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The Genetic Legacy of the Pre-Colonial Period in
Contemporary Bolivians
Patricia Taboada-Echalar
1.
, Vanesa A
´lvarez-Iglesias
1.
, Tanja Heinz
1.
, Laura Vidal-Bralo
1
,
Alberto Go
´mez-Carballa
1
, Laura Catelli
3
, Jacobo Pardo-Seco
1
, Ana Pastoriza
1
,A
´ngel Carracedo
1
,
Antonio Torres-Balanza
2
, Omar Rocabado
2
, Carlos Vullo
3,4
, Antonio Salas
1
*
.
1Unidade de Xene
´tica, Instituto de Ciencias Forenses and Departamento de Anatomı
´a Patolo
´xica e Ciencias Forenses, Facultade de Medicina, Universidade de Santiago
de Compostela, Galicia, Spain, 2Instituto de Investigaciones Forenses, Fiscalı
´a General del Estado Plurinacional de Bolivia, La Paz, Bolivia, 3Equipo Argentino de
Antropologı
´a Forense, Co
´rdoba, Argentina, 4Laboratorio de Inmunogene
´tica y Diagno
´stico Molecular, Co
´rdoba, Argentina
Abstract
Only a few genetic studies have been carried out to date in Bolivia. However, some of the most important (pre)historical
enclaves of South America were located in these territories. Thus, the (sub)-Andean region of Bolivia was part of the Inca
Empire, the largest state in Pre-Columbian America. We have genotyped the first hypervariable region (HVS-I) of 720
samples representing the main regions in Bolivia, and these data have been analyzed in the context of other pan-American
samples (.19,000 HVS-I mtDNAs). Entire mtDNA genome sequencing was also undertaken on selected Native American
lineages. Additionally, a panel of 46 Ancestry Informative Markers (AIMs) was genotyped in a sub-set of samples. The vast
majority of the Bolivian mtDNAs (98.4%) were found to belong to the main Native American haplogroups (A: 14.3%, B:
52.6%, C: 21.9%, D: 9.6%), with little indication of sub-Saharan and/or European lineages; however, marked patterns of
haplogroup frequencies between main regions exist (e.g. haplogroup B: Andean [71%], Sub-Andean [61%], Llanos [32%]).
Analysis of entire genomes unraveled the phylogenetic characteristics of three Native haplogroups: the pan-American
haplogroup B2b (originated ,21.4 thousand years ago [kya]), A2ah (,5.2 kya), and B2o (,2.6 kya). The data suggest that
B2b could have arisen in North California (an origin even in the north most region of the American continent cannot be
disregarded), moved southward following the Pacific coastline and crossed Meso-America. Then, it most likely spread into
South America following two routes: the Pacific path towards Peru and Bolivia (arriving here at about ,15.2 kya), and the
Amazonian route of Venezuela and Brazil southwards. In contrast to the mtDNA, Ancestry Informative Markers (AIMs) reveal
a higher (although geographically variable) European introgression in Bolivians (25%). Bolivia shows a decreasing autosomal
molecular diversity pattern along the longitudinal axis, from the Altiplano to the lowlands. Both autosomes and mtDNA
revealed a low impact (1–2%) of a sub-Saharan component in Bolivians.
Citation: Taboada-Echalar P, A
´lvarez-Iglesias V, Heinz T, Vidal-Bralo L, Go
´mez-Carballa A, et al. (2013) The Genetic Legacy of the Pre-Colonial Period in
Contemporary Bolivians. PLoS ONE 8(3): e58980. doi:10.1371/journal.pone.0058980
Editor: Carles Lalueza-Fox, Institut de Biologia Evolutiva - Universitat Pompeu Fabra, Spain
Received December 23, 2012; Accepted February 12, 2013; Published March 20, 2013
Copyright: ß2013 Taboada-Echalar 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: The research leading to these results has received funding from the People Program (Marie Curie Actions) of the European Union’s Seventh Framework
Program FP7/2007-2013/ under REA grant agreement nu290344, and the grants from the ‘‘Ministerio de Ciencia e Innovacio
´n’’ (SAF2008-02971) and from the Plan
Galego IDT, Xunta de Galicia (EM 2012/045) given to A.S. 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: antonio.salas@usc.es
.These authors contributed equally to this work.
Introduction
The Republic of Bolivia is located in central-south America. It is
bordered by Peru to the West, Chile to the southwest, Paraguay
and Argentina to the South, and Brazil to the North and East.
Before the arrival of the Europeans, the Andean region of the
country was an important part of the Inca Empire, the largest state
in Pre-Columbian America, although the Inca civilization arose
from the highlands of Peru in the early 13
th
century. The
Spaniards discovered the silver mines of Potosı
´in 1544 and soon
began enslaving Natives as workers in the mines. The Spanish
Empire conquered the region in the XVI century, and, during the
colonial period, this territory was called Upper Peru. In the XVII
century, the Spanish began bringing in African slaves in high
numbers to help work in the mines, an institution that would last
until abolition in 1826 (the independence of the country would
arrive in 1825).
Nowadays, Bolivia is politically divided into nine departments
and its geography varies from the high mountains in the Andes
(West) to the eastern lowlands (Llanos), situated within the
Amazon Basin.
