I Dissertation zur Erlangung des Akademischen Grades eines Doktors der Naturwissenschaften -Dr. rer. nat.- im Fachbereich 2 (Biologie/Chemie) der Universität Bremen vorgelegt von Katrin Bruder Bremen, Mai 2006 Erster Gutachter: Prof. Dr. G.O. Kirst Zweiter Gutachter: Prof. Dr. Ulrich Bathmann Tag und Ort des öffentlichen Kolloquiums: 11. September 2006, Universität Bremen II Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe. Die entnommenen Stellen aus benutzten Werken wurden wörtlich oder inhaltlich als solche kenntlich gemacht Katrin Bruder Contents III Table of Contents Summary ................................................................................................................................... 1 Zusammenfassung.................................................................................................................... 3 1. Introduction .......................................................................................................................... 5 1.1 Diatom systematics .......................................................................................................... 5 1.2 Some problematic genera ................................................................................................. 6 1.2.1 Navicula .................................................................................................................... 6 1.2.2 Pinnularia and Caloneis ............................................................................................ 7 1.3 Molecular phylogenetics .................................................................................................. 8 1.3.1 Nuclear-encoded rRNA genes................................................................................... 8 1.3.1.1 Small subunit rRNA gene ................................................................................ 10 1.3.1.2 Large subunit rRNA gene ................................................................................ 10 1.3.2 Plastid-encoded protein-coding genes..................................................................... 10 1.3.2.1 rbc L gene.......................................................................................................... 11 1.3.3 Gene combination ................................................................................................... 11 1.3.4 Molecular phylogenies of diatoms .......................................................................... 12 1.4 Aims of this study .......................................................................................................... 13 2. Materials and Methods ...................................................................................................... 15 2.1. Cultures ......................................................................................................................... 15 2.2. DNA Methods ............................................................................................................... 21 2.2.1 DNA isolation ......................................................................................................... 21 2.2.2. PCR ........................................................................................................................ 21 2.2.3. Sequencing ............................................................................................................. 22 2.3. Sequence Analysis......................................................................................................... 22 2.4. Microscopy.................................................................................................................... 26 2.4.1 Purification of the frustules..................................................................................... 26 2.4.2. Slide preparation .................................................................................................... 26 3. Results ................................................................................................................................. 27 3.1 Molecular data................................................................................................................ 27 3.1.1 SSU rRNA gene ...................................................................................................... 27 Contents IV 3.1.2 LSU rRNA gene...................................................................................................... 36 3.1.3 rbc L gene................................................................................................................. 43 3.1.4 Gene combination ................................................................................................... 51 3.2 Morphological support for molecular data..................................................................... 55 3.2.1 Navicula sensu stricto ............................................................................................. 55 3.2.2 Amphora .................................................................................................................. 56 3.2.3 Pinnularia and Caloneis .......................................................................................... 65 3.2.4 Stauroneis, Craticula and Navicula integra ............................................................ 72 3.2.5 Gomphonema .......................................................................................................... 75 3.2.6 Placoneis and Navicula hambergii ......................................................................... 82 3.2.7 Cymbella .................................................................................................................. 82 3.2.8 Navicula brockmannii ............................................................................................. 84 3.2.9 Varieties of Mayamaea atomus ............................................................................... 84 4. Discussion............................................................................................................................ 92 4.1 Comparison of the gene trees ......................................................................................... 92 4.1.1 Phylogenies based on the AlgaTerra cultures ......................................................... 93 4.1.1.1 Phylogenies based on SSU rDNA sequences................................................... 93 4.1.1.2 Phylogenies based on LSU rDNA sequences .................................................. 94 4.1.1.3 Phylogenies based on rbc L gene sequences..................................................... 95 4.1.1.4 Phylogenies based on the combined dataset .................................................... 96 4.1.1.5 General results of the analyses of the AlgaTerra cultures................................ 97 4.1.2 Phylogenies based on enlarged datasets................................................................ 100 4.1.2.1 Phylogenies based on SSU rDNA sequences................................................. 100 4.1.2.2 Phylogenies based on LSU rDNA sequences ................................................ 102 4.1.2.3 Phylogenies based on rbc L gene sequences................................................... 103 4.1.3 General relationships of the genera....................................................................... 103 4.2 Relationships within the genera ................................................................................... 105 4.2.1 Navicula sensu stricto ........................................................................................... 105 4.2.2 Amphora ................................................................................................................ 105 4.2.3 Caloneis and Pinnularia ........................................................................................ 107 4.2.4 Navicula integra .................................................................................................... 109 4.2.5 Gomphonema ........................................................................................................ 110 4.2.6 Placoneis and Navicula hambergii ....................................................................... 111 Contents V 4.2.7 Cymbella ................................................................................................................ 112 4.2.8 Navicula brockmannii ........................................................................................... 112 4.2.9 Varieties of Mayamaea atomus ............................................................................. 113 References ............................................................................................................................. 114 Appendix ............................................................................................................................... 126 Acknowledgements............................................................................................................... 141 Summary 1 Summary The recent taxonomy of diatoms is basically based on investigations of valve morphology, cell components and life cycle (e.g., Round et al. , 1990). But the development of PCR has facilitated the use of DNA sequences for inferring phylogenies. Based on morphology, the taxonomy of the family Naviculaceae ( sensu Krammer & Lange-Bertalot, 1986) has been highly changed (e.g., Round et al. , 1990), but little work