Darwin's 'mystery of mysteries': the role of sexual selection in plant speciation.

Sexual selection is considered one of the key processes that contribute to the emergence of new species. While the connection between sexual selection and speciation has been supported by comparative studies, the mechanisms that mediate this connection remain unresolved, especially in plants. Similarly, it is not clear how speciation processes within plant populations translate into large-scale speciation dynamics. Here, we review the mechanisms through which sexual selection, pollination, and mate choice unfold and interact, and how they may ultimately produce reproductive isolation in plants. We also overview reproductive strategies that might influence sexual selection in plants and illustrate how functional traits might connect speciation at the population level (population differentiation, evolution of reproductive barriers; i.e. microevolution) with evolution above the species level (macroevolution). We also identify outstanding questions in the field, and suitable data and tools for their resolution. Altogether, this effort motivates further research focused on plants, which might potentially broaden our general understanding of speciation by sexual selection, a major concept in evolutionary biology.

Epigenetic targeting of transposon relics: beating the dead horses of the genome?

Transposable elements (TEs) have been seen as selfish genetic elements that can propagate in a host genome. Their propagation success is however hindered by a combination of mechanisms such as mutations, selection, and their epigenetic silencing by the host genome. As a result, most copies of TEs in a given genome are dead relics: their sequence is too degenerated to allow any transposition. Nevertheless, these TE relics often, but not always, remain epigenetically silenced, and if not to prevent transposition anymore, one can wonder the reason for this phenomenon. The mere self-perpetuating loop inherent to epigenetic silencing could alone explain that even when inactive, TE copies remain silenced. Beyond this process, nevertheless, antagonistic selective forces are likely to act on TE relic silencing. Especially, without the benefit of preventing transposition, TE relic silencing may prove deleterious to the host fitness, suggesting that the maintenance of TE relic silencing is the result of a fine, and perhaps case-by-case, evolutionary trade-off between beneficial and deleterious effects. Ultimately, the release of TE relics silencing may provide a 'safe' ground for adaptive epimutations to arise. In this review, we provide an overview of these questions in both plants and animals.

Eunuchs or Females? Causes and Consequences of Gynodioecy on Morphology, Ploidy, and Ecology of Stellaria graminea L. (Caryophyllaceae).

Plant speciation results from intricate processes such as polyploidization, reproductive strategy shifts and adaptation. These evolutionary processes often co-occur, blurring their respective contributions and interactions in the speciation continuum. Here, relying on a large-scale study, we tested whether gynodioecy triggers the divergent evolution of flower morphology and genome between sexes, and contributes to the establishment of polyploids and colonization of ecological niches in Stellaria graminea. We found that gynodioecy in S. graminea leads to flower morphology divergence between females and hermaphrodites, likely due to sexual selection. Contrary to our expectations, gynodioecy occurs evenly in diploids and tetraploids, suggesting that this reproductive strategy was not involved in the establishment of polyploids. Both diploid and tetraploid females have a larger genome size than hermaphrodites, suggesting the presence of sex chromosomes. Finally, ecology differs between cytotypes and to a lesser extent between sexes, suggesting that the link between environment and presence of females is indirect and likely explained by other aspects of the species' life history. Our study shows that gynodioecy leads to the consistent evolution of sexual traits across a wide range of populations, cytotypes and environments within a given species, and this likely contributes to the phenotypic and genetic distinctiveness of the species from its sister clades.

Early evolution of reproductive isolation: A case of weak inbreeder/strong outbreeder leads to an intraspecific hybridization barrier in Arabidopsis lyrata.

