Comparative morphological, epidermal, and anatomical studies of Pinus roxburghii needles at different altitudes in the North-West Indian Himalayas

Abstract

The aim of the present study was to understand the ecological adaptation of Pinus roxburghii Sarg. in the North-West Himalayan region. P. roxburghii needles showed morphological, epidermal, and anatomical variation at different altitudes. Needle length was negatively correlated with altitude. Stomatal characters like stomatal density, stomatal index, and guard cell lengths were found to be affected by environmental factors and showed a direct correlation with altitude. The results showed that potential conductance index was dependent on the climatic conditions of the habitat. The anatomical properties of needles exhibited variation from lower to higher elevation, especially in the number and position of resin ducts.

Full text

65
http://journals.tubitak.gov.tr/botany/
Turkish Journal of Botany
Turk J Bot
(2013) 37: 65-73
© TÜBİTAK
doi:10.3906/bot-1110-1
Comparative morphological, epidermal, and anatomical studies of Pinus roxburghii
needles at dierent altitudes in the North-West Indian Himalayas
Satyendra Prakash TIWARI*, Pradeep KUMAR, Deepika YADAV, Devendra Kumar CHAUHAN
Sahni Palaeobotany Laboratory, Department of Botany, University of Allahabad, Allahabad, India
* Correspondence: sptiwariau@yahoo.co.in
1. Introduction
Chir pine (Pinus roxburghii Sarg.) is the dominant tree species
in the North-West region of the Indian Himalayas. It is a
hard pine of lower elevation, occurring between altitudes of
500 and 2500 m and is extensively distributed from Bhutan
to Afghanistan. Chir pine grows gregariously, oen forming
pure formation in xerophytic and well-lit environments
(Mehra, 1988). P. roxburghii is an economically valuable
species, balancing the ecosystem of the Indian mountains.
e plants show microhabitat-related morphological and
anatomical features at dierent altitudes.
Körner (2007) proposed the altitude-related theory
of biological phenomenon, which adversely aected the
plant communities like reduction in plant species number
(Nagy, 2003), plant productivity (Luo et al., 2004), body
or organ size trends (Fabbro & Körner, 2004), plant
physiology and morphology (Hoch & Körner, 2003), gene
ecology (Reisch et al., 2005), and life history characteristics
(Klimes, 2003).
Leaf traits are oen aected by the ecosystem’s
characteristics, as they are directly exposed to the
environment. In vascular plants the stomata of leaves
are the most important physiological apparatus for
photosynthesis and transpiration. e development
of stomata (about 400 million years ago) is therefore
considered a key event in the evolution of advanced land
plants (Hetherington & Woodward, 2003). Stomatal
dierentiation and development are determined by genetic
factors (He et al., 1998). Stomatal initiation is controlled
by the CO2 HIC gene, setting stomatal number during
leaf formation (Gray et al., 2000). Stomatal parameters are
specic for a particular species but are aected by multiple
ecological factors, including altitude gradient (Beerling
& Kelly, 1996), atmospheric CO2 concentration (Van de
Water et al., 1994), temperature, light, and irradiance
(Lockheart et al., 1998). Environmental eects on stomatal
density, stomatal conductance, and stomatal index have
been widely studied in living as well as in fossil plants
(Woodward & Bazzaz, 1988; Royer et al., 2001; Kouwenberg
et al., 2003). δ13 C and stomatal density are popular tools
for determining palaeoatmospheric CO2 level (Beerling et
al., 1995). Morphological and anatomical features of Pinus
needles also depend on abiotic factors (Fahn & Bemayoun,
1976; Schoettle & Rochelle, 2000). Physical factors like
growth, altitude, decrease in air temperature, atmospheric
pressure, increasing precipitation, and wind velocity aect
plant growth (Friend & Woodward, 1990; Körner, 2007). At
very high elevation sites severe environmental conditions
become severe for plant development and growth. In the
present study we describe morphological, epidermal,
and anatomical variations observed in the needles of P.
roxburghii growing at dierent altitudes. e paper also
mentions ecological adaptation adopted by P. roxburghii
plants in response to stressed environmental conditions.
Abstract: e aim of the present study was to understand the ecological adaptation of Pinus roxburghii Sarg. in the North-West
Himalayan region. P. roxburghii needles showed morphological, epidermal, and anatomical variation at dierent altitudes. Needle length
was negatively correlated with altitude. Stomatal characters like stomatal density, stomatal index, and guard cell lengths were found to
be aected by environmental factors and showed a direct correlation with altitude. e results showed that potential conductance index
was dependent on the climatic conditions of the habitat. e anatomical properties of needles exhibited variation from lower to higher
elevation, especially in the number and position of resin ducts.
Key words: Ecological adaptation, Pinus, resin duct, stomatal density, stomatal index
Received: 01.10.2011 Accepted: 11.09.2012 Published Online: 26.12.2012 Printed: 22.01.2013
Research Article

