New Genes Beat Extinction

N o one knows how many species share the planet with us. E. O. Wilson, the great Harvard biologist and among the most qualified people on earth to make the estimate, has guessed that there are between 10 and 100 million. In his words, "We don't know, not even to the nearest order of magnitude." Wilson and several other biologists who specialize in the subject recently estimated that mankind knew of about 1.4 million species. Thus, by the reckoning of the some of the world's most eminent biologists, humans are only dimly aware of between 1 and 10% of the current cast of species on earth. We know virtually nothing about most of them.

Why? There are two reasons. Many live and die in places that most humans rarely even think about: the soil, the ocean depths, and the forest canopy, especially the canopies of the rapidly dwindling rain forests. As living creatures go, humans are big, and we tend to think mostly about the other big species that we routinely interact with—farm animals, the garden plants and song birds about our homes, the fish we catch. These constitute a tiny fraction of the total. The most enthusiastic amateur naturalist can probably identify fewer than 1000 species. But the vast majority of species, whatever their true number, are exceedingly small. Of the 1.4 million known species, more than half are insects, spiders, centipedes, or related organisms with jointed exoskeletons—hardly a favorite group among humans. The next largest group is the flowering plants. Of the 90-99% of the species that we may not even be aware of, the vast majority are microorganisms, mostly a riot of bacteria that are invisible to us.

Are they important? Unquestionably. Earth's great life cycles depend critically on them. Without insects to pollinate them, most flowering plants would rapidly become extinct. Bacteria and other soil organisms are nature's recyclers; in decomposing the dead of larger species, they reconstitute the topsoil that is constantly eroding. Without them there would be mass famine. Ironically, it is from among the handful of bacteria which we do know well that we have developed many of the antibiotics we use to subdue the otherwise often fatal infections caused by other bacteria.

There is good reason to think that, as man's expanding numbers consume the rain forests and pollute the waters, we are killing off species surely by the thousands, perhaps by the tens of thousands, every year. The logic that underlies this assertion is simple. Careful study of rain forest canopies, for example, yields new species on almost every expedition. For the most part, these are insects that over millions of years have worked out a little niche for themselves in an exceedingly complicated environment. Often it turns out that a newly discovered insect can survive only in a particular part of a particular species of tree because that is the niche in which its source of food evolved. Destroy the trees to make way for more fields for cows to support more human families and the organisms for which that species of tree is a world unto itself will perish.

Because they can see insects, committed field biologists and good amateur naturalists will continue to improve our inventory of them. For smaller species, about the importance of which to our own survival we know virtually nothing, it is highly likely that we will remain ignorant. We know of about 1000 viruses and 5000 bacteria, numbers that may constitute less than 1% of the actual diversity. Many of them may live in the guts of the insects that perish when third-world farmers struggling to feed their families cut down the last of a variety of a particular species of tree. They become extinct without ever having been recorded on mankind's global inventory.

What can we do to save the species which we know are endangered? The best approach is of course not to let them become so. The surest way to do that is to set aside large preserves, tracts of sufficient size that the larger species will have a fighting chance to survive. George Schaller, one of the world's great field biologists, has accomplished undreamed-of success in this regard. Among his many campaigns to influence governments to protect ecosystems, the greatest victory has been in China, which recently decided to protect an area in Mongolia that is nearly half as big as the eastern United States from human development. But many large species have recently become extinct, and many more are hanging from an evolutionary precipice. For them molecular genetics and reproductive biology may be the only rescue net.

Sexing the Spix's Macaw

What is the most endangered large animal species on earth? Possibly the Spix's macaw (Cyanopsitta spixii), a brilliantly blue colored, parrot-like bird native to Brazil. In early 1995 there were only 32 on the planet, most in the collections of exotic bird fanciers, and only one known in the wild. The Spix's macaw is sexually monomorphic; that is, males and females look alike. In the hope of bringing the species back from the edge of extinction, conservationists wanted to provide a mate for the last wild bird, but they did not know whether it was male or female. Ornithologists who had closely observed the wild bird's behavior thought it was a male, but could not be sure. Because it is the only remaining member of its species that has grown up in the wild, conservationists were extremely reluctant to risk harming it by capturing it for sexing. Molecular biologists provided a solution.

