Polyploidy in angiosperms is an influential factor to trigger apomixis, the reproduction of asexual seeds. Apomixis is usually facultative, which means that both sexual and apomictic seeds can be formed by the same plant. Environmental abiotic stress, e.g. light stress, can change the frequency of apomixis. Previous work suggested effects of stress treatments on meiosis and megasporogenesis. We hypothesized that polyploidy would alter the stress response and hence reproductive phenotypes of different cytotypes. The main aims of this research were to explore with prolonged photoperiods, whether polyploidy alters proportions of sexual ovule and sexual seed formation under light stress conditions. We used three facultative apomictic, pseudogamous cytotypes of the Ranunculus auricomus complex (diploid, tetraploid, and hexaploid). Stress treatments were applied by extended light periods (16.5 h) and control (10 h) in climate growth chambers. Proportions of apomeiotic vs. meiotic development in the ovule were evaluated with clearing methods, and mode of seed formation was examined by single seed flow cytometric seed screening (ssFCSS). We further studied pollen stainability to understand effects of pollen quality on seed formation. Results revealed that under extended photoperiod, all cytotypes produced significantly more sexual ovules than in the control, with strongest effects on diploids. The stress treatment affected neither the frequency of seed set nor the proportion of sexual seeds nor pollen quality. Successful seed formation appears to be dependent on balanced maternal: paternal genome contributions. Diploid cytotypes had mostly sexual seed formation, while polyploid cytotypes formed predominantly apomictic seeds. Pollen quality was in hexaploids better than in diploids and tetraploids. These findings confirm our hypothesis that megasporogenesis is triggered by light stress treatments. Comparisons of cytotypes support the hypothesis that ovule development in polyploid plants is less sensitive to prolonged photoperiods and responds to a lesser extent with sexual ovule formation. Polyploids may better buffer environmental stress, which releases the potential for aposporous ovule development from somatic cells, and may facilitate the establishment of apomictic seed formation.

Alpine plants grow in harsh environments and are thought to face occasional frost during the sensitive reproductive phase. Apomixis (asexual reproduction via seed) can be advantageous when sexual reproduction is disturbed by cold stress. Apomictic polyploids tend to grow in colder climates than their sexual diploid relatives. Whether cold temperatures actually induce apomixis was unknown to date.

High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic species occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown.

Hybridization and polyploidization are powerful evolutionary factors that are associated with manifold developmental changes in plants such as irregular progression of meiosis and sporogenesis. The emergence of apomixis, which is asexual reproduction via seeds, is supposed to be connected to these factors and was often regarded as an escape from hybrid sterility. However, the functional trigger of apomixis is still unclear. Recently formed di- and polyploid Ranunculus hybrids, as well as their parental species were analysed for their modes of mega- and microsporogenesis by microscopy. Chromosomal configurations during male meiosis were screened for abnormalities. Meiotic and developmental abnormalities were documented qualitatively and collected quantitatively for statistical evaluations.

Hybridisation and polyploidy are major forces contributing to plant speciation. Homoploid (2x) and heteroploid (3x) hybrids, however, represent critical stages for evolution due to disturbed meiosis and reduced fertility. Apomixis--asexual reproduction via seeds--can overcome hybrid sterility, but requires several concerted alterations of developmental pathways to result in functional seed formation. Here, we analyse the reproductive behaviours of homo- and heteroploid synthetic hybrids from crosses between sexual diploid and tetraploid Ranunculus auricomus species to test the hypothesis that developmental asynchrony in hybrids triggers the shift to apomictic reproduction. Evaluation of male and female gametophyte development, viability and functionality of gametes shows developmental asynchrony, whereas seed set and germinability indicate reduced fitness in synthetic hybrids compared to sexual parents. We present the first experimental evidence for spontaneous apospory in most hybrids as an alternative pathway to meiosis, and the appearance of functional apomictic seeds in triploids. Bypassing meiosis permits these triploid genotypes to form viable seed and new polyploid progeny. Asynchronous development causes reduced sexual seed set and emergence of apospory in synthetic Ranunculus hybrids. Apomixis is functional in triploids and associated with drastic meiotic abnormalities. Selection acts to stabilise developmental patterns and to tolerate endosperm dosage balance shifts which facilitates successful seed set and establishment of apomictic lineages.

Hybridization and polyploidization play important roles in plant evolution but it is still not fully clarified how these evolutionary forces contribute to the establishment of apomicts. Apomixis, the asexual reproduction via seed formation, comprises several essential alterations in development compared to the sexual pathway. Furthermore, most natural apomicts were found to be polyploids and/or hybrids. The Ranunculus auricomus complex comprises diploid sexual and polyploid apomictic species and represents an excellent model system to gain knowledge on origin and evolution of apomixis in natural plant populations. In this study, the second generation of synthetically produced homoploid (2x) and heteroploid (3x) hybrids derived from sexual R. auricomus species was analyzed for aposporous initial cell formation by DIC microscopy. Complete manifestation of apomixis was determined by measuring single mature seeds by flow cytometric seed screen. Microscopic analysis of the female gametophyte formation indicated spontaneous occurrence of aposporous initial cells and several developmental irregularities. The frequency of apospory was found to depend on dosage effects since a significant increase in apospory was observed, when both F1 parents, rather than just one, were aposporous. Other than in the F1 generation, diploid Ranunculus F2 hybrids formed BIII seeds and fully apomictic seeds. The results indicate that hybridization rather than polyploidization seems to be the functional activator of apomictic reproduction in the synthetic Ranunculus hybrids. In turn, at least two hybrid generations are required to establish apomictic seed formation.

