Early population differentiation in extinct aborigines from Tierra del Fuego-Patagonia Ancient mtDNA sequences and Y-Chromosome STR characterization.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 123:361–370 (2004) Early Population Differentiation in Extinct Aborigines From Tierra del Fuego-Patagonia: Ancient mtDNA Sequences and Y-Chromosome STR Characterization Jaume Garcı́a-Bour,1 Alejandro Pérez-Pérez,1 Sara Álvarez,2 Eva Fernández,1 Ana Marı́a López-Parra,2 Eduardo Arroyo-Pardo,2 and Daniel Turbón1* 1 Secció d’Antropologia, Departament de Biologia Animal, Universitat de Barcelona, E-08028 Barcelona, Spain Laboratorio de Biologı́a Forense, Departamento de Toxicologı́a y Legislación Sanitaria, Facultad de Medicina, Universidad Complutense, E-28040 Madrid, Spain 2 KEY WORDS mtDNA; Y-STR; ancient DNA; Fuegians; Tierra del Fuego; America 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 ampliﬁcations 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 ampliﬁed, 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 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 diversiﬁcation of the Fuegians right after their arrival at the southernmost extreme of South America. Am J Phys Anthropol 123: 361–370, 2004. © 2004 Wiley-Liss, Inc. 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 ﬁts the data better (Tarazona-Santos and Santos, 2002; Lell et al., © 2004 WILEY-LISS, INC. Grant sponsor: Spanish DGICYT; Grant number: PB97-0925; Grant sponsor: UCM; Grant number: PR48/01-9837. *Correspondence to: Daniel Turbón, Secció Antropologia, Departament de Biologia Animal, Facultad de Biologia, Universitat de Barcelona, E-08028 Barcelona, Spain. E-mail: firstname.lastname@example.org Received 14 January 2003; accepted 11 April 2003 DOI 10.1002/ajpa.10337 362 J. GARCÍA-BOUR ET AL. 2002), a differential pattern of genetic drift and gene ﬂow has been proposed to explain the Y-chromosome variability observed for haplogroup 18, deﬁned 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 ﬁrst 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 Paciﬁc coast and islands of Tierra del Fuego, intensively exploiting marine resources for food, whereas the Selk’nam (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 Selk’nam. 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 deﬁnitive 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, Selk’nam, Yamana, and Kaweskar) were analyzed from a larger sample (Lalueza et al., 1997). The samples that yielded signiﬁcant PCR ampliﬁcation 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 classiﬁcation 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 ampliﬁed 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 dentist’s 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) ﬁlled 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 37°C 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 ﬁnal volume of 300 l. Extraction controls without powdered sample were processed in parallel, to test for contamination during the extraction process. Amplification HVRI mtDNA ampliﬁcations were performed through a nested-PCR assay (with 30 PCR cycles per round), using the external primers L16154 5⬘AATACTTGACCACCTGT-3⬘ and H16400 5⬘-TTCAC- 363 ABORIGINES FROM TIERRA DEL FUEGO TABLE 1. mtDNA HVRI sequence polymorphisms in Fuegian-Patagonian sample studied1 Ethnic group Andérson 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 Aonikenk Aonikenk Kaweskar Kaweskar Kaweskar Kaweskar Selknam Selknam Selknam Yamana Yamana Yamana Yamana Yamana Aonikenk Aonikenk Kaweskar Kaweskar Kaweskar Kaweskar Kaweskar Kaweskar Kaweskar Yamana Haplotype 1 6 1 