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A Preliminary Phylogenetic Analyses of Schismatoglottis Ca!.Vpfrafa Complex Utilizing Nuclear ITS Region

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A Preliminary Phylogenetic Analyses of Schismatoglottis Ca!.Vpfrafa Complex Utilizing Nuclear ITS Region A Preliminary Phylogenetic Analyses of Schismatoglottis Ca!.vpfrafa Complex Utilizing Nuclear ITS Region Nurul Fatini Shahirah Binti Suharizan This report is submitted in partial fulfillment of the requirement for the degree of Bachelor of Science with Honours in Plant Resource Science and Management Faculty of Resource Science and Technology Universiti Malaysia Sarawak 2012 ACK1~OWLEDGEMENTS First of all, my special thanks to my supervisor, Dr Wong Sin Yeng for her guidance, support, advice and help to complete my Final Year Project. Thanks for giving me giving me an opportunity in doing the project and also for the valuable knowledge. Gratitude are also extended to M.Sc students who guided and helped me during laboratory work: Low Shook Ling, April Ting Pei Jen, Hoe Yin Chen. Special thanks to Nur Adilah bt Mohd Arif, my partner in labbratory work. To my family especially my beloved parents, Suharizan bin Abd Ghani and Fadhiwatty bt Abdul Wahab, thanks for your understanding and moral support. To my friends: Nur Aimi bt Abdullah Sani, Rofiah binti Junaidi, Syarifah Nur Liyana and Nur Alyani binti Badrol Hisyam, thank you for your encouragement and help. I IV TABLE OF CONTENTS Title.................................................................................... I Approval sheet. .... II Declaration............................................................................ III Acknowledgements.... .......... ................................ ......... ........... IV Tab-Ie of Contents. ................................................................ ... V List of Abbreviations................................................................ VII List of Tables and Figures....... .................. ....... ........................ ... VIII Abstract. .............................................................................. Chapter 1: Introduction.............................................................. 2 1.0 Introduction...................................................................... 2 l.l Problem statement...................................................... 4 1.2 Objectives................................................................ 4 Chapter 2: Literature Review........................................................ 5 2.1 Araceae................................................................... 5 2.2 Schismatoglottis calyptrata complex................................. 6 2.3 Phylogeny................................................................ 8 2.4 ITS Region............................................................... 9 Chapter 3: Material and Methodology............................................. 11 3.1 Samples collection....................................................... 11 3.2 DNA Extraction.......................................................... 13 v 3.3 PCR Amplification ...................................................... 14 3.4 Agarose Gel Electrophoresis ............................................. 14 3.5 PCR Purification And Sequencing ..................................... 15 3.6 Data Analyses .......................................................... 16 Chapter 4: Results and Discussion ................................................. 17 4.1 Presence ofDNA bands from amplification ...................... .. 18 4.2 Sequences alignment. ................................................. .. 19 4.3 Maximum Parsimony Analyses ........................................ 27 4.4 Maximum Likelihood Analyses ........................................ 29 I,: 4.5 Discussion................................................................... 31 H, !, ! "]' 4.5.1 Comparison ofPhylogenetic Analyses ........................... 31 4: I' .i 4.5.2 Morphology.......................................................... 33 I, ji 4.5.3 Geology............................................................... 36 11 I'.1 Chapter 5: Conclusions and Recommendations ...................................... 37 !l I' I~ " References................................................................................. 