The Genetic Analysis of Lasaea hinemoa: The Story of an Evolutionary Oddity KATHERINE LOCKTON A thesis submitted for the degree of Master of Science at the University of Otago, Dunedin, New Zealand 1 March 2019 ABSTRACT Lasaea is a genus of molluscs that primarily consists of minute, hermaphroditic bivalves that occupy rocky shores worldwide. The majority of Lasaea species are asexual, polyploid, direct developers. However, two Australian species are exceptions: Lasaea australis is sexual, diploid and has planktotrophic development, whereas Lasaea colmani is sexual, diploid and direct developing. The New Zealand species Lasaea hinemoa has not been phylogeographically studied. I investigated the phylogeography of L. hinemoa using mitochondrial and nuclear gene sequencing (COIII and ITS2, respectively). Additionally, I investigated population- level structuring around Dunedin using microsatellite markers that I developed. It was elucidated that the individuals that underwent genetic investigation consisted of four clades (Clade I, Clade II, Clade III and Clade IV). Clade I and Clade III dominated in New Zealand and support was garnered through gene sequencing and microsatellite analysis for these clades to represent separate cryptic species, with biogeographic splitting present. Clade II consisted of individuals that had been collected from the Antipodes Island. The Antipodes Island contained individuals from two clades (Clade I and Clade II), with Lasaea from the Kerguelen Islands being more closely related to individuals from Clade II than Clade I was to Clade II. This genetic distinction between Clade I and Clade II seemed to indicate transoceanic dispersal via the Antarctic Circumpolar Current (ACC) between the Kerguelen Islands and Antipodes Island. Clade IV clustered very distinctly from L. hinemoa, appearing to represent transoceanic dispersal by another Lasaea species. i ACKNOWLEDGEMENTS I would like to acknowledge the contributions of some truly invaluable people throughout the course of my master’s degree. First and foremost, I’d like to acknowledge the efforts of my supervisor Hamish Spencer; his support and guidance has been integral to this project, and I’ve appreciated his response time to emails and patience listening to my concerns. I’ve appreciated the support and advice of Tania King, Martyn Kennedy and Kirsten Donald in the lab, the bioinformatic wisdom of Ludo Dutoit, and the microsatellite guidance of Graham McCulloch. This project would have been impossible without the help of my many fieldwork assistants throughout New Zealand; particularly Luke Easton, Clare Cross, William Pearman, Tiffany Lo, Rachael Hudson and Yasmin Foster. My friends for their endlessly appreciated support, from listening to me in times of stress to catching up for social activities. Ellie Hay, for your advice with phylogenetic analysis and friendship. Amber Helliwell, for all help throughout the past two years, at the gym, providing a long-suffering soundboard or just socialising. Philip Greenspoon, for surviving me as an ‘office intruder’ and reading drafts of my work. Lastly, I’d like to thank my family for their endurance and patience these past two years. Harry and Jonathan for helping me sample throughout Taranaki, despite well expressed concerns regarding their limited distribution. Antonia, for being a bedrock of support (even if it was non-verbally, and frequently unexpressed). Cordelia, for her ability to make me laugh in stressful situations. My Mum, for her endless love and support in all aspects of my life. My Grandfather (Grandad) for nutritionally supporting throughout all of January 2019, and for ensuring that I had as many cushions to study with as I could ever want. Everyone I’ve mentioned, and many more people around the department every day have enabled me to work through the past two years complete this thesis, and you have my endless gratitude. ii TABLE OF CONTENTS ABSTRACT ...................................................................................................................... i ACKNOWLEDGEMENTS .............................................................................................ii TABLE OF CONTENTS ............................................................................................... iii LIST OF FIGURES .......................................................................................................... v LIST OF TABLES ....................................................................................................... viii 1. General Introduction .............................................................................................. 1 1.1 The genus Lasaea .............................................................................................................. 1 1.1a Lasaea ........................................................................................................................... 1 1.1b Asexual Reproduction in the genus Lasaea ................................................................... 2 1.1c Direct Development in the genus Lasaea ...................................................................... 5 1.1d Polyploidy in the genus Lasaea ..................................................................................... 8 1.2 Lasaea species relationships ............................................................................................. 9 1.3 Rationale for study ........................................................................................................... 10 1.4 Objectives ......................................................................................................................... 11 1.5 References ........................................................................................................................ 11 2. Nuclear and Mitochondrial Gene Sequencing of Lasaea hinemoa ................... 17 2.1 Abstract ............................................................................................................................ 17 2.2 Introduction ..................................................................................................................... 17 2.3 Methods ............................................................................................................................ 21 2.3a Sampling ..................................................................................................................... 21 2.3b DNA Extraction and PCR Conditions ........................................................................ 22 2.3c Sequence Processing ................................................................................................... 25 2.3d Phylogenetic Analysis ................................................................................................. 25 2.3e Population Genetics .................................................................................................... 29 2.4 Results .............................................................................................................................. 30 2.4a Sampling ..................................................................................................................... 30 2.4b DNA Sequencing and Phylogenetics ........................................................................... 31 2.4c Population Genetics Analyses ..................................................................................... 39 2.5 Conclusions ...................................................................................................................... 46 2.6 Limitations ....................................................................................................................... 46 2.7 References ........................................................................................................................ 48 3. Population Genetics of Lasaea hinemoa utilizing Microsatellite Analysis ....... 53 3.1 Abstract ............................................................................................................................ 53 iii 3.2 Introduction ..................................................................................................................... 53 3.3 Methods ............................................................................................................................ 56 3.3a Sampling ..................................................................................................................... 56 3.3b Development of Microsatellite Markers ..................................................................... 57 3.3c DNA Extraction and PCR Conditions ......................................................................... 59 3.3d Data Processing .......................................................................................................... 60 3.3e Phylogenetic Analysis ................................................................................................. 60 3.3f Formatting and Data Manipulation ............................................................................ 61 3.3g Population Genetic Analyses ...................................................................................... 61 3.4 Results .............................................................................................................................. 62 3.4a Phylogenetic Analysis ................................................................................................. 62 3.4b Formatting and Data Manipulation