TIWARI et al. / Turk J Bot
66
2. Materials and methods
Needles of P. roxburghii were collected from 3 dierent
altitudes (1215, 1350, and 1775 m) in the Kumaun region.
e Kumaun Mountains occupy the central sector of the
Himalayas from lat 28°44′ to 30°49′N and long 78°45′ to
81°1′E (Figure 1). is vast region has variable topography,
climate, soil, and vegetation. Besides the mountainous
forms, needles of the same species were collected from a
plant growing in the Department of Botany, University
of Allahabad, Allahabad, Uttar Pradesh, India. Allahabad
is situated in the upper Gangetic Plain of India. All the
climatic datasets of dierent altitudes were obtained from
the Indian Meteorological Department, New Delhi (Table
1). For the purpose of this study 5 trees were selected at
each site (98, 1215, 1350, and 1775 m). irty needles were
selected from well-grown shoots of each tree.
For morphological observations 20 needles were
randomly selected from each tree. e length was
measured by the conventional method. Micro-slides
were prepared by traditional methods and the technique
proposed by Eo (2012). Transverse sections were stained
with a combination of Safranin and Fastgreen (Johansen,
1940). All microscopic slides were examined under a
light microscope (Olympus CH20i) and electronic image
Figure 1. Location map showing plant collection site.
Table 1. Climatic dataset from all 4 elevations showing temperature, average rainfall, and relative humidity.
Altitude (m) Temperature (°C) Rainfall average (mm) Relative humidity (%)
Max (mean) Min (mean)
98 32.55 21.25 80.42 70
1215 28.27 15.22 648 85
1350 28.55 15.22 659 85
1775 25.62 12.35 1152 90
Temperature (maximum and minimum means of centigrade), average rainfall (millimetres), relative humidity (percentage).
81ʹ00ʹ40°
81ʹ00ʹ40°
77ʹ33ʹ29°
N
S
WE
77ʹ33ʹ29°
31ʹ33ʹ34°
31
ʹ33ʹ34°
28
ʹ38ʹ43° 28ʹ38ʹ43°
Legend
NEPAL
UTTAR PRADESH
HIMACHAL PRADESH
CHINA
Distt.
Almo
ra

