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Discerning the Origins of the Negritos, First Sundaland People:
Deep Divergence and Archaic Admixture
Timothy A. Jinam1, Maude E. Phipps2, Farhang Aghakhanian2, Partha P. Majumder3, Francisco Datar4,
Mark Stoneking5, Hiromi Sawai6, Nao Nishida6,7, Katsushi Tokunaga6, Shoji Kawamura8, Keiichi Omoto9, andNaruya Saitou1,*
1

Division of Population Genetics, National Institute of Genetics, Mishima, Japan

2

Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Sunway City, Selangor, Malaysia

3

National Institute of Biomedical Genomics, Kalyani, West Bengal, India

4

Department of Anthropology, University of the Philippines, Diliman, Quezon City, The Philippines

5

Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany

6

Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Japan

7

Department of Hepatic Disease, Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Chiba, Japan

8

Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan

9

Department of Anthropology, Faculty of Science, The University of Tokyo, Japan*Corresponding author: E-mail: saitounr@nig.ac.jp.
Accepted: July 9, 2017

Abstract
Human presence in Southeast Asia dates back to at least 40,000 years ago, when the current islands formed a continental shelf called
Sundaland. In the Philippine Islands, Peninsular Malaysia, and Andaman Islands, there exist indigenous groups collectively called
Negritos whose ancestry can be traced to the “First Sundaland People.” To understand the relationship between these Negrito
groups and their demographic histories, we generated genome-wide single nucleotide polymorphism data in the Philippine Negritos
and compared them with existing data from other populations. Phylogenetic tree analyses show that Negritos are basal to other Eastand Southeast Asians, and that they diverged from West Eurasians at least 38,000 years ago. We also found relatively high traces ofDenisovan admixture in the Philippine Negritos, but not in the Malaysian and Andamanese groups, suggesting independent introgression and/or parallel losses involving Denisovan introgressed regions. Shared genetic loci between all three Negrito groups could
be related to skin pigmentation, height, facial morphology and malarial resistance. These results show the unique status of Negrito
groups as descended from the First Sundaland People.
Key words: Negritos, Southeast Asia, admixture, population genetics, Denisovan.

Introduction
The question of how and when anatomically modern
humans made the journey out of Africa and into all corners of the world has been of great interest. It is widely
acknowledged that humans have been in Southeast Asia
(SEA) at least 40–50 thousand years ago (ka). Indeed, human remains found in Niah Cave in Borneo (Barker et al.
2007), Callao Cave in the Philippines (Mijares et al. 2010)
and Tam Pa Ling in Laos (Demeter et al. 2012) were dated
to approximately that time period. Then, the current
islands of Sumatra, Java and Borneo were connectedwith the Asian mainland, forming the landmass known
as Sundaland. A cluster of islands separate Sundalandfrom another landmass called Sahul, made up of what is
now New Guinea and Australia.
There are several human populations scattered throughout
SEA that are thought to be descendants of the “First
Sundaland People.” They are collectively known as Negritos
and are currently found in the Andaman Islands, Malay
Peninsula and several islands in the Philippines. They havebeen traditionally associated with a hunter-gathering lifestyle,and also exhibit physical features that are distinct from theirß The Author(s) 2017. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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non-Negrito neighbors, namely short stature, frizzy hair, anddark skin (Barrows 1910; Radcliffe-Brown 1922; Evans 1937).
These observations led to the idea that the Negritos might beclosely related to African Pygmies who also exhibit similar
phenotypes (Howells 1973). Alternatively, the similar phenotypes may have arisen due to adaptation to relatively similar
environmental conditions in Africa and Southeast Asia (Coon
1965), namely convergent evolution.
Early genetic studies that utilized various red blood cell
enzymes and serum proteins, led by one of us (K.O.), foundthat Philippine Negritos have closer affinities to Asia-Pacificpopulations than to African Pygmies (Omoto et al. 1978,
1981; Matsumoto et al. 1979; Horai et al. 1981). Later studiesof mitochondrial DNA (mtDNA) reported basal lineages inAndamanese and Malaysian Negritos that date back to theearliest migrations to SEA (Thangaraj et al. 2005; Hill et al.
2006; Jinam et al. 2012). Genome-wide Single Nucleotide
Polymorphisms (SNPs) studies added further insight to thegenetic diversity of Negritos. Using 50k SNPs, the HUGO
Pan-Asian SNP Consortium proposed a single wave of migration into SEA, with the Malaysian and Philippine Negritos as
the forerunners (The HUGO Pan-Asian SNP Consortium2009). Subsequent studies reported various demographic
and evolutionary factors that affected their genetic diversity.
These included admixture (Jinam et al. 2013), long term isolation and bottlenecks (Deng et al. 2014; Aghakhanian et al.
2015), and adaptation to malaria (Liu et al. 2015). However,compared with Malaysian Negritos, genome-wide studies involving the Philippine Negritos are relatively limited.
Here we generated approximately 1 million genome-wide
SNPs in four Negrito (Aeta, Agta, Batak, and Mamanwa) andthree non-Negrito groups (Tagalog, Visayan, and Manobo)
from the Philippines. By combining this newly generated
SNP data with previously published data of Andamaneseand Malaysian Negritos, as well as other neighboring populations in SEA, we sought to obtain a deeper view of thedemographic events that shaped the genetic diversity of the
Negritos. Specifically, we would like to find out when these
populations diverged; are there any genetic similarities among
the Negrito groups; and did admixture with archaic humans
have any impact on their genetic diversity?

