AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 123:361370 (2004)

Early Population Differentiation in Extinct Aborigines From Tierra del Fuego-Patagonia: Ancient mtDNA Sequences and Y-Chromosome STR Characterization
lvarez,2 Eva Ferna Jaume Garc a-Bour,1 Alejandro Pe rez-Pe rez,1 Sara A ndez,1 Ana Mar a Lo pez-Parra,2 2 1 Eduardo Arroyo-Pardo, and Daniel Turbo n *
Seccio dAntropologia, Departament de Biologia Animal, Universitat de Barcelona, E-08028 Barcelona, Spain Laboratorio de Biolog a Forense, Departamento de Toxicolog a y Legislacio n Sanitaria, Facultad de Medicina, Universidad Complutense, E-28040 Madrid, Spain
2 1

KEY WORDS

mtDNA; Y-STR; ancient DNA; Fuegians; Tierra del Fuego; America American origin. The analysis of both mtDNA and Y-STRs revealed DNA from Amerindian ancestry. The observed polymorphisms are consistent with the hypothesis that the ancient Fuegians are close to populations from southcentral Chile and Argentina, but their high nucleotide diversity and the frequency of single lineages strongly support early genetic differentiation of the Fuegians through combined processes of population bottleneck, isolation, and/or migration, followed by strong genetic drift. This suggests an early genetic diversication of the Fuegians right after their arrival at the southernmost extreme of South America. Am J Phys Anthropol 123: 361370, 2004. 2004 Wiley-Liss, Inc.

ABSTRACT Ancient mtDNA was succesfully recovered from 24 skeletal samples of a total of 60 ancient individuals from Patagonia-Tierra del Fuego, dated to 100 400 years BP, for which consistent amplications and two-strand sequences were obtained. Y-chromosome STRs (DYS434, DYS437, DYS439, DYS393, DYS391, DYS390, DYS19, DYS389I, DYS389II, and DYS388) and the biallelic system DYS199 were also amplied, Y-STR alleles could be characterized in nine cases, with an average of 4.1 loci per sample correctly typed. In two samples of the same ethnic group (Aonikenk), an identical and complete eight-loci haplotype was recovered. The DYS199 biallelic system was used as a control of contamination by modern DNA and, along with DYS19, as a marker of

The reconstruction of the biological history of aboriginal Amerindian populations has been widely debated in the literature for the last two decades. Attempts to reconstruct population dynamics in America have focused intensely on anthropological, odontological, linguistic, and more recently, genetic information. Beringia was the sole path towards America from Asia (Fiedel, 1992; Cavalli-Sforza et al., 1994; Crawford, 1998). However, strong controversy persists on the number and timing of migratory waves that moved in and southward into the continent. Archaeological data support two distinct dates for the initial settlement: a recent date of 12,000 BP according to the Clovis arrow points found at numerous sites (Hoeffecker et al., 1993; Szathmary, 1993), and an ancient one of 35,000 BP based on recently discovered lithic industries from Toca do Boqueirao da Pedra Furada, in Brazil (Meltzer et al., 1994), Pendejo Cave, New Mexico (Chrisman et al., 1996), and Meadowcroft Rock Shelter, in western Pennsylvania (Adovasio and Carlisle, 1988; Adovasio et al., 1990), not without some dispute (Dillehay, 1997). Little consensus is found among researchers supporting either a monophyletic, single-wave colonization, with a unique genetic stock for all Amerindian groups (Merriwether et al., 1995;

