Enzyme polymorphism, morphometric variation and population structure in a restricted endemic, Silene diclinis (Caryophyllaceae)

Silene diclinis is a dioecious perennial herb, restricted to a small area of the SE Spanish province of Valencia. Polyacrylamide gel electrophoresis and morphometric analysis were carried out on cultivated material derived from seed collected from the main population of S. diclinis at Játiva. Only two out of 26 inferred enzyme loci showed substantial variation. There was some indication of population subdivision in both. The esterase locus also showed homozygote excess within the population as a whole and within one subpopulation. Morphological characters showed significant differentiation between subpopulations.
Low allozyme variability characterizes populations of most endemic species that have been investigated. Isolation and historical events may have been as important as population size or edaphic specialization in causing this low variability. But S. diclinis resembles other outbreeding plants in showing population structure and homozygote excess, suggesting restricted within-population gene flow. Mechanisms that may restrict gene flow in S. diclinis include environmentally-induced differences in flowering time, poor seed dispersal and possibly short pollinator foraging distances. Morphological variability and the incidence of developmental abnormalities are speculatively linked with homozygosity. Storage of genetic diversity in space can occur in restricted as well as widespread species and sampling for gene banking should then be spatially representative. Silene diclinis is threatened by continued habitat loss and consequent genetic erosion; its long-term future may depend on the gene bank and the botanic garden.     

Nectar Dynamics and Sugar Composition in Flowers of Silene and Saponaria Species (Caryophyllaceae)

Abstract: Nectar production in Saponaria officincilis and in five species of Silene (S. ciba, S. dioica, S. noctiflora, S. nutans, S. vulgaris ) was examined during two consecutive years (May to July 1993, and May to June 1994) in the Botanical Garden of the University of Giessen. Nectar volume and sugar concentration were studied in relation to time of day, flower sex, flower age, and flowering stage. Nectar amount in all species studied (except S. dioica ) increased in the afternoon or in the evening until midnight (or until the early morning in S. nutans ). After midnight and until midday, nectar volume in non-visited flowers (except S. dioica ) decreased. Nectar volume in non-visited S. dioica flowers increased constantly with flower age, indicating a stable nectar secretion rate, possibly favouring both day- and night-active flower visitors. Even at the time of highest nectar secretion, all species studied presented several nectarless flowers. Sucrose dominance in the nectar of the nocturnal species S. nutans and Saponaria officinalis fits well with the general syndrome of flowers pollinated by hawkmoths. The syndrome also applies to the nocturnal but regularly selfing, S. noctiflora . The more generalis-tic species S. dioica and S. vulgaris , which are regularly visited by bumblebees as well as nocturnal moths, secreted hexose-domi-nant nectar. Unexpectedly, Silene alba , the only nocturnal species that strictly excluded day-active flower visitors by closing flowers during the day, also secreted hexose-dominant nectar. In some cases, nectar volumes and nectar concentration differed significantly between hermaphroditic, male, and female flowers. Female flowers of S. alba, S. dioica , and S. nutans contained significantly less concentrated nectar than male or hermaphroditic ( S. nutans ) ones. In S. noctifiora and S. vulgaris the difference was not statistically significant but nectar concentration did show the same tendency.     

Multiple nuclear gene phylogenetic analysis of the evolution of dioecy and sex chromosomes in the genus Silene

In the plant genus Silene, separate sexes and sex chromosomes are believed to have evolved twice. Silene species that are wholly or largely hermaphroditic are assumed to represent the ancestral state from which dioecy evolved. This assumption is important for choice of outgroup species for inferring the genetic and chromosomal changes involved in the evolution of dioecy, but is mainly based on data from a single locus (ITS). To establish the order of events more clearly, and inform outgroup choice, we therefore carried out (i) multi-nuclear-gene phylogenetic analyses of 14 Silene species (including 7 hermaphrodite or gynodioecious species), representing species from both Silene clades with dioecious members, plus a more distantly related outgroup, and (ii) a BayesTraits character analysis of the evolution of dioecy. We confirm two origins of dioecy within this genus in agreement with recent work on comparing sex chromosomes from both clades with dioecious species. We conclude that sex chromosomes evolved after the origin of Silene and within a clade that includes only S. latifolia and its closest relatives. We estimate that sex chromosomes emerged soon after the split with the ancestor of S. viscosa, the probable closest non-dioecious S. latifolia relative among the species included in our study.     

