CHAPTER 6
99
Chapter 6
Assessment of genetic diversity through micro-
morphometric markers
6.1 Introduction
6.2 Materials and Methods
6.3 Results
6.4 Discussion
6.1 Introduction
relatively scarce.
Cladiastics
Cladistics is a method of
classifying species of
organisms into groups
called clades, which consist
of an ancestor organism
and all its descendant only.
For example, birds,
dinosaurs, crocodiles, and
all descendants (living or
extinct) of their most
recent common ancestor
form a clade. In terms of
biological systematics, a
clade is a single "branch"
on the "tree of life", a
monophyletic group.
Cladists use cladograms
diagrams which show
ancestral relations between
species to represent the
monophyletic relationships
of species, termed sister
group relationships. This is
interpreted as representing
phylogeny, or evolutionary
relationships.
Source: Wikipedia
CHAPTER 6
Interesting patterns of species
nature have mostly been revealed through many
microstructures, which may be essential for elucidating
relationship between acquired morphology and the
endurance of flowering plants. In response to continuous
selection pressure during species diversification, variations
at interspecific level occur, and are responsible for varied
phenotypes. The evolution of such morphological variability
is mostly regulated by genetic components and their
interactions with environment.
A good example of such variability is the pollen
grains. The type of pollen grain of any given taxon is
characteristic and consistent suggesting high degree of
genetic regulation (Hemsley et al. 2000). Therefore, study of
differential pollen features from genetic perspective is
essential to enhance our understanding of the ecological
significance of variations in pollen morphologies. The cluster
of mature pollen grains comprised of a complex str
called pollinium having complete cell walls containing sperm
cells inside. The pollinium cell walls are uniquely developed
over time with constructions and elaborated surface layers,
though vary subtly in surface stratification among species
(Lumaga et al. 2006). At maturity, the pollen surface has
outer multilayered exine wall interrupted by openings called
aperture; ii) an inner single or multilayered intine; and
pollen coat, composed of lipids and proteins that fills the
sculptured cavities of exine. The pollen heterogeneity of
Dendrobium species may have promising systematic utility as
proposed earlier (Pridgeon 1999). Since all living
species are epiphytes, thus making it difficult to observe
pollination and hence, information on pollination biology is
is a method of
classifying species of
organisms into groups
, which consist
of an ancestor organism
and all its descendant only.
For example, birds,
dinosaurs, crocodiles, and
scendants (living or
extinct) of their most
recent common ancestor
form a clade. In terms of
biological systematics, a
clade is a single "branch"
on the "tree of life", a
cladograms –
diagrams which show
ral relations between
to represent the
elationships
of species, termed sister-
group relationships. This is
interpreted as representing
phylogeny, or evolutionary
Source: Wikipedia
100
Interesting patterns of species-level diversity in
nature have mostly been revealed through many
microstructures, which may be essential for elucidating
relationship between acquired morphology and the
sponse to continuous
selection pressure during species diversification, variations
at interspecific level occur, and are responsible for varied
phenotypes. The evolution of such morphological variability
is mostly regulated by genetic components and their
A good example of such variability is the pollen
grains. The type of pollen grain of any given taxon is
characteristic and consistent suggesting high degree of
. 2000). Therefore, study of
differential pollen features from genetic perspective is
essential to enhance our understanding of the ecological
significance of variations in pollen morphologies. The cluster
of mature pollen grains comprised of a complex structure
having complete cell walls containing sperm
cells inside. The pollinium cell walls are uniquely developed
over time with constructions and elaborated surface layers,
though vary subtly in surface stratification among species
. 2006). At maturity, the pollen surface has i) an
outer multilayered exine wall interrupted by openings called
an inner single or multilayered intine; and iii) a
pollen coat, composed of lipids and proteins that fills the
vities of exine. The pollen heterogeneity of
species may have promising systematic utility as
proposed earlier (Pridgeon 1999). Since all living Dendrobium
species are epiphytes, thus making it difficult to observe
tion on pollination biology is
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101
The seeds of orchids are generally microscopic in nature and numerous; in some
species over a million per capsule having a simple embryo and often lacking endosperm
(Dressler 1993; Molvary and Chase 1999; Clements 2003). The seeds are wind dispersed and
exhibit a great deal of varied morphological characters in size, form and structure, for
example, seed size varies from 150 µm to 6000 µm (Molvray and Kores 1995). The seeds are
protected with seed coat often showing marked thickenings and sculpturing. The thickenings
are thin in terrestrial orchids whereas thick in epiphytes (Vij et al. 1992). The testa cells and
embryos of different species may vary significantly in size, shape, colour, and volume (Arditti
et al. 1979; Arditti et al. 1980; Swamy et al. 2004). The number of cells contributing to testa
formation also varied greatly among species, ranging from 2-20 in number and most often
studied through scanning electron microscopy (Arditti et al. 1979; Molvray and Kores 1995).
Changes in seed morphology brought about through evolution had been important as a
source of systematic character to circumscribe sub generic groups or hypothetical
relationships among species within a genus (Mathews and Levins 1986; Ness 1989; Larry
1995). However, seed micro-specifics in orchid systematics had restricted exploration as
taxonomic and climatic preferences. Since seed coat ornamentation and sculpturing are
considered as non-specific to reveal the taxonomic relationship at inter-specific level,
comparative knowledge of seed micromorphology in respect to their climatic preferences is
yet rare in orchids (Vij et al. 1992; Jeeja and Ansari 1994; John and Jack 1998; Lumga et al.
2006).
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102
6.2 Materials and methods
6.2.1 Collection of micromorphometric data from pollinia
In the present study, both qualitative (3) and quantitative (9) micrmorphometric
traits (all together 12 variables) from pollinia were recorded (Table 6.1). Qualitative traits
were recorded either on the basis of presence (recorded 1 for presence and 0 for absence)
or magnitude (small 1, medium 2, and large 3) of expression. Variables were measured up to
4th decimal point of millimetre (mm).