About 10.2 million people live in Bolivia (Instituto Nacional de
Estadı
´stica of Bolivia; INE; http://www.ine.gob.bo/). The country
harbors a great cultural diversity. The number of individual
languages known in Bolivia is 45; of those, 37 are living languages,
one is a second language without mother-tongue speakers, and
seven have no known speakers [1,2]. The main language spoken
today in Bolivia is Spanish, but there are also other important pre-
Hispanic languages, such as Quechua (inherited from the Incan
Empire; spoken by .1.6 million inhabitants), and Aymara (1.3
PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e58980
million inhabitants). The Quechua-speaking peoples inhabit
mostly the (Sub)Andean valleys of Cochabamba and Chuquisaca
and some mountain regions in Potosı
´and Oruro, while Aymara is
mainly spoken in the high plateau (Altiplano) of the departments of
La Paz, Oruro and Potosı
´(the area around Lake Titicaca). There
are also other ethnic groups in the East, mainly living in the Llanos
(including the Bolivian Amazon areas) e. g. Chiquitanos
(.110,000 inhabitants), Guaranı
´es (.78,300 inhabitants; living
mainly on the border with Paraguay), Moxen˜os (.76,000
inhabitants); these groups mainly occupy the departments of
Santa Cruz, Beni, Pando and Tarija. The statistics vary slightly
according to the different sources (see e.g. also [3])
The ethnic composition of Bolivia includes a great diversity of
cultures but most indigenous peoples have assimilated a ‘mestizo’
culture. The Amerindian population accounts for approximately
55%; the remaining population is believed to be admixed with
Europeans and Africans. There are more than 30 ethnic groups in
Bolivia, the largest being Quechua- (about 1,500,000) and
Aymara-speaking (25%). Several pseudo-ethnic terms are com-
monly used in Bolivia to self-designate their ancestries, such as
‘Mestizos’ (considered to be a mixture of Native Bolivians and
Europeans), ‘Blancos’ (‘Whites’; considered to be descendants of
Europeans or ‘Criollos’), ‘Afro-Bolivians’, Asians, and others
(mainly Europeans from Germany, France, Italy, Portugal, and
a minority of people coming from neighboring countries such as
Argentina, Brazil, Chile, Colombia, etc.). The ‘Afro-Bolivians’ are
descendants of African enslaved people, live mainly in the
department of La Paz, and are concentrated in the provinces of
Nor Yungas and Sud Yungas. Asian individuals are mainly
Japanese (about 14,000) and Chinese (4,600).
Analysis of mtDNA variation has been used to explore
demographic patterns in different regions of America, helping to
unravel the origin of different ethnic groups, and the impact of
modern migrations [4–9]. Some South American regions have
received more attention in the literature than others; e.g.
Argentina [10–12], Brazil [13,14], Colombia [15–17], etc.
However, limited genotyping efforts have been dedicated to
Bolivians, and those have mainly focused on analysis of a few
Native American Bolivians. Bert et al. [18] analyzed the
mitochondrial DNA diversity in three different populations from
the Llanos de Moxos (n= 54) located in the lowlands of the
Amazonian basin; the data indicated a higher genetic diversity in
this locality than that observed in other American populations. In
2004, Dornelles et al. [19] analyzed a sample of Ayorea (n= 91)
individuals living in two Bolivian and one Paraguayan (neighbor-
ing) communities; the data suggested the effect of strong genetic
drift in this population, significantly reducing the amount of
variability. More recently, Corella et al. [20] analyzed several
small samples from the Bolivian Piedmont, including Chimane,
Moseten, Aymara and Quechua (n= 100); the results suggest high
genetic diversity in the area and high levels of inter-group
variability. Afonso-Costa et al. [21] analyzed 111 individuals
collected from La Paz, and the data was analyzed in a forensic
context. Finally, Gaya`-Vidal et al. [22] analyzed a sample of
Aymaras and Quechuas from Bolivia (n= 189); according to the
authors, the data support a past common origin of the Altiplano
populations in the ancient Aymara territory with independent
although related histories with the Peruvian Quechuas.
Autosomal SNPs have been analyzed in only few studies.
Galanter et al. [23] analyzed a panel of ancestry informative
markers (AIMs) in a collection of pan-American samples, including
a few from Bolivia. These authors found that most of the Bolivians
have a main Native American component (ranging from 90% to
98%), with the exception of the Yungas province, showing a
dominant African nature. Recently, Watkins et al. [24] carried out
a genome-wide analysis of 28 Bolivians, and although the results
agreed with Galanter et al. in that Bolivian genomes have
predominant Native American features, they estimated a higher
European component (12%) in these peoples.
The goal of the present study is to explore the variability of the
Bolivian populations globally, including non-Native Bolivians from
rural and urban populations, as well as those representing main
departments and ecological regions (from the high mountains to
the Llanos), and comparing them to previous studies that were
carried out on a more local scale and to Native populations. Here,
we have carried out the largest sampling of Bolivian populations to
date (n= 720). The data have been meta-analyzed jointly with
previously available data from Bolivia and a large pan-American
database. Whole genome sequencing was also carried out on
selected mtDNAs in order to investigate Native American
branches that have not been investigated before. In addition,
AIMs have been genotyped in order to infer patterns of main
continental ancestries that have contributed to the recent history of
the country.
Materials and Methods
Sample collection
A total of 720 samples were recruited for the present study.
They represent the three main regions of the country: a) Andean
or Altiplano (n= 240); b) Sub-Andean (n= 204); and c) the
Llanos (n= 276) which to a large extent corresponds to the
Bolivian Amazonian Basin. These samples were collected in the
following Bolivian departments: Beni (n= 102), Chuquisaca (n=
89), Cochabamba (n= 103), La Paz (n= 253), Pando (n= 90),
and Santa Cruz (n= 83). Birthplaces and other geographic
information are given in Table S1. Additional samples (n= 490)
were collected from the literature: La Paz (n= 110; [21]),
Chimane (n= 10), Moseten (n= 10), Aymara (n= 10) and
Quechua (n= 16) [20], Ayoreo (n= 91; [19]), and Trinitario (n
= 12), Yuracare (n= 15), Ignaciano (n= 15), and Movima (n=
12), all of which are around the small area of Llanos de Moxos
[18], Aymara (n= 96) and Quechua (n= 93) [22].
A subset of the total samples (n= 178), representing the
departments of La Paz (n= 105), and Chuquisaca (n= 73), were
genotyped for a panel of 46 ancestry informative markers (AIMs)
in Heinz et al. [25]. Additional Bolivian samples selected from the
total were genotyped de novo (n= 420) and merged with those that
were previously genotyped.
Ethics statement
Written informed consent was obtained from all sample donors.
Analysis of mtDNA sequences was approved by the institutional
review boards of Santiago de Compostela (Spain). Moreover, the
study conforms to the Spanish Law for Biomedical Research (Law
14/2007- 3 of July).
DNA sequencing of the control region and entire
genomes
Samples were PCR amplified and sequenced for HVS-I region,
as described previously [26]. Entire genome sequencing was done
as described in [27,28].
We have followed the phylogenetic approach to scan the
sequences as an a posteriori sequence quality control using the
principles described in [29–31]. This filter was also applied to the
data collected from the literature.
