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Climate change is rapidly altering the distribution of suitable habitats for many species as well as their pathogenic microbes. For many pathogens, including vector‐borne diseases of humans and agricultural pathogens, climate change is expected to increase transmission and lead to pathogen range expansions. However, if pathogens have a lower heat tolerance than their host, increased warming could generate so‐called thermal refugia for hosts. Predicting the outcomes of warming on disease transmission requires detailed knowledge of the thermal tolerances of both the host and the pathogen. Such thermal tolerance studies are generally lacking for fungal pathogens of wild plant populations, despite the fact that plants form the base of all terrestrial communities. Here, we quantified three aspects of the thermal tolerance (growth, infection, and propagule production) of the naturally occurring fungal pathogen Microbotryum lychnidis‐dioicae, which causes a sterilizing anther‐smut disease on the herbaceous plant Silene latifolia. We also quantified two aspects of host thermal tolerance: seedling survival and flowering rate. We found that temperatures >30°C reduced the ability of anther‐smut spores to germinate, grow, and conjugate in vitro. In addition, we found that high temperatures (30°C) during or shortly after the time of inoculation strongly reduced the likelihood of infection in seedlings. Finally, we found that high summer temperatures in the field temporarily cured infected plants, likely reducing transmission. Notably, high temperatures did not reduce survival or flowering of the host plants. Taken together, our results show that the fungus is considerably more sensitive to high temperatures than its host plant. A warming climate could therefore result in reduced disease spread or even local pathogen extirpation, leading to thermal refugia for the host. [ABSTRACT FROM AUTHOR]

The oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), known as oxi-mCs, garners significant interest in plants as potential epigenetic marks. While research in mammals has established a role in cell reprogramming, carcinogenesis, and gene regulation, their functions in plants remain unclear. In rice, 5hmC has been associated with transposable elements (TEs) and heterochromatin. This study utilizes Silene latifolia , a dioecious plant with heteromorphic sex chromosomes and a genome with a large proportion of TEs, which provides a favourable environment for the study of oxi-mCs in individual sexes. Notably, we detected surprisingly high levels of oxi-mCs in S. latifolia comparable with mammals. Nuclei showed enrichment in heterochromatic regions, except for 5hmC whose signal was homogeneously distributed. Intriguingly, the same X chromosome in females displayed overall enrichment of 5hmC and 5fC compared with its counterpart. This fact is shared with 5mC, resembling dosage compensation. Co-localization showed higher correlation between 5mC and 5fC than with 5hmC, indicating no potential relationship between 5hmC and 5fC. Additionally, the promoter of several sex-linked genes and sex-biased TEs clustered in a clear sex-dependent way. Together, these findings unveil a hypothetical role for oxi-mCs in S. latifolia sex chromosome development, warranting further exploration. [ABSTRACT FROM AUTHOR]

The anther‐smut host–pathogen system has provided extensive insights into the evolutionary ecology of disease resistance, transmission modes, host shifts, pathogen specialization, and disease evolution in metapopulations. It also has led to unexpected insights into sex ratio distorters, sex chromosome evolution, and transposable elements in fungi. In addition, anther‐smut disease played a major role in Linnaeus' germ theory and the correspondence on parasitic castration between Darwin and Becker, one of the first female botanists. Here, we explicitly highlight some of the realities in the process of science, using an unusual autobiographical approach to describe how we came to collaborate on this system in the 1980s. Using perspectives from our different career stages, we present a surprising narrative that could not be deduced from merely reading the published papers. While our work was grounded in previous ecological and evolutionary theory, it was the product as much of empirical failures and intellectual roadblocks, as the result of a progressive scientific method. Our experiences illustrate not only the "human dimension of science" but more importantly show that linear sequences of hypothesis testing do not necessarily lead to new study systems and new ideas. We suggest there is a need to re‐evaluate the scientific method in ecology and evolution, especially where the challenge is to engage in a productive dialog between natural history and theory. [ABSTRACT FROM AUTHOR]

Introduction: Plants undergo various natural changes that dramatically modify their genomes. One is polyploidization and the second is hybridization. Both are regarded as key factors in plant evolution and result in phenotypic differences in different plant organs. In Silene, we can find both examples in nature, and this genus has a seed shape diversity that has long been recognized as a valuable source of information for infrageneric classification. Methods: Morphometric analysis is a statistical study of shape and size and their covariations with other variables. Traditionally, seed shape description was limited to an approximate comparison with geometric figures (rounded, globular, reniform, or heart-shaped). Seed shape quantification has been based on direct measurements, such as area, perimeter, length, and width, narrowing statistical analysis. We used seed images and processed them to obtain silhouettes. We performed geometric morphometric analyses, such as similarity to geometric models and elliptic Fourier analysis, to study the hybrid offspring of S. latifolia and S. dioica. Results: We generated synthetic tetraploids of Silene latifolia and performed controlled crosses between diploid S. latifolia and Silene dioica to analyze seed morphology. After imaging capture and post-processing, statistical analysis revealed differences in seed size, but not in shape, between S. latifolia diploids and tetraploids, as well as some differences in shape among the parentals and hybrids. A detailed inspection using fluorescence microscopy allowed for the identification of shape differences in the cells of the seed coat. In the case of hybrids, differences were found in circularity and solidity. Overal seed shape is maternally regulated for both species, whereas cell shape cannot be associated with any of the sexes. Discussion: Our results provide additional tools useful for the combination of morphology with genetics, ecology or taxonomy. Seed shape is a robust indicator that can be used as a complementary tool for the genetic and phylogenetic analyses of Silene hybrid populations. [ABSTRACT FROM AUTHOR]

