Y-chromosome diversity suggests southern origin and Paleolithic backwave migration of Austro- Asiatic speakers from eastern Asia to the Indian subcontinent OPEN

Abstract

Analyses of an Asian-specific Y-chromosome lineage (O2a1-M95)—the dominant paternal lineage in Austro-Asiatic (AA) speaking populations, who are found on both sides of the Bay of Bengal—led to two competing hypothesis of this group's geographic origin and migratory routes. One hypothesis posits the origin of the AA speakers in India and an eastward dispersal to Southeast Asia, while the other places an origin in Southeast Asia with westward dispersal to India. Here, we collected samples of AA-speaking populations from mainland Southeast Asia (MSEA) and southern China, and genotyped 16 Y-STRs of 343 males who belong to the O2a1-M95 lineage. Combining our samples with previous data, we analyzed both the Y-chromosome and mtDNA diversities. We generated a comprehensive picture of the O2a1-M95 lineage in Asia. We demonstrated that the O2a1-M95 lineage originated in the southern East Asia among the Daic-speaking populations ~20–40 thousand years ago and then dispersed southward to Southeast Asia after the Last Glacial Maximum before moving westward to the Indian subcontinent. This migration resulted in the current distribution of this Y-chromosome lineage in the AA-speaking populations. Further analysis of mtDNA diversity showed a different pattern, supporting a previously proposed sex-biased admixture of the AA-speaking populations in India. There is a broad consensus that modern humans originated in Africa and then migrated to Asia along a coastal route by way of the Indian subcontinent as early as 60 thousand years ago (KYA) 1–7. However, the later dispersion of this ancestral population across Asia is far less clear. Linguistic analyses have grouped Asian populations across eight language families in eastern

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SCIENTIFIC RepoRts | 5:15486 | DOI: 10.1038/srep15486
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Y-chromosome diversity suggests
southern origin and Paleolithic
backwave migration of Austro-
Asiatic speakers from eastern Asia
to the Indian subcontinent
Xiaoming Zhang1,*, Shiyu Liao2,*, Xuebin Qi1,*, Jiewei Liu1,8, Jatupol Kampuansai5,
Hui Zhang1, Zhaohui Yang3,4, Bun Serey6, Tuot Sovannary6, Long Bunnath6, Hong Seang
Aun6, Ham Samnom7, Daoroong Kangwanpong5, Hong Shi3,4 & Bing Su1,4
Analyses of an Asian-specic Y-chromosome lineage (O2a1-M95)—the dominant paternal lineage in
Austro-Asiatic (AA) speaking populations, who are found on both sides of the Bay of Bengal—led to
two competing hypothesis of this group’s geographic origin and migratory routes. One hypothesis
posits the origin of the AA speakers in India and an eastward dispersal to Southeast Asia, while
the other places an origin in Southeast Asia with westward dispersal to India. Here, we collected
samples of AA-speaking populations from mainland Southeast Asia (MSEA) and southern China, and
genotyped 16 Y-STRs of 343 males who belong to the O2a1-M95 lineage. Combining our samples
with previous data, we analyzed both the Y-chromosome and mtDNA diversities. We generated a
comprehensive picture of the O2a1-M95 lineage in Asia. We demonstrated that the O2a1-M95 lineage
originated in the southern East Asia among the Daic-speaking populations ~20–40 thousand years
ago and then dispersed southward to Southeast Asia after the Last Glacial Maximum before moving
westward to the Indian subcontinent. This migration resulted in the current distribution of this
Y-chromosome lineage in the AA-speaking populations. Further analysis of mtDNA diversity showed
a dierent pattern, supporting a previously proposed sex-biased admixture of the AA-speaking
populations in India.
