All Creatures Great and Small

As pollution, habitat loss, global warming, and disease increase the number of threatened and endangered species, attempts to protect them are more crucial than ever. That’s where the Frozen Zoo comes in.

By Alisa Opar

On a sunny spring afternoon, the San Diego Zoo is teeming with shorts-clad tourists of all ages. While most visitors gravitate toward the pandas, giraffes, and gorillas, one little boy seems particularly taken with the Javan bantengs, a species of endangered Southeast Asian wild cattle that can grow to be seven feet long and weigh nearly a ton. Asked which one is his favorite, the child sizes up each of the animals before settling on a male with a dark blue-black coat grazing closest to him. It happens to be the spitting image of another banteng that died in 1980, and the resemblance is more than superficial: The four-year-old animal at the zoo is its clone.

The banteng wouldn’t be alive if it weren’t for a satellite of the San Diego Zoo located 35 miles north of the city, in Escondido. It too houses an impressive collection of exotic animals, but there are no tourists milling about here. Tucked in a corner room on the first floor of the zoo’s Center for Conservation Research, its inhabitants—not entire animals, but samples of their sperm, eggs, embryos, tissue, and other cells—are cryogenically preserved. Inside one of its thousands of vials, which are stored on tall racks and kept in huge cylindrical stainless-steel freezers, are cells from the banteng that scientists preserved 27 years ago.

Welcome to the Frozen Zoo, perhaps the world’s largest repository of genetic samples from endangered species. For the past three decades, scientists have relied on its collection to carry out a variety of critical conservation and research efforts. And with the number of endangered species on the rise, its leaders hope to one day stockpile samples from virtually every type of animal on Earth.

When geneticist Oliver Ryder and his colleagues began collecting samples for the Frozen Zoo 31 years ago, they had no idea how essential these actions would be to saving endangered species. The project was the brainchild of pathologist Kurt Benirschke, who led the Frozen Zoo until Ryder took over 15 years ago. These days, Ryder sports a neat salt-and-pepper beard, wears a button-down shirt and slacks, and socks with his sandals. His serious demeanor drops occasionally when, without changing his facial expression, he cracks a joke to lighten the mood. But he’s all business as he explains that he began freezing cells because of all their practical uses. Zoos often held only one animal of an extremely rare species, he says, and when that creature died, scientists lost the opportunity to study it. Ryder was also interested in studying the chromosomes of various animals to determine how closely related they were to each other (a science that was cutting-edge at the time). So he and his colleagues decided to freeze and stockpile viable cells at every opportunity to save them for later studies.

Over time, advances in technology allowed the preserved cells to be used for a wider variety of research and conservation activities. Today, the Frozen Zoo is a multinational research facility that stores samples from more than 7,200 animals representing some 675 species. Hundreds of scientists across the globe rely on its resources in their work to save endangered and threatened animals, and for a host of other purposes. The facility is run out of the department of Conservation and Research for Endangered Species (CRES), a center funded by the nonprofit Zoological Society of San Diego, along with grants from many other institutions.

Although the cells can be used for cloning, the zoo’s purpose isn’t to create carbon copies of endangered or extinct animals. In fact, it’s quite the opposite. “What we’re trying to conserve is the genetic diversity of species,” says Ryder, which gives animals the best chance of survival in the wild.

As he walks through the labs adjacent to the Frozen Zoo, Ryder explains how samples from, say, a bush buck on the African savannah come to “live” in this frigid repository. Scientists collect samples from animals in zoos and in the wild (often the tab of skin that’s removed when an animal is tagged), and send them to the Frozen Zoo to be deposited. The cells are divided, processed, placed in vials, and then frozen in liquid nitrogen, which preserves them for an estimated 10,000 years. When researchers need a sample, they remove it from the freezer and thaw out the cells.

The zoo’s workings are comparable to a bank—researchers deposit and withdraw samples as needed. “It’s not a mausoleum,” Ryder says, lifting a rack from one of the freezers with gloved hands and getting enveloped in the mist formed from the nitrogen vapor condensing water in the air. “This isn’t supposed to be just a place for the DNA of disappearing species. It’s a tool to prevent extinctions.”

