The alpines of the high altitude mountains are known for their hostile environment, which include fluctuations in air temperature, total atmospheric pressure, heat and UV radiation, and plants growing there encounter interacting stresses such as dehydration and low temperature (Körner 2007, Shepherd & Griffiths 2006). Altitudinal gradients in high altitude mountain ecosystem further influenced plant growth and reproduction, as well as the resource availability of such as water, heat and nutrients (Körner 2003). High altitudes mountains ecosystems' biological diversities have long been investigated by ecologists, which are most fragile habitats and are also rich depositories of the species (Kumar and Sharma 2016). R. anthopogon is especially interesting due to its altitudinal range and capacity to tolerate the year's major environmental fluctuations. As a result, it is anticipated to have adequate systems to deal with the extreme circumstances of high altitudes. Hence, we investigated how an altitudinal gradient influenced the eco-physiological and biochemical characteristics of this evergreen dwarf shrub. R. anthopogon grows on the highest tops of the mountains in the study area, which is why three study sites were chosen. Site I is the starting location for the R. anthopogon occurrence, Site III is the peak of the mountain summit, the top resort of the R. anthopogon in the study region, and Site II is the intermediate site between the two.
Community composition and phyto-sociological study of R. anthopogon
Quantitative data of the phyto-sociological parameters are the major players in determining the status of a plant community and its ecosystem patterns (Bhat et al. 2020). The phyto-sociological study of R. anthopogon showed variation with altitude. In this study we found that the several herbaceous plant species are associated with R. anthopogon and the maximum number of herbaceous species associated with this species was reported at higher elevation. The vegetation analysis shown that R. anthopogon was associated with 30 species (falling into 27 genera and 20 families). The dominant herbaceous species associated with R. anthopogon in the study sites encompassed Tenaxia cachemyriana, Acomastylis elata, Viola biflora, Trachydium roylei, shrub species Rhododendron campanulatum and Juniperus communis. These species were stated earlier also as the dominant plant species in sub-alpine and alpine region of Garhwal Himalaya (Chandra et al. 2018, Jamloki et al. 2021) and Nepal Himalaya (Sharma et al. 2020). Altitude is a key physiographic attribute that extremely impacts the distribution, structure of plant species and growth forms, and slight modification in altitude sharply changes the topographic and climatic conditions (Kharakwal et al. 2005). The density of R. anthopogon was found maximum at higher elevation (3570 m asl) while minimum at lower elevation (3370 m asl).The importance value index (IVI) of this species showed its dominant nature at higher elevation where it forms pure dense patches and support the favourable habitat for the growth and development of other herbaceous plant species (Sharma et al. 2020). The variance in species diversity between the communities at different altitudes usually arises from variation in the quality of the site (Denslow, 1980). In this study, we observed that the different diversity indices such as Shanon- Wiener diversity index and Simpson index of dominance showed a variation with altitude. Shannon Wiener diversity values parallel to present study reported by the many investigators in the Himalayan range. However, the present study diversity values (2.28 to 2.50) and Simpson diversity values (0.86 to 0.95) suggest a dominant nature of only few species at higher altitudes. The value of Simpson diversity values in the study sites indicated the dominance of one or few species i.e., Trachydium roylei, Tenaxia cachemyriana, Carex setosa, Sibbaldia cuneata, (Jamloki et al. 2021).