Nomenclature of mtDNA variants are referred against the
revised Cambridge Reference sequence or rCRS [32,33], and
Genetic Variability of Contemporary Bolivians
PLOS ONE | www.plosone.org 2 March 2013 | Volume 8 | Issue 3 | e58980
haplogroup nomenclature follows Phylotree Build 15 (www.
phylotree.org; [34]); see also [35]. For the sake of inter-population
comparisons and population summaries, we make the simplifica-
tion that all A, B, C, and D haplotypes correspond to the Native
American branches and not to the East Asian ones [36], given that
specific mtDNA SNPs were not available. Therefore, along the
text and figures, we used haplogroup labels according to the level
of phylogenetic resolution used in the present study; e.g. B4 instead
of B2; although it is most likely that all B4 belong to the Native
American haplogroup A2. Table S1 provides however the most
accurate haplogroup classification according to the level of
phylogenetic resolution obtained in the present study. Entire
genomes generated in the present study are publicly accessible via
GenBank with accession numbers KC503925 to KC503933.
AIM-INDEL genotyping
Bolivian samples were genotyped for 46 INDEL markers [37] in
a multiplex PCR amplification and capillary electrophoresis
process. Each AIM-Indelplex PCR amplification was performed
with 5 ml 2x Quiagen Multiplex PCR Master Mix, 10x Primer
Mix, and 0.5 ml DNA (concentration between 0.5 – 5 ng/ml) in a
final volume of 10 ml. PCR thermocycling conditions were: initial
temperature of 95uC for 15 min; 28 cycles at 94uC for 30 sec,
60uC for 90 sec, and 72uC for 60 sec; final step at 72uC for 60 min.
Following amplification, 0.8 ml PCR product was added to 11.5 ml
Hi-Di Formamide (Applied Biosystems) and 0.3 ml Liz-500 Size
Standard (Applied Biosystems). DNA fragments were separated
according to size using a 3130 Genetic Analyzer (Applied
Biosystems) and were analyzed with GeneMapper (Applied
Biosystems).
Statistical analysis and molecular dating
Haplotype (H) and nucleotide (p) diversities, and mean number
of pairwise differences (M) were calculated using DnaSP v.5
software [38]. Arlequin 3.5.1.2 [39] was used to compute
AMOVA (Analysis of Molecular Variance) and the significance
of the covariance components associated with different levels of
genetic structure were tested on haplotype frequencies applying a
non-parametric permutation procedure. Population pairwise F
ST
values, between/within population average nucleotide pairwise
differences, and Nei’s inter-population distances, were also
computed using Arlequin 3.5.1.2 [39].
Diversity indices, phylogeographic inferences and inter-popula-
tion comparisons were carried out using the sequence range 16090
to 16365, since this is the most commonly reported segment in the
literature. Problematic variation located around 16189, which was
usually associated with length heteroplasmy, e.g., 16182C or
16183C, was ignored.
Fisher’s exact test and Pearson’s chi-square test were undertak-
en using the R package (http://www.r-project.org/); a significant
nominal value of a= 0.05 was considered.
Maximum parsimony trees were built for the complete genomes
obtained in the present study and those collected from the
literature. For each cluster, the time to the most recent common
ancestor (TMRCA) was calculated by computing the averaged
distance (r) of all the haplotypes in a clade to the respective root
haplotype. Heuristic estimates of the standard error (s) were
calculated from an estimate of the genealogy, as done in [40].
Hotspot mutations (16182C, 16183C and 16519) were excluded
from calculations (as usual). The corrected evolutionary rate
proposed by Soares et al. [41] was used to convert mutational
distances into years. The TMRCA values obtained in the present
study could be slightly over-estimated as indirectly suggested by
estimates obtained in the literature regarding the entrance of the
first Paleo-Indians into the American continent [4,5,9]. Therefore,
the TMRCA values obtained here should be validated using a
larger number of entire genomes.
Analysis of population structure was undertaken using the
software STRUCTURE 2.3.4 [42–44]. Both, burn-in and Markov
Chain Monte Carlo (MCMC) repetition were set to a length of
100,000. Parameters were selected as indicated in Heinz et al.
[25]. Furthermore, the reference samples obtained from the
Human Genome Diversity Cell Line Panel, HGCP-CEPH [45],
were used to assist in clustering; this panel constitutes a collection
of 556 reference samples representing four main continents: Africa
(n= 105), Europe (n= 158), America (n= 64), and East Asia (n
= 229). Each run was repeated five times from K=2to
K= 7. Structure Harvester (http://taylor0.biology.ucla.edu/
structureHarvester/) was used to estimate optimal Kvalues.
CLUMPP 1.1.2 [46] and Distruct 1.1 [47] were used to prepare
data for visualization as bar plot representations. R 2.13.0 [48],
together with the SNPassoc package [49], was used to run two-
and three-dimensional Principal Component Analysis (PCA). We
compared and evaluated the results obtained from both
approaches.
Snipper [50] (http://mathgene.usc.es/snipper/) was used to
make four-way predictions of ancestral origin (Africa, Europe, East
Asia, and Native Americans) of Bolivian profiles. SNP data
collected from HapMap populations were also used as training sets
(http://hapmap.ncbi.nlm.nih.gov). Prediction was based on max-
imum likelihood.
Results
Mitochondrial DNA molecular diversity in Bolivia
Several diversity indices have been computed considering
different hierarchical levels (Table 1). When analyzed by main
regions, all seemed to show similar diversity values; with the
Department of La Paz harboring the lowest haplotype diversity,
and Chuquisaca being the region with the lowest nucleotide
diversity. The highest haplotype diversity was found in the North
(Pando) but the highest nucleotide diversity is observed in the
South (Santa Cruz). Therefore, there is no obvious correlation
between departments and molecular diversity as measured by way
of statistical summary indices. When examining the diversity by
rural and urban populations, it was observed that rural
populations harbor higher nucleotide and haplotype diversity
than urban populations.
The most apparent geographic pattern was found when
examining molecular diversity values by main ecological region.
The Andean followed by the Sub-Andean regions had substantial
lower diversity values than the Llanos. Therefore, the diversity was
found to increase longitudinally, from the high mountains to the
lowlands of the Llanos.
