The effects of inbreeding, genetic dissimilarity and phenotype on male reproductive success in a dioecious plant.

Pollen fate can strongly affect the genetic structure of populations with restricted gene flow and significant inbreeding risk. We established an experimental population of inbred and outbred Silene latifolia plants to evaluate the effects of (i) inbreeding depression, (ii) phenotypic variation and (iii) relatedness between mates on male fitness under natural pollination. Paternity analysis revealed that outbred males sired significantly more offspring than inbred males. Independently of the effects of inbreeding, male fitness depended on several male traits, including a sexually dimorphic (flower number) and a gametophytic trait (in vitro pollen germination rate). In addition, full-sib matings were less frequent than randomly expected. Thus, inbreeding, phenotype and genetic dissimilarity simultaneously affect male fitness in this animal-pollinated plant. While inbreeding depression might threaten population persistence, the deficiency of effective matings between sibs and the higher fitness of outbred males will reduce its occurrence and counter genetic erosion.

Comparative population genetic structure in a plant-pollinator/seed predator system.

Comparative analyses of spatial genetic structure of populations of plants and the insects they interact with provide an indication of how gene flow, natural selection and genetic drift may jointly influence the distribution of genetic variation and potential for local co-adaptation for interacting species. Here, we analysed the spatial scale of genetic structure within and among nine populations of an interacting species pair, the white campion Silene latifolia and the moth Hadena bicruris, along a latitudinal gradient across Northern/Central Europe. This dioecious, short-lived perennial plant inhabits patchy, often disturbed environments. The moth H. bicruris acts both as its pollinator and specialist seed predator that reproduces by laying eggs in S. latifolia flowers. We used nine microsatellite markers for S. latifolia and eight newly developed markers for H. bicruris. We found high levels of inbreeding in most populations of both plant and pollinator/seed predator. Among populations, significant genetic structure was observed for S. latifolia but not for its pollinator/seed predator, suggesting that despite migration among populations of H. bicruris, pollen is not, or only rarely, carried over between populations, thus maintaining genetic structure among plant populations. There was a weak positive correlation between genetic distances of S. latifolia and H. bicruris. These results indicate that while significant structure of S. latifolia populations creates the potential for differentiation at traits relevant for the interaction with the pollinator/seed predator, substantial gene flow in H. bicruris may counteract this process in at least some populations.

Ancestral and monophyletic presence of diplostigmaty in Sebaea (Gentianaceae) and its potential role as a morphological mixed mating strategy.

* Diplostigmaty, the presence of a primary (apical) stigma and secondary (mid-stylar) stigmas along the style, is only known from the genus Sebaea (Gentianaceae). Early work indicated that the secondary stigmas provide a mechanism of autogamy, suggesting that it might ensure reproductive assurance. * Here, we test the monophyly of this unique morphological trait. Using Bayesian methods, we infer a nuclear DNA phylogeny for 96 accessions, including c. 50% of the species from the genus Sebaea. With this phylogeny, we infer the distribution of ancestral states on critical nodes using parsimony and likelihood methods. * The inferred nrDNA phylogeny shows that the genus Sebaea is divided in two statistically well-supported clades, A and B, consistent with recent estimates. The most recent ancestor (MRCA) of clade A, except the most basal species (Sebaea pusilla), is resolved as diplostigmatic. No reversal to a single stigma is observed within this clade. * We suggest that diplostigmaty is evolutionarily stable through time. We also discuss why this reproductive system is not found elsewhere than in Gentianaceae and the potential advantage of diplostigmaty as a stable mixed mating strategy.

Effects of pollination timing on seed paternity and seed mass in Silene latifolia (Caryophyllaceae).

