PHYLOGENETIC AND SYSTEMATIC STUDIES OF THE AMORPHOPHALLUS ... and systematic studies of... · PHYLOGENETIC AND SYSTEMATIC STUDIES OF THE AMORPHOPHALLUS SPECIES IN SARAWAK ... Phylogenetic

PHYLOGENETIC AND SYSTEMATIC STUDIES OF THE AMORPHOPHALLUS SPECIES IN SARAWAK

AizatJapar

Master of Science (plant Systematics)

2013

rusat Khidmat M.aklumat Akademik UNIVERSlTI MALAYSIA SARAWAK

Phylogenetic and Systematic Studies of the Amorphophal/us Species in Sarawak

,.....

P.KHIDMAT MAKLUMAT AKADEMIK

111111"Im1111111111000245972

Aizat bin Japar

A Thesis Submitted In Fulfillment of the Requirement of Master of Science

(Plant Systematic)

Department of Plant Science and Environmental Ecology

Faculty of Resource Science and Technology

University Malaysia Sarawak

November 2013

DECLARATION

I hereby declare that this thesis is based on my original work except for quotations and

citations which have been duly acknowledged. I also declare that this thesis has not been

previously or concurrently submitted for any other degree of qualification to any other

university or institution ofhigher learning.

(Aizat bin Japar)

(Postgraduate Student No.: 09021459)

111

DEDICATION

My dedication goes to my dearest family members especially my father, Encik Japar Din,

my mother Puan Hasiah Razak, brothers and sister for their supports and inspirations

given in completing this thesis successfully.

IV

I

ACKNOWLEDGEMENT

Bismillahirrahmanirrahim. I would like to express my grateful thanks to Allah S.W.T for

giving me opportunity, health, knowledge and protection in completing my postgraduate

studies. First and foremost, I offer my sincerest gratitude to my supervisors, Professor Dr Isa

Ipor, Professor Dr. Cheksum @ Supiah Tawan, Professor Madya Dr. Hairul Azman @ Amir

Harnzah Roslan for their wonderful guidance, dedication throughout this project and time in

reviewing the thesis manuscript. Not forgotten, Mr Qammil Muzammil and all lecturers from

Department of Plant Science and Environmental Ecology for their advice and aid during this

study.

Numerous thanks to my colleagues and acquaintances especially Miss Haniza Razali, Mr

Frankie Lanying, Miss Norma Mamat, Miss Norazima, Miss Norhana, Miss Nabella, Miss

Jong Jen and others for supporting and lending their help in completing this thesis. To my

senior Miss Qistina, Miss Angeline Simon and others who kindly spared their time and

knowledge. My sincere appreciation to all of laboratory assistants of the FRST faculty,

especially Mr Hidir Marzuki, Mr Najib Fardos, Mr Sekudan Tedong, Mr Salim Arip and Mr

Nurfazillah for their assistance.

I would also like to thank Universiti Malaysia Sarawak (UNIMAS) especially Centre of

Graduate Studies and Faculty of Resource Science and Tec!mology, for the opportunity and,

providing Zamalah scholarship and fmancial grant.

v

ABSTRACT

(A systematic study was conducted on ten species of Amorphophallus in Sarawak based

on morphology, ecology and genomic sequences data. The objectives of this study were;

(I) to document morphological characteristics and ecological data of ten selected

Amorphophallus species in Sarawak; (2) to find a suitable method of DNA extraction (3)

to reconstruct phylogenetic tree and examine the phylogenetic relationship between the

Amorphophallus speciej) Materials to be used as herbarium specimen and DNA samples

were collected, from localities around Sarawak such as Kuching, Sri Aman, Padawan and

Mulu. Random amplified polymorphic DNA technique was applied and genetic profiles

were obtained for the selected species generated using primers OPC-06, OPD-05 and

OPD-20. Dendrogram created using the unweighted pair group method with arithmetic

averages provide enough information for the analysis of genetic diversity. Phylogenetic

trees for Amorphophallus species were reconstructed using two genomic sequence, rbcL

genes and trnL-F intergenic spacer. The sequenced data was analyzed by four different

methods; Bayesian, maximum likelihood, maximum parsimony and neighbor joining. The

out-groups for the phylogenetic trees are from genus Arisaema, DiefJenbachia, and

Bognera. The reconstructed phylogenetic trees were successful to resolve phylogenetic

relationship for several species ofAmorphophallus.

