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IV.2 Ferula jaeschkeana Vatke. (Apiaceae) IV.2a Study Area: During the present study, populations of this species were located in Sonamarg in Kashmir, Drass in Ladakh and Kishtwar in Jammu regions of J&K state. Different sites surveyed during the present study along with their space coordinates are listed in table IV. 21. Table IV. 21 Different study sites along with their space coordinates S. No. Study site 1. 2. Drass proper (D) Bhimbat (B) Altitude (m amsl) 3122.8 3079 Space coordinates 3. 4. Morpochu (MP) Mushko Valley (MV) 3111.4 3218.5 34˚ 25΄ N 75˚ 44΄ E 34˚ 26΄ N 75˚ 39΄ E 5 6. 7. 8. Trungian (T) Tiasbu (TB) Goshan (G) Sonamarg (S), Kashmir 3125.8 3162 3140 2739.1 34˚ 25΄ N 75˚ 44΄ E 34˚ 25΄ N 75˚ 45΄ E 34˚ 25΄ N 75˚ 45΄ E 34˚ 17΄ N 75˚ 90΄ E 9. Kishtwar (K), Jammu 2289 33˚ 18 ΄ N 75˚ 47΄ E 34˚ 25΄ N 75˚ 45΄ E 34 25΄ N 75˚ 48΄ E IV.2b Habitat and ecology: At different study sites, plants of this species grow on a variety of soil types, ranging from rocky slopes with scanty soil to crop fields to the humus rich forest floor. Except in Kishtwar population, F. jaeschkeana plants grow on mountain slopes or fields exposed to sunlight (Figs. 43a to h). Plant is tolerant to extremely low atmospheric temperatures, which falls to sub-zero during winters at all sites. In Drass it may fall up to -60ºC. Snowfall is a regular feature of all the habitats of the species. 57 IV.2c Associated plant species: In Drass valley, only herbaceous vegetation characteristic of alpine regions was found. Some important species growing in the area are Dactylorhiza hatagirea (Orchidaceae), Lotus corniculatus, Astragalus rhizanthes and Trifolium repens (Papillionace), Taraxacum officinale (Asteraceae), Gentiana (Gentianaceae), Potentilla sp. (Rosaceae), Hyoscyamous niger algida (Apocynaceae), Lindelofia stylosa (Boraginaceae) and Prangos pabularia (Apiaceae). These herbaceous species were found to occur frequently in grasslands throughout the Drass region but did not grow in close proximity of F. jaeschkeana. However, Prangos pabularia, was always present in close association with F. jaeschkeana in all the populations located in Drass region (Figs. 43g to h). In Kishtwar, 2 distinct populations of F. jaeschkeana were found growing at the floor of a mixed forest of semi evergreen type. Major flora of the forest includes Quercus baloot, Pinus roxburghii, Acer caesium, Juglans regia, Olea ferruginea, Pyrus pashia, Ficus palmata, Pistacia integerrima, and Indigofera hebepetala. IV.2d Plant phenotype: Ferula jaeschkeana Vatke. is a tall perennial herb that grows upto an average height of 113.65 cm (Fig. 44a). It is the tallest native herb of the habitat. The plant perennates through a thick and massive rhizome that grows upto 5 - 6 feet deep in the soil and helps the plant to survive through the extreme cold conditions of its habitat (Fig. 44b). Aerial stems appear during the flowering season that extends between May and July. These stems are upright, green and herbaceous and glabrous (Fig. 44c). With the progression of flowering season, colour of the stem changes from green to yellow. These aerial stems secrete a thick milky fluid from 58 the site of any mechanical injury or damage caused by insects. This fluid changes to brown sticky gum like substance upon exposure to air (Fig. 44d). The aerial stem is unbranched except at the apex. Leaves are both cauline and basal. The basal leaves arising from the underground stem are 41.9 cm long and 45 cm broad on an average. These leaves spread on ground all around the plant. Morphometric detail of the species is listed in table IV. 22. IV.2e Floral – morphology: Peduncles or inflorescence bearing axis arise from the axil of each cauline leaf during the flowering season. Number of peduncles borne by an aerial stem varies from 7 - 16. Peduncles are about 26 cm long on an average. At the stage of peak flowering, the average expansion of an inflorescence is 14 cm. Inflorescences continue expansion and further lax as the flowering season progresses and the plants move from flowering to the fruiting stage. inflorescences Each of the young is enclosed by a 2 - 18 cm long sheathy enclosure derived from the base of the subtending leaf. It terminates in a pinnatisect lamina 20.2 cm long and 19.07 cm broad at the tip of the sheath (Fig. 45). Inflorescence emerges out of this structure by longitudinal splitting of the sheath (Fig. 46) due to elongation of peduncle. Inflorescence is a compound umbel (Fig. 47). One to five primary rays or pedicels arise from each primary umbel (1 st order umbels), each of which bear secondary umbels (2nd order umbels). 