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Indigenous populations of Malaysia known as Orang Asli (OA) show huge morphological, anthropological and linguistic diversity. However, the genetic history of these populations remained obscure. We performed a high density array genotyping using over 2 million SNPs in 3 major groups of Negrito, Senoi and Proto-Malay. Structural analyses indicated that although all OA groups are genetically closest to East Asian (EA) populations, they are substantially distinct. We identified a genetic affinity between Andamanese and Malaysian Negritos which may suggest an ancient link between these two groups. We also showed that Senoi and Proto-Malay may be admixtures between Negrito and EA. Formal admixture tests provided evidence of gene flow between Austro-Asiatic speaking OAs and populations from Southeast Asia and South China which suggest a widespread presence of these people in SEA before Austronesian expansion. Elevated linkage disequilibrium (LD) and enriched homozygosity found in OAs reflect isolation and bottlenecks experienced. Estimates based on Ne and LD indicated that these populations diverged from East Asians during the late Pleistocene (14.5 to 8 YBP). The continuum in divergence time from Negritos to Senoi and Proto-Malay in combination with ancestral markers provides evidences of multiple waves of migration into SEA starting with the first Out-of-Africa dispersals followed by Early-train and subsequent Austronesian expansions. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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Unravelling the Genetic History of Negritos and Indigenous
Populations of Southeast Asia
Farhang Aghakhanian
1
, Yushima Yunus
2
, Rakesh Naidu
1
, Timothy Jinam
3
,AndreaManica
4
,
Boon Peng Hoh
2
, and Maude E. Phipps
1,
*
1
Jeffrey Cheah School of Medicine and Health Sciences, Monash University (Malaysia), Selangor, Malaysia
2
Institute of Medical Molecular Biotechnology, Faculty of Medicine, Universiti Teknologi MARA, Selangor, Malaysia
3
Division of Population Genetics, National Institute of Genetics, Mishima, Japan
4
Evolutionary Ecology Group, Department of Zoology, University of Cambridge, United Kingdom
*Corresponding author: E-mail: maude.phipps@monash.edu.
Accepted: April 9, 2015
Abstract
Indigenous populations of Malaysia known as Orang Asli (OA) show huge morphological, anthropological, and linguistic diversity.
However, the genetic history of these populations remained obscure. We performed a high-density array genotyping using over 2
million single nucleotide polymorphisms in three major groups of Negrito, Senoi, and Proto-Malay. Structural analyses indicated that
although all OA groups are genetically closest to East Asian (EA) populations, they are substantially distinct. We identified a genetic
affinity between Andamanese and Malaysian Negritos which may suggest an ancient link between these two groups. We also
showed that Senoi and Proto-Malay may be admixtures between Negrito and EA populations. Formal admixture tests provided
evidence of gene flow between Austro-Asiatic-speaking OAs and populations from Southeast Asia (SEA) and South China which
suggest a widespread presence of these people in SEA before Austronesian expansion. Elevated linkage disequilibrium (LD) and
enriched homozygosity found in OAs reflect isolation and bottlenecks experienced. Estimates based on N
e
and LD indicated that these
populations diverged from East Asians during the late Pleistocene (14.5 to 8 KYA). The continuum in divergence time from Negritos to
Senoi and Proto-Malay in combination with ancestral markers provides evidences of multiple waves of migration into SEA starting
with the first Out-of-Africa dispersals followed by Early Train and subsequent Austronesian expansions.
Key words: Negritos, Senoi, Proto-Malay, population genetics, SNPs.
Introduction
The events and period of prehistoric peopling of Southeast
Asia (SEA) have been controversial. Human remains from
archeological sites such as Callao Cave in Philippines (Mijares
et al. 2010) and Niah Cave in Malaysia (Barker et al. 2007)
suggest that SEA was populated by anatomically modern
humans approximately 50–70 kilo years ago (KYA). In 2009,
a large-scale genome-wide study by the HUGO-Pan Asia con-
sortium showed that all East Asians and Southeast Asians
originated from a single wave Out-of-Africa” via a southern
coastal route (HUGO Pan-Asia SNP Consortium 2009).
Thereafter, two models have been proposed to explain sub-
sequent migrations involved in shaping todays SEA popula-
tions. The Out-of-Taiwan model refers to the Austronesian
language expansion that occurred around 5,000–7,000
years before the present. This replaced the pre-existing
Australoid people with Austronesian agriculturists (Diamond
and Bellwood 2003; Bellwood 2005). In the long period be-
tween the first initial Out-of- Africa and the recent “Out-of-
Taiwan” migrations, recent genetic studies on mitochondrial
DNA (mtDNA) suggest an Early Train wave of migration during
the late Pleistocene to early Holocene (Hill et al. 2006, 2007;
Soares et al. 2008; Karafetetal.2010; Jinam et al. 2012).