TIWARI et al. / Turk J Bot
67
analysis equipment (Leica DM 2500 and Motic 2.0 Image
Plus).
Stomatal parameters, like guard cell lengths of 15
stomata, were measured at 40× (resolution 648 × 486, 1296 ×
97) from each of the needles from all 4 elevations. Stomatal
density was determined by the method of Hultine and
Marshall (2001). irty needles were selected randomly for
the stomatal count. e epidermis of the leaf was separated
by maceration and stomatal count made from the middle
part of the needles at 10× and 20× with the help of a Motic
2.0 Image Plus camera. e stomatal density, stomatal index,
and potential conductance index were calculated with the
help of the formulae given below as equations 1–3:
SD = SC/NL × NW ........................................................... (1)
SI = SC × 100/SC + nEC .............................................. (2)
PCI = (guard cell length)2 × SD × 10–4 ....................... (3)
PCI = Potential conductance index, SD = Stomatal
density, SI = Stomatal index SC = Stomatal counts, NL
= needle length, Nw = needle width, nEC = number of
epidermal cells.
All the statistical analysis was performed with the help
of SPSS v. 10.0 and STATISTICA 11 soware; graphs were
prepared using Origin 6.1.
3. Results
3.1. Morphological analysis
In this study, the needle morphology of Pinus roxburghii
was aected by the altitude gradient. Length of needles
from dierent elevations was measured and the needle
length was negatively correlated (r = 0.9635, P = 0.0364)
with altitude. Needle length at 98 m (NL= 29.98 cm) was
2 times greater than that at 1775 m (NL= 15.14 cm) (Table
2). Needle length was less variable at medium elevation,
showing about 15% decrease from 1215 m to 1350 m
compared to that of lower to higher elevation (20% needle
length decrease from 98 m to 1215 m, 24% decrease from
1350 m to 1775 m).
3.2. Epidermal analysis
Epidermis of Pinus roxburghii needles showed highly
contrasting characters at dierent elevations. e distance
between 2 stomatal rows signicantly decreased in needles
of plants from lower to high altitudes. At an elevation of
98 m the needles showed 13.61 rows of nonstomatiferous
cells, which decreased to 4.80 rows of nonstomatiferous
cells at an elevation of 1775 m. It was highly correlated (r
= 0.9815) with altitude. Stomatal density increased due to
decreases in the number of nonstomatiferous rows, as they
are inversely correlated to each other. Stomatal density
showed a positive correlation (r = 0.8284, P = 0.1716) with
altitude (Figure 2). Stomatal density increased 59.46%
from lower altitude to higher altitude. Stomatal index also
showed a positive correlation (r = 0.8689, P = 0.1310) with
altitude. e length of guard cells in needles of P. roxburghii
was also aected by a change in elevation and it increased
with altitude (Figure 3). Stomatal conductance depended
on both stomatal density and size of stomatal aperture
(Holland & Richardson, 2009). Potential conductance
index was also measured and found to be signicantly
correlated (r = 0.8637, P = 0.1365) with altitude.
3.3. Anatomical analysis
Anatomical characters of Pinus roxburghii needles varied
with altitude. Transverse sections of needles from all
4 sites were studied. ey showed a thick cuticle and
well-developed mesophyll tissue. e hypodermis was
commonly 2–4 layered, showing less variation at 98, 1215,
and 1350 m altitudes but showing much variation at higher
elevations. e layer of hypodermis decreased and became
single layered (Figure 4). As P. roxburghii is a hard pine,
the vascular bundles numbered 2 and were situated close
to each other, but needles from higher elevations showed
vascular bundles located opposite each other.
Table 2. Variations in stomatal density, stomatal index, nonstomatiferous cells, needle length, potential conduct index, guard cell length,
position of resin duct, and number of resin ducts.
Altitude
(m)
Stomatal density
Pore/mm2
(mean ± SD)
Stomatal
index
NSCs between
stomata (mean)
Nl (cm)
mean ± SD PCI
Guard cell
length (µm)
mean ± SD
Position of
resin duct
Number of
resin ducts
98 29.0 ± 2.127 4.208 13.614 29.98 ± 3.511 4.30 38.51 ± 2.444 EX 3
1215 37.75 ± 4.632 6.404 9.266 23.70 ± 2.101 7.71 45.22 ± 3.234 EX, SM 0, 1, 2
1350 43.40 ± 6.707 7.451 7.466 20.15 ± 1.889 9.57 46.96 ± 2.593 EX, M 2
1775 71.55 ± 8.140 12.451 4.800 15.14 ± 0.900 17.71 49.76 ± 2.351 M, ED 3
Stomatal density and guard cell length (data are mean of 15 replicates and standard deviation); NSC: Nonstomatiferous cells; Nl: Needle
length; PCI: Potential conduct index.