4 - 15 secondary rays or secondary pedicels arising from these secondary umbels bear the umblets (3rd order umbels) which ultimately produce pedicels bearing the flowers (Fig. 47). Number of flowers per umblet varies from 3 - 34. Involucral 59 Table IV. 22 Morphometric detail of F. jaeschkeana S. No. Character Average value 1. Plant height 2. No. of peduncles per plant 3. Length of peduncle (cm) 4. Length of basal leaves (cm) 5. Width of basal leaves (cm) 6. Length of petiole in basal leaves (cm) 7. Length of cauline leaves (cm) 8. Width of cauline leaves (cm). 9. Length of the sheath derived from the basal part of cauline leaves (cm) * - Mean ± SD; ** - Range; n - sample size 113.6 ± 27.3* (64.5 - 170) ** n = 40 11 ± 2.3 (7 - 16) n = 38 26 ± 12.1 (6 - 59) n = 80 41.9 ± 13.2 (17.5 - 78) n = 26 45 ± 17.8 (20 - 84.2) n=26 17.2 ± 6 (9.5 - 31) n = 20 20.3 ± 8 (9 - 46) n = 80 19.07 ± 10.8 (2 - 19) n = 80 7.3 ± 2.2 (2 - 18) n = 80 bracts are absent in this species, but at the level of 1st and 2nd order umbels a pair of simple leaf like bracts is present. Both hermaphrodite and staminate flowers were found in the species. IV.2f Variation in sex expression: Depending upon the pattern of distribution of hermaphrodite and staminate flowers in a plant, 2 types of plants were observed in all the populations: 60 f(i) Andromonoecious or Type A plants: Plants of this type were profusely branched and had a specific distribution pattern of the bisexual and staminate flowers. On each of the flowering peduncle in these plants, the central umbel was hermaphrodite, but 2 - 3 secondary umbels were staminate (Fig. 48a). In some plants of this type however, one or two lateral umbels bear hermaphrodite flowers and an exceptionally elongated pedicel arises from the lateral umbels and produces another umbel bearing only the staminate flowers (Fig. 48b). f(ii) Hermaphrodite or Type B plants: Plants of this type were not as profusely branched as type A plants (Fig. 49a). All the umbels on such plants had only hermaphrodite flowers. Occasionally however, some staminate flowers or flowers with rudimentary female parts were found in some of the umbels of these plants (Fig. 49b). Their distribution however, did not follow any specific pattern on the plant. Frequency of occurrence of each type of plants in different populations surveyed is given in the table IV. 23. Flowers are ebracteate, pedicellate, actinomorphic, pentamerous and epigynous. Stylopodium is present on top of the ovary. In each hermaphrodite flower perianth is uniseriate and petaloid. Petals are white with a greenish tinge in bud, polypetalous arising from just beneath the stylopodial disc. Each of the five petals is linear - lanceolate 2 mm long and 1.1 mm broad with an acute apex that is tightly incurved over anthers in the bud. 61 Table IV. 23 Frequency of type A and type B plants in different populations S. No. Population 1. 2. 3. 4. 5. 6. 7. 8. Drass -1 Tiasbu Goshan-1 Drass-2 Drass-3 Drass-4 Goshan-2 Trungian Total average Total no. of flowering individuals per quadrate (10 m x 10 m) 07 25 16 66 11 19 04 13 161 Percentage of Percentage of type A plants type B plants 57.1% 44% 37.5% 86.3% 90.9% 25% 84.2% 84.6 63.7% 42.8% 56% 62.5% 13.6% 9.1% 75% 15.8% 15.4% 36.2% Androecium consists of five stamens, rarely six, which arise alternately to the petals from beneath the stylopodial disc. Staminal filaments remain coiled in buds and anthers lie at the margin of the disc (Fig. 50a), but they unroll sequentially with opening of the flowers (Fig. 50b). Anthers are bilobed, basifixed and extrorse; dehisce by longitudinal slits and orient upwards in mature stamens. Gynoecium consists of an inferior ovary that is bicarpellary, syncarpous and bilocular. The two ovarian locules are indistinguishable in flower but become separate in mature