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POPULATION STUDY OF PAPHIOPEDILUM STONEI HOOK.
(ORCHIDACEAE) FROM LIMESTONE AREAS OF NORTHERN WEST OF
SARAWAK
MAS IZZATY BTE MAZLAN
Bachelor of Science with Honours
(Plant Resource Science and Management)
June 2012
Faculty of Resource Science and Technology
i
POPULATION STUDY OF PAPHIOPEDILUM STONEI HOOK. (ORCHIDACEAE)
FROM LIMESTONE AREAS OF NORTHERN WEST OF SARAWAK
MAS IZZATY BTE MAZLAN
This final year project report is submitted in partial fulfillment of the requirement for
The Degree Bachelor of Science with Honours
(Plant Science Resource and Management)
Department of Plant Science and Environmental Ecology
Faculty of Resource Science and Technology
UNIVERSITI MALAYSIA SARAWAK
2012
ii
APPROVAL SHEET
Name of Candidate : Mas Izzaty Bte Mazlan
Matric number : 23974
Title of Dissertation : Population Study of Paphiopedilum stonei Hook.
(Orchidaceae) From Limestone Areas of Northern West of
Sarawak
……………………………………..
(Prof. Dr. Cheksum@Supiah Tawan)
Supervisor
………………………………………
(Mr. Qammil Muzzammil Abdullah)
Co-supervisor
……………………………………
(Dr. Siti Rubiah Zainudin)
Coordinator of Plant Resource Science and Management
Faculty of Resource Science and Technology
iii
DECLARATION
I declare that no portion of the work referred to this dissertation has been submitted in
support of an application for another degree of qualification of this or any other
university or institution of higher learning.
……………………………………………
(Mas Izzaty Bte Mazlan)
Programme of Plant Resource Science and Management
Department of Plant Resource Science and Environmental Ecology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
iv
ACKNOWLEDGEMENTS
Alhamdulillah, I am very thankful to Allah who gave me strength in completing
this project. The special thank goes to my helpful supervisor, Prof. Dr.
Cheksum@Supiah Tawan and my co-supervisor, Mr. Qammil Muzzammil Abdullah.
The supervision and support that they gave truly help the progression and smoothness of
this project. The co-operation is much indeed appreciated.
My grateful thanks also go to Mr. Mohd Syafiq Sahat, Mr Hidir Marzuki and Mr
Mohd Safri for their contribution during the field and laboratory works. Not forgetting,
great appreciation goes to the rest of my friends Noor Ain binti Che Ahmad and Angela
anak George who helped me from time to time during the project. The whole project
really brought us together to appreciate the true value of friendship and respect of each
other.
Great deals appreciated go to the contribution of my faculty - Faculty of
Resource Science and Technology (FSTS). I am also would like to thankful the
Coordinator of Plant Resource Science and Management, Dr. Siti Rubiah Zainudin and
all the staff of FSTS.
Last but not least I would like to thank my beloved family especially my mother,
Hjh Rahmah binti Hj Ishak and my father Hj Mazlan bin Daud who had encouraged and
gave ideas and moral support throughout the project.
