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Multicellular organisms, such as plants and animals, have independently evolved several times over the last hundreds of millions of years. The evolution of multicellularity has significantly shaped modern ecosystems, yet its origins remain largely unknown. Due to the ancient history and the small size scale of early multicellular organisms, few intact fossils have been preserved. To uncover the origins of large and complex life, researchers have turned to alternative methods such as phylogenetic modeling, experimental evolution, and theoretical frameworks. While these approaches have provided novel insights in the early steps of multicellular evolution, few studies have considered the role of adaptation in these novel life cycles. This thesis addresses the gap in our knowledge by employing mathematical modeling and computer simulations to study adaptation in novel multicellular life cycles. The first paper investigates the effects of unicellular reproduction modes, such as budding or binary fission, on the spread of growth rate mutations. It demonstrates that unicellular history significantly influences the adaptation rate, with budding cells exhibiting greater sensitivity to the spatial distribution of mutations. In Paper II, the role of multicellular reproduction mode for the adaptation of altruistic and selfish mutations is explored. Specifically, the study examines how adaptation is affected when the filaments are exposed to a size-based selective pressure. It reveals that while the adaptation of altruistic mutations is favored by large offspring, the spread of selfish mutations depends on both offspring size and selection strength. While Papers I and II assume deterministic life cycle structures at the multicellular level, paper III investigates the evolution of life cycle regulation when cells use internal information. The model demonstrates that when cells only have access to a limited amount of information, there is significant variation in the types of life

The velocity of the gliding movement of filamentous cyanobacteria on a solid surface usually has a strong temperature dependency, and the higher the temperature, the faster the speed. Former studies on this phenomenon were conducted using filamentous cyanobacteria with straight morphology. We examined the velocity of the gliding movement of Arthrospira platensis NIES-39 along its longitudinal axis to see if the same was true for this cyanobacterium with helicoid trichomes. Experimental results showed little temperature dependency in the velocity in a wide temperature range in this cyanobacterium. However, when we examined the velocity using mutants with straight trichomes, their velocity was strongly affected by temperature, like other formerly analyzed filamentous cyanobacteria. This result indicates that the helicoid morphology of A. platensis trichomes confers temperature compensation to their migration velocity, enabling them to keep a relatively constant velocity under various temperatures. Migration of wild-type trichomes is considerably suppressed compared to the straight-trichome mutants on solid media. The temperature compensation in the locomotion of this organism appears to be established as part of such a suppression. It was also found that the velocity of this cyanobacterium depended on the trichome length when they were atypically short (<250 µm); the shorter the trichomes, the slower the gliding movement tended to be. This result indicates that the coordinated action of a high number of cells constituting the trichome is required for efficient gliding movement.

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.

Regulated cellular aggregation is an essential process for development and healing in many animal tissues. In some animals and a few distantly related unicellular species, cellular aggregation is regulated by diffusible chemical cues. However, it is unclear whether regulated cellular aggregation was part of the life cycles of the first multicellular animals and/or their unicellular ancestors. To fill this gap, we investigated the triggers of cellular aggregation in one of animals’ closest unicellular living relatives—the filasterean Capsaspora owczarzaki. We discovered that Capsaspora aggregation is induced by chemical cues, as observed in some of the earliest branching animals and other unicellular species. Specifically, we found that calcium ions and lipids present in lipoproteins function together to induce aggregation of viable Capsaspora cells. We also found that this multicellular stage is reversible as depletion of the cues triggers disaggregation, which can be overcome upon reinduction. Our finding demonstrates that chemically regulated aggregation is important across diverse members of the holozoan clade. Therefore, this phenotype was plausibly integral to the life cycles of the unicellular ancestors of animals.

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.

The emergence of animals from their unicellular ancestors is a major evolutionary event. Thanks to the study of diverse close unicellular relatives of animals, we now have a better grasp of what the unicellular ancestor of animals was like. However, it is unclear how that unicellular ancestor of animals became the first animals. To explain this transition, two popular theories, the choanoblastaea and the synzoospore, have been proposed. We will revise and expose the flaws in these two theories while showing that, due to the limits of our current knowledge, the origin of animals is a biological black swan event. As such, the origin of animals defies retrospective explanations. Therefore, we should be extra careful not to fall for confirmation biases based on few data and, instead, embrace this uncertainty and be open to alternative scenarios. With the aim to broaden the potential explanations on how animals emerged, we here propose two novel and alternative scenarios. In any case, to find the answer to how animals evolved, additional data will be required, as will the hunt for microscopic creatures that are closely related to animals but have not yet been sampled and studied.

Determining how adaptive possibilities do or do not become evolutionary realities is central to understanding the tempo and mode of evolutionary change. Some of the simplest evolutionary landscapes arise from underdominance at a single locus where the fitness valley consists of only one less-fit genotype. Despite their potential for rapid evolutionary change, few such examples have been investigated. We capitalized on an experimental system in which a significant evolutionary shift, the transition from uni-to-multicellularity, was observed in asexual diploid populations of Saccharomyces cerevisiae experimentally selected for increased settling rates. The multicellular phenotype results from recessive single-locus mutations that undergo loss-of-heterozygosity (LOH) events. By reconstructing the necessary heterozygous intermediate steps, we found that the evolution of multicellularity involves a decrease in size during the first steps. Heterozygous genotypes are 20% smaller in size than genotypes with functional alleles. Nevertheless, populations of heterozygotes give rise to multicellular genotypes more readily than unicellular genotypes with two functional alleles, by rapid LOH events. LOH drives adaptation that may enable rapid evolution in diploid yeast. Together these results show discordance between the phenotypic and genotypic multicellular transition. The evolutionary path to multicellularity, and the adaptive benefits of increased size, requires initial size reductions.

The prevalence of multicellular organisms is due in part to their ability to form complex structures. How cells pack in these structures is a fundamental biophysical issue, underlying their functional properties. However, much remains unknown about how cell packing geometries arise, and how they are affected by random noise during growth - especially absent developmental programs. Here, we quantify the statistics of cellular neighborhoods of two different multicellular eukaryotes: lab-evolved 'snowflake' yeast and the green alga Volvox carteri. We find that despite large differences in cellular organization, the free space associated with individual cells in both organisms closely fits a modified gamma distribution, consistent with maximum entropy predictions originally developed for granular materials. This 'entropic' cellular packing ensures a degree of predictability despite noise, facilitating parent-offspring fidelity even in the absence of developmental regulation. Together with simulations of diverse growth morphologies, these results suggest that gamma-distributed cell neighborhood sizes are a general feature of multicellularity, arising from conserved statistics of cellular packing.

