Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants.

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

The relation of meiotic behaviour to hybridity, polyploidy and apomixis in the Ranunculus auricomus complex (Ranunculaceae).

BACKGROUND: 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. RESULTS: Allopolyploids showed significantly higher frequencies of erroneous microsporogenesis than homoploid hybrid plants. Among diploids, F2 hybrids had significantly more disturbed meiosis than F1 hybrids and parental plants. Chromosomal aberrations included laggard chromosomes, chromatin bridges and disoriented spindle activities. Failure of megasporogenesis appeared to be much more frequent in than of microsporogenesis is correlated to apomixis onset. CONCLUSIONS: Results suggest diverging selective pressures on female and male sporogenesis, with only minor effects of hybridity on microsporogenesis, but fatal effects on the course of megasporogenesis. Hence, pollen development continues without major alterations, while selection will favour apomixis as alternative to the female meiotic pathway. Relation of investigated errors of megasporogenesis with the observed occurrence of apospory in Ranunculus hybrids identifies disturbed female meiosis as potential elicitor of apomixis in order to rescue these plants from hybrid sterility. Male meiotic disturbance appears to be stronger in neopolyploids than in homoploid hybrids, while disturbances of megasporogenesis were not ploidy-dependent.

Winter Nights during Summer Time: Stress Physiological Response to Ice and the Facilitation of Freezing Cytorrhysis by Elastic Cell Wall Components in the Leaves of a Nival Species.

Ranunculus glacialis grows and reproduces successfully, although the snow-free time period is short (2-3 months) and night frosts are frequent. At a nival site (3185 m a.s.l.), we disentangled the interplay between the atmospheric temperature, leaf temperatures, and leaf freezing frequency to assess the actual strain. For a comprehensive understanding, the freezing behavior from the whole plant to the leaf and cellular level and its physiological after-effects as well as cell wall chemistry were studied. The atmospheric temperatures did not mirror the leaf temperatures, which could be 9.3 °C lower. Leaf freezing occurred even when the air temperature was above 0 °C. Ice nucleation at on average -2.6 °C started usually independently in each leaf, as the shoot is deep-seated in unfrozen soil. All the mesophyll cells were subjected to freezing cytorrhysis. Huge ice masses formed in the intercellular spaces of the spongy parenchyma. After thawing, photosynthesis was unaffected regardless of whether ice had formed. The cell walls were pectin-rich and triglycerides occurred, particularly in the spongy parenchyma. At high elevations, atmospheric temperatures fail to predict plant freezing. Shoot burial prevents ice spreading, specific tissue architecture enables ice management, and the flexibility of cell walls allows recurrent freezing cytorrhysis. The peculiar patterning of triglycerides close to ice rewards further investigation.

Chasing the Apomictic Factors in the Ranunculus auricomus Complex: Exploring Gene Expression Patterns in Microdissected Sexual and Apomictic Ovules.

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.

Epigenetic Patterns and Geographical Parthenogenesis in the Alpine Plant Species Ranunculus kuepferi (Ranunculaceae).

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.

A little bit of sex prevents mutation accumulation even in apomictic polyploid plants.

