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Key Message: This study, using multi-omics combined with physiologic assays, found that calcium-ion signaling can regulate phenolic acid accumulation in R. chrysanthum leaves in response to UV-B stress. UV-B stress is a severe abiotic stress capable of destroying cellular structures and affecting plant growth. Rhododendron chrysanthum Pall. (R. chrysanthum) is a plant that has been exposed to high levels of UV-B radiation for an extended period, leading to the development of adaptive responses to mitigate UV-B stress. As such, it serves as a valuable experimental material for studying plant resilience to UV-B stress. We utilized R. chrysanthum as the experimental material and subjected it to UV-B stress. We conducted a comprehensive analysis of the changes in R. chrysanthum under both control and UV-B stress conditions using multi-omic and physiologic assays. Our aim was to investigate the molecular mechanism underlying R. chrysanthum's resistance to UV-B stress, with a focus on calcium-ion signaling. UV-B stress was found to impact the photosynthesis of R. chrysanthum by decreasing the maximum photosynthetic efficiency of photosystem II, reducing Fm, and increasing F0. In addition, the composition of numerous phenolic acid compounds was significantly altered. Genes and proteins related to calcium signaling showed significant differences, with some proteins (CML, CPK1, CRK3, ATP2C, ERG3, CAR7) being modified by acetylation. The correlation between genes and proteins involved in calcium signaling and phenolic compounds suggested that calcium signaling may play a role in regulating the accumulation of phenolic compounds under UV-B stress to help R. chrysanthum adapt. This study examines the impact of calcium-ion signaling on the accumulation of phenolic acid compounds, offering insights for future research on the molecular mechanisms underlying plant resilience to UV-B stress., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The effect of global climate change on plant-pollinator interaction is not limited to changes in phenology and richness within communities but also includes the spatial mismatch caused by the inconsistency of geographical distribution changes. Subsequently, the pollinator interaction network may be remodeled or even disrupted. In this study, we simulated the suitable habitat niche of 15 Rhododendron species and their eight pollinator species as well as their overlapping versus geographical mismatch under the current and three future climate change scenarios in 2090s, using MaxEnt. Results showed that the suitable habitat of all Rhododendron species would decrease in 2090s. In particular, 10, 8, and 13 Rhododendron-pollinator assemblages would have a reduced spatial match region under the climate change scenarios, mainly due to the contraction of the suitable habitat of Rhododendron species. The results provide novel insights into the response of plant-pollinator interactions to global warming, useful to prioritize conservation actions of alpine plant ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Background: Rhododendron chrysanthum Pall. (R. chrysanthum) is a plant that lives in high mountain with strong UV-B radiation, so R. chrysanthum possess resistance to UV-B radiation. The process of stress resistance in plants is closely related to metabolism. Lysine acetylation is an important post-translational modification, and this modification process is involved in a variety of biological processes, and affected the expression of enzymes in metabolic processes. However, little is known about acetylation proteomics during UV-B stress resistance in R. chrysanthum., Results: In this study, R. chrysanthum OJIP curves indicated that UV-B stress damaged the receptor side of the PSII reaction center, with a decrease in photosynthesis, a decrease in sucrose content and an increase in starch content. A total of 807 differentially expressed proteins, 685 differentially acetylated proteins and 945 acetylation sites were identified by quantitative proteomic and acetylation modification histological analysis. According to COG and subcellular location analyses, DEPs with post-translational modification of proteins and carbohydrate metabolism had important roles in resistance to UV-B stress and DEPs were concentrated in chloroplasts. KEGG analyses showed that DEPs were enriched in starch and sucrose metabolic pathways. Analysis of acetylation modification histology showed that the enzymes in the starch and sucrose metabolic pathways underwent acetylation modification and the modification levels were up-regulated. Further analysis showed that only GBSS and SSGBSS changed to DEPs after undergoing acetylation modification. Metabolomics analyses showed that the metabolite content of starch and sucrose metabolism in R. chrysanthum under UV-B stress., Conclusions: Decreased photosynthesis in R. chrysanthum under UV-B stress, which in turn affects starch and sucrose metabolism. In starch synthesis, GBSS undergoes acetylation modification and the level is upregulated, promotes starch synthesis, making R. chrysanthum resistant to UV-B stress., (© 2024. The Author(s).)

Flowering exhibits a significant relationship with environmental stimuli and changes. Effect of photoperiodism and vernalization have been well studied in flowering phenology; however, the effect of soil temperature on flowering is less explored which is one of the major factors of vegetation growth in alpine ecosystem. This study thus focuses on the effects of soil and air temperature on flowering response of Rhododendron arboreum Sm., a Himalayan tree species, which is also an indicator of spring initiation in high altitude regions. To monitor the flowering pattern, we employed automated phenocam, which was set up at 3356 masl in Tungnath (Indian Alpine region of Uttarakhand) for time-lapse photography of timberline ecotone. Soil and air temperature were recorded continuously at the timberline ecotone. Three years (2017 to 2020) of datasets were used for the present study. The phenocam observations displayed an interesting event in the year 2019-2020 with complete absence of flowering in R. arboreum population at Tungnath timberline ecotone. From the soil temperature data, an increase in winter (Dec-Jan, during which floral buds form) soil temperature, by > 1 °C, and no accumulation of freezing degree-days were found for the year 2019-2020. Air temperature however did not display any relationship with the failure of flowering, ruling out aerial chilling or frost injury of floral buds. From the results, a possible relationship between soil temperature and flowering can be suggested pointing towards necessary root apex vernalization stimulus in shallow rooted Rhododendrons. However, the dependency of flowering in Rhododendrons on winter soil temperature further requires continuous monitoring and more observations to make concrete inferences., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Cold acclimation (CA) is a well-known strategy employed by plants to enhance freezing tolerance (FT) in winter. Global warming could disturb CA and increase the potential for winter freeze-injury. Thus, developing robust FT through complete CA is essential. To explore the molecular mechanisms of CA in woody perennials, we compared field and artificial CAs. Transcriptomic data showed that photosynthesis/photoprotection and fatty acid metabolism pathways were specifically enriched in field CA; carbohydrate metabolism, secondary metabolism and circadian rhythm pathways were commonly enriched in both field and artificial CAs. When compared with plants in vegetative growth in the chamber, we found that the light signals with warm air temperatures in the fall might induce the accumulation of leaf abscisic acid (ABA) and jasmonic acid (JA) concentrations, and activate Ca 2+ , ABA and JA signaling transductions in plants. With the gradual cooling occurrence in winter, more accumulation of anthocyanin, chlorophyll degradation, closure/degradation of photosystem II reaction centers, and substantial accumulation of glucose and fructose contributed to obtaining robust FT during field CA. Moreover, we observed that in Rhododendron 'Elsie Lee', ABA and JA decreased in winter, which may be due to the strong requirement of zeaxanthin for rapid thermal dissipation and unsaturated fatty acids for membrane fluidity. Taken together, our results indicate that artificial CA has limitations to understand the field CA and field light signals (like short photoperiod, light intensity and/or light quality) before the low temperature in fall might be essential for complete CA., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Owing to the high leaf mass per area, alpine evergreen sclerophyllous Rhododendron have low values of mesophyll conductance (g m ). The resulting low chloroplast CO 2 concentration aggravates photorespiration, which requires a higher ATP/NADPH ratio. However, the significance of photorespiration and underlying mechanisms of energy balance in these species are little known. In this study, eight alpine evergreen sclerophyllous Rhododendron species grown in a common garden were tested for their g m , electron flow to photorespiration, and energy balancing. Under saturating light, g m was the most limiting factor for net photosynthesis (A N ) in all species, and the species differences in A N were primarily driven by g m rather than stomatal conductance. The total electron flow through photosystem II (ETRII) nearly equaled the electron transport required for Rubisco carboxylation and oxygenation. Furthermore, blocking electron flow to photosystem I with appropriate inhibitors showed that electron flow to plastic terminal oxidase was not observed. As a result, these studied species showed little alternative electron flow mediated by water-water cycle or plastic terminal oxidase. By comparison, the ratio of electron transport consumed by photorespiration to ETRII (J PR /ETRII), ranging from 43%∼55%, was negatively correlated to A N and g m . Furthermore, the increased ATP production required by enhanced photorespiration was regulated by cyclic electron flow around photosystem I. These results indicate that photorespiration is the major electron sink for dissipation of excess excitation energy in the alpine evergreen sclerophyllous Rhododendron species. The coordination of g m , photorespiration and cyclic electron flow is important for sustaining leaf photosynthesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Biodiversity loss and variation in species responses to climate and land use change have been found across broad taxonomic groups. However, whether species from the same taxonomic group with distinct geographical ranges will respond differently is poorly understood. The aim of this study is to predict the potential impacts of future climate and land use change on the distribution of narrow- and wide-ranging Rhododendron species, and estimate their relative contribution in China. We applied the presence-only ecological niche model MaxEnt to predict the distribution of 10 narrow-ranging and 10 wide-ranging Rhododendron species for the year 2070, using three general circulation models and three scenarios of climate and land use change. We measured the predicted distribution change of each species using change ratio, distance and direction of core range shifts, and niche overlap using Schoener's D. We found that the distribution areas of six narrow-ranging species would decrease, of which one species would go extinct. The remaining four narrow-ranging species would experience range expansion. Distribution of all the wide-ranging Rhododendron species would decrease. All Rhododendrons will shift to the northwest. We conclude that Rhododendron species generally will be negatively affected by the climatic and land use change expected in 2070 from the three scenarios evaluated in this study, but some narrow-ranging species may be positively influenced. Narrow-ranging Rhododendron species are more vulnerable compared to wide-ranging Rhododendron species. This study demonstrated that the effects of climate and land use change on alpine and subalpine plant species is species-specific, thereby strengthening our understanding of the impacts of climate and land use change on plant distribution., (Copyright © 2018. Published by Elsevier B.V.)