Diversity was particularly low in some ethnic groups compared
to general urban and rural populations. For instance, the Ayoreo
(from Bolivia and Paraguay) and the Aymara had extremely low
diversity values compared to the average values observed in Native
Bolivians and Bolivians in general (Table 1), a fact that is most
likely due to strong founder effect in the case of the Ayoreo [19],
while in the Aymara discussed by Corella et al. [20] it was most
likely due to the small sample size analyzed (the values were
compared with those obtained with the Aymara population from
Gaya´-Vidal et al. [22] using a larger sample size). Our sample
from general rural and urban Beni showed significantly higher
diversity values than those observed from other Native American
groups from the same department (e.g. the Piedmont populations
Genetic Variability of Contemporary Bolivians
PLOS ONE | www.plosone.org 3 March 2013 | Volume 8 | Issue 3 | e58980
of Moseten, Chimane, etc [20], the Llanos populations analyzed in
[18]; see Table 1).
There are only few haplotypes in Bolivia that appears more
than two or three times in the whole dataset, again indicating the
high mtDNA diversity of the country (Figure S1 and Figure
S2). When evaluating the differentiation between departments, it
was again observed that comparisons of other departments with
Santa Cruz were the ones that displayed the highest F
ST
values
(Figure S3). The influence of this department is also indirectly
observed when studying F
ST
values between the main ecological
regions, indicating that the comparisons between Llanos and the
other two areas showed the largest values (Figure S4).
Finally, Figure S5 and Figure S6 show the expected (virtual)
heterozygosity in the main departments and in the main regions,
respectively; the dispersion of values indicated the existence of
substantial mtDNA diversity between departments.
Phylogeography
The mtDNA Native American component predominates in the
Bolivian population (98.4%); most of the variation can be classified
into one of main Native American haplogroups, (A: 14.3%, B:
52.6%, C: 21.9%, D: 9.6%), Figure 1.
There were remarkable differences in haplogroup frequencies
between the main Bolivian departments (Figure 1). For instance,
the department of La Paz had the following haplogroup
composition, A: 10%, B: 71%, C: 12%, and D: 7%, which was
in sharp contrast with the composition observed in the department
of e.g. Beni, A: 12%, B: 30%, C: 48%, and D: 10%. The
haplogroup distribution found by Afonso-Costa et al. [21] in a
smaller sample from La Paz was slightly different to that found in
the Bolivians from La Paz studied here, but both samples agreed
regarding the high proportion of haplogroup B in this area. This
geographic pattern mirrors in reality the location of the
departments at different altitudes; thus, the most important
differences are observed between Andean and Sub-Andean
populations versus Llanos. For instance, haplogroup B is the
predominant haplogroup in the Altiplano (71%), which decreases
to 61% in Sub-Andean populations and 32% in Llanos (Figure
1).
As expected, Bolivia shares more haplotypes with South
America than with Meso and North America (Table 2; Table
Table 1. Diversity indices in Bolivian mtDNAs and main American and African regions.
Populations groups Reference n
kSH p
M
Department
Beni Present study 102 63 71 0.98260.002 0.0247060.00130 6.818
Chuquisaca Present study 89 57 59 0.97160.009 0.0158860.00099 5.718
Cochabamba Present study 103 71 77 0.98160.007 0.0185560.00086 6.677
La Paz Present study 253 121 81 0.96060.007 0.0186460.00086 5.106
Pando Present study 90 54 60 0.98360.006 0.0197760.00068 7.117
Santa Cruz Present study 83 49 46 0.97860.006 0.0261860.00097 7.226
All Bolivia Present study 720 306 134 0.97660.002 0.0223760.00041 6.130
Rural
vs
. Urban
Rural Present study 189 108 92 0.98060.004 0.0244060.00069 6.709
Urban Present study 531 240 121 0.97560.003 0.0222560.00049 6.098
Regions
Andean Present study 240 116 78 0.96160.007 0.0185660.00088 5.086
Sub-Andean Present study 204 118 90 0.97360.006 0.0221560.00083 6.092
Llanos Present study 276 141 100 0.98260.003 0.0245860.00059 6.759
Native groups
Aymara-speakers (Beni) [20] 10 5 9 0.66760.163 0.0049960.00251 1.800
Moseten (Beni) [20] 10 8 18 0.95660.059 0.0176160.00209 6.356
Quechua-speakers (Beni) [20] 16 7 17 0.69260.124 0.0109460.00358 3.950
Chimane (Beni) [20] 10 8 16 0.93360.077 0.0142260.00212 5.133
Aymara-speakers [22] 96 39 48 0.95660.009 0.0164560.00142 4.523
Ayoreo [19] 91 8 10 0.47360.061 0.0062660.00104 2.260
Ignaciano [18] 15 11 23 0.93360.054 0.0177360.00237 6.400
Movina [18] 12 8 12 0.89460.078 0.0081460.00174 2.940
Quechua-speakers [22] 93 40 48 0.94660.012 0.0199760.00118 5.471
Trinitario [18] 12 11 22 0.98560.040 0.0188460.00212 6.803
Yuracare [18] 15 11 22 0.95260.040 0.0185260.00151 6.686
All Native 380 114 84 0.94660.006 0.0220160.00043 6.008
The indices were computed using the common segment of the HVS-I region from position 16090 to 16365.
NOTE: n= sample size; K= number of different haplotypes; S= number of segregating sites; H= haplotype diversity; p= nucleotide diversity; M= average number
of pairwise differences (mismatch observed mean).
doi:10.1371/journal.pone.0058980.t001
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S2), although these values have to be interpreted with care due to
the different sample sizes. Note however that the largest sample
size is in North America, but Bolivia only shares 18% of the
haplotypes with this region but 49% with other South American
populations (Table 2). It is also worth mentioning that 48
haplotypes are shared between the three American regions;
therefore, of the 68 haplotypes shared with North America, most
of them are also shared with South America (Table 2, Table S2).
A large amount of haplotypes has been only observed in Bolivia
(250 out of 383 different haplotypes; 65%).
There are eight mtDNAs that most likely belong to haplogroup
C1d (all carry the characteristic transition 16051); there is another
mtDNA that could be allocated to the northern South American
cluster C1d2a, which does not carry transition 16091 but carries
transition 16209 instead. C1d has been reported as one of the
founding Paleo-Indian mtDNA lineages of the American continent
[5] and is therefore found along the double continent. As has been
previously described [5], C1d did not necessarily follow a main
Pacific coastal route once it reached South America; this would
explain why this lineage appears more in the Llanos than in the
Andean region.