Abstract The anther‐smut host–pathogen system has provided extensive insights into the evolutionary ecology of disease resistance, transmission modes, host shifts, pathogen specialization, and disease evolution in metapopulations. It also has led to unexpected insights into sex ratio distorters, sex chromosome evolution, and transposable elements in fungi. In addition, anther‐smut disease played a major role in Linnaeus' germ theory and the correspondence on parasitic castration between Darwin and Becker, one of the first female botanists. Here, we explicitly highlight some of the realities in the process of science, using an unusual autobiographical approach to describe how we came to collaborate on this system in the 1980s. Using perspectives from our different career stages, we present a surprising narrative that could not be deduced from merely reading the published papers. While our work was grounded in previous ecological and evolutionary theory, it was the product as much of empirical failures and intellectual roadblocks, as the result of a progressive scientific method. Our experiences illustrate not only the “human dimension of science” but more importantly show that linear sequences of hypothesis testing do not necessarily lead to new study systems and new ideas. We suggest there is a need to re‐evaluate the scientific method in ecology and evolution, especially where the challenge is to engage in a productive dialog between natural history and theory.

IntroductionPlants undergo various natural changes that dramatically modify their genomes. One is polyploidization and the second is hybridization. Both are regarded as key factors in plant evolution and result in phenotypic differences in different plant organs. In Silene, we can find both examples in nature, and this genus has a seed shape diversity that has long been recognized as a valuable source of information for infrageneric classification.MethodsMorphometric analysis is a statistical study of shape and size and their covariations with other variables. Traditionally, seed shape description was limited to an approximate comparison with geometric figures (rounded, globular, reniform, or heart-shaped). Seed shape quantification has been based on direct measurements, such as area, perimeter, length, and width, narrowing statistical analysis. We used seed images and processed them to obtain silhouettes. We performed geometric morphometric analyses, such as similarity to geometric models and elliptic Fourier analysis, to study the hybrid offspring of S. latifolia and S. dioica.ResultsWe generated synthetic tetraploids of Silene latifolia and performed controlled crosses between diploid S. latifolia and Silene dioica to analyze seed morphology. After imaging capture and post-processing, statistical analysis revealed differences in seed size, but not in shape, between S. latifolia diploids and tetraploids, as well as some differences in shape among the parentals and hybrids. A detailed inspection using fluorescence microscopy allowed for the identification of shape differences in the cells of the seed coat. In the case of hybrids, differences were found in circularity and solidity. Overal seed shape is maternally regulated for both species, whereas cell shape cannot be associated with any of the sexes.DiscussionOur results provide additional tools useful for the combination of morphology with genetics, ecology or taxonomy. Seed shape is a robust indicator that can be used as a complementary tool for the genetic and phylogenetic analyses of Silene hybrid populations.

The evolution of a non-recombining sex-specific region is a key step in sex chromosome evolution. Suppression of recombination between the (proto-) X- and Y-chromosomes in male meiosis creates a non-recombining Y-linked region (NRY), while the X-chromosome continues to recombine in females. Lack of recombination in the NRY defines its main properties—genetic degeneration and accumulation of repetitive DNA, making X and Y chromosomes very different from each other. How and why recombination suppression on sex chromosomes evolves remains controversial. A strong difference in recombination rates between the sexes (heterochiasmy) can facilitate or even cause recombination suppression. In the extreme case—complete lack of recombination in the heterogametic sex (achiasmy)—the entire sex-specific chromosome is automatically non-recombining. In this study, I analyse sex-specific recombination rates in a dioecious plant Silene latifolia (Caryophyllaceae), which evolved separate sexes and sex chromosomes ~11 million years ago. I reconstruct high-density RNAseq-based genetic maps including over five thousand genic markers for the two sexes separately. The comparison of the male and female maps reveals only modest heterochiasmy across the genome, with the exception of the sex chromosomes, where recombination is suppressed in males. This indicates that heterochiasmy likely played only a minor, if any, role in NRY evolution in S. latifolia, as recombination suppression is specific to NRY rather than to the entire genome in males. Other mechanisms such as structural rearrangements and/or epigenetic modifications were likely involved, and comparative genome analysis and genetic mapping in multiple Silene species will help to shed light on the mechanism(s) of recombination suppression that led to the evolution of sex chromosomes. [ABSTRACT FROM AUTHOR]

Evolution of a non‐recombining sex‐specific region on the Y (or W) chromosome (NRY) is a key step in sex chromosome evolution, but how recombination suppression evolves is not well understood. Studies in many different organisms indicated that NRY evolution often involves several expansion steps. Why such NRY expansions occur remains unclear, although it is though that they are likely driven by sexually antagonistic selection. This paper describes a recent NRY expansion due to shift of the pseudoautosomal boundary on the sex chromosomes of a dioecious plant Silene latifolia. The shift resulted in inclusion of at least 16 pseudoautosomal genes into the NRY. This region is pseudoautosomal in closely related Silene dioica and Silene diclinis, indicating that the NRY expansion occurred in S. latifolia after it speciated from the other species ~120 thousand years ago. As S. latifolia and S. dioica actively hybridise across Europe, interspecific gene flow could blur the PAR boundary in these species. The pseudoautosomal genes have significantly elevated genetic diversity (π ~ 3% at synonymous sites), which is consistent with balancing selection maintaining diversity in this region. The recent shift of the PAR boundary in S. latifolia offers an opportunity to study the process of on‐going NRY expansion. [ABSTRACT FROM AUTHOR]

