Mitochondrial DNA Polymorphisms in Chilean Aboriginal Populations: Implications for the Peopling of the Southern Cone of the Continent

AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 113:19–29 (2000) Mitochondrial DNA Polymorphisms in Chilean Aboriginal Populations: Implications for the Peopling of the Southern Cone of the Continent MAURICIO L. MORAGA,1 PAOLA ROCCO,1 JUAN F. MIQUEL,2 FLAVIO NERVI,2 ELENA LLOP,1 RANAJIT CHAKRABORTY,3 FRANCISCO ROTHHAMMER,1 AND PILAR CARVALLO1* 1Programa de Gene´tica Humana, Instituto de Ciencias Biome´dicas, Facultad de Medicina, Universidad de Chile, Santiago 7, Chile 2Departamento de Gastroenterologıá, Facultad de Medicina, Pontificia Universidad Cato´lica de Chile, Santiago, Chile 3Human Genetic Center, University of Texas School of Public Health, Houston, Texas 77225 KEY WORDS Amerindian mtDNA haplogroups; sequence analysis; peopling of the southern cone ABSTRACT The mitochondrial DNAs (mtDNAs) from individuals belonging to three Chilean tribes, the Mapuche, the Pehuenche, and the Yaghan, were studied both by RFLP analysis and D-loop (control region) sequencing. RFLP analysis showed that 3 individuals (1.3%) belonged to haplogroup A, 19 (8%) to haplogroup B, 102 (43%) to haplogroup C, and 113 (47.7%) to haplogroup D. Among the 73 individuals analyzed by D-loop sequencing, we observed 37 different haplotypes defined by 52 polymorphic sites. Joint analysis of data obtained by RFLP and sequencing methods demonstrated that, regardless of the method of analysis, the mtDNA haplotypes of these three contemporary South American aborigine groups clus- tered into four main haplogroups, in a way similar to those previously described for other Amerindians. These results further revealed the absence of haplogroup A in both the Mapuche and Yaghan as well as the absence of haplogroup B in the Yaghan. These results suggest that the people of Tierra del Fuego are related to tribes from south-central South America. Am J Phys Anthropol 113:19–29, 2000. © 2000 Wiley-Liss, Inc. The analysis of mitochondrial DNA vari- and Central America. Of those studies con- ation has been extensively used in the re- ducted with South American groups, almost cent past, for genetic characterization of all have involved populations from the contemporary aboriginal populations of the northern region of the continent (Torroni et Americas. The primary goals of this analy- al., 1993; Easton et al., 1996; Santos et al., sis have been to determine the origins, re- 1996). Most of the data generated from lationships, and migrational patterns of these studies were obtained through RFLP New World populations. In this respect, the study of the frequency distribution of the four founding Amerindian haplogroups, Grant sponsor: FONDECYT; Grant number: 198-1111; Grant sponsor: Ministero degli Affari Esteri d’Italia; Grant number: suggested by Schurr et al. (1990) and de- 1091-G224/ICU/CILE; Grant sponsor: NIH; Grant number: GM fined by Torroni et al. (1992), has proven 41399. *Correspondence to: Pilar Carvallo, Departamento de Biologıá useful. Celular y Molecular, Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Casilla 114-D, Santiago, Chile. The majority of these studies have fo- E-mail: [email protected] cused on aboriginal populations from North Received 23 November 1998; accepted 28 March 2000. © 2000 WILEY-LISS, INC. 20 M.L. MORAGA ET AL. mapping, rather than by D-loop sequencing, To expand our knowledge about the ex- although some D-loop sequence data from tent of mtDNA variation in southern South these populations are available for analysis. America and to test the hypothesis of a sin- Similarly, the mtDNAs taken from southern gle wave of migration to the southern part of South American groups were primarily South America, we performed RFLP and characterized by RFLP analysis (Merri- sequence analysis on three Chilean aborig- wether et al., 1995; Lalueza et al., 1997). inal populations (the Pehuenche, the Mapu- Thus, there remains limited D-loop se- che, and the Yaghan). quence information from groups inhabiting MATERIALS AND METHODS the southern part of South America. Studies of South Amerindians using Population samples RFLP and D-loop sequence analysis have The samples include three different Chil- revealed a concordance between the distri- ean aboriginal populations: 105 Pehuenche bution of haplogroups A–D, and haplo- from the community of Trapa Trapa in the groups (lineage clusters) I–IV as defined by Province of Bio Bio, 8th region (37° 43Ј S; Horai et al. (1993), based on the nucleotide 71° 16Ј W), 111 Mapuche from the Huapi polymorphisms occurring in the D-loop of Island, Province of Valdivia, 10th region each haplogroup (Torroni et al., 1993; Eas- (40° 15Ј S; 72° 25Ј W), and the 21 last sur- ton et al., 1996; Santos et al., 1996). How- viving Yaghan from Puerto Williams, Na- ever, Santos et al. (1996) found that 7% of varino Island, Province of Antartica, 12th individuals belonging to tribes from the region (55° S; 67° 40Ј W). The three groups Brazilian Amazon Region comprise unusual analyzed correspond to Amerinds showing a haplogroups