Faculty of Resource Science and Technology

GENETIC ANALYSES OF SEA URCHIN IN MALAYSIAN BORNEO

Nursyuhaida Md Shahid

Master of Science

(Aquatic Science)

2013

GENETIC ANALYSES OF SEA URCHIN IN MALAYSIAN BORNEO

NURSYUHAIDA MD SHAHID

A thesis submitted

in fulfillment of the requirements for the degree of

Master of Science

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2013

i

DECLARATION

No portion of the work referred to in this dissertation has been submitted in support of an

application for another degree qualification of any other university or institution of higher

learning.

___________________________

NURSYUHAIDA MD SHAHID

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ii

ACKNOWLEDGEMENTS

First and foremost, I would like to thank God for giving me strength, peace of mind,

constant spirit and the blessings that had kept me working continuously throughout this project.

Sample collections have been partly funded by Ministry of Higher Education research

grant FRGS/07(02)/759/2010(45) awarded to Dr. Siti Akmar Khadijah Ab Rahim. Appreciation

goes to Sarawak Forestry Cooperation for the permission to conduct this study and collection

samples at Satang Island, NCCD.907.4.4(Jld.7)-62 and Park Permit N0.15/2012.

I would like to express my sincere gratitude and appreciation to my supervisor, Dr.

Ruhana Hassan, for her kind help, dedicated guidance, encouragement and moral support which

had made this interesting project starts and ends as planned. I would like to express gratitude to

my co-supervisor, Dr. Siti Akmar Khadijah Ab Rahim for her advice and useful discussions and

also to Mr. Raymie Nurhassan for all his kind help during sample collection. My sincere

appreciation goes to Dr. Ramlah Zainudin for her guidance and valuable advice on some

difficulties that I had encountered during data analysis. I would like to extend my gratitude to

Noor Syafiq Ishak, my beloved friends Nur Najiha Ahmad, Farah Adibah Esa, Siti Ratna

Mustafa, Nor Yasmin Kassim, Nurhartini Kamalia binti Yahya, Liyana Ismail and my lab mates

Mohd Khairulazman Sulaiman and Mohd Izwan Zulaini Abd Ghani for their kind assistance,

companionship and support that had helped me throughout this project.

Last but not least, all my love and greatest appreciation goes to my beloved father, Mr.

Md Shahid Ismail and my mother, Mrs. Salmi Marzuki for their love, continuous encouragement

and support in every aspect. Thank you.

iii

CONFERENCE PROCEEDING/ SEMINARS

Nursyuhaida Md Shahid & Ruhana Hassan. 2012. Relationships of Diadema setosum originated

from Satang Island, Sarawak based on PCR-RAPD profile and COI gene sequence analysis.

Extended abstract accepted for oral presentation at the 12th Symposium of the Malaysian Society

of Applied Biology 2012, Universiti Malaysia Terengganu , Kuala Terengganu, Terengganu. 1-3

June 2012.

Nursyuhaida Md Shahid & Ruhana Hassan. 2012. Relationships of Diadema setosum originated

from Satang Island, Sarawak based on PCR-RAPD profile, 16S rRNA gene and COI gene

sequence analysis. Proceeding of Universiti Malaysia Terengganu 12th Symposium of the

Malaysian Society of Applied Biology 2012, Kuala Terengganu, Terengganu. 1-3 June 2012.(In

press).

Nursyuhaida Md Shahid & Ruhana Hassan. 2012. Relationships among Sea urchin Diadema

setosum based on 16S rRNA gene sequence analysis. Extended abstract accepted for poster

presentation at the 4th Regional Conference on the Natural Resources in the Tropics, Universiti

Malaysia Sarawak , Kuching, Sarawak. 19-20 September 2012.

Nursyuhaida Md Shahid & Ruhana Hassan. 2012. Relationships among Sea urchin Diadema

setosum based on 16S rRNA gene sequence analysis. Poster presented at the 4th Regional

Conference on the Natural Resources in the Tropics, Universiti Malaysia Sarawak , Kuching,

Sarawak. 19-20 September 2012.

