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IV.1 Valeriana wallichii DC. (Valerianaceae) IV.1a Study area: During the present investigation, fifteen natural populations of this species were located in two north-western Himalayan states, Jammu and Kashmir and Uttrakhand, in India. Detail of study sites is given in table IV. 2. IV.1b Habitat and ecology: At all the sites surveyed during last 4 years, plants of this species were found growing as a part of ground vegetation on the moist and shady mountain slopes facing north and north-west. The habitat is humus rich and covered with the litter of higher trees and shrubs present at the canopy (Figs.1a to f). IV.1c Population Structure: Populations of this species were found to be disjunct and heterogenous with regard to the sex expression. Individuals with three different kinds of sex expression were found in every population. These were, a) Hermaphrodite plants (H type) with bisexual flowers (Fig. 2), b) Female plants (F type) with only pistillate flowers (Fig. 3) and c) Gynomonoecious plants (GM type) bearing both functional bisexual as well as male sterile flowers in the same inflorescence (Fig. 4). These sexual types are similar in all the features except for the flower type and size and thus can be distinguished only during flowering period (Table IV. 3). Number of individuals of each type was calculated in each population and their proportion at different sites is given in table IV. 4. Nearly all the plants in a population irrespective of their size bloom during the flowering season. An individual plant consists of several offshoots arising from a single rhizome giving it a clumped appearance (Fig. 5a). 29 Table IV. 2 In situ study sites of Valeriana wallichii S. No. Study site 1. Almora,Uttrakhand (AM) Altitude (m amsl) 1870 Space coordinates 2. Chakrata,Uttrahand (CR) 1781 33˚ 33' N 74˚ 24' E 3. Patnitop, J& K (PT) 2072.1 33˚ 05' N 75˚ 19' E 4. Bakori, J&K (BK) 1637 33˚ 21' N 74˚ 31' E 5. Budhal, J&K (BD) 1781 33˚ 22' N 74˚ 38' E 6. Bafliaz, J&K (BF) 1566.7 33˚ 21' N 74˚ 21' E 7. Noorichamb, J&K (NC) 1834.6 33 36' N 74˚ 25' E 8. Kund, J&K (K) 2159.6 33˚ 33' N 74˚ 23' E 9. Cha, J&K (C) 2440.4 33˚ 33' N 74˚ 24' E 10. Tungwali, J&K (TW) 2858.9 33˚ 34' N 74˚24' E 11. Sapanwali, J&K (SW) 2263.7 33˚ 33' N 74˚ 23' E 12. Azamtabad, J&K (AB) 2124.6 33˚ 33' N 74˚ 23' E 13. Thajwas, J&K (TG) 3108 34˚ 16' N 75˚ 17' E 14. Manyal Gali, J&K (MG) 1903 33˚ 33' N 74˚ 22' E 15. Dera Ki Gali, J&K (DKG) 2126.6 33˚ 35' N 74˚ 21' E 29˚ 37' N 79˚ 32' E IV.Id Associated plant species: Valeriana wallichii did not show a specific association with any particular plant species in nature. Although different herbaceous species were found coexisting with Valeriana populations at different sites. Some of the commonly found species were Viola canascence, Plantago lanceolata, Taraxacum officinale, Galium aparine. Micromeria biflora, Ttrifolium repens, Trifolium dubium, Potentila sp. Geranium 30 ocellatum, Gentiana argentea, Rannunculus laetus, Ranunculus muricatus. However, all the habitats of V. wallichii were moist and humus rich and mosses and ferns form the dominant vegetation there. Table IV. 3 Floral morphometry of Hermaphrodite and Female flowers of Valeriana wallichii S. No. Character 1. No. of flowers inflorescence 2. Length x width of bract (mm) 3. Length x width bracteole (mm) Length x width corolla (mm) 4. per of of 5. Stigma size (µ) 6. Size of stigmatic papillae (µ) Length of style (mm) 7. 8. Size of ovary (l x w) (mm) 9. Size of anthers (l x w) (mm) 10. Length of stamen (mm) Average value H flowers F flowers 383.8 ± 2 38.5* 385.4 ± 382.6* (130 - 1469)** (67 - 2162) ** n = 57 n = 38 3.9 ± 0.5 x 0.5 ± 0.09 3.92 ± 0.4 x I ± 0.1 n = 31 n = 25 3.2 ± 0.46 x 0.5 ± 0.09 3 ± 0.2 x 0.7 ± 0.2 n = 31 n = 30 6.4 ± 1.3 x 5.6 ± 0.9 3.2 ± 0.5 x 2 ± 0.4 (4.5 - 10.5) x (4 - 8) n = 80 n = 80 267.9 ± 65.14 x 474 ± 170 312.8 ± 65.8 x 546.3 ± 183.3 n = 60 n = 34 37.2 ± 9.2 x 16 ± 3.5 55.75 ± 14.2 x 19.7 ± 5.6 n = 70 n = 70 6.16 ± 0.9 3.4 ± 0.4 (4.5 - 8) (2 - 5) n = 80 n = 46 2.17 ± 0.5 x 0.96 ± 0.2 1.84 ± 0.5 x 1 ± 0.4 (1 - 3) x (0.5 - 2) n = 40 n = 40 1007.69 ± 48.3 x 965.38 ± 260.2 ± 39.18 x 192.6 ± 47.8 48.5 n = 15 n = 26 5.73 ± 0.69 (5 - 7) n = 30 *- Mean ± SD; ** - Range; n – Sample size 31 Table IV. 4 Proportion of each sex type in natural populations of V. wallichii S. No. Population Total individuals 104 H - type plants A B 62 59.6% F - type plants A B 38 36.5% 1. NC 2. BK 115 56 48.6% 56 48.6% 3 2.6% 3. BD-1 442 223 50.4% 204 46.1% 15 3.3% 4. BD-2 263 132 50.1% 122 46.3% 9 3.4% 5. MG 160 110 68.7% 47 29.3% 3 1.8% 6. BF 633 355 56.0% 269 42.4% 9 1.4% 7. DKG 178 111 62.3% 63 35.3 2.2% 1,895 1,049 55.3% 799 42.1% 47 Grand total GM - type plants A B 4 3.8% 4 2.4% Population codes: NC - Noorichamb; BK - Bakori; BD - Budhal; MG - Manyal Gali; BF - Bafliaz; DKG - Dera Ki Gali. A = No. of individuals; B = Proportion of each type in the population IV.1e Plant phenotype: Valeriana wallichii is an evergreen herb. The sporophytic body consists of an underground rhizome and leafy offshoots arising from it (Fig. 5a). With the onset of flowering, peduncles arise from these offshoots (Fig. 5b). Rhizome is 0.5 - 6.2 cm long, with irregular annulations and