Rhododendron arboreum inhabits the Himalayan climate otherwise detrimental to many species, though the underlying survival mechanism remains unclear. Such temperate species have an inherent endurance towards freezing temperature which is prerequisite for an initiation and transition to flowering phase. Orchestrating the molecular architecture is vital towards managing distinct abiotic signals. To determine the molecular factors directing growth, development, and tolerance under environmental extremes in the species, the high-throughput transcriptome and metabolome data from vegetative as well as cold-acclimatized flowering season tissues were generated. Firstly, the de novo assembly pertaining to the foliar and floral tissues comprising of 157,427 unigenes was examined for a comparative analysis. 4149 of 12,577 transcripts observed with a significant fluctuating expression corresponded to seasonal retorts. Following the interactive network, 525 genes were distinguished as the epicenters of sense, response, and tolerance. Secondly, liquid chromatography coupled to mass spectrometry was adopted to profile the extent of metabolite richness across the tissues of two seasons. Taking into account the formula-based mappings offered by MetaboSearch tool, 421 unique ions obtained were annotated to 173 KEGG compounds, especially secondary metabolites. Moreover, by integrating the transcript and metabolite annotations, it was found that right from active metabolism, signaling, development, and their regulations, supplementary response to abiotic/biotic stimuli was induced. A multifaceted response displayed during flowering not only sponsored the climatic encounters but brought the shift from vegetative to reproductive growth. Overall, this comprehensive approach following transcriptome and non-targeted metabolome elucidated the contribution of genetic and metabolic factors in environmental responses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Pollen grains can be dispersed singly or variously aggregated in groups. Whether the evolution of pollen aggregation is driven by the pollinator remains unexplored. We hypothesize that an extensive pollen aggregation is favored under a scarcity of pollinators. Variation in pollen aggregation by viscin threads in 13 Rhododendron species was measured as it is related to pollen removal in a visit. Visitation rates of functional pollinator groups that vary in their grooming behavior were investigated in each species. Pollen deposited on stigmas in the field was also sampled. Seven Rhododendron species were infrequently pollinated by low-intensity grooming animals, including birds, butterflies and moths. The other six species were more frequently pollinated by bees with a high intensity of pollen grooming. Bird- and Lepidoptera-pollinated species produced longer pollen-connecting threads that connected more pollen grains. Phylogenetically independent contrast analysis of the 13 species showed that pollinator visitation frequency was negatively related to amounts of pollen removal per visit but not to stigmatic pollen loads. The finding of interspecific patterns in pollen removal related to pollinator visitation frequency suggests pollinator-mediated selection on pollen packaging strategies, supporting the hypothesis of floral evolution via pollen export., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Higher elevations and, early as well as late phase of growing season are expected to be more stressful for plants in high altitudes. The present study was carried out on Rhododendron anthopogon D. Don, an evergreen shrub of Himalaya to understand variation in eco-physiological and biogeochemical traits due to combined effect of elevation gradient and growing season. We conducted our study at Rohtang, India (32°22'04″ N 77°15'17″ E) and undertook random sampling of leaves at four elevations (3200 m, 3600 m, 4000 m and 4250 m), and three time periods (late June, early August and late September) during growing season. We assessed 12 eco-physiological and biogeochemical variables and analysed results through ANOVA and multivariate analysis. It was found that leaf relative water content, nitrogen percentage (N%), carbon/nitrogen ratio (C/N ratio), total chlorophyll, malondialdehyde equivalents and proline content varied along two gradients (factors) with their interaction being statistically significant. Variance partitioning analysis of studied traits revealed that both factors contribute significantly, with 'season' component ranging between 55.75 % and 94.03 % for most of the parameters, whereas, 'elevation' component contributed more for leaf area, N% and C/N ratio (48.08 %-75.03 %). Our results suggest that eco-physiology of R. anthopogon is significantly influenced by interaction of seasonal variations coupled with elevation gradient. The study highlights the importance of examining both seasonal and elevational gradients in understanding plant adaptation strategies. Overall, our findings revealed that plasticity in eco-physiological and biogeochemical traits underline the wide distribution of R. anthopogon in the high altitudes., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The perennial evergreen woody shrub, Rhododendron tomentosum, confers associational resistance against herbivory and oviposition on neighbouring plants through passive adsorption of some of its constitutively emitted volatile organic compounds (VOCs). The adsorption process is dependent on transport of VOCs in the air. In polluted atmospheres, the VOCs may be degraded and adsorption impeded. We studied the effect of elevated ozone regimes on the adsorption of R. tomentosum volatiles to white cabbage, Brassica oleracea, and the oviposition of the specialist herbivore Plutella xylostella on the exposed plants. We found evidence for adsorption and re-emission of R. tomentosum volatiles by B. oleracea plants. Ozone changed the blend of R. tomentosum volatiles and reduced the amount of R. tomentosum volatiles recovered from B. oleracea plants. However, plants exposed to R. tomentosum volatiles received fewer P. xylostella eggs than control plants exposed to filtered air irrespective of whether R. tomentosum volatiles mixed with ozone. Ozone disrupts a volatile mediated passive plant-to-plant interaction by degrading some compounds and reducing the quantity available for adsorption by neighbouring plants. The change, however, did not affect the deterrence of oviposition by P. xylostella, suggesting that aromatic companion plants of Brassica crops may confer pest-deterring properties even in ozone-polluted environments., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

With more than 80% of flowering plant species specialized for animal pollination, understanding how wild pollinators utilize resources across environments can encourage efficient planting and maintenance strategies to maximize pollination and establish resilience in the face of environmental change. A fundamental question is how generalist pollinators recognize "flower objects" in vastly different ecologies and environments. On one hand, pollinators could employ a specific set of floral cues regardless of environment. Alternatively, wild pollinators could recognize an exclusive signature of cues unique to each environment or flower species. Hoverflies, which are found across the globe, are one of the most ecologically important alternative pollinators after bees and bumblebees. Here, we have exploited their cosmopolitan status to understand how wild pollinator preferences change across different continents. Without employing any a priori assumptions concerning the floral cues, we measured, predicted, and finally artificially recreated multimodal cues from individual flowers visited by hoverflies in three different environments (hemiboreal, alpine, and tropical) using a field-based methodology. We found that although "flower signatures" were unique for each environment, some multimodal lures were ubiquitously attractive, despite not carrying any reward, or resembling real flowers. While it was unexpected that cue combinations found in real flowers were not necessary, the robustness of our lures across insect species and ecologies could reflect a general strategy of resource identification for generalist pollinators. Our results provide insights into how cosmopolitan pollinators such as hoverflies identify flowers and offer specific ecologically based cues and strategies for attracting pollinators across diverse environments., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Premise of the Study: Although much is known about the anatomy of idioblasts, the relationship of leaf idioblasts to leaf physiology is poorly known. Our goal was to understand the relationship between idioblast abundance and leaf water relations. Tropical epiphytic and shrub Rhododendron species were the model system., Methods: Leaf succulence and idioblast metrics of 61 plants representing 17 species were compared with stomatal and water relation metrics. Correlation, ANOVA, and regression were used to understand which water relation traits were best aligned with the proportion of the leaf occupied by idioblasts for epiphytic and shrub species., Key Results: Idioblast volume per leaf area varied from 1.4-9.5 mm 3 ·cm -2 among accessions, and an index of stomatal area per leaf area varied from 0.08-3.3. Succulence, stomatal, and water relation metrics varied significantly among species. Total idioblast volume was negatively correlated with leaf succulence metrics, and positively correlated with relative water content at the turgor loss point. Idioblasts were better related to capacitance in thin leaves. All idioblast metrics were significantly greater in epiphytic species., Conclusions: Leaf idioblasts can have a significant effect on leaf-lamina water relations. Idioblasts likely function as a water buffering system for thin leaves in tropical Rhododendron species. The association of idioblast prominence with leaf water buffering is greatest for epiphytic species with thin leaves. Thus, the proliferation of leaf idioblasts may have been an important innovation for Rhododendron species diversification into tropical epiphytic habitats., (© 2017 Botanical Society of America.)