Multicellular life forms have evolved many times in our planet, suggesting that this is a common evolutionary innovation. Multiple advantages have been proposed for the emergence of multicellularity (MC). In this paper, we address the problem of how the first precondition for multicellularity, namely ‘stay together’ might have occurred under spatially limited resources exploited by a population of unicellular agents. Using a minimal model of evolved cell–cell adhesion among growing and dividing cells that exploit a localized resource with a given size, we show that a transition occurs at a critical resource size separating a phase of evolved multicellular aggregates from a phase where unicellularity (UC) is favoured. The two phases are separated by an intermediate domain where both UC and MC can be selected by evolution. This model provides a minimal approach to the early stages that were required to transition from individuality to cohesive groups of cells associated with a physical cooperative effect: when resources are present only in a localized portion of the habitat, MC is a desirable property as it helps cells to keep close to the available local nutrients.

During the evolution of multicellularity, the unit of selection transitions from that of the single cell to that of the integrated multicellular organism. Cellular specialization mediates this transition, as the evolution of differentiated cell types leads to fitness becoming a property of the multicellular organism. How does cellular specialization evolve? While we know that genetic changes result in the development of new cell types, it remains unclear whether the development of a new cell type can be ancestrally plastic prior to coming under developmental-genetic control via genetic assimilation. We use the volvocine green algae as a model system to address the overarching question of whether the plastic development of a new cell type preceded its fixation via genetic assimilation. Previous research on the evolution of cellular differentiation in this clade has determined that a gene necessary for somatic cell development is present in species with and without soma and that an ancestral stress response was co-opted during the evolution of somatic differentiation. This raises the possibility that somatic cells may have originated as a plastic trait prior to coming under developmental-genetic control. Here, I show that Eudorina species previously characterized as undifferentiated develop a small proportion of cells resembling soma following exposure to cold shock, an environmental stressor. We also find that the offspring of cold-shocked colonies (but not the grand-offspring) also develop somatic-like cells. We show that these cells are morphologically consistent with the somatic cells seen in closely related species that are obligately differentiated. We find that somatic-like cells in cold-shocked colonies are controlled by cell-level, temporal regulation while the production of somatic-like cells in the offspring of cold-shocked colonies are controlled by group-level, developmental regulation induced by the maternal environment. We propose that cell-level contro

Metazoans function as individual organisms but also as “colonies” of cells whose single-celled ancestors lived and reproduced independently. Insights from evolutionary biology about multicellular group formation help us understand the behavior of cells: why they cooperate, and why cooperation sometimes breaks down. Current explanations for multicellularity focus on two aspects of development which promote cooperation and limit conflict among cells: a single-cell bottleneck, which creates organisms composed of clones, and a separation of somatic and germ cell lineages, which reduces the selective advantage of cheating. However, many obligately multicellular organisms thrive with neither, creating the potential for within-organism conflict. Here, we argue that the prevalence of such organisms throughout the Metazoa requires us to refine our preconceptions of conflict-free multicellularity. Evolutionary theory must incorporate developmental mechanisms across a broad range of organisms—such as unusual reproductive strategies, totipotency, and cell competition—while developmental biology must incorporate evolutionary principles. To facilitate this cross-disciplinary approach, we provide a conceptual overview from evolutionary biology for developmental biologists, using analogous examples in the well-studied social insects.

Multicellular life forms have evolved many times on our planet, suggesting that this is a common evolutionary innovation. Multiple advantages have been proposed for the emergence of multicellularity (MC). In this paper, we address the problem of how the first precondition for MC, namely ‘stay together’, might have occurred under spatially limited resources exploited by a population of unicellular agents. Using a minimal model of evolved cell–cell adhesion among growing and dividing cells that exploit a localized resource with a given size, we show that a transition occurs at a critical resource size separating a phase of evolved multicellular aggregates from a phase where unicellularity (UC) is favoured. The two phases are separated by an intermediate domain where both UC and MC can be selected by evolution. This model provides a minimal approach to the early stages that were required to transition from individuality to cohesive groups of cells associated with a physical cooperative effect: when resources are present only in a localized portion of the habitat, MC is a desirable property as it helps cells to keep close to the available local nutrients.

Atmospheric oxygen at the level of present-day Earth (21%) is generally regarded as permissive for the development and survival of complex multicellular life, such as animals. The hypothesis that oxic conditions are conducive for multicellular life has subsequently been applied to the search for life elsewhere in the universe, with planets exhibiting atmospheric oxygen of a similar level to Earth being considered as candidates for habitability. However, an opposing view states that in fact hypoxia (1–5% oxygen) is a prerequisite for tissue renewal, and therefore also for complex multicellularity. Hypoxia is present in many facets of life, such as during early mammalian embryogenesis as well as in niches like bone marrow, where populations of undifferentiated stem cells are maintained. This view implies that oxic conditions are indeed challenging for the evolution of complex multicellularity, and furthermore questions the association between oxic conditions and the potential for multicellular life on exoplanets. Here, I review, test and discuss the association between hypoxic conditions and the evolution of complex multicellular life. Through an extensive study of the available literature, I review how hypoxia and cellular hypoxia-response machineries play a vital role in many stages of metazoan development, regulating the transcription of multiple genes and maintaining cell stemness during mammalian embryogenesis. I perform experiments to mimic the development of an hypoxic sedimentary niche on an exoplanet, and test how sediment grain size alters the depth of the hypoxic zone within these sediments. The results demonstrate that, in the absence of microorganisms, the hypoxic niche expands to greater depths within coarser sediments than in finer sediments. Where microorganisms were present within this sediment, the depth of the hypoxic niche was significantly reduced. Microbial respiration rapidly turned the sediment anoxic. Therefore, I propose that the absence of m