Materials and Methods
Sample Information
Peripheral blood samples from the Philippines were collectedby the Japanese–Philippine joint study team headed by one ofus (K. O.), from 1975 to 1985. The DNA were extracted andpurified using the phenol–chloroform method, and were preserved in freezers; now stored at Department of Integrated
Biosciences, Graduate School of Frontier Sciences, theUniversity of Tokyo. A total of 150 individual samples fromfour Negrito groups and three non-Negrito groups were selected for genome-wide SNP typing using the Affymetrix 6.0

2014

array. The sample information is listed in supplementary table
S1 in the Supplementary Material online. The geographicallocations of these populations are depicted in supplementary
figure S1 in the Supplementary Material online. This study has
been approved by the ethics committee of the University ofTokyo (15-223) and the National Institute of Genetics, Japan.

Data Quality Checks
The SNP genotypes, typed at Department of Human Genetics,
Graduate School of Medicine, the University of Tokyo, werecalled using Affymetrix Birdseed v2 algorithm, with a more
stringent confidence score cutoff of 0.1 instead of the default
0.5. In total, genotypes for 868,261 autosomal SNPs wereobtained. Further SNP quality filtering was done using PLINK
software (Purcell et al. 2007) to omit SNPs with <95% genotyping call rate. SNPs that deviated from the Hardy–Weinberg
equilibrium (P < 11010) were also removed from each population separately, before merging the data again.
Closely related pairs of individuals were identified in each
population using KING software (Manichaikul et al. 2010). To
keep as many individuals as possible, only one individual fromeach pair that had first degree relations (estimated kinshipcoefficient >0.25) were removed; in total 25 individuals
were omitted.

