AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 139:284294 (2009)

Pre-Hispanic Mesoamerican Demography Approximates

the Present-Day Ancestry of Mestizos Throughout the
Territory of Mexico
Rodrigo Rubi-Castellanos,1 Gabriela Martnez-Cortes,1 Jose Francisco Munoz-Valle,2
Antonio Gonzalez-Martn,3 Ricardo M. Cerda-Flores,4 Manuel Anaya-Palafox,5
and Hector Rangel-Villalobos1
1
Instituto de Investigacion en Genetica Molecular, Centro Universitario de la Cienega, (CUCienega-UdeG),
Ocotlan, Jalisco, Mexico
2
Instituto de Investigacion en Reumatologa y del Sistema Musculo-Esqueletico,
Centro Universitario de Ciencias de la Salud (CUCS-UdeG), Guadalajara, Jalisco, Mexico
3
Departamento de Zoologa y Antropologa Fsica, Universidad Complutense de Madrid (UCM),
28040 Madrid, Spain
4
Departamento de Genetica de Poblaciones y Bioinformatica, Centro de Investigacion Biomedica
del Noreste (CIBN-IMSS), Monterrey, Nuevo Leon, Mexico
5
Laboratorio de Genetica Forense, Instituto Jalisciense de Ciencias Forenses (IJCF), Tlaquepaque,
Jalisco, Mexico

KEY WORDS STRs; CODIS; Mexican; Mestizo; admixture; STRUCTURE

ABSTRACT Over the last 500 years, admixture
among Amerindians, Europeans, and Africans, princi-
pally, has come to shape the present-day gene pool of
Mexicans, particularly Mestizos, who represent about
93% of the total Mexican population. In this work, we
analyze the genetic data of 13 combined DNA index sys-
tem-short tandem repeats (CODIS-STRs) in 1,984 unre-
lated Mestizos representing 10 population samples from
different regions of Mexico, namely North, West, Cen-
tral, and Southeast. The analysis of molecular variance
(AMOVA) test demonstrated low but significant differ-
entiation among Mestizos from different regions (FST 5
0.34%; P 5 0.0000). Although the spatial analysis of
molecular variance (SAMOVA) predicted clustering
Mestizo populations into four well-delimited groups, the
main differentiation was observed between Northwest
when compared with Central and Southeast regions. In
addition, we included analysis of individuals of Amerin-
dian (Purepechas), European (Huelva, Spain), and
African (Fang) origin. Thus, STRUCTURE analysis was
performed identifying three well-differentiated ances-
tral populations (k 5 3). STRUCTURE results and
admixture estimations by means of LEADMIX software
in Mestizo populations demonstrated genetic heteroge-
neity or asymmetric admixture throughout Mexico,
displaying an increasing North-to-South gradient of
Amerindian ancestry, and vice versa regarding the
European component. Interestingly, this distribution of
Amerindian ancestry roughly reflects pre-Hispanic
Native-population density, particularly toward the
Mesoamerican area. The forensic, epidemiological, and
evolutionary implications of these findings are dis-
cussed herein. Am J Phys Anthropol 139:284294,
2009. V 2009 Wiley-Liss, Inc.
C

The admixture process presupposes the contact of an-
cestral populations that have previously been in relative
isolation from each another and generated hybrid popu-
lations; identification of the admixture process requires
recognition of modern populations that represent, as
closely as possible, the genetic diversity of parental pop-
ulations in this hybrid. Population structure describes
the presence of individuals or subpopulations with sig-
nificant differences in admixture components (Jobling
et al., 2004), which is important for the following diverse
purposes: admixture and association mapping; evolution-
ary studies; forensic casework; medical risk prediction;
and wildlife management (Sans, 2000; Liu et al., 2005).
In particular, there has been increasing interest about
admixed populations in the biomedical field, because it is
possible to make use of recent admixture for mapping
genes underlying ethnic variation in disease risk
(Martnez-Marignac et al., 2007).
In Latin America, hybrid populations have been gener-
ated since the time of European contact with the New
World in 1492. The Americas were first colonized by
Native Americans who arrived in the last 15,00025,000
years according to molecular data (Schurr and Sherry,
2004; Kemp et al., 2007; Tamm et al., 2007; Achilli et al.,
Additional Supporting Information may be found in the online
version of this article.
Grant sponsor: CONACyT 48710.
Rodrigo Rubi-Castellanos and Hector Rangel-Villalobos contrib-
uted equally to this work.
*Correspondence to: Hector Rangel-Villalobos, Instituto de Inves-
tigacion en Genetica Molecular, Universidad de Guadalajara, CP
47810 Ocotlan, Jalisco, Mexico. E-mail: hrangel@cuci.udg.mx
Received 25 May 2008; accepted 24 October 2008
DOI 10.1002/ajpa.20980
Published online 12 January 2009 in Wiley InterScience
(www.interscience.wiley.com).