BACKGROUND: In the absence of sex and recombination, genomes are expected to accumulate deleterious mutations via an irreversible process known as Muller's ratchet, especially in the case of polyploidy. In contrast, no genome-wide mutation accumulation was detected in a transcriptome of facultative apomictic, hexaploid plants of the Ranunculus auricomus complex. We hypothesize that mutations cannot accumulate in flowering plants with facultative sexuality because sexual and asexual development concurrently occurs within the same generation. We assume a strong effect of purging selection on reduced gametophytes in the sexual developmental pathway because previously masked recessive deleterious mutations would be exposed to selection. RESULTS: We test this hypothesis by modeling mutation elimination using apomictic hexaploid plants of the R. auricomus complex. To estimate mean recombination rates, the mean number of recombinants per generation was calculated by genotyping three F1 progeny arrays with six microsatellite markers and character incompatibility analyses. We estimated the strength of purging selection in gametophytes by calculating abortion rates of sexual versus apomictic development at the female gametophyte, seed and offspring stage. Accordingly, we applied three selection coefficients by considering effects of purging selection against mutations on (1) male and female gametophytes in the sexual pathway (additive, s = 1.000), (2) female gametophytes only (s = 0.520), and (3) on adult plants only (sporophytes, s = 0.212). We implemented recombination rates into a mathematical model considering the three different selection coefficients, and a genomic mutation rate calculated from genome size of our plants and plant-specific mutation rates. We revealed a mean of 6.05% recombinants per generation. This recombination rate eliminates mutations after 138, 204 or 246 generations, depending on the respective selection coefficients (s = 1.000, 0.520, and 0.212). CONCLUSIONS: Our results confirm that the empirically observed frequencies of facultative recombination suffice to prevent accumulation of deleterious mutations via Muller's ratchet even in a polyploid genome. The efficiency of selection is in flowering plants strongly increased by acting on the haplontic (reduced) gametophyte stage.

Climatic differentiation in polyploid apomictic Ranunculus auricomus complex in Europe.

BACKGROUND: Polyploidy and apomixis are important factors influencing plant distributions often resulting in range shifts, expansions and geographical parthenogenesis. We used the Ranunculus auricomus complex as a model to asses if the past and present distribution and climatic preferences were determined by these phenomena. RESULTS: Ecological differentiation among diploids and polyploids was tested by comparing the sets of climatic variables and distribution modelling using 191 novel ploidy estimations and 561 literature data. Significant differences in relative genome size on the diploid level were recorded between the "auricomus" and "cassubicus" groups and several new diploid occurrences were found in Slovenia and Hungary. The current distribution of diploids overlapped with the modelled paleodistribution (22 kyr BP), except Austria and the Carpathians, which are proposed to be colonized later on from refugia in the Balkans. Current and historical presence of diploids from the R. auricomus complex is suggested also for the foothills of the Caucasus. Based on comparisons of the climatic preferences polyploids from the R. auricomus complex occupy slightly drier and colder habitats than the diploids. CONCLUSIONS: The change of reproductive mode and selection due to competition with the diploid ancestors may have facilitated the establishment of polyploids within the R. auricomus complex in environments slightly cooler and drier, than those tolerated by diploid ancestors. Much broader distribution of polyploid apomicts may have been achieved due to faster colonization mediated by uniparental reproductive system.

Effects of cold treatments on fitness and mode of reproduction in the diploid and polyploid alpine plant Ranunculus kuepferi (Ranunculaceae).

Background and Aims: 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. Methods: We tested experimentally in climate cabinets for effects of low temperatures and repeated frost on phenology, fitness and mode of reproduction in diploid and tetraploid cytotypes of the alpine species Ranunculus kuepferi. The reproduction mode was determined via flow cytometric seed screening (FCSS). Key Results: Diploids produced the first flowers earlier than the tetraploids in all treatments. Cold treatments significantly reduced the fitness of both cytotypes regarding seed set, and increased the frequency of apomictic seed formation in diploids, but not in tetraploids. Over consecutive years, the degree of facultative apomixis showed individual phenotypic plasticity. Conclusions: Cold stress is correlated to expression of apomixis in warm-adapted, diploid R. kuepferi, while temperature-tolerant tetraploids just maintain facultative apomixis as a possible adaptation to colder climates. However, expression of apomixis may not depend on polyploidy, but rather on failure of the sexual pathway.

Herb Hydraulics: Inter- and Intraspecific Variation in Three Ranunculus Species.

