The relationship between pollen grain size and progeny gender in dioecious Silene latifolia (Caryophyllaceae)

Summary Experiments and observations conducted during the past 90 years have provided conflicting evidence concerning the existence of a size difference between pollen grains containing an X chromosome (female-determining) and those containing a Y chromosome (maledetermining) in dioecious Silene latifolia. Were such a size difference to exist, this might explain, at least in part, the observation that X-bearing pollen tubes reach the ovary more quickly, on average, than Y-bearing pollen tubes. We tested for such a size difference by separating pollen collected from single anthers into three size classes: small, large, and random. Fruit set (number of pollinated flowers that set fruit) and seed set (number of seeds per capsule) did not differ for these three pollination treatments. Progeny sex ratios resulting from these three pollen size classes also did not differ significantly. Thus, pollen grain size is not affected by which of the two sex chromosomes is present. Our experiment is the first direct test of this relationship. Based on our results, size differences should not be invoked to explain competitive differences in male- and female-determining microgametophytes.     

Sexual dimorphism in nuclear DNA content and floral morphology in populations of Silene latifolia (Caryophyllaceae)

The evolution of flower size has become a major focus of plant population biology. In order to gain insight into the causal basis for flower-size variation, we have explored the relationship between nuclear DNA content, flower size, and cell size within and among populations of the dioecious plant Silene latifolia. We found significant variation among populations for both DNA content and flower size, with a consistent sexual dimorphism within all populations (males have a bigger genome, but smaller flowers). The overall correlation between DNA content and flower size was negative, especially within males. The cell dimensions of calyx and petal cells were not significantly different between the sexes, indicating that females have bigger flowers because they contain more cells. These findings are discussed in the context of nucleotype theory, which predicts a slower growth rate (division rate) for cells with greater DNA content. This leads to the suggestion that males have smaller flowers because of the relatively slow rate of cell division due to their larger genome. It would be of great interest to know whether associated effects on flower size of changes in genome size of the type investigated in the present study can be generalized to other species.     

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.     

Genetic structure of hybrid zones between Silene latifolia and Silene dioica (Caryophyllaceae): evidence for introgressive hybridization

Natural hybrid zones provide a valuable tool to study introgressive hybridization, because they can contain a wide variety of genotypes that result from many generations of recombination. Here we used molecular markers and morphological variation to describe the structure of two natural hybrid zones between Silene latifolia and Silene dioica in the Swiss Alps. Populations in both hybrid zones consisted of few intermediate hybrids and were dominated by backcross hybrids. The latter were also found in the parental populations at the margins of the hybrid zones. Out of 209 amplified fragment length polymorphism (AFLP) markers scored in 390 individuals, only 7 (3.3%) were species specific. These results indicate that introgression between S. dioica and S. latifolia is extensive, and that hybrid zones act as bridges to gene flow between these two species. Analysis of linkage disequilibrium identified few populations in which hybridization is ongoing, whereas in most populations linkage disequilibrium has eroded. Where hybridization is ongoing, strong changes in species-specific marker frequencies and morphological traits were observed. Plastid introgression into the hybrid zone was found to be bidirectional, but only the S. latifolia plastid haplotype was found in a nuclear S. dioica background. This unidirectional plastid introgression from S. latifolia into S. dioica is most likely due to pollen-flow from S. dioica onto S. latifolia, and results in plastid capture. Comparisons between the molecular and the morphological hybrid indices revealed that morphology in this study system is useful for identifying hybrids, but not for detailed analysis of hybrid zone structure.     

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.Key words: Reproduction, reproductive success, pollen, siring success, microsatellite DNA, paternity, pollen tube growth, seed mass, Silene alba, stigma wilting     

Sex-specific physiology and source-sink relations in the dioecious plant Silene latifolia

Differences in reproductive demands between the sexes of dioecious plants could cause divergence in physiology between the sexes. We found that the reproductive effort of female Silene latifolia plants increased to more than twice that of male plants or female plants that were prevented from setting fruit by lack of pollination after 4 weeks of flowering. Whole-plant source/sink ratios of pollinated females were significantly lower than those of males or unpollinated females because of investment in fruit. We hypothesized that these differences in source/sink ratio between the sexes and within females, depending on pollination, would lead to differences in leaf photosynthetic rates. Within females, we found that photosynthetic capacity was consistent with measurement of whole-plant source/sink ratio. Females that were setting fruit had 30% higher light-saturated photosynthetic rates by 28 days after flowering than females that were not setting fruit. Males, however, had consistently higher photosynthetic rates than females from 10 days after flowering onwards. Males also had approximately twice the dark respiration rates of fruiting females. We found that female reproductive structures are longer-lived and contribute more carbon to their own support than male reproductive structures. Despite the higher rates of leaf dark respiration and lower calyx photosynthetic rates, males fix more carbon than do females. We conclude that females have a sink-regulated mechanism of photosynthesis that allows them to respond to variations in fruit set. This mechanism is not, however, sufficient to explain why male S. latifolia plants have higher rates of photosynthesis, higher source/sink ratios, and lower reproductive allocation, but fail to grow larger than female plants.     