5 6 1 6 1 8 7 1 6 1 8 9 1 6 2 0 9 1 6 2 2 3 1 6 2 4 1 1 6 2 5 0 1 6 2 8 6 1 6 2 9 1 1 6 2 9 4 1 6 2 9 6 1 6 2 9 8 1 6 3 0 4 1 6 3 1 1 1 6 3 1 5 1 6 3 1 8 1 6 3 2 5 1 6 3 2 7 1 6 3 3 9 1 6 3 4 2 1 6 3 6 2 C C C C C C C C C C C C C C D D D D D D D D D D G 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 A 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T T 䡠 䡠 䡠 䡠 䡠 䡠 T T T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 䡠 䡠 C C T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C C 䡠 䡠 䡠 䡠 䡠 䡠 C C C 䡠 T T T T T T T T T T T T T T T T T T T T T T T T A 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 G 䡠 䡠 䡠 䡠 䡠 䡠 䡠 G G 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C T 䡠 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 T T T 䡠 䡠 䡠 䡠 C T 䡠 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T T T 䡠 䡠 䡠 䡠 T 䡠 C C C C C C 䡠 C 䡠 C C 䡠 C C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C C 䡠 䡠 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 A 䡠 G 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T 䡠 C C C C C C C C 䡠 C C C C C C C 䡠 䡠 䡠 䡠 C 䡠 C C C 䡠 T T T T T T T T 䡠 T T T T T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 C 䡠 䡠 T 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C 䡠 䡠 䡠 䡠 䡠 䡠 䡠 䡠 C C C C 1 Haplotype lineages obtained are concordant with expected haplogroup classiﬁcation, 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 ﬁnal PCR product of 240 bp. One microliter of extracted DNA was ampliﬁed 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 (Garcı́a-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 ampliﬁcation of alleles (Williams et al., 2002), and are thus more suitable for ancient DNA studies. Ampliﬁcations were performed using ﬂuorescent-labeled primers. In all instances, 40 cycles of PCR ampliﬁcation were performed. Ampliﬁcation 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 ampliﬁed following Schultes et al. (1999), and STRs for DYS434, DYS437, and DYS439 were ampliﬁed as reported by Ayub et al. (2000). Allelic sizes are shown in Table 3. All ampliﬁed products were ﬁrst 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-speciﬁc ampliﬁcation (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 ampliﬁed at separate laboratories (Anthropology at Universitat de Barcelona (UB), and Forensic Biology at Universidad Complutense (UCM)). At the UB lab, one male re- 364 J. GARCÍA-BOUR ET AL. 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 ampliﬁcations and sequencing. A female researcher (S.L.R.) carried out all Y-chromosome ampliﬁcations 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 ampliﬁcation experiment. Only results with a lack of ampliﬁcation 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 speciﬁcity 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-Amerindianspeciﬁc 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 ampliﬁed mtDNA HVRI region for the Fuegian sample. All sequences obtained were easily 365 ABORIGINES FROM TIERRA DEL FUEGO 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 Amerindian C⫹D Amerindian A⫹B Circumpolar South African Other Haplogroup X Fueguian Amerindian C⫹D Amerindian A⫹B 0.01621 0.01597 0.01556 0.02880 0.02955 0.02147 0.02437 0.04091 0.02471 0.04153 0.02347 0.02860 0.02385 0.02982 Circumpolar South African 0.02180 0.04270 0.01731 0.04460 0.01117 0.02498 0.02884 0.02301 0.03070 0.04136 0.03849 Other Haplogroup X 0.02297 0.02784 0.00405 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 366 J. GARCÍA-BOUR ET AL. Fig. 3. Median network plot (Network 126.96.36.199, 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 afﬁnities 