In preprints: exciting new insights into the lives of our closest relatives

The transition from unicellular organisms to multicellular animals is one of the biggest events in our evolutionary history. Unsurprisingly, how this occurred has been the subject of enormous interest and, indeed, spirited debate for more than 150 years, with a myriad of hypotheses set forth (reviewed by Brunet and King, 2022). In the past two decades, this field has undergone a renaissance with an enormous expansion in our understanding of the closest relatives of animals, the unicellular holozoans. This began with omics approaches, which revealed surprisingly high genetic conservation between animals and their relatives (King et al., 2008; Suga et al., 2013; Brunet and King, 2017; Sebe-Pedros et al., 2017), while the development of refined culture systems and functional tools has revealed the conservation of many cellular and molecular processes, previously thought to be restricted to animals, within these lineages (for example Brunet et al., 2019; Dudin et al., 2019). One particularly intriguing discovery has been that many unicellular holozoan lineages possess facultative multi-cellular stages within their lifecycles. These multicellular states are either derived clonally (via serial cell division) (Fairclough et al., 2010) or via aggregation (Sebé-Pedrós et al., 2013). Whether the ancestor of animals had such a multicellular life stage and how that may relate to the evolution of definitive animal multicellularity is unclear. The formation of these multicellular states is usually environmentally triggered, unlike the intrinsic regulatory mechanisms driving animal development (Alegado et al., 2012), and cells in those multicellular states are generally thought to be equivalent, with no evidence for spatial cell differentiation or patterning. Now, two preprints from emerging groups in the field have shattered many of these previous assumptions, shedding new light on the mechanism underlying multicellularity in two unicellular holozoan lineages.

In the first, Ros-Rocher, Reyes-Rivera and colleagues (Ros-Rocher et al., 2024 preprint) investigated the life history of Choanocea flexa, a choanoflagellate, the closest relatives of animals. The authors recently discovered C. flexa and described a life history with a multicellular stage known as a sheet colony that displays light-induced contractile cell behaviours (Brunet et al., 2019). Now, they seek to understand the formation of these sheet colonies and found they can originate from a single cell by clonal division, similar to all other known examples of choanoflagellate multicellularity (Fairclough et al., 2010; Leadbeater, 2015). To their surprise, however, they also find that sheet colonies can form by aggregation. This type of mixed clonal-aggregative multicellularity was hitherto undescribed in any species. The authors next investigate how this unique life history may be related to the ecology of the organisms. C. flexa is unique because, unlike other choanoflagellate research organisms (and indeed many unicellular holozoan models), it can be readily found in the field and its ecology can, therefore, be studied. The authors find that C. flexa resides in a challenging environment with extreme salinity fluctuations and periods of complete desiccation. Combining field measurements with lab experiments, they show that C. flexa has a cyst-like stage that forms during desiccation, in addition to its unicellular and colonial life stages. They propose a model for a life cycle that is tightly linked to the environment and switches to cysts during periods of desiccation and then to multicellular sheets during periods of low salinity. They further propose that this mixed clonal-aggregate type of multicellularity reflects the need to rapidly grow colonies during periods of low salinity to maximise feeding. Together, this work shows, for the first time, a holozoan that can form a multicellular stage through a mixed clonal/aggregative process and calls strongly into question the previous assumption that these types of multicellularity are mutually exclusive. It also shows how flexible these lifecycles can be and hints that transitions between these types of multicellularity may be more common than previously thought and could occur frequently in permissive environments.

Similar to C. flexa, many other unicellular holozoans have multicellular phases in their lifecycles, such as choanoflagellate rosettes (Fairclough et al., 2010) and filasterean aggregates (Sebé-Pedrós et al., 2013). In all these cases, the transition to multicellularity is facultative and environmentally triggered. However, there is no evidence for different cell fates within those multicellular structures, although different cell morphologies have been reported in Salpingoeca rosetta colonies (Laundon et al., 2019). This has led to the hypothesis that cell differentiation within a multicellular structure, as well as internal programmes directing the development of those fates, are specific to animals. In their preprint, Olivetta and colleagues (Olivetta et al., 2024 preprint) turn this on its head, providing evidence for directed cell differentation in the ichthyosporean Chromosphaera perkinsii. The Ichthyosporea are the sister group to animals, choanoflagellates and filastereans. The best-studied species [e.g. Sphaeroforma arctica (Dudin et al., 2019) and Creolimax fragrantissima (Suga and Ruiz-Trillo, 2013)], also have multicellular stages, although they are transient. These species grow as a multi-nucleated syncytium or coenocyte before briefly cellularising before releasing single cells (Dudin et al., 2019; Suga and Ruiz-Trillo, 2013; Sebé-Pedrós et al., 2017). C. perkinsii, on the other hand, has a different life history that the authors now describe in detail, building on their previous work (Shah et al., 2023 preprint). Using synchronised cell populations, they show that the C. perkinsii life cycle begins as a single cell, which grows without divisions for ∼65 h. It then begins to undergo coupled nuclear/cytoplasmic divisions and, after ∼30 h, releases hundreds of cells. In a stunning observation, the authors note that the life cycle is highly stereotypic, and they see that the first divisions are reproducibly asymmetric – something commonly seen in animal embryos. The transcriptomic signature of the different life stages indicates large-scale changes in gene expression. The combination of these findings leads to the conclusion that the C. perkinsii lifecycle is regulated via some intrinsic mechanisms, similar to animals. At the end of the lifecycle, the colony releases cells of different types, broadly characterised as mitotic or flagellated. The authors search for the origin of these different cells and, surprisingly, they find that they form within the colony before release. It also appears that flagellate differentiation may have some spatial specificity within the organism. Together, these observations provide the first evidence for potential spatial cell differentiation in unicellular holozoans. There are many questions arising from this exciting work. How is this developmental programme orchestrated? What drives both development progression and differentiation? Is there really spatial control over cell differentiation in the colonies and, if so, how is that regulated? Answering these complex questions will likely further advance our understanding of the similarities and differences to animal development.

Together, these two preprints push the boundaries of our understanding of what is possible within our closest unicellular relatives. They brilliantly highlight the need for a broader taxon sampling within all unicellular holozoan groups to fully reveal the diversity of mechanisms they employ during their life cycles. Only once we have a complete understanding of the genome, cell and ‘developmental’ biology of a diverse range of unicellular holozoans will we be able to understand which aspects of animal development are restricted to animals and which were present in their unicellular ancestors. As highlighted recently, it may well be impossible to ever fully understand the ancestors of animals and the path evolution took (Ruiz-Trillo et al., 2023) but, by dissecting the mechanistic basis of life-history transitions in multiple unicellular holozoan groups, we will likely discover – at the very least – the capability of those ancestors.