Genetic Evidence Supports the Multiethnic Character of Teopancazco, a Neighborhood Center of Teotihuacan, Mexico (AD 200-600)
Genetic Evidence Supports the Multiethnic Character of Teopancazco, a Neighborhood Center of Teotihuacan, Mexico (AD 200-600)
Brenda A. Álvarez-Sandoval 0 1
Linda R. Manzanilla 0 1
Mercedes González-Ruiz 0 1
Assumpció Malgosa 0 1
Rafael Montiel 0 1
0 1 Laboratorio Nacional de Genómica para la Biodiversidad, Unidad de Genómica Avanzada, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional , Irapuato, Guanajuato , Mexico , 2 Instituto de Investigaciones Antropológicas, Universidad Nacional Autónoma de México , Mexico City, Mexico, 3 Unitat d'Antropologia , Departamento de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autònoma de Barcelona , Bellaterra, Barcelona , Spain
1 Editor: David Caramelli, University of Florence , ITALY
Multiethnicity in Teopancazco, Teotihuacan, is supported by foreign individuals found in the neighborhood center as well as by the diversity observed in funerary rituals at the site. Studies of both stable and strontium isotopes as well as paleodietary analysis, suggest that the population of Teopancazco was composed by three population groups: people from Teotihuacan, people from nearby sites (Tlaxcala-Hidalgo-Puebla), and people from afar, including the coastal plains. In an attempt to understand the genetic dynamics in Teopancazco we conducted an ancient DNA (aDNA) analysis based on mtDNA. Our results show that the level of genetic diversity is consistent with the multiethnicity phenomenon at the neighborhood center. Levels of genetic diversity at different time periods of Teopancazco's history show that multiethnicity was evident since the beginning and lasted until the collapse of the neighborhood center. However, a PCA and a Neighbor-Joining tree suggested the presence of a genetically differentiated group (buried at the Transitional phase) compared to the population from the initial phase (Tlamimilolpa) as well as the population from the final phase (Xolalpan) of the history of Teopancazco. Genetic studies showed no differences in genetic diversity between males and females in the adult population of Teopancazco, this data along with ample archaeological evidence, suggest a neolocal post-marital pattern of residence in Teopancazco. Nevertheless, genetic analyses on the infant population showed that the males are significantly more heterogeneous than the females suggesting a possible differential role in cultural practices by sex in the infant sector. Regarding interpopulation analysis, we found similar indices of genetic diversity between Teopancazco and heterogeneous native groups, which support the multiethnic character of Teopancazco. Finally, our data showed a close genetic relationship between Teopancazco and populations from the “Teotihuacan corridor” and from Oaxaca and the Maya region, in agreement with previous archaeological evidence.
Funding: This work was supported by Consejo
Nacional de Ciencia y Tecnología (www.conacyt.gob.
mx) grant No. CB-2008-01-105481 to RM and
Consejo Nacional de Ciencia y Tecnología (www.
conacyt.gob.mx) fellowship (Reg. No. 300750) to
BAA-S. The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
The ancient city of Teotihuacan, located in the Central Mexican highlands (Fig 1), can be
considered the most influential civilization of the Classic period of Mesoamerica (AD 200–600). In
the period of its greatest splendor, Teotihuacan supported a population of more than 100,000
inhabitants from different places of origin, constituting a multiethnic civilization [1, 2].
The population of Teotihuacan was organized in different social and spatial units; one of
them was the multiethnic neighborhood center headed by intermediate elites . Teopancazco
represents one of these centers located at the southeast of the Ciudadela . Different
construction phases have been found in this site: Tlamimilolpa (AD 200–300) was the initial phase of
the Teopancazco’s history; previous stable and strontium 87/86 isotopes studies, and a
paleodietary analysis of burials from this phase [4–8] have reported evidence that the population of
this phase was composed mainly of local people and of foreigners from sites belonging to the
“Teotihuacan corridor to the Gulf Coast” (Puebla-Hidalgo-Tlaxcala) , having limited
contact with other distant populations. By the end of Tlamimilolpa (AD 300–350), differential
rituals were found which suggested the end of a constructive phase and/or drastic social changes
originated by population rearrangements (Transitional phase) [1, 10–12]. During this time,
individuals intentionally decapitated were considered foreigners, according with previous
isotopic studies, coming from low altitudes and some others as foreigners coming from the
“Teotihuacan corridor to the Gulf Coast” [4–9]. The latter period of the Teopacazco´s history (AD
350–550) is called Xolalpan (although there is also a later Metepec construction phase), and
has been also characterized by previous isotopic studies [4–9] by the presence of people from
the “Teotihuacan corridor” sites, from the coastal plains, reverse migrants, and local people,
suggesting demographic changes during this phase in Teopancazco.
