Variation in Growth Dynamics and Reproductive Allocation in Ajuga Species across Different Altitudes

Ajuga is a medicinally important genus of the Himalayan region. This study was carried out on the phenotypic variability and reproductive allocation in two species of genus Ajuga, A. bracteosa and A. parviflora growing in variable habitats of Kashmir valley. Our findings clearly displayed significant divergence among sites which reveals a definite impact of altitude on morphological and reproductive features of the species under study. In A. bracteosa the plants growing at low altitudes are comparatively taller and produce more number of leaves, greater leaf dimensions and inflorescence number. But in case of A. parviflora a more or less reverse trend was seen. Principal component analysis (PCA) revealed that the habitat of Kangan and Dachigam proved relatively better for the growth of A. bracteosa and Brinal 2 and Gogaldara for A. parviflora. Maximum resources were allocated to the growth and development of the leaves followed by stem, root tubers and inflorescence in both the species. The reproductive effort was higher (40.48 ± 1.59) for the high altitude Betab valley population in case of A. bracteosa and was lowest for the high altitude Brinal 2 population (45.88 ± 1.37) in case of A. parviflora. The regression analysis revealed a positive correlation between branch length and root length, branch length and apical leaf length in both the species and a negative correlation between branch length and number of branches per plant in both the species. Our results are very useful to introduce the species into cultivation and developing strategies for conservation.


Introduction
Plants are unable to escape the hazards of environment in which they grow because of their sessile habit. To cope with this, many plants undergo variation in one or more morphological characters in response to both abiotic (e.g., climate and weather) and biotic (e.g., grazing and competition) factors of the environment as an adjustment to resource availability. This adjustment to environment is generally referred as phenotypic plasticity [1]. Recently more attention has been given to the acclimation of morphological, biochemical and physiological traits of plants along an altitudinal gradient [2][3][4].
Abiotic factors have a significant effect on the morphological variability, despite the fact that it may be due to ontogenic programming of the plant as well. Abiotic variables such as water stress, mineral nutrient deficiency and geographical position may have an impact on the overall plant morphology. For example, latitude is negatively correlated with plant height [5], while altitude may also affect the leaf morphology and plant height [6,7]. The plant morphological parameters such as height, leaf area and petiole length vary with different altitudes [8], with a progressive decrease in plant height with an increase in altitude. Many plants have smaller leaves at high altitudes [9][10][11]. These morphological adaptations are presumably associated with decrease in temperature as well as nutrient and water limitations [10,12]. It may also be an adaptive strategy against the hazardous impact of strong wind that normally blows at high altitude, thereby improving photosynthetic activities of plant [8,13].
The genus Ajuga L. consists of about 40-50 species [14] mostly distributed in the north temperate zone of world [15] also from Europe to Asia and Australia [16]. Ajuga bracteosa Wall ex Benth. belongs to family Lamiaceae and is commonly called as Jan e adam. It is perennial, erect or ascending hairy herb, often prostrate with oblanceolate or sub-spathulate leaves [17]. Various crude extracts of this plant have shown activities including antidiabetic, antioxidant, antibacterial, diuretic, stimulant, astringent, rheumatism, febrifuge, headache, earache and is also effective in jaundice, malarial fever and hypertension [18,19]. Ajuga parviflora is an annual or short lived perennial herb [20]. It has been used as an astringent and for the treatment of swollen wounds, diarrhea, rheumatism, fever, eye trouble and for the diseases of bladder [21]. They also have the cancer chemo-preventive, hypoglycemic and hypotensive effects [22]. The present study was devised for the first time to understand the variation in growth characteristics and changes in allocation patterns in relation to the environmental conditions in two related species Ajuga bracteosa and A. parviflora growing at different altitudes of Kashmir Himalaya.

Morphological characterization
The studies were carried out by selecting ten mature flowering individuals randomly from each population in order to observe the various morphological parameters of the species. The populations were analyzed for morphological traits like root length, branch length, number of branches per plant, number of leaves per plant, basal leaf length and breadth, apical leaf length and breadth, inflorescence number per plant and number of flowers per inflorescence. The plants were measured in situ and most of these parameters were recorded at flowering stage in all the populations. We used linear regression analysis to determine the correlation between various morphological parameters across the altitudinal gradient. Principal component analysis (PCA) was carried out to analyze the morphological traits in relation to habitat dynamics and to infer the coherence between different vegetative and reproductive parameters.  A positive correlation between root length and branch length, branch length and apical leaf length and a negative correlation between number of branches per plant and branch length was observed in both the species (Figure 3). Principal component analysis (PCA, Figure 4) of all morphological characters across the study sites reveals that the major differences between populations were due to size characteristics in both the species.
In case of A. bracteosa, PCA reveal that the high altitude populations are non-favorable to most of the vegetative and reproductive traits indicating better growth conditions at lower altitudes. In case of A. parviflora, a reverse trend was observed.
In case of A. bracteosa, apical leaf breadth, Inflorescence number per plant and basal leaf breadth were found to be favoring the Kangan population. Basal leaf length, apical leaf length, branch

Resource allocation
Ten mature and healthy flowering plants from all the natural populations were harvested for the study of resource partitioning in different parts of a plant. The plants were fragmented into individual parts such as root, branches, leaves and inflorescence.
Using an electronic balance, fresh weight (weighing as fresh) and dry weight (after oven-drying for 48 h at 80°C) of the plants were determined following Kawano and Masuda [23]. Reproductive effort (RE) was calculated from the estimates of dry weight or biomass allocated to reproductive and vegetative structures following Yaqoob and Nawchoo [24].