The rich ethnological diversity that exists in Peninsular
Malaysia provides a great opportunity to study SEA prehistory.
The current Malaysian population comprises three major
ethnic groups including Malay, Chinese, and Indians. In addi-
tion to these groups, Peninsular Malaysia is home to other
ethnicities including several minor indigenous communities
collectively known as “Orang Asli” (OA) or Original
People.” Making up approximately 0.6% of Malaysian popu-
lation, OA has been classified into three groups, namely
GBE
ß The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
1206 Genome Biol. Evol. 7(5):1206–1215. doi:10.1093/gbe/evv065 Advance Access publication April 14, 2015
Negrito (Semang), Senoi, and Proto-Malay (aboriginal Malay)
based on linguistic, physical, and anthropological characteris-
tics. Each OA group could be further subdivided into six sub-
groups based on their lifestyle and geographical location.
Malaysian Negritos are Austro-Asiatic (AA) speakers and
inhabit in northern parts of Peninsular Malaysia. The tradition
of these hunter-gatherers involves northern Aslian dialect of
AA language, egalitarianism, and patrilineal descent system.
On the basis of their hunter-gathering lifestyle and physical
characteristics including their small body size, dark skin pig-
mentation, cranio-facial morphology, and frizzy hair,
Malaysian Negritos traditionally are grouped with other
Negrito communities in South Asia and SEA such as
Andaman islanders, Mani in Thailand, Philippine Negritos,
and other phenotypically similar populations in Papua New
Guinea and Australia. These similarities have led to the general
idea that all Negrito populations of SEA and Oceania origi-
nated from a common ancestral group which entered SEA
during the earliest human dispersals into Asia (Endicott
2013). However, genetic studies have provided mixed evi-
dence. Although a genetic affinity between Andaman is-
landers, Malaysian and Philippine Negritos was detected by
some authors (Jinam et al. 2012; Chaubey and Endicott
2013), several mtDNA (Endicott et al. 2003; Thangaraj et al.
2005; Wang et al. 2011), Y chromosome (Delfin et al. 2011;
Scholes et al. 2011), and autosomal (HUGO Pan-Asia SNP
Consortium 2009) studies indicate that Negrito populations
are closer to their neighboring non-Negrito communities.
Senoi, who are AA speakers, make up the largest group
among the OA populations. They traditionally practice slash-
and-burn farming and their phenotypic features are interme-
diate between Australoid and Mongoloid people. The origin
of the Senoi is obscure; however, based on archeological and
limited genetic studies, they have been linked with AA agri-
culturists from mainland SEA or South China who arrived
in Peninsular Malaysia in the mid-Holocene (Hill et al. 2006).
Proto-Malays exhibit Mongoloid feature and speak
Austronesian dialects. They are taller, fairer, and may have
straighter hair. These are the agriculturists and fishermen
who are believed to have settled in coastal areas of Malaysia
during the Austronesian (out-of-Taiwan) expansion.
Previous studies of these Malaysian populations have relied
on relatively small sample sizes and low density genetic mar-
kers, limiting the power of the analysis. Here, we provide a
more comprehensive insight and better estimate of diver-
gence time for populations in SEA, by leveraging on larger
sample sizes on very high-density Illumina HumanOmni 2.5
BeadChip arrays. We first investigated how distinct OAs are
from other Asian populations, quantifying genetic structure
within the Asian continent. We also examined linkage disequi-
librium (LD) decay and runs of homozygosity (ROH) to study
population history and consanguinity. Finally, we examined
gene flow between OA population and other populations in
East Asian (EA) and estimated the divergence time for these
populations to elucidate events involved in the peopling
of SEA.
Materials and Methods
Ethics Statements, Sample Collection, and Genotyping
This study was approved by the Ministry of Health Malaysia
under National Medical Research Registry MNDR ID #09—
23-3913, JAKOA (Department of Orang Asli Development,
Government of Malaysia) and Monash University Human
Research Ethics Committee.
Following consultation with JAKOA officers in the various
districts in different states, courtesy visits were made to OA
community elders and the rationale of the study and the pro-
cedure of sample collection explained. Once they had agreed
and informed their communities, field visits were carried out.
Individuals who provided informed consent and also answered
questionnaires were included.