To gain a better understanding of cold acclimation in rhododendron and in woody perennials in general, we used the 2D-DIGE technique to analyze the rhododendron proteome during the seasonal development of freezing tolerance. We selected two species varying in their cold acclimation ability as well as their thermonasty response (folding of leaves in response to low temperature). Proteins were extracted from leaves of non-acclimated (NA) and cold acclimated (CA) plants of the hardier thermonastic species, R. catawbiense (Cata.), and from leaves of cold acclimated plants of the less hardy, non-thermonastic R. ponticum (Pont.). All three protein samples (Cata.NA, Cata.CA, and Pont.CA) were labeled with different CyDyes and separated together on a single gel. Triplicate gels were run and protein profiles were compared resulting in the identification of 72 protein spots that consistently had different abundances in at least one pair-wise comparison. From the 72 differential spots, we chose 56 spots to excise and characterize further by mass spectrometry (MS). Changes in the proteome associated with the seasonal development of cold acclimation were identified from the Cata.CA-Cata.NA comparisons. Differentially abundant proteins associated with the acquisition of superior freezing tolerance and with the thermonastic response were identified from the Cata.CA-Pont.CA comparisons. Our results indicate that cold acclimation in rhododendron involves increases in abundance of several proteins related to stress (freezing/desiccation tolerance), energy and carbohydrate metabolism, regulation/signaling, secondary metabolism (possibly involving cell wall remodeling), and permeability of the cell membrane. Cold acclimation also involves decreases in abundance of several proteins involved in photosynthesis. Differences in freezing tolerance between genotypes can probably be attributed to observed differences in levels of proteins involved in these functions. Also differences in freezing tolerance may be attributed to higher levels of some constitutive protective proteins in Cata. than in Pont. that may be required to overcome freeze damage, such as glutathione peroxidase, glutamine synthetase, and a plastid-lipid-associated protein.

Background: Rhododendron chrysanthum Pall., an endangered species with significant ornamental and medicinal value, is endemic to the Changbai Mountain of China and can also serve as a significant plant resource for investigating the stress tolerance in plants. Proteomics is an effective analytical tool that provides significant information about plant metabolism and gene expression. However, no proteomics data have been reported for R. chrysanthum previously. In alpine tundra, the abiotic stress will lead to a severe over-accumulation of reactive oxygen species (ROS). Many alpine plants overcome the severe stresses and protect themselves from the oxidative damage by increasing the ratio and activity of antioxidant enzymes., Results: In our study, wild type and domesticated Rhododendron chrysanthum Pall. were used as experimental and control groups, respectively. Proteomics method combined with biochemical approach were applied for the stress tolerance investigation of R. chrysanthum at both protein and molecular level. A total of 1,395 proteins were identified, among which 137 proteins were up-regulate in the experimental group. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidases (APXs), and glutathione peroxidase (GPX) were significantly higher and the expression of APXs and GPX were also increased in the experimental group. Moreover, the interaction network analysis of these enzymes also reveals that the antioxidant enzymes play important roles in the stress resistance in plants., Conclusions: This is the first report of the proteome of Rhododendron chrysanthum Pall., and the data reinforce the notion that the antioxidant system plays a significant role in plant stress survival. Our results also verified that R. chrysanthum is highly resistant to abiotic stress and can serve as a significant resource for investigating stress tolerance in plants., Reviewers: This article was reviewed by George V. (Yura) Shpakovski and Ramanathan Sowdhamini.

In this study, 17 anatomical structure indexes of 15 Rhododendron cultivars were mea-sured by scanning electron microscope (SEM). Leaf anatomical structure indexes were screened via coefficient of variation, analysis of correlation and hierarchical cluster analysis, and comprehensive evaluation on heat resistance for each cultivar was conducted by the subordinate function. The results showed that the leaves of Rhododendron cultivars were typical bifacial leaf and the epidermal anticlinal walls showed slightly sinuate. The stomata only distributed in the lower epidermis and the shape was ruleless. The anatomical structure indexes all reached a significant level difference among 15 cultivars (P<0.01), except for lower epidermis thickness (P<0.05). Thickness of lamina corneum, stomatal density, stomatal width, the thickness palisade tissue and looseness of leaf spongy tissue were the main factors related to the hardness, while other indexes, such as stomatal length, stoma aperture, stomatal opening, length and thickness of upper epidermis, length and thickness of lower epidermis, thickness of spongy tissue, the ratio of the palisade tissue to spongy tissue, tightness of leaf palisade tissue, leaf thickness and media thickness didn't show much effect on heat resistance. There were some differences among 15 cultivars in heat resistance, and the order was Rhododendron 'Song Jiang Da Tao Hong' > Rhododendron 'Zhuang Yuan Hong' > Rhododendron 'Lv Se Guang Hui' > Rhododendron 'Fen Zhen Zhu' > Rhododendron 'Wai Guo Hong' > Rhododendron 'Lan Yin' > Rhododendron 'Bi Zhi' >Rhododendron 'Da He Zhi Chun' > Rhododendron 'Guo Qi Hong' > Rhododendron 'Yu Ling Long' > Rhododendron 'Hong Shan Hu' > Rhododendron 'Ning Bo Hong' > Rhododendron 'Tao Ban Zhu Sha' > Rhododendron 'Ai Ding Bao' > Rhododendron 'Liu Qiu Hong'. According to the heat hardiness, the cultivars could be divided into 4 groups: R. 'Song Jiang Da Tao Hong', R. 'Zhuang Yuan Hong' and R. 'Lv Se Guang Hui' with high heat resistance, R. 'Fen Zhen Zhu', R. 'Wai Guo Hong', R. 'Lan Yin', R. 'Bi Zhi', R. 'Da He Zhi Chun', R. 'Guo Qi Hong' and R. 'Yu Ling Long' with medium heat resistance, R. 'Hong Shan Hu', R. 'Ning Bo Hong', R. 'Tao Ban Zhu Sha' and R. 'Ai Ding Bao' with lower heat resistance, R. 'Liu Qiu Hong' without heat resistance. However, the accurate heat hardiness evaluation of Rhododendron still needs to consider other factors, including morphological structure, physiological and biochemical indicators and genetic factor of heat resistance, the harmfulness of Rhododendron, and the recovery state after being injured by high temperature.

