Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal Route for the Peopling of the Americas

Article (PDF Available)inThe American Journal of Human Genetics 82(3):583-92 · April 2008with43 Reads
DOI: 10.1016/j.ajhg.2007.11.013 · Source: PubMed
Abstract
It is well accepted that the Americas were the last continents reached by modern humans, most likely through Beringia. However, the precise time and mode of the colonization of the New World remain hotly disputed issues. Native American populations exhibit almost exclusively five mitochondrial DNA (mtDNA) haplogroups (A-D and X). Haplogroups A-D are also frequent in Asia, suggesting a northeastern Asian origin of these lineages. However, the differential pattern of distribution and frequency of haplogroup X led some to suggest that it may represent an independent migration to the Americas. Here we show, by using 86 complete mitochondrial genomes, that all Native American haplogroups, including haplogroup X, were part of a single founding population, thereby refuting multiple-migration models. A detailed demographic history of the mtDNA sequences estimated with a Bayesian coalescent method indicates a complex model for the peopling of the Americas, in which the initial differentiation from Asian populations ended with a moderate bottleneck in Beringia during the last glacial maximum (LGM), around approximately 23,000 to approximately 19,000 years ago. Toward the end of the LGM, a strong population expansion started approximately 18,000 and finished approximately 15,000 years ago. These results support a pre-Clovis occupation of the New World, suggesting a rapid settlement of the continent along a Pacific coastal route.
ARTICLE
Mitochondrial Population Genomics Supports
a Single Pre-Clovis Origin with a Coastal Route
for the Peopling of the Americas
Nelson J.R. Fag undes,
1,2,7
Ricardo Kanitz,
1,7
Roberta Eckert,
1
Ana C.S. Valls,
1
Mauricio R. Bogo,
1
Francisco M. Salzano,
2
David Glenn Smith,
3
Wilson A. Silva Jr.,
4
Marco A. Zago,
4
Andrea K. Ribeiro-dos-Santos,
5
Sidney E.B. Santos,
5
Maria Luiza Petzl-Erler,
6
and Sandro L. Bonatto
1,
*
It is well accepted that the Americas were the last continents reached by modern humans, most likely through Beringia. However, the
precise time and mode of the colonization of the New World remain hotly disputed issues. Native American populations exhibit almost
exclusively five mitochondrial DNA (mtDNA) haplogroups (A–D and X). Haplogroups A–D are also frequent in Asia, suggesting a north-
eastern Asian origin of these lineages. However, the differential pattern of distribution and frequency of haplogroup X led some to
suggest that it may represent an independent migration to the Americas. Here we show, by using 86 complete mitochondrial genomes,
that all Native American haplogroups, including haplogroup X, were part of a single founding population, thereby refuting multiple-
migration models. A detailed demographic history of the mtDNA sequences estimated with a Bayesian coalescent method indicates
a complex model for the peopling of the Americas, in which the initial differentiation from Asian populations ended with a moderate
bottleneck in Beringia during the last glacial maximum (LGM), around ~23,000 to ~19,000 years ago. Toward the end of the LGM,
a strong population expansion started ~18,000 and finished ~15,000 years ago. These results support a pre-Clovis occupation of the
New World, suggesting a rapid settlement of the continent along a Pacific coastal route.
Introduction
In the complex history of human migrations, it is widely
accepted that the New World continents were the ones col-
onized most recently by Homo sapiens, most likely from
Asia through Beringia.
1
A popular model for the peopling
of the Americas suggests that the archaeological remains
known as the Clovis complex (thought to be the oldest un-
equivocal evidence of humans in the Americas) represent
the people that first colonized the continent after a late-
glacial migration through the ice-free corridor that sepa-
rated the Laurentide and Cordilleran ice sheets.
1
However,
the recently re-evaluated age of the Clovis sites to only
between about 12.7 and 13.2 thousand years ago (kya)
2
and the confirmed human presence at the Monte Verde
site located in southern South America around 14.5 kya
3
challenge this Clovis-first model and call for alternative
hypotheses. Because the earlier date for Monte Verde im-
plies that peopling of the Americas south of Beringia oc-
curred before the ice-free corridor was formed, a first migra-
tion along the Pacific coast may have been a viable route.
4
Unfortunately, archaeological verification of this scenario
is very difficult because most of the late Pleistocene coast
is currently underwater; the sea level has risen more than
120 m since the end of the last glacial maximum (LGM).
5
The maternally inherited mitochondrial DNA (mtDNA)
has been widely used to understand the peopling of the
Americas. Since the first studies, it has been found that
extant Native American populations exhibit almost ex-
clusively five mtDNA haplogroups (A–D and X)
6
classified
in the autochthonous haplogroups A2, B2, C1, D1, and
X2a.
7
Haplogroups A–D are found all over the New World
and are frequent in Asia, supporting a northeastern Asian
origin of these lineages.
6,8
This distribution, together
with the similar coalescence time for these haplogroups,
was used to suggest a single-migration model.
9–12
How-
ever, a different pattern of diversification and distribution
of haplogroup B found in some studies led some authors to
hypothesize that it could represent a later and separate
migration from the joint arrival of haplogroups A, C, and
D.
13
The history of haplogroup X is more elusive; it is pres-
ently found in the New World at a relatively low fre-
quency
14
and only in North America,
15
it is rare in West
Eurasians, and it is almost absent in Siberia.
16
In addition,
some have claimed that Native American haplogroup X is
less diverse and has a younger coalescence time than hap-
logroups A–D
17
. These differential features have been cited
to argue that haplogroup X represents an independent
migration to the Americas from Asia or even Europe.
17
More specifically, it has been used to support a putative
connection between the European Solutrean and the
American Clovis lithic technologies.
18
This so called ‘Solu-
trean hypothesis’ proposed the colonization of North
America by Europeans through the North Atlantic, even
1
Faculdade de Biocie
ˆ
ncias, Pontifı
´
cia Universidade Cato
´
lica do Rio Grande do Sul, Porto Alegre, RS, 91619-900, Brazil;
2
Departamento de Gene
´
tica, Univer-
sidade Federal do Rio Grande do Sul, Porto Alegre, RS, 91501-970, Brazil;
3
Molecular Anthropology Laboratory, Department of Anthropology, University of
California, Davis, Davis, CA 95616, USA;
4
Faculdade de Medicina, Universidade de Sa
˜
o Paulo, Ribeira
˜
o Preto, SP, 14049-900, Brazil;
5
Departamento de
Patologia, Universidade Federal do Para
´
,Bele
´
m, PA, 66075-970, Brazil;
6
Departamento de Gene
´
tica, Universidade Federal do Parana
´
, Curitiba, PR, 81531-990,
Brazil
7
These authors contributed equally to this work.
*Correspondence: slbonatto@pucrs.br
DOI 10.1016/j.ajhg.2007.11.013. ª2008 by The American Society of Human Genetics. All rights reserved.
The American Journal of Human Genetics 82, 1–10, March 2008 1
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
though this interpretation is heavily debated (e.g.,
19
). All
the five founding haplogroups have been shown to be
present in Native Americas in pre-Columbian times.
12,20
In general, the studies on mtDNA control-region varia-
tion have been taken to support a pre-Clovis migration,
between ~20 and 30 kya, before the LGM, for the single
(or the most ancient) migration.
6,21
However, the uncer-
tainties about and range around these dates are very large.
One cause for this variation is the limited information
content of the mtDNA control region, which is also too
divergent to allow reliable substitution-rate estimation by
comparison with the chimpanzee.
22
Alternatively, the
complete coding region of the mtDNA is being increas-
ingly used to circumvent these limitations in studies of
human migrations (e.g.,
22,23
) but has not been used so
far for studying the origin of Native Americans.
Another frequent controversy is about the size of the
founding population during the peopling of the Americas.