Merging with Other Samples
The resulting 782,966 autosomal SNP from the Philippines
were merged with five populations from Malaysia and thePhilippines which were also genotyped using Affymetrix 6.0
arrays (Reich et al. 2011), including two Negrito groups (Jehai
and Mamanwa) and three non-Negrito Austronesian populations (Manobo, Bidayuh, and Temuan). In addition, three
HapMap (International HapMap Consortium 2005) populations (Han Chinese (CHB), Caucasians (CEU) and Yorubans(YRI)) that were genotyped using Affymetrix 6.0 arrays weremerged with the Philippine and Malaysian data sets, resulting
in a total of 519,832 SNPs.
The above data set was further merged with two
Andamanese groups (Jarawa and Onge) (Basu et al. 2015) and
two Malaysian Negrito groups (Bateq and Kintaq) (Aghakhanian
et al. 2015). The number of overlapping SNP loci was 112,845.
We further merged the data with the Human Genome Diversity
Panel (HGDP) SNP data generated with the Affymetrix Human
Origins array (Patterson et al. 2012). The number of available
SNPs after merging was 44,960. The various data sets used for
subsequent analyses are listed in supplementary table S2 in the
Supplementary Material online.

Data Analysis
Principal Component Analysis (PCA) was performed using thesmartpca program from the EIGENSOFT package (Patterson
et al. 2006) to infer relationships at the individual level. In

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addition, ADMIXTURE analysis (Alexander et al. 2009) was
done to assess population substructure and admixture within
individuals. We also performed population level phylogenetic
analysis by constructing Neighbor Joining trees (Saitou andNei 1987) from Nei’s standard genetic distances (Nei 1972)
computed using PHYLIP (http://evolution.genetics.washing
ton.edu/phylip.html). One thousand bootstrap replicates of
the input data were generated to assess the robustness ofthe tree branching patterns. Networks were constructed using the Neighbor-Net algorithm (Bryant and Moulton 2004)
implemented in Splitstree software (Huson and Bryant 2006).
To assess possible geneflow events between populations, weused the treemix software (Pickrell and Pritchard 2012). The
robustness of treemix estimates was tested using 1,000 bootstrap replicates of the input data, generated using perl scripts
developed and used for Jomon ancient genome analysis(Kanzawa-Kiriyama et al. 2017).
We also performed a formal test for admixture using the
D-statistics method from the Admixtools software package(Patterson et al. 2012). The f4 ratio test from the same software
package was used to estimate the proportion of Denisovan
admixture in the various Negrito groups, assuming the tree
topology in supplementary figure S2 in the Supplementary
Material online. We further applied the RD(x) statistic (Qinand Stoneking 2015) to verify Denisovan introgression, taking
into account Neanderthal ancestry in various Southeast Asian
populations. The RD(x) statistics is defined as the ratio of two f4(or D) statistics: f4(Yoruban, Denisovan; French, x)/f4(Yoruban,
Neanderthal; French, x), where x is the test population. Values>1 imply Denisovan ancestry in population x.
Pairwise allele sharing distances (ASD) (Gao and Martin2009) were calculated from the genome sequences ofDenisovan (Meyer et al. 2012), Altai Neanderthal (Prüfer
et al. 2014), a Papuan individual (Green et al. 2010), an
Australian Aborigine (Rasmussen et al. 2011), three Aeta
(Philippine Negrito) individuals (Pagani et al. 2016) and five
Han Chinese (The 1000 Genomes Project Consortium 2015),
using a perl script. The position of the three Aeta individuals
from Pagani et al. (2016) in the PCA plot (supplementary fig.
S3, Supplementary Material online) suggests that they may be
more admixed than some Aeta individuals from our data set.
SNP loci with missing genotypes were omitted and the total
number of SNPs used for this analysis was 753,276. We defined allele sharing (AS) as 1ASD and calculated this statistic
for nonoverlapping blocks of 1,000 SNPs. For each block, we
took the ratio of ASDenisovan/ASNeanderthal in Papuan, Australian
Aborigine, and Aeta separately. Blocks with ratios >1 are putatively enriched with Denisovan ancestry while the rest are
set to zero AS with Denisovan. We further subtracted
ASDenisovan-CHB values from each putative Denisovan shared
block to get a clearer signal of Denisovan ancestry.
To estimate divergence times between pairs of populations, we used the R-package NeON (Mezzavilla andGhirotto 2015) which implements a method based on

