The microbes of the trickster who shake the tree of life

Every mythology needs a good crook, and there are few better than the Nordic god Loki. He fuels trouble and insults other gods. He is elusive, anarchic and ambiguous. In other words, it is the perfect identity for a group of microbes – the Lokiarchaeota – that rewrites a fundamental story about the first roots of life.

These undisciplined microbes belong to a class of unicellular organisms called archaea, which look like microscopic bacteria but are as distinct in some ways as the human being. The Lokis, as they are sometimes called, were discovered by sequencing the seabed mud DNA collected near Greenland.¹. With a few related microbes, they urge biologists to reconsider one of the greatest events in the history of life on Earth: the appearance of eukaryotes, the group of organisms including all plants, animals, fungi and others.

The discovery of archaea in the late 1970s led scientists to suggest that the tree of life has long been divided into three main trunks, or "domains". A trunk gave birth to modern bacteria; an archée. And the third product of eukaryotes. But debates quickly broke out on the structure of these trunks. According to a model of "three domains", archaea and eukaryotes diverged from a common ancestor. But a two-domain scenario suggested that eukaryotes diverged directly from a subgroup of archaea.

The arguments, though sometimes inflamed, eventually stagnate, says microbiologist Phil Hugenholtz of the University of Queensland in Brisbane, Australia. Then, the Lokis and their relatives came in as a "breath of fresh air," he said, and relaunched the file of a two-estate tree.

These newly discovered archaea possess genes considered characteristic of eukaryotes. And an in-depth analysis of the body's DNA suggests that modern eukaryotes belong to the same group of Archaeans. If so, virtually all the complex life – from green algae to blue whales – originally came from Archaea.

But many scientists are not convinced. The construction of evolutionary trees is a complex and controversial work. And no one has yet published evidence showing that these organisms can be grown in the laboratory, which makes them difficult to study. The debate is always resentful. Supporters on both sides are "very hostile to each other and 100% think there is nothing wrong with the other side," says Hugenholtz. Some refuse to express an opinion, for fear of offending their older colleagues.

The challenge is a deeper understanding of the biological leap that produced eukaryotes: "The most important thing that has happened since the beginning of life," according to Evolution biologist Patrick Keeling at the University of British Columbia. Columbia in Vancouver, Canada. Where they come from "is one of the most fundamental questions to understand the nature of biological complexity," he says. To answer this question, "we must determine who is related to whom".

Two becomes three

For scientists half a century ago, life on Earth was divided into two categories: eukaryotes, living beings whose cells contain internal structures wrapped in a membrane, such as a nucleus; and prokaryotes, unicellular organisms that usually lack internal membranes. Bacteria were the only prokaryotes that biologists knew. Then, in 1977, evolutionary biologist Carl Woese and his colleagues described the archeology as a third distinct form of life – a form of life that goes back billions of years.². Life, said Woese, should be divided into three categories rather than two.

He was not without his detractors. In the 1980s, evolutionary biologist James Lake of the University of California at Los Angeles proposed that eukaryotes be sisters of the archaea that he called the eocytes, which means dawn cells.^3,4. The idea has evolved in the two-domain scenario.

Lake and Woese fought fiercely for their competing models, culminating in a legendary scream match in the mid-1980s. Subsequently, Woese "did not want to meet Jim Lake," says microbiologist Patrick Forterre at the same time. Institut Pasteur in Paris. Lake does not dispute acrimony. "It was really a debate and there was a lot of politics," he says. Woese died in 2012.

Today, the discussion on the origin of eukaryotes has matured. Many people on both sides agree that the origin of eukaryotes probably involved a stage called endosymbiosis. This theory, defended by the late biologist Lynn Margulis, argues that a simple host cell alive a lifetime ago swallowed a bacterium and that both have formed a mutually beneficial relationship. These captive bacteria eventually evolved into mitochondria – the cellular substructures that produce energy – and the hybrid cells became what are now called eukaryotes.