The initial results showing the existence of few founder hap-
logroups for the mtDNA and Y chromosome suggested
a strong population bottleneck,
6
although this interpreta-
tion was not supported by further mtDNA studies.
21
How-
ever, a recent analysis of several genomic loci, including
mtDNA, suggested that the Americas could have been
founded by as few as 80 effective individuals, and even the
largest values in the credible interval only comprise a few
hundred effective individuals.
24
On the other hand, the
study of other single genetic systems does not seem to
support much loss of genetic diversity during the initial
settlement of the continent;
25–28
instead, it concludes that
a moderate-intensity bottleneck is the best scenario. Another
recent genomic study using exclusively autosomal intergenic
markers also suggested moderate values, with the Native
American founding population consisting of around 500
effective individuals (95% confidence interval 74–1332).
29
In this study, we analyze 86 mtDNA genomes (58 of them
new) belonging to all five major Native American hap-
logroups (A2, B2, C1, D1, and X2a) to provide a better
understanding of the timing and mode of the peopling of
the New World. Our analysis suggests a complex scenario
for this migration, in which the founding population
underwent a moderate bottleneck during the LGM to
expand along the continent toward the end of the LGM,
around 18 kya, probably via a Pacific coastal route. Further-
more, we support a model in which all mtDNA haplogroups
were present in this expansion, thus refuting multiple-
migration scenarios such as the Solutrean hypothesis.
Material and Methods
Subjects
DNA samples were obtained from 58 individuals from South and
North American native populations and most have been collected
directly by some of the authors (F.M.S., S.E.B.S., M.A.Z., or D.G.S.).
Table 1 provides further details on the individuals studied. All
ethical guidelines were followed, as stipulated by the institutions
involved in the study.
PCR, Sequencing, and Contig Assembling
Given the low quantity of some of our DNA samples, we per-
formed a genomic preamplification protocol by using the
GenomiPhi kit (GE Healthcare) on these. The PCR amplifications,
using primers covering the entire mitochondrial genome, were
performed as described elsewhere.
30
Sequencing reactions cover-
ing the entire mitochondrial genome for both strands
30
were
read in a MegaBACE 1000 (GE Healthcare) with the ET Termina-
tors cycle sequencing kit. Chromatograms were assembled in indi-
vidual genomes with the Phred-Phrap-Consed package.
31,32
After
an initial visual inspection for low-quality regions in the assembly,
we aligned the contigs generated for every individual to each other
and to the corrected Cambridge reference sequence (rCRS)
33,34
and checked all variable positions in the original chromatograms.
Possible phantom mutations were again verified in the chromato-
grams and, whenever needed, resequenced from a new PCR prod-
uct.
35
Although some mtDNAs have a partial sequence already
published,
36
the whole genomes were mostly resequenced to
ensure maximum quality.
Additional Data
To the 58 genomes obtained here, we added 28 complete mtDNA
genomes published throughout the literature (see Table 1). This
makes a dataset of 86 complete mtDNA genomes characterized
from mainly Native American individuals. We have deliberately
restricted our analysis to the populations known as ‘Amerinds,’
leaving aside people from Eskimo-Aleuts and Na-Dene
´
linguistic
groups. We
11
and others (reviewed in
6
) have already demon-
strated that the latter two were part of the single founding popu-
lation that gave origin to all Native Americans. However, there is
also evidence
6
that the Eskimo-Aleuts and Na-Dene
´
diverged
from Amerinds > 10 kya and underwent independent population
contractions and re-expansions around the circumartic region.
Methods such as Bayesian skyline plot, neutrality tests, etc., are
only applicable to a group of populations that share the same
demographic history. We have therefore not incorporated the
haplogroup D2 in our study, because it is only found in Aleutians
and in a few other Beringian populations in low frequency.
37
Two
large-scale databases (ref.
38
and GenBank accession numbers
DQ282387–DQ282487) encompassing Native American mtDNAs
have not been used in the main analyses because they consist of
data from nonnative individuals. However, to check the robust-
ness of our conclusions, we also performed most of our analyses
by adding these nonnative individuals to generate a dataset of
244 mtDNA genomes comprising all available sequences from
the five Native American haplogroups. The differences between
the results of the native (86 sequences) and those of the 244 se-
quence datasets are very small in all analyses. Therefore, our re-
sults with the 86 dataset are robust and authentically represent
present-day mtDNA diversity in ‘Amerinds.’
Data Analysis
All statistical analyses were done with the slowly evolving mtDNA
coding region (positions 577–16022) only. Control-region
sequence was used to confirm haplogroup assignment. To check
for mutations separating Native American and Old World hap-
logroups, we compared our sequences with sequences belonging
to Asian (haplogroups A–D) and European (haplogroup X) individ-
uals available in the literature.
16,22,39–47
Basic diversity statistics,
neutrality tests, and mismatch distributions were calculated with
Arlequin 3.11.
48
2 The American Journal of Human Genetics 82, 1–10, March 2008
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
Maximum-likelihood phylogenetic trees were constructed with
PAUP* 4.0
49
under the HKYþG evolutionary model, assuming an
alpha parameter of 0.12.
23
The assumption of a molecular clock
was tested with the PAML package
50
under the HKYþG model,
assuming an alpha parameter of 0.12. For the Amerind dataset
(n ¼ 86), the null hypothesis of a molecular clock cannot be re-
jected (p ¼ 0.13). Median-joining networks
51
were constructed
with the program Network 4.1.0.2, and the time to most recent
common ancestor (TMRCA) for each haplogroup was then calcu-
lated on the basis of r with a rate of 1.26 3 10
8
substitutions
per site per year
39
for the mtDNA coding region.
The TMRCAs for each Native American mtDNA haplogroup
with an external calibration point were estimated with the soft-
ware r8s 1.7
52
as follows. A maximum-likelihood tree estimated
in PAUP* as described above with 100 bootstrap replications was
optimized with the Langley-Fitch model and the Powell algorithm
with the optimal smoothing value (S ¼ 1) obtained by a cross-
validation procedure. We calibrated our estimates by assuming
that the Pan and Homo lineages had separated from each other
completely by 6 million yr ago and added 500 ky for lineage
sorting.
23,39
This procedure avoids the assumption of a substitu-
tion rate known a priori. This tree was constructed with sequences
available in GenBank from Pan (D38113, D38116, X93335) and an
assorted set of 40 mtDNA sequences belonging to other hap-
logroups, also including Asians from haplogroups A–D, that
were used to break long branches to improve phylogenetic recon-
struction.
