How Beach Life Favors Blond Mice Sand Hills of Nebraska (Science, 28 August, p 1095) “We’re finally at the point where we can start to identify the genes responsible for phenotypic variation,” says Hoekstra And while working in Arizona, she says she In June, at a meeting in Cold Spring Harpicked up far too many “presents” bulging with bor, New York, Hoekstra described the third an angry rattlesnake Fortunately, this trap of the three genes responsible for coat-color weighs too little to have a snake inside, and no variation in Peromyscus mice and laid out her deadly spiders are expected view of the order in which mutations leading In a line of about 100 traps, Hoekstra to paler mice occurred “We’re trying to retrieves eight mice; her companions turn up reconstruct the evolutionary path, genetic step four more, not a bad take for a full-moon by genetic step,” she says “Understanding night, when mice tend to be less active how characters evolve is a critical question, The mice are part of a project started years and she is bringing a significant contribution,” ago to figure out the genetic changes that says developmental geneticist Claude Desplan underlie adaptations these animals make to the of New York University He adds that her work world around them Biologists have long mar- demonstrates that “one can really identify veled at how oldfield mice living on beaches evolving traits.” are much paler than those living inland, and Hoekstra and her team are part of a Hoekstra is searching for pigment genes genomics explosion in natural history studresponsible for the color variation ies “This is an example of work She’s combining molecular, devel… merging the ‘green’ and opmental, genetic, and ecological ‘white’ side of biology, in which approaches, including putting sciencemag.org we learn about trait evolution thousands of clay decoys on from the biochemical levels Podcast interview beaches to test the effects of coat within cells to how those traits with author color on predation risk and map- Elizabeth Pennisi are selected for or against in natping genes and testing pigment ural populations,” says Hans protein function in cell cultures “We’re attack- Ellegren, an evolutionary biologist at Upping the system from all sides,” says Hoekstra sala University in Sweden Mark McKone, a On this trip, Hoekstra and her team are biologist at Carleton College in Northfield, looking not just at coat-color variation but Minnesota, agrees: The work “could be a also at variation in burrow-building Most model for how to approach evolution in the deer mice build short, shallow burrows; old- postgenomic period,” when genetic inforfield mice go for deeper, longer ones Back mation and tools are more readily available in the lab, Harvard graduate student Evan Kingsley is trying to pin down the genetics New tools, classic model of tail length: Mice in forests have longer Hoekstra’s team represents the latest genertails Recently, Hoekstra postdoc Catherine ation of researchers tracking down genes Linnen described a genetic change under- that underlie so-called quantitative traits lying light-colored deer mice that match the such as height or body mass, which— FREEPORT, FLORIDA—It’s a hot, sticky July night here in western Florida, but to Hopi Hoekstra, it feels like Christmas Eve Hoekstra, a Harvard University evolutionary biologist, and her field crew have set out more than 400 small metal boxes, throwing a handful of sunflower seeds into each box before setting it on the ground, usually next to a mound of sand representing the debris from a mouse burrow When she inspects these live animal traps the following morning, she says it will be like “unwrapping presents.” Her eagerness is palpable “You’re going to be blown away by this field,” graduate student Jesse Weber had told Hoekstra when they first drove down a sand road into the Lafayette Creek Wildlife Management Area, a 13-square-kilometer expanse of overgrown fields kept open in part by controlled burns Never before had Weber and Harvard postdoc Vera Domingues seen such a dense concentration of burrows dug by the oldfield mice, Peromyscus polionotus, that they study By 7:30 the next morning, Hoekstra, Domingues, Weber, and Harvard undergraduate Diane Brimmer are making their way from trap to trap, sidestepping fire ant hills, prickly pear, and thorny vines while keeping an eye out for pygmy rattlers Typically, the trapdoors are still ajar, and at most a grasshopper or two jumps out into Hoekstra’s face as she empties the sunflower seeds But three traps down the line, the door is