v
ABBREVIATIONS
ANOVA : Analysis of Variance
CITES : Convention in Trade on Endangered Species of Wild Fauna and
Flora
CaCO3 : Calcium Carbonate
CaMg : Calcium Magnesium
CH2CCH2(HO)2 : Gluteraldehyde
Co : Cobalt
DNA : Deoxyribonucleic acid
GPS : Global Positioning System
H2O2 : Hydrogen peroxide
HUMS : Herbarium of Universiti Malaysia Sarawak
Na2HPO4 : Disodium phosphate
Na2HPO4. H2O : Dihydrogen Sodium Phosphate
SEM : Scanning Electron Microscope
SiO2 : Silicon dioxide
UNIMAS : Universiti Malaysia Sarawak
vi
TABLE OF CONTENTS
TITLE PAGES
TITLE PAGE i
APPROVAL SHEET ii
DECLARATION iii
ACKNOWLEGEMENTS iv
ABBREVIATIONS v
TABLE OF CONTENTS vi
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF APPENDICES xi
ABSTRACT
xii
ABSTRAK
xiii
1.0 INTRODUCTION 1
1.1 General Introduction
1.2 Problem Statement
1.3 Objectives
1
3
3
2.0 LITERATURE REVIEW
2.1 Order Asparagales
2.2 Family Orchidaceae
2.2.1 Orchidaceae
2.2.2 Genus Paphiopedilum
2.3 Ecology
2.4 Paphiopedilum stonei
2.5 Scanning Electron Misroscope
2.6 Stomata
4
4
5
5
7
8
9
10
10
vii
3.0 MATERIALS AND METHODS
3.1 Study Sites
3.2 Plot establishment along the Trail
3.3 Specimen collection
3.4 Scanning Electron Misroscope
3.4.1 Sample fixation
3.4.2 Washing
3.4.3 Dehydration
3.4.4 SEM
3.5 Data Analysis
3.5.1 Morphological characteristics of P. stonei
3.5.2 Leaves Measurement
3.5.3 Mean and standard deviation of three selected study sites
11
11
14
15
16
16
17
17
17
19
19
19
20
4.0 RESULTS AND DISCUSSION
4.1 Morphological characteristic description
4.2 Measurement of P. stonei leaves
4.3 Density of Paphiopedilum stonei
4.4 Mean and Standard deviation for three study areas
4.2.1 Mean value for Bau, Padawan and Serian areas
4.4.2 Standard deviation for Bau, Padawan and Serian areas
4.5 Anatomy of the Leaf
4.5.1 Type of Stomata
4.5.2 Length and Width of Stomata
21
21
27
29
42
42
42
45
46
49
5.0 CONCLUSIONS AND RECOMMENDATION 50
6.0 REFFERENCES 52
7.0 APPENDICES 55
viii
LIST OF TABLES
TABLES
PAGES
Table 1: The mean of leaf length for each plot in three studies areas, Bau,
Padawan and Serian
28
Table 2: Mean and Standard deviation for Bau limestone area 43
Table 3: Mean and Standard deviation for Padawan limestone area 43
Table 4: Mean and Standard deviation for Serian limestone area 43
Table 5: One way ANOVA analysis 44
Table 6: Leave Anatomy Description 45
ix
LIST OF FIGURES
FIGURES PAGES
Figure 1: Limestone hills of Bau, Padawan and Serian studies areas 11
Figure 2: Various studies area of the limestone in Bau, Padawan and Serian 12
Figure 3: Example of Trails and Marking Points of P. stonei in the studies sites 14
Figure 4: Plot size for 10m×10m 14
Figure 5: Measurement of guard cell and stomata 18
Figure 6: Comparison of morphological between P. kolopakingii and P. stonei 22
Figure 7: The flowering plant in greenhouse and plants of P. stonei that
Growing in its natural habitat at a limestone hill in Simpang
Boring, Bau
24
Figure 8: Paphiopedilum Stonei Hook 26
Figure 9: The percentage of P. stonei that found in three studies sites 29
Figure 10: Population of P. stonei at Bau limestone area 31
Figure 11: Other types of orchids that found during field survey 33
Figure 12: Population of P. stonei at Padawan limestone areas 34
Figure 13: Population of P. stonei at Serian limestone area
Figure 14: The total distribution of P. stonei in three studies areas Bau,
Padawan and Serian
35
37
x
Figure 15: The ordinal directions of P. stonei that found in Bau, Padawan and
Serian
38
Figure 16: The elevation of P. stonei that found in Bau, Padawan and Serian 40
Figure 17: Picture of stomata from three types of Paphiopedilum species 47
xi
LIST OF APPENDICES
APPENDICES
PAGES
Appendix 1: The density of P. stonei for each plot in three study areas
Bau, Padawan and Serian
55
Appendix 2: Number of P. stonei in the ordinal directions 56
Appendix 3: Altitude for population of P. stonei at Bau limestone area 57
Appendix 4: Altitude for population of P. stonei at Padawan limestone
area
58
Appendix 5: Altitude for population of P. stonei at Serian limestone area 59
xii
Population study of Paphiopedilum stonei Hook. (Orchidaceae) from limestone
areas of Northern West of Sarawak
Mas Izzaty Bte Mazlan
Plant Resource Science and Management Programme
Department of Plant Science and Environmental Ecology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Limestone is very unique habitat; it is a place for many endemic plant species. Among them are Orchids,