Nursyuhaida Md Shahid & Ruhana Hassan. 2012. Genetic diversity among Sea urchin Diadema

setosum from Satang Island, Sarawak and Mantanani Island, Sabah based on 16S rRNA gene

analysis. Paper accepted for oral presentation at the Aquatic Colloqium 2012, Universiti

Malaysia Sarawak , Kuching, Sarawak. 26 September 2012.

iv

ABSTRACT

Echinoidea is the well known class in Phylum Echinodermata, with over 900 extant species.

They play significant roles in maintaining the health of coral reef ecosystems and the associated

organisms. Genus Diadema (Echinoidea: Diadematidae) have been reported to be the most

widespread and ecologically important shallow water genera of tropical sea urchins. Distinctions

in their distributions and ecology among species of genus Diadema, are complicated to elucidate

due to complexity in making reliable identifications. Genus Diadema had involved in many

debates, particularly on the specific status of the sympatric species, Diadema setosum and

Diadema savignyi. Therefore, phylogenetic relationships of Genus Diadema in Malaysian

Borneo have been conducted using 16S rRNA gene analysis. Monophyletic clade of genus

Diadema with respect to the outgroup was revealed with significant bootstrap support. Two

monophyletic clades with deep split separating D. setosum (Clade I) and D. savignyi (Clade II)

were observed, with significant bootstrap support and high genetic variation recorded (9.85%).

The results suggested that D. setosum and D. savignyi are two distinct entities based on

phylogenetic analysis and unique morphological characteristics observed. The results also

suggest that D. setosum and D. savignyi are sympatric species based on their distribution and

overlapping ranges in Malaysian Borneo. Genetic structure and extent of variation among D.

setosum from Malaysian Borneo (Satang Island, Sarawak; Mantanani Island, Sabah; Kudat,

Sabah and Larapan Tengah, Sabah) were later conducted using 16S rRNA gene analysis to test

whether the population of D. setosum in Malaysian Borneo is panmictic or structured. Lack of a

significant relationship between net nucleotide divergences (Da) and geographic distance was

revealed indicates no associations between genetic and geography of D. setosum in Malaysian

v

Borneo. No distinct geographical clades could be observed among D. setosum populations and

minimum-spanning network (MSN) revealed sharing of haplotypes among populations. High

number of migrants per generation (Nm = 2.14 to 17.20) with low nucleotide subdivision (Nst =

0.03 to 0.19) and estimate of population subdivision (FST = 0.03 to 0.19) were recorded, except

for Mantanani Island population. Mantanani Island population showed low level of gene flow

(Nm = 0.13 to 0.69) and significant genetic differentiation ΦST values suggesting slight genetic

isolation and subdivision from Larapan Tengah and Kudat populations. Gene flow occurrence

among D. setosum populations suggested population expansion and that D. setosum population

in Malaysian Borneo is panmictic. D. setosum as a highly dispersive species that possessed long

planktonic stage might explain the gene flow occurrence among populations which further

facilitated by historical events, prevailing surface current and impact of ballast water. Whereas,

slight genetic isolation of Mantanani Island population occurred due to dispersal restriction of D.

setosum larvae influenced by the historical events on the isolation of South China Sea from Sulu-

Celebes Sea, water flow restrictions in the connecting straits, prevailing South China Sea

Southwest monsoon and the formation of gyres off the Borneo coast. All findings from this study

are based on limited samples thus future studies should involve more specimens collected from a

wider geographical area in order to further understand the genetic structure and phylogenetic

relationships of sea urchin in Malaysian Borneo.

Keywords: D. setosum, D. savignyi, 16S rRNA gene, sympatric species, population expansion.

vi

Analisis Genetik Landak Laut di Kepulauan Borneo, Malaysia

ABSTRAK

Kelas Echinoids merangkumi lebih 900 spesies landak laut dan ia adalah kelas yang paling

dikenali di dalam Filum Echinodermata. Landak laut memainkan peranan penting dalam

mengekalkan kestabilan ekosistem terumbu karang serta organisma yang berkaitan dengannya.

