Conservation Genetics of Saproxylic Beetles by Max Blake SN: 129004813 Supervisor: Prof Paul Shaw ~ 1 ~ ~ 2 ~ ‘They tell us that we lost our tails Evolving up from little snails I say it's all just wind in sails’ Devo – 1978 "Whenever I hear of the capture of rare beetles, I feel like an old war-horse at the sound of a trumpet" Charles Darwin ~ 3 ~ Contents Acknowledgments ............................................................................................................................... 7 Declaration .......................................................................................................................................... 9 Abstract ............................................................................................................................................. 11 1 Biodiversity conservation and saproxylic beetles 10 1.1 Invertebrate Conservation .......................................................................................................... 14 1.2 Saproxylic organisms and tree-rot .............................................................................................. 15 1.3 Objectives and methods of Conservation Genetics .................................................................... 18 1.3.1 Genetic Variation ................................................................................................................. 18 1.3.2 Molecular Taxonomy ........................................................................................................... 24 1.3.3 Historical processes shaping species genetic diversity ........................................................ 25 1.3.4 Morphological modelling and morphometrics .................................................................... 29 1.3.5 Species Distribution Modelling ............................................................................................ 30 1.4 Study Species .............................................................................................................................. 31 1.5 Aims and Objectives .................................................................................................................... 40 2 Development of microsatellite markers and a preliminary study of nuclear and mitochondrial DNA diversity in the Stag Beetle (Lucanus cervus L.) 43 2.1 Introduction. ............................................................................................................................... 49 2.2 Materials and methods ............................................................................................................... 50 2.2.1 Sample collection and DNA extraction ................................................................................ 50 2.2.2 Identification of microsatellites ........................................................................................... 50 2.2.3 Microsatellite genotyping and statistical analysis ............................................................... 52 2.2.4 mtDNA sequencing and analysis .......................................................................................... 52 2.3 Results ......................................................................................................................................... 53 2.4 Discussion .................................................................................................................................... 54 3 Trichius phylogeography and taxonomy 57 3.1 Introduction ................................................................................................................................ 64 3.2 Methods ...................................................................................................................................... 66 3.2.1 Sample Collection ................................................................................................................ 66 3.2.2 DNA extraction ..................................................................................................................... 66 3.2.3 PCR & Sequencing Protocols ................................................................................................ 67 3.2.4 Statistical analysis of COI sequence data ............................................................................. 68 ~ 4 ~ 3.2.5 Statistical analysis of Wingless ............................................................................................. 69 3.2.6 Morphological analysis ........................................................................................................ 70 3.3 Results ......................................................................................................................................... 72 3.3.1 mtDNA .................................................................................................................................. 72 3.3.2 Wingless ............................................................................................................................... 74 3.3.3 Morphological Data ............................................................................................................. 75 3.4 Discussion .................................................................................................................................... 76 4 Geometric morphometrical and morphological variation across European Gnorimus 102 4.1 Introduction .............................................................................................................................. 110 4.2 Methods .................................................................................................................................... 112 4.2.1 Sample Collection .............................................................................................................. 112 24..2 Phenotypic Scoring ............................................................................................................. 113 4.2.3 Landmark Geometric Morphometric Analysis ................................................................... 114 4.3 Results ....................................................................................................................................... 116 4.3.1 Phenotypic Scoring Analysis ............................................................................................... 116 4.3.1.1. Differences between species, subspecies and sexes ..................................................... 116 4.3.1.2 Male leg shape ................................................................................................................ 118 4.3.2 Landmark Geometric Morphometric Analysis ................................................................... 118 4.3.2.1 Dorsal Gnorimus nobilis against G. variabilis .................................................................. 118 4.3.2.2 Ventral Gnorimus nobilis against G. variabilis ................................................................ 119 4.3.2.3 Dorsal shape of Gnorimus nobilis subspecies ................................................................. 119 4.4 Discussion .................................................................................................................................. 120 5 Systematics and phylogeography of Gnorimus in Europe 139 5.1 Introduction .............................................................................................................................. 148 5.2 Methods .................................................................................................................................... 152 5.2.1 Sampling Protocol .............................................................................................................. 152 5.2.2 DNA Extraction Protocol .................................................................................................... 152 5.2.3 mtDNA genotyping and statistical analysis ........................................................................ 153 5.2.5 Nuclear gene sequencing and analysis .............................................................................. 156 5.3 Results ....................................................................................................................................... 158 5.3.1 Mitochondrial data ............................................................................................................ 158 5.3.1.1 COI sequences ................................................................................................................. 158 5.3.1.2 Control Region ................................................................................................................ 160 ~ 5 ~ 5.3.4 Heat Shock Protein ............................................................................................................. 162 5.3.5 Orco .................................................................................................................................... 162 5.4 Discussion .................................................................................................................................. 163 5.4.1 MtDNA diversity and phylogeographic patterns in European Gnorimus .......................... 163 5.4.2 Nuclear gene diversity and allele conservation across Gnorimus ..................................... 167 5.5 Conclusions ............................................................................................................................... 171 DNA Sampling of rare