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 afﬁnities, 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 ABORIGINES FROM TIERRA DEL FUEGO TABLE 3. Ranges of allelic sizes of Y-chromosome markers studied involve PCR amplification of relatively short ancient DNA fragments1 System Allelic range (bp) DYS434 DYS437 DYS439 DYS390 DYS391 DYS393 DYS19 DYS389I DYS389II DYS199 110–122 186–202 238–258 203–227 279–291 119–131 186–202 145–169 259–291 201–241 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 ﬁrst PCR product of 241 bp is reampliﬁed to yield a second 201-bp fragment. yielded ampliﬁcation for Y-STRs DYS390 and DYS391, which may be due to the lack of preservation of DNA fragments or to a lesser efﬁciency of ampliﬁcation 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 ampliﬁcations 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 ampliﬁcation. 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 ampliﬁed samples, and haplotype DYS19*14/DYS199*T is present in 2 cases. Both haplotypes are absent outside America, and strongly support a South American afﬁnity 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- 367 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 sufﬁce 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 diversiﬁcation of the Fuegian lineages, and time estimates of lineage divergence (using Network 188.8.131.52, 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 ﬂow. 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 ampliﬁed 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 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 León (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.) Site KAW KAW SEL SEL EUR AON YAM KAW KAW AON YAM KAW AON YAM YAM YAM KAW KAW KAW KAW KAW Root Root Root Root Present RM3 RM1 RM3 RM3 LM1 LM2 LM1 LI2 ?C ?C Root Root Root Root Root Root Ethnic groups Sample mt-DNA D C D D C C C D C C C D C C D D D D C C T BP ⬎400 ⬎100 ⬎100 ⬎100 ⬎100 ⬎100 ⬎100 ⬎100 ⬎100 ⬎100 ⬎200 ⬎200 ⬎200 ⬎200 ⬎200 ⬎200 ⬎200 ⬎200 ⬎200 ⬎200 ⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫺ ⫺ ⫹⫹⫹ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ C DYS199 T 13 13 9 9 9 14 13 13 9 14 13 8 8 8 8 10 13 12 13 13 13 13 10 10 11 11 10 10 10 10 10 10 26 26 26 26 27 12 14 12 12 12 12 12 12 12 DYS19 DYS434 DYS437 DYS439 DYS393 DYS389I DYS389II DYS388 Sex aﬁliation (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 ampliﬁcations. C, canine; I, incisor; M, molar; R, right; L, left; AON, Aonikenk (populations from Patagonia, north Magallanes strait); KAW: Kaweskar (Alakaluf); YAM: Yamana; SEL, Selk’nam (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. 1 F71 F74 F85 F89 F1 F5 F10 F11 F14 F18 F26 F27 F34 F35 F46 F57 F59 F67 F68 F69 Label 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. ABORIGINES FROM TIERRA DEL FUEGO Fig. 4. DYS199 typing of some Fuegian/Patagonian samples, most of which show positive ampliﬁcation of allele T. Simultaneous ampliﬁcation of alleles C and T (as in F14 and F18) indicate contamination from PCR ampliﬁcations. 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 afﬁnities found between the Fuegians and the modern Amerindians discard a Paleolithic, pre-Amerindian origin for the Fuegians, but other markers studied sustain an early diversiﬁcation 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 Cientı́ﬁcas de Ushuaia (Argentina) for kindly providing the samples to D.T. LITERATURE CITED Adovasio JM, Carlisle RC. 1988. The Meadowcroft Rockshelter. Science 239:713–714. Adovasio JM, Pedler DR. 1997. Monte Verde and the antiquity of humankind in the Americas. Antiquity 71:573–580. Adovasio JM, Donahue J, Stuckenrath R. 1990. The Meadowcroft Rockshelter radiocarbon chronology 1975–1990. Am Antiq 55: 348 –354. Ayub Q, Mohyuddin A, Qamar R. 2000. Identiﬁcation and characterisation of novel human Y-chromosomal microsatellites from sequence database information. Nucleic Acids Res 28:8. Bailliet G, Rothhammer F, Carnese FR, Bravi CM, Bianchi NO. 1994. Founder mitochondrial haplotypes in Amerindian populations. Am J Hum Genet 54:27–33. 