Bonatto and Salzano, 1997; Stone and Stoneking, 1998), or a multiple-wave hypothesis, with at least four major mtDNA founding lineages (Torroni et al., 1993), including as many as 13 distinct demes (Bailliet et al., 1994; Foster et al., 1996, 1997; Bianchi et al., 1997). The little Y-chromosome polymorphism variability observed within Native American populations (Torroni et al., 1994; Pena et al., 1995; Underhill et al., 1996; Bianchi et al., 1997) supports both the single migration wave hypothesis (Santos et al., 1999; Karafet et al., 1999) and a two-wave colonization (Lell et al., 2002). Although there is some dispute as to which model ts the data better (Tarazona-Santos and Santos, 2002; Lell et al.,
Grant sponsor: Spanish DGICYT; Grant number: PB97-0925; Grant sponsor: UCM; Grant number: PR48/01-9837. *Correspondence to: Daniel Turbo n, Seccio Antropologia, Departament de Biologia Animal, Facultad de Biologia, Universitat de Barcelona, E-08028 Barcelona, Spain. E-mail: turbon@ub.edu Received 14 January 2003; accepted 11 April 2003 DOI 10.1002/ajpa.10337

2004 WILEY-LISS, INC.

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2002), a differential pattern of genetic drift and gene ow has been proposed to explain the Y-chromosome variability observed for haplogroup 18, dened by a C-T transition for the DYS199 system (TarazonaSantos et al., 2001). The aborigines from Tierra del Fuego, nowadays extinct, may play a key role in the understanding of the colonization of the American continent. They were traditionally considered the descendants of the rst Paleoindian settlers in America, given some plesiomorphic traits and some homoplasias in their skulls, which they share with Australian Aborigines (Lahr, 1994, 1995). Clear evidences of Fuegian settlement are found in Monte Verde 12,000 B.P. (Dillehay and Collins, 1988, Adovasio and Pedler, 1997; Meltzer et al., 1997) and in the Beagle Channel 12,000 10,000 B.P. (Martinic, 1992; Piana et al., 1992). The Fuegians occupied the southern extreme of South America and included at least four distinct ethnic groups. The Kaweskar (also referred to as Alakaluf) and the Yamana lived on the Pacic coast and islands of Tierra del Fuego, intensively exploiting marine resources for food, whereas the Selknam (usually referred to as Ona), who lived on Isla Grande, south of the Estrecho de Magallanes, were terrestrial hunters of guanaco (Lama guanaco) and coruro (Spalacopus cyanus). The Aonikenk (or Tehuelche), who lived in Patagonia, north of the Estrecho de Magallanes, were closely related to the Selknam. The Fuegian groups were strongly adapted to harsh, cold environmental conditions and consumed a diet rich in animal proteins and fat, with nil consumption of plant foods, which were scarce, seasonal, and with a low calorie content. At the beginning of the twentieth century, the Fuegians went extinct due to overpopulation and competition for land use, especially after the arrival of European colonists. Yamana descendants still inhabit the Navarino island, and a small Kaweskar group lives in Puerto Eden. However, both groups most probably result from population admixture, since the Navarino island, once a shelter for the three Fuegian groups, was recently populated with Chilean workers (Chilotes) for the wood industry. Some genetic analyses of modern Amerindian populations suggested that the Fuegians were related to tribes from south-central Chile and Argentina (Rothhammer et al., 1986; Llop, 1996; Moraga et al., 2000), whereas mtDNA haplogroup analyses on extinct Fuegians indicate that they descend from a distinct Paleoindian migration, lacking haplogroups A and B (Lalueza et al., 1997). The recovery and analysis of ancient DNA, both mitochondrial and nuclear, from extinct Fuegians may be extremely useful for understanding the human colonization of America (Merriweather et al., 1996; Williams et al., 2002), as has been the case for Europe (Gerstenberger et al., 1999; Schultes et al., 1999). Since the reconstruction of the population history of aboriginal humans from Tierra del Fuego seems to be controversial, their genetic background and variability

