The Effect of Breeding System on Polymorphism in Mitochondrial Genes of Silene

Gynodioecy is a breeding system characterized by the co-occurrence of hermaphrodite and female individuals, generally as the result of nuclear–cytoplasmic interactions. The question remains whether the genetic factors controlling gynodioecy are maintained in species over long evolutionary timescales by balancing selection or are continually arising and being replaced in epidemic sweeps. If balancing selection maintains these factors, then neutral cytoplasmic diversity should be greater in gynodioecious than hermaphroditic species. In contrast, epidemic sweeps of factors controlling gynodioecy should decrease cytoplasmic diversity in gynodioecious relative to hermaphroditic species. We took a comparative approach in which we sequenced two mitochondrial genes, cytochrome b (cob) and cytochrome oxidase (cox1), for multiple populations of several hermaphroditic, gynodioecious, and dioecious species in the genus Silene. Breeding system was predictive of polymorphism. Gynodioecious species harbor many old haplotypes while hermaphroditic and dioecious species have little to no nucleotide diversity. The genealogical structure of neither gene departed from neutral expectations. Taken together, our results suggest that balancing selection acts on cytoplasmic male-sterility factors in several gynodioecious species in the genus.     

Genetic determination of male sterility in gynodioecious Silene nutans

Gynodioecy, the coexistence of female and hermaphrodite plants within a species, is often under nuclear–cytoplasmic sex determination, involving cytoplasmic male sterility (CMS) genes and nuclear restorers. A good knowledge of CMS and restorer polymorphism is essential for understanding the evolution and maintenance of gynodioecy, but reciprocal crossing studies remain scarce. Although mitochondrial diversity has been studied in a few gynodioecious species, the relationship between mitotype diversity and CMS status is poorly known. From a French sample of Silene nutans, a gynodioecious species whose sex determination remains unknown, we chose the four most divergent mitotypes that we had sampled at the cytochrome b gene and tested by reciprocal crosses whether they carry distinct CMS genes. We show that gynodioecy in S. nutans is under nuclear–cytoplasmic control, with at least two different CMSs and up to four restorers with epistatic interactions. Female occurrence and frequency were highly dependent on the mitotype, suggesting that the level of restoration varies greatly among CMSs. Two of the mitotypes, which have broad geographic distributions, represent different CMSs and are very unequally restored. We discuss the dynamics of gynodioecy at the large-scale meta-population level.     

Functional characterization of gynodioecy in Fragaria vesca ssp. bracteata (Rosaceae)

Background and Aims

Gynodioecy is a phylogenetically widespread and important sexual system where females coexist with hermaphrodites. Because dioecy can arise from gynodioecy, characterization of gynodioecy in close relatives of dioecious and sub-dioecious species can provide insight into this transition. Thus, we sought to determine whether Fragaria vesca ssp. bracteata, a close relative to F. chiloensis and F. virginiana, exhibits the functional and population genetic hallmarks of a gynodioecious species.

Methods

We compared reproductive allocation of females and hermaphrodites grown in the greenhouse and estimated genetic diversity (allelic diversity, heterozygosity) and inbreeding coefficients for field-collected adults of both sexes using simple sequence repeat (SSR) markers. We estimated mating system and early seed fitness from open-pollinated families of both sex morphs.

Key Results

Under greenhouse conditions, females and hermaphrodites allocated similarly to all reproductive traits except flower number, and, as a consequence, females produced 30 % fewer seeds per plant than hermaphrodites. Under natural conditions, hermaphrodites produce seeds by self-fertilization approx. 75 % of the time, and females produced outcrossed seeds with very little biparental inbreeding. Consistent with inbreeding depression, seeds from open-pollinated hermaphrodites were less likely to germinate than those from females, and family-level estimates of hermaphrodite selfing rates were negatively correlated with germination success and speed. Furthermore, estimates of inbreeding depression based on genetic markers and population genetic theory indicate that inbreeding depression in the field could be high.