ere is a broad consensus that modern humans originated in Africa and then migrated to Asia along a
coastal route by way of the Indian subcontinent as early as 60 thousand years ago (KYA)1–7. However, the
later dispersion of this ancestral population across Asia is far less clear. Linguistic analyses have grouped
Asian populations across eight language families in eastern Asia and South Asia: Altaic, Sino-Tibetan
1State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of
Sciences, Kunming 650223, China. 2School of Life Sciences, Anhui University, Hefei 230039, China. 3Institute of
Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China. 4Yunnan
Key Laboratory of Primate Biomedical Research, Kunming 650500, China. 5Department of Biology, Faculty of
Science, Chiang Mai University, Chiang Mai 50200, Thailand. 6Department of Geography and Land Management,
Royal University of Phnom Penh, Phnom Penh 12000, Cambodia. 7Capacity Development Facilitator for Handicap
International Federation and Freelance Research, Battambang 02358, Cambodia. 8Kunming College of Life
Science, University of Chinese Academy of Sciences, Beijing 100101, China. *These authors contributed equally to
this work. Correspondence and requests for materials should be addressed to H.S. (email: shih@kmust.edu.cn) or
B.Su. (email: sub@mail.kiz.ac.cn)
Received: 18 May 2015
Accepted: 28 September 2015
Published: 20 October 2015
OPEN

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(ST, split into Han and Tibeto-Burman (TB) sub-branches), Daic, Hmong-Mien (HM), Austro-Asiatic
(AA), Austronesian (AU), Dravidian (DR) and Indo-European (IE). With wide distribution in mainland
China and Siberia, both Altaic and ST form two northern language families, DR and IE comprise the two
main language families of the Indian subcontinent, while Daic, HM, AA and AU make up the southern
language families that are primarily distributed in southern China and Southeast Asia.
Trying to use linguistic families to map out the origin and migration patterns of human populations
in Asia has resulted in far less consensus. For example, of the southern language families, AA has a some-
what unique geographic distribution, with a wide distribution not only in southern China and Southeast
Asia, but also in India. Subsequently, AA is the eighth largest language family in the world in terms of
population size (104 millions)8 with two major branches: Munda in eastern, northeastern and central
India and Mon-Khmer, which stretches from northeastern India to the Andaman-Nicobar islands, Malay
Peninsula and vast Mekong delta in MSEA. AA is the rst language of many ethnic groups in Cambodia,
Vietnam, Laos, ailand, Burma and Malaysia, and serves as the main ocial language in Cambodia and
Vietnam. Taking these realities into account, decades of research has resulted in long-standing debate
about the geographic origin and prehistoric migratory route of the AA-speaking populations.
Similarly, analysis of genetic data to characterize the origin and migration history of AA-speaking
populations has led to two rival hypotheses9–15. Data from the maternal lineage (mtDNA) makes a clear
distinction between Munda-speakers in India and Mon-Khmer speakers in Southeast Asia, with a lack
of shared mtDNA haplogroups9,15–17. By contrast, data from the paternal lineage (Y-chromosome) indi-
cates a shared Asian-specic haplogroup (O2a1-M95) between the AA speakers from India (66.44% on
average) and from Southeast Asia (56.55% on average)9,10,12,13,18. Given the relatively young age (< 10
KYA) of the O2a1-M95 lineage estimated from the Y-chromosome short tandem repeats (Y-STRs) var-
iation in India, the migratory route of the AA speakers would likely begin in Southeast Asia and then
move to India11,12. However, the high mtDNA haplotype diversity in Munda-speaking populations14,15
and an independent estimate of an old coalescence age (~65 KYA) of the O2a1-M95 lineage in the Indian
AA-speaking populations10 suggests an Indian origin followed by a dispersal to Southeast Asia, possibly
before the Last Glacial Maximum (LGM, 19.0–26.5 KYA)19. is latter hypothesis seems to cope better
with the more widely agreed upon costal migration of modern humans from Africa to Asia by way of
the Indian subcontinent.
While both theories have certain peculiar merits, neither has dealt well with the large discrepancy
of the estimated ages of the O2a1-M95 lineage from dierent studies. One explanation for the marked
dierences in the estimate may be limited samplings of the AA speakers in India and/or dierent gen-
otyping approaches10,12. Fortunately, a recent study with a more extensive sampling of the AA speakers
in India and a few samples from Southeast Asia9 has claried some of these inconsistencies. rough
a genome-wide screening of 610K autosomal sequence variations and uniparental loci, Chaubey et al.
demonstrated an older coalescent time (average 22.4 ± 4.9 KYA) of the O2a1-M95 lineage in Southeast
Asia than that in India (average 15.9 ± 1.6 KYA), lending greater credence to the proposed westward
migration of the AA speakers from Southeast Asia to India. Chaubey et al. also proposed a sex-specic
admixture of the AA-speaking immigrants with local India populations by showing a dierent pattern
in the mtDNA lineage9.
