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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).
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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
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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
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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
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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
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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.
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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
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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.
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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
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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