Data analysis
ANOVA was used to test for differences between populations for all the morphological characters measured using the SPSS 16.0 software. Tukey's multiple comparison of means was used to compare all populations and the differences between individual means were deemed to be significant at p ≤ 0.05.

Growth characteristics
Present investigation revealed a wide range of suitable habitats for the growth of A. bracteosa and A. parviflora (Figure 2). A. bracteosa thrives best in the open rocky slopes and landslide areas; while as A. parviflora prefers moist or rocky shady slopes in association with coniferous forests. Ajuga bracteosa is perennial, erect or ascending hairy herb, often prostrate, oblanceolate

Reproductive allocation at flowering
The partitioning of resources is non-uniform among different parts of the plants of A. bracteosa (Table 3). There was a remarkable difference in total above ground dry weight biomass and dry weight of different vegetative structures among the plants of studied populations, growing at different altitudes. Maximum resources were allocated towards the growth and development of leaves (0.572 ± 0.12 g to 0.659 ± 0.260 g) followed by root (0.400 ± 0.072 g to 0.514 ± 0.047 g) and branches (0.340 ± 0.041 g to 0.624 ± 0.057 g) while the least resources were allocated towards inflorescence (0.091 ± 0.222 g to 0.208 ± 0.030 g). A somehow similar trend was followed by the plant parts of A. parviflora (Table 4). Maximum resources were allocated towards the growth and development of leaves (0.369 ± 0.057 g to 0.521 ± 0.224 g) followed by branches (0.173 ± 0.034 g to 0.437 ± 0.085 g) and root (0.131 ± 0.009 g to 0.413 ± 0.048 g) while the least resources were allocated towards inflorescence (0.087 ± 0.014 g to 0.499 ± 0.130 g). In the present study, considerable phenotypic variability was observed within and across the individuals of different populations of A. bracteosa and A. parviflora growing along the altitudinal gradient in the Kashmir valley. The morphological characteristics and resource allocation patterns showed both inverse as well as direct correlation with the altitude. Plant height decreased with an increase in altitude, an adaptive phenomenon to protect the plant against the severe conditions at higher altitudes [25,26]. The harsh climatic conditions as well as the shorter growing season results in the overall slow growth rate, which in turn enables the plants to efficiently utilize the available resources [27]. The reduced plant height at higher altitude prevents the damaging effects caused by the strong winds prevalent at high elevations and keeps the leaves closer to warmer soil to enhance its photosynthetic efficiency [13]. However, the decrease in plant height is compensated by a concomitant increase in the number of branches in the plants growing at higher altitudes. The present study revealed that the plants growing at low altitudes are comparatively taller and produce more number of leaves. Our results are supported and confirmed by the observations of Johnson and Cook [28], Hickman [29] and Bresson et al. [30] who have also observed a reduction in length, breadth and area of leaves with an increase in altitude. However, in this study, leaf dimensions show a contrasting trend being greater in low altitude populations in case of A. bracteosa and in high altitude populations in case of A. parviflora. The reproductive effort also shows a contrasting trend and in case of A. bracteosa, plants growing at higher altitudes had more reproductive effort than the plants growing at lower elevations. These findings are in conformity with Fabbro and Korner [31] and Molina-Montenegro et al. [32] but in case of A. parviflora, a reverse trend was observed.

Conclusions
We propose that the heterogeneity of the environmental conditions is the main source of phenotypic variation of two Ajuga species. Our observation revealed a wide range of suitable habitats for the growth of A. bracteosa and A. parviflora.
With increased altitude, a significant decrease is found in the phenotypic characteristics of A. bracteosa, thus it seems that these sites endure a larger environmental stress. But in case of A. parviflora a reverse trend was seen. Increasing altitude resulted in a decrease in the allocation of biomass to reproductive structures in the form of decreasing dry weight. However, future studies should explore the phenotypic plasticity in more detail in plants growing at contrasting environments in order to evaluate precisely the contribution of the phenotypic plasticity to the phenotypic variation in different Ajuga species.

Acknowledgements
The author acknowledges UGC for providing fellowship under FIP scheme.  Table 3 Allocation of resources (mean ± SD) in the form of dry weight (g) toward different plant parts in Ajuga bracteosa across the study sites.  Table 4 Allocation of resources (mean ± SD) in the form of dry weight (g) toward different plant parts in Ajuga parviflora across the study sites. * Means labelled with the different small letters indicate that they significantly differ from each other among different populations