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Brazilian frog believed ‘extinct’ for 50+ years, found with eDNA testing

  • A Brazilian frog species, Megaelosia bocainensis, thought to have gone extinct in 1968 has been found with eDNA testing, which picks up the traces of environmental DNA that are left behind by living organisms in soil, water and air.
  • The missing frog’s eDNA was detected in the Atlantic Forest biome in Parque Nacional da Serra da Bocaina, its last known habitat in São Paulo state, Brazil.
  • The researchers used metabarcoding — a form of rapid DNA sequencing — in order to monitor entire communities, rather than only specific rare target species.
  • The innovative highly sensitive eDNA sampling technique provides a valuable tool for conservation scientists to evaluate the status of threatened species and to confirm the presence of species that are difficult to monitor and often go undetected using traditional methods.
Phasmahyla guttata, also known as the spotted leaf frog, is endemic to the Atlantic Forest, which is where Megaelosia bocainensis went missing 52 years ago, and where its eDNA was recently found. However, scientists have yet to see M. bocainensis in the flesh. Image by Leo Malagoli.

Scientists in Brazil have found evidence of a frog that was previously believed to have become extinct more than 50 years ago, marking a significant step forward for the innovative environmental DNA (eDNA) testing technique.

Fieldwork targeted at detecting traces of particular endangered and low-population species living in the Atlantic Forest and the Cerrado savanna grasslands uncovered the continued existence of Megaelosia bocainensis, a frog known for its rounded snout and granular skin that had not been seen since 1968 in its São Paulo state Atlantic Forest habitat.

Researchers, including scientists from Cornell University, singled out 30 frogs across 6 sites for analysis, of which there were 13 species that had totally disappeared, 12 species that had disappeared locally, and five species that had become hard to find. The study, published in the journal Molecular Ecology, found a total of seven species — including a number that had either declining populations or had disappeared entirely from local sightings — through use of eDNA surveys.

“The technique has been used for many years,” says Carla Martins Lopes, lead author of the paper and a researcher at São Paulo State University in Brazil. “But the difference with our study is that we have applied the technique on such a huge scale of species. For conservation it’s really helpful, because by using environmental DNA you don’t need to directly see the species and it’s not an invasive process — you don’t need to manipulate the animal and you don’t need to disturb the environment — and it’s cheap.”

Carla Martins Lopes performs eDNA sampling. Image by Célio Haddad.

The research team hiked into Brazilian sampling sites to do the eDNA surveys, which pick up the traces of DNA that are left behind by living organisms in soil, water and air. They collected up to 60 liters of stream or pond water at a time using a special filter to capture and preserve the DNA, which was then extracted and genetically sequenced in the lab to create “metabarcodes” of information.

Key to that analysis was a step in which the researchers removed genetic material from humans, pigs, chickens, and other locally common organisms found in their water samples, until finally they isolated the frog DNA.

But since Megaelosia bocainensis had seemingly vanished from the Parque Nacional da Serra da Bocaina 52 years ago, there were no tissues from which to extract DNA in order to compare it with today’s eDNA. The frog is, in fact, only known from a single museum specimen collected in 1968, and is currently listed by the IUCN Red List as Data Deficient, with the entry lacking a photo.

However, the researchers, who drew on local museum collections, did have the sequences for all the sister species in the genus Megaelosia and they also knew the ranges of those sister species and M. bocainensis — that all helped them pinpoint the missing frog. Though no one, to this point, has actually seen it.

Vitreorana eurygnatha, a small glass-frog found in the Atlantic Forest. Image by Celio F. B. Haddad.

Jos Kielgast, program officer of the IUCN amphibian specialist group and an eDNA expert at the University of Copenhagen, who was not involved in the study, believes the findings are “remarkable” because the researchers used metabarcoding — a form of rapid DNA sequencing — in order to monitor entire communities, rather than only specific rare target species.

“Using eDNA for monitoring wildlife has the potential to be a game changer,” says Kielgast. The research team’s “findings warrant optimism and potential for action to ensure that these species persist in a fragile Brazilian environment. However, at the moment it remains a subject for basic science innovation and research and not a substitute for traditional monitoring methods applied by governments and in other official frameworks.”

Joel Corush, a postdoctoral research fellow at Wayne State University’s Department of Biological Sciences, who was also not involved in the study, believes the highly sensitive eDNA sampling technique provides a valuable tool for conservation scientists to evaluate the status of threatened species and to confirm the presence of species that are difficult to monitor and often go undetected using traditional methods.

“Studies like this, that include data deficient or endangered species, provide critical knowledge on species that we can’t easily study,” says Corush, who has been studying eDNA since 2012, including fieldwork into the detection of invasive Asian carp in the U.S. Great Lakes. “When you’re planning conservation and you have better data, you can better prioritize areas and protect,” he says.

The IUCN Red List entry page for the long missing Megaelosia bocainensis. Screenshot on October 2, 2020.

Corush notes, however, that it’s important to continue traditional sampling, in order for scientists to understand fundamental information including the behaviors of any given species. “The processes complement each other well, and neither technique tells the full story,” he adds. “But the use of eDNA is a perfect case of finding the conservation community using creative tools to analyze the environment. While the methods are still developing, it’s a rare positive for biodiversity.”

Previous research involving eDNA has successfully uncovered evidence of the rare golden tree frog (Phytotriades auratus) on the island of Trinidad in the West Indies and white sharks (Carcharodon carcharias) in southern California. But recent progress in understanding how temperature, light and weather affect eDNA has improved the quality of data obtained. In 2020, the technique has been used by the University of Arizona to analyze the sewage system on college campuses to assess the potential prevalence of coronavirus.

The prospect is that eDNA will also help battle diseases in the natural environment. Lopes and her team are also currently analyzing eDNA from more recent fieldwork in Paraná and Rio Grande do Sul, in the south of Brazil, attempting to glean information about the spread of Batrachochytrium dendrobatidis, also known as Bd, a fungal disease that has pushed some amphibian species to near extinction.

“This could prove crucial in building knowledge about the spread of the disease” says Lopes.

Banner image: Researcher Carla Martins Lopes performs eDNA sampling. Image by Célio Haddad.

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Phasmahyla exilis, also known as the mottled leaf frog, is found in the state of Espírito Santo. Brazil is known for its vast array of spectacularly colored frog species. Image by João Luiz Gasparini.