The Sister Chromatid Division of the Heteromorphic Sex Chromosomes in Silene Species and Their Transmissibility towards the Mitosis.

Young sex chromosomes possess unique and ongoing dynamics that allow us to understand processes that have an impact on their evolution and divergence. The genus Silene includes species with evolutionarily young sex chromosomes, and two species of section Melandrium, namely Silene latifolia (24, XY) and Silene dioica (24, XY), are well-established models of sex chromosome evolution, Y chromosome degeneration, and sex determination. In both species, the X and Y chromosomes are strongly heteromorphic and differ in the genomic composition compared to the autosomes. It is generally accepted that for proper cell division, the longest chromosomal arm must not exceed half of the average length of the spindle axis at telophase. Yet, it is not clear what are the dynamics between males and females during mitosis and how the cell compensates for the presence of the large Y chromosome in one sex. Using hydroxyurea cell synchronization and 2D/3D microscopy, we determined the position of the sex chromosomes during the mitotic cell cycle and determined the upper limit for the expansion of sex chromosome non-recombining region. Using 3D specimen preparations, we found that the velocity of the large chromosomes is compensated by the distant positioning from the central interpolar axis, confirming previous mathematical modulations.

Inbreeding in a dioecious plant has sex- and population origin-specific effects on its interactions with pollinators.

We study the effects of inbreeding in a dioecious plant on its interaction with pollinating insects and test whether the magnitude of such effects is shaped by plant individual sex and the evolutionary histories of plant populations. We recorded spatial, scent, colour, and rewarding flower traits as well as pollinator visitation rates in experimentally inbred and outbred, male and female Silene latifolia plants from European and North American populations differing in their evolutionary histories. We found that inbreeding specifically impairs spatial flower traits and floral scent. Our results support that sex-specific selection and gene expression may have partially magnified these inbreeding costs for females, and that divergent evolutionary histories altered the genetic architecture underlying inbreeding effects across population origins. Moreover, the results indicate that inbreeding effects on floral scent may have a huge potential to disrupt interactions among plants and nocturnal moth pollinators, which are mediated by elaborate chemical communication.

Destroying habitats can reduce the size of local populations of many plants and animals. For plants, a smaller population means a greater chance of inbreeding, where individual plants that are closely related to each other mate and produce offspring. Inbreeding often results in offspring that are weaker than their parents which can reduce the plant's chance of survival. Many plants rely on animals to help them to breed. For example, bees carry pollen ­ containing the male sex cell ­ to other flowers which then fertilize the plant to produce seeds. Flowers use a wide range of attributes to attract animals such as their colour, scent and providing them with food. However, inbreeding may alter these characteristics which could make it harder for inbred plants to reproduce, meaning that populations would end up shrinking even faster. To test this theory, Schrieber et al. studied flowers from white campions which use moths to breed. Inbred plants had smaller and fewer flowers, and had a different smell. In particular, they produced less of a chemical scent that is known to attract moths at night. Schrieber et al. then tracked moths visiting a mixed population of inbred and control plants. Fewer moths visited the inbred flowers, particularly the ones that were female. This shows that inbreeding may accelerate population loss and extinction by making flowers less attractive to animals. This work highlights the impact habitat destruction has on plants and shows how species can decline rapidly as populations shrink. This could help to support conservation efforts and inform ecology models to better understand our effect on the environment.

The effects of pollination, herbivory and autonomous selfing on the maintenance of flower colour variation in Silenelittorea.

