Rare protozoan from sludge in Norwegian lake does not fit on main branches of tree of life

Biologists all over the world have been eagerly awaiting the results of the genetic analysis of one of the world's smallest known species, hereafter called the protozoan, from a little lake 30 kilometer south of Oslo in Norway.

When researchers from the University of Oslo, Norway compared its genes with all other known species in the world, they saw that the protozoan did not fit on any of the main branches of the tree of life. The protozoan is not a fungus, alga, parasite, plant or animal.

"We have found an unknown branch of the tree of life that lives in this lake. It is unique! So far we know of no other group of organisms that descend from closer to the roots of the tree of life than this species. It can be used as a telescope into the primordial micro-cosmos," says an enthusiastic associate professor, Kamran Shalchian-Tabrizi, head of the Microbial Evolution Research Group (MERG) at the University of Oslo.

His research group studies tiny organisms hoping to find answers to large, biological questions within ecology and evolutionary biology, and works across such different fields as biology, genetics, bioinformatics, molecular biology and statistics.

World's oldest creature

Life on Earth can be divided up into two main groups of species, prokaryotes and eukaryotes. The prokaryote species, such as bacteria, are the simplest form of living organisms on Earth. They have no membrane inside their cell and therefore no real cell nucleus. Eukaryote species, such as animals and humankind, plants, fungi and algae, on the other hand do.

The family tree of the protozoan from the lake near Ås starts at the root of the eukaryote species.

"The micro-organism is among the oldest, currently living eukaryote organisms we know of. It evolved around one billion years ago, plus or minus a few hundred million years. It gives us a better understanding of what early life on Earth looked like.," Kamran says to the research magazine Apollon.

How they move

The tree of life can be divided into organisms with one or two flagella. Flagella are important when it comes to a cell's ability to move. Just like all other mammals, human sperm cells have only one flagellum. Therefore, humankind belongs to the same single flagellum group as fungi and amoebae.

On the other hand it is believed that our distant relatives from the family branches of plants, algae and excavates (single-celled parasites) originally had two flagella.

The protozoan from Ås has four flagella. The family it belongs to is somewhere between excavates, the oldest group with two flagella, and some amoebae, which is the oldest group with only one flagellum.

"Were we to reconstruct the oldest, eukaryote cell in the world, we believe it would resemble our species. To calculate how much our species has changed since primordial times, we have to compare its genes with its nearest relatives, amoebae and excavates," says Shalchian-Tabrizi.

Caught with a tasty morsel

The protozoan is not easy to spot. It lives down in the sludge at the bottom of a lake.

It is 30 to 50 micrometres long and can only be seen with a microscope. When Professor Dag Klaveness of MERG wants to catch the protozoan he sticks a pipe down into the lakebed, removes a column of sludge and pours a bile green algae mixture over it.

The algae are such tempting morsels for the small protozoa that they swim up.

"We can then pick them out, one by one, with a pipette," says Klaveness.

There are not many of them. And the University of Oslo biologists have not found them anywhere else other than in this lake.

"We are surprised. Enormous quantities of environmental samples are taken all over the world. We have searched for the species in every existing DNA database, but have only found a partial match with a gene sequence in Tibet. So it is conceivable that only a few other species exist in this family branch of the tree of life, which has survived all the many hundreds of millions of years since the eukaryote species appeared on Earth for the first time."

Not very sociable

The protozoan lives off algae, but the researchers still do not know what eats the protozoan. Nor do they know anything about its life cycle. But one thing is certain:

"They are not sociable creatures. They flourish best alone. Once they have eaten the food, cannibalism is the order of the day," notes Klaveness.

The protozoan has a special cell indentation. It looks like a groove.

"The species has the same intracellular structure as excavates. And it uses the same protuberances as amoebae to catch its food. This means that the species combines two characteristics from each family branch of the main eukaryote groups. This further supports the hypothesis that the species from this lake belongs to a primordial group. Perhaps it descended from the antecedents of both the excavates and amoeba?" asks Shalchian-Tabrizi.