Climate change is producing modifications in the intensity and frequency of rainfall in some regions of the planet. According to predictions, annual rainfall distribution in Western Europe will result in an increase in episodes of drought, thereby negatively affecting nutrient availability. Since high mountain systems will be particularly vulnerable, the physiological and nutritional responses to changes in summer rainfall were monitored over the course of two consecutive summers on three species, which are representative of subalpine forests: birch (Betula pendula Roth.), rhododendron (Rhododendron ferrugineum L.) and mountain pine (Pinus uncinata Mill.). Birch was especially susceptible to scarce precipitation showing alterations in leaf morphology and a decline in net photosynthesis (A) due to stomatal closure, which led to photoinhibition and to early leaf senescence as shown by the photosynthetic nitrogen-use efficiency (PNUE), carbon/nitrogen (C/N) ratio, foliar N and 13 C isotope discrimination (Δ 13 C) results. The Δ 13 C of the soluble fraction is a good estimator of intrinsic water-use efficiency in this species. Rhododendron and mountain pine had sclerophyllous leaves, as indicated by leaf mass per area, Δ 13 C, PNUE and C/N results. Rhododendron was particularly affected by short periods of scarce rainfall, which negatively affected gas exchange and photochemistry, and reduced the remobilization of leaf N and P. Mountain pine was the most tolerant species since alterations of gas exchange, photochemistry and Δ 13 C were not observed. Its highest investment of N in RuBisCo and highest potassium, iron and magnesium leaf concentration contributed to the highest A rates observed., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Information on the photosynthetic process and its limitations is essential in order to predict both the capacity of species to adapt to conditions associated with climate change and the likely changes in plant communities. Considering that high-mountain species are especially sensitive, three species representative of subalpine forests of the Central Catalan Pyrenees: mountain pine (Pinus uncinata Mill.), birch (Betula pendula Roth) and rhododendron (Rhododendron ferrugineum L.) were studied under conditions associated with climate change, such as low precipitation, elevated atmospheric [CO2 ] and high solar irradiation incident at Earth's surface, in order to detect any photosynthetic limitations. Short-term high [CO2 ] increased photosynthesis rates (A) and water use efficiency (WUE), especially in birch and mountain pine, whereas stomatal conductance (gs ) was not altered in either species. Birch showed photosynthesis limitation through stomatal closure related to low rainfall, which induced photoinhibition and early foliar senescence. Rhododendron was especially affected by high irradiance, showing early photosynthetic saturation in low light, highest chlorophyll content, lowest gas exchange rates and least photoprotection. Mountain pine had the highest A, photosynthetic capacity (Amax ) and light-saturated rates of net CO2 assimilation (Asat ), which were maintained under reduced precipitation. Furthermore, maximum quantum yield (Fv /Fm ), thermal energy dissipation, PRI and SIPI radiometric index, and ascorbate content indicated improved photoprotection with respect to the other two species. However, maximum velocity of carboxylation of RuBisco (Vcmax ) indicated that N availability would be the main photosynthetic limitation in this species., (© 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Premise of the Study: Biotic and abiotic pressures affect the beginning and end of phenologies differently, but little is known about how these temporal components may be determined by phylogeny. We tested for phylogenetic signal in the phenological components and related traits among Himalayan Rhododendron species., Methods: We constructed a phylogeny with trnL-trnL-trnF, atpH-I, RPB2-I (3F-4R), and ITS 4-5, and examined it in combination with trait data recorded for a nine-species assemblage on Mt. Yulong, Yunnan, China., Key Results: Uniquely among phenological traits, 'last flowering day' had a significant phylogenetic signal. Last flowering day was latest in the clade with the smallest fruits. A similar association between the end of flowering and reproductive investment existed in data from Flora of China (Wu et al., 2005) for 160 Yunnan Rhododendron species, for which last flowering month was correlated with fruit size., Conclusions: Phylogenetic determinants of last flowering day and fruit size may be driven by limited time for fruit development before the onset of cold temperatures in autumn-a temporal niche that only small-fruited species can occupy. This strategy is analogous to 'fast and cheap' spring ephemerals. More may be gained from late-phenology studies, both within and among species and across seasons., (© 2016 Botanical Society of America.)

Mountain areas are particularly sensitive to climate change. Species distribution models predict important extinctions in these areas whose magnitude will depend on a number of different factors. Here we examine the possible impact of climate change on the Rhododendron ferrugineum (alpenrose) niche in Andorra (Pyrenees). This species currently occupies 14.6 km2 of this country and relies on the protection afforded by snow cover in winter. We used high-resolution climatic data, potential snow accumulation and a combined forecasting method to obtain the realized niche model of this species. Subsequently, we used data from the high-resolution Scampei project climate change projection for the A2, A1B and B1 scenarios to model its future realized niche model. The modelization performed well when predicting the species's distribution, which improved when we considered the potential snow accumulation, the most important variable influencing its distribution. We thus obtained a potential extent of about 70.7 km(2) or 15.1% of the country. We observed an elevation lag distribution between the current and potential distribution of the species, probably due to its slow colonization rate and the small-scale survey of seedlings. Under the three climatic scenarios, the realized niche model of the species will be reduced by 37.9-70.1 km(2) by the end of the century and it will become confined to what are today screes and rocky hillside habitats. The particular effects of climate change on seedling establishment, as well as on the species' plasticity and sensitivity in the event of a reduction of the snow cover, could worsen these predictions.

It has recently been proposed that microrefugia played an important role in species survival during past climate change events. However, the current distributions of microrefugia remain largely unknown. Wind-hole sites are areas affected by preferential flows of cool air generated in interstitial spaces created by rock fragments or colluvia. Alpine plant species occurring in lowland wind-hole sites isolated from alpine zones may be relicts of the last glacial period. Hokkaido, northern Japan, is known to contain many wind-hole sites in which alpine plant species can occur. Here we surveyed 55 wind-hole sites in the Kitami region, eastern Hokkaido, and observed two alpine plant species (lingonberry, Vaccinium vitis-idaea, and Labrador tea, Rhododendron groenlandicum ssp. diversipilosum var. diversipilosum) in 14 wind-hole sites. Statistical modeling showed that wind-hole sites are likely to occur in areas with high maximum slope angles and volcanic rock cover, and concave surfaces. Our predictions of wind-hole site distributions suggest that such topographic conditions are common in our study area, and that many undiscovered wind-hole sites exist. Ignoring microhabitats may greatly underestimate species distributions in topographically complex regions, and dispersed cool spots may also function as stepping stones and temporal habitats for cold-adapted species. Because these localized unique habitats usually occur in economically unproductive sites, identifying and protecting potential microrefugia (cool spots) would be a robust and cost-effective mitigation of climate change impacts.

Although many angiosperms are serviced by flying pollinators, reports of wings as pollen vectors are rare. Flame azalea (Rhododendron calendulaceum) is visited by diverse insects, yet previous observations suggested that only butterfly wings may transfer pollen to stigmas. We used an experimental approach to determine whether butterfly wings are the primary vehicle of pollination in flame azalea. Over two seasons of observations, only butterflies (Papilio glaucus and Speyeria cybele) contacted both anthers and stigmas, yet because of differences in wing-flapping behavior, P. glaucus transferred pollen most efficiently. In contrast, bee species specialized either on pollen or nectar but did not contact both anthers and stigmas. A field experiment revealed that flowers excluding butterflies experienced almost complete fruit failure, whereas fruit set in open flowers did not differ from those that were hand pollinated. Additionally, butterflies had 56-fold more azalea pollen on their wings than bodies, while azalea stigmas bore both pollen and wing scales. These results suggest that plants with many visitors contacting reproductive organs may still specialize on a single guild of visitors for pollination and that wing-borne pollen transfer is a key mode of flame azalea pollination.

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., (© 2014 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Invasive alien plants can compete with native plants for resources, and may ultimately decrease native plant diversity and/or abundance in invaded sites. This could have consequences for native mutualistic interactions, such as pollination. Although invasive plants often become highly connected in plant-pollinator interaction networks, in temperate climates they usually only flower for part of the season. Unless sufficient alternative plants flower outside this period, whole-season floral resources may be reduced by invasion. We hypothesized that the cessation of flowering of a dominant invasive plant would lead to dramatic, seasonal compositional changes in plant-pollinator communities, and subsequent changes in network structure. We investigated variation in floral resources, flower-visiting insect communities, and interaction networks during and after the flowering of invasive Rhododendron ponticum in four invaded Irish woodland sites. Floral resources decreased significantly after R. ponticum flowering, but the magnitude of the decrease varied among sites. Neither insect abundance nor richness varied between the two periods (during and after R. ponticum flowering), yet insect community composition was distinct, mostly due to a significant reduction in Bombus abundance after flowering. During flowering R. ponticum was frequently visited by Bombus; after flowering, these highly mobile pollinators presumably left to find alternative floral resources. Despite compositional changes, however, network structural properties remained stable after R. ponticum flowering ceased: generality increased, but quantitative connectance, interaction evenness, vulnerability, H'2 and network size did not change. This is likely because after R. ponticum flowering, two to three alternative plant species became prominent in networks and insects increased their diet breadth, as indicated by the increase in network-level generality. We conclude that network structure is robust to seasonal changes in floral abundance at sites invaded by alien, mass-flowering plant species, as long as alternative floral resources remain throughout the season to support the flower-visiting community.

In potted azalea (Rhododendron simsii hybrids) the broad mite Polyphagotarsonemus latus (Banks) is considered a severe pest with an important economic impact. Although chemical control is available, permitted acaricides are limited and have a restricted number of applications. Therefore, growers have a keen interest in alternative control measures. Recently, research on the behaviour and population dynamics of P. latus on azalea leaf disks stored at different temperatures indicated that survival and reproductive capacity of broad mite is reduced drastically when temperature drops below 7°C. In Flanders, storage of azalea plants at 3°C is common practice to pause flower development (in function of the date that plants have to be ready for sale) before forcing them to flower in a heated greenhouse. Hence, an experiment was set-up to verify and quantify the effect of cold storage of azalea on broad mite infection. Azalea plants were infected with P. latus and stored at 3°C for 2, 3 or 4 weeks. Then, plants were transferred to a heated greenhouse for 2 weeks to check whether surviving female broad mites were still able to reproduce. The number of P. latus on azalea was assessed before cold treatment, immediately after treatment, and 2 weeks after transfer to the heated greenhouse. Results confirmed that cold storage can play a role in broad mite control as the P. latus population was significantly reduced (up to 90%) immediately after treatment. A further decrease in the number of P. latus during storage in the heated greenhouse indicated that cold treatment during 4 weeks had also an effect on the reproduction capacity of P. latus. We conclude that cold storage of azalea plants (at least 4 weeks at maximum 3°C) should be considered as an additional and alternative control method for P. latus at the end of the azalea production cycle.