Morphological characteristics of R. anthopogon along an altitudinal gradient
Leaf size is the most significant factor in acclimatisation and adaptation to harsh environmental conditions, and reduction in leaf size is the key feature in most stresses in alpine plants, whether trees, herbs, or shrubs (Paudel et al. 2019, Liu et al. 2020). Low temperature, high light intensity, and wind speed are important factors influencing leaf form and size in alpine plants, and leaf trait diversity along altitude is an adaptive strategy to deal with environmental problems (Liu et al. 2020, Li et al. 2020). The leaf length and area of R. anthopogon decreased with increasing elevation in this study as well. Low temperatures, other climatic extremes including smaller growing season impede plant development and growth at high altitudes (Körner 2012). Plant organ size reduction with increasing elevations is helpful in regions with chilling temperature, high wind speed and heavy snowfall, since it helps the plant to reduce damage of the tissues while retaining strong aerodynamic and thermal resistance (Körner 2003, 2012). We found considerable variation and trends in leaf area (LA), leaf thickness, specific leaf area (SLA), and leaf dry matter content (LDMC) over the elevation gradient in our study. However, Rathore et al. (2018) found no differences in SLA and LDMC along altitudinal gradients in the R. anthopogon population in Himachal, India. Reductions in leaf area and SLA at higher elevations were too the adaptations to lower temperature, higher light intensities, and other associated stress factors, as detected in some alpine evergreens viz. Nothofagus menzisii (Körner et al. 1986) and Metrosideros polymorpha (Tang and Oshawa 1999). We observed that the leaf thickness of R. anthopogon increased with elevation in our study. This demonstrates that smaller and thicker leaves are in general at higher elevations; increases in leaf thickness also account for enhanced mechanical strength to endure stressful circumstances such as cold temperature and heavy wind at higher altitude (Lütz 2010). Leaf thickness rises together with altitudinal gradients, which is consistent with previous research (Körner 2003, Zhang et al. 2014, Liu et al. 2020). Thicker leaves allow better leaf adaptation in harsh environments; the thicker the leaves, the better the buffer between inner and outside leaf temperatures, which contributes to sustaining normal physiological activity in plants at higher altitudes when temperatures are low (Liu et al. 2020). Thicker leaves at high altitudes can help protect against the damage caused by high-level ultraviolet irradiation (Ma et al. 2012) and allow for increased water storage (Guo et al., 2017). Petiole length decreased with elevation gradients in R. anthopogon in our study; however petiole diameter did not decrease as much as length, indicating that petiole became thicker and shorter as altitude increased. It could be an adaptation for plants in more exposed alpine beds, because shorter petioles allow plants to arrange their leaves more compactly and provide better temperature homeostasis.
Physio-Biochemcal properties of R. anthopogon in the study area
Temperature extremes caused by seasonal and often diurnal freeze-thaw cycles with hot days and cold nights, water desiccation, particularly observed by perennials and evergreens in the alpine highlands during winters, can kill plants if they are not properly acclimated (Körner 2007). Plants must develop cold hardiness and resistance in order to acclimate or adapt in such hostile environments, which requires morphological as well as biochemical and physiological adjustments to the various plant biochemical and physiological processes. Soluble sugar and soluble protein are important in plant osmoregulation (Cui et al. 2018), and their accumulation in cells increases cellular fluidity, stabilises cell membranes, and decreases osmotic potential (Basu et al. 2007). In this investigation, soluble carbohydrates and soluble protein appear to play an essential role in cold acclimation in R. anthopogon. We found that the soluble starch and protein content of R. anthopogon increased along with the altitudinal gradient in both seasons studied, which was consistent with previous research in alpine floras (Rathore et al. 2018, Cui et al. 2018), but that soluble sugar positively correlated with altitude in summer but decreased with altitude in winter. However, as elevation increased, the chlorophyll content decreased while the carotenoids increased. Higher elevations floras do have higher concentration of carotenoids, which protects plants from UV radiation and oxidative stress. Carotenoids are also lipophilic antioxidants that may detoxify many kinds of reactive oxygen species (ROS).
