POPULATION GENETIC STRUCTURE of MALAYAN TAPIR (Tapirus Indicus Desmarest) in PENINSULAR MALAYSIA LIM QI LUAN FS 2019 55

POPULATION GENETIC STRUCTURE OF MALAYAN TAPIR (Tapirus indicus Desmarest) IN PENINSULAR MALAYSIA

LIM QI LUAN

FS 2019 55 POPULATION GENETIC STRUCTURE OF MALAYAN TAPIR (Tapirus indicus Desmarest) IN PENINSULAR MALAYSIA UPM

LIM QI LUAN

COPYRIGHTThesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of © Science March 2019 COPYRIGHT

All material contained within the thesis, including without limitation to text, logos, icons, photographs and all other artwork, is copyright material of Universiti Putra Malaysia unless otherwise stated. Use may be made of any material contained within the thesis for non-commercial purposes from the copyright holder. Commercial use of the material may only be made with the express, prior, written permission of Universiti Putra Malaysia.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

POPULATION GENETIC STRUCTURE OF MALAYAN TAPIR (Tapirus indicus Desmarest) IN PENINSULAR MALAYSIA

LIM QI LUAN

March 2019 UPM

Chair: Geetha Annavi, PhD Faculty: Science

The Malayan tapir (Tapirus indicus Desmarest) is an endangered fauna listed in the International Union for Conservation of Nature (IUCN) Red List with estimated population size at less than 2,000 individuals in Peninsular Malaysia. Despite the existing conservation programme and ecological information about this species, the population genetic structure of the Malayan tapir in Peninsular Malaysia is still not well-known, largely due to a lack of available genetic markers. The lack of such information may impede the on-going efforts for its conservation and management. The works presented here aimed to develop genetic markers for the investigation of population genetic structure of the Malayan tapir in Peninsular Malaysia. Forty-one microsatellite markers comprising of seven random amplified microsatellite (RAM)-isolated and 34 cross-amplification microsatellite markers, obtained from literature and National Center for Biotechnology Information (NCBI) database, were screened with polymerase chain reaction (PCR), sequencing and fragment analysis in 67 Malayan tapirs. Eight polymorphic markers were successfully developed and used in the population genetic structure analysis. Using K-means clustering algorithm, five clusters were inferred among the wild samples (N = 57), which showed a complex population structure probably comprising multiple continuous populations that also experiencing considerably restricted gene flow due to isolation by geographical barriers especially mountain ranges. Mitochondrial control region sequences in Peninsular Malaysia samples (N = COPYRIGHT44; including two samples from Singapore Zoo) revealed two clades that might be established during the late Pleistocene. One of the clades was exclusive in Peninsular Malaysia samples in comparison with the Thailand samples from a previous study. However, the geographical distribution of the clades did not show a clear population structure. A total of 12 novel haplotypes were detected. © Both the markers suggested low to moderate genetic diversity in the Malayan tapir studied. In addition, a universal sex-typing method based on the sex-

determining region Y and zinc finger gene (as positive control) was tested. A preliminary assessment of sex ratio was conducted using the data extracted from the tapir datasheets obtained from the Department of Wildlife and National Parks, Sungai Dusun Wildlife Conservation Centre and Zoo Negara; and aided with the developed sex-typing marker for those biological samples with unknown sex. Overall, there was no significant bias towards either sex. Nevertheless, in the wild-born tapirs, the sex ratio seemed to favour females and the opposite was observed in the captive-born tapirs. From 2004 to 2015, there seemed to be an increase in the male proportion but no extreme ratio was found. Combined with microsatellite data, there was no sex-biased dispersal detected in a spatial autocorrelation analysis that might shape the population structure of the Malayan tapir observed. A major limitation in all these studies was the sampling bias where, across Peninsular Malaysia, more samples were sampled from the Selangor-Negeri Sembilan-Pahang regions and only a few were representatives of the populations from the north forest complexes. UPM

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

STRUCKTUR POPULASI GENETIK TAPIR MALAYA (Tapirus indicus Desmarest) DALAM SEMENANJUNG MALAYSIA

LIM QI LUAN

Mac 2019 UPM

Pengerusi: Geetha Annavi, PhD Fakulti: Sains

Tapir Malaya (Tapirus indicus Desmarest) adalah sejenis haiwan terancam yang tersenarai dalam Senarai Merah Kesatuan Antarabangsa untuk Pemuliharaan Alam Sekitar (IUCN) dengan saiz populasi dianggar kurang daripada 2,000 ekor di Semenanjung Malaysia. Walaupun terdapat program pemuliharaan dan maklumat ekologi tentang spesies ini, struktur genetik populasi Tapir Malaya di Semenanjung Malaysia masih belum dikaji sepenuhnya, sebahagian besarnya disebabkan oleh kekurangan penanda genetik yang sedia ada. Kekurangan maklumat sedemikian boleh menghalang usaha berterusan untuk pemuliharaan dan pengurusannya. Kajian ini bertujuan untuk menguji penanda-penanda genetik dan seterusnya memakainya untuk mengkaji struktur genetik populasi Tapir Malaya di Semenanjung Malaysia. Empat puluh satu penanda mikrosatelit sedia ada di literatur dan data National Center for Biotechnology Information (NCBI) yang terdiri daripada tujuh penanda RAM (Random Amplified Microsatellite) dan 34 penanda mikrosatelit yang diperoleh daripada spesies tapir lain telah diuji dengan reaksi berantai polimerase (PCR), penjujukan dan analisis genotip pada 67 Tapir Malaya. Lapan penanda polimorfik berjaya dikembangkan dan digunakan dalam analisis struktur genetik populasi tapir. Kaedah gugusan K-means telah mencadang kewujudan lima kluster genetik Tapir Malaya di antara populasi tapir liar (N = 57). Taburan kluster-kluster tersebut di Semenanjung Malaysia turut menunjukkan struktur populasi yang kompleks dan berkemungkinan COPYRIGHTterdiri daripada beberapa populasi berterusan yang juga mengalami aliran gen terhad mungkin disebabkan oleh faktor pengasingan geografi seperti banjaran gunung. Analisis terhadap jujukan nukleotida control region mitokondria dalam sampel Semenanjung Malaysia (N = 44 termasuk dua sampel daripada Zoo Singapura) telah mencadang kewujudan dua klad pada zaman Pleistosen © Akhir. Salah satu daripada klad tersebut adalah eksklusif dalam sampel Semenanjung Malaysia berbanding dengan sampel dari Thailand dalam kajian

