Untersuchungen zur Phylogeographie und Systematik von Asselspinnen (Arthropoda: Pycnogonida) des Südpolarmeeres Studies on the phylogeography and systematics of sea spiders (Arthropoda: Pycnogonida) of the Southern Ocean Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie und Biotechnologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum angefertigt im Lehrstuhl für Evolutionsökologie und Biodiversität der Tiere vorgelegt von Lars Christian Dietz aus Duisburg Referent: Prof. Dr. Ralph Tollrian Korreferent: Prof. Dr. Dominik Begerow Dissertation Lars Dietz ERKLÄRUNG Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und bei keiner anderen Fakultät eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um sechs in Wort und Bild völlig übereinstimmende Exemplare. Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde. Bochum, den _____________________________________ 1 Dissertation Lars Dietz Inhaltsverzeichnis Curriculum Vitae 3 1) General Introduction 4 2) Publikation I: Morphological and genetic data clarify the 21 taxonomic status of Colossendeis robusta and C. glacialis (Pycnogonida) and reveal overlooked diversity 3) Publikation II: Evidence from morphological and genetic data 60 confirms that Colossendeis tenera Hilton, 1943 (Arthropoda: Pycnogonida), does not belong to the Colossendeis megalonyx Hoek, 1881 complex 4) Publikation III: Regional differentiation and extensive 82 hybridisation between mitochondrial clades of the Southern Ocean giant sea spider Colossendeis megalonyx 5) Publikation IV: Assessing demographic responses to past 125 climate change in Southern Ocean sea spiders 6) Publikation V: Pallenopsis patagonica (Hoek, 1881) – a species 151 complex revealed morphology and DNA barcoding, with description of a new species of Pallenopsis by Wilson, 1881 7) Publikation VI: Exploring Pandora’s box: Potential and pitfalls of 177 low coverage genome surveys for evolutionary biology 8) General Discussion 201 9) Summary 217 10) Zusammenfassung 220 11) Acknowledgements 223 2 Dissertation Lars Dietz Curriculum Vitae von Lars Christian Dietz Geburt am 22.1.1987 in Duisburg Schulische Ausbildung: 1993-1996: Grundschule Am Knappert, Duisburg 1996-2005: Reinhard-und-Max-Mannesmann-Gymnasium, Duisburg Abitur am 9.6.2005 Studium: Studium der Biologie und Biotechnologie in Bochum seit Wintersemester 2005 Erwerb des Diploms am 17.11.2010 am Lehrstuhl für Evolutionsökologie und Biodiversität der Tiere (Thema: „Multi-Gen-Analyse zur Phylogenie ausgewählter Pantopoda“) Promotionsstudium seit 18.10.2011 Beschäftigung als wissenschaftlicher Mitarbeiter 1.2.2011 – 31.9.2014 (Ruhr-Universität Bochum) Beschäftigung als wissenschaftliche Hilfskraft 1.10.2014 – 31.1.2015 (Ruhr-Universität Bochum) 3 Dissertation Lars Dietz 1) General Introduction 1.1 The Southern Ocean ecosystem The Southern Ocean is the ocean around the continent of Antarctica (Fig. 1). While there are contrasting definitions of the borders of the Southern Ocean, it is - for biological purposes - most meaningful to define the Antarctic Convergence (also called the Southern Polar Front) as the northern border of the Southern Ocean (e.g. Dell 1972). The region south of the convergence, including land and ocean, is known as the Antarctic and the region to the north of it is called the Subantarctic. The Antarctic convergence, where the warm water masses from the north and the cold ones from the south meet, isolates the Southern Ocean ecosystem from those of other oceans. In the convergence zone there is a strong eastward current, the Antarctic Circumpolar Current (ACC) driven by westerly winds. Most of the Southern Ocean is deep sea, as the Antarctic continental shelf is rather narrow. However, the shelf extends to greater depths than those of other continents (up to 1000 m; Domack 2007). The shelf fauna is considerably better known than that of the deep sea, and most of the samples investigated in this dissertation originated on the shelf. The Antarctic continent, and therefore also its shelf area, is subdivided into the smaller West Antarctica and the larger East Antarctica, which are separated by the Ross and Weddell Seas. The Antarctic Peninsula, with adjacent islands, extends from West Antarctica to the north, and is the best-sampled region of Antarctica (Griffiths et al. 2011). The islands of the Scotia Arc (South Georgia, South Sandwich, South Orkneys, South Shetlands) are situated in the Atlantic sector of the Southern Ocean between South America and the Antarctic Peninsula. Other islands in the Antarctic zone include Bouvet Island in the South Atlantic and the islands of the Kerguelen Plateau in the