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.     

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.     

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.     

A genetic method to resolve gender complements investigations on sex ratios in Rumex acetosa

The perennial dioecious weed, Rumex acetosa, possesses sex chromosomes (XX in females, XY1Y2 in males). Yet, the operational sex ratios are female-biased. Until now, sex ratio studies in R. acetosa, as in most plants, have relied on data obtained at sexual maturity. To resolve gender among the seeds and nonflowering plants of R. acetosa, a genetic method involving a DNA marker located on both Y chromosomes has now been developed and applied. The results suggest that the sex ratios are about 1 : 1 in the whole seed pool, but that a significant female bias develops by the time of flowering. Since the age of sexually mature plants is two years or more, the time frame during which the female bias present at sexual maturity develops can be several years. It appears that male seeds germinate at a lower rate and males suffer from a greater mortality during the years-long lifespan of R. acetosa. However, there are no considerable sex-related differences in vegetative vigour.     

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.     

ZW,XY, and yet ZW: Sex chromosome evolution in snakes even more complicated

Snakes are historically important in the formulation of several central concepts on the evolution of sex chromosomes. For over 50 years, it was believed that all snakes shared the same ZZ/ZW sex chromosomes, which are homomorphic and poorly differentiated in “basal” snakes such as pythons and boas, while heteromorphic and well differentiated in “advanced” (caenophidian) snakes. Recent molecular studies revealed that differentiated sex chromosomes are indeed shared among all families of caenophidian snakes, but that boas and pythons evolved likely independently male heterogamety (XX/XY sex chromosomes). The historical report of heteromorphic ZZ/ZW sex chromosomes in a boid snake was previously regarded as ambiguous. In the current study, we document heteromorphic ZZ/ZW sex chromosomes in a boid snake. A comparative approach suggests that these heteromorphic sex chromosomes evolved very recently and that they are poorly differentiated at the sequence level. Interestingly, two snake lineages with confirmed male heterogamety possess homomorphic sex chromosomes, but heteromorphic sex chromosomes are present in both snake lineages with female heterogamety. We point out that this phenomenon is more common across squamates. The presence of female heterogamety in non‐caenophidian snakes indicates that the evolution of sex chromosomes in this lineage is much more complex than previously thought, making snakes an even better model system for the evolution of sex chromosomes.     

Centromere organization and UU/V sex chromosome behavior in a liverwort

In 1917, sex chromosomes in plants were discovered in a liverwort with hetermorphic U and V chromosomes. Such heteromorphy is unexpected because, unlike the XY chromosomes in diploid-dominant plants, in haploid-dominant plants the female U and the male V chromosomes experience largely symmetrical potential recombination environments. Here we use molecular cytogenetics and super-resolution microscopy to study Frullania dilatata, a liverwort with one male and two female sex chromosomes. We applied a pipeline to Illumina sequences to detect abundant types of repetitive DNA and developed FISH probes to microscopically distinguish the sex chromosomes. We also determined the phenotypic population sex ratio because biased ratios have been reported from other liverworts with heteromorphic sex chromosomes. Populations had male-biased sex ratios. The sex chromosomes are monocentric, and of 14 probes studied (eight satellites, five transposable elements and one plastid region), four resulted in unique signals that differentiated the sex chromosomes from the autosomes and from each other. One FISH probe selectively marked the centromeres of both U chromosomes, so we could prove that during meiosis each U chromosome associates with one of the opposite telomeres of the V chromosome, resulting in a head-to-head trivalent. The similarity of the two U chromosomes to each other in size and in their centromere FISH signal positions points to their origin via a non-disjunction event (aneuploidy), which would fit with the general picture of sex chromosomes rarely crossing-over and being prone to suffer from non-disjunction.     

Sex without sex chromosomes: genetic architecture of multiple loci independently segregating to determine sex ratios in the copepod Tigriopus californicus

Sex‐determining systems are remarkably diverse and may evolve rapidly. Polygenic sex‐determination systems are predicted to be transient and evolutionarily unstable, yet examples have been reported across a range of taxa. Here, we provide the first direct evidence of polygenic sex determination in Tigriopus californicus, a harpacticoid copepod with no heteromorphic sex chromosomes. Using genetically distinct inbred lines selected for male‐ and female‐biased clutches, we generated a genetic map with 39 SNPs across 12 chromosomes. Quantitative trait locus mapping of sex ratio phenotype (the proportion of male offspring produced by an F2 female) in four F2 families revealed six independently segregating quantitative trait loci on five separate chromosomes, explaining 19% of the variation in sex ratios. The sex ratio phenotype varied among loci across chromosomes in both direction and magnitude, with the strongest phenotypic effects on chromosome 10 moderated to some degree by loci on four other chromosomes. For a given locus, sex ratio phenotype varied in magnitude for individuals derived from different dam lines. These data, together with the environmental factors known to contribute to sex determination, characterize the underlying complexity and potential lability of sex determination, and confirm the polygenic architecture of sex determination in T. californicus.     

