Updated Three-Stage Model for the Peopling of the Americas

Updated Three-Stage Model for the Peopling of the Americas

Connie J. Mulligan1*, Andrew Kitchen1, Michael M. Miyamoto2

1 Department of Anthropology, University of Florida, Gainesville,
Florida, United States of America, 2 Department of Zoology, University
of Florida, Gainesville, Florida, United States of America

Background

We re-assess support for our three stage model for the peopling of the
Americas in light of a recent report that identified nine non-Native
American mitochondrial genome sequences that should not have been
included in our initial analysis. Removal of these sequences results
in the elimination of an early (i.e. ~40,000 years ago) expansion
signal we had proposed for the proto-Amerind population.
Methodology/Findings

Bayesian skyline plot analysis of a new dataset of Native American
mitochondrial coding genomes confirms the absence of an early
expansion signal for the proto-Amerind population and allows us to
reduce the variation around our estimate of the New World founder
population size. In addition, genetic variants that define New World
founder haplogroups are used to estimate the amount of time required
between divergence of proto-Amerinds from the Asian gene pool and
expansion into the New World.
Conclusions/Significance

The period of population isolation required for the generation of New
World mitochondrial founder haplogroup-defining genetic variants makes
the existence of three stages of colonization a logical conclusion.
Thus, our three stage model remains an important and useful working
hypothesis for researchers interested in the peopling of the Americas
and the processes of colonization.

Citation: Mulligan CJ, Kitchen A, Miyamoto MM (2008) Updated Three-
Stage Model for the Peopling of the Americas. PLoS ONE 3(9): e3199.
doi:10.1371/journal.pone.0003199

Editor: Henry Harpending, University of Utah, United States of America

Received: August 4, 2008; Accepted: August 11, 2008; Published:
September 17, 2008

Copyright: © 2008 Mulligan et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source
are credited.

Funding: This study was supported by a grant from the National Science
Foundation to CJM (BSR-0518530) and by funds from the Department of
Zoology, University of Florida to MMM. The funders had no role in
study design, data collection and analysis, decision to publish, or
preparation of the manuscript.

Competing interests: The authors have declared that no competing
interests exist.

* E-mail: mulligan@xxxxxxxxxxxxxx
Introduction

We recently published a three stage model for the peopling of the
Americas [1]. Specifically, we proposed that a recent, rapid expansion
into the Americas was preceded by a long period of population
stability in greater Beringia by the proto-Amerind population after
divergence from their ancestral Asian population. We used two
complementary coalescent methods, Bayesian skyline plot [2] and
isolation-by-migration [3] analyses, to estimate past population
growth patterns in Native American populations and to estimate a New
World founder effective population size. We explicitly incorporated
archaeological, geological, and paleoecological constraints into our
analyses to enhance the anthropological relevance of the results and
to provide a comprehensive model for the initial settlement of the
Americas.

Fagundes et al. [4] have published a re-analysis of the data we used
in developing our three stage model for the peopling of the Americas
[1]. Specifically, they identified nine mitochondrial coding region
sequences that we assumed were Native American sequences, but instead
are likely to derive from Asian or European individuals. Fagundes et
al. are correct in this assessment, i.e. five sequences were
reclassified as Asian after their publication as Native American
sequences [5] and four sequences were mistakenly included in our
original study. The effect of removing these sequences from the
Bayesian skyline plot analysis is that the suggestion of an early
expansion event in the skyline plot is no longer apparent, a finding
that we have reconfirmed by re-running our original dataset without
these nine genomes. It appears that the non-Native American sequences
introduced additional variation that created an expansion signal that
does not exist in an analysis of only Native American sequences.

In light of these facts, we have now analyzed the largest dataset of
Native American mitochondrial coding genomes using publicly available
sequences (n = 148; [6]) in a Bayesian skyline plot analysis. We also
provide an estimate for the duration of the period of population
isolation required for the generation of New World founder haplogroup-
defining variants. As in our previous analysis, we evaluate the
significance of our results in concert with other non-genetic data.
Results

