In der Arbeit wird der Einsatz kalorimetrischer Methoden zur Bestimmung der katalytischen Aktivität von Enzymimmobilisaten gezeigt. Für die Untersuchung sphärischer Immobilisate wurde ein Strömungskalorimeter eingesetzt. Es wurde ein mathematisches Modell entwickelt, mit dem die kinetischen Parameter des immobilisierten Enzyms aus der gemessenen Temperaturdifferenz berechnet werden können. Durch Untersuchungen an Modellimmobilisaten wurde gezeigt, dass das Strömungskalorimeter im Impulsbetrieb zum schnellen Screening der Aktivität geeignet ist. Am Beispiel der Enzymimmobilisierung in Sol-Gel-Schichten wurde der Einsatz der Impulsmethode für die Optimierung von Immobilisierungsvorschriften gezeigt. Zur Untersuchung flächiger Immobilisate wurde ein IC-Kalorimeter eingesetzt. Um kinetische Aussagen zu erzielen, wurde das IC-Kalorimeter hinsichtlich Empfindlichkeit und dynamischen Verhaltens charakterisiert. Es konnte gezeigt werden, dass die kalorimetrisch und photometrisch bestimmten Geschwindigkeitskonstanten für die Immobilisate übereinstimmen.

Root processes – decomposition, production, and mortality – are intrinsic parts of ecosystem carbon cycling and yet have been understudied in the context of biodiversity-ecosystem functioning relationships. In a long-term grassland diversity experiment (the Jena Experiment), I investigated (1) whether and how plant diversity affected root decomposition and (2) how plant diversity enhanced standing root biomass and influenced root turnover. Plant diversity may affect root decomposition via three non-mutually exclusive pathways: (1) root litter quality, (2) soil biota, and/or (3) soil abiotic conditions. In Chapter 3, via three decomposition experiments, I demonstrate that plant species richness negatively affects root decomposition via the root litter quality pathway and the soil environment pathway (including soil biotic and abiotic conditions). The presence of grasses negatively affects root decomposition while the presence of legumes positively affects root decomposition. In Chapter 4, I further explored the pathways driving the relationship between plant diversity and root decomposition using structural equation modeling. The final structural equation model suggests that root chemistry is a universal pathway for explaining the effects of plant diversity on root decomposition and that Oribatida are also involved in root decomposition. Most importantly, I directly show that different measures of plant diversity influence root decomposition via different pathways. In Chapter 5, I shift my focus to root production and mortality which collectively determine standing root biomass. I monitored in situ root dynamics biweekly for one growing season using minirhizotrons. I show that plant species richness consistently enhances standing root length throughout the observational period. However, plant species richness did not affect rates of root length production, mortality, or turnover. Only root lifespan increased with increasing plant species richness. The lack of significant diversity effect on root length-based measures may be due to the age of the studied communities. In mature grasslands, plant species richness may only have a minor effect on root turnover and one growing season may be too short to detect such a small effect. The results of this dissertation collectively provide new insights into the relationship between plant species richness and soil carbon stock in mature grasslands. Based on the new paradigm of soil carbon formation, plant species richness may enhance soil carbon stock through a greater input of partially decomposed root residuals (reduced root decomposition) and a higher input of root exudates (increased standing root biomass).:ACKNOWLEDGEMENTS .......................................................................................III 1 GENERAL INTRODUCTION ............................................................................... 1 1.1 Biodiversity-Ecosystem Functioning Research in the Context of Global Biodiversity Loss ……........................................................................................... 1 1.2 Biodiversity-Ecosystem Functioning Relationships .............................................................................................................................. 2 1.3 Effects of Plant Diversity on Decomposition ................................................... 6 1.4 Effects of Plant Diversity on Root Turnover .................................................. 10 1.5 Scope and Contents of this Dissertation ...................................................... 14 2 GENERAL METHODS ......................................................................................17 2.1 The Jena Experiment ................................................................................... 17 2.2 Root Decomposition Experiments ................................................................ 18 2.3 Applying Structural Equation Modeling to Understand the Diversity-Decomposition Relationships ............................................................................. 22 2.4 In-situ Monitoring of Root Turnover Using Minirhizotrons ............................. 25 3 PLANT SPECIES RICHNESS NEGATIVELY AFFECTS ROOT DECOMPOSITION IN GRASSLANDS ................................................................................................27 4 ROOT CHEMISTRY AND SOIL FAUNA, BUT NOT SOIL ABIOTIC CONDITIONS EXPLAIN THE EFFECTS OF PLANT DIVERSITY ON ROOT DECOMPOSITION..44 5 THE EFFECTS OF PLANT SPECIES RICHNESS ON ROOT TURNOVER IN EXPERIMENTAL GRASSLANDS...........................................................................74 6 GENERAL DISCUSSION..................................................................................105 6.1 Summary of Contents in this Dissertation.................................................... 105 6.2 Advancing Our Understanding of Root Decomposition in BEF Research.... 106 6.3 Time Matters for the Diversity–Root Turnover Relationship......................... 112 6.4 Outlook........................................................................................................ 117 REFERENCES...................................................................................................120 SUMMARY .........................................................................................................137 ZUSAMMENFASSUNG .......................................................................................142 LIST OF PUBLICATIONS....................................................................................148 AUTHOR CONTRIBUTION STATEMENTS..........................................................150 DECLARATION OF INDEPENDENT WORK........................................................159 CURRICULUM VITAE .........................................................................................160