closed and Hoekstra senses something inside At past field sites, she’s had to worry about lethal spiders crawling in, positioned to nab any unsuspecting hand 1330 11 SEPTEMBER 2009 Online VOL 325 SCIENCE Published by AAAS www.sciencemag.org CREDITS (LEFT TO RIGHT): SHAWN CAREY/MIGRATION PRODUCTIONS; J B MILLER/FLORIDA PARK SERVICE A young evolutionary biologist tackles the genetic complexity of a classic case of adaptation in mice Downloaded from www.sciencemag.org on June 29, 2010 NEWSFOCUS Lighten up Several genes transformed mainland mice (left) into paler beach mice that blend in better with their environment unlike, say, eye color—vary by degree and are influenced by multiple genes It is painstaking work Researchers home in on such genes through intensive breeding studies combined with careful analysis of trait characteristics: spots, stripes, and so on for coat color; depth, length, and angle for burrowing behavior They correlate the traits with specific markers in genetic maps to pinpoint stretches of DNA known as quantitative trait loci (QTLs) that contain the genes of interest “This is done well in insects but is much more difficult in mammals,” says Desplan Over the past 20 years, several studies have identified QTLs in mammals, but few have managed to narrow the search to specific Her animal of choice is a textbook case of adaptation Peromyscus mice are distant relatives of house mice For more than a century, researchers had observed them in the wild, describing their looks and behaviors In 1909, light-colored P polionotus were discovered on Florida’s barrier islands, a sharp contrast to dark-brown, gray-bellied mainland mice of the same species Some 6000 years ago, dark oldfield mice moved into these newly formed beaches and islands Today, eight subspecies of these light-colored P polionotus exist on Florida’s coasts In the late 1920s, natural historian Francis Sumner guaranteed P polionotus a place in the textbooks when he drove from Florida’s Gulf Coast inland 150 kilometers collecting mice in eight places along the way, noting a correlation between soil and mouse color When he started, he was convinced that humidity caused the variation in color light areas of their bodies, traits duly noted for each individual This variation indicated that more than one gene was involved, but because the second generation still contained some mice that looked like the parents, Hoekstra knew that relatively few genes were important “It wasn’t one, it wasn’t 100,” Hoekstra recalls So she decided to go after them all Weber and Cynthia Steiner, now at the San Diego Zoo Institute for Conservation Research in California, developed and applied a set of more than 100 microsatellite markers, small pieces of variable DNA located across the genome They correlated the markers with the presence or absence of the various color pattern traits That work yielded three hot spots—QTLs—that seemed to determine what the mice looked like The researchers looked at the sequences of the house mouse and rat genomes for pigmentrelated genes at those locations and found DISTRIBUTION OF BEACH AND MAINLAND MICE Mainland mouse Lafayette Creek mice Santa Rosa Island beach mouse Anastasia Island beach mouse Alabama beach mouse LOCATION Perdido Key beach mouse Choctawhatchee beach mouse St Andrew beach mouse Pallid beach mouse* *extinct subspecies Southeastern beach mouse CREDIT: ADAPTED FROM C STEINER ET AL., MOL BIOL EVOL 26, 35 (2009), FIG Mouse of a different color Mice from different locales have evolved site-specific coat colors, except those at Lafayette Creek, which have a variety of pelt patterns genes, let alone identify mutations that result in changes such as coat color The discovery in 2005 by David Kingsley of Stanford University in Palo Alto, California, and colleagues that a change in the ectodysplasin gene led to the loss of armor in freshwater sticklebacks (Science, 25 March 2005, p 1928) “got the field excited,” says Hoekstra It was the first QTL study using natural populations to come up with a gene that was not already suspected to be involved and, later, to pin down its mutation Hoekstra hopes to go into more detail with her mouse studies Whereas Kingsley focused on the gene with the biggest effect, she is searching for several genes “If we identify multiple genes and understand the interactions between those genes, we can also learn something new about evolutionary processes,” she explains By the project’s end, he was more convinced that genetics caused the differences, driven by selection for camouflage “It’s one of