Gynomonoecy in Silene italica (Caryophyllaceae): Sexual Phenotypes in Natural Populations

Abstract: Some gynodloecious species present not only hermaphrodite and female individuals but also gynomonoecious individuals that produce both female and perfect flowers. In a study of natural populations of Silene italica , we found that gynomonoecious individuals were common in all populations (from 13 to 39%) and that their frequency was independent of the frequency of females. No relationship was found between the number of flowers a plant produced and its sex. The majority of gynomonoecious individuals showed a low proportion of female flowers but all proportions were found. Female flowers of these individuals were well-developed flowers that set fruit as often as female flowers on pure females. The percentage fruit set was characteristic of the type of flowers (around 56% for perfect flowers and 76% for female flowers) whatever the sex of the individual plant. The determination of sex is not known but data indicate that environmental conditions could be responsible for a shift between the hermaphrodite and the gynomonoecious states rather than between the female and the gynomonoecious states.     

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.     

Reproduction and progeny of Silene latifolia (Caryophyllaceae) as affected by atmospheric CO2 concentration

Sexual dimorphism in plants has been known for over two millennia. However, little is known about how male and female reproduction of dioecious species will respond to anthropogenic environmental perturbations. Using growth chambers, the effects of CO(2) enrichment on male and female reproduction in Silene latifolia were examined and whether parental CO(2) environment affected progeny germination and sex ratio. Reproduction of male and female S. latifolia was enhanced by a similar magnitude at elevated CO(2). Over the growing season, males produced 16 times as many flowers as females did fruits per plant, but no difference in reproductive biomass between genders was observed at ambient or elevated CO(2). Germination of seeds produced by plants grown at different CO(2) concentrations was significantly different. Female seeds from higher CO(2)-grown plants tended to emerge earlier than those from ambient-CO(2)-grown plants, but emergence of male seeds was little affected. Overall, seeds from elevated-CO(2)-grown plants had 20% higher germination and were more female-biased than those from ambient-CO(2)-grown plants. Because of the enhanced reproduction and more female-biased progeny under elevated CO(2), the population structure of this cosmopolitan weedy species will likely be altered in a future environment.     

Climate and clinal variation in seed morphology of the white campion, Silene latifolia (Caryophyllaceae)

Silene latifolia (white campion) is widespread in W Eurasia and shows clinal variation in seed morphology. Two extensive parapatric races, within which seed morphology changes gradually with geographic distance, are separated by a transition zone where the morphological gradient is steeper. A sample of seeds from across the species' native range was scored for morphological characters and analysed using detrended correspondence analysis ordination and multiple regression, in order to simplify mapping of the variation pattern and to investigate statistical relationships with climatic variables. The transition zone, as defined by ordination scores, follows the convolutions of the 0C isotherm for January mean temperature along its entire length from N Europe to the Middle East. January temperature proved to be a highly significant predictor of seed morphology even after the removal of simple spatial trends. Although the origins of comparable large-scale clines have often been interpreted in historical terms, the result reported here supports a role for climatic selection in maintaining the existing variation pattern.     

Maturation timing of stamens and pistils in the dioecious plant Silene latifolia

The dioecious plant Silene latifolia depends on nocturnal insects for pollination. To increase the chance of cross-pollination, pollen grains seem to be released and stigmas seem to be receptive simultaneously at night. We divided the floral development of S. latifolia into 1–20 stages, and determined the timetables of male and female function. The corolla of both male and female flowers opens at sunset (1900 hours) and closes at sunrise (0900 hours). To investigate the period of the reproductive phase of male and female function, we measured the germination rate on a pollen medium and the pollen germination rate on stigma during the period when stamens and stigmas were viable in the timetable. Male flowers had early- and late-maturing stamens that had the highest pollen viability, germination rate and pollen tube growth at midnight (0000 hours) at 1 day after flowering (DAF) and 0000 hours at 2 DAF. In contrast, female flowers maintained a germination rate of nearly 100 % from 1800 hours at 1 DAF to 1200 hours at 3 DAF. These results suggested that S. latifolia transferred the matured pollen grains from male flowers to female flowers only at night.     

Within/between population crosses reveal genetic basis for siring success in Silene latifolia (Caryophyllaceae)

Divergence at reproductive traits can generate barriers among populations, and may result from several mechanisms, including drift, local selection and co-adaptation between the sexes. Intersexual co-adaptation can arise through sexually antagonistic co-evolution, a timely hypothesis addressed in animals but, to our knowledge, not yet in flowering plants. We investigated whether male and female population of origin affected pollen competition success, offspring fitness and sex ratio in crosses within/between six genetically differentiated populations of the white campion, Silene latifolia. Each female was crossed with pollen from one focus male from the same population, and pollen from two focus males from two distinct populations, both as single-donor and two-donor crosses against a fixed tester male with a 2-h interpollination interval (n = 288 crosses). We analysed paternity with microsatellite DNA. Male populations of origin significantly differed for siring success and in vitro pollen germination rates. In vitro pollen germination rate was heritable. Siring success also depended on sex ratio in the female family of origin, but only in between-population crosses. In some female populations, two-donor crosses produced less female-biased sex ratios compared with single-donor crosses, yet in other female populations the reverse was true. Offspring sex ratio varied with donor number, depending on the female population. Within/between population crosses did not differ significantly in seed set or offspring fitness, nor were siring success and offspring fitness significantly correlated. Altogether this suggests reproductive divergence for traits affecting pollen competition in S. latifolia.     