Intraspecific flower colour variation has been generally proposed to evolve as a result of selection driven by biotic or abiotic agents. In a polymorphic population of Silene littorea with pink- and white-flowered plants, we studied pollinators, analysed flower colour perception and tested for differences in pollinator visitation. We also experimentally analysed pollinator limitation in fruit and seed set, and the degree of autonomous selfing. The incidence of florivory and leaf herbivory was compared over 3-4 years. Silene littorea is mainly pollinated by bees and butterflies. Pollinators preferred pink flowers, which did not show pollinator limitation. On the contrary, white flowers showed pollinator limitation in fruit set. White-flowered plants had less floral display and higher levels of florivory than pink plants. Flower colour morphs of S. littorea can reproduce in the absence of pollinators by autonomous selfing, setting 20% and 12% of fruit and seeds in the pink morph and 27% and 20% in the white morph, respectively. Fruit set of white flowers produced by autonomous selfing did not differ from open-pollinated flowers. In conclusion, S. littorea is pollinated by insects of different orders that more frequently visit pink flowers, which is reflected in pollinator limitation of fruit set in white flowers. Moreover, this species has a mixed mating system in which both colour morphs can reproduce in the absence of pollinators by autonomous selfing, although white flowers mainly produce fruits by autogamy. We suggest that reproductive assurance by autonomous selfing helps to maintain flower colour polymorphism in this population.

Differentially Expressed Genes Shared by Two Distinct Cytoplasmic Male Sterility (CMS) Types of Silene vulgaris Suggest the Importance of Oxidative Stress in Pollen Abortion.

Cytoplasmic male sterility (CMS), encoded by the interacting mitochondrial and nuclear genes, causes pollen abortion or non-viability. CMS is widely used in agriculture and extensively studied in crops. Much less is known about CMS in wild species. We performed a comparative transcriptomic analysis of male sterile and fertile individuals of Silene vulgaris, a model plant for the study of gynodioecy, to reveal the genes responsible for pollen abortion in this species. We used RNA-seq datasets previously employed for the analysis of mitochondrial and plastid transcriptomes of female and hermaphrodite flower buds, making it possible to compare the transcriptomes derived from three genomes in the same RNA specimen. We assembled de novo transcriptomes for two haplotypes of S. vulgaris and identified differentially expressed genes between the females and hermaphrodites, associated with stress response or pollen development. The gene for alternative oxidase was downregulated in females. The genetic pathways controlling CMS in S. vulgaris are similar to those in crops. The high number of the differentially expressed nuclear genes contrasts with the uniformity of organellar transcriptomes across genders, which suggests these pathways are evolutionarily conserved and that selective mechanisms may shield organellar transcription against changes in the cytoplasmic transcriptome.

An asexual flower of Silene latifolia and Microbotryum lychnidis-dioicae promotes sex-organ development.

Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia, respectively. Microbotryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae. To assess this hypothesis, an asexual mutant without a gynoecium or stamen was infected with M. lychnidis-dioicae. A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of flower bud development as well as in the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of gynoecium development was not released. M. lychnidis-dioicae would have a function similar to stamen-promoting factor (SPF), since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. An infection experiment also revealed that a deletion on the Y chromosome of the asexual mutant eliminated genes for maturation of tapetal cells because the tapetal cells did not mature in the asexual mutant infected with M. lychnidis-dioicae.

Structural, physiological and genetic diversification of Silene vulgaris ecotypes from heavy metal-contaminated areas and their synchronous in vitro cultivation.

MAIN CONCLUSION: Results provide significant comparison of leaf anatomy, pigment content, antioxidant response and phenolic profile between individuals from miscellaneous populations and describe unified cultivation protocols for further research on stress biology. The plant communities growing on heavy metal-polluted areas have attracted considerable attention due to their unique ability to tolerate enormous amounts of toxic ions. Three ecotypes of Silene vulgaris representing calamine (CAL), serpentine (SER) and non-metallicolous (NM) populations were evaluated to reveal specific adaptation traits to harsh environment. CAL leaves presented a distinct anatomical pattern compared to leaves of SER and NM plants, pointing to their xeromorphic adaptation. These differences were accompanied by divergent accumulation and composition of photosynthetic pigments as well as antioxidant enzyme activity. In CAL ecotype, the mechanism of reactive oxygen species scavenging is based on the joint action of superoxide dismutase and catalase, but in SER ecotype on superoxide dismutase and guaiacol-type peroxidase. On the contrary, the concentration of phenylpropanoids and flavonols in the ecotypes was unchanged, implying the existence of similar pathways of their synthesis/degradation functioning in CAL and SER populations. The tested specimens showed genetic variation (atpA/MspI marker). Based on diversification of S. vulgaris populations, we focused on the elaboration of similar in vitro conditions for synchronous cultivation of various ecotypes. The most balanced shoot culture growth was obtained on MS medium containing 0.1 mg l-1 NAA and 0.25 mg l-1 BA, while the most abundant callogenesis was observed on MS medium enriched with 0.5 mg l-1 NAA and 5.0 mg l-1 BA. For the first time, unified in vitro protocols were described for metallophytes providing the opportunity to conduct basic and applied research on stress biology and tolerance mechanisms under freely controlled conditions.