The protozoan was discovered as early as 1865, but it is only now that, thanks to very advanced genetic analyses, researchers understand how important the species is to the history of life on Earth.

Breeding enormous quantities of the protozoan

Dag Klaveness has, together with research fellow Jon Bråte, managed to breed large quantities of the species. No one has done this before. Klaveness has spent the last 40 years specialising in breeding organisms that are difficult to breed or that are difficult to isolate from other species.

Breeding is important if we want to analyse the creature's genes. More than just a few are needed for a genetic test. Researchers have needed to breed large quantities. The work is demanding and has taken many months.

The protozoan's favourite food is green algae, but since both the protozoan and the green algae are eukaryote species, i.e. species with real cell nuclei, it is easy to confuse the genes of the protozoan with those of its food in the gene sequencing. Therefore, Klaveness has chosen to feed the protozoan with blue green bacteria, which are genetically very different to the protozoan. Blue green bacteria are not exactly its favourite dish, but the protozoan can only choose between eating or dying.

Blue green bacteria are prokaryotes, i.e. species without membranes or real cell nuclei. This allows the researchers to differentiate between the genes of the protozoan and its food in the gene sequencing.

Klaveness has a number of vats of the protozoan in the laboratory. The algae mixture sinks to the bottom. The protozoan dives down when it wants to eat.

In optimum conditions they divide every second day. However, with blue green bacteria on the menu, which is just as boring as if you only got carrots for several months and nothing else, the protozoan grows much more slowly.

When the protozoa have reproduced enough, they are centrifuged out and gene sequenced. The genes are then compared with equivalent gene sequences from other species. "We have gene sequenced 300,000 parts of the genome (the total genetic material), but we still do not know how large the genome is. We are currently only looking for the most important parts," explains Kamran Shalchian-Tabrizi.

Traces from primordial times

The problem is that DNA sequences change a lot over time. Parts of the DNA may have been wiped away during the passing of the years. Since the protozoan is a very old species, an extra large amount of gene information is required.

"It is often the case with such ancient organisms that features they share in common with other known species have been wiped away from the DNA sequence because of long-term mutations. You can compare it with tarmacing. If you tarmac a road enough times, you will no longer see the cobblestones. Therefore, you have to collect large gene sequences to find common traces from prehistoric times."

Research fellow Sen Zhao was responsible for the extensive, statistical calculations. In order to calculate the family link they have used information from the research group's own Bioportal in cooperation with the high performance computing group at the University of Oslo.

Resolving evolutionary mysteries

Kamran Shalchian-Tabrizi explains that the tree of life can provide fundamental answers to great evolutionary mysteries.

"In order to understand what a species is today, we have to understand how they have changed genetically. The tree of life allows us to explain cellular change processes by connecting the genome and morphology (appearance) with its way of life."

Among other things, Shalchian-Tabrizi wants to use the protozoan to investigate when photosynthesis arose among eukaryote organisms. Photosynthesis takes place in chloroplast.

Chloroplasts were originally free-living, blue green bacteria. If the researchers find genetic residues of these bacteria in the protozoan from Ås, this may indicate that photosynthesis arose earlier than supposed.

"There are many likely scenarios, but we still do not know the answer," acknowledges Shalchian-Tabrizi.

The researchers also want to question when other characteristics arose, e.g. mitochondria, which are the energy motors of our cells.

Purifying drinking water in Japan

In recent years researchers have found some apparently matching examples of the protozoan from Ås in Japan and South East Asia. A researcher from Japan arrived in Oslo with a glass of the species solely so that Klaveness could breed them.

"We are now going to gene sequence these organisms, because it is not certain that the genes are the same, even if the morphology is similar," says Klaveness.

The Japanese hope that the protozoan can be used to purify drinking water by removing toxic, blue green bacteria.