To answer the question, Richard Griffiths and Bela Tiwari, who work in the Department of Zoology at Oxford University, developed a molecular method to sex birds from the tiny amounts of DNA that can be extracted from the tips of molted feathers. In contrast to humans, in birds it is the females that have different sex chromosomes. In humans the female has two X chromosomes and the male has an X and a Y; in birds the females have one W and one Z chromosome and the males have two Z chromosomes. In 1993 Griffiths and Tiwari isolated a highly conserved gene (called C-W) which seems to be found on the W chromosome in virtually all but one group of bird species. They also knew of a second, closely related bird gene called C-2 that is located on a non-sex chromosome.

To develop a DNA test to sex the last wild Spix's macaw, they had to learn more about the nature of the C-W gene in macaws. To help them, a company called Stratagene made a genomic library from the DNA of a close relative, the hyacinth macaw, which is not endangered. A genomic library is simply a collection of bacterial colonies, each of which includes within it a little bit of the chopped-up DNA of an organism's entire set of genes. The bacterial colonies act as tiny, locked bookcases that protect the information until the researcher wants it. Unlike regular libraries, the books in genomic libraries are created without a card catalog. Fortunately, the molecular biologist has ways to rapidly scan the library to find the book (gene) of interest.

Griffiths and Tiwari used their chicken C-W gene as a probe to find its equivalent in the hyacinth macaw DNA. They next amplified 104-base-pair stretches of DNA from the C-W and C-2 genes in the Spix's macaw. There are just a few nucleotide differences between the sequences, but it is enough so that they can be easily distinguished. The C-W sequence includes a spot that can readily be cut by a special enzyme into two pieces of different length. The Z chromosome has no comparable sequence. Because both male and female Spix's macaws have two copies of the C-2 DNA which is not on either W or Z, it should be present regardless of sex. But only a female will have C-W DNA; a male, which does not have a W chromosome, will not show evidence of the C-W gene.

It took two years for field biologists to collect just three feathers molted by the wild Spix's macaw. Griffiths and Tiwari extracted DNA from the tips of these hard-won feathers and compared it to the DNA from five birds of known sex that are in captivity. All the tests on the known males yielded a single DNA band, as did the test on the wild bird. It is a male.

In the hope that this would be the result, conservationists had for five years been training a captive female Spix's macaw to live in the wild, even giving it lessons in how to find and eat natural foods. Late in 1995, they released her into the jungle less than 100 yards from her potential mate. I wish the story had a happy ending, but it does not. Less than a week later the female disappeared, and is presumed dead. The one encouraging development is that the local people, who over the decades destroyed much of the Spix's macaw's habitat, have been moved by the campaign to save the bird from extinction, and are working with the conservationists. Thanks to captive breeding programs, there are now 39 birds, and more releases into the wild are planned.

DNA Testing in the Zoo

Many of the world's major zoos now turn to molecular genetics to help them reassess breeding programs to preserve endangered species. This sometimes leads to surprising findings and bitter controversy. Why? It is difficult and expensive to acquire the founder stock, and if a zoo purchases an animal that is later shown not to be what it was represented to be, it will not be able to use it. In 1995 Dr. Terry Maples, director of Zoo Atlanta, stopped a plan to breed a pair of rare Sumatran tigers when tests suggested that the big cat he had purchased was probably the offspring of a cross in the wild between a Sumatran and the nonendangered Bengal tiger. At a 1995 meeting of the American Association for the Advancement of Science, Maples opined that the Sumatran cat had been "polluted" by Bengal genes. What then is the big cat that is the offspring of such a mating? According to the standard view among evolutionary biologists, if the Suma-tran and the Bengal freely (even if infrequently) breed in the wild, they are members of the same species. Why then should Dr. Maples have halted the breeding program? Who decides the boundaries that define species and what constitutes a subspecies (or local variety) that is worth conserving?

India is home to dwindling populations of both Asiatic lions and Siberian tigers. Recent study of the DNA of 28 of the last 350 lions on the Indian subcontinent (many of which live in the Gir National Park in Gujarat) brought good news. The population appears to be sufficiently genetically diverse to have a decent chance of survival. DNA analysis of two wild Indian tigers brought less welcome news. They were found to carry gene variants usually only seen in Siberian tigers, which means that those two "species" now mate in the wild. Since there are many more Siberian tigers, in a few generations the wild Indian tiger will likely be superceded by a form that is much closer to the Siberian tiger, and a few decades hence, there will be no Indian tigers.

