0 Ghent University Faculty of Sciences, Department of Biology Phycology Research group Diversity of the marine red alga Portieria in the Philippines, an integrative approach Dioli Ann Payo Promotor: Prof. Dr. O. De Clerck Thesis submitted in partial fulfillment Co-Promotors: Prof. Dr. H. Calumpong of the requirements for the degree of Dr. F. Leliaert Doctor (PhD) of Sciences (Biology) 26 September 2011 i EXAM COMMITTEE ______________________________ Members of the reading committee Dr. Line Le Gall (Muséum National d'Histoire Naturelle, Paris) Prof. Dr. Ludwig Triest (Vrije Universiteit Brussel) Dr. Yves Samyn (Koninklijk Belgisch Instituut voor Natuuurwetenschappen) Members of the examination committee Prof. Dr. Dominique Adriaens (Chairman Pre-Defense, Ghent University) Prof. Dr. Koen Sabbe (Chairman Public Defense, Ghent University) Prof. Dr. Olivier De Clerck (Promotor, Ghent University) Prof. Dr. Hilconida Calumpong (Co-Promotor, Silliman University, Philippines) Dr. Frederik Leliaert (Co-Promotor, Ghent University) Prof. Dr. Annemieke Verbeken (Ghent University) Dr. Heroen Verbruggen (Ghent University) _______________________________________________________ The research reported in this thesis was funded by the Flemish Interuniversity Council (VLIR) and the Global Taxonomy Initiative, Royal Belgian Institute of Natural Sciences. This was performed at the Phycology Research Group (www.phycology.ugent.be) and at the Institute of Environment & Marine Sciences, Silliman University, Philippines. ii iii ACKNOWLEDGEMENTS First of all, I would like to acknowledge the people who made it possible so I could start with this PhD project on Portieria. It all started from Prof. Olivier De Clerck‘s discussion with Prof. John West about this alga. Next thing that happened was the endorsement of Prof. West and Prof. Lawrence Liao of my application for VLIR PhD scholarship. Prof. Dr. Eric Coppejans has supported my PhD application and has always welcomed me in his lab since 2003 when I started to work on Dictyota. A good number of Prof. Coppejans‘ Portieria collections had been included in this work. I would also like to thank my co-promoter in the Philippines Prof. Dr. Hilconida Calumpong, for her confidence in me. Despite of not knowing me in 2005, she accepted my proposal and supported my PhD scholarship application. Prof. Calumpong‘s support on this research ranged from advice on experimental design and field work, reserving an exclusive room and an outdoor space for my experimental work at the Silliman laboratory, and reviewing my manuscript drafts. Prof. Calumpong also gave me opportunities for exposure by inviting me to present during the Friday laboratory meetings. I would also like to mention the help extended by her entire staff, especially Jacinta Lucañas, Julieta Echaves and Mrs. Pacita Raterte for lab equipment and research fund concerns, and the generally friendly lab atmosphere created by her entire staff and PhD students. Annalie Candido, Rio Naguit, Wilson Tisera, Amida Diwata Macansantos also helped to collect Portieria at Apo Island, Bunaken Island, Indonesia, and Spratley Islands. Zaccharias “Nong Iyas” Generoso has been a great assistance in the field, in my grazing experiments and who entertained me with his amusing field research stories in the old days. I am also indebted to Abner Bucol who assisted me for two years even during rough seas in my monthly surveys in Siquijor and Siaton and during my first trip to Batanes. Apart from allowing me to hire their boats, keen fishermen from Siquijor suggested good sites for Portieria. Some friends from college and NGO days also got dragged into this affair of collecting Portieria, like Rey Ladiao, Florabel Fumar, Bernie Caliñajan, William Villaver, Peddy Caet and Nonoy of Liloan, Cebu. Thanks are also due to Basco, Batanes Mayor Manuel Viola who forwarded my letter of intent to collect to the Municipal Environment Officer, Francis Dominggo, who in turn assisted during the Batanes collections. Generous phycologists from different institutions have also enriched the number of my samples such as Alison Sherwood, Tom Cowling, Max Hommersand, Willem Prud’homme Van Reine, Stefano Draisma, Tom Schils, Diane and Mark Littler. David Reid Wiseman generously sent a copy of his PhD thesis of 1973 on Ochtodes and Chondrococcus. iv In Ghent, despite of all the scheduling difficulties and the technical troubles, Renaat Dasseville made it possible that I could use the GC-MS. I am also indebted to him for the GC-MS knowledge that he imparted. Joannamel Colo, the Ecomama thesis student I supervised, performed the traditional measurements on Portieria and also a portion of the chemical work. Frederic Lynen of the chemistry department also welcomed me in his lab and allowed me to use HPLC