Cable bacteria form centimeter-long, multicellular filaments than can consist of ten thousands of cells. They evolved a unique energy metabolism that involves co-operation among cells that separately perform oxidation of the electron donor (sulfide, H2S) and reduction of the electron acceptor (oxygen, O2). This division of labor is facilitated via long-range electrical currents that run from cell to cell along a network of conductive fibers. This research provides further insights into the metabolism of these peculiar organisms. It was discovered that only the cells that oxidize sulfide have the capacity for growth whereas the cells that reduce oxygen serve to dispense electrons as quickly as possible without any growth. Thus, these oxygen-reducing cells appear to provide a kind of “community service” to the filament by ensuring an electron current without any capacity for growth. However, access to oxygen is essential for the survival of the filament and it appears to pace the cycles of growth and cell division among a filament where cell division is synchronized among cells within a filament. Cells residing in the oxic zone are believed to (temporarily) rely on storage compounds of which polyphosphate (poly-P) is most ubiquitously found in cable bacteria. Poly-P is an inorganic biopolymer that consists of tens to hundreds of phosphate residues linearly linked together by high-energy phosphoanhydride bonds. We found poly-P activity in almost all cable bacteria cells and it appears to have an essential role in the metabolic regulation of cable bacteria.

The evolution of multicellularity paved the way for significant increases in biological complexity. Although multicellularity has evolved many times independently, we know relatively little about its origins. Directed evolution is a promising approach to studying early steps in this major transition, but current experimental systems have examined only a subset of the possible evolutionary routes to multicellularity. Here we consider egalitarian routes to multicellularity, in which unrelated unicellular organisms evolve to become a multicellular organism. Inspired by microbial syntrophies and lichens, we outline three such routes from a system of different species to an interdependent relationship that replicates. We compare these routes to contemporary experimental systems and consider how physical structure, the threat of invasion, division of labour and co-transmission affect their evolution.

Early multicellular organisms must gain adaptations to outcompete their unicellular ancestors, as well as other multicellular lineages. The tempo and mode of multicellular adaptation is influenced by many factors including the traits of individual cells. We consider how a fundamental aspect of cells, whether they reproduce via binary fission or budding, can affect the rate of adaptation in primitive multicellularity. We use mathematical models to study the spread of beneficial, growth rate mutations in unicellular populations and populations of multicellular filaments reproducing via binary fission or budding. Comparing populations once they reach carrying capacity, we find that the spread of mutations in multicellular budding populations is qualitatively distinct from the other populations and in general slower. Since budding and binary fission distribute age-accumulated damage differently, we consider the effects of cellular senescence. When growth rate decreases with cell age, we find that beneficial mutations can spread significantly faster in a multicellular budding population than its corresponding unicellular population or a population reproducing via binary fission. Our results demonstrate that basic aspects of the cell cycle can give rise to different rates of adaptation in multicellular organisms.

Multicellularity has evolved numerous times across the tree of life. One of the most fundamental distinctions among multicellular organisms is their developmental mode: whether they stay together during growth and develop clonally, or form a group through the aggregation of free-living cells. The five eukaryotic lineages to independently evolve complex multicellularity (animals, plants, red algae, brown algae, and fungi) all develop clonally. This fact has largely been explained through social evolutionary theory’s lens of cooperation and conflict, where cheating within non-clonal groups has the potential to undermine multicellular adaptation. Multicellular organisms that form groups via aggregation could mitigate the costs of cheating by evolving kin recognition systems that prevent the formation of chimeric groups. However, recent work suggests that selection for the ability to aggregate quickly may constrain the evolution of highly specific kin recognition, sowing the seeds for persistent evolutionary conflict. Importantly, other features of aggregative multicellular life cycles may independently act to constrain the evolution of complex multicellularity. All known aggregative multicellular organisms are facultatively multicellular (as opposed to obligately multicellular), allowing unicellular-level adaptation to environmental selection. Because they primarily exist in a unicellular state, it may be difficult for aggregative multicellular organisms to evolve multicellular traits that carry pleiotropic cell-level fitness costs. Thus, even in the absence of social conflict, aggregative multicellular organisms may have limited potential for the evolution of complex multicellularity.

[EN] Eukaryotes encompass an unprecedented diversity of forms, sizes and lifestyles. However, tackling the real breadth of that diversity is a challenging task. In the last decades, the assessment of biodiversity has seen substantial progress due to the incorporation of culture-independent techniques based on Next Generation DNA Sequencing. This thesis is a composition of three independent projects that implement these techniques aiming to expand our understanding of the extant eukaryotic biodiversity. In the first project, we obtain the nuclear and mitochondrial genomes of Txikispora philomaios, an uncultured unicellular parasite, using a metagenomic approach. We define the phylogenetic position of T.philomaios that branches within Filasterea, a group of unicellular eukaryotes that is closely related to animals and is key to elucidate the transition to animal multicellularity. Despite T.philomaios possessing a reduced genome in comparison to other Filasterea, it has a complete flagellar toolkit and its genome encodes many proteins that are related to multicellularity in animals. In the second project, we seek undescribed molecular diversity inside the phylum Platyhelminthes, one of the most diverse and biomedically important animal phyla. To this end, we analyze global metabarcoding data of the 18S rDNA gene from marine and freshwater habitats. Our results show that a large part of the molecular diversity of Platyhelminthes remains undocumented and identify freshwater environments as potential reservoirs for novel species of flatworms. Finally, in the third project, we investigate the molecular novelty, the taxonomic composition and the structure of the eukaryotic community in Sanabria Lake by sequencing the V4 hypervariable region of the 18S rDNA gene. We show to which extent the choice of the analytical methods (ASVs or OTUs) affects the final results and conclusions of a biodiversity survey. Altogether, our results broaden our perspective of eukaryotic diversity, [ES] Los organismos eucariotas abarcan una diversidad inmensa de formas, tamaños y estilos de vida. Sin embargo, abordar la verdadera amplitud de esa diversidad es una tarea laboriosa. Durante las últimas decadas, los estudios de biodiversidad han experimentado un progreso importante gracias a la incorporación de técnicas basadas en la secuenciación del ADN. Esta tesis es una composición de tres proyectos independientes donde estas técnicas se aplican con el objetivo de ampliar nuestro conocimiento de la biodiversidad eucariota. En el primer proyecto, obtenemos el genoma nuclear y el genoma mitocondrial de un parásito a partir de un metagenoma eucariota. Definimos la posición filogenética de este nuevo organismo que se posiciona junto con Filasterea, un grupo de organismos unicelulares que está estrechamente relacionado con los animales y es clave para estudiar la transición a la multicelularidad animal. El análisis del contenido génico muestra que el nuevo organismo posee un genoma reducido en comparación con los otros Filasterea. A pesar de eso, su genoma codifica un flagelo completo y muchas proteínas que están relacionadas con la multicelularidad en los animales. En el segundo proyecto, buscamos diversidad molecular no descrita dentro de los platelmintos, uno de los filos animales más diversos e importantes desde un punto de vista biomédico. Con este fin, analizamos datos globales de metabarcoding del gen 18S del ADN ribosomal procedentes de hábitats marinos y de agua dulce. Nuestros resultados muestran que gran parte de la diversidad molecular de los platelmintos sigue sin estar documentada e identifican los habitats de agua dulce como puntos donde buscar nuevas especies de platelmintos. Por último, en el tercer proyecto, investigamos la novedad a nivel molecular, la composición taxonómica y la estructura de la comunidad eucariota del lago Sanabria, un lago oligotrófico. Secuenciamos la región hipervariable V4 del gen 18S del ADN ribosmal y demostramos cómo la