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.     

Dynamic sex chromosomes in Old World chameleons (Squamata: Chamaeleonidae)

Much of our current state of knowledge concerning sex chromosome evolution is based on a handful of ‘exceptional’ taxa with heteromorphic sex chromosomes. However, classifying the sex chromosome systems of additional species lacking easily identifiable, heteromorphic sex chromosomes is indispensable if we wish to fully understand the genesis, degeneration and turnover of vertebrate sex chromosomes. Squamate reptiles (lizards and snakes) are a potential model clade for studying sex chromosome evolution as they exhibit a suite of sex‐determining modes yet most species lack heteromorphic sex chromosomes. Only three (of 203) chameleon species have identified sex chromosome systems (all with female heterogamety, ZZ/ZW). This study uses a recently developed method to identify sex‐specific genetic markers from restriction site‐associated DNA sequence (RADseq) data, which enables the identification of sex chromosome systems in species lacking heteromorphic sex chromosomes. We used RADseq and subsequent PCR validation to identify an XX/XY sex chromosome system in the veiled chameleon (Chamaeleo calyptratus), revealing a novel transition in sex chromosome systems within the Chamaeleonidae. The sex‐specific genetic markers identified here will be essential in research focused on sex‐specific, comparative, functional and developmental evolutionary questions, further promoting C. calyptratus’ utility as an emerging model organism.     

REPLICATED ORIGIN OF FEMALE‐BIASED ADULT SEX RATIO IN INTRODUCED POPULATIONS OF THE TRINIDADIAN GUPPY (POECILIA RETICULATA)

There are many theoretical and empirical studies explaining variation in offspring sex ratio but relatively few that explain variation in adult sex ratio. Adult sex ratios are important because biased sex ratios can be a driver of sexual selection and will reduce effective population size, affecting population persistence and shapes how populations respond to natural selection. Previous work on guppies (Poecilia reticulata) gives mixed results, usually showing a female‐biased adult sex ratio. However, a detailed analysis showed that this bias varied dramatically throughout a year and with no consistent sex bias. We used a mark‐recapture approach to examine the origin and consistency of female‐biased sex ratio in four replicated introductions. We show that female‐biased sex ratio arises predictably and is a consequence of higher male mortality and longer female life spans with little effect of offspring sex ratio. Inconsistencies with previous studies are likely due to sampling methods and sampling design, which should be less of an issue with mark‐recapture techniques. Together with other long‐term mark‐recapture studies, our study suggests that bias in offspring sex ratio rarely contributes to adult sex ratio in vertebrates. Rather, sex differences in adult survival rates and longevity determine vertebrate adult sex ratio.     

环境决定爬行动物性别研究的进展

爬行动物的性别决定机制有两种,一种是由环境决定性别,另一种是异型性染色体决定性别。前者,在爬行动物中具有普遍性;未发现有异型性染色体的爬行动物,其性别由环境因子决定。剧烈的环境条件,可能压倒基因型性别决定。H-Y抗原,可检测未发现异型性染色体决定性别物种的遗传决定型。     

Microsatellite (GATA)n reveals sex-specific differences in Papaya

Papaya, an economically important fruit plant, is polygamous in nature. The sex of dioecious papaya plants can be deduced only after they attain reproductive maturity (6–8 months). Normally, 50% of the population in a field is composed of unfruitful male plants and almost 45% of these have to be uprooted at the flowering stage. This unnecessary cultivation of unwanted males leads to wastage of resources, which can be avoided if the sex of the plant is determined at juvenile stage. Morphological and cytological studies conducted so far have failed to differentiate between the various sex forms of papaya. Its dioecious nature, occasional sex-reversal of male flowers and the absence of a heteromorphic pair of sex chromosomes make papaya an interesting system to study sex determination at the molecular level. In the present study, highly informative microsatellite and minisatellite probes were employed to identify sex-specific differences in papaya. Among these, only the microsatellite probe (GATA)₄ demonstrated sex-specific differences in all the cultivars analysed. The diagnostic potential of this microsatellite marker was exploited to sex papaya plants at the seedling stage. This study also indicates that the genetic material of the X and Y chromosomes of papaya is diverging in a sex-specific manner and hence they are in the process of differentiation. Received: 26 February 1999 / Accepted: 25 March 1999     

