The ancient Maya city of Chichén Itzá in Mexico’s Yucatán Peninsula has long been associated with human sacrifice, with hundreds of bones unearthed from temples, a sacred sinkhole and other underground caverns.

A long-held misconception is that the victims were often young and female — an impression that has stuck in the contemporary imagination and become hard to dislodge even as more recent research has suggested that both men and women were among those sacrificed as well as children. A study published Wednesday in the journal Nature adds unexpected detail to that more complex picture. 

The new analysis, based on ancient DNA from the remains of 64 people who archaeologists believe had been ritually sacrificed and then deposited in an underground chamber, found the victims were all young boys, many of whom were closely related.

“There were two big moments of surprise here,” said lead study author Rodrigo Barquera, a researcher in the department of archeogenetics at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany.

“We were thinking, influenced by traditional archaeology that we would find, a non-sex-biased burial or mostly girls,” he said.

“And the second one (was) when we found out that some of them were related and there were two sets of twins.”

Analysis can only reveal so much

The lurid notion that the Maya only sacrificed young women or girls is largely a myth that originated from early and romantic accounts of Chichén Itzá’s sacred sinkhole, or cenote, said Rubén Mendoza, an archaeologist and professor in the department of social sciences and global studies at California State University, Monterey Bay. He wasn’t involved in the study but is an editor of a new book on ritual sacrifice in Mesoamerica. 

“This characterization of Maya sacrifice was catapulted to the forefront through media depictions of young maidens (aka virgins) being hurled to their deaths at the Sacred Well,” he said via email.

However, the mystery of exactly whom the Maya sacrificed has been hard to untangle because it’s impossible to identify the sex of a child’s skeleton by analyzing bones alone.

While the pelvis and a few other bones can reveal whether the skeleton was an adult male or female, the tell-tale differences only emerge during puberty and, even among adults, natural variation can make accurate identification difficult.

This difficulty makes genetic analysis particularly valuable, said study co-author Christina Warinner, John L. Loeb Associate Professor of the Social Sciences and Anthropology at Harvard University and a group leader at the Max Planck Institute for Evolutionary Anthropology. But the impact of ancient DNA, which has revolutionized archaeology in Europe and higher latitudes, has been more limited in tropical areas because DNA degrades more easily in warm conditions. However, recent advances in ancient DNA technology are expanding its reach, she said.

“We’re getting better and better at retrieving even very small amounts of DNA. And suddenly, we now have the ability to do these large-scale genomic studies and apply ancient DNA as a tool to help us understand the past in Mesoamerica,” Warinner said. “I am so excited about that because this is an area of the world which has this incredibly rich history.”

Boys younger than 6 were sacrificed

The team behind the new study was able to extract and sequence ancient DNA from 64 out of around 100 individuals, whose remains were found scattered in a water chultún — an underground storage chamber discovered in 1967 about 400 metres (437 yards) from the sacred sinkhole in Chichén Itzá.

With radiocarbon dating, the team found that the underground cavern was used for 500 years, although most of the children whose remains the team studied were interred there between AD 800 and 1,000 — during the height of Chichén Itzá’s political power in the region.

All the children were boys, who had been drawn from the local Maya population at that time, according to the DNA analysis, and at least a quarter of them were closely related to at least one other child in the chultún. The group also included two pairs of twins as well as siblings and cousins. Most of the boys were between 3 and 6 years old when they died.

Analysis of variants or isotopes of carbon and nitrogen in the bones also suggested that the related children had similar diets. Together, according to the authors, these findings suggested that related male children were likely selected in pairs for ritual sacrifices linked to the chultún.

“It is surprising to me to see family members, given the enormous time breadth of the deposit, which by radiocarbon dates is now confirmed to have been used over a time span of 500 years, during which these bodies slowly accumulated,” said Vera Tiesler, a bioarcheologist and professor at the Autonomous University of Yucatán, in an email. She wasn’t involved in the research.

While the study authors believe this finding reveals the only known burial of sacrificed male children, Tiesler said that the ancient Maya ritual calendar was complex, likely with different “victim profiles” for different religious occasions throughout the year and time cycles.

How twins were identified

To avoid sampling the same child twice, the team used the same bone from each child — the petrous bone in the base of the skull.

“Since each child only has one of those, you can be sure that we didn’t double sample any individuals,” Warinner said. “And that’s actually what allowed us to identify identical twins.”

Twins hold a special place in the origin stories and spiritual life of the ancient Maya, Warinner added, particularly a story called the “Hero Twins” in which two brothers descend into the underworld to avenge their father’s death.

It’s not clear how or exactly why the children were sacrificed, but sacrificial methods in use at the time included decapitation and removal of the heart.

“I think we have to remember that death, and everything that these rituals imply, were completely different to us, because we have a very different view of the world than the one that they had,” Barquera said. “For them, it was not losing a child, not losing one of their kids, but an opportunity given by whatever forces to be part of this special burial.”

Connections to present day

Warinner said the study was the first time that genetic material recovered from ancient Maya remains was detailed enough to be sequenced, providing a richer picture of who the victims were and to whom they were — and are — related.

The team compared the ancient DNA with that of 68 residents of the present-day Maya community of Tixcacaltuyub. The researchers found the two shared a close genetic signature.

“They were super happy to learn that they were related to the people that once inhabited Chichén Itzá,” Barquera said.

The team also showed how the residents’ immune systems had been shaped by the biological consequences of diseases that European colonizers brought. The researchers found the local Indigenous population today has genetic variants that may have protected them against salmonella infection, thought to be the pathogen behind the devastating 1545 cocoliztli epidemic.

María Ermila Moo-Mezeta, a Mayan coauthor of the study and research professor at the Autonomous University of Yucatán, said the new analysis was significant for her, as a professor of Indigenous origin, to preserve the “historical memory of the Mayan people.”

It was fascinating to learn how past suffering had left a stamp on the immune system of present-day Maya communities, Tiesler added.

“This study is decisively new; a starting point for further, more specific inquiries about the convoluted trajectory of the Maya,” she said.