How animals evolved from a single-celled ancestor, transitioning from a unicellular lifestyle to a coordinated multicellular entity, remains a fascinating question. Key events in this transition involved the emergence of processes related to cell adhesion, cell–cell communication and gene regulation. To understand how these capacities evolved, we need to reconstruct the features of both the last common multicellular ancestor of animals and the last unicellular ancestor of animals. In this review, we summarize recent advances in the characterization of these ancestors, inferred by comparative genomic analyses between the earliest branching animals and those radiating later, and between animals and their closest unicellular relatives. We also provide an updated hypothesis regarding the transition to animal multicellularity, which was likely gradual and involved the use of gene regulatory mechanisms in the emergence of early developmental and morphogenetic plans. Finally, we discuss some new avenues of research that will complement these studies in the coming years.

Multicellularity is a key evolutionary innovation, leading to coordinated activity and resource sharing among cells, which generally occurs via the physical exchange of chemical compounds. However, filamentous cable bacteria display a unique metabolism in which redox transformations in distant cells are coupled via long-distance electron transport rather than an exchange of chemicals. This challenges our understanding of organismal functioning, as the link among electron transfer, metabolism, energy conservation, and filament growth in cable bacteria remains enigmatic. Here, we show that cells within individual filaments of cable bacteria display a remarkable dichotomy in biosynthesis that coincides with redox zonation. Nanoscale secondary ion mass spectrometry combined with 13C (bicarbonate and propionate) and 15N-ammonia isotope labeling reveals that cells performing sulfide oxidation in deeper anoxic horizons have a high assimilation rate, whereas cells performing oxygen reduction in the oxic zone show very little or no label uptake. Accordingly, oxygen reduction appears to merely function as a mechanism to quickly dispense of electrons with little to no energy conservation, while biosynthesis and growth are restricted to sulfide-respiring cells. Still, cells can immediately switch roles when redox conditions change, and show no differentiation, which suggests that the “community service” performed by the cells in the oxic zone is only temporary. Overall, our data reveal a division of labor and electrical cooperation among cells that has not been seen previously in multicellular organisms.

In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.

Bacteria regulate the life histories of diverse eukaryotes, but relatively little is known about how eukaryotes interpret and respond to multiple bacterial cues encountered simultaneously. To explore how a eukaryote might respond to a combination of bioactive molecules from multiple bacteria, we treated the choanoflagellate Salpingoeca rosetta with two sets of bacterial cues, one that induces mating and another that induces multicellular development. We found that simultaneous exposure to both sets of cues enhanced multicellular development in S. rosetta, eliciting both larger multicellular colonies and an increase in the number of colonies. Thus, rather than conveying conflicting sets of information, these distinct bacterial cues synergize to augment multicellular development. This study demonstrates how a eukaryote can integrate and modulate its response to cues from diverse bacteria, underscoring the potential impact of complex microbial communities on eukaryotic life histories.IMPORTANCE Eukaryotic biology is profoundly influenced by interactions with diverse environmental and host-associated bacteria. However, it is not well understood how eukaryotes interpret multiple bacterial cues encountered simultaneously. This question has been challenging to address because of the complexity of many eukaryotic model systems and their associated bacterial communities. Here, we studied a close relative of animals, the choanoflagellate Salpingoeca rosetta, to explore how eukaryotes respond to diverse bacterial cues. We found that a bacterial chondroitinase that induces mating on its own can also synergize with bacterial lipids that induce multicellular "rosette" development. When encountered together, these cues enhance rosette development, resulting in both the formation of larger rosettes and an increase in the number of rosettes compared to rosette development in the absence of the chondroitinase. These findings highlight how synergistic interactions among bacterial cues

Cell fusion takes place in all domains of life and contributes greatly to the formation of complex multicellular structures. In particular, many fungi, such as the filamentous Neurospora crassa, rely on conspecific somatic cell fusion to drive the unicellular-to-multicellular transition and formation of the interconnected mycelial syncytium. This can, however, lead to the transmission of infectious elements and deleterious genotypes that have a negative impact on the organismal fitness. Accumulating evidence obtained from natural populations suggests that N. crassa has evolved various self/non-self or allorecognition systems to avoid fusion between genetically non-identical spores or hyphae at all costs. Here we present an overview of the recent advances made in the field of fungal allorecognition, describe its genetic basis, and comment on its evolutionary meaning. These data pinpoint the multilayered complexity of the cooperative social behaviors undertaken by a model eukaryotic microbe.

Inspired by the patterns of multicellularity in choanoflagellates, the closest living relatives of animals, we quantify the biophysical processes underlying the morphogenesis of rosette colonies in the choanoflagellate Salpingoeca rosetta We find that rosettes reproducibly transition from an early stage of 2-dimensional (2D) growth to a later stage of 3D growth, despite the underlying variability of the cell lineages. Our perturbative experiments demonstrate the fundamental importance of a basally secreted extracellular matrix (ECM) for rosette morphogenesis and show that the interaction of the ECM with cells in the colony physically constrains the packing of proliferating cells and, thus, controls colony shape. Simulations of a biophysically inspired model that accounts for the size and shape of the individual cells, the fraction of ECM, and its stiffness relative to that of the cells suffices to explain our observations and yields a morphospace consistent with observations across a range of multicellular choanoflagellate colonies. Overall, our biophysical perspective on rosette development complements previous genetic perspectives and, thus, helps illuminate the interplay between cell biology and physics in regulating morphogenesis.

