Dictyostelids (Amoebozoa) are a group of social amoebae consisting of approximately 150 species, which are found in terrestrial habitats worldwide. They are divided into eight major clades based on molecular phylogeny, and within these clades are many species complexes. Some species are seemingly cosmopolitan in distribution, while others are geographically restricted. In this thesis dictyostelids were recovered from high latitude habitats (soils in Sweden and Iceland) as well as from the soles of shoes. Morphological characters and DNA sequence analyses were used to identify isolates that were recovered and delimit new species, as well as to investigate the monophyly of Dictyostelium aureostipes. Nine species were reported from Northern Sweden and four from Iceland. Among the isolates recorded in Sweden were two new species, described as D. barbibulus and Polysphondylium fuscans. P. fuscans was among the four species recovered from footwear, contributing evidence for anthropogenic transport of dictyostelids. Ecological patterns were assessed using linear regression and generalized linear models. The ecological analyses of dictyostelids recovered from Iceland indicate that these organisms are most frequently found in soils of near-neutral pH, but also exhibit a species richness peak in moderately acidic soils. These analyses indicate that in Iceland dictyostelid species richness decreases with altitude, and in the northern hemisphere the species richness increases with decreasing latitude. A three-region analysis of the D. aureostipes species complex indicated that this species is in fact made up of at least five phylogenetically distinct clades, and in light of this the group is in need of taxonomic revision. These results indicate that the dictyostelid species richness is higher than previously known, especially in high-latitude regions, and that even seemingly well-defined species may harbour cryptic diversity. Presently, species ranges may be expanding via anthropogenic dispersal but despite this, the dictyostelids are found to exhibit biogeographic trends well known from macroorganisms, such as a latitudinal gradient of species richness.

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Here, I elaborate on the hypothesis that complex multicellularity (CM, sensu Knoll) is a major evolutionary transition (sensu Szathmary), which has convergently evolved a few times in Eukarya only: within red and brown algae, plants, animals, and fungi. Paradoxically, CM seems to correlate with the expansion of non-coding DNA (ncDNA) in the genome rather than with genome size or the total number of genes. Thus, I investigated the correlation between genome and organismal complexities across 461 eukaryotes under a phylogenetically controlled framework. To that end, I introduce the first formal definitions and criteria to distinguish ‘unicellularity’, ‘simple’ (SM) and ‘complex’ multicellularity. Rather than using the limited available estimations of unique cell types, the 461 species were classified according to our criteria by reviewing their life cycle and body plan development from literature. Then, I investigated the evolutionary association between genome size and 35 genome-wide features (introns and exons from protein-coding genes, repeats and intergenic regions) describing the coding and ncDNA complexities of the 461 genomes. To that end, I developed ‘GenomeContent’, a program that systematically retrieves massive multidimensional datasets from gene annotations and calculates over 100 genome-wide statistics. R-scripts coupled to parallel computing were created to calculate >260,000 phylogenetic controlled pairwise correlations. As previously reported, both repetitive and non-repetitive DNA are found to be scaling strongly and positively with genome size across most eukaryotic lineages. Contrasting previous studies, I demonstrate that changes in the length and repeat composition of introns are only weakly or moderately associated with changes in genome size at the global phylogenetic scale, while changes in intron abundance (within and across genes) are either not or only very weakly associated with changes in genome size. Our evolutionary correlations are robust to: different phylogenetic regression methods, uncertainties in the tree of eukaryotes, variations in