Being a multicellular organism, with cells specialized in one or few functions is a high-risks high-reword evolutionary strategy; on the one hand, they can get bigger and influence their surroundings more easily, but this comes at the expense of more inefficient metabolisms, and less adaptability.

The first form of life must have been a single-cell organism, so how and why did a cell "decide" to become a higher organism? The birth of multicellularity on our planet is still not fully understood but one of the hypotheses relies on the beneficial effect of having a bigger size, with respect to predation.

And here comes today's paper: "De novo origins of multicellularity in response to predation" from Matthew D. Herron and other colleagues, published in Scientific Report in 2019. This study aimed to recreate this evolutionary adaptation in the lab, starting with a unicellular organism, and forcing it to develop multicellularity under stress.



Methods

Three different model organisms were used in this experiment:
  1. Chlamydomonas reinhardtii: This is the tested organism, it's an alga that lives as a single bi-flagellate cell around 10 μm in size, it can use both autotrophic and heterotrophic metabolisms, making its growth easy to control. When subjected to stress its sexual phase is triggered, leading to the formation of gametes that can fuse together creating a new zygote. In this stage, the cell doesn't have flagella and remain "dormant" until good environmental condition arise. Finally, from the zygote 4 or 8 flagellated cells are released, and the cycle continues.
  2. Paramecium tetraurelia: This is the predator cultured together with C. reinhardtii, it's a unicellular ciliate, bigger in size (50-300 μm) that feeds on smaller organisms, conducting them into its buccal overture, to be finally incorporated and digested.
  3. Brachionus calyciflorus: A different ciliate of the group "rotifers", similar in size and feeding behavior to P. tetraurelia. This species has been used to test the effectiveness of the multicellular trait against predation, ensuring that only the size difference was tested, and other C. tetraurelia- specific defenses that may be developed by the algae, did not interfere with the measure.

Figure 1: The three organisms used in the study (not in scale), from left to right: Chlamydomonas reinhardtii, Paramecium tetraurelia and Brachionus calyciflorus. Sources: protist.i.hosei.ac.jp, Wikipedia and aquaportail.com 


The experiment's set-up was simple, five populations of C. reinhardtii were cultured in the presence of the predator, and after a few hundred generations, over the course of 50 weeks, the different strains were tested for the development of multicellular structures.
To check the development of multicellularity, direct microscope observations were done, coupled with cell counting, DAPI nuclei staining, and time-lapse video footage of the life cycle.

To verify the effectiveness of multicellular structures against predation, the researchers used a spectrophotometric analysis, measuring the difference in absorbance of culture in the presence and absence of rotifers. The decrease in absorbance of the culture over time was used to measure the rate of predation in the different strains and compare it to the control population.


Results

How many multicellular strains did develop?

Out of the first 5 populations, 2 developed simple multicellular structures, and after a round of isolation and selection, 5 final multicellular strains were isolated. After observation of the colonies, the researchers found 3 main new life cycles (see Fig 2).
The observed structures were described in detail, focusing on the number of cells per cluster, their origin, and their behavior during reproduction. It was also found that some strains developed even multicellular propagules.

Figure 2. Scheme of the developed life cycles. Normal C. reinhardii cycle, with the release of vegetative cells from the developed zygote (A), production of multicellular clusters, with the release of both propagules and vegetative cells (B), vegetative cells released directly by the cluster (C), reproduction only by propagules (D).


Which advantage did these multicellular structures give to the algae?

Observing the rate at which absorbance decreased in the presence of B. calcyflorus, it was found that the control (unicellular) strains, had on average a 2.5 times greater rate of predation by rotifers. With such a big difference in predation rates, it's plausible that over the course of the experiment, small initial differences in the size of the first clusters were highly favored with respect to free-swimming cells.


Are these structures truly multicellular organisms?

As shown by the different developed life cycles, the structures do not arise from a simple aggregation of cells; clusters are encapsulated in a single cell wall coming from the first cell in the cluster meaning that those cells originated from the division of a single cell. 
This behavior is also preserved in strains, as the multicellular structures appear even in the absence of the predator, and are conserved over different generations.

These cell clusters can be also referred to as "palmelloid" a type of structure that arises from the failing of daughter cells to fully separate, and their ability to produce substances that help adhesion with other cells in the cluster.
Similar behavior was also found in other species like algae of the genre Chlorella, bacteria, and yeasts like Saccharomyces, that were subjected either to predation, or by the selecting pressure of sedimentation (cells that aggregate or form multicellular structures, settled at the bottom of the container, and only free-swimming cells are discarded from the upper phase).


Conclusions

The experiment showed how the selective pressure given by the presence of a single-cell predator can favor the development of multicellular structures in a unicellular species.
This also shows how plastic organisms can be with their metabolism or behavior, in this case, the formation of palmelloids is a consequence of keeping a temporary structure already present in the alga life cycle, making it the predominant phase.

What I really enjoyed about this work was the fairly simple set-up and the clear purpose, I'm not familiar with unicellular algae and it was nice to see some of the protocols commonly applied for these model organisms. It was also pretty interesting to recognize some of the protozoa species cited in this article since I'm getting more familiar with some ciliates commonly found in water environments and wastewater treatment plants.