Table 6.1 Micromorphometric traits from pollinia used for measuring genetic
distances among species of Dendrobium
Qualitative trait description Code Quantitative trait description Code 1. Pollinia color PoC 4. Pollinia length PoL 2. Pollinia shape PoS 5. Pollinia width PoW 3. Ttetrad type TeT 6. Ratio of PoL & PoW PoL/PoW 7. Tetrad length TeL 8. Tetrad width
9. Ratio of TeL & Tew TeW TeL/TeW
10.Pollen polar axis PpA 11.Pollen equatorial axis PeA 12.Ratio of PpA & PeA PpA/PeA
6.2.2 Collection of micromorphometric data from seed
In the present study, seeds were collected from at least 5 pods per population and
measurements were taken from 20 seeds per pod. Wild forms were evaluated for seed
characters in the field at the time of collection and/or in the laboratory. Both qualitative (2)
and quantitative (13) morphological characteristics (all together 15 variables) of all collected
species were recorded using previously reported seed traits descriptors (Table 6.2). Among
the quantitative morphometric traits (variables), seed volume, embryo volume and free air
space were measured according to formulae provided by Arditii et al. (1980). States of shape
characters were identified according to the plant identification terminology (Harris and
Harris 2001).
Table 6.2 Micromorphometric traits from seed used for measuring genetic distances
among species of Dendrobium
Qualitative trait description Code Quantitative trait description Code 1. Seed color PoC 3. Seed length SeL 2. Seed shape PoS 4. Seed width SeW 5. Ratio of SeL & SeW RSLW 6. Seed volume SeV 7. Embryo length
8. Embryo width EML EMW
6.2.3 Light Microscope (LM) study of pollinia
Five pollinia with three replications were observed for shape; measurements for
pollinia length and breadth were recorded digitally with the help of a stereoscopic
microscope (hund WETZLAR 1021471) with epi
study the pollens, pollinia were placed on a clean
needle, and then observed under
recorded digitally at 40X of magnification using Leica
recorded for length and width of pollenaria, spore tetrad and polar and equatorial axis of
pollen. Total 18 pollens were studied for each individual species and standard deviation
were calculated. Pollinia
axis and length of equatorial axis (
also observed (Figure 6.2
Figure 6.1: Schematic diagram of studying
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9.Ratio of EML & EMW 10.Embryo volume 11.Ratio of SEV & EmV 12.Testa cell length 13.Testa cell width 14.Ratio of TCL & TCW 15.Free air space
Light Microscope (LM) study of pollinia
Five pollinia with three replications were observed for shape; measurements for
pollinia length and breadth were recorded digitally with the help of a stereoscopic
WETZLAR 1021471) with epi-ilumination (hund WETZLAR FLQ 150). To
study the pollens, pollinia were placed on a clean glass slide and crushed with
needle, and then observed under the Leica DM 2500 microscope. M
ly at 40X of magnification using Leica-Quin softwere. Measurements were
recorded for length and width of pollenaria, spore tetrad and polar and equatorial axis of
pollen. Total 18 pollens were studied for each individual species and standard deviation
inia shape was determined by calculating the value of length of polar
length of equatorial axis (Figure 6.1). Pollen and spore tetrad characteristics were
Figure 6.2).
: Schematic diagram of studying micromorphometric traits from pollinia
103
RELW EmV ROV TCL TCW RTLW ASP
Five pollinia with three replications were observed for shape; measurements for
pollinia length and breadth were recorded digitally with the help of a stereoscopic
lumination (hund WETZLAR FLQ 150). To
slide and crushed with the help of a
. Measurements were
softwere. Measurements were
recorded for length and width of pollenaria, spore tetrad and polar and equatorial axis of
pollen. Total 18 pollens were studied for each individual species and standard deviations
shape was determined by calculating the value of length of polar
Pollen and spore tetrad characteristics were
micromorphometric traits from pollinia
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104
Figure 6.2: Schematic diagram of studying micromorphometric traits from pollen and spore
tetrad
6.2.4 Light Microscope (LM) study of seed
Seeds were stained with safranin and spread on a slide with a drop of water and
covered with a cover slip. Twenty seeds were observed for each species and measurements
were recorded digitally using Lica DM 2500 microscope and Lica-Quin softwere. The color of
the seeds were observed and described in subjective terms with the help of optical
microscope. The following parameters were studied under Light microscope: seed- color,
shape, length, breadth, volume; testa cells- number in longitudional axis, shape, length,
breadth; embryo- length, breadth, volume; and percentage of free air space present (Figure
6.3). The seed and embryo volumes were calculated following the method of Arditti et al.
(1980).
6.2.5 Scanning Electron Microscope (SEM) study of pollinia
The dissected pollinia were directly mounted on copper stubs using double side
sticky tape. The specimens were then sputter coated with gold-palladium alloy. These were
examined and photographed under JEOL-35 JSM CF scanning electron microscope at an
accelerating voltage of 15 kv to study the pollinium surface structure, aperture, shapes, and
exine sculpturing of pollen grain (Figure 6.1).
6.2.6 Scanning Electron Microscope (SEM) study of seed
Seeds were mounted on aluminium copper stubs using double adhesive tape. The
samples were then sputter-coated with gold palladium alloy for five minutes and
photographed on a JEOL-35 JSMCT-SEM at an accelerating Voltage of 15-20 KV. Detailed
seed coat (testa cells) surface studies were conducted by observing under SEM. The
considered parameters were seed coat sculpturing and thickenings (Figure 6.3).
Figure 6.3: Schematic diagram of the quantitative morphometric traits used in this
study
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105
6.2.7 Data analysis
The mean data of quantitative traits for all the species together in three subsequent
years were measured and then subjected to further analyses. The variability among all
analyzed species was exhibited with medians and percentiles and plotted as box plots. Mean
values were calculated upto 4th decimal point for each species along with standard error.
Correlation analysis had been performed among different characters (variables) to observe
the trend among these variables. Multivariate analysis was done by numerical taxonomic
techniques using the procedure of cluster analysis.
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106
6.3 RESULTS
6.3.1 Pollinia micro-morphology
6.3.1.1 Measuring genetic diversity through qualitative markers
6.3.1.1.1 Pollinaria morphology: In Dendrobium species, the pollinaria has multiples
of four-cell pollen clustered as bi-lobed, mostly smooth and kidney shaped, however with
marked variations (Figure 6.4). The pollinaria morphology in any particular section was
discrete in shape across members. It was fusiform, slightly curved or comma shaped, with
the inside surface slightly flattened. In most species, the two parts of each pair which cling
together are approximately of the same size. The variations in the shapes were distinct and
consistent among investigated species and are summarized below (Figure 6.4).