Agrobacterium rhizogenes-mediated transformation of a dioecious plant model Silene latifolia.

Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.

Ecological divergence plays an important role in strong but complex reproductive isolation in campions (Silene).

New species arise through the evolution of reproductive barriers between formerly interbreeding lineages. Yet, comprehensive assessments of potential reproductive barriers, which are needed to make inferences on processes driving speciation, are only available for a limited number of systems. In this study, we estimated individual and cumulative strengths of seven prezygotic and six postzygotic reproductive barriers between the recently diverged taxa Silene dioica (L.) Clairv. and S. latifolia Poiret using both published and new data. A combination of multiple partial reproductive barriers resulted in near-complete reproductive isolation between S. dioica and S. latifolia, consistent with earlier estimates of gene flow between the taxa. Extrinsic barriers associated with adaptive ecological divergence were most important, while intrinsic postzygotic barriers had moderate individual strength but contributed only little to total reproductive isolation. These findings are in line with ecological divergence as driver of speciation. We further found extensive variation in extrinsic reproductive isolation, ranging from sites with very strong selection against migrants and hybrids to intermediate sites where substantial hybridization is possible. This situation may allow for, or even promote, heterogeneous genetic divergence.

Characterization of the mating system of a native perennial tetraploid herb, Silene stellata.

PREMISE OF THE STUDY: Nursery pollination systems can range from obligate to facultative. In a system where generalists provide substantial pollination service, an important question is whether the cost of seed predation outweighs the benefit provided by the nursery pollinator to cause the plant to evolve toward more generalized pollination. Using a facultative system native to North America, we tested whether nursery pollinator vs. strictly mutualistic generalists affect mating-system parameters of the host plant and explored the implications for long-term coevolution. METHODS: We used paternity analyses with 11 microsatellite markers to characterize the mating system of Silene stellata when pollination service is primarily through the nursery pollinator Hadena ectypa and generalist moths. KEY RESULTS: Our experimental population of S. stellata was predominantly outcrossing (average outcrossing rate t = 0.83), and mating-system parameters were similar between pollinator groups. We detected significant correlations in both selfing and outcrossed paternity at the fruit and maternal family level, corresponding to limited pollen dispersal (mean = 3.9 m). Among individuals, variation in anther-stigma separation was positively associated with outcrossing rate, which suggests the importance of herkogamy in preventing selfing. CONCLUSIONS: Correlated paternity suggests that seeds from the same fruit and/or plants are sired by a limited number of pollen donors, resulting from low pollen dispersal and potential male-male competition. The similar mating-system parameters of the two pollinator groups suggest that selection for higher outcrossing in S. stellata is likely to be through floral design rather than through increased pollinator specialization with H. ectypa.

Genomic imprinting mediates dosage compensation in a young plant XY system.

Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced expression and eventual loss of Y genes1. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2-4. However, the early steps in the evolution of dosage compensation remain unknown, and dosage compensation is poorly understood in plants5. Here, we describe a dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males, but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno6 for the evolution of X inactivation in mammals.

Genetic architecture of traits associated with reproductive barriers in Silene: Coupling, sex chromosomes and variation.

