Y-linked haplotypes in Amerindian chromosomes from Mexican populations: Genetic evidence to the dual origin of the Huichol tribe

Legal Medicine 8 (2006) 220–225 www.elsevier.com/locate/legalmed

Brief communication

Y-linked haplotypes in Amerindian chromosomes from Mexican populations: Genetic evidence to the dual origin of the Huichol tribe L.A. Pa´ez-Riberos a, J.F. Mun˜oz-Valle b, L.E. Figuera c, I. Nun˜o-Arana a, L. Sandoval-Ramı´rez c, A. Gonza´lez-Martı´n d, B. Ibarra c, H. Rangel-Villalobos a,* a

Laboratorio de Gene´tica Molecular, Centro Universitario de la Cie´nega, Universidad de Guadalajara (CUCie´nega-UdeG), Carretera Ocotla´n-Tototla´n Km. 3.3., A.P. 106, 47810 Ocotla´n, Jalisco, Me´xico b Laboratorio de Inmunologı´a, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Jalisco, Me´xico c Divisio´n de Gene´tica, Centro de Investigacio´n Biome´dica de Occidente (CIBO-IMSS), Guadalajara, Jalisco, Me´xico d Departamento de Zoologı´a y Antropologı´a Fı´sica, Universidad Complutense de Madrid (UCM), Madrid, Spain Received 4 October 2005; received in revised form 21 January 2006; accepted 27 February 2006 Available online 21 June 2006

Abstract We studied six Y-linked short tandem repeats (Y-STRs) to describe the internal diversity of the Amerindian haplogroup Q-M3 in 129 males from eight Mexican populations. The low gene diversity in the Huichol tribe demonstrated the effects of genetic drift, attributable to geographic isolation and founder effect. The presence of two principal paternal lineages supported the historical and anthropometric records, which indicate that Huichols were formed by the fusion of two ancestral Mexican tribes. Moreover, genetic distances and close relationships of haplotypes between Huichols and Tarahumaras were in agreement with their linguistic affiliation. The high genetic diversity of the Pure´pechas and wide distribution of haplotypes along the constructed network-joining tree suggest that the present genetic composition was influenced by Pure´pecha dominance in western Mesoamerica. The Y-haplotypes shared between populations suggest that, among the Amerindian tribes studied herein, the paternal genetic pool of Nahuas could have contributed more importantly to the European-admixed population, the Mexican-Mestizos. q 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Y-chromosome; STRs; Amerindian; Mexican population; Mestizo; Mexican tribes

1. Introduction The study of markers in the non-pseudoautosomal region of the Y-chromosome demonstrates our ability to elucidate the history of human populations. In America, the binary marker M3 (transition C/T) distinguishes Amerindian Y-chromosomes [1]. Moreover, Y-linked STRs, with a higher mutation rate permit us to analyze the internal diversity of the Amerindian haplogroup known as Q-M3 [2]. It is particularly noteworthy that there are few Y-chromosome studies that analyze the origin and genetic relatedness among Mexican populations [3]. In Mexico, Mestizos are * Corresponding author. Tel.: C52 392 92 500 26x8363; fax: C52 392 92 530 99. E-mail address: [email protected] (H. Rangel-Villalobos).

1344-6223/$ - see front matter q 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2006.02.003

the most widely distributed population living in both urban and rural regions throughout the country; using language as a selection criterion, these constitute nearly 90% of the total population [4]. In addition to the admixed Spanish-speaking Mestizo population, there are more than 60 well-defined Mexican Amerindian tribes [5]. Unfortunately, Mexican tribes are diminishing due to ongoing absorption into Mestizo society; thus, the opportunity for analyzing this Amerindian component in order to accept or discard the hypothesis proposed by other disciplines such as linguistics, ethnology, and archeology is at stake. In this work, we analyzed the internal diversity of Amerindian Y-chromosomes (Q-M3) from six Mexican populations by means of six Y-linked short tandem repeats (Y-STRs) (DYS19, DYS389-I, DYS390, DYS391, DYS392, and DY393). We discussed genetic evidence

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from a historical perspective; in particular, we found evidence favoring a dual paternal origin of the Mexican Huichol tribe.

