Molecular phylogenetic analyses of Ecdysozoa and Haemosporida Dissertation submitted by Janus Borner to the University of Hamburg with the aim of achieving a doctoral degree (Dr. rer.nat.) at the Faculty of Mathematics, Informatics and Natural Sciences Department of Biology Hamburg, 2017 Gutachter der Dissertation: Prof. Dr. Thorsten Burmester Prof. Dr. Iris Bruchhaus Prof. Dr. Arndt von Haeseler Datum der mündlichen Prüfung: 07.09.2017 Danksagung Mein Dank gilt Prof. Dr. Thorsten Burmester für die ausgezeichneten Forschungsbedingungen, für das Vertrauen und die Freiheit, die ich bei der Umsetzung von eigenen Projekten genossen habe, für die Unterstützung in allen Lebenslagen, und für die enorme Geduld, die ich am Ende reichlich in Anspruch genommen habe. Genauso danke ich Prof. Dr. Iris Bruchhaus, auf deren Hilfe und Engagement ich stets zählen konnte, und die viele wichtige Kollaborationen für mich in die Wege geleitet hat. Ganz besonders möchte ich mich bei Maria Machola bedanken, der guten Seele des Labors, die mir über die Jahre immer wieder bei der Laborarbeit geholfen hat, und deren vorbildliche Organisation mir das ein oder andere Mal den Tag gerettet hat. Herzlich bedanken möchte ich mich auch bei Jenny Tiede, die im Rahmen ihrer Bachelorarbeit wichtige Daten zum Haemosporida-Projekt beigetragen hat. Christian Pick danke ich für die viele Hilfe am Anfang meiner Doktorarbeit und dafür, dass er den Büro-Alltag nie hat langweilig werden lassen. Dafür danke ich auch meinen aktuellen Büro-Kollegen Andrej und Conny. Ich möchte mich ganz herzlich bei allen Mitgliedern der Arbeitsgruppe bedanken für die angenehme Arbeitsatmosphäre, die lustigen Partys und die gegenseitige Unterstützung. Vielen Dank auch an die ehemaligen Kollegen, die ich manchmal vermisst habe, und zu deren Reihe ich mich nun geselle. Danke Sven, Mariana, Marco, Julia, Jessi, Sammy und Co.! Ich danke meinen Eltern, die mich immer und in allen Dingen unterstützt haben. Vielen Dank an dieser Stelle auch an meine Großmutter! Ganz besonders danke ich meiner Freundin, Annika Herwig, die mich mit viel Geduld (und Ungeduld) auf den langen letzten Metern begleitet hat. Mit Dir erscheinen mir alle Aufgaben etwas leichter (Doktorarbeiten ausgenommen), alle Horizonte etwas weiter, alle Tage etwas heller, und alle Morgen etwas früher. ;) Table of Contents 1 Introduction ............................................................................................................................................2 1.1 Molecular phylogenetics ....................................................................................................................2 1.2 The phylogeny of Ecdysozoa .............................................................................................................4 1.2.1 Myriapoda.....................................................................................................................................7 1.2.2 Chelicerata ....................................................................................................................................8 1.3 The phylogeny of Apicomplexa .........................................................................................................9 1.3.1 Haemosporidian relationships ................................................................................................... 10 1.4 Publications in chronological order ................................................................................................. 13 2 Discussion............................................................................................................................................. 14 2.1 Bioinformatic approaches in phylogenetics..................................................................................... 14 2.1.1 Bioinformatic pipelines for the generation of phylogenetic datasets ......................................... 14 2.1.2 Data mining of public databases for parasite contamination ..................................................... 17 2.1.3 Automated primer design for phylogenetic datasets ................................................................. 19 2.2 Phylogeny of Ecysozoa with focus on Arthropoda ......................................................................... 21 2.2.1 The deep phylogeny of Ecdysozoa ............................................................................................. 21 2.2.2 Myriapod relationships............................................................................................................... 24 2.2.3 Chelicerate relationships............................................................................................................. 26 2.2.4 Dating the arthropod tree ........................................................................................................... 