We use a Bayesian skyline plot to visually illustrate changes in
Native American female effective population size (Ne) over time.
Bayesian skyline plots assume a single migration event, which makes
the approach ideal for questions concerning the peopling of the
Americas since it is generally agreed that there was a single
migration [7]. Our new skyline plot (Fig. 1) strongly supports a large
population expansion (~1.8 orders of magnitude, or 80-fold) that
occurred ~16–12 thousand years ago (kya). This timing suggests an
entry to the New World that was coincident with the retreat of the
North American ice sheets, i.e. the opening of an ‘ice-free corridor’
~17–14 kya [8], [9]. Immediately before this expansion, there is a
small drop in effective population size ~17–16 kya (this is an
insignificant change, as judged by the overlap in 95% confidence
intervals at the beginning and end of the population decrease),
possibly corresponding to a population bottleneck prior to entry to
the Americas. Before 17 kya, the skyline plot is flat with no evidence
of the early (~40 kya) population expansion we reported previously
[1]. The absence of an early expansion signal in the skyline plot may
simply indicate that divergence of proto-Amerinds from the Asian gene
pool was not accompanied by significant population growth. These
results are highly consistent with our earlier analysis of only 20
Native American mitochondrial coding genomes [10], in support of
theoretical expectations by Felsenstein [11] that increasing sample
size is an inefficient way to improve the accuracy of maximum
likelihood estimations from coalescent analyses of population genetic
data.
thumbnail

Figure 1. Bayesian skyline plot of 148 Native American mitochondrial
coding genome sequences.

The curve plots median Nef with 95% credible intervals indicated by
light gray lines. The shaded gray box highlights the significant
increase of Nef during the colonization of the Americas 16–12 kya. The
blue box depicts the calculated time required for the generation of
New World defining mitochondrial variants and its shaded region
represents the variation in these estimates, i.e. 7–15 thousand years
before entry to the New World (see Table 1). The green arrow
identifies the date of the Yana River site of human occupation in
western Beringia [21].
doi:10.1371/journal.pone.0003199.g001

Our new analysis (with non-Native American sequences eliminated and
more Native American sequences added) shows a larger population
increase (80-fold vs 16-fold) over a smaller period of time (16–12 kya
vs 16–9 kya) relative to our previous analysis that inadvertently
included non-Native American sequences [1]. The non-Native American
sequences likely introduced additional variation that artificially
increased Ne prior to the expansion. Thus, we can estimate a new Ne
for the New World founding population of 1,800 (this number is
multiplied by two since the skyline plot only estimates the female
effective population size). This number is closer to our previous
isolation-with-migration (IM)-based estimate of 1,200 [1] and thus
reduces the variation around our estimate of the size of the founding
population to ~1,000–2,000 effective individuals.

Prior to entry to the New World, we propose a period of isolation. A
valid question remains - How long was the period of isolation? In the
absence of a biphasic skyline plot, we can calculate first
approximations of the time necessary to generate the defining variants
for the New World mithochondrial founding haplogroups. All New World
mitochondrial sequences cluster in five monophyletic clades,
representing founding haplogroups that are differentiated from non-New
World haplogroups by the presence of specific, defining genetic
variants. The variants that occur on the branch leading to each New
World founding haplogroup represent variation that evolved prior to
expansion into the Americas whereas variation within each founding
haplogroup, i.e. nucleotide diversity within a haplogroup, represents
variation that evolved after entry to the Americas – we are interested
in the variation that occurred prior to entry into the Americas. There
is strong consensus on the number of New World founding haplogroup-
defining variants, including both coding and non-coding hypervariable
regions I and II (HVRI+II) variants [5], [12]. However, there is a
wide range of substitution rates that have been estimated for both
coding and non-coding variants [13]–[17]. Fagundes et al. [4], [18]
tend to favor the slower substitution rates whereas we generally favor
the faster substitution rates, particularly for coding variants since
a faster rate (~1.7×10−8 substitutions/site/year) has been confirmed
using two independent approaches [13], [16]. However, to be complete
since there is ongoing debate about the correct calculation of
substitution rates most recently [19], [20], we present a series of
estimates based on coding and HVRI+II variants using both fast and
slow substitution rates (Table 1). As is evident from the
calculations, there is a wide range of estimates for the time
necessary to generate the New World defining variants, i.e. averages
range from ~6,000 to ~25,000 years. By averaging across coding and non-
coding variants and including fast and slow substitution rates, we
report a range of ~7–15 thousand years. This estimate suggests that
Amerind ancestors may have experienced a period of isolation lasting
at least 7–15 thousand years prior to their expansion into the
Americas (see the blue box in Fig. 1).
thumbnail

