SYSTEMATICS AND EVOLUTION OF THE RATTAN GENUS KORTHALSIA BL. (ARECACEAE) WITH SPECIAL REFERENCE TO DOMATIA A thesis submitted by Salwa Shahimi For the Degree of Doctor of Philosophy School of Biological Sciences University of Reading February 2018 i Declaration I can confirm that is my own work and the use of all material from other sources have been properly and fully acknowledged. Salwa Shahimi Reading, February 2018 ii ABSTRACT Korthalsia is a genus of palms endemic to Malesian region and known for the several species that have close associations with ants. In this study, 101 new sequences were generated to add 18 Korthalsia species from Malaysia, Singapore, Myanmar and Vietnam to an existing but unpublished data set for calamoid palms. Three nuclear (prk, rpb2, and ITS) and three chloroplast (rps16, trnD-trnT and ndhF) markers were sampled and Bayesian Inference and Maximum Likelihood methods of tree reconstruction used. The new phylogeny of the calamoids was largely congruent with the published studies, though the taxon sampling was more thorough. Each of the three tribes of the Calamoideae appeared to be monophyletic. The Eugeissoneae was consistently resolved as sister to Calameae and Lepidocaryeae, and better resolved, better supported topologies below the tribal level were identified. Korthalsia is monophyletic, and novel hypotheses of species level relationships in Korthalsia were put forward. These hypotheses of species level relationships in Korthalsia served as a framework for the better understanding of the evolution of ocrea. The morphological and developmental study of ocrea in genus Korthalsia included detailed study using Light and Scanning Electron Microscopy for seven samples of 28 species of Korthalsia, in order to provide understanding of ocrea morphological traits. The new phylogenetic hypothesis allowed homology of ocrea types to be tested. Ancestral reconstructions revealed that the inflated ocreas are not homologous; based on these reconstructions, it is suggested that ocreas that provide domatia for ants evolved more than once. Despite their non-homology, ocreas are remarkably uniform developmentally. New time calibrated trees estimated the divergence time for the Korthalsia crown node to be 19.89 MYR and ocrea to appear 17.06 MYR consistent with the published estimates for other groups. A taxonomic treatment of the ant Korthalsia species was based on material gathered from four herbaria and during the two field trips. Ten species were recognised in the treatment. Descriptions, keys and distribution maps to all species are presented. iii ACKNOWLEDGEMENTS In the name of Allah, Most Gracious, Most Merciful First of all, I would like to express my sincere gratitude to my both supervisors, Prof. Julie Hawkins (University of Reading) and Dr. William Baker (Royal Botanic Garden, Kew) for all continuous support, patience, motivation, advice and immense knowledge. I am immensely grateful to both of them for their guidance and for always believing in me. I am grateful to the staff of Royal Botanic Gardens, Kew for the access to their facilities. I would like to thank Dr. Paula Rudall, Chrissie Prychid and Maria Conejero, staff of Jodrell Laboratory for their advice and assistance with anatomical work. In Reading, my thanks goes to Dr. Stephen Ansel for his amazing help in the lab. Special thanks to Dr. John Dransfield and Palm Group at Kew for the lively discussion and friendship. In particular, I would like to thank Dr. Wolf Eiserhardt for his advice and for helping me analyses the data. My appreciation also extended to my officemates, Mazhani, Yati, Natali, Marco, Estevão, Deniz, Sawai, Paul, Naila, Riyadh, Aseel, Rahmah, and Lina for giving me motivation and helps in numerous ways. I would like to thank Dr. Asyraf Mansur and Dr. Rahmad Zakaria from University Science Malaysia, Dr. Saw Leng Guan and Dr. Richard Chung from Forest Research Institute Malaysia (FRIM) for providing me an excellent assistant during my field work. My thanks goes to Dr. David Middleton, Ms Serena Lee and Ms Bazilah for their hospitality during my visit to Singapore Herbarium and Singapore Botanic Garden, and Dr. Adrian Loo for helping me collected the specimens. To my dearest friend Azi Jamaludin, thank you for being you. If not for your advice and encouragement, I wouldn’t be here. To Ahmad Naqib and Kak Roziana I am forever be grateful for your help that you give me in many ways. I owed you guys a lot. To my scholarship sponsor the Ministry of Higher Education Malaysia (MOHE) and also my employer, University Malaysia Terengganu, thank you so much for believing in me and making it possible for me to complete my Ph.D in the University of Reading. Last but not least, I would like to express my gratitude and dedicate this thesis to my beloved family, who shown their tremendous support, unconditional love and infinite prayers. Thank you all. iv CONTENTS ABSTRACT .................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................. iv CONTENTS ...................................................................................................................... v LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ......................................................................................................... ix CHAPTER 1 ..................................................................................................................... 1 GENERAL INTRODUCTION ......................................................................................... 1 1.1 Overview ................................................................................................................. 1 1.2 Malesian region ....................................................................................................... 1 1.3 Palms ....................................................................................................................... 3 1.3.1 Palm classification and phylogeny ................................................................... 5 1.3.2 Subfamily Calamoideae .................................................................................... 8 1.3.3 Genus Korthalsia ............................................................................................ 10 1.4 Ant-plant relationships .......................................................................................... 12 1.4.1 Ant and rattan associations ............................................................................. 13 1.4.2 Ants and Korthalsia ........................................................................................ 15 1.5 Aims and thesis structure ...................................................................................... 17 CHAPTER 2 ................................................................................................................... 18 A PHYLOGENETIC FRAMEWORK OF THE CALAMOIDEAE .............................. 18 2.1 Introduction ........................................................................................................... 18 2.1.1 Objectives ........................................................................................................... 20 2.2 Material and methods ............................................................................................ 21 2.2.1 Taxon sampling .............................................................................................. 21 2.2.2 DNA isolation ................................................................................................. 21 2.2.3 DNA amplification and purification ............................................................... 21 2.2.4 DNA sequencing ............................................................................................ 30 2.2.5 Sequence assembly and alignment ................................................................. 31 2.2.6 Phylogenetic analyses ..................................................................................... 31 2.2.6.1 Maximum Likelihood .............................................................................. 31 2.2.6.2 Bayesian Inference ................................................................................... 31 v 2.3 Results ................................................................................................................... 33 2.3.1 Monophyly of the tribes and inter-tribal relationships ................................... 33 2.3.2 Monophyly of the subtribes ............................................................................ 39 2.3.3 Relationship between the subtribes ................................................................ 40 2.3.4 Species relationship with Calamus and Korthalsia ........................................ 41 2.3.4.1 Delimitation and subdivision of Calamus ............................................... 41 2.4 Discussion ............................................................................................................. 42 2.4.1 Evaluating higher-level classification ............................................................ 42 2.4.2 New insights into the relationships among subtribes and between genera .... 44 2.4.3 Species relationships within genera: Calamus and Korthalsia ...................... 45 CHAPTER 3 ..................................................................................................................