Purpose: Thallium (Tl) is one of the most toxic elements known and its contamination is an emerging environmental issue associated with base metal (zinc-lead) mining wastes. This study investigated the nature of Tl tolerance and accumulation in Silene latifolia, which has so far only been reported from field-collected samples. Methods: Silene latifolia was grown in hydroponics at different Tl concentrations (0, 2.5, 5, 30 and 60 μM Tl). Elemental analysis with Inductively coupled plasma atomic emission spectroscopy (ICP-AES) and laboratory-based micro-X-ray fluorescence spectroscopy (μ-XRF) were used to determine Tl accumulation and distribution in hydrated organs and tissues. Results: This study revealed unusually high Tl concentrations in the shoots of S. latifolia, reaching up to 35,700 μg Tl g−1 in young leaves. The species proved to have exceptionally high levels of Tl tolerance and had a positive growth response when exposed to Tl dose rates of up to 5 μM. Laboratory-based μXRF analysis revealed that Tl is localized mainly at the base of the midrib and in the veins of leaves. This distribution differs greatly from that in other known Tl hyperaccumulators. Conclusions: Our findings show that S. latifolia is among the strongest known Tl hyperaccumulators in the world. The species has ostensibly evolved mechanisms to survive excessive concentrations of Tl accumulated in its leaves, whilst maintaining lower Tl concentrations in the roots. This trait is of fundamental importance for developing future phytoextraction technologies using this species to remediate Tl-contaminated mine wastes. [ABSTRACT FROM AUTHOR]

Dioecious plants possess diverse sex determination systems and unique mechanisms of reproductive organ development; however, little is known about how sex-linked genes shape the expression of regulatory cascades that lead to developmental differences between sexes. In Silene latifolia , a dioecious plant with stable dimorphism in floral traits, early experiments suggested that female-regulator genes act on the factors that determine the boundaries of the flower whorls. To identify these regulators, we sequenced the transcriptome of male flowers with fully developed gynoecia, induced by rapid demethylation in the parental generation. Eight candidates were found to have a positive role in gynoecium promotion, floral organ size, and whorl boundary, and affect the expression of class B MADS-box flower genes. To complement our transcriptome analysis, we closely examined the floral organs in their native state using field emission environmental scanning electron microscopy, and examined the differences between females and androhermaphrodites in their placenta and ovule organization. Our results reveal the regulatory pathways potentially involved in sex-specific flower development in the classical model of dioecy, S. latifolia. These pathways include previously hypothesized and unknown female-regulator genes that act on the factors that determine the flower boundaries, and a negative regulator of anther development, SUPERMAN - like (SlSUP). [ABSTRACT FROM AUTHOR]

Abstract Genetic covariance between two traits generates correlated responses to selection, and may either enhance or constrain adaptation. Silene latifolia exhibits potentially constraining genetic covariance between specific leaf area (SLA) and flower number in males. Flower number is likely to increase via fecundity selection but the correlated increase in SLA increases mortality, and SLA is under selection to decrease in dry habitats. We selected on trait combinations in two selection lines for four generations to test whether genetic covariance could be reduced without significantly altering trait means. In one selection line, the genetic covariance changed sign and eigenstructure changed significantly, while in the other selection line eigenstructure remained similar to the control line. Changes in genetic variance–covariance structure are therefore possible without the introduction of new alleles, and the responses we observed suggest that founder effects and changes in frequency of alleles of major effect may be acting to produce the changes.

We study the effects of inbreeding in a dioecious plant on its interaction with pollinating insects and test whether the magnitude of such effects is shaped by plant individual sex and the evolutionary histories of plant populations. We recorded spatial, scent, colour, and rewarding flower traits as well as pollinator visitation rates in experimentally inbred and outbred, male and female Silene latifolia plants from European and North American populations differing in their evolutionary histories. We found that inbreeding specifically impairs spatial flower traits and floral scent. Our results support that sex-specific selection and gene expression may have partially magnified these inbreeding costs for females, and that divergent evolutionary histories altered the genetic architecture underlying inbreeding effects across population origins. Moreover, the results indicate that inbreeding effects on floral scent may have a huge potential to disrupt interactions among plants and nocturnal moth pollinators, which are mediated by elaborate chemical communication.

