Administrative Seat: University of Sfax, Tunisia University of Messina, Italy National School of Engineers of Sfax Department of Chemical, Biological, Biological Engineering Department Pharmaceutical and Environmental Sciences Unité de Biotechnologie des Algues Doctorate in Applied Biology and Doctorate in Biological Engineering Experimental Medicine – XXIX Cycle DNA barcoding identification of the macroalgal flora of Tunisia Ramzi MILADI Doctoral Thesis 2018 S.S.D. BIO/01 Supervisor at the University of Sfax Supervisor at the University of Messina Prof. Slim ABDELKAFI Prof. Marina MORABITO TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................... 3 ABSTRACT ..................................................................................................................... 6 1. INTRODUCTION ...................................................................................................... 8 1.1. SPECIES CONCEPT IN ALGAE ..................................................................................... 9 1.2. WHAT ARE ALGAE? ................................................................................................. 11 1.2.1. CHLOROPHYTA ....................................................................................................... 12 1.2.2. RHODOPHYTA ........................................................................................................ 13 1.3. CLASSIFICATION OF ALGAE ..................................................................................... 15 1.4. SPECIES DELIMITATION BY A DNA BARCODING APPROACH .................................. 16 1.4.1. ALGAE IDENTIFICATION USING DNA BARCODING .................................................. 17 1.4.2. DNA BARCODING APPLICATIONS ........................................................................... 18 1.4.3. LIMITATIONS OF BARCODING ................................................................................. 19 1.5. SPECIFIC BIODIVERSITY OF MARINE FLORA IN TUNISIA ........................................ 21 1.5.1. GEOGRAPHICAL POSITION ...................................................................................... 21 1.5.2. INVENTORY ............................................................................................................ 21 1.5.3. ALIEN SEAWEED INTRODUCTION ............................................................................ 22 1.5.4.1. Natural introduction. .......................................................................................... 23 1.5.4.2. Anthropogenic introduction ............................................................................... 23 2. AIM OF THE THESIS ............................................................................................. 26 3. MATERIALS AND METHOS ................................................................................ 28 3.1. SPECIMEN COLLECTION AND PRESERVATION ......................................................... 29 3.1.1. COLLECTING LOCATIONS ON THE TUNISIAN COASTS ............................................. 29 3.1.2. PRESERVATION ...................................................................................................... 31 3.2. MOLECULAR TECHNIQUES ...................................................................................... 32 3.2.1.1. DNA EXTRACTION PROTOCOL FOR RED ALGAE (WIZARD ® DNA CLEAN UP SYSTEM, PROMEGA) ........................................................................................................................ 32 DNA EXTRACTION RECIPES APPENDIX 2. ........................................................................ 33 3.2.1.2. DNA EXTRACTION PROTOCOL FOR GREEN ALGAE (CTAB) ..................................... 33 3.2.2.1. POLYMERASE CHAIN REACTION ............................................................................ 35 4. DNA BARCODING SHADES LIGHT ON NOVEL RECORDS IN THE TUNISIAN RED ALGAL FLORA (SUBMITTED) .................................................. 39 5. A DNA BARCODING SURVEY OF ULVA (CHLOROPHYTA) IN TUNISIA REVEALS THE PRESENCE OF THE OVERLOOKED ALIEN U. OHNOI (ACCEPTED FOR PUBLICATION). ...................................................................... 120 6. CONCLUSIONS ..................................................................................................... 154 1 7. REFERENCES ........................................................................................................ 157 APPENDIX 1. LIST OF SPECIMENS ..................................................................... 179 A.1.1. PROJECT: TUGRE - GREENS OF TUNISIA (PHYLUM CHLOROPHYTA) ............ 180 A.1.2. PROJECT: TURED - REDS OF TUNISIA (PHYLUM RHODOPHYTA) ................... 