Plant mating systems are driven by several pre-pollination factors, including pollinator availability, mate availability and reproductive traits. We investigated the relative contributions of these factors to pollination and to realized outcrossing rates in the patchily distributed mass-flowering shrub Rhododendron ferrugineum. We jointly monitored pollen limitation (comparing seed set from intact and pollen-supplemented flowers), reproductive traits (herkogamy, flower size and autofertility) and mating patterns (progeny array analysis) in 28 natural patches varying in the level of pollinator availability (flower visitation rates) and of mate availability (patch floral display estimated as the total number of inflorescences per patch). Our results showed that patch floral display was the strongest determinant of pollination and of the realized outcrossing rates in this mass-flowering species. We found an increase in pollen limitation and in outcrossing rates with increasing patch floral display. Reproductive traits were not significantly related to patch floral display, while autofertility was negatively correlated to outcrossing rates. These findings suggest that mate limitation, arising from high flower visitation rates in small plant patches, resulted in low pollen limitation and high selfing rates, while pollinator limitation, arising from low flower visitation rates in large plant patches, resulted in higher pollen limitation and outcrossing rates. Pollinator-mediated selfing and geitonogamy likely alleviates pollen limitation in the case of reduced mate availability, while reduced pollinator availability (intraspecific competition for pollinator services) may result in the maintenance of high outcrossing rates despite reduced seed production., (© 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Global warming leads to increasing irregular and unexpected warm spells during autumn, and therefore natural chilling requirements to break dormancy are at risk. Controlled cold treatment can provide an answer to this problem. Nevertheless, artificial cold treatment will have consequences for carbon reserves and photosynthesis. In this paper, the effect of dark cold storage at 7 °C to break flower bud dormancy in the evergreen Rhododendron simsii was quantified. Carbohydrate and starch content in leaves and flower buds of an early ('Nordlicht'), semi-early ('M. Marie') and late ('Mw. G. Kint') flowering cultivar showed that carbon loss due to respiration was lowest in 'M. Marie', while 'Mw. G. Kint' was completely depleted of starch reserves at the end of cold treatment. Gene isolation resulted in a candidate gene for sucrose synthase (SUS) RsSus, which appears to be homologous to AtSus3 and had a clear increase in expression in leaves during cold treatment. Photosynthesis measurements on 'Nordlicht' and the late-flowering cultivar 'Thesla' showed that during cold treatment, dark respiration decreased 58% and 63%, respectively. Immediately after cold treatment, dark respiration increased and stabilised after 3 days. The light compensation point followed the same trend as dark respiration. Quantum efficiency showed no significant changes during the first days after cold treatment, but was significantly higher than in plants with dormant flower buds at the start of cold treatment. In conclusion, photosynthesis stabilised 3 days after cold treatment and was improved compared to the level before cold treatment., (© 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Background: Decreases in mate and/or pollinator availability would be expected to affect the selective pressure on plant mating systems. An increase in self-fertilization may evolve to compensate for the negative effects of pollination failure. However, the benefit of selfing in variable pollination environments depends on the relative fitnesses of selfed and outcrossed progeny. We investigated the potential for selfing to provide reproductive assurance over the lifetime of a long-lived perennial species and its variation between plant patches of various sizes. Patch size is likely to affect mate and pollinator availabilities, thereby affecting pollination success and the rate of selfing. We estimated fruit and seed set, reproductive assurance, self-compatibility, the multilocus patch selfing rate and lifetime inbreeding depression in natural patches of Rhododendron ferrugineum (Ericaceae), a mass-flowering species characterized by considerable patch size variation (as estimated by the total number of inflorescences)., Results: Open seed set declined linearly with increasing patch size, whereas pollinator-mediated seed set (emasculated flowers) was not significantly affected. Progeny array analysis indicated that the selfing rate declined with increasing patch size, consistent with greater reproductive assurance in small sparse patches than in large, dense patches. However, fruit set and adult fitness decreased with decreasing patch size, with an estimated mean lifetime inbreeding depression of 0.9 (obtained by comparing F values in adults and progenies)., Conclusions: Lifetime inbreeding depression strongly counteracts the advantage of reproductive assurance due to selfing in this long-lived species. The poor fitness of selfed offspring should counteract any evolution towards selfing, despite its potential to alleviate the negative consequences of pollen limitation. This study highlights the need to estimate lifetime inbreeding depression, together with mating system and pollination parameters, if we are to understand the actual benefit of selfing and avoid the overestimation of reproductive assurance.

Responses by flowering plants to climate change are complex and only beginning to be understood. Through analyses of 10,295 herbarium specimens of Himalayan Rhododendron collected by plant hunters and botanists since 1884, we were able to separate these responses into significant components. We found a lack of directional change in mean flowering time over the past 45 y of rapid warming. However, over the full 125 y of collections, mean flowering time shows a significant response to year-to-year changes in temperature, and this response varies with season of warming. Mean flowering advances with annual warming (2.27 d earlier per 1 °C warming), and also is delayed with fall warming (2.54 d later per 1 °C warming). Annual warming may advance flowering through positive effects on overwintering bud formation, whereas fall warming may delay flowering through an impact on chilling requirements. The lack of a directional response suggests that contrasting phenological responses to temperature changes may obscure temperature sensitivity in plants. By drawing on large collections from multiple herbaria, made over more than a century, we show how these data may inform studies even of remote localities, and we highlight the increasing value of these and other natural history collections in understanding long-term change.

Fragmentation of natural vegetation creates one of the largest threats to plant-pollinator interactions. Although fragmentation impacts on plant populations have been explored in many, mainly herbaceous, species, the response of wild mass-flowering species is poorly known. Here, we studied 28 heathland patches dominated by the mass-flowering shrub Rhododendron ferrugineum, each presenting different R. ferrugineum floral display sizes (total inflorescence number per patch) and patch isolation (median distance to the three nearest patches). We assessed the impacts of these two factors on (i) heathland patch visitor assemblage (considering R. ferrugineum versus surrounding community) and (ii) R. ferrugineum flower visitation rate and pollen transfer limitation (comparing seed set from emasculated to pollen-supplemented flowers). We found that diversity and abundance of bees visiting R. ferrugineum in heathland patches significantly decreased with decreasing R. ferrugineum floral display, while overall visitor density per patch and flower visitation rate increased. Moreover, a decrease in massive floral display and increase in patch isolation resulted in reduced visitor density in the surrounding community. Even in patches with few individuals, we found disproportionate visitor abundance in R. ferrugineum compared to the surrounding community. Finally, pollen transfer limitation in R. ferrugineum was neither affected by visitation rate nor by patch attributes. By disproportionally attracting pollinators from co-flowering species, and probably promoting geitonogamous pollen transfer, the mass-flowering trait appears adequate to compensate, in terms of conspecific pollen transfer, for the decrease in visitor diversity and abundance and in mate availability, which usually result from population fragmentation., (© 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.)

It is postulated that leaf thermonasty (leaf curling) in rhododendrons under sub-freezing temperatures is caused by water redistribution due to extracellular freezing. We hypothesize that aquaporins (AQPs), the transmembrane water-channels, may be involved in regulating water redistribution and thus leaf curling. Our experimental system includes two Rhododendron species with contrasting leaf curling behavior whereby it was observed in R. catawbiense but not in R. ponticum. We compared leaf movements and the expression of two AQPs, i.e. R. catawbiense/ponticum plasma-membrane intrinsic protein 2 (Rc/RpPIP2;1 and Rc/RpPIP2;2), in the two species under freezing-rewarming and dehydration-rehydration cycles. To determine the relationship between extracellular freezing and leaf-curling, we monitored leaf-curling in R. catawbiense with or without controlled ice-nucleation. Our data indicate that extracellular freezing may be required for leaf curling. Moreover, in both species, PIP2s were up-regulated at temperatures that fell in ice-nucleation temperature range. Such up-regulation could be associated with the bulk-water efflux caused by extracellular freezing. When leaves were frozen beyond the ice-nucleation temperature range, PIP2s were continuously down-regulated in R. catawbiense along with the progressive leaf curling, as also observed for RcPIP2;2 in dehydrated leaves; as leaves uncurled during re-warming/rehydration, RcPIP2 expression was restored. On the other hand, R. ponticum, a non-curling species, exhibited substantial up-regulation of RpPIP2s during freezing/dehydration. Taken together, our data suggest that RcPIP2 down-regulation was associated with leaf curling. Moreover, the contrasting PIP2 expression patterns combined with leaf behavior of R. catawbiense and R. ponticum under these two cycles may reflect different strategies employed by these two species to tolerate/resist cellular dehydration., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Flowering phenology of tree rhododendron (Rhododendron arboreum Sm.) was monitored in situ along elevation gradients in two distinct ecological settings. Observations were carried out in Gaoligong Nature Reserve (GNR) in China and in the Kanchenjunga Conservation Area (KCA) in Nepal. Using the crown density method, flowering events of the selected species were recorded. Flowering duration and synchrony were determined within each site and along the elevation gradient in each study area. Our observations showed high synchrony throughout the elevation gradient, especially for peak flowering. Mean 15-day soil temperature, soil parameters (soil moisture, nitrogen, organic matter and pH), age of the observed trees, and site characteristics (litter cover, canopy cover, inclination) were related to mean initial and peak flowering dates using partial least squares regression (PLS). Results differed between the two sites, but winter temperature was the most important variable affecting the regression model for both initial flowering and peak flowering at both sites. After temperature, soil moisture was the most important variable for explaining initial flowering dates. The distribution of tree rhododendron indicates that it is able to grow in a wide range of habitats with different environmental conditions. The recent trend of rising winter-spring temperature and the detected bloom-advancing effect of high temperatures during this period suggest that tree rhododendron might expand its distributional range in response to global warming.