the best studies of intraspecific variation,” says Hoekstra Giants in evolutionary biology, including Ernst Mayr, Theodosius Dobzhansky, John Maynard Smith, J B S Haldane, and Sewall Wright, have cited the work as a classic example of adaptation Others followed Sumner, looking at various aspects of beach mice ecology, but they were unable to pin down the genetics Hoekstra saw an opportunity: “We now have the molecular tools to answer the questions that they were asking more than a half-century ago.” She and her colleagues bred dark and light mice, then generated 800 second-generation offspring These hybrid mice differed in their stripes and splotches and the extent of dark or www.sciencemag.org SCIENCE VOL 325 Published by AAAS Downloaded from www.sciencemag.org on June 29, 2010 NEWSFOCUS promising candidates One was Mc1r, which codes for a receptor protein in pigmentproducing cells Hoekstra was at first skeptical In her studies of black pocket mice on volcanic rock in Arizona, one version of that gene was responsible for the black mice and another for light mice; it was not clear how the gene might play a role in determining fine details such as nose blazes and tail stripes But not only did they prove that Mc1r was involved, they also found a single-base change that led to an amino acid mutation that dampened receptor activity (Science, 15 July 2005, p 374; July 2006, p 101) A second candidate gene, Agouti, panned out as well In this case, the versions of the gene in dark and light mice were identical; yet the gene in beach mice was much more active, leading to much more messenger RNA and presumably protein that reduced dark-pigment pro- 11 SEPTEMBER 2009 1331 NEWSFOCUS Mouse maven Hopi Hoekstra combines molecular and field expertise to study the genetics of wild mice 1332 laboratory mice, made for dirty-blond mice Corin was also active in the hair follicles of oldfield mice, Hoekstra reported in June at “Evolution: The Molecular Landscape” in Cold Spring Harbor The gene in light and dark mice was almost the same, but it was much more active in light mice Thus, as with Agouti, a change in regulation may be key to the change in coat color In the simplest scenario, the effect of these genes would be additive: Two “light” versions of the variable genes would lead to a paler mouse than one version would, and the 11 SEPTEMBER 2009 VOL 325 SCIENCE Published by AAAS palest mice would have “light” versions of all three But that’s not the case with Agouti, Corin, and Mc1r These genes have epistatic interactions: A “dark” Agouti version counters any lightening effect of a “light” Corin or Mc1R, for example These epistatic effects can dictate the order in which alleles in a population must pop up in order to be selected for and spread “You need to have the agouti allele first,” says Hoekstra, because the “light” versions of Corin or Mc1r would be invisible to selection if only the “dark” agouti were present www.sciencemag.org CREDIT: E PENNISI/SCIENCE duction, particularly in the cheeks, tail, and eyebrows, Hoekstra, Weber, and Steiner reported in 2007 They had a false start with the third region identified in the QTL studies Harvard graduate student Emily Jacobs-Palmer eventually ruled out several pigmentation genes, including a promising one called Kit that turned out to lie outside the QTL Then last year, Bruce Morgan of Harvard Medical School in Boston and his colleagues reported that mutating a gene called Corin, which was expressed in the hair follicles of Downloaded from www.sciencemag.org on June 29, 2010 Park She studied the biomechanics of invertebrates throughout the school year During that time, James Patton, curator of mammals at the Berkeley Museum of Vertebrate Zoology, got her hooked on four-legged furry creatures by taking her to trap gophers in Arizona And before starting graduate school, she spent months as shipboard mammalogist on a joint Japanese, Russian, and American expedition to collect animals in the Kuril Islands off Russia Her Ph.D dissertation at the University of Washington, Seattle, involved months of fieldwork in the Andes tracking down a sex chromosome polymorphism in mice Some females seemed to have both a big and a small X, which later proved to be a Y chromosome, even though these females were completely fertile, producing more young than the typical female with two X chromosomes “This was an oddball system,” Hoekstra recalls Afterward, “I got interested in more general questions.” Fascinated by the genetics underlying adaptation, Melding Mammals and Molecules to Track Evolution