Abstract Background S. latifolia is a model organism for the study of sex chromosome evolution in plants. Its sex chromosomes include large regions in which recombination became gradually suppressed. The regions tend to expand over time resulting in the formation of evolutionary strata. Non-recombination and later accumulation of repetitive sequences is a putative cause of the size increase in the Y chromosome. Gene decay and accumulation of repetitive DNA are identified as key evolutionary events. Transposons in the X and Y chromosomes are distributed differently and there is a regulation of transposon insertion by DNA methylation of the target sequences, this points to an important role of DNA methylation during sex chromosome evolution in Silene latifolia. The aim of this study was to elucidate whether the reduced expression of the Y allele in S. latifolia is caused by genetic degeneration or if the cause is methylation triggered by transposons and repetitive sequences. Results Gene expression analysis in S. latifolia males has shown expression bias in both X and Y alleles. To determine whether these differences are caused by genetic degeneration or methylation spread by transposons and repetitive sequences, we selected several sex-linked genes with varying degrees of degeneration and from different evolutionary strata. Immunoprecipitation of methylated DNA (MeDIP) from promoter, exon and intron regions was used and validated through bisulfite sequencing. We found DNA methylation in males, and only in the promoter of genes of stratum I (older). The Y alleles in genes of stratum I were methylation enriched compared to X alleles. There was also abundant and high percentage methylation in the CHH context in most sequences, indicating de novo methylation through the RdDM pathway. Conclusions We speculate that TE accumulation and not gene decay is the cause of DNA methylation in the S. latifolia Y sex chromosome with influence on the process of heterochromatinization.

Abstract Background The rise and fall of the Y chromosome was demonstrated in animals but plants often possess the large evolutionarily young Y chromosome that is thought has expanded recently. Break-even points dividing expansion and shrinkage phase of plant Y chromosome evolution are still to be determined. To assess the size dynamics of the Y chromosome, we studied intraspecific genome size variation and genome composition of male and female individuals in a dioecious plant Silene latifolia, a well-established model for sex-chromosomes evolution. Results Our genome size data are the first to demonstrate that regardless of intraspecific genome size variation, Y chromosome has retained its size in S. latifolia. Bioinformatics study of genome composition showed that constancy of Y chromosome size was caused by Y chromosome DNA loss and the female-specific proliferation of recently active dominant retrotransposons. We show that several families of retrotransposons have contributed to genome size variation but not to Y chromosome size change. Conclusions Our results suggest that the large Y chromosome of S. latifolia has slowed down or stopped its expansion. Female-specific proliferation of retrotransposons, enlarging the genome with exception of the Y chromosome, was probably caused by silencing of highly active retrotransposons in males and represents an adaptive mechanism to suppress degenerative processes in the haploid stage. Sex specific silencing of transposons might be widespread in plants but hidden in traditional hermaphroditic model plants.

The long‐term contamination that followed the nuclear disaster at Chernobyl provides a case study for the effects of chronic ionizing radiation on living organisms and on their ability to tolerate or evolve resistance to such radiation. Previously, we studied the fertility and viability of early developmental stages of a castrating plant pathogen, the anther‐smut fungus Microbotryum lychnidis‐dioicae, isolated from field sites varying over 700‐fold in degree of radioactive contamination. Neither the budding rate of haploid spores following meiosis nor the karyotype structure varied with increasing radiation levels at sampling sites. Here, we assessed the ability of the same M. lychnidis‐dioicae strains to perform their whole life cycle, up to the production of symptoms in the plants, that is, the development of anthers full of fungal spores; we also assessed their viability under experimental radiation. Fungal strains from more contaminated sites had no lower spore numbers in anthers or viability, but infected host plants less well, indicating lower overall fitness due to radioactivity exposure. These findings improve our understanding of the previous field data, in which the anther‐smut disease prevalence on Silene latifolia plants caused by M. lychnidis‐dioicae was lower at more contaminated sites. Although the fungus showed relatively high resistance to experimental radiation, we found no evidence that increased resistance to radiation has evolved in populations from contaminated sites. Fungal strains from more contaminated sites even tolerated or repaired damage from a brief acute exposure to γ radiation less well than those from non‐ or less contaminated sites. Our results more generally concur with previous studies in showing that the fitness of living organisms is affected by radiation after nuclear disasters, but that they do not rapidly evolve higher tolerance. [ABSTRACT FROM AUTHOR]

Genetic covariance between two traits generates correlated responses to selection, and may either enhance or constrain adaptation. Silene latifolia exhibits potentially constraining genetic covariance between specific leaf area (SLA) and flower number in males. Flower number is likely to increase via fecundity selection but the correlated increase in SLA increases mortality, and SLA is under selection to decrease in dry habitats. We selected on trait combinations in two selection lines for four generations to test whether genetic covariance could be reduced without significantly altering trait means. In one selection line, the genetic covariance changed sign and eigenstructure changed significantly, while in the other selection line eigenstructure remained similar to the control line. Changes in genetic variance–covariance structure are therefore possible without the introduction of new alleles, and the responses we observed suggest that founder effects and changes in frequency of alleles of major effect may be acting to produce the changes. [ABSTRACT FROM AUTHOR]

Premise: Water availability is an important abiotic factor, resulting in differences between plant species growing in xeric and mesic habitats. Species with populations occurring in both habitat types allow examination of whether water availability has acted as a selective force at the intraspecific level. Investigating responses to water availability with a dioecious species allows determination of whether males and females, which often have different physiologies and life histories, respond differently. Methods: An experiment varying water availability was performed under an outdoor rain‐out shelter using plants from two mesic and two xeric populations of the dioecious plant Silene latifolia. Early growth rate, flowering propensity, flower size, and specific leaf area were measured. At the end of the season, the plants were harvested, aboveground and root biomass were measured, and the total number of flowers and fruit produced were counted. Results: Compared to the two mesic populations, plants from the two xeric populations grew more slowly, were less likely to flower, took longer to flower, had thicker leaves, invested less in aboveground biomass and more in root biomass, produced fewer flowers and fruit, but were more likely to live. Many traits exhibited significant habitat type × treatment interactions. Compared to the xeric populations, males—but not females—from mesic populations had less root biomass and greatly reduced their flower production in response to low water availability. Conclusions: Mesic and xeric populations responded in ways congruent with water availability being a selective force for among‐population divergence, especially for males. [ABSTRACT FROM AUTHOR]

