University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies Legacy Theses 1999 Comparative morphology and evolutionary trends in the class gastropoda through three-dimensional tomography and DNA sequence analysis Uyeno, Theodore Akira Uyeno, T. A. (1999). Comparative morphology and evolutionary trends in the class gastropoda through three-dimensional tomography and DNA sequence analysis (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/21836 http://hdl.handle.net/1880/25358 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca THE UNIVERSITY OF CALGARY Comparative Morphology and Evolutionary Trends in the Class Gastropoda through Three-Dimensional Tomography and DNA sequence analysis by Theodore Akira Uyeno A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES M PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE. DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY,ALBERTA December, 1999 0 Theodore Akira Uyeno 1999 National Library Bibliotheque nationale 1+1 .ma,, du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. rue Wellington OttawaON K1AOW Ottawa ON KIA ON4 Canada Canada The author has granted a non- L'auteur a accorde une Licence non exclusive licence allowing the exclusive pennettant a la National Library of Canada to Bibliotheque nationale du Canada de reproduce, loan, distribute or sell reproduke, prster, distriiuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format electronique. The author retains ownership of the L'auteur conserve la propriete du copyright in this thesis. Neither the droit d'auteur qui protege cette these. thesis nor substantial extracts fkom it Ni la these ni des extraits substantiels may be p~tedor otherwise de celle-ci ne doivent &re imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT Comparative Morphology and Evolutionary Trends in the Class Gasttopoda through Three-Dimensional Tomography and DNA sequence analysis This study evaluated the validity of the recently developed multidisciplinary scheme for gastropod subclass phylogeny. The multidisciplinary scheme appeared to be more suitable than the longstanding, hlyentrenched classical scheme, which is seen currently in textbooks and many journals. In order to ascertain this, I desmibed and analyzed novel molecular and morphological characters as cladograms, which I then compared to the two phylogenetic schemes. I accumulated these characters using two techniques: t 8s ribosomal DNA sequencing and three-dimensional magnetic resonance microscopy (MRM). For this study, I sequenced DNA encoding the 18s rRNA subunit of seventeen previously uncharacterized gastropods in order to develop a more complete molecular survey of the Gastropoda. The result was a robust computer analyzed consensus tree. The MRM portion of my thesis dealt with the acquisition of the first 3D non-destructive models of the musculo-skeletal arrangements within the gastropod foot. The eight models generated represent a pan-class selection and were analyzed for and resulted in several morphological and functional trends regarding the tarsos and columellar musculature and their integration with each other. In short, the phyiogenetic analysis of these two datasets supported the new multidisciplinary scheme but with modifications. It also shows that the smoothly graded classical prosobranch- opisthobranch-pulmonate scheme is probably artificial. Additionally, both these techniques have been adapted for addition to the toolbox of malacologists and other biologists for uses that extend beyond ph ylogenetic studies. I am a lucky fellow.. Although this field of science is, in my opinion, of true value and fills most of its workers with a miraculous feeling of discovery and self worth, rising through the educational column is often a gaunt prospect with respect to personal finances and time. These obstructions, in my case, have been amply overcome with the help of my family both genetically related and adopted. These people, to whom I am in debt, I Iist below: My wife Angela for all her support through and help with this thesis. My Mom and Dad for help in reading through my thesis and growing me up right. My supe~sorDr. G. Bourne, who is my first mentor and good friend. My brother Keith for being a good brother and helping with the software. My best buddy Mike Newel for being a great lab mate and an invaluable third hand in many of the protocols, Professionally, I am in debt to many, many people. I look at the M.Sc. program, and life in general, as an opportunity to learn as much as you possibly can and there are people that have gone out of their way to grant me the chance