should be thoroughly examined to draw any denitive conclusion. The present paper contributes to the genetic characterization of mtDNA and Y-chromosome markers from extinct humans from Tierra del Fuego, and aims to depict a model for human migration on the American continent. MATERIALS AND METHODS The sample Fuegian individuals belonging to one of the four Fuego-Patagonian groups (Aonikenk, Selknam, Yamana, and Kaweskar) were analyzed from a larger sample (Lalueza et al., 1997). The samples that yielded signicant PCR amplication products for the HVRI mtDNA region were used for further mtDNA sequencing and Y-chromosome characterization. Prior analyses of these same samples by RFLPs (Lalueza et al., 1997) allowed an independent classication to one of the four major Amerindian haplogroups. All Fuegians were grouped only into either haplogroup C or D, none of them showing RFLPs indicative of haplogroups A or B. Clear mtDNA sequences were obtained for 24 of the 60 samples analyzed. Fuegian individuals for whom mtDNA was successfully amplied and sequenced were also analyzed for a group of Y-chromosome polymorphisms, and eventually 20 of the initial 24 samples were typed. The samples consisted of complete teeth or tooth roots from the skeletal collections held at several museums and institutions in Chile and Argentina. Most of the samples are dated to the end of the nineteenth century (100 200 BP), and may thus represent a single temporal slice. DNA extraction The external surface of all samples was removed with a dentists sand-blaster (Base 1 Plus, Dentalfarm) to eliminate both soil and exogenous DNA contaminants. Samples were ground in a cryogenic impact grinder (Freezer Mill, Spex 6700) lled with liquid nitrogen (LN2). Approximately 600 mg of the obtained powder were washed three times with 8 ml 0.5 M EDTA, pH 8.0, and incubated overnight at 37C in 10 ml of a lysis buffer solution (5 mM EDTA, 10 mM TRIS, 0.5% SDS, 50 g/ml Proteinase-K). Remaining tissues were removed by centrifugation, and DNA was extracted from the supernatant by a standard, high-volume phenol/chloroform protocol. The aqueous phase was concentrated by centrifugation dialysis using Centricon-30 microconcentrators (Amicon) and desalted with 15 ml of sterile water to a nal volume of 300 l. Extraction controls without powdered sample were processed in parallel, to test for contamination during the extraction process. Amplification HVRI mtDNA amplications were performed through a nested-PCR assay (with 30 PCR cycles per round), using the external primers L16154 5AATACTTGACCACCTGT-3 and H16400 5-TTCAC-

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TABLE 1. mtDNA HVRI sequence polymorphisms in Fuegian-Patagonian sample studied1 1 6 1 5 6 G A 1 6 1 8 7 C T T T T 1 6 1 8 9 T C C C 1 6 2 0 9 T C C C C 1 6 2 2 3 C T T T T T T T T T T T T T T T T T T T T T T T T 1 6 2 4 1 A G G G 1 6 2 5 0 C T 1 6 2 8 6 C T 1 6 2 9 1 C T 1 6 2 9 4 C T T T T T T 1 6 2 9 6 C T T T T T 1 6 2 9 8 T C C C C C C C C C C C 1 6 3 0 4 T C C C 1 6 3 1 1 T C 1 6 3 1 5 T C 1 6 3 1 8 A G 1 6 3 2 5 T C C C C C C C C C C C C C C C C C C 1 6 3 2 7 C T T T T T T T T T T T T T 1 6 3 3 9 C T

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Ethnic group Ande rson Researcher J.G.-B. F89 F14 F49 F67 F59 F26 F85 F83 F41 F34 F5 F18 F35 F46 F27 F1 F69 F74 F68 F71 F11 F10 F50 F57
1

Haplotype

1 6 3 4 2 T C C C

1 6 3 6 2 T C C C C C

Aonikenk Aonikenk Kaweskar Kaweskar Kaweskar Kaweskar Selknam Selknam Selknam Yamana Yamana Yamana Yamana Yamana Aonikenk Aonikenk Kaweskar Kaweskar Kaweskar Kaweskar Kaweskar Kaweskar Kaweskar Yamana

C C C C C C C C C C C C C C D D D D D D D D D D

Haplotype lineages obtained are concordant with expected haplogroup classication, according to prior RFLPs analyses. No European complete cross-contamination sequences were obtained, or any blank contaminations, and independent L and H DNA strand sequencies yielded identical results. All analyses were made by researchers of European origin. Hence, authenticity of ancient DNA sequences is greatly supported. Polymorphic sites labeled T (with grey shadow) at np 16,294 and 16,296 and C at np 16,304 in the researcher (J.G.-B.), are also present in some Fuegians (F67, F18, F74, F68, and F71). Although none of these samples share same haplotype combination as researcher, those three polymorphisms are not considered in genetic analysis, to minimize overestimation of polymorphism. Shaded Fuegian references, such as in F89, indicate samples for which Y-STRs could also be characterized.