Alpine ecosystems present extreme challenges to its inhabitants, resulting in the development of reactive oxygen species (ROS) diurnal and seasonal freeze-thaw cycles, high light intensities, increased UV radiation exposure, desiccation, and other factors all contribute to cellular damage and ROS generation in alpine plants (Trivedi and Nautiyal 2020). Although alpine flora protected very well from the ROS by enzymes, pigments like carotenoids and their higher level of the secondary metabolites (Germino 2014). The plant cell's lipid peroxidation by ROS can be assessed by detecting the Malondialdehyde (MDA) concentration, which is an indication of lipid peroxidation and oxidative stress (Hashim et al. 2020). The environment becomes harsher in alpines as altitude increases, and more oxidative stress is expected, yet R. anthopogon appears to be well buffered from ROS due to its lower lipid peroxidation or MDA activity as altitude increases. Lower MDA content as elevation increases indicate that plants have a high anti-oxidative ability to avoid lipid peroxidation, indicating greater stress resistance (Campos et al., 2003). Presence of the lower MDA concentration in plants from higher elevations also shown their stronger cold resistance capacities, as it is commonly considered that plants with higher lipid peroxidation are susceptible to cold (Rathore et al. 2018) This appears to be clear in the case of R. anthoogon in this study since as altitude increased, important antioxidants such as phenolics, carotenoids, and enzyme activity of the SOD and POD increased. Plants such as Leymus secalinus, an alpine plant of the Tibetan Plateau, similarly demonstrated a drop in MDA concentration as altitude increased (Cui et al. 2018). Several alpine flora showed the very high ROS protection such as Dryas octopetala, Rhododendron ferrugineum, and Vaccinium myrtillus with high phenolic content (Lefebvre et al. 2016) a study in Gaultheria trichophylla showed positive correlations between the altitudes and the phenolic content (Bahukhandi et al. 2017) such like the our study. The present study was observed that the several enzymatic activity of R. anthopogon such as SOD and POD increased with increasing elevation which favours their growth in such types of adverse climatic conditions. Antioxidant protection was proved to play important role in Rhododendron chrysanthum (Zhou et al. 2017) adaptation in alpine habitat in the same way higher SOD and POD activities are must for R. anthoogon existence in higher altitudes. The phenolic content of R. anthopogon was highest at higher elevations and lowest at lower elevations. A variety of plant species have shown increases in phenolic content after UV-B exposure (Chaves et al. 1997, Turunen et al. 1999).
The most notable finding in this study was the seasonal change in biochemical components, which was particularly noticeable for the lower altitude or Site II grown R. anthopogon plants. Plants of the same species demonstrated stronger freezing tolerance than their lower altitude counterparts in numerous alpine plant species investigations. Neuner et al. (2020) shown convincingly that elevation has a substantial effect on freezing tolerance in numerous alpine plant species. Sit III of the study area is present in near the peak of the mountain summit and hence exposed to more climatic extremes in the whole time of a year, to cope up those extremes plants are already well acclimatized. Lower altitude plants, on the other hand, are often less exposed to climate extremes in summer, but before autumn and winter, they require physio-biochemical adjustment to achieve freezing resistance (Sierra-Almeida et al. 2009, Neuner et al. 2020). In October, the pigments, total soluble proteins, total soluble sugar, total soluble starch, and total phenolic content were higher than in May. In the research area, the end of September and early October mark the beginning of winter and the period for evergreen plants to develop more freezing tolerance. The accumulation of osmolytes for osmotic adjustment is the most common low temperature adaptation of plants (Magaña Ugarte et al. 2019), and an increase in total soluble sugar is found in many alpine plants as winter approaches and other abiotic stress conditions. In their investigation of four Rhododendron species, Li et al. (2022) found the highest total soluble sugar levels in fall and winter in R. aganniphum, R. nyingchiense, R. wardii, and R. triflorum. In October, there was a higher accumulation of total soluble protein, and many research revealed that proteins have a role in the formation of freezing tolerance in plants. Physiological, biochemical, and proteomic investigation of the alpine plant Potentilla saundersiana indicates that proteins promoted abiotic acclimatisation to high-altitude conditions in plants and that protein-driven acclimation was altitude dependent (Ma et al. 2015). In our investigation, all pigments showed an increase in the onset of