This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Short, Katherine A Title: Life in the extreme when did tardigrades colonise Antarctica? General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. 1 Life in the Extreme: when did 2 Tardigrades Colonise Antarctica? 3 4 5 6 7 8 9 Katherine Short 10 11 12 13 14 15 A dissertation submitted to the University of Bristol in accordance with the 16 requirements for award of the degree of Geology in the Faculty of Earth 17 Sciences, September 2020. 18 Word Count 33252 i 19 Abstract 20 21 Antarctica has long been isolated from its nearest landmasses and has been at least partially 22 glaciated for the past ~35 million years. This long-term isolation combined with the fragmentation of 23 habitats creates a unique environment for the examination of biogeographic patterns. Previously it 24 was believed that the repeated full glaciation of Antarctica wiped out the pre-existing terrestrial 25 fauna and flora and that the continent was, therefore, subsequently re-colonised after periods of 26 glacial retreat, such as from the particularly extensive Last Glacial Maximum. However, recent 27 molecular studies of a variety of taxonomic groups have shown that much of the current terrestrial 28 life found on the continent is generally ancient and must have survived these glaciation events. The 29 main body of Antarctica (East or continental Antarctica) and West Antarctica and the Antarctic 30 Peninsula show differing geological histories and distinct biogeographic patterns in a variety of 31 invertebrates including mites, springtails and nematodes, indicating distinct evolutionary histories. 32 However, despite tardigrades being the most common and widespread invertebrate within 33 Antarctica there has been limited study of their Antarctic biogeography. This thesis set out to 34 investigate the evolutionary and biogeographic patterns of two eutardigrade species (Acutuncus 35 antarcticus and Mesobiotus furciger). At the initiation of this study, A. antarcticus was believed to be 36 a pan-Antarctic species, with a Gondwanan distribution, and M. furciger a globally common taxon 37 and a Southern hemisphere species with a widespread Antarctic distribution. Individuals were 38 sampled that originated from multiple locations across Antarctica. These individuals were then used 39 to document a range of morphometric characters and underwent molecular extraction targeting the 40 three gene regions of 18S, 28S and COX1. Further sequence data were added from GenBank. 41 Phylogenetic analyses using both Maximum Likelihood and Bayesian methods showed that both 42 species are likely to be species groups, with highly distinct lineages in the maritime and continental 43 Antarctic regions. The levels of divergence detected strongly support cryptic speciation. This 44 conclusion was further supported by morphological analyses, which identified significant differences 45 between material sampled from each of these regions. Ancestral state and molecular clock analyses 46 showed that both of these species groups had ancient Antarctic origins, requiring long-term regional 47 survival within Antarctica, consistent with patterns identified in other invertebrate taxa. 48 Morphological comparisons between individuals within single genetic clades obtained from different 49 habitats also identified significant differences, supporting phenotypic plasticity in response to 50 environmental conditions. This finding, not noted previously in tardigrades, has important 51 taxonomic implications, as species indentification relying on morphological characters alone may 52 now result in miss-identification. 53 54 55 56 57 58 59 60 61 62 ii 63 Author’s Declaration 64 65 I declare that the work in this dissertation was carried out in accordance with 66 the requirements of the University's Regulations and Code of Practice for 67 Research Degree Programmes and that it has not been submitted for any other 68 academic award. Except where indicated by specific reference in the text, the 69 work is the candidate's own work. Work done in collaboration with, or with the 70 assistance of, others, is indicated as such. Any views expressed in the 71 dissertation are those of the author. 72 73 74 SIGNED: ...Katherine A. Short......................................... DATE:..04/09/2020....... 75 76 77 78 79 80 81 82 83 84 85 iii 86 Table of Contents 87 LIST OF FIGURES…………………………………….………………………………………………………………………………………….ix 88 LIST OF TABLES ……………………………………….…………………………….………………………………………………………….xi 89 ACKNOWLEDGMENTS………………………………………………………….…………………………………………………………..xii 90 THESIS INTRODUCTION………………………………………………………………………………………………………………………1 91 - Summary………………………………………………………………………………………………………………………………1 92 - Geological history of Antarctica…………………………………………………………………………………………….4 93 - What makes a species and why is that important?...................................................................5 94 - Current described biogeography of Antarctica………………………………………………………………………6 95 - Tardigrades in Antarctica………………………………………………………………………………………………………8 96 - Study species- Acutuncus antarcticus and Mesobiotus furciger…………………………………………10 97 - Thesis chapters……………………………………………………………………………………………………………………12 98 CHAPTER 1. PHYLOGEOGRAPHIC ANALYSES OF TWO ANTARCTIC EUTARDIGRADE SPECIES 99 (ACUTUNCUS ANTARCTICUS AND MESOBIOTUS FURCIGER) SHOW ANCIENT, ANTARCTIC-SPECIFIC 100 LINEAGES…………………………………………………………………………………………………………………………………………14 101 - Abstract………………………………………………………………………………………………………………………………14 102 1.1. Introduction………………………..……….…………………………………………..…………………..…………..…..…..15 103 1.2. Methods………………………..……………………………………………………………………………………………………18 104 1.2.1. Material included.……………………..………………….………………………………………………18 105 1.2.2. Tardigrade extraction……………………………………………………………………….…..………20 106 1.2.3. RNA extraction……………………………………………………………………………………………..20 107 1.2.4. Sequence collations and database creation………………………………………………….21 108 1.2.5. Phylogenetic analysis……………………………………………………………………………………22 109 1.2.6. Molecular clock analysis……………………………………………………………………………….22 iv 110 1.2.7. Biogeographic analysis………………………………………………………………………………….23 111 1.3. Results………………………………………………………………………….......……………..………………………………..25 112 1.3.1. Mesobiotus furciger…………………..………………….……………………………………………..25 113 1.3.2. Acutuncus antarcticus…………………………………………………………………………………..26 114 1.3.3. Molecular clock analysis……………………………………………………………………………….28 115 1.3.4. Biogeographic analysis………………………………………………………………………………….29 116 1.4. Discussion……………………..……………..………………….......……………………………………………………………31 117 1.4.1. Mesobiotus furciger and Acutuncus antarcticus phylogeny…………………………..31 118 1.4.2. Antarctic history and biogeography………………………………………………………………33 119 CHAPTER 2. PAN-ANTARCTIC MOLECULAR ANALYSES OF THE ANTARCTIC EUTARDIGRADE 120 ACUTUNCUS ANTARCTICUS (HYPSIBIOIDEA) SHOW STRONG BIOGEOGRAPHIC PATTERNS AND DEEP 121 ‘INTRA-SPECIFIC’ DIFFERENTIATION…………………………………………………………………………………………………36 122 - Abstract…….....…………………………………………………………………………………………………………………….36 123 2.1. Introduction.............................................................................................................................37 124 2.2. Methods...................................................................................................................................39 125 2.2.1. Definition of Antarctic regions..........................................................................39 126 2.2.2. Collection and identification of material..........................................................39 127 2.2.3. DNA extraction, amplification and sequencing.................................................41 128 2.2.4. Sequence alignment and phylogenetic analysis...............................................42 129 2.2.5. Molecular clock analysis...................................................................................43 130 2.2.6. Haplotype network analysis..............................................................................44 131 2.3. Results...............................................................................................................................44 132 2.3.1. Phylogenetic analysis........................................................................................44 v 133 2.3.2. Molecular dating...............................................................................................45