Group 1: Asymmetric shape of pollinaria, due to unequal length of two pollinia, was
observed in D. terminale of the section Aporum. The two parts were closely attached to each
other throughout the length without having any distinction of lobes. Exine morphology was
observed to be of regular type.
Group 2: Spindle shape was observed in D. infundibulum, D. longicornu and D. williamsonii,
the representative members of section Formosae. The two parts were tapering towards
both ends, while tapering toward the upper part is maximum. The adjacent inner lobes were
longer than the outer lobes which were characteristic of this section.
Group 3: Round shape was observed in D. bensoniae, and D. wardianum, the representative
members of the section Dendrobium (clade I). Two parts were attached throughout and the
lobes were distinct. Two parts of the pollinaria and two lobes of each pollinium are equal in
size.
Group 4: Triangular shape was observed to be the characteristics of D. nobile and D.
ochreatum, the representative members of the section Dendrobium (clade II). Two equal
parts were distinct, whereas two lobes were marginally clear.
Group 5: Toe shape was observed in D. aphyllum, D. polyanthum and D. transparens, the
representative members of section Dendrobium (clade III). The two parts were attached
throughout the length and two lobes were parallelly distinct. The unequal length of the two
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107
lobes, create a notch at upper portion of the pollinaria and was found responsible for its
characteristic shape.
Group 6: Ovoid shape was observed to be the characteristic of D. moschatum and D.
fimbriatum, the representatives of the section Holochrysa. Both the parts were equally
tapering towards both the end. Sometimes lobes were not well distinguished.
Group 7: Kidney shape pollinaria were observed in D. densiflorum and D. thyrsiflorum, the
representative members of section Densiflora. The two parts were attached at the upper-
most and basal regions creating a stomata-type opening. Two lobes were well distinguished.
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108
Figure 6.4: Pollinia morphology as observed under light microscope with the aid of epi-
elumination
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109
6.3.1.1.2 Pollen ultra structure: All total 18 species representing 5 established
sections were represented in Figure 6.5. Scanning electron micrographs of different species
showed distinct characteristics in spape and ultasturucture. But there is a stunning similarity
between members of representative sections. As for example pollinaria of members of the
section Densiflora were kidney shaped while the members of the section formosae were
spindle shaped. Pollen shape was observed to be sub-prolate type and pollen aperture was
found to be colpate type in all the examined species. Pollen aperture shape and number
varies in different species of Dendrobium. Aperture with circular pore and elongated-narrow
furrows was observed in D. longicornu and D. williamsonii (section Formosae, Figure 6.5b).
Roundish aperture was found in D. aphyllum and D. polyanthum (section Dendrobium,
Figure 6.5c). Irregular shaped aperture was observed in D. fimbriatum and D. moschatum
(section Holochrysa, Figure 6.5d). Irregular and kidney shaped aperture was found in D.
densiflorum and D. thyrsiflorum (section Densiflora, Figure 6.5e). Mono-aperture (D.
aphyllum, D. densiflorum, D. devonianum, D. falconeri, D. moschatum, D. nobile, D.
thyrsiflorum and D. williamsonii), Mono- and Bi-aperture (D. densiflorum and D. falconerii),
Bi-aperture (D. chrysanthum), Bi- and tri-aperture (D. fimbriatum and D. ochreatum), tri-
aperture (D. longicornu), and mono-, bi- and tri-aperture (D. polyanthum) were observed in
different experimental species. The exine of pollen was found to be unsculptured and
granular bodies were found on the surface in all the examined species.
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110
Figure 6.5: SEM of pollen ultra-structure of different species from different section of the
genus Dendrobium
(d) Section: Holochrysa
(a) Section: Formosae
(b) Section: Dendrobium
(e) Section: Densiflorum
(c) Section: Aporum
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111
6.3.1.1.3 Comparative exine morphology: The outer pollen wall, called exine, is
structurally complex comprising several distinct layers with explicit organizational patterns.
These extraordinary surface features are important in the elucidation of the origin of the
morphological complexity and diversity that may be useful to understand the science of
palynology and its role in genetic diversity. The SEM of exine showed extreme variations in
the number, distribution, and surface architecture (Figure 6.6). Most often the surface of
pollen was psilate and unsculptured. However, a gradual transition from psilate to rugular or
scabrate pollen with several intermediary stages were observed. D. anceps from the section
Aporum and D. infundibulum, D. longicornu and D. williamsonii from the section Formosae
exhibited rugular type of exine morphology, whereas D. chrysotoxum, D. densiflorum and D.
thrysiflorum from the section Densiflora showed rugulate-scabrate type of exine
morphology. Members of the section Dendrobium also showed psilate type of exine
morphology with a range of variation. Species such as D. aphyllum and D. primulinum from
the Clade III showed psilate-scabrate type while another group of species from Clade I and II
showed psilate-perforate type of exine morphology.
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112
Figure 6.6: Scanning electron micrographs of different pollinaria showing gradual changes
6.3.1.1.4 Spore tetrads: The LM study of pollinia revealed that the pollen grains were
liberated as tetrad into two discrete types, which varied at inter-specific level irrespective of
their the sections. The representative pollen grains were (i) decussate type of tetrads that
were most common and observed in the majority of investigated species, for example, D.
fimbriatum and D. aphyllum; and (ii) T-shaped type that was observed in D. devonianum, and
D. williamsonii (Figure 6.7). In all cases, the pollen tetrad appeared to be angular,
tetrahedral to polygonal, and cohered with small, profuse exinal connections to make up the
pollinium. These finding were in accordance with
two Dendrobium species in the section Holochrysa,
highlighting the exine sculpturing and striated surface perforations (Arora
present study the tetrad types were mostly common among investigated species and thus
considered unsuitable to be the distinguishing character in orchids.
