Decoding the world’s largest animal genome

Let's travel back through time! We are in the Devonian period, around 420-360 million years ago. In a shallow shore area, something happened that would change life on our planet forever: a fish from the group of carnivorous finfish used its powerful, paired fins to pull itself out of the shallow water onto land and move its body across the muddy shore floor. It is in no hurry to return to the water. It can easily stay in the air because this fish already has lungs - just like us land vertebrates today.

This or something similar could have been the first landfall of a vertebrate - and thus one of the most important events in evolution. After all, all later land vertebrates - the so-called tetrapods - can be traced back to a fish. In addition to amphibians, reptiles and birds, these also include mammals, including humans. But one mystery remains: How was it that the fish of the carnivorous lineage were so well prepared for the conquest of the mainland?

A look at the living relatives

In order to find the solution to this mystery after all this time, the genetic material of the closest relatives of our Devonian ancestor that still exist today has now been analysed in order to draw conclusions about its appearance. Only three lines of these closest relatives - the lungfish - are still alive today: one in Africa, one in South America and one in Australia. Evolution seems to have forgotten them, because these ancient "living fossils" still look very much like their ancestors. As our genetic material, DNA, is made up of nucleobases, the sequence of which contains the actual genetic information, a comparative analysis of the lungfish genomes requires knowledge of their complete sequences.

It was already known that the genomes of lungfish are huge, but how gigantic they really are and what can be learnt from them was not yet clear. Accordingly, the sequencing of lungfish genomes was technically and bioinformatically very complex and complicated. However, an international research team led by Constance biologist Axel Meyer and Würzburg biochemist Manfred Schartl has now succeeded in completely sequencing the genome of the South American lungfish and an African representative. The previously largest genome sequence of the only Australian representative(Neoceratodus) had already been sequenced by the same team. The results of the current work were published in the scientific journal Nature.

Contribution of the LIB

Alexander Suh, Head of the Centre for Molecular Biodiversity Research at the LIB, contributed his expertise in the identification and classification of transposons to this study. These DNA segments can multiply in the genome and thus increase genome growth. "We not only found a large number of transposons in the lungfish, but also an astonishingly high diversity of different transposons," emphasises Alexander Suh. "Different transposons can replicate in different ways and have different effects on each other and on neighbouring genes. Therefore, this result illustrates the complexity of the gigantic genomes of lungfish."

Iker Irisarri, head of the Phylogenomics Department at the LIB, was involved in inferring the evolutionary relationships and divergence times between all major vertebrate strains, including all three living lungfish strains. This was the first time that only fully sequenced genomes were used in such analyses, allowing much more accurate conclusions to be drawn. The evolutionary reconstructions carried out by Iker Irisarri also show that the gigantic genomes of the lungfish began to expand more than 200 million years ago, even before the ancestor of all three lungfish living today. Genome expansion accelerated further in the lineage that led to the South American lungfish, which had the greatest rate of genome expansion ever recorded: the equivalent of a complete human genome every ten million years. Interestingly, these results are mirrored in the reconstruction of cell size evolution from the lungfish fossil record. "Considering the close relationship between cell and genome size, this coincidence is not surprising," explains Iker Irisarri. "Nevertheless, it is exciting to see how two completely independent data sources - the genome sizes determined by sequencing and the cell sizes measured from fossils - agree so well."

Very, very large - but why?

The genetic material of the South American lungfish in particular breaks all records in terms of size: "At over 90 gigabases (i.e. 90 billion bases), the DNA of the South American representative is the largest of all animal genomes and more than twice as large as the genome of the previous record holder, the Australian lungfish. 18 of the 19 chromosomes of the South American lungfish alone are each larger than the entire human genome with its almost three billion bases," says Meyer. So-called autonomous transposons are responsible for the fact that the lungfish genome has grown to this enormous size over time. These are DNA segments that "multiply" and then change their position in the genome - which in turn causes the genome to grow.

Although this also happens in other organisms, the research team's analyses have shown that the expansion rate of the genome of the South American lungfish is by far the fastest known: in the past, its genome has grown by the size of the entire human genome every ten million years. "And it continues to grow," reports Meyer. "We have found evidence that the transposons responsible are still active." They also discovered the mechanism for this gigantic genome growth: the extreme expansion is at least partly due to very low piRNA concentrations. This type of RNA is part of a molecular mechanism that normally limits the spread of transposons.

Surprisingly stable despite everything

Because transposons multiply, jump around in the genome and thus contribute to its growth, they can greatly alter and destabilise the genetic material of an organism. This is not always detrimental and can even be an important driving force of evolution. Sometimes these "jumping genes" also cause evolutionary innovations because they can cause changes in gene functions. It is therefore all the more surprising that the current study found no correlation between the enormous transposon excess and genome instability - the genome of the lungfish is unexpectedly stable and the gene arrangement is surprisingly conservative. This fact allowed the research team to reconstruct the original architecture of the chromosome set (karyotype) of the ancestral tetrapod from the sequences of the lungfish species still living today.

In addition, the comparison of the lungfish genomes enabled them to draw conclusions about the genetic basis of differences between the representatives still living today. The Australian lungfish, for example, still has the limb-like fins that once allowed its relatives to move on land. In today's other lungfish representatives from Africa and South America, these fins, which are similar in bone structure to our arms, have evolved back into filamentous fins over the last 100 million years or so. "In our study, we were also able to use experiments with CRISPR-Cas transgenic mice to reveal that this simplification of the fins is due to a change in the so-called shh signalling pathway," says Meyer.

During embryonic development in mice, for example, the shh signalling pathway controls the number and development of the fingers, among other things. The study results thus provide additional evidence for the evolutionary connection between the fin rays of bony fish and the fingers of terrestrial vertebrates. As scientists now have the complete genome sequences of all current lungfish families at their disposal thanks to the new study, genetic comparative studies such as this one will provide further insights into the carnivorous ancestors of land vertebrates in the future - and thus help to solve the puzzle of vertebrate landwalking.