Genus Diadema (Echinoidea: Diadematidae) adalah landak laut tropika yang paling meluas

taburan geografinya serta memainkan peranan penting di dalam genera landak laut air cetek,

namun perbezaan di antara spesies di dalam genera ini amat rumit untuk dikenalpasti. Genus

Diadema banyak terlibat dalam perdebatan berkenaan status spesifik Diadema setosum dan

Diadema savignyi. Oleh itu, hubungan filogenetik Genus Diadema di Kepulauan Borneo,

Malaysia telah dikaji dengan menggunakan analisis gen 16S rRNA. Genus Diadema membentuk

kumpulan monofiletik terpisah daripada Echinothrix calamaris dan Tripneustes gratilla dengan

sokongan bootstrap yang tinggi. Genus Diadema kemudiannya membentuk dua kumpulan

monofiletik mengasingkan D. setosum dan D. savignyi ke dalam kumpulan berbeza, dengan

sokongan bootstrap yang tinggi serta variasi genetik sebanyak 9.85%. Kajian ini menunjukkan

D. setosum dan D. savignyi adalah dua spesies landak laut yang berbeza dan mempunyai

karakter morfologi yang unik. Kajian ini juga menunjukkan dua spesies landak laut ini adalah

spesies simpatrik berdasarkan taburan geografi yang bertindih di Kepulauan Borneo, Malaysia.

Struktur populasi dan variasi genetik di antara D. setosum di Kepulauan Borneo, Malaysia

(Pulau Satang, Sarawak; Pulau Mantanani, Sabah; Kudat, Sabah and Larapan Tengah, Sabah)

telah dikaji menggunakan analisis gen 16S rRNA. Hasil kajian menunjukkan tiada hubungan

signifikan di antara genetik dengan geografi dan tiada perbezaan kumpulan mengikut geografi

vii

diilustrasi di dalam pokok filogenetik. Populasi D. setosum di Kepulauan Borneo, Malaysia

menunjukkan perkongsian haplotaip di antara populasi, jumlah migran per generasi yang tinggi

(Nm = 2.14 hingga 17.20), pembahagian nukleotida dan pembahagian populasi yang rendah (Nst

dan FST = 0.03 hingga 0.19), kecuali untuk populasi di Pulau Mantanani. Populasi di Pulau

Mantanani menunjukkan jumlah aliran gen yang rendah (Nm = 0.13 to 0.69) serta perbezaan

genetik yang signifikan membuktikan berlakunya sedikit pengasingan genetik di antara populasi

ini dengan Larapan Tengah dan Kudat. Kajian ini menunjukkan bahawa hubungan genetik yang

rapat di antara populasi D. setosum di Kepulauan Borneo, Malaysia berlaku disebabkan

kejayaan penyebaran larva di antara populasi, dengan bantuan morfologi larva dan peringkat

plaktonik yang panjang, perubahan geologi dan aspek hidrografi dan juga impak perlepasan air

balast dari kapal ke dalam laut. Sedikit perbezaan genetik di antara Pulau Mantanani dan

populasi lain dipengaruhi oleh faktor seperti perubahan geologi, sekatan aliran air di antara

laut, monsun barat daya di Laut China Selatan dan pembentukan gyres di luar pantai Borneo.

Kesemua penemuan dalam kajian ini hanya berdasarkan jumlah sampel yang terhad, oleh

kerana itu, kajian pada masa akan datang harus melibatkan jumlah sampel yang lebih besar dan

meliputi kawasan geografi yang lebih luas untuk lebih memahami struktur genetik dan hubungan

filogenetik di antara landak laut di Kepulauan Borneo, Malaysia.

