Impacts of habitat variability on the phenotypic traits of Aconitum violaceum ex Stapf. at different altitudes and environmental conditions in the Ladakh Himalaya, India

Aconitum violaceum Jacq. ex Stapf is endemic to the Northern Himalayan regions of India, Pakistan and Nepal. To adapt and endure the rigorous stressful situations, phenotypic features of a specific plant species vary across different climatic conditions and elevations. The aim of this study was to reveal the impact of altitude and environmental conditions on the morphological attributes of a threatened species A. violaceum . Plants at high elevations (Maan - man, 4010 m asl) were short; whereas the plants grown at ecological conditions of population - I (Khawous, 3220 m asl) showed better growth and development in all morphological attributes. It has been noticed that there is a considerable reduction in the plant height, rhizome, leaf dimension, flower number and leaf number at higher elevations. Regression analysis and Pearson ’ s correlation coefficient demonstrat-ed a positive relationship between plant height and all other morphological attributes. Principal component analysis (PCA) revealed that the habitat, ecological conditions and elevations of population - I (Khawous) and Population - II (Numsuru) were proved to be relatively better for the luxuriant growth and development of an endemic plant species A. violaceum . From the observations, it can be inferred that A. violaceum grows successfully at an elevation ranges between 3000 to 3500 m asl. This study provides a com-prehensive account of phenotypic variability concerning environmental conditions and suggested a suitable environment for sustainable cultivation of this important medicinal plant species in the Ladakh Himalaya.


Introduction
Phenotypic plasticity is the potential of a genotype to manifest alternative morphological, behavioural and physiological attributes in response to environmental conditions (1,2). To thrive under adverse environmental conditions, plants can either adjust to the local environmental circumstances genetically or respond to environmental variation through phenotypic plasticity (3,4). Phenotypic plasticity can play an essential role in evolution by altering or modifying the developmental pathways in plant bodies, thus leading to the creation of phenotypic diversity in nature (1). Variations in general growth forms of resource accumulating organs such as roots, tubers, rhizomes, leaves and flowers of a plant are essential to adjust the available resources (5). Plants acclimatize themselves under acute environ-mental conditions either by manifesting phenotypic malleability or by adapting genetically (6). Phenotypic flexibility is crucial for plants as they are stationary and must live in a constantly challenging environment (6,7). Phenotypic variability is believed to exist more regularly at higher elevations, enabling alpine plants to adjust to the temporally fluctuating environment (2,8). Although; plasticity is beneficial for the plant to survive under extreme climatic conditions; somehow it induces negative impacts on the fitness of plant species (9). With increasing elevations, plants must adapt to altering environmental conditions (10). Plants regulate important physiological processes such as gaseous exchange, photosynthetic rates and vegetative traits in response to climate fluctuations. Reducing plant height with respect to elevation disparity is the most noticeable change in plant morphology (10,11).
Aconitum is a large genus of family Ranunculaceae with over 300 species distributed in the temperate areas of the Northern Hemisphere; such as Asia, Central Europe and North America (12). A. violaceum Jacq. ex Stapf is an erect biennial herb attaining maximum height of 30 cm tall (13,14), endemic to the Northern Himalayas (15). In India, A. violaceum is randomly distributed in the alpine and subalpine regions of Jammu and Kashmir, Himachal Pradesh, Uttarakhand and Ladakh Union Territory. A. violaceum is known as Aconite, violet monkshood in English; Atis, Bishmool and Mohand in Kashmir region and Bova-nagpo, Jhimba, Yangtso in Ladakh region (16).

Materials and Methods
In the present study, a dynamic technique was used to understand the phenotypic variability under different environmental and altitudinal conditions. The survey was conducted during May 2019 to October 2021.