Despite the data contributions from Chaubey et al. and numerous other studies on AA speakers, AA
populations from MSEA and southern China continue to be under- sampled and represented. Similarly,
no other southern populations have been included in these analysis to date, in spite of the high frequency
of O2a1-M95 in certain populations, such as among Daic-speaking populations that have a ~45% fre-
quency20–23. Complicating these oversights, existing genomic analysis also suers from some deciencies.
For example, the Illumina Human Hap 610K Chips were developed by covering sequence variations iden-
tied in limited world populations, which in turn limits its power to detect genetic relationships among
the hypothetically ancient AA populations. Given the sampling, methodology and technical limitations
inherent in the existing literature, basic questions—where did the O2a1-M95 carrying AA-speakers orig-
inally emerge, or when did it begin expanding into Asia—remain unanswered.
In this study, we collected samples of 21 AA-speaking populations from Cambodia, ailand and
southern China (totally, 646 males)(Fig.1). For individuals belonging to the O2a1-M95 lineage (343 of
the 646)18, we conducted genotyping of 16 Y-STRs. We also collected published data of the O2a1-M95
carriers from 107 populations (2,510 O2a1-M95 out of 7,693 male individuals in total) covering all the
geographic distributions of the AA speakers as well as the other major language families in eastern Asia
and India. To date, this data marks the most comprehensive collection of data of O2a1-M95 diversity.
Our analysis showed that the O2a1-M95 lineage initially originated in the southern part of eastern Asia
among the Daic-speaking populations around 20–40 KYA, followed by a southward dispersal to the
heartland of MSEA ~16 KYA, and then a westward migration to India ~ 10 KYA. Furthermore, analysis
of more than 20,000 mtDNA sequences, including these AA populations and other Asian populations,
demonstrated that the maternal lineage has a dierent pattern from the Y-chromosome for these AA
populations, supporting the proposed sex-biased admixture of the AA immigrants with local people in
the Indian subcontinent.

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SCIENTIFIC RepoRts | 5:15486 | DOI: 10.1038/srep15486
Results
High O2a1-M95 frequencies in the AA populations from MSEA and southern China. e
O2a1-M95 lineage was reported to be highly prevalent in some AA populations in India, e.g., as high
as 67.53% and 74.00% respectively in Munda and Mon-Khmer populations9,10. We observed high
O2a1-M95 frequencies in AA populations not analyzed in previous studies from Cambodia (70.67%),
ailand (52.51%) and Southern China (30.00%) (Fig.2A, Table1 and supplementary Table S2)10–12,15.
In the Andaman-Nicobar islands, O2a1-M95 was also widespread (~45.18% on average) and is xed
(100%) in several populations, such as the Shompen and Onge9,10, likely due to a strong bottleneck eect
in these island populations, which is reected in other major Y-chromosome lineages (e.g. DE-YAP and
O3-M22)24–26. (Fig.2A and supplementary Table S2). Consistent with previous results, the collective data
shows that O2a1-M95 lineage is dominant in almost all AA populations, including those from MSEA
and southern China, making it an informative genetic marker for tracing the patrilineal prehistory of
the AA populations.
Dating the O2a1-M95 lineages of dierent Asian populations based on Y-STRs variations.