Duplicative transfer of a MADS box gene to a plant Y chromosome

Y chromosomes carry genes with functions in male reproduction and often have few other loci. Their evolution and the causes of genetic degeneration are of great interest. In addition to genetic degeneration, the acquisition of autosomal genes may be important in Y chromosome evolution. We here report that the dioecious plant Silene latifolia harbors a complete MADS box gene, SlAP3Y, duplicated onto the Y chromosome. This gene has no X-linked homologs but only an autosomal paralog, SlAP3A, and sequence divergence suggests that the duplication is a quite old event that occurred soon after the evolution of the sex chromosomes. Evolutionary sequence analyses using homologs of closely related species, including hermaphroditic Silene conica and dioecious Silene dioica and Silene diclinis, suggest that both SlAP3A and SlAP3Y genes encode functional proteins. Indeed, quantitative RT-PCR and in situ hybridization analyses showed that SlAP3A is expressed specifically in developing petals, but SlAP3Y is much more strongly expressed in developing stamens. The S. conica homolog, ScAP3A, is expressed in developing petals, suggesting subfunctionalization with evolution of male-specific functions, possibly due to evolutionary change in regulatory elements. Our results suggest that the acquisition of autosomal genes is an important event in the evolution of plant Y chromosomes.     

Reproduction and Pollination in Central European Populations of Silene and Saponaria Species

Flower morphology, flowering phenology, flower visitors, reproductive systems, and stigmatic receptivity of six species of Silene and Saponaria (Silene alba, S. dioica, S. vulgaris, S. nutans, S. noctiflora. Saponaria officinalis) were studied from April to October 1993 and from April to June 1994 in natural populations around Giessen in Hessen/Central Germany and, additionally, in individuals grown from seeds in the Botanical Garden of the University of Giessen. With the exception of Saponaria officinalis and S. noctiflora, all species were regularly visited and pollinated by crepuscular and nocturnal moths and hawkmoths, but only one species, S. alba, was exclusively pollinated by these night-active insects. The other species showed mixed pollination syndromes in which nocturnal and diurnal insects both promoted pollen transfer. Geitonogamy or even autogamy occurred in the gynodioecious and hermaphrodite species S. vulgaris, S. nutans, S. noctiflora, and Saponaria officinalis. Silene noctiflora, the only annual species, is pseudocleistogamous; the majority of its flowers did not open, and fruit set occurred after selfing in bud.     

Evolution of reproductive systems in the genus Silene

The genus Silene contains both hermaphrodite, gynodioecious and dioecious species, dioecy being represented in three sections of the genus. To locate the events of change of reproductive systems, we compared ITS sequences of 22 species of Silene chosen throughout the whole genus, and four putative outgroup species. Gynodioecy, which is the most common reproductive system within the genus Silene and in closely related genera such as Saponaria and Dianthus, is proposed to be ancestral in the genus. Dioecy has evolved at least twice: once in the section containing S. latifolia, and once in the clade containing S. otites and S. acaulis ssp. bryoides. Evolution towards hermaphroditism, associated with evolution of selfing has also occurred at least twice, in S. gallica and S. comica.     

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.     

Dynamics of drift, gene flow, and selection during speciation in Silene

The mechanics of speciation with gene flow are still unclear. Disparity among genes in population differentiation (F(ST)) between diverging species is often interpreted as evidence for semipermeable species boundaries, with selection preventing "key" genes from introgressing despite ongoing gene flow. However, F(ST) can remain high before it reaches equilibrium between the lineage sorting of species divergence and the homogenizing effects of gene flow (via secondary contact). Thus, when interpreting F(ST), the dynamics of drift, gene flow, and selection need to be taken into account. We illustrate this view with a multigenic analyses of gene flow and selection in three closely related Silene species, S. latifolia, S. dioica, and S. diclinis. We report that although S. diclinis appears to have evolved in allopatry, isolation with (bidirectional) gene flow between S. latifolia and S. dioica is likely, perhaps as a result of parapatric speciation followed by more extensive sympatry. Interestingly, we detected the signatures of apparently independent instances of positive selection at the same locus in S. latifolia and S. dioica. Despite gene flow between the species, the adaptive alleles have not crossed the species boundary, suggesting that this gene has independently undergone species-specific (diversifying or parallel) selection.     