2. Subjects and methods We analyzed a total of 129 unrelated Mexican individuals. Mestizo samples consisted of 31 males born in western Mexico. Amerindian samples originated from 98 native males, including Huichols (34) from the Sierra Madre Occidental in northern Jalisco and eastern Nayarit states, Pure´pechas (15) from the lacustrine region of Michoaca´n state, Tarahumaras (11) from mountains and canyons in the northern Mexican state of Chihuahua, Nahuas (24) from the highlands in southern Puebla state, and Tzotziles (4) from the state of Chiapas (Fig. 1). Additionally, we included QM3 haplogroups of Mayas (6) and Zuni Indians (4) [6]. Detailed anthropologic descriptions regarding Huichols, Pure´ pechas, and Tarahumaras have been previously described [7]. We collected Nahua samples from the municipalities of San Jose´ Miahuatla´n and Zoquita´n. Nahuas are the most widely distributed ethnic group in the country, with the states of Puebla and Veracruz having the highest Nahua populations. Linguistically, the Nahuas belong to the Nahua-Cuitlateco group, Yutonahua trunk, and Nahua family. We collected Tzotzil samples from the village of San Cristo´bal de las Casas, Chiapas. Furthermore, the Tzotziles belong to the Maya-Totonaco group, Mayense trunk, Mayense family, and Yax subfamily [5]. Prior to inclusion, all volunteers signed an informed consent form

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approved by the Ethical Research Committee at the CUCie´nega (University of Guadalajara, UdeG). DNA was extracted by standard phenol–chloroform and salt-out methods. We analyzed six Y-linked STRs: DYS19, DYS389-I, DYS390, DYS391, DYS392, and DYS393; primers and PCR conditions were previously reported [8]. Amerindian M3 polymorphism was analyzed according to the protocol reported by Lell et al. [1] with the MunI restriction enzyme (Life Technologies, Inc.). We used positive controls to confirm correct digestion, as well as negative PCR-controls to detect possible contamination. The amplified products were run on vertical polyacrylamide gels with 1!TBE buffer prior to silver staining. Nomenclature for allele STRs was based on repeat number. In this report, we analyze exclusively Y-chromosomes with the Amerindian marker M3; they were named along this manuscript as Q-M3, according to the recommendations of the Y-chromosome consortium [9]. We estimated allele frequencies by counting method, and gene diversities such as theoretical heterozygocity (h) for each Y-linked STR, as well as haplotype diversity (D) with its variance for total population. The relationship among populations was analyzed by (a) haplotypes shared among populations, (b) clustering diagrams using the neighbor-joining (NJ) method based on the coancestry coefficient [10], and (c) the network-joining tree between STR-haplotypes with reduced median (RM) algorithm. For this purpose, mutational weights were inversely correlated with the allelic variance of each STR. Finally, the RM network was purged of superfluous links with maximum parsimony (MP) algorithm [11]. For the RM tree, we measured ancestral node age in

Fig. 1. Geographical distribution of the Mexican tribes and Mestizo population.

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mutational events. It was converted into years using the mutation rate of 3.17!10K3 estimated by Kayser et al. [12] and generation time of 20 years. Analysis was performed with the ARLEQUIN 2000 software package [13], the GDA versio´n 1.1 program [14] and NETWORK 4.1.1.1 [15].

Table 1 Allele distribution and diversity parameters (%) of six Y-STRs of the Amerindian haplogroup Q-M3 in Mexican populations Y-STR allele DYS19 13 14 h DYS389-I 9 10 11 12 h DYS390 22 23 24 25 26 h DYS391 9 10 11 12 h DYS392 11 12 13 14 15 16 17 h DYS393 12 13 14 15 h

n

%

Gs.d.

