A fish on its way to new shores

Ice Age bones reveal how sticklebacks adapt to new habitats

Three-spined sticklebacks live in both salt and fresh water. When the glaciers melted at the end of the last ice age and new lakes were formed, sticklebacks from the sea found new habitats in them. Felicity Jones and her team at the Friedrich Miescher Laboratory of the Max Planck Society in Tübingen are investigating how the genome of the fish changes in the course of adaptation. 12,000-year-old stickleback bones provide insights into the early stages of this process.

Text: Elke Maier

The story begins with a chance find: in spring 2018, a team of geologists from the Norwegian Geological Survey (NGU) set off on a journey to the far north of Norway. The aim of the expedition was to collect drill and collect cores of the sediments from lakes near the coast in order to get a picture of the sea level fluctuations at the end of the last ice age. When geologist Anders Romundset of NGU filtered the sediment samples through a fine sieve in the laboratory, they found not only algal and plant remains in them. The millimetre sized bones and spines of fish were also hanging in the mesh.

The bones were so well preserved that the scientists knew immediately what they were dealing with: the three-spined stickleback (Gasterosteus aculeatus). The four to six centimetre long spiny fish can still be found in the sea and in the lakes of Scandinavia. Using the radiocarbon method, the researchers determined an age of around 12,000 years for the bones. This means they came from the time when large parts of northern Europe were still covered by thick ice sheets. At this time, the lakes were just starting to form by transitioning from a marine bay, to isolated freshwater lake. The retreat of the glacial ice sheets at the end of the last ice age allowed the land to slowly rise up above sea-level – over time some marine bays became isolated from the ocean and filled with freshwater. Embedded in the sediment at the bottom of the lake, they had survived the millennia.

A hodgepodge of DNA

The geologists handed over their find to Andrew Foote, evolutionary ecologist at the Norwegian University of Science and Technology in Trondheim. Foote, an expert on ancient DNA, seized the opportunity. In a special laboratory at the University of Copenhagen, he and colleague Tom Gilbert set out to search for remnants of the genetic material in the bones. One of the difficulties was that the samples not only contained stickleback DNA, but also pieces of DNA from other organisms - plants and bacteria, for example, that had lived in the same environment at the time. In the end, the fragments they were looking for made up only one per cent of this hodgepodge. Nevertheless, Foote managed to fish out and sequence the stickleback DNA with great effort.

For Felicity Jones, research group leader at the Friedrich Miescher Laboratory in Tübingen, the success of her Norwegian collaboration partner Foote was a stroke of luck. Together with her team, the Australian is investigating the basics of evolution. The scientists want to find out which molecular mechanisms ensure that organisms can adapt to new habitats or even form new species. Sticklebacks are ideal as model organisms for this: over the course of the last 10000-20000 generations, marine forms have invaded and adapted to numerous and diverse freshwater habitats (lakes, streams, swamps). In the temperate climate zones of the northern hemisphere, the animals are common in many bodies of water.

"The exciting thing for us is that sticklebacks have colonised new freshwater habitats from the sea many times independently," says Felicity Jones, who has done research in Scotland, New Zealand and the USA before coming to Tübingen. "This allows us to investigate the same questions in several parallel systems and thus rule out the possibility that the adaptive mutations we find are merely one-off chance events."  As fish adapted to their new environment, very similar changes in their shape, behaviour or physiology occurred repeatedly at different sites - a process known as parallel evolution. As in a huge field laboratory, the researchers can therefore use the sticklebacks to work out the fundamental molecular mechanisms that play a role when organisms adapt to a new environment.

In his major work "On the Origin of Species", Charles Darwin provided the first plausible explanation of how the diversity of life arose more than 160 years ago. According to this explanation, all present-day species are descended from common ancestors whose descendants have, in the course of millions of years, distributed themselves among the various habitats and split up into different lineages. The driving force behind this development is natural selection: Of all the descendants of a living being, those that are best adapted to their environment produce more offspring. They pass on their heritable characteristics to more individuals in the next generation.

Better than Darwin's finches

A famous example of Darwin's theory are the finches of the Galapagos archipelago. Starting from one ancestral species, the birds on the different islands have produced quite different beak shapes, depending on what food they used. Such a split is what evolutionary biologists call an adaptive radiation. "Sticklebacks are another example, only much better," says Felicity Jones, laughing. Because unlike the Galapagos finches, they do researchers the favour of performing the evolutionary spectacle of adaptive divergence on several stages at once – providing biological replicates of the evolutionary process.