To investigate whether our inferences were robust when the
assumption of a strict molecular clock was relaxed, we used the
Bayesian approach for the estimation of the coalescence times
53
Table 1. Individuals Used for the Analyses Whose mtDNAs
Were Obtained in this Work or Gathered from Literature
Hg ID
GenBank Accession
Number Tribe/Population Reference
A2 ACHE30 EU095194 Ache **
A2 WAI01 EU095195 Waiwai **
A2 WAI25 EU095196 Waiwai **
A2 ZOR02 EU095197 Zoro
´
**
A2 SURU01 EU095198 Suruı
´
**
A2 WPI167 EU095199 Waia˜pi **
A2 Y655 EU095200 Yanomama **
A2 PTJ03 EU095201 Poturujara **
A2 Y623 EU095202 Yanomama **
A2 KKT13 EU095203 Kayapo
´
/Kriketun **
A2 KTN130 EU095204 Katuena **
A2 GRC149 EU095205 Guarani/Rio das Cobras **
B2 ACHE78 EU095206 Ache **
B2 GAVI23 EU095207 Gavia˜o **
B2 POMO01 EU095208 Pomo/North California **
B2 WAI24 EU095209 Waiwai **
B2 XAV04 EU095210 Xavante **
B2 XAV12 EU095211 Xavante **
B2 1876 EU095212 Quechua **
B2 1880 EU095213 Quechua **
B2 1881 EU095214 Quechua **
B2 GRC169 EU095215 Guarani/Rio das Cobras **
B2 KBK23 EU095216 Kayapo
´
/Kubemkokre **
B2 KBK39 EU095217 Kayapo
´
/Kubemkokre **
B2 KKT01 EU095218 Kayapo
´
/Kriketun **
B2 KRC33 EU095219 Guarani/Rio das Cobras **
B2 KTN209 EU095220 Katuena **
B2 Y637 EU095221 Yanomama **
C1 WAI16 EU095222 Waiwai **
C1 ZOR19 EU095223 Zoro
´
**
C1 ZOR31 EU095224 Zoro
´
**
C1 1875 EU095225 Quechua **
C1 1878 EU095226 Quechua **
C1 ARL58 EU095227 Arara/Arara do Laranjal **
C1 PTJ68 EU095228 Poturujara **
C1 Y591 EU095229 Yanomama **
C1 Y650 EU095230 Yanomama **
C1 Y669 EU095231 Yanomama **
D1 GAVI12 EU095232 Gavia˜o **
D1 GAVI26 EU095233 Gavia˜o **
D1 SUR22 EU095234 Suruı
´
**
D1 WAI05 EU095235 Waiwai **
D1 ZOR23 EU095236 Zoro
´
**
D1 GRC131 EU095237 Guarani/Rio das Cobras **
D1 KTN18 EU095238 Katuena **
D1 PTJ01 EU095239 Poturujara **
D1 TYR04 EU095240 Tiryo
´
**
D1 TYR16 EU095241 Tiryo
´
**
X2a CHIP20 EU095242 W. Chippewa/NE **
X2a CHIP44 EU095243 W. Chippewa/NE **
X2a CHIP76 EU095244 W. Chippewa/NE **
X2a CHIP85 EU095245 W. Chippewa/NE **
X2a SAM2 EU095246 Chippewa/NE **
X2a SW097 EU095247 Chippewa/NE **
X2a JEM22 EU095248 Jemez/SE **
X2a JEM435 EU095249 Jemez/SE **
X2a JEM990 EU095250 Jemez/SE **
X2a SIOU59 EU095251 Siouan/SE **
A2 Na5A AY195786 Native American*
39
A2 N/A AF346971 Chukchi
22
A2 haplotype A AF382010 Canary
42
A2 AM17 DQ112832 Auca
47
Table 1. Continued
Hg ID
GenBank Accession
Number Tribe/Population Reference
B2 Na1B AY195749 Native American*
39
B2 N/A AF347001 Pima
22
B2 AM12 DQ112889 Mayan
47
B2 AM15 DQ112790 Colombian Indian*
47
B2 AM16 DQ112791 Colombian Indian*
47
C1 Na4C AY195759 Native American*
39
C1 haplotype C AF382009 Canary
42
C1 N/A AF347012 Warao
22
C1 N/A AF347013 Warao
22
C AM03 DQ112789 Colombian Indian*
47
C AM04 DQ112888 Mayan
47
C AM06 DQ112846 Navajo
47
D1 Na2D AY195748 Native American*
39
D1 N/A AF346984 Guarani
22
D1 AM01 DQ112772 Brazilian Indian*
47
D1 AM02 DQ112776 Brazilian Indian*
47
D1 AM07 DQ112871 Quechua
47
D1 AM08 DQ112872 Pima
47
D1 AM09 DQ112773 Brazilian Indian*
47
D1 AM10 DQ112774 Brazilian Indian*
47
D1 AM11 DQ112775 Brazilian Indian*
47
D1 AM14 DQ112843 Guarani
47
X2a NA22 N/A Ojibwa
7
X2a Na3X AY195787 Navajo
39
Hg denotes haplogroup, and ID indicates label in Figure 1. Individuals were
assigned to Hg C when no data for their control region were available.
*No further information available.
**This work.
The American Journal of Human Genetics 82, 1–10, March 2008 3
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
implemented in BEAST v1.4, which applies Markov Chain Monte
Carlo integration for parameter estimation over the space of all
equally likely trees. Population size dynamics through time (i.e.,
a Bayesian Skyline plot)
53
were also estimated with this approach
in BEAST. It is important to emphasize that in this method all
genomes were analyzed simultaneously without the assumption
of any phylogenetic structure a priori, such as the existence of hap-
logroups or the number of founding haplotypes. Estimations were
carried out assuming HKYþG model with the same rate used for
r time estimations but with log-normal relaxation allowed. The
analysis was run for 60 million iterations, with the first 10% dis-
carded as burn-in. Genealogies and model parameters were sam-
pled every 1,000 iterations thereafter.
Results
As expected, all mitochondrial genomes obtained here
grouped in the five known haplogroups, as shown in the
schematic trees in Figure 1. The diversity patterns within
each Native American haplogroup, including haplogroup
X, are remarkably alike. All haplogroups exhibit similar nu-
cleotidediversity values,aswellas a marked excessof low-fre-
quency variants that is characteristic of a strong and recent
population expansion as shown by significant negative
values for Tajima’s D and Fu’s Fs statistics (Table 2), and single
waves in the mismatch distribution graphics (Figure 2).
Figure 1. Schematic Tree from the Five Native American mtDNA Haplogroups with Sequences Obtained Here and Indicating the
Coding-Region Substitutions
Letters following positions indicate transversions, and the others are transitions. Transition 3552 in the X2a haplogroup was absent in the
individual Na3X from,
33
so it is not considered to be a marker for this haplogroup.
Table 2. Summary Statistics and Coalescence Times for the Five Native American Haplogroups
Haplogroup n S p (SD) % Tajima’s D Fu’s Fs r (95% CI)
a
Bayesian (95% CI)
A2 16 58 0.0512 (0.0282) 2.333** 9.897** 20,552 (14,953–26,151) 21,290 (16,550–28,130)
B2 21 72 0.0504 (0.0273) 2.468** 15.997** 20,307 (15,246–25,369) 22,140 (17,570–28,730)
C 17 44 0.0417 (0.0233) 2.097** 7.200** 17,227 (11,461–22,994) 20,680 (16,830–26,260)
D1 20 44 0.0484 (0.0263) 1.594** 7.280** 21,580 (13,263–29,896) 21,430 (16,850–28,730)
X2a 12 20 0.0304 (0.0180) 1.277* 2.410* 17,983 (6,056–29,910) 20,730 (16,100–29,000)
Average 19,530 21,254
Summary statistics and coalescence times were based on median-joining calculation (r) and on Bayesian estimation. n indicates number of sequences,
S indicates number of segregating sites, p indicates nucleotide diversity, and CI indicates confidence interval.
a
Estimated as r 5 2 3 standard deviation (SD); *p < 0.10; **p < 0.05.
4 The American Journal of Human Genetics 82, 1–10, March 2008
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
Using the standard substitution rate of 1.26 3 10
8
per site
per year for the mtDNA coding region,
39
all haplogroups
show coalescence times around 20 kya, with both a strict
(r) and a relaxed molecular clock method (Bayesian, Table
2), together with a close correspondence in the distribution
of coalescence times for each haplogroup (Figure 3). Addi-
tionally, similar values are found when we compute a maxi-
mum likelihood tree with an external calibration point (i.e.,
without assuming a predetermined substitution rate, Fig-
ure 4A). For each haplogroup, all Native American sequences
trace back to a single founder haplotype that can be distin-
guished from Old World haplogroups by the presence of
exclusive mutations (Figure 1) or, in the case of haplogroup
C, by specific control region sequence motifs, corroborating
the results from Bandelt et al.
7
We observed 2, 5, 0, 1, and 3
coding-region mutations which are markers for the five Na-
tive American haplogroups A–D, and X, respectively. The ac-
cumulation of these Native American specific mutations
possibly reflect the duration of the transition period after
the ancestral population divergence from Asians but before
the within-haplogroups diversification.