105 24

81.4 18.6 30.28

3.4 3.4

20 77 30 2

15.5 59.7 23.3 1.6 56.50

3.2 4.3 3.7 1.1

9 51 51 14 4

7 39.5 39.5 10.9 3.1 67.02

2.3 4.3 4.3 2.7 1.5

15 82 30 2

11.6 63.6 23.3 1.6 52.75

2.8 4.3 3.7 1.1

1 5 27 41 33 16 6

0.8 3.9 20.9 31.8 25.6 12.4 4.9 77.03

0.8 1.7 3.6 4.1 3.9 2.9 1.9

5 107 14 3

3.9 82.9 10.9 2.3 29.88

1.7 3.3 2.7 1.4

Diversity parameters Populationa

n (#)

D

p

Mestizo Huichol Pure´pecha Tarahumara Nahua Total pop.a

31 (25) 34 (15) 15 (14) 11 (9) 24 (19) 129 (74)

98.49G1.2 89.85G3.2 99.05G2.8 96.35G5.1 96.07G3.1 98.5G3.7

3.15 2.3 3.4 2.65 2.89 3.16

Gene diversity (h); haplotype diversity (D); sample size (n) and number of different haplotypes (#); average of pairwise differences (p). a Populations with sample size lesser than 10 individuals are not included.

3. Results and discussion Allele distribution and diversity parameters of the total sample number are presented in Table 1. This is the first report of Y-linked STRs in Mexican males from Amerindian origin (Q-M3); allele frequencies for all six Y-STRs were in agreement with previous reports of Amerindians [6] but different from Mexican-Mestizos for most Y-STRs (P! 0.05), excluding DYS390 [8]. Haplotype distribution, by population and pooled groups, is presented in Table 2; these data can be applied for personal identification purposes in ethnic communities, and to infer the Amerindian origin of biological samples found in forensic casework all around the country. It was interesting that Pure´pechas presented the highest diversity (p and D), also with regard to the admixed Mexican-Mestizos, whereas Huichols had the lowest (Table 1). In Huichols, results imply genetic drift, as previously suggested by autosomal markers [7], and are in agreement with his geographic and cultural isolation [16]. Among haplotypes shared between populations, haplotypes A17, A25, and A27 were the most widely distributed in three Mexican populations, respectively (Table 2). Based on STR allele frequencies, we inferred ancestral STRhaplotypes (DYS19-13, 389-I-10, 390-24/23, 391-10, 39214, and 393-13). The haplotype in bold (A24) was identical to the ancestral Amerindian haplotype 0A, previously obtained by Bianchi et al. [6]; in both studies, this was neither the most frequent nor the most geographically dispersed. However, due to its central position it was considered the ancestral node in the RM network for measuring age of Q-M3 (Fig. 2), which was estimated in 17,948.9G3958.1 years; this was in the middle of previous assessments [2,6], but in agreement with archeologic and anthropologic records [17]. Nahuas possessed an even distribution along the RM network and shared the major number of haplotypes (4) with Mestizos. In concurrence, genetic distances demonstrated Nahuas to be the closest Amerindian group to Mestizos in the NJ tree (Not shown). This suggests that, among the Amerindian tribes studied herein, the paternal genetic pool of Nahuas could have contributed more importantly to the admixed Mexican-Mestizos than the remaining tribes, which could be explained by the wide geographic distribution of Nahuas that includes 11 different Mexican states [18]. It was of particular note that Huichols presented two haplotypes not directly linked, with high frequencies (A44Z20.6% and A62Z23.5%). This could be the result of polygyny, previously common but no longer practiced [16] and genetic drift previously described with autosomal markers [7]. For the purposes of discussion, these frequent haplotypes (A44 and A62) were considered as two principal Huichol groups. The A44 group was derived from ancestral haplotype A25 and included principally Tarahumaras and Huichols (Fig. 2). The close relationship between

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Table 2 Haplotype distribution by population of six Y-STRs of the Amerindian haplogroup Q-M3 in Mexican populations IDa

DYS19, 389-I, 390, 391, 392, 393

Mestizo

Tarahumara

Huichol

Pure´pecha

Tzotzil

Nahua

Mayab

Zunib

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 A25 A26 A27 A28 A29 A30 A31 A32 A33 A34 A35 A36 A37 A38 A39 A40 A41 A42 A43 A44 A45 A46 A47 A48 A49 A50 A51 A52 A53 A54 A55 A56 A57 A58 A59 A60