Figure 6.7: Two types of spore tetrad observed in all experimental species. Only
representative photograp
6.3.1.2 Measuring genetic diversity through qua
6.3.1.2.1 Variability among quantitative traits
among different sections ranging from 0.74 µm to 2.75 µm with an average of 1.67 µm.
infundibulum (member of section Formosae) had the largest pollinium size, whereas
anceps (member of section Aporum) had the smallest pollinium size (Figure II; Table II).
ratio of polar axis length (µm) and equatorial axis length (µm) was calculated to determine
the pollen shape. When the ratio
considered as prolate shaped, and the ratio <1.33 was considered as sub
(Erdtman 1960). Based on this calculation, pollen from all investigated species were
observed to be sub-prolate shaped.
box plot analyses were perfor
ratio of TeL/TeW, PpA, PeA and ratio of PpA/PeA (
variability is distinct in pollen length with consider
pollen polar axis exhibited minimum variation. From box plot analyses, minimum and
maximum pollen lengths among investigated species were observed in
infundibulum, respectively. Other significantly variable parameters were ratios of PoL/PoW
CHAPTER 6
pollinium. These finding were in accordance with the previous SEM study
species in the section Holochrysa, D. moschatum and
highlighting the exine sculpturing and striated surface perforations (Arora
present study the tetrad types were mostly common among investigated species and thus
considered unsuitable to be the distinguishing character in orchids.
Two types of spore tetrad observed in all experimental species. Only
resentative photographs of spore tetrad from D. williamsonii and D. aphyllum
Measuring genetic diversity through quantitative markers
Variability among quantitative traits: The pollinia length varied significantly
nt sections ranging from 0.74 µm to 2.75 µm with an average of 1.67 µm.
(member of section Formosae) had the largest pollinium size, whereas
(member of section Aporum) had the smallest pollinium size (Figure II; Table II).
ratio of polar axis length (µm) and equatorial axis length (µm) was calculated to determine
the pollen shape. When the ratio was >1.33 or ranging between 1.33
considered as prolate shaped, and the ratio <1.33 was considered as sub
. Based on this calculation, pollen from all investigated species were
prolate shaped. To determine the variability across investigated species,
box plot analyses were performed for traits such as PoL, PoW, ratio of PoL/PoW, TeL, TeW,
ratio of TeL/TeW, PpA, PeA and ratio of PpA/PeA (Figure 6.8). It was evident that the
variability is distinct in pollen length with considerable overlapping among species
is exhibited minimum variation. From box plot analyses, minimum and
maximum pollen lengths among investigated species were observed in
, respectively. Other significantly variable parameters were ratios of PoL/PoW
113
previous SEM study which focused on
and D. gibsonii, mainly
highlighting the exine sculpturing and striated surface perforations (Arora 1986). In the
present study the tetrad types were mostly common among investigated species and thus
Two types of spore tetrad observed in all experimental species. Only
D. aphyllum are shown
The pollinia length varied significantly
nt sections ranging from 0.74 µm to 2.75 µm with an average of 1.67 µm. D.
(member of section Formosae) had the largest pollinium size, whereas D.
(member of section Aporum) had the smallest pollinium size (Figure II; Table II). The
ratio of polar axis length (µm) and equatorial axis length (µm) was calculated to determine
>1.33 or ranging between 1.33-2.00, those were
considered as prolate shaped, and the ratio <1.33 was considered as sub-prolate shaped
. Based on this calculation, pollen from all investigated species were
To determine the variability across investigated species,
med for traits such as PoL, PoW, ratio of PoL/PoW, TeL, TeW,
). It was evident that the
able overlapping among species, whereas
is exhibited minimum variation. From box plot analyses, minimum and
maximum pollen lengths among investigated species were observed in D. anceps and D.
, respectively. Other significantly variable parameters were ratios of PoL/PoW
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114
and TeL/TeW. Similarly, pollen length was found minimum and maximum in D. anceps and D.
infundibulum, respectively. However, minimum and maximum ratios of TeL/TeW were
examined in D. thrysiflorum and D. chrysanthum, respectively (Figure 6.8).
Figure 6.8: Variability of pollinia micromorphometric traits represented by box-plot analysis
(only few representative traits were shown)
6.3.1.2.2 Genetic diversity: PCA analysis was carried out in 17 species with 9 pollen
micromorphometric variables. The eigen values for the first three principal components (PC)
were shown in Table 6.3. Contribution of the individual traits (variables) in first three PC
was represented in Table 6.4. Genetic distences (Eucladian distances) among the studied
species as revealed by morphometric markers were presented in Table 6.5. Based on PCA
tri-plot, only distantly related sections such as Aporum and Formosae could be distinguished
from other sections (Figure 6.9). However, members of section Densiflora could not be
segregated from section Holochrysa and two clades of section Dendrobium. This observation
was well supported by the poor percentage values of total variability shown by principal
components. Therefore, it may be concluded that pollen micromorphometric parameters
can elucidate the phylogeny only for selected sections. The same picture is also evident from
cluster analysis (Figure 6.10).
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115
Table 6.3 Eigen values of the first three principal components
F1 F2 F3
Eigenvalue 4.262 2.054 1.483
% variance 47.353 22.817 16.474
Cumulative % 47.353 70.170 86.644
Table 6.4 Contribution of the individual traits (variables) in first three principal
components
F1 F2 F3
PoL 12.594 19.719 1.247
PoW 14.636 14.011 3.136
PoL/PoW 14.032 17.915 2.032
TeL 13.124 6.086 13.315
TcW 12.015 16.513 5.522
TcL/TcW 0.234 2.156 53.397
PpA 15.712 12.772 0.144
PeA 17.113 6.947 1.931
PpA/PeA 0.540 3.881 19.276
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116
Table 6.5 Genetic distances (Eucladian distances) among the studied species as revealed by pollinia micr-morphometric markers
D.
an
cep
s
D.
ap
hyllu
m
D.
chry
san
thu
m
D.
chry
soto
xum
D.
de
nsi
flo
rum
D.
de
vo
nia
nu
m
D.
falc
on
eri
i
D.
fim
bri
atu
m
D.
infu
nd
ibu
lum
D.
lon
gic
orn
u
D.
mo
sch
atu
m
D.
no
bile
D.
och
rea
tum
D.
pa
rish
ii
D.
pri
mu
lin
um
D.
thyrs
iflo
rum
D.