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terdahulu. Walau bagaimanapun, pertaburan geografi klad tidak menunjukkan struktur populasi yang jelas. Sejumlah 12 haplotip baharu telah diperoleh. Kedua-dua penanda genetik mencadangkan kepelbagaian genetik yang rendah atau sederhana dalam populasi Tapir Malaya yang dikaji. Di samping itu, kaedah mengenal pasti jantina berdasarkan gen penentuan seks pada kromosom Y dan gen “zinc finger” (sebagai kawalan positif) telah diuji. Penilaian awal nisbah jantina dilakukan menggunakan data yang diperoleh daripada Jabatan Hidupan Liar dan Taman Negara, Pusat Konservasi Hidupan Liar Sungai Dusun dan Zoo Negara, dan dibantu dengan kaedah mengenal pasti jantina tersebut untuk sampel biologi yang tidak diketahui jantina. Secara keseluruhannya, nisbah tapir jantan dan betina tidak berbeza secara signifikan. Walau bagaimanapun, dalam populasi tapir liar, nisbah jantina nampaknya memihak kepada betina dan keadaan sebaliknya didapati dalam populasi tapir yang dilahirkan dalam kurungan. Dari tahun 2004 hingga 2015, peningkatan kadar jantan didapati walaupun perbezaan nisbah jantan dan betina adalah tidak ketara. Apabila data mikrosatelit diuji mengikut kategori seks, tidak ada perbezaan didapati dalam taburan genetik antara tapir jantan dan betinaUPM yang mungkin membentuk struktur populasi Tapir Malaya. Satu kelemahan utama dalam semua kajian ini adalah pensampelan yang kurang merata di rantau Semenanjung Malaysia. Iaitu lebih banyak sampel telah diperoleh dari kawasan Selangor-Negeri Sembilan-Pahang tetapi hanya segelintir sampel diperoleh dari kawasan utara Semenanjung Malaysia yang dirangkumi kompleks hutan yang luas.

ACKNOWLEDGEMENTS

Along this journey, I have gained support from so many people who are big- hearted and helpful in so many ways that without them, I might not be able to complete this master thesis to fulfil the graduation requirement of a Master course with research from University Putra Malaysia (UPM), and to step forward for another chapter in life into the science of wildlife conservation.

First and foremost, I would like to give thanks to my supervisory committee: Dr Geetha Annavi, Dr Christina Yong Seok Yien, Dr Ng Wei Lun, and Prof. Dr Ahmad Ismail for their teachings and guidance in conducting research and writing scientific papers. Thanks to my family who are always there to support me financially and emotionally.

UPM I also want to thank Department of Wildlife and National Parks (PERHILITAN) personnel e.g. Dr Jeffrine Japning Rovie-Ryan and Ms Norsyamimi Rosli for their support and advice during my time at National Wildlife Forensic Laboratory (NWFL), PERHILITAN, where I conducted part of my research works. Regarding the samples, all the sampling procedures were approved by the Institutional Animal Care and Use Committee, UPM (ethical approval ref.: UPM/IACUC/AUP-R033/2016). I would like to thank PERHILITAN for their permission and provision of most of the samples used in this study (permit ref. NRE 600-2/2/21 JILID 2(42)). Thanks to Mdm Noor Azleen binti Mohd Kulaimi (PERHILITAN) for aiding in the collection of tapir samples from the NWFL. Many thanks to Dr Geetha Annavi and her students who were involved in collecting the Malayan tapir samples from Zoo Negara (also known as the National Zoo of Malaysia) and Sungai Dusun Wildlife Conservation Centre (Sungai Dusun WCC). I would like to thank Dr Donny Yawah (PERHILITAN), Dr Mat Naim Bin Haji Ramli (Zoo Negara) and Dr Kavitha Jayaseelan (Zoo Negara) for their assistance in collecting blood samples at the Sungai Dusun WCC and Zoo Negara respectively.

Financially, this project was supported by Ministry of Education’s Fundamental Research Grant Scheme (FRGS); project code: FRGS/1/2014/SG03/UPM/02/8); project vote no: 5524552, and Malaysian Nature Society Tapir Project Grant awarded to Dr Geetha Annavi. I also want to thank UPM for the Graduate Research Fellowship Fund and Ministry of Education for the MyBrainSc Scholarship that has supported me financially throughout the first two years of my master study. Additionally, I would like to COPYRIGHTtake this opportunity to thank various institutes on the materials used in this thesis. Maps containing geography layers used the following services: SRTM 1ARC, a product of USGS; and ASTER GDEM, a product of METI and NASA. The map was produced using Copernicus data and information funded by the European Union - EU-DEM layers. ©

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COPYRIGHT © This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows:

Geetha Annavi, PhD Senior Lecturer Faculty of Science Universiti Putra Malaysia (Chairman)

Christina Yong Seok Yien, PhD Senior Leturer Faculty of Science Universiti Putra Malaysia (Member)

Ahmad Ismail, PhD UPM Professor Faculty of Science Universiti Putra Malaysia (Member)

Ng Wei Lun, PhD Assistant Professor China-ASEAN College of Marine Sciences Xiamen University of Malaysia (Member)

______ROBIAH BINTI YUNUS, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

vii Declaration by graduate student

I hereby confirm that: • this thesis is my original work; • quotations, illustrations and citations have been duly referenced; • this thesis has not been submitted previously or concurrently for any other degree at any other institutions; • intellectual property from the thesis and copyright of thesis are fully-owned by Universiti Putra Malaysia, as according to the Universiti Putra Malaysia (Research) Rules 2012; • written permission must be obtained from the supervisor and the office of Deputy Vice-Chancellor (Research and Innovation) before thesis is published (in the form of written, printed or in electronic form) including books, journals, modules, proceedings, popular writings, seminar papers, manuscripts, posters, reports, lecture notes, learning modules orUPM any other materials as stated in the Universiti Putra Malaysia (Research) Rules 2012. The thesis has undergone plagiarism detection software. • there is no plagiarism or data falsification/fabrication in the thesis, and scholarly integrity is upheld as according to the Universiti Putra Malaysia (Graduate Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia (Research) Rules 2012. The thesis has undergone plagiarism detection software.