C 2009
V WILEY-LISS, INC.
 ADMIXTURE AND STRUCTURE ANALYSIS IN MEXICAN MESTIZOS
2008; Fagundes et al., 2008; Kitchen et al., 2008). In
Mexico, the process of European colonization began in
1519 when the Spaniards reached the Southeast region
of the country (presently the island of Cozumel), passing
through the state of Tabasco, and subsequently estab-
lishing the first town hall in the New World in the pres-
ent-day state of Veracruz (baptized in the Colony as Villa
Rica de la Vera Cruz). Subsequent events included the
conquest of the Mayas and Aztecs, the largest Amerin-
dian tribes at the time. After the conquest of Tenochti-
tlan, the Aztec capital city, nearly 85% of the Spanish
conquerors remained in the new state, christened The
New Spain partitioned certain parts of the territory and
formed several cities in this native land. The origin of
the Spanish conquerors was as follows: 35% from Anda-
lusia; nearly 50% from Leon, Extremadura, and Old and
New Castilla; 14% from other regions of Spain; and the
remaining 6% from outside of Spain, principally individ-
uals from Portugal and Genoa, Italy (Grunberg, 2004).
In addition to European ancestry in Mexico, African line-
ages emerged when the number of natives decreased
considerably in some regions; the Spaniards subse-
quently brought slaves from several regions of Africa,
including Cape Verde, Guinea, Angola, and the Congo,
principally, with Bantu, Congo, and Mandinga language
affiliations (Aguirre-Beltran, 1989). In fact, Spanish con-
querors imported slaves by means of contracts with Por-
tuguese slave traders, who initially exported Africans
through their West African port at Elmina in Ghana
(Douglas-Price et al., 2006). Additionally, different
genetic studies have described the possible origins of
these African people in America. For instance, mitochon-
drial DNA (mtDNA) analysis has suggested both West
and West-Central Africa as the most important suppliers
of African ancestry in Central and North America (Salas
et al., 2004). In Mexico, 30 - and 50 -haplotype analysis in
bA and bS chromosomes, respectively, has confirmed the
African roots of Mestizos from the Costa Chica region in
the states of Oaxaca and Guerrero in South-Central
Mexico (Magana et al., 2002). However, we must bear in
mind that African genes could have arrived in Mexico
from Spanish soldiers with Moorish ancestry (Aguirre-
Beltran, 1989) as a result of the Islamic occupation of
the Iberian Peninsula for nearly eight centuries (Gerard
et al., 2006). Moreover, several authors do not exclude
the existence of a certain degree of gene flow between
sub-Saharan and Mediterranean regions (Pereira et al.,
2005); this evidence can explain African contributions to
the Mexican gene pool that did not result directly from
slave trade.
The 500 years of admixture among Native Americans,
African slaves, and Europeans, primarily Spaniards, has
formed the majority of the contemporary population of
Mexico. This present-day Spanish-speaking population is
commonly referred to as Mestizo. In 1570, Mestizos con-
stituted \0.5%, but in 1810 they comprised nearly 40%
of the total Mexican population (Aguirre-Beltran, 1989)
and currently make up about 93% of the total popula-
tion, in addition to Mexican ethnic groups (INEGI,
2008). The National Institute of Anthropology defines a
Mestizo as a person born in Mexico, who has a Spanish-
derived last name, and has a family with Mexican ances-
tors who can be traced back to the third generation
(Sanchez-Serrano, 1996). However, the extent to which
several generations of intermarriage and interbreeding
among ancestral populations have determined their
actual genetic structure remains largely unknown. One
285
of the most accepted models for describing the present-
day biological diversity of Mexican Mestizos is the trihy-
brid, which presents different admixture components
depending on the geographical area (Lisker et al., 1996);
this model is represented in a tripolar diagram in which
the edges are very narrow, indicating that the number of
individuals who are genetically pure for any of the
three racial components is insignificant (Gorodezky
et al., 2001). Indeed, analysis of autosomal, uniparental
(mtDNA and Y-chromosome), and ancestry informative
markers (AIMs) in a rural Guerrero-state population,
allowed to suggest that heterogeneityalso described as
asymmetric admixture (Falush et al., 2003)is a major
characteristic of the Mexican population (Bonilla et al.,
2005). A similar analysis with different genetic systems
in population samples from Mexico City demonstrated
genetic stratification, which has been associated with ei-
ther socioeconomic or educational factors (Lisker et al.,
2004; Martnez-Marignac et al., 2007). In fact, different
levels of Amerindian, European, and African components
have been estimated in Mexican populations, ranging
from 27.6 to 94.5, 4.2 to 70.8, and 0.9 to 40.5%, respec-
tively (reviewed by Bonilla et al., 2005). Concurrently,
significant paternal heterogeneity has been demon-
strated by comparison of Y-STR haplotypes among three
Mestizo populations from North, West, and Central Mex-
ico, which has been attributable to the highest Amerin-
dian ancestry in Mexico City (Rangel-Villalobos et al.,
2008). Similarly, this genetic heterogeneity has also been
established in Hispanic populations from the United
States by means of autosomal DNA markers, due to a
diminishing West-East gradient of Native-American
ancestry (Bertoni et al., 2003). Contrariwise, D1S80,
HLA-DQA1, and STR loci analysis has displayed nondif-
ferentiation among three Mestizo populations from
North, West, and Central Mexico, suggesting relative
genetic homogeneity (Cerda-Flores et al., 2002a,b). Simi-
lar homogeneity has been demonstrated in Hispanic-
American populations (excluding those from the state of
Texas in the United States) by means of Y-STRs and
mtDNA (Kayser et al., 2003). Finally, a recent genome-
wide characterization of Latin American Mestizos has
displayed extensive variation in Native American and
European ancestry, principally (Wang et al., 2008).
Nevertheless, conclusions concerning admixture and
structure in Mexico are limited by the scarce number of
Mestizo populations analyzed from different geographi-
cal regions and the insufficiency of studies that include a
considerable number and/or type of DNA markers.
Ideally, noncoding or neutral genetic markers are
selected in an attempt to exclude the selection process as
an explanation for modern genetic-diversity patterns
(Kayser et al., 2003; Jobling et al., 2004). Among these
markers, microsatellites or short tandem repeats (STRs)
have proven useful for linkage analysis, forensics, pater-
nity cases, and anthropological studies. Some STR char-
acteristics, such as heterozygosity, abundance, polymor-
phism, mutation rate, and ease of polymerase chain
reaction (PCR) amplification, are important in the four
previously mentioned fields. Thus, STRs have become a
suitable tool in human population genetics, specifically
for population substructure evaluation (Bosch et al.,
2000; Bamshad et al., 2003). The relatively high muta-
tion rate of STRs allow one to use these markers to
address recent population history, rather than employing
binary markers (indels and single nucleotide polymor-
phisms [SNPs]), which are accumulated more slowly
American Journal of Physical Anthropology
286 R. RUBI-CASTELLANOS ET AL.
TABLE 1. Description of the Mexican Mestizo and Ancestral populations considered in this study
Population Abbr. Sample size Geographic region Reference
Mexican Mestizos
Chihuahua Chi 162 North-Central Martnez-Gonzalez et al., 2005
Nuevo Leon NL 143 Northeast Cerda-Flores et al., 2002a
Jalisco Jal 309 West Rubi-Castellanos et al., 2008
Mexico City Mex 242 Central Luna-Vazquez et al., 2005
Central Regiona CR 211 Central Hernandez-Gutierrez et al., 2005
Hidalgo Hid 106 Central Gorostiza et al., 2007
Puebla Pue 313 Central Rubi-Castellanos et al., 2008
Veracruzb Ver 130 East-Central This study
Campeche Camp 106 Southeast Sanchez et al., 2005
Yucatan Yuc 262 Southeast Rubi-Castellanos et al., 2008
Ancestral populations
Purepechab Ame 161 West-Central Mexico This study
Huelva, Spain Eur 114 South Spain Coudray et al., 2007
Fangs Afri 130 West Africa Calzada et al., 2005
a
Central Region includes the Mexican states of Morelos, Queretaro, Mexico City, and Puebla (Figure 1).
b
New STR population data available as electronic supplementary material (ESM).