Conclusions

The joint consideration of allocation and mating system suggests that compensation may be sufficient to maintain females given the current understanding of sex determination. Fragaria vesca ssp. bracteata exhibited similar sex morph-dependent patterns of mating system and genetic diversity, but less reproductive trait dimorphism, than its sub-dioecious and dioecious congeners.     

Nucleotide diversity in Silene latifolia autosomal and sex-linked genes

The plant Silene latifolia has separate sexes and sex chromosomes, and is of interest for studying the early stages of sex chromosome evolution, especially the evolution of non-recombining regions on the Y chromosome. Hitch-hiking processes associated with ongoing genetic degeneration of the non-recombining Y chromosome are predicted to reduce Y-linked genes'' effective population sizes, and S. latifolia Y-linked genes indeed have lower diversity than X-linked ones. We tested whether this represents a true diversity reduction on the Y, versus the alternative possibility, elevated diversity at X-linked genes, by collecting new data on nucleotide diversity for autosomal genes, which had previously been little studied. We find clear evidence that Y-linked genes have reduced diversity. However, another alternative explanation for a low Y effective size is a high variance in male reproductive success. Autosomal genes should then also have lower diversity than expected, relative to the X, but this is not found in our loci. Taking into account the higher mutation rate of Y-linked genes, their low sequence diversity indicates a strong effect of within-population hitch-hiking on the Y chromosome.     

Flower scent composition in night-flowering Silene species (Caryophyllaceae)

Floral scent of 13 night-flowering Silene species (Caryophyllaceae) was collected by headspace adsorption and analysed via gas chromatography and mass spectrometry. Benzenoids together with isoprenoids dominated the scent in all species. Among the benzenoids, benzaldehyde (Silene subconica 35.5%, Silene succulenta 23.1%, Silene sericea 15.6%, Silene vulgaris 12.2%, and Silene nutans 9.9%), methylbenzoate (Silene saxifraga 96.1%, S. succulenta 15.2%), benzyl acetate (Silene dichotoma 37.8%, S. nutans 30.1%, Silene italica 9.0%, and Silene latifolia 5.5%), or benzyl alcohol (Silene viscosa 36.1%) occur in the largest amounts. p-Cresol is only found in the floral scent of S. dichotoma (28.5%). Among the isoprenoids, monoterpenes occur in the largest amounts (myrcene 23% in Silene chlorantha, trans-β-ocimene 27.2% in S. nutans and 34.9% in S. sericea, fenchyl acetate 12.7% in S. chlorantha, β-linalool 40.5% in S. chlorantha and 14.5% in S. italica). Relatively high amounts of lilac compounds occur in S. latifolia (49.1%), Silene otites (35.7%), S. subconica (15.2%), and S. vulgaris (59.6%). Higher amounts of sesquiterpenes (isoprenoids) were only found in Silene vallesia with β-bourbonene and γ-muurolene.The vast majority of chemicals identified are common components of a wide array of scented angiosperm flowers. Nevertheless, the results conform most strongly with the findings in other night-blooming and/or moth-pollinated flowers. All investigated Silene species follow the general trend of floral scent compounds typical for moth-pollinated flowers, i.e. flowers having acyclic terpene alcohols (e.g. linalool), aromatic alcohols (benzyl alcohol, 2-phenylethanol) and esters derived from them, and small amounts of nitrogen-containing compounds.     

