Tiny fish bares all: New insights on evolution from study of sticklebacks

Steve Fisch

David Kingsley and his colleagues have sequenced the genomes of 21 threespine sticklebacks, giving an unprecedented view of the species' genetic diversity.

To the uninitiated, the tiny threespine stickleback might look like nothing more than a scruffy anchovy with an attitude. But this tough little fish, with its characteristic finny mohawk, is a darling of evolutionary biologists.

That’s because it exhibits some of the most recent, and most dramatic, adaptive changes of any animal alive today. Flourishing in fresh water or salty, appearing ponderously armored or slippery sleek, light-skinned or dark, this plucky pisces has made itself so uniquely at home in countless lakes, streams and oceans that early naturalists initially classified it as more than 50 separate species. It’s the ultimate changeling.

Now researchers at the Stanford University School of Medicine and the Broad Institute have analyzed the whole-genome sequence of 21 threespine sticklebacks chosen from geographic locations around the world. The findings, which will appear in the April 5 issue of Nature, better identify which regions of the genome are responsible for the stickleback’s many variations.

The scientists found that the animals — despite their different haunts — repeatedly developed the same traits through changes in similar regions of their genomes. Specifically, the researchers identified 147 regions that varied consistently among freshwater and marine sticklebacks. About 80 percent of the changes involved regulatory regions of the genome that control when, where and how genes are expressed.

“This addresses a classic debate in evolutionary biology,” said professor of developmental biology David Kingsley, PhD, the study’s senior author. “How do new traits evolve in natural populations? Do they arise through mutations in the coding regions of genes, which alter the structure and function of encoded proteins? Or are new traits the result of modifications in the regulatory regions of genes, which control where and when already-established proteins are expressed?”

The difference is stark: Mutations, or changes in the DNA sequence of a gene, can affect the structure of the protein that is made from that gene. Sometimes these changes can be advantageous, perhaps by increasing a protein’s ability to bind to an elusive yet critical biological target. But most often, they’re neutral or (if they disrupt the function of the protein entirely) even harmful.

Not every protein is expressed in every cell, however. The genes that encode them wink on and off throughout the body, like city lights, at distinct times and places. But their flickering isn’t random: it’s dictated by other, non-protein-coding sequences of DNA called regulatory regions. Often these surround the genes they control; sometimes they act at great distances. 

There are a lot of potential advantages to changing regulatory sequences, rather than the coding regions of genes. “Many genes work in multiple places in the body,” said Kingsley. “If you change their protein product, you simultaneously disrupt everything that gene does. In contrast, if you alter the regulatory switches that control where and when a gene is expressed, it may become possible to confine a change to one part of the body, or one developmental stage, for example, and avoid possible lethal consequences.”

The effect is like using a tool you know is effective in one application in a new way (let’s see if I can use a nail-pounding hammer to whack this board into place), rather than trying to craft an entirely new tool with no idea if it will work at all (there’s probably a good reason that you’ve never heard of the hamwrench).

The findings reverberate far beyond the watery domains of the tiny stickleback: They may help scientists understand how the whale lost its hind limbs when it returned to the sea, for example, or how early humans evolved variations in skin colors as they migrated across our green-blue planet.

“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, PhD, co-corresponding author of the paper and the scientific director of vertebrate genome biology at the Broad Institute of MIT and Harvard. “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.

 The stickleback’s claim to fame arises from the unique way it was dispersed at the end of the last Ice Age. When the glaciers melted, abundant flows of freshwater generated innumerable lakes and streams. As connections between ponds, streams and oceans later dried up, colonizing fish populations became stranded. Over the past 10,000 years (a blink of an evolutionary eye, really), the various stickleback populations developed traits suitable to their new environments.

“The threespine stickleback has evolved like many other animals,” said Kingsley. “But they’ve done it recently enough that they are ideally suited for study. The fact that they’ve evolved the same traits over and over again allowed us to search for those genes that control adaptation to marine or freshwater environments.”

For the study, the researchers picked one fish to serve as a reference for the entire threespine stickleback species. They sequenced the entire genome of that fish (a female from Bear Paw Lake in Alaska) to a high degree of accuracy and published the sequence for use by all stickleback researchers. But then they went one — well, actually 20 — steps further: they picked 10 pairs of marine and freshwater fish from locations around the world and sequenced their entire genomes as well.

Marine and freshwater sticklebacks exhibit some distinctive differences. The marine fish are heavily armored and slow, covered with plates and spines. They migrate long distances, eat small food particles found in open water and live most of their lives in salty environments. In contrast, the freshwater fish are lightly armored and fast, losing armor and shrinking spines, darting quickly for cover, eating larger food particles found on lake and stream bottoms, and living in environments with low levels of sodium, chloride and calcium. However the two types of fish can and do interbreed in areas where freshwater streams enter the salty ocean.

Comparing the genome of a fish that lives at the mouth of a stream with that of another several kilometers upstream can identify which regions in the genome carry important evolutionary changes that help the fish adapt to their unique environments.

“Essentially, we looked for those special places in the genome that are consistently different between marine and freshwater fish, every time the distinct types have evolved around the world,” said Kingsley. “We found an order of magnitude more of these adaptive regions than previously known because we were able to look all over the genome.”

Most of the 147 regions that the researchers identified were small — fewer than 5,000 base pairs — which allowed the scientists to pinpoint what genes or regulatory regions were affected. The changes involved genes that play roles in armor development, cellular signaling, behavior and metabolism, and control of kidney structure.

Finally, 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 researchers are now planning to sequence an additional 200 threespine sticklebacks to identify genes responsible for other variable traits, such as body size, growth rate and life span.

“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 Kingsley. “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.”

Postdoctoral scholar Felicity Jones, PhD, and former graduate student Yingguang Chan, (now at the Max Planck Institute for Evolutionary Biology in Germany) share first authorship along with the Broad Institute’s Manfred Grabherr, PhD, (now of the Science for Life Laboratory in Uppsala, Sweden) and Pamela Russell. Stanford researchers involved in the research include postdoctoral scholar Haili Zhang, PhD; graduate students Timothy Howes and Alex Pollen; and research assistant Shannon Brady; as well as former postdoctoral scholar Craig Miller, PhD; former graduate student Brian Summers, PhD; and former research specialist Anne Knecht, PhD.

The research was supported by the National Human Genome Research Institute, the Jane Coffins Childs Fund, the National Science Foundation, the National Defense Science and Engineering Graduate Fellowship program, the European Science Foundation, Stanford’s Bio-X program and the Howard Hughes Medical Institute.