One of the most successful conservation efforts aided by the Frozen Zoo is the California condor recovery project. The program is headed by Mike Wallace, a lanky, tanned wildlife biologist whose office is one floor above and around the corner from the Frozen Zoo. Today he is willing to chat, but only for a few minutes. He’s itching to drive south to Baja, Mexico, to scour a canyon where he’s pretty sure a pair of the endangered birds of prey is tending to an egg. His hunch proves right; the egg was the first to be laid by a California condor living in the wild in more than 60 years. And earlier this year, for the first time in nearly a century, a condor was spotted flying over San Diego.

When the condor preservation program launched in 1980, only 19 of the birds remained in the wild. Today, the condor population has swelled to nearly 300 birds, 135 of which live in the wild. “Condors are as rare and endangered as a species gets, and we came very close to losing them,” says Wallace. “With the Frozen Zoo, we basically have a duplicate population of condors. It’s a hedge for the future.”

The project began by bringing the 19 wild birds into captivity at the San Diego Zoo, where they joined eight others already there. Then scientists banked DNA samples from these 27 birds in the Frozen Zoo. Those samples have since been used countless times. One of the first steps to rebuilding the population required figuring out which birds should breed. By studying tissue samples, researchers discovered that the remnant population consisted of birds from three different clans, or families. So they devised a system to breed condors that were least genetically similar, which increases the chance that they’ll survive environmental changes and disease. “That way, each population we have is genetically whole,” says Wallace, so nearby birds can mate without the danger of inbreeding.

Even as the condor population continues to climb, the Frozen Zoo’s resources guide conservation efforts for the birds in other ways. Researchers have discovered, for instance, a genetic disorder that causes some chicks to develop shortened appendages, which makes most of them unable to hatch. With cell samples from the Frozen Zoo, scientists have created a genetic map of the birds and are using it to find the mutation that causes the disease. They then will design a genetic test to identify the carriers (the process is akin to a test for cystic fibrosis in humans). Once they find out which birds have the mutation, they can adjust their breeding plans accordingly. “In the space of three years, we’ve made progress on the condor genome that took 20 years in human genetics—at one ten-thousandth the cost,” says Ryder.

While Ryder says the Frozen Zoo’s contributions to species conservation and genetic research have been exhilarating, he still feels an immense obligation to grow its collection. “All this technology is becoming available to ask questions that weren’t answerable before, and the material we’re trying to save is disappearing,” he says. “It’d be a real shame to have a collection of really good materials from something that went extinct. So the Frozen Zoo, though it may be the largest of its kind, is a token effort. We have 675 species out of millions.”

Ryder has reason to be concerned. A number of studies indicate that extinction rates for many species are accelerating, spurred by loss of habitat, pollution, and disease. According to the World Conservation Union (IUCN), more than 16,000 species face extinction today; in many cases, the loss or decline of even a single species has a ripple effect on other species that depend on it. A 2004 paper in Science, for instance, estimated that 6,300 parasites, pollinators, and other species might be “coendangered” because the animals they rely on for survival are endangered. These threats will only worsen, scientists say, if global warming continues unchecked.

All these challenges are also compounded by the fact that researchers often only realize what’s happening to a species “almost when it’s too late,” Ryder says. In part, this is because despite all of our knowledge, scientists still don’t fully understand how ecosystems work, and they can’t always predict how environmental disturbances will affect a particular species. Though the Frozen Zoo can’t answer every question, the studies conducted with its resources give the people who draw up conservation plans—not only the scientific community, but also policy makers and a host of others—a better understanding of the animals they’re trying to protect and the environments they live in. Black rhinos, for instance, are found throughout Africa, but researchers only recently discovered that different geographic groups are quite genetically diverse. “That means that if we want to preserve genetic diversity as it occurs in nature, or as we think it occurs in nature, we shouldn’t be interbreeding those animals right now,” says Ryder.

 The threat of extinction is not the only reason Ryder feels compelled to add new samples as soon as possible. Environmental disturbances can cause species to change over time, so studying older samples and comparing them to new ones help scientists understand those changes and develop conservation strategies. “No one sees the Frozen Zoo as a substitute for keeping the natural world intact,” says Dan Wharton, head of the Central Park Zoo in New York City who is not affiliated with the Frozen Zoo. “But when you think of the potential options that are inherent in this kind of storage, really, it is the responsible thing to do right now, because there is so much uncertainty as to how many other conservation strategies are going to unfold.”