Wide archaeological evidence supporting the multiethnicity of Teopancazco has been
reported: the different burial practices found in this site, the large number of faunal elements
associated with the coast, foreign pottery from the Gulf Coast, cotton cloth, and foreign
materials processed in Teopancazco [12, 13]. This evidence supports the idea that this neighborhood
Fig 1. Map of Mexico in pre-Hispanic times showing the localization of Teotihuacan. The frontier
between Mesoamerica and Aridoamerica is shown.
center was one articulating Teotihuacan with the Gulf Coast suggesting the presence of
corridors of ally sites (i.e. the region of Puebla-Hidalgo-Tlaxcala proposed by García-Cook in 1981
), through which intermediate elites sponsored caravans from Teotihuacan to sites in
Veracruz, Guatemala, the “Bajío” region and Michoacán  in order to bring sumptuary goods
and specialized labor, mainly of foreign origin [12, 13] and ultimately producing the
multiethnic composition in the population of Teopancazco [1, 10, 11, 13, 15, 16–19]. Before our
research, no genetic studies had been conducted in order to assess the multiethnicity of
Teopancazco, characterized otherwise by ample archaeological evidence.
Regarding the population of Teopancazco characterized by sex, previous osteological
analyses showed an unusual higher proportion of adult males (48.38%) in relation to adult females
(ca. 15%) [9, 20]. Several studies have been conducted to detect differences in genetic diversity
between adult males and females due to social organization within a population; the differences
might be the result of post-marital residential patterns  taking into account that patrilocal
pattern has been the most common residence pattern in human civilizations . Spence 
reported a biodistance analysis of cranial, dental, and postcranial traits in human remains from
Teotihuacan showing a preference for patrilocality. Until now, no inferences about
post-marital residence patterns present in Teopancazco have been reported. On the other hand, Gallego
 and Alvarado-Viñas  reported that approximately 29% of the formal burials found in
Teopancazco were infants. The study of infants also has social significance, although
traditionally they have been neglected in archaeological interpretations mainly due to methodological
issues [25–27]. Currently, comparisons between genetic diversity in male and female infants
buried at Teopancazco have not yet been reported. These studies could help to elucidate
differences in genetic diversity in the infant population according to their sex in order to find out if
the differences can be explained by cultural practices, such as funerary rituals and to
understand their meaning in antiquity .
Our genetic study from Teopancazco is the first attempt to genetically characterize the
ancient population of Teopancazco assessing changes in genetic diversity through its history,
from the initial period to the final construction phase of the neighborhood center, as well as to
assess and compare genetic diversity by sex and by age at death of the Teopancazco individuals.
Likewise, our data allowed us to assess the genetic relationships between Teopancazco and
other Mesoamerican populations.
Materials and Methods
Sampling and Ethics Statement
The population under study came from Teopancazco, Teotihuacan, Central Mexico: Latitude
North: 19.67464167, Longitude West: 98.84532778. UTM: 2, 14, Q, 516213, 2175485,
“TEOPANCAZCO”. Bone and tooth samples were collected in several extensive excavation field
seasons (1997–2005) and provided by Dr. Linda R. Manzanilla, head of the project “Teotihuacan.
Elite and rulership. Excavations in Xalla and Teopancazco” (Last authorizations for the Project:
Official letter 401.B (4)19.2013/36/0579 of the National Institute of Anthropology and
History). Archaeological permits for Dr. Linda R. Manzanilla, director of the project “Teotihuacan.
Elite and rulerhip”, by the Consejo de Arqueología (Archaeological Council) of the Instituto
Nacional de Antropología e Historia (National Institute of Anthropology and History) are as
follows: First field season: C.A. 401-36/1077 October 16, 1997. 11th field season: C.A. 401-36/
0787 July 11, 2003. 12th field season: C.A. 401-36/0824 July 9, 2004. The sampling process was
carried out under strict criteria in order to avoid exogenous contamination. The human burial
remains from Teopancazco are kept in the Instituto de Investigaciones Antropológicas
(UNAM) under the custody of Dr. Linda R. Manzanilla. The samples are part of the Mexican
A subset of 50 samples from 46 different individuals buried at Teopancazco was sent by Dr.
Linda R. Manzanilla to the aDNA laboratory in LANGEBIO, CINVESTAV, Mexico, in order
to be analyzed (burials 1A, 1B, 2, 3, 4, 5, 10A, 16, 17, 35, 38, 39, 45, 46, 48, 49, 51, 55, 56, 59, 60,
61, 65, 67, 71, 78, 88, 89, 90, 91, 92, 96, 99, 100, 101, 102, 103, 105, 107, 108, 110, 111, 112, 116,
RT14239, and RT12805). Additionally, a subset of eleven samples (burials 1B, 2, 3, 4, 38, 45,
49, 60, 102, 103, and 105) was sent to the Universitat Autònoma de Barcelona (UAB), Spain, in
order to replicate results.
DNA extraction was carried out under strict conditions for the analysis of ancient DNA, in a
dedicated clean room, positively pressurized with ultra filtered and UV-irradiated air, with
separation of pre- and post-PCR areas. Laboratory equipment was treated with 30% bleach for
DNA contamination removal. The use of disposable filter-plugged pipette tips, tubes,
protective cloths, hair covers, laboratory gloves, surgical masks, glasses, and shoe protectors to
prevent contaminations was mandatory. Solutions and buffers were irradiated with ultraviolet
light. Negative extraction controls and negative PCR controls were always employed.
Buccal swab samples from the personnel related to samples handling were analyzed in order
to discard the possibility of contamination during excavations or laboratory procedures.