How the sticklebacks change in the course of adaptation is impressively shown in the formation of the bone plates on the sides of their bodies to protect themselves from predators. Individuals from the open sea, which can hardly hide from predators, rely on extensive armour plating for their defence. Their conspecifics in the lake, who find shelter in plant thickets, save themselves the material expense. They strip down and eventually have only a few bony plates at the front of their torso.

Evolutionary biology has made tremendous progress since Darwin's time. Thanks to modern analytical methods, researchers can now search the genome for the characteristic traces that evolution has left there. "Most of our understanding of how the genome functions comes from laboratory studies of inbred ‚model‘ organisms. In contrast, we know relatively little about how naturally occurring genetic differences influence the evolution of wild populations," says Felicity Jones.

Prehistoric bone find

The prehistoric bone find opened up completely new possibilities for the Tübingen researchers and their collaborators: For the first time, they had an ancestor of the freshwater stickleback in front of them, and this fish, which had lived around 12,000 years ago, also revealed genetic information. "To our knowledge, these are the oldest fish bones from which genome data have ever been obtained," says Felicity Jones. "They open a window into the past, so we can understand the type of genetic variation the animals carried when adapting to their new habitat."

The bony relics had been found in a sediment layer that marks the transition from salt to fresh water. The fish they came from had once lived in brackish water. When the glaciers melted towards the end of the Ice Age and a huge ice load fell away, the land mass gradually lifted. In the process, lakes near the coast were gradually separated from the sea. Sticklebacks, which were trapped in the isolated waters, managed to adapt to the new conditions and reproduce. In the different waters, many new types developed over time that differed from their relatives in the sea. From a purely external point of view, the fish varied in their body size and pigmentation, the length of their dorsal spines and the size and number of their bone plates.

To get an idea of what had changed at the genetic level, the researchers compared the genetic material of the Ice Age stickleback with that of its descendants. To do this, they analysed sticklebacks from two coastal lakes south of the town of Hammerfest. Ancient bones were found in both the lakes but the sequencing of ancient DNA was more successful for one lake than the other. In addition, the researchers also sequenced the genomes of marine sticklebacks from the same area.

Evolutionary building blocks

The comparison showed that the Ice Age fish was genetically very similar to its modern-day marine conspecifics: "The bones mainly contained gene variants that are advantageous for life in salt water," says Melanie Kirch, a doctoral student in Felicity Jones' research group, who analysed a large part of the genome data. However, variants were also found that already showed an adaptation to freshwater. Such gene variants are also found sporadically among today's marine sticklebacks. The researchers assume that the latter occasionally mate with conspecifics from freshwater, for example in the estuaries of rivers. As a result, freshwater gene variants repeatedly enter the marine population. For the marine sticklebacks, these variants are useless or even disadvantageous and therefore do not spread. When colonising new freshwater habitats, however, they prove to be wild cards: If evolution can access such ready-made building blocks, adaptation is possible in a very short time - in the case of sticklebacks within a few decades. If, on the other hand, the appropriate gene variants first have to arise by chance through mutation, millions of years can sometimes pass.

The genome comparison provided even more details on the development of freshwater sticklebacks. It showed that the fish from the two lakes were genetically less diverse than their ancestors from the sea. On the one hand, this was due to the fact that the isolated individuals that newly colonised the lakes at that time had only brought a small part of the gene variants with them that were found among the marine sticklebacks. On the other hand, some variants had disappeared from the gene pool in the newly established populations by chance over time - a process known as genetic drift. "By chance alone, even those variants that would be advantageous for life in freshwater were lost in this way," says Felicity Jones. Such severe genetic impoverishment, typical of small founder populations, is what biologists figuratively call a "genetic bottleneck".

Momentous bottleneck

For the freshwater sticklebacks, this bottleneck was momentous: genetic variation is the material from which evolution produces new adaptations. If many different gene variants are available, it can draw on the full resources. In the fish from the two lakes, however, the variation was greatly reduced: "We suspect they are not as well adapted to their habitat as they could be," Jones interprets the results.

In the future, Felicity Jones and her collaborator Andy Foote are enthusiastic about  further research on the evolutionary history of Scandinavia. One of their goals is to genetically evaluate even more prehistoric stickleback bones from younger sediment layers. "If we had bones not only from a single fish, but from several that lived hundreds of years apart, we could directly trace how the genome changed over time after the sticklebacks arrived in their new habitat," she says.