Multicellularity is a key evolutionary innovation, leading to coordinated activity and resource sharing among cells, which generally occurs via the physical exchange of chemical compounds. However, filamentous cable bacteria display a unique metabolism in which redox transformations in distant cells are coupled via long-distance electron transport rather than an exchange of chemicals. This challenges our understanding of organismal functioning, as the link among electron transfer, metabolism, energy conservation, and filament growth in cable bacteria remains enigmatic. Here, we show that cells within individual filaments of cable bacteria display a remarkable dichotomy in biosynthesis that coincides with redox zonation. Nanoscale secondary ion mass spectrometry combined with 13C (bicarbonate and propionate) and 15N-ammonia isotope labeling reveals that cells performing sulfide oxidation in deeper anoxic horizons have a high assimilation rate, whereas cells performing oxygen reduction in the oxic zone show very little or no label uptake. Accordingly, oxygen reduction appears to merely function as a mechanism to quickly dispense of electrons with little to no energy conservation, while biosynthesis and growth are restricted to sulfide-respiring cells. Still, cells can immediately switch roles when redox conditions change, and show no differentiation, which suggests that the “community service” performed by the cells in the oxic zone is only temporary. Overall, our data reveal a division of labor and electrical cooperation among cells that has not been seen previously in multicellular organisms., BT/Environmental Biotechnology

A major challenge in evolutionary biology has been to explain the variation in multicellularity across the many independently evolved multicellular lineages, from slime moulds to vertebrates. Social evolution theory has highlighted the key role of relatedness in determining multicellular complexity and obligateness; however, there is a need to extend this to a broader perspective incorporating the role of the environment. In this paper, we formally test Bonner's 1998 hypothesis that the environment is crucial in determining the course of multicellular evolution, with aggregative multicellularity evolving more frequently on land and clonal multicellularity more frequently in water. Using a combination of scaling theory and phylogenetic comparative analyses, we describe multicellular organizational complexity across 139 species spanning 14 independent transitions to multicellularity and investigate the role of the environment in determining multicellular group formation and in imposing constraints on multicellular evolution. Our results, showing that the physical environment has impacted the way in which multicellular groups form, highlight that environmental conditions might have affected the major evolutionary transition to obligate multicellularity.

Almost all animals undergo embryonic development, going from a single-celled zygote to a complex multicellular adult. We know that the patterning and morphogenetic processes involved in development are deeply conserved within the animal kingdom. However, the origins of these developmental processes are just beginning to be unveiled. Here, we focus on how the protist lineages sister to animals are reshaping our view of animal development. Most intriguingly, many of these protistan lineages display transient multicellular structures, which are governed by similar morphogenetic and gene regulatory processes as animal development. We discuss here two potential alternative scenarios to explain the origin of animal embryonic development: either it originated concomitantly at the onset of animals or it evolved from morphogenetic processes already present in their unicellular ancestors. We propose that an integrative study of several unicellular taxa closely related to animals will allow a more refined picture of how the last common ancestor of animals underwent embryonic development.

The question of how phenotypic and genomic complexity are inter‐related and how they are shaped through evolution is a central question in biology that historically has been approached from the perspective of animals and plants. In recent years, however, fungi have emerged as a promising alternative system to address such questions. Key to their ecological success, fungi present a broad and diverse range of phenotypic traits. Fungal cells can adopt many different shapes, often within a single species, providing them with great adaptive potential. Fungal cellular organizations span from unicellular forms to complex, macroscopic multicellularity, with multiple transitions to higher or lower levels of cellular complexity occurring throughout the evolutionary history of fungi. Similarly, fungal genomes are very diverse in their architecture. Deep changes in genome organization can occur very quickly, and these phenomena are known to mediate rapid adaptations to environmental changes. Finally, the biochemical complexity of fungi is huge, particularly with regard to their secondary metabolites, chemical products that mediate many aspects of fungal biology, including ecological interactions. Herein, we explore how the interplay of these cellular, genomic and metabolic traits mediates the emergence of complex phenotypes, and how this complexity is shaped throughout the evolutionary history of Fun, T.G. acknowledges support from the Spanish Ministry of Science and Innovation for grants ‘Centro de Excelencia Severo Ochoa’ and PGC2018‐099921‐B‐I00, cofunded by the European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423, and grants from the European Union's Horizon 2020 research and innovation programme under the grant agreement ERC‐2016‐724173, and the Marie Sklodowska‐Curie grant agreement No H2020‐MSCA‐IF‐2017‐793699. T.G.'s research group also receives support from a INB Grant (PT17/0009/0023 ‐ ISCIII‐SGEFI/ERDF)., Peer Reviewed, Postprint (published version)

Proper morphogenesis requires the regulated interplay between cellular behaviors and physical constraints. Studies of physiology and morphogenesis in protist and animal systems with respect to this interplay have led to important mechanistic insights. Comparatively little work, however, has studied how morphogenetic processes evolve in the context of physical constraints. Choanoflagellates, the closest living relatives of animals, can form multicellular colonies of various shapes and sizes. This diversity and the simplicity of multicellular forms in conjunction with their important phylogenetic position makes choanoflagellates an ideal system for studying the evolution of morphogenesis. Because most work has focused on genetics and genomics, little is known about the cellular and biophysical mechanisms underlying the regulation of multicellular form in choanoflagellatesHere, I quantify the biophysical processes underlying the morphogenesis of rosette colonies in the choanoflagellate Salpingoeca rosetta. I find that rosettes reproducibly transition from an early stage of 2D growth to a later stage of 3D growth, despite the underlying stochasticity of the cell lineages. I postulate that the extracellular matrix (ECM) exerts a physical constraint on the packing of proliferating cells, thereby sculpting rosette morphogenesis. Perturbative experiments coupled with biophysical simulations demonstrate the fundamental importance of a basally-secreted ECM for rosette morphogenesis. In addition, this yields a morphospace for the shapes of multicellular colonies, consistent with observations across a range of choanoflagellates. Overall, this biophysical perspective on rosette development complements previous genetic perspectives and thus helps illuminate the interplay between cell biology and physics in regulating morphogenesis.I also present the previously undescribed species Choanoeca flexa, a splash pool choanoflagellate that forms cup-shaped colonies. The colonies rapidly