GGAGGATGGTGG-3 and the internal primers L16158 5-CTTGACCACCTGTAGTA-3 and H16394 5-GAGGATGGTGGTCAAGG-3, providing a nal PCR product of 240 bp. One microliter of extracted DNA was amplied in 25 l reaction volume (20 mM Tris-HCl, pH 8.4, 50 mM KCl, 3 mM MgCl2, 0.18 mM dNTPs, 0.04% W-1 stabilizer (Gibco BRL), 0.6 M primers, and 0.05 units of Taq BRL polymerase). DNA recovered from agarose gels was sequenced automatically in an ABI-Prism 377 Analyzer using Dye Terminators (Applied Biosystems). Both strands were sequenced separately, and only consistent mutation points were considered (Garc a-Bour et al., 1998). In the same samples, the Y-chromosome STR markers of genetic systems DYS434, DYS437, DYS439, DYS390, DYS391, DYS393, DYS19, DYS389I, DYS389II, and DYS388 were studied. Y-chromosome markers do not produce unequal amplication of alleles (Williams et al., 2002), and are thus more suitable for ancient DNA studies. Amplications were performed using uorescent-labeled primers. In all instances, 40 cycles of PCR amplication were performed. Amplication protocols for DYS393, DYS391, DYS390, and DYS19 were carried out following Kayser et al. (1997) and as described by De Knjiff et al. (1997) for DYS388. The systems DYS389-I and DYS389-II were amplied following Schultes et al.

(1999), and STRs for DYS434, DYS437, and DYS439 were amplied as reported by Ayub et al. (2000). Allelic sizes are shown in Table 3. All amplied products were rst observed with ultraviolet light (UV) in 2% agarose gels with ethidium bromide staining, and then typed in an ALF-Sequencer (Pharmacia) with ladders and controls supplied by Dr. De Knijff for all systems except for DYS434, DYS437, DYS439, and DYS389-I/ II, which were typed with allelic ladders obtained in one of our laboratories (Complutense University), using the allele lengths reported by Ayub et al. (2000) and Schultes et al. (1999). The DYS199 single-nucleotide polymorphism (SNP) was typed by allele-specic amplication (Underhill et al., 1996). The C3 T transition at this locus, referred to as marker M3, delineates a major Native American founder haplotype (Underhill et al., 1996; Bianchi et al., 1997, 1998; Lell et al., 2002). For this system, amplicons were obtained after 38 cycles of PCR. Y-chromosome PCR typing was repeated twice for all systems, to obtain consistent results. CONTAMINATION CONTROLS DNA was extracted and amplied at separate laboratories (Anthropology at Universitat de Barcelona (UB), and Forensic Biology at Universidad Complutense (UCM)). At the UB lab, one male re-

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Fig. 1. UPGMA tree for all sequences analyzed (n 400), including Fuegian/Patagonian sequences and comparative Amerindian and Asiatic sample (total sample size of n 400). Kimura two-parameter distance was used in MEGA version 2.1 software package (43). Solid triangles are Fuegian C and D lineages; open squares are Amerindian X lineages; solid circles are South African lineages; grey diamonds are Asian lineages; and open diamonds are Asian Circumpolar lineages.

