Mechanisms governing sex-ratio variation in dioecious Rumex nivalis

Sex ratios of flowering individuals in dioecious plant populations are often close to unity, or are male biased owing to gender-specific differences in flowering or mortality. Female-biased sex ratios, although infrequent, are often reported in species with heteromorphic sex chromosomes. Two main hypotheses have been proposed to account for female bias: (1) selective fertilization resulting from differential pollen-tube growth of female- versus male-determining microgametophytes (certation); (2) differences in the performance and viability of the sexes after parental investment. Here we investigate these hypotheses in Rumex nivalis (Polygonaceae), a European alpine herb with female-biased sex ratios in which females possess XX, and males XY1Y2, sex chromosomes. Using field surveys and a glasshouse experiment we investigated the relation between sex ratios and life-history stage in 18 populations from contrasting elevations and snowbed microsites and used a male-specific SCAR-marker to determine the sex of nonflowering individuals. Female bias among flowering individuals was one of the highest reported for populations of a dioecious species (mean female frequency = 0.87), but males increased in frequency at higher elevations and in the center of snowbeds. Female bias was also evident in nonflowering individuals (mean 0.78) and in seeds from open-pollinated flowers (mean 0.59). The female bias in seeds was weakly associated with the frequency of male flowering individuals in populations in the direction predicted when certation occurs. Under glasshouse conditions, females outperformed males at several life-history stages, although male seeds were heavier than female seeds. Poor performance of Y1Y2 gametophytes and male sporophytes in R. nivalis may be a consequence of the accumulation of deleterious mutations on Y-sex chromosomes.     

Sexing pollen reveals female bias in a dioecious plant

* Information on angiosperm sex ratios has largely been restricted to surveys of flowering individuals. These often deviate from equality, with male bias more commonly reported. Female-biased sex ratios are concentrated in a few taxa and have been linked to the possession of heteromorphic sex chromosomes and bias introduced during the gametophytic stage of the life cycle. It has been proposed that differences in gamete quantity and quality could give rise to female bias, although there is no direct evidence with which to evaluate this possibility. * Here, we use flow cytometry to investigate microgametophytic 'sex ratios' in a flowering plant. We demonstrate that differences in DNA content between the sexes in Rumex nivalis, a species with heteromorphic sex chromosomes, make it possible to distinguish female- vs male-determining pollen nuclei. * We found a small but significant female bias in microgametophytes produced by males (mean 0.515) with significant variation among family means (range 0.463-0.586), and 18 of 22 families averaging > 0.50. * The observed female bias at the gametophytic stage of the life cycle is consistent with the direction of bias previously reported for seeds and vegetative and reproductive plants in wild populations of R. nivalis, but is insufficient to fully explain the degree of bias.     

Sex Determination by Sex Chromosomes in Dioecious Plants

Abstract: Sex chromosomes have been reported in several dioecious plants. The most general system of sex determination with sex chromosomes is the XY system, in which males are the heterogametic sex and females are homogametic. Genetic systems in sex determination are divided into two classes including an X chromosome counting system and an active Y chromosome system. Dioecious plants have unisexual flowers, which have stamens or pistils. The development of unisexual flowers is caused by the suppression of opposite sex primordia. The expression of floral organ identity genes is different between male and female flower primordia. However, these floral organ identity genes show no evidence of sex chromosome linkage. The Y chromosome of Rumex acetosa contains Y chromosome-specific repetitive sequences, whereas the Y chromosome of Silene latifolia has not accumulated chromosome-specific repetitive sequences. The different degree of Y chromosome degeneration may reflect on evolutionary time since the origination of dioecy. The Y chromosome of S. latifolia functions in suppression of female development and initiation and completion of anther development. Analyses of mutants suggested that female suppressor and stamen promoter genes are localized on the Y chromosome. Recently, some sex chromosome-linked genes were isolated from flower buds of S. latifolia.     