Kata kunci: D. setosum, D. savignyi, gen 16S rRNA, spesies simpatrik, perkembangan populasi

viii

TABLE OF CONTENTS

Page

DECLARATION i

ACKNOWLEDGEMENT ii

CONFERENCE PROCEEDINGS / SEMINARS iii

ABSTRACT iv

ABSTRAK vi

TABLE OF CONTENT viii

LIST OF TABLES xi

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xvi

LIST OF APPENDICES xvii

Chapter 1 : Introduction 1

1.0 Introduction 1

1.1 Objectives of study 4

1.2 Layout of Thesis 5

Chapter 2 : Literature Review 6

2.1 The Echinodermata and Genus Diadema 6

2.2 Morphological descriptions of all sea urchin species involved in this study 9

2.2.1 Diadema setosum Leske, 1778 10

ix

2.2.2 Diadema savignyi Michelin, 1845 11

2.2.3 Echinothrix calamaris Pallas, 1774 13

2.2.4 Tripneustes gratilla Linnaeus, 1758 14

2.3 Molecular studies on The Echinoids 16

Chapter 3 : Phylogenetic Analysis of Genus Diadema in Malaysian Borneo 20

3.1 Introduction 20

3.2 Materials and Methods 24

3.2.1 Sample Collection and Preservation 24

3.2.2 Total Genomic DNA Extraction using Modified CTAB protocol (Doyle &

Doyle, 1987)

26

3.2.3 Amplification of 16S rRNA gene sequences 27

3.2.4 DNA Purification and Sequencing Analysis 27

3.2.5 Data analysis 28

3.3 Result and Discussion 29

3.3.1 Total Genomic DNA Extraction and Optical Density Reading 29

3.3.2 Amplification of 16S rRNA gene 32

3.3.3 Sequencing Analysis 34

3.3.4 Genetic Divergences Analysis 36

3.3.5 Phylogenetic Analysis 38

3.4 Conclusion and Recommendations 44

x

Chapter 4 : Genetic Structure of D. setosum Populations in Malaysian Borneo 45

4.1 Introduction 45

4.2 Materials and Method 48

4.2.1 Sample Collection and Preservation 48

4.2.2 Data Analysis 50

4.3 Result and Discussion 52

4.3.1 Total Genomic DNA Extraction and Optical Density Reading 52

4.3.2 Amplification of 16S rRNA gene 59

4.3.3 Sequencing Analysis 61

4.3.4 Genetic Divergences Analysis 63

4.3.5 Population Genetic Analysis 65

4.4 Conclusion and Recommendation 84

Chapter 5 : Conclusion and Recommendation 86

REFERENCES 89

APPENDICES 101

xi

LIST OF TABLES

Caption Page

Table 2.1 Classification of Genus Diadema 8

Table 2.2 Classification of sea urchin species involved in this study 9

Table 3.1 Samples of D. setosum, D. savignyi, E. calamaris and T. gratilla analyzed

for DNA (16S rRNA) gene sequences variation with locality, GPS

reading and field voucher

25

Table 3.2 Optical density reading of Genomic DNA extraction products of Sea

urchin species involved in this study.

31

Table 3.3 BLAST results of all 16S rRNA gene sequences obtained in this

study.

35

Table 3.4 Genetic distance (%) based on 16S rRNA gene sequences analysis in this

study.

37

Table 4.1 Optical density reading of genomic DNA extraction products from Satang

Island, Sarawak

55

Table 4.2 Optical density reading of D. setosum genomic DNA extraction products

from Sabah

58

Table 4.3 Summary of BLAST results of D. setosum 16S rRNA gene sequences

obtained in this study

62

Table 4.4 Genetic Distance in percentage for 16S rRNA gene sequences obtained in

this study.

64

Table 4.5 Samples of D. setosum analyzed for DNA (16S rRNA) gene sequences

variation with locality, GPS reading, field voucher and identified

haplotype

66

Table 4.6 Measures of haplotypes and nucleotide diversity (π) within populations of

D. setosum analyzed by location. N: number of individuals.

67

Table 4.7 Measures of nucleotide diversity (π) and net nucleotide divergence among

populations of D. setosum analyzed by location.

68

xii

Caption Page

Table 4.8 Summary statistics of 16S rRNA gene sequence variation in four different

populations of D. setosum in Malaysian Borneo

73

Table 4.9 Measure of geographical population differentiation in D. setosum based

on an analysis of molecular variance approach with 16S rRNA gene

information.

75

Table 4.10 Genetic differentiation matrix of population calculated by ΦST. The p

values are shown in parenthesis.