Low X/Y divergence in four pairs of papaya sex-linked genes

Sex chromosomes in flowering plants, in contrast to those in animals, evolved relatively recently and only a few are heteromorphic. The homomorphic sex chromosomes of papaya show features of incipient sex chromosome evolution. We investigated the features of paired X- and Y-specific bacterial artificial chromosomes (BACs), and estimated the time of divergence in four pairs of sex-linked genes. We report the results of a comparative analysis of long contiguous genomic DNA sequences between the X and hermaphrodite Y (Y(h)) chromosomes. Numerous chromosomal rearrangements were detected in the male-specific region of the Y chromosome (MSY), including inversions, deletions, insertions, duplications and translocations, showing the dynamic evolutionary process on the MSY after recombination ceased. DNA sequence expansion was documented in the two regions of the MSY, demonstrating that the cytologically homomorphic sex chromosomes are heteromorphic at the molecular level. Analysis of sequence divergence between four X and Y(h) gene pairs resulted in a estimated age of divergence of between 0.5 and 2.2 million years, supporting a recent origin of the papaya sex chromosomes. Our findings indicate that sex chromosomes did not evolve at the family level in Caricaceae, and reinforce the theory that sex chromosomes evolve at the species level in some lineages.     

HALDANE'S RULE IS EXTENDED TO PLANTS WITH SEX CHROMOSOMES

Haldane's rule is an empirical phenomenon that has been observed in animals with sex chromosomes. The rule states that the heterogametic sex (XY or ZW) will be “absent, rare, or sterile” following hybridization between two species. Despite the near ubiquity of Haldane's rule in animal hybridizations, it has not been documented in organisms other than animals. Here, we show evidence for both rarity and sterility in hybrid male but not female offspring in crosses between three dioecious plant species from the genus Silene with heteromorphic (XY) sex chromosomes. Our results are consistent with Haldane's rule, extending its applicability to plants with sex chromosomes.     

Long-term sex reversal by oestradiol in amniotes with heteromorphic sex chromosomes

Oestradiol application during embryonic development reverses the sex of male embryos and results in normal female differentiation in reptiles lacking heteromorphic sex chromosomes, but fails to do so in birds and mammals with heteromorphic sex chromosomes. It is not clear whether the evolution of heteromorphic sex chromosomes in amniotes is accompanied by insensitivity to oestradiol, or if the association between oestradiol insensitivity and heteromorphic sex chromosomes can be attributable to phylogenetic constraints in these taxa. Turtles provide an ideal system to examine the potential relationship between oestradiol insensitivity and sex chromosome heteromorphy, since there are species with heteromorphic sex chromosomes that are closely related to species lacking heteromorphic sex chromosomes. We investigated this relationship by examining the long-term effects of oestradiol-17beta application on sex determination in Staurotypus triporcatus and Staurotypus salvinii, two turtle species with male heterogamety. After raising the turtles in the lab for 3 years, we found follicular and Müllerian duct morphology in oestradiol-treated turtles that was identical to that of untreated females. The lasting sex reversal suggests that the evolutionary transition between systems lacking heteromorphic sex chromosomes and those with heteromorphic sex chromosomes is not constrained by a fundamental mechanistic difference.     

Can the meiotic sex ratio explain the sex ratio bias in adult populations in the dioicous moss Drepanocladus lycopodioides?

Sex ratio variation is commonly observed in natural populations of many organisms with separate sexes and genetic sex determination, including bryophytes. Most bryophyte populations exhibit female-skewed expressed adult sex ratios, generally inferred from counts of sexually mature plants. For the rarely sexually reproducing perennial dioicous moss Drepanocladus lycopodioides, we showed that a female bias also exists in the genetic adult sex ratio, using a specifically designed molecular sex-associated marker. Here, we investigated whether the meiotic spore sex ratio contributes to the observed bias in genetic adult sex ratio in natural populations. Earlier attempts to study meiotic sex ratios have involved commonly cultivated ruderals that rapidly express sex in the laboratory. We established single-spore cultures from field-collected sporophytes from these populations and used the marker to assess the sex of individual sporelings. Spore germinability was (near) complete, and mortality among sporelings was virtually absent. The true meiotic sex ratio did not differ from equality, but strongly differed both from the observed genetic sex ratios in the natural adult populations, and from the European scale genetic sex ratio. We conclude that the biased population sex ratios in this species arise at life cycle stages after spore germination. Sexual dimorphism may selectively favour female proliferation during some phase of gametophyte development. Based on methodological progress, we successfully used a perennial study species with rare sexual reproduction, which significantly broadens the life history spectrum investigated in bryophyte sex ratio studies.     