BACKGROUND AND AIMS: Competition among genetically different pollen donors within one recipient flower may play an important role in plant populations, increasing offspring genetic diversity and vigour. However, under field conditions stochastic pollen arrival times may result in disproportionate fertilization success of the first-arriving pollen, even to the detriment of the recipient plant's and offspring fitness. It is therefore critical to evaluate the relative importance of arrival times of pollen from different donors in determining siring success. METHODS: Hand pollinations and genetic markers were used to investigate experimentally the effect of pollination timing on seed paternity, seed mass and stigmatic wilting in the the dioecious plant Silene latifolia. In this species, high prevalence of multiply-sired fruits in natural populations suggests that competition among different donors may often take place (at fertilization or during seed development); however, the role of variation due to pollen arrival times is not known. KEY RESULTS: First-arriving pollen sired significantly more seeds than later-arriving pollen. This advantage was expressed already before the first pollen tubes could reach the ovary. Simultaneously with pollen tube growth, the stigmatic papillae wilted visibly. Individual seeds were heavier in fruits where one donor sired most seeds than in fruits where both donors had more even paternity shares. CONCLUSIONS: In field populations of S. latifolia, fruits are often multiply-sired. Because later-arriving pollen had decreased chances of fertilizing the ovules, this implies that open-pollinated flowers often benefit from pollen carry-over or pollinator visits within short time intervals, which may contribute to increase offspring genetic diversity and fitness.

How does breeding system variation modulate sexual antagonism?

The study of sexually antagonistic (SA) traits remains largely limited to dioecious (separate sex), mobile animals. However, the occurrence of sexual conflict is restricted neither by breeding system (the mode of sexual reproduction, e.g. dioecy or hermaphroditism) nor by sessility. Here, we synthesize how variation in breeding system can affect the evolution and expression of intra- and inter-locus sexual conflicts in plants and animals. We predict that, in hermaphrodites, SA traits will (i) display lower levels of polymorphism; (ii) respond more quickly to selection; and (iii) involve unique forms of interlocus conflict over sex allocation, mating roles and selfing rates. Explicit modelling and empirical tests in a broader range of breeding systems are necessary to obtain a general understanding of the evolution of SA traits.

Benefits and costs to pollinating, seed-eating insects: the effect of flower size and fruit abortion on larval performance.

Plant-pollinator interactions are well-known examples of mutualism, but are not free of antagonism. Antagonistic interactions and defenses or counter-defenses are expected particularly in nursery pollination. In these systems, adult insects, while pollinating, lay their eggs in flowers, and juveniles consume the seeds from one or several fruits, thereby substantially reducing plant fitness. The outcome of such interactions will depend, for the plant, on the balance between pollination versus seed predation and for the larvae on the balance between the food and shelter provided versus the costs imposed by plant defenses, e.g., through abortion of infested fruits. Here, we examine the costs and benefits to the larvae in the nursery-pollination system Silene latifolia/Hadena bicruris. Using selection lines that varied in flower size (large- vs. small-flowered plants), we investigated the effects of variation in flower and fruit size and of a potential defense, fruit abortion, on larval performance. In this system, infested fruits are significantly more likely to be aborted than non-infested fruits; however, it is unclear whether fruit abortion is effective as a defense. Larger flowers gave rise to larger fruits with more seeds, and larvae that were heavier at emergence. Fruit abortion was frequently observed (ca. 40% of the infested fruits). From aborted fruits, larvae emerged earlier and were substantially lighter than larvae emerging from non-aborted fruits. The lower mass at emergence of larvae from aborted fruits indicates that abortion is a resistance mechanism. Assuming that lower larval mass implies fewer resources invested in the frugivore, these results also suggest that abortion is likely to benefit the plant as a defense mechanism, by limiting both resources invested in attacked fruits, as well as the risk of secondary attack. This suggests that selective fruit abortion may contribute to the stability of mutualism also in this non-obligate system.

Polyandry and female control: the red flour beetle Tribolium castaneum as a case study.

