After a lengthy experiment with tantalizing implications for origin-of-life studies, a research group in Japan has reported creating a test tube world of molecules that spontaneously evolved both complexity and, surprisingly, cooperation. Over hundreds of hours of replication, a single type of RNA evolved into five different molecular “species” or lineages of hosts and parasites that coexisted in harmony and cooperated to survive, like the beginning of a “molecular version of an ecosystem,” said Ryo Mizuuchi, the lead author of the study and a project assistant professor at the University of Tokyo.
Their experiment, which confirmed previous theoretical findings, showed that molecules with the means to replicate could spontaneously develop complexity through Darwinian evolution, “a critical step for the emergence of life,” the researchers wrote.
“We can provide the direct evidence; we can see what can actually happen” when a replicating molecule complexifies in a test tube, Mizuuchi said.
This was the first and probably most important step toward evolving a complex network of replicators in the lab, said Sijbren Otto, a professor of systems chemistry at the University of Groningen in the Netherlands who was not involved in the study. “With what is shown here, the path ahead becomes a lot clearer, and one becomes a lot more optimistic that this can actually work out.”
Joana Xavier, a computational biologist at University College London, hailed the work by Mizuuchi and his colleagues as a “great proof of concept” for how a minimal system can complexify. It’s “a very significant advance,” she said.
The Spawn of Spiegelman’s Monster
The roots of the new experiments reach back to the 1960s, when the molecular biologist Sol Spiegelman created what he called “the little monster” in his laboratory. Despite the overtones of Frankenstein in that label, his little monster was not green, square-browed, growling or even alive. It was a synthetic molecule that filled test tubes with copies of itself.
Spiegelman’s monster was a mutating strand of RNA based on a viral genome. The biologist had discovered he could indefinitely replicate it simply by heating and mixing it in the presence of nucleotide building blocks and a polymerizing enzyme called a replicase. He soon realized, however, that his molecules were getting smaller over time: The copies that shed unnecessary genes replicated more quickly, which improved their chances of being collected in samples and transferred to new test tubes for further replication. Just like living species, his molecules had started mutating and evolving under the pressure of natural selection to better survive inside their glass world.
These studies were the world’s first experimental demonstration of Darwinian evolution at the molecular level — “evolution by natural selection, survival of the fittest,” said Eugene Koonin, a National Institutes of Health distinguished investigator at the National Center for Biotechnology Information. “In those conditions, the fittest simply meant the fastest replicating.”
Spiegelman’s work inspired decades of further study, much of which was foundational to research on life’s origins and provided fuel for the RNA world hypothesis that life sprang from self-replicating RNA molecules. But those studies left unanswered a crucial question: Could a single molecular replicator evolve into a complex network of multiple replicators?
About a decade ago, when Norikazu Ichihashi was an associate professor of bioinformatic engineering at Osaka University in Japan, he set out to learn the answer by tweaking Spiegelman’s test tube world. “We tried to develop our system to be a little more lifelike,” Ichihashi said.
Ichihashi and his team developed an RNA molecule that encoded a replicase, which can make copies of RNA. But for the molecule to translate its own code, the scientists needed to add something more: ribosomes and other gene translation machinery that they borrowed from the common gut bacteria Escherichia coli. They embedded the machinery inside droplets and added them to a mixture of RNAs and raw materials.
Then came years of tedious mixing and waiting.
Their long-term experiment involved incubating their replication system at 37 degrees Celsius (the temperature of a human body or a hot summer’s day), adding new droplets with fresh translation systems, and stirring the mixture to induce replication. Every few days or so they analyzed RNA concentrations in the test tubes, and every week or so they froze samples from the latest mixture. Every half year or so, they sequenced large batches of the collected samples to see if the RNA had acquired new mutations and evolved into a new lineage.
Evolution in Test Tubes
After 215 hours and 43 rounds of replication, the researchers began to see interesting results, which they reported in the Proceedings of the National Academy of Sciences in 2016. The original RNA had been replaced by lineages of two other RNAs. One, which the researchers described as a “host,” could use its own replicase to copy itself, like the original molecules. The other lineage, a “parasite,” needed to borrow the gene expression machinery of the hosts.
When Ichihashi and his colleagues extended the experiment to 120 rounds of replication over 600 hours, they found that the host lineage had split into two separate host lineages, and one of the hosts had evolved two distinct parasites. But it wasn’t just the number of lineages that had increased; so had the complexity of their interactions. The hosts had acquired mutations that interfered with the ability of the parasites to hijack their replicative resources — but the parasites had also developed mutations that served as a defense against those obstacles. The hosts and parasites seemed to be coevolving.
The populations of parasites and hosts greatly fluctuated as they competed for the realm in “evolutionary arms races,” the scientists reported in 2020 in eLife. Each RNA lineage transiently rose to dominance, then lost its place to another one. “If one lineage dominated, then another lineage decreased,” said Ichihashi, who is now a professor at the University of Tokyo.