throughout evolutionary time (Sahoo and Kashyap,

2005). It is probable that the most widely used STRs are
those employed in forensic casework, particularly the 13
core loci of the combined DNA index systems (CODIS),
introduced by the US Federal Bureau of Investigation
(FBI); these 13 CODIS core loci have been analyzed in
different countries, resulting in CODIS-STR population
databases worldwide (Butler, 2006). Furthermore,
CODIS-STR population data have been utilized for struc-
ture inference and admixture estimation in different
populations (Bosch et al., 2000; Cerda-Flores et al.,
2002a; Sahoo and Kashyap, 2005).
In this article, we analyzed the genetic data of 13
CODIS-STRs in 1,984 individuals of 10 Mestizo popula-
tion samples from different regions of Mexico (North,
West, Central, and Southeast). For admixture and
population structure estimation in this country, we also
included STR data of three populations considered as an-
cestral: Mexican Amerindians; Spaniards from the South Fig. 1. Geographical location of the Mexican populations an-
of Spain; and West Africans. Results were fitted to the alyzed in this work. For discussion purposes, bold lines indicate
trihybrid model, involving Amerindian, European, and the limits of the Mesoamerican region (dotted lines). See Table
1 for further explanation of abbreviations.
African ancestries, to estimate significant variation in
admixture components throughout Mexico. This supported
genetic heterogeneity or asymmetric admixture among employed agricultural copper instruments, exceptional in
Mexican-Mestizo populations, which seems to be related the Mesoamerican area. Historical records show Purepe-
with pre-Hispanic Native-population demography. chas as one of the most important pre-Hispanic cultures:
the Purepechas came to control the majority of western
Mexico (70,000 km2) including the states of Michoacan,
Guerrero, Jalisco, and Colima. In point of fact, the Pure-
SUBJECTS AND METHODS pechas were one of the few groups who resisted Aztec
Population sample expansion before the Spanish Conquest (Michelet, 2001;
CDI, 2008). At the time of the conquest, the fall of the
We analyzed the STR genetic data of 2,389 individuals Aztec capital city (Tenochtitlan) gave rise-without oppo-
from 13 population samples, including 10 Mestizo popu- sition-to the subjugation and control of the Purepechan
lations from different regions of Mexico, and three pa- state; as a reward, this Amerindian tribe received a
rental populations (Table 1); the geographical location of great degree of autonomy that was not conferred on
each Mexican population sample is represented in other tribes (CDI, 2008; Haskell, 2003). Regarding the
Figure 1. Before study inclusion, all volunteers signed suitability of Purepechas as the Amerindian ancestor, we
an informed consent form, according to Helsinki Declara- can argue the following: 1) their high gene and Y-haplo-
tion Ethical Guidelines. type diversity, 2) their even distribution in a network
Each parental population was selected according to joining tree of Amerindian Y-haplotypes, and 3) their
reasons exposed as follows. With respect to the Purepe- null European paternal admixture (Paez-Riberos et al.,
chas, the tribe considered as the Amerindian ancestor, 2006). Taken together, this suggests that the Purepechas
these derive from a mixture of Chichimecas, Nahuas, have been the beneficiaries of different Amerindian
and pre-Tarascos established at Lake Patzcuaro in pres- ancestries, in agreement with their late conformation
ent-day Michoacan State at the end of the XII century. during the post-classical era, becoming a cosmopolitan
This tribe was noteworthy because the Purepechas pre-Hispanic state (Michelet, 2001). Accordingly, a high

