From Howard Hughes Medical Institute

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Wild populations of stickleback fish have evolved major changes in bony armor styles (shaded) in marine and freshwater environments. New research shows that this evolutionary shift occurs over and over again by increasing the frequency of a rare genetic variant in a single gene.

In a stunning example of evolution at work, scientists have now found that changes in a single gene can produce major changes in the skeletal armor of fish living in the wild.

The surprising results, bring new data to long-standing debates about how evolution occurs in natural habitats.

“This is one of the first cases in vertebrates where it’s been possible to track down the genetic mechanism that controls a dramatic change in skeletal pattern, a change that occurs naturally in the wild.”
David M. Kingsley

“Our motivation is to try to understand how new animal types evolve in nature,” said molecular geneticist David M. Kingsley, a Howard Hughes Medical Institute investigator at the Stanford University School of Medicine. “People have been interested in whether a few genes are involved, or whether changes in many different genes are required to produce major changes in wild populations.”

The answer, based on new research, is that evolution can occur quickly, with just a few genes changing slightly, allowing newcomers to adapt and populate new and different environments.

In collaboration with zoologist Dolph Schluter, at the University of British Columbia, Vancouver, Canada, and Rick Myers and colleagues at Stanford, Kingsley and graduate student Pamela F. Colosimo focused on a well-studied little fish called the stickleback. The fish—with three bony spines poking up from their backs—live both in the seas and in coastal fresh water habitats all around the northern hemisphere.

Sticklebacks are enormously varied, so much so that in the 19th century naturalists had counted about 50 different species. But since then, biologists have realized most populations are recent descendants of marine sticklebacks. Marine fish colonized new freshwater lakes and streams when the last ice age ended 10,000 to 15,000 years ago. Then they evolved along separate paths, each adapting to the unique environments created by large scale climate change.

“There are really dramatic morphological and physiological adaptations” to the new environments, Kingsley said.

For example, “sticklebacks vary in size and color, reproductive behavior, in skeletal morphology, in jaws and teeth, in the ability to tolerate salt and different temperatures at different latitudes,” he said.

Kingsley, Schluter and their co-workers picked one trait—the fish’s armor plating—on which to focus intense research, using the armor as a marker to see how evolution occurred. Sticklebacks that still live in the oceans are virtually covered, from head to tail, with bony plates that offer protection. In contrast, some freshwater sticklebacks have evolved to have almost no body armor.

“It’s rather like a military decision, to be either heavily armored and slow, or to be lightly armored and fast,” Kingsley said. “Now, in countless lakes and streams around the world these low-armored types have evolved over and over again. It’s one of the oldest and most characteristic differences between stickleback forms. It’s a dramatic change: a row of 35 armor plates turning into a small handful of plates – or even no plates at all.”

Using genetic crosses between armored and unarmored fish from wild populations, the research team found that one gene is what makes the difference.

“Now, for the first time, we’ve been able to identify the actual gene that is controlling this trait,” the armor-plating on the stickleback, Kingsley said

The gene they identified is called Eda , originally named after a human genetic disorder associated with the ectodysplasin pathway, an important part of the embryonic development process. The human disorder, one of the earliest ones studied, is called ectodermal dysplasia.

“It’s a famous old syndrome,” Kingsley said. “Charles Darwin talked about it. It’s a simple Mendelian trait that controls formation of hair, teeth and sweat glands. Darwin talked about `the toothless men of Sind,’ a pedigree (in India) that was striking because many of the men were missing their hair, had very few teeth, and couldn’t sweat in hot weather. It’s a very unusual constellation of symptoms, and is passed as a unit through families.”

Research had already shown that the Eda gene makes a protein, a signaling molecule called ectodermal dysplasin. This molecule is expressed in ectodermal tissue during development and instructs certain cells to form teeth, hair and sweat glands. It also seems to control the shape of – bones in the forehead and nose.

Now, Kingsley said, “it turns out that armor plate patterns in the fish are controlled by the same gene that creates this clinical disease in humans. And this finding is related to the old argument whether Nature can use the same genes and create other traits in other animals.”

