By Janet Fang

A Panamanian park has lost around 40 percent of its amphibian species in the past decade, with some dying out before biologists had even learned of their existence, according to research published July 19 in the Proceedings of the National Academies of Science. Combining genetics with nearly ten years of field surveys, biologists discovered 11 new species, only to find that five of them are already extinct in the area.

"We're losing things before we find them," says Andrew Crawford, an evolutionary geneticist at the University of the Andes in Bogotá, Colombia, and lead author of the study.

The disease chytridiomycosis, caused by the chytrid fungal pathogen Batrachochytrium dendrobatidis, threatens more than 2,800 amphibian species worldwide. Amphibians infected by the disease have skin several times thicker than normal, which affects their ability to breathe and the transfer of electrolytes.

Anticipating the arrival of B. dendrobatidis in El Copé, Panama, as a wave of infection advanced from the northwest of the country, co-author Karen Lips, a biologist at the University of Maryland in College Park, began leading field surveys in the 4-kilometer area in 1998. Her team set up transects, walking along 100-meter lines, marking down the species observed and collecting samples. The epidemic hit in 2004, enabling researchers to conduct a before-and-after comparison.

After sequencing toe clippings or liver samples from 297 dead specimens collected before the decline, the researchers found that the area was home to 63 known species. By using DNA barcoding--which involves sequencing standardized DNA marker fragments to match specimens with known species--Crawford and his co-workers identified a further 11 species that were previously unrecognized.

Thirty of these species are now extinct in the area, including "five that were wiped out before we even knew they were there," says Crawford. This brings the total loss of amphibian lineages to 41 percent. Naming a species that is already extinct was "pretty sobering," says Crawford.

Branching out

The researchers also looked at amphibian evolutionary history, which considers all the frogs, salamanders and caecilians that have existed over millions of years.

Conservationists use species as a category to describe what needs protecting. The International Union for Conservation of Nature, based in Gland in Switzerland, for example, has an authoritative list of threatened species. But, according to Crawford, it is not always the best measure of diversity, especially when the relationship between species is not very clear. "There are so many debates on whether something is one species or two," he says. "And rather than fight about it, we made an evolutionary tree and looked at what percentage of branches was lost."

Crawford and his team mapped out the history and relationships--such as their common ancestors and when they split from other species--of all the species they found in the area. They found that 33 percent of all the branches in this evolutionary tree are gone--El Copé has lost 33 percent of the total history of all its amphibians.

"Up until now, we've only had a very crude estimate of what is lost. It has just been, 'We've lost a lot of species,'" says Vance Vredenburg, a biologist from San Francisco State University in California, who recently published on the dynamics of the disease. Species could have separated from each other in the past 20 years or in the past 20 million years, he says.

"What they did was quantify it," adds Vredenburg. "They took what we knew and added significance."

Crawford and his colleagues also looked at where extirpations occurred in the amphibian tree. Previous studies found patterns suggesting that some lineages and groups are hit harder than others, but in El Copé, the disease hit across the entire range of evolutionary history, wiping out some lineages and not others in a random way.

What now?

"The fungus is moving fast," says Crawford, who is conducting rapid surveys in the easternmost province of Panama before the fungus hits the area.

Animals can be cured with anti-fungal solutions, but the problem is how to introduce healthy populations into the wild without causing re-infections. "There's a lot of hope, hypotheses and preliminary data, but no solution," says Crawford. In the meantime, herpetologists are attempting to preserve animals by removing them from their natural habitat.

Another conservation method being explored is the use of probiotics. Frogs and salamanders have symbiotic bacteria growing on their skin, defending them against the fungus. Vredenburg and his team are taking bacteria from healthy populations in the wild and culturing them in the lab--hoping to inoculate populations with heavy doses of their own beneficial bacteria, "to give them a big immune boost so that they have a fighting chance."

Amphibians are the oldest class of existing four-legged vertebrates, having been around for 300 million years. "Part of what's so alarming is that these long-term survivors are dropping off the face of the Earth right now," says Vredenburg.