American Journal of Physical Anthropology

 ADMIXTURE AND STRUCTURE ANALYSIS IN MEXICAN MESTIZOS
migration rate has been suggested for the Mesoamerican
area (Nm 5 24.76) within which the Purepecha region is
located (see Fig. 1), resulting in a tendency to homoge-
nize the Mesoamerican gene pool (Rangel-Villalobos
et al., 2008). In terms of European ancestry in Mestizos,
this gene pool in its majority includes South and Central
Spain, with a predominance of the Andalusian and Cas-
tilian regions, respectively. Indeed, Palos de la Frontera,
a southwestern Spanish port in the province of Huelva
that belongs to the autonomous community of Andalusia,
served as the principal port of Spanish transit to New
Spain during the Colonial period. Therefore, we are able
to set forth the argument that the influence of this
region in Spain, and particularly that of Huelva, on
Mexican Mestizos is relevant at present (Aguirre-
Beltran, 1989). With respect to the third component, the
cultural area responsible for African ancestry among
Mexican Mestizos at the beginning of the conquest was
the present Republic of Cape Verde, with principally
Mandinga language affiliations. Nonetheless, during the
XVII century, the Bantu language family-associated of
Congo, Angola, and Sao Tome and Prncipe areas became
more relevant. In addition to Sao Tome and Prncipe
area, Equatorial Guinea, and other Fang-dominated
countries (Bantu language family) were the objects of
slave trade during the Colonial period. Although data on
the origin of slaves in this area is limited, Fang influ-
ence on this geographical area presupposes an important
contribution to African ancestry in the Mexican Mestizo.
Laboratory analysis
DNA was extracted by different methods: i) from dried
blood spotted on FTA paper or buccal swabs by Chelex1
100, or ii) from fresh blood by salting-out and phenol-
chloroform methods. Amplifications were carried out in
an 8 lL volume containing 12 ng of DNA template fol-
lowing manufacturer recommendations for the AmpF/
1
STR IdentifilerTM PCR amplification kit (Applied Bio-
systems, Foster City, CA). Amplified products were ana-
lyzed by capillary electrophoresis using the ABI PrismTM
310 Genetic Analyzer. Results were analyzed utilizing
Genescan 3.1 and Genotyper software. Genotypes were
designed by comparison with allelic ladders provided
with the kit. Once profiles and databases were obtained
to ensure consistence comparison and discussion, we con-
sidered only data from the 13 CODIS-STRs comprising
D3S1358, vWA, FGA, D8S1179, D21S11, D18S51,
D5S818, D13S317, D7S820, D16S539, THO1, TPOX, and
CSF1PO.
Data analysis
Intrapopulational analysis. For each STR/population
datum, we checked genotype-frequency departures from
Hardy-Weinberg expectations and linkage disequilibrium
between pairs of loci by exact tests. Significance levels
were assessed by 5,000 bootstrap iteration 95% confi-
dence intervals (CI) employing Arlequin 3.1 software
(Excoffier, 2005). For published STR population data
used in this study, this analysis was verified from the
report in the literature.
Interpopulational comparison. We estimated pair-
wise differences and genetic distances (FST) among popu-
lation samples applying Bonferroni correction to evalu-
ate FST P values according to the number of pairwise
comparisons for each population (P \ 0.0056; 95% CI).
287
Genetic distances were represented in a multidimen-
sional scaling (MDS) plot with SPSS for Windows ver-
sion 10 software. Second, we performed spatial autocor-
relation analysis by means of the autocorrelation index
for DNA analysis (AIDA) to evaluate whether a certain
particular evolutionary scenario had shaped the actual
genetic component of Mexican Mestizos (Bertorelle and
Barbujani, 1995)
Admixture proportions and structure inference.
These were estimated for each Mexican-Mestizo popula-
tion using maximum likelihood estimation of admixture
following parameters recommended for LEADMIX soft-
ware (Wang, 2003). We utilized spatial analysis of molec-
ular variance (SAMOVA), which is similar to the tradi-
tional analysis of molecular variance (AMOVA), but that
is sufficiently powerful to define finely population groups
that are both geographically and genetically homogene-
ous, and groups sufficiently differentiated from each
other (Dupanloup et al., 2002). Furthermore, hierarchi-
cal analyses were corroborated with Arlequin 3.1 soft-
ware (Excoffier, 2005). Finally, we used STRUCTURE
software, a model-based clustering program, to estimate
the number (K) of clusters. The STRUCTURE program
determines posterior probability Pr(X/K) that fits with
the data employed (Falush et al., 2003). Many reviews
have been published recently on the pertinence of this
type of study, and on ascertaining which model is most
appropriate for inference (Rosenberg et al., 2005). After
removing all information concerning geographic label
(unsupervised), we chose the admixture model that pre-
supposes the majority of individuals possess a mixed
membership coefficient among the different groups
assessed. Moreover, the model of correlated frequencies
(F-model) was utilized, assuming constant genetic flow
among neighboring populations. Nonetheless, we previ-
ously cited evidence of asymmetric admixture in Mexico
(see the Introduction section); thus, we employed a sepa-
rate a value for each population to resolve problems in
these situations (Falush et al., 2003). We utilized a
35,000-iteration burn-in period followed by 3,500 itera-
tions; an increase in iteration number did not improve
results. For each K-cluster value from 1 to 13, 100 repli-
cates were carried out for a particular K. With the repli-
cates obtained, we used the cluster matching and permu-
tation program (CLUMPP) to measure the symmetric
similarity coefficient (SSC) among outcomes, identifying
modes for each K (Jakobsson and Rosenberg, 2007). Of
the three CLUMPP algorithms, we considered the Large-
KGreedy algorithm with 1,000 (2
K
8) and 200 (9

K
13) random input sequences. Next, we employed a
program for graphic display of population structure
(DISTRUCT 1.1) of STRUCTURE results (Rosenberg,
2004). Finally, we implemented a supervised clustering
method that identifies the genetic pool of parental popu-
lations within Mexican Mestizos, driving these popula-
tions to fit into one cluster (POPFLAG 5 1). A total of
25 repetitions were assessed, following identical settings
as in the unsupervised method.
RESULTS
Genotype distribution by locus and two-loci combina-
tion were in agreement with Hardy-Weinberg expecta-
tions for all STRs and Mexican-Mestizo populations.
Although some small P values were obtained for some
tests, Bonferroni correction suggested little evidence for
departures from independence, and the latter does not
American Journal of Physical Anthropology
288 R. RUBI-CASTELLANOS ET AL.

merit further discussion. The majority of comparisons

Yuc

displayed significant differentiation among populations

0.0000 0.00195 6 0.0014 0.99902 6 0.0002 0.26953 6 0.0154

(Table 2). The Central Region (CR) was the most differ-
entiated with respect to the majority of Mexican Mestizo
populations (P \ 0.0056), followed by Chihuahua,