winter in R. anthopogon, with Chl b showing the greatest increase. High altitude plants are characterised by a higher ratio of Chl a/b and a lower ratio of Chl/Car (Germino 2014) due to higher xanthophylls and other carotenoids content that also provides oxidative protection from non-radiative dissipation of excitation energy and showed altitudinal and seasonal variations (Gonzalez et al. 2007). Higher pigment and carbohydrate content in R. anthopogon leaves at the start of winter indicated that the plant might be physiologically active and continue out photosynthesis until late winter. Some evergreen alpine plants, such as Euonymus kiautschovicus and Mahonia repens, showed no winter photosynthetic down-regulation (Adams et al. 2002). The initial study period is May, which represents the beginning of summer or the beginning of the active growth period in the northern hemisphere's alpine eco-system, and October represents the end of the active growth period, which could be one reason for the higher pigments and primary metabolites at the end of the growing season so that the plant can gain as many benefits as possible The activity of most enzymes decreases over the winter due to the obvious temperature kinetics of the enzymes, which could explain why SOD and POD enzyme activities decreased in R. anthopogon in this study. Because of the decrease in overall physio-biochemical activity during the winter, ROS production is also reduced, resulting in lower MDA content. In winter, low antioxidant enzyme protection may be compensated by increasing total phenolic content to defend against potentially hazardous ROS and radiations in R. anthopogon.
Seed Germination
The seeds of R. anthopgon needed cold stratification to germinate, and hormonal treatment with GA3 improved germination even more. The strict requirement for cold stratification for germination in R. anthopogon seeds revealed that the species has physiological dormancy (PD) and that cold temperatures below 4 Ċ are required for breaking seed dormancy and increasing seed germination percentage by GA3, indicating that the PD in R. anthopogon seeds is intermediate to non-deep PD (Baskin and Baskin 2004). According to Baskin and Baskin (2004), PD dormancy is present in more than 70% of arctic-alpine floras, and cold stratification is the most efficient technique to overcome such dormancies. Cold stratification has also been demonstrated to serve a substantial influence in reducing the higher temperature requirements for seed germination by some Tundra species (Baskin and Baskin 2001). In various studies, Rhododendrons showed enhanced germination after cold stratification, such as in Rhododendron arboreum Smith, where germination increased significantly on stratification but declined as the stratification period progressed (Vipasha et al. 2017), and Rhododendron aureum, where germination percentage was also higher in moist chilled treated seeds (Shimono and Kudo 2005). The cold stratification requirement in nature in alpine areas is fulfilled by their snow fed beds, but due to the current climate change scenario, increasing soil temperature leads to earlier snow layer thinning, so the stratifying requirement of the seeds may not be completed, resulting in low propagation via seeds in some alpine flora in the near future (Briceño et al. 2015). As a result of the investigation, the failure of R. anthopogon to germinate without stratification may be a dangerous circumstance for the species.
GA3 pre-treatment has been shown to promote total seed germination in several floras with dormant or non-dormant seeds (Herranz et al. 2010), as well as to increase seed germination percentage and speed. In our study, GA3 was able to increase germination percentage by 20–25% in R. anthopogon and also accelerate germination pace with lower MGT, greater germination percentage on the 20th day, and earlier germination beginning. Other Rhododendron species, including R. niveum (Singh et al. 2010), R. purdomii (Zhao 2014), and R. aureum (Shimono and Kudo 2005), showed improved germination after GA3 treatment. Even low temperatures improved germination by increasing endogenous GA3 levels; GA3 improves seed germination by lowering endosperm resistance and encouraging radical development during germination (Baskin and Baskin 2001). On the basis of final seed germination percentages, the soil substratum was shown to be the most effective for R. antopogon seed germination; nevertheless, the difference between the substratums for seed germination features is not as pronounced. When cold pre-treated R. anthopogon seeds are treated with GA3 prior to germination, any substrate, such as filter paper, native soil, or MS media, can be used. As a result, the applicability and suitability of a specific substratum can be crucial on a necessity basis. For example, MS media is the best substratum for preparing disease-free sterile seedlings, while soil substratum is the best for growing plants in the field.