The ritual human sacrifice story is told by the ancient Maya ruins

On the discovery of a temple and underground chamber: no evidence of sacrifice from the ancient Maya in Chichén Itz or other cities of close relatives

“This was very, very astonishing,” says study co-author Oana Del Castillo-Chávez, a biological anthropologist at the Yucatán National Institute of Anthropology and History in Mérida, Mexico. She says that there is no evidence of sacrifice from Chichén Itz or other ancient Maya cities of close relatives.

The underground chamber and cave were found in the 1960’S near the Sacred Cenote. The remains had no signs of violence, but were found as part of a shrine.

In the hope of identifying the sex of the remains and to glean other genetic insights, Del Castillo-Chávez teamed up with immunogeneticist Rodrigo Barquera and palaeogeneticist Johannes Krause at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, and their colleagues. The team obtained ancient genome data from the skulls of 64 of the roughly 106 individuals buried at the chultún.

There is no reason why the children were selected for sacrifice. Isotopic analysis of their bones suggested that their plant-heavy diets — probably maize (corn) — were typical of ancient Maya. The isotopic profiles of related individuals suggested that they were raised the same way.

“Probably it was part of preparing them for this sacrifice,” says Barquera, who is from Mexico. “Death and sacrifice for them means something completely different to what it means to us. It was a big honor for them to be part of this.

Principal components of HLA association using the smartpca application: a comparison with the Bonferroni correction for precontact Chichén Itz and postcontact Tixcacaltuyub

An HLA allele that has become more than twice as common has been linked to protection against severe Salmonella infections. A previous study by Krause’s team has linked the bacterium Salmonella enterica sp. In the 16th century, millions of people were killed in a disease outbreak called the cocoliztli epidemic.

Smartpca (v.16000) from the Eigensoft package160 was used to calculate principal components (PCs) of variation in the dataset, using the options ‘lsqproject: YES’ and ‘shrinkmode: YES’. Initially, we projected the ancient individuals on PCs calculated on the genetic variation in 371 worldwide populations, to access the continental-level ancestries in the ancient individuals. We used variation from 172 Native American populations to project the ancient individuals on PCs.

We applied a development version of OptiType151 on sequence data from the immune-captured libraries, mapped against a custom HLA reference panel containing 1,025 alleles with ‘common’ or ‘intermediate’ CIWD 3.0 designation (, tag GRG). The number of possible best matches was set to infinity. In this way we obtained HLA class I and class II alleles for the genes HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1. Of the 882 raw allele calls, 72 (8.1%) with anomalous coverage patterns induced by reads cross-mapping to several loci were overruled in favour of secondary allele predictions. The frequencies and properties of the haplotypes were reported. and kinship when possible. We used Fisher exact tests and adjusted the P values to compare the frequencies of precontact Chichén Itz and postcontact Tixcacaltuyub. We use the FDR adjustment as, compared to methods such as the Bonferroni correction, the FDR approach controls for a low proportion of false positives, instead of ensuring the existence of absolutely no false positives, resulting in increased statistical power155. It has been argued74 that certain types of non-overlapping association between HLA loci may be a signature of pathogen selection. The ({f}{{\rm{adj}}}^{ }) metric74,75,156 was calculated for each pairwise combination of HLA loci in the dataset, using YCH and TIX haplotypes for which we had data for every HLA locus. We created 5,000 random permutations of the order of the alleles in one of the Y CH and TIX loci to provide a point of comparison. We generated distributions of possible ({f}_{{\rm{adj}}}^{ }) scores for each pair of loci in the dataset that would be obtained if the alleles at those loci were associated entirely at random. We then calculated the difference between the ({f}{{\rm{adj}}}^{ }) value calculated from each dataset for each pair of loci and the mean of the ({f}_{{\rm{adj}}}^{ }) scores calculated from the randomized data for that pair of loci, then divided that difference by the standard deviation of ({f}_{{\rm{adj}}}^{* }) calculated from the randomized data. The scoring system gave us the ability to rank the two pairs by how much non-overlapping they showed between the same alleles. We looked at the roles of the HLA-DRB1/3/4/5 and -DQA1/DQB1 all genes in resistance to S. enterica. proteins that have been previously reported to be highly immunogenic in humans (Supplementary Methods: ‘In silico binding prediction assays’) IedB Analysis Resource virtual machine image157,158 is implemented using NetMHCIIpan-4.0. The adjusted rank values were used to classify binders as strong or weak, with the weaker binders being classified as 10% or less strong.