branches irregularly (Fig. 5a). It emits a pleasant odour due to which the species derives its local name ‘Mushkbala’. Primary root is a tap root present at the tip of rhizome where as numerous fibrous roots arise throughout the length of the rhizome. Leaves arise directly from the rhizome in opposite decussate manner. These basal leaves are petiolate, petioles 2 – 19 cm long, leaf lamina is cordate-ovate with undulating margins and an acute apex. Leaves have unicostate reticulate venation. All the basal leaves arising from the rhizome are 32 similar except for the pair of pinnatisect leaves in whose axils the flowering peduncles arise during 2nd to 3rd week of November and mark the initiation of flowering. The plants collected from Chakrata and Almora in Uttrakhand state differed from the plants of other accessions in terms of their stoloniferous habit. In these plants some aerial shoots were produced during flowering season that grow horizontally over the ground and produce leafy offshoots aerially and adventitious roots below at the point of contact with soil (Fig. 6). These offshoots later established as independent plants when the stolon dries up. IV.If Flowering: Flowering season in this species extends from 2nd to 3rd week of November to 2nd week of May. Peak flowering time is between February to middle of March. The initiation and total duration of flowering phase was found to be dependent upon the local atmospheric temperature and other physical conditions. Low atmospheric temperature and other climatic parameters like snowfall in the habitat range were observed to delay the onset of flowering up to 2nd week of January. However, it was observed that a sudden increase in atmospheric temperature during peak flowering period accelerates the flowering leading to early onset of the fruiting in the 1st Week of April. IV.Ig Floral phenology and morphology: Inflorescences appear from the axil of the youngest pair of basal leaves during 2 ndto 3rd week of November. Peduncles elongate and reach up to an average height of 65.7 cm. These are unbranched and bear 1 - 2 pairs of pinnatisect leaves in opposite decussate manner (Figs. 7 to 8). Each of these leaves bear 3 - 7 leaflets. Peduncles have a purple tinge. Inflorescence is a complex dichasial cyme but last few flowers 33 are borne in helicoid manner (Figs. 9a to b). Average spread of the inflorescence is 10 cm. Flowers are bracteate, bracteolate, zygomorphic, epigynous and bisexual or pistillate (Figs. 10a to d). Bracts and bracteoles are linear with purple tip and margins. Calyx is represented by 10 - 13 infolded fleshy segments present at the top of ovary (Fig. 11). These segments unfold and are transformed into a feathery pappus like structure during fruit formation (Fig. 12). Glandular hair in triplets are present beween the calyx lobes or segments (Fig. 13). Corolla is white, gamopetalous, pentamerous and tubular with five equal lobes arising quincuncially from the corolla tube. A sac like projection is present at the base of corolla in which nectar is present (Fig. 14). The corolla tube is hairy inside. Three epipetalous stamens with dorsifixed anthers constitute the androecium. Anthers are tetralocular and dehisce by longitudinal slits. In pistillate flowers, rudimentary stamens with shriveled and empty anthers lie at the base of corolla tube (Fig. 15). In gynomonoecious plants, some of the male sterile flowers have anthers with non viable pollen (Fig. 16a to c), and some flowers are like those in female plants. Gynoecium consists of an inferior ovary which is unilocular having a single anatropous ovule with apical placentation. A single style arises from the top of ovary and terminates in a trifid stigma which is papillate and dry type (Fig. 17). Surface of ovary is marked with three longitudinal ridges. Pubescence over the ovarian surface may be dense, sparse or altogether lacking. Data on morphology is tabulated in table IV. 5. 34 Table IV. 5 Morphological characters in Valeriana wallichii S. No. Character Average value 1. Length of rhizome (cm) 2. Length of petiole (cm) 3. Length x width of basal leaves (cm) 4. Length x width of peduncle leaves (cm) 5. No. of peduncles per plant 5.4 ± 11.3* (0.5 - 6.2)** n = 40 9.16 ± 4.6 (2 - 19) n = 80 5.9 ± 2.38 x 4.18 ± 1.7 (1.8 - 12); (1.2 - 9.2) n = 80 4.65 ± 1.6 x 3.35 ± 1.2 (2.6 - 9.1) x (1.7 - 5.8) n = 37 12 ± 7 (2 - 37) n = 80 6. Height of peduncle (cm) 7. Diameter of inflorescence (cm) 40.7 ± 5.5 (5 - 65.7) n = 80 10 ± 5.5 (1.4 - 26) n = 80 * - Mean ± SD; ** - Range; n - Sample size IV.1h Floral biology: h (i) Anthesis and pattern of flowering in an inflorescence: Flowers begin to open between 0500 - 0600 h and continue opening throughout the day, with the maximum number of flowers opening between 1200 - 1400 h. Flowers in an inflorescence open in a specific order, from centre towards periphery. Inflorescence in this species is a compound dichasial cyme. Inflorescence axis branches up to several orders. As in a typical cymose inflorescence, a floral bud is present at each point of bifurcation of the floral axis. As shown in Fig. 9b the floral buds at branching points of the same order open simultaneously starting from the 35 bud present at the point of first