Organohalides have been abundantly utilized as pesticides and in industrial processes for the past 100 years, with over 30 000 sites in Europe still being contaminated today. Because of their recalcitrance, large quantities have accumulated in soils, sediments, and groundwater. Many organohalides can cause multiple adverse health effects, including neurological damage, congenital malformations, and a variety of human cancers. Fortunately, bacterial genera from a diverse range of phyla are capable of detoxifying these organohalides via anaerobic respiration, i.e., by using them as their terminal electron acceptor. These metabolic pathways involve a reductive dehalogenation reaction, during which a chlorine atom dissociates and thereby either immediately reduces the toxicity of the organohalide, or enables it to be further degraded by a broader range of organisms. Thus, organohalide-respiring bacteria can be used for the bioremediation of contaminated environments. To be able to support this application, fundamental research on these reactions and the metabolism of organohalide-respiring bacteria is a prerequisite. Many aspects of the physiology of organohalide-respiring bacteria are unresolved. Organohalide-respiring bacteria harbor up to 38 reductive dehalogenase homologous genes, which putatively encode the key enzymes of reductive dehalogenation. However, the regulation, protein-coding ability, the function of these enzymes as well as their interactions with other proteins has yet to be elucidated. Organohalide-respiring bacteria are difficult to study due to their slow growth, low biomass yields, oxygen sensitivity and genetic inaccessibility. The aim of this thesis was to circumvent these obstacles by introducing new methods for studying organohalide respiration and thereby enabling the formulation of informed predictions about the functions of reductive dehalogenases and the identity of their regulators. For this, obligate and facultative organohalide-respiring bacteria were assessed. To form a basis of the current research in the field, all available genomic, transcriptomic and proteomic literature on organohalide-respiring bacteria were reviewed and compared. Through combining quantitative expression data of hundreds of orthologs and subjecting them to statistical analyses, many new aspects of the metabolism of organohalide-respiring bacteria were uncovered. Especially notable were the unclear expression patterns of reductive dehalogenases and their accessory proteins. An important conclusion from this review was that shotgun proteomics is essential to reveal how many reductive dehalogenase proteins are produced in parallel, but this approach alone cannot clarify the function of these enzymes nor their underlying regulation processes. Therefore, the next chapter of this thesis aimed to extend and refine the standard proteomics approaches. First, proteomics conducted via mass spectrometry requires optimization of sample processing and analysis. Utilizing harsher conditions for protein extraction and digestion substantially improved proteome coverage compared to previous studies, especially of membrane proteins. The combination of this approach with a highly stringent statistical filtering procedure allowed a more detailed, reliable and thus more valid view of the proteome to be obtained from the model organism Sulfurospirillum halorespirans. The quantification of the putative protein histidine kinase provided the first evidence of its involvement in controlling organohalide respiration together with the putative response regulator, forming a complete two-component regulatory system. The quantification of the putative quinol dehydrogenase membrane subunit also supported its involvement in the organohalide respiratory chain of this genus. We observed that S. halorespirans undergoes the same type of peculiar memory-effect as Sulfurospirillum multivorans, that is, continuing to produce its complete dehalogenating machinery even after prolonged cultivation on a non-halogenated electron acceptor. To reveal the underlying mechanism, protein lysine acetylation was additionally measured, which is an important post-translational modification involved in many regulatory processes across all living organisms. Lysine acetylations are, e.g., known to alter the binding properties of DNA-interacting proteins like transcription factors or response regulators but have a range of other regulatory effects. In the first ‘acetylome’ study of an organohalide-respiring bacterium and an Epsilonproteobacterium, one-third of all S. halorespirans proteins were found to be acetylated at one point over the course of a long-term cultivation experiment. Interestingly, the putative response regulator of the two-component regulatory system described earlier was acetylated during the metabolic transition phase, after short-term adaptation to a non-halogenated electron acceptor. Another advancement of shotgun proteomics was its combination with thermal proteome profiling to elucidate substrate specificities of reductive dehalogenases and their regulators. The underlying principle behind thermal proteome profiling is to identify the interaction of a protein with a binding ligand through its impact on the thermal stability of the protein. The thermal stability of hundreds of proteins can be measured in parallel by a proteomics approach. Aliquots of protein extract are first incubated at different temperatures, and the non-denatured fraction of each protein is then quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), thus allowing the composition of melting curves of each protein to be determined. With this unbiased approach, unknown protein-ligand interactions can also be identified. In a proof-of-concept study on S. multivorans, we adapted the method to anaerobic conditions and showed that this technique is suitable for the detection of interactions between enzymes and their specific substrates. For example, a melting curve shift was detected when the tetrachloroethene reductive dehalogenase, PceA, bound to its known substrate, trichloroethene. Furthermore, the melting curve shift of the putative response regulator in the two-component regulatory system indicated at least an indirect interaction between it and trichloroethene, providing the first biochemical evidence of its role in organohalide respiration besides mere expression data. In conclusion, this work not only includes the first systematic analysis of all omics-based studies conducted to date but substantially advanced the methods for assessing organohalide-respiring bacteria by providing a more detailed picture of their physiology. Besides methodological advances, it was demonstrated that the two-component regulatory system interacts with halogenated compounds and that its post-translational modification might impact long-term downregulation of the organohalide respiratory apparatus in Sulfurospirillum spp. The insights into the involvement of the two-component regulatory system in the organohalide respiration of Sulfurospirillum spp. would not have been uncovered by using less complex standard shotgun proteomics measurements. In the future, our findings will help to further elucidate regulators and functioning of reductive dehalogenases also in other organohalide-respiring bacteria.:Summary 7 Zusammenfassung 10 1 Introduction 14 1.1 Halogenated compounds and the environment……………………...