searcher performed all DNA extractions and another (J.G.-B.) all mtDNA amplications and sequencing. A female researcher (S.L.R.) carried out all Y-chromosome amplications at UCM to minimize male contamination. During all assays, protective clothes, sterile gloves, and face-shields were worn. Working surfaces were thoroughly cleaned with bleach and 70% ethanol. All sterile, disposable laboratory materials used for cleaning, digesting, extracting, concentrating, and amplifying were previously irradiated with UV for 30 min. Control blanks were prepared in each extraction set, and two negative controls that contained all but DNA were also included in each amplication experiment. Only results with a lack of amplication in these negative controls were considered. As a further preventive control for system DYS393, the two female researchers in the laboratory were also typed, since Y-STR

primers are known to anneal to X-chromosome segments. Moreover, the allele specicity of system DYS199 was used to detect modern DNA contamination from European ancestry, since allele T is only present in autochthonous Native Americans. Cases with a double band (C and T) were considered contaminated results. The yields of non-Amerindianspecic mtDNA haplogroups were indicative of contamination. RESULTS mtDNA Ancient mtDNA was succesfully recovered from 24 skeletal samples from a total of 60 Fuegian individuals studied. Table 1 shows the variable positions within the amplied mtDNA HVRI region for the Fuegian sample. All sequences obtained were easily

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TABLE 2. Sequence diversity was estimated from within-groups mean nucleotide diversity (number in bold in diagonal) and between-groups means of groups considered (lower triangular matrix)1 Fueguian Fueguian Amerindian CD Amerindian AB Circumpolar South African Other Haplogroup X 0.01621 0.01597 0.02880 0.02437 0.04091 0.02347 0.02860 Amerindian CD 0.01556 0.02955 0.02471 0.04153 0.02385 0.02982 0.02147 0.02180 0.04270 0.02498 0.02884 0.01731 0.04460 0.02301 0.03070 0.01117 0.04136 0.03849 0.02297 0.02784 0.00405 Amerindian AB Circumpolar South African Other Haplogroup X

1 Within-groups mean nucleotid diversity is measured as arithmetic mean of all individual pairwise differences between taxa within groups, and average distance between two groups is arithmetic mean of all pairwise distances between taxa in intergroup comparisons. Sequence diversity of Fuegian sample studied is similar to that of Amerincian C D comparative group.

ascribed to the Amerindian haplogroups C or D, given their substitutions at np 16,223 T, 16,298 C, and 16,327 T, and 16,223T, 16,325C, and 16,362C, respectively (Torroni et al., 1993). As expected (Lalueza et al., 1997), none of the samples showed lineages characteristic of haplogroups A or B. Independent sequences were obtained for both DNA strands, and the results were consistent in all cases. Three single mutation points present in the researcher (J.G.-B.) were discarded for the sequence analyses to prevent overestimation of mtDNA polymorphisms. As a group, the 24 Fuegian sequences analyzed show 19 polymorphic sites belonging to 17 distinct lineages, 15 of which can be considered single sequences, not clustered with other non-Fuegian lineages. A comparative sequence database was built, including 358 Amerindians (339 with haplogroups A, B, C, or D and 19 with haplogroup X), 9 Circumpolar Inuit, 11 Asians (Han and Korean), and 22 South Africans. Figure 1 shows the UPGMA tree obtained, including all sequences and using the Kimura two-parameter distance with the MEGA version 2.1 molecular evolutionary software (Kumar et al., 2001). The African sequences form a clear outgroup, and while some Asian lineages cluster closer to the African stock, others do not. The Fuegian cluster scattered throughout the Amerindian stock by haplogroups, with some sequences showing a close-to-the-root position. As a whole, the Amerindian lineages show a great degree of genetic diversity (Table 2), larger than expected given the size of the sample studied. The overall within-group mean nucleotide diversity (d) of the Amerindian sample is 0.02541, most of which can be attributed to the high genetic diversity of the Amerindian A B lineages (d 0.02147), whereas the C D Amerindian lineages show lower diversity (d 0.01556), and the African group shows the smallest diversity (d 0.01117). Fuegians show a larger diversity value (d 0.01621) than C D Amerindians (Table 2), despite the small size of the Fuegian sample studied. This is mainly due to the fact that Fuegian lineages cluster closer to the root of their haplogroup subtree,