About 10,000 orangutans exist in the forests of Sumatra and Borneo, two large islands in Indonesia separated at one point by only about 50 miles of ocean. In Malay the word "orangutan" means "people of the forest." This reflects millennia of close contact. Until a century ago, the Indonesian people thought of these apes as primitive human relatives, a folklore that is easily understandable, especially if one spends time observing orangutans. It is not just that they look a lot like us; they also have behaviors like ours. They are smart too, capable, for example, of learning rudimentary sign language. Primatologists claim that they have taught individual orangutans to perform tasks as complex as picking locks, rowing boats, and making pancakes.

Fearful of the steady loss of habitat caused by the burgeoning human population, about 20 years ago the Indonesian government and a consortium of zoos in the United States set up crash breeding programs. At the time it was thought that orangutans from Borneo differed only slightly from those in Sumatra, that at most the two should be considered a pair of subspecies, and that forced crosses between them would be acceptable. This was largely because the two islands are thought to have been a single land mass until less than 100,000 (perhaps just 20,000) years ago. Conservation zoologists reasoned that not enough time had passed for them to evolve many distinct genetic differences. Furthermore, there was good evidence that for centuries humans had been transporting young orangutans, often as pets, between the islands, which suggests that the two groups had continued to mate until the quite recent past.

In the United States today there are about 80 orangutans that are the offspring of a Sumatran and a Bornean parent. All have been sterilized or placed on long-term birth control. Comparative DNA analysis of orangutans from each island indicates that they are much more different at the genetic level than was thought on the basis of comparing physical and behavioral differences. The most experienced observers can determine to which island group the adult males belong, and no one can correctly assign adult females. The Bornean males tend to have rounder faces and darker fur; the Sumatran males tend to have hair that is redder and more curly. Genetically, the differences are more dramatic. In one subspecies part of the chromosome number two (in each species chromosomes are arbitrarily numbered according to length, with the largest being designated as number one) is inverted compared to that of the other. Chromosomal inversions, in which a big chunk of DNA breaks off, flips, and rejoins the rest of the chromosome, are relatively common. Among closely related species, examining the differences in the number and shape of chromosomes, because they can be discerned under the microscope, was one of the earliest methods used to get some indication of evolutionary divergence. At the chromosomal level, the two subspecies of orangutan are more different from each other than are lions from tigers. This, and the fact that careful study of key proteins has discovered significant differences in the sequence of amino acids of which they are composed, has led many scientists to argue that the orangutans constitute two distinct species.

By painstakingly comparing samples of mitochondrial DNA from the two groups, scientists have concluded that the sequence differences are sufficiently great to suggest that they diverged at least 20,000, and possibly hundreds of thousands of, years ago. If correct, the longer estimate is crucially important, for it suggests that the two groups became reproductively isolated long before they were physically isolated by an ocean. This means that other forces had to be at work and, if true, is a powerful argument in favor of two distinct species. But opinion is not unanimous. One scientist, Dr. C. Cam Muir, a geneticist at Simon Fraser University in British Columbia, who studied the precise nucleotide sequence of five orangutan genes, concluded that the two groups were much too closely related to be called different species.

We are left with an odd situation. Over the last 30 years, we have decided that Sumatran and Bornean orangutans were no more different from each other than say Japanese are from Swedes. Realizing that they were on the brink of extinction in the wild, zoo programs have mated orangutans which are the descendants of animals born on different islands to each other. Now on the basis of DNA studies, some zoologists have decided that they have created a hybrid which, if it ever existed in nature, never took hold, and have decided that it should not reproduce. A few scientists have argued that this is a form of "primate racism" that recalls the 19th century obsession with creating a typology of humans in which northern Europeans were awarded the top spot. At the least, the sterilization of orangutans seems to be a bizarre feature of an effort to prevent extinction.