……….……. 14 1.2 Transformation of organohalides……………………..……………….…………….. 15 1.3 Reductive dehalogenation………………………..……………………………….…... 16 1.3.1 Dehalococcoides mccartyi……………………………………………….……… 18 1.3.2 Sulfurospirillum spp. …………………..………………………………..……... 20 1.4 Proteomics……………………..………………..…………………………………...….. 22 1.4.1 The principle of shotgun proteomics..………………..………………....……. 22 1.4.2 Protein lysine acetylations–an important post-translational modification…………………………………………………………...………… 24 1.4.3 Thermal proteome profiling..………………..………..……..………………... 28 1.5 Objectives..………………..……..………..………..………………..…………………. 29 2 Publications 31 2.1 Overview of publications..………..………………..………….………..…………….. 31 2.1.1 Publication 1..………..………….………..…….………..………………………. 31 2.1.2 Publication 2..………..…………..………..…….………..……………………… 31 2.1.3 Publication 3..………..…………….………..…..………..……………………… 32 2.1.4 Publication 4..………..…………..……….…..………..……………….……….. 32 2.2 Published articles..………..……………....…………..………..………………..……. 33 3 Discussion 88 3.1 The application of ‘omics’ to organohalide-respiring bacteria..………..………... 88 3.2 Parallel proteome and acetylome analysis..………..………………..…………….. 91 3.2.1 Specific challenges for the analysis of protein lysine acetylations………. 92 3.2.2 Insights into the metabolism of S. halorespirans..………..………………... 93 3.3 Protein interaction analysis by thermal proteome profiling..………..……......... 97 3.3.1 Other potential approaches to study protein-ligand-interactions..…….... 98 3.3.2 Potential of using thermal proteome profiling for organohalide- respiring bacteria..………..……….………..………….………..……………… 99 3.4 Conclusions and future perspectives..………..……………..………..…..………… 101 4 References 104 5 Appendix 118 5.1 Declaration of authorship..………..……………..………..……………………..…… 118 5.2 Author contribution of published articles..………..……………..……………….... 118 5.3 Curriculum vitae..………..………………..…………….………..…………………… 124 5.4 List of publications and conference contributions..………..……………...………. 124 5.5 Acknowledgements..………..………… ………..…………………..…………..…….. 127 5.6 Supplementary material..…………………..………..………………………….……. 128 5.6.1 Supplementary material for Publication 3..………..……..………..……….. 128 / Während der letzten einhundert Jahre wurden halogenierte organische Verbindungen großflächig in Industrie und Landwirtschaft eingesetzt, wodurch heute mehr als 30 000 Flächen in Europa kontaminiert sind. Aufgrund ihrer eingeschränkten Abbaubarkeit konnten sich riesige Mengen in Böden, Sedimenten und Grundwasser ausbreiten. Viele halogenierte organische Verbindungen können erhebliche nachteilige Auswirkungen auf die Gesundheit des Menschen haben, u.a. neurologische Schäden, Fehlbildungen und eine Vielzahl von Krebserkrankungen. Glücklicherweise sind bestimmte Bakterientypen unterschiedlicher Phyla in der Lage, diese Stoffe mittels anaerober Atmung, d.h. über deren Nutzung als terminalen Elektronenakzeptor, umzuwandeln. Diese reduktive Dehalogenierung, bei der ein Chlor-Rest abgespalten wird, vermindert die Toxizität der meisten Organohalide bzw. macht sie zugänglich für den Abbau durch ein breiteres Organismenspektrum. Demgemäß können Organohalid-atmende Bakterien für die Bioremediation kontaminierter Flächen genutzt werden. Voraussetzung für deren Einsatz ist jedoch das Verständnis der zugrundeliegenden biochemischen Reaktionen und des Metabolismus der Organohalid-Atmer. Viele Aspekte der Physiologie Organohalid-atmender Bakterien sind noch ungeklärt. Die Organismen besitzen bis zu 38 unterschiedliche Gene, die reduktive Dehalogenasen, die Schlüsselenzyme der Organohalid-Atmung, kodieren. Allerdings sind deren Regulation, Proteinkodierung, die Funktion der einzelnen Enzyme sowie deren Interaktionen mit anderen Proteinen noch unbekannt. Die Forschung an Organohalid-atmenden Bakterien wird durch deren langsames Wachstum, die geringen Zelldichten, die hohe Sensitivität gegenüber Sauerstoff und fehlende gentechnische Methoden erschwert. Ziel dieser Arbeit war es, die genannten Hindernisse mittels neuartiger Methoden an Organohalid-Atmern zu umgehen und damit Regulatoren und Funktionsweise der reduktiven Dehalogenasen zu bestimmen. Hierfür wurden sowohl obligate als auch fakultative Organohalid-atmende Bakterien herangezogen. Als Grundlage führte ich zunächst alle bisher durchgeführten Genomik-, Transkriptomik- und Proteomikstudien zu Organohalid-atmenden Bakterien zusammen. Hunderte zu Orthologen kombinierte und statistisch analysierte quantitative Expressionsdaten lieferten dabei ein umfassendes Bild vom Metabolismus der Organohalid-Atmer. Insbesondere die unklaren Expressionsmuster der reduktiven Dehalogenasen und ihrer akzessorischen Proteine wurden offenbar. Eine wichtige Erkenntnis des Review-Prozesses war, dass Standard-Proteomikansätze zwar unerlässlich sind, um beispielsweise die gleichzeitige Produktion mehrerer reduktiver Dehalogenasen offenzulegen, aber weder deren Funktionen noch Regulation aufklären können. Aus diesem Grund sollten im weiteren Verlauf dieser Arbeit die bisher genutzten Shotgun-Proteomikmethoden weiterentwickelt werden. Für eine umfassende Proteinanalyse mittels Massenspektrometrie müssen zunächst Probenaufarbeitung und Analyse optimiert werden. Durch die Verwendung harscherer Bedingungen bei Proteinextraktion und -verdau konnten wir die Proteomabdeckung, insbesondere unter Membranproteinen, im Vergleich zu früheren Studien erheblich verbessern. In Kombination mit einem sehr stringenten statistischen Filterprozess erlaubte dies einen detaillierten und validen Blick auf das Proteom des Modellorganismus Sulfurospirillum halorespirans. Die Quantifizierung der mutmaßlichen Protein-Histidinkinase ist der erste Beleg dafür, dass diese zusammen mit dem Regulationsprotein im Zweikomponentensystem an der Kontrolle der Organohalid-Atmung in Sulfurospirillum spp. beteiligt ist. Die quantifizierte Membranuntereinheit der Quinoldehydrogenase stützt die Annahme zu deren Beteiligung an der Atmungskette dieses Organismus. Wir konnten weiterhin zeigen, dass in S. halorespirans die gleiche außergewöhnliche Langzeitregulation wie in Sulfurospirillum multivorans wirksam ist, sodass auch nach langanhaltender Kultivierung auf nicht-halogenierten Substraten der komplette Organohalid-Atmungsapparat synthetisiert wird. Zur Aufklärung der zugrundeliegenden Regulation erweiterten wir unsere Analyse um Protein-Lysin-Acetylierungen, wichtige posttranslationale Modifikationen, die an verschiedensten regulatorischen Prozessen in allen Lebewesen beteiligt sind. Protein-Lysin-Acetylierungen beeinflussen z.B. die Wechselwirkungen zwischen Transkriptionsfaktoren oder Regulationsproteinen und der DNA, aber haben noch viele weitere regulatorische Effekte. In dieser ersten „Acetylom“-Studie an einem Organohalid-atmenden Bakterium bzw. einem Epsilonproteobacterium, konnten wir zeigen, dass ein