Plant sex chromosomes: molecular structure and function

Recent molecular and genomic studies carried out in a number of model dioecious plant species, including Asparagus officinalis, Carica papaya, Silene latifolia, Rumex acetosa and Marchantia polymorpha, have shed light on the molecular structure of both homomorphic and heteromorphic sex chromosomes, and also on the gene functions they have maintained since their evolution from a pair of autosomes. The molecular structure of sex chromosomes in species from different plant families represents the evolutionary pathway followed by sex chromosomes during their evolution. The degree of Y chromosome degeneration that accompanies the suppression of recombination between the Xs and Ys differs among species. The primitive Ys of A. officinalis and C. papaya have only diverged from their homomorphic Xs in a short male-specific and non-recombining region (MSY), while the heteromorphic Ys of S. latifolia, R. acetosa and M. polymorpha have diverged from their respective Xs. As in the Y chromosomes of mammals and Drosophila, the accumulation of repetitive DNA, including both transposable elements and satellite DNA, has played an important role in the divergence and size enlargement of plant Ys, and consequently in reducing gene density. Nevertheless, the degeneration process in plants does not appear to have reached the Y-linked genes. Although a low gene density has been found in the sequenced Y chromosome of M. polymorpha, most of its genes are essential and are expressed in the vegetative and reproductive organs in both male and females. Similarly, most of the Y-linked genes that have been isolated and characterized up to now in S. latifolia are housekeeping genes that have X-linked homologues, and are therefore expressed in both males and females. Only one of them seems to be degenerate with respect to its homologous region in the X. Sequence analysis of larger regions in the homomorphic X and Y chromosomes of papaya and asparagus, and also in the heteromorphic sex chromosomes of S. latifolia and R. acetosa, will reveal the degenerative changes that the Y-linked gene functions have experienced during sex chromosome evolution.     

Sex reversal and primary sex ratios in the common frog (Rana temporaria)

Sex reversal has been suggested to have profound implications for the evolution of sex chromosomes and population dynamics in ectotherms. Occasional sex reversal of genetic males has been hypothesized to prevent the evolutionary decay of nonrecombining Y chromosomes caused by the accumulation of deleterious mutations. At the same time, sex reversals can have a negative effect on population growth rate. Here, we studied phenotypic and genotypic sex in the common frog (Rana temporaria) in a subarctic environment, where strongly female‐biased sex ratios have raised the possibility of frequent sex reversals. We developed two novel sex‐linked microsatellite markers for the species and used them with a third, existing marker and a Bayesian modelling approach to study the occurrence of sex reversal and to determine primary sex ratios in egg clutches. Our results show that a significant proportion (0.09, 95% credible interval: 0.04–0.18) of adults that were genetically female expressed the male phenotype, but there was no evidence of sex reversal of genetic males that is required for counteracting the degeneration of Y chromosome. The primary sex ratios were mostly equal, but three clutches consisted only of genetic females and three others had a significant female bias. Reproduction of the sex‐reversed genetic females appears to create all‐female clutches potentially skewing the population level adult sex‐ratio consistent with field observations. However, based on a simulation model, such a bias is expected to be small and transient and thus does not fully explain the observed female‐bias in the field.     

Does the timing of attainment of maturity influence sexual size dimorphism and adult sex ratio in turtles?

The attainment of sexual maturity has been shown to affect measures of sexual size dimorphism (SSD) and adult sex ratios in several groups of vertebrates. Using data for turtles, we tested the model that sex ratios are expected to be male‐biased when females are larger than males and female‐biased when males are larger than females because of the relationship of each with the attainment of maturity. Our model is based on the premise that the earlier‐maturing sex remains smaller, on average throughout life, and predominates numerically unless the sexes are strongly affected by differential mortality, differential emigration, and immigration, or biased primary sex ratios. Based on data for 24 species in seven families, SSD and sex ratios were significantly negatively correlated for most analyses, even after the effect of phylogenetic bias was removed. The analyses provide support for the model that SSD and adult sex ratios are correlated in turtles as a result of simultaneous correlation of each with sexual differences in attainment of maturity (bimaturism). Environmental sex determination provides a possible mechanism for the phenomenon in turtles and some other organisms. © 2014 The Authors. Biological Journal of the Linnean Society published by John Wiley & Sons Ltd on behalf of The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 142–149.     