Reproductive strategies play a major role in plant speciation. Notably, transitions from outcrossing to selfing may lead to relaxed sexual selection and parental conflict. Shifts in mating systems can affect maternal and paternal interests, and thus parent-specific influence on endosperm development, leading to reproductive isolation: if selfing and outcrossing species hybridize, the resulting seeds may not be viable due to endosperm failure. Nevertheless, it remains unclear how the switch in mating systems can impact reproductive isolation between recently diverged lineages, that is, during the process of speciation. We investigated this question using Arabidopsis lyrata, which recently transitioned to selfing (10,000 years ago) in certain North American populations, where European populations remain outcrossing. We performed reciprocal crosses between selfers and outcrossers, and measured seed viability and endosperm development. We show that parental genomes in the hybrid seed negatively interact, as predicted by parental conflict. This leads to extensive hybrid seed lethality associated with endosperm cellularization disturbance. Our results suggest that this is primarily driven by divergent evolution of the paternal genome between selfers and outcrossers. In addition, we observed other hybrid seed defects, suggesting that sex-specific interests are not the only processes contributing to postzygotic reproductive isolation.

On the origin of the widespread self-compatible allotetraploid Capsella bursa-pastoris (Brassicaceae).

Polyploidy, or whole-genome duplication, is a common speciation mechanism in plants. An important barrier to polyploid establishment is a lack of compatible mates. Because self-compatibility alleviates this problem, it has long been hypothesized that there should be an association between polyploidy and self-compatibility (SC), but empirical support for this prediction is mixed. Here, we investigate whether the molecular makeup of the Brassicaceae self-incompatibility (SI) system, and specifically dominance relationships among S-haplotypes mediated by small RNAs, could facilitate loss of SI in allopolyploid crucifers. We focus on the allotetraploid species Capsella bursa-pastoris, which formed ~300 kya by hybridization and whole-genome duplication involving progenitors from the lineages of Capsella orientalis and Capsella grandiflora. We conduct targeted long-read sequencing to assemble and analyze eight full-length S-locus haplotypes, representing both homeologous subgenomes of C. bursa-pastoris. We further analyze small RNA (sRNA) sequencing data from flower buds to identify candidate dominance modifiers. We find that C. orientalis-derived S-haplotypes of C. bursa-pastoris harbor truncated versions of the male SI specificity gene SCR and express a conserved sRNA-based candidate dominance modifier with a target in the C. grandiflora-derived S-haplotype. These results suggest that pollen-level dominance may have facilitated loss of SI in C. bursa-pastoris. Finally, we demonstrate that spontaneous somatic tetraploidization after a wide cross between C. orientalis and C. grandiflora can result in production of self-compatible tetraploid offspring. We discuss the implications of this finding on the mode of formation of this widespread weed.

Disentangling the components of triploid block and its fitness consequences in natural diploid-tetraploid contact zones of Arabidopsis arenosa.

Hybrid seed inviability (HSI) is an important mechanism of reproductive isolation and speciation. HSI varies in strength among populations of diploid species but it remains to be tested whether similar processes affect natural variation in HSI within ploidy-variable species (triploid block). Here we used extensive endosperm, seed and F1 -hybrid phenotyping to explore HSI variation within a diploid-autotetraploid species. By leveraging 12 population pairs from three ploidy contact zones, we tested for the effect of interploidy crossing direction (parent of origin), ploidy divergence and spatial arrangement in shaping reproductive barriers in a naturally relevant context. We detected strong parent-of-origin effects on endosperm development, F1 germination and survival, which was also reflected in the rates of triploid formation in the field. Endosperm cellularization failure was least severe and F1 -hybrid performance was slightly better in the primary contact zone, with genetically closest diploid and tetraploid lineages. We demonstrated overall strong parent-of-origin effects on HSI in a ploidy variable species, which translate to fitness effects and contribute to interploidy reproductive isolation in a natural context. Subtle intraspecific variation in these traits suggests the fitness consequences of HSI are predominantly a constitutive property of the species regardless of the evolutionary background of its populations.

Hybrid seed incompatibility in Capsella is connected to chromatin condensation defects in the endosperm.