Drittel aller S. halorespirans-Proteine im Verlauf der Langzeitkultivierung mindestens einmal acetyliert wurden. Interessanterweise war auch das mutmaßliche Regulatorprotein des oben erwähnten Zweikomponentensystems während der metabolischen Umstellungsphase, d.h. nach Kurzzeitanpassung an den nicht-halogenierten Elektronenakzeptor, acetyliert. Eine zusätzliche Weiterentwicklung der klassischen proteomischen Messungen war deren Kombination mit Thermal Proteome Profiling, um Substratspezifitäten und Regulatoren von reduktiven Dehalogenasen zu bestimmen. Zugrundeliegendes Prinzip des Thermal Proteome Profiling ist die Identifikation eines Proteinbindungspartners über dessen Einfluss auf die Thermostabilität der Faltung eines Proteins. Die Thermostabilität tausender Proteine kann mit Hilfe eines Proteomikansatzes bestimmt werden. Hierfür werden extrahierte Proteine zunächst aufgeteilt und unterschiedlichen Temperaturen ausgesetzt. Die nicht-denaturierte Fraktion jedes Proteins kann mittels Flüssigchromatographie mit Tandemmassenspektrometrie-Kopplung (LC-MS/MS) quantifiziert und zu Schmelzkurven zusammengesetzt werden. Mit dieser Methode können auch unbekannte Protein-Liganden-Interaktionen identifiziert werden. In unserer Machbarkeitsstudie an S. multivorans konnten wir zeigen, dass die von uns modifizierte Technik auch zur Aufklärung von Enzym-Substrat-Interaktionen und sogar unter anaeroben Bedigungen eingesetzt werden kann. So konnte nachgewiesen werden, dass die Schmelzkurve der reduktiven Tetrachlorethen-Dehalogenase PceA durch Bindung ihres bekannten Substrates Trichlorethen signifikant verschoben wurde. Außerdem deutet die Verschiebung der Schmelzkurve des mutmaßlichen Regulatorproteins des Zweikomponentensystems zumindest auf eine indirekte Interaktion mit Trichlorethen hin und ist damit, abgesehen von bloßen Expressionsdaten, der erste biochemische Beleg für dessen Rolle bei der Organohalid-Atmung. Zusammenfassend beinhaltet diese Arbeit nicht nur die erste systematische Analyse und Kombination aller bisher verfügbaren „Omics“-Studien, sondern auch deren Weiterenwiclung für die Untersuchung organohalid-atmender Bakterien, wodurch ein detailliertes Bild von deren Physiologie geschaffen werden konnte. Neben den technischen Neuerungen konnte gezeigt werden, dass das Zweikomponentensystem von Sulfurospirillum sp. mit halogenierten organischen Verbindungen interagiert und dass dessen posttranslationale Modifikation die Langzeitreulation des Organohalid-Atmungsapparates beeinflussen könnte. Die Einblicke in die Beteiligung des Zweikomponentensystems an der Organohalidatmung in Sulfurospirillum sp. wären durch Nutzung von weniger komplexen Standard-Proteomikmethoden unentdeckt geblieben. In Zukunft können uns diese neu entwickelten Methoden dabei unterstützen, Funktionalität und Regulation von reduktiven Dehalogenasen in anderen Organohalid-Atmern aufzuklären.:Summary 7 Zusammenfassung 10 1 Introduction 14 1.1 Halogenated compounds and the environment……………………...……….……. 14 1.2 Transformation of organohalides……………………..……………….…………….. 15 1.3 Reductive dehalogenation………………………..……………………………….…... 16 1.3.1 Dehalococcoides mccartyi……………………………………………….……… 18 1.3.2 Sulfurospirillum spp. …………………..………………………………..……... 20 1.4 Proteomics……………………..………………..…………………………………...….. 22 1.4.1 The principle of shotgun proteomics..………………..………………....……. 22 1.4.2 Protein lysine acetylations–an important post-translational modification…………………………………………………………...………… 24 1.4.3 Thermal proteome profiling..………………..………..……..………………... 28 1.5 Objectives..………………..……..………..………..………………..…………………. 29 2 Publications 31 2.1 Overview of publications..………..………………..………….………..…………….. 31 2.1.1 Publication 1..………..………….………..…….………..………………………. 31 2.1.2 Publication 2..………..…………..………..…….………..……………………… 31 2.1.3 Publication 3..………..…………….………..…..………..……………………… 32 2.1.4 Publication 4..………..…………..……….…..………..……………….……….. 32 2.2 Published articles..………..……………....…………..………..………………..……. 33 3 Discussion 88 3.1 The application of ‘omics’ to organohalide-respiring bacteria..………..………... 88 3.2 Parallel proteome and acetylome analysis..………..………………..…………….. 91 3.2.1 Specific challenges for the analysis of protein lysine acetylations………. 92 3.2.2 Insights into the metabolism of S. halorespirans..………..………………... 93 3.3 Protein interaction analysis by thermal proteome profiling..………..……......... 97 3.3.1 Other potential approaches to study protein-ligand-interactions..…….... 98 3.3.2 Potential of using thermal proteome profiling for organohalide- respiring bacteria..………..……….………..………….………..……………… 99 3.4 Conclusions and future perspectives..………..……………..………..…..………… 101 4 References 104 5 Appendix 118 5.1 Declaration of authorship..………..……………..………..……………………..…… 118 5.2 Author contribution of published articles..………..……………..……………….... 118 5.3 Curriculum vitae..………..………………..…………….………..…………………… 124 5.4 List of publications and conference contributions..………..……………...………. 124 5.5 Acknowledgements..………..………… ………..…………………..…………..…….. 127 5.6 Supplementary material..…………………..………..………………………….……. 128 5.6.1 Supplementary material for Publication 3..………..……..………..……….. 128

In this paper Lewontin’s notion of “quasi-independence” of characters is formalized as the assumption that a region of the phenotype space can be represented by a product space of orthogonal factors. In this picture each character corresponds to a factor of a region of the phenotype space. We consider any region of the phenotype space that has a given factorization as a “type”, i.e. as a set of phenotypes that share the same set of phenotypic characters. Using the notion of local factorizations we develop a theory of character identity based on the continuation of common factors among different regions of the phenotype space. We also consider the topological constraints on evolutionary transitions among regions with different regional factorizations, i.e. for the evolution of new types or body plans. It is shown that direct transition between different “types” is only possible if the transitional forms have all the characters that the ancestral and the derived types have and are thus compatible with the factorization of both types. Transitional forms thus have to go over a “complexity hump” where they have more quasi-independent characters than either the ancestral as well as the derived type. The only logical, but biologically unlikely, alternative is a “hopeful monster” that transforms in a single step from the ancestral type to the derived type. Topological considerations also suggest a new factor that may contribute to the evolutionary stability of “types”. It is shown that if the type is decomposable into factors which are vertex irregular (i.e. have states that are more or less preferred in a random walk), the region of phenotypes representing the type contains islands of strongly preferred states. In other words types have a statistical tendency of retaining evolutionary trajectories within their interior and thus add to the evolutionary persistence of types.