branching of the axis and moving towards periphery i.e. buds at the successive orders of branching. Buds of any particular order open on the same day. The hermaphrodite flowers exhibit dichogamy and are protandrous (Figs. 18a to b). Anther dehiscence occurs simultaneously with the beginning of anthesis when a small opening is formed at the top of floral bud by separation of corolla lobes. All the 3 anthers dehisce simultaneously. Style remains concealed inside the flower at this time (Figs. 18c to d). A flower takes about 5 to 6 h to open completely when 5 corolla lobes are oriented almost perpendicular to the corolla tube and 3 stamens elongate and extend out of corolla. After remaining in this position for about three days, the fives petals as well as stamens droop downwards. A flower remains on the inflorescence for 5 to 6 days after anthesis and thereafter corolla tube gets detached from the top of ovary. On the other hand, in pistillate flowers emergence of stigma from a tiny pore formed at the top of corolla tube marks the beginning of anthesis (Fig. 19). The stigmatic lobes can be distinguished at this time (Figs. 19 to 20a). The longevity of female flowers on inflorescence is same as that of the hermaphrodite flowers. h (ii) Stigma receptivity: The time of stigma receptivity varies in female and hermaphrodite flowers. In female flowers, stigma is receptive as soon as it comes out of the corolla tube i.e. right at anthesis (Fig. 20b). Its three lobes and papillate nature can be observed under a microscope even at this stage (Figs. 21). As the flower opens completely, style elongates and stands erect in the middle of flower and three lobes of stigma also expand and become clearly differentiated. It retains 100% receptivity for 5 days and the decline in receptivity was observed on 6th day. Stigma also withers by this time. 36 However, if stigma is pollinated in between this period style dries within 5 - 6 hours of pollination. Stigma is densely papillate and wet. In hermaphrodite flowers, which are protandrous, stigma emerges out of corolla tube after two days of anthesis and anther dehiscence. At this time the three stigmatic lobes are indistinguishable (Fig.18d). By this time the stamens have become fully extended and directed away from the centre of the flower. After about 52 h, three lobes become distinct. Stigma receptivity commences on the 3rd day more precisely after 50 h of anthesis and is retained for 3 days with the peak receptivity on the 5th day of anthesis i.e. between 96 - 120 h. Stigma looses receptivity, withers and dries on 6th day. Stigma is papillate and dry. The pattern of stigma receptivity in female and hermaphrodite flowers is compared in table IV. 6. A continuous rise in the atmospheric temperature accompanies the progression of the flowering season From January to March. This rise also affects the rate of style elongation and time of onset of stigma receptivity. With moderately low atmospheric temperature during the initial phases of flowering season between Januarys to middle of March, rate of style elongation and receptivity follow the pattern described above. Some of the manual pollinations made during later periods of flowering season, showed that in some cases, emergence of style from the corolla tube and onset of stigma receptivity occurs early with in a period of 7 - 12 h after anthesis (Table IV. 7). It was experimentally studied during the late flowering period i.e. between 3rd week of March and 2nd week of April. 37 Table IV. 6 Status of stigma receptivity in hermaphrodite and pistillate flowers in V. wallichii S. No. 1. 2. 3. 4. 5. 6. 7. Time after anthesis ( Days) At anthesis Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Percentage of receptive stigmata H- Flowers F- flowers nil 9.3% 10% 66% 87.8% 100% 18% 100% 100% 100% 100% 100% 100% 25% h (iii) Pollen output, pollen viability, pollen size and pollen ovule ratio: A single flower of this species on an average produces 2,260 pollen whereas only a single ovule is present in each ovary. This results in a high pollen ovule ratio of 2260:1 for the species. Pollen grains are spherical in shape, with rough exine and three germ pores. Pollen grains are at two celled stage at the time of their release from the anthers. The average viability of pollen as determined by FDA test and 1% Acetocarmine was found to be 91.5 % and 91.9% respectively (Figs. 22a to b). However, some of the plants from different populations were found to have low pollen viability. Five such plants from three populations, Noorichamb and Patnitop in Jammu and Kashmir and Chakrata in Uttrakhand were analysed in detail. Comparison of pollen viability among the normal plants of each of these three populations to the abnormal plants i.e. plants showing low viability is shown in table IV. 7. As shown in the table IV. 7 the overall average for pollen viability of these abnormal plants was 51.8% (R= 36.9% - 62.9%) as compared to the species average of 91.5% and 91.9% in FDA and 1% Acetocarmine respectively (Figs. 23a to b). Pollen grains of the 38 species were also found to exhibit polymorphism with respect to size. It was noticed that within a single anther pollen grains corresponding to three classes of size were