But the researchers kept the experiments going, and by round 130, another host had evolved. By round 160, one of the parasites had disappeared; some rounds later, another parasite had appeared. By round 190, the researchers had hit on a new surprise: The huge dynamic swings in the population of each lineage had started giving way to smaller waves. This stabilization suggested that the lineages were no longer competing to replicate. Instead, they had started to interact as a network and cooperate in a state of quasi-stable coexistence.
Mizuuchi and Ichihashi, who did the experiments with Taro Furubayashi (who was a doctoral student in Ichihashi’s lab at the time and is now a research fellow at the University of Tokyo), were floored by the findings, which they reported in Nature Communications in March. They’re just “mere molecules,” Mizuuchi said. “It’s pretty unexpected.”
Cooperative Parasites Do Their Share
Koonin agrees that their findings are striking. Their “experimental setup is more elaborate, it’s more realistic, and the results are more complex and rich, but [it’s] fully compatible” with Spiegelman’s, he said. They watched a single type of molecule replicate and gather mutations under natural selection — but then went further by letting the divergent molecules evolve into a community under one another’s influence, just as communities of living cells, animals or people would. In the process, the researchers explored some of the rules governing what it takes for such complex communities to become stable and enduring.
Some of these results confirmed the predictions of earlier experimental studies of how complexity can arise in viruses, bacteria and eukaryotes, as well as some theoretical work. A study from Koonin’s lab, for instance, also suggested that parasites were inevitable in the emergence of complexity.
“Without parasites, this level of diversification is probably not possible,” Mizuuchi said. Evolutionary pressures that parasites and their hosts place on each other lead both sides to split into new lineages.
A more surprising fundamental principle that emerged was the critical role of cooperation. The five lineages belonged to different small networks of cooperation, and some were more cooperative than others. By round 228, for example, one of the three hosts had evolved into a “super cooperator” that could replicate itself and all the other lineages; the other two hosts could each replicate only themselves and one of the parasites.
Scientists have focused on studies of competition in evolution for so long that the role of cooperation “has been a bit overlooked,” Xavier said. “I think cooperation is also going to start having a major role, especially in origins, because there are so many things that have to come together in the right way.”
The cooperation among RNAs was focused entirely on replication in the system that Ichihashi, Mizuuchi and their colleagues observed. But the researchers hope that it will be possible to coerce the RNAs to evolve a completely different function too, such as a metabolic one, by adjusting the natural selection criteria inside the test tubes.
A Different Destiny
“Scientists like to entertain each other, and the best entertainment is a surprise,” said David Deamer, a research professor of biomolecular engineering at the University of California, Santa Cruz. He considers it a good paper but noted that what happened in the laboratory may not translate to what happened at the dawn of life.
Indeed, the scenario in Ichihashi’s lab could not reflect what played out at the start of life since the experiments depended on translation machinery from E. coli. “The quintessential problem with the origin of life is: How did protein synthesis itself begin?” said Charlie Carter, a professor of biochemistry and biophysics at the University of North Carolina School of Medicine.
But Koonin thinks that if researchers found a way to evolve complexity using truly self-replicating systems of molecules, they would see something very much resembling the networks depicted in the paper. “They, in the very least, beautifully illustrate the types of processes that likely occurred in the origin of life,” Koonin said.
To Otto, the study suggests that once you’ve solved the problem of accurate replication with molecules at this level of complexity, they will complexify further: The experiment “doesn’t show you how you got there, but once you’re there at this stage, it does chart the way ahead,” he said.
Carrying on with their work, Ichihashi and his colleagues wanted to see if they could re-create the same sustainable network in a separate experiment, so they extracted samples of the five lineages. This time, however, they found that while four of the lineages continued to replicate and survive for at least 22 more rounds, the fifth disappeared. “I don’t know why,” Ichihashi said. “It’s a very strange point.”
One possibility is that the system was even more complex than the researchers thought, and when they isolated the five lineages, they missed a sixth one critical for the survival of the lineage that disappeared. With theoretical models, Ichihashi’s group confirmed that the four remaining lineages could sustainably and interdependently replicate, and that knocking out any one of the four would lead to the extinction of at least one of the others. Their simulation also pointed to the counterintuitive discovery that knocking out one of the parasites would lead to the extinction of its host.
Meanwhile, the researchers have continued their main test tube experiments and are waiting to see whether their network will complexify further. They have also begun similar experiments that use DNA instead of RNA.
“We observed just the beginning” of how these communities of molecular replicators can evolve, Ichihashi said. “I think that they have a different destiny in the future — we cannot predict what happens.”
Correction: May 5, 2022
An earlier version of the “Evolution of a Complex Network” figure was missing some arrows indicating interactions among the hosts and parasites. It has been replaced.