181 APPENDIX 2. DNA PROTOCOLS .......................................................................... 183 A.2.1. DNA EXTRACTION RECIPES .............................................................................. 184 A.2.2. POLYMERASE CHAIN REACTION RECIPES ........................................................ 186 A.2.3. AGAROSE GEL ELECTROPHORESIS RECIPES ..................................................... 187 2 Acknowledgements Acknowledgements 3 Acknowledgements First and above all, I praise God, the almighty for providing me this opportunity and granting me the capability to proceed successfully. Undertaking this PhD has been a truly life- changing experience for me and it would not have been possible to do without the assistance and support that I received from many people. Foremost, I would like to express my sincere gratitude to my Tunisian supervisor, Prof.Slim Abdelkafi who has been a tremendous mentor for me. I’m grateful to him for accepting me into his group, for his constant support, motivation and allowing me to grow as a research scientist. I have been extremely lucky to have a supervisor who care so much about my work and I would never have been possible for me to take this work to completion without his support and encouragement. Special and heartily thanks to my Italian supervisor Prof. Marina Morabito for accepting me into her group, for her patience, motivation, enthusiasm and immense knowledge in DNA barcoding approach. It is with her supervision that this work came into existence. Her guidance helped me in all the time of research and correction of this thesis. As my supervisor, she has constantly forced me to remain focused on achieving my goal. I’m greatly indebted to all the persons at both the University of Sfax and the University of Messina who agreed to conclude the co-tutorship agreement that allowed me to compete for a joint PhD degree especially to Prof. Salvatore Cuzzocrea, Coordinator of the PhD in Applied Biology and Experimental Medicine at Messina. Furthermore, I am thankful to the funding sources EMMAG organization that allowed me to pursue my studies in Messina. Dr. Angela Garozzo at the foreign PhD Secretary and Dr. Francesca Pollicino at the Erasmus office were very helpful and remarkably kind with me during my entire stay in Messina. My sincere thanks also go to Dr. Antonio Manghisi for his advice and for acting as a mentor to me. I’m deeply grateful to him who gave me the opportunity to serve as a Teaching Assistant in his courses during my thesis. I gained a lot from his vast Biology knowledge and scientific curiosity. 4 Acknowledgements I would also like to thanks Prof. Giusi Genovese for her guidance, encouragement, friendship and support during the whole period of the study. I never forget the opportunity that she gave to me to work on evaluation of antibacterial activity of algae against pathogens relevant in aquaculture, to improve my personal skills. In my daily work, I have been blessed with a friendly and cheerful group of Professors, Technicians and fellow students in Messina and Tunisia. They made a cooperative atmosphere at work and also useful feedback and insightful comments on my work. I am especially grateful to Dr. Fakher Kamoun and Dr. Mohamed Ali Ayadi for their incredible support and encouragement. I would also like to thank my external referees for their interest in my work and for their very valuable comments and suggestions on this thesis. Finally, last but not the least, I would like to thank my family: the most special thanks go to my Mom, who took the lead to heaven before the completion of this work. Words cannot express how grateful I am to her for encouraging me to follow my dreams. Her prayer for me was what sustained me thus far. Also, this thesis is heartily dedicated to the memory of my father Sleim. I miss him every day, but I’m glad to know he saw this process through to its completion. I never forget the sacrifices that he made on my behalf. I warmly thank and appreciate my brother Najib for his material and spiritual support throughout my years of study. Also, I’m grateful to my brother Ahmed and my two lovely sisters Ola and Mejda for their encouragement and assistance in numerous ways. I can just say thanks for my two best friends of my life Heni and Houcine who helped me in algae collection and may Allah give them all the best in return. Thank you. Ramzi MILADI 5 Abstract Abstract 6 Abstract Tunisia has a key position in the Mediterranean and constitutes a transition area with a rich habitat diversity between eastern and western basins. The latest inventory of marine macrophytes dates back to 1987, updated in 1995. The target of this thesis was to carry on a molecular-assisted alpha taxonomy (MAAT) survey of macroalgae, which allowed