For each individual analyzed, a fragment of bone or a complete teeth, in a good state of
preservation (without fissures), was used. Samples were processed in a type II B2 biological security
hood located inside the clean room. The outer surface of the bone or teeth was UV-irradiated
for 15 min and a dental drill was used to remove 0.1 g of powdered material. Powdered samples
were added to 5 ml of extraction buffer (50 mM Tris-HCl pH 8.0, 0.425 M EDTA pH 8.0, 0.5%
SDS) and 50μl of Proteinase K solution (10 mg/ml). Samples were incubated at 37°C for 24 hrs,
followed by a phenol/chloroform DNA extraction . Aliquots of raw DNA extracts were
analyzed with a High Sensitivity DNA Assay Chip Kit on a Bioanalyzer 2100 (Agilent) to
quantify and assess the quality of extracted DNA. The result of this assay shows the distribution of
DNA fragment sizes in the extract, and helps to determine the DNA fragmentation pattern of
each sample. Highly degraded samples show a distribution with a peak of less than 100 bp (i.e.
most of the fragments are smaller than 100 bp) .
Ancient DNA extraction and mtDNA amplification and sequencing in the UAB were
performed as previously described .
DNA amplification and Haplogroup determination
Amplification and cloning of an HVRI segment (positions 16190–16339). A 149 base
pair (bp) portion located between positions 16190 and 16339 (numbered according to
Anderson et al. , of the D-loop region was PCR amplified with primers MT-F16190 5' CCCCATG
CTTACAAGCAAGT-3'  and MT-R16339 5'-GTGCTATGTACGGTAAATGG-3' . A
30 μL reaction contained 2 μL of aDNA extract, 1X buffer (150 mM Tris-HCl pH 8.0, 500 mM
KCl), 2.5 mM MgCl2, 200 μM each dNTPs, 0.4 μM each primer, and 0.12 U of Bioline
TaqDNAPolymerase (Biotaq). Thermal cycling was carried out under the following conditions: 5
minutes at 94°C; followed by 40 cycles of 0.5 minutes at 94°C, 0.5 minutes at 52°C, 0.5 minutes
at 72°C; and a final step of 7 minutes at 72°C. The PCR product was then cloned using a TOPO
TA Cloning kit and TOP10 competent cells (Invitrogen) following manufacturer’s instructions.
A minimum of 50 colonies were selected from each cloned sample for colony PCR using the
M13 primers. We then sequenced several clones and obtained between 3 and 11 clones per
sample that contained the transformed vector with the fragment of interest. DNA sequencing
was performed in both directions at the Genomic Services Unit in LANGEBIO, CINVESTAV.
Sequences obtained were aligned to the mtDNA reference sequence (rCRS)  using CLC
Sequence viewer v.6.5.1 (CLC bio, Aarhus, Denmark). To determine the mitochondrial
haplogroup we analyzed a set of diagnostic mutations characterizing haplogroups A, B, C and D,
between positions 16190–16339 . The haplogroups were confirmed using HaploGrep
software, which is supported by up-to-date knowledge of the mtDNA phylogeny , MitoMap
 and PhyloTree website .
A D-loop fragment (700 bp) was amplified and sequenced in order to discard possible
contamination from personnel related to samples handling.
Haplogroup characterization by High Resolution Melting analysis (HRM). We
developed an HRM method to determine the mitochondrial haplogroup of the ancient samples.
This analysis is based on the melting temperature (Tm) difference of the amplified DNA
fragments carrying diagnostic mutations located in the mitochondrial coding region belonging to
Native American Haplogroups A, C, D and X. Haplogroup B was identified by the presence of
a 9-pb deletion. Primers were designed to amplify short fragments (59–63 bp) and therefore
the analysis was efficient even for samples for which amplification of the D-Loop fragment
(149bp) was unsuccessful. Primers and conditions used are shown in Table 1. Fragments were
amplified with the LightCycler 480 Real-Time PCR Instrument using the LightCycler 480 High
Resolution Melting Master kit (Roche). The PCR reactions were performed in triplicate in a
total volume of 20 μl containing 2 μl of template DNA, 2 mM MgCl2 1X LightCycler
MasterMix (with dUTP instead of dTTP) and 0.2 μM of each primer. Thermal cycling was carried out
under the following conditions: 10 minutes at 95°C; followed by 80 cycles of 30 seconds at
95°C, 30 seconds at 60°C; and a final step of 45 seconds at 72°C. After PCR the HRM analysis
was performed as described . The curves obtained from each haplogroup amplicon were
compared with two references: one with the diagnostic mutation (positive control) and other
lacking it (negative control) (Table 1).
Intrapopulation analysis. Intrapopulation statistical analyses were carried out on the
haplogroup and the haplotype (sequence) level to assess if there were changes in genetic diversity
in a temporal scale through the history of the neighborhood center. Teopancazco was analyzed
as a whole, and with the samples divided into three different historical phases: Tlamimilolpa
(the initial period, AD 200–300), Transitional (characterized by a termination ritual with
decapitated males buried in the same pit, AD 300–350), and Xolalpan (the most active period
for exchange relations, AD 350–550) [9, 12, 38].