VI

Kajian Filogenetik dan Sistematik Terhadap Spesies Amorphophallus di Sarawak

ABSTRAK

Satu kajian sistematik telah dijalankan ke atas sepuluh spesies Amorphophallus terpilih

di Sarawak berdasarkan maklumat morfologi, ekologi dan jujukan gen. Matlamat utama

kajian ini adalah: (1) mencatat ciri-ciri morfologi dan maklumat ekologi tentang sepuluh

spesies Amorphophallus di Sarawak; (2) mencari teknik mengekstrak DNA yang sesuai

(3) membina pokok filogenetik dan menganalisis hubungan filogenetik antara spesies

A morpho phallus. Bahan-bahan untuk digunakan sebagai sampel herbarium dan gen

telah dikumpulkan daripada seluruh Sarawak seperti Kuching, Sri Aman, Padawan dan

Mulu. Teknik 'random amplified polymorphic DNA' telah diaplikasi dan profil genetik

betjaya diperoleh untuk spesis terpilih dengan menggunakan primer OPC-06, OPD-05

dan OPD-20. Dendogram direka dengan menggunakan 'un weighted pair group method

with arithmetic averages' telah menyediakan maklumat yang secukupnya untuk analisis

kepelbagaian genetik. Pokok filogenetik telah betjaya dibina semula untuk spesis

Amorphophallus dengan menggunakan jujukan kawasan kloroplas rbcL dan trnL-F. Data

jujukan dianalisis dengan menggunakan empat teknik; 'Bayesian', 'maximum likelihood',

'maximum parsimony' dan 'neighbor joining '. Kumpulan luaran untuk pokok filogenetik

tersebut adalah dari Arisaema, Dieffenbachia dan Bognera. Pokok filogenetik yang

dibina semula telah betjaya menyelesaikan hubunganfilogenetik untuk sebahagian spesis

Amorphophallus.

Vll

Pusat Khidmat Maklumat Akademik VNlVERSITI MAlAYSIA SARAWAK

TABLE OF CONTENTS

PAGE

Title Page ............. . .......................... . ........................................ .

Approval Sheet........................................................................... 11

Declaration. . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .... 111

Dedication. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . ... IV

Acknowledgement. . ........................................... . ........ .... ......... ..... v

Abstract ..................................................................................... VI

Abstrak . . ............................. . ...................................................... Vll

Table of Contents.. .. ........................................... ... ...... ......... ..... .... VllI-XI

List ofFigures .......................................... ............................. ....... xii-xvii

List ofTables ........... .. .................................................... ....... .. .... XVIll

Abbreviation.. ....................... . ..... ........................ .. .. ...... ...... . .... XXIX

CHAPTER ONE: INTRODUCTION

1.1 Introduction ........................................................................... 1-2

1.2 Problem Statement..... .. ............................ .. ............. ........... ..... 3-4

1.3 Objectives. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5

CHAPTER TWO: LITERATURE REVIEW

2.1 Family Araceae Juss. ... . ........................................ ... ................ 6-7

2.2 Genus Amorphophallus ............................................................ 8-11

2.2.1 Taxonomy...................................................... ......... . .. 8

2.2.2 Morphology............................. ... ................................ 8-9

2.2.3 Habitat and Distribution.............................................. ..... 9-11

V111

2.3 Food Crops and Uses............................................................... 11-12

2.4 DNA Extraction...................................................... .. ............. 13

2.5 Polymerase Chain Reaction (PCR) .............................. . ................ 14

2.6 Random Amplified Polymorphic DNA........................................... 14-15

2.7 Molecular Phylogenetics ................ . ........................................... 16

2.8 Chloroplast DNA (cpDNA) ........................................ ......... ....... 17-18

2.8.1 rbcL Genes.. ........ . ........ . .............. . ..... ............... ....... ... 19

2.8.2 trnL-F Intergenic Spacer Region........................................ 20

2.9 Previous works on Family Araceae and Genus Amorphophallus ............ 21-22

CHAPTER THREE: MATERlALS AND METHODS

3.1 Locations and Study Area.......................................................... 23

3.2 Collection ofHerbarium Specimens............................................. 24

3.3 Data Compilation and Herbarium Specimens Labeling................... .... 25

3.4 Comparison of Morphological Characteristics............................ . .... 25

3.5 Plant Genomic DNA................................................................ 26

3.5.1 Plant DNA Samples Collection ......................................... .. 26

3.5.2 Modified DNA Extraction Protocol C (Doyle and Doyle, 1990;

28-29Sharma et al., 2008) ...................................................... .

