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Brief communication Mitochondrial haplotype C4c confirmed as a founding genome in the Americas.

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Brief Communication: Mitochondrial Haplotype C4c
Confirmed as a Founding Genome in the Americas
Ripan S. Malhi,1* Jerome S. Cybulski,2 Raul Y. Tito,3 Jesse Johnson,4 Harold Harry,5 and Carrie Dan6
Department of Anthropology, Animal Biology and Institute for Genomic Biology, University of Illinois
Urbana-Champaign, IL
Canadian Museum of Civilization, Gatineau, Quebec, Canada
Department of Anthropology, University of Oklahoma, Norman
Department of Animal Biology, University of Illinois Urbana-Champaign, IL
Stswécemc/Xgat’temc Indian Band, British Columbia, Canada
Tk’emlups Indian Band, British Columbia, Canada
Native American; peopling of the Americas; mtDNA
Mitochondrial DNA analysis of 31 unrelated Shuswap speakers from a previously poorly sampled
region of North America revealed two individuals with
haplogroups rarely found in the Americas, C4c and C1d.
Comparison of the complete genomes of the two individuals with others found in the literature confirms that C4c
is a founding haplotype and gives insight into the evolution of the C1d haplotype. This study demonstrates the
importance of collecting and analyzing data from Native
North Americans when addressing hypotheses about the
peopling of the Americas. Am J Phys Anthropol 141:494–
497, 2010. V 2009 Wiley-Liss, Inc.
The evolutionary force of genetic drift has been used
to explain the patterns of genetic diversity among Native
American populations (Cavalli-Sforza et al., 1994). The
action of genetic drift can cause some haplotypes in low
frequency to become much more frequent in one or a few
populations based on effective population size. For example, the high frequency of mitochondrial DNA (mtDNA)
haplotype D4h3 in the Cayapa Indians of Ecuador (22%)
is likely a result of genetic drift (Rickards et al., 1999;
Kemp et al., 2007). The paucity of sampling from certain
geographic regions in the Americas combined with the
action of genetic drift on Native Americans suggests that
additional undocumented genetic diversity may exist in
living Native Americans. This undocumented genetic diversity may provide greater insight into the early population history of Native Americans.
Malhi and Cybulski have identified geographic regions
that are poorly sampled in North America (NSF BCS
No. 0745459). To fill this sampling gap, they began collaborating with the Shuswap-speaking community of
Stswécemc to analyze three ancient individuals (5,000
years before present) from the archaeological sites of
China Lake and Big Bar Lake in British Columbia,
Canada. A single individual from Big Bar Lake was
identified as haplogroup A, whereas two individuals
from China Lake, buried together, exhibited the substitution at nucleotide position (np) 10,400, characteristic
of haplogroup M, but these two individuals did not
exhibit the control region or coding region substitutions
for haplogroups C or D (Cybulski et al., 2007; Malhi
et al., 2007).
We subsequently analyzed the mitochondrial genomes
of 31 unrelated (at the grandparent-level) Shuswapspeakers from British Columbia to potentially identify
haplogroup M in living individuals that are similar to
the mtDNAs identified in the ancient China Lake individuals. Our results indicate that all individuals belong
to one of the five founding haplogroups (A, B, C, D, and
X). However, two individuals belong to haplogroups
infrequently observed in the Americas, C1d and C4c.
The whole genome analysis of these individuals confirms
C4c as a founding haplotype and provides insight into
the evolution of the C4c and C1d-founding haplotypes.
The information gained from the analysis of the Shuswap population underscores the importance of analyzing
populations from North America to learn about the early
population history of Native Americans.
C 2009
Cheek swabs and/or saliva samples and genealogical
information were collected from volunteers among individuals residing in Shuswap-speaking communities (see
Fig. 1). All samples were collected in accordance with
IRB protocol No. 07409 from the University of Illinois
Urbana-Champaign. DNA from the biological samples
was extracted using the method described in Miller et al.
(1998). The DNA samples were amplified using the
Qiagen Repli-g Whole Genome Amplification kit. Primers
were designed for regions of the mitochondrial genome
and used to identify subhaplogroups (Tamm et al., 2007;
Achilli et al., 2008). The hypervariable region I (HVRI)
for all individuals was sequenced, and the complete
Additional Supporting Information may be found in the online
version of this article.
