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Early population differentiation in extinct aborigines from Tierra del Fuego-Patagonia Ancient mtDNA sequences and Y-Chromosome STR characterization.

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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: turbon@ub.edu
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 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
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 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
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.
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