Fig. 2. Neighbor-joining (NJ) tree derived with ancient Fuegian/Patagonian sequences and C D Amerindian comparative sequences, along with Asian and African lineages used as outgroups. Kimura two-parameter distance was used in MEGA version 2.1 software package. Solid triangles are Fuegian C D lineages; open squares are Amerindian Na-Dene lineages; grey diamonds are Asian lineages; and solid circles are South African lineages.

especially in the D haplogroup. When analyzing the between-group mean nucleotide diversity, the smallest value is obtained when comparing Fuegians and

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Fig. 3. Median network plot (Network 3.1.1.1, Fluxus Tech. Ltd.) of Amerindian lineages included in Figure 3. Solid circles are Fuegian lineages; gray circles are Amerindian C lineages; and open circles are Amerindian D lineages. Fuegian references outside plot refer to those included in main C node. Mutated positions are not shown (short segments are indicative of one mutation difference). All lineages have identical weights.

Amerindians C D (d 0.01597), and the largest value when the Africans are compared with the other groups (d ranges from 0.04091 0.04460). The between-group nucleotide diversity between the Amerindians A B and C D is d 0.02955. Figure 2 shows the neighbor-joining (NJ), Kimura two-parameter tree obtained when including only the C and D haplogroups along with the Asian and African lineages used as outgroups, which cluster close to one another in the tree. The Fuegian C and D lineages are clearly separated from them, showing afnities with the other Amerindian lineages, despite the great diversity observed compared with that of the African and Asian samples. A median network plot of lineages C and D (Fig. 3) shows a

clear pattern of differentiation of the Fuegian lineages, some of them diverging from the major Amerindian groups in as many as four point mutations. However, in the NJ tree, some Fuegian D lineages show smaller genetic distances, and hence greater afnities, with some Na-Dene and Asian lineages than with some other Fuegian D sequences. Y-chromosome Y-STR alleles (Table 3) were characterized in nine cases, with an average of 4.1 loci per sample correctly typed. Table 4 shows the provenience and dating of each Fuegian sample, and summarizes the results obtained for the Y-chromosome STRs and SNP. None of the 20 Y-chromosome-typed samples

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TABLE 3. Ranges of allelic sizes of Y-chromosome markers studied involve PCR amplification of relatively short ancient DNA fragments1 System DYS434 DYS437 DYS439 DYS390 DYS391 DYS393 DYS19 DYS389I DYS389II DYS199 Allelic range (bp) 110122 186202 238258 203227 279291 119131 186202 145169 259291 201241

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1 However, poor preservation of ancient DNA may result in strong DNA fragmentation, and thus, lack of PCR products for some genetic systems studied. In DYS199 system, a rst PCR product of 241 bp is reamplied to yield a second 201-bp fragment.

yielded amplication for Y-STRs DYS390 and DYS391, which may be due to the lack of preservation of DNA fragments or to a lesser efciency of amplication of these systems in ancient samples. All other systems yielded positive results for at least one of the samples considered. Nine of the 20 samples studied (45%) showed positive amplications for at least one Y-chromosome system. Samples F10, F11, F26, F27, F34, F35, F57, F67, F68, F71, and F74 did not amplify for any Y-chromosome system (F57, F67, F68, and F74 had been previously sexed as females, based on osteological sex determination). All Y-chromosome haplotypes obtained had at least one mismatch locus when compared with the male researcher (J.G.-B.), which reduces the likelihood of cross-contamination. However, for the DYS199 system, samples F14 and F18 show both C and T alleles (Fig. 4), owing to exogenous contamination. Given that DYS199*T is not found outside America (Underhill et al., 1996) and that the two negative controls showed no band at all, a DYS199*C allele may have contaminated sample F14 during amplication. This may also hold true for F18, although the band intensities of alleles DYS199*T and DYS199*C are quite similar in this case. Samples F1 and F14 show identical alleles for all systems considered, both belonging to Aonikenk individuals, but with distinct datings. Haplotype DYS19*13/DYS199*T is represented in 4 of the 9 amplied samples, and haplotype DYS19*14/DYS199*T is present in 2 cases. Both haplotypes are absent outside America, and strongly support a South American afnity of the PCR products obtained and sustain the authenticity of the ancient DNA recovery. DISCUSSION Since ancient DNA fragments are not equally preserved within the archaeological context, the genetic picture derived from our analysis may be randomly distorted (Williams et al., 2002). However, the phylogenetic consistency of the results obtained, both for the Y-STRs and mtDNA sequences, strongly sup-