Somatic cell fusion and conspecific cooperation are crucial social traits for microbial unicellular-to-multicellular transitions, colony expansion, and substrate foraging but are also associated with risks of parasitism. We identified a cell wall remodeling (cwr) checkpoint that acts upon cell contact to assess genetic compatibility and regulate cell wall dissolution during somatic cell fusion in a wild population of the filamentous fungus Neurospora crassa. Non-allelic interactions between two linked loci, cwr-1 and cwr-2, were necessary and sufficient to block cell fusion: cwr-1 encodes a polysaccharide monooxygenase (PMO), a class of enzymes associated with extracellular degradative capacities, and cwr-2 encodes a predicted transmembrane protein. Mutations of sites in CWR-1 essential for PMO catalytic activity abolished the block in cell fusion between formerly incompatible strains. In Neurospora, alleles cwr-1 and cwr-2 were highly polymorphic, fell into distinct haplogroups, and showed trans-species polymorphisms. Distinct haplogroups and trans-species polymorphisms at cwr-1 and cwr-2 were also identified in the distantly related genus Fusarium, suggesting convergent evolution. Proteins involved in chemotropic processes showed extended localization at contact sites, suggesting that cwr regulates the transition between chemotropic growth and cell wall dissolution. Our work revealed an allorecognition surveillance system based on kind discrimination that inhibits cooperative behavior in fungi by blocking cell fusion upon contact, contributing to fungal immunity by preventing formation of chimeras between genetically non-identical colonies.

U tekstu ću se baviti analizom transfera adaptivne vrednosti tokom velikih prelaza u evoluciji i njegovog mesta u modelima selekcije na više nivoa. Cilj analize je da se pokaže kako evolucija socijalnog ponašanja može da objasni nastanak novih evolucionih individua., In this article I will analyze the transfer of fitness during the major transitions in evolution and its place in the multilevel selection models. The aim of the analysis is to show how social evolution can explain the evolutionary transitions in individuality.

Evolution is often deemed irreversible. The evolution of complex traits that require many mutations makes their reversal unlikely. Even in simpler traits, reversals might become less likely as neutral or beneficial mutations, with deleterious effects in the ancestral context, become fixed in the novel background. This is especially true in changes that involve large reorganizations of the organism and its interactions with the environment. The evolution of multicellularity involves the reorganization of previously autonomous cells into a more complex organism; despite the complexity of this change, single cells have repeatedly evolved from multicellular ancestors. These repeated reversals to unicellularity undermine the generality of Dollo's law. In this article, we evaluated the dynamics of reversals to unicellularity from recently evolved multicellular phenotypes of the brewers yeast Saccharomyces cerevisae. Even though multicellularity in this system evolved recently, it involves the evolution of new levels of selection. Strong selective pressures against multicellularity lead to rapid reversibility to single cells in all of our replicate lines, whereas counterselection favoring multicellularity led to minimal reductions to the rates of reversal. History and chance played an important role in the tempo and mode of reversibility, highlighting the interplay of deterministic and stochastic events in evolutionary reversals.

Extracellular matrix (ECM) is considered central to the evolution of metazoan multicellularity; however, the repertoire of ECM proteins in nonbilaterians remains unclear. Thrombospondins (TSPs) are known to be well conserved from cnidarians to vertebrates, yet to date have been considered a unique family, principally studied for matricellular functions in vertebrates. Through searches utilizing the highly conserved C-terminal region of TSPs, we identify undisclosed new families of TSP-related proteins in metazoans, designated mega-TSP, sushi-TSP, and poriferan-TSP, each with a distinctive phylogenetic distribution. These proteins share the TSP C-terminal region domain architecture, as determined by domain composition and analysis of molecular models against known structures. Mega-TSPs, the only form identified in ctenophores, are typically >2,700 aa and are also characterized by N-terminal leucine-rich repeats and central cadherin/immunoglobulin domains. In cnidarians, which have a well-defined ECM, Mega-TSP was expressed throughout embryogenesis in Nematostella vectensis, with dynamic endodermal expression in larvae and primary polyps and widespread ectodermal expression in adult Nematostella vectensis and Hydra magnipapillata polyps. Hydra Mega-TSP was also expressed during regeneration and siRNA-silencing of Mega-TSP in Hydra caused specific blockade of head regeneration. Molecular phylogenetic analyses based on the conserved TSP C-terminal region identified each of the TSP-related groups to form clades distinct from the canonical TSPs. We discuss models for the evolution of the newly defined TSP superfamily by gene duplications, radiation, and gene losses from a debut in the last metazoan common ancestor. Together, the data provide new insight into the evolution of ECM and tissue organization in metazoans.

Understanding how and why cells cooperate to form multicellular organisms is a central aim of evolutionary biology. Multicellular groups can form through clonal development (where daughter cells stick to mother cells after division) or by aggregation (where cells aggregate to form groups). These different ways of forming groups directly affect relatedness between individual cells, which in turn can influence the degree of cooperation and conflict within the multicellular group. It is hard to study the evolution of multicellularity by focusing only on obligately multicellular organisms, like complex animals and plants, because the factors that favour multicellular cooperation cannot be disentangled, as cells cannot survive and reproduce independently. We support the use of Saccharomyces cerevisiae as an ideal model for studying the very first stages of the evolution of multicellularity. This is because it can form multicellular groups both clonally and through aggregation and uses a family of proteins called 'flocculins' that determine the way in which groups form, making it particularly amenable to laboratory experiments. We briefly review current knowledge about multicellularity in S. cerevisiae and then propose a framework for making predictions about the evolution of multicellular phenotypes in yeast based on social evolution theory. We finish by explaining how S. cerevisiae is a particularly useful experimental model for the analysis of open questions concerning multicellularity.

