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The Reproductive Ecology of Icmadophila Splachnirima, Including Aspects of the Reproduction in Additional Members of Icmadophilaceae

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The Reproductive Ecology of Icmadophila Splachnirima, Including Aspects of the Reproduction in Additional Members of Icmadophilaceae The reproductive ecology of Icmadophila splachnirima, including aspects of the reproduction in additional members of Icmadophilaceae Dissertation submitted by Lars Rudolf Ludwig Graduate Biologist (German Diplom-Biologe, equals MSc) for the degree Doctor of Philosophy (PhD) in the Department of Botany, University of Otago, Dunedin, New Zealand. 18th May 2015 First submitted 1st October 2014 ii Contents List of Figures ........................................................................................................................................ vii List of Tables ......................................................................................................................................... xii Acknowledgements ................................................................................................................................. 1 Abstract ....................................................................................................................................... 2 General Introduction .................................................................................................................... 3 1. Lichen reproduction ............................................................................................................................ 3 1.1 Sexual reproduction of lichens .................................................................................................. 3 1.1.1 The sexual reproductive cycle ................................................................................................ 3 1.1.2 Mating Systems ...................................................................................................................... 5 1.2 Vegetative reproduction ........................................................................................................... 5 1.3 Reproductive and Life Strategies in Lichens .............................................................................. 6 2. Introduction to the lichen family Icmadophilaceae and the main study species Icmadophila splachnirima ............................................................................................................................................ 7 3. Relevance of Research ...................................................................................................................... 11 4. Thesis outline .................................................................................................................................... 12 5. Aims ................................................................................................................................................... 16 CHAPTER 1 - The Biogeography of Icmadophila splachnirima ...................................................... 17 1. Introduction ...................................................................................................................................... 17 2. Material & Methods .......................................................................................................................... 17 3. Results ............................................................................................................................................... 18 4. Discussion .......................................................................................................................................... 30 5. Summary ........................................................................................................................................... 31 CHAPTER 2 - A molecular-genetic reassessment of the phylogeny of the lichen genus Icmadophila Trevis. ....................................................................................................................................... 32 1. Introduction ...................................................................................................................................... 32 2. Methods ............................................................................................................................................ 33 2.1 Examined specimens ............................................................................................................... 33 2.2 A homemade DNA extraction and PCR purification kit ........................................................... 35 2.2.1. Composition of reagents for DNA extraction...................................................................... 36 2.2.2 DNA Extraction Procedure Part I – DNA raw extracts .......................................................... 36 2.2.3 DNA Extraction Procedure Part II – Spin-column purification of DNA raw extracts (and PCR products) ....................................................................................................................................... 37 2.3 Loci and Primer Selection ........................................................................................................ 37 2.4 PCR protocols and sequences ................................................................................................. 39 iii 2.5 Sequence alignments and phylogenetic analysis. ................................................................... 42 2.5.1 Sequence alignments ........................................................................................................... 42 2.5.2 Phylogenetic analyses .......................................................................................................... 42 3. Results ............................................................................................................................................... 44 3.1 Phylogenetic results ................................................................................................................ 44 4. Discussion .......................................................................................................................................... 49 4.1 The Phylogeny of Icmadophila ................................................................................................ 49 4.2 Phylogenetic relationships among the remaining genera of Icmadophilaceae ...................... 50 5. Summary ........................................................................................................................................... 51 CHAPTER 3 - Population-genetic analysis of Icmadophila splachnirima ........................................ 52 1. Introduction ...................................................................................................................................... 52 2. Material and Methods ...................................................................................................................... 54 2.1 Origin of analysed specimens ................................................................................................. 54 2.2 DNA Extraction ........................................................................................................................ 59 2.3 Examined loci .......................................................................................................................... 59 2.4 DNA sequence generation ...................................................................................................... 59 2.5 Population Genetic Sequence Analysis ................................................................................... 60 2.6 Molecular-genetic Identification of photobionts .................................................................... 61 3. Results ............................................................................................................................................... 61 3.1 Mycobiont of I. splachnirima .................................................................................................. 61 3.2 Photobiont of I. splachnirima .................................................................................................. 63 3.3 Photobiont identity of I. splachnirima and some other species ............................................. 69 4. Discussion .......................................................................................................................................... 71 5. Summary ........................................................................................................................................... 78 CHAPTER 4 - Characterisation of the mating type locus (MAT) and mating type analysis of Icmadophila splachnirima .......................................................................................................... 79 1. Introduction ...................................................................................................................................... 79 2. Material and Methods ...................................................................................................................... 82 2.1 Outline of the approach .......................................................................................................... 82 2.2 Design of degenerate primers for conserved motifs of MAT idiomorphs and potentially flanking loci ................................................................................................................................... 83 2.3 Amplification of short conserved portions of the MAT idiomorphs and potential flanking loci using degenerate primers ............................................................................................................. 86 2.4 Inverse
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