Scientists searching for genetic clues to vertebrate evolution have long been fascinated by the tiny marine stickleback fish, known for its ability to adapt and thrive in salty oceans or freshwater streams around the world. Now, a team of researchers from the Broad Institute and Stanford University has analyzed the entire genetic sequence of 21 threespine sticklebacks and identified which regions of the genome regulate their ability to adapt to such markedly different environments.

"Sticklebacks are essentially the 'Darwin's finches' of the sea - their adaptation to novel environments is clearly visible and is easily readable in the genome," said Kerstin Lindblad-Toh, co-senior author of the paper and the scientific director of vertebrate genome biology at the Broad Institute.

"And even though the exact genetic mutations will not translate from one species to the next, this work helps us develop an understanding of pathways that govern how organisms take shape." Lindblad-Toh is also a professor in comparative genomics at Uppsala University and director of Science for Life Laboratory Uppsala, a strategic research center.

Researchers identified 147 regions that varied consistently in marine-freshwater evolution. And while evolutionary differences depend on both protein-coding and regulatory changes in the genome, regulatory changes predominate.

"This work is exciting because it tells us what you would expect: Molecular adaptation happens largely by genetic regulation in the non-coding region of the genome," Lindblad-Toh said. Only about 1.5% of the genome codes for some 20,000 proteins. The function of non-coding regions - the so-called "dark matter" of the genome - is not fully understood.

"The fact that regulatory changes appear to predominate in adaptive evolution makes sense," she added. "If a protein was completely changed, it would probably have a different function in many different cell types - and that could be detrimental to the fish rather than help it adapt to a new environment."

When Charles Darwin was compiling his research on natural selection and evolution, he observed that closely related groups of finches in the Galapagos exhibited differences in beak structure, an adaptation to different climate conditions and food sources on different islands in the archipelago.

Much like Darwin's finches, the sticklebacks exhibit different traits in different environments - researchers found changes in outward appearance, skeletal structure, behavior, and metabolism. Saltwater sticklebacks are larger and plated with scaly armor and spikes.

The freshwater fish are smaller, faster, and sport less armor. These adaptations happened over a period of 10,000 years or so - an eyeblink in evolutionary time - after the glaciers of the Pleistocene era retreated, trapping some saltwater sticklebacks in freshwater lakes and streams created by the melting ice.

"We studied sticklebacks because you can see the timing of the adaptation very clearly, over 10,000 years instead of millions of years," Lindblad-Toh said. "You see signatures that tell you very specifically where in the genome the adaptive pressure has been acting."

For the study, the researchers picked one stickleback to serve as a reference genome for the entire species, sequencing the entire genome of a female from Bear Paw Lake in Alaska to nine-fold coverage for accuracy. Researchers then sequenced the genomes of 10 pairs of marine and freshwater fish from different waters all over the world.

"One of the great things about genomics in general is that by decoding the sequence of an organism, it becomes much easier to study a whole range of interesting problems, including what makes an animal look a particular way, evolve particular behaviors, cope with particular environments or adapt to different food sources, predators and diseases," said David Kingsley, a professor of developmental biology at Stanford University School of Medicine and co-senior author.

"With this paper, researchers will not only have access to a high-quality reference sequence, but that sequence will be annotated with information about what regions are important in particular environments, and what kind of changes are involved in the evolution of various traits. It's a big leap forward for the field."

Because most of the 147 regions identified in the study were small - less than 5,000 base pairs of DNA - scientists could pinpoint which genes or regulatory regions are affected. The researchers found that the 147 regions are not randomly located throughout the genome.

"Some sections of chromosomes are chock full of differences that are contributing to evolution," said Kingsley. "It is quite dramatic to look at the entire genome and see these powerful chunks being used over and over again."

The identification of locations that were used repeatedly in stickleback adaptation provided a rare opportunity to estimate the contribution made by protein-coding changes in the genome versus regulatory changes. Slightly more than 80% of the changes involved regulatory regions that control how genes are expressed.

"This paper puts the pieces together in a big way," said Federica Di Palma, assistant director of vertebrate genome biology at the Broad. "This study will likely reveal that genes controlling diversity and adaptation in sticklebacks also have relevance in human adaptation and disease.

For example, the same Ectodysplasin signaling pathway responsible for armor loss in freshwater sticklebacks also underlies human developmental disorders affecting hair, teeth, and exocrine glands. Trends are also emerging suggesting that we are more likely to find regulatory changes in these genes with complex expression patterns and functions."

The research team plans to broaden the study of stickleback populations, and has already selected another 200 fish to sequence in order to examine other traits, such as changes in behavior.

The findings appear in the April 5 issue of Nature.