Exploration, collection and documentation
Various habitats in Ladakh Himalaya (India) from Zojila to Zanskar in the West; Leh to Nubra in the East, were explored extensively and assessed to choose a specific study sites across the region. To investigate the phenotypic variability in A. violaceum along the altitudinal gradient and ecological conditions, 3 sites (populations) have been se-lected based on their accessibility, habitat structure and specific environmental conditions, along with 1 control site at Kashmir University Botanical garden (KUBG). Given due cognizance to the threat status, vigor mature flowering plants of A. violaceum was collected quite judiciously from different study sites. The specimens were identified and deposited in the Kashmir University Herbarium (KASH) under voucher number 3739-KASH. 10-15 individuals of the species were randomly selected from each study site and were marked with tags. The tagged individuals were monitored on regular basis from May 2019 to October 2021. Vegetation type, habitat, geo-coordinates, altitudes along with associated species of the selected sites were recorded. The vegetative propagules (rhizome and seeds) of the plant were judiciously collected and transplanted to KUBG (control site) to study the morphological variations.

Species morphology and phenotypic variability
The randomly selected individuals in the populations were observed continuously at regular intervals of 5-10 days. Variations in morphological traits like plant height, rhizome, petiole, leaf, pedicel dimensions and floral characters such as the dimension of flower whorls, follicles and seeds were recorded.

Statistical analysis
LSD and Tukey's test (one-way ANOVA) were done using IBM-SPSS software (version 23) to compare the significant level and the average ± standard error were computed using Microsoft Excel (office-10). Linear regression and Pearson's correlation coefficient analysis were performed to examine the correlation between various morphological attributes across different environmental conditions and elevations to find an ideal habitat for the better growth and development of this species. Principal component analysis (PCA) was carried out to assess the phenotypic traits in relation to different habitat conditions. Origin-Pro 2019b 64 software was used to analyze the PCA and R-software (version 4.1.2; 64 bit) was used to determine the correlation. To assess the variability in phenotypic features, data was collected for 10-15 plants from each population. The photographs were taken using a Canon DSLR (1300D), an Olympus (EPl-5) camera and a 1-plus 9-pro mobile camera.

Species morphology and phenotypic variability
The morphological characteristics of A. violaceum are enumerated in (Table 2), (Fig. 2. A-F). The phenotypic characters of A. violaceum showed significant variation at different altitudes and climatic conditions. This phenotypic variation was observed in 4 selected sites. Viz, Khawous (3220 m asl), Numsuru (3350 m asl), Maan-man, Drass (4020 m asl) and Kashmir University Botanical Garden (KUBG) Srinagar (1595 m asl) as a control site. The morphological     (Fig. 4). Principle Component Analysis (PCA) revealed that there is a gradual decrease in the size of morphological traits with the increase in altitude and vice versa (Fig. 5). The population growing at high elevation (Maan-man, Drass) showed a decrease in size of all phenotypic traits as compared to other populations, thus sepa- Our results were in accordance with the former observations (25). It has been stated that reducing plant height and leaf dimension along with altitude is beneficial for the plant species as it prevents them from strong winds (26).
It has been noticed that there is a tangled relationship between the environment and plants. With the change in altitudinal gradient, a range of environmental factors also vary which imposes a challenge to the plants, to adapt and survive under varying environmental and altitudinal gradients. Plants bring variations in their biochemical and morphological features to acclimate and overcome these stress circumstances (27). The change in morphological traits with varying elevation indicates plasticity and the fixed evolutionary changes (genetic) in these traits may lead to speciation (28).

Conclusion
The present study reveals that variations in the environmental conditions proved to have an enormous impact on the growth pattern and development of A. violaceum. The plants growing at altitudinal ranges between 3000-3220 m  asl (Population-I) were comparatively much more vigorous and diverse in all morphological characteristics. In addition, flower colours in population-I (khawous) were diverse (violet, blue, light white with blue veins, variegated); whereas the plant specimens grown at (KUBG) grew well in the beginning, however, as the summer temperatures rose, it gradually lost its vigorousness and dried out before reaching the flowering stage. The individuals of population -III (Maan-man, Drass) showed a decreased growth rate in all aspects of morphological traits such as plant height, leaf length, leaf width, number of flowers, rhizome length etc as compared to other population. This study aimed to find a suitable habitat for the establishment, development, cultivation, sustainable economic utilization and conservation of A. violaceum, a threatened endemic medicinal plant of the western Himalaya. It can be concluded that the ecology and elevation of population-I (khawous) are suitable for the growth and development of this threat-