DNA Sequence-Based Identification and Molecular Phylogeny Within Subfamily Dipterocarpoideae (Dipterocarpaceae) Dissertation Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) at Forest Genetics and Forest Tree Breeding, Büsgen Institute Faculty of Forest Sciences and Forest Ecology Georg-August-Universität Göttingen By Essy Harnelly (Born in Banda Aceh, Indonesia) Göttingen, 2013 Supervisor : Prof. Dr. Reiner Finkeldey Referee : Prof. Dr. Reiner Finkeldey Co-referee : Prof. Dr. Holger Kreft Date of Disputation : 09.01.2013 2 To My Family 3 Acknowledgments First of all, I would like to express my deepest gratitude to Prof. Dr. Reiner Finkeldey for accepting me as his PhD student, for his support, helpful advice and guidance throughout my study. I am very grateful that he gave me this valuable chance to join his highly motivated international working group. I would like to thank Prof. Dr. Holger Kreft and Prof. Dr. Raphl Mitlöhner, who agreed to be my co-referee and member of examination team. I am grateful to Dr. Kathleen Prinz for her guidance, advice and support throughout my research as well as during the writing process. My deepest thankfulness goes to Dr. Sarah Seifert (in memoriam) for valuable discussion of my topic, summary translation and proof reading. I would also acknowledge Dr. Barbara Vornam for her guidance and numerous valuable discussions about my research topic. I would present my deep appreciation to Dr. Amarylis Vidalis, for her brilliant ideas to improve my understanding of my project. My sincere thanks are to Prof. Dr. Elizabeth Gillet for various enlightening discussions not only about the statistical matter, but also my health issues. Many thanks are to Prof. Dr. Martin Ziehe for his support and recommendation to DAAD. I am also grateful to get acquainted with Alexandra Dolynska, who gave me enormous helps during my lab works. Without her help it would be difficult for me to finish my study on time. I would also like to acknowledge Gerold Dinkel for his help to solve computer problem. My thanks also go to Regina Berkeley, who always helps me with the administration matters during my study. I would like to acknowledge former students: Dr. Nga Phi Nguyen, Dr. Yanti Rachmayanti, and Dr. Hani Siti Nuroniah for the data and discussion. Many thanks to my roommates: Dr. Alexandra Kuchma and Dr. Yazar Minn, for their kindness, help and support during my stay in Germany. I also would like to thank all my friend in the Institute; Randy Villarin, Konstantina Kameubun, Dr. Rajendra K.C., Dr. Chunxia Chung, Dr. Marius Ekue, Markus Müller, Fitri Yola Amandita, and Crusty Estoque, for knowledge sharing and discussion. I need to acknowledge the Aceh government for granting me the scholarship, and Syiah Kuala University for allowing me to take study leave overseas. I would also like to acknowledge The German academic exchange service (DAAD) who gives me their best assistant during my study in Germany. 4 My sincerest thanks to all Acehnese student societies in Germany for their kind support, to my friends from Aceh in Göttingen for making me feel like home, especially to Maimun Rizal, Ikhlas Ali Amran , Rezky Syahrezal, Ichsan, Rita Khathir, Faradilla Fadlia and Febi Mutia for their help and support during my study. I also appreciate the Indonesian community for their support and warm friendship during my study in Göttingen. Many thanks to Dr. Siti Nurleily Marliana and Alex for proof reading this dissertation. I would like to deliver my sincere thanks to my best friends in Biology Department Syiah Kuala University: Betty Mauliya, Zumaidar, Yekki Yasmin, Fauziah and Leni Fitri, who always give support and caring during my study. My deepest gratitude to my mother (in memoriam), who has a dream about going to Germany, which finally drove me to this country, to my mother in law for her support and prayer throughout my study, and to my brothers and sisters for their love and caring. My sincere thanks to my sister Enny Rohainy and her husband Prof. Dr. Herry Suhardiyanto for their love and support in my life. I would also like to acknowledge the support and warm discussion with my niece, Asadatun Abdullah. Finally, I would like to express my profound gratitude to my husband, Eka Oktavianus, for his patient, love, and encouragement, as well as to my great children: Shofiya Assyifa, Adullah Abdul Aziz, and Najiya Assyifa, who showed me their brave, patient, and independent that made my study life easier. 5 Content CONTENT .......................................................................................................................................... 2 LIST OF FIGURES .......................................................................................................................... 9 LIST OF TABLES .......................................................................................................................... 10 1 INTRODUCTION ...................................................................................................... 11 1.1 Background of the study ------------------------------------------------------------------------------------ 11 1.2 The Requirement of Molecular Taxonomic Tools ------------------------------------------------------ 12 1.3 Molecular Identification Tools for Dipterocarps -------------------------------------------------------- 14 1.4 Rationale of the study --------------------------------------------------------------------------------------- 15 2 PLANT MOLECULAR SYSTEMATICS .................................................................... 16 2.1 DNA Sequence Data ---------------------------------------------------------------------------------------- 16 2.1.1 Nuclear DNA ................................................................................................................................. 17 2.1.2 Mitochondrial DNA ....................................................................................................................... 18 2.1.3 Chloroplast DNA ........................................................................................................................... 18 2.2 Molecular phylogenies of plants --------------------------------------------------------------------------- 21 2.3 DNA-based identification ---------------------------------------------------------------------------------- 23 2.4 DNA barcoding ---------------------------------------------------------------------------------------------- 23 2.5 Molecular taxonomic identification key ------------------------------------------------------------------ 25 2.6 The Dipterocarpaceae family------------------------------------------------------------------------------- 25 2.6.1 The subfamily Dipterocarpoideae .................................................................................................. 26 2.6.2 Tribe Shoreae ................................................................................................................................. 27 2.6.3 Current research on Dipterocarpaceae ........................................................................................... 28 3 MATERIAL AND METHODS ............................................................................... 29 3.1 Material ------------------------------------------------------------------------------------------------------- 29 3.1.1 Data mining for the selection of DNA regions ............................................................................... 29 3.1.2 Additional sequence information from leaf collections ................................................................. 29 3.2 Methods ------------------------------------------------------------------------------------------------------- 31 3.2.1 Laboratory methods ....................................................................................................................... 31 3.2.2 DNA sequence analysis .................................................................................................................. 33 6 4 RESULTS ..................................................................................................................... 37 4.1 DNA sequence characteristics ----------------------------------------------------------------------------- 37 4.1.1 psbC- trnS IGS region .................................................................................................................... 37 4.1.2 trnL intron region ........................................................................................................................... 38 4.1.3 matK region ................................................................................................................................... 38 4.1.4 rbcL region ..................................................................................................................................... 39 4.2 Molecular phylogeny based on four chloroplast regions ----------------------------------------------- 39 4.2.1 psbC- trnS IGS region .................................................................................................................... 39 4.2.2 trnL intron region ........................................................................................................................... 42 4.2.3 matK region ................................................................................................................................... 45 4.2.4 rbcL region ....................................................................................................................................