Previous data have established direct archaeological evidence that semisedentary forager-farmers in the Mesoamerican region existed during the millennia preceding the onset of sedentary agrarian life in the Maya area. The present study concerned the identification of the maternal genetic origin of skeletal remains discovered in the Puyil cave through sequence analysis of the mitochondrial DNA. The individuals pertained to the Archaic and Classic periods and the samples were found inside the Puyil cave located in Tabasco, Mexico, an area occupied by Zoque and Maya populations. The technologies used were next-generation sequencing and Sanger sequencing. The analysis was was made possible by the development of haplotype networks for comparing mitochondrial DNA sequences from ancient and contemporary civilizations from the American continent and Asia. The sequences displayed haplogroups A, A2, C1, C1c, and D4, while haplogroup B was absent. The haplotype networks showed a close genetic relationship with Maya populations from Mexico and Guatemala and with contemporary native populations from Bolivia, Brazil, Dominican Republic, China, and Asia. The analysis with ancient mitochondrial DNA study sequences showed evidence of Maya ancestors’ migration from North to South America and through the Caribbean islands. The results of this study confirm that a Maya ancestor of the ancient remains from the Puyil cave is shared with contemporary Maya and that expansion of the founding haplogroups A, C, and D came from Asia.

Cave studies are important for better understanding the origins of the pre-Hispanic Mexican populations and the relationships among them. Particularly in the Puyil cave, bone remains and cultural remains will help to reveal how an ancient population migrated. Among Mesoamerican cultures, caves had a significant meaning in their cosmogonic conception of the world. The cave of Puyil contains pre-Hispanic burials evidence with more than 40 mortuary remains, distributed in the small cavities, sills, and spaces of the floor surface that make up the chambers and galleries formed by carbonate rocks. The ornamental elements of the ritual objects that accompany the remains indicate burial practices of pre-Hispanic tradition. The distribution of the funeral deposits inside the Puyil cave suggests that there was a hierarchical deposition of osteological remains and that pre-Hispanic groups of different ethnicities have used this ceremonial and funerary place from the Archaic to the Classical Period, and that it has been used even in current times.