patterns of LD and allele frequencies in the genome(McEvoy et al. 2011). From the resulting pairwise estimatesof divergence times, we constructed phylogenetic trees using
the UPGMA method (Sokal and Michener 1958; see Saitou
2013 for the algorithm), because by definition divergencetime estimates should follow a constant rate of evolution.
In order to identify genetic loci that may be shared among
Negrito groups, we calculated pairwise Fst (Weir andCockerham 1984) between Andamanese, Malaysian andPhilippine Negritos and Han Chinese. We then identified
SNP sites that have low Fst among the three Negrito groups(Fst from 0 to 0.05) but high Fst between Negritos and Han
Chinese (>10-fold difference in Fst). The possible functions ofthese SNPs were examined using the Panther Gene Ontology(Thomas et al. 2003).

Results
Population Structure and Admixture
We first investigated the relationship between individuals by
PCA. Figure 1A shows that the first two principal components(PCs) separates the Andamanese, Malaysian Negritos andPhilippine Negritos into distinct clusters. If Papuans andMelanesians were included (supplementary fig. S4A,
Supplementary Material online), the Philippine Negritos werelocated between the Papuans and Malaysian Negritos alongPC2. When the Andamanese individuals were omitted, PC1
separates the Aeta, Agta, and Batak from the other populations
whereas PC2 separates the Mamanwa and Jehai from othergroups (fig. 1B). The Agta, Aeta, and Batak individuals form acomet-like pattern along PC1, which may indicate admixture
events. Similarly, the Mamanwa also showed the comet-likepattern along PC2. The PCA plot without Agta and Aeta (supplementary fig. S4B, Supplementary Material online) places the
Batak close to the non-Negrito Philippine groups, suggesting ahigh proportion of admixture. The Manobo and Mamanwa,
both living in northern Mindanao, have a high affinity as several
Manobo individuals clustered with the Mamanwa (fig. 1B andsupplementary fig. S4B, Supplementary Material online).
The results of ADMIXTURE analysis from k ¼ 2–7 areshown in figure 2. The cross-validation error assuming k ¼ 1
to k ¼ 9 number of clusters shows that k ¼ 7 has the lowest
error (supplementary fig. S5, Supplementary Material online).
The orange-colored component is highest in theAustronesian-speaking non-Negrito groups, with varying proportions in the four Philippine Negritos, suggesting admixture.
Among the Philippine Negrito groups, the Batak have thehighest proportion of this orange component, corresponding
well to their close proximity to the non-Negritos in the PCA
plot (fig. 1B). From k ¼ 6, the Mamanwa have their own genetic component (white), and at k ¼ 7, the Batek were differentiated from other populations (yellow). These observations
suggest that the Mamanwa and Batek have experienced asubstantial amount of long-term genetic drift. To verify the

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FIG. 1.—Principal component analysis plot of (A) Andamanese(Jarawa and Onge), Malaysian Negritos (Batek, Jehai, and Kintak), andPhilippine Negritos (Aeta, Agta, Mamanwa, and Batak) with nonNegrito Southeast Asians; (B) Malaysian and Philippine populations. MlyNN, Malaysian non-Negritos (Temuan and Bidayuh); Phil-NN, Philippine
non-Negritos (Tagalog and Visayan; Manobo was treated separately).

presence of admixture, we used the D-statistic (Patterson
et al. 2012). The results for D(Philippine Negrito,
Andamanese; French, x), are shown in supplementary figure
S6 in the Supplementary Material online. A negative Z-score
implies gene flow between the Philippine Negritos and population x; highly negative Z-scores were observed forPhilippine Negritos and Philippine non-Negritos, suggesting
gene flow tended to involve groups that are geographicallyclose. We classified individuals from Aeta, Mamanwa andManobo groups who have less than 60% of their corresponding ancestral component proportion based on ADMIXURE
result at k ¼ 6 as highly admixed. In total, 22 individuals
were omitted from subsequent population-based analyses.