The requirements of the water transport system of small herbaceous species differ considerably from those of woody species. Despite their ecological importance for many biomes, knowledge regarding herb hydraulics remains very limited. We compared key hydraulic features (vulnerability to drought-induced hydraulic decline, pressure-volume relations, onset of cellular damage, in situ variation of water potential, and stomatal conductance) of three Ranunculus species differing in their soil humidity preferences and ecological amplitude. All species were very vulnerable to water stress (50% reduction in whole-leaf hydraulic conductance [kleaf] at -0.2 to -0.8 MPa). In species with narrow ecological amplitude, the drought-exposed Ranunculus bulbosus was less vulnerable to desiccation (analyzed via loss of kleaf and turgor loss point) than the humid-habitat Ranunculus lanuginosus Accordingly, water stress-exposed plants from the broad-amplitude Ranunculus acris revealed tendencies toward lower vulnerability to water stress (e.g. osmotic potential at full turgor, cell damage, and stomatal closure) than conspecific plants from the humid site. We show that small herbs can adjust to their habitat conditions on interspecific and intraspecific levels in various hydraulic parameters. The coordination of hydraulic thresholds (50% and 88% loss of kleaf, turgor loss point, and minimum in situ water potential) enabled the study species to avoid hydraulic failure and damage to living cells. Reversible recovery of hydraulic conductance, desiccation-tolerant seeds, or rhizomes may allow them to prioritize toward a more efficient but vulnerable water transport system while avoiding the severe effects that water stress poses on woody species.

Application of heat stress in situ demonstrates a protective role of irradiation on photosynthetic performance in alpine plants.

The impact of sublethal heat on photosynthetic performance, photosynthetic pigments and free radical scavenging activity was examined in three high mountain species, Rhododendron ferrugineum, Senecio incanus and Ranunculus glacialis using controlled in situ applications of heat stress, both in darkness and under natural solar irradiation. Heat treatments applied in the dark reversibly reduced photosynthetic performance and the maximum quantum efficiency of photosystem II (Fv /Fm), which remained impeded for several days when plants were exposed to natural light conditions subsequently to the heat treatment. In contrast, plants exposed to heat stress under natural irradiation were able to tolerate and recover from heat stress more readily. The critical temperature threshold for chlorophyll fluorescence was higher under illumination (Tc (')) than in the dark (Tc). Heat stress caused a significant de-epoxidation of the xanthophyll cycle pigments both in the light and in the dark conditions. Total free radical scavenging activity was highest when heat stress was applied in the dark. This study demonstrates that, in the European Alps, heat waves can temporarily have a negative impact on photosynthesis and, importantly, that results obtained from experiments performed in darkness and/or on detached plant material may not reliably predict the impact of heat stress under field conditions.

Chloroplast protrusions in leaves of Ranunculus glacialis L. respond significantly to different ambient conditions, but are not related to temperature stress.

The occurrence of chloroplast protrusions (CPs) in leaves of Ranunculus glacialis L. in response to different environmental conditions was assessed. CPs occur highly dynamically. They do not contain thylakoids and their physiological function is still largely unknown. Controlled in situ sampling showed that CP formation follows a pronounced diurnal rhythm. Between 2 and 27 °C the relative proportion of chloroplasts with CPs (rCP) showed a significant positive correlation to leaf temperature (TL; 0.793, P < 0.01), while irradiation intensity had a minor effect on rCP. In situ shading and controlled laboratory experiments confirmed the significant influence of TL. Under moderate irradiation intensity, an increase of TL up to 25 °C significantly promoted CP formation, while a further increase to 37 °C led to a decrease. Furthermore, rCP values were lower in darkness and under high irradiation intensity. Gas treatment at 2000 ppm CO2/2% O2 led to a significant decrease of rCP, suggesting a possible involvement of photorespiration in CP formation. Our findings demonstrate that in R. glacialis, CPs are neither a rare phenomenon nor a result of heat or light stress; on the contrary, they seem to be most abundant under moderate temperature and non-stress irradiation conditions.

Two distinct plant respiratory physiotypes might exist which correspond to fast-growing and slow-growing species.