Inbreeding and enemy infestation are common in plants and can synergistically reduce their performance. This inbreeding ×environment (I × E) interaction may be of particular importance for the success of plant invasions if introduced populations experience a release from attack by natural enemies relative to their native conspecifics. Here, we investigate whether inbreeding affects plant infestation damage, whether inbreeding depression in growth and reproduction is mitigated by enemy release, and whether this effect is more pronounced in invasive than native plant populations. We used the invader Silene latifolia and its natural enemies as a study system. We performed two generations of experimental out‐ and inbreeding within eight native (European) and eight invasive (North American) populations under controlled conditions using field‐collected seeds. Subsequently, we exposed the offspring to an enemy exclusion and inclusion treatment in a common garden in the species’ native range to assess the interactive effects of population origin (range), breeding treatment, and enemy treatment on infestation damage, growth, and reproduction. Inbreeding increased flower and leaf infestation damage in plants from both ranges, but had opposing effects on fruit damage in native versus invasive plants. Inbreeding significantly reduced plant fitness; whereby, inbreeding depression in fruit number was higher in enemy inclusions than exclusions. This effect was equally pronounced in populations from both distribution ranges. Moreover, the magnitude of inbreeding depression in fruit number was lower in invasive than native populations. These results support that inbreeding has the potential to reduce plant defenses in S. latifolia, which magnifies inbreeding depression in the presence of enemies. However, future studies are necessary to further explore whether enemy release in the invaded habitat has actually decreased inbreeding depression and thus facilitated the persistence of inbred founder populations and invasion success. [ABSTRACT FROM AUTHOR]

Inbreeding and herbivory can interactively reduce the performance of flowering plants. Here, we investigated whether the magnitude of plant inbreeding depression increases under herbivory as a result of diminished leaf metabolic responses to herbivory in inbreds, which entails increased herbivore growth and feeding damage. We additionally explored whether genetic differentiation among native and invasive plant populations impacts the magnitude and direction of these inbreeding × herbivory interactions.Inbred and outbred plants from eight European (native) and eight North American (invasive) populations of Silene latifolia were exposed to two consecutive control or herbivory treatments (infestation with larvae of Mamestra brassicae). After the first treatment, leaf metabolic responses to herbivory were assessed with untargeted metabolic fingerprinting using UHPLC‐ESI+‐QTOF‐MS/MS. After the second herbivory treatment, herbivore growth and plant feeding damage were assessed. Moreover, plant performance traits were recorded 4 weeks after the second herbivory treatment.Metabolic fingerprinting revealed that changes in the pool sizes of various metabolic features in response to herbivory were less intense in inbred than outbred plants from native and invasive populations. While herbivore growth and feeding damage were largely unaffected by plant breeding status in invasive populations, herbivores consumed more biomass from, but showed reduced growth on, inbred than outbred native plants. The magnitude of inbreeding depression in plant performance traits was not affected by herbivory.Synthesis. Our findings suggest that inbreeding compromises herbivory‐induced metabolic defences in S. latifolia, and simultaneously reduces the nutritional quality of plants. The magnitude and direction of these inbreeding effects were shaped by genetic differentiation among native and invasive plants. These range‐specific responses provide novel insights into the role of inbreeding x herbivory interactions in invasion success. Inbreeding and herbivory can interactively reduce plant performance. Using native and invasive Silene latifolia populations, we found support that inbreeding compromises herbivory‐induced metabolic plant defences as well as plant nutritional values, which shapes herbivore growth and feeding damage. The magnitude and direction of these inbreeding effects were modified by genetic differentiation among native and invasive plants. These range‐specific responses highlight the need to investigate multiple populations with different evolutionary histories to gain a deeper understanding of inbreeding × herbivory interactions and provide novel insights into the role of these interactions in invasion success. [ABSTRACT FROM AUTHOR]

Highlights • In vitro regeneration of a model dioecious species Silene latifolia was significantly improved. • Stable genetic transformation was achieved by Agrobacterium rhizogenes. • Targeted mutagenesis with TALEN and CRISPR/Cas9 nucleases on plant sex chromosomes is demonstrated in protoplasts. Abstract Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia , based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

This research investigated whether anther smut disease caused by Microbotryum violaceum agg. on Silene baccifera in Europe is caused by a host‐specific lineage, or if it represents a host shift of the pathogen from a related species. Characterization of ITS sequences of anther smut from S. baccifera confirmed their strong similarity to the pathogen endemic on Silene latifolia. Cross‐inoculation studies showed that S. baccifera was susceptible to anther smut isolates from S. latifolia, S. dioica and S. vulgaris; conversely isolates from S. baccifera could cause disease on S. latifolia. In an experimental field study, spore transmission from diseased S. latifolia to healthy S. baccifera was rare relative to intraspecific transmission within S. latifolia. The distribution of anther smut in natural populations based on herbarium specimens indicated that disease occurrence on S. baccifera was very sporadic. These findings strongly suggest that anther smut disease on S. baccifera in Europe is usually a temporary host shift from the self‐sustaining populations of Microbotryum on other species. [ABSTRACT FROM AUTHOR]