genome size estimates, and randomly reduced datasets. Then, I investigated the correlation between the 35 genome-wide features and the cellular complexity of the 461 eukaryotes with phylogenetic Principal Component Analyses. Our results endorse a genetic distinction between SM and CM in Archaeplastida and Metazoa, but not so clearly in Fungi. Remarkably, complex multicellular organisms and their closest ancestral relatives are characterized by high intron-richness, regardless of genome size. Finally, I argue why and how a vast expansion of non-coding RNA (ncRNA) regulators rather than of novel protein regulators can promote the emergence of CM in Eukarya. As a proof of concept, I co-developed a novel ‘ceRNA-motif pipeline’ for the prediction of “competing endogenous” ncRNAs (ceRNAs) that regulate microRNAs in plants. We identified three candidate ceRNAs motifs: MIM166, MIM171 and MIM159/319, which were found to be conserved across land plants and be potentially involved in diverse developmental processes and stress responses. Collectively, the findings of this dissertation support our hypothesis that CM on Earth is a major evolutionary transition promoted by the expansion of two major ncDNA classes, introns and regulatory ncRNAs, which might have boosted the irreversible commitment of cell types in certain lineages by canalizing the timing and kinetics of the eukaryotic transcriptome.:Cover page Abstract Acknowledgements Index 1. The structure of this thesis 1.1. Structure of this PhD dissertation 1.2. Publications of this PhD dissertation 1.3. Computational infrastructure and resources 1.4. Disclosure of financial support and information use 1.5. Acknowledgements 1.6. Author contributions and use of impersonal and personal pronouns 2. Biological background 2.1. The complexity of the eukaryotic genome 2.2. The problem of counting and defining “genes” in eukaryotes 2.3. The “function” concept for genes and “dark matter” 2.4. Increases of organismal complexity on Earth through multicellularity 2.5. Multicellularity is a “fitness transition” in individuality 2.6. The complexity of cell differentiation in multicellularity 3. Technical background 3.1. The Phylogenetic Comparative Method (PCM) 3.2. RNA secondary structure prediction 3.3. Some standards for genome and gene annotation 4. What is in a eukaryotic genome? GenomeContent provides a good answer 4.1. Background 4.2. Motivation: an interoperable tool for data retrieval of gene annotations 4.3. Methods 4.4. Results 4.5. Discussion 5. The evolutionary correlation between genome size and ncDNA 5.1. Background 5.2. Motivation: estimating the relationship between genome size and ncDNA 5.3. Methods 5.4. Results 5.5. Discussion 6. The relationship between non-coding DNA and Complex Multicellularity 6.1. Background 6.2. Motivation: How to define and measure complex multicellularity across eukaryotes? 6.3. Methods 6.4. Results 6.5. Discussion 7. The ceRNA motif pipeline: regulation of microRNAs by target mimics 7.1. Background 7.2. A revisited protocol for the computational analysis of Target Mimics 7.3. Motivation: a novel pipeline for ceRNA motif discovery 7.4. Methods 7.5. Results 7.6. Discussion 8. Conclusions and outlook 8.1. Contributions and lessons for the bioinformatics of large-scale comparative analyses 8.2. Intron features are evolutionarily decoupled among themselves and from genome size throughout Eukarya 8.3. “Complex multicellularity” is a major evolutionary transition 8.4. Role of RNA throughout the evolution of life and complex multicellularity on Earth 9. Supplementary Data Bibliography Curriculum Scientiae Selbständigkeitserklärung (declaration of authorship)

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Biological cell populations, such as in tissues or microbial communities, are constantly subject to different sources of noise and variability. Despite this, multicellular systems are still able to function properly because cells coordinate with each other by communication. Using biological model systems to study this multiscalar process can be challenging because of their innate complexity. In this thesis, we address this challenge by building a synthetic multicellular system using bottom-up in vitro assembly approaches. Using this platform, we aim to study the effect of cell-to-cell communication