3.5.3 Genomic DNA Quantification .... .. ................. .................. '" 30

3.5.4 Genomic DNA Purification ............................................ .. 30

3.6 Polymerase Chain Reaction (PCR) Amplification........... ........... ....... 31

3.6.1 Random Amplification Polymorphic DNA (RAPD-PCR) ........... 31-32

3.6.2 Amplification of rbcL genes.......... . .................................. 33-34

IX

3.6.3 Amplification of trnL-trnF Intergenic Spacer (lGS).................. 34-35

3.7 Sequencing Analysis..................... .. ..................................... . ... 36-39

CHAPTER FOUR: MORPHOLOGY AND ECOLOGY RESULTS

4.1 Habitat and forest vegetation of the localities studies....................... ... 40-44

4.2 Morphological description ofA morpho phallus ... ... ... ... ... ... ... ... ... ... ... .... 45-57

4.3 Key to Amorphophallus species in Sarawak ..................................... 58

4.4 Results and Discussion

4.4.1 Tuber................ .................. ..................... ................. 59-60

4.4.2 Petiole......................................... ... ........ . ......... ........... 61-63

4.4.3 Leaflet.............................................. ....... ... .. .......... ... 64-65

4.4.4 Inflorescence........ .. .................... . ........... .. ................... 66-69

4.4.5 Life Cycle and Infructescence.............. ............................. 70-71

CHAPTER FIVE: MOLECULAR ANALYSES RESULTS

5.1 Plant Genomic DNA

5.1.1 Results and Discussion.. .................................... ........ . .. .. 72

5.1.1.1 DNA Extraction.. .......................................... .... 72-79

5.1.1.2 DNA Purification Analysis ................................ ... 80-82

5.2 Random Amplification Polymorphic DNA (RAPD-PCR) ........... .......... 83

5.2.1 Results and Discussion

5.2.1.1 PCR Optimization.... .. .................... ..... ...... . ....... 83-86

5.2.1.2 Primer Screening.......... . .......... .... ..................... 87-88

5.2.1.3 RAPD-PCR Amplification Result.......................... 89-92

5.2.1.4 Data Analysis........................ .. ....... . ......... .... .... 93-96

x

I

5.3 Amplification of rbcL Gene................................... .................. ... 97


5.3.1.1 PCROptimization .................... .......................... 97-100

5.3.1.2 Analysis ofrbcL Genes Sequence ........................ .. 101-103

5.3.1.3 Analysis of Phylogenetic Tree.... ................ ... ........ 104-109

5.4 Amplification of trnL-trnF Intergenic Spacer............. . ..................... 110


5.4.1.1 PCR Optimization ............................................. 110-113

5.4.1.2 Analysisof trnL-F Intergenic Spacer.................... ... 114-117

5.4.1.3 Analysis of Phylogenetic Tree .............................. 118-123

CHAPTER SIX: GENERAL DISCUSSION AND CONCLUSION

6.1 Habitat Distribution.......................................................... 124-125

6.2 Morphological Characteristics. ....................................... . ............ 126

6.3 DNA Extraction Protoco1........... ...... ..................... ... ........ ........... 127-128

6.4 RAPD Markers.......................................................... ... ........... 129-130

6.5 Phylogenetic Analysis........................................................... ... 130-133

6.6 Conclusion and Recommendation.............. .................................. 134-135

REFERENCES............. . ..................... ...... ......... .. . ....... ............... ......... 136-157

Appendix I ................................................................................... 158-159

Appendix II .................................................................................. 160-161

Appendix III ................................................................................ 162-163

Appendix VI ................................................................................ 164-169

Appendix V .................................................................................. 170-179

Xl

LIST OF FIGURES

Figure No. Titles Page

Figure 1 Map showing the location in Sarawak 23

Figure 2 The tubers ofAmorphophallus in Sarawak (AI: A. brachyphyllus; A2: A. 60

(AI-A9) borneensis; A3: A. eburneus; A4: A. pendulus; A5: A. infundibuliformis;

A6: A. ranchanensis; A7: A. julaihii; A8: A. angulatus and A9: A.

costatus).