The fact is that there is no definitive way to say that two closely related varieties belong to one or two species. In the wild, the key criterion is reproductive behavior. With what animal does the animal in question mate? Even here, the edges are fuzzy. Depending on the situation, an animal that almost always mates with its own kind may drift just a bit to mate with a member of a group that is very closely related (as in the case of the Indian tigers). The current obsession with defining the perimeter of the orangutan species is by its very nature an arbitrary, some might argue foolish, exercise. The fact is that we know very little about orangutan DNA, surely much less than we know about their bodies and their behaviors. We must be a bit wary of making too much of DNA differences, which may turn out sometimes to mean less than we currently think. One wonders if Atjeh, the 33-year-old hybrid orangutan at the National Zoo in Washington, will someday be able to get his vasectomy reversed.

Saving the Florida Panther

The Florida panther (P. concolor coryi) may be the world's most endangered subspecies of big cat. Surrounded by millions of their only predator, humans, by 1967 there were only about 30 of these big, gray-tinted cats teetering on the edge of extinction in the Big Cypress National Preserve in south Florida. For many generations, virtually all of the cubs have been born to matings between genetically close relatives, often siblings. To any veterinarian who examines one of these animals, the harmful consequences of inbreeding are immediately obvious. In addition to innocuous features like kinked tails and cowlicks, nearly all the adult males have crypt-orchidism, an inherited defect causing one or both testicles to fail to descend into the scrotum, which reduces their fertility. About 80% of the cubs have significant heart murmurs, and in recent years three of them have died of atrial septal defects (holes in the wall that separates the two upper chambers of the heart). Virtually all the Florida panthers are badly infected with parasites, probably the result of a genetically weakened immune system.

The plight of the Florida panther is even more desperate than that of the orangutan. In 1992 the World Conservation Union warned that unless there was a sustained effort to repopulate the species, it would be extinct by 2055. In response, in 1995 the U.S. Department of the Interior, working in conjunction with state authorities in Texas and the Florida Fish and Wildlife Conservation Commission, captured eight female Texas cougars and resettled them in the Big Cypress Swamp. Zoologists chose this closely related subspecies because until about 150 years ago its natural habitat overlapped that of the Florida panther. If they found mates among the remaining male Florida panthers, the offspring could constitute a desperately needed dose of new genes for the highly inbred group. Less inbred animals would not enter life with two genetic strikes against them.

At first the rescue operation looked like it would fail. Three of the eight females uprooted from the arid climate in West Texas died. But the remaining five did successfully mate, producing 17 healthy kittens. Many of them have now reproduced, in some cases with a Texas cougar and in other instances with a hybrid offspring from one of the other four litters. In late 1999 zoologists estimated that the population of panthers in the Big Cypress Swamp had grown to 50-70 adults and kittens, and it looked like a robust breeding population would soon be reestablished.

Once again, evolutionary purists have begun to argue that if these two subspecies breed well and expand the population of pumas, the program will not have saved the Florida panthers. Instead, it will have created yet a new variety of big cat in south Florida. In the words of David Maehr, an assistant professor of forestry at the University of Kentucky who once led the recovery effort, the crossbreeding "is spiraling out of control." He thinks that if it continues for even a few more years, the distinctive physical features of the Florida panther will vanish. Dr. Stephen O'Brien, one of the world's authorities on the genetics of cat species, strongly disagrees. He has shown that throughout the Americas the various subspecies of puma (known regionally as mountain lions, cougars, catamounts, and panthers) interbreed. Thus, it is highly likely that until human encroachment trapped them in a small habitat, Florida panthers routinely mated with members of sister subspecies. Indeed, some of the Florida panther's distinctive features probably arose only after it was trapped in the Big Cypress Swamp.

Conservationists hope to rebuild the panther population in Florida to 500 animals and to increase its range. Not surprisingly, as a few of the big cats have expanded their perimeter, people living in the region have protested. If the population remains under 500 and geographically contained to its current region, to counter the devastating effects of inbreeding conservationists will have to import new animals about every decade.