Table 1. Estimates of time necessary to generate the mitochondrial
genome variants that define New World founding haplogroups.
doi:10.1371/journal.pone.0003199.t001
Discussion

Our proposal for a three stage model for the peopling of the Americas
remains essentially unchanged despite the modifications to the skyline
plot described above. The three stages remain; 1) divergence of
Amerind ancestors from the Asian gene pool, 2) prolonged period of
isolation, lasting at least 7–15 thousand years, during which time
genetic variants specific to and present throughout the New World were
generated, and 3) rapid expansion into the Americas ~16 kya
concomitant with a large population increase. The existence of
mitochondrial New World-defining variants that are widespread
throughout the Americas has been noted in numerous publications most
recently [6], [12] and indicates that there must have been a period of
isolation during which time these variants arose. The idea of a period
of population isolation prior to expansion into the Americas was first
mentioned by Bonatto and Salzano [14] and most recently supported by
Tamm et al. [12]. Thus, divergence from the Asian gene pool and entry
into the Americas were separated by this period of isolation, making
the existence of three stages a logical conclusion.

In our previous study, we suggested that the period of isolation
occurred during occupation of greater Beringia [1]. The fact that
Beringia is now inundated may explain why no archaeological evidence
of human occupation has been found, although greater Beringia
encompasses such a vast territory that more terrestrial archaeological
sites may yet be discovered. The documentation of human occupation at
the Yana River site ~30 kya [21] provides independent support for the
presence of humans in greater Beringia as early as 30,000 years ago
[22] and strengthens our proposal of a Beringian occupation from ~30–
16 kya. Furthermore, multiple fossil sites document the presence of
large mammals in Alaska and Siberia [23]–[25]. Fossil pollen and plant
microfossils from eastern Beringia indicate a productive, dry
grassland ecosystem [26] suggesting the entire range of Beringia was
capable of supporting a large mammal fauna. Archaeological evidence
and ethnographic analogy both suggest that Amerind ancestors in
Beringia were skilled hunters who relied upon megafauna for sustenance
and likely extended their hunting ranges in response to demographic
changes in the large mammal population [27]. Thus, it is highly
probable that humans inhabited the central part of greater Beringia,
i.e. Beringia, for an extended period of time. In fact, the first
published Bayesian skyline plot focused on the Beringian steppe bison
(using 169 ancient DNA sequences and 22 modern sequences) and revealed
a sharp population decline beginning ~30 kya [2] leading us to suggest
that Beringian populations of humans may have been associated with the
decline in steppe bison.

In conclusion, our three stage model remains an important and useful
working hypothesis for researchers interested in the peopling of the
Americas and the processes of colonization. We believe that divergence
from the ancestral gene pool and expansion into a new territory were
not simultaneous events, as is often assumed in models of population
demographic history. Specifically, movement from Asia to the New World
was interrupted by an extended period of population isolation and
stability. Entry into the New World was mediated by a population of
1,000–2,000 effective individuals. The relevance of our model is due
to its reliance on a synthetic approach that combines genetic data
with multiple sources of anthropological and paleoenvironmental
information. As a working hypothesis, our model is predictive. In
particular, it predicts that key archaeological sites await discovery
under the Bering Sea.
Materials and Methods

A dataset of 148 human mitochondrial coding genomes was assembled from
the publicly available sequences used by Achili et al. [6] and then
aligned as described in Kitchen et al. [1]. Bayesian skyline plots [2]
of the aligned coding genomes were used to estimate changes in Amerind
Nef over time by providing highly parametric, piecewise estimates of
Nef. In these analyses, estimates of τ (Nef×generation time) were
converted to Nef by dividing by a generation time of 20 years,
following convention [3]. Using a generation time of 25 years
decreases Nef estimates by 20%, but does not affect the time
estimates. Skyline plots were generated using the program BEAST v1.4
(http://beast.bio.ed.ac.uk). These BEAST analyses relied on the same
coalescent and substitution models and run conditions as Kitchen et
al. [10]. Markov chains were run for 100,000,000 generations and
sampled every 2,500 generations with the first 10,000,000 generations
discarded as burn-in.

Author Contributions
Conceived and designed the experiments: CJM AK MM. Performed the
experiments: CJM AK. Analyzed the data: CJM AK MM. Wrote the paper:
CJM AK MM.
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