ports the authenticity of the ancient DNA recovered, whose preservation may depend on the taphonomic conditions in the dry, extremely cold environment of the archaeological Fuegian sites, in contrast with the poor preservation observed in tropical regions (Holland et al., 1993; Kumar et al., 2000). The lack of haplogroups A and B in the Fuegian sample has been attributed to the common origin of all Fuegian groups rather than to their independent loss through genetic drift (Lalueza et al., 1997). The short time of divergence and genetic isolation that can be claimed between the Fuegians and Amerindians may not sufce to produce a clear clustering of the Fuegians in the sequence trees (some distinct Y-chromosome haplotypes can be traced though in the Fuegians). Despite this, the median network plot (Fig. 3) succeeds in showing the diversication of the Fuegian lineages, and time estimates of lineage divergence (using Network 3.1.1.1, Fluxus Tech. Ltd.) are 5,264 1,641 years BP for the C lineages, and 34,054 10,090 years BP for the D lineages, taking as ancestral nodes the most frequent for each group of lineages and a mutation rate of 1 every 20,180 years (default value). The haplotype DYS199*T/DYS19*14/DYS389II*26/ DYS389I*10/DYS393*13 is observed in samples F1 and F14, and was only described in one modern sample from Tayacaja in the Peruvian Andes out of the 162 individuals studied from 12 South American populations (Tarazona-Santos et al., 2001; Lell et al., 2002). Tarazona-Santos et al. (2001) did not include populations south of northern Argentina. The Amerindian populations in the Amazonian region, the central Brazilian plateau, and the Chaco region may thus result from strong processes of genetic differentiation through genetic drift and low gene ow. The Fuegian populations may also have undergone severe geographical isolation and genetic drift. However, these alone do not explain the higher diversity of Fuegian lineages compared with Amerindian C D, nor the high proportion of single sequences within the Fuegian and the close-to-the-root position of some of their mtDNA lineages, as can be observed also in the median network of C and D lineages (Fig. 4). However, other markers, such as the DYS199*T allele in all amplied Fuegian samples, regardless of their ethnic group, as well as in most South Americans (Underhill et al., 1996; Tarazona-Santos et al., 2001) and in Asian populations (Lell et al., 2001), support a close relationship between these populations, perhaps with genesis of the M3 mutation in Beringia followed by a quick spread throughout the New World. Although few Y-STR data are available for some parts of the American continent, the presence of haplotypes DYS199*T/ DYS19*14 and DYS199*T/DYS19*13 in all four Fuego/Patagonian groups also suggests that the Fuegians are related to tribes from south-central Chile and Argentina (Rothhammer et al., 1986; Llop, 1996; Moraga et al., 2000). However, this model would require the loss of haplogroups A and B either

TABLE 4. Fuegian samples studied with indications on provenance, ethnic affiliation and dating.1 mtDNA and Y-Chr, STRs results are shown for each genetic system studied. DYS199 BP 14 13 14 13 9 9 8 8 13 12 13 9 8 13 13 mt-DNA T C DYS19 DYS434 DYS437 DYS439 DYS393 DYS389I DYS389II DYS388 10 10 10 10 26 27 26 12 12 12 12 Ethnic groups Sample