Previous studies have sampled few AA populations from MSEA and Southern China9,10,12. To ll the
sampling gap, we sampled a wide range of AA-speaking populations from Cambodia, ailand and
southern China18 and genotyped 16 Y-STRs loci for those samples belonging to the O2a1-M95 lineage
(Fig.1, Table1 and supplementary Table S1). Integrating these samples with the previous data, we dated
the O2a1-M95 lineages among dierent regional populations (Fig.3, supplementary Tables S3 and S4)
and observed that the O2a1-M95 lineage has the oldest time of most recent common ancestor (TMRCA)
among the populations in the southern part of mainland China and Taiwan (~20–40 KYA), most of
which are Daic speaking (Fig.3, Supplementary Tables S3 and S4). e average TMRCA for these Daic
and Austronesian populations from southern China is ~30 KYA, markedly older than those in MSEA
(~16 KYA), India (~10 KYA) or Island Southeast Asia (ISEA, ~11 KYA) (Fig.3, supplementary Tables
S3 and S4). e estimated coalescence ages for the AA speakers from MSEA, ISEA and India are similar
to those reported by Chaubey et al.9. At the same time, the estimated ages of O2a1-M95 lineages in the
Daic populations was consistent with the estimated ages of its sister lineages (O3-M122 and C-M130)
in the same geographic regions3,27, supporting the proposed antiquity of the Daic populations. ese
lines of evidence suggest that the O2a1-M95 lineage initially originated in the Daic populations living in
southern China, prior to a southward expansion to MSEA and later migrations to India and ISEA aer
the LGM (19.0–26.5 KYA)19.
Comparison of haplotype diversity of the O2a1-M95 lineages among dierent geographic
populations. In line with the estimated TMRCAs, the unbiased Y-STRs haplotype diversity of the
O2a1-M95 lineage are the highest in populations from southern China (~0.5017 on average), particu-
larly among the Daic populations, followed by those in MSEA (~0.3858), ISEA (~0.3680) and then India
(~0.3168) (Fig.2B and supplementary Table S5), which together match the proposed migratory routes
from southern China to MSEA, and then to ISEA and India. We further calculated the pairwise genetic
distances measured by Fst (supplementary Table S6) and constructed an un-rooted neighbor-joining (NJ)
tree based on the Y-STRs variations that showed populations clustered primarily along their respective
language families and not by geographic regions. is tree structure suggests a within language family
Figure 1. Geographic locations of the studied populations in Asia that contain the O2a1-M95 lineage.
Populations are color-coded based on their language families. e gure was modied from our previous
report43 using Microso Powerpoint 2011 (Microso Corporation, USA).

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genetic anity, though there were several interesting exceptions (Fig.4). e AA populations from India
clustered with the AA populations from Cambodia, not with the Dravidian and Indo-European speakers
from India. is grouping strongly supports the hypothesized shared genetic ancestry among the AA
populations, consistent with the previous observation by Chaubey et al.9. We also observed a lack of clear
geographic clustering in the Y-STRs based phylogenetic network of O2a1-M95 (Fig.2C), likely due to
continuous gene ows among the regional AA speakers9.
Interestingly, our analysis departed from several previous observations that had found a clear diver-
gence of the Andaman-Nicobar Island populations from the other AA speakers9. Here, we detected
shared Y-STRs haplotypes in these isolated island populations with some MSEA populations (Fig.2C),
which may not have been apparent in earlier studies due to a generalized under representation of MSEA
populations.
mtDNA diversity suggests a sex-biased migration of AA-speakers from MSEA to the Indian
subcontinent. To check the maternal side of the AA populations, we collected 21,470 mtDNA
sequences from 545 populations distributed in East Asia, Southeast Asia and South Asia (supplementary
Table S7) and analyzed the patterns of mtDNA diversity. Compared with the dominant occurrence of
the O2a1-M95 lineage (65.53% on average) and the high frequency (e.g., 44.57%) of other East Asian
specic lineages (NO, N, O, P and Q, supplementary Table S8) in the South Asia populations, we found
only ~16.46% mtDNA sequences belonging to the East Asian specic lineages (A, B, C, D, F, G, M9 and
M12) in South Asia (supplementary Figures S1, S2 and supplementary Table S9). PCA analysis using
mtDNA haplotype frequencies indicated a clustering pattern of geographic locations and not language
families, which is dierent from that of the Y-chromosome data. For example, AA and TB populations
from India clustered with Dravidian and Indo-European populations from India and not the other AA
populations from southern China and Southeast Asia (Fig.5). is discrepancy supports the notion that
the prehistoric migration of the AA-speakers from MSEA to India was likely sex-biased, conrming the
hypothetical sex-biased admixture of the India AA populations posited by Chaubey et al.9.