The Frozen Zoo is also pushing to diversify the types of species in its collection. For the most part, the samples stored there come from mammals, which regularly receive the most conservation dollars, due in no small part to their cute-factor. But in the past few years, the bank has been stocking up on bird and reptile samples. And this year, pending a grant, Ryder and veterinary pathologist Allan Pessier will begin adding more cells from amphibians, which are particularly vulnerable to extinction right now.

Pessier, who speaks softly and quickly and often flashes a shy smile, is admittedly biased when it comes to amphibians. “I prefer them to most humans,” he jokes. In a more serious tone, Pessier explains that amphibians, while not “cute and fuzzy like pandas,” play a vital role in the food chain by keeping insect populations in check. They’re also important indicators of environmental health because they absorb water and oxygen through their skin.

Amphibians are like the proverbial canary in the coal mine: If they start dying,

it’s a red flag that the environment they live in is changing.

Since 1980, more than 110 species of frogs, toads, salamanders, and newts have gone missing in the wild and are believed to be extinct, and populations of 435 species are in decline. Fully one-third of the more than 5,700 amphibian species known today are considered to be threatened, according to the IUCN. Not all of the problems are being caused by pollution and habitat degradation, however: One particular threat is an infectious disease called chytridiomycosis, caused by the chytrid fungus, which is found everywhere from the Australian outback to the Rocky Mountains to the rainforests of Central America. The disease has been causing sporadic deaths in some amphibian populations and complete mortality in others.

Pessier, who has studied the disease for more than 10 years, has seen up close the damage it has caused. Last year, he traveled to Panama and visited an area known as El Valle, which had clearly been hit by the fungus. “It took us twenty minutes to find one frog,” says Pessier. Yet in another region, about 100 miles away in a rainforest outside Panama City, Pessier and colleagues encountered hundreds of frogs from 20-odd species. “We were literally slipping on them, there were so many,” he says. “I had no idea what declines associated with the fungus really meant until I saw the differences between infected and uninfected areas of forest.”

Scientists are racing to determine the origin of the fungus and what makes it lethal to some frogs. But for now, the only line of defense is to bring infected frogs into captivity, treat them, and establish healthy captive colonies with the hopes of returning them to the wild. “The captive program assures that genetic diversity of these frogs is maintained despite declines and extinctions in the wild,” says Pessier. “It is an emergency measure, but necessary, since the frogs are disappearing so quickly.” Having cells from the skin of these frogs stored at the Frozen Zoo could enhance these measures dramatically, he says. By studying cell lines, researchers may be able to better understand how the fungus kills amphibians and determine ways to combat it.

The biggest challenge right now is growing the cells properly. The samples stored at the Frozen Zoo must be uncontaminated—that is, free of chemicals, fungi, and other contaminants that interfere with research and testing procedures. While a simple cleaning process to take a skin sample usually suffices, the moist, porous skin of frogs is an ecosystem unto itself; it hosts anti-fungal bacteria and other microorganisms, which makes getting a proper sample difficult. So researchers are working on methods for obtaining viable samples from adult amphibians that have died naturally—perhaps

Taking cells from inside their eyes. To date, they’ve grown cells from two species, American bullfrogs and African claw-toed frogs.

They hope studying these samples can lead to clues that will help them protect at-risk species.

It’s a start, but Ryder stresses that collecting a reserve of amphibian cells will require cooperation from scientists and governments around the world. To that end, Frozen Zoo researchers have begun working with a variety of conservation groups, some of which are bringing amphibians into captivity to protect them from the lethal fungus. Such efforts, explains Ryder as he gently places a rack of cylinders back into the freezer, are crucial for saving all species, not just amphibians. “Our job right now is to fill as many of these freezers as we can and to get as many other people to do the same and to protect it as a resource for the future,” says Ryder. That way, scientists may have the same success with other species as they’ve had with condors. “We’re still at the very early stages of learning how to employ the Frozen Zoo and being able to extract information from it. Who knows what we’ll be able to do in the future?”