Plasticity and the evolution of group-level regulation of cellular differentiation in the volvocine algae

Abstract

During the evolution of multicellularity, the unit of selection transitions from single cells to integrated multicellular cell groups, necessitating the evolution of group-level traits such as somatic differentiation. However, the processes involved in this change in units of selection are poorly understood. We propose that the evolution of soma in the volvocine algae included an intermediate step involving the plastic development of somatic-like cells. We show that Eudorina elegans, a multicellular volvocine algae species previously thought to be undifferentiated, can develop somatic-like cells following environmental stress (i.e. cold shock). These cells resemble obligate soma in closely related species. We find that somatic-like cells can differentiate directly from cold-shocked cells. This differentiation is a cell-level trait, and the differentiated colony phenotype is a cross-level by-product of cell-level processes. The offspring of cold-shocked colonies also develop somatic-like cells. Since these cells were not directly exposed to the stressor, their differentiation was regulated during group development. Consequently, they are a true group-level trait and not a by-product of cell-level traits. We argue that group-level traits, such as obligate somatic differentiation, can originate through plasticity and that cross-level by-products may be an intermediate step in the evolution of group-level traits.

Keywords: cellular differentiation; evolution of multicellularity; individuality; multilevel selection; plasticity; volvocine algae.

Figure 1.

Representative volvocine algae species and the Eudorina life cycle. (A) Unicellular Chlamydomonas reinhardtii CC124. (B) Undifferentiated Eudorina elegans UTEX 1201. (C) Soma-differentiated Pleodorina californica UTEX 198. (D) Germ-soma differentiated Volvox carteri Eve. (E) Asexual life cycle of E. elegans. During the reproductive phase, all cells in a colony undergo several cleavage divisions followed by inversion, giving rise to offspring colonies. The offspring colonies then hatch out of the parent colony. The deposition of extracellular matrix causes colonies to expand in size without the addition of new cells. (F) A volvocine phylogeny showing an ancestral state reconstruction of cellular differentiation. Pie charts at the nodes represent the posterior probabilities based on stochastic character mapping. Teal nodes and tips correspond to undifferentiated species and ancestral populations, yellow refers to obligate somatic differentiation, and purple represents obligate germ and somatic cells. The Eudorina clade is denoted with the bracket. The teal arrow points to the region of the phylogeny containing E. elegans UTEX 1201 (inferred from [17], which found that E. elegans UTEX 1201 is sister to E. elegans NIES 719). Figure modified from [18] with permission. (G) A summary of the three generations examined in this experiment. G1 is cold-shocked and the differentiated phenotype (when present, shown through the presence of a smaller cell) occurs as a direct response to the stress. G2 colonies are the offspring of G1, and the differentiated phenotype (when present) arises during development. G3 are the offspring of G2. Figure modified from [13] with permission. Created with BioRender.com/b07d532.

Figure 2.

Somatic-like cells develop in response to environmental stress. (A) More cold-treatment G1 and G2 colonies have somatic-like cells than do corresponding controls. The proportion of colonies with (light green) and without (dark green) somatic-like cells is shown for G1 and G2 cold-shocked and control treatments. The percentage of differentiated or undifferentiated colonies is shown on the bars. (B) The differentiated phenotype is not inherited. The number of colonies with (light green) and without (dark green) somatic-like cells is shown for G3 cold-treatment and control colonies, grouped by whether their G2 parent had somatic-like cells. The percentage of differentiated or undifferentiated colonies is shown on the bars. (C) The number of somatic-like cells per differentiated G1 and G2 colony. Box plots show the proportion of somatic-like cells per differentiated colony. The dots show the underlying data distribution. (D) Cells can become somatic-like in response to cold shock outside the colony context. The fates of single cells were tracked following their separation from the colony. The bars show the proportion of cold-shocked and control single cells that became somatic-like (light green), reproduced and gave rise to an offspring colony (dark green) or were large reproductive cells but did not reproduce and were most likely dead (khaki). A summary of the three generations from figure 1F is reproduced here for reference. Bar charts were generated in Python. Created with BioRender.com/b23e901.

Figure 3.

Summary of the plastic responses in response to cold stress. (A) The development of two G1 colonies following cold shock. All the undifferentiated cells (dark green) reproduce. In the left colony, one cell (small and light green) becomes somatic-like and does not reproduce. Since the environment directly induces G1 somatic-like cells, their differentiation is under cell-level control. (B) The development of four of the G2 offspring of the G1 colonies. Some of the G2 offspring colonies develop somatic-like cells (small and light green). Colonies with somatic-like cells can develop from either differentiated or undifferentiated G1 parents. Since G2 somatic-like cells arise during development in the offspring of cold-shocked colonies, their development is under group-level control. (C) Four of the G3 grand-offspring of G1 colonies. Three of the colonies are undifferentiated and one has a somatic-like cell. The differentiated colony was chosen randomly; the presence or absence of somatic cells in G2 does not affect the likelihood that G3 offspring will be differentiated.

Figure 4.

Plastic somatic-like cells are similar to the obligate somatic cells of other species. (A) Brightfield image of cold-shocked G1 E. elegans. (B) Cold-shocked G1 stained with FDA. Both the reproductive and somatic-like cells fluoresce, indicating they are alive. (C) Brightfield image of the G2 offspring of a cold-shocked colony. (D) A G2 cold-treatment colony stained with FDA. Both the reproductive and somatic-like cells fluoresce, indicating they are alive. (E) Brightfield image of a control colony. (F) A control colony stained with FDA. All cells fluoresce, indicating that they are alive. (G) The average cell diameters of G1 and G2 somatic-like cells are significantly different. Both G1 and G2 somatic-like cells are significantly smaller than reproductive cells. (H) The average cell diameters of E. elegans G1 and G2 somatic-like cells, P. starrii NIES 1362 somatic cells and V. carteri Eve somatic cells are shown. Somatic-like cell sizes do not differ significantly from the sizes of somatic cells. (I) A brightfield image of cold-shocked G1 E. elegans. The flagella of a somatic-like cell are visible. (J) A brightfield image of cold-shocked G2 E. elegans. The flagella of a somatic-like cell are visible. (K) A brightfield image of control E. elegans. The flagella of undifferentiated cells are visible.

Figure 5.

Plasticity may mediate the transition from cell-level, environmental regulation to group-level, developmental regulation of cellular differentiation. An environmental cue triggers a change in cellular state (the cessation of reproduction), shown by the change from dark green to light green in a unicellular individual. This is a cell-level response to the environment. The proposed intermediate stages discovered in this study are shown in the grey box. An environmental signal triggers a change in cellular state (from reproductive to somatic-like) in some cells in the group. This is a cell-level response. The environmental signal can also trigger a developmental change in some reproductive cells, as the development of the next generation is altered. This is a group-level response. Finally, we propose that environmental cell-level regulation was lost, and a change in the developmental signalling pathway led to the development of obligate somatic cells regardless of the environment. Figure modified from [13] with permission.