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DNA Detectives Hunt For Forgotten Frog

Frog on green branch
One of the frogs discovered via eDNA analysis. It hasn’t been seen since 1977, but it’s out there! Leo Malagoli

Loaded with dedication and innovation, a team of researchers from Cornell University hiked into the forests and grasslands of Brazil to try to figure out if they could find a frog that was thought to be extinct since 1968. Spoiler alert: they believe they did!

The frog Megaelosia bocainensis has been known only from a museum sample since 1968. But just because humans hadn’t seen one in over 50 years didn’t necessarily mean that the frogs were truly extinct, reasoned the researchers.

Brown frog on black background
1968 Museum sample of Megaelosia bocainensis Délio Baêta

So in carrying out their frog hunt, they hiked to Brazilian streams and ponds carrying battery packs, a small pump, and tubes. The setup was used to siphon off water through a special filter that could hold on to any environmental DNA (eDNA) that passed through. Once the filter was saturated, the capsule in which it was contained was sealed up to preserve it and brought back to the labs, where the work truly began.

Finer and Finer

You can imagine how many bits of eDNA might be floating in pond and river samples in areas full of life. So the researchers had to narrow things down. They initially searched for 30 target species where bocainensis and other missing or rare frogs were once seen: 13 were presumed extinct, 12 were no longer found in their original habitats but were still found elsewhere, and five had been abundant in the areas of the study, but had since become tough to find.

As part of the winnowing process, they had to eliminate DNA from other animals, including humans, chickens, and pigs. Once that was done, they were able to see which bits of frog DNA were floating in the samples.

“Now you’ve got a subset of genetic sequences that we know only belong to frogs, and then it’s step by step, going finer and finer until you get to the genus and species you are looking for,” said senior author Kelly Zamudio, the Goldwin Smith Professor of Ecology and Evolutionary Biology in Cornell’s College of Arts and Sciences.

Because there are no DNA samples from M. bocainensis in existence,  the researchers had to use a process of elimination. By comparing the DNA found in the water to the known DNA sequences of “sister frogs” in the Megaelosia family, they were able to narrow things down to one remaining sequence that didn’t match any of the other frogs, which they think has got to be Megaelosia bocainensis—although the researchers do acknowledge the limitation of their method as well as the need for sampling from nearby areas.

“We know there’s a Megaelosia there,” Zamudio said, “we just don’t know which one it is, but the only one that has ever been reported there historically is the one that went missing. Do we believe it? That’s how far the analysis can take us.”

On top of potentially confirming M. bocainensis, the team was also able to verify the existence of four of the declined species and two locally disappeared species, adding to the idea that analyzing eDNA is a valid and valuable approach when searching for species in the wild.

The research has been published in the peer-reviewed journal, Molecular Ecology.