In many eukaryotic genomes only a small fraction of the DNA codes for proteins, but the non-protein coding DNA harbors important genetic elements directing the development and the physiology of the organisms, like promoters, enhancers, insulators, and micro-RNA genes. The molecular evolution of these genetic elements is difficult to study because their functional significance is hard to deduce from sequence information alone. Here we propose an approach to the study of the rate of evolution of functional non-coding sequences at a macro-evolutionary scale. We identify functionally important non-coding sequences as Conserved Non-Coding Nucleotide (CNCN) sequences from the comparison of two outgroup species. The CNCN sequences so identified are then compared to their homologous sequences in a pair of ingroup species, and we monitor the degree of modification these sequences suffered in the two ingroup lineages. We propose a method to test for rate differences in the modification of CNCN sequences among the two ingroup lineages, as well as a method to estimate their rate of modification. We apply this method to the full sequences of the HoxA clusters from six gnathostome species: a shark, Heterodontus francisci; a basal ray finned fish, Polypterus senegalus; the amphibian, Xenopus tropicalis; as well as three mammalian species, human, rat and mouse. The results show that the evolutionary rate of CNCN sequences is not distinguishable among the three mammalian lineages, while the Xenopus lineage has a significantly increased rate of evolution. Furthermore the estimates of the rate parameters suggest that in the stem lineage of mammals the rate of CNCN sequence evolution was more than twice the rate observed within the placental amniotes clade, suggesting a high rate of evolution of cis-regulatory elements during the origin of amniotes and mammals. We conclude that the proposed methods can be used for testing hypotheses about the rate and pattern of evolution of putative cis-regulatory elements.

My thesis combines taxonomy, phylogenetics, divergence time estimations and ancestral area reconstructions, nomenclature, and other methods to shed light on the evolutionary processes that shaped the extant biodiversity of the buckthorn family (Rhamnaceae Juss.). In a first step, this thesis provides a major contribution to the taxonomic assessment and arrangement within the buckthorns. By describing the new genus Pseudoziziphus Hauenschild, and resurrecting four genera, this thesis highlights and compensates underestimated diversity. Simultaneously, it provides a phylogenetic framework based on the largest molecular data set in the family so far. In a second step, the taxonomic and phylogenetic framework was used to reconstruct solid divergence time estimates and ancestral area estimates for Rhamnaceae, with a focus on the ziziphoid lineage, and the genus complex Alphitonia Reissek ex Endl. sensu lato. By this, it was possible to unravel evolutionary processes within the history of Rhamnaceae and provide a biogeographic scenario for the ziziphoid lineages, including Alphitonia s.l.:General Introduction 1 Bibliography 19 Chapter 1 36 Phylogenetic relationships within the cosmopolitan buckthorn family (Rhamnaceae) support the resurrection of Sarcomphalus and the description of Pseudoziziphus gen. nov. 37 Bibliography 48 Appendix 1 50 Supplements 55 Chapter 2 56 Analysis of the cosmopolitan buckthorn genera Frangula and Rhamnus s.l. supports the description of a new genus, Ventia 57 Bibliography 67 Appendix 1 68 Supplements 71 Erratum 72 Chapter 3 74 The influence of the Gondwanan break-up on the biogeographic history of the ziziphoids (Rhamnaceae) 75 Bibliography 96 Appendix 1 105 Appendix 2 115 Supplements 117 Chapter 4 118 Biogeographic analyses support an Australian origin for the IndomalesianAustralasian wet forest-adapted tropical tree and shrub genus Alphitonia and its close allies (Rhamnaceae) 119 Bibliography 144 Appendix 1 153 Appendix 2 161 Appendix 3 166 General Conclusions 172 Bibliography 182 Appendix 186 Spatio-temporal evolution of Allium L. in the Qinghai-Tibet-Plateau region: Immigration and in situ radiation 187 Curriculum Vitae 200 Declaration of Independent work 207 Author contributions 208

Summary The localization of sound sources in humans is based on the binaural cues, interaural time and level differences (ITDs, ILDs) and the spectral cues (Blauert 1997). The ITDs relate to the timing of sound arrival at the two ears. For example, a sound located at the right side will arrive at the right ear earlier than at the left ear. The ILDs refer to the difference of sound pressure-level between the two ears. In the example mentioned above, if the sound located at the right has short wavelength then it will arrive at the right ear with higher sound-pressure than at the left ear. This is because a sound with short wavelength cannot bypass the head. In other words, the head creates an obstacle that diffracts the waves and that is why the sound arriving at the ear closer to the sound source will receive the sound with higher sound-pressure. Due to the association of each of the binaural cues with the wavelength of a sound, Rayleigh (1907) proposed the ‘duplex theory’ of sound source localization suggesting that on the azimuth, the ITDs is the main localization cue for low frequency sounds and the ILDs is the main localization cue for high frequency sounds. The spectral cues are based on the shape of the pinna’s folds and they are very useful for sound source localization in elevation but they also help in azimuthal localization (Schnupp et al. 2012). The contribution of the spectral cues on the azimuthal localization arises from the fact that due to the symmetrical position of the ears on the head, the binaural cues vary symmetrically as a function of spatial location (King et al. 2001). Whereas the ITDs have a very symmetrical distribution, the ILDs become more symmetrical the higher the sound frequency is. This way, there are certain locations within the left-frontal and left-posterior hemifield, as well as the right-frontal and the right-posterior hemifield that share the same binaural cues, which makes the binaural cues ambiguous and so the auditory system cannot depend solely on these for sound source localization. To resolve this ambiguity, our auditory system uses the spectral cues that help to disambiguate frontal-back confusion (King et al. 2001, Schnupp et al. 2012). The role of these cues in localizing sounds in our environment is well established. But their role in acoustic motion localization is not yet clear. This is the topic of the current thesis. The auditory localization cues are processed on the subcortical and cortical level. The ITDs and ILDs are processed from different neurons along the auditory pathway (Schnupp et al. 2012). Their parallel processing stages seem to converge at the inferior colliculus as evidence shows from cat experiments (Chase and Young 2005). But in humans, an electroencephalographic (EEG) study measuring the mismatch negativity (MMN; Schröger 1996) and a study using magnetoencephalographie (MEG; Salminen et al. 2005) showed that these cues are not integrated. One of the models of the spatial representation of sound sources is Jeffress’ place code (1948). This model suggests that each location of the azimuthal space is encoded differently, thus the name ‘place code’. Evidence in support of this model comes from studies on the cat (Yin and Chan 1990). However, arguments against this model come from studies in gerbils whose results showed that their subcortical neurons respond maximally to locations that are outside the physiological range based on the size of their heads (Pecka et al. 2008). An alternative model of auditory spatial encoding is the hemifield code (von Bekesy 1960). This model proposes that subcortical neurons are separated into two populations, one tuned to the left hemifield and another tuned to the right. Thus, the receptive field of the neurons is wide and the estimation of the sound source location is derived from the balance of activity of these two populations. Evidence from human studies support this model. Salminen and colleagues (2009) employed an adaptation paradigm during MEG recording. They presented sets of adaptor and probe stimuli that either had the same or different spatial location. Their results showed that the response to the probe