Phylogenetic Relationships
To investigate the relationships among Negritos, we constructed a Neighbor-Joining (NJ) tree (fig. 3A). This tree shows

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that following the split from Europeans, the Papuans/
Melanesians, Andamanese, Malaysian, and Philippine
Negritos all appear basal to other Southeast and East Asian
populations, in that branching order. The Neighbor-Net network in supplementary figure S7 in the Supplementary
Material online shows a relatively long split (in blue color)that groups the Andamanese and Malaysian Negritos whilea short split (in red color) groups the Andamanese andPhilippine Negritos together. There is another split that separates all three groups of Negritos (except Batak) from theremaining populations. These splits suggest some shared genetic components that may not be immediately evident fromother methods such as phylogenetic trees or PCA.
The treemix result assuming five gene flow events is shownin figure 3B. For visual clarity, only three gene flow events areshown. The gene flow from Denisovan to Papuan was observed 811 times out of 1,000 bootstrap replicates, and isconsistent with previous reports (Reich et al. 2011; Meyer
et al. 2012; Malaspinas et al. 2016). Interestingly, a separate
gene flow event from Denisovan to some Philippine Negritos(Agta and Aeta) was also inferred. Out of 1,000 bootstrap
replicates, this gene flow event was observed 733 times.
Assuming the migration weight is analogous to gene flow
proportion, the estimated gene flow from Denisovan to
Papuan and from Denisovan to Philippine Negritos were
4.6% and 1.4%, respectively. The gene flow event with thehighest migration weight was from the Malaysian nonNegritos to the Malaysian Negritos, at 47%. However, it
was only observed 69 times out of 1,000 bootstrap replicates.
In the remaining bootstrap replicates, the gene flow directions
were from various positions along the internal branches of theSoutheast/East Asian cluster towards Malaysian Negritos.
Other inferred events involved gene flows from the French
to Cambodians and from Malaysian Negritos to non-Negrito
Southeast Asians and Han Chinese (supplementary fig. S8,
Supplementary Material online). The topology of the treemix
output was different from the NJ tree which assumed no geneflow (fig. 3A). The low bootstrap probabilities suggest that thetreemix tree topology may not be reliable.

Denisovan Introgression
We estimated the proportion of the Denisovan ancestry in SEA
populations using the f4 ratio test, assuming the tree topology
in supplementary figure S2A in the Supplementary Material
online. If the positions of Han Chinese and Denisovan areswapped (supplementary fig. S2B, Supplementary Material online), the estimated Denisovan ancestry proportion remains thesame, but the Z-scores were very high (supplementary table S3,
Supplementary Material online). In either case, our results showthat the Papuans have the highest Denisovan genetic component, consistent with a previous result (Meyer et al. 2012).
Philippine Negritos also show a substantial proportion of
Denisovan components, particularly in the Aeta (fig. 4).

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FIG. 2.—Results of ADMIXTURE assuming k ¼ 2 to k ¼ 7. Each vertical line represents an individual and the different colors represent various ancestry
components. Negrito group labels are indicated in red text.

We further applied the RD(x) test (Qin and Stoneking 2015) toconfirm the presence of Denisovan introgression, taking into
account possible Neanderthal introgression (see “Materials
and Methods” section). Populations with RD(x) values >1 possibly carry Denisovan ancestry and supplementary figure S9 in
the Supplementary Material online shows that the Papuans,
Melanesians, and Philippine Negritos (Aeta) may indeed haveexperienced Denisovan gene flow.
To investigate whether these high proportions of Denisovan
ancestry in the Papuans and Aeta were at the same genetic loci,we calculated Denisovan allele sharing within nonoverlapping
blocks of 1,000 SNPs. The distribution of those blocks showsthat the highest allele sharing with Denisovans was in theAustralian Aborigine, followed by Papuan and Aeta (fig. 5A).
If only Papuans and Aeta are considered, 45.7% of those blocks
are present in both groups, while 40% and 14.3% are foundonly in Papuans and Aeta, respectively. The distribution ofblocks in chromosome 3 is an example of the different patternsof Denisovan allele sharing in Papuans and Aeta (fig. 5B).