Females of many animal species are polyandrous, and there is evidence that they can control pre- and post-mating events. There has been a growing interest in consequences of polyandry for male and female reproductive success and offspring fitness, and its evolutionary significance. In several taxa, females exhibit mate choice both before and after mating and can influence the paternity of their offspring, enhancing offspring number and quality, but potentially countering male interests. Studying female mating biology and in particular post-copulatory female control mechanisms thus promises to yield insights into sexual selection and the potential of male-female coevolution. Here, we highlight the red flour beetle Tribolium castaneum (Herbst), a storage pest, as a model system to study polyandry, and review studies addressing the effects of polyandry on male sperm competitive ability and female control of post-mating events. These studies show that the outcome of sperm competition in the red flour beetle is influenced by both male and female traits. Furthermore, recent advances suggest that sexual conflict may have shaped reproductive traits in this species.

Effects of inbred/outbred crosses on progeny sex ratio in Silene latifolia (Caryophyllaceae).

Sex ratio polymorphism has been extensively studied in Silene latifolia, but it is neither known whether inbreeding (which is likely to occur under field conditions) affects it, nor which of the proposed mechanisms (Y degeneration, X-linked drive) is more important. Both mechanisms predict reduced pollen performance. In this study, females were crossed with pollen from related and unrelated males in single-donor and two-donor crosses, and the sex ratio of offspring (n = 866, 60 crosses), sons'in vitro pollen germination and sex ratios in parental families were scored. Flowers receiving only unrelated pollen produced a significant excess of sons. Sex ratios were not significantly correlated between generations. Sons'in vitro pollen germination was significantly negatively correlated with the 'sex-ratio phenotype' of maternal grandfathers, but not of fathers. This generation leap may be consistent with X-linked determinants because Y-linked determinants alone cannot explain it (grandfathers, fathers and sons share the same Y chromosome). Further work is required, but inbreeding and limited dispersal may lead to local accumulation of biasing factors, a process potentially countered by conditional shifts to produce more sons in pure outbred crosses.

Intraspecific competition reveals conditional fitness effects of single gene polymorphism at the Arabidopsis root growth regulator BRX.

Intraspecific genetic variation for morphological traits is observed in many organisms. In Arabidopsis thaliana, alleles responsible for intraspecific morphological variation are increasingly being identified. However, the fitness consequences remain unclear in most cases. Here, the fitness effects of alleles of the BRX gene are investigated. A brx loss-of-function allele, which was found in a natural accession, results in a highly branched but poorly elongated root system. Comparison between the control accession Sav-0 and an introgression of brx into this background (brxS) indicated that, surprisingly, brx loss of function did not negatively affect fitness in pure stands. However, in mixed, well-watered stands brxS performance and reproductive output decreased significantly, as the proportion of Sav-0 neighbors increased. Additional comparisons between brxS and a brxS line that was complemented by a BRX transgene confirmed a direct effect of the loss-of-function allele on plant performance, as indicated by restored competitive ability of the transgenic genotype. Further, because plant height was very similar across genotypes and because the experimental setup largely excluded shading effects, the impaired competitiveness of the brx loss-of-function genotype likely reflects below-ground competition. In summary, these data reveal conditional fitness effects of a single gene polymorphism in response to intraspecific competition in Arabidopsis.

Sexual conflict over floral receptivity.

In flowering plants, the onset and duration of female receptivity vary among species. In several species the receptive structures wilt upon pollination. Here we explore the hypothesis that postpollination wilting may be influenced by pollen and serve as a general means to secure paternity of the pollen donor at the expense of female fitness. Taking a game-theoretical approach, we examine the potential for the evolution of a pollen-borne wilting substance, and for the coevolution of a defense strategy by the recipient plant. The model without defense predicts an evolutionarily stable strategy (ESS) for the production of wilting substance. The ESS value is highest when pollinator visiting rates are intermediate and when the probability that pollen from several donors arrives at the same time is low. This finding has general implications in that it shows that male traits to secure paternity also can evolve in species, such as plants, where mating is not strictly sequential. We further model coevolution of the wilting substance with the timing of stigma receptivity. We assume that pollen-receiving plants can reduce the costs induced by toxic pollen by delaying the onset of stigmatic receptivity. The model predicts a joint ESS, but no female counter-adaptation when the wilting substance is highly toxic. This indicates that toxicity affects the probability that a male manipulative trait stays beneficial (i.e., not countered by female defense) over evolutionary time. We discuss parallels to male induced changes in female receptivity known to occur in animals and the role of harm for the evolution of male manipulative adaptations.