Peripheral blood samples were collected from 169 individ-
uals belonging to Negrito (Jehai, Bateq, Kintaq, and Mendriq
subgroups), Senoi (MahMeri and CheWong subgroup), and
Proto-Malay (Seletar, Jakun, and Temuan subgroups) groups
(fig. 1). Genotyping was performed using Illumina Human
Omni 2.5 array (Illumina Inc., San Diego, CA).
Quality Control and Data Integration
Quality controls were applied to the data obtained from each
OA community separately to exclude problematic samples
and single nucleotide polymorphisms (SNPs). All SNPs that
failed the Hardy–Weinberg exact (HWE) test (P < 10
6)
and
displayed missing rates >0.05 across all samples in each pop-
ulation were removed. Additionally, samples with call rate
<0.99 were excluded. Gender concordance was examined
using PLINK v1.07 (Purcell et al. 2007) and samples with incon-
sistency between genotype results and questionnaire-reported
sex were excluded. In order to avoid analysis of close relatives,
unknown relatedness was measured between all pairs of
individuals within each population using PLINK’s (v1.07)
Identity-by-Descent estimation, PI_Hat. An upper cut-off
threshold of 0.375 was set to exclude first-degree relatedness
within each population. Finally, a principal component analysis
(PCA) using EIGENSOFT v3.0 (Patterson et al. 2006)wasper-
formed to remove outliers from each population across first
ten eigenvectors. In the final stage, all OA populations were
merged into one data set and pruned for SNPs that failed
HWE (P < 10
6) test
andmissingratesmorethan0.05across
all samples.
The OA genotype data were merged with data from
Human Genome Diversity Project (HGDP) (Li et al. 2008), 89
Malay individuals from Singapore Genome Variation Project
(SGVP) (Teo et al. 2009) and Onge and Jarawa Negritos from
Andaman islands were genotyped using Illumina Human
1.2M (SNP population data courtesy of P. Majumder and A.
Genetic History of Negritos and Indigenous Populations GBE
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Basu). After merging data sets (supplementary table S1,
Supplementary Material online), a total of 291,096 overlap-
ping autosomal SNPs remained for downstream analysis.
Population Structure Analysis
PCA was used to identify population structure across indige-
nous Malaysians. PCA analysis was performed on genotyped
data of OA combined with Andamanese Negritos, Oceanians,
South and East Asian populations in the HGDP, and Malays
from SGVP using EIGENSOFT v3.0. To balance sample sizes
across our populations, 30 Malay individuals were randomly
sampled from SGVP data set (which contains 89 individuals).
SNPs with r
2
> 0.5 were pruned out in order to avoid the
effects of excessive LD between SNPs. After this pruning a
total of 204,426 SNPs remained for analysis. Pairwise Fst dis-
tance between populations in same data set were calculated
using EIGENSOFT v3.0, and a Neighbor-net tree was con-
structed by SplitsTree v4 software (Huson and Bryant 2006).
ADMIXTURE v1.22, a clustering algorithm, was used on
pruned SNPs to estimate the ancestral population clustering
(Alexander et al. 2009).
PLINK v1.07 was used to estimate ROH in selected popu-
lations. PLINK takes 5,000 kb (50 SNPs) sliding windows across
the genome and allows for 1 heterozygous and 5 missing calls
in each window. To minimize the effects of LD on ROH, min-
imum ROH length was set to be 500 kb because it is unusual
for LD to extend beyond 500 kb. LD decay for each population
was calculated as r
2
using PLINK. Pairwise LD between all
possible SNPs was calculated and mean LD was measured in
bins of 5 kb.
TreeMix v1.12 (Pickrell and Pritchard 2012) was used to
explore the population relationships and migration events.
Same data set described above was used to estimate the
Maximum Likelihood tree with Yoruba as outgroup. We
used blocks of 200 SNPs (-k 200) to account for LD and mi-
gration edges added sequentially until the model explained
99% of variances. We estimated the D statistics using
ADMIXTOOLS (Patterson et al. 2012) to examine gene flow
between OAs and surrounding populations. Divergence time
between OA and EA was estimated using 399,971 shared
SNPs between our data and HapMap 3 (The International
HapMap 2005). Effective population size (N
e
) and divergence
time between OAs and Yoruba in Ibadan (YRI), Han Chinese in
Beijing (CHB), and Japanese in Tokyo (JPT) samples were esti-
mated according to the method suggested by McEvoy et al.