Dioecious plants in the Caryophyllaceae family are susceptible to infection by members of the anthericolous smut fungi. In our studies of the Silene latifolia/Microbotryum lychnidis-dioicae pathosystem, we were interested in characterizing the plant-pathogen interaction at the molecular level before and during teliosporogenesis. This takes place during floral bud development, and we hoped to capture the interaction by Illumina Next-Gen RNA-Sequencing. Using previous literature that documented the stages of the floral buds for S. latifolia, we examined the floral buds from plants grown and infected under growth chamber conditions, using the disserting microscope to determine the stage of floral buds based on the morphology. We compiled the information and determined the size of floral buds that correspond to the desired stages of development for tissue collection, for the purpose of RNA-sequencing. This offers a practical approach for researchers who require a large number of floral buds/tissue categorized by stages of development, ascertaining whether infected/uninfected buds are at comparable stages of development and whether this also holds true for male vs. female buds. We also document our experience in infecting the plants and some of the unusual morphologies we observed after infection.

This paper examines the rise and fall of Carl Linnaeus's ideas on living contagion, focusing on his work with plant smut diseases. Early in his career, Linnaeus named a plant altered by anther-smut disease as a separate species, but then, probably realizing it was a diseased specimen, demoted it to a variety. He later drew direct parallels between minute insects attacking plants and infectious diseases in humans, but did not yet draw an analogy to smut diseases. After Otto von Münchhausen had sent Linnaeus the first instalment of his book Der Hausvater (1764), Linnaeus realized smuts were contagious. He carried out his own investigations that appeared to confirm Münchhausen's conclusion that smut spores germinated to produce living and mobile animalcules. This cemented Linnaeus's view that animalcules caused contagion in human diseases, a view which he expressed forcefully, urging further studies. However, his results were questioned and discounted by others, especially John Ellis. An analysis of correspondence between Linnaeus and other microscopists shows that it is likely Linnaeus did actually see "animalcules" emerging from cereal grains. He was unaware that smut-like symptoms in wheat could also be caused by seed-gall nematodes in the genus Anguina. Linnaeus himself came to doubt the connection between fungi and contagion, and did not pursue these studies further. The presumption that Linnaeus was fanciful in his observations of animalcules may partly explain why his views had only a tangential impact on the germ-theory of disease, and why his insights remain unappreciated to this day. [ABSTRACT FROM AUTHOR]

Background: S. latifolia is a model organism for the study of sex chromosome evolution in plants. Its sex chromosomes include large regions in which recombination became gradually suppressed. The regions tend to expand over time resulting in the formation of evolutionary strata. Non-recombination and later accumulation of repetitive sequences is a putative cause of the size increase in the Y chromosome. Gene decay and accumulation of repetitive DNA are identified as key evolutionary events. Transposons in the X and Y chromosomes are distributed differently and there is a regulation of transposon insertion by DNA methylation of the target sequences, this points to an important role of DNA methylation during sex chromosome evolution in Silene latifolia. The aim of this study was to elucidate whether the reduced expression of the Y allele in S. latifolia is caused by genetic degeneration or if the cause is methylation triggered by transposons and repetitive sequences. Results: Gene expression analysis in S. latifolia males has shown expression bias in both X and Y alleles. To determine whether these differences are caused by genetic degeneration or methylation spread by transposons and repetitive sequences, we selected several sex-linked genes with varying degrees of degeneration and from different evolutionary strata. Immunoprecipitation of methylated DNA (MeDIP) from promoter, exon and intron regions was used and validated through bisulfite sequencing. We found DNA methylation in males, and only in the promoter of genes of stratum I (older). The Y alleles in genes of stratum I were methylation enriched compared to X alleles. There was also abundant and high percentage methylation in the CHH context in most sequences, indicating de novo methylation through the RdDM pathway. Conclusions: We speculate that TE accumulation and not gene decay is the cause of DNA methylation in the S. latifolia Y sex chromosome with influence on the process of heterochromatinization. [ABSTRACT FROM AUTHOR]

White campion (Silene latifolia , Caryophyllaceae) was the first vascular plant where sex chromosomes were discovered. This species is a classic model for studies on plant sex chromosomes due to presence of large, clearly distinguishable X and Y chromosomes that originated de novo about 11 million years ago (mya), but lack of genomic resources for this relatively large genome (∼2.8 Gb) remains a significant hurdle. Here we report S. latifolia female genome assembly integrated with sex-specific genetic maps of this species, focusing on sex chromosomes and their evolution. The analysis reveals a highly heterogeneous recombination landscape with strong reduction in recombination rate in the central parts of all chromosomes. Recombination on the X chromosome in female meiosis primarily occurs at the very ends, and over 85% of the X chromosome length is located in a massive (∼330 Mb) gene-poor, rarely recombining pericentromeric region (Xpr). The results indicate that the non-recombining region on the Y chromosome (NRY) initially evolved in a relatively small (∼15 Mb), actively recombining region at the end of the q-arm, possibly as a result of inversion on the nascent X chromosome. The NRY expanded about 6 mya via linkage between the Xpr and the sex-determining region, which may have been caused by expanding pericentromeric recombination suppression on the X chromosome. These findings shed light on the origin of sex chromosomes in S. latifolia and yield genomic resources to assist ongoing and future investigations into sex chromosome evolution. [Display omitted] • Genome sequencing reveals sex chromosome evolution in white campion • The sex-determining region initially evolved in a small, actively recombining region • The non-recombining region on the Y (NRY) likely evolved due to inversion on the X • Extensive pericentromeric recombination suppression facilitated NRY expansion Yue et al. describe the evolution of sex chromosomes in white campion, based on the chromosome-level genome assembly integrated with a genetic map. The sex-determining region (SDR) has likely evolved in an ∼15 Mb region, which stopped recombining due to X-linked inversion. SDR expansion was facilitated by extensive pericentromeric recombination suppression. [ABSTRACT FROM AUTHOR]