TIWARI et al. / Turk J Bot
68
Pinus roxburghii had 2–3 resin ducts per needle,
situated medianly and externally. At lower elevation (98
m) needles showed 3 resin ducts placed externally (Figure
4). At higher altitude (1215 m) the number of resin ducts
varied from 0 to 2. ese were situated slightly medianly
and externally. Needles of plants at 1350 m altitude had
2 resin ducts, placed medianly and slightly externally. At
higher elevation (1775 m), resin ducts numbered 3 but
were endonal in position (Figure 4), being just the opposite
of those at the lower elevation.
4. Discussion
ere are complex ecological factors aecting plant growth
at dierent altitudes, especially conifers, which are present
over a range of elevated regions in the world. Altitude
plays an important role in changing the physical factors,
decreasing total atmospheric pressure, and reducing
atmospheric temperature with implications for ambient
humidity (annual temperature decreases with elevation by
about 6.5 °C per km), increasing radiation under a cloudy
sky because of reduction in turbidity and a high fraction of
radiation at any given total radiation (Körner, 2007).
Changes in epidermis structure in needles are an eco-
physiochemical process. Environmental factors show a
direct response on stomatal pattern in the epidermis of
needles in the Kumaun Mountains. Epidermal features
such as stomatal density, stomatal index, guard cell length,
and potential conductance index (PCI) vary with altitudes.
e present study shows that stomatal parameters play a
signicant role in the adjustment of plants at dierent
altitudes. Stomatal arrangement responds to light intensity;
an increase in light intensity results in an increase in stomatal
index (Coupe et al., 2006). P. roxburghii, which grows on
the eastward slopes of the Kumaun Mountains, receives
more light intensity and light period than plants growing
on the westward slopes. e stomata of P. roxburghii are
more aected by the light intensity. Körner (1988, 1999)
suggested that the elevation changes the stomatal density
due to the eect of foliar light interruption. In the case of
the rst type of mountains, insulation increases at higher
elevation because shorter atmospheric path length reduces
scattering and absorption. In the case of the second type
of mountains, the frequency of cloud immersion increases
with elevation and insulation decreases with elevation.
e rst described mountains show stomatal density that
typically increases with elevation due to less scattering
of light or a higher transpiration rate. e North-West
Himalayan mountainous region has diverse topography;
it is considered the rst type of mountain system because
Figure 2. A- Negative correlation between needle length and altitude. B- Correlation between stomatal density (SD) and altitude.
0 200 400 600 800 1000 1200 1400 1600 1800 2000
14
16
18
20
22
24
26
28
30
32
34
Needle length (cm)
Altitude (m)
r = 0.9635
AB
0 200 400 600 800 1000 1200 1400 1600 1800
2000
20
30
40
50
60
70
80
Stomatal density (pore/mm
2
)
Altitude (m)
r = 0.8284
0 200 400 600 800 1000 1200 1400 1600 1800
2000
34
36
38
40
42
44
46
48
50
52
54
Guard cell length (µm)
Altitude (m)
r = 0.9965
Figure 3. Correlation between guard cell length of stomata and
altitude.

TIWARI et al. / Turk J Bot
69
the stomatal density of P. roxburghii increases with
altitude. Stomatal density is the major measure that
indicates gaseous changes. According to Woodward
(1987), stomatal density and stomatal index decrease with
increasing atmospheric CO2 level both in geological time
and under laboratory conditions. It is reported that CO2
can directly aect stomatal dierentiation (Lockheart et
al., 1998) and that stomatal density is negatively correlated
with atmospheric CO2 below 3000 m (Qiang et al., 2003)
because CO2 level decreases with increasing elevation.
Previous studies have shown increases in stomatal
density, with elevation acting as a limiting factor in
photosynthesis. Increases in stomatal density resulting in
increasing stomatal conductance should oset the decreases
in pCO2, but it is reported that such CO2 availability is
not a limiting factor (McElvain, 2004). Stomatal density
AB
10 µm
10 µm
10 µm 10 µm
CD
E
G
F
H
Figure 4. Transverse section of Pinus roxburghii needles. A- Needle section at 98 m, showing 3 external resin ducts, B- Needle section at
1215 m, resin duct absent, C- Needle section at 1350 m, 2 external resin ducts, D- Needle section at 1775 m, 3 resin ducts 1 medial and
2 endonal in position, E- Structure of a resin duct, F- External resin duct, G- Medial resin duct, H- Endonal resin duct.
Abbreviations: EC = epithelial cell, SC = sheath cell, MC = mesophyll cell, L = lumen of resin duct, scale bar = 10 µm.