Sex-ratio distortion driven by migration loads

The significance of migration load in driving the evolution of recipient populations has long been documented in population genetics, but its effects have not been linked to the formation of biased sex ratios in natural populations. In this study, we develop a single-locus model to demonstrate how the migration load can shape the primary and secondary sex ratios in dioecious plants where sexual dimorphism is determined by the sex chromosomes (the XX-XY or similar systems). Our results show that migration load can generate an array of sex ratios (from the female- to male-biased primary/secondary sex ratios), depending on the selection systems at the gametophyte and sporophyte stages and on the sex ratio in the migrating seeds. Ovule abortion and the purging of maladaptive genes from the immigrating pollen at the gametophyte stage can alter the primary sex ratio and indirectly alter the secondary sex ratio. The presence of maladaptive sex-linked genes from the migrating pollen and seeds of males facilitates the outcome of the female-biased secondary sex ratios, while the presence of maladaptive sex-linked genes from the migrating seeds of females can lead to the male-biased secondary sex ratios. The detrimental effects of the Y-chromosome from the migrating pollen and seeds can enhance the formation of female-biased primary and secondary sex ratios. These theoretical predictions highlight an alternative approach to the existing sex-ratio theories for interpreting the formation of biased sex ratios in the populations that are subject to the impacts of maladaptive genes from immigrants.     

Sexual conflict in action: An antagonistic relationship between maternal and paternal sex allocation in the tammar wallaby,Notamacropus eugenii

Sex ratio biases are often inconsistent, both among and within species and populations. While some of these inconsistencies may be due to experimental design, much of the variation remains inexplicable. Recent research suggests that an exclusive focus on mothers may account for some of the inconsistency, with an increasing number of studies showing variation in sperm sex ratios and seminal fluids. Using fluorescent in‐situ hybridization, we show a significant population‐level Y‐chromosome bias in the spermatozoa of wild tammar wallabies, but with significant intraindividual variation between males. We also show a population‐level birth sex ratio trend in the same direction toward male offspring, but a weaning sex ratio that is significantly female‐biased, indicating that males are disproportionately lost during lactation. We hypothesize that sexual conflict between parents may cause mothers to adjust offspring sex ratios after birth, through abandonment of male pouch young and reactivation of diapaused embryos. Further research is required in a captive, controlled setting to understand what is driving and mechanistically controlling sperm sex ratio and offspring sex ratio biases and to understand the sexually antagonistic relationship between mothers and fathers over offspring sex. These results extend beyond sex allocation, as they question studies of population processes that assume equal input of sex chromosomes from fathers, and will also assist with future reproduction studies for management and conservation of marsupials.     

COMPARATIVE ANALYSES OF SEX‐RATIO VARIATION IN DIOECIOUS FLOWERING PLANTS

Dioecious plant species commonly exhibit deviations from the equilibrium expectation of 1:1 sex ratio, but the mechanisms governing this variation are poorly understood. Here, we use comparative analyses of 243 species, representing 123 genera and 61 families to investigate ecological and genetic correlates of variation in the operational (flowering) sex ratio. After controlling for phylogenetic nonindependence, we examined the influence of growth form, clonality, fleshy fruits, pollen and seed dispersal vector, and the possession of sex chromosomes on sex‐ratio variation. Male‐biased flowering sex ratios were twice as common as female‐biased ratios. Male bias was associated with long‐lived growth forms (e.g., trees) and biotic seed dispersal and fleshy fruits, whereas female bias was associated with clonality, especially for herbaceous species, and abiotic pollen dispersal. Female bias occurred in species with sex chromosomes and there was some evidence for a greater degree of bias in those with heteromorphic sex chromosomes. Although the role of interactions among these correlates require further study, our results indicate that sex‐based differences in costs of reproduction, pollen and seed dispersal mechanisms and sex chromosomes can each play important roles in affecting flowering sex ratios in dioecious plants.     

