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Mesoamerica is a cultural and geographic region with a northern boundary adjacent to the American Greater Southwest, meanwhile its southeastern boundary includes the Maya area, which is adjacent to the Caribbean. These regions are of interest to analyze genetic structure, ancestry and gene flow between the ancient populations. We identified the mitochondrial DNA haplogroups and haplotypes in 19 colonial and 10 pre-Columbian Maya from Xcaret and 6 Paquimé individuals. We analyzed our data together with 603 ancient individuals, 95 colonial and 502 pre-Columbian. The results show clear genetic differences between Mesoamerica, American Greater Southwest and Caribbean regions. High frequency of haplogroup A in Paquimé and Mine Canyon and the distribution of their haplotypes in the networks suggest that these populations are probably genetically related with both, Mesoamerica and the American Greater Southwest. The genetic structure of the Maya is due to common ancestry and it was maintained by geographic isolation and gene flow mostly between Mayan populations. The Spanish conquest did not change this structure in the Maya from Xcaret, Quintana Roo. Although populations from Central Mexico are not genetically homogenous, they are clearly different to Maya. Teotihuacan and Cholula were contemporary cities that allied to control the region, however they show genetic differences that could be related with a distant common ancestry; they probably descended from the same group but separated very early, before their arrival to Central Mexico.