Disentangling the effects of mating systems and mutation rates on cytoplamic diversity in gynodioecious Silene nutans and dioecious Silene otites

Many flowering plant species exhibit a variety of distinct sexual morphs, the two most common cases being the co-occurrence of females and males (dioecy) or the co-occurrence of hermaphrodites and females (gynodioecy). In this study, we compared DNA sequence variability of the three genomes (nuclear, mitochondrial and chloroplastic) of a gynodioecious species, Silene nutans, with that of a closely related dioecious species, Silene otites. In the light of theoretical models, we expect cytoplasmic diversity to differ between the two species due to the selective dynamics that acts on cytoplasmic genomes in gynodioecious species: under an epidemic scenario, the gynodioecious species is expected to exhibit lower cytoplasmic diversity than the dioecious species, while the opposite is expected in the case of balancing selection maintaining sterility cytoplasms in the gynodioecious species. We found no difference between the species for nuclear gene diversity, but, for the cytoplasmic loci, the gynodioecious S. nutans had more haplotypes, and higher nucleotide diversity, than the dioecious relative, S. otites, even though the latter has a relatively high rate of mitochondrial synonymous substitutions, and therefore presumably a higher mutation rate. Therefore, as the mitochondrial mutation rate cannot account for the higher cytoplasmic diversity found in S. nutans, our findings support the hypothesis that gynodioecy in S. nutans has been maintained by balancing selection rather than by epidemic-like dynamics.     

'Junk' DNA and phenotypic evolution in Silene section Siphonomorpha.

One of the long-standing mysteries in genomic evolution is the observation that much of the genome is composed of repetitive DNA, resulting in inter- and intraspecific variation in nuclear DNA content. Our discovery of a negative correlation between nuclear DNA content and flower size in Silene latifolia has been supported by our subsequent investigation of changes in DNA content as a correlated response to selection on flower size. Moreover, we have observed a similar trend across a range of related dioecious species in Silene sect. Elisanthe. Given the presence of sex chromosomes in dioecious Silene species, and the tendency of sex chromosomes to accumulate repetitive DNA, it seems plausible that dioecious species undergo genomic evolution in ways that differ from what one might expect in hermaphroditic species. Specifically, we query whether the observed relationship between nuclear DNA content and flower size observed in dioecious Silene is a peculiarity of sex chromosome evolution. In the present study we investigated nuclear DNA content and flower size variation in hermaphroditic species of Silene sect. Siphonomorpha, as close relatives of the dioecious species studied previously. Although the nuclear DNA contents of these species were lower than those for species in sect. Elisanthe, there was still significant intra- as well as interspecific variation in nuclear DNA content. Flower size variation was found among species of sect. Siphonomorpha for petal claw and petal limb lengths, but not for calyx diameter. This last trait varies extensively in sect. Elisanthe, in part due to sex-specific selection. A negative correlation with nuclear DNA content was found across populations for petal limb length, but not for other floral dimensions. We conclude that impacts of nuclear DNA content on phenotypic evolution do manifest themselves in hermaphroditic species, so that the effects observed in sect. Elisanthe, and particularly in S. latifolia, while perhaps amplified by the genomic impacts of sex chromosomes, are not limited to dioecious taxa.     

GENETIC ARCHITECTURE OF ISOLATION BETWEEN TWO SPECIES OF SILENE WITH SEX CHROMOSOMES AND HALDANE'S RULE

Examination of the genetic architecture of hybrid breakdown can provide insight into the genetic mechanisms of commonly observed isolating phenomena such as Haldane's rule. We used line‐cross analysis to dissect the genetic architecture of divergence between two plant species that exhibit Haldane's rule for male sterility and rarity, Silene latifolia and Silene diclinis. We made 15 types of crosses, including reciprocal F₁, F₂, backcrosses, and later‐generation crosses, grew the seeds to flowering, and measured the number of viable ovules, proportion of viable pollen, and sex ratio. Typically, Haldane's rule for male rarity in XY animal hybrids is explained by interactions involving recessive X‐linked alleles that are deleterious when hemizygous (dominance theory), whereas sterility is explained by rapid evolution of spermatogenesis genes (faster‐male evolution). In contrast, we found that the genetic mechanisms underlying Haldane's rule between the two Silene species did not follow these conventions. Dominance theory was sufficient to explain male sterility, but male rarity likely involved faster‐male evolution. We also found an effect of the neo‐sex chromosomes of S. diclinis on the extreme rarity of some hybrid males. Our findings suggest that the genetic architecture of Haldane's rule in dioecious plants may differ from those commonly found in animals.     

C-banded karyotypes of two Silene species with heteromorphic sex chromosomes.

