Phylogeography, Species Distribution Modelling, Mitochondrial Genome Evolution and Conservation of the Fijian Frogs (Ceratobatrachidae )
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PHYLOGEOGRAPHY, SPECIES DISTRIBUTION MODELLING, MITOCHONDRIAL GENOME EVOLUTION AND CONSERVATION OF THE FIJIAN FROGS (CERATOBATRACHIDAE ) by Tamara Osborne-Naikatini A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Copyright © 2015 by Tamara Osborne-Naikatini School of Biological and Chemical Sciences Faculty of Science, Technology and Environment The University of the South Pacific August 2015 DECLARATION Statement by the Author I, Tamara Osborne-Naikatini, declare that this thesis is my own work and that to the best of my knowledge, it contains no material previously published, or substantially overlapping with material submitted for the award of any degree at any institution, except where due acknowledgement is made in the text. Signature ……………………………………. Date ……………………………. Name ……………………………………………………………………………………... Student ID No ……………………………………………………………………….... Statement by Supervisor The research in this thesis was performed under my supervision and to my knowledge is the sole work of the Ms. Tamara Osborne-Naikatini. Signature ……………………………………. Date ……………………………. Name ……………………………………………………………………………………... Designation ………………………………………………………………………….... Dedication “When I was a child, I spake as a child, I understood as a child, I thought as a child: but when I became a man, I put away childish things. For now we see through a glass, darkly; but then face to face: now I know in part; but then shall I know even as also I am known. And now abideth faith, hope, love, these three; but the greatest of these is love.” Corinthians 13 Verses 11-13 (Saint James Bible) This thesis is dedicated to my late father, William Osborne… Daddy this is for you. i Acknowledgements I owe much to all the kind people who have supported me throughout the many years it took to birth this manuscript. First and foremost to my supervisors: Professor Peter Lockhart, Ms. Patricia McLenachan, and Dr. Ralph Riley, without whom this thesis would not be what it is today. I am truly inspired by these amazing scientists, whose academic achievements have in no way made them less humble or understanding. Academics like Dr. Glenn Aguilar and Dr. Linton Winder whose assistance with data analysis and reviewing of several chapters has made this dissertation better. To the many generous and open-hearted people that I met whilst travelling around the Fijian islands chasing frogs, I will be forever grateful for your friendship and assistance. To the following villages who allowed me access to the forests and rivers on their lands and to the village communities that housed and fed me and my guides/ assistants, my lifelong appreciation: Viwa (Viwa Island); Viro, Tavea, Rukuruku, and Lovoni (Ovalau Island); Tavoro, Lavena, Somosomo, Qeleni, and Vuna (Taveuni Island); Lovu, Nuvukailagi, Nukuloa, Nawaikama, Sawaieke, and Malawai (Gau Island); Waisali, Nadi-i-cake, Driti, Nasealevu, Saqani, and Navonu (Vanua Levu); Vunisea, Nalidi, Wainamakutu, Navunibau, Nadarivatu, Navai, Naga, and Matokana (Viti Levu). Thanks and much love to my friends who kept me laughing and sane (in no particular order): Anna Sahai, Reena Suliano, Kelera Macedru-Buadromo, Scott Buadromo, Mere Valu, Elenoa Seniloli, Tuverea Tuamoto, Lote Daulako, Awei Bainivalu-Delaimatuku, Eleazar O’Connor, Maika Daveta and Nunia Thomas. To all those around me at work and on the various social media networks who supported me through the last dark days of writing, I am humbled by your support and I will never forget it. To my close and extended family, I owe you my unconditional love and my apologies for being absent when I was needed and for being distant while lost in my head. Especially, my children Liora and Tiana - thank you for giving your mother something else to obsess about. And of course, to my better half Mr. Naikatini, without whom I would have given up a long time ago. I could not even begin to describe how much I owe you but it’s safe to say that when it’s your turn I will repay you in full… and then some. ii Abstract The Fijian Cornufer (Subgenus Cornufer) species are the easternmost extent of a native amphibian species in the South Pacific, and are endemic to the Fijian archipelago. Both species are currently classified by the International Union for the Conservation of Nature (IUCN) as threatened. There is distinct genetic divergence between certain island populations, which would suggest that insular isolation has led to evolution of multiple, additional species. These characteristics along with traits that identify other Ceratobatrachid frogs (polymorphic colouration, terrestrial breeding, unique characteristics of larval development, calling patterns), make for a particularly