4]), all anabolic pathways to construct new cells remain under strong selection to preserve their function. Moreover, because some catabolic reactions use the same intermediates as anabolic pathways (a form of pleiotropy) [5], growth on alternative carbon sources may be nonetheless preserved. Thus, we wondered whether the physiology of E. coli might actually prove to be robust during long-term evolution on glucose alone.Over the first 2,000 generations, the LTEE lines gained more often than lost fitness across a range of different environments [6]. In addition, a high-throughput screen of cellular respiration (Biolog) for the best-studied clone from these lines showed 171 relative gains and only 32 losses [7]. Even these losses in substrate respiration did not translate to reduced fitness versus the ancestor; rather, the evolved clone was simply relatively worse in the foreign resources than in glucose [7]. Evidently, each of the five beneficial mutations found in this early clone was broadly beneficial and imparted few tradeoffs [8]. Generalists rather than specialists were the rule.Between 2,000 and 20,000 generations, fitness losses in foreign conditions became more obvious but not always consistent. Some lines became less fit than the ancestor in a dilute complex medium (LB) [9], all lines grew worse at high (>40°C) and low (<20°C) temperature [10], and all lines became sensitive to the resource concentration in their environment, even for glucose [9]. Did subsequent beneficial mutations cause these tradeoffs (antagonistic pleiotropy), or did other, neutral or slightly harmful mutations accumulate by drift (Figure 1)? We must consider the population genetic dynamics of these LTEE populations. The hallmark of neutral theory [11] is that mutations with no effect in the selective environment should become fixed in the population at the rate of mutation. For the ancestor of this experiment, the mutation rate is ∼10⁻³ per genome per generation [12],[13], so only a handful of neutral mutations would have fixed by the time tradeoffs became evident, and would not likely explain the early specialization.However, an important extension of neutral theory is that slightly harmful mutations—those whose effects are roughly the inverse of the population size or below, 1/N—can also be fixed by drift [14]. Millions of slightly deleterious mutations were produced in these populations, which cycled between 5×10⁶ and 5×10⁸ cells each day. Might these mutations account for tradeoffs over the first 10–20,000 generations? In small populations, the effect of these mutations can be substantial, which explains why bottlenecked populations may experience fitness declines or even the genome erosion frequently seen in bacterial endosymbionts [15]. But in the large LTEE populations, most deleterious mutations are weeded out by selection and only those with the slightest effects may accumulate over very long time scales. Thus, because these early losses tended to occur when adaptation in the selective environment was most rapid, and because the randomness and rarity of mutation accumulation should not produce parallel changes over these time scales, early specialization is best explained by antagonistic pleiotropy [9],[10].Later in the LTEE, elevated mutation rates began to evolve in certain lines, resulting in a fundamental change in the population genetic environment [16],[17] that should increase the rate of functional decay in unused, essentially neutral functions. These mutator populations tended to perform worse in multiple environments, and in theory should continue to specialize more rapidly by accelerated mutation accumulation. As a first test, we used Biolog plates to assay respiration on 95 different carbon sources over the first 20,000 generations [18]. Although mutators tended to exhibit a reduced breadth of function in this assay, the difference was not statistically significant [18]. Rather, a surprising number of losses of function were shared among replicate lines, and we took this parallelism as further support of antagonistic pleiotropy driven by selection for common sets of adaptive mutations.Here the LTEE offers its greatest advantage: more time, both for evolution and innovative research. Over subsequent generations, mutator lines should continue to accumulate greater mutational load by drift and hence become more specialized than lines retaining the low ancestral rate. Genomic sequences of the evolved lines now have confirmed this increased mutational load [3],[19] in the six of 12 lines that are now mutators [16]. In this issue, Leiby and Marx [20] have readdressed these questions by retracing old steps, applying the prior Biolog assays to lines spanning 50,000 generations of evolution, and by pioneering new high-throughput assays of fitness in many resources. Somewhat surprisingly, these methods disagree and challenge the reliability of Biolog data as a fitness proxy. As a proprietary measure of cellular respiration, it can demonstrate major functional shifts but is less reliable than growth rate as a fitness parameter.More importantly, Leiby and Marx provide clear evidence that niche breadth in the LTEE was shaped by both mutation accumulation and pleiotropy. Growth rates actually increased on several resources, and hence the pleiotropic effects of adaptation to glucose were synergistic, broadening functionality particularly over the first 20,000 generations, as well as antagonistic, producing fewer tradeoffs than previously thought [20]. Pleiotropic effects were also somewhat unpredictable: a sophisticated flux-balance analysis [21] of foreign substrates did not reveal more gains for resources similar to glucose or losses for dissimilar resources. Some early losses linked to selection (maltose, galactose, serine) [6] became complete, but also subtle gains of function for dicarboxylic acid metabolism, perhaps related to growth on metabolic byproducts, became amplified. The most striking pattern was that mutator populations became specialists, diminished for many functions owing to their greater mutational burden, and this only became evident after 50,000 generations in a single resource. These convergent functional losses were not caused by selection, as is often argued, but rather by an absence of selection in the face of mutational pressure. Mutational decay by genetic drift takes a long time, and it will take much longer for the non-mutator lines, it seems.Although Leiby and Marx [20] correctly emphasize the importance of truly long-term selection combined with deficient DNA repair to reveal effects of mutation accumulation, decay has been witnessed in other systems undergoing regular population bottlenecks over shorter time scales [22],[23]. Antagonistic pleiotropy can also reveal its effects much more rapidly than was seen in the LTEE, especially when selection discriminates among discrete fitness features in a heterogeneous environment, such as in the colonization of a new landscape [24],[25]. What this study uniquely illustrates is the unpredictability of pleiotropic effects of adaptation to a simple environment, which in turn shows how chance draws from a distribution of contending beneficial mutations may produce divergent outcomes, ranging from generalists to specialists. A sample of the first mutants competing to prevail in the LTEE system showed variable niche breadth [26] so perhaps we should not be surprised that the footprints of these large-effect mutations endure. Further study of the precise mechanisms by which different mutations produce more fit offspring will teach us more about the origins of diversity that beguile us. We can also gain a broader perspective on the longstanding tension between chance and necessity [27]—a motivator of the LTEE—by focusing more on what is unnecessary, such as how organisms grow in foreign environments. Often insight comes from studying at the margins of a problem, and here, the limits to the growth of these bacteria have allowed us to focus more on how exactly they have accomplished their most essential tasks.