13 09 22 10 13 13 13 09 22 10 13 14 13 09 22 10 14 13 13 09 23 09 13 13 13 09 23 10 13 13 13 09 24 09 13 13 13 09 24 09 13 15 13 09 24 09 14 15 13 09 24 11 17 14 13 09 25 09 13 13 13 09 25 10 13 13 13 10 23 09 13 14 13 10 23 10 13 13 13 10 23 10 13 14 13 10 23 10 14 12 13 10 23 10 14 13 13 10 23 10 15 13 13 10 23 10 16 14 13 10 23 11 14 13 13 10 23 11 15 13 13 10 23 11 16 14 13 10 24 10 11 13 13 10 24 10 12 13 13 10 24 10 14 13 13 10 24 10 15 13 13 10 24 10 16 13 13 10 24 10 17 13 13 10 24 11 13 13 13 10 24 11 14 13 13 10 24 11 14 14 13 10 24 11 16 13 13 10 24 11 16 14 13 10 24 11 17 13 13 10 25 10 13 13 13 10 25 10 14 13 13 10 25 10 16 13 13 10 25 11 14 13 13 10 25 11 15 13 13 10 26 10 14 13 13 11 22 09 15 13 13 11 22 09 16 13 13 11 22 10 15 12 13 11 22 10 15 13 13 11 23 10 15 13 13 11 23 11 13 13 13 11 23 11 15 12 13 11 23 11 15 13 13 11 23 12 15 13 13 11 24 10 14 13 13 11 24 10 16 13 13 11 24 11 13 13 13 11 24 11 14 13 13 11 24 11 16 13 13 11 25 10 16 13 13 11 25 11 14 14 13 11 26 10 14 13 13 12 24 10 15 13 14 09 24 09 13 15 14 09 24 10 16 13 14 09 24 11 15 13

0 1 0 0 0 0 0 0 2 1 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 2 1 1 1 1 1 1 0 1 2 0 0 0 3 0 0 0 0 0 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0

0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 2 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 1 0 0 0 0 0 0 0 3 0 0 3 1 0 0 2 0 0 0 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 7 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0

1 0 0 0 1 1 1 1 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 1 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0

0 0 0 1 0 1 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 1 5 0 2 1 1 0 0 0 1 0 1 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0

0 0 0 1 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0

0 1 0 1 0 1 0 2 0 2 0 1 0 1 0 1 0 2 0 2 0 1 0 1 0 3 0 2 0 2 0 4 0 3 0 1 0 2 1 3 0 1 0 1 0 5 0 3 0 3 0 3 1 3 0 1 0 2 0 1 0 2 0 1 0 1 0 1 0 2 0 1 0 1 0 2 0 3 0 1 0 1 0 1 0 2 0 9 0 1 0 1 0 1 0 1 0 2 0 1 0 1 0 2 0 1 0 1 0 1 0 1 0 1 0 1 1 1 1 1 (continued on next page)

Pooled pop.

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224 Table 2 (continued) IDa

DYS19, 389-I, 390, 391, 392, 393

Mestizo

Tarahumara

Huichol

Pure´pecha

Tzotzil

Nahua

Mayab

Zunib

Pooled pop.

A61 A62 A63 A64 A65 A66 A67 A68 A69 A70 A71 A72 A73 A74

14 10 23 09 14 13 14 10 23 10 14 13 14 10 23 10 15 13 14 10 23 12 15 13 14 10 24 10 13 13 14 10 24 10 14 14 14 10 24 10 15 13 14 10 24 10 16 13 14 10 24 11 13 13 14 10 25 11 13 13 14 11 22 09 16 13 14 11 24 10 14 12 14 12 25 10 14 13 13 09 23 09 13 14

0 0 0 0 1 2 1 0 1 1 0 0 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 8 0 1 0 0 0 0 0 0 1 0 0 0

1 0 1 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 1 0 0 0 1 0 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 8 1 1 1 2 1 1 1 1 1 1 1 1

Tarahumaras and Huichols was in agreement with the inferred genetic relatedness as displayed in the NJ tree (not shown), with their linguistic affiliation to the Uto-Aztecan family, and with previous results analyzing STRs and VNTRs [7]. The A62 group was derived from haplotype A24 (0A from Bianchi et al., [6]) and included the majority of Mexican tribes, suggesting that the A62 group is more ancestral than A44. Interestingly, tradition maintains that the Huichols came from Hicuripa, a place geographically located at North-Center of Mexico in the state of San Luis