Begonias, Aroids, Gensneroids and many more. Orchidaceae is the second largest family after Poaceae in
monocotyledon and it is one of the largest in the flowering plant kingdom, with recent estimates ranging
from 20 000 to 35 000 species that grouped into five subfamilies. Orchids are not only important for their
aesthetic value but also being used as ecological indicators. Paphiopedilum stonei Hook. also one of the
most highly prized of all species in the genus because it is very rare and beautiful. This has made them
vulnerable to habitat change and destruction. Although it is a protected species under the convention on
International Trade in Endangered species (CITES), the treat from collectors remains a problem. The
purposed of this documentation was to investigate the population of P. stonei in three selected areas at
Bau, Padawan and Serian. There was very little study on the density of P. stonei at the limestone habitat
because the population of slipper orchid was decreasing due the over collected by orchid hunters. This
study involved field works and laboratory works. A total of fourty - five sub plots had been set up in three
studies sites with 10 m x 10 m each Bau has the highest number of P. stonei with 63 individuals of 48.1
% from the total number recorded. Followed by Padawan and Serian with 39 and 29 individuals. The
comparison of population P. stonei between the three selected areas that had been analysed by using
ANOVA had been produced and concluded that mean number of P. stonei occurred in the three study
areas and was of significant difference (P = 0.002). All the dried sample have prepared for the SEM
(Scanning Electron Microscope) studies used the model JSM 6390LA in the FRST laboratory. The leaves
samples of 3 species of Paphiopedilum were examined. The stomata for P. stonei, P. bullenianum Rchb. f
and P. hookerae Rchb. f had been identified using scanning electron microscope (SEM).The SEM images
have been observed and analyzed. Three types of stomata were observed; Desmocytic, Pericytic and
Cyclocytic. This characteristic could be used to differentiate the three species and provide new taxonomic
information which has not been reported before.
Keywords: Paphiopedilum stonei, Slipper orchids, Orchidaceae, limestone, Sarawak
xiii
Population study of Paphiopedilum stonei Hook. (Orchidaceae) from limestone areas
of Northern West of Sarawak
Mas Izzaty Bte Mazlan
Plant Resource Science and Management Programme
Department of Plant Science and Environmental Ecology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRAK
Batu kapur merupakan habitat yang sangat unik dan ia adalah habitat untuk kebanyakkan spesies
tumbuhan yang endemik. Antaranya ialah Orkid, Begonias, Aroids, Gensneroids dan banyak lagi. Orkid
adalah famili kedua terbesar selepas Poaceae dalam monokotiledon dan ia adalah salah satu yang
terbesar di alam tumbuhan berbunga, dengan anggaran baru-baru ini terdiri daripada 20 000 hingga 35
000 spesies terdiri daripada lima sub famili. Orkid bukan sahaja penting untuk nilai estetika tetapi juga
digunakan sebagai penunjuk ekologi. Paphiopedilum stonei Hook. juga salah satu yang paling berharga
antara semua spesies di dalam genus kerana ia sangat unik dan cantik. Ini telah membuat mereka
terdedah kepada perubahan habitat dan kemusnahan. Walaupun ia adalah spesies yang dilindungi di
bawah Konvensyen Perdagangan Antarabangsa Spesies Terancam (CITES), ancaman dari pengumpul
masih merupakan satu masalah yang besar. Tujuan utama projek ini dijalankan ialah untuk untuk
menyiasat populasi P. stonei di tiga kawasan terpilih iaitu di Bau, Padawan dan Serian. Terdapat kajian
yang amat sedikit kepada kepadatan P. stonei di habitat batu kapur kerana populasi selipar orkid telah
berkurangan disebabkan telah diambil oleh pemburu orkid. Kajian ini melibatkan kerja-kerja lapangan
dan kerja makmal. Sebanyak empat puluh lima sub plot telah dijalankan dalam tiga tapak kajian dengan
ukuran 10 m x 10 m untuk setiap sub plot. Bau mempunyai bilangan P. stonei yang tertinggi dengan 63
individu iaitu sebanyak 48.1% daripada bilangan jumlah yang dicatatkan. Diikuti oleh Padawan dan
Serian dengan 39 dan 29 individu. Perbandingan populasi P. stonei di antara ketiga-tiga kawasan yang
terpilih yang telah dianalisis dengan menggunakan ANOVA dan hasilnya bahawa bilangan min untuk P.