(2011). To estimate LD, pairwise LD was calculated as r
2
using
PLINK v1.07. In order to minimize the effects of small sample
size, all individuals were pooled together in their respective OA
groups. Admixture time between OAs and EA was estimated
by rolloff package using 399,971 SNPs by HapMap3 and OAs.
Results
To understand population structure across Negritos, other OA
subgroups, and their relationship with neighboring popula-
tions in Asia and Oceania, a PCA was performed (fig. 2 and
supplementary fig. S1, Supplementary Material online). As
presented in figure 2A, the first component, which captures
FIG.1.Geographical location of Orang Asli communities recruited in this study.
Aghakhanian et al. GBE
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32% of total variation, clearly distinguishes South Asian pop-
ulations from those in the East. From PC2, the Onge and
Jarawa, both Negrito subgroups, clustered together and
were distinct from other populations. However, they ap-
peared closest to Papuans and Melanesians. The Malaysian
Negrito subgroups, while clustering closer to East Asians,
showed a tendency toward other Negrito subgroups in
Oceania and Andaman islands. The rest of OAs such as
Senoi and Proto-Malays as well as Singaporean Malays were
located between Malaysian Negritos and East Asian clusters
indicating that these groups might be admixed between these
two populations. However, both Senoi and Proto-Malay
groups lay closer to East Asians on PC4 suggesting that all
these populations may have a common origin.
Like PCA analysis, the results of Neighbor-net tree showed
that OAs are closest to EA populations. As evident in supple-
mentary figure S2, Supplementary Material online, all four
subgroups of Negritos formed a clade, while Senoi and
Proto-Malay were positioned at various points between
these two clades. The long branches observed in Bateq,
Jehai, Kintaq, CheWong, Seletar, and MahMeri suggest
strong drift in each of these populations. Interestingly,
Seletar located between Malaysian Negritos and Oceanians.
The tree also indicated genetic affinity between Andamanese
and Oceanians.
In order to determine critical ancestral components that
may have shaped the genetic architecture among the OAs,
we applied ADMIXTURE analysis. The results of ADMIXTURE
from K =2 to K = 12 are shown in figure 3. Each individual is
represented as a vertical bar and their corresponding ancestry
components are shown by different colors. Different colors
indicate different ancestry lineages. As presented, K =2 sepa-
rated Central-South Asia (red) and EA (yellow) and the latter
appears to be the major component in all OA groups. From
K = 3, Andamanese component (pink) appeared. This compo-
nent also presented considerably in Oceanians and in lesser
extent in Malaysian Negritos. At higher K =4 and K =5,
Negrito (dark green) and Oceanian (dark blue) components
appeared respectively. The best model which had the lowest
cross validation error suggests nine major ancestral groups
which gave rise to the 40 distinct populations included in
our study. At K = 9, all Negrito subgroups showed similar an-
cestral patterns. However, we observed small portions of
other ancestral components (shown in yellow and purple) in
some Negrito individuals (especially Mendriqs).
Results of ADMIXTURE at K = 9 also showed that two Senoi
subgroups had different ancestral patterns. The purple colored
ancestry component is highest in MahMeri, but also present in
the Proto-Malay and Malay. The CheWongs appear to have
MahMeri, Negrito, and East Asian components. At K =11,
CheWong appeared distinct.
Different patterns of ancestry were identified in Proto-
Malays. At K = 9, Jakun and Temuan had similar ancestral
components, but there was a unique substantial component
(shown in light blue) only present in the Seletar from K =6.
The ADMIXTURE results further support the uniqueness
of OAs.
To understand the relationship between our populations
and examine the gene flow between them, we used
TreeMix (fig. 4 and supplementary fig. S3, Supplementary
Material online). Using Yoruba as root, the graph that best
fits our data (99.4% of variances) inferred six migration
events. The tree topology was consistent with geographical
distribution of populations and with previously shown
Neighbor-net tree. Andamanese and Oceanians grouped to-
gether in a deep clade, while all OA groups formed a distinct
cluster. Focusing on migration events, a migration (migration
weight 0.37) directed from root Onge and Jarawa toward
FIG.2.PCA of Orang Aslis and surrounding populations.
Genetic History of Negritos and Indigenous Populations GBE
Genome Biol. Evol. 7(5):1206–1215. doi:10.1093/gbe/evv065 Advance Access publication April 14, 2015 1209
Malaysian Negrito root. The resulting tree also highlighted
another migration (0.39) from the root of Bateq and Jehai
to CheWong.