to population variability in a minimal and simplified context. To achieve this, we require a synthetic cell population with (i) quantifiable gene expression dynamics, (ii) customizable population variability, and (iii) intercellular communication. Having these characteristics will allow us to test different initial configurations of population variability and monitor population gene expression dynamics with and without cell-to-cell communication. To generate these synthetic cell populations, reconstituted cell-free expression systems (CFES) are encapsulated into monodisperse-sized liposomes using double-emulsion microfluidics. Both transcription and translation levels are simultaneously monitored and quantified to develop models of cell-free gene expression dynamics and differentiate between bulk and encapsulated formats. Population variability was then incorporated by combining different batches of cells to create distinct subpopulations or by using a two-inlet double-emulsion microfluidic device to generate single populations with a large dispersion of encapsulated DNA template. Lastly, genetic circuits based on the quorum sensing system of Vibrio fischeri are used to implement diffusion-mediated intercellular signalling. Quorum sensing gene circuits in Escherichia coli extract-based CFES were tested in bulk and phase transfer-generated synthetic cells. Together with these experimental systems, corresponding models of synthetic cell populations that can account for population variability and secrete-and-sensing communication are developed using mixed-effects models and moment dynamics. Overall, this work leverages CFES and microfluidic technologies to reproducibly generate a simplified in vitro model of multicellular systems that can be easily monitored spatiotemporally to study multi-scalar processes.:Preface Chapter 1 Bottom-up multicellular systems Chapter 2 Building blocks: cell-free expression and liposomes Chapter 3 Gene expression dynamics in synthetic cell populations Chapter 4 Variability and communication in synthetic cell populations Chapter 5 Modeling variability & communication in synthetic cell populations Summary and outlook Appendices Bibliography / Biologische Zellpopulationen, z.B. in Geweben oder mikrobiellen Gemeinschaften, sind ständig verschiedenen Quellen von Rauschen und Variabilität ausgesetzt. Trotzdem sind multizelluläre Systeme in der Lage, ordnungsgemäß zu funktionieren, weil sich die Zellen durch Kommunikation miteinander abstimmen. Die Verwendung biologischer Modellsysteme zur Untersuchung dieses multiskalaren Prozesses kann aufgrund ihrer angeborenen Komplexität eine Herausforderung darstellen. In dieser Arbeit gehen wir diese Herausforderung an, indem wir ein synthetisches multizelluläres System mit Hilfe von Bottom-up-in vitro-Assembly-Ansätzen aufbauen. Mit Hilfe dieser Plattform wollen wir die Auswirkungen der Kommunikation von Zelle zu Zelle auf die Populationsvariabilität in einem minimalen und vereinfachten Kontext untersuchen. Um dies zu erreichen, benötigen wir eine synthetische Zellpopulation mit (i) quantifizierbarer Genexpressionsdynamik, (ii) anpassbarer Populationsvariabilität und (iii) interzellulärer Kommunikation. Mit diesen Eigenschaften können wir verschiedene Ausgangskonfigurationen der Populationsvariabilität testen und die Genexpressionsdynamik der Population mit und ohne Zell-zu-Zell-Kommunikation beobachten. Um diese synthetischen Zellpopulationen zu erzeugen, werden rekonstituierte zellfreie Expressionssysteme (CFES) mit Hilfe der Doppelemulsions-Mikrofluidik in monodisperse Liposomen eingekapselt. Sowohl die Transkriptions- als auch die Translationsraten werden gleichzeitig überwacht und quantifiziert, um Modelle für die Dynamik der zellfreien Genexpression zu entwickeln und zwischen Bulk- und verkapselten Formaten zu unterscheiden. Die Variabilität der Populationen wurde dann durch die Kombination verschiedener Zellchargen zur Bildung unterschiedlicher Subpopulationen oder durch die Verwendung einer mikrofluidischen Doppelemulsionsvorrichtung mit zwei Einlässen zur Erzeugung einzelner Populationen mit einer großen Streuung der eingekapselten DNA-Vorlage einbezogen. Schließlich werden genetische Schaltkreise auf der Grundlage des Quorum-Sensing-Systems von Vibrio fischeri verwendet, um