The five mtDNAs belonging to D4h3a found in our Bolivian
samples most likely arrived at about the same time as C1d but
probably followed a different route. According to [4], D4h3a
appeared to spread along the Pacific coast. In agreement with this
hypothesis, the five D4h3a Bolivians were observed in the Andean
region, and three out of the five D4h3a lineages belong to the
Peruvian sub-clade D4h3a3.
According to expectations, no members of the Paleo-Indian
founder X2a, which has a North American distribution, have been
found in Bolivians. Also, in good agreement with Bodner et al. [9],
we did not find any members belonging to southern cone lineages
D1j and D1g, favoring the hypothesis that these two haplogroups
crossed the Andes at lower latitudes (Chile towards Argentina),
where they were most likely incubated.
Very recently, de Saint Pierre et al. [51] proposed two new
lineages, B2i2 and C1b13, which are thought to have originated in
the Southern Cone, and are closely associated with the Paleo-
Mapuche people in Chile and Argentina. Members of these two
lineages have only been sporadically found outside of Chile and
Argentina. Here, we only found one member of C1b13, and
according to the hypothesis formulated by de Saint Pierre et al.
[51], this mtDNA could have arrived in Bolivia from the South
instead of from the West.
Some Bolivian mtDNAs are particularly interesting given that
they appear at relatively high frequencies in this region and
neighboring areas but are absent in the rest of the continent. For
instance, the motif 16189 16217 16290 appears six times within
Figure 1. Map of Bolivia showing the location of the samples collected in the present study. The pie charts represent the distribution of
basal Native American haplogroup frequencies in the region.
doi:10.1371/journal.pone.0058980.g001
Genetic Variability of Contemporary Bolivians
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haplogroup B4 in different regions from Bolivia; it also appeared
in four Andean Peruvian Quechua individuals [52]. The motif
16173 16192 16223 16298 16325 16327 16346 (haplogroup C)
appears twelve times in Bolivia, and all of them in the Llanos.
Curiously, this motif was found also in another individual from La
Paz in [21] and another one from Chaco, in the North of
Argentina, close to the Bolivian Llanos [11].
Haplogroup L (L referring to all mtDNA branches excluding
macro-haplogroups M and N) mtDNAs represent the sub-Saharan
exclusively maternal genetic component of the country, which in
this case only accounts for 1% of the total mtDNA pool (Table
S1). The proportion of African recent ancestry in Bolivia is very
low compared to other South American countries that were more
influenced by the African slave trade [16,17,53]. There are only
seven different haplotypes belonging to some of the typical sub-
Saharan sub-clades. Individual #BNI19 belongs to L0a1b2, which
is spread in all of sub-Saharan Africa as well as in America, but
carries a distinctive transition (within L0a1) at position 16271
(which gets an score of nine in the mutation list of Soares et al.
[41]). The L1b (#BNI75) and L1c1a1 (#LaPaz467) profiles most
likely come from the West-Central African region. The L1c3b1a
and the L3f1b4a profiles appear in Cabinda, Angola and
Mozambique [54–57], but also in West-Central, in Gabon [58].
As expected [53,59], the most likely geographic origin of the
African Bolivian profiles is west-central Africa, but also encom-
passes the Southwest and the Southeast.
It is also curious that the European mtDNA component
represents less than 1% of the Bolivian population. With the level
of resolution of the HVS-I segment, it is not possible to assign
European lineages to a particular region in Europe; the four
sequences observed here belong to haplogroups H1af, K and X1.
Table 2. Shared haplotypes between Bolivia and other
American regions.
N
SH with Bolivia H H
1
North America 7551 68 1875 1085
Meso-America 2792 68 925 612
South-America 5727 187 1587 1009
Bolivia 1127 383 383 250
Only the segment 16024 to 16385 was considered for comparison. N= sample
size; SH = shared haplotypes; H= number of different haplotypes; H
1
=
number of unique haplotypes per region.
doi:10.1371/journal.pone.0058980.t002
Figure 2. Maximum parsimony tree of the main branches characterizing haplogroups A2 and B2, indicating the new branches
generated in the present study, namely, A2ah and B2o. The mutations are displayed along the branches: numbering is according to the rCRS
[71]: a mutation alone indicates a transition while an upper case letters as suffix (A, C, G, T) indicates a transversion; ‘‘+’’ and ‘‘d’’ refer to insertions and
deletions, respectively; ‘‘s’’ refers to a synonymous variant while non-synonymous variants are indicated by way of the amino acid change; a star
indicates a variant located in the ATP6 or ATP8 gene; a prefix @ indicates a back mutation; and underlined positions indicate parallel mutations.
Sample ID (see Table S3) for the entire genomes generated in the present study are as follows: #1: COBIJA577, #2: COBIJA604, #3: STACRUZ261, #4:
BENI86, and #5: STACRUZ221.
doi:10.1371/journal.pone.0058980.g002
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An Asian origin for the profile C16104T C16223T cannot be
disregarded given that two exact haplotype matches appeared in
China [60] but any in Europe (in our in-house database of more
than 26700 HVS-I profiles).
Entire genomes from Native American Bolivians
In order to investigate the Native American lineages in our
Bolivian samples further, we selected those showing a distinctive
pattern from the control region data. Thus, we chose two mtDNAs
carrying the tandem variants T16097C A16098G on top of the
sequence motif for haplogroup A2, and a group of six mtDNAs all
carrying transition G16145A on top of the B2 basal motif (Table
S3). Entire genome sequencing revealed some interesting
phylogeographic features of these mtDNAs.
The tandem transitions T16097C A16098G conform per se a
very distinctive motif that is found very rarely in mtDNA
databases. Within haplogroup A2, this motif alone (without any
other control or coding region variant) defines a novel branch of
the Native American phylogeny, here named as A2ah (Figure 2).
The two genomes show variability within this clade and, given its
very restrictive location within Bolivia, this most likely point to an
origin in Bolivia or the surrounding territories. We further
investigated A2ah in public databases of entire mtDNA genomes.