Divergence Time Estimates
We estimated divergence times using three data sets withdifferent number of SNPs: 110k, 290k and 480k

(supplementary table S2, Supplementary Material online) as
increasing the number of loci (L) decreases the number of
populations for which individuals sampled from the populations have on these L loci. Assuming a generation time of
30 years (Fenner 2005), the pairwise divergence times using
three data sets are listed in supplementary table S4 in the
Supplementary Material online and the UPGMA tree representing the 480k SNP data is shown in supplementary figure
S10 in the Supplementary Material online. The topology of
the UPGMA tree is similar to the NJ tree in figure 3A, with
the Negritos basal to other Southeast and East Asians. The
Negritos diverged from Europeans 30–38 ka, whereas the
split time of Malaysian and Philippine Negritos was 13–15 ka,
depending on the SNP data set used.

Shared Genetic Loci among the Negritos
In order to investigate shared genetic loci among theAndamanese, Malaysian and Philippine Negritos, we identified SNP loci that have low Fst values among the three Negrito
groups, but high Fst values between Negritos and nonNegritos (Han Chinese). Of the 112,845 SNPs, 4,313 met
the cutoff values (see “Materials and Methods” section). Of
these, 41% are located within genes. The biological processes

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FIG. 3.—(A) Neighbor-joining tree constructed from Nei’s standard genetic distance. (B) Maximum-likelihood tree generated using Treemix, assuming
five geneflow events, with only three shown for clarity. Numbers in red and blue text represent average migration weights and bootstrap supports forbranches out of 1,000 bootstrap replicates, respectively. Only bootstrap values >50% are shown.

(Adhikari et al. 2016). Details of those SNPs are listed in supplementary table S6 in the Supplementary Material online.

Discussion

FIG. 4.—Estimation of Denisovan ancestry in Southeast Asians using
the f4 ratio test. Proportion of Denisovan ancestry is represented by colored circles. Phil-NN, Philippine non-Negritos (Tagalog, Visayan, andManobo).

that are associated with those genes are listed in supplementary table S5 in the Supplementary Material online.
Interestingly, some of those genes were associated with certain phenotypes identified from genome-wide association
studies. OCA2 and SLC45A2 were associated with skin pigmentation (Stokowski et al. 2007), ACAN and ADAMTS17
with height variation (Wood et al. 2014), and PAX3, PREP,
and GRID1 were associated with facial and scalp features

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This study presents an encompassing view of early humanmigrations into Southeast Asia (SEA) by analyzing genomic
data of Negrito groups from three geographical regions.
Although most of these Negrito groups currently reside inrural and sometimes inaccessible areas, they are far from being genetic isolates, as shown by PCA, ADMIXTURE, andD-statistics analyses. A clear difference was observed between
the Mamanwa individuals from this sampling and the ones
collected fairly recently (<10 years ago) on the Neighbor-Net
network (supplementary fig. S11, Supplementary Material online). Recently sampled Mamanwa were closer to the
Manobo, a non-Negrito group. This suggests admixture has
been and probably still is occurring between the Mamanwa
and Manobo. Alternatively it may imply a bias in sampling ofindividuals that represent a population. The consequence of
this admixture is also reflected in mtDNA and Y-chromosomal
diversity of the Philippine Negritos (Delfin et al. 2011, 2014).
An early study in the 1980s based on classic genetic
markers hypothesized a dual origin of the Philippine Negrito
populations. Genetic distance and phylogenetic analyses indicated that the Mamanwa were distinct from the Aeta andGenome Biol. Evol. 9(8):2013–2022 doi:10.1093/gbe/evx118 Advance Access publication July 11, 2017