Nei’s genetic diversity indices  were calculated over haplogroup (Ĥa) and haplotype
(Ĥb) data. A one-way ANOVA was performed in order to compare Nei’s genetic diversity
indices at the haplogroup level among the three chronological groups analyzed. A two-tailed t-test
was performed in order to compare Nei’s gene diversities indices at the haplotype level among
the temporal groups analyzed. These analyses were carried out using GraphPad Prism software
v6.00 (GraphPad Software, La Jolla, CA USA, www.graphpad.com). An homogeneity test of
haplogroup frequency distributions was carried out between the analyzed populations using an
exact test of population differentiation , with 200,000 steps of the Markov chain and 5,000
dememorization steps, with α = 0.016 after Bonferroni correction. These analyses were carried
out using the Arlequin software v184.108.40.206 .
Infant population analysis. The sex of the infants was previously determined by real-time
PCR amplification of small fragments of the amelogenin gene, followed by High Resolution
Melting analysis (HRM) . Nei’s genetic diversity indices  were calculated over
haplogroup (Ĥa) data for both groups. Gene diversities were compared by a two-tailed t-test using
GraphPad Prism software v6.00 (GraphPad Software, La Jolla, CA USA, www.graphpad.com).
A homogeneity test of haplogroup frequency distributions was carried out by an exact test of
population differentiation  as described above.
Patterns of post-marital residence. The sex of the adult individuals from Teopancazco
was previously determined by osteological methods [20, 29]. Nei’s genetic diversity (Ĥa) 
was calculated over haplogroup frequencies for both female and male groups. Levels of genetic
diversity were compared by a two-tailed t-test using GraphPad Prism v6.00 (GraphPad
Software, La Jolla, CA USA, www.graphpad.com). A homogeneity test of haplogroup frequency
distributions was carried out by an exact test of population differentiation  as described
above. Genetic data were compared with archaeological evidence available in order to make
inferences about post-marital residential pattern.
Interpopulation analysis. Interpopulation statistical analyses were carried out on the
haplogroup and the haplotype (sequence) level. Mitochondrial haplogroup frequencies from all
reference populations are reported in S1 Table. Haplogroups frequencies from modern
Mesoamerican populations located in the geographic region called “the Teotihuacan corridor to the
Gulf Coast” (Hidalgo, Puebla and Veracruz), Oaxaca and Maya regions (Fig 2) [42–51] were
analyzed in order to evaluate possible genetic relationships with Teopancazco. An exact test of
population differentiation  was used as described above (α = 0.00036 after Bonferroni
correction). Comparisons in levels of genetic diversity (Ĥa) between Teopancazco and, Maya,
Otomi, Nahua populations [44–46, 49–53] and Lacandon populations  were carried out by
using a one-way ANOVA with a post-hoc Dunnett’s test.
A Principal Component Analysis (PCA) based on frequencies of mitochondrial
haplogroups was done with XLSTAT software v2014.1.03 (Addinsoft, www.xlstat.com). A Pima
population was used as external group (Aridoamerican population) due to its relatively higher
genetic distance to Mesoamerican populations .
Fig 2. Geographical locations of indigenous American populations used in the present study.
Haplogroup frequencies (S1 Table) were used to calculate Reynold’s genetic distances 
between all pairs of populations analyzed, and these distances were used to construct a
Neighbor-Joining tree . The analysis was done using SEQBOOT, GENDIST, NEIGHBOR, and
CONSENSE programs from the PHYLIP package v3.695 . The tree robustness was assessed
by bootstrapping (100 pseudoreplicates). A Maximum Likelihood tree was constructed from
the haplotype data using the TN93 model . A discrete gamma distribution was used to
model evolutionary rate differences among sites (4 categories, alpha parameter = 0.9821).
Nodal support was assessed by bootstrapping (1000 pseudoreplicates). Data used included the
16 sequences available from Teopancazco and sequences previously reported in GenBank 
and HVRBase++  from 9 Native Mexican populations (S2 Table). The tree was constructed
with MEGA software v6.1 .
We were able to recover mtDNA data from 29 out of the 46 available samples (63.04%). All
these 29 samples amplified for short fragments (59-63pb) of the coding region, however, only
16 of them (55.17%; 34.78% from the total samples) also amplified for the 149bp fragment of
the HVR-I. These 16 sequences have been deposited in GenBank (Accession numbers
KR813318-KR813333). No matches were detected between sequences recovered from skeletal
remains and from personnel related to samples handling (S3 Table).
We sent eleven samples (bone and teeth) to the Universitat Autònoma de Barcelona (UAB),
Spain, in order to replicate results. For two of the samples (burials 2 and 3) no amplification
was obtained. Nine samples were amplified; however, the sequences of five of them (burials 1B,
4, 38, 60, and 105) showed an unusual number of substitutions constituting haplotypes without
phylogenetic sense and therefore were considered artifacts. Reliable sequences were obtained
for only four of the eleven samples (36.36%). These sequences showed SNPs in agreement with
the SNPs previously determined in the LANGEBIO laboratory for those samples (burials 45,
49, 102, and 103). The percentage of efficiency (sequences) obtained in the UAB was similar to
the efficiency obtained in LANGEBIO (34.78%). This result confirms the poor DNA
preservation at the site.
The haplogroup determined for each of the amplified individuals (N = 29) is reported in
Table 2. All samples correspond to previously reported Native American haplogroups [62–65].