williu
mso
nii
D. anceps 1
D. aphyllum 0.793 1
D. chrysanthum 0.913 0.931 1
D. chrysotoxum 0.908 0.959 0.971 1
D. densiflorum 0.841 0.987 0.930 0.981 1
D. devonianum 0.883 0.976 0.946 0.993 0.995 1
D. falconerii 0.829 0.997 0.939 0.975 0.996 0.989 1
D. fimbriatum 0.760 0.992 0.883 0.939 0.986 0.969 0.990 1
D. infundibulum 0.662 0.977 0.842 0.901 0.960 0.933 0.967 0.986 1
D. longicornu 0.638 0.971 0.828 0.887 0.951 0.920 0.959 0.981 0.999 1
D. moschatum 0.883 0.977 0.980 0.995 0.983 0.989 0.986 0.952 0.922 0.910 1
D. nobile 0.784 0.998 0.914 0.957 0.991 0.978 0.997 0.997 0.983 0.978 0.971 1
D. ochreatum 0.908 0.946 0.962 0.998 0.973 0.989 0.964 0.925 0.888 0.872 0.988 0.944 1
D. parishii 0.771 0.972 0.884 0.959 0.987 0.978 0.979 0.978 0.972 0.965 0.959 0.981 0.958 1
D. primulinum 0.823 0.998 0.943 0.967 0.988 0.982 0.997 0.987 0.963 0.956 0.983 0.994 0.956 0.967 1
D. thyrsiflorum 0.800 0.990 0.900 0.959 0.995 0.984 0.994 0.997 0.976 0.969 0.966 0.996 0.950 0.987 0.989 1
D. williumsonii 0.663 0.979 0.844 0.900 0.960 0.932 0.968 0.987 1.000 0.999 0.923 0.984 0.886 0.970 0.966 0.976 1
In bold, significant values (except diagonal) at the level of significance alpha=0.050 (two-tailed test)
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117
Figure 6.9: Representative graphical presentation of principal component analysis where a
tri-plot (x, y, z) with first 3 PC was plotted with the help of pollinia traits (qualitative) only.
The numbers (1-34) indicating species serial number
Figure 6.10: Cluster dendrogram with first 3 PCs of all the variable traits under investigation
CHAPTER 6
118
6.3.1.3 Intra-specific genetic diversity
Intra-specific variation was observed in pollinia morphology, though no difference
was found at ultra-structure or exine morphology. This difference was prominent in different
varieties of D. aphyllum and D. nobile (Figure 6.11). In D. polyanthum distinct difference was
observed among two populations collected from distant places (Table 3.1).
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Figure 6.11: Intra-specific variation in pollinia micro-morphology,
6.3.1.4 Pollinia micromorphology and ecological adaptation
As observed in the present study, the pollen length was the most variable parameter
among all pollen characters studied. Hence, to investigate the rationale behind such
significant variation in pollen length (assuming that it is without any phylogenetic
implications), an effort was made to correlate the pollen length with an ecologically
important trait, spur-length, to reveal if both these traits have been evolved in response to
ecological adaptation. In
observed between these two traits, highlighting the fact that the pollen morphometric traits
are evolved more in response to ecological factors rather than in the course of species
evolution (Figure 6.12).
Figure 6.12: In corelation studies significant positive interaction (R
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icromorphology and ecological adaptation
As observed in the present study, the pollen length was the most variable parameter
among all pollen characters studied. Hence, to investigate the rationale behind such
significant variation in pollen length (assuming that it is without any phylogenetic
plications), an effort was made to correlate the pollen length with an ecologically
length, to reveal if both these traits have been evolved in response to
ecological adaptation. In the present study, a significantly positive correlation (R
observed between these two traits, highlighting the fact that the pollen morphometric traits
are evolved more in response to ecological factors rather than in the course of species
).
elation studies significant positive interaction (R2 = 0.81) was observed
between
PoL and CoL
120
As observed in the present study, the pollen length was the most variable parameter
among all pollen characters studied. Hence, to investigate the rationale behind such
significant variation in pollen length (assuming that it is without any phylogenetic
plications), an effort was made to correlate the pollen length with an ecologically
length, to reveal if both these traits have been evolved in response to
correlation (R2=0.816) was
observed between these two traits, highlighting the fact that the pollen morphometric traits
are evolved more in response to ecological factors rather than in the course of species
= 0.81) was observed
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121
6.3.2 Seed micro-morphology
6.3.2.1 Measurement of genetic diversity through qualitative markers
6.3.2.1.1 Seed morphology: Seeds of Dendrobium species are extremely small, being
less than 1 mm in size, with a wide variety of shapes (ellipsoid, oblongoid, ovoid, globose,
trigonous or tetragonous). The seed shape and color of mature seed coat of different species
of Dendrobium did not exhibit large variations. The colour of seeds exhibits a range of
different shades from yellow to brownish-yellow (Table 6.6).
Table 6.6 Qualitative seed traits of the experimental species of the genus
Dendrobium
Species name Seed shape Seed colour Testa cell
D. chrysanthum Fusiform Yellow Fusiform
D. chrysotoxum Fusiform Orange yellow Fusiform
D. crepidatum Fusiform Yellow Fusiform
D. denneanum Fusiform Yellow Fusiform
D. densiflorum Fusiform Orange yellow Fusiform
D. devonianum Fusiform Yellow Fusiform
D. farmer Fusiform Yellow Fusiform
D. fimbriatum Fusiform Yellow Fusiform
D. formosum Oval and twisted Orange yellow Fusiform
D. heterocarpum Fusiform Yellow Fusiform
D. hookereanum Fusiform Brownish yellow Fusiform
D. infundibulum Fusiform White Fusiform
D. moschatum Fusiform Orange yellow Fusiform
D. nobile Fusiform Yellow Fusiform
D. ochreatum Fusiform Yellow Fusiform
D. parishii Fusiform Yellow Fusiform
D. polyanthum Elliptic Golden yellow Fusiform
D. transparens Fusiform Yellow Fusiform
D. wardianum Fusiform Yellow Fusiform
D. williamsonii Elliptic Orange yellow Fusiform
6.3.2.1.2 Seed ultra structure: The SEM study revealed that in majority of the species
testa cells arrangements were simple with cells attached in a straight, longitudinal, head to
tail fashion. The Dendrobium seeds have elongated, thick medial testa cells with relatively
high anticlinal cell wall without an abrupt transition zone. SEM studies revealed that the
thickening of testa cells was well developed and strongly raised in all studied species.