Camp

Jalisco, and Yucatan states (Table 2). The main excep-
tion was Hidalgo, the Central population that presented
nonsignificant FST P values in all comparisons (P [
0.99). This result was explained to a great degree by the

0.0112 0.46777 6 0.0149 0.99902 6 0.0002

missing data of STR genotypes observed in this popula-
tion sample, rather than by a real nondifferentiation of
the Hidalgo population. Excluding Hidalgo and CR, we

Hid

identified two large regions that were based on the fol-
lowing nondifferentiations: i) North and West Region,
NL versus Chi, and Jal and ii) Central and Southeast
Region, Camp versus Pue, Ver, and Yuc; Ver versus Mex,

0.0002 0.99902 6 0.0002

and Pue, and Pue versus Mex. As observed, these simi-

TABLE 2. Pairwise FST P-values (below diagonal) among 10 Mexican Mestizo populations
larities involve a neighboring population or a population
from the same region. In addition, all comparisons

Ver

between populations from these two large regions were

Nonsignificant FST P values are underlined (Bonferroni correction assuming nine comparisons of each population; P [ 0.0056).
significant (P \ 0.0056). On confirmation of these two
large regions identified previously, the MDS plot with
Mexican Mestizos displayed two well-differentiated y-

0.0050
axis-limited clusters: North/West and Central/Southeast
(Fig. 2a). In a second MDS plot including ancestral popu-
lations, it was evident that Central-Southeast popula-

Pue

6
6
6
6

tions (Mex, Pue, Camp, Ver, and Yuc) were closer to the

0.03613
0.99902
0.10449
0.00000
Purepechas, the Amerindian tribe employed as ancestral
(Ame). Conversely, Northwest populations (Chi, NL, and
Jal) outlined another cluster relatively closer to the

0.0000
0.0000
0.0092
0.0000
0.0000
Andalusians, the southern Spanish population consid-
ered as the European ancestor (Eur) (Fig. 2b). Interest-
ingly, the CR was included in the Northwest cluster and
CR

6
6
6
6
6

was closer to the European ancestor; this was unex-

0.00000
0.00000
0.89551
0.00000
0.00000
pected, considering the geographical origin of this sam-
ple. Ancestral-component estimation in Mexican Mesti-
zos demonstrated that admixture proportions throughout
0.0000
0.0041
0.0026
0.0002
0.0010
0.0000
Mexico, with the exception of CR, displayed a clear
North-to-South increase of the Amerindian component
(38.375.7%), with a proportional decrease of the Euro-
Mex

6
6
6
6
6
6

pean component (50.38.5%), and vice versa. Conversely,

0.00000
0.01953
0.00586
0.99902
0.00098
0.00000

African ancestry remained relatively low and constant

across populations (9.418.5%) (see Fig. 3).
To explain whether relationships among Mexican-Mes-
0.0010
0.0000
0.0000
0.0000
0.0002
0.0000
0.0000

tizo populations adhere to a particular evolutionary sce-

nario (i.e., isolation by distance, etc.), we performed a
spatial autocorrelation analysis with AIDA software. The
Jal

6
6
6
6
6
6
6

correlograms generated demonstrated solely a random

0.00098
0.00000
0.00000
0.00000
0.99902
0.00000
0.00000

process, without an apparent geographic pattern to

explain these relationships (plot not shown).
The AMOVA test demonstrated a low but significant
0.0062
0.0000
0.0000
0.0000
0.0000
0.0002
0.0000
0.0000

differentiation among Mexican populations (FST 5

0.34%; P 5 0.0000), whereas the main source of varia-
tion was within individuals (FIT 5 99.97%), attributable
NL

6
6
6
6
6
6
6
6

to the CODIS-STR loci polymorphic genetic system (Ta-

0.08496
0.00000
0.00000
0.00000
0.00000
0.99902
0.00000
0.00000

ble 3). In addition, based on AMOVA tests, we inferred

that the best grouping of Mexican populations suggested
separation of populations into three well-differentiated
0.0026
0.0016
0.0000
0.0010
0.0000
0.0000
0.0014
0.0000
0.0000

groups (FCT 5 0.434%; P 5 0.00098), representing the

North/West (Chi, NL, Jal, and CR), Center (Mex, Hid,
Pue, and Ver), and Southeast (Camp and Yuc) (Table 3).
Chi

6
6
6
6
6
6
6
6
6

The internal consistency of these three groups was corro-

0.00977
0.00293
0.00000
0.00098
0.00000
0.00000
0.99707
0.00000
0.00000

borated by the low and nonsignificant differentiation of

the populations into groups (FSC 5 0.027%; P 5 0.086).
This clustering was in close agreement with the ances-
Camp

tral components estimated previously and with the tripo-

Mex

Pue

Hid

Yuc
Chi

Ver
NL

CR
Jal

lar diagram indicating larger European and Amerindian

American Journal of Physical Anthropology

 ADMIXTURE AND STRUCTURE ANALYSIS IN MEXICAN MESTIZOS 289

Fig. 2. Multidimensional scaling (MDS) plot based on genetic differentiation (FST) values between Mexican Mestizos and three
ancestral populations. A: Genetic relationships among Mexican Mestizos: North/West, and Central/Southeast regions are delimited
by y-axis identified by two circles (comments about the assignment of CR are addressed in Discussion section); B: Genetic relation-
ships including ancestral populations. The position of northwestern Mestizos can be observed toward the European ancestor,
whereas central-southeastern Mestizos are nearer the Amerindian. See Table 1 for further explanation of abbreviations. [Color fig-
ure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 3. Admixture components in 10 Mexican-Mestizo populations, representing Amerindian (dark brown blocks), European
(green blocks), and African (orange blocks) ancestries. See Table 1 for further explanation of abbreviations. [Color figure can be
viewed in the online issue, which is available at www.interscience.wiley.com.]