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FIG. 5.—(A) Distribution of Denisovan allele-sharing in Papuan, Australian Aborigine, and Aeta (Philippine Negrito) published genome sequences,calculated in 1,000 SNP blocks and (B) Example of Denisovan allele-sharing patterns in chromosome 3 for Papuan and Aeta.

other populations. Together with the fact that the mean stature of Mamanwas was taller than the Aeta, it was postulated
that while the Aeta originated from Sundaland, theMamanwa had roots in Wallacea, south of Mindanao(Omoto 1984). In this study, however, this hypothesis was
not substantiated by the genome-wide phylogenetic analyses.
Divergence time estimates using an LD based method
yielded similar results even when using different SNP data
sets. However, our divergence time estimates might possibly
be underestimated. In equations Ne ¼ 1/(4c) * [(1/r2)2] andT ¼ 2Ne*Fst (24), where Ne is effective population size, c isrecombination distance between loci, r2 is the measure of LD,
and T is divergence time in generations, the estimate of T
decreases with increasing LD (r2). However, large blocks of
SNPs in high LD in populations that have experienced extreme
genetic drift from severe bottlenecks and/or recent admixture,
may lead to an underestimation of T. Indeed our results suggest that Malaysian and Philippine Negritos did experience
admixture with their neighboring non-Negrito populations(figs. 1 and 2; supplementary fig. 6, Supplementary Material
online). We tried to mitigate the effects of recent admixture

by filtering out admixed individuals based on PCA andADMIXTURE results.
Both our NJ (fig. 3A) and UPGMA (supplementary fig. S10,
Supplementary Material online) trees show that after divergence from Europeans, the ancestral Asians subsequently split
into Papuans, Negritos and East Asians, implying a one-wave
colonization of Asia. This is in agreement with a previous
survey using a less dense SNP microarray (HUGO Pan-Asian
SNP Consortium 2009) and another study using genome
sequences of Andamanese individuals (Mondal et al. 2016).
This is in contrast to the study based on whole genome
sequences that suggested Australian Aboriginal/Papuan first
split from European/East Asians 60 ka, and later Europeans
and East Asians diverged 40 ka (Malaspinas et al. 2016).
This implies a two-wave migration into Asia (Reich et al.
2011), and other studies using uniparental loci and HLA genes
also seem to support that idea (Oppenheimer 2012; Di et al.
2015). Although our results appear to support the singlewave model, care should be taken in interpreting the NJ
and UPGMA trees, because they do not assume gene flow
after population differentiation. It may be possible that East