Many plants capture and kill insects but, until relatively recently, only carnivorous plants with digestive enzymes were known to gain directly from the nutrients of those insects. Recent studies show that some carnivorous plants lack digestive enzymes and have evolved digestive mutualisms with symbiotic insects that digest their prey for them. Rhododendron macrosepalum, a plant with sticky leaves that captures insects, has an association with symbiotic Mirid bugs that consume the insects captured. Here, we determine what the nature of the relationship is between Mirid and plant. We find that R. macrosepalum has no digestive enzymes of its own but that it does not seem to have the ability to absorb hemipteran faeces through its leaf cuticle. Naturally occurring levels of (15) N and (14) N were used to determine that R. macrosepalum gains no nitrogen through its association with the Mirid bugs and that it obtains all of its nitrogen from the soil. The Mirids, on the other hand, seem to obtain nitrogen from insects captured by the plant, as well as from plant tissues. The relationship between plant and Mirid is not a digestive mutualism but more likely an antagonistic relationship. This study adds to our understanding of how digestive mutualisms evolve and shows that insect capture alone, or in combination with a symbiotic insect relationship does not necessarily make a plant 'carnivorous'., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Hydraulic conductance of leaves (K(leaf)) typically decreases with increasing water stress and recent studies have proposed different mechanisms responsible for decreasing K(leaf) . We measured K(leaf) concurrently with ultrasonic acoustic emissions (UAEs) in dehydrating leaves of several species to determine whether declining K(leaf) was associated with xylem embolism. In addition, we performed experiments in which the surface tension of water in the leaf xylem was reduced by using a surfactant solution. Finally, we compared the hydraulic vulnerability of entire leaves with the leaf lamina in three species. Leaf hydraulic vulnerability based on rehydration kinetics and UAE was very similar, except in Quercus garryana. However, water potentials corresponding to the initial decline in K(leaf) and the onset of UAE in Q. garryana were similar. In all species tested, reducing the surface tension of water caused K(leaf) to decline at less negative water potentials compared with leaves supplied with water. Microscopy revealed that as the fraction of embolized xylem increased, K(leaf) declined sharply in Q. garryana. Measurements on leaf discs revealed that reductions in lamina hydraulic conductance with dehydration were not as great as those observed in intact leaves, suggesting that embolism was the primary mechanism for reductions in K(leaf) during dehydration., (Published 2011. This article is a U.S. Government work and is in the public domain in the USA.)

One major characteristic of invasive alien species is their occurrence at high abundances in their new habitat. Flowering invasive plant species that are visited by native insects and overlap with native plant species in their pollinators may facilitate or disrupt native flower visitation and fertilisation by forming large, dense populations with high numbers of flowers and copious rewards. We investigated the direction of such a proposed effect for the alien invasive Rhododendron ponticum in Irish habitats. Flower visitation, conspecific and alien pollen deposition, fruit and seed set were measured in a self-compatible native focal plant, Digitalis purpurea, and compared between field sites that contained different relative abundances of R. ponticum. Flower visitation was significantly lower at higher alien relative plant abundances than at lower abundances or in the absence of the alien. Native flowers experienced a significant decrease in conspecific pollen deposition with increasing alien abundance. Heterospecific pollen transfer was very low in all field sites but increased significantly with increasing relative R. ponticum abundance. However, lower flower visitation and lower conspecific pollen transfer did not alter reproductive success of D. purpurea. Our study shows that indirect interactions between alien and native plants for pollination can be modified by population characteristics (such as relative abundance) in a similar way as interactions among native plant species. In D. purpurea, only certain aspects of pollination and reproduction were affected by high alien abundances which is probably a result of high resilience due to a self-compatible breeding system. Native species that are more susceptible to pollen limitation are more likely to experience fitness disadvantages in habitats with high relative alien plant abundances.

Plants in nutrient-poor environments typically have low foliar nitrogen (N) concentrations, long-lived tissues with leaf traits designed to use nutrients efficiently, and low rates of photosynthesis. We postulated that increasing N availability due to atmospheric deposition would increase photosynthetic capacity, foliar N, and specific leaf area (SLA) of bog shrubs. We measured photosynthesis, foliar chemistry and leaf morphology in three ericaceous shrubs (Vaccinium myrtilloides, Ledum groenlandicum and Chamaedaphne calyculata) in a long-term fertilization experiment at Mer Bleue bog, Ontario, Canada, with a background deposition of 0.8 g N m(-2) a(-1). While biomass and chlorophyll concentrations increased in the highest nutrient treatment for C. calyculata, we found no change in the rates of light-saturated photosynthesis (A(max)), carboxylation (V(cmax)), or SLA with nutrient (N with and without PK) addition, with the exception of a weak positive correlation between foliar N and A(max) for C. calyculata, and higher V(cmax) in L. groenlandicum with low nutrient addition. We found negative correlations between photosynthetic N use efficiency (PNUE) and foliar N, accompanied by a species-specific increase in one or more amino acids, which may be a sign of excess N availability and/or a mechanism to reduce ammonium (NH(4)) toxicity. We also observed a decrease in foliar soluble Ca and Mg concentrations, essential minerals for plant growth, but no change in polyamines, indicators of physiological stress under conditions of high N accumulation. These results suggest that plants adapted to low-nutrient environments do not shift their resource allocation to photosynthetic processes, even after reaching N sufficiency, but instead store the excess N in organic compounds for future use. In the long term, bog species may not be able to take advantage of elevated nutrients, resulting in them being replaced by species that are better adapted to a higher nutrient environment.

In the European Alps, Rhododendron ferrugineum grows in silicate regions while Rhododendron hirsutum is restricted to limestone areas. At geologically mixed sites, also hybrids (Rhododendron × intermedium) can occur. We hypothesised that hydraulic properties would vary with the species' habitat requirements. Key hydraulic parameters (vulnerability to drought-induced embolism, stomata regulation) and related wood characteristics as well as diurnal courses of water potential (Ψ) and stomatal conductance were analysed on plants growing on a silicate, a limestone and a geologically mixed site. Highest embolism resistance[Ψ at 50% loss of conductivity (Ψ (50)), -3.24 ± 0.18 MPa] and the highest safety margin between the Ψ at stomata closure (Ψ (SC) at 10% of maximal leaf conductance) and Ψ (50) were observed in R. hirsutum at the limestone site (1.57 MPa). Like in R. ferrugineum, hydraulic parameters indicated less resistance at the geologically mixed site. Highest Ψ (50) (-1.95 ± 0.12 MPa), corresponding to wide conduits and a reduced conduit wall reinforcement, was found in R. × intermedium. Diurnal courses indicated a rapid stomata closure in response to low Ψ in R. hirsutum and R. × intermedium. The plasticity in drought adaptation of R. hirsutum corresponds to its ability to colonise dry limestone areas. In contrast, hydraulic limitations of R. × intermedium may explain restrictions to rather moist sites. This study provides insight into the role of xylem hydraulics and stomata regulation in shrub water relations, interspecific and site-specific differences in drought adaptation, as well as effects of hybridisation on plant hydraulics.