Population size and identity influence the reaction norm of the rare, endemic plant Cochlearia bavarica across a gradient of environmental stress.

Habitat degradation and loss can result in population decline and genetic erosion, limiting the ability of organisms to cope with environmental change, whether this is through evolutionary genetic response (requiring genetic variation) or through phenotypic plasticity (i.e., the ability of a given genotype to express a variable phenotype across environments). Here we address the question whether plants from small populations are less plastic or more susceptible to environmental stress than plants from large populations. We collected seed families from small (<100) versus large natural populations (>1,000 flowering plants) of the rare, endemic plant Cochlearia bavarica (Brassicaceae). We exposed the seedlings to a range of environments, created by manipulating water supply and light intensity in a 2 x 2 factorial design in the greenhouse. We monitored plant growth and survival for 300 days. Significant effects of offspring environment on offspring characters demonstrated that there is phenotypic plasticity in the responses to environmental stress in this species. Significant effects of population size group, but mainly of population identity within the population size groups, and of maternal plant identity within populations indicated variation due to genetic (plus potentially maternal) variation for offspring traits. The environment x maternal plant identity interaction was rarely significant, providing little evidence for genetically- (plus potentially maternally-) based variation in plasticity within populations. However, significant environment x population-size-group and environment x population-identity interactions suggested that populations differed in the amount of plasticity, the mean amount being smaller in small populations than in large populations. Whereas on day 210 the differences between small and large populations were largest in the environment in which plants grew biggest (i.e., under benign conditions), on day 270 the difference was largest in stressful environments. These results show that population size and population identity can affect growth and survival differently across environmental stress gradients. Moreover, these effects can themselves be modified by time-dependent variation in the interaction between plants and their environment.

Molecular and quantitative genetic differentiation in European populations of Silene latifolia (Caryophyllaceae).

BACKGROUND AND AIMS: Among-population differentiation in phenotypic traits and allelic variation is expected as a consequence of isolation, drift, founder effects and local selection. Therefore, investigating molecular and quantitative genetic divergence is a pre-requisite for studies of local adaptation in response to selection under variable environmental conditions. METHODS: Among- and within-population variation were investigated in six geographically separated European populations of the white campion, Silene latifolia, both for molecular variation at six newly developed microsatellite loci and for quantitative variation in morphological and life-history traits. To avoid confounding effects of the maternal environment, phenotypic traits were measured on greenhouse-reared F(1) offspring. Tests were made for clinal variation, and the correlations among molecular, geographic and phenotypic distances were compared with Mantel tests. KEY RESULTS: The six populations of Silene latifolia investigated showed significant molecular and quantitative genetic differentiation. Geographic and phenotypic distances were significantly associated. Age at first flowering increased significantly with latitude and exhibited a Q(st) value of 0.17 in females and 0.10 in males, consistent with adaptation to local environmental conditions. By contrast, no evidence of isolation-by-distance and no significant association between molecular and phenotypic distances were found. CONCLUSIONS: Significant molecular genetic divergence among populations of Silene latifolia, from the European native range is consistent with known limited seed and pollen flow distances, while significant quantitative genetic divergence among populations and clinal variation for age at first flowering suggest local adaptation.

High prevalence of multiple paternity within fruits in natural populations of Silene latifolia, as revealed by microsatellite DNA analysis.

Data on multiple paternity within broods has been gathered in several animal species, and comparable data in plants would be of great importance to understand the evolution of reproductive traits in a common framework. In this study, we first isolated and characterized six microsatellite loci from the dioecious plant Silene latifolia (Caryophyllaceae). The polymorphism of the loci was assessed in 60 individual females from four different populations. Two of the investigated loci showed a pattern of inheritance consistent with X-linkage. These microsatellite loci were highly polymorphic and therefore useful tools for parentage analysis. We then used four of the markers to determine paternity within naturally pollinated fruits in four European populations. This study revealed widespread multiple paternity in all populations investigated. The minimum number of fathers per fruit varied from one to nine, with population means ranging from 3.4 to 4.9. The number of fathers per fruit was not significantly correlated with offspring sex ratios. High prevalence of multiple paternity within fruits strongly suggest that pollen competition is likely to occur in this species. This may substantially impact male reproductive success and possibly contribute to increase female and offspring fitness, either through postpollination selection or increased genetic diversity. Wide variation in outcrossing rates may be an overlooked aspect of plant mating systems.

