PATTERNS OF GENOME SIZE DIVERSITY IN INVERTEBRATES: CASE STUDIES ON BUTTERFLIES AND MOLLUSCS A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by PAOLA DIAS PORTO PIEROSSI In partial fulfilment of requirements For the degree of Master of Science April, 2011 © Paola Dias Porto Pierossi, 2011 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 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-82784-0 Our file Notre reference ISBN: 978-0-494-82784-0 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'lnternet, 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. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. 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. 1+1 Canada ABSTRACT PATTERNS OF GENOME SIZE DIVERSITY IN INVERTEBRATES: CASE STUDIES ON BUTTERFLIES AND MOLLUSCS Paola Dias Porto Pierossi Advisor: University of Guelph T. Ryan Gregory This thesis investigates genome size relationships with organismal and ecological traits in butterflies and molluscs. Relationships between genome size and organismal-level traits were analyzed for 84 species of butterflies. All estimates were found to be within the hypothetical 2pg threshold for holometabolous insects, and positive correlations were observed with sperm length, habitat choices, and food preferences. In addition, correlations between genome size and ecological traits, such as habitat shifts and latitude were examined in 259 species of molluscs (gastropods and bivalves). While variation in DNA content was not associated with environmental shifts, within gastropods it was positively correlated with latitude. Conversely, in bivalves both environmental shifts and latitude were marginally correlated to DNA content. These results suggest that before a complete understanding of the evolution of the genome can be achieved, multiple levels of biological organization will need to be explored in different taxa. ACKNOWLEDGEMENTS First and foremost I would like to thank Dr. T Ryan Gregory, my advisor, for making all of this possible. When we met 6 years ago, I could never have imagined how this experience would shape my life. Your invaluable mentoring has inspired me to always dream bigger and aim higher. Thank you for all your advice and guidance. I would also like to express my sincere gratitude to my committee members, Dr. Sarah Adamowicz and Dr. Paul Hebert for their unfailing assistance throughout the creation and development of this thesis. Thanks are due to all members of the Gregory, Lynn, Crease, Hebert and Adamowicz labs, past and present. In particular, I would like to thank Tyler Elliott and Nick Jeffery for sharing this experience with me every step of the way and for putting up with my endless questions and requests. The completion of this thesis would not have been possible without many collaborations and partnerships. For that, I would like to thank Adrienne Brewster and the entire staff of the Cambridge Butterfly Conservatory, and Cheryl Tyndall and the staff of Niagara Butterfly Conservatory for supplying so many of the specimens used in this study; Michael Mucci and Tannis Slimmon for providing the adequate conditions for me to raise my butterflies; Dr. John Wilson, Dr. Vazrick Nazari, Jayme Sones and the staff at the Canadian Center for DNA Barcoding for their vital assistance in making sure my samples were properly barcode; Dr. Andre Martel, Dr. Eva Pip, Dr. Gerry Mackie, and Robert Forsyth for identifying all my molluscan specimens; and the staff at the Churchill Northern Study Center for their excellent hospitality and accommodations. You have all been instrumental in allowing me to conduct my research. Most importantly, I would like to thank all my friends and family for all of their love and support. I am especially indebted to my parents, Joceli Pierossi and Deborah Porto Pierossi for all the sacrifice you have made to provide the best opportunities for me. I am forever grateful to your continuous encouragement for me to pursue a higher level of education, and for believing I could do it when I didn't believe it myself. To my sister, Juliana Pierossi, thank you for your wisdom and guidance. You are much more than my big sister, you are the example I try to follow. Thank you for all the lessons you taught me along the way. Lastly, thanks to Callie