To further investigate gene flow between OAs and other
populations, we used D statistics (table 1 and supplementary
tables S2 and S3, Supplementary Material online). The com-
puted D statistics demonstrated significant gene flow be-
tween Andamanese and Malaysian Negritos but there was
no significant gene flow detected between Andamanese
and other OA groups. This suggests that an earlier gene
flow occurred before other OA groups arrived in Peninsular
Malaysia. The D statistics supported admixture between dif-
ferent OA groups, as gene flows between Negrito/Senoi,
Negrito/ Proto-Malays, and Senoi/Proto-Malays were evident.
We also traced admixture in AA-speaking OAs and those of
Mainland SEA and Lahu and Dai, ethnic groups from South
China.
Focusing on OAs in Malaysia, we determined inheritance of
parental genome components, and calculated ROH in all OA
groups against Malay from Singapore. Figure 5A shows the
distribution of ROH in these populations. As expected, all
Negrito groups generally showed long and high ROH com-
pared with other OA groups. This is indicative of small popu-
lation size or consanguinity. Interestingly, Seletar had the
longest ROH among all OA groups which may reflect higher
levels of autozygosity.
To further examine the genetic isolation and admixture
between OA groups, we calculated pairwise LD between all
autosomal SNPs. LD is the nonrandom association of two SNPs
and its decay can be affected by factors like drift, admixture,
and inbreeding. Figure 5B shows the LD decay in OA sub-
groups and Singaporean Malays. LD in all OA groups was
markedly higher even for long pairwise SNPs distances.
We estimated the divergence time (T) of OA groups and
Africans to be around 67 KYA assuming generation time of 25
years which is a good agreement with other reported estima-
tions of EA and African divergence previously (McEvoy et al.
2011; Pugach et al. 2013). Our results inferred earlier diver-
gence of Negritos from EA in 14–15 KYA which predate those
of Senoi (10–11 KYA) and Proto-Malay (8–9 KYA) (table 2).
Admixture time estimation between OA groups using
“rolloff” showed that the admixture date between Negrito
and Senoi to be around 40 generations which was older than
Negrito/Proto-Malay and Senoi/Proto-Malay admixture which
occurred around 20 generations before the present.
Discussion
Despite the rich ethnic diversity present in SEA, the region has
been underrepresented in large-scale international genome
data sets such as HAPMAP and 1000 Genome Project
(LuandXu2013). Diverse linguistic, morphological, and an-
thropological characteristics found in minor ethnic groups of
Malaysia, known as OA, offered a promising opportunity to
understand the populations of East Asia and SEA.
Our investigation has contributed substantially more data
and provided more comprehensive insight into the population
structure of diverse indigenous groups and their prehistoric
links to other populations in mainland SEA and East Asia.
Apparently, the OAs are genetically closer to EA populations
compared with those in South Asia or Oceania. However, our
results provided evidences supporting genetic affinity be-
tween Malaysian and Andamanese Negritos. Our results are
entirely consistent with other SNP studies suggesting link be-
tween Andamanes, Malaysian Negritos, and Melanesians
(Reich et al. 2011; Chaubey and Endicott 2013).
FIG.3.ADMIXTURE analysis of Orang Asli, Andamanese, South Asian, and East Asian ethnic groups from HGDP and Singaporean Malay.
Aghakhanian et al. GBE
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On a finer scale, Malaysia Negrito subgroups were clearly
different from EA populations. This distinct pattern may have
resulted from genetic drift. It is also conceivable that they had
longer periods of isolation from other inhabitants in the
region, as indicated by Fst and LD decay. The ancestral com-
ponent (dark green) belonging” to Malaysian Negritos
was also spread among Southeast Asian and Southern
Chinese populations. However, although Negritos
FIG.4.Treemix tree of Orang Asli subgroups, Negrito groups of Andaman Islands, and South and East Asian populations from HGDP.
Genetic History of Negritos and Indigenous Populations GBE
Genome Biol. Evol. 7(5):1206–1215. doi:10.1093/gbe/evv065 Advance Access publication April 14, 2015 1211
predominantly shared this ancestral component, the
Mendriq shared more portions of other ancestral components
with East Asians and Senoi. This suggests more recent
gene flow between them and their neighboring popula-
tions, most likely Malays. A similar observation was reported
in Jehai, a Negrito subgroup using a less SNP (Jinam et al.
2013).
The Senoi and Proto-Malay were closely related to EA,
either because they share relatively recent common ancestors
or because of recent gene flow. However, different patterns
emerged in Seletar and CheWong. The corresponding ances-
tral component of Seletar, a subgroup of Proto-Malay,
emerged at K = 6 in ADMIXTURE and Neighbor-net tree
showed an affinity to the Oceanian. Anthropological informa-
tion regarding origins of the Seletar is scarce and anecdotal.
