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).