present, small size (<30 μ), medium (30-50 μ) and large sized (50-60μ) (Table IV. 8) (Fig. 24). Table IV. 7 Pollen viability in different types of plants of Valeriana wallichii S. No. Population Plants with high pollen viability Plants with low pollen viability code Stainability FCR test Stainability PT 94% ± 5.5* (83.8 - 98.9%)** n = 10 90.9% ± 6.5 (80.4 - 97.6%) n = 13 i) PT20: 62.9% ± 38 (6.3 - 99.8%) n = 10 FCR test 1. ii) PT64: 55.62% ± 24.4 (11 - 92.4%) n = 10 53.8 ± 23.1 (7.1 - 9 4.9%) n = 12 i) CR7: 55.6% ± 6.5 (40.3 - 61.5%) n = 10 57.9% ± 4.3 (43.9 - 57.5) n = 10 ii) CR20: 36.9% ± 6.1 (31.2 - 47.8%) n = 10 46.6% ± 6.3 (39.9 - 56.9) n=6 i) NC9: 48.3% ± 1 4.6 (20.3 - 67.84%) n = 11 43.5% ± 11.9 (20.3 - 57.9) n = 10 2. CR 93.5% ± 2.3 (88.9 - 96.4%) n = 10 90.9% ± 4.6 (83 - 99.7%) n = 19 3. NC 92.7% ± 6 (78.8 - 98.8%) n = 12 91.8% ± 9.4 (67.7 - 100%) n = 20 * - Mean ± SD; ** - Range. n – Number of plants studied. Population codes: PT= Patnitop (Jammu and Kashmir); CR= Chakrata (Utrakhand); NC= Noorichamb (Jammu and Kashmir) 39 Table IV. 8 Frequency (%age) of pollen grains (per anther) of different sizes in Valeriana wallichii Parameter Mean Frequency Small pollen (%) 4.5* (0.5- 24.5) ** (n = 20) Medium pollen (%) 93.8 (72.9 - 100) (n = 20) Large pollen (%) 1.58 (0.3 - 7.5) (n = 20) *-Mean; **- Range; n-Sample size Besides size, difference in viability of these pollen was also observed which was 74%, 95.5% and 100% respectively in Acetocarmine and 55.8%, 84 % and 100% respectively in FDA (Fig. 24). Relative proportion of different kinds of pollen in 12 anthers taken from different plants is shown in the table IV. 9. Table IV. 9 Frequency (%age) of different types of PMCs in 12 different anthers Anther no. Frequency of Small PMCs (20 - 30μ) Frequency Frequency of of Medium Large PMCs PMCs (50 - 65 μ) (30 - 50μ) 1. 40.3% 50.8% 8.7% 2. 29.5% 64.7% 5.6% 3. 30.5% 65.5% 4% 4. 84.4% 15.5% 0% 5. 54.2% 42.8% 2.8% 6. 46.6% 52.5% 0.8% 7. 29.5% 64.7% 5.6% 8. 46.5% 44.8% 8% 9. 70.1% 28% 1.7% 10. 18.7% 75% 6.2% 11. 31.5% 67.6% 0.7% 12. 45.2% 48.4% 6.3% 40 h (iv) Pollination: Mode of pollination in this species is entomophilly. Anemophily was ruled out on the basis of results of hanging slide experiments. Also due to the large size and rough exine, pollen remains clinged to the anthers unless foraged by the insects. Conspicuous and dense inflorescences attract a number of insects. Flowers offer nectar and pollen as rewards to the visiting insects. Different species of bees, Apis florea, Apis cerana, Apis dorsata are the most common visitors (Figs. 25a to b). Apis florea makes its appearance during peak flowering season and keeps visiting the flowers till the season ends. Apis cerana and Apis dorsata were however, present right from the beginning till end of the flowering period. Besides these, house flies, ants (Fig. 26) and lady bird. Beetles are among the other insect species that were found to visit the inflorescences of Valeriana wallichii throughout the flowering season but their visitation rate decline towards the end of flowering season. Butterflies and bumble bees also make occasional visits to these flowers. The frequency of visits of insects, their foraging behavior and pollen load on different body parts has been listed in table IV. 10. Foraging behavior and pollen load on body parts of different insects proves Apis florea, Apis dorsata and Apis cerana to be the main pollinators of this species. However, inspite of their less frequent visits, house flies are also capable of pollinating the pistils. All these insects visit the inflorescences of both hermaphrodite. 41 Table IV. 10 Foraging behavior and pollen load on body parts of insects S. No . 1. Name insect of the Visiting duration Apis dorsata Through Very out the frequent season. 2. Apis cerana Early parts Frequent of season (Jan.-Feb.) 3. Apis florea 6. Musa domestica 7. Lady bird beetle Middle and later parts of the season (March April) Throughou t the season Later part of the season 8. Bombus sp. 9. Big black Ants Frequency of visits Very frequent Preferred target Nectar only Cephalothorax = 3,380 Abdomen = 6,397 Wings = 76 Nectar only Cephalothorax = 1917 Abdomen = 1894 Nectar and Cephalothorax pollen = 874 Abdomen = 486 Frequent Pollen only Frequent Aphids infesting the inflorescen ces. Nectar Middle of Occasional season Later part Frequent of the season Pollen load Aphids Cephalothorax = 311 Abdomen = 286 Nil Nil Nil and female plants during later period of the season, plants particularly floral parts of Valeriana wallichii become heavily infested with aphids which suck the sap through their piercing mouth parts (Figs. 27a to c). The primary target of ants and lady bird beetles visiting the inflorescences was to prey upon these aphids, pollen if available is their secondary choice (Fig. 28). Beetles, ants and Apis florea were also seen eating the floral parts like corolla and style during the later phase of flowering season. Pollinator activity also shows a considerable decline and almost stops with the rise in 42 atmospheric temperature as the flowering season advances. Pollen load on open pollinated stigmata from natural populations as well as from plants grown in nursery was studied to estimate the pollination