stonei antara ketiga-tiga kawasan kajian adalah tidak sama. Perbandingan bilangan P. stonei antara
ketiga-tiga kawasan terpilih telah dianalisis dengan menggunakan ANOVA dan perbezaan yang
signifikan (P =0,002). Semua sampel yang kering telah disediakan untuk dikaji dengan SEM
(Pengimbasan Mikroskop Elektron) menggunakan JSM 6390LA model di makmal FRST. Sampel daun
dari 3 spesies Paphiopedilum telah diperiksa oleh SEM. Stomata untuk P. stonei, P. bullenianum Rchb. f
dan P. hookerae Rchb.f telah dikenal pasti dengan menggunakan mikroskop imbasan elektron (SEM).
Imej SEM telah diperhatikan dan dianalisis. Tiga jenis stomata telah dijumpai; Desmocytic, Pericytic
dan Cyclocytic. Ciri-ciri ini digunakan untuk membandingkan ketiga-tiga spesies dan memberi maklumat
taksonomi yang baru di mana tidak pernah dilaporkan sebelum ini.
Kata kunci: Paphiopedilum stonei, orkid selipar, orkid, batu kapur, Sarawak
1
1.0 INTRODUCTION
1.1 General Introduction
Borneo is the third largest island in the world and lies on the Sunda shelf in the eastern
half of the Malesian region in South East Asia (MacDonald, 1956). Straddling the
equator between 108˚50΄ E to 119˚ 20΄ E, and from 7˚ 4΄ N to 4˚ 10΄ S, the island is
over 1300 km long and 950 km wide with an area of nearly 740,000 square kilometers.
Borneo is a centre of diversity of many edible and is truly an „island of fruits‟ but could
equally be referred to as the „Orchid Island‟ (Whitmore, 1984). van Steenis (1971)
described the largest family in the region is the Orchidaceae with 3000-4000 species,
comprising 12-16% of the flora. About 2500- 3000 orchid species that has estimated by
Lamb (1991) that are found in Borneo, equivalent to 10% of the world orchids, 10-12%
of the Malesian flora and 75% of the Malesian orchid flora. Among these 30-40% are
thought to be endemic to the island.
Stibig &Malingreau (2003) described that most species of plants in Borneo can be found
in its forest habitats such as mangrove, peat swamp, freshwater swamp forests, tropical
evergreen lowland forests, tropical lower montane rain forest, strand and coastal forest
and hill dipterocarp forests, and it is also home to the largest heath forests in Southeast
Asia. Among them, limestone habitat is unique with high endemism.
Limestone is a sedimentary rock consisting mainly of the mineral calcite (calcium
carbonate, CaCO3). The other common mineral in limestone is dolomite [CaMg (CO
3)].
2
Common impurities in limestone include chert (microcrystalline, cryptocrystalline
quartz or amorphous silica, SiO2), clay, organic matter and iron oxides (Banda et al.,
2004). Sabah and Sarawak have extensive limestone formations occurring in the forms
of numerous limestone hills and bedrock. Majority, limestone hills in Sarawak are
found in the Kuching division such as at Bau, Penrissen and Padawan – Serian areas
(Kiew et al., 2004).
