MOLECULAR PHYLOGENETICS OF TRICHONYMPHA CF. COLLARIS AND A PUTATIVE PYRSONYMPHID: THE RELEVANCE TO THE ORIGIN OF SEX by JOEL BRYAN DACKS B.Sc. The University of Alberta, 1995 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER'S OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1998 © Joel Bryan Dacks, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ~2—oc)^Oa^ The University of British Columbia Vancouver, Canada Date {X^ZY Z- V. /^P DE-6 (2/88) Abstract Why sex evolved is one of the central questions in evolutionary genetics. To address this question I have undertaken a molecular phylogenetic study of two candidate lineages to determine the first sexual line. In my thesis the hypermastigotes are confirmed as closely related to the trichomonads in the phylum Parabasalia and found to be more deeply divergent than a putative pyrsonymphid. This means that the Parabasalia are the first sexual lineage. From this I go on to infer that the ancestral sexual cycle included facultative sex. The relevance of these inferences is then examined with respect to the current theories on the origin of sex. ii Table of contents Abstract • ii List of Tables iv- List of Figures :v Acknowledgements vii CHAPTER I Introduction 1 General question 1 Theories on the origin of sex 4 Phylogenetics 8 Eukaryote phylogeny.... 13 Order of branches at the base of the eukaryotic tree..l4 Specific questions to be addressed 16 CHAPTER II Phylogenetic placement of the oxymonads 22 Introduction 22 Materials and Methods 28 Results 34 CHAPTER III Phylogenetic placement of the hypermastigotes 58 Introduction 58 Materials and Methods 64 Results 69 CHAPTER IV Discussion 92 Overview 92 Oxymonad project 92 Hypermastigote project ....96 The ancestral sexual cycle and its infered traits 101 • •• ill List of Tables Table # Title Page# Table 1 Guide to taxonomy of some organisms in this thesis 21 Table 2 Predicted fragment size from likely gut symbionts. 56 Table 3 Three classes of clones from PCR replicate 4 57 Table 4 Internal sequencing primers 90 Table 5 Organisms and accession numbers : Chapter Three 91 Table 6 Morphological traits : metamonads and P. lanterna 115 iv List of Figures Figure # Title : Page# Figure 1 Effect of sex on variance 17 Figure 2 "Monophyletic" and "Paraphyletic" diagramatically explained 18 Figure 3 The six to eight kingdom classification of Eukaryotes 19 Figure 4 Basal branches of the Eukaryotic tree 20 Figure 5 Isolation of Pyrsonympha sp. from R. hesperus hindgut 44 Figure 6 PCR products from replicates 1 and 2, Chapter Two 45 Figure 7 Sequence of putative pyrsonymphid 46 Figure 8 Top seven BLAST hits from the putative pyrsonymphid 47 Figure 9 Parsimony tree, Chapter Two 48 Figure 10 Neighbour joining distance tree, Chapter Two 49 Figure 11 Restriction analysis of clones PI 2, P7, P4, P9 50 Figure 12 PCR products from replicates 3 and 4, Chapter Two 51 Figure 13 Restriction digestion of clone from replicate 3, Chapter Two 52 Figure 14 Restriction digestion of clones from replicate 4, Chapter Two 53 Figure 15 BLAST results from sequence of clone L20 54 Figure 16 BLAST results from sequence of clone U39 55 Figure 17 Trichonympha cf. collaris 74 Figure 18 Sexual cycle of Trichonympha from Cryptocercus 75 Figure 19 Evolution of the Parabasalans 76 Figure 20 Isolation of T. cf. collaris from Z. angusticollis 77 Figure 21 Trichonympha sp. viewed using phase contrast microscopy 78 Figure 22 PCR products from amplification, Chapter Three 79 Figure 23 Trichonympha cf. collaris ssu rRNA gene sequence 80 Figure 24 BLAST results from Trichonympha cf. collaris sequence 81 Figure 25 T. cf. collaris and R. flavipes gut symbiont 2 ssu rRNA 82 V Figure 26 Neighbour joining tree, Chapter Three 83 Figure 27 Parsimony tree, Chapter Three 84 Figure 28 Trichonympha cf. collaris probed with universal probe 85 Figure 29 Trichonympha cf. collaris incubated with the no-probe control 86 Figure 30 T. cf. collaris and S. strix incubated with specific probe 87 Figure 31 Trichomonas and S. strix probed with universal probe 88 Figure 32 S. strix and Trichomonas incubated with the no-probe control 89 Figure 33 Maximum Likelihood tree of E.F.