efficiency in the species. It was observed that in case of natural populations 68% of stigmata carried pollen load. Out of 150 pistils scored during the time of peak flowering, 102 carried pollen load. Average number of pollen grains accumulated per stigma was 4 in case of female flowers and 5 in case of hermaphrodite flowers. Pollen load on stigmata from plants grown in nursery was analyzed during peak flowering in March with intense activity of pollinators and near the end of flowering period in April when insect activity declines. Table IV. 11 shows the pollen load on stigmata in case of female and hermaphrodite flowers during these phases of flowering season i.e. beginning of the flowering to its termination. Table IV. 11 Pollen load on stigmata during different phases of flowering season S. No. Phase period 1. of Percentage of stigmata carrying pollen load. Beginning flowering No. of Stigmata stigmata with scored pollen load H plants 112 89 2. Peak Flowering F H F plants plants plants 130 241 100 89 205 85 68.4% 85% 85% 3. Ending H F plants plants 171 78 117 6 68.4% 7.6% 43 79.4% h (v) Pollen- pistil interaction: Results of fluorescent microscopy of the pistils pollinated with self as well as cross pollen showed that, in both cases pollen tubes traverse the stylar tissue and reach the ovules without any hindrance (Figs. 29a to d). Pollen tubes enter the ovule through micropyle but in some pistils pollen tubes traversing through the funiculus were also observed. Rate of pollen tube growth and time required by them to reach the ovary was also determined. It was found that, in case of female flowers pollen tubes reached ovary in about 1.30 - 2 h whereas in hermaphrodites they took 3 - 5 h to reach the ovary. Rate of pollen tube growth in case of self or cross pollination was also compared by manually pollinating stigmata with self pollen in some flowers and with cross pollen in others on the same inflorescence and fixing these pistils after different intervals. Results showed that after the same time duration pollen tubes in cross pollinated pistils have moved a greater length than those in the self pollinated pistils. h (vi) Stylar movement: Style in the female as well as bisexual flowers of this species typically remains straight in the middle of the corolla tube (Fig. 30), but it was observed that as and when the frequency of pollinator visits to the inflorescences falls either due to rise in atmospheric temperature during later part of the season or due to continuous rains for 2 - 3 days during any part of season, styles in bisexual flowers assume peculiar curvature or bend so that the stigmata tend to approach the nearby anthers. Bent or bowed styles bring the stigma to a position close to the anthers of the same or nearby flowers of same inflorescence. In some cases, stigmata were observed even touching these anthers. Ideally under normal conditions stigma and anthers are separated by an average distance of 4 mm (3 - 5 mm), but it was observed that in styles undergoing 44 curvature this distance was reduced to 1 mm only. In some cases, it was also observed that styles instead of emerging straight from the corolla, tilt towards one side so that the stigma could receive the pollen fallen on the throat of corolla tube. Different kinds of movements observed in style are shown in Figs. 31a to h. To study the role of pollen limitation in inducing stylar movements, some inflorescences were put to artificially induced pollen stress by bagging them. It was found that frequency of bent styles increased in bagged inflorescences. In some flowers anthers were removed so as to check the effect of presence or absence of anthers on stylar movement. Frequency of bent styles was considerably low in such emasculated flowers (Table IV. 12) and whenever the bending was observed in such flowers, styles Table IV. 12 Frequency of bent styles under different experimental conditions Treatment Year No. of flowers observed Frequency of bent style 2010 1,714 ( 69 inflorescences) 26.13% 2011 632 (15 inflorescences) 27.3% 2012 1,012 (39 inflorescences) 39.3% 2010 885 (6 inflorescences) 61.6% 2011 152 (6 inflorescences) 32.8% 2012 113 (5 inflorescences) 39.8% 2011 123 (5 inflorescences) 10.56% 2012 46 (3 inflorescences) 13% Open pollination Bagged Inflorescences Flowers emasculated were found to be deflected towards the anthers of adjacent flowers. In some cases style showed curvature towards the staminal filaments also. The effect of different 45 manipulations i.e. bagging and emasculation on frequency of stylar bending is given in the table IV 12. In order to determine the time when the styles begin to assume a bend or curvature to approach the anthers, some flowers were tagged just at the time of anthesis and were observed at different time intervals. It was observed that styles emerge straight out of the corolla tube and remain so for about three days. If left unpollinated, these styles begin to move towards the nearby anthers on the third day of anthesis. This is also in coincidence with the onset of stigma receptivity. To check whether the curvature developed in styles was reversible or not, stigmata were manually pollinated with conspecific pollen in the styles that had developed a bend