Molecular evolution and in silico analysis of the mitochondrial genome of the hermaphroditic swan mussel, Anodonta cygnea (Bivalvia: Unionoida) by Emily Erin Chase B.Sc. Honours Acadia University 2015 Thesis submitted in partial fulfillment of the requirements for the Degree of Master of Science (Biology) Acadia University Fall Graduation 2017 © by Emily Erin Chase, 2017 This thesis by Emily Erin Chase was defended successfully in an oral examination on 22 September 2017 The examining committee for the thesis was: ________________________ Dr. Michael Robertson, Chair ________________________ Dr. T. Rawlings, External Examiner ________________________ Dr. M. Coombs, Internal Examiner ________________________ Dr. D.T. Stewart, Co-Supervisor ________________________ Dr. S. Breton, Co-Supervisor _________________________ Dr. Brian Wilson, Head/Director This thesis is accepted in its present form by the Division of Research and Graduate Studies as satisfying the thesis requirements for the degree Master of Science (Biology) …………………………………………. ii I, Emily Erin Chase, grant permission to the University Librarian at Acadia University to archive, preserve, reproduce, loan or distribute copies of my thesis in microform, paper, or electronic formats on a non-profit basis. I undertake to submit my thesis, through my University, to Library and Archives Canada and to allow them to archive, preserve, reproduce, convert into any format, and to make available in print or online to the public for non-profit purposes. I, however, retain the copyright in my thesis. ______________________________ Author ______________________________ Supervisor ______________________________ Date iii TABLE OF CONTENTS Title i Approval of thesis ii ii Library release form iii iii Table of contents iv List of tables vi List of figures vii Abstract viii Abbreviations and symbols ix Acknowledgements xiii Preface xiiv Chapter 1: Mitochondrial DNA, doubly uniparental 1 inheritance and freshwater mussels: A literature review Overview 1 Freshwater mussels and study species: Anodonta cygnea 5 The conservation status of freshwater mussels 8 Hermaphroditism and sexual strategies of freshwater mussels 10 Doubly uniparental inheritance in freshwater mussels 17 Mitochondrial DNA and evolutionary relationships among 24 FWMs Trends in the analysis of complete genomes of freshwater 25 mussels Summary and objectives 26 Tables 28 References 29 iv Chapter 2: The complete H-type mitochondrial genome of 49 freshwater mussel Anodonta cygnea (Bivalvia: Unionidae) Abstract 49 Introduction 50 Materials and Methods 56 Results 60 Discussion 68 Figures 79 Tables 85 References 89 Chapter 3: Relative evolutionary rates of sex-associated 101 mtDNA genomes in the order Unionoida (Bivalvia): A comparison among male-, female- and hermaphrodite- transferred mitochondrial genomes Abstract 101 Introduction 102 Materials and Methods 106 Results and Discussion 111 Figures 123 Tables 126 References 131 v LIST OF TABLES Table Abbreviated Caption Page Chapter 1 1 Standard genes found in mtDNA 28 2 Shared unassigned regions of mtDNA 28 Chapter 2 1 Species specific primers used to amplify 85 Anodonta cygnea mitochondrial genomes 2 Hermaphroditic (H-type) mtDNA shared 85 unassigned regions S1 GenBank accessible complete or near 86 complete mtDNA of order Unionoida S2 GenBank accessible cox1 sequences used 87 S3 GenBank accessible F-, H- and M-ORFs 88 used Chapter 3 1 Cox1 nucleotide divergence of F-, H- and 126 M-type groups 2 Overall cox1 nucleotide divergence of F-, 126 H- and M-type mitochondrial DNA 3 Tajima’s D nucleotide relative rates test 127 of cox1 sequences vi 4 Number of synonymous and 127 nonsynonymous substitutions between cox1 of F-, H- and M-type groups 5 Number of synonymous and 127 nonsynonymous substitutions between all protein coding genes of F-, H- and M- type groups 6 Purifying selection tests of cox1 within F- 128 and H-type mtDNA 7 Positive selection tests of cox1 within F- 128 and H-type mtDNA S1 GenBank accessible cox1 sequences used 129 S2 The ratio of nonsynonymous to 130 synonymous substitutions in F- and H- type mtDNA S3 Neutral selection tests of cox1 within F- 130 and H-type mtDNA LIST OF FIGURES Figure Abbreviated Caption Page Chapter 2 1 Gene order of Anodonta cygnea 79 mitochondrial DNA 2 Nucleotide and amino acid divergence of 80 Anodonta cygnea mitochondrial DNA 3 Example potential secondary structures in 81 shared unassigned region trnF-nad5 of Anodonta cygnea vii 4 Amino acid properties of H-ORFs 81 5 Bayesian Inference tree of F- and H-type 82 cox1 with transmembrane descriptions of F- and H-ORFs 6 Bayesian Inference tree of M-type cox1 83 with transmembrane descriptions of M- ORFs 7 NAD5 protein alignment of three bivalve 83 species with translocated Anodonta