Master of Science / Department of Biology / Bradley J. Olson / The evolution of multicellularity is a major transition in the morphological organization of organisms, however, the molecular mechanisms important for this transition in any taxa are currently not well understood. In most taxa, the molecular signature of the transition to multicellularity is obscured by nearly a billion years of divergence. In contrast, multicellularity evolved recently in the volvocine algae. As a result, the genomes of member species remarkably similar, suggesting the transition to multicellularity only requires the evolution of a few genes. The volvocine algae include members that span the range of morphological complexity from unicellular (e.g. Chlamydomonas reinhardtii) to undifferentiated multicellular (e.g. Gonium pectorale), to species with differentiated tissues (e.g. Volvox carteri). Corresponding to the morphological phenotypes, members of these organisms range from a simplified cell wall in the unicellular species into an expanded extracellular matrix important for multicellular group formation. To find genes important for multicellularity in undifferentiated multicellular Gonium pectorale, we performed a forward genetic screen for unicellular mutants. From this we identified two mutants, uc-1C7, where 99.6% of cells are unicellular and uc-1H7 where 95% of cells are unicellular. Both mutants were found to be sensitive to detergent lysis suggesting these mutants have defects in extracellular matrix assembly. Additionally, total cell wall extracts were prepared from uc-1C7 and wild-type strains. These were subjected to tandem mass spectrometry to identify which proteins were present in both lines, which demonstrated that the uc-1C7 mutant is missing major components of its cell wall. Using an antibody that is specific for Chlamydomonas cell walls, preliminary immunofluorescence tests show a reduced signal in the uc-1C7 and uc-1H7 mutants compared to wild-type. Our results point to two unicellular Gonium mutants that have defects in assembling a functional extracellular matrix. Because these mutants have a unicellular phenotype, it demonstrates that cell wall of Gonium is essential for undifferentiated colony formation.