77

Table 4.11 Measure of nucleotide subdivision (Nst), population subdivision (FST) and

gene flow (Nm) among populations of D. setosum in Malaysian Borneo

77

xiii

LIST OF FIGURES

Caption Page

Figure

2.1

Photograph showing morphological characteristics of D. setosum, the long

spine black sea urchin. (Photo credited to Mr. Raymie Nurhassan and Dr. Siti

Akmar Khadijah Ab Rahim)

10

Figure

2.2

Photograph showing morphological characteristics of D. savignyi, the black

sea urchin. (Photo credited to Mr. Raymie Nurhassan and Dr. Siti Akmar

Khadijah Ab Rahim)

12

Figure

2.3

Photograph showing morphological characteristics of E. calamaris, the

banded sea urchin. (Photo credited to Mr. Norhakimi Mohamad and Dr. Siti

Akmar Khadijah Ab Rahim)

13

Figure

2.4

Photograph showing morphological characteristics of T. gratilla, the

collector urchin. (Photo credited to Mr. Raymie Nurhassan and Dr. Siti

Akmar Khadijah Ab Rahim)

15

Figure

3.1

Map of Malaysian Borneo showing study locations and sample sizes of the

sea urchins collected for this study. n, sample size.

24

Figure

3.2

Gel photograph showing genomic DNA extraction products, representatives

of each Sea urchin species involved in this study. Total genomic DNA

extraction was conducted using modified CTAB protocol (Doyle and Doyle,

1987) and the extraction products were run on 1% Agarose gel in 1x TBE

buffer at 90volt for 1 hour.

29

Figure

3.3

Gel photograph showing all PCR products of D. savignyi, E. calamaris and

two representatives of T. gratilla samples. PCR products were run on 1%

Agarose gel in 1x TBE buffer at 90 volt for 1 hour.

33

Figure

3.4

A maximum Parsimony 50% majority rule consensus tree constructed using

16S rRNA gene sequences of D. setosum, D. savignyi and E. calamaris with

T. gratilla as the outgroup. Bootstrap values correspond to Maximum

Parsimony and Neighbour joining respectively. Tree length is 240 with

consistency index (CI) = 0.9292 and retention index (RI) = 0.9779

40

xiv

Caption Page

Figure

3.5

Bayesian inference of the 50% majority rule consensus tree of 16S rRNA

gene sequences of D. setosum, D. savignyi and E. calamaris with T. gratilla

with T. gratilla as the outgroup. Bootstrap values of maximum likelihood

and Bayesian posterior probabilities (BPPs) are accordingly indicated above

the branch nodes.

41

Figure

4.1

Map of Malaysian Borneo showing locations involved in sample collections

during this study. (Maps are adapted from Googlemap.com)

49

Figure

4.2

Gel photograph showing total genomic DNA extraction products of D.

setosum collected from Malaysian Borneo. Total genomic DNA extraction

was conducted using modified CTAB protocol (Doyle and Doyle, 1987) and

the extraction products were run on 1% Agarose gel in 1xTBE buffer at

90volt for 1 hour.

53

Figure

4.3

Gel photo showing PCR products of 16S rRNA gene of all D. setosum

samples collected from Satang Island, Sarawak. PCR products were run on

1% Agarose gel in 1X TBE buffer at 85 volt for 1 hour.

59

Figure

4.4

Gel photo showing PCR products of 16S rRNA gene of all D. setosum

samples collected from Sabah. PCR products were run on 1% Agarose gel in

1X TBE buffer at 85 volt for 1 hour.

60

Figure

4.5

Scatter plots showing the relationship of geographical distance and

percentage of net nucleotide divergences, Da between populations of D.

setosum. Regression coefficient: y = -0.000114, correlation coefficient = -

0.719102 and regression statistic p =0.961.

67

Figure

4.6

A maximum parsimony 50% majority rule consensus tree constructed of 16S

rRNA gene sequences of Malaysian Borneo D. setosum with E. calamaris

and T. gratilla as the outgroup. Bootstrap values above 50% are indicated

above branch correspond to Maximum Parsimony and Neighbour joining

respectively. Tree length is 191 with consistency index (CI) = 0.9424 and

retention index (RI) = 0.7925. L: bootstrap value less than 50%.