Haplogroup A is the most frequent (55%), haplogroups B and D are less represented (21% and
17% respectively), and haplogroup C has the lowest frequency (7%). Samples from
Teopancazco were divided into three different temporal periods: Tlamimilolpa (N = 10), the
Tlamimilolpa-Xolalpan Transition (N = 11), and Xolalpan (N = 8). The Tlamimilolpa and Xolalpan
periods showed a similar frequency of haplogroup A (~ 60%) and B (~10%) while the
frequencies in Transitional period were 50% and 30% respectively. Regarding haplogroup C, a twofold
increase in frequency was observed in the Tlamimilolpa and Transitional periods (20%), in
* Samples used in Nei’s genetic diversity estimation at haplotype level reported in Table 3.
16223T, 16260T, 16290T, 16319A
16223T, 16290T, 16319A
16223T, 16290T, 16319A
16223T, 16290T, 16319A
16223T, 16290T, 16319A
comparison to the Xolalpan phase, and, notably, the presence of haplogroup D was observed
only in the Tlamimilolpa and Xolalpan phases (i.e. it is absent in the Transitional period)
No significant differences in haplogroup frequencies among the three phases of
Teopancazco were detected (global exact test of population differentiation, p> 0.05). However, the
absence of haplogroup D in the Transitional period might be significant in relation to the
genetic structure of this population even if not statistically significant, as discussed below.
Haplogroup frequencies were used to estimate Nei’s genetic diversity for the three periods of the
Teopancazco´s history (Table 3). The levels of diversity (Ĥa) are similar in the three periods
analyzed: Tlamimilolpa 0.6444 ± 0.1518, Transitional 0.6909 ± 0.0861, and Xolalpan
0.6429 ± 0.1841, and the differences were not significant (one-way ANOVA, F (2, 26) = 0.381,
p> 0.05). However, the PCA (Fig 3) shows a closer proximity between Tlamimilolpa and
Xolalpan groups while a differentiation of the Transitional period can be observed. This result is
consistent with the observed topology in the Neighbor-Joining tree constructed from
haplogroup frequencies. (S1 Fig).
D-loop sequences from 16 individuals buried at Teopancazco (burials 2, 10A, 38, 45, 55, 56,
59, 60, 61, 89, 92, 103, 105, 108, 116, RT12805) were used to assess haplotype diversity
(Table 3) in the three historical periods. Higher levels of diversity were found in the
Tlamimilolpa and the Transitional periods with no significant differences among them (two-tailed
ttest, p> 0.05). Haplotype diversity in Xolalpan was not detected.
Infant population analysis. As we were able to determine sex in the infant population
from Teopancazco, we analyzed genetic diversity by sex in the infants (Table 4). As in the
adults, male infants showed higher genetic diversity (Ĥa) than female infants (0.8000 ± 0.2086
and 0.5238 ± 0.2086, respectively), and in this case the difference between them was significant
(two-tailed t-test, p< 0.05). However, no difference in haplogroup frequencies distributions
was detected between both groups (exact test of population differentiation, p> 0.05).
Post-marital residential pattern. We analyzed genetic diversity by sex in a total of 12
adult individuals (Table 4). An exact test of population differentiation failed to show a
significant difference in haplogroups frequencies between females and males (exact test of population
differentiation, p> 0.05). We also found a higher genetic diversity (Ĥa) in males
(0.7333 ± 0.1552; N = 6) than in females (0.5333 ± 0.1721; N = 6) nevertheless, no significant
difference in genetic diversity was found (two-tailed t-test, p> 0.05).
Differences in Nei’s genetic diversity among Teopancazco, Nahua, Otomi, and Maya
populations were statistically significant (one-way ANOVA, F (15, 680) = 385.6, p< 0.05). Pairwise
Nb Haplotype sample size.
Fig 3. Principal Component Analysis based on mitochondrial haplogroups frequencies. TEO
(Teopancazco), TEO-TLAM (Tlamimilolpa period), TEO-XOL (Xolalpan period), TEO-TRAN (Transitional
phase), PIMA (Pima, Aridoamerica), ZAP (Zapotec, Oaxaca), OTO (Otomi, Hidalgo), OTO I (Otomi, Hidalgo),
OTO II (Otomi, Hidalgo), NAH (Nahua, Veracruz), NAH I (Nahua, Veracruz), NAH II (Nahua, Puebla), NAH III
(Nahua, Hidalgo), MAY (Maya, Xcaret), MAY II (Maya, Yucatán), MAY III (Maya, Campeche), MAY IV (Maya,
Quintana Roo), TEPE (Tepehua, Hidalgo), HUA (Huastec, Hidalgo) MIXT (Mixtec, Oaxaca), MIXT II (Mixtec,
comparisons indicated that Teopancazco shows no significant differences to one of the
Maya population from Yucatán (MAY II) (Dunnett test, p = 0.1950), two Otomi populations
(OTO I and II, Dunnett test p = 0.6679 and 0.8777, respectively) and three Nahua populations
(NAH V, VII and IX, Dunnett test p = 0.6157, p = 0.9998 and p = 0.9494, respectively).
Nevertheless, pairwise comparisons indicated that Teopancazco’s diversity is significantly
different (p< 0.00036) from the diversity found in a Lacandon population, which is one of the
most isolated Mexican Native group and shows a lower diversity index (0.0426 ± 0.0403).