Figure 3 The petioles of ten Amorphophallus in Sarawak (B 1: A. ranchanensis; 63

(BI-BI0) B2: A. costatus; B3: A. hewittii; B4: A. pendulus; B5: A. eburneus; B6:

A. infundibuliformis; B7: A. borneensis; B8: A. angulatus; B9: A.

brachyphyllus and B 1 0: A. julaihii).

Figure 4 The leaflets of ten Amorphophallus in Sarawak (Cl: A. brachyphyllus; 65

(CI-CIO) C2: A. ranchanensis; C3: A. borneensis; C4: A. costatus; C5: A.

inlundibuliformis; C6: A. hewittii; C7: A. eburneus; C8: A. pendulus; C9:

A. angulatus and CIO: A.julaihii).

Figure 5 The inflorescence of ten Amorphophallus ill Sarawak (Dl: A. 67

(DI-DI0) irifundibuliformis; D2: A. brachyphyllus; D3: A. ranchanensis; D4: A.

costatus; D5: A. angulatus; D6: A. hewittii; D7: A. julaihii; D8: A.

pendulus; D9: A. eburneus and DI0: A. borneensis).

Figure 6 The infructescence of A morpho phallus ill Sarawak (El: A. 71

(EI-E4) irifundibuliformis; E2: A. borneensis; E3: A. julaihii and E4: A.

brachyphyllus).

Figure 7 Lane 1-7: DNA extracted from A. borneensis using Protocol A; lane 8 74

Xll

,..

14: DNA extracted fromA. borneensis using Protocol B; lane M: 100bp

ladder (Fermentas).

Figure 8 DNA extracted using protocol C. Lane 1-5: sample from A. costatus; 75

lane 6-10: sample from A. infundibuliformis; lane M: 100bp ladder

(Fermentas).

Figure 9 Lane 1-5: DNA extracted from silica dried leaves of A. angulatus; lane 77

6: 100bp ladder (Fermentas); lane 7-11: DNA extracted from fresh

leaves ofA.angulatus.

Figure 10 Agarose gel (1 %) electrophoresis of purified DNA samples. Lanes 1: A. 81

borneensis; 2: A. brachyphylus; 3: A. eburneus; 4: A. julaihii; 5: A.

hewittii; 6: A. ranchanensis; 7: A. costatus; 8: A. pendulus; 9: A.

angulatus; 10: A. infundibuliform is. Lane M is 100 bp DNA ladder

(F ermentas).

Figure 11 PCR products from different annealing temperature usmg A. 86

ranchanensis DNA samples. Lane 1-8 (34.0, 34.5, 35.5, 36.9, 38.0,40.3,

41.3, 42.0°C). Lane 9 represents negative control and M represents 100

bp ladder (F ermentas).

Figure 12 PCR products from different concentration of MgCh usmg A. 86

ranchanensis DNA samples. Lane 1-6 (1.5, 2.0, 2.5, 3.0, 3.5,4.0 mM).

Lane M represents 100 bp ladder (Fermentas).

Figure 13 RAPD-PCR amplification of twelve Amorphophallus specIes usmg 90

primer OPC-06. Lane MI: I kb ladder (Ferment as), (2: A.

brachyphyllus; 3: A. angulatus; 4: A. borneensis; 5: A. ranchanensis; 6:

A. pendulus; 7: A. hewitii; 8: A. julaihii; 9: A. costatus; 10: A.

xiii

infundibu lifonn is; 11: A. eburneus; 12: A. bornensis Kalimantan; 13: A.

hewitii Kalimantan). Lane 14 represents negative control and M2

represents 100 bp ladder (Fermentas).

Figure 14 RAPO-PCR amplification of twelve Amorphophallus speCIes usmg 91

primer OPD-20. Lane Ml: 1 kb ladder (Fermentas), (2: A.



infundibulifonnis; 11: A. eburneus; 12: A. bornensis Kalimantan; 13: A.


represents 100 bp ladder (Fermentas).