Ordinarily, “you wouldn’t look at that gene and say it’s an obvious candidate for dramatically changing skeletal structures in wild animals that end up completely viable and healthy,’ he said. ” Eda gene mutations cause a disease in humans, but not in the fish. So this is the first time mutations have been found in this gene that are not associated with a clinical syndrome. Instead, they cause evolution of a new phenotype in natural populations.”

The research with the wild fish also shows that the same gene is used whenever the low armor trait evolves. “We used sequencing studies to compare the molecular basis of this trait across the northern hemisphere,” said Kingsley. “It doesn’t matter where we look, on the Pacific coast, the East coast, in Iceland, everywhere. When these fish evolve this low-armored state they are using the same genetic mechanism. It’s happening over and over again. It makes them more fit in all these different locations.”

Because this trait evolves so rapidly after ocean fish colonize new environments, he added, “we wondered whether the genetic variant (the mutant gene) that controls this trait might still exist in the ocean fish. So we collected large numbers of ocean fish with complete armor, and we found a very low level of this genetic variant in the marine population.”

So, he said, “the marine fish actually carry the genes for this alternative state, but at such a low level it is never seen;” all the ocean fish remain well-armored. “But they do have this silent gene that allows this alternative form to emerge if the fish colonize a new freshwater location.”

Also, comparing what happens to the ectodysplasin signaling molecule when its gene is mutated in humans, and in fish, shows a major difference. The human protein suffers “a huge amount of molecular lesions, including deletions, mutations, many types of lesions that would inactivate the protein,” Kingsley said.

But in contrast, “in the fish we don’t see any mutations that would clearly destroy the protein.” There are some very minor changes in many populations, but these changes do not affect key parts of the molecule. In addition, one population in Japan used the same gene to evolve low armor, but has no changes at all in the protein coding region. Instead, Kingsley said, “the mutations that we have found are, we think, in the (gene’s) control regions, which turns the gene on and off on cue.” So it seems that evolution of the fish is based on how the Eda gene is used; how, when and where it is activated during embryonic growth.

Also, to be sure they’re working with the correct gene, the research team used genetic engineering techniques to insert the armor-controlling gene into fish “that are normally missing their armor plates. And that puts the plates back on the sides of the fish,” Kingsley said.

“So, this is one of the first cases in vertebrates where it’s been possible to track down the genetic mechanism that controls a dramatic change in skeletal pattern, a change that occurs naturally in the wild,” he noted.

“And it turns out that the mechanisms are surprisingly simple. Instead of killing the protein (with mutations), you merely adjust the way it is normally regulated. That allows you to make a major change in a particular body region – and produces a new type of body armor without otherwise harming the fish.”

Image: David Kingsley, HHMI at Stanford University, modified from Cuvier (1829).

From Howard Hughes Medical Institute

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Ocean sticklebacks are dark colored fish that often migrate into new environments. Multiple stickleback populations have evolved lighter gill and skin colors following colonization of new lakes and streams at the end of the last ice age. Ocean (upper) compared to freshwater creek (lower) sticklebacks, both collected near Vancouver, British Columbia. Scientists have identified a genetic change controlling rapid evolution of skin color in fish, and shown that the same mechanism also contributes to recent evolution of skin color in humans.

When humans began to migrate out of Africa about 100,000 years ago, their skin color gradually changed to adapt to their new environments. And when the last Ice Age ended about 10,000 years ago, marine ancestors of ocean-dwelling stickleback fish experienced dramatic changes in skin coloring as they colonized newly formed lakes and streams. New research shows that despite the vast evolutionary gulf between humans and the three-spined stickleback fish, the two species have adopted a common genetic strategy to acquire the skin pigmentation that would help each species thrive in their new environments.

The researchers, led by Howard Hughes Medical Institute investigator David Kingsley, published their findings in the December 14, 2007, issue of the journal Cell. Kingsley and first author Craig Miller are at the Stanford University School of Medicine, and other co-authors are from the University of Porto in Portugal, the University of British Columbia, the University of Chicago, and the Pennsylvania State University Further studies of stickleback, they say, may reveal other malleable pieces of genetic machinery both fish and humans have used for adaptation.

