Towards a molecular phylogeny of the Euglenozoa: analyses of ribosomal DNA sequences and deduced secondary structure elements Thesis submitted in partial fulfilment of the requirements of the degree Doctor rer. nat. of the Faculty for Mathematics and Natural Sciences Chair in Biology and Didactics of Biology – Zoology Bergische Universität Wuppertal, Germany submitted by Stefan Pärschke Wuppertal December 2015 Die Dissertation kann wie folgt zitiert werden: urn:nbn:de:hbz:468-20160128-144301-5 [http://nbn-resolving.de/urn/resolver.pl?urn=urn%3Anbn%3Ade%3Ahbz%3A468-20160128-144301-5] First reviewer: Prof. Dr. A. Preisfeld Second reviewer: Prof. Dr. H. Wägele Table of contents I Contents Contents ................................................................................................................................ I-IV 1 Abstract .................................................................................................................................. 1 2 Introduction ........................................................................................................................... 3 2.1 Characteristics of Euglenozoa ........................................................................................... 3 2.2 Biogeography of Euglenozoa ............................................................................................ 6 2.3 Euglenozoa in eukaryote systematics ................................................................................ 8 2.4 Phylogenetic relationships of Euglenozoa......................................................................... 9 2.5 Euglenozoan ribosomal DNA.......................................................................................... 12 2.6 Scope of this thesis .......................................................................................................... 13 3 Material ................................................................................................................................ 15 3.1 Organisms ........................................................................................................................ 15 3.1.1 Strains of euglenid flagellates .................................................................................... 15 3.1.2 Strains of diplonemids ............................................................................................... 15 3.1.3 Strains of Escherichia coli ......................................................................................... 15 3.2 Media ............................................................................................................................... 16 3.2.1 Media for euglenid flagellates ................................................................................... 16 3.2.2 Media for diplonemids ............................................................................................... 16 3.2.3 Media for E. coli ........................................................................................................ 16 3.3 DNA samples................................................................................................................... 16 3.4 Buffers and solutions ....................................................................................................... 17 3.5 Stock solutions................................................................................................................. 17 3.6 Oligonucleotides .............................................................................................................. 18 3.7 Suppliers of reagents ....................................................................................................... 19 3.8 Suppliers of enzymes ....................................................................................................... 20 3.9 Suppliers of kits ............................................................................................................... 20 Table of contents II 3.10 Suppliers of standards.................................................................................................... 20 3.11 Suppliers of consumables .............................................................................................. 21 3.12 Suppliers of laboratory equipment ................................................................................ 21 3.13 Databases and online tools ............................................................................................ 22 3.14 Computer software ........................................................................................................ 23 4 Methods ................................................................................................................................ 24 4.1 Isolation of DNA and RNA ............................................................................................. 24 4.1.1 Isolation of total DNA ............................................................................................... 24 4.1.2 Isolation of plasmid DNA .......................................................................................... 24 4.1.3 Isolation of total RNA ............................................................................................... 25 4.2 Polymerase chain reactions (PCRs) ................................................................................. 25 4.2.1 Standard PCR ............................................................................................................ 26 4.2.2 Colony PCR ............................................................................................................... 26 4.2.3 Reverse-Transcription-PCR ....................................................................................... 27 4.3 Agarose gel electrophoresis ............................................................................................. 27 4.4 Purification of PCR products........................................................................................... 27 4.5 Cloning ............................................................................................................................ 28 4.6 Preparation of vector DNA .............................................................................................. 28 4.7 Quantification of vector DNA ......................................................................................... 29 4.8 Sequencing ...................................................................................................................... 29 4.9 Assembly of ribosomal DNA sequences ......................................................................... 29 4.10 Alignments of sequence data ......................................................................................... 30 4.10.1 SSU rDNA sequences .............................................................................................. 30 4.10.2 LSU rDNA sequences ............................................................................................. 33 4.11 Datasets.......................................................................................................................... 35 4.12 Computer analyses......................................................................................................... 35 4.12.1 Statistical tests ......................................................................................................... 35 Table of contents III 4.12.2 Model testing ........................................................................................................... 36 4.12.3 Maximum likelihood analyses ................................................................................. 36 4.12.4 Bayesian inferences ................................................................................................. 36 4.12.5 Phylogenetic networks ............................................................................................. 37 4.12.6 Spectral analyses ...................................................................................................... 37 4.13 Secondary structure analyses ......................................................................................... 37 5 Results and Discussion ........................................................................................................ 38 5.1 Phylogenetic analyses of SSU rDNA sequences ............................................................. 38 5.1.1 Preliminary dataset .................................................................................................... 38 5.1.2 Marine versus freshwater Euglenozoa ....................................................................... 43 5.1.3 Combined dataset ...................................................................................................... 53 5.2 SSU rDNA nucleotide sequence analyses ....................................................................... 62 5.2.1 Base composition ....................................................................................................... 62 5.2.2 Identity matrix ........................................................................................................... 65 5.2.3 Secondary structure ................................................................................................... 66 5.2.4 SSU rDNA variable regions ...................................................................................... 72 5.3 Phylogenetic analyses of LSU rDNA sequences ............................................................