Oxygen has been assumed to be a vital trigger for the evolution of multicellular life forms on Earth, partly based on its power to promote substantial energy flux in cell respiration and partly as biosynthesis of compounds like collagen require oxygen. However, the co-evolution of large life and the Earth’s chemical environment is not well understood at present, and there is particular disagreement in the field about whether the Cambrian explosion of animal life forms was a chemical or biological event. Here, I discuss the evolution of multicellularity, divided in simple or complex forms, in light of the evolution of ocean water column chemistry in both the Proterozoic and the early Paleozoic. Even if the appearance of animals is confined to the Ediacaran, other fossil evidence of complex multicellularity can be argued to occur in the Paleo-, Meso- and Neoproterozic. These finds are, if anything, reason enough to keep searching for early experiments in complex multicellularity. In this search, we may have to expand our toolbox by looking at e.g. trace element aggregations and the isotopic composition of key elements.  Research over the last couple of years have accentuated that much of the interval between the Ediacaran and the Devonian was dramatic with transitional ocean chemistry at the same time that large forms of animal life experienced dynamic radiation and ecological expansion. Results presented here describe some aspects of this time, including geochemistry from Chengjiang and a mechanism for preserving non-mineralized Cambrian animals that was partly dependent on specific ocean chemistry. Also, geochemical proxies using iron and molybdenum are used to infer a Paleozoic atmosphere with less than 50% of present levels of oxygen. The possibility that the subsequent rise is due to terrestrial plants and linked to the appearance of large predatory fish is discussed. Finally, the first mass extinction in the end-Ordovician is linked to low oxygen concentrations in the water column. It appears that more than oxygen was critical to allow the radiation of large life forms on Earth, but that chemistry and tectonic activity were intimately intertwined to biology, in a dance of permitting and being determined by certain aspects of ecology. / Under lång tid har vi sett atmosfärens syrehalt som avgörande för att stora livsformer skulle börja utvecklas på jorden, delvis eftersom syre är ett energirikt bränsle men också för att det krävs vid sammansättningen av vissa ämnen som djur behöver, till exempel proteinet kollagen. Men, i själva verket, har vi inte lyckats reda ut detaljerna om hur utvecklingen av tidigt, stort liv och miljö satt samman, och om den kambriska explosionen framförallt var en biologisk eller kemiskt händelse. I den här avhandlingen diskuterar jag hur utvecklingen av flercellighet, då uppdelat i enkla och komplexa former, kan vara kopplad till hur havens kemi förändrats både i proterozoikum (2.5-0.5 miljarder år sedan) och paleozoikum (0.5-0 miljarder år sedan. Även om fossil från moderna djur dyker upp runt ediacaran och kambrium, så finns det långt äldre fossil som kan påvisa flercellighet. Dessa fossil ger, om inte annat, anledning att leta vidare efter fler spår av pre-kambrisk flercellighet och kanske kan vi utöka våra sökmetoder till att också tolka ansamlingar, eller isotopsammansättningar, av spårmetaller. Den kambriska explosinen av djurliv (med startskott för 543 miljoner år sedan) är ett etablerat begrepp, men den senaste årens forskning har satt fokus på att en längre period, från ediacaran till devon, var en dynamisk tid med skiftande havskemi, nya djurarter och experimentella ekologiska nätverk. I den här avhandlingen presenteras några resultat som belyser just denna övergångstid, som geokemin i Chengjiang som beskriver hur havets kemi skiftar från syrefritt till sulfatfritt till syrerikt, och hur djur utan skal och ben kunde bli bevarade genom att flera unika förhållanden sammanföll. En annan studie visar hur molybden använts för att påvisa att atmosfärens syrehalt, under den här perioden, var högst hälften av vår moderna nivå. Vi hävdar att stigningen som skedde i devon, delvis tack vare växternas intåg på land, och att stigningen kan speglas i att fiskar först då hade råd att jaga och växa sig stora. Slutligen visar jag också på hur det första stora massutdöendet kan vara sammankopplat med syrefria hav, snarare än kyla och mer syre än djuren klarade av. Ett komplext samspel mellan flera kemiska ämnen, utöver syre, tektonisk aktivitet och biologi ser ut att höra samman med den dramatiska uvecklingen för stora livsformer på jorden.

The evolution of multicellularity is an example of an evolutionary transition in individuality, in which a group of lower-level biological units (cells, in this case) emerges as a higher-level unit (the multicellular organism) with its own fitness, heritability and individuality. The volvocine green algae are a model system for the transition to multicellularity and for the evolution of cellular differentiation. Some of the developmental changes that collectively make up this transition have occurred more than once in the volvocine lineage; others have reverted from derived to ancestral states. The transition from cells to multicellular organisms began over 200 million years ago in this lineage, and the subsequent changes have been sporadic, with several important changes occurring early in the transition and some body plans remaining largely unchanged over long evolutionary time scales. Two suites of characters that differ among species within the genus Volvox have each evolved convergently or in parallel in lineages that diverged at least 175 million years ago. This complex history suggests that other origins of multicellularity may have involved important roles for cooperation, conflict and conflict mediation; parallel evolution of some traits; sporadic rather than constant change; and long-term coexistence of forms with different levels of complexity. Data from one species, Pleodorina starrii, support motility as a major selective pressure driving the the origins of cellular differentiation. Optimization of the proportion of soma in this species appears to be prevented by a constraint that prevents independent change in colonies with different numbers of cells. Finally, P. starrii presents an exceptionally high level of phenotypic variability, suggesting that the genotype-phenotype map has not completely shifted from the cell to the colony and that the transition to a new, higher-level individual in this species is incomplete.