*Correspondence to: Ripan S. Malhi, Assistant Professor, Department of Anthropology, University of Illinois Urbana-Champaign,
209F Davenport Hall, 607 Matthews Avenue, Urbana, IL 61801.
Received 26 June 2009; accepted 23 October 2009
DOI 10.1002/ajpa.21238
Published online 21 December 2009 in Wiley InterScience
Fig. 1. Approximate extent of Shuswap territory (dotted line; after Ignace (1998)] in northwestern North America.
mitochondrial genome was sequenced for individuals
that belong to C1d and C4c. Samples were amplified
using primer sets to generate 11 overlapping amplicons.
These overlapping amplicons were then sequenced using
33–35 primer pair sets (all primers are available upon
request from the corresponding author). All diagnostic
and unique mutations were confirmed with at least two
sequences. DNA sequences for HVRI for all individuals
and complete mitochondrial genome sequences for the
individuals belonging to C4c and C1d are available in
Nucleotide diversity was estimated for the complete
genomes, using the coding region (np 577–16,023) and
were also calculated for the HVRI (np 16,024–16,364) of
all the Shuswap population samples analyzed. The nucleotide diversity of the HVRI region of the Shuswap
was then compared to other nucleotide diversity estimates for the same genomic region from North American
populations reported in Hunley and Long (2005). Nucleotide diversity and standard error estimates were generated in MEGA 4.0 (Tamura et al., 2007). Estimates of
phylogenetic dispersion (q) and standard error (r) were
calculated for complete mitochondrial sequences using
np 577–16,023 as in Perego et al. (2009). Coalescence
times were calculated for C4c and C1d using the
sequence from Ijka 72 (Tamm et al., 2007) and sequences
compiled in Perego et al. (2009), respectively, using a
rate of 4,610 years per substitution and a rate of 7,650
years per synonymous transition (Perego et al., 2009).
It has become routine in the human mitochondrial
genome diversity literature to use the rho statistic (q) to
provide a chronological date for the time to most recent
common ancestor of mtDNA sequences in a clade (Cox,
2008). We use rho, in addition to nucleotide diversity, as
a tool in comparison with estimates made in previous
studies (Tamm et al., 2007; Perego et al., 2009). However, it should be noted that the rho statistic has
recently been shown to have a slight downward bias,
type I error rates, and a large asymmetric variance (Cox,
Of the 31 samples analyzed, 11 belonged to haplogroup
A2 (including one belonging to A2a), one to haplogroup
B2, one to haplogroup C4c, eight to haplogroup C1b, one
to haplogroup C1d, eight to haplogroup D1, and one to
haplogroup X2a.
The nucleotide diversity estimate for the HVRI of the
Shuswap is 0.0156 and comparable with populations
from the Northwest Coast of North America. The nucleotide diversity of the Shuswap is significantly higher than
in Arctic and Subarctic populations such as the Inuit
and Alaskan Athabascan (Table S1). Nucleotide diversity
and phylogenetic dispersion estimates for the C4c clade
are 0.000518 and 4.000, respectively (Table 1). The coalescence time estimate of the C4c clade is 18,440 years,
but these estimates contain a substantial degree of
uncertainty due to the small sample size for this clade
(N 5 2; Ijka and Shuswap individuals). Comparing
the two individuals that belong to haplogroup C4c
American Journal of Physical Anthropology
TABLE 1. Nucleotide diversity and phylogenetic dispersion for mitochondrial clades
All substitutions
Synonymous transitions
T (ky)
T (ky)
Old C1d
New C1d
18.4 6 6.5
9.7 6 2.5
15.2 6 4.8
11.5 6 6.6
7.6 6 2.5
9.6 6 2.8
Tamm et al., 2007; this study
Perego et al., 2009
Perego et al., 2009; this study
A rate of 4,610 years per substitution and 7,650 years per synonymous transition were used (Perego et al., 2009).
Fig. 2. Phylogeny of complete mtDNA sequences belonging to C1d and C4c. Mutations are transitions unless specified. Transversions are indicated by an A, G, C, or T after the nucleotide position. Insertions are indicated by an ‘‘i,’’ deletions are indicated by
a ‘‘d,’’ recurrent mutations are underlined, and mutations back to the rCRS nucleotide are designated by a ‘‘@.’’ The C stretch
length polymorphism in regions 303–315 was disregarded in the tree. Samples ‘‘SHU 01’’ and ‘‘SHU 03’’ were analyzed in this study.