fruits. A single anatropous ovule is pressent in each locule. Placentation is axile. Styles 2, separate throughout their length, arise just at the inner margins of the central ridge of the stylopodial disc. In a young flower, styles are adpressed in parallel orientation. As the flower matures, the two styles move outwardly, orient themselves at 180º and finally bend downwards (Figs. 51a to f). Stigma, globular and wet, turns prominently glistening and pink at receptivity (Fig. 51e). In some rare cases, 3 styles and tricarpellary ovary represent gynoecium, but one of the locules is rudimentary containing shriveled 62 ovule. In staminate flowers while rest of the structure remains same, gynoecium is completely lacking or rarely the rudiments of style and ovarian locules were observed which did not grow to maturity. Fruit is a cremocarp, 2.5 cm long, splitting up into 2 mericarps that remain attached by a Y- shaped carpophore. Four resin canals demarcated externally by longitudinal ridges are present on each mericarp (Figs. 52a to b). Seed is fused with the fruit wall. Dimensions of different floral parts and other parameters are given in the table IV. 24. IV.2g Population structure: In all the natural populations of F. jaeschkeana surveyed during the present investigation, it was found that, individuals both in flowering and non-flowering phase were present (Figs. 53 to 54). The non flowering individuals were either juvenile that had not attained the reproductive age, or mature plants that remained in vegetative phase throughout the season. Juveniles could be easily marked out from the mature plants in a population by lesser number (commonly a single pair) and smaller size of basal leaves as compared to a number of large sized basal leaves present in mature plants. No constancy was observed in abundance and periodicity of flowering in this species. During the period of study spanning across three years, it was observed that the flowering is more during one season, but relatively less during the following season. Some plants were marked that produced flowering aerial shoots during each year and were thus flowered annually, where as many plants produced flowering shoots only during alternate seasons. This was also confirmed by the presence of dried remains of the flowering axis of previous season on such plants. 63 Table IV. 24 Observations on floral morphometry in F. jaeschkeana S. No. Character Average value 1. Diameter of main umbel (cm) 2. No. of umbels per peduncle 3. No. of umblets per umbel 4. No. of flowers per umblet 5. 6. No. of flowers per plant Diameter of umblet (cm) 7. Length of umblet (cm) 8. length of floral pedicel (mm) 9. Length x width of petal (mm) 10. Length of stamen (mm) 11. Length of style (mm) 12. Size of stigma (μ) 13. Length x diameter of ovary (mm) 14.1 ± 6.8* (2.5 - 30)** n = 80 3 ± 1.2 (1 - 5) n = 23 8 ± 2.1 (4 - 15) n = 80 17 ± 5.3 (3 - 34) n = 80 4,488 1.4 ± 0.2 0.9 - 1.8) n = 28 1.5 ± 0.25 (1 - 1.9) n = 28 3.5 ± 0.7 (2 - 4.5) n = 30 2 ± 0.16 x 1 ± 0.2 (2 - 2.5) x (0.5 - 1.5) n = 17 4.3 ± 0.48 (3.9 - 5.5) N = 30 3 ± 0.3 (2 - 3) n = 30 420 ± 35 x 340 ± 47 (350 - 510) x (270 - 430) n = 20 1.5 ± 0.7 x 2.1 ± 0.3 (0.5 - 3) x (1.5 - 3) n = 32 * - Mean ± SD; ** - Range; n - sample size 64 IV.2h Flowering: Flowering initiates in the month of May after the plants have resumed vegetative growth after a long period of perennation through winters. It extends up to July. Aerial shoots bearing corn like inflorescences emerge during first week of May. Inflorescences come out of the enclosing sheaths and expose the flowers that start opening during 3rd week of May and continue opening till the end of July. h (i) Pattern or sequence of flowering: Pattern of flowering in the species was studied at different levels i.e. at the level of an individual plant, at the level of peduncle, at the level of umbel and finally at the level of umblet. In a plant flowering was found to be acropetal i.e. flowering began in lower most peduncles in a plant followed by the peduncles towards the apex of the plant in order of maturity. In each peduncle, central umbels were the first to bloom followed by the lateral umbels after 3 - 4 days, giving a centrifugal pattern of flowering. In an individual