The evolution of reproductive barriers and their underlying genetic architecture is of central importance for the formation of new species. Reproductive barriers can be controlled either by few large-effect loci suggesting strong selection on key traits, or by many small-effect loci, consistent with gradual divergence or with selection on polygenic or multiple traits. Genetic coupling between reproductive barrier loci further promotes divergence, particularly divergence with ongoing gene flow. In this study, we investigated the genetic architectures of ten morphological, phenological and life history traits associated with reproductive barriers between the hybridizing sister species Silene dioica and S. latifolia; both are dioecious with XY-sex determination. We used quantitative trait locus (QTL) mapping in two reciprocal F2 crosses. One to six QTLs per trait, including nine major QTLs (PVE > 20%), were detected on 11 of the 12 linkage groups. We found strong evidence for coupling of QTLs for uncorrelated traits and for an important role of sex chromosomes in the genetic architectures of reproductive barrier traits. Unexpectedly, QTLs detected in the two F2 crosses differed largely, despite limited phenotypic differences between them and sufficient statistical power. The widely dispersed genetic architectures of traits associated with reproductive barriers suggest gradual divergence or multifarious selection. Coupling of the underlying QTLs likely promoted divergence with gene flow in this system. The low congruence of QTLs between the two crosses further points to variable and possibly redundant genetic architectures of traits associated with reproductive barriers, with important implications for the evolutionary dynamics of divergence and speciation.

Transgenerational plasticity in Silene vulgaris in response to three types of stress.

The environment experienced by plants can influence the phenotype of their offspring. Such transgenerational plasticity can be adaptive when it results in higher fitness of the offspring under conditions correlated with those experienced by the mother plant. However, it has rarely been tested if such anticipatory parental effects may be induced with different environments. We grew clonal replicates of Silene vulgaris under control conditions and three types of stress (nutrient deficiency, copper addition and drought), which are known from natural populations of the species. We then subjected offspring from differently treated mother plants to each of the different stress treatments to analyse the influence of maternal and offspring environment on performance and several functional traits. Current stress treatments strongly influenced biomass and functional traits of the plants, mostly in line with responses predicted by the theory of functional equilibrium. Plant performance was also influenced by maternal stress treatments, and some effects independent of initial size differences remained until harvest. In particular, stressed mothers produced offspring of higher fitness than control plants. However, there was no evidence for treatment-specific adaptive transgenerational plasticity, as offspring from a mother plant that had grown in a specific environment did not grow better in that environment than other plants. Our results indicate that the maternal environment may affect offspring traits and performance, but also that this transgenerational plasticity is not necessarily adaptive.

Both life-history plasticity and local adaptation will shape range-wide responses to climate warming in the tundra plant Silene acaulis.

Many predictions of how climate change will impact biodiversity have focused on range shifts using species-wide climate tolerances, an approach that ignores the demographic mechanisms that enable species to attain broad geographic distributions. But these mechanisms matter, as responses to climate change could fundamentally differ depending on the contributions of life-history plasticity vs. local adaptation to species-wide climate tolerances. In particular, if local adaptation to climate is strong, populations across a species' range-not only those at the trailing range edge-could decline sharply with global climate change. Indeed, faster rates of climate change in many high latitude regions could combine with local adaptation to generate sharper declines well away from trailing edges. Combining 15 years of demographic data from field populations across North America with growth chamber warming experiments, we show that growth and survival in a widespread tundra plant show compensatory responses to warming throughout the species' latitudinal range, buffering overall performance across a range of temperatures. However, populations also differ in their temperature responses, consistent with adaptation to local climate, especially growing season temperature. In particular, warming begins to negatively impact plant growth at cooler temperatures for plants from colder, northern populations than for those from warmer, southern populations, both in the field and in growth chambers. Furthermore, the individuals and maternal families with the fastest growth also have the lowest water use efficiency at all temperatures, suggesting that a trade-off between growth and water use efficiency could further constrain responses to forecasted warming and drying. Taken together, these results suggest that populations throughout species' ranges could be at risk of decline with continued climate change, and that the focus on trailing edge populations risks overlooking the largest potential impacts of climate change on species' abundance and distribution.

Paradoxical effects of density on measurement of copper tolerance in Silene paradoxa L.