TABLE 3. AMOVA and SAMOVA test results among ent states (see Fig. 1), definition of a single geographical
10 Mexican-Mestizo populations with one, threea, and fourb groups origin of this population sample was rendered compli-
Among Among Within cated; thus, we excluded CR for the subsequent hierarch-
Number populations individuals individuals ical analysis performed with SAMOVA software. This
of groups (%) (%) (%) test predicted four well-delimited groups with a small
FST P FIS P FIT P
variation reduction within individuals (FIT 5 99.65%)
One 0.34 0.0000 20.30 0.81525 99.9 0.49462 and among groups (FCT 5 0.430%) (Table 3) as follows: i)
North-Central (Chi), ii) Northeast-West (NL, and Jal),
Among iii) Central-Southeast (Mex, Hid, Pue, Ver, and Camp),
populations Within and iv) the southeastern state of Yucatan (Yuc). It was
Among groups within groups individuals
evident that two populations from opposite poles
FCT P FSC P FIT P remained as independent groups (Chi and Yuc). This ob-
servation was in agreement with the MDS plot that
Three a
0.434 0.00098 0.027 0.08602 99.84 0.49365 demonstrated NL and Jal as closer to each other with
Fourb 0.430 0.00196 -0.082 0.66276 99.65 0.00000 respect to Chi (see Fig. 2a). Similarly, in another MDS
omitting CR plot including only Mestizos from the Central-Southeast
a region (zoom in), Yucatan (Yuc) remained isolated in a
1) Chi, NL, Jal, and CR; 2) Mex, Pue, Hid, and Ver; 3) Camp
different cluster (plot not shown).
and Yuc.
b
1) Chi; 2) NL and Jal; 3) Mex, Pue, Hid, Ver, and Camp; 4) For analysis of Mexican Mestizos with the STRUC-
Yuc (according to SAMOVA test). TURE program, clusters and memberships were identi-
fied using Andalusians (Europeans), Fangs (Africans),
and Purepechas (Mexican-Amerindians) as ancestral
ancestry in Northwest and Central-Southeast regions, populations. Unsupervised population-data analysis
respectively (Gorodezky et al., 2001). However, incorrect allowed to infer K 5 3 as best predictor of Mexican-Mes-
assignment of the CR within the Northwest group was tizo genetic structure, with each ancestral population
again evident. Because the source of CR includes differ- forming a distinctive cluster (Fig. 4a). With a 2
K
5,

American Journal of Physical Anthropology

290 R. RUBI-CASTELLANOS ET AL.

Fig. 4. Bar plot of the membership coefficient for each individual from Mexican-Mestizo populations. (A) Unsupervised and (B)
supervised model are represented. All blocks match among different K. Black lines separate individuals from different populations.
Label above the bar indicates ancestral population or geographical region, and label below the bar indicates the particular popula-
tion or state where the sample was obtained. See Table 1 for further explanation of abbreviations. [Color figure can be viewed in
the online issue, which is available at www.interscience.wiley.com.]

Amerindian was determined as a separated cluster; this Ancestral populations

is in agreement with the excessive similarity of individ-
ual genomes within the Americas compared with
genomes in other regions (Wang et al., 2007). Conversely,
any distinctive cluster was made up of different K val-
ues, and the membership coefficient of all populations
was divided among the different Ks assessed. Subse-
quently, we implemented a supervised analysis (POP-
FLAG 5 1) labeling Africans, Europeans, and Amerin-
dians as ancestors, compelling these to yield correspon-
dence of Mexican Mestizos to each ancestor (Fig. 4b).
This assessment allowed to observe a North-to-South
gradient for European and Amerindian components that
decreased and increased, respectively, with a relatively
constant African ancestry analogous to the unsupervised
analysis and to admixture estimates presented previ-
ously (Figs. 3 and 4a). As previously noted, the Central-
Region population sample (CR) was the exception to the
aforementioned ancestral-component gradient.
DISCUSSION
In recent years, Mexico has been the focus of attention
regarding its ancestral components, which have been
evaluated by classical blood markers, HLA genes, auto-
somal STRs, and most recently, by Y-STRs and AIMs
(Lisker et al., 1996; Gorodezky et al., 2001; Cerda-Flores
et al., 2002a,b; Bonilla et al., 2005; Martnez-Marignac
et al., 2007). In concordance with ethnohistory, these
studies have described three main ancestral components
in Mexican Mestizos: Amerindian; European; and Afri-
can. However, consistent admixture estimates and
STRUCTURE analysis in different Mexican Mestizo pop-
ulations obtained with a reliable and widely used meth-
odology (i.e., autosomal CODIS-STRs) had not been con-
ducted throughout the country. To our knowledge, this
report constitutes the first and largest study of Mestizos
that includes the most important regions of Mexico:
North; West; Central; and Southeast. This analysis con-
ducted evaluation of admixture components (trihybrid
model) and population structure in Mexico, including the
data of 13 CODIS-STR loci of 2,389 individuals from 10
different Mestizo population samples and from three
populations representing Amerindian, European, and
African ancestries.
The success of admixture studies resides in the choice
of ancestral populations in conjunction with evidence
from several fields (ethnohistory, archeology, etc.) to
obtain ancestors who are as similar as possible to the
past founders and conquerors of the nation, which will
aid in achieving a better explanation for the genetic
admixed-population composition observed at present
(Jobling et al., 2004). However, it is obvious that the
complexity of human population history renders it diffi-
cultor even impossibleto achieve an appropriate
selection of a representative parental population. The
suitability of Purepechas, Andalusians, and Fangs to be
considered as Amerindian, European, and African ances-
tors of Mexican Mestizos, respectively, was properly
explained in the Population sample section. In fact, the
appropriateness of ancestor selection seems to be con-
firmed by the congruency of our results, because these
formed particular and very distinctive clusters in the
analyses here performed, such as genetic distances
(MDS plot, Fig. 2b) and unsupervised population
STRUCTURE analysis (Fig. 4a).
Admixture estimates
Recently, admixture estimates have received attention
in particular areas, mainly association mapping, evolu-
tionary studies, and disease-risk prediction (Sans, 2000;
Liu et al., 2005; Wang et al., 2008). Notwithstanding
this, the sources promoting changes in estimates play a
key role in the analysis. If only 515% of total genetic
variation results from differences among human popula-
tions, it is evident that alleles common in one population
are also common in others. Within this context, we found
two related situations: i) the majority of genetic markers
are unaffected by admixture, and some authors suggest
selection of markers that exhibit sufficient allelic differ-
ences among parental populations (Halder and Shriver,
2003), and ii) erroneous or incomplete archeological and
ethnohistoric data complicate genetic-background assess-
ment (Jobling et al., 2004). Despite that in Mexico a
variable amount of African, European, and Amerindian
ancestry has been estimated by means of different
genetic systems (reviewed by Bonilla et al., 2005), we
implemented a maximum likelihood estimation of admix-

