Though molecular phylogenetics has been very successful in reconstructing the evolutionary history of species, some phylogenies, particularly those involving ancient events, have proven difficult to resolve. One approach to improving the resolution of deep phylogenies is to increase the amount of data by including multiple genes assembled from public sequence databases. Using modern phylogenetic methods and abundant computing power, the vast amount of sequence data available in public databases can be brought to bear on difficult phylogenetic problems. In this thesis I outline the motivation for assembling large multigene datasets and lay out the obstacles associated with doing so. I discuss the various methods by which these obstacles can be overcome and describe a bioinformatics solution, TaxMan, that can be used to rapidly assemble very large datasets of aligned genes in a largely automated fashion. I also explain the design and features of TaxMan from a biological standpoint and present the results of benchmarking studies. I illustrate the use of TaxMan to assemble large multigene datasets for two groups of taxa – the subphylum Chelicerata and the superphylum Lophotrochozoa. Chelicerata is a diverse group of arthropods with an uncertain phylogeny. When a set of mitochondrial genes is used to analyse the relationships between the chelicerate orders, the conclusions are highly dependent upon the evolutionary model used and are affected by the presence of systematic compsitional bias in mitochondrial genomes. Lophotrochozoa is a recently-proposed group of protostome phyla. A number of distinct phylogenetic hypotheses concerning the relationships between lophotrochozoan phyla have been proposed. I compare the phylogenetic conclusions given by analysis of nuclear and mitochondrial protein-coding and rRNA genes to evaluate support for some of these hypotheses.

One of the great unsolved evolutionary questions concerns the origin and phylogeny of the major animal phyla that appeared in the fossil record more than 540 million years ago, during the Cambrian explosion. Although new molecular information has been very useful, we still have little understanding about the origin of most of the phyla of bilaterians living today. The richly diverse fossil remains from this critical early Cambrian interval are particularly well exposed in China, where exceptionally-preserved fossil lophotrochozoans including brachiopods are particularly abundant. In particular the exceptionally-preserved Cambrian lophophorates from the Chengjiang and Guanshan Lagerstätten have offered new sources of critical palaeobiological data that have been shown to be important for understanding the early ecology and evolution of lophotrochozoans. This thesis comprises a detailed study of new, abundant, exceptionally-preserved material of five lophotrochozoan species from the Chengjiang and Guanshan Lagerstätten. Kuangshanotreta malungensis from Chengjiang is the earliest known example of an attached acrotretoid brachiopod representing the oldest evidence about the palaeoecology of the diverse yet, enigmatic acrotretoid linguliform stock that comprises an important component of the Cambrian evolutionary fauna. Eoglossa chengjiangensis from Chengjiang is the earliest known representative of the Glossellinae. Diandongia pista occurs abundantly both in the Chengjiang fauna and the younger Guanshan fauna, and it’s exceptionally well-preserved and strongly mineralized shells shows that it belongs within the Botsfordiidae. In contrast, the last two species from Chengjiang examined for this thesis - Yuganotheca elegans and Cotyledion tylodes belong within the stem of the Brachiopoda and the Lophotrochozoan entoprocts.

Left/right (L/R) asymmetries, differences in morphology between the otherwise mirrored left- and right-hand sides of the body, are found in animals across the Bilateria. For many years it was thought that the mechanisms for establishment of these asymmetries had evolved separately in the three superphyla that constitute the Bilateria, but the discovery in 2009 that the TGF-beta ligand Nodal shares a conserved role in the Deuterostomia and Lophotrochozoa has re-ignited debate and interest in this field. In this thesis, work examining the establishment and maintenance of L/R asymmetries in the lophotrochozoan superphylum is presented, aimed at uncovering the wider conservation of these pathways across the Bilateria. Illumina sequencing and a range of de novo assembly techniques were used to derive genomic and transcriptomic data respectively for two primary model organisms, the limpet Patella vulgata and the serpulid annelid Pomatoceros lamarckii. Additionally, collaborative work lead to the derivation of transcriptomes for two other mollusc species and the genome of the monogont rotifer Brachionus plicatilis. A range of analysis was performed on these novel resources and is detailed here, with particular reference to the transcription factor cassettes contained in these datasets. These sequence resources formed the basis for examination of the breaking of initial symmetry in these model organisms. Known read-outs of correct establishment of L/R asymmetry, the expression of genes Nodal and Pitx on the right of the body, were codified in the course of normal development in P. vulgata. Pharmacological inhibitors of genes implicated in the establishment of L/R asymmetry, particularly ATPase ion channels, were then applied to embryos. After development, markers of normal development were assayed for signs of bilateral inversion. Although radialised phenotypes were observed, it is unclear whether these are specifically the result of L/R asymmetry defects. The localisation of ATPase mRNA and serotonin, often posited as a small molecule potential morphogen, were also assayed, although no conclusions could be drawn as to a role in the establishment of L/R asymmetry for these molecules, counter to some evidence from vertebrates. Once symmetry is broken, the TGF-beta pathway is responsible for the communication, specification and maintenance of tissue identity across the L/R axis. The novel sequence resources described in this thesis provided a comprehensive window into this signalling cassette, and detailed here is a treatment of the TGF-beta pathway within the Lophotrochozoa. Ligand diversity has increased markedly in some clades, while signal transduction and regulatory steps are relatively unchanged. This work has increased our knowledge of lophotrochozoan biology and particularly the mechanisms underpinning the establishment of asymmetry in this under-researched clade, however, much remains to be discovered about the ultimate origin of asymmetry itself.

