Experimental evolution of an alternating uni- and multicellular life cycle in Chlamydomonas reinhardtii

Abstract

The transition to multicellularity enabled the evolution of large, complex organisms, but early steps in this transition remain poorly understood. Here we show that multicellular complexity, including development from a single cell, can evolve rapidly in a unicellular organism that has never had a multicellular ancestor. We subject the alga Chlamydomonas reinhardtii to conditions that favour multicellularity, resulting in the evolution of a multicellular life cycle in which clusters reproduce via motile unicellular propagules. While a single-cell genetic bottleneck during ontogeny is widely regarded as an adaptation to limit among-cell conflict, its appearance very early in this transition suggests that it did not evolve for this purpose. Instead, we find that unicellular propagules are adaptive even in the absence of intercellular conflict, maximizing cluster-level fecundity. These results demonstrate that the unicellular bottleneck, a trait essential for evolving multicellular complexity, can arise rapidly via co-option of the ancestral unicellular form.

Figure 1. Multicellular Chlamydomonas morphology.

(a) Multicellular C. reinhardtii (left) settle faster than a contemporary population undergoing settling selection that remained unicellular (right), forming a pellet after 20 min of settling on the bench (cultures shown are 72 h old). (b) Cells are held in place by a transparent extracellular matrix, indicated by arrows. (c) Motile propagules released from multicellular clusters (phase contrast microscopy); (d) the ancestral unicellular growth form. Note that c and d are phenotypically identical. (e) Cluster formation from a single cell. Cells form clusters by ‘staying together’ after mitotic reproduction, not aggregation of unrelated cells. All scale bars are 25 μm.

Figure 2. Multicellular Chlamydomonas alternate between motile unicells and sessile clusters.

(a) C. reinhardtii were evolved by serial transfer every 72 h. Swimming unicells are abundant during the first 24 h of culture, but are virtually absent during the latter 48 h of the culture cycle. Time points demarcated by different letters (A, B) are significantly different at the α=0.05 level (F_7,79=4.1, ANOVA; P=0.0008; differences between time points assessed with Tukey–Kramer HSD). Values shown in (a) are the mean±s.e.m. of 9 replicates per time point. (b) Flagella become active shortly after transfer to fresh medium, causing the cluster to convulse rapidly. Cluster movement was measured (relative to maximum displacement) using time-lapse microscopy. Maximum cluster movement was obtained after 4.25 hours, when the first motile unicellular propagules were released from the cluster. (c) Mean size of multicellular C. reinhardtii increased throughout the culture cycle. Cluster size remained constant during the dispersal phase (0–24 h), but increased nearly threefold during the growth phase (24–72 h). Error bars are the s.e.m. of 9 replicates per time point.

Figure 3. Life cycle evolution.

(a) Life cycle of multicellular C. reinhardtii. Shortly after settling selection and transfer to fresh medium, motile unicells disperse away from the parent cluster. These cells lose motility and develop into clusters prior to the next round of settling. (b) Our model predicts that smaller clusters should experience reduced survival, up to a cluster size of ~70 cells (survival is assured for larger clusters). (c) Shown are the number of surviving offspring a single 64-cell cluster is calculated to produce as a function of propagule size and the number of doublings propagules undergo prior to settling selection (dark blue: 1, red: 2, green: 3, purple: 4, light blue: 5, orange: 6). Despite the reduced survival of smaller clusters during settling selection, unicellular propagules maximize the number of surviving offspring a cluster can produce.