This work investigated if the assessment of tolerance to trace metals can depend on plant density in the experimental design. A non-metallicolous and a metallicolous populations of Silene paradoxa were hydroponically cultivated at increasing density and in both the absence (-Cu conditions) and excess of copper (+Cu conditions). In -Cu conditions, the metallicolous population showed a lower susceptibility to plant density in comparison to the non-metallicolous one, explained by a higher capacity of the metallicolous population to exploit resources. In +Cu conditions, an alleviating effect of increasing density was found in roots. Such effect was present to a greater extent in the non-metallicolous population, thus making the populations equally copper-tolerant at the highest density used. In shoots, an additive effect of increasing plant density to copper toxicity was reported. Its higher intensity in the metallicolous population reverted the copper tolerance relationship at the highest plant densities used. In both populations, a density-induced decrease in root copper accumulation was observed, thus concurring to the reported mitigation in +Cu conditions. Our work revealed the importance of density studies on the optimization of eco-toxicological bioassays and of metal tolerance assessment and it can be considered the first example of an alleviating effect of increasing plant number on copper stress in a metallophyte.

Pollination context alters female advantage in gynodioecious Silene vulgaris.

Gynodioecy, the co-occurrence of females and hermaphrodites, is arguably the most common angiosperm gender polymorphism in many florae. Females' ability to invade and persist among hermaphrodites depends, in part, on pollinators providing adequate pollination to females. We directly measured diurnal and nocturnal pollinators' contributions to female and hermaphrodite seed production in artificial populations of gynodioecious Silene vulgaris by experimentally restricting pollinator access. We found that female relative seed production in this system depended strongly on pollination context: females produced more than twice as many seeds as hermaphrodites in the context of abundant, nectar-collecting moths. Conversely, females showed no seed production advantage in the context of pollen-collecting syrphid flies and bees due to acutely hermaphrodite-biased visitation. We infer that variation in pollinator type, behaviour and abundance may be important for achieving the female relative fitness thresholds necessary for the maintenance of gynodioecy. Generally, our study illustrates how pollinator-mediated mechanisms may influence the evolution of breeding systems and associated suites of floral traits. Segments of a pollinator community may facilitate gynodioecy by selecting for plant characteristics that increase the attractiveness of both sexes to pollinators, such as nectar rewards. Conversely, discriminating visitors in search of pollen may restrict gynodioecy in associated plant lineages by reducing male steriles' fitness below threshold levels.

Non-coding RNA may be associated with cytoplasmic male sterility in Silene vulgaris.

Cytoplasmic male sterility (CMS) is a widespread phenomenon in flowering plants caused by mitochondrial (mt) genes. CMS genes typically encode novel proteins that interfere with mt functions and can be silenced by nuclear fertility-restorer genes. Although the molecular basis of CMS is well established in a number of crop systems, our understanding of it in natural populations is far more limited. To identify CMS genes in a gynodioecious plant, Silene vulgaris, we constructed mt transcriptomes and compared transcript levels and RNA editing patterns in floral bud tissue from female and hermaphrodite full siblings. The transcriptomes from female and hermaphrodite individuals were very similar overall with respect to variation in levels of transcript abundance across the genome, the extent of RNA editing, and the order in which RNA editing and intron splicing events occurred. We found only a single genomic region that was highly overexpressed and differentially edited in females relative to hermaphrodites. This region is not located near any other transcribed elements and lacks an open-reading frame (ORF) of even moderate size. To our knowledge, this transcript would represent the first non-coding mt RNA associated with CMS in plants and is, therefore, an important target for future functional validation studies.

Differential adaptation drives ecological speciation in campions (Silene): evidence from a multi-site transplant experiment.

In order to investigate the role of differential adaptation for the evolution of reproductive barriers, we conducted a multi-site transplant experiment with the dioecious sister species Silene dioica and S. latifolia and their hybrids. Crosses within species as well as reciprocal first-generation (F1 ) and second-generation (F2 ) interspecific hybrids were transplanted into six sites, three within each species' habitat. Survival and flowering were recorded over 4 yr. At all transplant sites, the local species outperformed the foreign species, reciprocal F1 hybrids performed intermediately and F2 hybrids underperformed in comparison to F1 hybrids (hybrid breakdown). Females generally had slightly higher cumulative fitness than males in both within- and between-species crosses and we thus found little evidence for Haldane's rule acting on field performance. The strength of selection against F1 and F2 hybrids as well as hybrid breakdown increased with increasing strength of habitat adaptation (i.e. the relative fitness difference between the local and the foreign species) across sites. Our results suggest that differential habitat adaptation led to ecologically dependent post-zygotic reproductive barriers and drives divergence and speciation in this Silene system.