was more reduced when the adaptor was located at the far left location and not when the adaptor and probe shared the exact same location. Also, an EEG study on auditory motion showed that sounds that move from central to lateral locations elicit higher amplitudes than when the move in the opposite direction (Magezi and Krumbholz 2010). The authors concluded that these results are based on the movement of the sound source towards the location of the maximal activity of the neurons (also in Salminen et al. 2012). The ability to detect moving objects is well-embedded into our nature. Whereas it enriches predators and prey with the skills to survive, in everyday life it enables us to interact with our environment. For example, the task of crossing a street (without traffic signs) safely is based on the encoding of visual and auditory features of moving vehicles. In the visual modality, the capability of the system to encode motion is based on motion-specific neurons (Mather 2011). In the auditory modality, the debate over whether these sensors exist is still ongoing. One theory on how the auditory system encodes motion is the ‘snapshot’ theory (Chandler and Grantham 1991, Grantham 1986). In a series of experiments, Grantham (1986) showed that auditory perception was not affected by features of motion such as velocity, but it was more sensitive on distance as a spatial cue. Thus, what he suggested is that the encoding of auditory motion is based on the mechanisms that encode stationary sounds. In other words, when a sound is moving it activates the neurons that correspond to the points that are located along the trajectory of that sound but in a serial manner. This way, the perception of auditory motion is based on ‘snapshots’ instead of processing motion as a complete feature. This mechanism of auditory motion processing corroborates with Jeffress’ place code (1948). Animal studies on monkeys (Ahissar et al. 1992) and owls (Wagner et al. 1994) showed that neurons responded similarly to moving and stationary sounds. Evidence against this theory come from a recent behavioural study that introduced velocity changes within acoustic motion and showed that participants were able to detect them (Locke et al. 2016). The authors concluded that if ‘snapshot’ theory would be true, then these detections of velocity change would not occur. Another theory of auditory motion was evolved that supports the motion-specific mechanisms in the brain (Warren et al. 2002, Docummun et al. 2004, Poirier et al. 20017). A human study using functional magnetic resonance imaging (fMRI) and positron-emission tomography (PET) showed evidence of a motion-specific cortical network that includes the planum temporale and the parietotemporal operculum (Warren et al. 2002). The authors suggested that these areas are part of a posterior processing stream that is responsible for analysis of auditory moving objects. Moreover, a recent primate fMRI study provided evidence of motion-specificity in the activity of the posterior belt and parabelt regions of the primary auditory cortex (Poirier et al. 2017). The authors contrasted cortical response to auditory motion with stationary and spectrotemporal sounds and found that the aforementioned cortical areas were only activated by moving sounds. All in all, the neuronal mechanism underlying auditory motion perception has been vaguely described. However, there is an increasing number of evidence that show that specialized motion areas and mechanisms exist in the cortex. To study how exactly these mechanisms function, it is important to know which aspects of the stimulus paradigm affect the response. Study 1. In this study, I focused on eliciting the cortical motion-onset response (MOR) in the freefield. This specific response is measured with EEG and it is elicited when a sound motion follows a stationary sound without any temporal gaps between them. The stationary part serves as an adaptive sound and the onset of motion provides a release-of-adaptation, which gives rise to the MOR. One of the focus was to investigate the effect on the MOR when the initial part is moving in space instead of being stationary. In addition, a secondary focus was the effect of the stimuli frequency on the MOR. I hypothesized that, due to the adaptation provided by the initial stimulus part, the motion response would be smaller after moving than after stationary adaptation. Also, I expected that the effects of frequency would follow the literature and since the motion response is a late response, the amplitude would be smaller after the high frequency than low frequency stimulus presentation. The results showed that the current paradigm did not elicit the MOR. Comparison of the current experimental settings with those used previously in the literature showed that the MOR is strongly depended on the adaptation time provided by the first part of the stimuli. Study 2. In this study, the stimulus characteristics were adapted after failing to elicit the response in the previous study. In addition, I employed an active instead of a passive paradigm, since data from the literature show that the motion response is strongly dependent on the allocation of attention on auditory motion. Thus, in this study, the elicitation of the MOR was successful. The current study examines the modulation of the MOR based on the frequency-range of sound stimuli. Higher amplitude on the motion response was expected after the presentation of stimuli with high frequency spectrum. Also, I studied the effects of hemifield presentation and the direction of motion on the MOR. The results showed that the early part of the motion response (cN1) was modulated by the frequency range of the sounds with stronger amplitudes elicited by stimuli with high frequency range. Study 3. This study is focused on analysis from data collected in the previous study. The focus, however, is on the effects of the stimulus paradigm on the MOR. I hypothesized that after the adaptation provided by an initial moving part, lower amplitude was expected in comparison to the stimuli with an initial stationary part. These responses were also analysed based on the effects of stimulus frequency. The results showed that the stimulus paradigm with the initial moving part elicited a response that resembles the MOR but has lower amplitude. In addition, the effects of stimulus frequency evident from the previous analysis apply here as well, with high frequency stimuli eliciting higher MOR amplitude than low frequency stimuli. Study 4. This study examined further the effects of stimuli characteristics on the MOR. Since the latency of the MOR in the previous study was a bit later than what is usually reported in the literature, the focus here was to test the effects of motion velocity and adaptation duration on the MOR. The results showed that faster velocity elicited higher amplitudes on the peak-to-peak comparison. Separate analysis on the MOR components, showed that this effect was based on higher cN1 amplitude. A separate analysis between the electrodes over the left and right hemisphere, showed that the peak-to-peak amplitude was stronger on the electrodes over the right hemisphere. Lastly, the strong adaptation created by the long duration of the initial stationary part provided abundant evidence of auditory motion, which led to the separation of the cP2 into its constituent parts. Study 5. This behavioural study focused on the effect of motion adaptation on the rear field to the presentation of motion in the frontal field. Thus, the presentation of adaptors and probes within the left-frontal and left-rear fields aimed at locations that share the same ITDs and ILDs. The disambiguation of auditory localization of motion is based on how these interaural cues interact with the spectral cues. A moving probe was presented in the left hemifield, following an adaptor that spanned either the same trajectory or a trajectory located in the opposite field (frontal/ rear). Participants had to indicate the direction of the probe. The results showed that performance was worse when adaptor and probe were sharing the same binaural cues, even if they were in different hemifields and their direction was opposite. But the magnitude of the adaptation effect when the pair was in different hemifields was smaller, thus showing motion-direction detection depends on the integration of interaural and spectral cues.

