These humble decomposers played a key role in some of the planet's earliest terrestrial ecosystems. Yet despite their remarkable history, major questions about their evolution remained unanswered.

Now, an international team led by Virginia Tech researchers has filled in one of the final gaps in the millipede family tree, shedding new light on the ancient creatures that helped prepare Earth for life on land.

The study, published in Current Biology, presents the first complete evolutionary history of all living millipede orders. By combining DNA data from modern species with physical evidence preserved in fossils, the researchers traced the origins of millipedes back nearly 460 million years, suggesting they existed long before the oldest millipede fossils discovered so far.

"Millipedes beat vertebrates onto land by more than 80 million years," said Paul Marek, the study's lead investigator and associate professor in the College of Agriculture and Life Sciences' Department of Entomology. "They really set the stage for later life on land, including humans and vertebrates."

Solving a Longstanding Millipede Mystery

For more than 100 years, scientists recognized the existence of two rare millipede groups, Siphoniulida and Siphonocryptida. However, because fresh specimens were unavailable for DNA analysis, researchers could not determine exactly where these groups belonged on the millipede family tree.

One group consists of millipedes less than a centimeter long that spend their lives underground. The other survives in only a handful of known locations.

"These last two were kind of like our white whales," Marek said.

To find them, researchers traveled to Los Tuxtlas in Mexico and the Canary Islands of Spain. There they collected Siphoniulus neotropicus and Hirudicryptus canariensis, two species whose DNA had never before been included in an evolutionary study.

"It took 10 people over a week just to find this one tiny 10-millimeter adult," said Luisa "Fernanda" Vasquez-Valverde M.S. '21, Ph.D. '24, the paper's first author and an assistant in Marek's lab. "Finding them in the field was hard because we were just seeing this little white nematode. We didn't know for sure it was a millipede until we looked under the microscope."

The team sequenced DNA from the two groups and compared hundreds of genes across 82 millipede species. They also incorporated evidence from 29 fossils. Together, these data allowed the researchers to determine where the mysterious groups fit within millipede evolution and when their lineages first emerged.

The project produced terabytes of genetic data and relied on Virginia Tech's Advanced Research Computing resources to reconstruct evolutionary relationships stretching back hundreds of millions of years.

The results showed that Siphonocryptida is not a separate millipede order as previously thought, but instead belongs within an existing lineage. Siphoniulida, meanwhile, was finally placed alongside its closest evolutionary relatives.

Millipedes on an Ancient Earth

The analysis indicates that millipedes may have originated nearly 460 million years ago, about 35 million years earlier than the oldest known millipede fossils and significantly earlier than previous estimates suggested.

"The biggest surprise was just how ancient some of these lineages turned out to be," Marek said.

At that time, Earth looked very different from today. According to Marek, millipedes were among the early pioneers of life on land, helping recycle nutrients by feeding on decaying organic material in some of the first terrestrial ecosystems.

"There were no vertebrates, no trees, no leaves, no flowering plants, no plants with seeds," Marek said. "Millipedes were feeding on decaying mosses, decomposed slime, and primordial gunk on the surface of the Earth."

The Origins of Millipede Chemical Defenses

The newly completed evolutionary tree also helped researchers pinpoint when one of the group's most notable adaptations appeared.

"They made the first chemical weapons," Marek said. "They're little chemical factories."

The study suggests these chemical defenses originated about 260 million years ago, offering the clearest evidence yet for when millipedes first evolved this capability.

Unsung Ecosystem Engineers

Today, millipedes remain among the world's most important detritivores. By breaking down dead plant material, they help recycle nutrients and support healthy ecosystems.

"It's really kind of puzzling that they have such an important function in the ecosystem, and yet they're so poorly known," Marek said.

Even with more than 14,000 described species worldwide, researchers believe tens of thousands of millipede species may still be undiscovered. Marek and his students have found new species in places ranging from Virginia Tech's Blacksburg campus to Los Angeles.

For scientists such as Vasquez-Valverde, that sense of exploration is part of the appeal.

"There is all this potential for discovery," she said. "It keeps me wondering what else we're going to find."

The research was funded by the National Science Foundation and involved scientists from the Field Museum of Natural History, Hampden-Sydney College, Universidad de La Laguna, Virginia Tech's School of Plant and Environmental Sciences, the Australian National Insect Collection, West Virginia University, and Universidad Autonoma del Estado de Hidalgo.