We found just one entire genome (JQ702082) that carries the
transition A16098G on top of the A2 motif but it differs
substantially from the two genomes found in Bolivia (apart from
lacking the transition T16097C); therefore, its inclusion within
A2ah should be considered to be very dubious. According to the
entire genome sequences available, it can be said that haplogroup
A2ah originated about 5,200 thousand years ago (kya) (although
with a large 95%CI: 0.1–10.5). We additionally investigated the
phylogeographic characteristics of these lineages by looking at the
abundant amount of data available in the literature on control
region segments. We only detected nine mtDNAs matching the
motif of A2ah. Four out of ten were observed in the data reported
by Behar et al. [61] but geographic information was not available.
Two other HVS-I mtDNAs were found in the ‘Hispanic’ North
American subset of the SWGDAM database [62]; three other
A2ah mtDNAs were observed in South America, two among the
Brazilian samples of Alves-Silva et al. [13], and one in the Toba
from Gran Chaco (North Argentina) [63]. In Bolivia, only four
A2ah members were found (0.5%), all of which were observed in
the Llanos (three in Santa Cruz and one in the Beni department).
In general, the HVS-I data suggest that this lineage could have
originated in Bolivia or some place in Central South America. The
two members observed in the ‘Hispanic’ samples from the
SWGDAM could perfectly represent recent immigrations into
Figure 3. Maximum parsimony tree of haplogroup B2b. The map on the top right inset indicates the geographic location of the B2b genomes
represented in the tree; circles are proportional to the sample size. The arrows indicate the two tentative migration routes (a and b in the map) of this
lineage (see main text) along the American continent. References for entire genomes are as follows (see Table S1 and S3): #6: BENI90, #7:
STACRUZ258, and #8: COBIJA110, #9 Mco-10 (present study, Table S1 and S3). For more information, see the legend of Figure 2.
doi:10.1371/journal.pone.0058980.g003
Table 3. AMOVA computed based on haplotype pairwise
differences of Bolivian populations (significant tests: 20,022
permutations; adjusted P-value,0.0000).
Within
Populations
Among
Populations
Rural vs. Urban 99.85 0.15
Andean vs. Sub-Andean vs. Llanos 93.15 6.85
Departments 93.44 6.56
Provinces 93.60 6.40
doi:10.1371/journal.pone.0058980.t003
Genetic Variability of Contemporary Bolivians
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USA. The motif T16097C A16098G was also observed in
Mainland Scotland (Western Islands; Isla of Skye) and two other
samples in [61], but none of the mtDNAs carried the variation
defining haplogroup A2.
A phylogenetic conflict exists when trying to reconstruct the
most parsimonious tree of the six B2 mtDNAs carrying transition
G16145A. Three of the genomes (#6, #7 and #8inFigure 3)
carried the synonymous transition G6755A, while three other
genomes (#1, #2 and #3) lacked G6755A but carried the non-
synonymous transitions T7270C and T16092C instead (Figure
2). Given that G16145A seems to have a much higher mutation
rate than G6755A (22 versus 2 mutational hits in Soares et al. [41],
and 24 versus 5 in Phylotree; respectively), we decided to resolved
the phylogeny as shown in Figure 2 and Figure 3.
Thus, the motif T7270C T16092C G16145A defines a new
branch of the Native American phylogeny named here as B2o,
which is represented by three Bolivian genomes (Figure 2). No
other entire genome was observed in public resources belonging to
B2o. Dating the phylogeny of B2o (Figure 2) indicates that this
haplogroup originated about 2.6 kya (95%CI: 6.8–13.2). Search-
ing control region databases, we observed only two HVS-I
candidate B2o sequences: one was observed in San Martin de
Pangoa in Peru´, a small town on the eastern slope of the Andes
inhabited by Quechua and Nmatsiguenga people [64], while the
other B2o sequence was observed in Guam (an island located in
the western Pacific Ocean, territory of USA). The latter, however,
carries the motif T16189C T16217C A16247G C16261T, which
is very common in the Micronesia, Australia, etc. [65,66] and
points to a different clade of the worldwide phylogeny, haplogroup
B4a. In Bolivia, we found seven B2o representatives (1%), most of
them in the Llanos (five in Pando and one in Santa Cruz
departments), and one in a Sub-Andean locality of La Paz.
Therefore, altogether the data suggest that this lineage has also
originated in Bolivia or some nearby region, most likely located in
the Andes.
The entire genomes of the three Bolivian mtDNAs carrying
G16145A but lacking T16092C, all share the transition G6755A.
By searching the databases on entire genomes for the transition
G6755A, we observed 10 additional mtDNAs belonging to this
branch. In reality, this clade had already been described in the
recent literature [6,7] and was named B2b. However, its internal
variation was never analyzed in detail. Figure 3 shows the
phylogeny of B2b and the geographical location where the entire
genomes were observed. Geographic and/or ethnic affiliation was
available for eleven entire genomes (including the Bolivian ones):
one Pomo (North California, USA) [8], one Mexican American
[67], two Venezuelans from Pueblo Llano [68], one Yanomama
(Brazil, Venezuela) [8], one Cayapa (Equador) [6], one Kayapo´/
Kubemkokre (South Amazonia, Brazil) [8], one Xavante (Brazil)
[8], and three Bolivians (present study). We additionally detected
one entire genome in our DNA databank in a Mataco Native from
North Argentina, which was added to the phylogeny of Figure 3
(#9). According to the phylogeny of B2b in Figure 3, B2b
appeared about 21.4 kya (95% CI: 4.4–26.7). The Bolivian sub-
clade B2b2 is much younger, approximately 15.2 kya (95%CI 4.4–
26.7). In the large HVS-I database, we only observed 14 B2b
candidates, almost overlapping the territories represented by the
entire genomes. In Bolivia, we counted seven B2b candidates: one
Andean (La Paz), four Sub-Andean (Cochabamba, La Paz and
Chuquisaca), and two in the Llanos (Beni and Santa Cruz).
Figure 4. Average continental ancestry of Bolivians analyzed using a panel of 46 AIMs. These values are obtained from the STRUCTURE
analysis and the optimal value K=4.
doi:10.1371/journal.pone.0058980.g004
Genetic Variability of Contemporary Bolivians
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AMOVA analysis of mtDNA profiles
AMOVA was undertaken considering different hierarchical
levels; by Department, Rural vs. Urban, and Andean vs. Sub-
Andean vs. Llanos. As expected, the within-population variation
accounted for most of the variance (ranging from 93–100%;
Table 3), independently of the population sub-division employed.