38 'I I, ~ !:, .. ,,[" 1i'·'·;'·1 1 VI LIST OF ABBREVIATIONS CI Consistency Index CIA Chlorofotm-Isoamyl Alcohol CTAB Cetyl Trimethylammonium Bromide DNA Deoxyribonucleic Acid EtBR Ethidium Bromide EtOH Ethanol HI Homoplasy Index ITS Internal Transcribed Spacer MP Maximum Parsimony i, ,I .: nrDNA Nuclear Ribosomal DNA 1 1: RAxML Randomized Axe1erated Maximum Likelihood I: RC Rescaled Consistency "I" ':1 I: RI Retention Index !! • TBR Tree Branch Swapping I 1 I VII LIST OF TABLES Table Page 3.0 Details on the taxa used for this study 12 4.0 Nucleotide sequences of the accessions 20 LIST OF FIGURES Figure Page 2.0 The three coding and two internal transcribed spacer regions of nuclear ribosomal DNA repeat unit ofa typical angiosperm 10 4.0 DNA Amplification before purification 17 4.1 DNA Amplification after purification 18 4.2 Maximum parsimony analysis of 50% majority consensus 28 4.3 Maximum likelihood analyses 30 4.4 (a): Leaf lamina of Schismatoglottis calyptrata from Kg Sikog, Padawan 34 (b): Inflorescence of Schismatoglottis calyptrata from Kg Sikog, Padawan 34 VIII A Preliminary Phylogenetic Analyses of Schismatoglottis calyptrata Complex Utilizing Nuclear ITS Region Nurul Fatini Shahirah bind Suharizan Plant Resource Science and Management Faculty of Resource Science and Technology Universiti Malaysia Sarawak ABSTRACT Sehismatoglottis ealyptrata complex (Schismatoglottideae: Araceae) consist of at least 40 species. This study focused on six accessions of Sehismatoglottis calyptrata complex from Kuching, Sibu and Selangor. Molecular investigation was carried out to resolve the relationship between the species investigated. The nuclear ITS region located in nuclear ribosomal DNA (nrDNA) were used for this study. DNA was extracted from dried leaf using CTAB method. Extracted DNA were amplified and purified before sending for sequencing at a commercial company. The sequences obtained were checked manually and aligned in BioEdit ver 7.0.5 together with an outgroup, Phi/onotion americanum. Phylogeny analyses were carried out in PAUP ver v.4.0b10 for maximum parsimony and RAxML for maximum likelihood. Maximum parsimony and maximum likelihood analyses showed strong and moderate bootstrap support (MP: 100%, ML: 43%) for the ingroups excluding Schismatoglottis calyptrata AR 3586. This study shows that all the species sampled from Sarawak are closely related based on the molecular evidence. Keywords: Araceae, Schismatoglottis calyptrata complex, molecular investigation, phylogenetic, ITS region ABSTRAK ~~~!Q&:lQJ'1l§....f.frJJ!1?1!1lli! kompleks (Schismatoglottis : Araceae) mempunyai sekurang-kurangnya 40 spesies. Kajian ini bertumpu terhadap enam sampel Schismatoglottis calyptrata kompleks daripada Kuching, Sibu dan Selangor. Kajian seeara molekular telah dijalankan bagi mengenalpasti hubungan an tara spesies yang terlibat. Nuklear ITS yang terletak di dalam nuklear ribosomal DNA (nrDNA) telah digunakan. DNA telah diekstrak daripada sampel daun menggunakan kaedah eTAB, DNA yang telah diekstrak kemudian diampliji dan puriji sebelum dihantar untuk aturan di syarikat komersial. Aluran yang diperoleh lelah disemak secara manual dan diatur menggunakan BioEdit ver 7.0.5 bersama-sama outgroup, Phi/onotion americanum. Analisis filogenetik dilakukan oleh PAUP ver vA.ObIO untuk membina maksimum parsimoni dan RAxML untuk maksimum persamaan. Maksimum parsimoni dan maksimum persamaan menunjukkan sokongan kuat dan sederhana (MP : I OO%, ML:43%) atas kesemua ingroup keeuali Schismatoglottis calvptrata AR 3586. Kajian ini menunjukkan bahawa semua sampel daripada Sarawak adalah berkait rapat berdasarkan kajian molekular. Kata kunci : Araceae, Schismatoglottis calvptrata kompleks, kajian molekular,jilogenetik, NuklearlTS 1 CHAPTER! INTRODUCTION 1.0 Introduction Araceae (aroids or keladi) comprises the fourth largest family of Monocotyledons after the orchids, grasses and sedges. Araceae is also one of the most important and diverse plant families in the Asian humid tropics. The richest and most diverse area is Central and South America (Neotropic ecozone), notably along the mountainous Pacific coast of northeastern South America (Mayo et aI., 1997). The next richest assemblage is in Southeast Asia, particularly the Malay Archipelago. The Malay Archipelago includes the major landmasses (West to East): Sumatera, the Malay Peninsula including Singapore, Jawa and Borneo. The Malay Archipelago forms part of the larger informally ecological region which is known as Malesia. Malesia consists of the Malay Archipelago, the remainder of Indonesia, almost of the Philippines, Timor Leste, and the island of New Guinea. Malesia comprises much of the Indomalayan ecozone, although from a species distribution standpoint it is necessary to include the western most islands of the tropical Pacific (Oceania ecozone) as far east as Vanuatu, together with the islands to the north and northwest of Australia. The next richest region for aroids are tropical Africa, followed by warm temperate Eurasia, India, Madagascar and the Seychelles, Southern Africa, and North America including northern most Mexico (Mayo et al., 1997) 2 Tribe Schismatoglottideae is a predominantly Indomalayan taxon comprising 13 genera of terrestrial, lithophytic or rheophytic herbs with a primary distribution centered on Borneo but with the total distribution ranging to South West Myanmar, South West China, and Vanuatu (Hay & Yuzammi, 2000; Wong et aI., 2010). Schismatoglottis
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