Testing for the Footprint of Sexually Antagonistic Polymorphisms in the Pseudoautosomal Region of a Plant Sex Chromosome Pair

The existence of sexually antagonistic (SA) polymorphism is widely considered the most likely explanation for the evolution of suppressed recombination of sex chromosome pairs. This explanation is largely untested empirically, and no such polymorphisms have been identified, other than in fish, where no evidence directly implicates these genes in events causing loss of recombination. We tested for the presence of loci with SA polymorphism in the plant Silene latifolia, which is dioecious (with separate male and female individuals) and has a pair of highly heteromorphic sex chromosomes, with XY males. Suppressed recombination between much of the Y and X sex chromosomes evolved in several steps, and the results in Bergero et al. (2013) show that it is still ongoing in the recombining or pseudoautosomal, regions (PARs) of these chromosomes. We used molecular evolutionary approaches to test for the footprints of SA polymorphisms, based on sequence diversity levels in S. latifolia PAR genes identified by genetic mapping. Nucleotide diversity is high for at least four of six PAR genes identified, and our data suggest the existence of polymorphisms maintained by balancing selection in this genome region, since molecular evolutionary (HKA) tests exclude an elevated mutation rate, and other tests also suggest balancing selection. The presence of sexually antagonistic alleles at a locus or loci in the PAR is suggested by the very different X and Y chromosome allele frequencies for at least one PAR gene.     

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.     

Asymmetrical conspecific seed-siring advantage between Silene latifolia and S. dioica

Background and Aims

Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible.

Methods

Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers.

Key Results

The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers.

Conclusions

The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.     

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.     

Sex ratio in populations of Silene otites in relation to vegetation cover,population size and fungal infection

Abstract. In contrast to populations of most dioecious Silene species (which usually are female-biased), populations of Silene otites have been frequently reported to be male-biased. We describe sex ratio variation in 34 natural S. otites populations in Central Germany in relation to vegetation cover, population size and fungal infection. The overall sex ratio was unbiased in 1994 and only slightly male-biased in 1995. Sex ratio varied among the populations from 26.6 % to 72.6 % females. The sex ratio of small populations varied strongly due to stochastic processes. Furthermore, we found that populations in habitats with high vegetation cover contained a higher percentage of females. Hermaphroditic plants, theoretically, could increase male bias as they only produce male or hermaphroditic offspring. Their frequency in the populations, however, was far too low to affect sex ratio. In 1994 12.1 % and in 1995 17.0 % of the plants were infected by the smut fungus Ustilago major. Disease incidence in the population was not related to sex ratio, suggesting equal susceptibility of males and females. The sex ratio of partially infected plants did not deviate from the population sex ratio, both under field conditions and in a greenhouse laboratory experiment. The results suggest that the frequently reported male bias in Silene otites populations is not a general pattern, but is mainly caused by environmental conditions.     

A new plant sex-linked gene with high sequence diversity and possible introgression of the X copy

We describe patterns of DNA sequence diversity in a newly identified sex-linked gene, SlX9/SlY9, in Silene latifolia (Caryophyllaceae). The copies on both sex chromosomes seem to be functional, and each maps close to the respective X- and Y-linked copy of another sex-linked gene pair, SlCypX/SlCypY. The Y-linked copy has low diversity, similar to what has been found for several other Y-linked genes in S. latifolia, and consistent with the theoretical expectations of hitch-hiking processes occurring on a non-recombining chromosome. However, SlX9 has higher diversity than other genes on the S. latifolia X chromosome. We evaluate the hypothesis of introgression from the closely related species S. dioica as an explanation for the high sequence diversity observed.     

An angiosperm-wide analysis of the gynodioecy–dioecy pathway

Background and Aims

About 6 % of an estimated total of 240 000 species of angiosperms are dioecious. The main precursors of this sexual system are thought to be monoecy and gynodioecy. A previous angiosperm-wide study revealed that many dioecious species have evolved through the monoecy pathway; some case studies and a large body of theoretical research also provide evidence in support of the gynodioecy pathway. If plants have evolved through the gynodioecy pathway, gynodioecious and dioecious species should co-occur in the same genera. However, to date, no large-scale analysis has been conducted to determine the prevalence of the gynodioecy pathway in angiosperms. In this study, this gap in knowledge was addressed by performing an angiosperm-wide survey in order to test for co-occurrence as evidence of the gynodioecy pathway.

