Regulation of sedimentation rate shapes the evolution of multicellularity in a unicellular relative of animals

Significant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits which control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves, are still poorly understood. Here, using isolates of the ichthyosporean Sphaeroforma genus - close unicellular relatives of animals with brief transient multicellular life stages - we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least two distinct physical mechanisms. We first find a dramatic >300x variation in sedimentation rate for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Using experimental evolution with sedimentation rate as a focal trait, we readily obtained fast settling S. arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal "clumping" multicellular variants with increased size and density. Additionally, density increases arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in four additional species from the Sphaeroforma genus, suggesting variation in these traits might be widespread in the marine habitat. By sequencing evolved isolates, we identified mutations in regulators of cytokinesis, plasma membrane remodelling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the evolution of multicellularity.