The Systematic Revision of the Freshwater Red Algal Order Thoreales (Nemaliophycidae, Rhodophyta) A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Emily T. Johnston August 2012 © 2012 Emily T. Johnston. All Rights Reserved. 2 This thesis titled The Systematic Revision of the Freshwater Red Algal Order Thoreales (Nemaliophycidae, Rhodophyta) by EMILY T. JOHNSTON has been approved for the Department of Environmental and Plant Biology and the College of Arts and Sciences by Morgan L. Vis Professor of Environmental and Plant Biology Howard Dewald Interim Dean, College of Arts and Sciences 3 Abstract JOHNSTON, EMILY T., M.S., August 2012, Environmental and Plant Biology The Systematic Revision of the Freshwater Red Algal Order Thoreales (Nemaliophycidae, Rhodophyta) Director of Thesis: Morgan L. Vis The freshwater red algal order Thoreales has a worldwide distribution and contains two genera, Nemalionopsis with two species, and Thorea with four to 11 species recognized by various researchers. The controversy surrounding the number of Thorea species stems from a lack of discrete morphological characters to define taxa and little molecular data generated to date to inform phylogenetic studies. Previous research focused on morphology, placed most taxa in synonymy with either T. violacea or T. hispida, but also recognized T. zollingeri and T. clavata as distinct, leaving four morphological species in Thorea. Within Nemalionopsis, N. shawii was recognized, but N. shawii forma caroliniana was synonymized with N. tortuosa. A recent molecular study hinted at greater genetic diversity within the Thoreales than shown by the morphologically circumscribed taxa. The goal of this research was to determine the number of phylogenetic species within the order and assess biogeographic trends. Specimens were collected in Europe, temperate and sub-tropical North America, the Caribbean, South America, Eastern Asia and South Pacific Islands. Sequence data for 40 specimens were generated for the rbcL (chloroplast) gene, the LSU (nuclear) gene, and the COI barcode (mitochondrial). Bayesian Inference and Maximum Likelihood analyses indicated a much greater number of species within the Thoreales than currently 4 recognized from morphological analyses. This research has uncovered four species within Nemalionopsis and ten in Thorea; however, more diversity is suspected as both genera are probably under-collected worldwide. The taxon, T. hispida, is a cosmopolitan species with little genetic variation among specimens from four continents. All other taxa within the Thoreales appear to have restricted geographic ranges. Approved: _____________________________________________________________ Morgan L. Vis Professor of Environmental and Plant Biology 5 Acknowledgments So many people have helped with this research, and without them this project would not have been possible. I would like to thank my lab mates for all their encouragement, support, and advice: Wayne Chiasson, Sam Drerup, Lauren Fuelling, Maria Eugenia Garcia, Jonathan Gray, Emily Keil, Daryl Lam, Eric Salomaki, Mariah Thrush, and Alex Vandenbroek. Numerous people have also assisted me in the field and provided me with places to stay during my fieldwork. Their help cannot be underestimated: I would like to thank Siripen Traichaiyaporn, Ae Wilailak and Athiraj Iamsamang who hosted me in Thailand, fed me, and prayed that I had a safe journey throughout Southeast Asia. I would like to thank Nurliah Buhari (Ocha) for her constant help in the field and her dedication to finding freshwater algae across Indonesia, no matter how long the days, and her constant sense of good humor. I would like to thank Lim Phaik-Eem and Dr. Yong Hoi Sen for their help in Malaysia getting to field sites across the southern portion of the peninsula. And I would to thank Fisher Saint Amour for his expert photographic assistance, his constant companionship in the field, and his patience with weeks of field work. I would like to thank Dave Hillis and Robert McCurdy for hosting me at the Independence Creek Nature Preserve, helping me find freshwater algae in the spring system, the amazing sweet potatoes and spring water, and their wonderful hospitality and company. I would like to thank Kathy Yule and her husband and for letting me stay in their home without any time limit, the fantastic food, the amazing stories, and the constant advice about 6 collecting in Malaysia. I would like to thank Dr. Yule’s students for taking me with them on their collecting trip so I could explore nearby wetlands. Many people have also provided me with specimens, searched extensively for specimens in locations I could not get to myself, or put me touch people who could help me: Rosa