Signature: ______Date: ______

Name and Matric No.: ______

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Declaration by Members of Supervisory Committee

This is to confirm that: • The research conducted and the writing of this thesis was under our supervision; • Supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature: ______Signature: ______Name of Name of Chairman of Member of UPM Supervisory Supervisory Committee: Committee: ______

Signature: ______Signature: ______Name of Name of Member of Member of Supervisory Supervisory Committee: Committee: ______

TABLE OF CONTENTS

Page ABSTRACT i ABSTRAK iii ACKNOWLEDGEMENTS v APPROVAL vi DECLARATION viii LIST OF TABLES xiv LIST OF FIGURES xviii LIST OF APPENDICES xxiv LIST OF ABBREVIATIONS xxv

CHAPTER 1 INTRODUCTION 1 UPM 2 LITERATURE REVIEW 4 2.1 Introduction to the Family Tapiridae 4 2.2 Malayan Tapir (Tapirus indicus Desmarest) 5 2.2.1 Nomenclature and Subspecies 5 2.2.2 Morphological Characteristics 6 2.2.3 Reproduction and Longevity 7 2.2.4 Nocturnal Behaviour and 7 Vocal Communication 2.2.5 Population Density, Distribution 8 and Range of Habitat 2.2.6 Foraging Pattern and Ecological Role 12 2.2.7 IUCN Status of Malayan Tapir and 13 Threats Faced by the Fauna 2.2.8 Malayan Tapir Conservation 13 Programmes 2.3 What is a Genetic Marker and What Can It Do? 15 2.3.1 Microsatellite as a Genetic Marker 16 2.3.2 Mitochondrial DNA Control Region 18 as a Genetic Marker 2.3.3 Sex Markers as Genetic Tools for 20 Sex-Identification 2.4 Principles of Conservation Genetics 22 2.4.1 What is Population Genetic 23 Structure? 2.4.2 Why Genetic Diversity Matter 23 in Species Conservation? COPYRIGHT2.5 Importance of Conservation Genetics for 24 Malaysian Fauna 2.5.1 Conservation Genetics as a Solution to 25 Conservation Questions 2.5.2 Conservation Genetics in the Case of 26 © Malayan Tapir 2.6 Previous Genetic Studies on Tapirs 26

3 DEVELOPMENT OF MICROSATELLITE MARKERS 28 AND SUBSEQUENT APPLICATION FOR INVESTIGATING GENETIC DIVERSITY AND POPULATION GENETIC STRUCTURE OF THE MALAYAN TAPIR IN PENINSULAR MALAYSIA 3.1 Introduction 28 3.2 Methodology 29 3.2.1 Descriptions for the Malayan Tapir 29 Samples Used in This Study 3.2.2 Extracting the DNA of Malayan Tapir 29 Samples 3.2.3 Development of Microsatellite Markers 30 and Multiplex PCR Design 3.2.4 Genotyping the Malayan Tapir Samples 38 in Fragment Analysis 3.2.5 Characterisation of Microsatellite 39 Markers UPM 3.2.6 Population Structure Analysis 41 3.3 Results 43 3.3.1 The Quality of Sample DNA 43 3.3.2 Development of Microsatellite Markers 44 and Multiplex PCR Design 3.3.3 Genotyping the Malayan Tapir Samples 56 in Fragment Analysis 3.3.4 Characterisation of Microsatellite 56 Markers 3.3.5 Revealing Population Genetic Structure 65 in the Malayan Tapir 3.4 Discussion 75 3.4.1 Development of Microsatellite Markers 75 3.4.2 Genetic Diversity of the Malayan Tapir 75 as Shown by the Microsatellites 3.4.3 Population Genetic Structure in the 76 Malayan Tapir 3.4.4 Conservation Implications of This Study 78 3.4.5 Underlying Assumptions and Errors in 79 This Study 3.5 Conclusion 79

4 GENETIC DIVERSITY, POPULATION GENETIC 81 STRUCTURE AND PHYLOGEOGRAPHIC OF THE MALAYAN TAPIR IN MALAY PENINSULA 4.1 Introduction 81 COPYRIGHT4.2 Methodology 82 4.2.1 PCR Amplification and Sequencing of 82 Mitochondrial DNA CR in the Malayan Tapir 4.2.2 Assembly and Alignment of the 83 © Mitochondrial DNA CR Sequences 4.2.3 Genetic Diversity Accessed by 83 Mitochondrial DNA CR

4.2.4 Population Genetic Structure and 84 Differentiation 4.3 Results 85 4.3.1 PCR Amplification and Sequencing of 85 Mitochondrial DNA CR, and Error Estimation 4.3.2 Sequence Variations Detected in PM 85 and TH Samples 4.3.3 Estimation of Genetic Diversity of PM 85 and TH Samples 4.3.4 Population Genetic Structure and 89 Phylogeographic Distribution of Two Clades of Malayan Tapir 4.3.5 AMOVA Tests for Population 94 Differentiation 4.4 Discussion 95 4.4.1 Low to Moderate Genetic Diversity in UPM95 the Malayan Tapir 4.4.2 Population Genetic Structure of the 96 Malayan Tapir in the Malay Peninsula 4.4.3 Inference of Population Demographic 98 History 4.4.4 Conservation Implications of This Study 100 4.4.5 Other Notes on Haplotypes of 101 Mitochondrial DNA CR versus CytB 4.5 Conclusion 102

5 MOLECULAR SEX-IDENTIFICATION AND 103 PRELIMINARY ESTIMATION OF WILD MALAYAN TAPIR SEX RATIO IN PENINSULAR MALAYSIA 5.1 Introduction 103 5.2 Methodology 104 5.2.1 PCR Amplification, Sequencing and 104 Nucleotide BLAST in NCBI Database 5.2.2 Characterisation of SRY and ZF by 106 Fragment Analysis 5.2.3 Sex-Typing Samples of Unknown Sex 106 5.2.4 Estimation of Sex Ratios and Testing for 107 Sex-Biased Dispersal 5.3 Results 108 5.3.1 Searching for Matching SRY and ZFX 108 Sequences in NCBI Database 5.3.2 Testing and Characterisation of Sex 108 COPYRIGHT Marker 5.3.3 Sex-Typing Samples of Unknown Sex 110 5.3.4 Overall Sex Ratios, Sex Ratios by 111 States or Biennium, and Sex-Biased Dispersal in the Malayan Tapir © 5.4 Discussion 113 5.4.1 Verification and Characterisation of 113 SRY and ZF Gene for Sex-Identification

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5.4.2 Marker Efficiency in Sex-Identification of 114 the Malayan Tapir 5.4.3 Estimation of Sex-Ratios and Sex- 115 Biased Dispersal 5.5 Conclusion 117

6 A GENERAL DISCUSSION ON THE MALAYAN 118 TAPIR POPULATION AS ASSESSED BY MICROSATELLITES AND MITOCHONDRIAL DNA CONTROL REGION, AND ON THE ASSESSMENT OF ITS SEX RATIO AND SEX-BIASED DISPERSAL IN PENINSULAR MALAYSIA 6.1 Integration of Genetic Markers for 118 Population Genetic Study 6.2 Inference of Genetic Diversity, Population 118 Genetic Structure and Sex-Biased Dispersal UPM 6.3 Molecular Assets for Future Studies 121

7 SUMMARY, CONCLUSIONS, AND 122 RECOMMENDATIONS FOR FUTURE RESEARCH 7.1 Summary and Conclusions 122 7.2 Recommendations 123