Background: The volvocine lineage, containing unicellular Chlamydomonas reinhardtii and differentiated multicellular Volvox carteri, is a powerful model for comparative studies aiming at understanding emergence of multicellularity. Tetrabaena socialis is the simplest multicellular volvocine alga and belongs to the family Tetrabaenaceae that is sister to more complex multicellular volvocine families, Goniaceae and Volvocaceae. Thus, T. socialis is a key species to elucidate the initial steps in the evolution of multicellularity. In the asexual life cycle of C. reinhardtii and multicellular volvocine species, reproductive cells form daughter cells/colonies by multiple fission. In embryogenesis of the multicellular species, daughter protoplasts are connected to one another by cytoplasmic bridges formed by incomplete cytokinesis during multiple fission. These bridges are important for arranging the daughter protoplasts in appropriate positions such that species-specific integrated multicellular individuals are shaped. Detailed comparative studies of cytokinesis between unicellular and simple multicellular volvocine species will help to elucidate the emergence of multicellularity from the unicellular ancestor. However, the cytokinesis-related genes between closely related unicellular and multicellular species have not been subjected to a comparative analysis. Results: Here we focused on dynamin-related protein 1 (DRP1), which is known for its role in cytokinesis in land plants. Immunofluorescence microscopy using an antibody against T. socialis DRP1 revealed that volvocine DRP1 was localized to division planes during cytokinesis in unicellular C. reinhardtii and two simple multicellular volvocine species T. socialis and Gonium pectorale. DRP1 signals were mainly observed in the newly formed division planes of unicellular C. reinhardtii during multiple fission, whereas in multicellular T. socialis and G. pectorale, DRP1 signals were observed in all division planes during embryogenesis. Conclusions: These results indicate that the molecular mechanisms of cytokinesis may be different in unicellular and multicellular volvocine algae. The localization of DRP1 during multiple fission might have been modified in the common ancestor of multicellular volvocine algae. This modification may have been essential for the re-orientation of cells and shaping colonies during the emergence of multicellularity in this lineage.

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 multicellularity plays in driving the evolution of sexual diversity seen across the eukaryotic tree and well as informs critical hypotheses on the evolution of anisogamous sex, among the most challenging problems in evolutionary theory.

Technology, nowadays, has given us huge computational potential, but computer sciences have major problems tapping into this pool of resources. One of the main issues is how to program and design distributed systems. Biology has solved this issue about half a billion years ago, during the Cambrian explosion: the evolution of multicellularity. The evolution of multicellularity allowed cells to differentiate and so divide different tasks to different groups of cells; this combined with evolution gives us a very good example of how massively parallel distributed computational system can function and be “programmed”. However, the evolution of multicellularity is not very well understood, and most traditional methodologies used in evolutionary theory are not apt to address and model the whole transition to multicellularity. In this thesis I develop and argue for new computational artificial life methodologies for the study of the evolution of multicellularity that are able to address the whole transition, give new insights, and complement existing methods. I argue that these methodologies should have three main characteristics: accessible across scientific disciplines, have potentiality for complex behaviour, and be easy to analyse. To design models, which possess those characteristics, I developed a model of genetic regulatory networks (GRNs) that control artificial cells, which I have used in multiple evolutionary experiments. The first experiment was designed to present some of the engineering problems of evolving multicelled systems (applied to graph-colouring), and to perfect my artificial cell model. The two subsequent experiments demonstrate the characteristics listed above: one model based on a genetic algorithm with an explicit two-level fitness function to evolve multicelled cooperative patterning, and one with freely evolving artificial cells that have evolved some multicelled cooperation as evidenced by novel measures, and has the potential to evolve multicellularity. These experiments show how artificial life models of evolution can discover and investigate new hypotheses and behaviours that traditional methods cannot.