The emergence and maintenance of multicellularity requires the coexistence of diverse cellular populations displaying cooperative relationships. This enables long-term persistence of cellular consortia, particularly under environmental constraints that challenge cell survival. Toxic environments are known to trigger the formation of multicellular consortia capable of dealing with waste while promoting cell diversity as a way to overcome selection barriers. In this context, recent theoretical studies suggest that an environment containing both resources and toxic waste can promote the emergence of complex, spatially distributed proto-organisms exhibiting division of labour and higher-scale features beyond the cell–cell pairwise interactions. Some previous non-spatial models suggest that the presence of a growth inhibitor can trigger the coexistence of competitive species in an antibiotic-resistance context. In this paper, we further explore this idea using both mathematical and computational models taking the most fundamental features of the proto-organisms model interactions. It is shown that this resource-waste environmental context, in which both species are lethally affected by the toxic waste and metabolic tradeoffs are present, favours the maintenance of diverse populations. A spatial, stochastic extension confirms our basic results. The evolutionary and ecological implications of these results are outlined.

The evolutionary transition from single-celled to multicellular life has occurred dozens of times, yet the evolution of the genetic and developmental mechanisms underlying these transitions is not well understood. The volvocine green algae provide an excellent model system to study questions about the evolution of multicellularity because different species within this group span a range from single-celled, to undifferentiated multicellularity, to multicellularity with two distinct cell-types. This dissertation focuses on how the genes and developmental mechanisms that control cellular differentiation in the volvocine green algae evolved. I show that a suite of transcription factors, known as the reg cluster, which are thought to be involved in the development of cellular differentiation, arose early in the evolution of multicellularity in this group and are present in many species that lack cellular differentiation as well (Appendix A). I then study the development and genetics of Volvox powersii, a species that represents an ideal model organism for studying the early stages of cellular differentiation and multicellular complexity because it is thought to possess an ancestor-like developmental program. I show that a morphological mutant of this species possesses a mutation in the rlsB reg cluster gene and I developed a novel transformation procedure to test the causality of this mutation (Chapter 3). I also used laser scanning confocal fluorescence microscopy to test hypotheses about how cell fate is determined in V. powersii. I find that cell size is unlikely to play a role in cell fate in V. powersii but provide evidence that chloroplast DNA nucleoid number may be involved in cellular differentiation (Chapter 4). This result suggests that differential inheritance of chloroplast nucleoids may be an early arising mechanism for cell fate specification in the volvocine algae. I also demonstrate a mutagenesis procedure of Volvox ferrisii, a species that unambiguously

Microbial cells within clonal populations can display different morphologies or carry out different tasks. This heterogeneity is beneficial at the population level and allows microbes to spread risk or separate incompatible activities. Heterogeneity is also evident in filamentous bacteria and fungi, which form mycelial networks consisting of interconnected hyphae. Here, heterogeneity is observed between clonal mycelial particles, between different zones of colonies, between adjacent hyphae and even between adjacent compartments of individual hyphae. In this review, we compare this multiscale heterogeneity in filamentous bacteria and fungi and discuss the underlying mechanisms. These mechanisms might provide targets to improve the exploitability of these organisms as cell factories in the biotech sector.

The role of homeodomain (HD) transcription factors during development in animals is well established since the identification of the homeobox gene clusters. In the kingdom Fungi homeodomain genes also play a crucial role during multicellular development. They were first identified in mating type loci, which regulate sexual development. Later, other HD genes were shown to be involved in fruiting body development in several members of Ascomycota and Basidiomycota. In this review we describe recent research on HD transcription factors in fungi. An evolutionary framework is provided by reanalyzing 222 previously published fungal genomes to identify potential functions of HD transcription factors in multicellular development and fructification.

Filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two interdependent cell types that exchange nutrients and regulators. Vegetative cells provide heterocysts with reduced carbon, and heterocysts provide vegetative cells with fixed nitrogen. Additionally, heterocyst differentiation from vegetative cells is regulated by inhibitors of differentiation produced by prospective heterocysts and heterocysts. Proteinaceous structures known as septal junctions join the cells in the filament. The SepJ protein is involved in formation of septal junctions in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SepJ bears extra-membrane and membrane (permease) domains and is located at the cell poles in the intercellular septa of the filament. Here we created Anabaena mutants that produce SepJ proteins altered in the permease domain. Some of these mutant SepJ proteins did not provide functions needed for Anabaena to form long filaments and (in some cases) differentiate heterocysts, identifying amino acids and amino acid stretches that are important for the structure or function of the protein. Some other mutant SepJ proteins fulfilled filamentation and heterocyst differentiation functions but failed to provide normal communication function assessed via the intercellular transfer of the fluorescent marker calcein. These mutant SepJ proteins bore mutations in amino acids located at the cytoplasmic face of the permease, which could affect access of the fluorescent marker to the septal junctions. Overall, the data are consistent with the idea that SepJ carries out multiple roles in the multicellular function of the Anabaena filament.

In a previous study we established forward genetics in the choanoflagellate Salpingoeca rosetta and found that a C-type lectin gene is required for rosette development (Levin et al., 2014). Here we report on critical improvements to genetic screens in S. rosetta while also investigating the genetic basis for rosette defect mutants in which single cells fail to develop into orderly rosettes and instead aggregate promiscuously into amorphous clumps of cells. Two of the mutants, Jumble and Couscous, mapped to lesions in genes encoding two different predicted glycosyltransferases and displayed aberrant glycosylation patterns in the basal extracellular matrix (ECM). In animals, glycosyltransferases sculpt the polysaccharide-rich ECM, regulate integrin and cadherin activity, and, when disrupted, contribute to tumorigenesis. The finding that predicted glycosyltransferases promote proper rosette development and prevent cell aggregation in S. rosetta suggests a pre-metazoan role for glycosyltransferases in regulating development and preventing abnormal tumor-like multicellularity.

