SYSTEMATIC STUDIES IN THE TYLOPHORINAE (ASCLEPIADEAE: ASCLEPIADOIDEAE: APOCYNACEAE) WITH REFERENCE TO PAKISTANI TAXA By SAYED AFZAL SHAH 03041411001 DEPARTMENT OF PLANT SCIENCES QUAID-I-AZAM UNIVERSITY ISLAMABAD, PAKISTAN 2018 SYSTEMATIC STUDIES IN THE TYLOPHORINAE (ASCLEPIADEAE: ASCLEPIADOIDEAE: APOCYNACEAE) WITH REFERENCE TO PAKISTANI TAXA BY SAYED AFZAL SHAH A THESIS SUBMITTED TO THE QUAID-I-AZAM UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN PLANT GENETICS AND GENOMICS DEPARTMENT OF PLANT SCIENCES QUAID-I-AZAM UNIVERSITY ISLAMABAD, PAKISTAN 2018 FOREIGN EVALUATORS OF THE THESIS Professor Hiroshi Tobe 1. Department of Botany, Faculty of Biology, Kyoto University; Kyoto, Japan Dr. David Goyder 2. Royal Botanical Gardens, Kew, UK He Who has, made for you the earth like a carpet spread out; has enabled you to go about therein by roads (and channels); and has sent down water from the sky." With it have We produced diverse pairs of plants each separate from the others (Quran, Surah Ta Ha, 53). DEDICATION To my late father (1946-1994) To my mother who always prayed for my success My late grandfather (1920s-2018) To my brothers, sisters, wife and uncles for their Understanding and support during my studies Acknowledgments I have no words to express my deepest sense of gratitude to the gracious, the greatest “Almighty Allah”, the all-powerful, the omnipresent, the most merciful and most compassionate, whose innumerable blessings enabled me to complete this difficult task. I also pay all the tributes to the Holy Prophet Muhammad (PBUH), the most perfect and exalted among and of ever born on the surface of the earth for enlightening our conscience. I extend wholehearted gratitude to my Supervisor Dr. Abdul Samad Mumtaz, for his inspirational guidance, keen interest, scholastic supervision, encouragement, valuable suggestions and direction right from the start till the end of the present study. I also wish to express deepest and sincerest thanks to my co-supervisor Dr. Amir Sultan (in charge National Herbarium, Pakistan) for his constant support, useful suggestions, valuable discussions, and provision of research material. I would also like to extend whole hearted gratitude to my host supervisor Dr. Jun Wen (National Museum of Natural History, Department of Botany NMNH, Smithsonian Institution; Washington DC, USA) for her excellent support, providing access to the excellent facilities at NMNH including Laboratory of Analytical Biology, laboratory of SEM, laboratory of Anatomy and National Herbarium of USA. I would like to extend my sincere appreciation to all the people and their representative institutions from where the research material was acquired and experimental work done viz., National Herbarium of Pakistan, and all the curators of the herbaria including Karachi University herbarium Pakistan, Herbarium of Islamabad, Quaid-i-Azam University, Pakistan Museum of Natural History, Pakistan Forest Institute, Hazara University, National Herbarium of USA (Smithsonian Institution), Missouri Botanical Garden (USA), New York Botanical Garden (USA), Grey Herbarium of Harvard University (USA), British Museum (UK), Royal Botanical Gardens, Kew, London (UK), Herbarium Gottingen (Germany) and Botanical Garden and Botanical Museum Berlin- Dahlem (Germany). Thanks are also extended to Mr. Johnson Gabriel for help with molecular studies, Mr. Scott Whittaker for kind help with SEM and Mr. Stanley Yankowski for help in light microscopy. I extend sincere thanks to Atta Ullah, Asad Ullah, Fazli Rahman, Barkat Ali, Baz Gul, Mujeeb ur Rahman, Imran Ahmad, Raees Khan, Murtaza Turi, Mahmud Ullah and Wahid Hussain for great help during field work. I am highly thankful to Miss. Surat un Nisa for her help in data compilation and making excellent plant illustrations. Mr. M. Salim is also acknowledged for his contribution to the plant illustrations. I am grateful to Dr. Sigrid Liede-Schumann, Dr. Tadashi Yamashiro and Dr. Zahid Ullah for their kind help, guidance and fruitful suggestions. I feel utmost pride in expressing my deepest sense of gratitude to my best friends M. Maqsood Alam, Iftikhar Ali, Hussain Badshah, Zain ul Abedin, M. Ilyas, Javed Iqbal, Amir Shahbaz, M. Usman, Imran Ali, Sana Ullah, Raees Khan, Riaz Ahmad Khan, Ijaz Aziz, Wasim Akhtar, Abbas Ullah, Zia ul Islam, Nasar Khan, Waqar Ahmad and Abdullah for their excellent company during my stay at university. I would also like to thank all the past and present members of the Laboratory of Genetics and Genomics, and colleagues of the department of Plant Sciences and Quaid-i-Azam University. I extend sincere appreciation to my colleagues Xu Su, Yousheng Chen, Xu Yuan, AJ Harris, Zhumei Ren, Xiodan Xu, Zhang Wei and Jioyo Feng at National Herbarium