Absence of the 9-bp Deletion of Mitochondrial DNA in Pre-HispanicInhabitants of Argentina

Dario A. Demarchi, Graciela M. Panzetta-Durtari, Sonia Colantonio, Alberto J. Marcellino

Human Biology, Volume 73, Number 4, August 2001, pp. 575-582 (Article)

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Absence of the 9-bp Deletion of Mitochondrial DNA in Pre-Hispanic Inhabitants of Argentina

DARÍO A. DEMARCHI,1,3 GRACIELA M. PANZETTA-DUTARI,2 SONIA E. COLANTONIO,1,3

AND ALBERTO J. MARCELLINO1,3

Abstract We investigated the incidence of the Region V mitochondrialDNA 9-base-pair (bp) deletion from human remains recovered from severalarchaeological sites and contexts throughout Argentina. Of the 34 samplesanalyzed, 24 yielded DNA extractions that gave clear amplification results.All of the individuals carried two repeats of the 9 bp, one of which has beenshown to be deleted in some individuals of Asian origin and defines mito-chondrial lineage B. Although most of the modern Amerindian groups in theregion exhibit the deletion in high frequencies, the absence of the 9-bp dele-tion among ancient populations of South America seems to be the rule ratherthan the exception, as was reported by several studies involving extinct pop-ulations. The evidence gathered until now suggests that the earliest settlers ofthis region of South America did not carry mitochondrial lineage B.

Mitochondrial DNA (mtDNA) has been used extensively as a tool for the recon-struction of human evolutionary history because of its maternal inheritance, lackof recombination, and high mutation rate relative to nuclear DNA (Brown et al.1979; Giles et al. 1980). Although most of the initial research has involved con-temporary populations, in the last years an increasing number of investigatorshave been publishing data from archaeological remains, providing a temporalcomponent to such studies (Rogan and Salvo 1990; Horai et al. 1991; Merri-wether et al. 1994; Fox 1996; Monsalve et al. 1996; Parr et al. 1996a; O’Rourkeet al. 1996, 2000; Stone and Stoneking 1993, 1998; Carlyle et al. 2000).

The Region V 9-base-pair (bp) deletion of mtDNA has been used to esti-mate the number of independent lineages that occur in the New World and totrace probable population migrations (Schurr et al. 1990; Torroni et al. 1993).

1Cátedra de Antropología Biológica y Cultural, Facultad de Ciencias Exactas, Físicas y Naturales, Universi-dad Nacional de Córdoba, Córdoba, Argentina.

2Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba,Córdoba, Argentina.

3Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, Buenos Aires, Ar-gentina.

Human Biology, August 2001, v. 73, no. 4, pp. 575–582.Copyright © 2001 Wayne State University Press, Detroit, Michigan 48201-1309

KEY WORDS: ADNA, AMERINDIANS

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This deletion involves the loss of one of two copies of a 9-bp sequence betweenthe lysine tRNA and COII genes. A single Asian origin accounts for most of the 9-bp deletions observed in Asians, Amerindians, and Pacific Islanders (Schurr etal. 1990; Ballinger et al. 1992). MtDNA screening of African populations demon-strated that an identical 9-bp deletion occurred in sub-Saharan Africa, being fre-quent in some Africam Pygmy populations (Vigilant et al. l991; Chen et al. 1995).Torroni et al. (1995) also found the deletion in an ltalian population. Phylogenet-ic analyses of Asian, European, and African control region sequences associatedwith the 9-bp deletion have indicated that they are not closely related and there-fore had separate origins. Although these studies have demonstrated that the 9-bpdeletion is not Asian specific, as Wrischnik et al. (1987) initially suggested, it hashad long and independent histories in Africa and Asia.

In this study, we investigated the incidence of the 9-bp deletion from hu-man remains recovered from several archaeological sites and contexts throughoutArgentina, in an additional attempt to understand the process of settlement of thisregion of South America.