The origin of the carbon atoms in CO2 respired by leaves in the dark of several plant species has been studied using 13C/12C stable isotopes. This study was conducted using an open gas exchange system for isotope labeling that was coupled to an elemental analyzer and further linked to an isotope ratio mass spectrometer (EA-IRMS) or coupled to a gas chromatography-combustion-isotope ratio mass spectrometer (GC-C-IRMS). We demonstrate here that the carbon, which is recently assimilated during photosynthesis, accounts for nearly ca. 50% of the carbon in the CO2 lost through dark respiration (Rd) after illumination in fast-growing and cultivated plants and trees and, accounts for only ca. 10% in slow-growing plants. Moreover, our study shows that fast-growing plants, which had the largest percentages of newly fixed carbon of leaf-respired CO2, were also those with the largest shoot/root ratios, whereas slow-growing plants showed the lowest shoot/root values.

Plant population differentiation and climate change: responses of grassland species along an elevational gradient.

Mountain ecosystems are particularly susceptible to climate change. Characterizing intraspecific variation of alpine plants along elevational gradients is crucial for estimating their vulnerability to predicted changes. Environmental conditions vary with elevation, which might influence plastic responses and affect selection pressures that lead to local adaptation. Thus, local adaptation and phenotypic plasticity among low and high elevation plant populations in response to climate, soil and other factors associated with elevational gradients might underlie different responses of these populations to climate warming. Using a transplant experiment along an elevational gradient, we investigated reproductive phenology, growth and reproduction of the nutrient-poor grassland species Ranunculus bulbosus, Trifolium montanum and Briza media. Seeds were collected from low and high elevation source populations across the Swiss Alps and grown in nine common gardens at three different elevations with two different soil depths. Despite genetic differentiation in some traits, the results revealed no indication of local adaptation to the elevation of population origin. Reproductive phenology was advanced at lower elevation in low and high elevation populations of all three species. Growth and reproduction of T. montanum and B. media were hardly affected by garden elevation and soil depth. In R. bulbosus, however, growth decreased and reproductive investment increased at higher elevation. Furthermore, soil depth influenced growth and reproduction of low elevation R. bulbosus populations. We found no evidence for local adaptation to elevation of origin and hardly any differences in the responses of low and high elevation populations. However, the consistent advanced reproductive phenology observed in all three species shows that they have the potential to plastically respond to environmental variation. We conclude that populations might not be forced to migrate to higher elevations as a consequence of climate warming, as plasticity will buffer the detrimental effects of climate change in the three investigated nutrient-poor grassland species.

Agrochemicals in field margins--assessing the impacts of herbicides, insecticides, and fertilizer on the common buttercup (Ranunculus acris).

The effects of herbicide, insecticide, and fertilizer inputs on the common buttercup Ranunculus acris in field margins were studied in an experimental field study. The test design allowed us to investigate the single and combined effects of repeated herbicide, insecticide, and fertilizer applications in successive growing seasons. To assess the effects of the agrochemical applications on R. acris, plant community assessments were carried out and a photodocumentation of the flowering intensity was performed over two years. In addition, the authors conducted a monitoring survey of R. acris in field margins in the proximity of the study site. In the field experiment, R. acris plant density decreased significantly with treatments including fertilizer. The herbicide caused a sublethal effect by reducing flower intensity by 85%. In the long run, both effects will result in a decline of R. acris and lead to shifts in plant communities in field margins. This was confirmed by the monitoring survey, where R. acris could hardly be observed in field margins directly adjacent to cereal fields, whereas in margins next to meadows the species was recorded frequently. Besides the implications for the plants, the sublethal effects may also affect many flower-visiting insects. The results indicate that the current risk assessment for nontarget plants is insufficiently protective for wild plant species in field margins and that consideration of sublethal effects is crucial to preserve biodiversity in agricultural landscapes.

Stomata are less responsive to environmental stimuli in high background ozone in Dactylis glomerata and Ranunculus acris.