DNA sequence diversity in genes in the partially sex-linked pseudoautosomal region ( PAR) of the sex chromosomes of the plant Silene latifolia is higher than expected from within-species diversity of other genes. This could be the footprint of sexually antagonistic ( SA) alleles that are maintained by balancing selection in a PAR gene (or genes) and affect polymorphism in linked genome regions. SA selection is predicted to occur during sex chromosome evolution, but it is important to test whether the unexpectedly high sequence polymorphism could be explained without it, purely by the combined effects of partial linkage with the sex-determining region and the population's demographic history, including possible introgression from Silene dioica. To test this, we applied approximate Bayesian computation-based model choice to autosomal sequence diversity data, to find the most plausible scenario for the recent history of S. latifolia and then to estimate the posterior density of the most relevant parameters. We then used these densities to simulate variation to be expected at PAR genes. We conclude that an excess of variants at high frequencies at PAR genes should arise in S. latifolia populations only for genes with strong associations with fully sex-linked genes, which requires closer linkage with the fully sex-linked region than that estimated for the PAR genes where apparent deviations from neutrality were observed. These results support the need to invoke selection to explain the S. latifolia PAR gene diversity, and encourage further work to test the possibility of balancing selection due to sexual antagonism. [ABSTRACT FROM AUTHOR]

• In order to investigate the role of differential adaptation for the evolution of reproductive barriers, we conducted a multi-site transplant experiment with the dioecious sister species Silene dioica and S. latifolia and their hybrids. • Crosses within species as well as reciprocal first-generation (F1) and second-generation (F2) interspecific hybrids were transplanted into six sites, three within each species' habitat. Survival and flowering were recorded over 4 yr. • At all transplant sites, the local species outperformed the foreign species, reciprocal F1 hybrids performed intermediately and F2 hybrids underperformed in comparison to F1 hybrids (hybrid breakdown). Females generally had slightly higher cumulative fitness than males in both within- and between-species crosses and we thus found little evidence for Haldane's rule acting on field performance. The strength of selection against F1 and F2 hybrids as well as hybrid breakdown increased with increasing strength of habitat adaptation (i.e. the relative fitness difference between the local and the foreign species) across sites. • Our results suggest that differential habitat adaptation led to ecologically dependent postzygotic reproductive barriers and drives divergence and speciation in this Silene system. [ABSTRACT FROM AUTHOR]

Mechanisms of suppression of pistil primordia in male flowers and of stamen primordia in female flowers differ in diclinous plants. In this study, we investigated how cell death and cell cycle arrest are related to flower organ formation in Silene latifolia. Using in situ hybridization and a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, we detected both cell cycle arrest and cell death in suppressed stamens of female flowers and suppressed pistils of male flowers in S. latifolia. In female flowers infected with Microbotryum lychnidis-dioicae, developmental suppression of stamens is released, and cell cycle arrest and cell death do not occur. Smut spores are formed in S. latifolia anthers infected with M. lychnidis-dioicae, followed by cell death in the endothelium, middle layer, tapetal cells and pollen mother cells. Cell death is difficult to detect using a fluorescein isothiocyanate-labeled TUNEL assay due to strong autofluorescence in the anther. We therefore combined a TUNEL assay in an infrared region with transmission electron microscopy to detect cell death in anthers. We show that following infection by M. lychnidis-dioicae, a TUNEL signal was not detected in the endothelium, middle layer or pollen mother cells, and cell death with outflow of cell contents, including the nucleoplast, was observed in tapetal cells. [ABSTRACT FROM AUTHOR]

In dioecious plants, males and females often show distinct morphological, physiological and life history traits as result of their different demands for reproduction. Such sexual dimorphism is likely to be accentuated under stressful conditions, such as that imposed by exposure to heavy metals. However little is known about the response of dioecious plants to stress by heavy metals. Here we use the dioecious herb Silene latifolia to investigate the growth and reproduction of males and females growing in soil polluted with either Cu or Cd. We also examined whether the sexes differed in the patterns of metal accumulation in their tissues. Patterns of biomass allocation to reproduction, roots, leaves and shoots (stem and leaves) were compared in male and female S. latifolia plants that were harvested after growing for 14 weeks in different soil conditions (non-polluted soil, Cu-polluted soil and Cd-polluted soil). In addition, patterns of metal accumulation between the sexes were also compared post-harvest by analysing the metal content in their tissues. Overall, metals decreased plants’ total dry mass allocated to leaves and to roots- particularly in males. Females accumulated more Cu in their tissues. However, this did not seem to correspond with females performing worse (in terms of growth and reproduction) than males when growing in soil polluted with this metal. Despite males and females having similar levels of Cd in their tissues, males seemed to have lower tolerance to this metal, as indicated by a lower total and leaf dry mass than females, and also by a lower number of flowers when growing in Cd-polluted soil. We also found contrasting differences in female seed production due to heavy metals, with number of seeds (but not mass) decreasing with Cu and increasing with Cd. Our results indicate the presence of sexual dimorphism in response to heavy metals, with the sexes differing in both patterns of accumulation and tolerance. [ABSTRACT FROM AUTHOR]