There is a paucity of information about this community. It is
plausible that Seletar might have experienced a recent
bottleneck as suggested by the long stretches of LD in their
genome. The low levels of mtDNA diversity (Jinam et al. 2012)
also provide support for the likelihood of a bottleneck in this
population. ADMIXTURE and TreeMix results from CheWong
suggest that they are intermediate between Negritos and
Senois. Because CheWong appeared distinct at K =11, it
can be inferred that their ancestors experienced one or possi-
bly more admixture events in the past, and later became iso-
lated from founding populations. The argument for
CheWongs to be admixed is supported by several factors.
First, the cultural practices of CheWong are more similar to
other Senoi rather than Negritos, while their language is
northern Aslian, similar Negrito dialects. Physically, they
appear to have intermediate phenotypes between Negrito
and Senoi. The genetic evidence presented here for the first
time may reduce disagreement among various anthropolo-
gists who study tribes in SEA (Benjamin 2013).
Table 1
Computed D Statistic Results Showing Gene Flow between Negrito and Other Populations in SEA
Group D Score Z Score
a
Group D score Z score
D (Jehai, Yoruba; Han, X) D (Jehai, Yoruba; Japanese, X)
Temuan 1.16 10
02
10.578 Temuan 1.82 10
02
15.328
Jakun 1.44 10
02
11.125 Jakun 2.10 10
02
15.438
Seletar 2.00 10
03
1.397 Seletar 8.80 10
03
5.923
MahMeri 9.30 10
03
6.931 MahMeri 1.60 10
02
11.455
CheWong 3.47 10
02
21.149 CheWong 4.11 10
02
24.359
Malay 6.00 10
04
0.744 Malay 7.40 10
03
7.999
Cambodian 2.40 10
03
2.464 Cambodian 9.20 10
03
8.482
Lahu 6.10 10
03
5.612 Lahu 1.30 10
02
10.602
Dai 7.80 10
03
8.537 Dai 1.46 10
02
13.701
D (Bateq, Yoruba; Han, X) D (Bateq, Yoruba; Japanese, X)
Temuan 1.02 10
02
9.277 Temuan 1.56 10
02
12.926
Jakun 1.44 10
02
10.388 Jakun 1.98 10
02
13.604
Seletar 1.80 10
03
1.25 Seletar 7.30 10
03
4.733
MahMeri 7.90 10
03
5.84 MahMeri 1.33 10
02
9.404
CheWong 3.63 10
02
19.949 CheWong 4.14 10
02
22.355
Malay 2.00 10
04
0.206 Malay 5.40 10
03
5.496
Cambodian 1.70 10
03
1.635 Cambodian 7.20 10
03
6.351
Lahu 4.60 10
03
4.032 Lahu 1.02 10
02
8.124
Dai 6.70 10
03
7.09 Dai 1.23 10
02
11.212
D (Kintaq, Yoruba; Han, X) D (Kintaq, Yoruba; Japanese, X)
Temuan 1.02 10
02
9.504 Temuan 1.65 10
02
14.175
Jakun 1.26 10
02
9.693 Jakun 1.88 10
02
13.683
Seletar 1.80 10
03
1.213 Seletar 8.10 10
03
5.271
MahMeri 8.20 10
03
6.342 MahMeri 1.44 10
02
10.695
CheWong 3.40 10
02
20.837 CheWong 4.00 10
02
23.709
Malay 0.00 0.056 Malay 6.40 10
03
6.985
Cambodian 2.00 10
03
2.089 Cambodian 8.30 10
03
7.879
Lahu 5.20 10
03
4.788 Lahu 1.16 10
02
9.756
Dai 7.20 10
03
7.749 Dai 1.36 10
02
12.934
a
Absolute Z score >3 shows significant gene flow between populations.
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The extent of ROH which are identical segments of an in-
dividual’s genome inherited from each parent may be indica-
tive of historical events such as bottlenecks, isolation, and
consanguinity within populations. Our findings of markedly
longer ROH in Negritos, who are the smallest OA group and
fast dwindling, may be due to their small population size and
isolation after an early divergence. Given that marriages be-
tween siblings and cousins are generally prohibited in current
Negrito communities, inbreeding is unlikely to have occurred,
although we cannot discount this entirely (Benjamin 2013).
They traditionally live in small groups composed of few fam-
ilies; so maintaining a small population over time may have
resulted in enriched ROH among them. This parallels some
African forager communities that have same lifestyle as
hunter-gatherer Negritos (Petersen et al. 2013; Patin et al.