cygnea partial NAD5 annotated S1 Predicted tRNA structures of H-type 84 mitochondrial DNA of Anodonta cygnea Chapter 3 1 Relative rates analysis group set up 123 methodology 2 Unionid cox1 maximum likelihood tree 124 3 Unionid cox1 maximum likelihood 125 unrooted trees of groups ABSTRACT viii Doubly uniparental inheritance is a fascinating phenomenon that still holds many mysteries. Two major components currently being explored are (1) novel proteins encoded by the mitochondrial DNA of male and female dioecious species (M- and F- type respectively) and of hermaphroditic species (H-type), and (2) the generally fast rate of evolution of these types of mitochondrial DNA and their different relative rates. These two components are explored herein and prefaced with a thorough literature review of freshwater mussels (order Unionoida), which includes the study species, Anodonta cygnea. Complete mitochondrial DNA of A. cygnea is sequenced and an in silico analysis is conducted on the subject and novel open reading frames (ORFs) of other members of order Unionoida. Relative rates of cox1 mitochondrial DNA within each mitotype are also explored. The complete mitochondrial DNA of A. cygnea consists of 13 protein coding genes, 2 rRNA, and 22 tRNA, and is 15,607 base pairs long, and contains a translocated portion of nad5. Based on in silico analysis, we conclude that the number and topology of transmembrane domains in F-ORFs are maintained across species, and that these contain signal peptides with corresponding signal cleavage sites. We suggest that H-type mitochondrial DNA is evolving at a slower rate than the F-type and discuss these results in the context of the arenas hypothesis. ABBREVIATIONS AND SYMBOLS ix ATP Adenosine atp6, ATP synthase triphosphate ATP6 subunit 8 atp8, ATP synthase BI Bayesian ATP8 subunit 8 Inference bp Base pairs C-TM-E Cellular- transmembrane domain- extracellular topology cox,1 Cytochrome cox2, Cytochrome COX1 oxidase subunit I COX2 oxidase subunit II cox3, Cytochrome Cytb, Cytochrome b COX3 oxidase subunit cob, III COB DNA Deoxyribonucleic DUI Doubly acid uniparental inheritance E- Extracellular- f-orf, F- Female specific TM-C transmembrane ORF open reading domain-celluar frame topology x F-type Female mitotype FWM Freshwater mussel h-orf, Hermaphroditic H-type Hermaphroditic H- specific open mitotype ORF reading frame ITS Internal IUB International transcribed Union of spacer Biochemistry LR- Long range LUR Longest PCR polymerase chain unassigned region reaction m-orf, Male specific ML Maximum M- open reading Likelihood ORF frame M- Male mitotype mtDNA Mitochondrial type deoxyribonucleic acid mt Mitochondrial n non-synonymous substitutions nd difference in Nd total difference in number of non- number of non- xi synonymous synonymous substitutions substitutions nad2, NADH nad,1 NADH NAD2 dehydrogenase NAD1 dehydrogenase subunit 2 subunit 1 nad4, NADH nad3, NADH NAD4 dehydrogenase NAD3 dehydrogenase subunit 4 subunit 3 nad,5 NADH nad4L, NADH NAD5 dehydrogenase NAD4L dehydrogenase subunit 5 subunit 4L ORF Open reading nad6, NADH frame NAD6 dehydrogenase subunit 6 rRNA Ribosomal ROS Reactive oxygen ribonucleic acid species rrnS Small ribosomal rrnL Large ribosomal subunit subunit ribosomal ribosomal ribonucleic acid ribonucleic acid s synonymous sd difference in substitutions number of xii synonymous substitutions sd total difference in SMI Strictly maternal number of inheritance synonymous substitutions SCS Signal cleavage SUR Shared site unassigned region SP Signal peptide tRNA Transfer ribonucleic acid TM Transmembrane trnC Cysteine domain trnA Alanine trnE Glutamic acid trnD Aspartic acid trnG Glycine trnF Phenylalanine trnI Isoleucine trnH Histidine trnL2 Leucine 2 trnL1 Leucine 1 trnN Asparagine trnM Methionine trnQ Glutamine trnP Proline trnS1 Serine 1 trnR Arginine trnT Threonine trnS2 Serine 2 trnW Trytophan trnV Valine trnY Tyrosine xiii ACKNOWLEDGEMENTS I would like to thank the following people and groups for their support, contribution, encouragement and effort towards this project and my experience at Acadia University: Brent Robicheau Dr. Anna Redden Dr. Donald Stewart Dr. David MacKinnon Dr. Sophie Breton Krista Mills NSERC Research and Graduate Studies Acadia University Sarah Veinot The Acadia Biology Department xiv PREFACE Chapters 2 and 3 of this thesis were written with the intent of being published in peer reviewed journals, thus, I have chosen to retain the more inclusive pronouns “we” and “our” for those chapters. Co-authors on these publications are specified at the beginning of these chapters. Chapters 2 and 3 also include a brief introduction, which will have overlap with the literature review in Chapter 1. xv Chapter 1 Mitochondrial DNA, doubly uniparental inheritance