ANOLIS SEX CHROMOSOMES ARE DERIVED FROM A SINGLE ANCESTRAL PAIR

To explain the frequency and distribution of heteromorphic sex chromosomes in the lizard genus Anolis, we compared the relative roles of sex chromosome conservation versus turnover of sex‐determining mechanisms. We used model‐based comparative methods to reconstruct karyotype evolution and the presence of heteromorphic sex chromosomes onto a newly generated Anolis phylogeny. We found that heteromorphic sex chromosomes evolved multiple times in the genus. Fluorescent in situ hybridization (FISH) of repetitive DNA showed variable rates of Y chromosome degeneration among Anolis species and identified previously undetected, homomorphic sex chromosomes in two species. We confirmed homology of sex chromosomes in the genus by performing FISH of an X‐linked bacterial artificial chromosome (BAC) and quantitative PCR of X‐linked genes in multiple Anolis species sampled across the phylogeny. Taken together, these results are consistent with long‐term conservation of sex chromosomes in the group. Our results pave the way to address additional questions related to Anolis sex chromosome evolution and describe a conceptual framework that can be used to evaluate the origins and evolution of heteromorphic sex chromosomes in other clades.     

Sex chromosomes evolved from independent ancestral linkage groups in winged insects

The evolution of a pair of chromosomes that differ in appearance between males and females (heteromorphic sex chromosomes) has occurred repeatedly across plants and animals. Recent work has shown that the male heterogametic (XY) and female heterogametic (ZW) sex chromosomes evolved independently from different pairs of homomorphic autosomes in the common ancestor of birds and mammals but also that X and Z chromosomes share many convergent molecular features. However, little is known about how often heteromorphic sex chromosomes have either evolved convergently from different autosomes or in parallel from the same pair of autosomes and how universal patterns of molecular evolution on sex chromosomes really are. Among winged insects with sequenced genomes, there are male heterogametic species in both the Diptera (e.g., Drosophila melanogaster) and the Coleoptera (Tribolium castaneum), female heterogametic species in the Lepidoptera (Bombyx mori), and haplodiploid species in the Hymenoptera (e.g., Nasonia vitripennis). By determining orthologous relationships among genes on the X and Z chromosomes of insects with sequenced genomes, we are able to show that these chromosomes are not homologous to one another but are homologous to autosomes in each of the other species. These results strongly imply that heteromorphic sex chromosomes have evolved independently from different pairs of ancestral chromosomes in each of the insect orders studied. We also find that the convergently evolved X chromosomes of Diptera and Coleoptera share genomic features with each other and with vertebrate X chromosomes, including excess gene movement from the X to the autosomes. However, other patterns of molecular evolution--such as increased codon bias, decreased gene density, and the paucity of male-biased genes on the X--differ among the insect X and Z chromosomes. Our results provide evidence for both differences and nearly universal similarities in patterns of evolution among independently derived sex chromosomes.     

Effects of soil properties and clonal growth on the apparent sex ratio of the flowering stems of the dioecious clonal shrub Aucuba japonica var. borealis growing in an evergreen coniferous secondary forest

In dioecious plants, the frequencies of flowering stems in each sex produced through clonal growth provide important information on the potential for reproductive success in the populations. However, apart from the light environment in their habitat, the factors affecting the flowering of each sex have not been well documented in shrub species that can maintain their populations in shady environments. In this research, we investigated seven soil variables and the flowering of stems of Aucuba japonica var. borealis in patches of this dioecious clonal shrub with different apparent stem sex ratios in an evergreen coniferous secondary forest with a shady forest floor. Of the 53 patches examined, 52 contained stems with flowers. Flowering stem ratios exhibited a positive relationship with available soil phosphate but a marginal negative relationship with exchangeable magnesium, whereas soil water content was associated with a female-biased flowering sex ratio. Stem density tended to be negatively related to flowering stem ratios in patches containing stems with female flowers but not males. While the results demonstrate that abundant amounts of certain nutrients and moisture in soils promote the production of flowers and/or a bias toward femaleness in patches, it is suggested that antagonistic effects of cations in the soil can inhibit the flowering of both sexes. In addition, the trade-off between sexual reproduction and clonal propagation in the females may amplify the variations in flowering in the population.