Haplogroup frequencies were used to evaluate genetic similarities among Teopancazco and
three groups of populations: the “Teotihuacan corridor to the Gulf Coast” (Hidalgo, Tlaxcala,
Puebla and Veracruz), Oaxaca and Maya region. No significant differences among populations
were found (global exact test of population differentiation, p> 0.05). This result is in agreement
with the PCA (Fig 3), which shows genetic proximity between all populations, while the
differentiation of the Pima (Aridoamerican population) is also evident. The topology of the
Neighbor-Joining tree constructed with haplogroup frequencies from all populations also supports
the observed genetic proximity between Teotihuacan (Teopancazco) and the Mesoamerican
populations (S1 Fig).
Comparisons at haplotype level were assessed in order to elucidate genetic relationships
among individuals from Teopancazco and from nine Native Mexican populations (S3 Table).
A Maximum Likelihood tree shows that the individuals buried in Teopancazco were closer to
individuals from Tepehuan, Zapotec, Maya, and Mixtec populations regardless of the period in
which they were buried (S2 Fig).
The multidisciplinary project “Teotihuacan. Elite and rulership. Excavations in Xalla and
Teopancazco”, headed by Linda R. Manzanilla, has produced relevant information supporting the
multiethnicity of Teopancazco, a neighborhood center located at the southeast of Teotihuacan
[9–13, 19]. However, before our research, comprehensive genetic studies aimed to better
understand the multiethnicity in this site had not been carried out. In this work, we
characterized the mitochondrial variability in Teopancazco, estimated levels of genetic diversity,
assessed changes in genetic composition in time, analyzed the genetic diversity by sex and by
age at death of Teopancazco’s individuals, and inferred the genetic relationships between
Teopancazco and Mesoamerican populations.
Genetic analyses of the Teopancazco population from the initial to the later phases of its
history allowed us to assess if there were changes in genetic diversity and composition in
accordance to previous isotopic and paleodietary studies as well as archaeological evidence [4–9].
These data suggest that the population of the initial phase of Teopancazco (Tlamimilolpa, AD
200–350) was composed mainly by local people and by foreigners from sites belonging to the
“Teotihuacan corridor to the Gulf Coast” . The evidence of limited contact with other
distant populations suggests a lower genetic diversity during this time in comparison to the final
phase of the Teopancazco history (the Xolalpan phase), characterized by the possible
expansion of exchange routes between Teotihuacan and Mesoamerica. However, Nei’s genetic
diversity at haplogroup (Ĥa) and haplotype level (Ĥb) in this phase (0.644 ± 0.1518 and
1.000 ± 0.1265, respectively), was similar to the Xolalpan phase, suggesting that the population
from Teopancazco was heterogeneous since the beginning. At the end of the Tlamimilolpa
phase (AD 300–350), differential burial rituals were found, in which some intentionally
decapitated individuals, mainly males, were considered foreigners coming from low altitudes and
some others coming from the “Teotihuacan corridor to the Gulf Coast” according with
previous isotopic analyses [7–9]. The increased presence of foreign people in this phase could have
implied an increase in genetic diversity. As a result, we should even expect a differential genetic
composition of the population of this phase in relation to the populations from the other
periods analyzed. In agreement with these expectations, the haplogroup frequencies in the
Transitional period showed a different pattern in comparison with the data observed in Tlamimilolpa
and Xolalpan times. In the Transitional phase, the diversity at haplogroup (Ĥa) and haplotype
(Ĥb) levels (0.690 ± 0.0861 and 0.9643 ± 0.0772, respectively) was higher than those in the
initial phase in the history of Teopancazco (Tlamimilolpa), although the differences between both
periods were not significant (p> 0.05). However, the absence of haplogroup D in the
Transitional period might be indicative of a differential genetic structure of this sample, in agreement
with the differential burial ritual applied to these individuals. Further evidence for the genetic
differentiation of the Transitional group can also be observed in both the PCA plot and the
Neighbor-Joining tree (Fig 3 and S1 Fig). Future studies increasing sample size or the amount
of sequence information obtained might shed further light into this issue. The last part of
Teopacazco´s history (the Xolalpan phase) was characterized, according with isotopic studies, by
the presence of people from several origin sites, suggesting demographic changes caused by
during this phase in Teopancazco . The expansion of exchange routes in this period might
have increased the genetic variability; however, we found no significant differences (p> 0.05)
in genetic diversity (Ĥa) or haplogroup frequencies between this period and the Tlamimilolpa
or Transitional periods. These data suggests that the genetic diversity levels were constant from
the initial phases of Teopancazco until the last part of the neighborhood history (Fig 4), and
that the possible increase of trade with foreign populations previously proposed had no effect
in mitochondrial genetic variability of Teopancazco’s population. The concurrent presence of
haplogroup D also reinforces the idea of genetic continuity at the haplogroup level between
Tlamimilolpa and Xolalpan times, contrasting with the discontinuity observed in the
Transitional group, which lacks haplogroup D and that is separated from Tlamimilolpa and Xolalpan
in both the PCA plot and the Neighbor-Joining tree, as mentioned above. Regarding the
haplotype data, we found high levels of genetic diversity in the Tlamimilolpa and Transitional phases
with no significant differences between them (p> 0.05). These data also support the
multiethnic character of Teopancazco. Genetic diversity at haplotype level in Xolalpan was not
determined because the three haplotypes obtained were identical.