Hybridization of closely related plant species is frequently connected to endosperm arrest and seed failure, for reasons that remain to be identified. In this study, we investigated the molecular events accompanying seed failure in hybrids of the closely related species pair Capsella rubella and C. grandiflora. Mapping of QTL for the underlying cause of hybrid incompatibility in Capsella identified three QTL that were close to pericentromeric regions. We investigated whether there are specific changes in heterochromatin associated with interspecific hybridizations and found a strong reduction of chromatin condensation in the endosperm, connected with a strong loss of CHG and CHH methylation and random loss of a single chromosome. Consistent with reduced DNA methylation in the hybrid endosperm, we found a disproportionate deregulation of genes located close to pericentromeric regions, suggesting that reduced DNA methylation allows access of transcription factors to targets located in heterochromatic regions. Since the identified QTL were also associated with pericentromeric regions, we propose that relaxation of heterochromatin in response to interspecies hybridization exposes and activates loci leading to hybrid seed failure.

Genetic basis and timing of a major mating system shift in Capsella.

A crucial step in the transition from outcrossing to self-fertilization is the loss of genetic self-incompatibility (SI). In the Brassicaceae, SI involves the interaction of female and male specificity components, encoded by the genes SRK and SCR at the self-incompatibility locus (S-locus). Theory predicts that S-linked mutations, and especially dominant mutations in SCR, are likely to contribute to loss of SI. However, few studies have investigated the contribution of dominant mutations to loss of SI in wild plant species. Here, we investigate the genetic basis of loss of SI in the self-fertilizing crucifer species Capsella orientalis, by combining genetic mapping, long-read sequencing of complete S-haplotypes, gene expression analyses and controlled crosses. We show that loss of SI in C. orientalis occurred < 2.6 Mya and maps as a dominant trait to the S-locus. We identify a fixed frameshift deletion in the male specificity gene SCR and confirm loss of male SI specificity. We further identify an S-linked small RNA that is predicted to cause dominance of self-compatibility. Our results agree with predictions on the contribution of dominant S-linked mutations to loss of SI, and thus provide new insights into the molecular basis of mating system transitions.

Winter-dormant shoot apical meristem in poplar trees shows environmental epigenetic memory.

Trees have a long lifespan and must continually adapt to environmental pressures, notably in the context of climate change. Epigenetic mechanisms are doubtless involved in phenotypic plasticity and in stress memory; however, little evidence of the role of epigenetic processes is available for trees growing in fields. Here, we analyzed the possible involvement of epigenetic mechanisms in the winter-dormant shoot apical meristem of Populus × euramericana clones in memory of the growing conditions faced during the vegetative period. We aimed to estimate the range of genetic and environmentally induced variations in global DNA methylation and to evaluate their correlation with changes in biomass production, identify differentially methylated regions (DMRs), and characterize common DMRs between experiments. We showed that the variations in global DNA methylation between conditions were genotype dependent and correlated with biomass production capacity. Microarray chip analysis allowed detection of DMRs 6 months after the stressful summer period. The 161 DMRs identified as common to three independent experiments most notably targeted abiotic stress and developmental response genes. Results are consistent with a winter-dormant shoot apical meristem epigenetic memory of stressful environmental conditions that occurred during the preceding summer period. This memory may facilitate tree acclimation.

Paternally expressed imprinted genes associate with hybridization barriers in Capsella.