Mitotic metaphase chromosomes of Silene latifolia (white campion) and Silene dioica (red campion) were studied and no substantial differences between the conventional karyotypes of these two species were detected. The classification of chromosomes into three distinct groups proposed for S. latifolia by Ciupercescu and colleagues was considered and discussed. Additionally, a new small satellite on the shorter arm of homobrachial chromosome 5 was found. Giemsa C-banded chromosomes of the two analysed species show many fixed and polymorphic heterochromatic bands, mainly distally and centromerically located. Our C-banding studies provided an opportunity to better characterize the sex chromosomes and some autosome types, and to detect differences between the two Silene karyotypes. It was shown that S. latifolia possesses a larger amount of polymorphic heterochromatin, especially of the centromeric type. The two Silene sex chromosomes are easily distinguishable not only by length or DNA amount differences but also by their Giemsa C-banding patterns. All Y chromosomes invariably show only one distally located band, and no other fixed or polymorphic bands on this chromosome were observed in either species. The X chromosomes possess two terminally located fixed bands, and some S. latifolia X chromosomes also have an extra-centric segment of variable length. The heterochromatin amount and distribution revealed by our Giemsa C-banding studies provide a clue to the problem of sex chromosome and karyotype evolution in these two closely related dioecious Silene species.     

A rapid means of sex identification inSilene latifolia by use of flow cytometry

Sex identification in dioecious plants using nonflowering material would have broad applications in both basic and applied research. We present a method using flow cytometry for diagnosing the sex of the dioecious speciesSilene latifolia Poiret (Caryophyllaceae) by means of sexual differences in nuclear DNA content and base-pair composition. Males have a significantly larger genome, attributable to the known sex-chromosome heteromorphism. Males and females also differ in the AT/GC composition, attributable to differences in non-recombining portions of the sex chromosomes. The two measures enable assignment of individuals to sex with a combined error rate of 9%. These results forS. latifolia indicate useful directions for future research into sex diagnostics for other dioecious species.     

Inbreeding Depression and Genetic Rescue in a Plant Metapopulation

While migration of individuals has been shown to increase the persistence of small isolated populations through a process known as the "rescue effect," the demographic effects that pollen-mediated gene flow may have in plant populations are not known empirically. This study investigates the role that inbreeding depression plays in newly colonized populations of a common, dioecious, weedy species, Silene alba. Experimental greenhouse studies presented here show that S. alba displays high levels of inbreeding depression (expressed as lowered germination success) in progeny produced with inbreeding coefficients of 0.125 (half-sib mated), 0.250 (full-sib mated), and 0.375 (second-generation sib mated). In addition, it is shown that the degree of inbreeding depression in 12 natural colonies varies with the degree of isolation from other established populations. Significantly, data from experimental populations showed that gene flow into patches comprised of full sibs was higher than those observed into patches comprised of unrelated individuals and may serve to mitigate the effects of inbreeding depression. It is suggested that population connectivity through pollen-mediated gene flow may have substantial effects on the persistence of isolated colonies and on the spatial structure of a metapopulation in general.     

The inter-specific hybrid Silene latifolia x S. viscosa reveals early events of sex chromosome evolution

The dioecious plant species Silene latifolia has a sex determination mechanism based on an active Y chromosome. Here, we used inter-specific hybrids in the genus Silene to study the effects of gene complexes on the Y chromosome. If the function of Y-linked genes has been maintained in the same state as in the hermaphrodite progenitor species, it should be possible to substitute such genes by genes coming from a related hermaphrodite species. In the inter-specific hybrid, S. latifolia x S. viscosa, anthers indeed develop far beyond the early bilobal stage characteristic of XX S. latifolia female plants. The S. viscosa genome can thus replace the key sex determination gene whose absence abolishes early stamen development in females (loss of the stamen-promoting function, SPF), so that hybrid plants are morphologically hermaphrodite. However, the hybrids have two anther development defects, loss of adhesion of the tapetum to the endothecium, and precocious endothecium maturation. Both these defects were also found in independent Y-chromosome deletion mutants of S. latifolia. The data support the hypothesis that the evolution of complete gender dimorphism from hermaphroditism involved a major largely recessive male-sterility factor that created females, and the appearance of new, dominant genes on the Y chromosome, including both the well-documented gynoecium-suppressing factor, and two other Y specific genes promoting anther development.     