American Journal of Physical Anthropology

 ADMIXTURE AND STRUCTURE ANALYSIS IN MEXICAN MESTIZOS
ture with the LEADMIX program, including a number of
population samples that are extensively distributed
throughout the Mexican territory and utilizing informa-
tive markers widely employed for human identification
purposes. Finally, our admixture estimates were concord-
ant with previous estimates achieved with different
genetic systems evaluated by means of ethnohistoric
records (Bonilla et al., 2005).
Comparisons with other genetic systems show that our
estimates of ancestry components for western Mestizos
(European, 53.2%; Amerindian, 30.8%; and African,
15.9%) were very close to those reported by binary Y-
linked loci (European, 6064%; Amerindian, 2521%;
and African, 15%). Moreover, and in agreement with
our present results, European and Amerindian predomi-
nance also was inferred, particularly in North and Cen-
tral Mestizos, respectively (Rangel-Villalobos et al.,
2008). Although unfortunately an exact paternal Mexi-
can-Mestizo contribution from all regions of the country
does not exist, available Y-linked data also suggest the
decreasing European North-to-South gradient that was
clearly observed in this study. Conversely, the maternal
counterpart (mtDNA) suggests relative homogeneity in
Mexican Mestizos because of the predominance of Amer-
indian haplogroups (A, B, C, and D), considering avail-
able data from the North (89.1%), West (82%), Central
(90.5%), and Southeast (98%) (Green et al., 2000; Bonilla
et al., 2005; Sandoval et al., 2006; Martnez-Marignac
et al., 2007). At present, the maternal composition of
Mexican-Mestizo populations deserves further research.
In fact, it is expected that a larger admixture impact can
be found in paternal ancestry, considering that Spanish
males were preferentially carried out during the Con-
quest of the New World; their mating with indigenous
females, this could explain the effect known as asymmet-
ric gene flow, which has been widely described in Latin-
American populations (Batista-Dos-Santos et al., 1999;
Carvajal-Carmona et al., 2000; Mesa et al., 2000;
Seielstad, 2000; Campos-Sanchez et al., 2006; Wang
et al., 2008).
Regarding the African component, the northern state
of Nuevo Leon exhibited the highest level (18.5%), three
times greater than previous estimates (5.02 6 2.82%)
(Cerda-Flores et al., 2002a). Explanations concerning dif-
ferences in these ancestry estimates could include the
following: 1) the parental populations selected, 2) the
statistical approaches employed for determining ances-
tral proportions, 3) the amount of loci employed (13 vs.
10), and 4) a population sample bias, especially in sub-
structured populations. However, the latter explanation
does not apply in this case because both studies utilized
the same STR database. Although AIMs analysis could
resolve these components to a profound degree, our
study provides a robust landscape of ancestry through-
out the Mexican territory, whichever genetic system is
employed.
Considering previous descriptions of higher African
ancestry in different regions along the Mexican littorals
(Lisker et al., 1996; Gorodezky et al., 2001), we expected
to find highest African ancestry levels in the coastal
state of Veracruz (see Fig. 1); however, this was not
observed (17.2%). The majority of these proposals have
arisen from historical records describing the African con-
tribution (Aguirre-Beltran, 1989), and some of these
have received support from genetic studies, for instance,
the Costa Chica region in the states of Oaxaca and Guer-
rero (Magana et al., 2002). Although the increase of Afri-
291
can ancestry could be true in particular regions (such as
in Costa Chica-region villages), our results suggest that
this is not necessarily true for population samples from
larger cities (such as the sample studied herein). In fact,
preliminary results detecting African Y-chromosomes
(xYAP) in the Costa Chica region have also failed to
detect higher African ancestry (Rangel-Villalobos et al.,
2008). Alternatively, gene flow over time of non-African
individuals into these communities (the diluting effect)
could explain their current genetic composition. A simi-
lar effect has been suggested in the Colombian popula-
tion of Antioquia, where ongoing admixture with Span-
ish males (but not with native females) has increased
the European nuclear ancestry of this Latin-American
population (Bedoya et al., 2006).
As demonstrated in the majority of Latin-American
populations, modern Mexican Mestizos basically present
trihybrid ancestry; namely, Amerindian, European, and
African. However, our estimates indicate different levels
for each ancestry throughout the country, demonstrating
genetic heterogeneity or asymmetric admixture, which
has been previously described in Mexico (Lisker et al.,
1996; Gorodezky et al., 2001) and in Latin-American
Mestizos (Sans, 2000; Wang et al., 2008). Although addi-
tional minor ancestries could exist in Mexican popula-
tion (i.e., Asian), previous results in Latin American
Mestizos suggest these would be poorly represented
(Wang et al., 2008); thus, the purpose of this study was
not identification and characterization of these minor
ancestries. Nonetheless, we obtained unexpected results
in the CR, which presented closer similarities to North
and West populations (Figs. 2 and 3) and significant dif-
ferentiation with regard to Central and Southeast popu-
lations, which are geographically closer to-or even
included in-the CR region (see Fig. 1). The main point
that could explain this contradictory result lies in the or-
igin of the CR sample, which was obtained from the Uni-
versidad Panamericana, a private educational institution
that offers paternity testing services whose population
sample comprises individuals from middle to upper socio-
economic strata (Hernandez-Gutierrez et al., 2005; per-
sonal communication). In fact, both higher socioeconomic
and educational status have been associated with higher