diffusionsvermittelte interzelluläre Signalübertragung zu implementieren. Quorum-Sensing-Genkreisläufe in CFES auf der Basis von Escherichia coli-Extrakten wurden in synthetischen Zellen getestet, die durch Bulk- und Phasentransfer erzeugt wurden. Zusammen mit diesen experimentellen Systemen wurden entsprechende Modelle synthetischer Zellpopulationen entwickelt, die die Populationsvariabilität und die Sekretions- und Sensing-Kommunikation mit Hilfe von Mixed-Effects-Modellen und Momentendynamik berücksichtigen können. Insgesamt nutzt diese Arbeit CFES- und Mikrofluidik-Technologien, um reproduzierbar ein vereinfachtes in vitro-Modell multizellulärer Systeme zu erzeugen, das leicht raum-zeitlich überwacht werden kann, um multiskalare Prozesse zu untersuchen.:Preface Chapter 1 Bottom-up multicellular systems Chapter 2 Building blocks: cell-free expression and liposomes Chapter 3 Gene expression dynamics in synthetic cell populations Chapter 4 Variability and communication in synthetic cell populations Chapter 5 Modeling variability & communication in synthetic cell populations Summary and outlook Appendices Bibliography

La bactérie Myxococcus xanthus forme des structures multicellulaires appelées corps fructifères pour résister à des conditions de carence nutritive. La formation de ces structures implique un système chimiotactique particulier, le système Frz, qui régule le changement de direction des cellules, provoqué par la relocalisation simultanée des deux appareils de motilité (A) et (S) d’un pôle à l’autre de la cellule. Au cours de ma thèse, j’ai travaillé sur la connexion entre le système chimiotactique Frz et ses protéines cibles MglAB dans le contrôle de l’inversion de la polarité. L’axe de polarité des cellules est établi par MglA, une petite protéine G de la famille Ras, qui constitue un embranchement vers la régulation des deux appareils de motilité au pôle avant, et son inhibiteur MglB localisé au pôle arrière. Nous avons montré qu’en interagissant directement et spécifiquement avec le cytosquelette, MglA contrôle l’assemblage et le désassemblage de la machinerie de motilité A. Par une approche évolutive, nous avons élucidé l’architecture modulaire du système Frz et l’implication de quatre domaines régulateurs pour connecter le système Frz aux protéines MglAB, filtrer et amplifier le signal. Nous proposons un mécanisme d’inversion de la polarité dans lequel l’action indépendante de deux RRs à chaque pôle de la cellule perturbe les interactions entre une petite protéine G et son inhibiteur apparenté pour convertir un axe de polarité stable en un oscillateur biochimique. La régulation de la direction de mouvement chez M. xanthus pourrait donc constituer un cas émergent de couplage entre des régulateurs de type procaryotes et eucaryotes. / The bacterium Myxococcus xanthus forms multicellular structures called fruiting bodies to resist to starvation conditions. Fruiting body formation implies a chemosensory-like system, the Frz system which regulates directional changes through the simultaneous pole-to-pole relocalization of two motility systems, (A) and (S). During my PhD, I have worked on the connection between the Frz chemosensory-like system and the downstream regulators MglA and MglB in the control of polarity inversion. The cell polarity axis is established by (i) a Ras-like small G protein, MglA, which constitutes a branch node in the regulation of A and S motility systems at the leading cell pole, and (ii) its cognate inhibitor MglB that localizes at the lagging cell pole. We showed that MglA interacts directly and specifically with the cytoskeleton to promote assembly and disassembly of the A-motility machinery. Using an evolutionary approach, we elucidated the modular architecture of the Frz system and the implication of four regulatory domains to (i) connect the Frz system to the MglAB proteins, (ii) filter and (iii) amplify the signal. We now propose a mechanism for polarity inversion in which the independent action of two response regulators at each cell pole perturbs the interactions between a small-G-protein and its cognate inhibitor to trigger the conversion of a stable polarity axis into a biochemical oscillator. The regulation of directional movement in M. xanthus is an interesting emergent coupling between prokaryotes and eukaryotes regulators.