Figure 15 RAPO-PCR amplification of twelve Amorphophallus species usmg 92

primer OPO-05. Lane Ml: 1 kb ladder (Fermentas), (2: A.



infundibulifonnis; 11: A. eburneus; 12: A. bornensis Kalimantan; 13: A.


represents ]00 bp ladder (Fermentas).

Figure 16 Oendogram often Amorphophallus species generated by UPGMA based 96

on RAPD marker.

Figure 17 PCR products from different annealing temperature. Lane 1-8; 48.0, 99

48.7, 49.9, 51.7, 54.1, 56.0, 57.2, 58.0 (OC). Lane 9 represents negative

control and M represents 100 bp ladder (Fermentas).

Figure 18 PCR products from different concentration of MgC1. Lane 1-5; 3.5, 3.0, 99

2.5, 2.0, 1.5 (mM). Lane 6 represents negative control and M represents

XIV

100bp ladder (Fermentas).

Figure 19 PCR products obtained using the optimized parameters. Lane 1: A. 100



infundibuf!formis; 10: A. eburneus; 11: A. bornensis Kalimantan; 12: A.

hewitii Kalimantan. Lane M represents 100 bp ladder (Fermentas).

Figure 20 Bayesian 50% rule-majority consensus tree obtained from the analysis 105

of rbcL data. Numbers below the branches are Bayesian posterior

probabilities.

Figure 21 Phylogenetic tree obtained from the analysis of rbcL data through the 106

maximum likelihood (ML) method. Numbers below branch indicate

percentage of bootstrap values estimates from 1000 bootstrap replicates.

Only bootstrap values >50% are shown and the groups are Arisaema

amurense.

Figure 22 The strict consensus of the 33 most parsimonious trees based on the 108

nucleotide sequences of the rbcL genes through maximum parsimony

(MP). Numbers on branches indicating percentage of bootstrap values

estimated from 1000 bootstrap replicates. Only bootstrap values >50%

are shown and the outgroup is Arisaema amurense.

Figure 23 Neighbor joining (NJ) tree based on Kimura's two-parameter distance. 109

Numbers below branch indicate percentage ofbootstrap values estimates

from 1000 bootstrap replicates. Only bootstrap values >50% are shown

and this tree is rooted with A. amurense.

Figure 24 PCR products from different annealing temperature. Lane 1-8: 56.0, 112

xv

56.5, 57.5, 58.9, 60.0, 62.4, 63.4, 64.0 0c. Lane 9 represents negative

contro 1 and M represents 1 OObp ladder (F errnentas).

Figure 25 peR products from different concentration ofMgCh. Lane 1-6: 1.5, 2.0, 113

2.5, 3.0, 3.5, 4.0 mM. Lane 7 represents negative control and M

represents 100bp ladder (Ferrnentas).

Figure 26 PCR products obtained using the optimized parameters. Lane 1: A. 113

brachyphyllus; 2: A. angulatus; 3: A. bomeensis; 4: A. ranchanensis; 5:


infundibuliformis; 10: A. eburneus; 11: A. bomensis Kalimantan. Lane

12 represents negative control and M represents 100 bp ladder

(Ferrnentas).

Figure 27 Bayesian 50% rule-majority consensus tree obtained from the analysis 119

of trnL-F data. Numbers below the branches are Bayesian posterior

probabilities. The out groups are DiefJenbachia aglaonematifolia and

Bognera recondita.

Figure 28 Phylogenetic tree obtained from the analysis of trnL-F data through the 120

maximum likelihood (ML) method. Numbers below branch indicate

percentage of bootstrap values estimates from 1000 bootstrap replicates.

Only bootstrap values >50% are shown and the groups are

DiefJenbachia aglaonematifolia and Bognera recondita.

Figure 29 The strict consensus of the 2 1 most parsimonious trees based on the 122

nucleotide sequences of the trnL-F lOS genes through maximum

parsImony (MP). Numbers on branches indicating percentage of

bootstrap values estimated from 1000 bootstrap replicates. Only

XVI

bootstrap values >50% are shown and the out group are DieJJenbachia

aglaonematifolia and Bognera recondita.

Figure 30 Neighbor joining (NJ) tree based on Kimura's two-parameter. Numbers 123

below branch indicate percentage of bootstrap values estimates from

1000 bootstrap replicates. Only bootstrap values >50% are shown and

the out group are DieJJenbachia aglaonematifolia and Bognera

recondita.

xvii

LIST OF TABLES

Table Titles Page

Table 1

Table 2

TabJe3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Species name, accession number and locality of collected 27

samples.