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Moreover, testa cells in selected species of temperate region, such as D. chrysanthum, D.
hookerianum were rather longitudinally folded (Figure 6.13). Anticlinal walls frequently
appeared to be strongly raised whereas the outer periclinal surface was generally sunken or
concave providing a tessellate appearance to the surface of a seed. However, the cell lumens
in majority of species were almost obliterated due to extensive development of the cell wall
thickenings. The observations on Dendrobium species suggested that the testa cells
arrangement may vary significantly within the genera.
6.3.2.1.3 Comparative Testa cell morphology: The seed coat is reticulate with
polygonal cells, which range from more or less isodiametric to tangentially elongated, being
sometimes irregular. Dendrobium seeds usually have a smooth membranous outer periclinal
wall, although they can sometimes have a fibrillar aspect due to epicuticular waxes. Scanning
electron microscopy based observations indicated that there were distinct and recognizable
morphological seed types (Figure 6.13).
The testa cells in Dendrobium species were observed to be long, sub-quadrate,
oblong, sub-elliptical or irregular in outline and fusiform in shape. The testa cells had
discrete length and width parameters in species from temperate and sub-tropical regions;
however, species from tropical region had overlapping dimensions. Their size may also be
uniform but often varied within the areas such as the median region and on the chalazal
end. The micropylar and the chalazal cells were much shorter and stouter than medial cells
which enveloped the embryo in all species. The medial cells were elongated in length and
encased the embryos in the seeds. The number of testa cell per seed was examined highest
in temperate species and lowest in tropical species with an exception of D. transparens
which was a sub-tropical species with higher number of testa cells.
Interestingly, in a single seed, some cells were sculptured with bead-like structures
and some cells were without any sculpture, for example, D. aphyllum, D. primulinum (syn. D.
polyanthum) (section Dendrobium) and D. formosum (section Formosae).
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Figure 6.13: Seed ultra structure showing testa cell morphology of some selected
experimental species showing incongruency with morphometric sections
6.3.2.2 Measurement of genetic diversity through quantitative markers
6.3.2.2.1 Variability among quantitative traits: Thirteen seed micromorphometric
variables including seed micromorphology and embryo related characters were examined
across all collected species. To represent the variability across species, box plots against all
variables were analyzed (Figure 6.14). Among all studied micromorphometric traits, seed
length was observed to show maximum variability whereas testa cell width exhibited
minimum variation. Statistical analyses revealed that variation in percent free air-space in
seeds of Dendrobium species had a significant variation among all species studied from
different climatic regions. Data suggested that species from temperate region had the
highest % free air-space followed by species from sub-tropical and tropical regions. Some of
the species which grow in the transit climatic zones also showed an overlapping pattern in
terms of free air-space (Figure 6.14).
Figure 6.14: Boxplots showing variability of representative traits (variables) at inter-specific
level
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125
6.3.2.2.2 Genetic diversity: PCA analysis was carried out in all the experimental
species with 13 seed micro-morphometric variables. The eigen values for the first three
principal components (PC) were shown in Table 6.7. Contribution of the individual traits
(variables) in first three PC was represented in Table 6.8. Genetic distances (Eucladian
distances) among the studied species as revealed by morphometric markers were presented
in Table 6.9. Principal component analysis did not resulted into any cluster equivalent with
presently established sections (Figure 6.15). In cluster analysis with first three PC, no proper
clusters were found (Figure 6.16).
Table 6.7 Eigen values of the first three principal components
F1 F2 F3
Eigenvalue 4.887 2.718 2.331
% variance 37.590 20.905 17.933
Cumulative % 37.590 58.495 76.428
Table 6.8 Contribution of the individual traits (variables) in first three principal
components
Traits F1 F2 F3
SL 1.045 16.232 12.391
SW 13.127 6.573 1.772
SL/SW 12.750 1.320 6.546
SV 13.725 5.121 2.052
TcL 0.147 17.531 12.629
TcW 0.299 0.633 25.036
TcN 1.021 0.055 7.959
EmL 17.335 0.356 2.627
EW 15.433 0.096 0.658
EL/EW 0.199 0.094 26.346
EV 16.025 1.484 1.790
SV/EV 5.451 24.320 0.094
AS 3.442 26.185 0.100
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Table 6.9 Genetic distances (Eucladian distances) among the studied species as revealed by seed micr-morphometric markers
D
. ch
rysa
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D.
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D.
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D.
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D.
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D.
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D.
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D.
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D.
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um
D.
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D.
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D.
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D.
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D. chrysanthum 1
D. chrysotoxum 0.993 1
D. crepidatum 0.978 0.964 1
D. denneanum 0.992 0.981 0.992 1
D. densiflorum 0.991 0.995 0.961 0.973 1
D. devonianum 1.000 0.993 0.979 0.992 0.992 1
D. farmer 0.968 0.976 0.928 0.937 0.991 0.971 1
D. fimbriatum 0.997 0.995 0.979 0.988 0.996 0.998 0.979 1
D. formosum 0.970 0.979 0.933 0.941 0.993 0.973 0.999 0.981 1
D. heterocarpum 0.924 0.940 0.882 0.885 0.965 0.929 0.988 0.944 0.987 1
D. hookereanum 1.000 0.993 0.977 0.993 0.991 1.000 0.967 0.997 0.969 0.923 1
D. infundibulum 0.967 0.975 0.930 0.936 0.989 0.970 0.998 0.978 0.999 0.985 0.966 1
D. moschatum 0.987 0.996 0.944 0.964 0.997 0.988 0.989 0.991 0.991 0.962 0.987 0.987 1
D. nibile 0.996 0.996 0.972 0.991 0.985 0.994 0.956 0.991 0.960 0.908 0.996 0.955 0.985 1
D. ochreatum 0.988 0.980 0.975 0.991 0.964 0.985 0.926 0.979 0.931 0.865 0.988 0.928 0.962 0.993 1
D. parishii 1.000 0.993 0.980 0.993 0.992 1.000 0.969 0.998 0.971 0.928 1.000 0.968 0.987 0.995 0.986 1
D. polyanthum 0.999 0.994 0.979 0.992 0.992 0.999 0.970 0.999 0.972 0.930 0.999 0.968 0.988 0.994 0.984 1.000 1
D. transparens 0.987 0.969 0.993 0.997 0.966 0.987 0.930 0.983 0.933 0.880 0.987 0.928 0.953 0.980 0.982 0.989 0.988 1
D. wardianum 0.993 0.982 0.992 1.000 0.974 0.992 0.939 0.989 0.943 0.888 0.993 0.938 0.966 0.991 0.991 0.994 0.993 0.997 1
D. williamsonii 0.981 0.987 0.940 0.955 0.996 0.983 0.996 0.988 0.996 0.977 0.980 0.992 0.996 0.972 0.945 0.982 0.983 0.948 0.957 1
In bold, significant values (except diagonal) at the level of significance alpha=0.050 (two-tailed test)
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Figure 6.15: Representative graphical presentation of principal component analysis where a
tri-plot (x, y, z) with first 3 PC was plotted with the help of seed traits (qualitative) only. The
numbers (1-34) indicating species serial number
Figure 6.16: Cluster dendrogram with the first 3 PC shows clustering of species into
definable sections
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6.3.2.3 Intra-specific genetic diversity
No significant differences were observed under LM or SEM in case of all the studied
varieties. Therefore, seed micro-morphometric markers were not considered for resolving
genetic diversity at intra-specific level.