of USA. Finally, I must specially acknowledge the Higher Education Commission of Pakistan for funding the six month visit to Smithsonian Institution without which this project would not have been completed in its current shape. Sayed Afzal Shah About this thesis This thesis comprises seven chapters Chapter one is a general introduction of the family Apocynaceae, Asclepiads (Asclepiadoideae), Tylophorinae and the study area (Pakistan). Chapter two reviews the existing literature on systematics of Tylophorinae Chapter 3–6 contain the results of taxonomic revision, palynology, molecular phylogeny and phytogeography respectively. Each of these chapters are written in the form of a manuscript comprising abstract, introduction, materials and methods, results and discussion. Chapter three is taxonomic revision hence, not discussed. Chapter seven comprise a combine discussion (synthesis) of all the chapter 3–6 and future recommendations. The bibliography was arranged in Endnote X6. The format of bibliography is followed after Plant Pathology. The National Assembly of Pakistan passed a constitutional amendment on 24th May 2018 seeking the merger of Federally Administered Tribal Areas (FATA) into Khyber Pakhtunkhwa (KP) province. The FATA merger bill was also approved by provincial assembly of KP on 27th May 2018. Therefore, the name FATA is not used in this thesis. The names of the FATA agencies are recognized in KP. CONTENTS CHAPTER ONE: INTRODUCTION ................................................................................... 1 1.1 Apocynaceae—an introduction ............................................................................... 1 1.2 Origin and classification history of Apocynaceae .................................................. 1 1.3 Sub-family Asclepiadoideae (the asclepiads) ......................................................... 2 1.4 The subtribe Tylophorinae—an introduction ......................................................... 4 1.5 Economic importance .......................................................................................... 5 1.6 Pakistan—the study area ......................................................................................... 6 1.6.1 Geography, environment and climate .............................................................. 6 1.6.2 Mountain ranges and floristic elements ........................................................... 7 1.7 Problem elucidation ................................................................................................ 8 1.8 Aims and objectives of the study ............................................................................ 9 References ....................................................................................................................... 12 CHAPTER TWO: LITERATURE REVIEW ..................................................................... 21 2.1 The systematics of subtribe Tylophorinae ............................................................ 21 2.1.1 Vincetoxicum: disentangling from Cynanchum and linking to Tylophora .... 21 2.1.2 The advent of molecular data in systematics of Tylophorinae ...................... 23 2.1.3 The intra-Tylophorinae phylogenetic relationships ....................................... 24 2.2 The taxonomy of Tylophorinae ............................................................................ 26 2.3 Biogeography of Tylophorinae ............................................................................. 26 2.4 Pollinaria and pollination biology ......................................................................... 27 2.4.1 Pollinarium morphology ................................................................................ 27 2.4.2 The pollination systems ................................................................................. 27 2.5 Characterization of seed surface ........................................................................... 28 2.6 Allozyme loci and microsatellite markers ............................................................ 28 2.7 Prediction of potential climatic niches .................................................................. 29 2.8 References ............................................................................................................. 32 CHAPTER THREE: TAXONOMIC REVISION OF PAKISTANI TYLOPHORINAE ... 37 3.1 Abstract ................................................................................................................. 37 3.2 Introduction ........................................................................................................... 37 3.3 Materials and methods .........................................................................................