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 Berkeley researchers Vance Vredenburg and Tate Tunstall collect data on infected frogs at Kings Canyon National Park. (Vance T. Vredenburg photo)

New findings in the Sierra's fungus/frog faceoff
Killer fungus could be a greater threat to the mountain yellow-legged frog's survival than previously realized

| 22 August 2007

A deadly fungus that has decimated populations of mountain yellow-legged frogs in the Sierra Nevada can likely be spread by sexual reproduction, seriously complicating efforts to save the frogs from extinction, according to a new genetic analysis led by Berkeley researchers.

The dramatic decline of the species over the past several decades has been attributed to the introduction of non-native predatory fish in some areas and to chytridiomycosis, a quickly spreading disease caused by the waterborne fungus Batrachochytrium dendrobatidis.

The study, recently published in the Proceedings of the National Academy of Sciences, suggests that the frog-killing fungus may play the bigger role in the frog's demise because of the pathogen's ability to spread over long distances and possibly persist in the environment as a consequence of sexual reproduction, according to the researchers.

"This group of fungi, when it reproduces sexually, can create spores that can last for a decade," says John Taylor, a professor of plant and microbial biology and principal investigator of the study. "That could make this pathogen a harder problem to defeat. As a resistant spore, the fungus could be transported by animals, including humans or birds, or lie dormant in an infected area until a new host comes along."

Biologists are still determining exactly how this fungus, identified in 1998, kills the amphibians it infects, but most believe that the pathogen disrupts the frogs' ability to absorb water through their skin.

In the western United States the fungus has been spreading quickly, moving west to east across the Sierra Nevada at a pace of about a mile per year, according to the researchers. Tens of thousands of mountain yellow-legged frogs in hundreds of sites have virtually disappeared in the wake of the pathogen's emergence in the area.

The researchers set out to determine which of two competing hypotheses for the origin of chytridiomycosis is more plausible. If the fungus were recently introduced to an area, the researchers would expect to find a single genotype that had spread by clonal reproduction. If, however, the fungus is endemic to a region, they would expect to find diverse genotypes resulting from a long history of association that provides enough time for isolates to diverge through mutation and genetic recombination. The animals in the area would, under those circumstances, normally be resistant to the pathogen's destructive effects because they would have co-evolved together. However, biologists theorize, changes to the environment - from global warming to pollution from agricultural chemicals - could make native frog populations susceptible to a pathogen with which they've previously co-existed.

According to the study, neither epidemic spread nor endemism alone explains the decline of these frogs. "We found sites dominated by a single fungal genotype, which suggests a recent spread of the pathogen through clonal reproduction," says lead author Jess Morgan, who was a post-doctoral researcher working with Taylor at the time the study was conducted. "But this study also provides the first evidence of genetic recombination in B. dendrobatidis, which results in multiple, related genotypes and signals that sexual reproduction is occurring."

The findings could help explain the global spread of this pathogen, which has also been found in South America, Australia, Europe, and Africa, say the researchers. While human-assisted spread is possible, the fungus has infected amphibians in pristine areas too remote for human activity.

"Up until now, people thought the movement of this pathogen was mainly via infected frogs, so such measures as restrictions on the pet trade were put in place," says Morgan. "If, in fact, this fungus produces resistant spores, people could be unwittingly transferring this pathogen around the world from dirt on our shoes or car tires. But spores could also hitchhike on the feathers of birds for quick transport across mountain ranges."

Moreover, if resistant spores remain in lakes where previous populations of frogs have succumbed to chytridiomycosis, attempts to repopulate the lakes with healthy frogs will likely fail. A number of such efforts conducted by researchers from Berkeley and UC Santa Barbara, at remote lakes in the Sierra Nevada where previous frog populations had been wiped out by chytridiomycosis, have met just that fate: Out of 10 reintroduction attempts over the past four years, seven have failed.

A challenging search for spores

Although genetic testing should be able to detect the spores, scientists do not know where to look for them. In addition, blindly testing environmental samples has thus far failed to yield evidence of the spores.

Infected frogs can be treated with fungicides to remove the pathogen, but researchers say it is not a practical long-term solution since the amphibians would remain susceptible to re-infection if returned to the same lake.

To conduct the study, researchers collected two species of mountain yellow-legged frogs from six sites in the Sierra Nevada (three in the southern portion of the range, and three in the north), cultured fungi samples from 100 of the amphibians, and arranged for sequencing of the pathogen's genome. They then compared genetic markers for the Sierra Nevada B. dendrobatidis samples with fungi collected from other regions around the world.

"The genotypes of our fungi in the Sierra are not that different from genotypes found around the world," says Taylor. "That means there must be someplace else on Earth where this fungus is endemic. One would guess that the frogs living where the ancestral population of this fungus is located would not be affected that badly. We could then try to determine the mechanisms those frogs use to resist the pathogen."

The study also found that two sites near Tioga Pass Road in Yosemite National Park contained two similar populations of fungi. Because the lakes where the frogs were found are 40 kilometers apart, the evidence is strong that movement of the fungi between the two locations was somehow assisted by humans, say the researchers.

"If we confirm that spores are a factor, then there may be precautions we can take to contain their spread," says Morgan. "This could involve cleaning shoes before moving from one infected site to another. Some fungi produce spores during certain times of the year. If that is the case with this fungus, we could consider restricting public access to infected sites during those times."

As the U.S. Fish and Wildlife Service considers listing the mountain yellow-legged frog as an endangered species, biologists are racing to find ways to stanch the spread of the frog-killing fungus.

"This frog used to be the most abundant amphibian, and perhaps the most abundant vertebrate, in the whole Sierra Nevada," says Knapp. "Over the past 30 years it has disappeared from up to 95 percent of its historic range, and its absence is impacting other organisms. Garter snakes that used to prey on these frogs are now declining. The frog's decline is leading to an unraveling of a high-elevation ecosystem."

The study was part of a larger project on chytridiomycosis and the mountain yellow-legged frog led by co-author Cheryl Briggs, a Berkeley associate professor of integrative biology.