REFERENCES 125 APPENDICES 151 BIODATA OF STUDENT 205

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LIST OF TABLES

Table Page

3.1 List of 78 Malayan tapirs in Category A and their referred 151 identities (ID), types of samples, PERHILITAN reference numbers (PERHILITAN ref. no.), individual names, sample collection year, sex, locations (from Table 3.2), year caught or born, gDNA concentrations (X: not measured). No. 1-68: wild- born or assumed to be wild-born. No. 69-78: captive-born. √ - DNA of biological samples used in Chapter 3 (SSR), Chapter 4 (CR) and Chapter 5 (SexID). Bold and underlined sex symbols (M and F) dictates sexes of samples that were identified in Chapter 5. Category B lists down extra eight Malayan tapirs used only for analysis in Chapter 5 but do not have available biological samples UPM

3.2 Extract of entries from PERHILITAN tapir datasheet and 156 assumption of sampling locations based on the entries. a – sampling locations partially deduced from the name of the individual; b – sampling locations deduced from other provided information

3.3 Description of samples used in this study 30

3.4 Forward (F) and reverse (R) primers selected from published 31 literature or designed from GenBank-deposited microsatellite sequences for cross-amplification in the Malayan tapir. The loci with name prefixes Tte, Tter and TtGT were developed from the lowland tapir, Tba for the Baird’s tapir and TpGT for the mountain tapir

3.5 Forward (F) and reverse (R) primers designed for seven 34 microsatellite loci isolated from random amplified microsatellite (RAM) genomic sequences (Q. L. Lim et al. 2018)

3.6 Touchdown profile for singleplex polymerase chain reaction 35

3.7 General polymerase chain reaction profile 35

3.8 Tails selected for the three-primer method of polymerase 37 COPYRIGHTchain reaction. Each tail was assigned a fluorescent dye and colour: 6-FAM (blue), HEX (green), and ROX (red) © 3.9 Touchdown profile for multiplex polymerase chain reaction 38

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3.10 Quantity of four types of samples and proportion of the 44 samples that were genotyped, range of concentration and mean yield ± standard deviation (SD) of gDNA samples that were extracted and genotyped (N = 72)

3.11 List of markers that did not pass the screening at agarose gel, 184 sequencing or fragment analysis levels. PCR profile used for the markers and their results were described

3.12 BLAST result of the sequences against Tapiridae (taxid: 185 9799), which were amplified with cross-amplification microsatellite primers in the Malayan tapir (Tapirus indicus)

3.13 Eighteen screened microsatellite loci and the detected repeat 47 motifs compared to the reported repeat motifs UPM 3.14 Primer dimer and hairpin formation that were detected in all 48 possible three-primer systems of the 24 microsatellite markers

3.15 Modified forward (F) and reverse (R) primers of 24 selected 49 microsatellite markers for the three-primer polymerase chain reaction, target motifs, selected dyes and tails (T), allele size (bp) detected in one Malayan tapir (TAP13) and all genotyped samples (N = 67). Underlined sequences are the attached tail sequences

3.16 Dimers detected in Multiplex Manager 1.2 186

3.17 Volumes of primer mix for the 23 markers (10 μM normal 54 primer, 2.5 μM modified primer, and 10 μM dye-labelled tail) in 10 μL PCR reactions prepared for the three multiplex panels

3.18 Genotypes of 67 Malayan tapirs at 10 loci that carry more than 168 one allele

3.19 Genotype error rate per allele as estimated in 11 replicate 187 samples

3.20 Estimation of error rate per allele in 22 microsatellite markers 57

3.21 Scored genotypes of TAP13 under different amount of gDNA 190

3.22 Allele frequencies in 10 marker loci (allele number > 1) in 59 COPYRIGHTWILD (N = 57), CAPTIVE (N = 10), and WIL+CAPTIVE (N = 67)

3.23 Counts and frequencies of all genotypes of eight microsatellite 63 © markers in 28 male and 34 female Malayan tapirs

3.24 Genetic diversity indices and Hardy-Weinberg (HW) exact test 65 of eight microsatellite loci characterised in WILD (N = 57)

3.25 Genepop output for linkage disequilibrium test for WILD (N = 190 57)

3.26 Pairwise group matrix of Fst values for total genetic distance 73 estimated with eight microsatellite markers in five clusters of the Malayan tapirs. Fst values are shown below the diagonal, while numbers of effective migrants per generation (Nm) are shown above the diagonal

3.27 Exact Hardy-Weinberg test and global tests across population, 191 across locus, or across all loci and populations 3.28 Global tests for linkage disequilibrium using Fisher’s methodUPM 192 across five clusters (N = 57)

3.29 Genetic diversity indices as determined with eight 193 microsatellite markers in five clusters (N = 57) identified by K- means clustering

3.30 Probability test for linkage disequilibrium of eight microsatellite 193 markers in five clusters (N = 57) identified by K-means clustering. Only pairs with p-values < 0.5 were shown

4.1 Forward (F) and reverse (R) primers of the mitochondrial 83 control region (CR)

4.2 The output of jModelTest for the best-fit substitution model 194 from among 88 models

4.3 Genetic diversity indices computed for Malayan tapir 87 populations in Peninsular Malaysia (PM), Thailand (TH) and both (PM+TH)

4.4 Distribution of 23 haplotypes detected in 81 Malayan tapirs 197 comprising PM (N = 44) and TH (N = 37) samples

4.5 Mitochondrial control region (1237 bp) revealed 23 haplotypes 88 and their frequencies (N) based on 38 variable sites (32 parsimony variable sites and 6 singletons) in 81 Malayan tapirs. The haplotypes Ti-1 (A, B, C), Ti-2 (A, B, C, D, E), and COPYRIGHTTi-3 (A, B, C) were identified in the previous study (Muangkram, Amano, et al., 2016). Red sites are singletons

4.6 Nineteen haplotypes inferred from 32 parsimony variable sites 198 © in the mitochondrial control region sequences (1237 bp) that were found in Peninsular Malaysia and Thailand captivity

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4.7 Analysis of Molecular Variance (AMOVA) based on 95 mitochondrial control region comparing Malayan tapir populations in Peninsular Malaysia (PM) and Thailand (TH), and between clades in the PM+TH (N = 81), PM (N = 44) and its subset of tapirs with known locations (N = 34)

5.1 List of all Malayan tapirs used for the multiplex amplification of 105 sex-determining region Y gene and zinc finger gene and for sex-typing

5.2 Primers of sex-determining region Y (SRY) and zinc finger 105 (ZF) gene

5.3 Sex-identification of 18 Malayan tapirs by co-amplification of 110 sex-determining region Y and zinc finger gene. Only bands or peak patterns that were consistent in two out of three replicates of polymerase chain reaction were accepted forUPM sex-typing a sample

