The University of Adelaide DOCTORAL THESIS INVESTIGATING THE INTERNAL AND EXTERNAL ECOLOGY OF SIX SUBTERRANEAN DIVING BEETLE SPECIES FROM THE YILGARN REGION OF CENTRAL AUSTRALIA Josephine Charlotte Anne Hyde A thesis submitted in the fulfilment of the requirements for the degree of Doctor of Philosophy in the Australian Centre for Evolutionary Biology and Biodiversity in the School of Biological Sciences, Faculty of Sciences, The University of Adelaide 26th June 2018 TABLE OF CONTENTS Abstract .................................................................................................................. 7 Thesis Declaration ............................................................................................... 10 Acknowledgments ............................................................................................... 11 Chapter 1: General Introduction .......................................................................... 13 Groundwater Dependent Ecosystems (GDEs) ................................................ 13 Subterranean groundwater systems in Australia ............................................. 14 The Yilgarn Region of Western Australia ....................................................... 15 Exemplar calcrete aquifers: Sturt Meadows and Laverton Downs ................. 15 Speciation within calcrete aquifers.................................................................. 17 Microbiome research ....................................................................................... 18 References ....................................................................................................... 22 Chapter 2: Diversity patterns of subterranean invertebrate fauna in calcretes ofthe Yilgarn Region, Western Australia ..................................................................... 29 Abstract ........................................................................................................... 32 2.1 Introduction ............................................................................................... 33 2.2 Materials and methods .............................................................................. 34 2.2.1 Data Analysis ..................................................................................... 37 2.3 Results ....................................................................................................... 39 2.5 Discussion ................................................................................................. 45 Acknowledgements ......................................................................................... 47 References ....................................................................................................... 48 Chapter 3: Diversity and geographic differences in viromes of the gut of Australian subterranean groundwater diving beetles ........................................... 52 Abstract ........................................................................................................... 55 3.1 Introduction ............................................................................................... 56 3.2 Results ....................................................................................................... 58 3 3.2.1 Viral Metagenomics overview ........................................................... 58 3.2.2 Viral results by habitat affiliation ....................................................... 60 3.2.3 Viral results by host species ............................................................... 61 3.2.4 Viral results by developmental stage ................................................. 62 3.3 Discussion .................................................................................................. 66 3.4 Conclusions ................................................................................................ 69 3.5 Experimental Procedures ........................................................................... 69 3.5.1 Specimen collection ........................................................................... 70 3.5.2 DNA extraction and sequencing ........................................................ 71 3.5.3 Taxonomic analyses ........................................................................... 72 3.5.4 Statistical analysis .............................................................................. 72 Acknowledgments ........................................................................................... 73 References ........................................................................................................ 74 Supplementary Tables ..................................................................................... 79 Chapter 4: Characterising the microbiome of Australian subterranean diving beetles ................................................................................................................ 110 Abstract .......................................................................................................... 113 4.1 Introduction ............................................................................................. 114 4.2 Experimental Procedures ......................................................................... 115 4.2.1 Specimen collection: ........................................................................ 116 4.2.2 DNA extraction and sequencing: ..................................................... 116 4.2.3 Taxonomic and Functional Analyses ............................................... 116 4.2.4 Statistical analysis ............................................................................ 118 4.3 Results ...................................................................................................... 118 4.4 Discussion ................................................................................................ 125 4.5 Conclusion ............................................................................................... 128 4 Acknowledgements ....................................................................................... 128 References ..................................................................................................... 130 Supplementary Figures .................................................................................. 135 Supplementary Tables ................................................................................... 136 Chapter 5: The first complete mitochondrial genomes of subterranean dytiscid diving beetles (Limbodessus and Paroster) from calcrete aquifers of Western Australia ............................................................................................................ 148 Abstract ......................................................................................................... 151 5.1 Introduction ............................................................................................. 152 5.2 Materials and methods ............................................................................ 153 5.2.1 Specimen collection ......................................................................... 153 5.2.2 DNA extraction and sequencing ...................................................... 154 5.2.3 Analysis and annotation ................................................................... 154 5.2.4 Sequence analysis ............................................................................ 155 5.3 Results and discussion ............................................................................. 155 5.3.1 Genome organisation ....................................................................... 155 5.3.2 Overlapping genes, intergenic regions, and skewness ..................... 161 5.3.3 Protein-coding genes ........................................................................ 162 5.3.4 Transfer RNAs ................................................................................. 162 5.3.5 Ribosomal RNAs ............................................................................. 163 5.3.6 Control region .................................................................................. 163 5.4 Conclusion ............................................................................................... 164 Acknowledgements ....................................................................................... 164 References ..................................................................................................... 165 Supplementary Figures .................................................................................. 170 Supplementary Tables ................................................................................... 173 Chapter 6: General Discussion .......................................................................... 179 5 6.1 Introduction .............................................................................................. 179 6.2 Summary of thesis ................................................................................... 179 6.3 Contributions of this research .................................................................. 180 6.4 Limitations ............................................................................................... 184 6.5 Future Work ............................................................................................. 184 6.6 Conclusion ..............................................................................................