Hybrid seed lethality is a widespread type of reproductive barrier among angiosperm taxa1,2 that contributes to species divergence by preventing gene flow between natural populations3,4. Besides its ecological importance, it is an important obstacle to plant breeding strategies 5 . Hybrid seed lethality is mostly due to a failure of the nourishing endosperm tissue, resulting in embryo arrest3,6,7. The cause of this failure is a parental dosage imbalance in the endosperm that can be a consequence of either differences in parental ploidy levels or differences in the 'effective ploidy', also known as the endosperm balance number (EBN)8,9. Hybrid seed defects exhibit a parent-of-origin pattern3,6,7, suggesting that differences in number or expression strength of parent-of-origin-specific imprinted genes underpin, as the primary or the secondary cause, the molecular basis of the EBN7,10. Here, we have tested this concept in the genus Capsella and show that the effective ploidy of three Capsella species correlates with the number and expression level of paternally expressed genes (PEGs). Importantly, the number of PEGs and the effective ploidy decrease with the selfing history of a species: the obligate outbreeder Capsella grandiflora had the highest effective ploidy, followed by the recent selfer Capsella rubella and the ancient selfer Capsella orientalis. PEGs were associated with the presence of transposable elements and their silencing mark, DNA methylation in CHH context (where H denotes any base except C). This suggests that transposable elements have driven the imprintome divergence between Capsella species. Together, we propose that variation in transposable element insertions, the resulting differences in PEG number and divergence in their expression level form one component of the effective ploidy variation between species of different breeding system histories, and, as a consequence, allow the establishment of endosperm-based hybridization barriers.

Changes in the epigenome and transcriptome of the poplar shoot apical meristem in response to water availability affect preferentially hormone pathways.

The adaptive capacity of long-lived organisms such as trees to the predicted climate changes, including severe and successive drought episodes, will depend on the presence of genetic diversity and phenotypic plasticity. Here, the involvement of epigenetic mechanisms in phenotypic plasticity toward soil water availability was examined in Populus×euramericana. This work aimed at characterizing (i) the transcriptome plasticity, (ii) the genome-wide plasticity of DNA methylation, and (iii) the function of genes affected by a drought-rewatering cycle in the shoot apical meristem. Using microarray chips, differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were identified for each water regime. The rewatering condition was associated with the highest variations of both gene expression and DNA methylation. Changes in methylation were observed particularly in the body of expressed genes and to a lesser extent in transposable elements. Together, DEGs and DMRs were significantly enriched in genes related to phytohormone metabolism or signaling pathways. Altogether, shoot apical meristem responses to changes in water availability involved coordinated variations in DNA methylation, as well as in gene expression, with a specific targeting of genes involved in hormone pathways, a factor that may enable phenotypic plasticity.

Endosperm-based hybridization barriers explain the pattern of gene flow between Arabidopsis lyrata and Arabidopsis arenosa in Central Europe.

Based on the biological species concept, two species are considered distinct if reproductive barriers prevent gene flow between them. In Central Europe, the diploid species Arabidopsis lyrata and Arabidopsis arenosa are genetically isolated, thus fitting this concept as "good species." Nonetheless, interspecific gene flow involving their tetraploid forms has been described. The reasons for this ploidy-dependent reproductive isolation remain unknown. Here, we show that hybridization between diploid A. lyrata and A. arenosa causes mainly inviable seed formation, revealing a strong postzygotic reproductive barrier separating these two species. Although viability of hybrid seeds was impaired in both directions of hybridization, the cause for seed arrest differed. Hybridization of A. lyrata seed parents with A. arenosa pollen donors resulted in failure of endosperm cellularization, whereas the endosperm of reciprocal hybrids cellularized precociously. Endosperm cellularization failure in both hybridization directions is likely causal for the embryo arrest. Importantly, natural tetraploid A. lyrata was able to form viable hybrid seeds with diploid and tetraploid A. arenosa, associated with the reestablishment of normal endosperm cellularization. Conversely, the defects of hybrid seeds between tetraploid A. arenosa and diploid A. lyrata were aggravated. According to these results, we hypothesize that a tetraploidization event in A. lyrata allowed the production of viable hybrid seeds with A. arenosa, enabling gene flow between the two species.

Endosperm-based postzygotic hybridization barriers: developmental mechanisms and evolutionary drivers.