or curvature towards anthers and observed for any change in curvature. It was found that of the 60 flowers selected for this experiment, in 32, styles tend to become straight after a period of 40 minutes to 20 h and the remaining dried. IV.1i Reproductive output: Reproductive output was determined by estimating percentage fruit set under open pollinated conditions in plants from natural populations as well as from plants grown in nursery under ex situ conditions. Plants from natural populations showed an average fruit set of 42%. Under ex situ conditions percentage age fruit set in female plants was 71% and in hermaphrodite plants it was 64.5%. Manually self pollinated flowers on hermaphrodite plants set 76.5% fruit and manually cross pollinated flowers from these plants produced 78% fruit. In case of female flowers the percentage fruit set in manual pollination was 85%. Inflorescences bagged for unassisted selfing showed 20.5% fruit set. Six female plants were grown in isolation away from the main populations growing in nursery. In such plants, fruit set was only 22.5% significantly lower than the plants grown amid 46 the hermaphrodite plants in nursery. Percent fruit set under different conditions and in different kinds of plants is listed in table IV. 13. IV.1j Fruit dispersal: Fruit in this species is a small, ovate achene with a crown of feathery pappus (Fig. 32) that is derived from the modification of calyx segments which vary from 10 - 13 in numbers. Two parallel ridges running along the length of fruit and converging at the base and top just under the pappus are also present on one side of the fruit (Fig. 33). The fruit may bear dense or sparse pubescence in the form of stiff hair between the ridges or may be glabrous. Soon after ripening fruits get detached from the base leaving a scar in the axil of the subtending bracteole. Pappus helps the detached fruits to slide down the plant and also get drifted away by wind. Due to large fruit size relative to pappus, fruits are unable to get dispersed to long distances and were observed to settle in close vicinity of the parent plant i.e. within a distance of 50 cm. Big black ants were also seen carrying the seeds. Fruits are indehiscent and as such seeds are never liberated from fruit wall and germinate directly on getting favorable conditions. 47 Table IV. 13 Reproductive output under natural and different experimental conditions S. No. Kind of treatment No. of No. of flowers fruits under formed observation 1. Open pollination in situ conditions Open H plants pollination under ex situ F plants conditions Manual cross H plants pollination F plants 3,647 1,533 No. of Percent plants to fruit set which the flowers belonged 21 42..03 % 17,241 11,125 53 64.5% 13,347 10,239 28 76.7% 711 555 10 78% 862 731 5 84.8% Manual self Pollination Unassisted Selfing Female plants grown in isolation 936 742 10 79.2% 8,271 1, 698 17 20.5% 1,039 234 5 22.5% 2. 3. 4. 5. 6. IV.Ik Seed germination and seed to seed cycle: In this species seeds do not undergo any period of dormancy and germinate soon after falling on a suitable substratum. In nature it was found that seeds fallen from parent plants start germinating when the plant itself is still producing new flowers. This was also confirmed by seed germination experiments carried under lab conditions (Figs. 34a to b). Seed germination began in the month of March and continued upto August. A small proportion of seeds also germinated in the month of October but no germination occurred thereafter during November and December (Table IV. 14). Different media or substrata were used for germinating the seeds but the best results were obtained in sand, garden soil and manure mixed in 1:1:1 ratio. Germination was epigeal i.e. radical emerges first and elongation of hypocotyl leads to the elevation of 48 cotyledonary leaves. Seed germination is initiated within 4 - 8 days of sowing the seeds. Seed germination experiments were conducted by taking seeds of different origin i.e. the ones produced from female and hermaphrodite plants under open pollination and also seeds developed through manual selfing and crossing. Results of these experiments conducted during different months of the year are summarized in table IV. 15. Table IV. 14 Seed germination during different times of year Activity No. of seeds put for germination No. of seeds that germinated % germination Mar. 150 Apr. 207 May 100 June 200 Jul. 201 Aug. 139 Sep. 31 Oct. 46 Nov. 29 Dec. 59 116 157 73 129 171 76 9 2 0 0 77.3% 75.8% 73% 64.5% 85% 54.6% 29% 4.3% 0 0 Table IV. 15 Results of seed germination experiments for different kinds of seeds S. No. 1. 2. 3. 