69

xv

Caption Page

Figure

4.7

Bayesian inference of the 50% majority rule consensus tree of 16S rRNA

gene sequences of D. setosum with E. calamaris and T. gratilla as the

outgroup. Bootstrap values of maximum likelihood and Bayesian posterior

probabilities (BPPs) are accordingly indicated above the branch nodes.

70

Figure

4.8

Minimum-spanning network generated by Network 4.6.10 (Bandelt et al.,

1999) illustrating the relationships of D. setosum in Malaysian Borneo. Each

circle represents a haplotype and the diameter scales to haplotype frequency.

Note that yellow circle indicates Mantanani haplotypes, pink circles indicate

Satang haplotypes, green circles indicate Larapan Tengah haplotypes and

Blue circle indicates Kudat haplotypes. Red circle indicates missing

haplotype (not in the sequences understudy or hypothetical haplotypes) and

bold number on the line connecting haplotypes indicate number of

mutational steps.

71

Figure

4.9

Mismatch distribution of D. setosum samples from Malaysian Borneo. The

green lines represent the observed and orange lines represent the expected

distributions for sudden expansion model.

74

xvi

LIST OF ABBREVIATIONS

Abbreviation Full term

bp Base pair

CTAB Cetyltrimethyl ammonium bromide

ddH2O Deionized distilled water

dNTP Deoxy-nucleotide triphosphates

DNA Deoxyribonucleic Acid

EDTA EthyleneDiamine Tetra-Acetic Acid

g Gram

L Liter

MgCl2 Magnesium Chloride

µl Microliter

µm Micrometer

µM Micromolar

ml Mililiter

mM Milimolar

mg Milligram

min Minute

M Molar

ng Nanogram

nm nanometer

rRNA Ribosomal Ribonucleic Acid

rpm Rotation per minute

NaCl Sodium Chloride

TBE Tris-Bromide-ethylenediaminetetraacetic

UV Ultra violet

U Unit

V Volt

xvii

LIST OF APPENDICES

Caption Page

APPENDIX A List of aligned sequence of 16S rRNA gene for Genus Diadema 101

APPENDIX B List of aligned sequence of 16S rRNA gene for all urchin species in

Chapter 3

103

APPENDIX C Genetic distance (%) based on 16S rRNA gene sequences analysis of

all sea urchin species involved in Chapter 3

105

APPENDIX D List of aligned sequence of 16S rRNA gene for Diadema setosum 106

1

1.0 Introduction

The Echinoids are recognized to be the well known class among the Echinodermata with

over 900 extant species (Littlewood & Smith, 1995) which play significant roles in maintaining

the health of coral reef ecosystems, thus providing certain margin of protection towards reef

fishes and other diverse associated organisms. The feeding habits of the Echinoids had exerted a

major role in shaping the benthic communities particularly in the temperate rocky bottom area

(Sala et al., 1998).

Sea urchins are widely distributed across Indo-Pacific region, Austria, Japan and the

Mediterranean Sea while in Malaysia region, sea urchins are abundance in every coastal areas

but mainly confine in Sabah water and Tioman Island (Markos et al., n.d). Most of the Echinoids

are not listed as endangered as they can be found abundantly throughout the globe. According to

International Union for Conservation of Nature (IUCN, 2012), the Echinoids have not been

assessed for the IUCN red list for endangered species, only one species namely Echinus

esculentus Linnaeus (1758), the European edible sea urchin have been classified as near

threatened.

Diadema setosum (Leske, 1778) or known as the long spine black sea urchin is extensively

distributed in Malaysia and commonly found in coral reef ecosystem. This species is believed to

be the oldest known species of the genus Diadema which formed basal branch in the cladogram

analysis of the Echinoids (Lessios et al., 2001) and morphologically characterized with

hemicyclic apical system, green bands of iridophores down the midlines of interambulacra with

orange anal ring and blue spots on the genital plates (Leske, 1778).