Other than an area for biodiversity conservation, limestone areas provide high levels of
radiation and endemism in both plant and animal species, including the little studied
subterranean fauna of caves (Bandarin, 2001). A wide array of flora and fauna
composition is present in limestone areas. Fauna compromising both vertebrates and
invertebrates can be found in this type of habitat. Interestingly, some of them have
developed some specialized morphological features to adapt to the environment. Plant
species, such as timber trees, mosses and other ornamental trees such as orchids, thrive
well in limestone environment (Lapis et al., 2004).
According to Kiew (1991) generally limestone areas are very rich in orchids and high
number of endemic species such as slipper orchid and jewel orchids. All the Bornean
slipper orchids are considered rare, their colonies usually in small patches and low
number of density. The limestone cliffs and hills of northern west of Sarawak are the
home of P. stonei, one of the most highly prized of all species in the genus. Highly
demand of P. stonei have continued to be made since its discovery and introduction in
1862 by Messrs. Low and Co. (Cribb, 1997).
3
Currently the deforestation of large tracts of lowland and lower montane forest in
Borneo is probably the greatest long-term threat to these plants. Protection of some of
the best populations within National Parks and Forest Reserves cannot entirely remove
the threat posed by collectors. Of the twelve species in Borneo, only five received
protection, two species in the Kinabalu Park, two and one in Gunung Mulu National
Park and Bako National Park respectively. The international trade in wild-collected
species slipper orchids has been somewhat curtailed by the placing of all
Paphiopedilum species on Appendix I of CITES (Convention in Trade on Endangered
Species of Wild Fauna and Flora) in 1988 (Cribb, 1997).
There is very little study on the density of P. stonei at the limestone habitat because the
population of slipper orchid is decreasing duly to the over collected by the orchid
hunters. Their attractive flowers and relatively rare have made these plants highly
attractive to orchid collectors and growers. Therefore this project aimed:
a) To investigate and compare density of P. stonei from limestone areas of Northern
West of Sarawak.
b) To compare morphological characteristics of P. stonei between the three selected
areas.
c) To distinguish the leaf anatomy characteristics between the selected species.
4
2.0 LITERATURE REVIEW
2.1 Order Asparagales
The Asparagales is a widely distributed order of monocotyledenous flowering plants,
consisting 29 families with 26,000 species. Among the families that included in order
Asparagales are Amarylidaceae, Irridaceae, Asparagaceae, Alliaceae, Hypoxidaceae,
Orchidaceae, Lanariaceae and Boryaceae. Most of the members are produce a tight
cluster of leaves from the base of the plant. Some members produce a stout trunk with
age, which bears the cluster of leaves and in a few cases leaves are produced along the
stem. Flowers are often produced at the end of a long stem and seeds of many species
are covered with a black or bluish layer of phytomelanin, although this is missing in the
case of seeds from Orchidaceae family. Orchidaceae is considered the largest family in
the order Asparagales with an estimation more than 20,000 over species (Lapis et al.,
2004).
Despite the diversity of this Order, DNA analysis has confirmed the relationships of
plants within the Asparagales, which used to be included in and has recently been
separated from the Liliales. Previously, many of the families were plaeed into to the old
order Liliales, a very large order containing almost all monocots with colourful tepals
and without starch in their endosperm. DNA sequence analysis indicated that the
species of Liliales are having superior ovary while Asparagales with inferior ovary. The
boundaries of the Asparagales and of its families have undergone a series of changes in
recent years; future research may lead to further changes and ultimately greater stability
(Kubitzki, 2006).
5
An economic point of view (Charles, 2004) the order Asparagales is the second
importance order within the monocots after the order Poales (which includes grasses
and cereals). Most of the species are used as food and flavourings (e.g. onion, garlic,
leek, asparagus, vanilla), as cut flowers (e.g. freesia, gladiolus, iris, orchids), and as
garden ornamentals (e.g. day lilies, lily of the valley, Agapanthus).