umbel also flowering progressed from peripheral umblets to the central ones. In each umblet flowers at the periphery open first followed by the central flowers. IV.2i Floral biology: 2i (i) Time and pattern of anthesis and anther dehiscence: The mature inflorescences could be differentiated from the younger inflorescences by the light yellow colour of the sheath enclosing them. Such a mature inflorescence emerges out of the sheath through a longitudinal slit formed on its adaxial side (Fig. 55a). It takes about 3 days for the inflorescence to emerge completely from the sheath and colour of inflorescence also changes from green to light yellow. Anthesis begins in peripheral flowers of the inflorescence between 9 65 - 10 am and continues up to 5 pm. Flowers are protandrous and the beginning of anthesis in a flower is marked by extension or straightening of filaments of 2 or 3 stamen out of 5 (Fig. 55b). The two styles appear as a protuberance at the time of anthesis (Fig. 55c). The staminal extension is followed by the unrolling of the petals which gradually turn downwards towards the pedicel. After 24 hours of beginning of anthesis, anther dehiscence begins in the first extended stamens. Meanwhile the other stamens also extend and within 40 - 45 hours of anthesis all the anthers dehisce. Male phase that starts at the anthesis with staminal elongation ends within 50 - 52 hours with the shedding of stamens. Nectar is secreted and collected in abundance over the stylopodial disc throughout this period. By the time stamens extend fully, styles elongate and start moving in opposite directions horizontally. Petals and stamens are shed after 52 hours i.e. on the third day of anthesis but the outward movement of the two styles continues for 7 - 8 days after which they turn dark green with reddish brown stripes and stigmata also turn brown and styles take a downwards bend towards the pedicel. The progression and sequence of flowering within a plant is synchronized. At any point of time, all the flowers on all the umblets of an umbel of same order are at more or less same stage of floral maturation. Moreover in andromonoecious plants anther dehiscence in staminate umbels closely coincides with the stage of stigma receptivity that a hermaphrodite umbel of preceding order is passing through (Fig. 56). During the course of opening of flowers, the colour of all the floral parts including peduncles changes from green at bud stage, to yellow at anthesis till the styles continue to move and red after the fertilization is completed and fruit set is started. 66 2i (ii) Stigma receptivity: Stigma becomes receptive after about 94 hours of anthesis. Receptive stigmata appear knob like and glisten with exudates. Peak receptivity was observed between 94 - 170 hours of anthesis after which it declined and after about 195 hours stigma turn brown and non- receptive. Examination of stigmata from pistils collected randomly from different populations for pollen load under open pollination conditions revealed that 94.2% of pistils had pollen load on stigmata with an average of 22 pollen (Fig. 57; Table IV. 25). Percentage germination per stigma was 81% on an average. This indicated an efficient pollination mechanism operating in this species. Pollen tubes traverse through the stylar tissue and reach the ovary whereupon one of them enters the ovule through the micropyle (Figs. 58 to 59). 2i (iii) Pattern of Nectar secretion: Nectar secretion in F. jaeschkeana was observed to be occurring in two phases, first during the male phase coinciding with the stamen elongation and anther dehiscence (Figs. 60 to 61) and second during the female phase at about the same time when the stigma becomes receptive (Fig. 62). Nectar secretion stops during the gap period of about 36 - 40 hours that spans between the end of the male phase and onset of stigma receptivity. In staminate flowers, nectar secretion stops completely after all the stamens have shed and flowers start drying soon after. But in hermaphrodite flowers, nectar secretion is resumed gradually as the stigma approaches the stage of receptivity and reaches to maximum at the stage of peak receptivity which comes after about 90 - 92 hours of anthesis and about 36 hours after