Die vorliegende Arbeit befasst sich mit Aspekten der frühen Entwicklung der Phoronida („Hufeisenwürmer“). An drei Arten wird der Furchungsprozess untersucht (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). Dies erfolgt sowohl mithilfe der 4D-Mikroskopie als auch anhand von immunocytochemischen Markierungen der Mitosespindeln und konfokaler Laser-Scanning-Mikroskopie. Verschiedene morphologische Merkmale des Furchungsprozesses werden quantitativ erfasst und innerhalb sowie zwischen den Arten verglichen. Die Ergebnisse zeigen eine weitgehend übereinstimmende Furchung bei P. pallida und P. muelleri Embryonen: Ab dem dritten Zellzyklus teilen sich die Blastomeren meist schräg – und alternierend dextral und sinistral – zur animal-vegetativ Achse. Dieses Muster zeigt überraschende Übereinstimmungen mit dem Muster der Spiralfurchung. Dies kann als morphologische Unterstützung molekular-phylogenetischer Befunde einer Stellung der Phoronida innerhalb der Spiralia/Lophotrochozoa interpretiert werden. Die Furchung bei P. vancouverensis unterscheidet sich von der Furchung der anderen beiden Arten; sie weist jedoch auch Unterschiede zu einer Radiärfurchung auf. Generell zeigt die Furchung aller drei Arten einen gewissen Grad an Variabilität. Anhand von in-vivo Einzelzellmarkierungen untersucht die Studie darüber hinaus das Schicksal der Blastomeren früher P. pallida Embryonen bis zu späten Gastrulationsstadien. Diese Analysen zeigen, dass die ersten beiden Furchungsteilungen durch die spätere Achse Blastoporus-Apikalplatte, jedoch in keinem konstanten Orientierungsverhältnis zur Ebene der Bilateralsymmetrie der Gastrula verlaufen. Dies unterscheidet sich von der Situation, wie sie von spiralfurchenden Tieren bekannt ist. Die Unterschiede und die beobachtete Variabilität des Furchungsprozesses werden im Licht unterschiedlicher Mechanismen der Spezifizierung von Zellschicksalen und Körperachsen bei verschiedenen Taxa der Spiralia und den Phoronida diskutiert. / This study addresses aspects of the early development of Phoronida (“horseshoe worms”). The cleavage process is analyzed for three species (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). These investigations are performed using 4D-microscopy as well as immunocytochemical stainings of the mitotic spindle apparatuses in combination with confocal laser-scanning microscopy. Different morphological features of the cleavage process are quantified and compared within as well as between the species. The results reveal a highly consistent cleavage of P. pallida and P. muelleri embryos: from the third cell cycle onward, the blastomeres divide mostly obliquely – and alternatingly dextral and sinistral – with respect to the animal-vegetal axis. This cleavage pattern shows surprising correspondences to the pattern of spiral cleavage. The finding can be interpreted as morphological support for recent molecule-based phylogenies, which indicate a position of Phoronida within the Spiralia/Lophotrochozoa clade. The cleavage of P. vancouverensis differs from the cleavage in the other two species; however, it also shows differences to a radial cleavage pattern. In all three species, the cleavage process also involves some degree of variability. Furthermore, the study traces the cell fates of early P. pallida embryos up to the state of late gastrulation, by the use of fluorescent in-vivo single cell markings. These analyses reveal that the first two cleavage divisions both pass through the later axis blastopore-apical plate of the gastrula, yet they do not pass in a constant relationship with respect to the later plane of bilateral symmetry. This differs from the situation known from spiral cleaving animals. The differences and the encountered variability of the cleavage process are discussed with respect to different mechanisms of the specification of cell fates and body axes in different taxa of the Spiralia and the Phoronida.

The evolutionary origin and the phylogeny of higher metazoan taxa is still under debate although considerable progress has been made in the past 20 years. Metazoa represents a monophyletic group of highly diverse animals including Bilateria, Cnidaria, Porifera, Ctenophores, and Placozoa. Bilateria comprises the majority of metazoans and consists of three major clades: Deuterostomia, Spiralia (= Lophotrochozoa sensu lato), and Ecdysozoa, whereas the sister group taxa Spiralia and Ecdyzozoa form the monophyletic clade Protostomia. Molecular data have profoundly changed the view of the bilaterian tree of life. One of the main questions concerning bilaterian phylogeny is the on-going debate about the evolution of complexity in Bilateria. It was assumed that the last common ancestor of Deuterostomia, Ecdysozoa and Spiralia had a segmented and coelomate body organization resembling that of an annelid. On the contrary, the traditional view is the evolution of Bilateria from a simple body organization towards more complex forms, assuming that the last common ancestor of Bilateria resembles a platyhelminth-like animal without coelomic cavities and segmentation. To resolve this question, it is necessary to unravel the phylogenetic relationships within Bilateria. By using mitogenomic and phylogenomic data, this thesis had a major contribution to clarify phylogenetic relationships within problematic metazoan taxa: (1) the phylogeny of Deuterostomia, (2) the questionable monophyly of Platyzoa, and first assumptions concerning the phylogeny of Gnathostomulida, Gastrotricha and Polycladida, (3) phylogenetic relationships within annelid taxa, especially Terebelliformia, Diurodrilidae, and Syllidae, with new insights into the evolution of mitochondrial gene order, and (4) new insights into the evolution of annelids, especially the interstitial ones, as well as the colonization of the interstitial realm.