she spent her postdoc trapping black mice on ancient Arizona volcanoes and tracking down the gene responsiSelf-described as a bubbly California girl, Hopi Hoekstra entered the Uni- ble for the change In these field studies, she developed a yen for her versity of California, Berkeley (UCB), not thinking about being a scientist camp meal of choice: cold SpaghettiOs and mini meat balls straight from Her goal was to become the U.S ambassador to the Netherlands—both her the can, with a Miller Light parents are Dutch—and an accomplished collegiate volleyball player Then She considers herself a molecular person: “We’re interested in the molshe got her first summer job: Dressed in white, she hiked the Berkeley Hills ecules that are important to the organism,” she says Yet she also knows just east of campus, a tick target for researchers assessing where and when just how much cornmeal it takes when skinning a mouse to ensure the pelt hikers were most susceptible to attacks by Lyme disease–transmitting ticks won’t be greasy and that shrews have fragile skin that’s hard to pull off “It still makes me itch just to think about it,” she says The breadth of projects include an analysis of shrew venom proteins But the experience made Hoekstra itch for more fieldwork and, even- and a collaboration on a genetic study of mice in Bulgaria that seem to tually, a life as a biologist Two years ago, she moved from the University cooperate to build large mounds that they coinhabit to get through of California, San Diego, to Cambridge, Massachusetts, as a Harvard Uni- harsh winters versity evolutionary biologist She is also currently curator of mammals at “Being able to be a molecular biologist and be comfortable with the Harvard’s Museum of Comparative Zoology Although only in her mid- whole organism—few people that as well as Hopi, and that’s where 30s, “Hopi has rapidly made herself a name in the evolutionary biology progress [in the field] will be made,” says Mark McKone, a biologist at community,” says Hans Ellegren of Uppsala University in Sweden Her Carleton College in Northfield, Minnesota “When you put [her research] honors include a young investigator award from the Arnold and Mabel together, it’s more than the sum of its parts.” Beckman Foundation and prizes from her professional societies and her Hoekstra doesn’t get out into the field much anymore Instead, she universities “She’s just about one of the deepest thinkers in the area,” lives vicariously through her students and postdocs, with the goal of spendsays Carlos Bustamante of Cornell University, who adds that her beach ing time at least once with each of them in the field “When they have a mice experiments “are beautifully thought out and designed.” really good day, they call and leave a message,” she says, or send a photo She traces her professional roots back to her UCB experience, where from their phones, such as an image of 44 traps stacked up against a brick she managed to research almost year-round, even as an undergraduate wall, signaling that their trapping yielded a bonanza “They just send a picOne summer, she analyzed pack rat middens in Yellowstone National ture [without words] because they know I know what it means.” –E.P CREDIT: E PENNISI/SCIENCE Burrowing in Weber has taken on an even more challenging project: using these mice to look at the genetics underlying burrowing behavior “It’s pathbreaking work on the evolution of behavior in a natural environment,” says field biologist Peter Grant of Princeton University “QTL studies are widespread in general but rare in behavior studies of organisms in nature.” Unlike coat color, almost nothing is known about genes that might guide burrowing Yet oldfield mice and their sister species, deer mice, differ dramatically and, it seems, consistently in the burrows they build The latter tend to knock off their digging less than 10 centimeters down Oldfield mice shovel down meter, even 2, hollow out a nest chamber, and then excavate an escape tunnel that tends to shoot directly back up to just below Here in Freeport, he’s doing some ground-truthing He catches the mice in the burrows so he can correlate their DNA with the tunnels’ dimensions He picks what looks like a freshly dug hole, shovels out some dirt, then drops to his knees to scoop the sand and clay away with his hands until he sees a round, light-colored spot in the wall of the hole His f inger easily pokes through it, revealing it to be a plug of sand blocking the burrow tunnel Alternating between shoveling and scooping and probing the tunnel with a