INTRASPECIFIC GENETIC VARIATION, POPULATION STRUCTURE, AND SPECIES DIVERSITY IN THE CARCHESIUMPOLYPINUMSPECIES COMPLEX A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by ELENI GENTEKAKI In partial fulfilment of requirements for the degree of Doctor of Philosophy December, 2009 © Eleni Gentekaki, 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Your Tile Votre reference ISBN: 978-0-494-64519-2 Our file Notre reference ISBN: 978-0-494-64519-2 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'Internet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. •+• Canada ABSTRACT INTRASPECIFIC GENETIC VARIATION, POPULATION STRUCTURE, AND SPECIES DIVERSITY IN THE CARCHESIUM POLYPINUM SPECIES COMPLEX Eleni Gentekaki Advisor: University of Guelph, 2009 Professor Denis H. Lynn Currently, the distribution of eukaryotic protists is a matter of intense debate. One side of the argument states that because of their small size, protists have the ability to disperse across geographic boundaries. Consequently, geographically structured populations and genetic variation are not expected. The other side argues that some of the bigger protists have limited dispersal and are therefore endemic with limited gene flow. The purpose of this thesis was to investigate the intraspecific genetic diversity of the ciliate Carchesium polypinum (Subclass: Peritrichia) and determine whether that diversity mapped onto the geography of the regions sampled. Sampling was carried out mostly in river basins of Southwestern Ontario, but samples were also obtained from British Columbia, North Carolina, and England. Three nuclear markers - internal transcribed spacer (ITS) 1, ITS2 and the hypervariable region of the large subunit rRNA (28S) - and one mitochondrial gene - cytochrome c oxidase subunit I {cox-1) - were used for the population-level analyses. This is the first time that the cox-1 gene is used on a ciliate other than Tetrahymena and Paramecium. The results indicated that C. polypinum harbors a large degree of genetic diversity; however, in most cases this diversity is not geographically partitioned. This is one of the first times that population genetics analyses are employed on protists. Furthermore, quantitative as well as qualitative analyses, based on both morphological characters and gene sequences of the small subunit rRNA (18S), uncovered robust evidence that C. polypinum is a cryptic species complex with no fewer than six members. ACKNOWLEDGEMENTS Completing my doctoral research and writing this thesis has been an adventurous journey, one that I would have never been able to complete without the help of some wonderful people. Dr. Lynn, when I first came to the department and we had our first meeting and you were telling me all about the research "holes" in the field, while pulling out thick volumes and old monographs, I felt that this was going to be a good experience. And was it ever! I have learned that in order to produce good quality work, a helpful and stimulating environment is required. Good ideas become great when they are developed and nurtured through discussions and when one is given freedom and encouragement to pursue them. But most importantly, I have learned that a strong work ethic and hard work are elemental for good science. You have been an outstanding mentor and I feel privileged to have worked with you. The members of my advisory committee, Drs. Elizabeth Boulding, Jingzhong Fu, and Patrick Boerlin, have been exemplary. Thank you for all your helpful suggestions and also for showing interest in my work. I appreciated your open door policy and the fact that you made me feel comfortable to just show up, ask questions, and discuss issues. Dr. Crease, even though you were not a member of my advisory committee you were always willing to see me and discuss problems that came up. Dr. Aggie Fernando, thank you for everything. I would not be here today writing this, if not for you. I owe you this and you will forever have my gratitude and respect. Rarely does one meet in the workplace someone who becomes a life long friend. Dr. Michaela Struder-Kypke you have been a mentor and a friend at the same time. You i are an amazing teacher and you taught me everything I know in phylogenetics. You have also been an incredible friend, there to listen to the good but also the bad. You are awesome and wonderful and I thank you for everything. Dr. John Clamp (NCCU, North Carolina) has been my collaborator for the past few years. Thank you so much for inviting me to North Carolina and making collection of samples in the area possible. I enjoyed our wonderful and enlightening discussions on these amazing organisms, the peritrichs. I have learned a lot from you. Drs. Chris Lobban (University of Guam) and Alan Warren (The Natural History Museum, UK), Laura Sanderson, and Chandni Kher have provided me with invaluable samples for my work and for that I am thankful. For this reason, I am also thankful to the Guelph wastewater treatment plant. Dr. Foissner (University of Leipzig) has provided me with constant feedback regarding morphology and taxonomy-related questions and for that, I am very grateful. In the background of every success there lurk the unseen heroes that keep all the strings going: Angela Holliss, Elizabeth Holmes, and Jeff Gross sequenced all of my samples in record time. Thank you all, you went above and beyond, taking a personal interest in my work and doing everything possible to make things work for me. You are not only outstanding professionals but also wonderful people. Mary Anne Davis, you are the guardian angel of graduate students, you keep things going for us all and you work so hard for arranging and organizing everything relating to our defenses. 11 A graduate student experience would not be the same without the friends that listen to your worries when things are not going so well or when they go great: Tasos Tsaousis, Chandni Kher, Vazrick Nazari, Beth Claire, John Wilson, Jill Smith, Chitchai Chantangsi, Megan Noyes, Chandler Andrews, Christy Carr, Erin Corstorphine, Kevin Kerr, you have enhanced my experience by far. I enjoyed sharing the woes and worries of our research, the uncertainty of the future but also our success stories. You have been great friends and I will miss you all. Last but not at all least I am indebted to my husband and daughter, the two most important people in my life. I did it, you guys!!!! I was able to do this, because of you. Without your constant support, encouragement, and patience I would have never made it. Thank you, you mean the world to me. iii TABLE OF CONTENTS CHAPTER ONE General Introduction INTRODUCTION 2 Why study protists 2 Alveolates 4 Phylum Ciliophora 5 The peritrichs 6 Phylogeography 8 Species identification in protists 15 Phylogeny of sessilid peritrichs 19 Research objectives 24 CHAPTER TWO High genetic diversity but no population structure of the peritrichous ciliate Carchesium polypinum in the Grand River basin (North America) inferred from nuclear and mitochondrial markers INTRODUCTION 27 MATERIALS AND METHODS 28 RESULTS 33 DISCUSSION 38 CHAPTER THREE Evidence for cryptic speciation in Carchesium polypinum Linnaeus, 1758 (Peritrichia) inferred from mitochondrial, nuclear, and morphological markers INTRODUCTION 53 MATERIALS AND METHODS 54 RESULTS 58 DISCUSSION 67 CHAPTER FOUR Spatial genetic variation among isolates of the peritrichous ciliate Carchesium polypinum in three river basins in Ontario, Canada INTRODUCTION 85 IV MATERIALS AND METHODS 87 RESULTS 90 DISCUSSION 97 CHAPTER FIVE A revised molecular phylogeny of the order Sessilida (subclass Peritrichia Ehrenberg, 1867) inferred using the small subunit (SSU) rDNA INTRODUCTION 116 MATERIALS AND METHODS 118 RESULTS 120 DISCUSSION 122 CHAPTER SIX Concluding discussion CONCLUSIONS 130 REFERENCES LITERATURE CITED 133 APPENDICES Appendix 1 153 Appendix 2 155 Appendix 3 156 Appendix 4 157 Appendix 5 160 Appendix 6 161 v LIST OF TABLES Table l-l...Peritrich sequences of 18S available in GenBank 22 Table 2-l...Intraspecific genetic divergence and standard errors of the cox-1 gene among five clades of Carchesium polypinum 35 Table 2-2... AMOVA analysis of the cox-1 gene sequences of Carchesium polypinum.31 Table 3-l...Intraspecific genetic divergence and standard errors of the cox-1 gene within five clades of Carchesium polypinum 60 Table 3-2..