European ancestry (Lisker et al., 2004; Gonzalez-
Burchard et al., 2005). Higher educational status, partic-
ularly in Mexico City, has been demonstrated, which
increases European admixture proportions significantly
(odds ratio [OR] 5 9.4; 95% CI 3.822.6) (Martnez-
Marignac et al., 2007). In conclusion, contradictory
results in the CR grouping are probably explained by a
sampling bias, this an outcome of stratification demon-
strated in Mestizos from Mexico. Considering this
information, we could anticipate that these population
samples involving higher socioeconomic or educational
status that were obtained from the Central (or even the
Southeast) region will probably exhibit a similar Euro-
pean ancestry to that of samples obtained from the
North and West regions of Mexico.
Structure and asymmetric admixture
In addition to the ancestry gradients discussed previ-
ously, results allowed clustering of Mexican-Mestizo pop-
ulations in different regions. In fact, SAMOVA analysis
identified four groups as best-data predictors (Table 3).
Discussion can arise concerning grouping (or separating)
populations from the same region, the limited number of
American Journal of Physical Anthropology
292 R. RUBI-CASTELLANOS ET AL.
populations and STR loci analyzed herein, population
structure by socioeconomic and educational status as
previously discussed, and considering that ancestral
components at present could be observed as a cline
throughout the Mexican Republic. Notwithstanding this,
differentiation between Central/Southeast and North/
West regions was clearly shaped, demonstrating an
asymmetric admixture. The apparent difference between
Chihuahua (North) and Yucatan (Southeast) depicts the
contrasting genetic composition among Mestizos. This
asymmetry in Mexican Mestizos plays a key role in the
design of epidemiological studies, particularly for case-
control assays in which, considering the present results,
similar ancestry must be demonstrated in both case and
control population samples to avoid flawed gene-disease
associations (Xu and Shete, 2005; Choudhry et al., 2006).
Our results are in agreement with admixture conclu-
sions with ancestry informative markers (AIMs)
obtained recently in Mestizos from Mexico City
(Martnez-Marignac et al., 2007; Wang et al., 2008).
Similarly, in terms of human identification purposes in
Mexican Mestizos, results compel us to employ the STR
database of the relevant population when DNA matching
is found in forensic casework or paternity testing. In
addition, when the appropriate STR database is not
available, our results could allow inferring the best-
nearby-population for DNA profile interpretation.
Taken together, supervised and unsupervised STRUC-
TURE analysis, admixture estimates, genetic differentia-
tion pattern (FST P values), and MDS plot indicated that
the Amerindian membership of Mestizos decrease to-
ward the northern region, simultaneously increasing
their European membership, with a relatively stable
African membership throughout the country. This pat-
tern probably reflects a complex admixture process that
involved several historic factors including warfare, epi-
demical diseases, slave trading, Spanish-immigration
density, Native-population density, etc. (Schurr and
Sherry, 2004). Regarding population density, the approxi-
mate Native-population size of Mexico and the Carib-
bean in contact with Spaniards has been estimated at
25 million inhabitants (Cook and Borah, 1971); these
Native-Americans were mainly concentrated in Mesoa-
merica, where important chiefdoms and states had been
formed and/or destroyed, and included, for example, the
Olmec, Teotihuacan, Toltec, Maya, and Aztec civiliza-
tions, among many others. These civilizations can be
classified as agricultural societies, which possess denser
and larger human populations than foraging societies
(Fiedel, 1992). Human depopulation by warfare and epi-
demic diseases (principally smallpox) in this area
reduced the Native population to nearly 3% of its origi-
nal size in 1623 (Cook and Borah, 1971). Although some
differences in the depopulation rate have been described,
as in the Tlaxcalteca tribe that was allied with Span-
iards during the Conquest, the depopulation process
affected all Native-American populations in general
(Crawford, 1998). Nonetheless, Native groups recuper-
ated their original sizes in 1810, principally because of
their higher growth rates with respect to Europeans and
Africans (Aguirre-Beltran, 1989). As a result, a rough
correspondence can be observed between the higher pre-
Hispanic population density of Mesoamerica and contem-
porary higher Amerindian ancestry in Central and
Southeast regions of Mexico. In brief, although many
and important historic and demographic factors have
been involved, we suggest that pre-Hispanic Native-pop-
American Journal of Physical Anthropology
ulation density has operated as the main factor for
shaping Amerindian ancestry in present-day Mexican-
Mestizo populations. Recently, a similar conclusion has
been obtained for Latin American Mestizos across the
continent based on genome-wide admixture analysis
(Wang et al., 2008).
CONCLUSIONS
Analysis based on CODIS-STRs in Mexican Mestizos
confirmed their admixture pattern comprises the trihy-
brid model. An ancestral-component gradient was estab-
lished, increasing European toward the North and Amer-
indian to the Southeast, whereas the African component
remained relatively constant. Two principal clusters
were formed by Mestizos in the Northwest when com-
pared with those in the Central-Southeast. Finally, we
propose that the present ancestral composition of Mexi-
can Mestizos geographically throughout the country cor-
responds to the pre-Hispanic Native-population density
that involves the Mesoamerican region in particular.
ACKNOWLEDGMENTS
The authors thank the anonymous reviewers who pro-
vided helpful comments to improve the manuscript.
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