For her doctoral dissertation, Yale’s Nathalie Alomar decided to study a small amphibian that appeared to have eluded the forces of evolution.

She found that there is more to its evolution than meets the eye.

In a new study, Alomar and a team of scientists report that the story of the common woodland salamander — long considered a classic example of “evolutionary stasis,” meaning that it has evolved into many species without changing its overall structure much at all — is more complicated than previously believed.

For the research, the team collected nearly 300 individual woodland salamanders — which are from the genus Plethodon, and ubiquitous in forests of the eastern United States — representing 30 distinct species. And while these animals looked quite similar, a subsequent laboratory analysis found that aspects of their physiology differed substantially.

Specifically, traits such as resistance to water loss, metabolic rate, and cold tolerance varied widely among the species, who appear to have evolved rapidly and differently largely in response to the climate of their habitats, they found.

The findings, published in The Proceedings of the National Academy of Sciences of the United States of America, challenge the widespread notion that evolutionary diversity can be judged primarily by an organism’s appearance.

Critically, they found, important physiological adaptations can occur even when body shape remains relatively unchanged. The project was part of a wider research program led by Martha Muñoz, an associate professor of ecology and evolutionary biology in Yale’s Faculty of Arts and Sciences.

“Many Plethodon species can look almost indistinguishable from one another, even when they come from very different regions,” said Alomar, a Ph.D. student in Yale’s Graduate School of Arts and Sciences. “But in the lab, I started noticing subtle differences, and it was exciting to see those qualitative impressions reflected in the quantitative patterns we found in the data.”

For the study, the researchers used specialized equipment to measure the salamanders’ skin resistance to water loss, metabolic rate, heat and cold tolerance, preferred temperatures, body size, limb proportions, and skull shape. They then combined these measurements with climate data and evolutionary analyses to determine how traits changed over time.

The results showed that physiological traits were strongly influenced by climate, and that internal physiology evolved faster than external, visible traits, the researchers say. Among the findings: heat tolerance changed relatively little among species, while how quickly the salamanders dried out when deprived of moist conditions varied more widely.

Appearance among the species was also found to not be completely static, however. Some subtle body and skull dimensions had evolved, the researchers said.

“This study was all about detecting the hidden inner workings of organisms,” said Muñoz.

“I’ve always been fascinated by what goes on under the hood,” she said. “These salamanders have a body plan that’s changed little for tens of millions of years. From an evolutionary biologist’s perspective, it’s a puzzle. It turns out that the diversity was there all along. It just wasn’t obvious from what meets the eye.”

In the work, researchers collected salamander specimens throughout Connecticut, as far south as Georgia, and as far west as the Ozark Mountains. “Over the course of four years, we visited nine states, and a small army of students participated in the field work,” Muñoz said.

Collecting the salamanders turned out to be nocturnal work.

“During the day, an Appalachian forest is familiar: trees, leaf litter, logs, and rocks,” Alomar said. “But after dark, especially after rain, the forest completely changes. Salamanders are suddenly everywhere, walking across the forest floor like they own the place. For animals that are lungless, wet-skinned, and so tied to cool, moist conditions, watching them thrive in their own environment was unforgettable.”

Up next, the researchers will zoom in on the salamanders’ evolutionary trajectory.

“How did they evolve across North America? What’s their deep history? What happened to them over the past glacial cycles?” Muñoz said. “We’ve uncovered the pattern. Now we want to understand the history that produced it.”

The Yale team included researchers from Yale Institute for Biospheric Studies and the Yale Peabody Museum. Other contributors included scientists from the University of Alabama and the University of North Carolina.