The reconstruction of reticulate evolutionary histories in plants is still a major methodological challenge. Sequences of the ITS nrDNA are a popular marker to analyze hybrid relationships, but variation of this multicopy spacer region is affected by concerted evolution, high intraindividual polymorphism, and shifts in mode of reproduction. The relevance of changes in secondary structure is still under dispute. We aim to shed light on the extent of polymorphism within and between sexual species and their putative natural as well as synthetic hybrid derivatives in the Ranunculus auricomus complex to test morphology-based hypotheses of hybrid origin and parentage of taxa. We employed direct sequencing of ITS nrDNA from 68 individuals representing three sexuals, their synthetic hybrids and one sympatric natural apomict, as well as cloning of ITS copies in four representative individuals, RNA secondary structure analysis, and landmark geometric morphometric analysis on leaves. Phylogenetic network analyses indicate additivity of parental ITS variants in both synthetic and natural hybrids. The triploid synthetic hybrids are genetically much closer to their maternal progenitors, probably due to ploidy dosage effects, although exhibiting a paternal-like leaf morphology. The natural hybrids are genetically and morphologically closer to the putative paternal progenitor species. Secondary structures of ITS1-5.8S-ITS2 were rather conserved in all taxa. The observed similarities in ITS polymorphisms suggest that the natural apomict R. variabilis is an ancient hybrid of the diploid sexual species R. notabilis and the sexual species R. cassubicifolius. The additivity pattern shared by R. variabilis and the synthetic hybrids supports an evolutionary and biogeographical scenario that R. variabilis originated from ancient hybridization. Concerted evolution of ITS copies in R. variabilis is incomplete, probably due to a shift to asexual reproduction. Under the condition of comprehensive inter- and intraspecific sampling, ITS polymorphisms are powerful for elucidating reticulate evolutionary histories.

Apomixis, the asexual reproduction via seeds, is associated to polyploidy and hybridization. To identify possible signatures of apomixis, and possible candidate genes underlying the shift from sex to apomixis, microarray-based gene expression patterns of live microdissected ovules at four different developmental stages were compared between apomictic and sexual individuals of the Ranunculus auricomus complex. Following predictions from previous work on mechanisms underlying apomixis penetrance and expressivity in the genus, gene expression patterns were classified into three categories based on their relative expression in apomicts compared to their sexual parental ancestors. We found evidence of misregulation and differential gene expression between apomicts and sexuals, with the highest number of differences detected during meiosis progression and emergence of aposporous initial (AI) cells, a key developmental stage in the ovule of apomicts where a decision between divergent reproductive pathways takes place. While most of the differentially expressed genes (DEGs) could not be annotated, gene expression was classified into transgressive, parent of origin and ploidy effects. Genes related to gametogenesis and meiosis demonstrated patterns reflective of transgressive and genome dosage effects, which support the hypothesis of a dominant factor controlling apomixis in Ranunculus and modulated by secondary modifiers. Three genes with probable functions in sporogenesis and gametogenesis development are identified and characterized for future studies.

Meiosis, the key step of sexual reproduction, persists in facultative apomictic plants functional to some extent. However, it still remains unclear how and why proportions of reproductive pathways vary under different environmental stress conditions. We hypothesized that oxidative stress mediates alterations of developmental pathways. In apomictic plants we expected that megasporogenesis, the stage directly after meiosis, would be more affected than later stages of seed development. To simulate moderate stress conditions we subjected clone-mates of facultative apomictic Ranunculus auricomus to 10 h photoperiods, reflecting natural conditions, and extended ones (16.5 h). Reproduction mode was screened directly after megasporogenesis (microscope) and at seed stage (flow cytometric seed screening). Targeted metabolite profiles were performed with HPLC-DAD to explore if and which metabolic reprogramming was caused by the extended photoperiod. Prolonged photoperiods resulted in increased frequencies of sexual vs. aposporous initials directly after meiosis, but did not affect frequencies of sexual vs. asexual seed formation. Changes in secondary metabolite profiles under extended photoperiods affected all classes of compounds, and c. 20% of these changes separated the two treatments. Unexpectedly, the renowned antioxidant phenylpropanoids and flavonoids added more to clone-mate variation than to treatment differentiation. Among others, chlorophyll degradation products, non-assigned phenolic compounds and more lipophilic metabolites also contributed to the dissimilarity of the metabolic profiles of plants that had been exposed to the two different photoperiods. The hypothesis of moderate light stress effects was supported by increased proportions of sexual megaspore development at the expense of aposporous initial formation. The lack of effects at the seed stage confirms the basic assumption that only meiosis and sporogenesis would be sensitive to light stress. The concomitant change of secondary metabolite profiles, as a systemic response at this early developmental stage, supports the notion that oxidative stress could have affected megasporogenesis by causing the observed metabolic reprogramming. Hypotheses of genotype-specific responses to prolonged photoperiods are rejected.