The cheetah, the world's fastest land animal, probably once came as close to extinction as the Florida panther is today. DNA studies have yielded evidence that the species passed through an evolutionary bottleneck, which means that all of today's animals are closely related. Studies of genetic variation among the big cats indicate that they have less than 10% of the genetic diversity expected in a species. Cheetahs are so inbred that it is very likely that a skin graft from any one to any other will take, indicating immunological identity! It also means that the species is in constant peril of being wiped out by a viral or other infection. In fact, cheetahs recently had a close brush with the feline infectious peritonitis virus, an agent that causes only mild symptoms in a tiny fraction of the house cats it infects. In them the virus is deadly; all the infected animals became seriously ill, and 60% died. Unfortunately, there are few places on earth where one can attempt to restore a natural cheetah population in the manner that is being attempted with the panther in Florida. Major zoos have been trying to breed cheetahs in captivity, but have had little success. Sometime in the 21st century, we will have to launch breeding programs for the cheetah as intensive as those currently under way for the Florida panther or we will lose that wonderful animal from the planet.

The rescue of the whooping crane from the brink of extinction pro vides good evidence that the effort to save the Florida panther can succeed. In 1941 there were only 22 whooping cranes left in the world. Since that time continuous and caring intervention by man, largely through the work of the International Crane Foundation headquartered in Baraboo, Wisconsin, has brought the population to about 240. DNA testing will soon play an important part in the continual rebuilding of the whooping crane population. Conservationists plan to study the DNA of the young adults and arrange matings that maximize the chance for genetic diversity in offspring. This should result in higher survival rates and a more robust population.

Why take on the quixotic task of trying to save nearly extinct primates, cats, birds, or some of our even more distant relatives? If neither reverence for life nor an aesthetic appreciation of nature is enough, there is a much more powerful rationale with which to persuade the doubters who would prefer to pave paradise and put up a parking lot. The world's biota, the sum total of living things, contain within their DNA information that will, if preserved, studied, and understood, offer humans dazzling new knowledge about disease and powerful new medicines to fight the infections that have always and will always plague humans. Every time the evolutionary light winks out for a species as seemingly inconsequential as an insect with a restricted range in the Brazilian rain forest canopy, we may have lost our chance to find a wonderful agent in the fight against cancer or AIDS. Of the world's ten most prescribed pharmaceuticals, nine derive from natural sources.

The black plague killed off more than a quarter of the population in Europe in the 14th century. Until it gradually attenuated, syphilis killed millions of Europeans and Asians as it spread across the globe in the 16th century. Measles and smallpox, introduced into the Americas and Polynesia by explorers, also killed millions. In the 19th century, tuberculosis, the white plague, was the number one cause of death among adults on both sides of the Atlantic. Although we have no idea what they were, over the millennia untold numbers of plagues have wiped out huge fractions of various animal and plant species. My point is that within the genomes of all living species there are molecular secrets indicating why some individuals survived to reconstitute the population. These data can provide genetic information with which to meet similar new attacks in the future. A compelling example is the discovery that a small number of humans carry a variant gene that renders them highly resistant to developing AIDS. The HIV virus can infect them, but cannot cause the full-blown disease. Study of this gene may point us in an important new direction in drug development.

The pharmaceutical industry has long screened countless compounds from nature to find the few that will become important new drugs. They have not even scratched the surface of biodiversity. Although there is great current interest in developing an in-house approach (called combinatorial chemistry) to generating important new compounds to treat disease, it would be unwise to ignore the wealth still hidden in nature. Companies such as Shaman Pharmaceuticals, a small botanical prospecting company in California, have the right idea. Their scientists are trying to draw upon the knowledge of indigenous peoples about locally used medicines to find important new compounds.

There are also many secrets to be found by studying the genomes of organisms that one is trying to protect. A strain of wild mouse in California was recently found to be highly resistant to a viral infection that was devastating other strains. Closer study showed that at some point in the past, the resistant strain had successfully integrated a portion of the viral DNA into its genome, making it essentially immune. As many as 3,000,000 DNA base pairs in our genome may be the "genetic fossils" of viruses that have infected our species or the primate from which it arose over the last 10 million years. It may be that the secret to halting the spread of future plagues lies in the analysis of human DNA sequences to retrace our ten-million-year struggle with viruses and bacteria.

Five cloned piglets, Millie, Christa, Alexis, Carrel, and Dotcom, created by the scientists at PPL Therapeutics. (Courtesy of PPL Therapeutics Ltd.)

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