The nature of the engulfing cell is the place where the two sides diverge. As adherents of the three domains say, the engulfer was an ancestral microbe, now extinct. According to Forterre, it was a "proto-eukaryote" – "neither a modern archeon nor a modern eukaryote". In this model, there were several major splits at the beginning of the evolution. The first was produced billions of years ago, when primitive organisms gave birth to both a bacterium and a group of extinct microbes. This latter group diverged in Archaea and the group that became eukaryotic.

In the two-domain world, however, a primitive organism has given rise to bacteria and archaea. And the body that finally swallowed the fateful bacteria was an archeon. This would make all eukaryotes a sort of archaic branch that surpasses – or, as some scientists call it, a "secondary domain" (see "Domains under discussion").

Scrambled messages

Without a machine to trace germs, sorting these hypotheses is extremely difficult. The fossil record of early eukaryotes is rare and the examples can be impenetrable. Scientists must instead rely on the documents inscribed in the genomes of modern organisms, themselves scrambled by the passage of time. "We are trying to solve something that happened probably a few billion years ago, using modern sequence data," says computer evolution biologist Tom Williams. from the University of Bristol, UK. This is not an easy task.

Current gene sequencing technologies have advanced the debate. Until recently, scientists who sought to identify bacteria or archaea in a particular habitat had to grow organisms in the laboratory. Researchers can now assess microbial diversity in a water or soil sample by capturing DNA and analyzing it using mathematical tools, a technique called meta-genomics. In 2002, scientists knew two categories (or phyla) of archaea. Today, thanks to metagenomics, the number of groups has exploded.

Evolutionary scientists quickly took advantage of the growing wealth. Using the latest powerful modeling techniques, they created an evolutionary tree forest detailing family relationships between archaea. The results, in many cases, place eukaryotes in the archaic ranks.

"In our opinion, the weight of evidence has actually shifted to a two-domain tree, the ocyte," Williams says. But for some, the debate was still brief on the data.

Then, in 2015, a group led by Thijs Ettema, an evolution microbiologist from the University of Uppsala in Sweden at the time, published DNA sequences of Lokiarchaeota, found in sediment dredged five years earlier.¹. In two years, the Etema team and other researchers announced the discovery of three new Archaean phyla linked to Lokis.^5,6. The set of new phyla was named Asgard after the kingdom of the Nordic gods.

The Asgard arches are small, but they have proved powerful. They relaunched the debate on the real number of areas of life. And they provide enticing insights into the nature of the cells that gave birth to early eukaryotes – at least to supporters of two domains.

As their namesake, Lokiarchaeota and their parents escape the easy description. They are undoubtedly archaea, but their genomes include a set of genes similar to some genes found in eukaryotes. Loki DNA, for example, contains genetic instructions for actins, proteins that form a skeletal-like framework in eukaryotic cells. The genes seemed so out of place that the researcher who spotted them was worried about the contamination. "I said, Hmm, how is that possible? Is it possible that it really acts from an archaic genome? ", Recalls Anja Spang, microbiologist of evolution, from the Royal Netherlands Institute for Research on the Sea in Texel.

Evolutionary modeling has reinforced the close connection between Asgard archaea and eukaryotes. The trees built by the Ettema team place all eukaryotes in the Asgard group.^5,7.

Today, many researchers are using data from these archaea to formulate a better picture of the eukaryotic precursor. He may have had some typical eukaryotic features before taking the mitochondrial predecessor. "There were probably very primitive membrane biology processes going on," says Ettema.

According to an analysis published this year⁷, the ancestor of Asgard Archaea is probably fed with carbon-based molecules, such as fatty acids and butane. This diet would have generated by-products that could feed the partner bacteria. Such food-sharing agreements – common to microbes – could have evolved into a more intimate relationship. An archeon could be snuggled next to his bacterial partner to facilitate nutrient exchanges, ultimately leading to the ultimate hug.

However, such scenarios still raise doubts. The main among those who are not convinced is Forterre. After reviewing the newspaper Asgard, he and his colleagues published an exhaustive rebuttal⁸ work.

Deceptive markers?

In a charge that infuriates Ettema, Forterre and his group suggested that some eukaryotic-type sequences found in Lokis were the result of contamination. A Loki protein called elongation factor 2, for example, was "probably contaminated with eukaryotic sequences," the Forterre team wrote in his review. Forterre now says he is uncertain about it.