Methods

Data from different sources were compiled to obtain (to our knowledge) the largest dataset on gynodioecy available, with 275 genera that include at least one gynodioecious species. This dataset was combined with a dioecy dataset from the literature, and a study was made of how often dioecious and gynodioecious species could be found in the same genera using a contingency table framework.

Key Results

It was found that, overall, angiosperm genera with both gynodioecious and dioecious species occur more frequently than expected, in agreement with the gynodioecy pathway. Importantly, this trend holds when studying different classes separately (or sub-classes, orders and families), suggesting that the gynodioecy pathway is not restricted to a few taxa but may instead be widespread in angiosperms.

Conclusions

This work complements that previously carried out on the monoecy pathway and suggests that gynodioecy is also a common pathway in angiosperms. The results also identify angiosperm families where some (or all) dioecious species may have evolved from gynodioecious precursors. These families could be the targets of future small-scale studies on transitions to dioecy taking phylogeny explicitly into account.     

Inbreeding effects on progeny sex ratio and gender variation in the gynodioecious Silene vulgaris (Caryophyllaceae)

In gynodioecious species, sex expression is generally determined through cytoplasmic male sterility genes interacting with nuclear restorers of the male function. With dominant restorers, there may be an excess of females in the progeny of self-fertilized compared with cross-fertilized hermaphrodites. Moreover, the effect of inbreeding on late stages of the life cycle remains poorly explored. Here, we used hermaphrodites of the gynodioecious Silene vulgaris originating from three populations located in different valleys in the Alps to investigate the effects of two generations of self- and cross-fertilization on sex ratio and gender variation. We detected an increase in females in the progeny of selfed compared with outcrossed hermaphrodites and inbreeding depression for female and male fertility. Male fertility correlated positively with sex ratio differences between outbred and inbred progeny, suggesting that dominant restorers are likely to influence male fertility qualitatively and quantitatively in S. vulgaris. We argue that the excess of females in the progeny of selfed compared with outcrossed hermaphrodites and inbreeding depression for gamete production may contribute to the maintenance of females in gynodioecious populations of S. vulgaris because purging of the genetic load is less likely to occur.     

Night life on the beach: selfing to avoid pollinator competition between two sympatric Silene species

Background and Aims Evolution of autonomous selfing may be advantageous because it allows for reproductive assurance. In co-flowering plants competing for pollinators, the least common and/or attractive could suffer pollen limitations. Silene niceensis and S. ramosissima are taxonomically related species sharing the same habitat, although S. ramosissima is less abundant and has a more restricted distribution. They also have the same a priori nocturnal pollinator syndrome, and show an overlapping flowering phenology. The aim of this study was to investigate whether a selfing strategy in S. ramosissima allows it to avoid pollinator competition and/or interspecific pollen transfer with S. niceensis, which would thus enable both species to reach high levels of fruit and seed set.Methods The breeding system, petal colour, flower life span and degree of overlap between male and female phases, floral visitor abundance and visitation rates were analysed in two sympatric populations of S. niceensis and S. ramosissima in southern Spain.Key Results Autonomous selfing in S. ramosissima produced very high fruit and seed set, which was also similar to open-pollinated plants. Silene niceensis showed minimum levels of autonomous selfing, and pollen/ovule ratios were within the range expected for the breeding system. In contrast to S. niceensis, flower life span was much shorter in S. ramosissima, and male and female organs completely overlapped in space and time. Upper surface petals of both species showed differing brightness, chroma and hue. Flowers of S. niceensis were actively visited by moths, hawkmoths and syrphids, whereas those of S. ramosissima were almost never visited.Conclusions The findings show that different breeding strategies exist between the sympatric co-flowering S. niceensis and S. ramosissima, the former specializing in crepuscular–nocturnal pollination and the latter mainly based on autonomous selfing. These two strategies allow both species to share the restricted dune habitat in which they exist, with a high female reproductive success due to the absence of pollinator competition and/or interspecific pollen flow.     