and GC-MS machines during the exploratory period of my Portieria extracts. I am also sincerely thankful to all the members of the Phycology Research Group for the generosity with their time, knowledge and all the help of any form extended to me during my PhD years. My DNA extraction, PCR and DNA sequencing work had been greatly assisted by Caroline Vlaeminck and Sofie D’hondt. Klaas Pauly and Heroen Verbruggen have also contributed to my Portieria samples. Special thanks are also due to the members of the brainstorming team who helped shape the direction of the molecular aspect of this research composed of Heroen Verbruggen, Frederik Leliaert and Olivier De Clerck. I greatly appreciate the administrative help provided by Christelle Vankerckhove but most of all her kindness, warmth and motherly demeanor. I shall also thank Agniezka Lipinska, Frédérique Steen, and Ana Tronholm, my lab roommates, for bringing fun and the wonderful and comforting conversations. I greatly appreciate all the kindness and help in the analyses and manuscript writing of my co-promoter Frederik Leliaert, from whom I learned a lot. I admire his effective way of transferring knowledge. Finally, I am greatly indebted to my promoter, Olivier De Clerck for believing that I can do a PhD, for all these learning opportunities (too many to mention) that he made possible for me, the supervision since my master‘s thesis period (which resulted to a best thesis award from VLIZ), during the thesis proposal writing and for always pushing me to do better. I appreciate the keenness he gave in every aspect of my work and also that ―hands-on‖ training. I leave my sincerest thanks to these promoters and will take with me all the lessons I have learned from them here in Ghent. I thank my thesis committee for the academic dedication exercised on reviewing the pre-defense version of this manuscript. It has made me realize the important role of a critique committee in improving the quality of an academic work. Portieria research would not have been possible without the PhD scholarship provided by the Flemish Interuniversity Council (VLIR) from 2006 to 2010. VLIR also funded my scholarship for a master‘s degree in Ecological Marine Management at the Free University of Brussels (VUB) from 2002 to 2004. The Global Taxonomy Initiative under the Royal Belgian Institute of Natural Sciences came to the rescue through Dr. Yves Samyn by providing fund for my trip back to and living allowance in Belgium in 2011. v Claire Van Maercken, my landlady of four years in Ghent, deserves sincere appreciation for a service to foreign students, homely accommodation and the conversations. My family gave inspiration and extended much needed help during the sampling and labwork in Silliman. -D.A. Payo- September 2011 Ghent, Belgium This academic work was performed in collaboration with Silliman University – Institute of Environmental and Marine Studies, Filipino biologists and locals of sampling sites in the Philippines. Prior informed consent was obtained from the mayor and the municipal environment natural resources officer of Basco. Portieria species have no economic value and are not classified by CITES (IUCN) as threatened or endangered. Export permit has been granted by the Bureau of Fisheries and Aquatic Resources. All other Indo-Pacific specimens have been generously donated by phycologists from different institutions. vi vii CONTENTS Exam Committee ...................................................................................................... ii Acknowledgements ................................................................................................. iv Contents .................................................................................................................. viii Abbreviations and Acronyms ............................................................................... xii List of Tables .......................................................................................................... xiv List of Figures ........................................................................................................ xvi 1 General introduction........................................................................................ 1 1.1 Marine biodiversity and secondary metabolites ............................................. 1 1.2 Portieria: a taxonomic perspective ..................................................................... 5 1.3 Portieria hornemannii .............................................................................................. 7 1.4 Cryptic species diversity ................................................................................... 10 1.4.1 Species delimitation approaches ......................................................