Potosı´. A fraction of this tribe survived by migrating to the Nayarit Mountains guided by the mythical leader Majakuagy, where they fused with local tribes like Teules, Tepehuanos, Coras, Cascanes, etc. forming the actual Huichols [16]. This account is supported by the existence of two basic physical phenotypes and the presence of individuals with different cephalic indexes, brachycephalous and dolichocephalous. It has been interpreted that the primitive group was brachycephalous, whereas the dolichocephalous was the more advanced group who brought

Fig. 2. Network joining tree by reduced median algorithm of Y-STR haplotypes (Q-M3) from Mexican-Mestizos and Amerindian tribes. The size of the circles corresponds to the haplotype frequency, and the lengths of the links are proportional to the number of mutations between haplotypes. The large circles indicate the two principal Y-linked lineages in Huichols.

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religion, arts, and agriculture to the present Huichol culture [16]. In agreement with these historical records, our results reveal the presence of two principal Y-chromosome lineages in Huichols, one ancestral derived from A24 (A62 group) and one more recent lineage derived from A25 (A44 group). The presence of North-Central tribes in group A44 (Tarahumaras, Zuni Indians, and Pure´pechas, principally) suggests that this Y-lineage represents the Northmigrating group who arrived more recently at the Nayarit Mountains. Although our results support the dual origin of these Mexican tribes, this observation should be confirmed with a larger Huichol sample size. The origin of the Pure´pechas involves admixture among Chichimecas, Nahuas, and pre-Tarascos, but migrations from Peru along the Pacific coast have been involved; there is linguistic, archeologic, and ethnographic evidence that supports this point of view. Linguistically, Pure´pechas are an isolated group without direct relationships with other Mesoamerican tribes; they have been related with the South American Quechua–Aymara tribes and the Zuni Indians from the southern area of the US [19]. Historical records show Pure´pechas as one of the most important Pre-Hispanic cultures that came to control the majority of western Mexico (70,000 km2) including the states of Michoaca´n, Guerrero, Jalisco, and Colima [20]. In point of fact, they were one of the few groups who resisted the Aztec expansion prior to the Spanish conquest [21]. This predominance of Pure´pechas in Mesoamerica could be related to the even distribution of haplotypes along the RM network (Fig. 2) and with the highest genetic diversity for both Y-linked (this report) and autosomal markers [7]. In conclusion, our results provided information with respect to (a) forensic potential of Y-STRs in Mexican males of Amerindian origin, (b) the genetic relationships among the studied Mexican populations, (c) the dual origin of the Huichols, (d) the genetic impact of the Pre-Hispanic importance of the Pure´pecha group, and (e) the manner in which wide geographic distribution of Nahuas could have influenced the present genetic pool of Mestizos. Acknowledgements We thank the QFBs and Vania Navarro, Cristina Camacho and Alejandra Moreno for their technical assistance. This work was supported by the grant no. 33949 of the Consejo Nacional de Ciencia y Tecnologı´a (CONACyT) to H.R.-V. We also thank CONACyT for the Doctoral fellowship to L.A.P.-R.

References [1] Lell JT, Brown MD, Schurr TG, Sukernik RI, Starikovskaya YB, Torroni A, Moore LG, Troup GM, Wallace DC. Y chromosome polymorphisms in Native American and Siberian populations: identification of Native American Y chromosome haplotypes. Hum Genet 1997;100:536–43.