Two mesotrophic grassland species, Ranunculus acris and Dactylis glomerata were exposed to a range of ozone treatments (16.2-89.5 ppb 24 h mean) and two watering regimes under naturally fluctuating photosynthetically active radiation (PAR), vapour pressure deficit (VPD) and temperature. Stomatal conductance was measured throughout the experiments, and the combined data set (>1000 measurements) was analysed for effects of low and high ozone on responses to environmental stimuli. We show that when D. glomerata and R. acris were grown in 72.6-89.5 ppb ozone the stomata consistently lose the ability to respond, or have reduced response, to naturally fluctuating environmental conditions in comparison to their response in low ozone. The maximum stomatal conductance (g(max)) was also significantly higher in the high ozone treatment for D. glomerata. We discuss the hypotheses for the reduced sensitivity of stomatal closure to a changing environment and the associated implications for ozone flux modelling.

Frost resistance of reproductive tissues during various stages of development in high mountain plants.

Frost resistance of reproductive vs aboveground vegetative structures was determined for six common European high alpine plant species that can be exposed to frosts throughout their whole reproductive cycle. Freezing tests were carried out in the bud, anthesis and fruit stage. Stigma and style, ovary, placenta, ovule, flower stalk/peduncle and, in Ranunculus glacialis, the receptacle were separately investigated. In all species, the vegetative organs tolerated on an average 2-5 K lower freezing temperatures than the most frost-susceptible reproductive structures that differed in their frost resistance. In almost all species, stigma, style and the flower stalk/peduncle were the most frost-susceptible reproductive structures. Initial frost damage (LT10) to the most susceptible reproductive structure usually occurred between -2 and -4°C independent of the reproductive stage. The median LT50 across species for stigma and style ranged between -3.4 and -3.7°C and matched the mean ice nucleation temperature (-3.7 ± 1.4°C). In R. glacialis, the flower stalk was the most frost-susceptible structure (-5.4°C), and was in contrast to the other species ice-tolerant. The ovule and the placenta were usually the most frost-resistant structures. During reproductive development, frost resistance (LT50) of single reproductive structures mostly showed no significant change. However, significant increases or decreases were also observed (2.1 ± 1.2 K). Reproductive tissues of nival species generally tolerated lower temperatures than species occurring in the alpine zone. The low frost resistance of reproductive structures before, during and shortly after anthesis increases the probability of frost damage and thus, may restrict successful sexual plant reproduction with increasing altitude.

Evaluating contemporary pollen dispersal in two common grassland species Ranunculus bulbosus L. (Ranunculaceae) and Trifolium montanum L. (Fabaceae) using an experimental approach.

Pollen flow is a key biological process that connects plant populations, preventing genetic impoverishment and inbreeding. Pollen-mediated long-distance dispersal (LDD) events are especially important for plant species in increasingly fragmented landscapes. Patterns of pollen dispersal were directly estimated and dispersal kernels modelled in an experimental population of Ranunculus bulbosus and Trifolium montanum to determine the potential for LDD. Eight and 11 microsatellite markers were used for R. bulbosus and T. montanum, respectively, to run a likelihood-based paternity analysis on randomly chosen offspring (Ntotal = 180 per species) from five maternal plants. High rates of selfing were found in R. bulbosus (average 45.7%), while no selfing was observed in T. montanum. The majority (60%) of mating events occurred at very short distances: the median of the observed dispersal distances was 0.8 m in both species, and the average distances were 15.9 and 10.3 m in R. bulbosus and T. montanum, respectively. Modelling the pollen dispersal kernel with four different distribution functions (exponential-power, geometric, 2Dt and Weibull) indicated that the best fit for both species was given by a Weibull function. Yet, the tail of the T. montanum pollen dispersal kernel was thinner than in R. bulbosus, suggesting that the probability for LDD is higher in the latter species. Even though the majority of pollen dispersal occurred across short distances, the detection of several mating events up to 362 m (R. bulbosus) and 324 m (T. montanum) suggests that pollen flow may be sufficient to ensure population connectivity in these herb species across fragmented grasslands in Swiss agricultural landscapes.

Patterns of genetic variation across altitude in three plant species of semi-dry grasslands.