Fig 4. Overview of the genetic history of Teopancazco, Teotihuacan. No significant differences were
observed in genetic diversity indices at haplogroup and haplotype levels between the three periods analyzed,
genetic diversity at the haplotype level was estimated only for the Tlamimilolpa and Transitional periods.
Pvalues of the statistical comparisons are shown inside the arrows.
Previous studies had reported that approximately 29% of the formal burials found in
Teopancazco were infants , and no genetic data about them have been reported. Thus, our
analysis of genetic sex determination represents the first attempt to analyze this population sector.
After the analysis of 16 infant individuals, we found an equal proportion of male and females
. Genetic information from the infant sector can help to understand the role that infants
played in ceremonial events within a population (i.e. human sacrifices), and in particular to
infer if there were differential burial pattern between sexes. For this reason, we compared, for
the first time, genetic diversity indices (Ĥa) between infant males and females in Teopancazco.
Our results indicated that there is a significantly higher genetic diversity in male infants
compared to female infants (0.8000 ± 0.2086 and 0.5238 ± 0.2086, respectively; p< 0.05). This
means the female group was more homogeneous than the male group, as observed for the
adult population (described above). In order to find out if the differences in genetic diversity
between sexes can be explained by cultural practices, such as funerary rituals, we then analyzed
when and where the infants were buried. In the Transitional period, six perinatal individuals
were placed in a flexed position on top of a large pit (surrounded by other small pits)  with
adult heads (mainly male) each inside a vessel. Sex determination showed that four of these
perinatal individuals were females (burials 45, 49, 56, and 61), while one was male and the
other one was not sampled. The females were set to the western corners of the pit, while the
male and the infant of undetermined sex to the east. This evidence suggests a possible relation
between burial orientation and sex in infants found in this specific termination ritual. Genetic
data suggest that females buried at this ritual belonged to a genetically homogeneous group.
Three of the infant females of the termination ritual (burials 45, 56, and 61) belonged to
mitochondrial haplogroup A, while the last one (burial 49) belonged to haplogroup B. Sequence
data indicated no relationships at haplotypic level between the females. This is the first time
that hypotheses based on genetic data in relation to infant sacrifices in Mesoamerican
populations are proposed, and warrants further investigations.
Teopancazco was a neighborhood center characterized by an unusual higher proportion of
adult males (48.38%) in relation to adult females (10–15%) [9, 20]. Before our research, no
genetic data had been addressed in order to compare genetic diversity between males and
females and elucidate the patterns of post-marital residence of people buried in this
neighborhood center. Thus, we analyzed genetic diversity in individuals grouped by sex, in groups of
similar sample size and we compared genetic data with archaeological evidence in order to
suggest a possible post-marital residence pattern. Nei’s genetic diversity (Ĥa) was higher in males
than in females (0.7333 ± 0.1552 and 0.5333 ± 0.1721, respectively); however, this difference
between them was not significant (p> 0.05). Likewise, the two groups were not different in
haplogroup frequencies (p> 0.05). These data suggest that females were as heterogeneous as
males, which is a characteristic of a neolocal pattern of post-marital residence, the
establishment of a new couple in an independent place of residence away from the relatives of either
spouse . Sample sizes are low (see Table 3) and a lack of power in the statistical test could
explain the lack of significance in the observed differences in genetic diversity. However, in a
neolocal population, foreign people with high degrees of mobility are correlated with exchange
systems [66–68], a characteristic observed in Teopancazco, especially in the Xolalpan phase. In
addition, it has been proposed that when an elite controls resources and property, the
remaining population follows a pattern of neolocal residence [69, 70], and it has been also proposed
that an intermediate elite ruled Teopancazco as well as other neighborhood centers in
Teotihuacan [10, 12, 18]. Furthermore, the neolocal hypothesis may be accurate only if we can
assume that the adults married in Teopancazco and the couple was also buried in Teopancazco,
and the different non-Teotihuacan burials belonging to females and males found in
Teopancazco supports this assumption (i.e. burial 102, a ritualized burial from a foreigner as indicated
by the isotopic analysis). Thus, Teopancazco had many of the characteristics that could have
promoted a neolocal population, contrary to the patrilocality pattern proposed by Spence .
Levels of mitochondrial genetic diversity found in Teopancazco, at haplogroup
(0.6404 ± 0.0738) and haplotype level (0.9167 ± 0.0643), supported the heterogeneous
character of the site, previously proposed based on evidence indicating trade and migration with
different areas and populations. Our genetic data shows genetic diversity levels similar to
populations that can be considered heterogeneous due to their high genetic fluxes with
Mesoamerican populations [51–52, 71].