2.2 Family Orchidaceae
According to Lamb (1991), the orchid family is probably the largest in the Flowering
Plant Kingdom containing more than of 20,000 species. Most classifications of orchids
have concentrated upon features of the flowers, especially of the sexual parts, to provide
clues as to relationships. Dressler (1990) has added much additional information from
vegetative morphology, anatomy, cytology, and micromorphology to substantiate his
classification and divides the orchid into five subfamilies: Apostasioideae,
Spiranthoideae, Orchidoideae, Epidendroideae and Cypripedioideae.
2.2.1 Subfamilies in Orchidaceae
The Apostasioideae, comprising the genera Apostasia and Neuwiedia are usually
considered to be most primitive orchids. Both genera are found in Borneo. They are
terrestrials with plicate leaves, an erect spicate many-flowered inflorescence. The
column bears a style to which two or stamens are partially fused at the base and have
root-stem tuberoids, an endomycorrhizal association and dust- like seeds (Dressler,
1981).
6
Anderson and Keong (1982), reported that the Spiranthoideae are well presented in
Borneo and include the pretty jewel orchids that are such a feature of the flora of the
forest floor. Genera such as Anoectochilus, Cystorchis, Dossinia, Ludisia and Zeuxine
are all included in this subfamily which is characterized by the dorsal erect anther which
is subequal to the rostellum, and usually by the creeping fleshy rhizome.
The example of species in Orchidoideae includes Alphyllorchis, Corybas, Habenaria
and Peristylus. They possess root- stem tuberoids, sectile pollinia and an anther firmly
attached by its base to column. The Epidendroideae characterized by hard discrete
pollinia in an apically attached anther. It is not only the largest subfamily but also the
most diverse and has been divided into two subfamilies by some authors. Those genera
with lateral inflorescences, and an anther with reduced partitions, superposed pollinia,
viscidia and stipe have been included in the Vandoideae. This distinction is, however,
far from clear-cut and Dressler (1981) has concluded that the Vandoideae should
therefore be included in the Epidendroideae.
The Cypripedioideae have also, at times, been treated as a separate family. They are
mainly terrestrial but a few are epiphytic. The stems may be short or long and the leaves
either plicate or conduplicate. The flowers are quite distinctive with a large dorsal sepal
and usually fused lateral sepals. The lateral petals are spreading or pendulous and range
from subcircular to elongate and ribbon-like. The lip is always slipper-shaped, hence
their popular name of “Slipper Orchids”. The column is short with two lateral ventral
fertile anthers and a large sterile, usually shield-shaped.anther or staminode. The stigma
is stalked and ventral. The Cypripedioideae do, however, have dust-like seeds, a
7
mycorrhizal association, and a column with fused stamens and style. Paphiopedilum is
the solely genus occurred in Borneo (Dressler, 1990).
2.2.2 Genus Paphiopedilum
Slipper orchids are remarkable group within the family Orchidaceae. They comprise five
genera: Selenipedium, Phragmipedium and Mexipedium, tropical American;
Cypripedium with a north temperate distribution in both the Old and New World; and
Paphiopedilum found only in tropical Asia and the adjacent islands from Sumatra
across to the Philippines and Solomon Islands. Altogether there are less than 150
species of slipper orchid and about half of these belong in Paphiopedilum, 47 in
Cypripedium, about 12 in Phragmipedium, six in Selenipedium with a single
Mexipedium species making up the numbers (Cribb, 1997).
Borneo is well endowed with orchids (Wood & Cribb, 1994), having one of the richest
orchid floras in the world. Amongst these can be found twelve species of slipper orchid,
all belonging to the genus Paphiopedilum. Slipper orchids are characterized by their
curious flowers and gain their common name given to the group is Lady‟s Slippers. The
flowers of slipper orchids are their most distinctive feature. As in all orchids the ovary
is inferior, which has borne below the sepals and other floral segments. In most Bornean
species the ovary is hairy but in P. rothschildianum, P. stonei, P. kolopakingii and P.
supardii it is glabrous.