The mtDNA haplogroups were determined mapping reads to the revised Cambridge reference sequence147. To confirm the corresponding haplogroups we used Haplo Grep2 and HAPLOFIND 149. The Y-haplogroup assignments had pileups of reads for each individual which mapped to YChr and were listed on the ISOGG Y-DNA Haplogroup Tree. We then manually assigned Y-Chr haplogroups for each individual based on the most downstream SNP retrieved after evaluating the presence of upstream mutations along the Y-Chr haplogroup phylogeny (Supplementary Fig. 6). Full mtDNA haplogroups and Y-Chr genotypes can be found in Supplementary Tables 9 (YCH) and 10 (TIX).

Using an in-solution capture approach based on modified immortalized probe sequences136, target immunity genes sequences, mtDNA, Y chromosome or a panel of 1,237,207 SNPs were enriched from the total DNA in the sequencing libraries32,33,137,138,139,140. After enrichment, captured library pools were paired-end sequenced on the Illumina Hiseq 4000 (Illumina) platform with 75 cycles providing about 20 million reads per assay.

The libraries were identified with the double- 100 l reactions using the PfuTurbo DNA Polymerase by Agile Technologies and the AccuPrime Pfx DNA Polymerase byThermo Fisher Scientific. The indexed products for each library were pooled, purified over silica columns using the MinElute PCR Purification Kit (QIAGEN N.V.), eluted in 44 µl of TET and again qPCR quantified, now using the IS5 and IS6 primers. The amplification of the product in 4 100 l reactions using Herculase II Fusion DNA Polymerase followed the manufacturer’s specifications with 0.3 M of each IS5/IS6 primer. A total of 2 ml of the conditioned product were diluted 1:10 and quantified using the Agilent 2100 Bioanalyzer DNA 1000 protocol (Agilent Technologies). Independent, equimolar (10 mM final concentration) pools of YCH libraries, TIX libraries and extraction and libraries blanks were then prepared for shotgun sequencing on the Illumina HiSeq 4000 Systems platform (Illumina)134,135. YCH libraries were sequenced to 5 million reads depth, whereas TIX libraries were sequenced to 11 million reads depth and then all of them were analysed to obtain basic quality control parameters using nf-core/eager v.2.3.4 (ref. 31).

For YCH, the bone powder was decalcized and digested by an overnight incubatee in a buffer of 37 C. The High PureViral Nucleic Acid kit Large Volume was used to purify the genes from the supernatant. The DNA was eluted in 100 l. Tris, 1 mM EDTA and 0.05% Tween) and frozen at −20 °C until library preparation128. The modern participants had their peripheral blood collected in a blood collection tube and then the DNA was removed using a kit from Zymo Research Corporation. Because our protocols are optimized for short-length aDNA and to avoid potential bias through laboratory methods, we sheared the DNA extracted from modern individuals using ultrasonic DNA shearing to an average length comparable to that of aDNA. Therefore, 50 μl of a 50 ng μl−1 dilution of each of the modern DNA samples were sheared to an average fragment length of 150 base pairs using a Covaris M220 Focused ultrasonicator (Covaris).