Sanderson for keeping me sane and making this experience a little more enjoyable. I dedicate this thesis to all of you. "Ofuturo sefaz agora E cada erro e uma vitoria Pois a derrota nao existe Nao ha conquista sem labuta A vida e uma infinita luta Onde so perde quern desiste." - Douglas Rafael ii TABLE OF CONTENTS LIST OF FIGURES viii LIST OF TABLES xi CHAPTER ONE: AN OVERVIEW OF GENOME SIZE RESEARCH 1 INTRODUCTION 2 Defining Genome Size 2 A Historical Account of Genome Size 2 The C-value Enigma 3 Genome Size Evolution in Animals 4 Genome Size Evolution in Invertebrates 5 A Hierarchical Approach to Genome Size Correlations 6 OBJECTIVES 8 CHAPTER TWO: CASE STUDY ONE: INVESTIGATING ORGANISMAL LEVEL PATTERNS OF GENOME SIZE DIVERSTIY IN BUTTERFLIES 12 INTRODUCTION 13 Genome Size Diversity in the Lepidoptera and Other Insects 13 Genome Size and Development in Insects 14 Genome Size Diversity within Butterflies 16 Genome Size and Phenotypic Properties 17 QUESTIONS AND PREDICTIONS 20 METHODS 22 Source of Specimens 22 Specimen Identification 24 Acquiring Genome Size Data 24 Feulgen Image Analysis Densitometry 25 Flow Cytometry 26 iii Other Parameters 27 Cellular Parameters 27 Morphological Parameters 28 Developmental Parameters 28 Ecological Preference Parameters 29 Data Analysis 30 RESULTS 31 Feulgen Image Analysis Densitometry and Flow Cytometry 31 Butterfly versus "Moths" 32 Within Butterfly Comparisons 33 Cellular Parameters 33 Morphological Parameters 33 Developmental Parameters 34 Ecological Preference Parameters 34 DISCUSSION 35 Feulgen Image Analysis and Flow Cytometry 35 Sperm and Haemolymph 35 FIA and FCM 37 "Moths" and Butterflies 37 Temperate "Moths" and Tropical Butterflies 38 Diurnal and Nocturnal Lifestyles 39 Within-Butterfly Comparisons 40 Cellular Parameters 40 Morphological Parameters 41 Developmental Parameters 41 iv Ecological Preference Parameters 42 QUESTIONS AND PREDICTIONS REVISITED 43 FUTURE DIRECTIONS 45 CHAPTER THREE: CASE STUDY TWO: AN EXAMINATION OF ECOLOGICAL PATTERNS IN GENOME SIZE DIVERSITY IN GASTROPOD AND BIVALVE MOLLUSCS 76 INTRODUCTION 77 Molluscs and Genome Size 79 Big Genomes for Land Pioneers? 80 QUESTIONS AND PREDICTIONS 82 METHODS 83 Source of Specimens 83 Collection Protocol 84 Genome Size Estimation 85 Compiled Data 86 Ecological Parameters 86 Phylogenetic Trees 88 Statistical Analysis 90 RESULTS 91 Overview of Genome Sizes in Bivalves and Gastropods 91 Bivalves 91 Gastropods 91 Genome Size and Shifts into Terrestrial Habitats in Gastropods 92 Genome Size and Shifts into Freshwater Habitats in Gastropods 94 Genome Size and Shifts into Freshwater Habitats in Bivalves 95 Genome Size and Thermal Regimes in Marine Gastropods and Bivalves 96 v DISCUSSION 97 Genome Size and Shifts into Terrestrial Habitats in Gastropods 97 Genome Size and Shifts into Freshwater Habitats in Gastropods 97 Genome Size and Shifts into Freshwater Habitats in Bivalves 98 Genome Size and Thermal Regimes in Marine Gastropods and Bivalves 99 QUESTIONS AND ANSWERS REVISITED 101 FUTURE DIRECTIONS 102 SUMMARY 128 CONCLUSION 129 REFERENCES 131 APPENDICES 146 Appendix 2.1 Flow Cytometry Histograms 147 Appendix 2.2 Images of wing scales 151 Appendix 2.3 Biological and ecological parameters analysed in the butterflies 155 Appendix 3.1 All genome size estimates obtained from the Animal Genome Size Database 171 Appendix 3.2 Mean genome size estimates for the species measured in multiple studies 185 Appendix 3.3 Genome size comparisons between estimates measured in this study and those compiled from the Animal Genome Size Database 187 Appendix 3.4 Ecological parameters used in the analysis of the molluscs 188 Appendix 3.5 Supplementary material in the construction of the phylogenetic trees 200 VI Appendix 3.6 Summary of all the data analysed by the three-way ANOVA 207 Appendix 3.7 Barcoding tree of gastropods 209 VII LIST OF FIGURES CHAPTER ONE Figure 1.1 Distribution of genome sizes across the major groups of animals 10 Figure 1.2 Conceptual framework of the different levels of biological organization .... 11 CHAPTER TWO Figure 2.1 Distribution of genome sizes across major insect orders 47 Figure 2.2 Phylogeny of all major superfamilies within Lepidoptera 48 Figure 2.3 Types of cells measured through Feulgen Image Analysis Densitometry ...