Chapter 6 Assessment of genetic diversity through micro...

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

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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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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



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


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



lumination (hund WETZLAR FLQ 150). To

slide and crushed with the help of a

. Measurements were

softwere. Measurements were


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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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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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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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

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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


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).


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


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


>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

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m

D.

chry

san

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D.

chry

soto

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D.

de

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flo

rum

D.

de

vo

nia

nu

m

D.

falc

on

eri

i

D.

fim

bri

atu

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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

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D.

pri

mu

lin

um

D.

thyrs

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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

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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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119

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




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



).

elation studies significant positive interaction (R2 = 0.81) was observed

between

PoL and CoL

120




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



= 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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122

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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123

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124

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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126

Table 6.9 Genetic distances (Eucladian distances) among the studied species as revealed by seed micr-morphometric markers

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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127

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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128

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

CHAPTER 6

among all studied species from different geographical regimes sho

=0.168) with the phylogeny deduced earlier based on seed quantitative


direct signatures of adaptation according to climatic preferences rather than their selection

Seed ultra structure showing testa cell morphology of some selected


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


rences rather than their selection

Seed ultra structure showing testa cell morphology of some selected


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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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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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.

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