Source: Ancient genomes reveal insights into ritual life at Chichén Itzá

Chichén Itzá, Yucatán, Mexico: an iconic archaeological site of the Terminal Classic Period. Ancient samples collected in the excavations of a cascading cave

Chichén Itzá was one of the largest and most influential Maya cities of the Terminal Classic Period and today stands as an iconic archaeological site among the most representative of pre-Hispanic monumental architecture in the Americas (Fig. 1). The architecture of the site consists of several different styles, with some of the structures resembling those found in Central Mexico (Teotihuacan, Tula) and in the Puuc and Chenes regions (northeastern and central Yucatan, respectively) of the Maya lowlands107. The Maya Puuc style represents the second phase of the Florescent period in the Yucatan Peninsula (ad 750–800)107,108. The city has been known to have been a chief regional political centre in the first half of the nineth century and stayed that way until the first half of the eleventh century. The ancient samples used in this study belonged to individuals whose skeletons were recovered from the archaeological excavations of a chultún (cistern) connected to a natural cave within the archaeological site of Chichén Itzá, Yucatán, Mexico. The Middle Preclassic to the Late Classic- Postclassic periods (ad 1000–1600) were the periods in which Chultns were used. The sampled skeletons, which have been studied using anthropometry and osteology guidelines at the Centro INAH Yucatán, were recovered between April and June 1967, during the construction of a new runway close to El Castillo (‘The Castle’, the main basament of the archaeological site and a building with special calendrical significance4,111) and 300 m northeast to the Sacred Cenote of Chichén Itzá. The Florescent style of construction that the chultn was built in would indicate that it was built around the late or Terminal Classic periods of Chichén Itz, for example. Many of the bones were in aatomic position in the chamber and cave. The many skeletons that were found, as well as the positions of them being covered by limestone powder and bark, are mostly consistent with a mixed secondary/primary burial with periodic clearance, which could be related to an important cultural offering. The minimum of 106 skeletons of children and infants were not apparent, but only included the skulls of the southern portion of the cave and the northern portion of the chamber. Many ceramic and clay objects, as well as animal bone remains, were also found in the cave4. The ceramic styles and the archaeological context indicate that the chultún was used during the Florescent period, contemporary to the use of another ritual cave near Chichén Itzá known as Balankanché (ad 860 ± 130)4,112. A radiocarbon test on a sample of the bark covering the bones from the chultún burial has also suggested a use date of around ad 920 ± 60 (ref. 107). Our 14C dates suggest that the ritual burial took place between 600 and 1100. According to the age determination by tooth eruption method113, most (about 50%) of the subadult individuals were found to belong to the age group of 3–6 years old4. The fact that the age of the children differs from the age group normally related to death in children associated with infectious diseases according to palaeodemography19,113,114,115 further supports the interpretation of this burial as a sacrificial offering4.

According to the report issued by The Curt-Engelhorn-Centre for Archaeometry (Mannheim, Germany), the portions of the petrous bone of 26 YCH samples were pretreated and analysed using a standardized procedure. Collagen was extracted from the bone samples (approximately 1 g, using a modified version of the Longin method118), purified by ultrafiltration (fraction greater than 30 kD) and freeze-dried. The CO2 was analysed using an analyser. The CO2 was then converted catalytically to graphite and analysed using a MICADAS-type AMS system (Ionplus AG). The isotopic ratios 14C/12C and 13C/12C of samples, calibration standard (oxalic acid II), blanks and control standards were measured simultaneously in the AMS system. 14C-ages can be normalized to 13C with atypical uncertainty of 2 and is also used for Oxcal.

Source: Ancient genomes reveal insights into ritual life at Chichén Itzá

Towards a Co-responsible Model of Health in Tixcacaltuyub: A Community-Aware Approach

The community of Tixcacaltuyub self-identifies as a Mayan community and has been in a years-long cooperative relationship with the Chemistry and Nursing Faculties of UADY, Mérida, Yucatán, following projects investigating the relationship between health and lifestyle in the community. J.C.L.R., M.E.M.M. and J.C.T.R., together with C. Tzec-Puch, have been working in collaboration with the community to communicate results of clinical tests and to develop health interventions in Tixcacaltuyub. As a result of these interventions, healthcare barriers and opportunities have been identified and work is now underway with the active participation of the community to implement a co-responsible model of healthcare. In the spring of 2023, J.C.L.R. and R.B traveled to Tixcacaltuyub to take the results of the genetic findings with them and to collect feedback from community members and participants. The feedback from these engagements was incorporated into the final manuscript. The Spanish translation of the main manuscript was included in our strategies for outreach and making results available to a broader audience.

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