-a sequences 112 Figure 34 Simplified Eukaryotic tree correlated with frequency of sex 113 Figure 35 Hypothetical obligate sexual cycle in a unicellular organism 114 VI Acknowledgements I would like to thank my committee members for their time and good advice. As well, I want to thank the people at Interior Pest Control for providing Reticulitermes specimens. Thanks also to Ema Chao and Hong Zhang for providing DNA for my PCR positive controls. There are many additional people around the university that I would like to thank for their help, academic and otherwise. You all know who you are and I am greatful to each of you. I would like to thank Andrew Roger for being the evolutionary post- doc that I never had. I want to thank Tamara Hartson for her drawings of Trichonympha and for making sure that I knew there was always room in front of her fire place and a glass of wine available when I really needed it. I want to thank my friends from the department, especially Patrick Carrier and Andris Maclnns, and from home, especially Ron Odagaki, Chris Eskiw and Ryan McKay. Your support has meant a lot to me. I would like to thank the members of the Redfield lab with whom I worked. Your advice and support has been very important to me over the past three years. Especially I would like to thank Leah Macfadyen for taking the time to proof read my thesis and to Laura Bannister and Shaun Cordes for making sure I reached the end of my thesis with my mental state intact. I want to thank my supervisor, Rosie Redfield, under whose training I have become a much better scientist. And finally I want to thank my family. vii Chapter I: Introduction General question : How did sex first evolve? Sexual reproduction is pivotal for so many organisms. Songs are written about it. Salmon swim incredible distances to die for it. Plants develop elaborate coloring and fragrances to encourage it, but this is not so for all of life. Many organisms reproduce effectively and prolifically without sex. Why would this be? What are the advantages of a sexual system? How did sex first evolve and why? Biologists have asked these questions time and time again. I will make my contribution to this debate by addressing the question of when sex originally evolved. Sexual reproduction is costly. In order for it to evolve, an advantage to sex must exist that outweighs its price. This advantage would allow sexual organisms to successfully compete with asexual ones. Some costs (Trivers, 1972), such as males and parental care, are unlikely to have bearing on the origin of sex. However fundamental costs, such as the additional energy and genetic machinery required to participate in sex and the attraction of mates for outcrossing species, are relevant to questions of its origins. A great deal of theoretical work has been done to examine the origin and evolution of sex (Maynard Smith, 1971), (Maynard Smith, 1978), (Bell, 1982), (Hamilton, Axelrod and Tanese, 1990). Models have shown how sex might be advantageous for reproducers with certain traits in given environments. These models are mathematically robust but may not be biologically relevant, as traits and environments are assumed in each model. Although some studies have been done that test specific assumptions, the majority of these assumptions still need to be examined. 1 Rather than testing individual models, I will use phylogenetics to infer traits of the ancestral sexual population. If it is assumed that sex evolved only once, then the divergence that occurred immediately after that can be thought to have produced two lineages. One is the line that gave rise to the majority of the eukaryotes, eventually leading to higher animals and plants. The other lineage diverged away from that line and gave rise to its own modern descendants. This second lineage will be referred to as the deepest diverging sexual lineage and it is this line that I am interested in. I will determine the modern descendants of the deepest diverging sexual lineage. By comparing their sexual cycles with those of more recently diverged organisms, I will infer aspects of the ancestral cycle. Models of the origin of sex will then be re-examined in light of these inferences. Sex: Definition and Evolution Sex must be defined before its evolution can be discussed. A sexual cycle, in the most elemental sense, can be defined as a cycle of reproduction in which two cells, designated gametes, fuse. A single cell results with double the chromosome content, or ploidy, of each of the individual cells. This process, called syngamy, is followed by the process of meiosis where the chromosome content is reduced back down to its halved state. In most organisms this occurs in two steps. Prior to the first step the DNA content is doubled such that chromosomes have two copies, each referred to as a chromatid.