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.

The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions.