Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Time travelling weevils: Unravelling the evolutionary past of Hadramphus using historical and modern DNA A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University by Emily D Fountain Lincoln University 2013 Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy. Abstract Time travel weevils: Unravelling the evolutionary past of Hadramphus using histrorical and modern DNA by Emily D Fountain Museum specimens are of increasing importance to the study of ecology, evolution, phylogenetics and conservation biology. In this thesis I explore the use of museum specimens to analyse population changes and declines in the endangered weevil genus Hadramphus over the past 125 years. Molecular phylogenetics aids our understanding of species taxonomy by including the neutral evolutionary history of a species’ genetic code rather than relying on morphological characteristics alone. The phylogeny of the genus Hadramphus was obtained using the mitochondrial gene cytochrome c oxidase subunit I (COI) and the ribosomal nuclear internal transcribe spacer 2 (ITS2). A multi-locus multi-species coalescent approach was used for building a species tree from the gene trees. Hadramphus pittospori was highly divergent from the other three species in the genus, suggesting the current genus is paraphyletic. A study of the phylogeography of Megadromus antarcticus was conducted using COI and ITS2. A Bayesian skyline plot was calculated to determine if the effective population size of M. antarcticus has changed. The population of M. antarcticus showed no decline, and diverged during the Pleistocene. The recent divergence fits with the biogeography of the Canterbury Plains which were heavily affected by the glacial cycles during the Pleistocene. The critically endangered Hadramphus tuberculatus was last sampled in 1922 and presumed extinct until its rediscovery in 2004. After severe range reduction from loss of habitat and the introduction of mammalian predators, the only known remaining population is at Burkes Pass Scenic Reserve. From 2009-2011, the reserve was surveyed for the weevil using visual searches and pitfall traps. Mark recapture was applied to determine population size, movement, and survivability. Analysis of the mark recapture data showed a large year to year ii variation in the estimated population sizes from 2009 to 2011. The data also showed that the weevil was highly mobile throughout the reserve, covering distances up to 190 metres. DNA from pinned museum specimens of Hadramphus tuberculatus has also been collected so that populations can be compared over time to map the loss of genetic diversity for the species. Non-destructive methods were used to extract DNA from specimens of H. tuberculatus collected from 1890-1922. Primers were designed to amplify short fragments of the COI mitochondrial gene (135 bp). Sequences were aligned and compared to COI sequences obtained from modern samples. Preliminary results suggest variation between modern and historic samples and a loss of nucleotide diversity during the past 100 years. A reduced representation library of genomic DNA for H. tuberculatus and H. spinipennis was built for next generation sequencing (454) to find single nucleotide polymorphism (SNP) for population genetic studies using both historic and modern samples. A method of SNP genotyping using high resolution melting (HRM) for both modern and historical weevil specimens continues to be investigated. Keywords: Hadramphus, weevil, phylogeography, phylogeny, historical DNA, next- generation sequencing, high resolution melting, single nucleotide polymorphism, Megadromus antarcticus, mark recapture, conservation, island restoration iii Acknowledgements There are many people who deserve my gratitude for their assistance and/or simple friendly conversation throughout my PhD project. First, I would like to thank my supervisors Adrian Paterson and Rob Cruickshank. Working with both of you has been enjoyable and I learned so much. I commend your support in various projects throughout the years both within in my doctoral work and outside my PhD. Also, I greatly appreciate the humour (and at times the sarcasm) that was brought to our interactions. I could never forget to thank my advisor, the lovely John Marris. I apologise for never fully being able to pronounce “Antarctica” to your standard, but I will never stop striving for excellence. You have inspired me to aim for continued work in museums. My appreciation goes to the people who have provided funding and support for this PhD, particularly the Miss EL Hellaby Indigenous Grasslands Research Trust for the extensive support over the years and the financial support provided by Mohamed bin Zayed Species Conservation Fund, Brian Mason Scientific and Technical Trust, Environment Canterbury (ECan), and the Department of Conservation (DoC). I would like to send my deepest gratitude to my mother, Diane Simaska. If it was not for her support emotionally, mentally, and financially this PhD never would have even started. Mom, you have been an inspiration and have set the best example for me to never give up and to believe that it is never too late to achieve what I want. I love you and this thesis is dedicated to you. I also want to thank my sister, Rachel Fountain, for all the quick messages of support and funny pictures to make me laugh and for listening to me when I needed it. Also, I would like to acknowledge Ben Wiseman, not just because he asked me to, but because he really does deserve it. You have been a tremendous help in the field, lab, and throughout my writing up this thesis. I think you may be one of the few who will ever read my thesis more than once! Thank you; I owe you chocolate. To Vikki Smith, you have been an amazing supporter, comma remover, bowl cake partner, and friend. Thanks for your help in the field and for editing my work and understanding why “this” is a legitimate word. It only seems appropriate to thank Andrew Pugh next; thanks for the laughs and answering my many questions. And after this I will say, “A-Team, my stats would never have been completed without you guys!” iv I acknowledge Mike Bowie for your help in the field with Hadramphus tuberculatus and with the prickly Aciphylla plants. Thanks to Phil Cochrane, who was Chuck Norris of the lab and then moved onto the real world to become my ECan go to man; you are awesome. I want to thank Agate Ponder-Sutton for being the best pet mathematician anyone could ever have, baking fantastic muffins, going for walks, and being a supportive friend over the years. Also, I want to acknowledge Jagoba Malumbres-Olarte for always being there to listen to me (even if it was just my loud voice carrying through the office wall), never failing to entertain, help out, offer advice, and sound the most amazing, endless whistle. Cameron Rossouw, you deserve so much of my gratitude for the cups of coffee, the field assistance, the super crazy computer, the harassment, the listening, and so much more you have provided over the years. You are a best friend and I could not have done it without you. Just a quick shout out to all the others who have helped in the field at some point: Sam Brown, Sam Rohland, Jamie Cooper, Katherine Hill, and Steve Pawson. Thanks also to Hamish Patrick for the weevil hunting and collecting, also, the molecular lab B513 and all the people who have been in and out of there over the years offering advice, help, and amusement. Finally, I want to thank my bunny, Piglet. He has done his job to perfection by enduring the extreme cuddles, hugs and attention to lower my stress and make writing this thesis easier. He has travelled with me to uni, out to get coffee, and to go to the store just so I can feel happier when I was stressed out and frustrated. Love you, Mr. Pig! v Table of Contents Abstract....................................................................................................................................... ii Acknowledgements ..................................................................................................................... iv Table of Contents ....................................................................................................................... vi List of Tables .............................................................................................................................. ix List of Figures ............................................................................................................................ xi Chapter 1 Context of Thesis ........................................................................................................ 1 Chapter 2 Introduction................................................................................................................ 4 2.1 The Genera Hadramphus and Lyperobius ...................................................................................