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Nicholaus Wade, New York Times, August 8, 1999

Leaf-cutting ants and their fungus farms are a marvel of nature and perhaps the best known example of symbiosis, the mutual dependence of two species.

But the textbook accounts, it turns out, do not tell even half the story. From research in the past five years the ants' symbiosis has emerged as far more intricate than it appears, involving not two but at least four species, their lives knotted together in a ruthless yet highly successful struggle for survival.

The ants and their agriculture have been extensively studied over the years, but the recent research has uncovered intriguing new findings about the fungus they cultivate, how they domesticated it and how they cultivate it and preserve it from pathogens.

For example, the fungus farms, which the ants were thought to keep free of pathogens, turn out to be vulnerable to a devastating mold, found nowhere else but in ants' nests. To keep the mold in check, the ants long ago made a discovery that would do credit to any pharmaceutical laboratory.

The fact that there was still so much to learn about leaf-cutter ants and their agriculture, biologists say, only underlines how much remains to be discovered about the world's plants and animals.

Ants invented agriculture 50 million years before people did, and the leaf-cutters, members of a large family called the attine ants, practice the most sophisticated example of it.

They grow their fungus, a kind of mushroom, in underground chambers that can reach the size of a football.

A single leaf-cutter nest may contain a thousand such chambers, embedded in an underground metropolis up to 18 feet deep, and support a society of more than a million ants.

These ant communities are the dominant plant-eaters of the Neotropics, the region comprising South and Central America, Mexico and the Caribbean. Biologists believe some 15 percent of the leaf production of tropical forests disappears down the nests of leaf-cutter ants.

In the nest the leaves are shredded and inoculated with the fungus, which digests them and is in turn eaten by the ants.

The ants' achievement is remarkable -- the biologist Edward O. Wilson has called it "one of the major breakthroughs in animal evolution" -- because it allows them to eat, courtesy of their mushroom's digestive powers, the otherwise poisoned harvest of tropical forests whose leaves are laden with terpenoids, alkaloids and other chemicals designed to sicken browsers.

So precious is their particular strain of fungus that the ants' virgin queens, before their nuptial flight, secrete a mouthful with which to seed the garden of their new nest. The worker castes they produce are so tailored to the craft of fungus gardening that they come in made-to-fit sizes -- large ants to saw off leaves, medium ones to shred them and miniature workers to seed them with fungus and clean off all alien growths.

Fungus growing seems to have originated only once in evolution, because all gardening ants belong to a single tribe, the descendants of the first fungus farmer. There are more than 200 known species of the attine ant tribe, divided into 12 groups, or genera. The leaf-cutters use fresh vegetation; the other groups, known as the lower attines because their nests are smaller and their techniques more primitive, feed their gardens with detritus like dead leaves, insects and feces.

A question that has long perplexed ant biologists is whether the funguses cultivated by attine ants are all descended from a single ancestor, just as the ants are. The issue was hard to settle because the ants' gardening habits prevent the fungus from forming mushrooms, the spore-bearing stage by which mycologists tell one fungus from another.

In 1994 a team of four biologists, Ulrich G. Mueller and Ted R. Schultz from Cornell University and Ignacio H. Chapela and Stephen A. Rehner from the United States Department of Agriculture, analyzed the DNA of ant funguses. The common assumption that the funguses are all derived from a single strain, they found, was only half true.

The leaf-cutters' fungus was indeed descended from a single strain, propagated clonally, or just by budding, for at least 23 million years. But the lower attine ants used different varieties of the fungus, and in one case a quite separate species, the four biologists discovered.

After further study, three of the biologists, Dr. Mueller, Dr. Rehner and Dr. Schultz, reported last year that funguses grown by lower attine ants fell into four groups of varieties, as if the ants had domesticated wild funguses at least four times in evolutionary history. Two of those occasions must have been quite recent because the biologists identified free-living counterparts for two of the four fungus groups they found in the ants' gardens. A single variety of fungus is grown in each nest, but most lower attine species cultivate at least two of the four fungal lineages, suggesting that varieties are exchanged among species every so often, the biologists concluded.

What evolutionary force could be driving these two patterns of fungus gardening, the pure clone cultivation of the leaf-cutters and fungus exchange program of the lower attines? The answer, or part of it, has been divined by Cameron R. Currie, a Ph.D. student in a climate no wild attine ever reached, the University of Toronto. Trained as an entomologist, Currie was attracted to the ants because of his interest in symbiosis and in the cheaters who take advantage of that mutualism.

The pure strain of fungus grown by the leaf-cutters, it seemed to him, resembled the monocultures of various human crops, that are very productive for a while and then succumb to some disastrous pathogen, such as the Irish potato blight. Monocultures, which lack the genetic diversity to respond to changing environmental threats, are sitting ducks for parasites. Currie felt there had to be a parasite in the ant-fungus system. But a century of ant research offered no support for the idea. Textbooks describe how leaf-cutter ants scrupulously weed their gardens of all foreign organisms. "People kept telling me, 'You know the ants keep their gardens free of parasites, don't you?' " Currie said of his efforts to find a hidden interloper.

But after three years of sifting through attine ant gardens, Currie discovered they are far from free of infections.