Materials and Methods

A total of 34 specimens from five archaeological sites throughout Argenti-na were studied. Eighteen desiccated tissue samples came from Pampa Grande (at1650 m of altitude, in the province of Salta), a site identified with the Candelariaculture, dating between 1000 and 1500 years before the present (yBP) (Gonzalez1972). Eight desiccated tissue samples and three tooth samples were gatheredfrom several locations in the Puna region (Andean Plateau, at around 3600 m ofaltitude) of the province of Jujuy. Based on their archaeological context, this se-ries might be dated between 500 and 900 yBP. Three tooth samples belong to TresArroyos (province of Buenos Aires), one of the oldest archaeological sites in Ar-gentina, dating to around 7000 yBP. Two other tooth samples were analyzed, onefrom the province af Córdoba, in the middle of the country, and the other from theprovince of Chubut, in the Patagonian region, both dating approximately 500 to900 yBP.

DNA was isolated from 40 to 80 mg of powdered teeth (powdered bydrilling), or dried soft tissue (cut into small pieces with a sterile scalpel). Thesamples were incubated in 1 mL 5M GuSCN, 0.1 M Tris HCI (pH 6.4), 0.02 MEDTA (pH 8.0), and 1.3% Triton X-100 overnight at 60°C (Paar et al. 1996b).Subsequently, solid tissue remains were pelleted by centrifugation and nucleicacids were isolated from supernatants employing two different commercial kits:Isoquick (Orca Research), and Wizard PCR (Promega).

Amplifications were done on a total volume of 25 µL using the primers andunder the conditions described by Stone and Stoneking (1993), and 1–5 µL of an-cient DNA extract. The PCR product was electrophoresed in a gel composed of2% Metaphor and 2% SeaKem (FMC), stained with ethidium bromide and visu-alized under UV light. The presence of the 9-bp deletion was investigated by run-

Absence of 9-bp Deletion in Ancient Amerindians / 577

ning the ancient samples together with contemporary Amerindian samples pre-senting the deletion.

Special care was taken to reduce the possibility of contamination of ancientsamples with modern DNA. Disposable laboratory coats, gloves, filter tips, dedi-cated pipetmen, and disposable laboratory ware were used throughout the analy-ses. Benches and equipment were frequently treated with a 20% bleach solutionand alcohol. The extractions were carried out under sterile conditions in thebioanthropology laboratory, in which no previous work with DNA had been per-formed. For each extraction procedure, a blank containing only reagents was in-cluded. This negative extraction was subsequently submitted to amplification as anegative control, in order to facilitate detection of the introduction of moderncontaminants.

All PCR reagents (except the Taq polymerase) were aliquoted and irradiat-ed with UV light for 30 min. Negative controls of amplification with all reagentsminus the DNA extract were included in each amplification, in order to provide aconstant check on the purity of the PCR reagents. Positive controls of amplifica-tion used were ancient samples of DNA that already had shown positive amplifi-cation, to avoid manipulation of modern DNA during the PCR. Reactions wereprepared in a laminar flow cabinet previously purged with UV light, located in aseparate room.

Results and Discussion

The appearance of the DNA extracts after extraction with the IsoQuick kitvaried significantly. Extracts from teeth appeared mostly clear and ready for am-plification. On the other hand, extracts from dried soft tissue (muscle and skin,principally) were invariably brown in color, most of them showing inhibitory ac-tivity for PCR amplification even after several dilutions (1/l0 to 1/50). This prob-lem was solved through purification of the DNA extracts with the Wizard PCR kit(Promega), after which the brown contaminants were removed. This method in-volves the use of resin-based minicolumns that simultaneously concentrate andpurify DNA. This method is designed to trap PCR products between 100 bp and10 kilobase pairs (kb), which is especially useful for aDNA purification because itsafeguards against contamination with modern DNA molecules, generally above10 kb (Yang et al. l998).

Out of the 34 samples analyzed, 24 yielded DNA extract that gave clear am-plification results (Table 1). The remaining extracts either did not contain DNA,inhibited amplification, or gave ambiguous amplifications. To confirm the initialresults, multiple independent extractions and amplifications were conducted.None of the negative controls of extraction and amplification yielded evidence ofcontamination with modern DNA.