225

[2] Ruiz-Linares A, Ortı´z-Barreintos D, Figueroa M, Mesa N, Mu´nera JG, Bedoya G, Ve´lez ID, Garcı´a LF, Pe´rez-Lezaun A, Bertranpetit J, Feldman MW, Goldstein DB. Microsatellites provide evidence for Y chromosome diversity among the founders of the New World. Proc Natl Acad Sci USA 1999;96:6312–7. [3] Torroni A, Chen Y, Semino OA, Santachiara-Beneceretti AS, Scott CR, Lott MT, Winter M, Wallace DC. mtDNA and Y-chromosome polymorphisms in four Native American populations from Southern Mexico. Am J Hum Genet 1994;54:303–18. [4] Ferna´ndez P, Serrano E. The indigenous population of Mexico as counted by the 1990 and 1995 censuses (CONAPO/DAF-INAH). Meeting of the Mexican Demography Society; 1996. [5] Scheffler L. Los Indı´genas Mexicanos. Me´xico D.F.: Editorial Panorama; 1999. [6] Bianchi NO, Catanesi CI, Bailliet G, Martinez-Marignac VL, Bravi CM, Vidal-Rioja LB, Herrera RJ, Lo´pez-Camelo JS. Characterization of ancestral and derived Y-chromosome haplotypes of New World native populations. Am J Hum Genet 1998;63:1862–71. [7] Rangel-Villalobos H, Rivas F, Sandoval L, Garcı´a-Carvajal ZY, Cantu´ JM, Figuera LE. Genetic variation among four Mexican populations (Huichol, Purepecha, Tarahumara and Mestizo) revealed by two VNTRs and four STRs. Hum Biol 2000;72:983–95. [8] Rangel-Villalobos H, Jaloma-Cruz AR, Sandoval L, Velarde-Fe´lix JS, Gallegos-Arreola MP, Figuera LE. Y-chromosome haplotypes for six STRs in a Mexican population. Arch Med Res 2001;32:232–7. [9] The Y chromosome Consortium. A nomenclature system for the tree of human Y-chromosomal binary haplogroups. Genome Res 2002;12: 339–48. [10] Reynolds J, Weir B, Cockerham C. Estimation of the concestry coefficient: basis for a short term genetic distance. Genetics 1983;105: 767–79. [11] Bandelt HJ, Forster P, Sykes BC, Richards MB. Mitochondrial portraits of human populations. Genetics 1995;141:743–53. [12] Kayser M, Roewer L, Hedman M, Henke L, Brauer S, Kru¨ger C, Krawczak M, Nagy M, Dobosz T, Szibor R, De Knijff P, Sajantilla A. Characteristics and frequency of germline mutations at microsatellite loci from the human Y-chromosome, as revealed by direct observation in father/son pairs. Am J Hum Genet 2000;66:1580–8. [13] Scheneider MT, Roessli D, Excoffier L. ARLEQUIN version 2000: a software for population genetic analysis. Geneva: Genetics and Biometry Laboratory, University of Geneva; 2000. [14] Lewis PO, Zaykin D. Genetic Data Analysis: computer program for the analysis of allelic data. Version 1.0 (d16c). Free program distributed by the authors over the internet from http://lewis.eeb. uconn.edu/lewishome/software.html. [15] Polzin T, Daneshmand SV. NETWORK 4.1.1.1. Steiner algorithm; Fluxus Technology Ltd. 2004. [16] Diguet L. Por tierras occidentales entre sierras y barrancas. Centro de estudios mexicanos y centroamericanos de la embajada de Francia en Me´xico-Instituto Nacional Indigenista, Me´xico D.F; 1992. [17] Crawford MH. The origin of Native Americans. Cambridge: Cambridge University Press; 1998. [18] Mansferrez E, Ba´ez-Cubero L. Nahuas de la Sierra norte de Puebla, Etnografı´a contempora´nea de los pueblos indı´genas de Me´xico, regio´n Oriental. Me´xico, D.F.: Instituto Nacional Indigenista (INI); 1995. p. 167–206. [19] Argueta-Villamar A. Los Pure´pechas, Etnogra´fia contempora´nea de los pueblos indı´genas de Me´xico, regio´n Centro. Me´xico D.F:: Instituto Nacional Indigenista (INI); 1995. p. 217–89. [20] Michelet D. La zona occidental en el poscla´sico. In: Manzanilla L, Lo´pez-Lujan L, editors. Historia Antigua de Me´xico, vol. III: El horizonte poscla´sico, Me´xico D.F.: Porrua, INAH e IIA (UNAM); 2001. p. 161–98. [21] Solı´s-Olguin F. Aztecas. Arqueologı´a Mexicana, edicio´n especial 13. Edit. Raı´ces, CONACULTA, INAH. 2003, p. 10–5.