2014).
The longest ROH observed in Seletar may best be explained
by the occurrence of a population bottleneck. In contrast,
other Proto-Malay groups had shorter and fewer ROH com-
pared with Seletar reflecting their larger outbred communi-
ties. LD in Negritos was generally higher compared with other
OA groups, a likely consequence of their isolation. The LD
patterns from our results are similar to those reported for
other isolated groups in Africa and Europe (Gross et al.
2011; Esko et al. 2013; Patin et al. 2014).
The Negrito divergence time is consistent with archeologi-
cal findings regarding the advent of Hoabinhian culture in
Mainland SEA (Bellwood 2007). The genetic evidence sup-
ports the view that Malaysian Negritos are descendants of
Hoabinhian hunter-gatherers who occupied northern parts
of Peninsular Malaysia during late Pleistocene. These hunter-
gatherers later interacted with Senoi agriculturists during early
Holocene era. It may have been these agriculturists who may
have introduced AA-based Aslian languages to Negritos. This
time frame also coincides with the Early Train migrations from
north to south approximately 10–30 KYA (Jinam et al. 2012).
However, our time estimation on LD decay can be affected by
any bottleneck experienced by these groups. It has been
shown that bottlenecks may result in overestimations of LD
in populations which consequently result in underestimation
of Ne and divergence time. Nevertheless, there are some chal-
lenges associated with our investigation. The ascertainment
bias that may be present may affect LD estimation. The con-
siderable difference between Negrito/Senoi and Negrito/
Proto-Malay admixture date may suggest that the migration
of Senoi ancestors to the Malaysian peninsular occurred earlier
than those of Proto-Malays. The latter are believed to be a part
of Out-of-Taiwan Austronesian expansion. However, our ad-
mixture time estimation seems to be much earlier than arche-
ological reports. In the absence of better analytical methods,
our analysis relied on rolloff which may reflect only the most
recent admixture event, rather than anything earlier.
To circumvent inaccuracy and further refine divergence
times, we performed D statistics to trace ancient admixture
within different OA groups and between OAs and other
FIG.5.(A) Runs of homozygosis in Orang Aslis and Malay from SGVP and (B) pattern of linkage disequilibrium decay in Orang Asli groups and SGVP
Malay.
Table 2
Divergence Time (KYA) Estimation between OA Groups and YRI, CHB,
and JPT
YRI CHB JPT
Negrito 66.8 14.5 14.6
Senoi 67.5 10 11
Proto-Malay 66.9 8.2 9.2
YRI 72 72
Genetic History of Negritos and Indigenous Populations GBE
Genome Biol. Evol. 7(5):1206–1215. doi:10.1093/gbe/evv065 Advance Access publication April 14, 2015 1213
populations in EA. Interestingly, we report gene flow
between AA-speaking OAs and Mainland Southeast Asia
(MSEA) and Southern Chinese populations. Existence of
Negrito ancestral components in some MSEA has been
reported by previous studies (HUGO Pan-Asia SNP
Consortium 2009).
In summary, we have demonstrated that the current OA
while related, are genetically distinct. The Negritos are very
different both phenotypically and genetically. The detailed re-
sults we have obtained lead us to speculate that their ances-
tors contributed significant ancestral genetic components
probably during the late Pleistocene to the populations of
East Asia and SEA. The continuum in divergence times from
Negritos to Senois to Proto Malays coupled with the language
transitions provide support to a narrative of at least three
major human migrations starting with Out of Africa, then
the Early Train followed by Out-of-Taiwan Austronesian
expansion.
Supplementary Material
Supplementary figures S1–S3 and tables S1–S3 are available
at Genome Biology and Evolution online (http://www.gbe.
oxfordjournals.org/).
Acknowledgments
This research was supported by Monash University (Malaysia)
Cardiometabolic Research Strength (CMR Fund No:
5140035), and Ministry of Science, Technology and
Innovation Grant (No: 100-RM1/BIOTEK 16/6/2 B)awarded
to M.E.Phipps and co-investigators. We thank the OA com-
munities of Malaysia for their participation and cooperation
and Department of Orang Asli Development. We are espe-
cially grateful to Dr Analabha Basu and Prof. Partha Majumder
from the National Institute of Biomedical Genetics, India, for
sharing their precious Onge and Jarawa SNP data sets with us
for this analysis. This has helped to shed light on global Negrito
populations. We acknowledge the technical assistance of Prof.