Numerous studies have focused on the drivers of diversity and stability of communities, especially under global change. However, multi-dimensionality of ecosystems due to biotic components (e.g predation, competition and adaptive dynamics) and abiotic factors (e.g. disturbances, resource dynamics and their distinct attributes) cause context-dependent outcomes and challenge the predictions. There are still controversies around complex community dynamics under varying regimes, however, finding mechanistical explanations will illuminate the fate of multispecies assemblages. Using model microbial communities, consisting of bacterial prey and protist predator, combined with simulation modelling and advanced statistics, this thesis investigated the impact of imposed disturbances (i.e. increased dilution rates that simulate density-independent mortality as press or pulse disturbances) (i) on transient recovery dynamics of a simple microbial food web, and (ii) on bacterial abundance, diversity and community structure in the absence or presence of a protist predator. In addition, this thesis questioned the impacts of species interactions and rapid trait shifts, as a response to predation and competition, on the community dynamics and stability. Our results revealed that the predator suffered more from disturbances over longer time periods. Reduced predation pressure caused a transient phase of prey release during and even after disturbances. Recovery time depended on the strength and duration of disturbances, however, coupling to an alternative resource increased the chance of fast recovery and stabilized the communities. In multi-species prey communities, bacterial abundance, diversity, and community composition were more affected by predation than by the disturbances and resource dynamics. Predator abundance, on the other hand, was strongly affected by the type of disturbance imposed. Importantly, community attributes had differential sensitivities, as reflected by their different response and recovery dynamics. Prey community dynamics varied more temporally andwere less stable under predation stress, while prey diversity increased significantly. Predation rapidly induced anti-predation traits, which altered population dynamics of both prey and predator. More importantly, predator and the resistant prey, in turn, elevated the number of direct cause-effect relationships between the community members. Our findings are not limited to the studied system and can be used to understand the dynamic response and recovery potential of many natural predator-prey or host-pathogen systems. They can be used as a base for future studies to illuminate the debates on the future communities.:Summary Zusammenfassung 1 Scope and Outline 2 General Introduction 2.1 Context dependency of community dynamics 2.2 Ecological disturbances 2.2.1 Transient dynamics and stability 2.2.2 Catastrophic shifts 2.3 Species interactions and evolutionary dynamics under environmental change 2.3.1 Species interactions and coexistence 2.4 Eco-evolutionary dynamics 2.5 Community assembly mechanisms 2.6 Dealing with complexities 2.6.1 Microbial model systems as a tool in ecology 2.6.2 Correlation, causation and the future of predictions 2.7 Aims of this study 3 Community Dynamics under Disturbances 3.1 Transient recovery dynamics of a predator-prey system 4 Interactions of Community Drivers 4.1 Interactions between predation and disturbances shape prey communities 5 Species Interactions and Evolutionary Dynamics Shaping Communities 5.1 Summary 5.2 Introduction 5.2.1 Predator-Prey Dynamics and Community Stability 5.2.2 Causal inferences 5.3 Aim of the study 5.4 Methods 5.4.1 Organisms 5.4.2 Microcosm experiments and estimation of species abundances 5.4.3 Statistical analysis 5.5 Results 5.5.1 Community dynamics 5.5.2 Dynamics of prey diversity and community stability 5.5.3 Causal links between the species dynamics 5.6 Discussion 5.7 Synopsis 6 General Discussion 6.1 Communities under disturbances: Predator{ prey dynamics 6.2 Temporal species dynamics and community assembly Synthesis and Outlook 7.1 Increasing complexity of species interactions 7.2 Going further from causal links 7.3 Metacommunities References 8 Appendix 8.1 Declaration of the authorship 8.2 Author contributions of published articles 8.3 List of publications and conference contributions 8.4 Acknowledgments 8.5 Supplementary material for Chapter 3 8.6 Supplementary material for Chapter 4 8.7 Supplementary material for Chapter 5

Inner speech has been implicated in Vygotsky´s theory of cognitive development. Converging evidence suggests that language skills as well as self-directed speech, in particular inner speech, is important for cognitive functions. However, the role of self-directed speech (including inner speech) in the relation between language skills and cognition have remained unclear. Despite a growing body of studies on the neural substrate of inner and overt speech, evidences of the neural differences between inner and overt speech have remained diffuse. The present dissertation aimed at filling the gap by investigating inner speech and its process of internalization with behavioral and neuroscientific methods. One study addressed the role of self-directed speech in the interplay between language skills and cognition by using the Tower of London task in children. Two studies addressed the neural correlates of the differences between inner and overt speech in adults and children during a picture-naming task by simultaneously applying the electroencephalography and functional near-infrared spectroscopy. The results of the first study confirmed that the internalization of speech is related to children´s cognition although it cannot explain the relation between language skills and problem-solving. The second study showed that inner speech is not overt speech without articulation, even more it is that prior phonological processes are downregulated. Moreover, the