AMOVA carried out by departments and ecological region
provided the highest values of among-population variance
(ranging from 10.48 to 11.58% of the total variance). The values
are similar given the correlation that exists between these two
classification criteria. The rural vs. urban division indicates
virtually no correlation among group subdivision. The analysis
carried out by province did not increase the among-population
variance, indicating again that the main factor influencing within-
population variation was the different altitudes in the country.
Continental ancestry in Bolivians
A panel of 46 AIMS was genotyped in Bolivians in order to infer
their main continental ancestry. Data from main continental
reference samples (Africa, East Asia, Europe and America) were
used as classification sets.
Analysis of ancestry was carried out using STRUCTURE.
Figure 4 summarizes the average continental ancestries observed
in Bolivians under different grouping schemes, while Figure 5
shows the STRUCTURE bar-plots. The analysis showed that, on
average, 71% of the component in the total Bolivian sample is
Native American, followed by 25% of European ancestry. When
examining the ancestry by departments, La Paz was the region
that showed the highest Native American ancestry (79%) (Figure
4and Figure 5), and, therefore, the lowest European component
(19%). On the other side is the department of Santa Cruz, with
57% of Native American ancestry and 39% European. The
African component was very low in all of the departments,
showing the highest value in the department of Pando (2.5%), in
the North. When examining by ecological regions, it was also
evident that the Native American component is higher in the
mountainous West (80%), and decreases progressively eastward:
sub-Andean (70%) and Llanos (64%). The differences are less
apparent when examining rural vs. urban areas (74% and 69% of
Native American component, respectively).
The STRUCTURE bar-plot of Figure 5 indicates the
ancestral membership for each individual in the source population
and the Bolivian samples. It clearly shows that most of the
individuals have a Native American ancestry, with only a few
exceptions. The high European component of Santa Cruz
compared to other departments is also evident in this bar-plot,
and is also evident in the Andeans. When looking at the most
extreme values, we found that there are only a few that have
European ancestry above 50% in most of the departments. It is,
however, rare to see individuals with high African membership;
the highest values correspond to 23% in one individual from La
Paz and another one from Pando (17%).
The membership of individuals into the East Asian cluster is
significantly higher in some individuals, as is also evident in the
STRUCTURE plots. However, this perhaps mirrors the fact that
the separation between the Native American component and the
Figure 5. STRUCTURE analysis of Bolivians based on the 46 AIMs panel genotyped in the present study. Each bar plot represent
analysis using different grouping schemes for the Bolivian samples. Only the results for the optimal K= 4 are represented (see complete analysis in
Text S1).
doi:10.1371/journal.pone.0058980.g005
Genetic Variability of Contemporary Bolivians
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East Asian one is not perfect. The overlap between Native
Americans and East Asians can be observed more clearly in the
PCA analysis (see below). This fact has been already observed in
Pereira et al. [37]. Thus, most of the East Asian component would
be captured by the Native American cluster in cases using a three-
group structure analysis (Native American, African, and Europe).
The PCA agrees well with the results observed in STRUC-
TURE. The Bolivian profiles group mainly with the Native
American ones from HapMap, with only some samples showing a
projection towards the European cluster (Figure 6). The minor
African component detected in the STRUCTURE analysis seems
not to be relevant in the PCA; only one sample show some affinity
to the African cluster; it is in fact the same sample with a 23%
African ancestry in STRUCTURE. The PCA carried out by
departments (Text S1) showed that the department of La Paz
(Andes) was more tightly grouped than the profiles from other
departments; in the other pole are the departments of Santa Cruz
and Pando, showing more dispersed patterns (Llanos). This west-
east pattern is more evident when observing the PCA by main
regions (Figure 6): the Andean profiles are more tightly grouped
in one pole of the plot, while the profiles from Llanos are in the
other pole and are more dispersed, with the sub-Andean profiles
occupying an intermediate position between East and West. This
dispersion is also mirrored when examining the standard deviation
(SD) of the Native American membership values in the main
regions: (i) Andean: mean = 79.7%, SD = 0.086%; (II) sub-
Andean: mean = 69.6%, SD = 0.09; and (iii) Llanos: mean
64.2%, SD = 0.137% (Table S4); given that the department of
La Paz is basically an Andean department, it also shows the lowest
SD (0.85%).
Finally, measuring ancestry using AIMS depends on many
factors, such as the use of different panels of SNPs (not only
different SNPs, but also different numbers of them), different
classification test samples, sample sizes, etc. Twelve of the samples
genotyped in the present study for the 46-indel panel were also
genotyped in Galanter et al. [23], thereby offering a good
opportunity to investigate the ability of different AIM panels to
estimate percentages of continental ancestry. As shown in Figure
7, the ancestry memberships obtained using Indels lead to a
decreased Native American ancestry (and proportional increased
Figure 6. PCA of Bolivian profiles based on the 46-AIMs panel genotyped in the present study. Each figure shows different grouping
schemes mainly aiming to show the within-Bolivian diversity.
doi:10.1371/journal.pone.0058980.g006
Genetic Variability of Contemporary Bolivians
PLOS ONE | www.plosone.org 10 March 2013 | Volume 8 | Issue 3 | e58980
European ancestry) compared to the estimates obtained using the
LACE panel (on average, the difference is about 15%). Given the
way in which the LACE panel was designed (in order to balance
the informative content of the different AIMS included in the
panel), it seems logical to believe that the Indels panel would tend
to over-estimate the proportion of European ancestry in Bolivians.
Note, however, that the genome-wide study of Watkins et al. [24]
carried out in few Bolivians estimated a higher European
component (12%) compared to the LACE. Further genome-wide
studies on a larger Bolivian sample will allow better estimates of
continental ancestries to be obtained.
Discussion
Bolivia shows a mainly Native American mtDNA component
(98%). Only 1.5% of the profiles have Sub-Saharan mtDNA
ancestry. The impact of Europeans in the Bolivian mtDNA pool is
minimal (0.5%), which also contrasts with other South American
locations [11,27]. Although the Native American mtDNA
component predominates in the country, there is a highly diverse
and geographic stratification in the country. In a large geograph-
ical scale, the largest difference corresponds to Llanos vs. the
Andean and Sub-Andean regions. The political definition of the
departments overlap quite well with the main latitudes observed in
the country, which would explain the correspondence observed
when carrying out different statistical analysis. Molecular diversity
is extremely low in some indigenous group compared to urban and
other rural populations, suggesting the existence of important
episodes of isolation and genetic drift in Native communities.