5.4 List of 18 Malayan tapirs from PERHILITAN database that 199 were of unknown sex. √ - Presence of amplicon. × - No amplification. Numbers under the headings of three trials denote the detected fragment size of the gene

5.5 The number of male (N = 16) and female (N = 31) Malayan 111 tapirs by state or region. The proportion of male tapirs is not calculated in the regions where there was only one tapir occurrence

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LIST OF FIGURES

Figure Page

2.1 Morphology of Tapirus indicus with Notes on the Physical 7 Characteristics (Edited Image; Nash, n.d.).

2.2 Mountain Ranges in Peninsular Malaysia. Regions 9 Highlighted in Green Shows the Extant Ranges of the Malayan Tapir According to the International Union for Conservation of Nature (IUCN) Red List (Traeholt et al., 2016). The Red Curve Indicates the Mountain Range: 1 – Kedah-Singgora Range, 2 – Bintang Range, 3 – Keledang Range, 4 – Titiwangsa Range, 5 – Benom Range, 6 – Tahan Range, and 7 – Pantai Timur Range. Alphabets Show the Locations of A – Belum Forest Complex, B – Taman NegaraUPM Forest Complex, C – Pahang Peat Swamp Forest, and D – Endau-Rompin Forest Complex. Image Created in QGIS 2.18 (QGIS Development Team, 2018) Using Base Map of Peninsular Malaysian Downloaded from the Database of Global Administrative Areas (GADM, Available on http://gadm.org) and Malayan Tapir Range Data Downloaded from the IUCN Website (https://www.iucnredlist.org/) and Edited Using Inkscape 0.92.3 (Harrington, 2018).

2.3 Predicted Distribution of the Malayan Tapir (Tapirus indicus) 10 in Peninsular Malaysia Using Maximum Entropy (MaxEnt) Model. Potentially Suitable Tapir Habitat (with ≥ 0.5 Logistic Value) Covers 69% of Forests in Peninsular Malaysia. Map Adapted from Clements et al. (2012).

3.1 Images of Gel Electrophoresis for 25 Microsatellite Markers 45 That were Successfully Amplified in Polymerase Chain Reaction and Yielded Single Band on 2% Agarose Gel. (A) Eighteen Cross-Amplified Markers: 1 – Tter3, 2 – Tter4, 3 – Tter7, 4 – Tter9, 5 – Tte12, 6 – Tter13, 7 – Tter14, 8 – Tter17, 9 – Tter18, 10 – TtGT021, 11 – TtGT048, 12 – TtGT070, 13 – TtGT137, 14 – TtGT215, 15 – Tba20, 16 – Tba 23, 17 – Tba25. (B) Seven Microsatellite Markers Developed from Random Amplified Markers: 1 – Ti13D2, 2 – Ti17J1, 3 – Ti17L1, 5 – Ti22M1, 6 – Ti22G1, 8 – Ti28D1, and 9 – Ti26I2. L – DNA Ladder with 50 bp (13 Bands) or COPYRIGHT100 bp (10 Bands) HyperLadderTM (Bioline, Germany). bp – Base Pair.

3.2 Peak Patterns of 24 Microsatellite Markers Obtained in 53 © Fragment Analysis in One Sample TAP13.

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3.3 Three Multiplex Panels, MP1, MP2 and MP3, Comprising a 54 Total of 23 Markers in Multiplex Manager. Range of Allele Size was Estimated from the Detected Size ± 15 bp Detected in One Sample of Malayan Tapir, TAP13. The Image was Edited to Enlarge the Font Size.

3.4 Gel Electrophoresis of the Products of Multiplex Polymerase 55 Chain Reactions (PCRs) According to Three Multiplex Panels (MP1, MP2 and MP3). Top Down, the Multiplex PCR Products are: (A) Tube 1—Ti26I2, TtGT021, Ti17L1 and Tter9; Tube 2—Ti13D2 and Tter18; Tube 3—TtGT215 and TtGT048; Tube 4—Ti28D1, Ti22G1 and Tte12; and Tube 5— Tter13 and TtGT137; (B) Tube 1—Tter4, Ti22M1 and Tter7; Tube 2—TtGT070 and Ti17J1; and Tube 3—Tter17 and Tba25; (C) Tube 1—Tter3, Tube 2—Tter14 and Tube 3— Tba23. L – DNA Ladder with 50 bp (13 Bands) or 100 bp (10 Bands) HyperLadderTM (Bioline, Germany). bp – Base Pair. UPM

3.5 Genotype Accumulation Curves for Eight Microsatellite 60 Markers Characterised in 67 Malayan Tapirs (Left) and 57 Malayan Tapirs (Right). Graph Plotted by Random Sampling of One to Seven (1 to N – 1) Loci Without Replacement for 1,000 Iterations (Horizontal Axis) and Counting the Number of Multilocus Genotypes (MLGs) Observed (Vertical Axis). Red Dash Line Indicates 90% of Unique MLGs Observed. Best-Fit Curve (Blue) was the Trend.

3.6 Distribution of Multilocus Genotypes (MLGs) of Eight 61 Microsatellite Markers in WILD+CAPTIVE (Top) and WILD (Bottom). Bars were Plotted for MLG Count (Vertical Axis) Against Unique MLGs (Horizontal Axis).

3.7 Graphs of Delta K Generated in Structure Harvester using 66 Output of STRUCTURE in WILD Dataset (N = 57) Which was Analysed Using Eight Microsatellite Markers. Inferred Number of Clusters (K) = 2 from Admixture and Correlated Allele Frequency Model (Top); and from Admixture and Independent Allele Frequency Model (Bottom).

3.8 Bar Blot Illustrating the Bayesian Genetic Structure for K = 2 66 and K = 3 in WILD Dataset (N = 57). Different Colours Represent Proportion of Membership to Each Inferred COPYRIGHTGenetic Cluster. 3.9 Principal Coordinate Analysis (PCoA) Showing the Genetic 67 Relationship Estimated with Eight Microsatellite Markers in 67 Malayan Tapirs in Different Dimensions: Principal © Coordinate Axis 1 against Axis 2 (A), Axis 2 against Axis 3 (B), and Axis 1 against Axis 3 (C).

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3.10 Bayesian Information Criterion (BIC) for Number of Clusters 68 (K) between 1 and 15.

3.11 Cumulative Variance Explained by a Number of Principal 179 Component (PC) Retained.

3.12 Discriminant Analysis of Principal Component (DAPC) Cross- 180 validation. (A) The Proportion of Successful Outcome Prediction at Various Number of Retained Principal Components Analysis (PCA) Axes Run at 30 Replicates from PCA 1 to 16. PCA = 4 to 12 were Selected for Second Run. (B) Second Run of Cross-Validation with 1,000 Replicates for a Range of Principal Component Analysis (PCA) Axes Retained, PCA = 4 to 12.