BACKGROUND: Environmental gradients caused by altitudinal gradients may affect genetic variation within and among plant populations and inbreeding within populations. Populations in the upper range periphery of a species may be important source populations for range shifts to higher altitude in response to climate change. In this study we investigate patterns of population genetic variation at upper peripheral and lower more central altitudes in three common plant species of semi-dry grasslands in montane landscapes. METHODOLOGY/PRINCIPAL FINDINGS: In Briza media, Trifolium montanum and Ranunculus bulbosus genetic diversity, inbreeding and genetic relatedness of individuals within populations and genetic differentiation among populations was characterized using AFLP markers. Populations were sampled in the Swiss Alps at 1800 (upper periphery of the study organisms) and at 1200 m a.s.l. Genetic diversity was not affected by altitude and only in B. media inbreeding was greater at higher altitudes. Genetic differentiation was slightly greater among populations at higher altitudes in B. media and individuals within populations were more related to each other compared to individuals in lower altitude populations. A similar but less strong pattern of differentiation and relatedness was observed in T. montanum, while in R. bulbosus there was no effect of altitude. Estimations of population size and isolation of populations were similar, both at higher and lower altitudes. CONCLUSIONS/SIGNIFICANCE: Our results suggest that altitude does not affect genetic diversity in the grassland species under study. Genetic differentiation of populations increased only slightly at higher elevation, probably due to extensive (historic) gene flow among altitudes. Potentially pre-adapted genes might therefore spread easily across altitudes. Our study indicates that populations at the upper periphery are not genetically depauperate or isolated and thus may be important source populations for migration under climate change.

Reduced soil water availability did not protect two competing grassland species from the negative effects of increasing background ozone.

Two common (semi-) natural temperate grassland species, Dactylis glomerata and Ranunculus acris, were grown in competition and exposed to two watering regimes: well-watered (WW, 20-40% v/v) and reduced-watered (RW, 7.5-20% v/v) in combination with eight ozone treatments ranging from pre-industrial to predicted 2100 background levels. For both species there was a significant increase in leaf damage with increasing background ozone concentration. RW had no protective effect against increasing levels of ozone-induced senescence/injury. In high ozone, based on measurements of stomatal conductance, we propose that ozone influx into the leaves was not prevented in the RW treatment, in D. glomerata because stomata were a) more widely open than those in less polluted plants and b) were less responsive to drought. Total seasonal above ground biomass was not significantly altered by increased ozone; however, ozone significantly reduced root biomass in both species to differing amounts depending on watering regime.

Effect of temperature on the progamic phase in high-mountain plants.

Progamic processes are particularly temperature-sensitive and, in lowland plants, are usually drastically reduced below 10 °C and above 30 °C. Little is known about how effectively sexual processes of mountain plants function under the large temperature fluctuations at higher altitudes. The present study examines duration and thermal thresholds for progamic processes in six common plant species (Cerastium uniflorum, Gentianella germanica, Ranunculus alpestris, R. glacialis, Saxifraga bryoides, S. caesia) from different altitudinal zones in the European Alps. Whole plants were collected from natural sites shortly before anthesis and kept in a climate chamber until further processing. Flowers with receptive stigmas were hand-pollinated with allopollen and exposed to controlled temperatures between -2 and 40 °C. Pollen performance (adhesion to the stigma, germination, tube growth, fertilisation) was quantitatively analysed, using the aniline blue fluorescence method. Pollen adhesion was possible from -2 to 40 °C. Pollen germination and tube growth occurred from around 0 to 35 °C in most species. Fertilisation was observed from 5 to 30-32 °C (0-35 °C in G. germanica). The progamic phase was shortest in G. germanica (2 h at 30 °C, 12 h at 5 °C, 24 h at 0 °C), followed by R. glacialis (first fertilisation after 2 h at 30 °C, 18 h at 5 °C). In the remaining species, first fertilisation usually occurred after 4-6 h at 30 °C and after 24-30 h at 5 °C. Thus, mountain plants show remarkably flexible pollen performance over a wide temperature range and a short progamic phase, which may be essential for successful reproduction in the stochastic high-mountain climate.