How Beach Life Favors Blond Mice

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.. Biologists have lo

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 f ield 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

morn-ing, 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

Man-agement Area, a 13-square-kilometer expanse

of overgrown fields kept open in part by

con-trolled 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

under-graduate Diane Brimmer are making their

way from trap to trap, sidestepping fire ant

hills, prickly pear, and thorny vines while

keep-ing an eye out for pygmy rattlers Typically, the

trapdoors are still ajar, and at most a

grass-hopper 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

And while working in Arizona, she says she picked up far too many “presents” bulging with

an angry rattlesnake Fortunately, this trap weighs too little to have a snake inside, and no deadly spiders are expected

In a line of about 100 traps, Hoekstra retrieves eight mice; her companions turn up four more, not a bad take for a full-moon night, when mice tend to be less active

The mice are part of a project started 6 years ago to figure out the genetic changes that underlie adaptations these animals make to the world around them Biologists have long mar-veled at how oldfield mice living on beaches are much paler than those living inland, and Hoekstra is searching for pigment genes responsible for the color variation

She’s combining molecular, devel-opmental, genetic, and ecological approaches, including putting thousands of clay decoys on beaches to test the effects of coat color on predation risk and map-ping genes and testing pigment protein function in cell cultures “We’re attack-ing the system from all sides,” says Hoekstra

On this trip, Hoekstra and her team are looking not just at coat-color variation but also at variation in burrow-building Most deer mice build short, shallow burrows; old-field mice go for deeper, longer ones Back

in the lab, Harvard graduate student Evan Kingsley is trying to pin down the genetics

of tail length: Mice in forests have longer tails Recently, Hoekstra postdoc Catherine Linnen described a genetic change under-lying light-colored deer mice that match the

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

In June, at a meeting in Cold Spring Har-bor, New York, Hoekstra described the third

of the three genes responsible for coat-color

variation in Peromyscus mice and laid out her

view of the order in which mutations leading

to paler mice occurred “We’re trying to reconstruct the evolutionary path, genetic step

by genetic step,” she says “Understanding how characters evolve is a critical question, and she is bringing a significant contribution,” says developmental geneticist Claude Desplan

of New York University He adds that her work demonstrates that “one can really identify evolving traits.”

Hoekstra and her team are par t of a genomics explosion in natural history

stud-ies “This is an example of work

… merging the ‘g reen’ and

‘white’ side of biology, in which

we learn about trait evolution from the biochemical levels within cells to how those traits are selected for or against in nat-ural populations,” says Hans Ellegren, an evolutionary biologist at Upp-sala University in Sweden Mark McKone, a biologist at Carleton College in Northfield, Minnesota, agrees: The work “could be a model for how to approach evolution in the postgenomic period,” when genetic infor-mation and tools are more readily available

New tools, classic model

Hoekstra’s team represents the latest gener-ation of researchers tracking down genes that underlie so-called quantitative traits such as height or body mass, which—

How Beach Life Favors Blond Mice

A young evolutionary biologist tackles the genetic complexity of a

classic case of adaptation in mice

Online

Podcast interview with author Elizabeth Pennisi.

sciencemag.org

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

com-bined with careful analysis of trait

character-istics: spots, stripes, and so on for coat color;

depth, length, and angle for burrowing

behavior They correlate the traits with

spe-cific markers in genetic maps to pinpoint

stretches of DNA known as quantitative trait

loci (QTLs) that contain the genes of

inter-est “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

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,

Califor-nia, 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

evolution-ary processes,” she explains

Her animal of choice is a textbook case of

adaptation Peromyscus mice are distant

rela-tives 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 collect-ing mice in eight places along the way, not-ing a correlation between soil and mouse color When he started, he was convinced that humidity caused the variation in color

By the project’s end, he was more con-vinced that genetics caused the differences, driven by selection for camouflage “It’s one of the best studies of intraspecific vari-ation,” 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 opportu-nity: “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

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 impor-tant “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 pigment-related genes at those locations and found

promising candidates One was Mc1r, which

codes for a receptor protein in pigment-producing cells Hoekstra was at first skepti-cal In her studies of black pocket mice on vol-canic 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; 7 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 presum-ably protein that reduced dark-pigment

pro-Lighten up Several genes transformed mainland

mice (left) into paler beach mice that blend in better

with their environment.

DISTRIBUTION OF BEACH AND MAINLAND MICE

LOCATION

Mainland mouse

Pallid beach mouse*

Southeastern beach mouse

Anastasia Island beach mouse

Perdido Key beach mouse

Alabama beach mouse

Lafayette Creek mice

St Andrew beach mouse Choctawhatchee

beach mouse

Santa Rosa Island beach mouse

*extinct subspecies

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.

