Cell Physiological Investigations of Zygnema circumcarinatum (SAG698-1a and 698-1b) in the Aspect of their Capability of Conjugation and Comparison to Field Samples Masterarbeit zur Erlangung des Akademischen Grades eines Masters of Science (M.Sc.) an der Leopold Franzens Universität Innsbruck Institut für Botanik/ Abteilung Zellbiologie Studienrichtung: Biologie Studienzweig: Botanik betreut von Mag. Dr., assoz. Prof. Holzinger Andreas und assoz. Prof. Roach Thomas Duncan, MSc PhD verfasst und eingereicht von Charlotte Permann Innsbruck, August 2020 Acknowledgements First of all, I wish to express my appreciation towards Mag. Dr., assoz. Prof. Holzinger Andreas for giving me the opportunity to work on this master thesis and being part of his research group. I want to thank him for his excellent guidance, support and advice during my graduation. My thanks also go to Mag. Sabrina Obwegeser B.Sc. M.Sc., without which I would not have been able to complete this research. Special thanks to Monika Huber B.Sc. and Gregor Pichler B.Sc. M.Sc., for not only giving me professional advice and support, but for more importantly making workdays more fun and enjoyable. At his point I also thank my family, especially my parents Edgar and Verena for the financial and mental supporting they gave me. I also want to thank my siblings Stephan and Florine and my partner Pascal. List of Contents 1. Introduction ..................................................................................................................................................... 1 1.1 Algae .......................................................................................................................................................... 1 1.2 Taxonomy .................................................................................................................................................. 1 1.3 Habitat ....................................................................................................................................................... 5 1.4 Cytology ..................................................................................................................................................... 6 1.4.1 Vegetative Cell Form .......................................................................................................................... 6 1.4.2 Chloroplast ......................................................................................................................................... 6 1.4.3 Cell Wall .............................................................................................................................................. 6 1.4.4 Nucleus ............................................................................................................................................... 7 1.4.5 Mougeotia .......................................................................................................................................... 7 1.4.6 Spirogyra ............................................................................................................................................. 7 1.4.7 Zygnema ............................................................................................................................................. 8 1.5 Vegetative Reproduction ........................................................................................................................... 9 1.6 Conjugation ............................................................................................................................................. 10 2. Material and Methods ................................................................................................................................... 14 2.1 Algal Strains and Cultivation .................................................................................................................... 14 2.2 Field Samples ........................................................................................................................................... 14 2.3 Induction of Conjugation of Zygnema circumcarinatum ......................................................................... 18 2.4 Induction of Germination of Field Sampled Zygospores ......................................................................... 18 2.5 Measurements of Cell - and Mucilage Sheath Size/ Chloroplast Count ................................................ 19 2.6 Microscopy .............................................................................................................................................. 19 2.6.1 Magnification and Resolution .......................................................................................................... 20 2.6.2 Light (Optical) Microscopy ................................................................................................................ 21 2.6.3 Differential Interference Contrast Microscopy ................................................................................ 22 2.6.4 Fluorescence Microscopy ................................................................................................................. 23 2.6.5 Confocal Laser Scanning Microscopy ............................................................................................... 24 2.6.6 Polarizing Microscopy ....................................................................................................................... 25 2.6.7 Scanning Electron Microscopy.......................................................................................................... 26 2.6.8 Transmission Electron Microscopy ................................................................................................... 28 2.6.9 Microscopes Used in this Study ........................................................................................................ 30 2.7 Calcofluor White Staining ........................................................................................................................ 31 2.8 Glutaraldehyde Osmium Tetroxide Chemical Fixation for TEM .............................................................. 32 2.9 Measurement of the Relative Electron Transport Rate .......................................................................... 34 2.10 High Performance Liquid Chromatography ........................................................................................... 38 3. Results ........................................................................................................................................................... 39 3.1 Zygnema circumcarinatum SAG698-1a/ SAG698-1b ............................................................................... 39 3.1.1 Induction of Conjugation .................................................................................................................. 39 3.1.2 Light Microscopy and Measurements .............................................................................................. 39 3.1.3 Confocal Laser Scanning Microscopy ............................................................................................... 44 3.1.4 Transmission Electron Microscopy ................................................................................................... 44 3.1.5 Light Requirements of Photosynthesis ............................................................................................. 46 3.1.6 Pigment Analysis by High Pressure Liquid Chromatography ............................................................ 48 3.2 Field Collected Zygospores ...................................................................................................................... 49 3.2.1 Light Microscopy & Measurements ................................................................................................. 49 3.2.2 Scanning and Transmission Electron Microscopy ............................................................................ 54 4. Discussion ...................................................................................................................................................... 55 4.1 Zygnema circumcarinatum ...................................................................................................................... 56 4.2 Field Sampled Zygnematophyceae Zygospores....................................................................................... 64 5. Summary ........................................................................................................................................................ 71 6. Bibliography ................................................................................................................................................... 73 7. Eidesstattliche Erklärung ............................................................................................................................... 83 List of Tables Tab. 1 Origin of the taken field samples; SRR slow runnig rivulet; Po pond ..................................................... 15 Tab. 2 Components of BBM Medium (Bischoff and Bold, 1963) + Vitmamins and C-Medium (Gauch, 1966)..17 Tab. 3 Magnification and resolution capabilities (Murphy, 2001); SEM Scanning Electron Microscopy, TEM Transmission Electron Microscopy .................................................................................................................... 20 Tab. 4 Terminology of fluorescence (Consalvey et al., 2005; Maxwell