Figure 2. Frequency distribution, Uh diversity and phylogenetic structure of the O2a1-M95 lineages
among Asian populations. Contour map shows the frequency (A) and Y-STRs Uh diversity (B) of lineage
O2a1-M95 in Asia. Colored dots indicate the geographic locations of the analysed populations that
correspond with Fig.1; Bars indicate the frequency and Uh diversity spectrum respectively. (C) Phylogenetic
network of Y-STRs haplotypes among O2a1-M95 populations generated from the following 14 Y-STRs:
DYS19, DYS389 I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448,
DYS458, DYS635 and GATA H4; Circles size is proportional to the number of samples. e contour maps
were generated using Surfer10 (Golden Soware Inc., Golden, USA), and the network was constructed using
the Network package 4.6.1.3 (www.uxus-engineering.com).

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Discussion
roughout the previous studies, the two primary competing conceptions on the origin and prehistoric
migratory pattern of the AA populations have le considerable debate, in part due to not including a
wider geographic sampling. Here, we tested these rival hypotheses by systematically collecting AA sam-
ples from MSEA and southern China, and observed high frequencies of the O2a1-M95 lineages across all
the studied AA populations. is broader survey conrmed that this Y-chromosome lineage represents
a genetic signature of all AA populations, and can serve as an eective genetic marker for tracing the
prehistoric movements and origins of these populations.
NO. Population Region L ocation Linguistic Family Sub-Branch N
O2a1-M95
Counts %
1 Brao Cambodia Ratanakri Austro-Asiatic West Bahnaric 37 24 64.86
2Jarai Cambodia Ratanakri Austronesian Chamic 45 34 75.56
3 Kachac Cambodia Ratanakri Austro-Asiatic North Bahnaric 17 13 76.47
4 Khmer Cambodia Kratie Austro-Asiatic Khmer 34 18 52.94
5 Kravet Cambodia Ratanakri Austro-Asiatic West Bahnaric 24 12 50.00
6 Kreung Cambodia Ratanakri Austro-Asiatic West Bahnaric 22 14 63.64
7Kuy Cambodia Stung Treng Austro-Asiatic Katuic 37 34 91.89
8 Lao Cambodia Stung Treng Daic Kadai 27 14 51.85
9Lun Cambodia Ratanakri Austro-Asiatic West Bahnaric 13 12 92.31
10 Mel Cambodia Kratie Austro-Asiatic Monic 19 15 78.95
11 Phnong Cambodia Kratie Austro-Asiatic South Bahnaric 26 20 76.92
12 Stieng Cambodia Kratie Austro-Asiatic South Bahnaric 12 8 66.67
13 Tom pou n Cambodia Ratanakri Austro-Asiatic South Bahnaric 51 37 72.55
14 Kraol Cambodia Ratanakri Austro-Asiatic South Bahnaric 1 1 100%
14 Blang ailand Chiang Rai Austro-Asiatic Waic 7 5 71.43
15 Htin ailand Nan Austro-Asiatic Mal-Phrai 35 30 85.71
16 Lawa ailand Chiang Mai Austro-Asiatic Waic 41 14 34.15
17 Palaung ailand Chiang Mai Austro-Asiatic Palaung-Riang 16 3 18.75
18 Mon ailand Chiang Mai Austro-Asiatic Monic 2 0 0
19 Bulang China Yunnan Austro-Asiatic Wai c 55 17 30.91
20 Wa China Yunnan Austro-Asiatic Palaung-Riang 57 5 8.77
21 De’ang China Yunnan Austro-Asiatic Wai c 68 13 19.12
Tot a l 646 343 53.10
Table 1. Sampled populations from MSEA and southern China.
Figure 3. Comparison of coalescence ages of the O2a1-M95 lineages among dienent geographic
populations. e age of each geographic or linguistic group was calculated by taking the average of
respective populations from supplementary Table S3.

