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This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Conserving the biodiversity of Kuwait through DNA barcoding the flora A thesis submitted for the degree of Doctor of Philosophy Mansour Abdullah Institute of Molecular Plant Sciences School of Biological Sciences University of Edinburgh & Royal Botanic Garden Edinburgh July 2017 Declaration: I hereby declare that the work contained in this thesis is my own, unless otherwise acknowledged and cited. This thesis has not in whole or part been previously presented for any degree. Mansour Abdullah 25th July 2017 i Abstract Biodiversity across the globe is threatened. Rapid surveying and monitoring techniques are required to understand the origin of the threats to biodiversity and to enable conservation actions to be undertaken. Kuwait is an arid desert country with a small flora of only 402 species. This flora is endangered by environmental factors, overgrazing, and human activities. DNA barcoding the flora and using Next Generation Sequencing (NGS) technologies allowed us to identify plants to species level, conduct a molecular taxonomic revision, and distinguish plant diversity found in soil environmental DNA samples. After investigating the discriminatory power of five commonly used DNA markers from plastid (matK, rbcL, trnH-psbA, trnL) and a nuclear genome (ITS2) on four largest genera of the flora using phylogenetics reconstruction tree based methods, two barcoding markers (rbcL and ITS2) were assigned to build a DNA reference library of the flora. Furthermore, the DNA reference library was tested to identify the plant diversity found below-ground level and comparing it with that above-ground, using environmental soil samples collected from both species rich and poor habitats in Kuwait by applying high-throughput sequencing methods. The DNA database provided in this study could be used as a reference library for the identification process and contribute towards the future of molecular taxonomy, biodiversity and ecological research in Kuwait. ii Acknowledgments I would like to give my sincere thanks and gratitude to my supervisors, Catherine Kidner, Tony Miller and Pete Hollingsworth, who have been supportive throughout my project and providing invaluable knowledge, skills and assistance which added a lot to my academic experience. It was a great privilege to have worked under their supervision. I am grateful to CMEP-RBGE, especially Sabina Knees, Sophie Neale and Alan Forrest for their kind support and professional guidance throughout my project. Special thanks to STS-RBGE, Michelle Hart, Laura Forrest, Ruth Hollands and Frieda Christie for expert advice, training and support in the lab. I would like to express my appreciation and thanks to the RBGE Herbarium staff, especially David Harris, for the support, guidance and for processing my herbarium specimens and digitizing. Many thanks to Stephan Helfer, for guiding me through the permit process to import my environmental samples. I owe my deepest gratitude to K.T. Mathew for his kindness, encouragement and support during my field visits and sampling specimens from Kuwait University Herbarium. Many thanks to all staff members and Ph.D. students at RBGE and UoE who have given help and support throughout my academic years. Special thanks to the Government of Kuwait and PAAET for financial support and funding my Ph.D. at the University of Edinburgh. I am eternally grateful to my kind parents, Fauzia and Taleb, for the support and prayers of encouragement and faith throughout my entire life. Many thanks to my sister, Alia, and brothers, AbdulNasser, Mohammad, Manaf and Ali for the motivation, inspiration and the support they always provided. Infinite thanks to my wife, Eman and my sons, Adam and Jassem for their support and encouragement and being patient while I’m away pursuing further studies. Many thanks to all my cousins and friends for their spiritual support, especially Ali Dashti, Musaed Abdullah, Mohammad Al-Mousa, Meshal Abdullah, Taher Al Baghli, Hasan Al-Abdullah, Ahmad Al-Hasan, Essa Buhamad. Above all, I thank God ‘Allah’, the most gracious and the most merciful, for providing me with knowledge, strength and energy to complete my Ph.D. iii Table of Contents Declaration…………………………………………………………………………...i Abstract…..…………………………………………………………………………..ii Acknowledgements……...………………………………………...…………………iii Chapter 1 - Introduction 1.1 Aims of the thesis and chapter overview ............................................................. 1 1.2 Geography, Topography and Geology, and Soils of Kuwait ................................ 3 1.2.1 Geography .................................................................................................... 3 1.2.2 Topography and Geology ............................................................................. 5 1.2.3 Soils ............................................................................................................. 7 1.3 Climate ............................................................................................................. 10 1.4 Phytogeography ................................................................................................ 11 1.5 Vegetation of Kuwait ........................................................................................ 13 1.5.1 Distribution of the vegetation ..................................................................... 14 1.5.2 Present vegetation ....................................................................................... 15 1.5.2.1 Changes to plant communities .............................................................. 17 1.6 Conservation and threats ................................................................................... 18 1.6.2 Natural regeneration of vegetation .............................................................. 19 1.6.3 Active restoration of vegetation .................................................................. 20 1.6.4 Biological soil crusts .................................................................................. 21 1.6.5 Conservation through protected areas ......................................................... 22 1.7 Natural resources and land management ........................................................... 25 Chapter 2 - Checklist of the flora of Kuwait 2.1 Current knowledge of the flora of Kuwait ......................................................... 29 2.1.2 Botanical Exploration of Kuwait ................................................................ 31 2.2 Methods ............................................................................................................ 33 2.3 Results .............................................................................................................. 36 2.3.1 Floristic analyses of the flora of Kuwait ...................................................... 48 2.3.2 Native and naturalised vascular plants of Kuwait ......................................... 52 iv 2.4 Discussion ........................................................................................................ 54 2.4.1 Native plants ............................................................................................... 54 2.4.2 Cultivated plants.......................................................................................... 55 2.4.3 Weedy plants ............................................................................................... 56 2.4.4 Conclusion .................................................................................................. 56 Chapter 3 - Choice of gene regions 3.1 Introduction ...................................................................................................... 58 3.2 Materials and methods ...................................................................................... 62 3.2.1. Sampling material ...................................................................................... 62 3.2.2 DNA extraction, amplification and sequencing ............................................ 64 3.2.3 Sequence editing, alignment and molecular analysis .................................... 67 3.3 Results .............................................................................................................. 69 3.3.1 DNA recoverability, amplification and sequences success. .......................... 69 3.3.2 Sequence quality and alignment................................................................... 71 3.3.3 BLASTn searches ........................................................................................ 73 3.3.4 Species discrimination ................................................................................. 75 3.4 Discussion .......................................................................................................
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