3.13 Scatter Plot of Discriminant Analysis of Principal 68 Components (DAPC) with Number of Genetic Clusters (A) UPMK = 4 and (B) K = 5 for WILD (N = 57). Each Cluster is Represented by Different Colours and Inertia Ellipses. Compared to K = 4, More Overlapping between the Ellipses of Clusters was Observed When K = 5.

3.14 Bar Plot Illustrating the Membership Probabilities of 57 70 Malayan Tapirs at Different Number of Genetic Clusters, K = 4 and K = 5. Colours Represent Different Clusters.

3.15 Geographic Distribution of 43 Malayan Tapirs Assigned to 72 Four (K =4) or Five (K = 5) Genetic Clusters Identified by K- Means Clustering.

3.16 Distribution of Five Clusters around the Titiwangsa Range, 73 Benom Range, Pantai Timur Range and Endau-Rompin National Park. The Dark Shade Areas are the Hill Shades.

3.17 Mantel Test for Relationship between the Matrices of 74 Pairwise Genetic and Geographic Distances under 999 Permutations. Correlation Coefficient (R) Obtained was 0.105 (p = 0.098), and Coefficient of Determination (R2) was 0.0109. Isolation-By-Distance was Insignificant in the Dataset (N = 43).

3.18 Correlogram Showing the Genetic Autocorrelation Coefficient 75 (r) as the Function of Distance. Upper (U) and Lower (L) COPYRIGHTConfidence Limits Bound the 95% Confidence Interval about the Null Hypothesis of No Spatial Structure in the Dataset (N = 43) as Determined by 999 Permutations. Distance Classes 15-30 km and 30-45 km Showed Significant Spatial Structure © (p < 0.05). Correlogram was Significant as Shown by Heterogeneity Test (Omega = 97.878, p = 0.001).

4.1 A Median-Joining (MJ) Network of 81 Malayan Tapirs from 90 Peninsular Malaysia Inclusive of Two Samples from Singapore (N = 44; Blue), and Thailand (N = 37; Yellow). Nodes Represent 23 Haplotypes. Sizes of Nodes are Proportional to the Number of Individuals Included in the Haplotypes. A Total of 38 Variable Sites Without Considering Gaps were Used for the Network Calculation. Ti-1(A, B, C), Ti-2(A, B, C, D, E) and Ti-3(A, B, C) were Identified in the Previous Study (Muangkram, Amano, et al., 2016). The Small Red Dots Represent the Unobserved Sequences. Bars Represent Nucleotide Substitutions.

4.2 A Neighbour-Joining (NJ) Tree of 81 Malayan Tapirs from 91 PM (N = 44; Prefix ‘TAP’) and TH Datasets (N = 37; Prefix ‘Ti-’) Representing the Topology of the 23 Haplotypes Identified with Mitochondrial Control Region. Lowland Tapir (Tapirus terrestris) was Included as an Outgroup. The FinalUPM Dataset for Tree Construction Has Included 1226 Positions. Bootstrapping (1,000 Replicates) Values > 50% were Shown Next to the Branches as Percentage. Labels Shown on the Right are the Haplotypes.

4.3 A Maximum Likelihood (ML) Tree Constructed Based on 92 Mitochondrial Control Region of 81 Malayan Tapirs from PM (N = 44; Prefix ‘TAP’) and TH Datasets (N = 37; Prefix ‘Ti-’). Lowland Tapir (Tapirus terrestris) was Included as an Outgroup. The Final Dataset for Tree Construction Has Included 1226 Positions. The Larger Tree is the Midpoint- Rooted Subtree of the ML Tree Drawn at the Bottom Left Corner. The Branch Lengths were Drawn to Scale, and the Scale Bar Carries the Unit of Number of Substitutions Per Site. Bootstrapping (1,000 Replicates) Values > 50% were Shown Next to the Branches as Percentage. Subtree Labels on the Right are the Haplotypes.

4.4 The Phylogeographic of 34 Samples of Malayan Tapirs from 93 PM Dataset That Made Up 12 Haplotypes (H_9 Not Included as the Only Representative TAP64 was from Unknown Location) As Shown in the Simplified Neighbour-Joining (NJ) Topology Tree Constructed with Mitochondrial Control Region (1266 Positions). Haplotypes in Black, Belong to Clade I, were Recorded Only in Thailand Samples and were Not Shown. Haplotypes in Blue Also Belong to Clade I, While COPYRIGHTHaplotypes in Red Belong to Clade II. 4.5 Test Statistic (z) Showing the Degree of Correlation between 94 Two Matrices: The Genetic Distances and Geographic © Distances of 34 Malayan Tapir. Line in the Centre is the Observed Z-Statistic (z = 429.6) with a Two-Sided P-Value = 0.710 Estimated from 10,000 Permutations, Indicating That

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the Two Distance Matrices Did Not Correlate Significantly.

4.6 Test Statistic (z) Showing the Degree of Correlation between 183 Two Matrices: The Genetic Distances and Geographic Distances of 33 Malayan Tapir. Line in the Centre is the Observed Z-Statistic (z = 377.3) with a Two-Sided P-Value = 0.766 Estimated from 10,000 Permutations, Indicating That the Two Distance Matrices Did Not Correlate Significantly.

4.7 A Neighbour-Joining (NJ) Tree Showing the Phylogenetic 97 Relationships of Malayan Tapirs Based on Partial Mitochondrial DNA Cytochrome B Gene. NJ Tree Adapted from Rovie-Ryan et al. (2008).

4.8 Comparative Phylogenetic Relationship of Malayan Tapirs 99 Based on Mitochondrial Cytochrome b (CytB) Haplotypes with Proposed Names Ti-1 and Ti-2 (Thailand) and TiUPM-3 (Thailand/Malaysia), Ti-4 (Unknown Origin), Ti-5 and Ti-6 (Malaysia), and Ti-7 (Sumatra). The Blue Horizontal Line Refers to the Number of Nucleotide Substitutions. Areas in Yellow Indicates the Current Distribution of the Malayan Tapir Modified from the IUCN Red List of Threatened Species (www.iucnredlist.org). Map Adapted from Muangkram et al. (2013).

5.1 Amplification of the SRY Gene and Zinc Finger (ZF) Gene in 109 22 Malayan Tapirs. (A) The Male Tapirs Yielded Amplicons of SRY Gene at ~200 bp and ZF Gene at ~450 bp. The Female Tapirs Yielded Only Amplicons of ZF Gene at ~450bp. ZF Gene Acts as a Positive Control for the Polymerase Chain Reaction (PCR). (B) Sex-Identification in 11 Females and 11 Males by Multiplexing the Primers of SRY Gene and ZF Gene in PCR, and the Products were Visualised on 2% Agarose Gel. All the Males and Females Show Consistent Sex-Specific Band Patterns. L - 50bp Ladder, N - Negative Control, M - Male, F - Female.