The natural hybridization that occurs between two sympatric species of Rhododendron subgenus Hymenanthes in Yunnan, China, was investigated. In field observations, it was noted that the putative hybrids between R. delavayi Franch. and R. cyanocarpum (Franch.) Franch. ex W.W. Sm. had intermediate morphologies. On the basis of morphology, chloroplast DNA (trnL-rpl32) and nuclear DNA (waxy), hybrids and parental species were identified. Hybridization occurred in both directions, but was asymmetrical, with R. delavayi as the major maternal parent. Reciprocal hand pollination treatments showed that either species, as pollen donor or pollen receiver, could produce fruits. It was noted that fruit set varied among treatments. The same pollinators (bumblebees) were shared in both parental species. From these results, we conclude that individuals with intermediate morphologies are indeed of hybrid origin from natural hybridization between R. cyanocarpum and R. delavayi. Furthermore, we presume the hybridization at the study site could have been initiated by habitat disturbance in the 1950s, and we may hence witness the early stages of hybrid swarm formation.

The ability to determine winter frost resistance of woody plants is limited for two reasons: (1) assessment of frost damage in midwinter is extremely difficult because results obtained by the currently available viability assays deviate greatly and (2) equipment that allows plants to be frozen at controlled freezing and thawing rates to below the midwinter frost resistance of most Northern Hemisphere woody plants is unavailable. To overcome these limitations, we developed a novel low-temperature freezing system (LTFS) that makes it possible to conduct in situ freezing experiments in midwinter with full control of cooling and thawing rates down to -70 degrees C. Frost resistance can be determined unequivocally by the regrowth test. The LTFS was tested on various, mostly subalpine, woody plants. Results obtained demonstrate the importance of conducting frost tests in situ. In needles of Picea abies (L.) Karst., frost injuries were not visible immediately after the frost test but took several weeks to develop fully. The low-freezing temperatures attained and the small control oscillations (typically +/-0.1 K) of the LTFS during cooling permitted in situ detection of low-temperature freezing exotherms in xylem of Quercus robur L. and in buds of P. abies and Rhododendron ferrugineum L., all of which showed supercooling. With the LTFS, the effects of low temperatures on plants can be specified directly by in situ assessment and regrowth tests.

Hybrids between the acid-loving species Rhododendron ferrugineum and the basic soil species Rhododendron hirsutum occur on soils of intermediate pH in the European Alps. Material from two hybrid zones approximately 500 m apart, and also nearby populations of each parent species, was surveyed for presence/absence of 31 random amplified polymorphic DNA markers that distinguish parents. Based on morphological assessment, the material comprised 51 putative hybrids, 18 putative R. ferrugineum individuals and 26 putative R. hirsutum plants. RAPD data were analysed using a Bayesian approach implemented by the program newhybrids, and also by principal coordinates analysis. The identity of all R. ferrugineum plants examined was confirmed; however, of the putative R. hirsutum individuals examined, two were certainly and 11 possibly hybrid derivatives. Among all hybrid derivatives examined, about half were designated as F1s or a similar class, otherwise backcrosses to R. hirsutum appeared to be common whereas other hybrid classes were rare and backcrosses to R. ferrugineum possibly absent. Despite this, artificially generated seed of F2 class and backcrosses in each direction showed greater viability than one parent (R. hirsutum). Introgression from R. ferrugineum was also detected in a population that from morphology appeared to contain only R. hirsutum. Hence, the direction of backcrossing might be highly asymmetric within hybrid zones, causing unidirectional gene flow from R. ferrugineum into R. hirsutum. Conversely, the rarity of backcrosses to R. ferrugineum, F2s and later hybrid generations, which might be due to phenology effects and habitat-mediated selection, could play a part in restricting gene flow towards R. ferrugineum.

The early light-induced proteins (ELIPs) are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting Chl a/b-binding proteins. Recent evidence from physiological and genetic (mutant) studies supports a photoprotective function for ELIPs, particularly when green tissues are exposed to high light intensities at suboptimal temperatures. Broad-leaved evergreens belonging to genus Rhododendron are often exposed to a combination of low temperatures and high light in their natural habitat as the understory plants in deciduous forests and, therefore, are expected to employ photoprotective strategies during overwintering phase. Here we report analysis and characterization of previously identified ELIP expressed sequence tags (ESTs) from winter-collected Rhododendron catawbiense leaves. 5' or 3' rapid amplification of complementary DNA ends (RACEs) coupled with bioinformatic analyses were used to identify seven unique ELIPs from the 40 ESTs and were designated as RcELIP1-RcELIP7. Phylogenetic analysis revealed separate clustering of ELIP homologs from lower plants, monocots and eudicots (including RcELIPs) and further indicated an evolutionary divergence of ELIPs among angiosperms and gymnosperms. To gain insights into the cold acclimation (CA) physiology of rhododendrons, relative and absolute quantitative expression of RcELIPs was examined during seasonal CA of R. catawbiense leaves using real time reverse transcriptase-polymerase chain reaction. All seven RcELIPs were distinctly upregulated during the CA. It is postulated that RcELIPs expression constitutes an adaptive response to cold and high light in winter-adapted rhododendron leaves and perhaps plays a key role in the protection of photosynthetic apparatus from these stresses.

Four in vitro experiments were set up to verify the colonization potential of ectomycorrhizal (EcM) Cenococcum geophilum FR. (strain CGE-4), saprotrophic Geomyces pannorum (LINK) SIGLER & CARMICHAEL (GPA-1) and a frequent root-associated, potentially ericoid mycorrhiza (ErM)-forming Meliniomyces variabilis Hambleton & Sigler (MVA-1) in roots of Rhododendron and Vaccinium. A typical ErM fungus, Rhizoscyphus ericae (Read) Zhuang & Korf (RER-1), was included for comparison. All fungal strains intracellularly colonized rooted Vaccinium microcuttings: GPA-1 occasionally produced hyphal loops similar to ErM, MVA-1 and RER-1 exhibited a typical ErM colonization pattern. CGE-4 hyphae grew vigorously on and around newly formed roots and rarely penetrated turgescent rhizodermal cells forming intracellular loose loops. Rooting of Rhododendron sp. microcuttings was not promoted by any fungal strain except CGE-4, which also promoted the most vigorous growth of Rhododendron ponticum L. seedlings. The widespread EcM fungus C. geophilum has a potential to colonize non-EcM roots and support their development which may influence overall growth of ericaceous plants. As shown for G. pannorum, structures resembling ErM may be formed by fungi that are to date not regarded as ericoid mycorrhizal.

In alpine ecosystems, microscale variation in snowmelt timing often causes different flowering phenology of the same plant species and seasonal changes in pollinator activity. We compared the variations in insect visitation, pollen dispersal, mating patterns, and sexual reproduction of Rhododendron aureum early and late in the flowering season using five microsatellites. Insects visiting the flowers were rare early in the flowering season (mid-June), when major pollinators were bumblebee queens and flies. In contrast, frequent visitations by bumblebee workers were observed late in the season (late July). Two-generation analysis of pollen pool structure demonstrated that quality of pollen-mediated gene flow was more diverse late in the season in parallel with the high pollinator activity. The effective number of pollen donors per fruit (N(ep)) increased late in the season (N(ep) = 2.2-2.7 early, 3.4-4.4 late). However, both the outcrossing rate (t(m)) and seed-set ratio per fruit were smaller late in the season (t(m) = 0.89 and 0.71, seed-set ratio = 0.52 and 0.18, early and late in the season, respectively). In addition, biparental inbreeding occurred only late in the season. We conclude that R. aureum shows contrasting patterns of pollen movement and seed production between early and late season: in early season, seed production can be high but genetically less diverse and, during late season, be reduced, possibly due to higher inbreeding and inbreeding depression, but have greater genetic diversity. Thus, more pollinator activity does not always mean more pollen movement.

We have previously analysed expressed sequence tags (ESTs) from non-acclimated (NA) and cold-acclimated (CA) Rhododendron leaves, and identified highly abundant complementary DNAs (cDNAs) possibly involved in cold acclimation. A potentially significant, but relatively unexplored, application of these EST data sets is the study of moderately abundant cDNAs, such as those picked only 1-3 times from each Rhododendron EST library containing approximately 430 ESTs. Using statistical tests and Northern blots, we established that the probability of differential expression of moderately abundant cDNAs based on the EST data is, indeed, a reasonably accurate predictor of their 'true' upregulation or downregulation as 11 out of 13 cDNAs (85%) studied fit this criterion. The analyses also revealed four aspects of cold acclimation in Rhododendron leaf tissues. Firstly, the concomitant upregulation of long-chain acyl-coenzyme A (acyl-CoA) synthetase, CTP:cholinephosphate cytidylyltransferase and delta-12 fatty acid desaturase in CA leaf tissues suggests that phospholipid biosynthesis and desaturation are important components of cold hardening in Rhododendron. Secondly, upregulation of plastidic nicotinamide adenine dinucleotide phosphatemalic enzyme (NADP-ME) in CA tissues suggests that malate is an important source of acetyl-CoA used for fatty acid biosynthesis during cold acclimation. Thirdly, down-regulation of plasma membrane intrinsic protein (PIP)2-1 aquaporin and upregulation of gated outward rectifying K+ channel (GORK) in CA tissues may be associated with the protection of overwintering leaves from freeze-induced cellular dehydration. Fourthly, upregulation of coumarate 3-hydroxylase may be associated with cell wall thickening in CA tissues. Physiological implications of these results, which reveal potentially novel regulations of cold acclimation in overwintering woody evergreens, are discussed. This work highlights the importance of also investigating low/moderately abundant ESTs (in addition to highly abundant ones) in genomic studies, in that it offers an effective strategy for identifying stress-related genes, especially when large-scale cDNA sequencing/microarray studies are not possible.