29 2.3 Phylogeny of Apicomplexa with focus on Haemosporida .............................................................. 33 2.3.1 Parasite contaminations help illuminate the deep phylogeny of Apicomplexa......................... 33 2.3.2 The phylogeny of haemosporidian parasites based on nuclear gene data ................................ 35 3 References ............................................................................................................................................. 38 4 Declaration of own contribution to the published manuscripts ....................................................... 51 5 Eidesstattliche Versicherung................................................................................................................ 52 1 1 Introduction 1.1 Molecular phylogenetics Phylogenetics is the study of the evolutionary history and relationships among living or extinct organisms (Wägele, 2001; Brown, 2002; Storch & Welsch, 2003; Reece et al., 2011). In general, phylogenetic reconstruction is based on the comparison of homologous characters between organisms. Tree inference then aims to find the phylogeny that best explains the distribution of character states among taxa. Before large molecular datasets became available, phylogenetics relied on comparative morphology. While morphological data can be highly informative for answerin g phylogenetic questions, the amount of described characters is often insufficient for analysis via mathematical methods (Brown, 2002). Additionally, the definition of what constitutes a homologous morphological character is dependent on human interpretation and therefore subjective in nature (Graur & Li, 2000). The advent of nucleotide sequencing techniques has enabled researchers to employ molecular sequence data for phylogenetic reconstruction and has challenged many traditional views on evolutionary relationships across the tree of life. The classification of all living organisms into three domains (Bacteria, Archaea, and Eukarya) was based on molecular data, which showed that Archaea, initially described as extremophile bacteria, represent an entirely new group of organisms that are genetically distinct from both bacteria and eukaryotes (Woese et al., 1990). Within Mammalia, a new superorder called Afrotheria was erected comprising elephants (Proboscidea), sea cows (Sirenia), hyraxes (Hyracoidea), aardvark (Tubulidentata), elephant shrews (Macroscelidea), and golden moles and tenrecs (Afrosoricida). These orders share few common morphological traits and were previously considered members of other established groups of mammals, including ungulates and insectivores. Nevertheless, molecular data unequivocally revealed that Afrotheria constitute an ancient group of mammals that evolved in Africa, presumably while the continent was isolated through plate tectonics (Springer et al., 1997; Madsen et al., 1997; Stanhope et al., 1998). Another example of the profound effect that molecular phylogenetics had on our view of evolutionary relationships are the protostomes. Based on sequence data, Protostomia have been divided into Ecdysozoa, including arthropods and nematodes, and Lophotrochozoa, including annelids and molluscs (Aguinaldo et al., 1997), thus 2 refuting the sister group relationship of Arthropoda and Annelida, which morphologists considered to be one of the best-supported relationships among animal phyla (e.g., Westheide & Rieger, 1996; Brusca & Brusca, 2003). Molecular sequence data can provide enormous amounts of phylogenetic information because each nucleotide or amino acid position can be considered as an independent character. Though individual positions only contain limited phylogenetic signal (or none at all), the combined information from hundreds or thousands of positions can be sufficient to reconstruct a well-resolved phylogeny. Due to the high costs and technical challenges initially associated with nucleotide sequencing, early molecular phylogenetic studies were usually limited to analyses of single genes (e.g., Woese et al., 1990; Irwin et al., 1991; Ruvolo et al., 1991). However, for the analysis of deep phylogenetic relationships, the amount of sequence data available from single genes may not be sufficient as nucleotide substitutions accumulate in the sequences over evolutionary time and stochastic noise may drown out the phylogenetic signal contained in data from a single gene (Saitou & Nei, 1986; Walsh et al., 1999). This issue can be overcome by employing supermatrices that contain concatenated data from multiple genes. It is important to note in this context that the phylogenetic history of an individual gene (gene tree) is not necessarily congruent with the branching