5.2 A Chromatogram Showing the Peaks of Sex-Identifying Sex- 110 Determining Region Y (SRY) and Zinc Finger Gene (ZF) from a Male Malayan Tapir. The Scored Fragment Sizes were 244 bp for SRY and 464 bp for ZF. In Female, the Peak for SRY is Absent. DB - Dye Blob. COPYRIGHT5.3 The Numbers of Male and Female Malayan Tapirs and the 112 Sex Ratios (Male/[Male+Female]) Per Biennium for Years 2004-2015 (N = 54). One Male Tapir was Present in the © Biennium 2016-2017 and was Not Shown in the Graph. 5.4 The Numbers of Male and Female Malayan Tapirs and the 112 Sex Ratios (Male/Male+Female) Per Year (2004-2017). Total

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Tapirs = 55.

5.5 Correlogram Showing the Genetic Autocorrelation Coefficient 113 (r) As the Function of Distance. Heterogeneity Test between the Correlograms of Two sexes (N = 43) was Insignificant (Omega = 24.2, p = 0.536).

6.1 Distribution of Two Clades That were Detected by 120 Mitochondrial DNA Control Region in 44 Malayan Tapir Samples Over Five Clusters Detected by Eight Microsatellite Markers.

UPM

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LIST OF APPENDICES

Appendix Page

A Details of Malayan Tapir Samples 154

B1 Genotype Scoring for 23 Microsatellite Markers 162

B2 Genotypes of 67 Malayan Tapirs at 10 Loci with More 171 Than One Allele

C1 QIAamp® DNA Mini Kit 174 C2 DNA Quantification UPM176 C3 Gel Electrophoresis 176

C4 pGEM®-T Easy Vector and Colony PCR 177

C5 Wizard®SV Gel and PCR Clean-Up System 178

C6 R Script for Genotype Cumulative Curve 179

C7 R Script for Discriminant Analysis of Principal Components 180 (DAPC)

C8 R Script for Mantel Test 181

D1 DAPC Cross-Validation 182

D2 Mantel Test 186

E Table of Results 187

F1 Full or Partial Sequences of 25 Microsatellite Loci 203

F2 Mitochondrial DNA Control Region Sequences 206

F3 SRY and ZFX Sequences 207

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LIST OF ABBREVIATIONS

+G (including) Gamma distribution +I (including) Invariant site λ Number of substitutions per site per year π Nucleotide diversity 2n Diploid number 3’ End of a linear DNA strand 5’ Beginning of a linear DNA strand 6-FAM 6-carboxyfluorescein (a fluorescent dye for fragment analysis) A Adenine AMOVA Analysis of Molecular Variance AU Action unit BIC Bayesian Information Criterion bp Base pair UPM C Cytosine CLUMPAK Cluster Markov Packager Across K CR Control region CSB Conserved sequence block CytB Cytochrome b delta G Gibbs energy DAPC Discriminant analysis of principal component dN Number of substitutions per site DNA Deoxyribonucleic acid DBS Dried blood spot PERHILITAN Department of Wildlife and National Parks ESU Evolutionarily significant unit ETAS Extended termination associated sequences F Fixation index Fst Fixation index (inbreeding coefficient) G Guanine GADM Database of Global Administrative Areas gDNA Genomic DNA H_ Name prefix for haplotype Hd Haplotype diversity He Expected heterozygosity HKY Hasegawa, Kishino & Yano model Ho Observed heterozygosity HEX A type of fluorescent dye for fragment analysis HWE Hardy-Weinberg equilibrium I Shannon’s information index COPYRIGHTIBD Isolation-by-distance IUCN International Union for Conservation of Nature K Number of genetic clusters K-means Unsupervised learning algorithm for clustering observations K80 Kimura 2-parameter model © LD Linkage disequilibrium LIZ500 Dye-labelled size standard with 16 fragments up to 500 bp

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MaxEnt Maximum entropy MCMC Markov chain Monte Carlo MLG Multilocus genotype MJ Median-joining ML Maximum likelihood MP Multiplex panel mtDNA Mitochondrial deoxyribonucleic acid MU Management unit MYA Million years ago N Count or number Na Allele number Ne Effective allele number NCBI National Center for Biotechnology Information NJ Neighbour-joining Nm Number of effective migrants per generation PC Principal component PCA Principal component analysis UPM PCoA Principal coordinate analysis PCR Polymerase chain reaction PIC Polymorphic information content PM Peninsular Malaysia RAM Random amplified microsatellite RNA Ribonucleic acid ROX A type of fluorescent dye for fragment analysis SD Standard deviation SNP Single nucleotide polymorphism SRY Sex-determining region Y SSR Simple sequence repeat STR Short tandem repeat T Thymine Taq Thermus aquaticus tD Divergence time TH Thailand mtDNA CR dataset Ta Annealing temperature Tm Melting temperature uHe Unbiased heterozygosity UTR Untranslated region WCC Wildlife Conservation Centre WGRB Wildlife Genetic Resource Bank X X chromosome Y Y chromosome ZF Zinc finger ZFX X-linked zinc finger gene COPYRIGHTZFY Y-linked zinc finger gene

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CHAPTER 1

INTRODUCTION

Genetic variation, which lays the basis for genetic diversity and thus genetic structure, is one of the three levels of biodiversity i.e. genetic diversity, species diversity and ecosystem diversity, that deserve to be conserved. Genetic diversity is often correlated to population fitness and surviving capacity of the population through adaptation (Barrett & Schluter, 2008; Reed & Frankham, 2003). International Union for Conservation of Nature (IUCN) ranking of a species as ‘Vulnerable’, ‘Endangered’, or ‘Critically Endangered’ is based on its statistics of population decline and range loss, which in turn implies a significant extinction risk faced by the concerned species as well as suggests a reduced genetic diversity by inbreeding and drift in the threatened populations (Rivers, Brummitt, Lughadha, & Meagher, 2014; Willoughby et al., 2015)UPM.

The Malayan tapir (Tapirus indicus Desmarest) is one of the endangered faunas listed in the IUCN Red List (Traeholt et al., 2016). The Malayan tapir is found in Southeast Asia including Thailand, Sumatra and Peninsular Malaysia. Its population in Peninsular Malaysia is declining due to threats such as habitat loss, habitat fragmentation and road kills. In Malaysia, the setting up of the Malayan Tapir Conservation Centre and the development of the Malayan Tapir Action Plan are among the efforts to conserve this species. The conservation efforts include captive breeding management and operations such as the rescue of displaced or injured tapirs, and reintroduction or translocation of the tapirs from one population to another (Magintan, Traeholt, & Karuppanannan, 2012; Mahathir et al., 2014).