The influence of demography and local mating environment on sex ratios in a wind‐pollinated dioecious plant

Negative frequency‐dependent selection should result in equal sex ratios in large populations of dioecious flowering plants, but deviations from equality are commonly reported. A variety of ecological and genetic factors can explain biased sex ratios, although the mechanisms involved are not well understood. Most dioecious species are long‐lived and/or clonal complicating efforts to identify stages during the life cycle when biases develop. We investigated the demographic correlates of sex‐ratio variation in two chromosome races of Rumex hastatulus, an annual, wind‐pollinated colonizer of open habitats from the southern USA. We examined sex ratios in 46 populations and evaluated the hypothesis that the proximity of males in the local mating environment, through its influence on gametophytic selection, is the primary cause of female‐biased sex ratios. Female‐biased sex ratios characterized most populations of R.  hastatulus (mean sex ratio = 0.62), with significant female bias in 89% of populations. Large, high‐density populations had the highest proportion of females, whereas smaller, low‐density populations had sex ratios closer to equality. Progeny sex ratios were more female biased when males were in closer proximity to females, a result consistent with the gametophytic selection hypothesis. Our results suggest that interactions between demographic and genetic factors are probably the main cause of female‐biased sex ratios in R. hastatulus. The annual life cycle of this species may limit the scope for selection against males and may account for the weaker degree of bias in comparison with perennial Rumex species.     

Pollination intensity influences sex ratios in dioecious Rumex nivalis, a wind-pollinated plant

Determining the mechanisms governing sex-ratio variation in dioecious organisms represents a central problem in evolutionary biology. It has been proposed that in plants with sex chromosomes competition between pollen tubes of female- versus male-determining microgametophytes (certation) causes female-biased primary sex ratios. Experimental support for this hypothesis is limited and recent workers have cast doubt on whether pollen-tube competition can modify sex ratios in dioecious plants. Here we investigate the influence of variation in pollination intensity on sex ratios in Rumex nivalis, a wind-pollinated alpine herb with strongly female-biased sex ratios. In a garden experiment, we experimentally manipulated pollination intensity using three concentric rings of female recipient plants at different distances from a central group of male pollen donors. This design enabled us to test the hypothesis that increasing pollen load size, by intensifying gametophyte competition, promotes female-biased sex ratios in R. nivalis. We detected a significant decline in pollen load at successive distance classes with concomitant reductions in seed set. Sex ratios of progeny were always female biased, but plants at the closest distance to male donors exhibited significantly greater female bias than more distant plants. The amount of female bias was positively correlated with the seed set of inflorescences. Hand pollination of stigmas resulted in approximately 100-fold higher stigmatic pollen loads than wind-pollinated stigmas and produced exceptionally female-biased progenies (female frequency = 0.96). Our results are the first to demonstrate a functional relation between stigmatic pollen capture, seed set, and sex ratio and suggest that certation can contribute towards female-biased sex ratios in dioecious plants.     

The true sex ratio in European Pseudocalliergon trifarium (Bryophyta: Amblystegiaceae) revealed by a novel molecular approach

Dioecious plants, including many bryophytes, rarely exhibit discernible sexual dimorphism before sexual maturity. Because many species and populations of dioecious bryophytes do not express their sex, it remains mostly unresolved whether expressing individuals reflect the ratios of genetically male and female plants. The present study assesses the population sex ratio of the wetland moss Pseudocalliergon trifarium in central and northern Europe. For the first time in a bryophyte, we estimate the sex ratio in a population by assessing directly both expressing and non‐expressing plants. Expressed gender ratio was assessed from herbarium specimens. Single shoots from non‐expressing specimens were sexed using a recently developed molecular sex marker. On the basis of the female and male frequencies in these two data sets and the overall proportion of expressing specimens, we estimate the European population sex ratio to be 1.93 : 1 (female/male). Expressed, non‐expressed, and population sex ratios are not significantly different from each other, suggesting that gender differences in rates of sex expression cannot account for the female bias. Earlier studies of P. trifarium failed to reveal gender‐specific growth rates or pre‐zygotic reproductive costs. Gender differences at the spore to protonemal stage, in mortality, or niche preferences could potentially explain the uneven sex ratio. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 132–140.     