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

Har-vard 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

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

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 coun-ters 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

selec-tion if only the “dark” agouti were present C

Self-described as a bubbly California girl, Hopi Hoekstra entered the

Uni-versity of California, Berkeley (UCB), not thinking about being a scientist

Her goal was to become the U.S ambassador to the Netherlands—both her

parents are Dutch—and an accomplished collegiate volleyball player Then

she got her first summer job: Dressed in white, she hiked the Berkeley Hills

just east of campus, a tick target for researchers assessing where and when

hikers were most susceptible to attacks by Lyme disease–transmitting ticks

“It still makes me itch just to think about it,” she says

But the experience made Hoekstra itch for more fieldwork and,

even-tually, a life as a biologist Two years ago, she moved from the University

of California, San Diego, to Cambridge, Massachusetts, as a Harvard

Uni-versity evolutionary biologist She is also currently curator of mammals at

Harvard’s Museum of Comparative Zoology Although only in her

mid-30s, “Hopi has rapidly made herself a name in the evolutionary biology

community,” says Hans Ellegren of Uppsala University in Sweden Her

honors include a young investigator award from the Arnold and Mabel

Beckman Foundation and prizes from her professional societies and her

universities “She’s just about one of the deepest thinkers in the area,”

says Carlos Bustamante of Cornell University, who adds that her beach

mice experiments “are beautifully thought out and designed.”

She traces her professional roots back to her UCB experience, where

she managed to do research almost year-round, even as an undergraduate

One summer, she analyzed pack rat middens in Yellowstone National

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 3 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 Washing-ton, 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 After-ward, “I got interested in more general questions.”

Fascinated by the genetics underlying adaptation, she spent her postdoc trapping black mice on ancient Arizona volcanoes and tracking down the gene responsi-ble for the change In these field studies, she developed a yen for her camp meal of choice: cold SpaghettiOs and mini meat balls straight from the can, with a Miller Light

She considers herself a molecular person: “We’re interested in the mol-ecules that are important to the organism,” she says Yet she also knows just how much cornmeal it takes when skinning a mouse to ensure the pelt won’t be greasy and that shrews have fragile skin that’s hard to pull off

The breadth of projects include an analysis of shrew venom proteins and a collaboration on a genetic study of mice in Bulgaria that seem to cooperate to build large mounds that they coinhabit to get through harsh winters

“Being able to be a molecular biologist and be comfortable with the whole organism—few people do that as well as Hopi, and that’s where progress [in the field] will be made,” says Mark McKone, a biologist at Carleton College in Northfield, Minnesota “When you put [her research]

together, it’s more than the sum of its parts.”

Hoekstra doesn’t get out into the field much anymore Instead, she lives vicariously through her students and postdocs, with the goal of spend-ing time at least once with each of them in the field “When they have a really good day, they call and leave a message,” she says, or send a photo from their phones, such as an image of 44 traps stacked up against a brick wall, signaling that their trapping yielded a bonanza “They just send a pic-ture [without words] because they know I know what it means.” –E.P.

Melding Mammals and Molecules to Track Evolution

Mouse maven Hopi Hoekstra combines molecular and field expertise to study

the genetics of wild mice.

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

pat-terns “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

uni-formly 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

Burrowing in

Weber has taken on an even more challenging

project: using these mice to look at the

genet-ics underlying burrowing behavior “It’s

path-breaking work on the evolution of behavior in

a natural environment,” says field biologist

Peter Grant of Princeton University “QTL

known about genes that might guide burrow-ing 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 1 meter, even 2, hollow out a nest cham-ber, and then excavate an escape tunnel that tends to shoot directly back up to just below

the surface The mice plug up the burrow about 15 centimeters from the entrance, seal-ing themselves safely in underground

Back in the lab, Weber has filled 10 boxes, each 122 cm by 152.5 cm by 92.5 cm tall, with 1.5 tons of premium playground sand

He has crossed oldfield with deer mice, then crossed their offspring back with either par-ent, and he’s looking at what sorts of burrows these backcrossed progeny dig The distribu-tion of burrow sizes in this second generadistribu-tion will provide a rough indication of how many genes are involved in determining burrow-ing behavior Weber squirts household insu-lating foam from a spray can down the bur-rows The foam expands to fill the nest and passageways and hardens to provide a three-dimensional model of the burrow So far he’s tested 200 mice and has partially filled the attic of the Museum of Comparative Zoology with casts of their burrows

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 shovel-ing and scoopshovel-ing and probshovel-ing the tunnel with

a long, flexible, plastic tube (sprinkler tubing), he excavates the tunnel, eventually breaking into a widened area filled with 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 half-meter, then bends her legs slightly, hands on her knees She looks like the volleyball player she used to be, expecting a ser ve, 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 bush to bush after the mouse until finally Weber has it in hand The other is long gone

While Weber measures the size and shape of the burrow, Hoekstra measures the sacrificed mouse, then dissects out its liver

to save for DNA tests, removes the skin to mount the pelt for future studies of the color patter n, and saves the skeleton for the museum’s collections The sun sets bright red in front of her, and the full moon is a big white ball in the sky behind her

Weber and Hoekstra seem tired but con-tent The bur rows they’ve dug up were deeper and longer than usual; shoveling heavy, wet sand was tough going They’ve been up since before dawn and have an evening of setting traps ahead of them

“But once in a while, it’s good if it’s hard,” Hoekstra says “Then you appreciate it when it’s easy.”

–ELIZABETH PENNISI

Bagging burrows The beach mice field crew measures a mouse burrow after making a cast of its tunnels.