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Asians and Negritos appear close in the phylogenetic tree as aresult of a long-term admixture. The method applied in thetreemix software (Pickrell and Pritchard 2012) may be suitablefor inferring gene flow events within a phylogenetic tree, but
the tree topology assuming gene flow has very low bootstrap
support (fig. 3B).
We did not observe any direct links between the different
Negrito groups and the African Pygmies (Biaka) (fig. 3B andsupplementary fig. S12, Supplementary Material online). This
is in agreement with previous results (Omoto et al. 1978,
1981; Basu et al. 2015) and suggests that observed morphological similarities among the Negritos and African pygmies
are more likely due to convergent evolution. The NeighborNet network (supplementary fig. S7, Supplementary Material
online) suggests possible common links among all three
Negrito groups (except highly admixed Batak in the Palawan
Island), which are not obvious in methods like PCA andADMIXTURE (figs. 1 and 2). Previous SNP analyses suggested
a link between the Andamanese and Malaysian Negritos(Chaubey and Endicott 2013; Aghakhanian et al. 2015), but
studies that have shown links among all the three Negrito
groups are few (Reich et al. 2011). We identified 4,313
SNPs that could probably represent genomic regions thatare shared among the common ancestors of the three
Negrito groups. Some of these regions may be related tothe common phenotypes—such as skin pigmentation, height,
and facial morphology—apparent in all Negritos (Stokowski
et al. 2007; Wood et al. 2014; Adhikari et al. 2016). These
SNPs are also found in IL4 and CDH13 genes which are related
to malarial resistance and were candidates for positive selection in the Malaysian Negritos (Liu et al. 2015). These observed patterns may have resulted from natural selection oralternatively via genetic drift, and further analyses are required
to confirm or exclude either case.
Previous studies have reported that Papuans, Melanesians,
and Australian Aboriginal retain high proportions ofDenisovan ancestry, ranging from 3% to 6% (Reich et al.
2011; Meyer et al. 2012; Malaspinas et al. 2016). Here we
report that the Aeta have the highest proportion of Denisovan
ancestry among the four Philippine Negrito groups but
Andamanese and Malaysian Negritos show very low signals(<1%) of Denisovan introgression. To explain these observations, we propose four possible scenarios of Denisovan introgression (supplementary fig. S13, Supplementary Material
online). Two scenarios (differing by invoking either a single
wave or two waves of migration to Asia) involve a single introgression event occurred in the common ancestor of
Papuans and Negritos, followed by parallel losses inAndamanese, Malaysian Negritos and East Asians (supplementary fig. S13A and B, Supplementary Material online).
The differences in the distribution of Denisovan blocks between Papuans and Philippine Negritos (fig. 5) may be attributed to genetic drift or subsequent gene flow from
populations that initially lacked Denisovan ancestry, such as

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non-Negrito Austronesian-speaking groups (supplementary
fig. S6, Supplementary Material online).
Alternatively, independent episodes of Denisovan admixture may have occurred in Papuan and Philippine Negrito
lineages (supplementary fig. S13C and D, Supplementary
Material online). If the Denisovan admixture took place inthe common ancestor of Papuans and Aeta, it must haveoccurred after 50 ka, which is our estimate of PapuanEuropean divergence. Indeed, Malaspinas et al. (2016) estimated that the Denisovan admixture happened 44 ka in the
Papuan/Australian Aborigine common ancestor. Althoughmultiple episodes of Denisovan introgression appears to be
the most parsimonious model, parallel loss of Denisovan ancestry in multiple lineages could also occur via genetic drift or
purifying selection if the Denisovan genetic components aredeleterious, as has been argued for Neanderthal introgression(Sankararaman et al. 2014).
In summary, we demonstrated that the Negritos ofAndaman Islands, Malay Peninsula, and Philippine Islands represent one of the earliest branches of anatomically modern
humans to have reached SEA, befitting the term the “First
Sundaland People” instead of “Negritos.” The interactions
they had with the environment, the pre-existing archaic
humans in the region, and much later with agriculturalist
migrants from the Asian mainland have all shaped their current genetic and cultural diversity.

Supplementary Material
Supplementary data are available at Genome Biology and
Evolution online.

Acknowledgments
This study was supported by JSPS KAKENHI Grant number
15K18622 awarded to T.A.J. and Grant-in-Aid (Grant no.
23247040) from JSPS to K.T. and to N.S. M.E.P. acknowledges the Ministry of Science, Technology and Innovations,
Malaysia for research grant 100-RM1 Biotek 16/6/2B andMonash University Malaysia for TMBG grant. M.S. acknowledges support from the Max Planck Society. P.P.M. expresses
gratitude to the Department of Biotechnology, Governmentof India, for financial support. We also thank Shogo Misawa
and Momoki Hirai for blood sampling in the Philippines,
Masako Mizuguchi for preparing perl scripts, and all the participants who provided samples and information for thisstudy. We also thank Asian DNA Repository Consortium foragreeing the use of these precious DNA samples. The SNP
genotype data used in this study are available upon request
to the corresponding author, along with an appropriate approval of human genomic DNA research ethics committee of
institutions to which researchers involved in the data analyses
belong.

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Discerning the Origins of the Negritos

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