7,11,21,54
Because
the standard rate for these mutations is equivalent to one
substitution per 5138 years,
39
it suggests that this transition
period took several thousand years, likely > 5ky.Allthe
above results strongly suggest a scenario in which all five
haplogroups were part of a single founding population
that ultimately led to the peopling of the whole American
continent.
To get a more realistic picture of the complex demo-
graphic history associated with the colonization of the
New World we applied the Bayesian skyline plot ap-
proach
53
to the whole Native American mtDNA genome
sample, noting that this estimate is unaffected by the dif-
ferent proposals for the actual number of founder lineages
among present day Native American mtDNAs.
6
The sky-
line plot (Figure 4B) identifies a moderate effective popula-
tion size reduction between ~23–19 kya reaching a mini-
mum of ~1000 women followed by a strong (~100-fold)
and rapid size expansion beginning ~19–18 kya and end-
ing ~16–15 kya. It is noteworthy that the time of the pop-
ulation reduction correlates very well with the LGM (23–
18 kya) while the expansion dates are in excellent agree-
ment with the end of the LGM, dated around 19–17
kya.
55,56
The Bayesian skyline plot of each haplogroup sep-
arately showed a similar pattern of population expansion
(data not shown).
Discussion
Overall, the Native American mtDNA genomic data
suggests the following scenario for the peopling of the
Americas. The transition period between the separation
of the Native American mtDNA haplogroups from their
Asian ancestors and the start of their diversification and
expansion into the Americas was estimated at > 5000
years. Adding this time to our estimates for the expansion
indicates that the beginning of the divergence of the
Native American founder population from its Asian ances-
tral population probably predates the LGM. There is evi-
dence of human settlements in the artic around 30 kya.
57
Therefore, it is possible that the precursors of Native Amer-
ican populations represent a human group that pioneered
colonization of northeast Asia before the LGM, during a
period of amelioration in the climatic conditions.
58
Although it was not possible to determine where in north-
east Asia this population stayed during this long period of
isolation, Beringia represents the best candidate for that lo-
cation, at least for the moderate bottleneck period (~20
kya) before the expansion. Toward the end of the Pleisto-
cene, Beringia was mostly exposed, and even though
Figure 2. Mismatch Distributions from Native American Hap-
logroups
The frequency of the number of differences between all pairs of
mtDNA genomes compared within each haplogroup.
Figure 3. Bayesian Estimation of TMRCA Density from Native
American Haplogroups
Relative density of age estimated by BEAST
53
in kya for each hap-
logroup.
The American Journal of Human Genetics 82, 1–10, March 2008 5
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
archaeological evidence for human presence in Beringia
around the LGM is controversial, the first evidence of hu-
man settlements around this area predates the LGM.
57
There is also strong evidence that the Beringian environ-
ment could sustain human populations during at least
part of the LGM, being considered both climatically and
ecologically a glacial refugium.
59
mtDNA results further
suggest that during the LGM the Native American found-
ing population experienced a reduction in size (with a min-
imum of around 1000 women) that lasted for 3000–4000
yr and may have been linked to the deteriorating condi-
tions of Beringia at the LGM.
60
In addition, the Beringian
refugium was ecologically isolated to the west and physi-
cally isolated to the east by the glaciers that seem to have
effectively blocked the way to America until near the end
of the LGM; this may help to explain why the population
stayed in Beringia for such a long time before expanding
south.
We estimate that beginning ~19–18 kya and ending
~16–15 kya (i.e., toward the end of the LGM), the Native
American founding population experienced a significant
demographic growth process that is most likely associated
with an extensive range expansion and may mark the
beginning of the effective colonization of the New World
south of Beringia. Given that the opening of the ice-free
corridor is dated not earlier than ~14 kya, our results
strongly support an alternative route for this expansion,
most likely along the western coast of North America.
61,62
Recent data have shown that this coastal route was largely
ice free by ~19 kya and that the environment improved
rapidly, being capable of supporting bears as of
~15 kya.
63
Interestingly, the end of the intense expansion
period coincides with the age of the southern South Amer-
ican Monte Verde site, ~14.5 kya.
3
The strong and rapid
population growth suggested by our data is consistent
with a model in which humans have traveled the > 13,000
km along the coast from Alaska to the southern tip of
Chile in a few thousand years.
64
All of the above age es-
timates were calculated with the standard mtDNA cod-
ing-region substitution rate that was presented by Mish-
mar et al.
39
and was used in the great majority of the
recent mtDNA studies. A new rate using exclusively synon-
ymous substitutions was recently suggested.
47
We have ap-
plied this approach to our dataset, and the only changes in
the results are the time estimates, which were in general ~5
ky more recent that those presented here. Because there are
some questions concerning this rate and its application
(e.g.,
65
), and because it has been used in just a few studies
to date, for the sake of comparison we presented here only
the age estimated with the rate of Mishmar et al.
39
This model could help explain why some of the earliest
known sites are in coastal South America whereas more
recent sites are more frequently situated inland. Associated
with the end of the ice age, sea level rose rapidly between
~18 and ~10 kya, inundating most of North America’s
Pacific coast, which was exposed during the earliest expan-
sion southward.
5
Some of the earliest sites might occur
along the much larger South American western coastal
plain because large portions of its prehistoric coastline
are still exposed.
66
The human dispersal from the coast
into the interior of the continent, perhaps driven by grow-
ing population density, depletion of coastal resources, and
rising sea levels,
4
was probably delayed by the need to cross
the mountain ranges and change living strategies and
technologies from those associated with coastal adapta-
tions. Interestingly, a similar model was proposed for the
first colonization of Asia, ~65 kya.
23
The moderate population reduction found here was also
supported by recent results from biparental loci
26,27,29
and
by some earlier results from both mtDNA
9
and nuclear
Figure 4. Phylogenetic Tree and Bayesian Skyline Plot for Na-
tive American mtDNAs
(A) Maximum-likelihood tree from 80 Native American mtDNA cod-
ing-region haplotypes. The time axis (in kya) was estimated with
a parametric molecular-clock model calibrated with the assumption
of human versus chimpanzee divergence at 6.5 million yr ago.
Branches with bootstrap support < 0.5 were collapsed.
(B) mtDNA Bayesian skyline plot showing the Native American pop-
ulation size trend with a log-normal relaxed clock with the standard
substitution rate of 1.26 3 10
8
sites/yr and a generation time of
25 yr. The y axis is the effective number of females. The thick solid
line is the median estimate and the thin lines (blue) show the 95%
highest posterior density limits estimated with 60 million chains.
Approximate dates for the LGM, Monte Verde, and Clovis sites are
shown in the middle panel. The time axis is limited to < 30 kya.
6 The American Journal of Human Genetics 82, 1–10, March 2008
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
data
25
but contradicts more extreme bottleneck hypothe-
ses such as that suggested by Hey.
24
In addition, Hey esti-
mated the time for the peopling of the Americas at only
~7 kya, about half of the age for the Monte Verde site.
Even though the estimates of Hey for the timing of the
New World colonization are broad enough to include in
the confidence interval dates as old as 15 and 30 kyr, the
point estimates are in clear contrast to our data. Such
differences may be explained by the different demographic
models assumed by these studies, by dataset composition,
and by differences in values from key parameters (e.g.,
generation time, date for human versus chimp divergence,
uncorrected distances for mutation-rate estimates).
Our results strongly support the hypothesis that
haplogroup X, together with the other four main mtDNA
haplogroups, was part of the gene pool of a single Native
American founding population; therefore they do not
support models that propose haplogroup-independent
migrations, such as the migration from Europe posed by
the Solutrean hypothesis.
18
We infer that haplogroup X
experienced a more limited expansion in intensity than
the former four haplogroups, and this is compatible with
its current very limited distribution.