Detecting de novo mitochondrial mutations in angiosperms with highly divergent evolutionary rates

Although plant mitochondrial genomes typically show low rates of sequence evolution, levels of divergence in certain angiosperm lineages suggest anomalously high mitochondrial mutation rates. However, de novo mutations have never been directly analyzed in such lineages. Recent advances in high-fidelity DNA sequencing technologies have enabled detection of mitochondrial mutations when still present at low heteroplasmic frequencies. To date, these approaches have only been performed on a single plant species (Arabidopsis thaliana). Here, we apply a high-fidelity technique (Duplex Sequencing) to multiple angiosperms from the genus Silene, which exhibits extreme heterogeneity in rates of mitochondrial sequence evolution among close relatives. Consistent with phylogenetic evidence, we found that Silene latifolia maintains low mitochondrial variant frequencies that are comparable with previous measurements in Arabidopsis. Silene noctiflora also exhibited low variant frequencies despite high levels of historical sequence divergence, which supports other lines of evidence that this species has reverted to lower mitochondrial mutation rates after a past episode of acceleration. In contrast, S. conica showed much higher variant frequencies in mitochondrial (but not in plastid) DNA, consistent with an ongoing bout of elevated mitochondrial mutation rates. Moreover, we found an altered mutational spectrum in S. conica heavily biased towards AT→GC transitions. We also observed an unusually low number of mitochondrial genome copies per cell in S. conica, potentially pointing to reduced opportunities for homologous recombination to accurately repair mismatches in this species. Overall, these results suggest that historical fluctuations in mutation rates are driving extreme variation in rates of plant mitochondrial sequence evolution.     

EVOLUTION OF SEX DETERMINATION SYSTEMS WITH HETEROGAMETIC MALES AND FEMALES IN SILENE

The plant genus Silene has become a model for evolutionary studies of sex chromosomes and sex‐determining mechanisms. A recent study performed in Silene colpophylla showed that dioecy and the sex chromosomes in this species evolved independently from those in Silene latifolia, the most widely studied dioecious Silene species. The results of this study show that the sex‐determining system in Silene otites, a species related to S. colpophylla, is based on female heterogamety, a sex determination system that is unique among the Silene species studied to date. Our phylogenetic data support the placing of S. otites and S. colpophylla in the subsection Otites and the analysis of ancestral states suggests that the most recent common ancestor of S. otites and S. colpophylla was most probably dioecious. These observations imply that a switch from XX/XY sex determination to a ZZ/ZW system (or vice versa) occurred in the subsection Otites. This is the first report of two different types of heterogamety within one plant genus of this mostly nondioecious plant family.     

Presence of two mitochondrial genomes in the mytilid Perumytilus purpuratus: Phylogenetic evidence for doubly uniparental inheritance

This study presents evidence, using sequences of ribosomal 16S and COI mtDNA, for the presence of two mitochondrial genomes in Perumytilus purpuratus. This may be considered evidence of doubly uniparental mtDNA inheritance. The presence of the two types of mitochondrial genomes differentiates females from males. The F genome was found in the somatic and gonadal tissues of females and in the somatic tissues of males; the M genome was found in the gonads and mantle of males only. For the mitochondrial 16S region, ten haplotypes were found for the F genome (nucleotide diversity 0.004), and 7 haplotypes for the M genome (nucleotide diversity 0.001), with a distance Dxy of 0.125 and divergence Kxy of 60.33%. For the COI gene 17 haplotypes were found for the F genome (nucleotide diversity 0.009), and 10 haplotypes for the M genome (nucleotide diversity 0.010), with a genetic distance Dxy of 0.184 and divergence Kxy of 99.97%. Our results report the presence of two well-differentiated, sex-specific types of mitochondrial genome (one present in the male gonad, the other in the female gonad), implying the presence of DUI in P. purpuratus. These results indicate that care must be taken in phylogenetic comparisons using mtDNA sequences of P. purpuratus without considering the sex of the individuals.