In last month's issue of the Proceedings of the National Academy of Sciences, he and two colleagues, Dr. Mueller and David Mairoch, isolated several alien organisms, particularly a family of parasitic molds called Escovopsis.

Escovopsis turns out to be a highly virulent pathogen that can devastate a fungus garden in a couple of days. It blooms like a white cloud, with the garden dimly visible underneath. In a day or two the whole garden is enveloped. "Other ants won't go near it and the ants associated with the garden just starve to death," Dr. Rehner said. "They just seem to give up, except for those that have rescued their larvae." The deadly mold then turns greenish-brown as it enters its spore-forming stage.

Evidently the ants usually manage to keep Escovopsis and other parasites under control. But with any lapse in control, or if the ants are removed, Escovopsis will quickly burst forth.

Although new leaf-cutter gardens start off free of Escovopsis, within two years some 60 percent become infected. The discovery of Escovopsis's role brings a new level of understanding to the evolution of the attine ants. "In the last decade, evolutionary biologists have been increasingly aware of the role of parasites as driving forces in evolution," Dr. Schultz said. There is now a possible reason to explain why the lower attine species keep changing the variety of fungus in their mushroom gardens, and occasionally domesticating new ones -- to stay one step ahead of the relentless Escovopsis.

Interestingly, Currie found that the leaf-cutters had in general fewer alien molds in their gardens than the lower attines, yet they had more Escovopsis infections. It seems that the price they pay for cultivating a pure variety of fungus is a higher risk from Escovopsis. But the leaf-cutters may have little alternative: they cultivate a special variety of fungus which, unlike those grown by the lower attines, produces nutritious swollen tips for the ants to eat.

Discovery of a third partner in the ant-fungus symbiosis raises the question of how the attine ants, especially the leaf-cutters, keep this dangerous interloper under control. Amazingly enough, Currie has again provided the answer.

"People have known for a hundred years that ants have a whitish growth on the cuticle," said Dr. Mueller, referring to the insects' body surface. "People would say this is like a cuticular wax. But Cameron was the first one in a hundred years to put these things under a microscope. He saw it was not inert wax. It is alive."

Currie discovered a specialized patch on the ants' cuticle that harbors a particular kind of bacterium, one well known to the pharmaceutical industry, because it is the source of half the antibiotics used in medicine. From each of 22 species of attine ant studied, Cameron and colleagues isolated a species of Streptomyces bacterium, they reported in Nature in April.

The Streptomyces does not have much effect on ordinary laboratory funguses. But it is a potent poisoner of Escovopsis, inhibiting its growth and suppressing spore formation. It also stimulates growth of the ants' mushroom fungus. The bacterium is carried by virgin queens when they leave to establish new nests, but is not found on male ants, playboys who take no responsibility in nest-making or gardening.

Because both the leaf-cutters and the lower attines use Streptomyces, the bacterium may have been part of their symbiosis for almost as long as the Escovopsis mold.

If so, some Alexander Fleming of an ant discovered antibiotics millions of years before people did.

Even now, the ants are accomplishing two feats beyond the powers of human technology. The leaf-cutters are growing a monocultural crop year after year without disaster, and they are using an antibiotic apparently so wisely and prudently that, unlike people, they are not provoking antibiotic resistance in the target pathogen.

In a loose team, the four biologists involved in the new findings are seeking to understand the deeper intricacies of the extraordinary system. Dr. Schultz, now at the Smithsonian Institution in Washington, D.C., is a specialist in the evolutionary relationships of ants, and Dr. Rehner, of the University of Puerto Rico, is an expert on fungi. Dr. Mueller, now at the University of Texas at Austin, is an ecologist Together with Dr. Currie, they hope to unravel the relationships woven between the four members of the symbiosis -- the attine ants, their mushroom, Escovopsis and the Streptomyces bacterium. There are doubtless other members to be discovered.

The four researchers are pleased but not much surprised to have discovered so much new about an already well studied system.

"It may be one of the best studied symbioses in biology but that is a sad reflection on how little we know in general," Dr. Schultz said.

Discovery of the deadly Escovopsis fungus could change the correlation of forces between ants and people. In the 10,000 years since the ants have had to deal with human agriculture, a blink of an eye in their 50 million year farming history, the ants have generally prevailed. They thrive in disturbed ground, and so have usually benefited from the clearing of forests.

For the most part, people and the attine empire have co-existed peacefully: the two species live on different scales and in separate spaces, people above ground and attines below it. But attines are a serious agricultural pest in much of the tropics. Currie, having shown that nests in the lab are devastated with a squirt of Escovopsis spores, suggests the fungus might prove a useful way of controlling attine nests.

There seems little objection to applying Escovopsis nest by nest. But Dr. Schultz expressed horror at indiscriminately wiping out all fungus-growing ants, many species of which are harmless.

That an Escovopsis-like discovery could tip the scales too far toward the human side was foreseen by the Dr. Wilson and his colleague Bert Holldoumlbler. In their book "The Ants," published in 1990, they wrote that biologists need to search for "the weak points" in the ants' social system: "The goal, however, should be intelligent management of their populations and never their complete eradication. Our advantage -- and responsibility -- lies in the fact that we can think about these matters and they cannot."
Matt Brennan