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 Results and Discussion
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 Results and Discussion
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.
brachyphyllus; 3: A. angulatus; 4: A. borneensis; 5: A. ranchanensis; 6:
A. pendulus; 7: A. hewitii; 8: A. julaihii; 9: A. costatus; 10: A.
infundibulifonnis; 11: A. eburneus; 12: A. bornensis Kalimantan; 13: A.
hewitii Kalimantan). Lane 14 represents negative control and M2
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.
brachyphyllus; 3: A. angulatus; 4: A. borneensis; 5: A. ranchanensis; 6:
A. pendulus; 7: A. hewitii; 8: A. julaihii; 9: A. costatus; 10: A.
infundibulifonnis; 11: A. eburneus; 12: A. bornensis Kalimantan; 13: A.
hewitii Kalimantan). Lane 14 represents negative control and M2
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
brachyphyllus; 2: A. angulatus; 3: A. borneensis; 4: A. ranchanensis; 5:
A. pendulus; 6: A. hewitii; 7: A. julaihii; 8: A. costatus; 9: A.
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:
A. pendulus; 6: A. hewitii; 7: A. julaihii; 8: A. costatus; 9: A.
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
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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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