Iekhsan Othman, Mr C.S. Chui, Ms T.Y. Tee, Ms U. Zulaiha, Dr
M.S. Kadir, and Dr A. Kadir. We are also grateful to the staff
from the Institute of Medical Molecular Biotechnology,
Universiti Teknologi MARA, for their participation in the
sample recruitment. The SNP genotype data (devoid of any
personal identification and anonymized) used in the popula-
tion analyses will be made freely available on request to the
corresponding author.
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Supplementary resource (1)

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17 The Maniq of southern Thailand is one of the last remaining practicing hunter-gatherer communities in 18 the world. However, our knowledge on their genetic origins and demographic history is still largely 19 limited. We present here the genotype data covering ~2.3 million SNPs of eleven unrelated Maniq 20 individuals. Our analyses reveal the Maniq to be closely related to the Semang populations of Malaysia 21 (Malay Negritos), who altogether carry an Andamanese-related ancestry linked to the 22 ancient Hòabìnhian hunter-gatherers of Mainland Southeast Asia (MSEA). Moreover, the Maniq 23 possess ~35% East Asian-related ancestry, likely brought about by recent admixture with surrounding 24 agriculturist communities in the region. In addition, the Maniq exhibit one of the highest levels of
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The primary focus of this article is on the so-called negritos of Peninsular Malaysia and southern Thailand, but attention is also paid to other parts of Southeast Asia. I present a survey of current views on the "negrito" phenotype-is it single or many? If the phenotype is many (as now seems likely), it must have resulted from parallel evolution in the several different regions where it has been claimed to exist. This would suggest (contrary to certain views that have been expressed on the basis of very partial genetic data) that the phenotype originated recently and by biologically well-authenticated processes from within the neighboring populations. Whole-genome and physical-anthropological research currently support this view. Regardless of whether the negrito phenotype is ancient or recent-and to the extent that it retains any valid biological reality (which is worth questioning)-explanations are still needed for its continued distinctiveness. In the Malay Peninsula, a distinctive "Semang" societal pattern followed by most, but not all, so-called negritos may have been responsible for this by shaping familial, breeding, and demographic patterns to suit the two main modes of environmental appropriation that they have followed, probably for some millennia: nomadic foraging in the forest, and facultative dependence on exchange or labor relations with neighboring populations. The known distribution of "negritos" in the Malay Peninsula is limited to areas within relatively easy reach of archaeologically authenticated premodern transpeninsular trading and portage routes, as well as of other non-negrito, Aslian-speaking populations engaged in swidden farming. This suggests that their continued distinctiveness has resulted from a wish to maintain a complementary advantage vis-à-vis other, less specialized populations. Nevertheless, a significant degree of discordance exists between the associated linguistic, societal-tradition, and biological patterns which suggests that other factors have also been at play.
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Southeast Asia houses various culturally and linguistically diverse ethnic groups. In Malaysia, where the Malay, Chinese, and Indian ethnic groups form the majority, there exist minority groups such as the "negritos" who are believed to be descendants of the earliest settlers of Southeast Asia. Here we report patterns of genetic substructure and admixture in two Malaysian negrito populations (Jehai and Kensiu), using ~50,000 genome-wide single-nucleotide polymorphism (SNP) data. We found traces of recent admixture in both the negrito populations, particularly in the Jehai, with the Malay through principal component analysis and STRUCTURE analysis software, which suggested that the admixture was as recent as one generation ago. We also identified significantly differentiated nonsynonymous SNPs and haplotype blocks related to intracellular transport, metabolic processes, and detection of stimulus. These results highlight the different levels of admixture experienced by the two Malaysian negritos. Delineating admixture and differentiated genomic regions should be of importance in designing and interpretation of molecular anthropology and disease association studies.
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The indigenous inhabitants of the Andaman Islands were considered by many early anthropologists to be pristine examples of a "negrito" substrate of humanity that existed throughout Southeast Asia. Despite over 150 years of research and study, questions over the extent of shared ancestry between Andaman Islanders and other small-bodied, gracile, dark-skinned populations throughout the region are still unresolved. This shared phenotype could be a product of shared history, evolutionary convergence, or a mixture of both. Recent population genetic studies have tended to emphasize long-term physical isolation of the Andaman Islanders and an affinity to ancestral populations of South Asia. We reexamine the genetic evidence from genome-wide autosomal single-nucleotide polymorphism (SNP) data for a shared history between the tribes of Little Andaman (Onge) and Great Andaman, and between these two groups and the rest of South and Southeast Asia (both negrito and non-negrito groups).
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