The sample ‘‘IJKA 72’’ was analyzed in Tamm et al. (2007). All other samples were compiled in Perego et al. (2009). Two sequences
matched sample ‘‘129.’’ The control regions for samples ‘‘345’’ and ‘‘AM03’’ were not sequenced and, therefore, the presence of np
16,051 in these samples is assumed in the figure.
demonstrates that substitutions at np 14,433 and 15,148
define the C4c clade (see Fig. 2).
The nucleotide diversity and phylogenetic dispersion
estimates for the C1d clade increased substantially with
the inclusion of the Shuswap individual belonging to
C1d (Table 1). The Shuswap individual lacks the substitution at np 7,697, which along with np 16,051, has been
used to define the C1d clade (Achilli et al., 2008).
The observation that all Shuswap individuals analyzed
in this study belong to one of the five known founding
haplogroups suggests that the haplotype M, observed at
China Lake 5,000 years ago, within Shuswap territory,
is either extinct or in very low frequency in the Americas. However, the Shuswap exhibit a high-genetic diversity compared to other populations analyzed in northern
American Journal of Physical Anthropology
regions of North America. This is in agreement with a
study of genome-wide autosomal microsatellite variation
among Native Americans (Wang et al., 2007), in which a
trend of decreasing heterozygosity from north to south
was observed in the Americas. Wang et al. (2007) attributed the observed pattern in heterozygosity to a ‘‘serial
founder effect.’’ However, it is possible that the observed
pattern of decreasing heterozygosity in the Americas is
also shaped by gene flow of Native North Americans
with Northeast Asians as inferred by Tamm et al.
The majority of complete mitochondrial genomes analyzed in the Americas to date are from South America
(Tamm et al., 2007; Achilli et al., 2008; Fagundes et al.,
2008; Perego et al., 2009). The few complete mitochondrial genomes that have been analyzed from North
American individuals are mainly from those that belong
to haplogroup X2a or where the geographic, cultural,
and linguistic origin associated with a Native American
population is unknown for many of the samples (Herrnstadt et al., 2002; Perego et al., 2009). We demonstrate
how the analysis of complete mitochondrial genomes
from previously unanalyzed populations in North America can provide important insight into the evolution of
founding haplotypes. We sequenced the complete mitochondrial genome of a Shuswap individual that belongs
to haplogroup C4c. Before this analysis, only one complete mitochondrial genome belonging to C4c was
sequenced in the Ijka of Columbia (Tamm et al., 2007).
The presence of this haplotype in only one South American tribe may have been a result of undocumented historical migration from Asia. However, the possibility of
undocumented historical migration is much less likely
now that mtDNA haplotype C4c has been identified in
the Shuswap of North America. The estimated coalescence date for the two mitochondrial genomes that
belong to haplogroup C4c is 18,440 6 6,520 years before
present (ybp). This date suggests an early split of the
Shuswap and Ijka mitochondrial genomes and the early
date combined with the large geographic distance
between the Shuswap and Ijka confirms haplotype C4c
as a founding haplotype in the Americas.
We defined ‘‘Shuswap 03’’ as belonging to haplogroup
C1d based on substitutions that define haplogroup C1
and the substitution at np 16,051 in the mitochondrial
genome. The lack of np 7,697 in this individual is either
the result of back mutation or the mitochondrial genome
of ‘‘Shuswap 03’’ evolved before the substitution at np
7,697 in the C1d clade. Another possibility is that this
mitochondrial genome belongs to an independent C1*
haplotype, and the substitution at np 16,051 is a result
of homoplasy. Although np 16,051 is found in HVRI, it
does not exhibit a high frequency of recurrent mutation
(Stoneking, 2000). Therefore, the mitochondrial genome
of ‘‘Shuswap 03’’ likely evolved before the substitution at
np 7,697 that defines all other members of the C1d
clade. Including the ‘‘Shuswap 03’’ in the C1d clade
increases the coalescence date for the C1d clade from
9,700 ybp to 15,210 ybp and brings the coalescence date
for C1d closer to what is observed in the clades of other
founding haplogroups (Perego et al., 2009). Additional
whole mitochondrial genome sequencing from samples in
the northern regions of North America will likely provide important information into the early population history of Native Americans and the evolution of the founding haplotypes of the Americas.
We are grateful to the participants of this study, the
associate editor, and Toomas Kivisild for helpful comments and suggestions.
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