study showed that the brain differentiates between inner and overt speech even when no linguistic and motoric process is necessary. The results of the third study showed less differences between inner and overt speech in children indicating that inner speech is not yet adult-like. Even more it seems that inner speech represents linguistically the same like overt speech in the children´s brain.:Table of Content Danksagung VIII Bibliographische Darstellung X Zusammenfassung XI Summary XVI Theoretical Part 1 1 Introduction and Overview 2 1.1 Introduction 2 1.2 Theoretical Background 4 1.3 Development of Inner Speech 15 1.4 Behavioral Significance of Inner Speech 17 1.4.1 The Link between Inner Speech and Cognition 18 1.4.2 The Link between Inner Speech and Language 22 1.4.3 The Link between Language Skills and Cognition 23 1.5 Psycholinguistic and Biological Significance of Inner Speech 25 1.5.1 Speech Production Model 27 1.5.2 Differences between Inner and Overt Speech in the Brain 32 1.6 Overview of the Goals and Research Questions 42 2 Study Designs and Methodologies 46 2.1 Behavioral Methods 46 2.1.1 Tower of London 46 2.2 Neuroscientific Methods 51 2.2.1 Electroencephalography 52 2.2.1.1 Physiological Principles of the EEG 52 2.2.1.2 Technical Principles of the EEG 55 2.2.1.3 EEG Frequency Bands 57 2.2.1.4 Event-related Brain Potentials (ERPs) 58 2.2.2 Functional Near-Infrared Spectroscopy 59 2.2.2.1 Physical Principles of the fNIRS 59 2.2.2.2 Physiological Principles of the fNIRS 65 2.2.2.3 Technical Principles of the fNIRS - Measurement Setup 66 Empirical Part 68 Preliminary Note 69 3 Study 1 - The role of self-directed speech in problem-solving 70 3.1 Introduction 70 3.1.1 The Association between Language Skills and Executive Functions 71 3.1.2 The association between language skills and use of self-directed speech 72 3.1.3 The role of self-directed speech in Executive Functions 73 3.1.3.1 Quantitative and qualitative analysis of self-directed speech 73 3.1.3.2 Dual-task method 74 3.1.3.3 Triggering self-directed speech 75 3.2 The present study 76 3.3 Material and Method 78 3.3.1 Participants 78 3.3.2 General procedure 79 3.3.3 Measures 79 3.3.3.1 Language Skills 79 3.3.3.2 Receptive and Expressive Vocabulary 79 3.3.3.3 Expressive Grammar 80 3.3.3.4 Receptive Grammar 80 3.3.3.5 Non-verbal intelligence (IQ) 81 3.3.3.6 Complex EF (problem-solving ability) 81 3.3.4 Coding speech 84 3.3.4.1 Quantity 85 3.3.4.2 Quality: Internalization 85 3.3.4.3 Quality: Spatial language 86 3.3.4.4 Quality: Planning function 86 3.3.4.5 Quality: Grammatical completeness 87 3.3.4.6 Interrater reliability 87 3.4 Results 88 3.4.1 Preliminary analyses 88 3.4.1.1 Factor analysis 88 3.4.1.2 Bivariate Correlations between all study measures 88 3.4.2 Main analyses 90 3.4.2.1 H1: Relation between language skills and problem-solving 90 3.4.2.2 RQ: Role of self-directed speech 91 3.4.2.2.1 RQ1: Mediation effect of self-directed speech 91 3.4.2.2.2 RQ2: Susceptibility to articulatory suppression 95 3.4.2.2.3 RQ3: Prompting self-directed speech 96 3.5 Discussion 99 3.5.1 Limitations and Next Steps 105 3.5.2 Conclusion 107 4 Study 2 - Differences between inner and overt speech in adults 108 4.1 Introduction 108 4.2 Material and Method 114 4.2.1 Participants 114 4.2.2 Material 114 4.2.3 Tasks and Procedure 115 4.3 NIRS/EEG Data Recordings 117 4.3.1 fNIRS Data Recording 117 4.3.2 EEG Data Recording 118 4.4 Data analyses 119 4.4.1 fNIRS Data Analyses 119 4.4.2 EEG Data Analyses 120 4.5 Results 121 4.5.1 fNIRS results 121 4.5.2 EEG results 122 4.6 Discussion 127 4.7 Conclusions 131 5 Study 3 - Differences between inner and overt speech in children 133 5.1 Introduction 133 5.2 Material and Method 138 5.2.1 Participants 138 5.2.2 Material 139 5.2.3 Tasks and Procedure 139 5.3 NIRS/EEG Data Recordings 141 5.3.1 fNIRS Data Recording 141 5.3.2 EEG Data Recording 143 5.4 Data analyses 143 5.4.1 fNIRS Data: Analyses 143 5.4.2 EEG Data Analyses 144 5.5 Results 145 5.5.1 fNIRS results 145 5.5.2 EEG results 146 5.6 Discussion 146 5.7 Conclusion 150 6 General Discussion 151 6.1 What role does self-directed and inner speech play in the relation between language and cognition in children? 152 6.1.1 Quantitative and qualitative analyses of self-directed speech 152 6.1.2 Articulatory suppression of self-directed speech 155 6.1.3 Triggering self-directed speech 158 6.1.4 Conclusion 159 6.2 What distinguishes inner speech from external (overt) speech in adults? 160 6.2.1 Preparation phase 161 6.2.2 Execution phase 162 6.2.3 Conclusion 164 6.3 What distinguishes inner speech from external (overt) speech in children? 164 6.3.1 Preparation phase 165 6.3.2 Execution phase 167 6.3.3 Conclusion 169 6.4 Limitations and Future Research 169 6.5 Conclusion 176 7 References 179 Appendices 212 Coding Manual of Self-Directed Speech 213 Coding scheme 213 Coding Speech 214 Quantity of Private Speech 214 Quality of Private Speech 216 Speech Structure 216 Content Functions 218 Syntactic Features 221 Publications and Conference Contributions 222 Selbstständigkeitserklärung 225

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical cell division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Making use of extended time-lapse, multi-photon microscopy and a careful three-dimensional analysis of cell division orientation, we show that SDO is required for neural rod midline formation during neurulation, but dispensable for body axis elongation during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the non-canonical Wnt receptor Frizzled 7 (Fz7), and that interfering with cell division orientation leads to severe defects in neural rod midline formation, but not body axis elongation. These findings suggest a novel function for Fz7 controlled cell division orientation in neural rod midline formation during neurulation. They also shed new light on the field of cell division orientation by uncoupling it from tissue elongation.