14
Outside America,
haplogroup X has always been found in small frequencies.
In Europe, it usually makes up less than 5% of mtDNA
diversity.
16
In Siberia, it has been described in only a few
populations,
16,46,67
none of which currently inhabit
eastern Siberia. It is likely that this haplogroup is absent
in eastern Siberian populations because of drift effects,
which impact rare variants more strongly. Thus, its proba-
bility of being lost through random effects would be high.
In support for this hypothesis, we note that current Sibe-
rian and Native American sequences belonging to the
haplogroup X are distantly related,
67
suggesting that the
intermediate lineages have been lost. Finally, it is notewor-
thy that haplogroup X is not the only one of the Native
American haplogroups that is more frequent in the New
World than in Siberia; haplogroups A and B also show
this pattern.
46
In the Americas, a likely explanation for the obser vation
that haplogroup X has a much more restricted distribution
would be that if we assume it was relatively rare in the
founding population, then it could have been lost by
successive founder effects and genetic drift as the expan-
sion wave moved southward. Actually, it was recently
shown
68
that the probability that an allele (e.g., a found-
ing haplotype) survives and expands spatially and in fre-
quency by ‘surfing’ on the wave of a range expansion
depends on its presence in the wave of expansion, which
in turn depends largely on its proximity to the edge of
the wave. Therefore, using this framework, one could con-
ceive that haplogroup X may have ‘failed’ to expand sim-
ply as a result of its location in the expansion wave and/or
its low initial frequency. A similar explanation may be used
to account for the existence of other similarly rare hap-
logroups in the Americas, such as the ‘cayapa’ subha-
plogroup D,
69
as well as the distribution of some rare Y
chromosome haplogroups,
70
without the need to postu-
late independent colonization events. In addition, the
existence of additional, rare founding haplotypes agrees
well with the moderate bottleneck estimated here. Such
strong and old demographic expansion inferred from our
data might also indicate that this was the most important
time frame in which major changes in haplogroup compo-
sition could occur. Interestingly, two studies with ancient
DNA samples scattered over most of the Holocene sug-
gested regional continuity in the frequency of mtDNA
haplogroups,
71,72
indicating that in these populations drift
has not played a major role in more recent times.
The fact that the five most common Native American
mtDNA haplogroups display similar diversity patterns
strongly indicates that they have not been much affected
by natural selection. Because human mtDNA does not
recombine, directional selection upon a specific substitu-
tion would favor the haplotype in which this variant
occurs, mimicking a demographic expansion. It is very
unlikely that in all haplogroups specific variants that
would be favored by natural selection with similar in-
tensity would have occurred by chance and at a similar
time. Therefore, our results strongly indicate that the diver-
sity pattern in Native American mtDNA results from a
demographic expansion in the founding population in
which all founding haplotypes were present.
Our detailed demographical model for the earlier settle-
ment of the Americas has implications for explaining some
of the high level of disagreement that has been found
among studies from different disciplines, especially in
relation to an ‘exact’ date for the peopling of the New
World. Our results indicate that, strictly speaking, we will
probably never be able to pinpoint a single and precise
date for the entering of the Americas, because it occurred
when Asia and America were not divided but were
connected by the huge land mass of the subcontinent
Beringia, and because it lasted several millennia, beginning
with the isolation from the Asian ancestors and ending
with the population size and range expansion into the
continent. Under our model, three periods that may define
a date for the peopling of the Americas can be delineated:
(1) the colonization of Beringia (because about half of it
was ‘America’ at that time) by the founding population;
(2) the movement out of Beringia—characterized by the
fast colonization of the continental Pacific coastal
plain—south of the ice sheets; and (3) the more recent
and more extensive colonization of inland continental
masses. Furthermore, the probability of coalescence of
mtDNA lineages within a population and the chance of
finding ancient archeological evidence go in opposite
directions. Consider, for example, period 1 in Beringia:
The many millennia of isolation followed by reduced
population size accelerated the coalescence of mtDNA
lineages into the haplogroups founding haplotypes (conse-
quently determining their ages), but these conditions,
aggravated by the fact that most of Beringia is now under-
water, make it more difficult to find good archaeological
The American Journal of Human Genetics 82, 1–10, March 2008 7
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
evidence of this period. On the contrar y, period 3 should
present a much higher probability of finding archaeologi-
cal sites, but it was very difficult to distinguish from the
previous period by the conventional methods of histori-
cal demography. Therefore, perhaps some of the heated
debates about dissimilar ‘colonization dates’ inferred by
different disciplines may actually reflect the estimation of
distinct periods as described above. More specifically, the
generally more recent colonization dates estimated from
extensive archaeological sites from inland North America
(e.g.,
2
) may reflect the third, latest event, whereas the
usually more ancient coalescence dates from mtDNA
haplogroups
6
actually reflect the earliest isolation of the
founding population in Beringia. Finally, the intermediate
genetic dates usually estimated from population expan-
sion times as well as the fact the most ancient archeological
sites have been found in the coast may reflect the early and
fast settlement along the coast of the continent. Our
results emphasize the necessity that the increasing amount
of population genetic data should be analyzed with
methods that provide more realistic pictures for similarly
complex evolutionary histories.
While our manuscript was under review, another study
on mtDNA genomic diversity of Native American
haplogroups has been published by Tamm et al.
73
Their
paper also suggests that the major Native American
mtDNA haplogroups were part of a single demographic
event, dating back to ~15 kya, after a period of isolation
in Beringia, supporting our previous model.
11
Our study
differs from the one of Tamm et al. in several issues (e.g.,
Tamm et al. do not date the age of haplogroup X expansion
in the Americas, and they use a synonymous substitution
rate
47
that furnishes slightly younger dates), but they pro-
vide similar conclusions about the colonization process.
Acknowledgments
Grant support was from the Brazilian Conselho Nacional de
Desenvolvimento Cientı
´
fico e Tecnolo
´
gico and the Fundac¸a
˜
ode
Amparo a Pesquisa do Rio Grande do Sul (S.L.B.) and by a CAPES
scholarship (N.J.R.F.). We are also grateful to Institutos do Mile
ˆ
nio
and Programas de Apoio a Nu
´
cleos de Excele
ˆ
ncia for extra support
(F.M.S.) and to the National Institutes of Health (D.G.S.). Research
was developed with help from CENAPAD-SP supercomputer
center. Thanks to Cladinara R. Sarturi, Ronaldo R. Ferreira, Luana
Cardoso-Silva, Renata Schmitt, Andre
´
Schnorr, Gabrielle D. Salton,
Mariana Magalha
˜
es, and Marina O. Favarini for technical help,
and to Kim Hill, A. Magdalena Hurtado, Ramiro Barrantes, and
Luis Rodriguez-Delfin for sample donations, as well as to all indi-
viduals who, by contributing their own samples, made this study
possible. We thank Claudio Bravi for help with checking muta-
tions and Eduardo Eizirik and three anonymous reviewers for their
suggestions.
Received: August 15, 2007
Revised: November 13, 2007
Accepted: November 29, 2007
Published online: February 28, 2008
Web Resources
Accession numbers and URLs for data presented herein are as
follows:
GenBank, http://www.ncbi.nlm.nih.gov/Genbank/
Network, http://www.fluxus-engineering.com/sharenet.htm
BEAST, http://beast.bio.ed.ac.uk/
Accession Numbers
The 51 sequences reported in this paper have been deposited in
GenBank with the accession numbers EU095194–EU095251.
References
1. Fagan, B.M. (2004). The Great Journey: The Peopling of
Ancient America (Gainesville, FL: University Press of Florida).
2. Waters, M.R., and Stafford, T.W. Jr. (2007). Redefining the age
of Clovis: Implications for the peopling of the Americas.
Science 315, 1122–1126.