Archaeological evidence suggests strong relationships between Teopancazco and several
Mesoamerican regions, mainly with ally sites towards the coastal region of the Gulf of Mexico
through the presence of a “Teotihuacan corridor” (Tlaxcala-Hidalgo-Puebla) originally
suggested by García-Cook . These relationships are represented by the presence of similar
architecture elements, burial rituals, and pottery similar to Teotihuacan’s [72–74]. Likewise,
previous isotopic analyses reported the presence in Teopancazco of possible migrants from
Oaxaca, Puebla, Hidalgo and Veracruz [7, 8]. A possible relationship with Maya populations
was based on information about a possible long-distance gene flow originated by bride
exchange, practiced between Teotihuacan and Mayan elites , as well as trading
relationships among Maya region and populations from Central Mexico via the Gulf Coast [76–77]. In
order to elucidate if trade connections were reflected in genetic relationships, we compared
Teopancazco with populations located in the “Teotihuacan corridor to the Gulf Coast”,
Oaxaca, Veracruz and the Maya regions. Our genetic data show a genetic homogeneity between
Teopancazco and all populations analyzed (global exact test of population differentiation,
p> 0.05), represented also in a PCA plot (Fig 3) which shows a clear close relationship among
all the Mesoamerican populations included, in agreement with the genetic homogeneity
reported previously in Mesoamerica . The closer genetic relationships were found between
Teopancazco and populations from the “Teotihuacan corridor” (OTO, OTO I, HUA, TEPE,
NAH II and III) and populations from Veracruz (NAH), in agreement with the idea that this
region should be related to Teopancazco in geographical and trade terms. Maya populations
from Quintana Roo (MAY IV) and Yucatán (MAY III); a Nahua population from Veracruz
(NAH I), and Zapotec and Mixtec populations from Oaxaca (ZAP and MIXT and MIXT II)
were the second group of populations more genetically related to Teopancazco in agreement
with the increase of geographical distance from Teotihuacan. On the contrary, the more distant
genetic relationships were found between Teopancazco and the Maya population from Xcaret
(MAY), an Otomi population from a mountain region of Hidalgo (OTO II), and a Pima
population (PIMA), suggesting that these populations were the most isolated in genetic and
geographical terms from Teopancazco. It is noteworthy that a Maya population from
Campeche (MAYIII) was genetically the most closely related to Teopancazco, even more than
populations from the corridor. However, no presence of Mayan elements in Teopancazco has been
reported. This pattern of relationships can also be observed in a Neighbor-Joining tree based
on haplogroup frequencies (S1 Fig) and in a Maximum Likelihood tree based on haplotype
data (S2 Fig), which shows the genetic proximity of Teopancazco to Maya, Tepehua, Mixtec,
and Zapotec populations.
Our results show that the genetic relationships between Teopancazco and populations from
the corridor are closer than the relationships with Maya and Oaxaca regions, in agreement
with the idea that most of the emigrants from Teotihuacan did not travel very far . Our
results are also in agreement with the idea that the Teotihuacan state expanded throughout the
Basin of Mexico, creating a large sustaining hinterland for the support of its large urban center,
by means of trade, political and possible human networks  that could imply an effect in the
population’s genetic diversity and could be the main reason for the multiethnic character of
In summary, our results imply that the population of Teopancazco was heterogeneous at
mtDNA level since the initial phase of the history of Teopancazco until its abandonment,
although the presence of a possible genetically differentiated group, buried during the
Transitional phase between Tlamimilolpa and Xolalpan has been detected, in agreement with the
peculiar way in which they were ritualized. Regarding the post-marital residence pattern in
Teopancazco, our genetic data, compared with archaeological evidence, suggest the presence of
a neolocal pattern. Genetic analysis of the infant population from Teopancazco allowed us to
put forward hypotheses about the termination ritual found in the Transitional period. We
found similar indices of genetic diversity between Teopancazco and other heterogeneous
civilizations such as Maya, Otomi and Nahua, which support the multiethnic character of
Teopancazco. Our data also suggest a close genetic relationship between Teopancazco and populations
from the “Teotihuacan corridor to the Gulf Coast” and from Oaxaca, in agreement with
previous archaeological evidence.
S1 Fig. Neighbor-Joining tree using Reynold´s genetic distances calculated between all
pairs of populations analyzed using haplogroups frequencies. TEO (Teopancazco),
TEO-TLAM (Tlamimilolpa period), TEO-XOL (Xolalpan period), TEO-TRAN (Transitional
phase), PIMA (Pima, Aridoamerica), ZAP (Zapotec, Oaxaca), OTO (Otomi, Hidalgo), OTO I
(Otomi, Hidalgo), OTO II (Otomi, Hidalgo), NAH (Nahua, Veracruz), NAH I (Nahua,
Veracruz), NAH II (Nahua, Puebla), NAH III (Nahua, Hidalgo), MAY (Maya, Xcaret), MAY II
(Maya, Yucatán), MAY III (Maya, Campeche), MAY IV (Maya, Quintana Roo), TEPE
(Tepehua, Hidalgo), HUA (Huastec, Hidalgo) MIXT (Mixtec, Oaxaca), MIXT II (Mixtec, Oaxaca).
We thank Hilda E. Ramos Aboites and Christian E. Martinez Guerrero for their technical
support and Dr. Sean Rovito for language editing of the manuscript.
Conceived and designed the experiments: BAA-S MGR AM RM. Performed the experiments:
BAA-S MGR. Analyzed the data: BAA-S LRM MGR AM RM. Contributed reagents/materials/
analysis tools: LRM AM RM. Wrote the paper: BAA-S RM.
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in patrilocal populations than in matrilocal populations. PNAS. 2005; 102:7476–7480. doi: 10.1073/
pnas.0409253102 PMID: 15894624
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