Indian Ocean. The Antarctic benthic fauna is strongly differentiated from that of other oceans (e.g. Aronson 2007). Particularly notable is the rarity of taxa with planktotrophic larval stages such as decapods, bivalves and teleost fish, while groups without such stages (pycnogonids, isopods, amphipods, some echinoderms) are much more diverse than in other oceans (Clarke & Johnston 2003). Within these groups, a large percentage of species is endemic to the Southern Ocean (35-90% in different taxa according to Arntz et al. 1997, Aronson et al. 2007, see also Fig. 2). There is also strong regional differentiation between various geographical regions of the Southern Ocean, especially between the Antarctic shelf, the surrounding islands, and the deep sea (e.g. Griffiths et al. 2011). 4 Dissertation Lars Dietz Fig. 1: The Southern Ocean, with the Polar Front and the main island groups marked. From Moles et al. (in press) Fig. 2. Percentage of species endemic to the Southern Ocean within different benthic invertebrate groups, from Aronson et al. (2007). Bars show Antarctic species number as a percentage of worldwide species number. Numbers are absolute Antarctic species numbers. 5 Dissertation Lars Dietz In the past, the diversity of Antarctic invertebrate species could only be assessed by the morphological determination of specimens, whose number was often limited due due the difficulties of conducting fieldwork in the Antarctic. Yet, in many groups, the presence of morphologically similar taxa (Lecointre et al. 2013) together with a high within-species variability for some taxa (e.g. the pycnogonid Colossendeis megalonyx; Child 1995 has led to confusion and disagreements concerning the synonymy of species. Molecular data as another line of taxonomic evidence may contribute to address these questions. 1.2 Molecular taxonomy and DNA barcoding In recent years, molecular data has increasingly been used for species-level taxonomy. It has been suggested that a single gene could be used as a “universal barcode” to identify species. In animals, the mitochondrial gene Cytochrome C Oxidase subunit 1 (CO1) has been proposed as the standard barcode fragment (Hebert et al. 2003) due to the fact that in most cases there is a significant gap (“barcoding gap”) between intra- and interspecific genetic distances in CO1. As a mitochondrial DNA (mtDNA) marker which is abundant within cells, relatively simple to amplify and for which several primer pairs have been developed that bind at conserved sites (e.g. Folmer et al. 1994) it is now commonly used throughout the animal kingdom. However, mtDNA is inherited only in the maternal line in almost all animals, meaning that it reflects only a single ancestral lineage. Therefore, mtDNA can be misleading in some cases: First, mtDNA from one species may cross into other related species by hybridization and will be inherited in all descendants of the hybrid in the maternal line (introgressive hybridization, see Toews & Brelsford 2012 for a review). The introgressed mtDNA may also become fixed within the species, especially if it has an adaptive advantage, leading to incongruence between phylogenetic trees based on mitochondrial and nuclear data. Secondly, sex-biased dispersal (i.e. one sex has a greater capability of dispersal than the other) also leads to incongruence between mitochondrial and nuclear data. Thirdly, due to its mode of inheritance and haploidy, mtDNA also generally has a lower effective population size than the nuclear genome (1/4 if the sex ratio is 1:1; Zink & Barrowclough 2008), unless females are much more numerous than males and therefore mutations become fixed more quickly. Because of these problems, mtDNA sometimes gives only an incomplete or misleading picture of population history (Ballard and Whitlock 2004). Therefore, for a detailed investigation of taxonomic questions, an integrative taxonomic approach should be used, incorporating several different datasets such as mtDNA, nuclear DNA and morphological characters. Next-generation sequencing methods such as 454 pyrosequencing (Metzker 2010) increasingly make it possible to generate large amounts of sequence data even for non- 6 Dissertation Lars Dietz model species. These techniques may be useful for phylogenetic and population genetic purposes. As an example, it is possible to generate complete mitochondrial genome sequences from next-generation sequencing data (Dietz et al. 2011, 2015). However, it would be useful to assess how many mitochondrial data, microsatellite markers and other types of data are to be expected in next-generation data from a given taxon. Also, large