3. Dillehay, T.D. (1997). The Archaeological Context and
Interpretation, Volume 2: Monte Verde, a Late Pleistocene
Settlement in Chile (Washington, DC: Smithsonian Institute
Press).
4. Dixon, E.J. (2001). Human colonization of the Americas:
Timing, technology and process. Quaternary Science Reviews
20, 277–299.
5. Clark, P.U., and Mix, A.C. (2002). Ice sheets and sea level of the
last glacial maximum. Quaternary Science Reviews 21, 1–7.
6. Schurr, T.G. (2004). The peopling of the New World: Perspec-
tives from molecular anthropology. Annu. Rev. Anthropol. 33,
551–583.
7. Bandelt, H.J., Herrnstadt, C., Yao, Y.G., Kong, Q.P., Kivisild, T.,
Rengo, C., Scozzari, R., Richards, M., Villems, R., Macaulay, V.,
et al. (2003). Identification of Native American founder
mtDNAs through the analysis of complete mtDNA sequences:
Some caveats. Ann. Hum. Genet. 67, 512–524.
8. Merriwether, D.A., Hall, W.W., Vahlne, A., and Ferrell, R.E.
(1996). MtDNA variation indicates Mongolia may have been
the source for the founding population for the New World.
Am. J. Hum. Genet. 59, 204–212.
9. Merriwether, D.A., Rothhammer, F., and Ferrell, R.E. (1995).
Distribution of the four-founding lineage haplotypes in
Native Americans suggests a single wave of migration for the
New World. Am. J. Phys. Anthropol. 98, 411–430.
10. Forster, P., Harding, R., Torroni, A., and Bandelt, H.-J. (1996).
Origin and evolution of Native American mtdna variation: A
reappraisal. Am. J. Hum. Genet. 59, 935–945.
11. Bonatto, S.L., and Salzano, F.M. (1997). A single and early
migration for the peopling of the Americas supported by
mitochondrial DNA sequence data. Proc. Natl. Acad. Sci.
USA 94, 1866–1871.
12. Stone, A.C., and 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.
13. Starikovskaya, Y.B., Sukernik, R.I., Schurr, T.G., Kogelnik,
A.M., and Wallace, D.C. (1998). MtDNA diversity in Chukchi
and Siberian Eskimos: Implications for the genetic history of
ancient Beringia and the peopling of the New World. Am.
J. Hum. Genet. 63, 1473–1491.
8 The American Journal of Human Genetics 82, 1–10, March 2008
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
14. Smith, D.G., Malhi, R.S., Eshleman, J., Lorenz, J.G., and
Kaestle, F.A. (1999). Distribution of mtDNA haplogroup X
among Native North Americans. Am. J. Phys. Anthropol.
110, 271–284.
15. Dornelles, C.L., Bonatto, S.L., Freitas, L.B., and Salzano, F.M.
(2005). Is haplogroup X present in extant South American In-
dians? Am. J. Phys. Anthropol. 127, 439–448.
16. Reidla, M., Kivisild, T., Metspalu, E., Kaldma, K., Tambets, K.,
Tolk, H.-V., Parik, J., Loogva
¨
li, E.L., Derenko, M., Malyarchuk,
B., et al. (2003). Origin and diffusion of mtDNA haplogroup X.
Am. J. Hum. Genet. 73, 1178–1190.
17. Brown, M.D., Hosseini, S.H., Torroni, A., Bandelt, H.-J., Allen,
J.C., Schurr, T.G., Scozzari, R., Cruciani, F., and Wallace, D.C.
(1998). MtDNA haplogroup X: An ancient link between
Europe/Western Asia and North America? Am. J. Hum. Genet.
63, 1852–1861.
18. Stanford, D., and Bradley, B. (2002). Ocean trails and prairie
paths? Thoughts about Clovis origins. In The Pleistoscene
Colonization of the New World, N. Jablonski, ed. (San Fran-
cisco, CA: Memoirs of the California Academy of Sciences),
pp. 255–271.
19. Straus, L.G., Meltzer, D.J., and Goebel, T. (2005). Ice age
Atlantis? Exploring the Solutrean-Clovis ‘connection’. World
Archaeol. 37, 507–532.
20. Malhi, R.S., and Smith, D.G. (2002). Haplogroup X confirmed
in prehistoric North America. Am. J. Phys. Anthropol. 119,
84–86.
21. Bonatto, S.L., and Salzano, F.M. (1997). Diversity and age of
the four major mtDNA haplogroups, and their implications
for the peopling of the New World. Am. J. Hum. Genet. 61,
1413–1423.
22. Ingman, M., Kaessmann, H., Pa
¨
a
¨
bo, S., and Gyllensten, U.
(2000). Mitochondrial genome variation and the origin of
modern humans. Nature 408, 708–713.
23. Macaulay, V., Hill, C., Achilli, A., Rengo, C., Clarke, D.,
Meehan, W., Blackburn, J., Semino, O., Scozzari, R., Cruciani,
F., et al. (2005). Single, rapid coastal settlement of Asia
revealed by analysis of complete mitochondrial genomes.
Science 308, 1034–1036.
24. Hey, J. (2005). On the number of New World founders: A
population genetic portrait of the peopling of the Americas.
PLoS Biol. 3, e193.
25. Kidd, J.R., Black, F.L., Weiss, K.M., Balazs, I., and Kidd, K.K.
(1991). Studies of three Amerindian populations using nuclear
DNA polymorphisms. Hum. Biol. 63, 775–794.
26. Heller, A.H., Salzano, F.M., Barrantes, R., Krylov, M., Benevo-
lenskaya, L., Arnett, F.C., Munkhbat, B., Munkhtuvshin, N.,
Tsuji, K., Hutz, M.H., et al. (2004). Intra and intercontinental
molecular variability of an Alu insertion in the 3
0
UTR of the
LDLR gene. Hum. Biol. 76, 591–604.
27. Battilana, J., Cardoso-Silva, L., Barrentes, R., Hill, K., Hur-
tado, A.M., Salzano, F.M., and Bonatto, S.L. (2007). Molec-
ular variability of the 16p13.3 region in Amerindians and
its anthropological significance. Ann. Hum. Genet. 71,
64–76.
28. Battilana, J., Fagundes, N.J.R., Heller, A.H., Goldani, A., Freitas,
L.B., Tarazona-Santos, E., Munkhbat, B., Munkhtuvsin, N.,
Krylov, M., Benevolenskaya, L., et al. (2006). Alu insertion
polymorphisms in Native American and related Asian popula-
tions. Ann. Hum. Biol. 33, 142–160.
29. Fagundes, N.J.R., Ray, N., Beaumont, M., Neuenschwander, S.,
Salzano, F.M., Bonatto, S.L., and Excoffier, L.E. (2007). Statisti-
cal evaluation of alternative models of human evolution.
Proc. Natl. Acad. Sci. USA 104, 17614–17619.
30. Rieder, M.J., Tayler, S.L., Tobe, V.O., and Nickerson, D.A.
(1998). Automating the identification of DNA variations using
quality-based fluorescence re-sequensing: Analysis of the hu-
man mitochondrial genome. Nucleic Acids Res. 26, 967–973.
31. Ewing, B., Hillier, L., Wendl, M.C., and Green, P. (1998).
Base-calling of automated sequencer traces using phred. I.
Accuracy assessment. Genome Res. 8, 175–185.
32. Gordon, D., Abajian, C., and Green, P. (1998). Consed:
a graphical tool for sequence finishing. Genome Res. 8,
195–202.
33. Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H.,
Coulson, A.R., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe,
B.A., Sanger, F., et al. (1981). Sequence and organization of
the human mitochondrial genome. Nature 290, 457–465.
34. Andrews, R.M., Kubacka, I., Chinnery, P.F., Lightowlers, R.N.,
Turnbull, D.M., and Howell, N. (1999). Reanalysis and
revision of the Cambridge reference sequence for human
mitochondrial DNA. Nat. Genet. 23, 147.