because of their lack of corre- percentage of admixture between 2–13% spondence between RFLP and sequencing (Llop et al., 1993, 1994). The geographic loca- analysis. There is also extensive informa- tions of the subjects are shown in Figure 1. tion referring to RFLP analysis in these populations. In addition, Bailliet et al. DNA extraction and PCR amplification (1994) described a subdivision of the classi- Total DNA was prepared from peripheral cal Amerindian haplogroups A, C, and D on blood lymphocytes (Gustincich et al., 1991). the basis of the presence or absence of the DNA amplification of the four polymorphic HaeIII np 16517 site. This division has led mtDNA regions, defining the four Amerin- to the creation of haplotypes A1/A2, C1/C2, dian haplogroups, was carried out by using and D1/D2. However, the HaeIII np 16517 the following specific primers: site has been shown to be highly polymorphic in African (Chen et al., 1995), Euro- Haplogroup A: pean (Torroni et al., 1996), and Asian (Ball- L604 GTAGCTTACCTCCTCAAAGCAA inger et al., 1992) populations, indicating H708 AGGGTGAACTCACTGGAACG that it may not be useful for distinguishing Haplogroup B: within Native American haplogroups A, C, L8214 CACAGTTTCATGCCCATCGT and D. Similarly, Forster et al. (1996) also H8294 ATGCTAAGTTAGCTTTACAGTGG postulated subdivisions of founding haplo- Haplogroup C: types, using D-loop sequence data. In this L13232 CGCCCTTACACAAAATGACATCAA case, they divided haplotypes A1 and A2 by H13344 GGAGCACATAAATAGTATGGC aC3T transition at np 16111 and defined Haplogroup D: D1 and D2 haplotypes by the presence or L5120 CCTAACTACTACCGAATTCCTA absence of the 16271 T3C transition, with H5255 ATTCTTCGATAATGGCCCATTTG this transition being present only in North American Indian mtDNAs. Hence, there PCR was performed in a final volume of would appear to be specific nucleotide 50 ␮l, containing 300 ng genomic DNA, 1 U changes in the D-loop, which distinguish Taq DNA polymerase (Promega), 25 pmoles between North-Central and South Ameri- of each primer, 200 nM of each deoxinucle- can populations. otide, and the appropriate buffer. Samples MTDNA POLYMORPHISMS IN ABORIGINAL CHILEANS 21 ers L29, GGTCTATCACCCTATTAACCAC; and H16401, CACCATCCTCCGTGAAATCA, labeled at the 5Ј end with ␥-32P-ATP. DNA sequence analysis was performed by electro- phoresis on high-resolution 6% polyacryl- amide gels, followed by autoradiography. Data analysis Sequence alignment was performed by using the Clustal W computer program (Thompson et al., 1994). Substitutions and deletions were inferred from direct sequence comparisons. The phylogenetic trees were obtained through two different methods: neighbor joining (Saitou and Nei, 1987), using the Clustal W program, and parsimony analysis (Swofford and Olson, 1990). Maximum parsimony trees were generated through random addition of sequences using the tree bisection and reconnection (TBR) algorithm, with the PAUP (Swofford, 1993) computer program. RESULTS RFLP analysis Fig. 1. Map of Chile, showing the geographic loca- tion of the three Chilean tribes included in the present All individuals analyzed showed one of study. the characteristic haplogroups, A, B, C, or D. As shown in Table 1, haplogroups C and D were the most frequent in the three pop- were processed under the following PCR ulations, appearing at very similar frequen- conditions: 1 cycle at 95°C for 5 min, fol- cies in all of them. Haplogroup A was lowed by 30 cycles at 95°C for 1 min, 55°C present in the Pehuenche at a very low fre- for 1 min, and 72°C for 1 min, and finally quency (2.8%), while both haplogroups A one cycle at 72°C for 5 min. Haplotypes A, C, and B were absent in the Yaghan group. We and D were analyzed by restriction diges- tested the gain of the HaeIII site at position tion, using HaeIII for haplotype A, HincII 16517 only in individuals belonging to hap- for haplotype C, and AluI for haplotype D. logroup B, since this is a specific marker to The resulting restriction fragments and differentiate Amerindians from non-Amer- the PCR product including region V, defin- indian B individuals (Torroni et al., 1992, ing haplotype B, were analyzed by electro- 1993). The gain of the HaeIII site was phoresis on 3% NuSieve-Agarose (2:1) (FMC present in all individuals exhibiting haplo- BioProducts) gels. PCR amplification of the group B, including 8 Mapuche and 11 Pehu- D-loop region was carried out with two enches. specific primers, spanning a region of A3ϫ 4 contingency table analysis sug- 1,021 bp: L15997, CACCATTAGCACCCA- gests that in terms of haplogroup frequen- AAGCT; and H408, CTGTTAAAAGTGCAT- cies, these three populations cannot be sta- ACCGCCA. tistically distinguished (chi-square ϭ 6.78, exact P-value ϭ 0.32 with 10,000 permuta- Direct sequencing of PCR products tions). However, in terms of haplotype di- DNA sequencing was carried out by us- versity (Nei, 1978) defined by these four ing the PCR dsDNA Cycle Sequencing haplogroups, the Yaghan have the lowest System (Life Technologies), with the prim- diversity (52.4%), and the Pehuenche the 22 M.L.

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