Stable transfection in the protist Corallochytrium limacisporum allows identification of novel cellular features among unicellular relatives of animals

The evolutionary path from protists to multicellular animals remains a mystery. Recent work on the genomes of several unicellular relatives of animals has shaped our understanding of the genetic changes that may have occurred in this transition. However, the specific cellular modifications that took place to accommodate these changes remain unclear. Functional approaches are now needed to unravel how different cell biological features evolved. Recent work has already established genetic tools in three of the four unicellular lineages closely related to animals (choanoflagellates, filastereans, and ichthyosporeans). However, there are no genetic tools available for Corallochytrea, the lineage that seems to have the widest mix of fungal and metazoan features, as well as a complex life cycle. Here, we describe the development of stable transfection in the corallochytrean Corallochytrium limacisporum . Using a battery of cassettes to tag key cellular components, such as nucleus, plasma membrane, cytoplasm and actin filaments, we employ live imaging to discern previously unknown biological features of C. limacisporum . In particular, we identify two different paths for cell division—binary fission and coenocytic growth—that reveal a non-linear life cycle in C. limacisporum . Additionally, we found that C. limacisporum is binucleate for most of its life cycle, and that, contrary to what happens in most eukaryotes, nuclear division is decoupled from cell division. The establishment of these tools in C. limacisporum fills an important gap in the unicellular relatives of animals, opening up new avenues of research with broad taxon sampling to elucidate the specific cellular changes that occurred in the evolution of animals. ### Competing Interest Statement The authors have declared no competing interest.