369 Bianchi NO, Bailliet G, Bravi CM. 1997. Origin of Amerindian Y-chromosomes as inferred by the analysis of six polymorphic markers. Am J Phys Anthropol 102:79 – 89. Bonatto SL, Salzano FM. 1997. A single and early migration for the peopling of the Americas supported by mitochondrial DNA sequence data. Proc Natl Acad Sci USA 94:1866 –1971. Cavalli-Sforza LL, Menozzi P, Piazza A. 1994. The history and geography of human genes. Princeton: Princeton University Press. Chrisman D, MacNeish RS, Mavalwala J, Savage H. 1996. Late Pleistocene human friction skin prints from Pendejo Cave, New Mexico. Am Antiq 61:357–376. Crawford MH. 1998. The origin of the Native Americans. Cambridge: Cambridge University Press. De Knijff P, Kayser M, Caglià A. 1997. Chromosome Y microsatellites: population genetic and evolutionary aspects. Int J Legal Med 110:134 –149. Dillehay TD. 1997. Monte Verde: a late pleistocene settlement in Chile. Volume 2. The archaeological context and interpretation. Washington, DC: Smithsonian Institution Press. Dillehay TD, Collins M. 1988. Early cultural evidence from Monte Verde in Chile. Nature 332:150 –152. Fiedel SJ. 1992. Prehistory of the Americas, 2nd ed. Cambridge: Cambridge University Press. Foster P, Harding R, Torroni A. 1996. Origin and evolution of Native American mtDNA variation: a reappraisal. Am J Hum Genet 59:935–945. Foster P, Harding R, Torroni A. 1997. Replay to Bianchi and Bailliet. Am J Hum Genet 61:246 –247. Garcı́a-Bour J, Pérez-Pérez A, Prats E, Turbón D. 1998. Secuercias de mtDNA de Aborı́genes de Tierra del Fuego-Patagonia y el origen de los Fueguinos. Anales del Instituto de la Patagonia 26:69 –75. Gerstenberger J, Hummel S, Schultes T, Hack B, Herrmann B. 1999. Reconstruction of a historical genealogy by means of STR analysis and Y-haplotyping of ancient DNA. Eur J Hum Genet 7:469 – 477. Hoeffecker JF, Powerds RW, Goebel T. 1993. The colonization of Beringia and the peopling of the New World. Science 259:46 – 53. Holand MM, Fifher DL, Mitchel LG, Rodrı́guez WC, Canik JJ, Merril CR, Weebn VW. 1993. Mitochondrial DNA sequence analysis of human skeletal remains: identiﬁcation of remains from the Vietnam War. J Forensic Sci 38:542–553. Karafet TM, Zegura SL, Posukh O, Osipova L, Bergen A, Long J, Goldman D, Klitz W, Harihara S, de Kniff P, Weibe W, Grifﬁths C, Templeton AR, Hammer ME. 1999. Ancestral Asian source(s) of New World Y-chromosome founding haplotypes. Am J Hum Genet 64:817– 831. Kayser M, Caglià A, Corach D. 1997. Evaluation of Y-chromosomal STRs: a multicenter study. Int J Legal Med 110:125–133. Kumar FS, Naﬁdze I, Walimbe FR, Stoneking M. 2000. Brief communication: discouraging prospect for ancient DNA from India. Am J Phys Anthropol 113:129 –133. Kumar S, Tamura K, Jakobsen IB, Nei M. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244 –1245. Lahr MM. 1994. The multiregional model of modern human origins: a reassessment of its morphological basis. J Hum Evol 26:23–56. Lahr MM. 1995. Patterns of modern humans diversiﬁcation: implications for Amerindian origins. Yrbk Phys Anthropol 38: 163–198. Lalueza C, Pérez-Pérez A, Prats E, Cornudella L, Turbón D. 1997. Lack of founding Amerindian mitochondrial DNA lineages in extinct aborigines from Tierra de Fuego-Patagonia. Hum Mol Genet 6:41– 46. Lell JT, Sukernik RI, Starikovskova YB, Su B, Lin L, Schurr TG, Underhill PA, Wallace DC. 2002. The dual origin and Siberian afﬁnities of Native American Y-chromosomes. Llop E. 1996. Genetic composition of Chilean aboriginal populations: HLA and other genetic marker variations. Am J Phys Anthropol 101:325–332. 370 J. GARCÍA-BOUR ET AL. Martinic M. 1992. Historia de la Región Magallánica. Fondo Nacional de Ciencia y Tecnologı́a. Universidad de Mafallanes. Santiago, Chile: Alfabeta Impresores. Meltzer DJ, Adovasio JM, Dillehay TD. 1994. On a Pleistocene human occupation at Pedra Furada, Brazil. Antiquity 68:695– 714. Meltzer DJ, Grayson DK, Ardila G, Barker AW, Dincauze DF, Haynes CV, Mena F, Nunez L, Stanford DJ. 1997. On the Pleistocene antiquity of Monte Verde, Southern Chile. Am Antiq 62:69 – 663. Merriwether DA, Rothhammer F, Ferrell RE. 1995. Distribution of four founding lineage haplotypes in Native Americans suggests a single wave of migration for the New World. Am J Phys Anthropol 98:411– 430. Moraga LM, Rocco P, Miquel JF, Nervi F, Llop E, Chakraborty R, Rothhammer F, Carvallo P. 2000. Mitochondrial DNA polymorphisms in Chilean aboriginal populations: implications for the peopling of the Southern Cone of the continent. Am J Phys Anthropol 113:19 –29. Pena SDJ, Santos FR, Bianchi NO. 1995. A major founding Ychromosome haplotype in Amerindians. Nat Genet 11:15–16. Piana EL, Vila A, Orquera L, Estevez J. 1992. Chronicles of Onashaga: archaeology in the Beagle Chanel. Antiquity 66: 771–783. Rothhammer F, Silva C, Cocilovo J, Quevedo S. 1986. Una hipótesis sobre el poblamiento de Chile basada en el análisis multivariado de medidas craneométricas. Rev Chung 16 –17:115– 118. Santos FR, Pandya A, Tyler-Smith C, Pena SDJ, Schanﬁeld M, Leonard WR, Osipova L, Crawford MH, Mitchel RJ. 1999. The central Siberian origin for Native American Y-chromosomes. Am J Hum Genet 64:619 – 628. Schultes T, Hummel S, Herrmann B. 1999. Ampliﬁcation of Ychromosomal STRs from ancient skeletal material. Hum Genet 104:164 –166. Stone AC, Stoneking M. 1998. mtDNA analysis of a prehistoric Oneota population: implications for the peopling of the New World. Am J Hum Genet 62:1153–1170. Szathmary EJ. 1993. Genetics of aboriginal North Americans. Evol Anthropol 1:202–220. Tarazona-Santos E, Santos FR. 2002. The peopling of the Americas: a second major migration? Am J Hum Genet 70:1377– 1381. Tarazona-Santos E, Carvalho-Silva BR, Peptener D, Luiselli D, de-Stefano GF, Martı́nez-Labarga C, Rickard O, Tayler-Smith C, Pena SDJ, Santos FR. 2001. Genetic differentiation in South Amerindian is related to environmental and cultural diversity: evidence from the Y-chromosome. Am J Hum Genet 60:1485– 1496. Torroni A, Schurr TG, CabellM F, Brown MD, Neel JV, Larsen M, Smith DG, Vullo CM, Wallace DC. 1993. Asian afﬁnities and continental radiation of the four founding Native American mtDNAs. Am J Hum Genet 53:536 –590. Torroni A, Chen Y-S, Semino O, Santachiara-Benerecetti AS, Scott CR, Lott ML, Winter M, Wallace DC. 1994. mtDNA and Y-chromosome polymorphisms in four Native American populations from southern Mexico. Am J Hum Genet 54:303–318. Underhill PA, Jin L, Zemans R. 1996. A pre-Columbian Y-chromosome-speciﬁc transition and its implications for human evolutionary history. Proc Natl Acad Sci USA 93:196 –200. Williams SR, Chagnon NA, Spielman RS. 2002. Nuclear and mitochondrial genetic variation in the Yanomanó: a test case for ancient DNA studies of prehistoric populations. Am J Phys Anthropol 117:246 –259.