While habitat protection and maintenance, and at one point, conservation intervention by humans are important to maintain the Malayan tapir population, population genetic diversity should not be overlooked in the conservation biology of the Malayan tapir. Examination of the amount of genetic diversity and its distribution pattern over a geographic area can provide valuable insights into the population genetic structure—number of subpopulations, genetic variation within the subpopulations, and the degree of gene flow between them, as well as make inference to the factors and demographic processes that shaped the genetic structure of the population (Allendorf, Luikart, & Aitken, 2013; Chakraborty, 1993). Understanding the Malayan tapir population genetic structure has important management and conservation implications for the COPYRIGHTspecies, for example, identifying population management units that may be genetically distinct from each other will help authorities such as Department of Wildlife and National Parks (PERHILITAN) in Malaysia to take caution when making decisions on reintroduction, translocation and breeding. Furthermore, the genetic data and information can be used for long-term monitoring © programme for both wild and captive Malayan tapir populations. Prior information on population genetic structure will also allow wildlife

conservationist to design their experiment or research plan for testing more sophisticated hypotheses using subpopulations as groups.

Population studies of the Malayan tapir based on conventional methods such as camera trapping and radiometry (e.g. Rayan et al., 2012; Traeholt & Sanusi, 2009; K. D. Williams, 1979) to study its population distribution, population density, home range etc. did not include the information on population genetic structure, which requires molecular or genetic markers to explore. The genetic approach offers advantages in term of grasping population information at the molecular level that is inaccessible by conventional methods, yet with proper research design and assessments, it can reveal similar population information obtained from the latter techniques and even more. For example, while both approaches can be used to estimate population size and density (Janečka et al., 2011), population structure, in the sense of distribution of individuals in a geographical area, can be more readily assessed by evaluating distribution pattern of their genetic diversity using samples from various sources,UPM rather than employing ecological field techniques to track down movement of a number of individuals. Genetic markers such as nuclear microsatellite markers (Pinho, Gonçalves da Silva, Hrbek, Venticinque, & Farias, 2014) and mitochondrial deoxyribonucleic acid (mtDNA) markers (Muangkram, Amano, et al., 2016) are popular tools for estimating genetic diversity within or among subpopulations.

Estimation of sex ratio and sex-biased dispersal, which are among the factors that influence population genetic structure, can be achieved with sex- identification markers e.g. sex-determining region Y (SRY) and zinc finger (ZF) gene for identifying sexes of collected samples (Pelizzon, da Silva Carvalho, Caballero, Manoel Galetti Junior, & Sanches, 2017; Quaglietta, Fonseca, Hájková, Mira, & Boitani, 2013). However, studies with these markers i.e. microsatellite, mtDNA, and sex-identification markers are still few or largely lacking in the Malayan tapir, if not totally absent, especially for populations residing in Peninsular Malaysia and Sumatra. Only a few genetic research on the Malayan tapir population, whether captive or wild, were conducted in the past decade. These projects focused mainly on the mtDNA genes e.g. cytochrome b (CytB) gene and control region (CR) to assess genetic diversity, phylogenetic or phylogeographic relationships in Thailand (Muangkram, Amano, et al., 2016; Muangkram et al., 2013) and Japan zoos (Ogata, Watanabe, & Ogawa, 2009), and in Peninsular Malaysia (Rovie-Ryan et al., 2008). Others include research that only aimed to reconstruct phylogenetic relationships among members of Tapiridae (Ashley, Norman, & Stross, 1996; de Thoisy et al., 2010). COPYRIGHT

As such, more genetic markers need to be developed to lay the ground for further studies in the population structure and diversity for the Malayan tapir, as well as using the genetic information for improving in-situ and ex-situ © conservation management. Therefore, the aim of this thesis and the research works performed within it was to fill the gap in the knowledge by developing and using genetic markers to provide novel insights into the population genetic

structure of the Malayan tapir in Peninsular Malaysia, to account for the current situation where the population genetic structure of Malayan tapir has remained not well-understood despite its ‘Endangered’ status. The objectives were:

1. to screen and characterise 41 microsatellite markers in the Malayan tapir, which includes seven novel microsatellite marker loci isolated from the Malayan tapir by random amplified microsatellite (RAM) markers and 34 microsatellite marker loci developed for the lowland tapir, Baird’s tapir, and mountain tapir;

2. to assess the genetic diversity and population genetic structure of the Malayan tapir in Peninsular Malaysia using the tested polymorphic microsatellite markers;

3. to assess the genetic diversity, population genetic structure and phylogenetic relationships of the Malayan tapir populationUPM in Peninsular Malaysia using mtDNA CR and with the inclusion of Thailand captive samples.

4. to verify and characterise the SRY/ZF sexing method in Malayan tapir for sex-typing samples of unknown sex, which are to be included for a preliminary assessment of sex ratio in the wild and captive-born Malayan tapir populations in Peninsular Malaysia, and to detect spatially sex-biased dispersal in relation to microsatellite data and geographical distances in the wild population.

Chapter 1, as has been described in this chapter, introduces the main research subject of this project—the Malayan tapir, clarifies the problems faced by the fauna and the main aim of this thesis in contributing valuable genetic tools and information for the conservation of the Malayan tapir. Chapter 2 gives a review of the subjects relevant to this project. Chapter 3 describes the development of microsatellite markers and its use to assess and clarify the population genetic structure of the Malayan tapir in Peninsular Malaysia. While Chapter 3 investigates population genetic structure in the Malayan tapir using biparentally inherited microsatellite markers, Chapter 4 investigates the population genetic structure using maternally inherited mtDNA CR. Other than population structure, the chapter also investigates phylogenetics and genetic diversity of Malayan tapir in Peninsular Malaysia by including the mtDNA CR sequences of the Malayan tapir kept in Thailand. Chapter 5 describes the development of a sex- typing method for samples of unknown sex. The sex data was then used to estimate sex ratio in the wild- and captive-born Malayan tapirs in Peninsular COPYRIGHTMalaysia. In addition, data from microsatellite markers and the sex data were combined to look for population structure caused by the differential in dispersal in different sexes. Chapter 6 provides a general discussion on the results obtained through Chapter 3 to Chapter 5. Lastly, Chapter 7 gives a recap of all the works conducted for objectives in Chapter 3 through Chapter 5 and the © conclusions made and provides recommendations on what can be researched in the future to widen the knowledge about the ecology and genetics of the Malayan tapir.

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