The evolution of animals from their unicellular ancestors was a major transition in evolutionary history that enabled the diversity and complexity of extant animals. Due to the lack of a clear fossil record of the progenitors of animals, we know relatively little about the first animals and the developmental events that predicated their evolution. My doctoral research utilized Salpingoeca rosetta, a model choanoflagellate and one of the closest living relatives of animals, to investigate the molecular changes that might have led to the origin of multicellularity in animals. S. rosetta facultatively forms multicellular “rosettes” through serial cell division in a process reminiscent of early animal embryogenesis. To determine the genetic underpinnings of rosette development in S. rosetta, I performed a forward genetic screen for rosette defect mutants (Chapter 2). I identified a new class of mutants that aggregate promiscuously into amorphous clumps of cells, but that do not develop into orderly rosettes. Two clumpy mutants, named Jumble and Couscous, mapped to lesions in genes encoding predicted glycosyltransferases. The mutations in the jumble and couscous genes were shown to disrupt glycosylation patterns at the basal pole of the extracellular matrix (ECM). The only previously identified gene required for rosette formation, rosetteless, was found to encode a protein that localizes at the basal pole of cells in the ECM-filled center of rosettes. Thus, all three genes known to be required for rosette development in S. rosetta play a role in establishing the ECM of rosettes and implicate the ECM in the regulation of multicellular development.Beyond the specific genes required for cell adhesion and cell signaling, animal multicellular development relies on transcriptional regulation of specific genes for cell differentiation. To examine the role of transcriptional regulation, I generated an improved S. rosetta genome assembly that allows for better annotation of regul

All biological systems follow the rules and constraints imposed during their evolution. Current-day gene phenotypes such as gene expression, gene essentiality, gene function and protein localization are linked with the time of evolutionary emergence of genes. In cancer, tumours rely on cellular processes that date back to unicellular ancestors (e.g., cell replication, glycolysis), while dysregulating key pathways linked to the emergence of multicellularity, suggesting that the transition from unicellularity to multicellularity left vulnerabilities in cells that act as guiding principles during cancer development. Therefore, in this thesis I integrate genomics, systems biology and evolutionary biology to investigate fundamental principles of tumourigenesis related to the evolutionary history of genes using gene expression and somatic mutation information across multiple tumour types. First, I coupled the evolutionary age of genes and cellular processes with their expression levels in tumour and normal samples, and found that tumours consistently activate genes from unicellular ancestors while switching off genes related to multicellularity. These consistent patterns were supported by a mutual exclusivity between the activity of genes and transcriptional networks of unicellular and multicellular ancestors, which promoted convergent evolution towards a state of loss of multicellularity. Second, I investigated how somatic mutations disrupted gene regulatory networks. Genes that emerged together with early metazoans were enriched in point mutations and copy- number alterations, indicating that gene innovations that took place at the onset of multicellularity play a fundamental role in cancer development. Importantly, the uncoupling of regulatory networks of unicellular and multicellular ancestors was mostly due to point mutations in gene regulators linking these networks. On the other hand, copy-number aberrations were directly involved in the activation and inactivation

Filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two interdependent cell types that exchange nutrients and regulators. Vegetative cells provide heterocysts with reduced carbon, and heterocysts provide vegetative cells with fixed nitrogen. Additionally, heterocyst differentiation from vegetative cells is regulated by inhibitors of differentiation produced by prospective heterocysts and heterocysts. Proteinaceous structures known as septal junctions join the cells in the filament. The SepJ protein is involved in formation of septal junctions in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SepJ bears extra-membrane and membrane (permease) domains and is located at the cell poles in the intercellular septa of the filament. Here we created Anabaena mutants that produce SepJ proteins altered in the permease domain. Some of these mutant SepJ proteins did not provide functions needed for Anabaena to form long filaments and (in some cases) differentiate heterocysts, identifying amino acids and amino acid stretches that are important for the structure or function of the protein. Some other mutant SepJ proteins fulfilled filamentation and heterocyst differentiation functions but failed to provide normal communication function assessed via the intercellular transfer of the fluorescent marker calcein. These mutant SepJ proteins bore mutations in amino acids located at the cytoplasmic face of the permease, which could affect access of the fluorescent marker to the septal junctions. Overall, the data are consistent with the idea that SepJ carries out multiple roles in the multicellular function of the Anabaena filament.

Over 600 million years ago, animals evolved from a unicellular or colonial organism whose cell(s) captured bacteria with a collar complex, a flagellum surrounded by a microvillar collar. Using principles from evolutionary cell biology, we reason that the transition to multicellularity required modification of pre-existing mechanisms for extracellular matrix synthesis and cytokinesis. We discuss two hypotheses for the origin of animal cell types: division of labor from ancient plurifunctional cells and conversion of temporally alternating phenotypes into spatially juxtaposed cell types. Mechanistic studies in diverse animals and their relatives promise to deepen our understanding of animal origins and cell biology.

During the evolution of multicellularity from unicellular ancestors, cells transition from being evolutionary individuals to components of more complex, multicellular evolutionary individuals. The volvocine green algae provide a powerful model system for understanding the genetic and morphological changes that underlie and are caused by the evolution of multicellularity. This dissertation concerns the role of cell cycle regulation, cellular differentiation, and sexual traits during the evolution of multicellularity. While some of these are shown to be causally important in the origins of multicellularity (Appendix B), others are driven by the evolution of multicellularity (Appendix D). We provide a review of recent mathematical models on the evolution of multicellularity, which are found to focus heavily on the later, subsequent stages of the evolution of multicellular complexity. We found that many of these models assume multicellular ancestors and instead evolve cellular differentiation, bringing attention to a gap in our understanding of the events in the initial stages of the evolution of multicellularity. We show that a focus on the early stages of the evolution of multicellularity reveals a powerful and critical role for regulation of the cell cycle at the origins of multicellularity (Appendix B). We further find that the genetic basis for cellular differentiation evolved sometime after the evolution of cell cycle regulation. We find that while the genetic basis for cellular differentiation evolved after cell cycle regulation, it also evolved earlier than previously predicted in the volvocine green algae, suggesting an important role in undifferentiated species (Appendix C). Lastly, having elucidated the origins and evolution of multicellularity, we find that multicellularity causes the evolution of sexual traits including anisogamy, internal fertilization, and subsequently sexual dimorphism (Appendix D). This work emphasizes the important role that multicellu

Philosophy & Theory in Biology: vol. 9, (dlps) 6959004.0009.003, http://hdl.handle.net/2027/spo.6959004.0009.003, This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. Please contact mpub-help@umich.edu to use this work in a way not covered by the license.