Elevational divergence and clinal variation in floral color and leaf chemistry in Silene vulgaris.

PREMISE OF STUDY: Environmental heterogeneity over a species range can lead to divergent selection among populations, leading to phenotypic differences. The plant flavonoid pathway controls key reproductive and defense-related traits and responds to selection and environmental stressors, allowing for hypotheses about phenotypic divergence across environmental gradients. We hypothesized that with increasing elevation, more flavonoids would be produced as a response to increased UV radiation and that plants would be better defended against herbivores. METHODS: We measured floral color, flavonoids, and herbivory in natural populations of Silene vulgaris (Caryophyllaceae) along elevational transects in the French Alps. We correlated phenotypes with environmental variables and calculated genotypic divergence (FST) to compare with phenotypic divergence (PST). KEY RESULTS: We found significant phenotypic variation in S. vulgaris along elevational gradients. Strong positive correlations were observed between floral color, leaf non-anthocyanidin flavonoid concentration, and elevation. Floral anthocyanin and leaf non-anthocyanidin flavonoid phenotypes negatively covaried with temperature and precipitation seasonality. Comparisons of PST to FST provided evidence for stabilizing selection on floral color among transects and divergent selection along the elevational gradient. CONCLUSIONS: Flavonoid production increases along elevational gradients in S. vulgaris, with clinal variation in calyx anthocyanins and increasing leaf non-anthocyanin flavonoid concentrations. Despite the photoprotective and antiherbivore properties of some flavonoids, flavonoid production in flowers and leaves was correlated with population microclimatic variables: temperature and precipitation. Taken together, the results suggest that different flavonoid groups are targeted by selection in different tissues and provide evidence for divergent patterns of selection for flavonoids between high and low elevations.

The effects of stress intensity and stress type on inbreeding depression in Silene vulgaris.

Inbreeding depression (ID) is generally assumed to increase under stressful conditions, but a number of studies have found the opposite pattern, that is that crossed offspring were more capable of exploiting benign conditions. Alternatively, the phenotypic variation hypothesis predicts that not stress intensity, but enhanced phenotypic variation in an environment leads to increased ID. We subjected inbred and crossed offspring of Silene vulgaris to drought, simulated herbivory, copper contamination, and two levels of nutrient deficiency and shade. In contrast to the predominant expectation, most stress treatments decreased inbreeding depression. With increasing nutrient limitation, ID decreased strongly, whereas under increasing shade ID did not change. These differences may be due to purging in the population of origin where conditions are nutrient-poor and dry, but not shaded. In contrast to the greenhouse experiment, ID was higher in a field site than in a more benign common garden. However, the predictions of the phenotypic variation hypothesis were met in both the greenhouse and the field versus garden experiment. The results suggest that there may be no general relationship between ID and stress intensity, but specific effects of stress type and the novelty and variability of the environment.

Post-floral Erection of Stalks Provides Insight into the Evolution of Fruit Orientation and Its Effects on Seed Dispersal.

That stalks reorient after flowering to face upwards is a common phenomenon in many flowering plants, indicating the potential importance of fruit orientation on seed dispersal. But this idea has not been subject to an empirical test. We examined this hypothesis by analysing the evolutionary correlation between fruit orientation and other characters and by investigating the effects of fruit orientation on seed dispersal. We found that 1) in a sub-alpine plant community, upward fruit orientation strongly correlates with fruits that act as seed containers, which are often of dry type and are dispersed by non-animal vectors; 2) as exemplified by the Campanulaceae s. str., fruit orientation strongly correlates with dehiscence position. Upwardly-oriented capsules dehisce at the apex, whereas pendent ones dehisce at the base, in both cases ensuring that seeds are released from an upright position; 3) in manipulation experiments on Silene chungtienensis, upward fruits (the natural state) exhibit much greater dispersal distances and more dispersive pattern than pendent ones, and have a more even distribution of dispersal direction than horizontal ones. Our results suggest that fruit orientation may have important function in seed dispersal, which may be the reason why the phenomenon that stalk erection after flowering occurs widely.