AMOVA carried out at Bolivian populations to different
hierarchical levels allowed a better understanding of the spatial
geographic patterns of variability in large regions. The results
agree that most of the variation accounts within populations, but
that the major differentiation occurs between the Andes and the
Llanos. In addition, some mtDNA phylogeographic features
indicate the presence of common lineages between different
Andean regions in Peru and Bolivia, most likely testifying for the
common demographic past during the Inca’s Empire period. This
continuity in the Andean region was also observed in the Aymaras
and Quechuas analyzed by Gaya´-Vidal et al. [22].
By way of sequencing the entire genome of nine mtDNAs (eight
from Bolivia and one from a northern Argentinean Native
Mataco), and compiling a large amount of data from the
literature, we could shed light on three Native American lineages:
A2ah, B2o and B2b. The three haplogroups are rare in Bolivia
(ranging from 0.5 and 1%) and are very rare continentally. The
three clades show significant diversity within the Bolivian country,
indicating that they most likely evolved locally after their arrival
from other neighboring regions. The clade for which there are
more data available (literature and present study) is B2b. This
haplogroup most likely arose in North California; however, as
suggested by its TMCRA (,21.4 kya; see also [67]) and its
phylogeographic characteristics, it could have been originated
even in the north most region of the American continent,
constituting a new minor Paleo-Indian founder. The data also
indicate that B2b traveled South, most likely following the Pacific
coastline (the main route followed by the First Americans)
[4,5,9,69]. It probably entered the South American sub-continent
following two different paths: (i) travelling further south following
the Pacific side, then reaching Equador, Peru, and Bolivia; and (ii)
following firstly an eastwards direction, and secondly southwards
crossing the Amazon basin in Venezuela and then Brazil (or firstly
Figure 7. Percentages of main continental ancestry inferred using different AIMs: the 46-Indels panel analyzed in the present study
versus
the 446-SNP LACE panel analyzed in [23].The information is only available for 12 individuals from Bolivia. The vertical bars only highlight
the difference of ancestry observed using different panels for each individual
doi:10.1371/journal.pone.0058980.g007
Genetic Variability of Contemporary Bolivians
PLOS ONE | www.plosone.org 11 March 2013 | Volume 8 | Issue 3 | e58980
bordering the Atlantic coast southwards). At the very least, these
lineages arrived in northern Argentina, but it is likely that further
sampling will probably detect B2b in the most southern edge of the
continent. The data indicate that the Bolivian sub-lineage B2b2
arrived in this region about 15,000 years ago.
Ancestry analyses carried out on a panel of AIMs indicated that
Bolivians have a substantial European ancestry (although a
possibility exists that this proportion could be over-estimated; see
above), which varies substantially between departments, thus
indicating a differential regional impact of the European
Colonialism in Bolivia. The African component is very low (in
agreement with the mtDNA) as compared to other South
American populations, such as the Caribbean coast [17,70],
Colombia [16], and Brazil [13], but is comparable to others such
as Argentina [10,11,27]. It must be taken into account that the
present study did not include samples from the Yungas, a small
province located within the department of La Paz that seems to
concentrate the main African component in Bolivians. The
African component of the Yungas people is not only inferred
from their distinguishable African cultural and biological features,
but also from the genetic inferences made using panels of AIMs
analyzed in a small group of people from this region [23].
The present study represents the largest and most comprehen-
sive analysis of genetic variation investigated to date in rural and
urban Bolivians from the point of view of mtDNA and autosomal
data. The results indicated that even those Bolivians that did not
self-identify as belonging to a particular Native America ethnic
group still preserved a main Native American genetic character in
their genomes. Deep analyses of selected mtDNA genomes also
indicate the presence of lineages that appears to be autochthonous
to these peoples; provided that these lineages have not been
identified in large American databases. The Native American
component of Bolivians is significantly higher when observing the
mtDNA instead of the autosomes. This is a common feature in
other South and Central American regions (e.g. Argentina [10],
Panama [2])where the maternal Native American component has
been found to be much better preserved in the maternal specific
genome, a fact that is generally explained by the higher proportion
of European males arriving to America than females.
Supporting Information
Figure S1 Frequency of different haplotypes (alleles in
figure) by department.
(TIFF)
Figure S2 Frequency of different haplotypes (alleles in
figure) by ecological region.
(TIFF)
Figure S3 Fst values between departments.
(TIFF)
Figure S4 Fst values between ecological regions.
(TIFF)
Figure S5 Expected (virtual) heterozygosity by depart-
ments.
(TIFF)
Figure S6 Expected (virtual) heterozygosity by main
ecological regions.
(TIFF)
Table S1 Mitochondrial DNA sequencing data for the
Bolivian samples analyzed in the present study. Note that
for a large proportion of sequences the range is generally larger
than what it is usually considered to be HVS-I and HVS-II.
(XLSX)
Table S2 Shared haplotypes between Bolivia and other
American locations.
(XLSX)
Table S3 Information on entire genome sequences
obtained in the present study and collected from the
literature and GenBank.
(XLSX)
Table S4 Continental ancestry values (standard devia-
tions in brackets) obtained using STRUCTURE for the
Bolivian samples averaged through five different itera-
tions (see Material and Methods for more information).
The minimum and the maximum values are also given.
(XLSX)
Text S1 Additional PCA and structure analyses carried
out in the Bolivian samples based on 46 AIMs.
(DOCX)
Acknowledgments
We would like to thank the donors for their participation in the present
project. The authors have declared that no competing interests exist.
Author Contributions
Draft the manuscript: AS. Agreed on the final version of the manuscript:
PTE VA
´I TH LVB AGC LC JPS AP ATB OR CV AS. Conceived and
designed the experiments: AS. Performed the experiments: PTE VA
´ITH
LVB AGC LC AP. Analyzed the data: PTE VA
´I TH LVB AGC JPS AS.
Contributed reagents/materials/analysis tools: A
´C ATB OR CV AS.
Wrote the paper: AS.
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