Andreu, Julie K. Backus, Brittany A. Bianco, Bethany J. Blakely, Rossano Bolpagni, Chad M. Carroll, Dawn T. DeColibus, Kyatt and Rainbo Dixon, Kandai Doi, Garett D. Fruchey, Nicole M. Howard, Janina Kwandrans, Maggie M. Molnar, Orlando Necchi Jr., Emily A. Nebgen, Claude Payri, Josh A. Ryan, Siripen Traichaiyaporn, Robert Verb, Hereon Verbruggen, John West, Brian Whitton, and Xie Shulian. I would especially like to thank Mayalen Zubia, who has searched, and still searches, relentlessly for Thorea violacea on La Réunion. I would also like to thank everyone who provided me with advice, publications, and helped me through some difficult questions in my research: Austin Bowden-Kerby, Takeaki Hanyuda for sharing his knowledge of Thorea, R. James Hickey for his advice on species concepts, Ray Holton for his advice on collecting sites in New York, Shigeru Kumano, Orlando Necchi Jr. for advice on morphology, Thorea in general, many publications, and being available for questions whenever I needed to ask someone, Curt Pueschel for generously providing me with reprints, advice on collecting in New York, and support of and sincere interest in my project, Gary Saunders, for collecting information, constant advice on primers and PCR methodology, and even use of his unpublished primers, and John West, for advice and humor about freshwater reds and Thoreales from the South Pacific. 7 I would also like to thank my committee members: Harvey Ballard, who has always supported me, and believed I was capable of anything, and Shawn Kuchta, for his support, guidance, and always being just an email away when I had a question. Working in the Environmental and Plant Biology Department at Ohio University has also been an amazing experience and so many people have supported me and encouraged me. I would like to thank Martha Bishop for all her assistance with practical matters in the lab and always being there to listen. I would like to thank Dr. Cantino for his belief in me and encouraging me to become a systematist, setting me off on this crazy path. I would like to thank Connie Pollard for all her help with the little details that, if left incomplete, would have made my research impossible. And I would like to thank Dr. Wyatt for always being available to answer my questions, no matter how small they were, and for assistance in becoming a better writer. I would like to thank my friends and family members, who provided constant support, encouragement, and advice, understood when I didn’t return phone calls for weeks, and often provided many hours of free baby-sitting so I could continue my research: Leah Bound, Paul Bound, Jeremy Brenner, Melissa Brooks, Patty Contreras, Christine Far, Julie Johnston, Paul Johnston, and Scott Johnston. I would like to thank Joe Richards, for his unfailing support and belief in me. His encouragement has meant the world to me. And I would like to thank Fisher Saint Amour, despite that he thinks I have the most boring job in the world; he is the entire reason I went back to school. 8 I would like to thank the following herbariums for use of their collections: the Floyd Bartley Herbarium, the New York Botanical Garden, the Paris Museum of Natural History, and the University of Guam. I would also like to thank my funding sources that made this research possible in a very real way: the Phycological Society of America, Ohio University, and the National Science Foundation (RedToL). I would like to thank the Northeast Algal Society for the student book award Freshwater Red Algae of the World, which has been an invaluable tool in my research. And finally, I would like to thank my advisor, Morgan Vis, who has always believed in me, pushed me to work harder, encouraged me to pursue graduate school when I doubted myself, and been a friend and mentor. 9 Table of Contents Page Abstract ............................................................................................................................... 3 Acknowledgments............................................................................................................... 5 List of Tables .................................................................................................................... 10 List of Figures ................................................................................................................... 11 Introduction ....................................................................................................................... 12 Material and Methods ....................................................................................................... 20 Specimen Collection and Preservation ........................................................................