From the agarose gel electrophoresis results it was observed that all indi-viduals carried two repeats of the 9-bp sequence, one of which has been shown to

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Table 1. Specimens Studied that Yielded Amplifiable DNA

Location Sample Type N Date yBP ProvinceArgentinian

Region

Pampa Grande Soft tissue 9 ∼1000–1500 Salta NorthwestPuna Soft tissue 7 ∼500–900 Jujuy NorthwestPuna Tooth 3 ∼500–900 Jujuy NorthwestPampa de Olaen Tooth 1 ∼500–900 Cordoba CentralTres Arroyos Tooth 3 ∼7000 Buenos Aires South CentralUnknown Tooth 1 ∼500–900 Chubut Patagonia

be deleted in some individuals of Asian or Asian-derived populations and definesmitochondrial lineage B.

Although in general the 9-bp deletion presents in high frequencies in con-temporary South Amerindians, its absence among ancient human remains recov-ered throughout South America has been the rule rather than the exception.Among a total of 122 individuals from extinct populations in several surveys(Table 2), only one sample, a mummy from Colombia in the northern extreme ofSouth America, exhibited the Region V deletion (Monsalve et al. 1996). It isworth noting that the identification of this sample (designated as MO in thatstudy) is ambiguous, since its control region sequence sample exhibited two ofthe polymorphisms (16111T and 16290T ) that typically occur in haplogroup Aand none that are usually seen in those from haplogroup B. Therefore, the se-quencing analysis suggests that this sample belongs to haplogroup A rather thanto B, although the restricted fragment length polymorphism (RFLP) screeningshowed it to lack the +HaeIII 663 mutation that defines lineage A.

On the other hand, Riveiro Dos Santos et al. (1996) analyzed 18 ancientDNA samples from Brazil dating from 500 to 4000 yBP, and identified one 4000-year-old sample that, based on its control region sequence motif, belongs to hap-logroup B. This constitutes the only clear finding of haplogroup B mtDNA in pre-Hispanic remains from Sonth America, but implies that haplogroup B had beenbrought to South America at least around 4000 yBP.

Table 2. Surveys on Extinct South American Populations in which the 9-bp DeletionWas Screened

Survey N DNA Source Argentinian Region

Present study 24 Tooth and tissue ArgentinaRogan and Salvo (1990) 12 Tissue Northern ChileHorai et al. (1991) 5 Tissue Northern ChileMerriwether et al. (1994) 15 Tissue Northern ChileMonsalve et al. (1996)a 8 Tissue ColombiaFox (1996) 58 Tooth T. Fuego–Patagonia

a. One specimen presented the deletion.

Absence of 9-bp Deletion in Ancient Amerindians / 579

The lack of the Region V deletion in the most southern part of South Amer-ica (Tierra del Fuego and Patagonia) is concordant with the patterns observed inmodern populations living in that region. Moraga et al. (2000) reported that thecontemporary Yaghan, in the most southern part of Chile, present an mtDNA hap-logroup pattern similar to that obtained by Fox (1996) for extinct groups fromTierra del Fuego–Patagonia. All specimens from the Tierra del Fuego–Patagoniaarea lack haplogroups A and B, and the authors suggest that they are most proba-bly descendants of one common ancestral Paleoindian population (Moraga et al.2000).

In contrast, the absence of the 9-bp deletion of mitochondrial DNA amongpre-Hispanic inhabitants of the central Andes region (from where 19 of the 24specimens analyzed here belong) is indeed unexpected, since modern nativegroups living in that region present the highest frequency of that deletion amongAmerindian populations. For example, Dipierri et al. (1998) found a frequency of0.65 for the deletion among native Andeans of the province of Jujuy in north-western Argentina. In the northern part of Chile, across the Andes and at approx-imately the same latitude, the deletion was observed at an even higher frequency:0.68 among the Aymara, and 0.72 among the Atacameño (Merriwether et al.1995).

Rothhammer and Bianchi (1995) observed that the mtDNA lineage B ex-hibits a marked range of frequencies in South America, and that low or high Bfrequencies are not confined to a specific geographic area or linguistic group.These authors suggest that since effective population size is smaller when the mi-tochondrial genome is considered, the possibility of a regional random loss of Bis highly probable. Wars, epidemics, and famine may have played an importantrole in producing this effect (Rothhammer and Bianchi 1995).

However, before reaching any conclusion based on a purely genetic expla-nation, it is necessary to examine the historic and cultural processes operating onthe genetic pool of the populations. Most of the native population that today in-habits northwestern Argentina (along the Andean plateau, intermontane corridors,and high valleys of the Andes), are Quechua or Aymara speakers, with an econo-my based principally on llama herding and subsistence agriculture. The majorityof these native Andeans live in small communities, where the nuclear family isthe basic social unit.