Silver enhances stamen development in female white campion (Silene latifolia [Caryophyllaceae])

Sex expression in the dioecious plant white campion (Silene latifolia Poiret subsp. alba) appears to be insensitive to exogenous applications of auxins, cytokinins, gibberellic acid, and ethylene; however, silver thiosulfate (Ag(2)S(2)O(3)), an ethylene inhibitor, enhanced stamen development in female white campion. In wild-type females, stamen development is arrested before the microspore mother cells are formed. In contrast, stamens of Ag(2)S(2)O(3)-treated females completed meiosis and produced microspores. Stamen development for these females was incomplete, however, and pollen did not mature. Ag(2)S(2)O(3) stimulated stamen development to the same extent in asexual white campion mutants that retained a Y chromosome but had lost Y-linked genes needed for early stages of stamen development. Although Ag(2)S(2)O(3) can inhibit ethylene signaling, the enhancement of stamen development in female white campion cannot be explained as a loss of ethylene response because no other ethylene inhibitor tested (1-methylcyclopropene, trans-cyclooctene, aminoethoxyvinylglycine, and cobalt chloride) caused stamens to develop in female plants. In addition, application of other metal ions could not enhance stamen development. Therefore, the effect we observed on female white campion was specifically caused by silver ions but not by their action on ethylene signaling.     

Efficient molecular sexing in dioecious Silene latifolia and S. dioica and paternity analysis in F(1) hybrids

Two polymerase chain reaction-based assays have been developed that work in combination with an efficient DNA extraction protocol to rapidly and reliably determine sex in the dioecious plant species Silene latifolia and S. dioica. In addition, one of the assays allows assessing paternity in the F(1) generation of intra- and interspecific matings involving the two species.     

Evidence for degeneration of the Y chromosome in the dioecious plant Silene latifolia

The human Y--probably because of its nonrecombining nature--has lost 97% of its genes since X and Y chromosomes started to diverge [1, 2]. There are clear signs of degeneration in the Drosophila miranda neoY chromosome (an autosome fused to the Y chromosome), with neoY genes showing faster protein evolution [3-6], accumulation of unpreferred codons [6], more insertions of transposable elements [5, 7], and lower levels of expression [8] than neoX genes. In the many other taxa with sex chromosomes, Y degeneration has hardly been studied. In plants, many genes are expressed in pollen [9], and strong pollen selection may oppose the degeneration of plant Y chromosomes [10]. Silene latifolia is a dioecious plant with young heteromorphic sex chromosomes [11, 12]. Here we test whether the S. latifolia Y chromosome is undergoing genetic degeneration by analyzing seven sex-linked genes. S. latifolia Y-linked genes tend to evolve faster at the protein level than their X-linked homologs, and they have lower expression levels. Several Y gene introns have increased in length, with evidence for transposable-element accumulation. We detect signs of degeneration in most of the Y-linked gene sequences analyzed, similar to those of animal Y-linked and neo-Y chromosome genes.     

Karyological analysis of an interspecific hybrid between the dioecious Silene latifolia and the hermaphroditic Silene viscosa.

The genus Silene is a good model for studying evolution of the sex chromosomes, since it includes species that are hermaphroditic and dioecious, while maintain a basic chromosome number of 2n = 24. For some combinations of Silene species it is possible to construct interspecific hybrids. Here, we present a detailed karyological analysis of a hybrid between the dioecious Silene latifolia as the maternal plant and a related species, hermaphroditic Silene viscosa, used as a pollen partner. Using genomic probes (the genomic in situ hybridization (GISH) technique), we were able to clearly discriminate parental genomes and to show that they are largely separated in distinct nuclear domains. Molecular GISH and fluorescence in situ hybridization (FISH) markers document that the hybrid genome of somatic cells was strictly additive and stable, and that it had 12 chromosomes originating from each parent, including the only X chromosome of S. latifolia. Meiotic analysis revealed that, although related, respective parental chromosomes did not pair or paired only partially, which resulted in frequent chromosome abnormalities such as bridges and irregular non-disjunctions. GISH and FISH markers clearly document that the larger genome of S. latifolia and its largest chromosome component, the X chromosome, were mostly employed in chromosome lagging and misdivision.     

Gender lability in trioecious Silene acaulis (Caryophyllaceae)

Gender expression of individual cushions of Silene acaulis was shown to vary between years. Fifteen of thirtnine (= 38%) cushions monitored changed gender expression between 1993 and 1995. Cushions dominated by female flowers were shown to be more stable in gender expression than cushions dominated by male or hermaphrodite flowers.     

Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia

Background

Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes.

Results

Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30 % of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome.

Conclusions

Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20 %.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1698-7) contains supplementary material, which is available to authorized users.