The endosperm is a nourishing tissue that serves to support embryo growth. Failure of endosperm development will ultimately cause embryo arrest and seed lethality, a phenomenon that is frequently observed upon hybridization of related plant species or species that differ in ploidy. Endosperm-based interspecies or interploidy hybridization barriers depend on the direction of the hybridization, causing nonreciprocal seed defects. This reveals that the parental genomes are not equivalent, implicating parent-of-origin specific genes generating this type of hybridization barrier. Recent work revealed that endosperm-based hybridization barriers are rapidly evolving. In this review, we discuss the developmental mechanisms causing hybrid seed lethality in angiosperms as well as the evolutionary forces establishing endosperm-based postzygotic hybridization barriers.

Non-reciprocal Interspecies Hybridization Barriers in the Capsella Genus Are Established in the Endosperm.

The transition to selfing in Capsella rubella accompanies its recent divergence from the ancestral outcrossing C. grandiflora species about 100,000 years ago. Whether the change in mating system was accompanied by the evolution of additional reproductive barriers that enforced species divergence remained unknown. Here, we show that C. rubella and C. grandiflora are reproductively separated by an endosperm-based, non-reciprocal postzygotic hybridization barrier. While hybridizations of C. rubella maternal plants with C. grandiflora pollen donors resulted in complete seed abortion caused by endosperm cellularization failure, the reciprocal hybridization resulted in the formation of small seeds with precociously cellularized endosperm. Strikingly, the transcriptomic response of both hybridizations mimicked respectively the response of paternal and maternal excess hybridizations in Arabidopsis thaliana, suggesting unbalanced genome strength causes hybridization failure in both species. These results provide strong support for the theory that crosses between plants of different mating systems will be unbalanced, with the outcrosser behaving like a plant of increased ploidy, evoking a response that resembles an interploidy-type seed failure. Seed incompatilibity of C. rubella pollinated by C. grandiflora followed the Bateson-Dobzhansky-Muller model, involving negative genetic interaction of multiple paternal C. grandiflora loci with at least one maternal C. rubella locus. Given that both species only recently diverged, our data suggest that a fast evolving mechanism underlies the post-zygotic hybridization barrier(s) separating both species.

Evolution and function of epigenetic processes in the endosperm.

The endosperm is an ephemeral tissue surrounding the embryo that is essential for its development. Aside from the embryo nourishing function, the endosperm serves as a battlefield for epigenetic processes that have been hypothesized to reinforce transposable element silencing in the embryo. Specifically, global DNA demethylation in the central cell may serve to produce small RNAs that migrate to egg cell and embryo to induce de novo DNA methylation. The Polycomb Repressive Complex 2 (PRC2) is particularly targeted to DNA hypomethylated regions, possibly alleviating the negative effects associated with loss of DNA methylation in the endosperm. The functional requirement of the PRC2 in the endosperm can be bypassed by increasing the maternal genome dosage in the endosperm, suggesting a main functional role of the endosperm PRC2 in reducing sexual conflict. We therefore propose that the functional requirement of an endosperm PRC2 was coupled to the evolution of a sexual endosperm and mechanisms enforcing transposon silencing in the embryo. The evolutionary consequences of this scenario for genome expansion will be discussed.

Epigenetic mechanisms of postzygotic reproductive isolation in plants.

Hybrid incompatibility is generally viewed as a consequence of negative epistatic interactions between alleles that do not cause negative fitness effects in their parents. Substantial evidence in support of the model has accumulated over recent years. Nevertheless, there is evidence that hybrid incompatibility can have an epigenetic basis and results from deregulated small RNAs (sRNAs), causing changes in DNA methylation and transposable element (TE) activation. Epigenetically regulated loci can impact on the expression of nearby located genes. Alteration of dosage-sensitive gene expression builds hybridization barriers in the endosperm; however, it may also offer an explanation for transgressive effects in plant hybrids. In this review we highlight recent advances that illuminate the role of epigenetic pathways in establishing hybrid incompatibility in plants.