Sex ratio of some long-lived dioecious plants in a sand dune area

In dioecious plants the fraction of males among flowering plants in the field (the secondary sex ratio) is the result of the fraction of males in the seeds (the primary sex ratio) and the subsequent survival and age at first reproduction of the two genders. It has been assumed that survival and age at first reproduction are the main determinants of biased secondary sex ratio but, especially for long-lived perennials, few data are available. We address this issue for natural populations of four long-lived perennials in a dune area. In Asparagus officinale and Bryonia dioica, the secondary sex ratio was unbiased. In Salix repens the secondary sex ratio was female-biased (0.337). Hippophae rhamnoides populations were male-biased; the average sex ratio of flowering plants was 0.658, while the fraction of males varied between 0.39 near the sea to 0.84 at the inland side of the dunes. The primary sex ratio was estimated by germinating seeds and growing plants under favourable conditions with minimal mortality. In S. repens the primary sex ratio in seeds was variable among mother plants and was, on average, female-biased (0.289). This is close to the secondary sex ratio, suggesting that the female bias already originates in the seed stage. In Hippophae rhamnoides the primary sex ratio was slightly male-biased (0.564). We argue that in this species, apart from the primary sex ratio, higher mortality and a later age at first reproduction for females contribute to the strong male bias among flowering plants in the field.     

Fluorescent Banding Pattern and Species-Specific DNA Marker in Rumex thyrsiflorus Fingerh.

The object of this work was to analyze the karyotype structure of Rumex thyrsiflorus using differential fluorescent methods of chromosome staining (C-banding/DAPI and CMA(3)/DA/DAPI) and molecular sex markers. The results obtained were compared with data on the structure of the sex chromosomes and autosomes in R. acetosa, a model species in studies of sex determination and sex chromosome evolution in plants with an XX/XY(1)Y(2) system. A high level of similarity was found in the sex chromosome structure of the 2 species, along with small differences in their autosomal complexes. It suggests that differentiation of these 2 closely related species was not accompanied by major structural changes within their sex chromosomes. Molecular tests, however, revealed differences in the composition of male-specific repetitive sequence RAYSII, occurring in the Y(1) chromosome. Amplification of this sequence showed the presence of a single product (～700 bp) in R. acetosa and of 2 products (～600 bp and ～700 bp) in R. thyrsiflorus. The longer product (～700 bp) was also revealed in R. arifolius, another species closely related to R. acetosa. The shorter DNA fragment, characteristic of R. thyrsiflorus, differed from the common product by of a large indel with a length of 110 bp. This fragment may serve as a species-specific molecular marker useful in taxonomical and population studies as well as in further research on the sex chromosome differentiation in R. thyrsiflorus.     

Seed sex ratio in dioecious plants depends on relative dispersal of pollen and seeds: an example using a chessboard simulation model

Two principles are important for the optimal sex ratio strategy of plants. (1) Sib mating. Because seed dispersal is restricted, sib mating may occur which selects for a female bias in the seed sex ratio. (2) Local resource competition (LRC). If a plant produces pollen its nuclear genes are dispersed in two steps: first through the pollen and then, if the pollen is successful in fertilizing an ovule on another plant, through the seed. If the plant produces an ovule, its genes are dispersed only through the seed. By making pollen instead of ovules the offspring of a single plant is then spread out over a wider area. This reduces the chance that genetically related individuals are close together and need to compete for the same resource. The effect is the strongest if pollen is dispersed over a much wider area than seeds. Less LRC for paternally vs. maternally derived offspring selects for a male bias in sex allocation. We study the above‐mentioned opposite effects in dioecious plants (with separate male and female individuals), with maternal control over the sex ratio (fraction males) in the seeds. In a two‐dimensional spatial model female‐biased sex ratios are found when both pollen and seed dispersal are severely restricted. If pollen disperses over a wider area than seeds, which is probably the common situation in plants, the seed sex ratio becomes male‐biased. If pollen and seeds are both dispersed over a wide area, the sex ratio approaches 0.5. Our results do not change if the offspring of brother–sister matings are less fit because of inbreeding depression.     