TIWARI et al. / Turk J Bot
70
and size are considered as eco-physiological parameters
because they conjugately inuence stomatal conductance.
According to Körner and Cochrane (1985), stomatal
density did not reect variations in stomatal conductance
under an integrated inuence of specic environment
at higher altitude. Guard cell length in needles of P.
roxburghii increases with altitude. Potential conductance
index is signicantly correlated from all elevation sites
because stomata at higher altitude were not open entirely
under severe environmental conditions such as low
temperature and irradiation. Enhanced UV-B radiation at
higher altitude limits stomatal opening normally and leads
to decreases in stomatal conductance, but in the present
study potential conductance index increased with altitude.
Stomatal conductance might be constrained at higher
altitude by low air and soil temperatures because they
inhibit stem sap ow, which increases the water potential
gradient and induces partial stomatal closure, leading to
decreased stomatal conductance. Water availability is an
important factor from lower altitudes to higher altitudes
that aects stomatal size and stomatal conductance.
Stomatal traits change with elevation due to response of
water availability rather than CO2 signicance (Beerling &
Kelly, 1996). Stomatal characters are modied by climatic
changes, which is also a signicant nding in the present
study.
In the present study, Pinus roxburghii showed
morphological traits negatively correlated with altitude.
Length of needle decreased with higher elevations. Eect
of altitude on morphology has been studied in Pinus
sylvestris L. (James et al., 1994), Pinus pumila Regel.
(Kajimoto, 1993), and Pinus contorta Douglas ex Louden
(Schoettle, 1990). ey all have shown reduced leaf length,
shoot growth, and leaf production per year with increasing
elevation. Leaf structure is modied according to need in
nature. Leaf morphology is aected by amount of δ13 C
and drought. Generally air becomes drier with increasing
elevation, but the diusion coecient of water vapour in air
also increases at higher elevation; both phenomena aect
the needle’s morphology. A sharp increase in needles’ δ13 C
suggested a strong capacity for CO2 assimilation, resulting
in rapid plant growth. Length of needles also varies due
to other environmental gradients like seasonal variation
(Armstrong et al., 1988) and temperature. e present
study reveals that the length of P. roxburghii needles is
sensitive to the limiting factors in any given environment
and shows morphological changes.
Anatomical properties like leaf structure, leaf shape,
and cell distribution also change together with leaf
function for adaptation to severe ecological conditions.
Chir pine needles from all 4 elevation sites showed
interesting anatomical variation. Epidermal cells were
smaller and narrower in plants at higher elevation sites
than in those at lower elevation. Number of hypodermal
cell layers decreased from lower to higher altitudes.
e mesophyll cells showed a similar arrangement but
dierentiation in size because CO2 concentration aected
mesophyll cell structure and development (Lin et al.,
2001). Leaf thickness increased due to well-developed
mesophyll tissue. Elevation also aected light intensity,
because the photosynthetic rate changed. Needle anatomy
is also inuenced by soil resource enrichment or nutrient
availability in soil.
Transverse sections of Pinus needles were cut to study
the structure and position of resin ducts. Resin ducts are
a characteristic feature of conifers, occurring in vascular
tissues and ground tissues of all plant organs. Resin ducts
are also useful in the identication of species. e role of
resin ducts in classication of Pinus is more appreciated
compared to other polyphyletic traits, which are oen
treated as ambiguous. In the resin duct of Pinus, the duct
cavity is surrounded by a thin layer of unliginied cells,
which are termed epithelial cells. Outside these are one or
more layers of cells with relatively thick unlignied walls,
termed sheath cells. In P. roxburghii, resin ducts have a
wide cavity, surrounding a layer of 6–7 thin and delicate
epithelial cells. e sheath cells may vary in number from
8 to 12 and have thick walls (Figure 4). According to Napp-
Zinn (1966) and Biswas and Johri (1997), 4 types of resin
ducts are present in the needles of Pinus: 1) ducts in contact
with the endodermis (i.e. endonal); 2) ducts in contact
with the hypodermis (i.e. external); 3) ducts present in the
middle of mesophyll layers (i.e. median); 4) ducts inside
the bundle sheath (i.e. septal) (Figure 4). Most Pinus
species contain 1 or 2 types of resin ducts in the needle.
Needles of P. roxburghii generally show 2–3 external or
medial resin ducts (septal resin duct is not seen) (Figure
4). Variation in the arrangement pattern of resin ducts
in P. roxburghii at dierent altitudes was studied here for
the rst time. e number of resin ducts, however, does
not vary from lower to higher elevation sites. e middle
elevation (1215, 1350 m) sites show much variation (0, 1,
2). e most signicant feature is the position of the resin
duct, which varied from external (98, 1215 m) to medial
(1215, 1350 m) to endonal (1775 m) at dierent elevations
(Figure 4). e number of resin canals in P. roxburghii is
controlled genetically because it does not vary in number
but variation is seen in the position of the mesophyll. e
change in position may be attributed to the change in
altitude gradient and climatic factors.
Variations in resin duct patterns and changes in
the number and size of mesophyll cells are reportedly
aected by altitude (Sheue et al., 2003). Variations in the
distribution and number of resin ducts in the plant body

