Ancient genomes reveal insights into ritual life at Chichén Itzá

Chichén Itzá, a major Mayan city in Yucatán, Mexico (AD 600-1000), remains a focus of archaeological study. Despite extensive research, questions persist about the social and cultural uses of its ceremonial spaces and the genetic links between its inhabitants and other Mesoamerican groups. This study addresses these gaps by analyzing the genomes of individuals from a subterranean mass burial near the Sacred Cenote, a significant ritual site. The burial, discovered in 1967, contained the remains of over 100 subadults, interred in a repurposed chultún (underground cistern) connected to a cave—features often associated with water, rain, child sacrifice, and the Mayan underworld. Previous research suggested child sacrifice for agricultural cycles or offerings to the rain god Chaac, with conflicting accounts regarding the sex and origins of the sacrificed individuals. This study aims to clarify these aspects using a combined bioarchaeological and genomic approach, comparing the ancient remains with those of modern-day Maya inhabitants of nearby Tixcacaltuyub. Community engagement played a crucial role in this research.

Existing literature on Chichén Itzá highlights its prominence during the Late and Terminal Classic periods, its complex ceremonial structures (e.g., El Castillo), and the extensive evidence of ritual killing, both in physical remains and monumental art. Studies on the Sacred Cenote previously suggested predominantly female victims, but more recent osteological analyses reveal both male and female remains. Isotopic studies have indicated both local and non-local origins for some individuals. However, details about the child sacrifice practices and the ceremonial use of subterranean features as mass graves remain unclear. The role of twins in Mayan mythology and their representation in the Popol Vuh is also well-documented, offering a rich context for interpreting the findings of this study. Previous genetic studies in the region have provided insights into ancient population structures and migration patterns, laying the groundwork for this more detailed analysis of the Chichén Itzá population.

The research involved a multi-faceted approach. Sixty-four subadult individuals from the Chichén Itzá chultún (YCH) were sampled for ancient DNA (aDNA) analysis, focusing on left petrous bones to avoid repeated sampling. Radiocarbon dating established a time range of AD 500-900. DNA extraction and library preparation were conducted using UDG-treated libraries for ancient samples and non-UDG-treated libraries for 68 modern-day individuals from Tixcacaltuyub (TIX). Sequencing depth was approximately 5-11 million reads. Stringent quality control measures were implemented to minimize contamination (less than 5%). Data analysis focused on 1.2 million ancestry-informative SNPs, the mitochondrial genome (mtDNA), and a panel of immune genes. Pairwise mismatch rate (PMR) analysis was used to assess kinship, while stable isotope analysis (δ¹³C and δ¹⁵N) of bone collagen provided insights into dietary patterns. Population genetic analyses, including principal component analysis (PCA) and admixture analysis, examined genetic relationships with present-day and ancient American populations. Outgroup FST statistics (using Mbuti as an outgroup) and qpWave were employed to assess genetic affinities. Locus-specific branch length (LSBL) analysis identified genomic regions under selection in both ancient and modern populations. Finally, analysis of non-overlapping associations between HLA alleles and in silico binding prediction assays explored pathogen-driven selection.

The genetic analysis revealed that all 64 individuals from the Chichén Itzá chultún were male. Remarkably, two sets of monozygotic twins and nine other pairs of close relatives were identified within the burial, indicating a significant overrepresentation of close kinship compared to expected random occurrence. Stable isotope analysis demonstrated dietary similarity among related individuals, suggesting shared upbringing and potentially simultaneous sacrifice. Population genetic analyses demonstrated long-term genetic continuity between the ancient Chichén Itzá population and modern-day Maya from Tixcacaltuyub, except for differences in immune-related genes. Tixcacaltuyub individuals showed evidence of European and African admixture following the colonial period, particularly evident in the Y chromosome, indicating sex-biased admixture. Genome-wide selection analyses revealed enrichment of genes involved in lipid metabolism in both ancient and modern populations, possibly reflecting adaptive responses to dietary changes. Crucially, the study found strong signals of selection at the HLA class II locus, specifically an increase in HLA-DR4 alleles in modern Maya, linked to resistance against *Salmonella enterica*, a pathogen associated with the 1545 cocoliztli pandemic. In silico binding prediction assays supported the role of HLA-DRB1*04:07 in *Salmonella* resistance.

The findings provide compelling evidence for ritual sacrifice of closely related male children at Chichén Itzá, potentially linked to the significant role of twins in Mayan mythology. The presence of twins and close relatives in the burial suggests intentional selection based on kinship, possibly reflecting specific ritual practices. The genetic continuity between ancient and modern Maya, despite post-colonial admixture, underscores a long-standing genetic legacy in the region. The selection signatures in immune-related genes, particularly at the HLA class II locus, strongly suggest adaptation to infectious diseases introduced during the colonial period, potentially driven by the cocoliztli pandemic. The dietary data, while showing overall similarity to other Classic period populations, hints at potential social status differences reflected in dietary variations. Further research could explore the specific ritual context of these sacrifices, investigate the precise dietary differences, and examine other potential selective pressures shaping the genome of this population.

This study provides a unique glimpse into the ritual life and genetic history of the Classic Maya at Chichén Itzá. The discovery of multiple sets of twins and other close relatives in a mass burial of male children points to ritual practices influenced by Mayan mythology. The genomic data reveals long-term genetic continuity in the region, with evidence of post-colonial adaptation to introduced pathogens. Future research should focus on integrating these genetic insights with archaeological and anthropological data to gain a more comprehensive understanding of Mayan culture and its interaction with disease.

The study is limited by the sample size of the ancient DNA analysis (64 individuals out of an estimated 106) and the focus on subadults. The inability to definitively link all dietary variations to specific factors (social status, breastfeeding, etc.) is another limitation. The study also focuses on a specific burial context, and the findings may not be entirely generalizable to all ritual practices at Chichén Itzá. Finally, the conclusions about pathogen selection are based on correlational evidence and could benefit from further investigation.