La communication intercellulaire se produit non seulement chez les eucaryotes, mais aussi chez certaines bactéries. Un tel exemple est la cyanobactérie filamenteuse Anabaena sp. PCC 7120, capable de former des hétérocystes suite à une carence en azote combiné. Un filament d'Anabaena est coordonné comme une unité multicellulaire; comment les cellules communiquent-elles le long de chaque filament et comment échangent-elles des ressources nutritionnelles demeurent des mécanismes encore mal élucidés. Des études récentes ont démontré que des molécules de petites tailles peuvent être échangées entre les cytoplasmes à travers des jonctions intercellulaires. De plus, le périplasme semble être continu le long de chaque filament, avec une membrane extérieure commune pour toutes les cellules. Toutefois, il n’est pas déterminé si le "périplasme continu" peut servir comme une route alternative pour les échanges moléculaires le long des filaments.Dans cette étude, la propriété du périplasme chez Anabaena a été évaluée par le suivi du mouvement de protéines fluorescentes (GFP ou iLOV) en utilisant des techniques microscopiques. Les protéines fluorescentes ont été exportées vers l'espace périplasmique, soit d'un hétérocyste soit d’une cellule végétative. Nous avons pu montrer que ces protéines fluorescentes restent dans le périplasme de la cellule d’origine, et que la GFP peut diffuser librement, mais seulement dans le périplasme d'un hétérocyste ou d’une cellule végétative. Ainsi, bien que le périplasme semble être continu le long du filament, une barrière intercellulaire semble exister pour empêcher la libre diffusion des protéines à la taille de ~27 kDa (GFP) ou ~13 kDa (iLOV). La couche de peptidoglycane pourrait constituer cette barrière et nous estimons que la limite pour la diffusion à travers cette barrière se situe entre 0.53 et 13 kDa.En parallèle, les voies métaboliques des cellules végétatives et des hétérocystes ont été comparées en utilisant une approche transcriptomique. L'expression différentielle des gènes impliqués dans le métabolisme nous permet d’appréhender la nature des métabolites pouvant être échangées entres ces deux types cellulaires. / Cell-cell communication occurs not only in eukaryotes but also in bacteria. One such example is the filamentous cyanobacterium Anabaena sp. PCC 7120, which is able to differentiate a specialized cell type named heterocyst upon nitrogen deprivation. A filament of Anabaena is coordinated as a multicellular unity; how the cells along each filament communicate and exchange resources are not yet fully understood. Recent studies demonstrated that small molecules can be rapidly exchanged from cytoplasm to cytoplasm through intercellular junctions. In addition, the periplasm appears to be continuous along each filament, with a shared outer membrane for all cells. However, whether the ‘continuous periplasm’ serves as an alternative route for molecular exchanges along the filament remains unknown. In this study, the property of periplasm in Anabaena was assessed by monitoring the movement of fluorescent proteins (GFP or iLOV) using microscopic techniques. Fluorescent proteins were exported to the periplasmic space of either a heterocyst or a vegetative cell and their diffusion was tested. We found that both GFP and iLOV remains in the producing cells, and at least GFP could diffuse freely in the periplasm of a heterocyst or a vegetative cell but failed to cross cell borders. Thus although periplasm appears to be continuous along the filament, barriers exist to prevent free diffusion of proteins up to the size of ~27 kDa (GFP) or ~13 kDa (iLOV). One candidate as diffusion barrier in the periplasm may be the peptidoglycan and we estimate the limit for diffusion of the barrier in the range between 0.53 to 13 kDa. In parallel, the biosynthetic pathways operating in vegetative cells and heterocysts were compared using oligonucleotide microarray. Differential expression of the genes involved in amino acids metabolism give clues as to which nitrogen-containing compounds might serve as the transfer vehicle in cell-cell exchanges.