the end of male phase. To understand the pattern of nectar secretion, flowers 67 in different stages of development, growing in nature were observed in different populations. In these flowers, stage of development i.e. initiation of anthesis and staminal elongation, anther dehiscence, shedding of stamens, beginning of female phase were correlated with the presence or absence of nectar over the stylopodial disc. Number of flowers observed at each of these stages and status of nectar secretion in them is given in the table IV. 25. In another experiment, 100 flowers on 5 different umbels on 3 plants were tagged before anthesis to monitor the changes in nectar secretion with different stages of floral phenology. All these umbels were bagged to ensure that insects do not consume the nectar and pattern of nectar secretion in original form could be observed. In all of these flowers it was found that, nectar secretion begins as soon as 2 - 3 stamens start elongating. It continues subsequently during the course of elongation of remaining stamens and anther dehiscence. Nectar secretion in all except 2 flowers stopped after all stamens had been shed. Stylopodial discs of these flowers remained dry and devoid of nectar for about 30 h when the two styles continue moving outwards. Nectar secretion again started after this period and after about 36 h nearly all the flowers had plenty of nectar over the stylopodium. In another set of observations flowers were manually emasculated and nectar secretion observed in them. Here also it followed the same pattern. 2i (iv) Pollen output, shape, pollen ovule ratio and viability: A single plant of F. jaeschkeana produces an average of 4,488 flowers and each flower produces 18,281 pollen grains on an average. Pollen of the species were rod or dumb-bell shaped with 2 germ pores that were present on the 2 lateral sides of a ridge in the middle of the pollen (Fig. 63). Pollen ovule ratio in the species 68 was found to be 9,141:1. Pollen viability of the species as estimated through stainability and FCR tests with 1% acetocarmine and FDA respectively was 83.7% and 75.13% (Table IV. 26). Some plants in natural populations were detected with very low pollen viability averaging only 37% (Figs. 65a to b). Table IV. 25 Status of nectar secretion during different stages of flower development in F. jaeschkeana S. No. 1. 2. 3. 4. 5. 6. Stage of flower development Mature buds just before anthesis Flowers undergoing stamen elongation and anther dehiscence i.e. male phase in progress All stamens shed and stigma not receptive i.e. transition phase Styles widely separated and stigma glistening, knob like and receptive Post fertilization stage i.e. ovaries enlarged and turned red No. of flowers Status of nectar observed 53 Absent 705 Present 344 66 Present in 6 flowers, absent in rest Present 11 Absent Staminate flowers with all 63 the stamens shed. Absent 69 Table IV. 26 Showing average values of various parameters of floral biology and pollination biology in F. jaeschkeana S. No Parameter Average value 1. Pollen count per flower. 18, 281 ± 4060.3* (8875 - 25,300)** n = 22 2. 3. 4. No. of ovules per flower Pollen ovule ratio Pollen size (μ) 2 9,141:1 32.5 ± 0.4 x 21.5 ± 0.8 n = 40 5. Pollen viability in FDA 6. 7. 8. 9. 75.13% ± 19.3 (23.4 - 91.5) n = 16 Pollen viability in acetocarmine 83.7% ± 4.7 (77.2 - 90.4) n = 58 Percentage of stigma with 94.2% pollen load in open pollination. n = 140 Average pollen load per stigma 22 ± 15.9 (1 - 58) n = 60 Percentage pollen germination 81% ± 26 on stigma in open pollination. (7.6 - 100) n = 61 * - Mean ± SD; ** - Range; n – Sample size IV.2j Pollination type: Anemophily was ruled out in the species by conducting hanging slide experiments in nature (Figs. 66a to b). Pollination was entomophillous in the species. Different types of insects, including 3 - 4 species of Apis, ants and beetles visited these plants in great frequency (Figs. 67a to k). Insects displayed different foraging behaviour and had different preferences for nectar or pollen as their feeding material. Several species of Apis are the most common visitors. Apis cerana is a very frequent visitor. It moves very quicky between plants in a population and also among the umbels