long, flexible, plastic tube (sprinkler tubing), he excavates the tunnel, eventually breaking Bagging burrows The beach mice field crew measures a mouse burrow into a widened area filled with after making a cast of its tunnels nesting material “This nest is gigantic,” he says He confers with Hoekstra about where she should stand in anticipation of mice emerging from the invisible escape hatch She shifts to the right a halfmeter, then bends her legs slightly, hands on her knees She looks like the volleyball player she used to be, expecting a serve, except she’s looking down, not up Weber pokes the tubing in a little farther Suddenly, two heads pop up about 20 centimeters to Hoekstra’s right She dives to clamp her gloved hands over the heads But as she peeks through her f ingers, one dashes out between her legs, and the other heads full speed in the opposite direction Both she and Weber pursue that one, darting from the surface The mice plug up the burrow bush to bush after the mouse until finally about 15 centimeters from the entrance, seal- Weber has it in hand The other is long gone ing themselves safely in underground While Weber measures the size and Back in the lab, Weber has filled 10 boxes, shape of the burrow, Hoekstra measures the each 122 cm by 152.5 cm by 92.5 cm tall, sacrificed mouse, then dissects out its liver with 1.5 tons of premium playground sand to save for DNA tests, removes the skin to He has crossed oldfield with deer mice, then mount the pelt for future studies of the color crossed their offspring back with either par- pattern, and saves the skeleton for the ent, and he’s looking at what sorts of burrows museum’s collections The sun sets bright these backcrossed progeny dig The distribu- red in front of her, and the full moon is a big tion of burrow sizes in this second generation white ball in the sky behind her will provide a rough indication of how many Weber and Hoekstra seem tired but congenes are involved in determining burrow- tent The burrows they’ve dug up were ing behavior Weber squirts household insu- deeper and longer than usual; shoveling lating foam from a spray can down the bur- heavy, wet sand was tough going They’ve rows The foam expands to fill the nest and been up since before dawn and have an passageways and hardens to provide a three- evening of setting traps ahead of them dimensional model of the burrow So far he’s “But once in a while, it’s good if it’s hard,” tested 200 mice and has partially filled the Hoekstra says “Then you appreciate it attic of the Museum of Comparative Zoology when it’s easy.” with casts of their burrows –ELIZABETH PENNISI www.sciencemag.org SCIENCE VOL 325 Published by AAAS 11 SEPTEMBER 2009 Downloaded from www.sciencemag.org on June 29, 2010 By backcrossing the second-generation mice with their parents and f iguring out which version of each of the three genes the offspring had, Hoekstra’s team was able to tease out the interactions among the genes The light-mouse version of Corin lightens the coat only when the light-mouse versions of both of the other genes are also present, Hoekstra reported Thus, it is likely that genetic change in Corin occurred after the changes to Mc1r and Agouti Meanwhile, Domingues and graduate student Lynne Mullen are trying to track down the exact base changes involved in the Agouti and Corin regulatory regions Working with postdoc Brant Peterson, they are figuring out a way to sequence 200,000-base chunks surrounding each of these genes in multiple individuals They plan to scan for differences that correlate with coat color patterns “We will probably see lots of differences,” says Hoekstra “The question is, ‘What are the important ones?’ ” The work Domingues is doing here might help answer that question The landscape is dotted with spots of white sand sparsely broken up by vegetation amid fields solidly covered with low bush and plants, and in a few places, meter-tall trees have taken hold When local fish and wildlife managers first directed her to this spot, Domingues expected the mice to be uniformly dark, but quite a few had beachlike features Hoekstra and Domingues eagerly discuss the pelage of each catch How far a dark stripe extends down the tail, the expanse of white on the cheeks, the presence of a nose blaze all matter, as they signal something interesting going on in the genetics of these supposed-to-be-dark mainland mice Domingues plans to try to pin down the genes—and mutations—involved in all the variation she sees, using the three genes implicated in beach mouse paleness as a jumping-off point 1333