Microgametophyte population sizes and plant reproductive output in the insect-pollinated Prunella grandiflora (Lamiaceae).

Intraspecific variation in pollen deposition and number of pollen tubes per style is rarely quantified, but is essential for assessing the occurrence of pollen limitation and pollen competition and their evolutionary implications. Moreover, pollen deposition, pollen tube growth, and the fate of fertilized ovules are rarely distinguished in field studies. Here we present such a study in eight natural populations of Prunella grandiflora. We quantified microgametophyte population sizes and inferred pollen limitation when the number of fertilizable ovules exceeded pollen tubes, and assessed seed set and fate after open pollination. Two and three populations had on average significantly fewer pollen grains and pollen tubes per flower, respectively, than the fixed number of fertilizable ovules per fruit, while one population experienced significant pollen competition. Style length was positively correlated with the number of pollen tubes. While pollen availability was very variable, seed abortion was significantly less frequent in denser populations, and in one population the proportion of well-developed seeds was significantly, positively correlated with the number of pollen tubes in the style. Less pollen deposition, lower numbers of pollen tubes reaching the base of the style, lower pollen quality and therefore increased abortion of fertilized ovules can all reduce seed set in natural P. grandiflora stands. Substantial intraspecific variability implies that microgametophyte competition also occurs in this species. Finally, style morphology may affect pollen receipt.

Natural genetic variation in Arabidopsis: tools, traits and prospects for evolutionary ecology.

BACKGROUND: The model plant Arabidopsis thaliana (Arabidopsis) shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, the potential of Arabidopsis for molecular genetic analysis of this natural variation has increased dramatically in recent years. SCOPE: Advanced genomics has accelerated molecular phylogenetic analysis and gene identification by quantitative trait loci (QTL) mapping and/or association mapping in Arabidopsis. In particular, QTL mapping utilizing natural accessions is now becoming a major strategy of gene isolation, offering an alternative to artificial mutant lines. Furthermore, the genomic information is used by researchers to uncover the signature of natural selection acting on the genes that contribute to phenotypic variation. The evolutionary significance of such genes has been evaluated in traits such as disease resistance and flowering time. However, although molecular hallmarks of selection have been found for the genes in question, a corresponding ecological scenario of adaptive evolution has been difficult to prove. Ecological strategies, including reciprocal transplant experiments and competition experiments, and utilizing near-isogenic lines of alleles of interest will be a powerful tool to measure the relative fitness of phenotypic and/or allelic variants. CONCLUSIONS: As the plant model organism, Arabidopsis provides a wealth of molecular background information for evolutionary genetics. Because genetic diversity between and within Arabidopsis populations is much higher than anticipated, combining this background information with ecological approaches might well establish Arabidopsis as a model organism for plant evolutionary ecology.

Time after time: flowering phenology and biotic interactions.

The role of biotic interactions in shaping plant flowering phenology has long been controversial; plastic responses to the abiotic environment, limited precision of biological clocks and inconsistency of selection pressures have generally been emphasized to explain phenological variation. However, part of this variation is heritable and selection analyses show that biotic interactions can modulate selection on flowering phenology. Our review of the literature indicates that pollinators tend to favour peak or earlier flowering, whereas pre-dispersal seed predators tend to favour off-peak or later flowering. However, effects strongly vary among study systems. To understand such variation, future studies should address the impact of mutualist and antagonist dispersal ability, ecological specialization, and habitat and plant population characteristics. Here, we outline future directions to study how such interactions shape flowering phenology.