on a plant. It 70 makes repeated visits to plants. Lateral and ventral sides of abdomen are brushed against anthers and stigma while collecting nectar from the disc. Apis florea is also very frequent but comparatively slower than Apis cerana in movements. It also makes repeated visits to several plants in a population and also all the umbels in a plant. It keeps its abdomen elevated and inclined towards the flower while resting on the margin of the disc. While collecing nectar, nearly all parts of its body come in contact with anthers and stigma. A species of Bombus (Fig. 67e) visits the inflorescences frequently. It lands on the margins of the disc and then moves over it, pollen baskets touch anthers. Its head also brushes against anthers and stigma while licking nectar from the discs. It visits nearly all the umbels with open flowers. Another species of Apis is a frequent visitor and forages the flowers for both nectar and pollen. Beetles (Figs. 67g & i) and Big black ants (Fig. 67j), are also present on the inflorescences. Ants move around the umbels and lick the nectar from disc. They remain clinging to pedicels and styles for collecting nectar. Beetles are the sluggish visitors that hardly move between the plants. They consume nectar and after it has dried they start eating floral parts like disc and styles. They also form net and enclose the whole inflorescences in it (Figs. 68a to e). House flies were also observed on the inflorescences in some populations. Foraging behaviour and effectiveness of these insects in carrying out pollination is summarized in table IV. 27. 71 IV.2k Reproductive output: Fruit set recorded from open pollinated plants from different populations was 92.4% whereas in manually self and cross pollinated flowers it was 91.2% and 97.5% respectively. The seed set was found to be 100% in all the fruits studied (Table IV. 28). Table IV. 27 Foraging behavior and pollen load on body of some frequent pollinators visiting F. jaeschkeana inflorescences S. No. Name of the Frequency insect of visits Apis cerana Very frequent Preferred target 2. Bombus sp Frequent Nectar only 3. Apis florea Very frequent Nectar and pollen 1. Pollen and nectar Pollen load on different body parts Head = 75 thorax = 75 abdomen = 8231 wings = 31 hind legs = 84 head = 26 Thorax = 8 Abdomen = 1936 Wings = 8 Hind legs =13 Head = 192 Thorax = 77 Abdomen = 4379 Wings = 49 Hind legs = 113 Table IV. 28 Percentage fruit set after different pollination treatments in F . jaeschkeana Kind treatment Open pollination MCP MSP of No. of flowers under observation 1,992 80 80 No. of No. of plants Percentage fruits under observation fruit set formed 1,841 10 92.4% 78 73 5 5 97.5% 91.2% 72 IV.2l Fruit and seed dispersal: Fruit is an indehiscent mericarp and the two cremocarps can’t dehisce on their own in nature. The most powerful means of fruit and seed dispersal was the mechanical action of wind upon the dried fruiting peduncles. Moreover manual intervention was also observed as an important means of dispersal as the dried peduncles were being collected and carried by natives to distant places for storage as fuel wood for winters (Fig. 69). IV.2m Seed germination: All attempts to raise the seeds under ex situ conditions failed. However, seed germination under natural conditions observed to be fairly good as predicted by counting the number of juvenile plants in different populations that appeared during each flowering season. IV.2n Herbivory: Towards the end of flowering season, the plants of this species become heavily infested by a species of beetle which enclose the whole inflorescences by forming a mesh (Figs. 68a to e). The adult female beetle lays eggs inside the ovaries most of which are fertilized by this time. When seed set occurs, larvae hatch out of these eggs and feed on developing embryos and cotyledons. This causes a huge damage to the seeds (Fig. 70) and reduces the reproductive output of the species. IV.2o Pollen Mother Cell meiosis: Pollen mother cells meiosis was regular in the species. Eleven bivalents were observed at diplotene which had interstitial as well as terminal chiasmata (Figs. 71a to b). The average chiasmata frequency calculated at diplotene stage from 50 different 73 pollen