interesting branch of the anuran tree of life. In this thesis I review the conservation status of the Fijian frogs synthesising geo-spatial and genetic analyses. The geo-spatial analyses indicate a need to re-assess the conservation status of the Fijian tree frog (Cornufer vitiensis), and for a systematic reappraisal of the Fijian ground frog (Cornufer vitianus). Novel characterisations of genome structure were generated. The complete mitochondrial genomes for both Fijian Ceratobatrachids were sequenced, showing a unique gene order for Neobatrachian frogs. This provides empirical data which may further current understanding of molecular evolution in neobatachrian lineages. The mitochondrial and nuclear data enable the identification of Integrated Operational Taxonomic Units (IOTUs) amongst island populations of both species. All of the genetic markers indicated that the Taveuni Island populations are divergent, possibly sub-species. Populations of Cornufer on Vanua Levu Island are likely source populations for the other islands in the Fiji group, and could well be the founding population of a putative Cornufer colonizing ancestor. Conservation efforts directed towards the Taveuni and Vanua Levu Island populations of Cornufer would inevitably safeguard two levels of genetic distinctiveness: ancestral genotypes with a possible evolutionary history of hybridization (and the capacity for generating transgressive phenotypes), as well as a divergent population of C. vitianus. iii Abreviations cytb cytochrome b oxidase DNA Dioxyribose nucleic acid IOTU Intergrated operational taxonomic unit IUCN International Union for the Conservation of Nature mtDNA Mitochondrial DNA nDNA Nuclear DNA RNA Ribose nucleic acid rRNA Ribosomal RNA SDM Species distribution model iv TABLE OF CONTENTS Dedication i Acknowledgements ii Abstract iii Abbreviations iv Table of Contents v List of Figures ix List of Tables xi Chapter One - General Introduction 1-21 1.1 The Ceratobatrachids of Fiji 2 1.1.1 Cornufer in the Pacific 2 1.1.2 The Fiji ground frog, Cornufer vitianus 3 1.1.3 The Fiji tree frog, Cornufer vitiensis 11 1.1.4 The taxonomic status of the Fiji frogs 13 1.2 Molecular Systematics of Anurans 13 1.2.1 Evolutionary relationships, molecular taxonomy and historical biogeography 13 1.2.2 Use of genetics in conservation of endangered anurans 16 1.2.2.1 Mitochondrial and nuclear markers used in phylogeography 16 1.2.2.2 Phylogeographic analyses: tools to discern population history and connectivity 18 1.3 Thesis Structure and Aims 20 Chapter Two - Fieldwork and DNA Preparation 22-28 2.1 Field Sites and Sampling Logistics 23 v 2.1.1 Field sites 23 2.1.2 Sampling effort 23 2.2 DNA Collection and Extraction 26 2.2.1 Toe-clipping strategy 26 2.2.2 Storage of tissue samples 26 2.2.3 DNA extraction protocol 26 2.2.4 Other methods used 27 2.2.5 Storage of extracted DNA samples 28 Chapter Three - Spatial Analyses of Abundance and Distribution 29-55 3.1 Introduction 29 3.2 Methods 32 3.2.1 Location and count data for frog populations 32 3.2.2 GIS layers and analyses 35 3.2.3 OpenModeller analyses 35 3.2.4 ArcGIS analyses 37 3.3 Results 38 3.3.1 Spatial analyses of frog distribution and abundance data 38 3.3.2 Spatial analyses of Species Distribution Models (SDMs) 40 3.4 Discussion 41 3.4.1 Broad-scale habitat preferences indicated by ArcGIS 41 3.4.2 Species distribution modelling for the Fiji Frogs 46 Chapter Four - Mitochondrial Gene Order and Evolution 56-82 4.1 Introduction 57 4.2 Methods 59 4.2.1 Sequencing of mitochondrial genomes of Fiji frogs 59 4.2.2 Long Range PCR and ABI3730 sequencing 59 4.2.3 Illumina sequencing of three frog genomes 59 4.2.4 Taxon sampling from GenBank genome sequences 61 4.2.5 Sequence alignments and data partitions 61 4.2.6 Phylogenetic reconstruction 62 4.2.6.1 PHYML trees 62 4.2.6.2 Divergence time estimates 62 vi 4.3 Results 63 4.3.1 Mitochondrial gene order in Fijian frogs 63 4.3.2 Phylogenetic relationships recovered 63 4.3.3 Molecular evolution of Neobatrachian mitochondrial genomes 72 4.3.4 Divergence time estimates for Fijian Frogs 72 4.4 Discussion 73 4.4.1 Molecular evolution and phylogeny of anuran mitogenomes 73 4.4.2 Phylogenetic reconstruction with anuran mitogenomes 77 4.4.3 Taxonomic implications from sequence analyses 78 4.4.4 Divergence of Cornufer spp. based on mitogenome sequences 79 Chapter Five - Phylogenetics and Population Structure 83-120 5.1Introduction 84 5.2 Methods 87 5.2.1 Mitochondrial marker development and sequencing 87 5.2.2 Nuclear markers obtained from reduced representation 87 Illumina sequencing 5.2.3 Alignments, splitsgraphs and model determination 88 5.2.4 Maximum Likelihood analyses 91 5.2.5 BEAST analyses 91 5.3 Results 92 5.3.1 Phylogeographic structure in 12SrRNA and Cytb genes of Fijian Ceratobatrachids 92 5.3.2 Phylogeographic structure of novel