Label

Site

13

10 10 11

F1 F5 F10 F11 F14 F18 F26 F27 F34 F35 F46 F57 F59 F67 F68 F69

AON YAM KAW KAW AON YAM KAW AON YAM YAM YAM KAW KAW KAW KAW KAW

RM3 RM1 RM3 RM3 LM1 LM2 LM1 LI2 ?C ?C Root Root Root Root Root Root

400 100 100 100 100 100 100 100 100 100 200 200 200 200 200 200 D C D D C C C D C C C D C C D D

12

12

F71 F74 F85 F89 13 13

Cerro Johnny 6785, Patagonia Isla navarino 849, Navarino Caverna 3N, Puerto Natales Caverna 3N, Puerto Natales Punta Delgada 26839 Canasaca Oeste 6788, Isla Hoste Baeriswyl Bay, close to Puerto Hambre Punta Leo n (Chubut, Argentina) South to Gable Island MT-IG794 Almanza, A-795/1 8008 (420), MHN *M, Pto. Harberton 8005 (415), MHN *F, Isla Grande 8622 (423), MHN *M?, Pto. Harris, 8607 (440), MHN *F, Pto. Harris, Dawson 8612 (442), MNN *F, Pto. Harris, Dawson 8800 (443), MHN *F, Pto. Harris, Dawson 422, MHN Dawson 8618 (439), MHN *F, Pto. Harris, Dawson (A) R.79.3.9, Porvenir museum (A) R.79.3.10, Porvenir museum Researcher (J.G.-B.) KAW KAW SEL SEL EUR Root Root Root Root Present 200 200 200 200 D D C C T 9 8 10 13

10 10 11

26 26

12 14 12

Sex aliation (F, female; M, male), whenever indicated, was determined based on osteological criteria. Aproximate datings (years BP) were available from archaeological information of each burial (200 BP indicates minimum dating, though burial would not be much older than date indicated). Whole teeth (M1, M2, or M3 molars, either from upper or lower jaws, right or left sided) or tooth roots were selected for analysis. mtDNA haplotypes for each sample are known (C or D). Data for Y-STR markers indicate alleles obtained for each sample. A single allele is expected at each locus. Not all analyses produced PCR amplications. C, canine; I, incisor; M, molar; R, right; L, left; AON, Aonikenk (populations from Patagonia, north Magallanes strait); KAW: Kaweskar (Alakaluf); YAM: Yamana; SEL, Selknam (from Isla Grande). For DYS199 SNP, means a strong signal in agarose gel, a weaker one, and indicates absence of signal. Blanks indicate that no characterization was possible for that sample and marker.

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Fig. 4. DYS199 typing of some Fuegian/Patagonian samples, most of which show positive amplication of allele T. Simultaneous amplication of alleles C and T (as in F14 and F18) indicate contamination from PCR amplications. C is negative control; R is researcher of European origin (J.G.-B.).

once in the common Fuegian-Amerindian ancestor or independently in all four Fuego-Patagonian groups. Again, genetic drift alone is unlikely to be responsible for the lack of two major Amerindian haplotypes in all four ethnics groups. Population bottleneck and/or early migration processes, followed by genetic drift by early isolation in Tierra del Fuego, may rather account for the molecular variability observed. Genetic drift could have produced the observed Fuegian vatiability alone, were the dates of the samples more spread in time. The presence of aboriginal populations in Tierra del Fuego dates back to 10,000 years BP, and the archaeological record shows a clear cultural Fuegian differentiation in the Beagle Channel at least 6,000 BP (Piana et al., 1992). The close afnities found between the Fuegians and the modern Amerindians discard a Paleolithic, pre-Amerindian origin for the Fuegians, but other markers studied sustain an early diversication of the Fuegians soon after the colonization of the South American continent. ACKNOWLEDGEMENTS This study was funded by the Spanish DGICYT to D.T. (PB97-0925) and by the UCM to E.A.-P. (PR48/ 01-9837). We also thank Magallanes University (Chile), the Museo Nacional de Historia Natural (Chile), and the Centro Austral de Investigaciones Cient cas de Ushuaia (Argentina) for kindly providing the samples to D.T. LITERATURE CITED
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