Importance of Silene latifolia ssp. alba and S. dioica (Caryophyllaceae) as host plants of the parasitic pollinator Hadena bicruris (Lepidoptera, Noctuidae)

With regards to pollination there exist several mutualistic relationships between Hadena -species and Caryophyllaceae. As mutualists have both negative and positive effects on their partners, mutualism is often betoken as reciprocative exploitation which may shift to parasitism if the exploitation of one partner becomes prevalent.
Several Silene - and Saponaria -species are considered to be larval host plants of Hadena bicruris . Although Silene latifolia ssp. alba and S. dioica are frequently cited as hosts of the seed eating larvae, field and laboratory observations at Ulm were suggestive for only S. latifolia ssp. alba being a suitable host. Records of the oviposition behavior of H. bicruris made it evident that in fact a considerable number of eggs could be found in planted stands of both species. On the other hand, phenological data of the flowering periods and of the oviposition behavior of H. bicruris showed that S. latifolia ssp. alba is clearly preferred for oviposition if host selection is possible due to contemporaneous flowering of individuals of both plant species growing at close range. In addition, the flowering periods of S. latifolia ssp. alba and the periods of moth activity overlap to a large extent. This is not the case in S. dioica . Feeding experiences first indicated that the caterpillars may not prefer one of the species to the other, but comparison of the pupal weight of the animals reared on fruits of exclusively one of the species showed that the seeds of S. latifolia ssp. alba were more profitable for nutrition than those of S. dioica ; the pupal weight of animals reared on seeds of the former species significantly exceeded that of animals reared on seeds of the latter one. The question arises if the symbiosis of H. bicruris and its hosts constitutes a stable situation or if an evolutionary shift to mutualism or parasitism will take place.     

Euchromatic domains in plant chromosomes as revealed by H4 histone acetylation and early DNA replication.

Using specific polyclonal antisera raised against acetylated isoforms of histone H4, we have analyzed their distribution in the dioecious plant Silene latifolia (syn. Melandrium album) possessing heteromorphic sex chromosomes. Our previous studies on this species have shown that one of the two X chromosomes in homogametic female cells is heavily methylated and late replicating, as a possible consequence of dosage compensation. Here we report that there are no detectable differences in intensity and distribution of H4 acetylation between these two X chromosomes. In S. latifolia only distal-subtelomeric chromosome regions, on both the sex chromosomes and autosomes, display strong signals of H4 acetylation at N-terminal lysines 5, 8, and 12. These acetylated domains correspond to the very early replicating distal chromosome regions as revealed by 5-bromodeoxyuridine pulses followed by the indirect immunofluorescence microscopy. The distribution of H4 acetylated at lysine 16 was uniform along the chromosomes. The unique distal-subtelomeric H4 acetylation signals were also observed in three other Silene species (S. vulgaris, S. pendula, and S. chalcedonica), but not in two non-related plant species tested (Allium cepa and Nicotiana tobacum). The presented data as well as our recent studies on the structure of S. latifolia chromosome ends indicate that Silene species possess the specific distal-subtelomeric location of euchromatin, gene-rich regions on chromosomes.     

Reproductive isolation mechanisms among four closely-related species of Conospermum (Proteaceae)

The effectiveness of geographical isolation, ecological isolation, temporal isolation, mechanical isolation, ethological isolation, cross-incompatibility, hybrid inviability, hybrid sterility and hybrid breakdown as practical barriers to gene flow in the field between Conospermum taxifolium, C. ericifolium, C. ellipticum and C. longifolium has been quantified. The barriers to gene flow between C. ericifolium and C. ellipticum are completely effective, owing to their allopatric distributions. The barriers to gene flow between C. taxifolium and these two species are only partially effective, as their ecological separation breaks down in intermediate habitats, and partially-fertile F₁ plants can grow in the areas of overlap. The barriers to gene flow between C. longifolium and the other three species are almost completely effective, as cross-incompatibility is very high and the F₁ plants are female-sterile.     

The sex ratlo in a dioecious endemic plant,Silene dielinis

Silene diclinis is a rare Spanish relative of the well-studied dioecious S. dioica and S. latifolia (=S. alba, S. pratensis). The main wild population and its cultivated derivatives showed female biased sex ratios. The ratios did not differ significantly between subpopulations or between the wild and cultivated samples, but they did deviate significantly from the expectation of a 1:1 male to female ratio. The characteristically biased sex ratio may be an incidental property of the XY sex determination system, with X-containing (female determining) pollen competing more effectively on the style than Y-containing grains.