Primer code, primer sequence, nucleotide length and O+C 32

content of primers used ill the Random Amplified

Polymorphic DNA analysis.

The sequence and nucleotide length ofrbcL primer. 33

The sequence and nucleotide length of trnL-F lOS 35

Length and accession number of the sequence obtained. 39

Localities and forest vegetation of each species used in this 43-44

study.

Summary of results between three modified DNA extraction 76

protocol.

DNA yield and purity. 82

Optimization ofRAPD-PCR reaction parameters. 84

Primers sequences tested in RAPD analysis. -: negative 88

amplification or no reproducible patterns; +: positive

amplification.

Similarity matrix for Jaccard's coefficient for 10 95

Amorphophallus species based on 153 bands obtained with 3

RAPD primers.

The optimized parameters for PCR amplification using rbcL 97

XV111

pnmer.

Table 13 Pairwise genetic distance (%) between the Amorphophallus 102

species based on rbcL gene sequenced.

Table 14 Nucleotide composition based on partial rbcL sequences. 103

Table 15 The optimized parameters for peR amplification using tmL- 110

F primer

Table 16 Nucleotide composition based on partial tmL-F intergenic 115

spacer sequences.

Table 17 Pairwise distance (%) among the Amorphophallus based on 116-117

tmL-F lGS sequenced.

Table 18 List ofhabitats for the ten selected Amorphophallus species. 125

XIX

CI

ABBREVIATIONS

~l

bp

cpDNA

CTAB

CIA

CaC12

DNA

dNTP

EDTA

EtBr

Ethanol

INDELS

rnIv

mg/ml

mM

ng

NJ

OD

PAUP

PCR

pmoV~1

RC

Micro Litre

Base pair

Consistency index

Chloroplast DNA

Hexadecyltrimethyl Ammonium Bromide

Chloroform isoamyl alcohol

Calcium chloride

Deoxyribonucleic Acid

Deoxynucleotide Triphosphate

Ethylene Diamintetra Acetic Acid

Ethidium bromide

Ethyl-alcoho I

Insertions/Deletions

Mass per Vo lume

Miligram per Millilitre

Milimolar

Nanogram

Neighbour Joining

Optical Density

Phylogenetic Analysis Using Parsimony

Polymerase Chain Reaction

Picomole per Microlitre

Rescaled Consistency Index

xxix

rONA

RI

RNA

TAE

Taq. Pol.

TBR

TRIS

trnL

trnF

UV

V

v/v

ribosomal DNA

Retention Index

Ribonucleic Acid

Tris; Acetic Acid; EDT A

Thermus aquaticus Super Therm DNA Polymerase

tree-bisection-reconnect ion

2-amino-2-(hydroxymethyl) -1 ,3-propanediol

Transfer RNA gene for Leucine

Transfer RNA gene for Phenylalanine

Ultra violet

Volts

Volume per Volume

xxx

CHAPTER ONE

INTRODUCTION

1.1 Introduction

Sarawak:, covering 124, 500 square kilometres and situated in the North West of Borneo

Island is the largest state in the Malaysian Federation. As part of the third largest island in

the world, Sarawak was blessed with an extraordinary array of geographical variations.

Lies in a region ofconstant rainfall and high temperature throughout the year (Mackinnon

et ai., 1997), combine with unique geology, this island have give birth to the development

of such incredible diversity in flora and fauna (Rautner, 2005). There are different types

of forest such as limestone, mangrove, peat swamp, freshwater swamp forest, heath

(kerangas) forest, lowland and hill dipterocarp forest. They are well known for housing

spectacular diversity of flora, up to 15,000 different flowering plants (Mackinnon et ai.,

1997). This includes the Aroids, one ofthe richest herbaceous groups in Borneo.

A great number of Aroid species are believed to exist, but have yet to be described and

previously unknown to man (Boyce, 2004). Some of them are unique species and has

been identified as endemic plant, found nowhere else besides Borneo like

Amorphophallus species. Amorphophallus species is a perennial herb plant with an

underground storage organ tuber. The uniqueness of Amorphophallus species lies at the

gigantic inflorescences which emerge from underground for every species has its own

size, shape and color (Boyce, 2004). This structure has been fascinated by many people

and it has great potential to be commercialized as an ornamental plant or ecotourism

attraction. Currently, there are around 16 Amorphophallus species have been found and

identified in Borneo (lpor et aI., 2007a).