Chapter 1: Introduction

2

Furthermore, D. setosum play a major role in the benthic ecology and the bio-erosion of the

coral reef as it has been recognized to be one of the highest contributors to bio-erosion and it

shows the highest herbivory rates among sea urchins species (Carrerio-Silva & McClanahan,

2001). The history of mass mortality of sea urchin that occurred around the 1980s (Lessios,

1995) showed that sea urchin population reduction or the excessive population number may pose

threat toward ecological balance especially within the coral reef ecosystems.

Research on evolutionary path, phylogenetic relationship and speciation of echinoids have

been particularly challenging due to high dispersal potential conferred by the planktonic larvae

and also difficulties in finding the barriers to gene flow in the wide ocean (Palumbi, 1994).

Genetic variation of a particular population and their evolutionary path are affected by gene flow

or the exchange of alleles (Hardy and Vekemans, 1999). For benthic sessile organism like D.

setosum, the planktonic larval stage is a crucial stage for connectivity and gene flow between

populations, considering that passive drift with ocean currents may assist their dispersal potential

over large geographical area (Cowen and Sponaugle, 2009).

Molecular studies have advanced understanding on marine invertebrates biology and their

evolutionary pattern in which the application of this indirect research methods provide better

insights into their taxonomy, population structure and species management (Smith and Wayne,

1996). There are many molecular studies that have been conducted on sea urchin using various

mtDNA genes for example Littlewood and Smith (1995), Janies (2001), Lessios (2001), Sponer

and Roy (2002), Stockley et al. (2005), Cowen and Sponaugle (2009) and Pérez-Portela et al.

(2010). However, there is still lack of studies conducted using 16S rRNA gene and up to January

2013 no molecular data on sea urchin species from Malaysia can be seen in GenBank.

3

Therefore, this study is designed to assess phylogenetic relationships of Genus Diadema in

Malaysian Borneo and to observe genetic structure and extent of variation between and within D.

setosum populations using 16S rRNA gene analysis. The genetic structure of D. setosum in

Malaysian Borneo is assessed to determine whether Malaysian Borneo has panmictic or

structured sea urchin population. This study also challenges the knowledge on either the seas

indeed contain present and past barriers to gene flow because the seas are after all connected and

planktonic larvae have high dispersal capabilities and able to delay metamorphosis for a long

period of time. Thus, this information is valuable for sustainable management of this species,

associated organisms and their habitats.

4

1.1 Objectives of study

The objectives of this study are;

(i) To re-construct molecular phylogeny of Genus Diadema in Malaysian Borneo based on 16S

rRNA gene information.

(ii) To determine phylogenetic relationship among D. setosum in Malaysian Borneo (Satang

Island, Sarawak; Mantanani Island, Sabah; Kudat, Sabah and Larapan Tengah, Sabah) and

extent of their genetic variation.

(iii) To determine whether Malaysian Borneo has panmictic or structured sea urchin population

and to identify factors that may influence the dispersal abilities of D. setosum larvae.

5

1.2 Layout of the Thesis

This thesis comprises five chapters. Chapter one is the general introduction on the

Echinodermata and description on the overall study whereas, chapter two comprises literature

review on the Echinodermata, classification of Genus Diadema, morphological

characteristics of sea urchin species that involved in this study and molecular studies

conducted on the Echinoderm.

In chapter three, 16S rRNA gene information was used to reconstruct the phylogenetic

relationship of Genus Diadema in Malaysian Borneo and to test the genetic variation and the

mode of speciation between D. setosum and D. savignyi in Malaysian Borneo.

The phylogenetic relationships, genetic structure and genetic variation among D.

setosum samples from four locations in Malaysian Borneo (Satang Island, Sarawak;

Mantanani Island, Sabah; Kudat, Sabah and Larapan Tengah, Sabah) based on 16S rRNA

gene analysis is discussed in chapter four. Moreover, factors that influence the dispersal

capabilities of D. setosum larvae for instance larval morphology and planktonic stage

duration, historical associations between seas, hydrographical aspects and impact of ballast

water are also highlighted in this chapter.

Finally, chapter six provides the conclusions that can be drawn from the findings of

each chapter and also provide recommendation for future studies.