35. Bandelt, H.J., Quintana-Murci, L., Salas, A., and Macaulay, V.
(2002). The fingerprint of phantom mutations in mitochon-
drial DNA data. Am. J. Hum. Genet. 71, 1150–1160.
36. Silva, W.A., Bonatto, S.L., Holanda, A.J., Ribeiro-dos-Santos,
A.K., Paixa
˜
o, B.M., Goldman, G.H., Abe-Sandes, K., Rodri-
guez-Delfin, L., Barbosa, M., Pac¸o-Larson, M.L., et al. (2002).
Mitochondrial genome diversity of Native Americans sup-
ports a single early entry of founder populations into America.
Am. J. Hum. Genet. 71, 187–192.
37. Derbeneva, O.A., Sukernik, R.I., Volodko, N.V., Hosseini, S.H.,
Lott, M.T., and Wallace, D.C. (2002). Analysis of mitochon-
drial DNA diversity in the Aleuts of the Commander Islands
and its implications for the genetic history of Beringia. Am.
J. Hum. Genet. 71, 415–421.
38. Herrnstadt, C., Elson, J.L., Fahy, E., Preston, G., Turnbull,
D.M., Anderson, C., Ghosh, S.S., Olefsky, J.M., Beal, M.F.,
Davis, R.E., et al. (2002). Reduced-median-network analysis
of complete mitochondrial DNA coding-region sequences
for the major African, Asian, and European haplogroups.
Am. J. Hum. Genet. 70, 1152–1171.
39. Mishmar, D., Ruiz-Pesini, E., Golik, P., Macaulay, V., Clark,
A.G., Hosseini, S., Brandon, M., Easley, K., Chen, E., Brown,
M.D., et al. (2003). Natural selection shaped regional mtDNA
variation in humans. Proc. Natl. Acad. Sci. USA 100, 171–
176.
40. Levin, B.C., Cheng, H., and Reeder, D.J. (1999). A human
mitochondrial DNA standard reference material for quality
control in forensic identification, medical diagnosis, and
mutation detection. Genomics 55 , 135–146.
41. Finnila
¨
, S., Lehtonen, M.S., and Majamaa, K. (2001). Phyloge-
netic network for European mtDNA. Am. J. Hum. Genet. 68,
1475–1484.
42. Maca-Meyer, N., Gonza
´
lez, A.M., Larruga, J.M., Flores, C., and
Cabrera, V.C. (2001). Major genomic mitochondrial lineages
delineate early human expansions. BMC Genet. 2, 13.
43. Ingman, M., and Gyllensten, U. (2003). Mitochondrial
genome variation and evolutionary history of Australian
and New Guinean aborigines. Genome Res. 13, 1600–1606.
44. Kong, Q.-P., Yao, Y.-G., Sun, C., Bandelt, H.-J., Zhu, C.-L., and
Zhang, Y.-P. (2003). Phylogeny of East Asian mitochondrial
DNA lineages inferred from complete sequences. Am.
J. Hum. Genet. 73, 671–676.
The American Journal of Human Genetics 82, 1–10, March 2008 9
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
45. Tanaka, M., Cabrera, V.M., Gonzalez, A.M., Larruga, J.M.,
Takeyasu, T., Fuku, N., Guo, L.J., Hirose, Y., Fujita, Y., Kurata,
K., et al. (2004). Mitochondrial genome variation in
eastern Asia and the peopling of Japan. Genome Res. 14,
1832–1850.
46. Starikovskaya, E.B., Sukernik, R.I., Derbeneva, O.A., Volodko,
N.V., Ruiz-Pesini, E., Torroni, A., Brown, M.D., Lott, M.T.,
Hosseini, S.H., Huoponen, K., et al. (2005). Mitochondrial
DNA diversity in indigenous populations of the southern
extent of Siberia, and the origins of Native American
haplogroups. Ann. Hum. Genet. 69, 67–89.
47. Kivisild, T., Shen, P., Wall, D., Do, B., Sung, R., Davis, K.,
Passarino, G., Underhill, P.A., Scharfe, C., Torroni, A., et al.
(2006). The role of selection in the evolution of human
mitochondrial genomes. Genetics 172, 373–387.
48. Excoffier, L., Laval, G., and Schneider, S. (2005). Arlequin
(version 3.0): An integrated software package for population
genetics data analysis. Evol. Bioinform. Online 1, 47–50.
49. Swofford, D.L. (2003). PAUP*. Phylogenetic Analysis Using
Parsimony (*and other methods) (Sunderland, MA: Sinauer).
50. Yang, Z. (1997). PAML: A program package for phylogenetic
analysis by maximum likelihood. Comput. Appl. Biosci. 13,
555–556.
51. Bandelt, H.J., Forster, P., and Ro
¨
hl, A. (1999). Median-joining
networks for inferring intraspecific phylogenies. Mol. Biol.
Evol. 16, 37–48.
52. Sanderson, M.J. (2003). R8s: inferring absolute rates of
molecular evolution and divergence times in the absence of
a molecular clock. Bioinformatics 19, 301–302.
53. Drummond, A.J., Rambaut, A., Shapiro, B., and Pybus, O.G.
(2005). Bayesian coalescent inference of past population
dynamics from molecular sequences. Mol. Biol. Evol. 22,
1185–1192.
54. Szathmary, E.J. (1993). mtDNA and the peopling of the
Americas. Am. J. Hum. Genet. 53, 793–799.
55. Yokoyama, Y., Lambeck, K., Deckker, P.D., Johnston, P., and
Fifield, L.K. (2000). Timing of the last glacial maximum from
observed sea-level minima. Nature 406, 713–716.
56. Schaefer, J.M., Denton, G.H., Barrell, D.J.A., Ivy-Ochs, S.,
Kubik, P.W., Andersin, B.G., Phhillips, F.M., Lowell, T.V., and
Schluchter, C. (2006). Near-synchronous interhemispheric
termination of the last glacial maximum in mid-latitudes.
Science 312, 1510–1513.
57. Pitulko, V.V., Nikolsky, P.A., Girya, E.Y., Basilyan, A.E.,
Tumskoy, V.E., Koulakov, S.A., Astakhov, S.N., Pavlova, E.Yu.,
and Anisimov, M.A. (2004). The Yana RHS site: Humans in
the Arctic before the last glacial maximum. Science 303,
52–56.
58. Vasil’ev, S.A. (2000). The Siberian mosaic: Upper Palaeolithic
adaptations and change before the Last glacial Maximum. In
Hunters of the Golden Age, W. Roebroecks, M. Mussi, J.
Svoboda, and K. Fennema, eds. (Leiden, Netherlands: Univer-
sity of Leiden), pp. 173–195.
59. Brubaker, L.B., Anderson, P.M., Edwards, M.E., and Lozhkin,
A.V. (2005). Beringia as a glacial refugium for boreal trees
and shrubs: New perspectives from mapped pollen data.
J. Biogeogr. 32, 833–848.
60. Elias, S.A. (2001). Beringian paleoecology: Results from the
1997 workshop. Quaternary Science Reviews 20, 7–13.
61. Fladmark, K.R. (1979). Routes: Alternate migration corridors
for early man in North America. Am. Antiq. 44, 55–69.
62. Dixon, E.J. (1993). Quest for the Origins of the First Americans
(Albuquerque, NM: University of New Mexico Press).
63. Kelly, R.L. (2003). Maybe we do know when people first came
to North America; and what does it mean if we do? Quaternary
Int. 109–110, 133–145.
64. Surovell, T.A. (2003). Simulating coastal migration in New
World colonization. Curr. Anthropol. 44, 580–591.