With P-V techniques, this paper determined and analyzed the water parameters of main tree species in the water conservation forest of Qilian Moutains. The results showed that the test water parameters varied with tree species, reflecting the complicacy of the drought-resistance mechanism of trees. Among 10 parameters, /phi pi100 - phi pi0/, RCV, ROWC0 epsilon(max) could reflect most information of drought resistibility. Based on their drought resistibility, the test tree species could be classified into 4 groups, i.e., high drought resistibility (Picea crassifolia and Rhododendron thymifolium ), sub-high drought resistibility (Sabina przeualwkii, Rhododendron anthopogonoides, Rhododendron capitaturn and Rhododendron przewalskii), low drought resistibility (Populus cathyana), and inferior drought resistibility (Poentilla fruticosa, Siraea salicifolia and Betula albosinensis). The relationships between predawn leaf water potential and soil water content could be well modeled by hyperbola function, power function or exponent function. Meanwhile, the test tree species could be also grouped as high potential delay dehydration species (Betula albosinensis and Rhododendron przeualskii), sub-high potential delay dehydration species (Picea crassifolia, Rhododendron thymifolium and Rhododendron capitatum), inferior potential tolerance species (Sabina przewalskii), and low potential tolerance species (Potentilla fruticosa, Spiraea salicifolia and Rhododendron anthopogonoides), based on their drought-resistance mechanism.

Many alpine plants are predominantly outcrossing, thus plant reproductive success is highly dependent on effectiveness of pollinators. How pollinators transfer pollen from one flower to another is of great interest in understanding the genetic structure in plant populations. We studied (1) the role and effectiveness of insect visitors for pollination, and (2) their contribution as pollen vectors for gene dispersal in a Rhododendron ferrugineum population. Various insect visitors were recorded, including Hymenoptera, Diptera, Coleoptera, and Lepidoptera. The most frequent and effective insects were honey bees and bumblebees. Muscid flies were considered as important pollinators, particularly due to their relatively high visitation rate. Syrphid flies, Formicidae, and Coleoptera were ineffective in transporting pollen, while the effectiveness of Lepidoptera and Empididae was negligible. A fluorescence labelling experiment revealed that pollen dispersal was restricted (0 - 2 m) in a dense R. ferrugineum stand and decreased in a leptokurtic fashion. This might lead to geitonogamous self-pollination that could explain the close relationship between individuals found in genetic studies of R. ferrugineum. However, some pollen grains may travel 40 - 45 m, which implies the occurrence of cross-pollination through the foraging activities of bumblebees and honey bees.

We studied variations in water relations and drought response in five Himalayan tree species (Schima wallichii (DC.) Korth. (chilaune) and Castanopsis indica (Roxb.) Miq. (dhale katus) at an elevation of 1400 m, Quercus lanata Smith (banjh) and Rhododendron arboreum Smith (lali gurans) at 2020 m, and Quercus semecarpifolia Smith (khasru) at 2130 m) at Phulchowki Hill, Kathmandu, Nepal. Soil water potential at 15 (Psi(s15)) and 30 cm (Psi(s30)) depths, tree water potential at predawn (Psi(pd)) and midday (Psi(md)), and leaf conductance during the morning (g(wAM)) and afternoon (g(wPM)) were observed from December 1998 to April 2001, except during the monsoon months. There was significant variation among sites, species and months in Psi(pd), Psi(md), g(wAM) and g(wPM), and among months for all species for Psi(s15). Mean Psi(pd) and Psi(md) were lowest in Q. semecarpifolia (-0.40 and -1.18 MPa, respectively) and highest in S. wallichii (-0.20 and -0.63 MPa, respectively). The minimum Psi value for all species (-0.70 to -1.79 MPa) was observed in March 1999, after 4 months of unusually low rainfall. Some patterns of Psi(pd) were related to phenology and leaf damage. During leafing, Psi(pd) often increased. Mean g(wAM) and g(wPM) were highest in Q. semecarpifolia (172 and 190 mmol m(-2) s(-1), respectively) and lowest in C. indica (78 and 74 mmol m(-2) s(-1), respectively). Soil water potential (Psi) at 15 cm depth correlated with plant Psi in all species, but rarely with g(wAM) and not with g(wPM). Plant Psi declined with increasing elevation, whereas g(w) increased. As Psi(pd) declined, so did maximal g(w), but overall, g(w) was correlated with Psi(pd) only for R. arboreum. Schima wallichii maintained high Psi, with low stomatal conductance, as did Castanopsis indica, except that C. indica had low Psi during dry months. Rhododendron arboreum maintained high Psi(pd) and g(w), despite low soil Psi. Quercus lanata had low g(w) and low Psi(pd) in some months, but showed no correlation between tree Psi and g(w). Quercus semecarpifolia, which grows at the highest elevation, had low soil and plant Psi and high g(w).

Frost resistance and ice nucleation temperatures of leaves, from bud swelling until after full expansion, were measured in situ for five major woody timberline species with recently developed field freezing equipment. Frost resistance determined in situ on leaves of attached twigs was significantly higher than values determined on detached leaves in laboratory tests (e.g., the temperature at which incipient frost damage was observed (LTi) was 1.2 degrees C higher for detached leaves than for attached leaves of Picea abies (L.) Karst.). Frost resistance of leaves of all species changed significantly during shoot expansion (e.g., changes of 7.2 and 11 degrees C for Rhododendron ferrugineum L. and Larix decidua Mill., respectively). Expanding leaves (between 0 and 60% of full expansion) were the most sensitive to frost, with LTi values ranging from -3.4 degrees C in R. ferrugineum to -6.3 degrees C in L. decidua. Among the studied species, P. abies and R. ferrugineum were the most frost sensitive throughout the shoot elongation period. In situ freezing patterns of leaves of attached twigs also differed from those of leaves of excised twigs. During leaf expansion, two distinct freezing exotherms were always registered in situ. The first freezing event (E1, high-temperature exotherm) was recorded at -1.5 +/- 0.2 degrees C and reflected extracellular ice formation. Exposure of leaves to temperatures at which E1 occurred was, in all cases, noninjurious. The low-temperature exotherm (E2) mostly coincided with frost damage, except for some stages of leaf expansion in R. ferrugineum and P. abies, indicating that in situ freezing exotherms were not accurate estimators of frost damage in these species.

Isolating barriers between interbreeding sympatric or parapatric interfertile species are maintained by processes that occur within their hybrid zones. Although the effects of intrinsic selection on hybrid fitness are well known, less is understood about extrinsic fitness variation. At Tiryal Dag, northeast Turkey, Rhododendron x sochadzeae (R. ponticum x caucasicum) forms large populations in which neither segregation nor backcrossing occur, in habitats intermediate between those of its parents. Using single-copy species-specific random amplified polymorphic DNA and inter simple sequence repeat markers, it was determined that most or all R. x sochadzeae plants are F1s, and that there are many separate genets present. Hand pollination and germination experiments showed that R. x sochadzeae plants can produce viable seed of F2s or backcrosses in either direction. Furthermore, adult backcrosses have been observed in habitats atypical for R. x sochadzeae. From this, all non-F1 hybrid derivatives appear to be eliminated in the hybrid zone at Tiryal Dag as a result of postgermination selection. This absence of post-F1 hybrid derivatives apparently prevents introgression. This type of hybrid population is here termed an F1-dominated hybrid zone (F1DZ), and also occurs in Encelia. The observed dominance of F1s within a narrow habitat range is best explained by habitat-mediated superiority of F1s over all other genotype classes. Therefore, habitat-mediated selection against the second hybrid generation might be preventing interspecific gene flow in R. x sochadzeae. F1DZ formation is postulated to require the formation of F1s in quantity, habitat-mediated superiority in F1s, and highly specific habitat conditions.