Archaeological and historical evidence suggests that these native Ameri-cans have little in common with the populations that inhabited this part of SouthAmerica around 1000 years ago. Several different groups inhabited the north-western region of Argentina by that time. The high valleys of the province ofSalta (where the Pampa Grande site is located) were inhabited by sedentary, agri-culturist groups, known in general as Diaguitas, who shared the Kaka (or kakana)language (Canals Frau 1986). Contemporarily, the Quebrada de Humahuaca andthe western Puna regions were inhabited by the Omaguaca Indians, a groupingthat included different groups such as the Purmamarca, Ocloya, Osa, Fiscara,Tiliar, and Jujuy (Canals Frau 1986). These groups, like the Diaguitas, exhibited

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advanced development of agricultural practices and pottery manufacture, as wellas considerable political integration (Canals Frau 1986).

Substantial changes in the history of those populations started during thesouthern expansion of the Inca Empire, from Peru and Bolivia (a.d. 1480–1530).During this short period, a number of mitimáes groups (populations previouslysubdued by the Empire) from outside the region were transplanted into locationsof varying ecological and climatic conditions along northwestern Argentina. Forexample, the use of the Puna region by relocated pastoral populations is known asone of “exclusive dedication” (Murra 1972). The Churumata, Paypaya, and otherChicha Indians (Quechua speakers) were some of these relocated populations(Vignati 1931; Krapovickas 1978).

Later, Spanish colonization increased the gene flow and depopulationprocesses through wars and new geographic relocations of entire indigenous pop-ulations. Although the exact extent of the replacement and depopulation is un-known, it is most likely that these dramatic and long-term demographic events re-sulted in the substantial modification of the gene pool of the inhabitants ofnorthwestern Argentina.

This evolutionary scenario contrasts markedly with the pattem of mtDNAhaplogroup variation observed in North America. O’Rourke et al. (2000) foundthat the distribution of mtDNA haplogroups in ancient samples is concordant withthe pattern observed in modern populations living in the same geographic region,which suggests that regional differentiation in mtDNA variation in native NorthAmericans is relatively stable, temporally and geographically. It is also interest-ing to note that the 9-bp deletion is present in almost all the ancient North Amer-ican populations screened for this marker (O’Rourke et al. 2000), with the excep-tion of the ancient Aleuts. The deletion is also absent in all contemporarypopulations of the North American Arctic, Aleuts as well as Inuits.

Regarding the five remaining samples analyzed (see Table 1), which comefrom the central and southern part of Argentina, it is even more premature to at-tempt any conclusion given the small number of individuals screened and thewide time span and large geographic area they represent. However, there are notmany reasons to expect a sample carrying the deletion, considering that its inci-dence decreases markedly in populations at higher latitudes in South America(Moraga et al. 2000).

Based on the current evidence, it is our interpretation that the earliest set-tlers of this region of South America did not carry mitochondrial lineage B. Al-though the absence of haplogroup B in a particular population could be a conse-quence of genetic drift, it is unreasonable to expect that several largely isolatedgroups could simultaneously lose the same lineage during their respective popu-lation histories. The high incidence of the deletion among the current inhabitantsof northwestem Argentina might be the result of different evolutionary events.The introduction of transplanted populations from outside the region (who mighthave introduced the 9-bp deletion), followed by depopulation and relocation ofthe original population, could have derived from founder effect followed by ge-

Absence of 9-bp Deletion in Ancient Amerindians / 581

netic drift and resulted in the substantial modification of the gene pool of the pop-ulation. Of course, the existence of gene flow with Aymara and Atacameño popu-lations from over the Andes cannot be underestimated.

Acknowledgments This research was supported by a grant from the Consejo Na-cional de Investigaciones Científicas y Técnicas de la Republica Argentina (CONICET),CONICOR, and SECyT (Universidad Nacional de Córdoba). Our gratitude to Luis Patritofor his support. We are also most grateful to Michael H. Crawford and two anonymous re-viewers for comments and corrections on earlier versions of this paper.

Received 1 August 2000; revision received 8 March 2001.

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