How loss of genetic exchanges (recombination) evolves between sex chromosomes is a long-standing question. Suppressed recombination may evolve when a sexually antagonistic (SA) polymorphism occurs in a partially sex-linked 'pseudoautosomal' region (or 'PAR'), maintaining allele frequency differences between the two sexes, and creating selection for closer linkage with the fully sex-linked region of the Y chromosome in XY systems, or the W in ZW sex chromosome systems. Most evidence consistent with the SA polymorphism hypothesis is currently indirect, and more studies of the genetics and population genetics of PAR genes are clearly needed. The sex chromosomes of the plant Silene latifolia are suitable for such studies, as they evolved recently and the loss of recombination could still be ongoing. Here, we used RAD sequencing to genetically map sequences in this plant, which has a large genome (c. 3 gigabases) and no available whole-genome sequence. We mapped 83 genes on the sex chromosomes, and comparative mapping in the related species S. vulgaris supports previous evidence for additions to an ancestral PAR and identified at least 12 PAR genes. We describe evidence that recombination rates have been reduced in meiosis of both sexes, and differences in recombination between S. latifolia families suggest ongoing recombination suppression. Large allele frequency differences between the sexes were found at several loci closely linked to the PAR boundary, and genes in different regions of the PAR showed striking sequence diversity patterns that help illuminate the evolution of the PAR. [ABSTRACT FROM AUTHOR]

Around six percent of flowering species are dioecious, with separate female and male individuals. Sex determination is mostly based on genetics, but morphologically distinct sex chromosomes have only evolved in a few species. Of these, heteromorphic sex chromosomes have been most clearly described in the two model species – Silene latifolia and Rumex acetosa . In both species, the sex chromosomes are the largest chromosomes in the genome. They are hence easily distinguished, can be physically separated and analyzed. This review discusses some recent experimental data on selected model dioecious species, with a focus on S. latifolia . Phylogenetic analyses show that dioecy in plants originated independently and repeatedly even within individual genera. A cogent question is whether there is genetic degeneration of the non-recombining part of the plant Y chromosome, as in mammals, and, if so, whether reduced levels of gene expression in the heterogametic sex are equalized by dosage compensation. Current data provide no clear conclusion. We speculate that although some transcriptome analyses indicate the first signs of degeneration, especially in S. latifolia , the evolutionary processes forming plant sex chromosomes in plants may, to some extent, differ from those in animals. [ABSTRACT FROM AUTHOR]

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%. [ABSTRACT FROM AUTHOR]

Fungal invasions are increasingly recognized as a significant component of global changes, threatening ecosystem health and damaging food production. Invasive fungi also provide excellent models to evaluate the generality of results based on other eukaryotes. We first consider here the reasons why fungal invasions have long been overlooked: they tend to be inconspicuous, and inappropriate methods have been used for species recognition. We then review the information available on the patterns and mechanisms of fungal invasions. We examine the biological features underlying invasion success of certain fungal species. We review population structure analyses, revealing native source populations and strengths of bottlenecks. We highlight the documented ecological and evolutionary changes in invaded regions, including adaptation to temperature, increased virulence, hybridization, shifts to clonality and association with novel hosts. We discuss how the huge census size of most fungi allows adaptation even in bottlenecked, clonal invaders. We also present new analyses of the invasion of the anther-smut pathogen on white campion in North America, as a case study illustrating how an accurate knowledge of species limits and phylogeography of fungal populations can be used to decipher the origin of invasions. This case study shows that successful invasions can occur even when life history traits are particularly unfavourable to long-distance dispersal and even with a strong bottleneck. We conclude that fungal invasions are valuable models to contribute to our view of biological invasions, in particular by providing insights into the traits as well as ecological and evolutionary processes allowing successful introductions. [ABSTRACT FROM AUTHOR]

Dimorphic mating-type chromosomes in fungi are excellent models for understanding the genomic consequences of recombination suppression. Their suppressed recombination and reduced effective population size are expected to limit the efficacy of natural selection, leading to genomic degeneration. Our aim was to identify the sequences of the matingtype chromosomes (a1 and a2) of the anther-smut fungi and to investigate degeneration in their nonrecombining regions. We used the haploid a1 Microbotryum lychnidis-dioicae reference genome sequence. The a1 and a2 mating-type chromosomes were both isolated electrophoretically and sequenced. Integration with restriction-digest optical maps identified regions of recombination and nonrecombination in the mating-type chromosomes. Genome sequence data were also obtained for 12 other Microbotryum species. We found strong evidence of degeneration across the genus in the nonrecombining regions of the mating-type chromosomes, with significantly higher rates of nonsynonymous substitution (dN/dS) than in nonmating-type chromosomes or in recombining regions of the mating-type chromosomes. The nonrecombining regions of the mating-type chromosomes also showed high transposable element content, weak gene expression, and gene losses. The levels of degeneration did not differ between the a1 and a2 mating-type chromosomes, consistent with the lack of homogametic/heterogametic asymmetry between them, and contrasting with X/Y or Z/W sex chromosomes. [ABSTRACT FROM AUTHOR]