But he and his colleagues continue to criticize the evolving Asgard trees. Even master tree builders recognize that it is difficult to understand the links that united living organisms two billion years ago. Biologists reconstruct these relationships by modeling how a particular "marker" – usually a protein or gene – has changed over time in organisms of interest.

The Forterre group says the Ettema team has unwittingly chosen deceptive markers to build their tree. Forterre and his group performed their own tree analysis using two large proteins as markers because, because of their size, large proteins are more likely to record the desired information. The result was a tree of three domains.

Ettema says the two beacons used by Forterre are insufficient to track events that occurred so long ago – a criticism echoed by other scientists. And when Etema's team tried to replicate Forterre's discovery, even with the two proteins used, the result was still a two-domain tree, he says. Ettema did not publish the results.

Ettema attributes some differences to the disciplinary history. "Patrick Forterre is a brilliant scientist in his field," he says, but with the Lokis, "he has gone a little beyond his expertise." Forterre says he has phylogenetic skills and his co-authors have more.

Nevertheless, all the two-domain fans do not give up the Forterre trees. Williams, for example, builds a tree using the latest analysis tools and incorporates new varieties of araceae. He hopes this effort will help him understand some of Forterre's results.

Norm Pace, of the University of Colorado, Boulder, a microbiologist at the University of Colorado, has also developed some of the essential methods for placing microbes on the tree of life. Pace says that over a very long time, some markers will undergo difficult changes to follow. Ettema and others use statistical methods to account for this stealthy change, but Pace rejects them. "Ettema and his colleagues say they can calculate invisible changes. I claim that it's a stupid hypothesis, "says Pace. But the methods are widely used. Ettema takes into account the fact that scientists can use various tests to determine if such changes affect their data.

Other scientists reserve their judgment: "Trees change," is a common refrain. Keeling says that he is "totally on the fence". And Hugenholtz agrees that "the jury is absent," although both scientists say they think evidence for two areas is growing.

While waiting for tree rustling to take hold, researchers are turning to other sources of data that may support a two-domain tree. Bacteria and eukaryotes have a set of lipids in their cell membranes, while archaeal membranes contain a different set. A mixture of both has been thought to be unstable. This "lipid fracture" was a sore point for proponents of both fields, because it implies that if eukaryotes came from archaea, they would have had to abandon the use of archaeal lipids to produce bacterial versions.

But the lipid fracture is not so important anymore. Last year, Dutch researchers succeeded in developing bacteria with cell membranes containing both archaeal and bacterial lipids.⁹. Scientists have also discovered bacteria in the Black Sea with genes for making both types of lipids.^ten. According to microbiologist Laura Villanueva, of the Royal Netherlands Institute for Research at Sea, a member of the team that studied Black Sea bacteria, microbes could have introduced such membranes during the transition of archaic to eukaryotes.

However, Asgard archers' analyzes, including Lokis, remain limited. "What people really expect, is the isolation of a member of these lineages," says Simonetta Gribaldo, an evolution microbiologist at the Pasteur Institute. "We have to catch them, we have to cultivate them."

Some have a slow metabolism and are slow to multiply – "exactly what you do not want if you try to grow an organism," says Ettema. Only a few scientists even admit to having tried. The microbiologist Christa Schleper of the University of Vienna, who is trying to cultivate the Asgard, calls this project "the craziest project for which I asked for money".

As elusive as microbes may be, a team has captured what it claims to be the first images of Asgard organisms. The images of one type show rounded cells, each containing a cluster of compacted DNA that resembles the characteristic that defines all eukaryotes, a nucleus. According to the microbiologist Rohit Ghai of the Biology Center of the Czech Academy of Science of České Budějovice, the images are "intriguing" but are not conclusive. He is co-author of the pre-print containing the images.¹¹.

The overall picture is not yet clear. In Nordic legends, Loki often wreaks havoc – and then puts everything back in order. While the Lokiarchaeota and their relatives are emerging from the shadows, supporters of two areas wish that they settle the longstanding debate about the origin of a complex life. But it could take a while. "When we discovered the Asgard archeology, we thought it would convince everyone," laughs Spang. "This was not the case."