65. Bandelt, H.J., Kong, Q.P., Richards, M., and Macaulay, V.
(2006). Estimation of mutation rates and coalescence times:
Some caveats. In Human Mitochondrial DNA and the Evolu-
tion of Homo Sapiens, H.J. Bandelt, V. Macaulay, and M.
Richards, eds. (Berlin: Springer), pp. 47–90.
66. Dillehay, T.D. (1999). The late Pleistocene cultures of South
America. Evol. Anthropol. 7, 206–216.
67. Derenko, M.V., Grzybowski, T., Malyarchuk, B.A., Czarny, J.,
Miscicka-Sliwka, D., and Zakharov, I.A. (2001). The presence
of mitochondrial haplogroups X in Altaians from South
Siberia. Am. J. Hum. Genet. 69, 237–241.
68. Klopfstein, S., Currat, M., and Excoffier, L. (2006). The fate of
mutations surfing on the wave of a range expansion. Mol.
Biol. Evol. 23, 482–490.
69. Kemp, B.M., Mahli, R.S., McDonough, J., Bolnick, D.A.,
Eshleman, J.A., Rickards, O., Martinez-Labarga, C., Johnson,
J.R., Lorenz, J.G., Dixon, E.J., et al. (2007). Genetic analysis
of early Holocene skeletal remains from Alaska and its implica-
tions for the settlement of the Americas. Am. J. Phys. Anthro-
pol. 132, 605–621.
70. Tarazona-Santos, E., and Santos, F.R. (2002). The peopling of
the Americas: A second major migration? Am. J. Hum. Genet.
70, 1377–1380.
71. O’Rourke, D.H., Hayes, M.G., and Carlyle, S.W. (2000).
Spatial and temporal stability of mtDNA haplogroup
frequencies in native North Americans. Hum. Biol. 72,
15–34.
72. Kaestle, F.A., and Smith, D.G. (2001). Ancient mitochondrial
DNA evidence for prehistoric population movement: The
Numic expansion. Am. J. Phys. Anthropol. 115, 1–12.
73. Tamm, E., Kivisild, T., Reidla, M., Metspalu, M., Glenn Smith,
D., Mulligan, C.J., Bravi, C.M., Rickards, O., Martinez-Labarga,
C., Khusnutdinova, E.K., et al. (2007). Beringian standstill and
spread of Native American founders. PLoS ONE 2, e829.
10 The American Journal of Human Genetics 82, 1–10, March 2008
Please cite this article in press as: Fagundes et al., Mitochondrial Population Genomics Supports a Single Pre-Clovis Origin with a Coastal
Route for the Peopling of the Americas, The American Journal of Human Genetics (2008), doi:10.101 6/j.ajhg.2007.11.013
    • "To access the demographic changes through time in SEA populations associated with the haplogroups studied , we obtained BSPs (Drummond et al. 2005; Fagundes et al. 2008) using BEAST (version 1.7.5) with a relaxed molecular clock (lognormal in distribution across branches and uncorrelated between them) using a mutation rate of 2.6186 × 10 −8 mutations per site per year for the wholemtDNA genome (Soares et al. 2012) and the HKY model of nucleotide substitutions with gamma-distributed rates, assuming a generation time of 25 years. In addition, we forced the larger subclades into monophyly to obtain a tree structure that was directly comparable with the remaining analyses (Fagundes et al. 2008). We visualised the plots with Tracer v1.3, and inferred the increment ratio by calculating the number of times that the effective population size increased during specific periods. "
    Full-text · Article · Apr 2016 · American Journal of Human Biology
    • "To access the demographic changes through time in SEA populations associated with the haplogroups studied , we obtained BSPs (Drummond et al. 2005; Fagundes et al. 2008) using BEAST (version 1.7.5) with a relaxed molecular clock (lognormal in distribution across branches and uncorrelated between them) using a mutation rate of 2.6186 × 10 −8 mutations per site per year for the wholemtDNA genome (Soares et al. 2012) and the HKY model of nucleotide substitutions with gamma-distributed rates, assuming a generation time of 25 years. In addition, we forced the larger subclades into monophyly to obtain a tree structure that was directly comparable with the remaining analyses (Fagundes et al. 2008). We visualised the plots with Tracer v1.3, and inferred the increment ratio by calculating the number of times that the effective population size increased during specific periods. "
    [Show abstract] [Hide abstract] ABSTRACT: There has been a long-standing debate concerning the extent to which the spread of Neolithic ceramics and Malay-Polynesian languages in Island Southeast Asia (ISEA) were coupled to an agriculturally-driven demic dispersal out of Taiwan 4000 years ago (4 ka). We previously addressed this question by using founder analysis of mitochondrial DNA (mtDNA) control-region sequences to identify major lineage clusters most likely to have dispersed from Taiwan into ISEA, proposing that the dispersal had a relatively minor impact on the extant genetic structure of ISEA, and that the role of agriculture in the expansion of the Austronesian languages was therefore likely to have been relatively minor. Here we test these conclusions by sequencing whole mtDNAs from across Taiwan and ISEA, using their higher chronological precision to resolve the overall proportion that participated in the “out-of-Taiwan” mid-Holocene dispersal as opposed to earlier, postglacial expansions in the Early Holocene. We show that, in total, about 20% of mtDNA lineages in the modern ISEA pool result from the “out-of-Taiwan” dispersal, with most of the remainder signifying earlier processes, mainly due to sea-level rises after the Last Glacial Maximum. Notably, we show that every one of these founder clusters previously entered Taiwan from China, 6-7 ka, where rice-farming originated, and remained distinct from the indigenous Taiwanese population after the subsequent dispersal into ISEA.
    Full-text · Article · Feb 2016
    • "For the time being, we cannot assess unambiguously the number of founder events, but our findings confirm the importance of microevolutionary processes, especially in Baja California and in Tierra del Fuego, as well as a combination of random and non-random processes (cold adaptation, recurrent gene flow, more recent arrival) in the Circum- Arctic area. Different interpretations also exist for genetic data, yielding support for a single origin of the first Americans (Chatters et al., 2014; Fagundes et al., 2008; Goebel et al., 2008) or dual ancestry (Raghavan et al., 2013) or recurrent gene flow (Ray et al., 2010). This underlines the need to incorporate both genetic and craniometric data from a maximum of populations in order to assess more precisely the effects of gene flow, phenotypic adaptation and the number of dispersal events. "
    [Show abstract] [Hide abstract] ABSTRACT: Objectives: Craniofacial variation in past and present Amerindians has been attributed to the effect of multiple founder events, or to one major migration followed by in situ differentiation and possibly recurrent contacts among Circum-Arctic groups. Our study aims to: (i) detect morphological differences that may indicate several migrations; (ii) test for the presence of genetic isolation; and (iii) test the correlation between shape data and competing settlement hypotheses by taking into account geography, chronology, climate effects, the presence of genetic isolation and recurrent gene flow. Methods: We analyzed a large sample of three-dimensional (3D) cranial surface scans (803 specimens) including past and modern groups from America and Australasia. Shape variation was investigated using geometric morphometrics. Differential external gene flow was evaluated by applying genetic concepts to morphometric data (Relethford-Blangero approach). Settlement hypotheses were tested using a matrix correlation approach (Mantel tests). Results: Our results highlight the strong dichotomy between Circum-Arctic and continental Amerindians as well as the impact of climate adaptation, and possibly recurrent gene flow in the Circum-Arctic area. There is also evidence for the impact of genetic isolation on phenetic variation in Baja California. Several settlement hypotheses are correlated with our data. Conclusions: The three approaches used in this study highlight the importance of local processes especially in Baja California, and caution against the use of overly simplistic models when searching for the number of migration events. The results stress the complexity of the settlement of the Americas as well as the mosaic nature of the processes involved in this process. Am. J. Hum. Biol., 2016. © 2016 Wiley Periodicals, Inc.
    Article · Feb 2016
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