tdearn the skills I hold so dear: My committee, who I've noticed is much more interactive and helpful than most: Dr. Bourne, Dr. Walker & Dr. Syed. I would also like to thank Dr. Syme for serving as my external examiner and Dr. Phillips, who was originally on my committee and who came to my aid countless times whenever I had a computer visualization prob tern or needed critical reading. Those who taught me about sequencing: Bob Winkfein (who went out of his way to do most of the sequencing), Dr. Honda and the Barnfield Marine Station. The guys at the Center for in Vivo Microscopy at the Duke University Medical Center, especially Dr. Brad Smith (now at U of Michigan at Ann Arbor), Mr. Gary Collier and Dr. Al Johnson for technical guidance, microscope time and critical readings. My 3D rendering guru, and one author of the great computer program Sterecon, Mr. Mike Marko of the Wadsworth Institute in Albany, New York. I'd also like to thank my department and Dr. Tim Buell of the Learning Commons for indispensable funding received for my research. Those people who helped me out here and there with administrative stuff and a plethora of small jobs: Karen & Wilma, Dr. Barclay, Shanna, and many others. Lastly, although my time with them was brief, I would like to thank Dr. Bill Kier of the University of North Carolina at Chapel Hill, Dr. John Gosline of the University of British Columbia and Drs. Vogel and Wainwright of Duke University for giving me much perspective and hope for the firture. DEDICATION I dedicate this thesis to my family and especially to my late grandparents, all of whom believed that "Education is the Great Separator of Men and Gentlemen". TABLE OF CONTENTS Section Page... ABSTRACT Ul ACKNOWLEDGMENTS DEDICATION TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES EPIGRAPH CHAPTER ONE: INTRODUCTION CHAPTER TWO: LITERATURE REVIEW 2.1 Topics covered 2.2 Overview of the spectrum of techniques 2.2.1 Spectrum and selection of imaging techniques 2.2.1.1 Gross dissection and whole organism imaging 2.2.1.2 Destructive sectioning 2.2.1.3 Optical sectioning 2.2.1 -4 Magnetic resonance sectioning 2.2.2 Spectrum of genetic techniques 2.2.2.1 Random Amplified Polymorphic DNA 2.2.2.2 Restriction Fragment Length Polymorphisms 2.2.2.3 Amplified Fragment Length Polymorphisms 2.2.2.4 Variable Number of Tandem Repeat markers 2.2.2.5 DNA sequencing 2.2.2.6 Gene selection 2.3 The development of phylogenies of the ctass Gastropods 2.3.1 The state and history of current gastropod schemes 2.32 Past research in gastropod phylogeny 2.3.2.1 On the geological evidence pertaining to gastropod phylogeny 2.3 -2.2 On molecular phylogeny research 2.3.2.3 011 research pertaining to morphological phylogeny 2.3 2.4 On the research based on ontogeny and embryology Section Page CHAPTER THREE: THE GASTROPODS OF THESE STUDIES CHAPTER FOUR: PHYLOGENIES USING DNA SEQUENCE ANALYSIS 4-1 Introduction 4.2 Material and methods 4.2.1 Protocols 4.22 Andysis 4.3 Results 4.4 Discussion 4.4.1 Interesting elements of the phylogenetic tree 4.4.2 Technical matters 4.4.2.1 High molecular weight DNA 4.4.2.2 Hyperactive gastropod DNAses 4.5 Summary and concluding remarks 1.5.1 Molecular conclusions for the phylogeny of the subclass taxa 4.5.2 Summary CHAPTER FIVE: TOMOGRAPHY AND SNAIL MORPHOLOGY 5.1 Introduction 5.1 .1 Characters of structure and hction 5.1 -2 Theory and mechanics of magnetic resonance microscopy 5.1 -3 Identification of structures 5.1.4 Organisms of study 5.1.5 Swey of the large subclass taxa 5.1.6 The evolution of form and fimction 5.2 Materids and methods 5.2-1 ProtocoIs 5.2-1.1 MRM 5.2.1.2 Histology 522Analysis 5.3 Results 5.4 Discussion 5.5 Summary and concluding remarks Section Page CHAPTER SIX: CONCLUSION 96 6.1 Conclusions based on the concatenation of morphological and molecular 96 phylogenetic trees 6.2 Comparison of the summary tree found by this research to other trees. 97 6.3 Future directions 99 LITERATURE CITED APPENDIX I: Whole snail MRM Images of the front, top and left side I. 1. t CalZiostoma canaliculatum (Opaque view) I. 1.2 Calliostorna canaliculcltum (Transparent view) L2. L Diaulula sandiegensis (Opaque view) 1.2.2 Diaulula sandiegmis (Transparent view) 1.3. L Halioris rufescem (Opaque view) 1.3.2 Haliotis rufescenr (Transparent view) 1.4.1 L-vnznaea stagnalis (Opaque view) 14.2 L_vmnaea stagnalis (Transparent view) L5. I Marks corniiarieris (Opaque view) 1.5.2 Marisa coman'etis (Transparent view) 1.6.1 Nucella ostrina (Opaque view) 1.6.2 Nucello osrrina (Transparent view) 1.7.1 Pomacea brrigesi (Opaque view) 1.72 Pomacea bn'dgesi (Transparent view) 1.8.