The focus of this project is Amorphophallus species which can be found in Sarawak.

There are ten ofthem; A. angulatus Hett. & A. Vogel, A. brachyphllus Hett., A. eburneus

Bogner, A. hewitti Aldrew., A. borneensis (EngJ.) EngJ. & Gehrem, A. infundibuliformis

Hett., A Dearden & A. Vogel, A. pendulus Bogner, A. julaihii lpor, Tawan & Boyce, A.

ranchanensis lpor, Tawan & Simon and Amorphophallus costatus Hett (Ipor et al.,

2007a). The distribution of these species spread around Sarawak. Their habitat can be

either on steep slopes, forest margin and limestone area. Among all of them, three species

which are endemic to limestone area in Sarawak are A. brachyphylus, A. eburneus and A.

julaihii.

2

1.2 Problem Statement

Amorphophallus (Family Araceae) is a tropical fleshy herb plants occurring in Africa,

Madagascar, India, continental South East Asia and West Malesia (Mayo et al., 1997;

Wiart, 2000). This plant produces an underground tuber from which the single leaflet

arises, followed by unique and variable size of inflorescence (lpor et al., 2006). Currently,

the genus is undergoing revision (Hetterscheid: in prep.) which will include pollen

morphology (van der Ham et al., 1998), odour biochemistry and pollination biology (Kite

& Hetterscheid, 1997; Kite et ai., 1998) and molecular data (Grob et ai., 2002, 2004).

Recent and on-going taxonomic studies of the Amorphophallus include the introduction

of new species from Asia, Africa and Madagascar (Hetterscheid & Ittenbach, 1996;

Ittenbach & Lobin, 1997; Bogner, 2003; Ipor et al., 2004) and ecological studies (lpor et

al., 2006; Sulaiman & Shunmugam, 201 0).

The identification and classification in plant systematic usually become problematic at the

lower taxonomic level (genus and species) because of similar morphology (Brinegar,

2009). There have been cases where researchers were in disagreement in placing of

certain taxa because of the morphology. From the perspective of evolution, these

hierarchical units (genus and species) have only recently diverged from common

ancestors and their DNA has had much less time to accumulate mutations (Brinegar,

2009). Two Amorphophallus species in Sarawak, A. eburneus and A. brachyphllus which

are endemic to limestone area are the example ofplants which have similar morphology.

Another example is A. hewitti and A. borneensis which are also difficult to identify unless

they produce flower. The similarities between those species lead to the possibility that

3

they might have close genetic relationship and genetic analysis can be the key to

differentiate them.

Several studies have been done to reveal the phylogenetic relationship within the genus

Amorphophallus using molecular data. Grob et at. (2004) presented a phylogeny of

Amorphophallus based on chloroplast and nuclear sequences (rbcL, matK, trnL, FLint2)

from 46 Amorphophallus species, two Pseudodracontium species and six outgroups.

Recently, Sedayu et al. (2010) presented morphological character evolution of 69

Amorphophallus species based on a combined phylogenetic analysis of trnL, rbcL and

LEAFY second intron sequences. Only a few species from Borneo were included in those

studies and not much infonnation was presented about the phylogenetic relationship

among Amorphophallus species in Borneo. Currently, there are 16 indigenous species

recorded are endemic to Borneo (Ipor et al., 2007a). Genetic analysis needs to be done to

determine the relationship and relatedness between those species. Random amplified

polymorphic DNA (RAPD) markers was applied to measure the genetic diversity among

the samples. Genomic sequences from trnL-F intergenic spacer (lGS) and rbcL region of

chloroplast DNA were obtained through direct sequencing for phylogenetic analysis. This

study is hopefully can provide infonnation regarding the evolutionary patterns and

identification ofAmorphophallus species.

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PHYLOGENETIC AND SYSTEMATIC STUDIES OF THE AMORPHOPHALLUS ... and systematic studies of... · PHYLOGENETIC AND SYSTEMATIC STUDIES OF THE AMORPHOPHALLUS SPECIES IN SARAWAK ... Phylogenetic