TIWARI et al. / Turk J Bot
71
are also aected by several genetic and environmental
factors, such as height and age of the tree, nutrition,
sunlight, radiation, temperature, wind, freezing, re,
insect attack, and phytohormones (Helmers, 1943; Fahn
& Bemayoun, 1976). ere is no signicant change in the
number of resin ducts with increasing elevation. Matziris
(1984) reported that the number of resin ducts increases
with increasing latitude. e evolutionary signicance of
the number of resin ducts is not well understood, but the
relationship between them and the resistance of genotypes
to specic insect attack has been reported (Overhulsen
& Cara, 1981). us it is clear that altitude aects the
position and number of resin ducts. Physiochemical
changes adapted by plants may also have an eect but this
requires detailed study.
5. Conclusion
Pinus roxburghii, a common wild species of conifer in the
North-West Himalayan region, grows at a wide range of
altitudes. It is the principle resin-producing species in
India. e present study reveals that P. roxburghii is mainly
aected by the altitude gradient or climatic factors. Trees
native of lower elevations can be distinguished from trees of
higher elevations on the basis of epidermal, morphological,
and anatomical characters of needles. Stomatal density,
stomatal index, and potential conductance index
show signicant variation at dierent altitudes due to
environmental factors such as CO2 concentration, light
intensity, and water availability (Figure 5). Needle length
is also aected by ecological factors; they are signicantly
negatively correlated with altitudes. e most interesting
A B
CD
Figure 5. Array and distribution of stomata on epidermis of Pinus roxburghii needles. A- at 98 m height, B- at 1215 m height, C- at 1350
m height, D- at 1775 m height. Scale bar = 20 µm.

TIWARI et al. / Turk J Bot
72
feature shown, however, is the variation in the position of
the resin duct (external, medial, and endonal) at dierent
elevations, which is a genetically controlled characteristic
in plant parts. Trees at lower elevation, with external resin
ducts, can be distinguished from higher elevation trees,
having medial and endonal resin ducts. us, we can
conclude that altitude and environmental factors can aect
the physiochemical process of P. roxburghii in the Indian
Himalayan region.
Acknowledgements
e authors are highly grateful to Prof Nupur Bhowmik
for going through the manuscript and giving her advice
about completing this paper. We are indebted to the
Head of Department for providing laboratory facilities.
e authors are also thankful to Prof Manju Sahney for
her valuable suggestions and all other members of the
Palaeobotany and Morphology laboratory.
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