6.3.2.4 Seed micromorphology and ecological adaptation
Testa cells had a significant variation in their length as well as in orientations;
however, different species could easily be grouped based on different phytogeographical
regions irrespective to their genetic relationship (Figure 6.17).
Group 1: In temperate species, the testa cell walls were smooth and without reticulations as
characterized in D. chrysanthum (section Dendrobium) and D. hookeranum (section
Holochrysa). Since the members of two different morphological sections were available in
this region, it could indeed be ascertained that such traits are specific to the species of
temperate region.
Group 2: In sub-tropical species testa cell wall thickenings were most prominent and may be
characterized into two morphotypes. First, the raised testa cell walls of species such as D.
densiflorum (section Densiflora), and D. nobile (section Dendrobium) were sculptured with
bead-like structures giving an appearance of “beads on strings”.
Group 3: In another group of sub-tropical species, the testa cell walls in D. parishii (section
Dendrobium), and D. williamsonii (section Formosae) were covered with cottony-white
substances. Both types of testa cell walls are characteristic features of the concerned
species, however, these are phylogenetically distant species belonging to different
morphological sections. These results, therefore, indicated that such characters have been
evolved as per the climatic preferences of species rather than during speciation.
Group 4: In the species of tropical region, the testa cells were raised with or without bead-
like structure as characterized in D. crepidatum (section Dendrobium) and D. farmeri
(section Densiflora).
To explore the potential seed characters modulated through ecological adaptation, a
novel comparative analysis of seed micromorphometry was performed in twenty species of
the genus Dendrobium (Orchidaceae) from well-defined altitude based phytogeographical
realms i.e. temperate, subtropical and tropical regions (Figure 6.18). The studied characters
included seed volume, free air space, seed coat ornamentation of the periclinal walls and
seed coat sculpturing and those could easily be used to distinguish groups of species based
on defined climatic regions. However, genetic relatedness derived with these characters
among all studied species from different geographical regimes sho
correlation (R2=0.168) with the phylogeny deduced earlier based on seed quantitative
characters. Based on these results, therefore, it may be concluded that such traits are the
direct signatures of adaptation according to climatic prefe
during speciation.
Figure 6.17: Seed ultra structure showing testa cell morphology of some selected
experimental species showing congruency with altitude wise species distribution
Figure 6.18: Non-significant correlat
Dendrobium proves their altiutude based climat
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among all studied species from different geographical regimes sho
=0.168) with the phylogeny deduced earlier based on seed quantitative
characters. Based on these results, therefore, it may be concluded that such traits are the
direct signatures of adaptation according to climatic preferences rather than their selection
Seed ultra structure showing testa cell morphology of some selected
experimental species showing congruency with altitude wise species distribution
significant correlation between tropical and sub-tropi
proves their altiutude based climatic preference during speciation
129
among all studied species from different geographical regimes showed non-significant
=0.168) with the phylogeny deduced earlier based on seed quantitative
characters. Based on these results, therefore, it may be concluded that such traits are the
rences rather than their selection
Seed ultra structure showing testa cell morphology of some selected
experimental species showing congruency with altitude wise species distribution
tropical species of
ic preference during speciation
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130
6.4 Discussion
6.4.1 Genetic diversity and pollen micro-morphomology
In orchid pollen grains, the ultrastructural features of the exine are initiated through
the deposition of the polysaccharide material, called primexine. Subsequently, the germinal
aperture are formed in positions on the surface through endoplasmic reticulum mediated
blocking of the deposition of primexine precursor (Heslop-Harrison 1968; Hesse 2000;
Lumga et al. 2006). However, the pattern formation is still unclear, though clues are there
for least characterized species-specific morphogens. Since Dendrobium species showed
significant variation in pollen surface stratification, general trends are needed to be
established for pattern formation on pollen surface that may clarify our understanding for
pattern formation process in biological systems.
It has been speculated that the prominent exine morphologies in Orchidoideae are
evolved from primitive tectate–perforate to the contemporary intectate form, through
disintegration from tectate–imperforate in Epidendroideae (Heslop-Harrison 1968; Burns-
Balogh and Hesse 1988; Hesse 2000; Lumga et al. 2006). Such exine ultrastructures are
certainly capable of developing an evolutionary hierarchy in orchids. For example, on the
basis of exine ultrastructure, members of the section Formosae may be considered as the
most advanced because of their regulate type and smooth topology. Williams and Broome
(1976) explained that the smooth surface of advanced orchids is the result of the fusion of
extosexine into a definite tectum; and progressive members may even lack detailed exine
sculpturing (Arora 1986; Lumga et al. 2006). The varied exine morphologies were well
reflected among investigated species of Dendrobium, and thus highlight their importance in
establishing the species relatedness.