“The genetic mechanisms that can produce these changes may be so constrained that evolution will tend to use the same sorts of genes in different organisms.”
David M. Kingsley

The stickleback has become a premier model organism for studying evolution because of its extraordinary evolutionary history, said Kingsley. “Sticklebacks have undergone one of the most recent and dramatic evolutionary radiations on earth,” he said. When the last Ice Age ended, giant glaciers melted and created thousands of lakes and streams in North America, Europe, and Asia. These waters were colonized by the stickleback’s marine ancestors, which subsequently adapted to life in freshwater. “This created a multitude of little evolutionary experiments, in which these isolated populations of fish adapted to the new food sources, predators, water color, and water temperature that they found in these new environments,” Kingsley explained.

Among those adaptations were new colorations that helped the fish camouflage themselves, distinguish species, and attract mates in their new environments. Until now, however, scientists had not understood what genetic factors drove the changes in skin pigmentation.

Human populations have also undergone pigmentation changes as they have adapted to life in new environments. The ecological reasons for those changes may be quite different from the forces driving the evolution of pigmentation in sticklebacks, said Kingsley. As human populations migrated out of Africa into northern climates, the need for darker pigmentation necessary to protect against the intense tropical sun diminished. With skin that was more transparent to sunlight, humans were better able to produce sufficient vitamin D in their new climate.

To begin to understand the genetic basis of skin pigmentation changes in fish, Kingsley and his colleagues crossed stickleback species that had different pigmentation patterns and used genetic markers and the recently completed sequence map of the fish’s genome to search for the mechanism regulating stickleback pigmentation. They searched for chromosome segments in the offspring that were always associated with inheritance of dark or light gills and skin. Through detailed mapping of one such segment, Kingsley and his colleagues found that a gene called Kitlg (short for “Kit ligand”) was associated with pigmentation inheritance. Kitlg was an excellent candidate for regulating pigmentation because mutant forms of the corresponding gene in mice produce changes in fur color, said Kingsley.

The Kitlg gene is involved in a variety of biological processes, including germ cell development, pigment cell development, and hematopoiesis. Light-colored fish have regulatory mutations that reduce expression of the Kitlg gene in gills and skin, but that do not reduce the gene’s function in other tissues. “By altering expression of this gene in one particular place in the body, the fish can fine tune the level of expression of that factor in some tissues but not others,” said Kingsley. “That lets evolution produce a big local effect on a trait like color while preserving the other functions of the gene.”

Humans also have a Kitlg gene, and Kingsley and his colleagues wondered if it played a role in regulating the pigmentation of human skin. One clue they had came from previous research by other groups that had revealed that the human Kitlg gene has undergone different changes among different human populations, suggesting that it is evolutionarily significant.

Kingsley and his colleagues tested whether the different human versions of the Kitlg gene are associated with changes in skin color. Humans with two copies of the African form of the Kitlg gene had darker skin color than people with one or two copies of the new Kitlg variant that is common in Europe and Asia.

“Although multiple chromosomal regions contribute to the complex trait of pigmentation in both fish and humans, we have identified one gene that plays a central role in color changes in both species,” said Kingsley.

“Since fish and humans look so different, people are often surprised that common mechanisms may extend across both organisms,” he said. “But there are real parallels between the evolutionary history of sticklebacks and humans. Sticklebacks migrated out of the ocean into new environments about ten thousand years ago. And they breed about once every one or two years, giving them five thousand to ten thousand generations to adapt to new environments.”

Although modern humans arose in Africa, they are thought to have migrated out of Africa in the last 100,000 years. “Humans breed about once every 20 years, giving them about 5,000 generations or so to emerge from an ancestral environment and colonize and adapt to new environments around the world,” Kingsley added. “So despite the difference in total years, the underlying process is actually quite similar. Whether it be fish or humans, there were small migrating populations encountering new environments and evolving significant changes in some traits in a relatively short time. And the genetic mechanisms that can produce these changes may be so constrained that evolution will tend to use the same sorts of genes in different organisms.”

Kingsley and his colleagues are now exploring the genetic basis of other evolved traits in the stickleback that could find a parallel in humans. “And given the degree to which evolutionary mechanisms appear to be shared between populations and organisms, we’re optimistic about finding the particular genes that underlie other recent adaptations to changing environments in both fish and humans,” he said.

Photo: Frank Chan, Craig Miller, and David Kingsley; Stanford University