Life on Earth could have existed already 3.8 Ga ago, and yet, more complex, multicellular life did not evolve until over three billion years later, about 700 Ma ago. Many have searched for the reason behind this apparent delay in evolution, and the dominating theories put the blame on the hostile Precambrian environment with low oxygen levels and sulphide-rich oceans. There are, however, doubts whether this would be the full explanation, and this thesis therefore focuses on a new hypothesis; the levels of the redox sensitive element arsenic increased in the oceans as a consequence of the change in weathering patterns that followed the upcome of free oxygen in the atmosphere at about 2.4 billion years ago. Given its toxicity, this could have had negative effects upon the life of the time. To test the hypothesis, 66 samples from drill cores coming from South Africa and Gabon with ages between 2.7 and 2.05 Ga were analysed for their elemental composition, and their arsenic content were compared with carbon isotope data from the same samples. These confirmed that a rise in arsenic concentration following the upcome of free oxygen in the atmosphere and the onset of oxidative weathering of continental sulphides. Arsenic, which is commonly found in sulphide minerals, was weathered together with the sulphide and delivered into the oceans, where it in the Palaeoproterozoic increased to over 600% compared to the older Archaean levels, at least locally. Iron had the strongest control over the arsenic levels in the anoxic (ferruginous and sulphidic) oceans, probably due to its ability to remove arsenic through adsorption. During oxygenated conditions, sulphur instead had the strongest influence upon arsenic, likely because of the lack of dissolved iron. The highest arsenic levels were found in samples recognised as coming from oxygenated conditions, although this might be due to the oxygenation state of arsenic affecting its solubility. Arsenic is toxic already at low doses, especially if the necessary arsenic detoxification systems had not yet evolved. However, the lack of correlation between arsenic and changes in δ13C indicated that the increase of arsenic did not affect the primary production between 2.7 and 2.05 Ga. Thus, whether arsenic could have affected the evolution of life during the Mesoproterozoic remains to be shown.

The transition to multicellularity is a major step in the evolution of complex life. The first steps in this transition are poorly understood because multicellularity evolved long ago, and transitional forms have been lost to extinction. Previous studies developed a novel microbial model system in which simple multicellularity could be evolved de novo (Ratcliff et al., 2012). By evolving our snowflake yeast to undergo sexual reproduction we hypothesized that sex created variation in key multicellular traits, which spurs multicellular adaptation. In our 'snowflake yeast' model system, two traits are of central importance: cluster size, and programmed cell death (apoptosis). Apoptosis previously evolved to regulate cluster size, by acting as break points within clusters, allowing them to modify the size and number of multicellular propagules they produce. In prior experiments, this only develops after yeast have evolved to form large clusters. Prior experiments in the lab demonstrated that the longer snowflake yeast have been evolving, the greater the fitness benefit provided by sex. Here we examine whether this is due to sex creating greater amounts of diversity in the traits of post-sex offspring in more highly evolved multicellular yeast, allowing post-recombination offspring to 'fine tune' their multicellular traits. By using flow cytometry, we collected data on our multicellular traits. By gathering the biomass mean of the cluster size in each population and staining the cells with propidium iodide to determine the apoptotic tendencies of our cells we were able to compare our outcomes to the pre-sex ancestor, and we determined there was no increase in variation. Although apoptosis did not have an increase in variation due to sex, it created a variation in cluster size; the variation was seen in the population W8. This still supports our hypothesis that sex creates variation in multicellular traits, which allows for rapid adaptation.

To maintain high average fitness, populations must effect selection against the deleterious mutations that continuously arise de novo. Theoretical models of mutation-selection balance predict that the maximum tolerable mutation rate is much lower for organisms growing in colonies than for those in well-mixed liquid media due to drift imposed by competition for position along the growing colony front. Simplifying assumptions made in these models, including the irreversibility and fixed fitness cost of mutations, do not strictly hold in extant species. To explore the applicability of these models in natural contexts, we have constructed a yeast strain which undergoes recombinase-mediated irreversible gene excision at a single locus with tunable fitness cost, but also possesses the random genomic mutation profile characteristic of yeast. We find that several theoretical predictions hold for our strain, including the dependence of maximum tolerable mutation rate on growth condition and selective coefficient. These results constitute the first direct biological test of mutation-selection balance theory.