4. Kind of seeds No. of seeds put No. of seeds for germination germinated Open pollinated from H 610 403 plants Open pollinated from F 582 457 plants From MCP of H plants 198 155 From MSP of H plants 212 145 Percentage germination 66% 78.5% 78.2% 68.3% Although percentage seed germination was found to be fairly good up to October and is completely lost thereafter, best results were obtained between March and August. A total of 1,295 seeds were tried for germination between March and December. They showed an overall germinability of 56.7%. However, the seed germination was found to be 72.4% between March and August. It was found that germination percentage was higher (78.5%) in female seeds as compared to 66% germination in seeds obtained from hermaphrodite flowers. Further, 78% of the seeds obtained from 49 manual cross pollination germinated whereas the germination percentage in case of self pollination seeds was 68% only. Seeds lose their viability in a period of seven months as they showed no germination during the following year. Seedling survival was almost negligible up to June perhaps due to high atmospheric temperature. Seedlings produced during July and August showed some percentage of survival. The plants thus established successfully entered the reproductive phase during the flowering period that followed. Thus the seed to seed cycle for the species is one year. In one observation, 100 seeds collected from female plants after open pollination were put to germination, 87 germinated. Out of these only 17 reached the maturity while the rest succumbed. All the seventeen entered the flowering phase and interestingly turned out to be hermaphrodite. IV.1l Pollen mother cell meiosis: Pollen mother cells meiosis in this species was found to be unusual and interesting. The diploid chromosome number was found to be 32. During meiosis 16 regular IIs were organized but these were found to undergo precocious separation at late diplotene to diakinesis as a result of which instead of IIs, 32 Is were observed at metaphase - I stage. These 32 Is remained uniformly distributed in the cell. Inspite of precocious disjunction, anaphase - I segregation was normal and each of the two poles received 16 chromosomes at the end of first anaphase. Anaphase – II was also normal and each of the 16 chromosomes split up into chromatids which segregate normally and move to the four poles. Tetrahedral tetrads were produced at the completion of meiotic division. Except for terminal xta in some bivalents in few pmcs at early diplotene, chiasmata could not be observed at diplotene, diakinesis or metaphase stage. However, on an average chiasmata frequency calculated at late pachytene to 50 early diplotene stage was found to be 5.4 i.e. 5 xta per cell and as such the recombination index for the species was calculated as 21.4. This kind of meiosis was predominant and found in the plants producing pollen of high viability that is around 91% when tested through Acetocarmine and FDA. Different stages of meiosis in this species are shown in Figs. 35a to f. Another interesting feature of the meiosis was that pollen mother cells varied in their size and could be distinguished as small (20 – 30 μ), medium (30 – 50 μ) and large (50 – 65 μ) type while in majority of these cell types chromosome number was invariable (Figs. 36a to d). But some large pmcs varied from small and medium sized pmcs in their ploidy level. In these pmcs 64 chromosomes were observed at metaphase – I and 32:32 segregation at anaphase - I (Figs. 37a to b). While the origin of these large sized pmcs is to be worked out in detail, in some anthers fusion of pmcs at pachytene stage was observed. The frequency of different kinds of pmcs as calculated in 12 anthers from 5 plants belonging to 3 populations is given in table IV. 8. Detail of the pmcs differing in ploidy level at different stages of meiosis in two anthers studied is given in table IV. 16. Table IV. 16 Chromosomal constitution of pmcs at different stages of meiosis in Valeriana wallichii Total no. of dividing pmcs Diplotene Metaphase - I Anaphase - I With 32 II With 16 II With 32 II With 16 II With 16:16 segregation With 32:32 segregation 78 0 7 4 8 42 3 14 85 0 81 0 1 1 2 0 51 Anaphase - II Few plants (n = 5) in 3 populations produced high frequency of sterile pollen (av. 51.8%). Pollen mother cell meiosis of these plants was also studied. It turned out to be another interesting observation. Pollen mother cells meiosis in these plants was found to follow a different and unusual mechanism than that occurring in the normal plants of the species. In this case 16 bivalents didn’t separate at diplotene (Fig. 38a) as expected of this species. Instead they remain associated for unusually long period extending even beyond anaphase – I (Figs. 38b to f). Thus bivalents fail to disjoin at anaphase - I and instead of 16:16 segregation of chromosomes in the form of univalents, each pole of the cell received 8 IIs after the Ist meiotic division was over. Laggards and bridges were also observed in these cells at anaphase - I. During the 2nd meiotic division, 8 bivalents present at each pole arrange themselves at the equatorial plate at the respective ends and bivalents finally undergo disjunction during anaphase – II. The 8 chromosomes after reaching the 4 poles of pmc undergo a final segregation to have 16 chromatids at each of these poles (Figs. 38g to h). Laggards were observed both at anaphase - I and - II. In certain pmcs stickiness among chromosomes was observed (Figs. 38i to m). The low pollen viability was observed in plants undergoing this deviant meiotic pathway (Figs. 39a to b). IV.1m Analysis of genetic diversity of the species using RAPD markers: Total genomic DNA extracted following the CTAB method of Doyle and Doyle (1990) with minor modifications was checked