Marine threespine sticklebacks haven't morphologically changed in an estimated 10 million years, but their freshwater offshoots show no signs of slowing down. These 5-cm-long, freshwater fish have undergone a recent evolutionary change, variably losing their calcified body armor and retractable pelvic and dorsal spines. Remarkably, isolated marine and freshwater sticklebacks can be hybridized in the laboratory, a fact that is allowing researchers to analyze the genetics behind their natural diversification.

"Despite all of the interest in how evolution really works, and despite all we know about the genetic pathways that build tissues, we have surprisingly few real examples where traits in natural populations are understood at the molecular level," says David Kingsley, a recent convert to stickleback research at Stanford University. Kingsley and colleagues have made the link, finding that a single gene might control pelvic armor loss in freshwater sticklebacks.

It's a finding that goes against a longstanding belief...


Isolated freshwater populations of the threespine stickleback, Gasterosteus aculeatus, have evolved repeatedly from marine sticklebacks, most recently around 20,000 years ago.1 Marine sticklebacks are prone to do this because they breed in freshwater, and on occasion become trapped in lakes. "The marine fish is a reasonably uniform ancestor, and it has invaded fresh water innumerable times ... this has set up a natural experiment," says Bell.

As with many fish, phenotypic diversity is broad. Most marine sticklebacks have bony plates on their sides running from head to tail and spikes protruding from behind some fins. These defenses are thought to provide protection from predators. But, many freshwater sticklebacks have lost this heavy armor. It's an adaptation to a lake environment without predators that is observed again and again, says Bell. "There are other systems like it, but none of them has phenotypic variation that is as conspicuous."

It was partly this obvious, natural variation that sparked Kingsley to take up sticklebacks as a model. His laboratory had been studying skeletal formation in mice since the 1980s, using chromosome mapping to clone trait genes. But in the summer of 1998, he and Catherine Peichel wondered if a system existed to uncover genes controlling natural traits. They could not use the mouse, owing to its long history as a laboratory animal, or the zebrafish, because of its natural habitat of life in a mud puddle.

But the effort involved in bringing genetics and genomics to a new system seemed worth it for the stickleback, says Kingsley. "The great thing about the sticklebacks is they have morphological, physiological, host-parasite, and so many other changes."


In the fall of 1998, Kingsley and Peichel enlisted the help of Dolph Schluter, an ecologist at the University of British Columbia. Three years later they reported that morphologically distinct stickleback populations could be crossed using in vitro fertilization.2 " [The 2001 paper] showed we could build a genome-wide linkage map in sticklebacks, and that some of the interesting differences in the literature could be mapped to particular chromosome regions," says Kingsley. Unfortunately, the original cross contained too few progeny to enable mapping of trait genes.

Such an undertaking would require a much bigger cross, including F2 progeny of the F1 hybrids, one that playfully came to be known as "the big cross," says Schluter. The big cross, completed over two years in Schluter's laboratory, refers to hybrids made between two of the most extreme phenotypes known: a heavily armored marine stickleback and the defenseless benthic population from Paxton Lake, British Columbia. It was the big cross that really set things in motion, comments Schluter.

Michael Shapiro, Kingsley, Schluter, and their colleagues made use of the big cross and genome-wide linkage mapping to search for genes involved in pelvic armor reduction of freshwater sticklebacks.3 They found that F1 hybrids all showed pelvic armor akin to their marine parent, whereas F2 progeny showed a 3:1 Mendelian ratio of unaffected to reduced pelvic armor. As they searched for chromosome regions, they found one major region apparently controlling this trait.

Shapiro and colleagues localized the effect to a chromosome region containing the Pitx1 gene.3 In mice, Pitx1 is required for hindlimb development and left-right limb symmetry. The big-cross F2 progeny showed left-right asymmetry in their pelvic spines, similar to phenotypes observed in Pitx1 knockout mice, but they exhibited no Pitx1 mutations. Instead, these fish showed site-specific regulatory mutations.

Susan Foster, associate professor at Clark University, Worcester, Mass., cautions that the work does not definitively show that Pitx1 is the gene responsible for pelvic reduction, and that other genes close to Pixt1 also may contribute. Nevertheless, the results do reveal "that loci identified in the laboratory, which have dramatic effects on development, also play a role in naturally occurring variation," says Foster, something that has never been experimentally verified until now.

In a complementary study, Nicholas Cole, Cheryll Tickle, and colleagues from the University of Dundee, UK, found that Pitx1, and its downstream target Tbx4, were differently expressed in sticklebacks with and without pelvic spines.4 This finding further supports the idea that regulatory mutations in key developmental genes may drive adaptive skeletal changes in nature.


Courtesy of William Cresko

The mid-section of stickleback fish highlighting variation in the pelvic structure. Fish have been cleared with trypsin and their bones stained with Alizarin red. A and B show the complete form of the structure, with robust girdle and stout spines. B shows only the pelvic structure dissected away from the body of the fish. Panals C through F show fish that have absent (C and E) or reduced (D and F) pelvic structures. In pelvic-reduced fish, only a small remnant of bone remains, as can be seen in the lateral (C and D) and ventral (E and F) views of these stickleback. Pictures of the fish were taken with head to the left and caudal fin to the right.

In another study, Pamela Colosimo, Kingsley, and others used the big cross to examine variation in bony lateral plates.5 After measuring the plate size, number, and pattern, the research team analyzed the genetic loci that control these phenotypes, finding that one chromosome region could account for 75% of the variation observed. This work has broken new ground and inspired others to try similar experiments, says Cliff Tabin, professor at Harvard Medical School.

John Postlethwait, professor at the University of Oregon, is carrying out similar stickleback crosses that point towards the same conclusions. William Cresko, Postlethwait, Bell, and others recently examined bony armor loss in three geographically isolated freshwater populations of Alaskan stickleback.6 They found large losses in body armor among the freshwater fish as compared to their marine ancestors, and upon crossing these fish, found that a single factor appeared to control the armor loss.

"It came as bit of a surprise to find that, in the case of sticklebacks, the factor controlling the number of plates and the presence or absence of the pelvis were simple Mendelian traits, since the normal idea is that evolution changes by small steps," says Postlethwait. Foster thinks the work is impressive, adding, "I don't think people really expected to see such large-effect genes playing a role in nature."


Overall, the stickleback appears to be gaining momentum as a general model. Recently, the National Institutes of Health established the Center of Excellence in Genomic Sciences at Stanford, aimed at creating genomic resources for fish. Further, a stickleback physical genome map, expressed sequence tag (EST) collection, and bacterial artificial chromosome (BAC) libraries are already available. The NIH has recently approved a program to sequence the stickleback genome.

Peichel suggests that such genomic resources will allow genetic investigation of other evolutionary questions. She recently set up her own lab, at the Fred Hutchinson Cancer Research Center in Seattle, to study the genetic basis of behavior using sticklebacks. "There has been a lot of work on mating behavior and aggression in sticklebacks. I want to take the genetic tools we have developed to try and get at the actual genes that control these behaviors," says Peichel.

Foster says that her lab is already observing the reevolution of pelvic armor in sticklebacks that 10 years ago didn't have any; the reappearance is likely due to the artificial introduction of trout into their territory. With the advent of molecular tools, the genes driving such processes can now be identified, giving a perspective on vertebrate evolution that other systems can't provide. A truly unique opportunity arises, says Bell. " [Sticklebacks] evolve within short enough time intervals that we can study species-level evolution within the professional lifetime of an investigator."

David Secko dsecko@the-scientist.com

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