mother cells was found to be 13 and thus the recombination index for this species is 24. Bivalents had only terminal chiasma at metaphase stage (Figs. 71c to d) Anaphase - I segregation was normal with 11 Is moving towards each pole (Fig. 71e). Segregation during anaphase - II was also normal (Fig. 71f). However, in some plants with low pollen viability, cytomixis and some other anomalies were observed. Chromatin material was observed to be moving between the adjoining pollen mother cells at different stages of meiosis (Figs. 72b to e). This inter pollen mother cell migration of chromatin resulted in some cells having increased chromatin material and some others with no chromatin at all. Also observed chromosome laggards and bridges during PMC meiosis (Figs. 73 to 75). As a result of these anomalies, such plants produced non functional pollen in high frequency (Fig. 76). IV.2p Analysis of genetic diversity in Ferula jaeschkeana using ISSRs: Total genomic DNA was extracted from leaf samples following the CTAB method (Doyle and Doyle, 1990) with some modifications (Sharma et al., 2013) and checked for its quality and quantity on 0.8% agarose gel (Fig. 77). Accordingly dilutions were made and subjected to primer screening. Total 20 random ISSR primers were screened and 16 selected on the basis of robustness of amplification, clarity and scorability of banding patterns, were employed for diversity analysis. Among 39 genotypes from 6 populations, 90 bands were generated of which 47 to 66 (52.22% to 73.33%) were polymorphic within populations. The range of bands generated per primer was 4 - 9, in the size range of 290 - 2050 bp, with the average of 5.6 bands per primer. Analysis of ISSR data revealed lowest frequency of polymorphic bands (52.22%) in MushkoValley (MV) population while Bhimbat (B) population showed the highest 74 polymorphism (73.33%) (Fig. 78; Table IV. 29). Shannon information index (I) and Nei’s gene diversity (H) were lowest for MV population (H = 0.1833; I = 0. 2760) and highest for B population (I = 0.4381; H = 0.3007) (Table IV. 29). Shannon index based analysis calculated the total species diversity (HT) 0.2906, average diversity within populations (HS) 0.2277 and among populations (GST) 0.2164 (Table IV. 30). The value of gene flow (Nm) was 1.8106. Mushko Valley and Taisbu showed highest (0.9426) while Mushko Valley and Drass populations showed least genetic identity (0.8563) (Table IV. 31). Neighbour joining tree showed two major clusters (Fig. 79), one comprising Bhimbat and Sonamarg populations and the other cluster having rest of the 4 populations. IV.2q Ex situ conservation: All our attempts to raise the plants under ex situ conditions in the Lead Botanic Garden of the University failed. The plants deposited in the garden didn’t survive even for one season. Seeds of the species collected from natural populations were subjected to chilling treatment to simulate freezing conditions which species encounters in nature. They were then subjected to germination in farm yard manure. Not even a single seedling was raised. 75 Table IV.29 Polymorphic percent, Nei’s genetic diversity (H) and Shannon’s information index (I) for ISSR in F. jaeschkeana Population H (mean) 0.2331 I (mean) H(SD) I (SD) Morpochu (MP) Polymorphic % 63.3 0.3482 0.1952 0.2810 Mushko Valley (MV) 52.2 0.1833 0.2760 0.1949 0.2821 Tiasbu (TB) 53.3 0.2043 0.3024 0.2045 0.2946 Drass (D) 61.1 0.2436 0.3576 0.2052 0.2948 Bhimbhat (B) 73.3 0.3007 0.4381 0.1948 0.2758 Sonamarg (S) 56.6 0.2012 0.3039 0.1897 0.2777 Table IV. 30 Shannon’s estimates of genetic diversity within and among populations of F. jaeschkeana HT HS 0.2906 T Proportion of Proportion of diversity within diversity among populations populations (GST) 0.7836 0.2164 0.2277 Table IV. 31 Nei’s Unbiased Measures of Genetic Identity and Genetic distance of 6 populations of F. Jaeschkeana Pop ID MP MV TB D B S MP 0.0000 0.9577 0.9477 0.9273 0.9039 0.9356 MV 0.0432 0.0000 0.9602 0.8779 0.8982 0.9402 TB 0.0537 0.0406 0.0000 0.9008 0.9274 0.9337 D 0.0754 0.1302 0.1045 0.0000 0.9334 0.9327 B 0.1011 0.1074 0.0754 0.0689 0.0000 0.9485 S 0.0665 0.0617 0.0686 0.0697 0.0529 0.0000 Nei's genetic identity (above diagonal) and genetic distance (below diagonal). 76