Sex ratio and gender stability in the dioecious plants of Israel

The hypothesis that in dioecious plants genders are usually stable and that the sex ratio is 1:l was tested in the dioecious species of Israel. The flora of Israel comprises some 2500 wild species, among which 43 are dioecious, one gynodioecious and one androdioecious. Forty-one out of these 45 species were examined during 1995-97. These species were examined in 120 populations comprising 7019 individuals, at 81 different localities. The majority of the species (33/41) manifested the expected 1:1 sex ratio. Of the eight species deviating from this ratio, a significant bias was repeatedly scored in five of them. Three other species of the willow family manifested sometimes strongly biased sex ratios or even monomorphic (unisexual) stands, apparently as a result of extensive vegetative cloning. The stability of the male and female morphs was also examined in the 41 species. Only eight individuals (from seven species) out of the 7019 individuals surveyed (0.1%) were found to be bisexual. In addition, in 27 populations belonging to 17 species, 909 males were tagged and revisited in fruiting time, and only three of them (0.3%) were found to set fruits. In addition not a single case of gender reversal between seasons was found. The field survey suggests that in dioecious plants sex determination and expression are usually effectively maintained. The very slight deviations from the 1:1 sex ratio and from the distinct male and female morphs suggest that sexual dimorphism in dioecious plants is usually stabile.     

Excessive variation in Y chromosomal DNA in Rumex acetosa (Polygonaceae)

Rumex acetosa is one of the few angiosperms that possesses sex chromosomes. The same types of abundant repetitive sequences cover both heterochromatic Y chromosomes present in males. The aim of this study was to investigate genetic variation in paternally inherited Y chromosomal DNA and in maternally inherited cpDNA, and to find out whether the examined genomic regions are suited to a phylogeographic study in R. acetosa. DNA sequence polymorphisms present in the 850-bp heterochromatic segment on the Y chromosomes were compared to variation in the 409-bp long chloroplast section (trnL- trnF spacer) in R. acetosa originating from several European locations and from the Altai mountains in Russia. A great amount of genetic variation was detected within the Y chromosomal region while only four chloroplast genotypes were detected. Although the chloroplast haplotypes possessed some geographic pattern, no clear phylogeographic pattern was detected based on the variable Y chromosomes. The mean Y chromosomal nucleotide diversity among all samples equaled 6.6 %, and the mean proportion of polymorphic sites per individual equaled 8.2 % among SNP sites and 1.7 % among all sites investigated. The high number of substitutions detected in the Y chromosomal DNA shows that this heterochromatic sequence has a high mutation rate. The diversity pattern indicates that gene flow via pollen is extensive and it blurs any geographical pattern in the Y chromosomal variation. The high number of repeats and uncertainty concerning the extent of recombination between the two Y chromosomes impair the usability of the Y chromosomal segment for phylogeographic or population genetic studies.     

X chromosome drive in a widespread Palearctic woodland fly,Drosophila testacea

Selfish genes that bias their own transmission during meiosis can spread rapidly in populations, even if they contribute negatively to the fitness of their host. Driving X chromosomes provide a clear example of this type of selfish propagation. These chromosomes have important evolutionary and ecological consequences, and can be found in a broad range of taxa including plants, mammals and insects. Here, we report a new case of X chromosome drive (X drive) in a widespread woodland fly, Drosophila testacea. We show that males carrying the driving X (SR males) sire 80–100% female offspring and possess a diagnostic X chromosome haplotype that is perfectly associated with the sex ratio distortion phenotype. We find that the majority of sons produced by SR males are sterile and appear to lack a Y chromosome, suggesting that meiotic defects involving the Y chromosome may underlie X drive in this species. Abnormalities in sperm cysts of SR males reflect that some spermatids are failing to develop properly, confirming that drive is acting during gametogenesis. By screening wild‐caught flies using progeny sex ratios and a diagnostic marker, we demonstrate that the driving X is present in wild populations at a frequency of ~ 10% and that suppressors of drive are segregating in the same population. The testacea species group appears to be a hot spot for X drive, and D. testacea is a promising model to compare driving X chromosomes in closely related species, some of which may even be younger than the chromosomes themselves.