Certainly pollen exine sculpture patterns could produce phylogenetic information at
the generic and subtribal levels (Chesselet and Linder 1993; Lumga et al. 2006). However, in
some orchid subtribes, such as in Disinae, pollen exine sculpture patterns were found to be
highly variable allowing the distinction at inter-specific level or, as Oryciinae, it is
substantially uniform for taxonomic resolution (Chesselet and Linder 1993). However, it is
more difficult to trace evolutionary tendencies within plant groups having similar pollination
strategies (Wang et al. 2003). Nevertheless the present study suggests that this was not the
case for the genus Dendrobium. All representatives from the sections Aporum and Formosae
exhibited similar rugular type of exine morphology, and exhibited close relatedness to the
ITS2 based reference phylogenetic tree. In the reference phylogenetic tree, members of
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131
clade 1 and clade 2 of section Dendrobium showed psilate type and psilate-scabrate type of
exine morphology, respectively. The members of sections Holochrysa and Densiflora were
subsequent to the the section Dendrobium where exine type was observed to be rugulate-
scabrate type with several intermediate stages. Inspite of such large variation in exine
morphology, a phylogenetic trend of exine evolution within the genus Dendrobium was
apparent.
Pridgeon (1999) studied the orchid palynology and suggested that the pollen
heterogeneity of Orchidaceae may have promising systematic utility. The suggested pollen
heterogeneity was well reflected in the present study through box-plot analysis where at
inter-specific level pollen length varied maximum and pollen polar axis varied minimum. As
noted elsewhere, the main limitation in recognizing a phylogenetic signal in pollen
characters of orchids depends on the influence of ecological factors such as differences in
pollination strategies, in spite of evolutionary affinities among taxa. An attempt to elucidate
the relationship between spur length of orchid flowers and pollen ultrastrcuture emphasized
its role in orchid pollination biology (van der Cingel 1995; Pridgeon et al. 2001). Since the
species of Dendrobium are mostly epiphytic, the knowledge of pollination biology in this
system is still limited.
6.4.2 Genetic diversity and seed micro-morphology
Species from temperate region had the highest ratio followed by those of sub-
tropical and tropical regions; and high correlation was observed between two traits. This
data showed incongruence with phylogenetic relationship derived from other numeric seed
micromorphometric traits as well as rITS-DNA sequences (Chattopadhyay et al. 2010).
Hence, genetically related species had different seed volume/embryo volume ratio
highlighting the preference of any particular species for a climatic region as their prime
habitat. However, as an exception, D. transparens was observed to have the lowest embryo
volume and lowest seed volume/embryo volume ratio making it most buoyant among all
studied species. This improbability is very prominent as this species is distributed throughout
climatic regions as well as in some other parts of the country depicting this adaptive change
in buoyancy for optimal seed dispersal.
Previously seed characters had been considered as distinguishing characters in
species identification and their subsequent evolution into other morphotypes with adaptive
strategy (Chattopadhyay et al. 2010; Vij et al. 1992). However, in the present study no
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132
significant contribution of these traits was observed in establishing the positive correlation
with different altitude based phytogeographic regions of the species. In parallel, seed
length/width ratios also exhibited a differential variation among all studied species. The
patterns of seed growth during maturation suggested that the seed elongated as a result of
an elongation in testa cell and not in numbers, thus increases buoyancy of seed beneficial
for wind dispersion (Arditti et al. 1980). Although the relative degree of truncation of the
seeds has been utilized as a good taxonomic parameter for species identification, yet it could
not distinguish species from different climatic regions (Arditti et al. 1980). Therefore, seed
length, width and their ratio were also observed to be non-significant in relation to
phytogeographical distribution of the species.
Earlier, it had been hypothesized that during speciation any variation in the volume
of embryos in orchid seeds is due to their increased length rather than their width (Arditti et
al. 1979). This may be due to the factor that the orchid seeds are extremely small and thin,
having ellipsoid to oblongoid shapes and could only lead to elongated embryo cells.
These observations revealed the simple form of testa cells arrangements, however,
in orchid simpler forms were suggested to be typical to terrestrial taxa, and spiral from
epiphytic taxon (Vij et al. 1992). In the present study, it was also ascertained that structure
“beads on strings” is a quite common and interesting feature across morpho-sections of
species studied. A gradual transition of this structure through reduction of beads number
was observed in species of tropical region (D. formosum from section Formosae) to species
of sub-tropical region (D. infundibulum from section Formosae; and D. transparens, and D.
wardianum from section Dendrobium) to temperate region species (D. chrysanthum from
section Dendrobium; and D. clavatum and D. hookerianum from section Holochrysa).
However it could also be assumed that “beads on strings” structure was originally developed
in the sub-tropical region members and gradually became obsolete into tropical and
temperate region species. Occasional presence of these beads in D. farmeri (temperate
region species) and D. wardianum (tropical region species) supported this assumption. Since
these structures were not restricted within members of any defined section and therefore,
could be concluded that such traits are not related to speciation, but evolved according to
climatic preferences of a species. These data will provide support to the hypothesis for the
adaptation of phylogenetically related species in different climatic regions during their
adaptive diversification.
The results of the present study highlight, to certain extent, the importance of the
morphology of orchid seeds in relation to their ecological adaptations required for the
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133
dispersal or to the establishment of new plants. Thus, due to their small size, Dendrobium
seeds can be regarded as ‘dust-seeds’ that are mainly dispersed by wind with parasitic or
hemiparasitic representatives in the family Orchidaceae. The great distance traversed by
orchid seeds is generally attributed to its light weight and buoyancy due to high percentage
of air space. This assumption is well reflected in Dendrobium species where the embryo
volume and percentage air space is directly related to the climatic preferences of the species
reflected through ultra-structure of the seeds. The lower seed volume and higher
percentage of free air space in the temperate species suggested an ecological adaptation for
proper dispersal in relatively low atmospheric pressure. The ultra structural features also
suggested a gradual expansion towards tropical and temperate regions from the subtropical
region. Fusiform tessellate seeds is a characteristic feature of the genus Dendrobium. In
temperate species the testa cell walls were smooth and without reticulations; in sub-tropical
species testa cell wall thickenings were either with bead-like structures or covered with
cottony white substances; whereas in tropical species the testa cell walls ornamentation is
not that prominent and may be with or without bead like structure. Therefore, it is clear that
the seed volume, free air space, and seed coat ornamentation in Dendrobium species are
directly related to climatic preference of the species rather than its phylogeny.