8
2.2.3 Ecology
Slipper orchids can be found in three situations in Borneo: on the ground, growing as
terrestrially; and on rocks as lithophytes. Five species, P. bullenianum, P. dayanum, P.
javanicum, P. hookerae, and P. lawrenceanum are truly terrestrial, growing in shallow
leaf litter and well-drained soil on ridges and steep slopes. Only one species, the
widespread P. lowii, is epiphytic. The remaining six species are litophytic, or very
rarely terrestrial or epiphytic. In the most extreme cases of P. sanderianum, P.
philippinense, P. supardi and P. stonei, the roots are strongly attached to the rock
substrate allowing the plants to grow on almost vertical cliffs and ledges. The other two
species, P. rothschildianum and P. kolopakingii, grow on ledges on steep slopes and
cliffs (Atwood & William 1978).
According to Braem (1988), slipper orchids can be found on a variety of substrates, all
nutrient- poor or infertile soil. Paphiopedilum bullenianum grows in peat-swamp forest
at the bases of small trees on slight peat hummocks or on the thin layer of peat in the
Kerangas forest. The condition there must be acidic. In contrast P. stonei, P.
sanderianum, P. philippinense, P. supardi grows on limestone rock where the pH is
either alkaline or more often more or less neutral.
The terrestrial species with tessellated or mottled leaves grow in deep shade on the forest
floor, whereas the plain-leaved species grow in dappled shade of open woodland, often
on cliffs and just below ridge-tops. North and north-east facing slopes are preferred by
some species such as P. stonei, P. sanderianum and P. rothschildianum.
9
Flowering occurs at the end of the rains in most Bornean slipper orchids. February to
April is the best season to see slipper orchids in flower but flowering can also occur in
September to November. Flowers last for between four to eight weeks.
2.3 Paphiopedilum stonei Hook.
The limestone cliffs and hills of western Sarawak are the home of P. stonei. Sir William
Hooker described it in December of that year in Curtis’s Botanical Magazine, the
forerunner of the present Kew Magazine, naming it for Mr Stone, the gardener of John
Day of Tottenham, London. It is a striking orchid readily recognized by its boldly
striped dorsal sepal, pink porrect lip, slender arcuate untwisted petals and distinctive
staminode. Its closest ally is probably P. kolopakingii, but that has more and smaller
ochreous flowers which are also less boldly marked with deep maroon (Cribb, 1983).
Paphiopedilum stonei has a very limited distribution and it‟s frequently collected by
nature lovers or individual for sale that caused inevitably depletion in the wild. It is
somewhat protected for limestone areas are not easily accessible and one hopes that
sufficient colonies survive where man cannot strip them. This species grows on sheer
cliffs at between 60-700 m elevations, lightly shaded by the crowns of tree growing at
the base of the cliffs. However, even sheer cliffs, one of its favored habitats, do not
seem to be sufficient to protect it as some irresponsible collectors will by all means to
collect this species for it fetches good prices in the illegal market (Cribb, 1997).
10
2.4 Scanning Electron Microscope (SEM)
The Scanning Electron Microscope (SEM), nowadays become most popular as
supportive tool in plant systematic studies. The SEM produces images of high
resolution, which means that closely spaced features particularly that cannot be
observed through naked eyes can be examined at high magnification. The combination
of higher magnification, larger depth of focus, greater solution and ease of sample
observation makes the SEM one of the most heavily used instruments in research areas
today. There are few steps of treatment the biological sample for SEM scanning which
is cleaning the sample, fixation, dehydration and drying and lastly coating process
(Klesd, 2012).
In Fixation process, chemical fixation of the sample to prevent autolysis and provide
stabilization of the sample state closet to the natural are caused for tissue and cells. The
used solution contained fixation (gluteraldehyde or formaldehyde) and buffer (sodium
cocodylate) for ensuring optimal condition like osmorality and Ph. The Ph of the
fixation should be as possible to the natural environment of cells and tissue fluids.
2.5 Stomata
Stomata is a pore in the epidermis encircled by two guard cells; often used to describe
both the pore itself and the two guard cells that surround it and regulate its size and thus
the rate of gas exchange. Some stomata have additional cells adjacent to the guard cells,
which are distinct from other epidermal cells in shape or wall thickness. These are the
subsidiary cells (Oparka & Turgeon 1999).