for its quality and quantity on 0.8% agarose gel (Fig. 40). Accordingly dilutions were made and subjected to primer screening. Total 80 random decamer primers (OPA, OPC, OPD and OPJ series of Operon, Technologies) were tested, of which 18 selected on the basis of robustness of 52 amplification, clarity and scorability of banding patterns, were employed for diversity analysis. Among 251 genotypes from 11 populations, 188 bands were generated of which 82 to 157 (44.5% to 83.5%) were polymorphic within populations. The range of bands generated per primer was 9 - 12, in the size range of 280 – 350 bp, with the Table IV. 17 Polymorphic percent, Nei’s genetic diversity (H) and Shannon’s information index (I) for RAPD in Valeriana wallichii Accessions *H (mean) 0.3097 0.2900 *I (mean) * H (SD) * I (SD) Almora (AM) Bafliaz (BF) Polymorphic % 83.51 76.06 0.4569 0.4255 0.1817 0.1974 0.2501 0.2746 Buddhal (BD) Cha (C) Chakrata (CR) Kund (K) Noorichamb (NC) Patnitop (PT) Sappanwali (SW) Thajwas (TG) Tungwali (TW) 82.45 82.98 83.51 81.91 79.79 79.79 79.79 44.15 79.26 0.3077 0.3322 0.3315 0.3101 0.3103 0.3048 0.2988 0.1757 0.2928 0.4522 0.4825 0.4820 0.4541 0.4548 0.4460 0.4396 0.2557 0.4323 0.1893 0.1862 0.1841 0.1905 0.1866 0.1944 0.1899 0.2138 0.1905 0.2593 0.2557 0.2542 0.2628 0.2599 0.2682 0.2641 0.3037 0.2639 average of 10.4 bands per primer. Analysis of RAPD data revealed Thajwas glacier populations to have the lowest frequency of polymorphic bands (44.15%), while Almora and Chakrata had the highest polymorphism (83.51%) (Table IV. 17). Shannon information index and Nei’s gene diversity were lowest for Thajwas population (H = 0.175; I = 0.255) and highest for Cha population (H = 0.331; I = 0.482) (Table IV. 17). Shannon index based analysis calculated the total species diversity (HT) 0.4060, average diversity within populations (HS) 0.2967 and among population (GST) 0.2579 (Table IV. 18). 53 The species has 74.21% of the diversity within population. AMOVA showed similar results with 67.33% variation existing within populations (Table IV. 19). However, the variation among populations though much lower was statistically significant. Table IV. 18 Shannon’s estimates of genetic diversity within and among populations of Valeriana wallichii HT HS 0.4060 0.2967 Proportion of diversity within populations 0.7421 Proportion of diversity among populations(GST) 0.2579 Table IV. 19 Analysis of molecular variance for 11 populations of Valeriana wallichii __________________________________________________________ Source of Sum of Variance Percentage variation d.f. squares components of variation P __________________________________________________________ Among populations 10 3218.370 12.92029 32.67 <0.001 Within populations 242 6444.761 26.63124 67.33 <0.001 ___________________________________________________________ Total 252 9663.130 39.55154 ___________________________________________________________ The FST distance between populations ranged from 0.1957 (between Kund and Cha) to 0.4711 (between Thajwas and Sappanwali) (Table IV. 20). Overall, Thajwas population had the greatest distance from other populations followed by Noorichamb and Patnitop. Neighbour joining tree showed two major clusters, one comprising Chakrata and Almora populations and the other all the other populations (Fig. 41). Thajwas population was the most differentiated from the rest (Figs. 41 to 42). The 54 Mantel test revealed non significant correlation between genetic and physical distances (r = -0.01376). Table IV. 20. FST distances between 11 populations of Valeriana wallichii AM CR BF BD C K NC PT SW TG TW AM CR BF BD C K NC PT SW TG TW 0.0000 0.2906 0.3062 0.3396 0.3080 0.3043 0.3163 0.3041 0.3117 0.4380 0.3447 0.0000 0.3689 0.3193 0.3096 0.3621 0.3158 0.3609 0.3582 0.4338 0.3469 0.0000 0.3229 0.3235 0.3679 0.3199 0.2990 0.2795 0.4430 0.3280 0.0000 0.3076 0.3369 0.2453 0.3696 0.3141 0.4394 0.3510 0.0000 0.1957 0.3163 0.2773 0.3077 0.2956 0.2561 0.0000 0.3187 0.3293 0.3456 0.3510 0.2284 0.0000 0.2889 0.2798 0.4533 0.3349 0.0000 0.3366 0.4519 0.3264 0.0000 0.4711 0.3664 0.0000 0.4168 0.0000 IV.1n Ex situ conservation: In view of the status and magnitude of threat which the species is facing in nature, a number of measures were tried for its conservation under ex situ conditions. These include, raising plants ex situ from seeds and seedlings collected from 11 populations of the species from two Indian states, J&K and Uttrakhand. The seedlings collected and deposited in Lead Botanic Garden (LBG) of the University were at 5 - 6 leaf stage. Seeds were germinated in experimental pots and seedlings thus raised then transferred to Beds in LBG. The plants raised in Botanic Garden performed better than the in situ plants (Table IV. 13). Fruit set in female and hermaphrodite plants under ex situ conditions was 71% and 64.5% respectively where as the fruit set under in situ plants was 42%. There was hence a significant increase in the reproductive out of the species under ex situ conditions. The seedlings raised from seeds under ex situ conditions flowered in the 55 following flowering season indicating the time taken by Valeriana wallichii to complete its seed to seed cycle. It came almost to one year (Figs. 42a to b). 56