The exposure to environmental stress can trigger epigenetic variation, which may have several evolutionary consequences. Polyploidy seems to affect the DNA methylation profiles. Nevertheless, it abides unclear whether temperature stress can induce methylations changes in different cytotypes and to what extent a treatment shift is translated to an epigenetic response. A suitable model system for studying these questions is Ranunculus kuepferi, an alpine perennial herb. Diploid and autotetraploid individuals of R. kuepferi were exposed to cold (+7°C day/+2°C night; frost treatment −1°C cold shocks for 3 nights per week) and warm (+15°day/+10°C night) conditions in climate growth chambers for two consecutive flowering periods and shifted from one condition to the other after the first flowering period. Methylation-sensitive amplified fragment-length polymorphism markers were applied for both years, to track down possible alterations induced by the stress treatments. Patterns of methylation suggested that cytotypes differed significantly in their profiles, independent from year of treatment. Likewise, the treatment shift had an impact on both cytotypes, resulting in significantly less epiloci, regardless the shift's direction. The AMOVAs revealed higher variation within than among treatments in diploids. In tetraploids, internally-methylated loci had a higher variation among than within treatments, as a response to temperature's change in both directions, and support the hypothesis of temperature stress affecting the epigenetic variation. Results suggest that the temperature-sensitivity of DNA methylation patterns shows a highly dynamic phenotypic plasticity in R. kuepferi, as both cytotypes responded to temperature shifts. Furthermore, ploidy level, even without effects of hybridization, has an important effect on epigenetic background variation, which may be correlated with the DNA methylation dynamics during cold acclimation.

Plant life in alpine habitats is shaped by harsh abiotic conditions and cold climates. Phenotypic variation of morphological characters and reproduction can be influenced by temperature stress. Nevertheless, little is known about the performance of different cytotypes under cold stress and how epigenetic patterns could relate to phenotypic variation. Ranunculus kuepferi, a perennial alpine plant, served as a model system for testing the effect of cold stress on phenotypic plasticity, reproduction mode, and epigenetic variation. Diploid and autotetraploid individuals were placed in climate growth cabinets under warm and cold conditions. Morphological traits (height, leaves and flowers) and the proportion of well-developed seeds were measured as fitness indicators, while flow cytometric seed screening (FCSS) was utilized to determine the reproduction mode. Subsequently, comparisons with patterns of methylation-sensitive amplified fragment-length polymorphisms (AFLPs) were conducted. Diploids grew better under warm conditions, while tetraploids performed better in cold treatments. Epigenetic patterns were correlated with the expressed morphological traits. Cold stress reduced the reproduction fitness but did not induce apomixis in diploids. Overall, our study underlines the potential of phenotypic plasticity for acclimation under environmental conditions and confirms the different niche preferences of cytotypes in natural populations. Results help to understand the pattern of geographical parthenogenesis in the species.

Polyploidization and the shift to apomictic reproduction are connected to changes in DNA cytosine-methylation. Cytosine-methylation is further sensitive to environmental conditions. We, therefore, hypothesize that DNA methylation patterns would differentiate within species with geographical parthenogenesis, i.e., when diploid sexual and polyploid apomictic populations exhibit different spatial distributions. On natural populations of the alpine plant Ranunculus kuepferi, we tested differences in methylation patterns across two cytotypes (diploid, tetraploid) and three reproduction modes (sexual, mixed, apomictic), and their correlation to environmental data and geographical distributions. We used methylation-sensitive amplified fragment-length polymorphism (methylation-sensitive AFLPs) and scored three types of epiloci. Methylation patterns differed independently between cytotypes versus modes of reproduction and separated three distinct combined groups (2x sexual + mixed, 4x mixed, and 4x apomictic), with differentiation of 4x apomicts in all epiloci. We found no global spatial autocorrelation, but instead correlations to elevation and temperature gradients in 22 and 36 epiloci, respectively. Results suggest that methylation patterns in R. kuepferi were altered by cold conditions during postglacial recolonization of the Alps, and by the concomitant shift to facultative apomixis, and by polyploidization. Obligate apomictic tetraploids at the highest elevations established a distinct methylation profile. Methylation patterns reflect an ecological gradient rather than the geographical differentiation.

Alpine habitats are shaped by harsh abiotic conditions and cold climates. Temperature stress can affect phenotypic plasticity, reproduction, and epigenetic profiles, which may affect acclimation and adaptation. Distribution patterns suggest that polyploidy seems to be advantageous under cold conditions. Nevertheless, whether temperature stress can induce gene expression changes in different cytotypes, and how the response is initialized through gene set pathways and epigenetic control remain vague for non-model plants. The perennial alpine plant Ranunculus kuepferi was used to investigate the effect of cold stress on gene expression profiles. Diploid and autotetraploid individuals were exposed to cold and warm conditions in climate growth chambers and analyzed via transcriptome sequencing and qRT-PCR. Overall, cold stress changed gene expression profiles of both cytotypes and induced cold acclimation. Diploids changed more gene set pathways than tetraploids, and suppressed pathways involved in ion/cation homeostasis. Tetraploids mostly activated gene set pathways related to cell wall and plasma membrane. An epigenetic background for gene regulation in response to temperature conditions is indicated. Results suggest that perennial alpine plants can respond to temperature extremes via altered gene expression. Tetraploids are better acclimated to cold conditions, enabling them to colonize colder climatic areas in the Alps.

Chapter 2 -Effects of temperature treatments on cytosine-methylation profiles of diploid and autotetraploid plants of the alpine species Ranunculus kuepferi (Ranunculaceae) Abstract………………………………………………………………………………….30 2.1 Introduction…………………………………………………………………31 2.2 Materials and Methods…………………………………………………….34 2.3 Results……………………………………………………………………….37 2.4 Discussion…………………………………………………………………...45 2.5 References…………………………………………………………………...50 2.6 Supporting Information……………………………………………………58 3. Chapter 3 -Phenotypic responses, reproduction mode and epigenetic patterns under temperature treatments in the alpine plant species Ranunculus kuepferi (Ranunculaceae)