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e Y-chromosome data collected in this study does not support an Indian origin of the O2a1-M95 lin-
eage, but instead shows that O2a1-M95 carriers in India originated in southern China and then migrated
from MSEA to India around 10 KYA aer the LGM. Moreover, our broader analysis that included Daic
speaking populations from southern China showed that this population possessed the most diversied
O2a1-M95 lineage with an average coalescence age of ~30 KYA, making it the oldest of all known
O2a1-M95 carrying populations, and thereby supporting an initial origin of this Y-chromosome lineage
in Daic speakers who migrated southward to MESA and a later westward to India ~10 KYA. During the
preparation of this manuscript, Arunkumar et al. published a similar analysis of O2a1-M95 in Asian pop-
ulations28, and their data also favored an east-to-west migration although the estimated age of migration
was much younger than ours due to dierent mutation rates and methods used for age estimation. Our
analysis of mtDNA diversity suggests that aer dispersal to India, the O2a1-M95 carrying populations
widely absorbed the local maternal gene pool. In contrast to the well-known earliest migration of mod-
ern humans from Africa to eastern Asia by way of the Indian subcontinent, our data illustrates a back
wave and sex-biased migration of the AA speakers from MSEA to India aer the LGM, hinting at a far
more complex prehistory of Paleolithic human populations.
Figure 4. NJ-tree constructed of Y-STRs variations among dierent language family populations.
Dierent linguistic families are shown using dierent colors. Branch length values are indicated above the
branch.
Figure 5. Map of principal component analysis (PCA) among Asian populations. Populations of East
Asia and South Asia were grouped respectively by geograpghic region and language family. AA and TB-
speaking populations closely clustered with DR anf IE populations in the lower le. e rst and the second
components explain 15.25% and 7.10% of the genetic variance, respectively.

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Materials and Methods
Genotyping and data collection. For the 343 male samples that belong to the O2a1-M95 lineage
from our previous study (Table1)18, we genotyped 16 Y-STRs (DYS19/394, DYS388, DYS389 I, DYS389
II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS458, DYS461, DYS635
and GATA H4) with the methods described previously29,30. DYS389I (DYS389cd) was subtracted from
DYS389II and renamed 389ab because DYS389II contains the repeat number of DYS389I. To dissect the
origin and migratory patterns of the O2a1-M95 lineage, we collected all available O2a1-M95 Y-STRs
data, which covers 107 geographic populations (up to 2,510 samples carrying O2a1-M95) from East Asia,
Southeast Asia and South Asia9,12,20–24,26,31–38 (Fig.1, supplementary Tables S1 and S2).
Data analysis. We estimated the time of most recent common ancestor (TMRCA) of the O2a1-M95
lineage using Y-STRs variation in each population as described previously, with a 25-year generation
time and a mutation rate of 6.9 × 10−4 12,39 (supplementary Table S3). For comparison, when calculating
the ages we used three sets of loci for each population: a) the actual number of loci in the corresponding
references, b) a 7-loci set (DYS19, DYS389 I, DYS389 II, DYS390, DYS391, DYS392 and DYS393) and c)
a 6-loci set (DYS19, DYS389 I, DYS390, DYS391, DYS392 and DYS393), and the results from dierent
calculations are very similar for most populations (supplementary Table S4). e mean TMRCAs of a
geographic region are the average of its populations (Fig.3). We also estimated the unbiased haplotype
diversity of every population using GenAlEx 6.3. When estimating the age and diversity, O2a1-M95 pop-
ulations with less than 10 samples were either excluded or merged to other closely related populations.
In total, the coalescence ages and diversity of the O2a1-M95 lineages from 105 Asian populations were
calculated (supplementary Tables S3 and S5).
A median-joining network, resolved with the MP algorithm, was constructed using the Network
package 4.6.1.3 (www.uxus-engineering.com). e O2a1-M95 variance isofrequency maps based on fre-
quency and unbiased haplotype diversity were generated using Surfer10 (Golden Soware Inc., Golden,
USA), following the Kriging procedure. Average number of pairwise dierence of Y-STRs for the studied
populations was calculated using the Arlequin 3.540, and NJ-tree was constructed with MEGA 6.041.
We performed principal component analysis (PCA) based on the frequencies of mtDNA haplogroups
according to the method developed by Richards et al.42 with MVSP 3.13.
To compare the paternal and maternal gene pool between populations from East Asia and South Asia,
we analyzed ~21,470 mtDNA sequences among these populations published previously (Supplementary
Table S7).
References
1. Macaulay, V. et al. Single, rapid coastal settlement of Asia revealed by analysis of complete mitochondrial genomes. Science 308,
1034–1036, doi: 10.1126/science.1109792 (2005).
2. Su, B. et al. Y-Chromosome evidence for a northward migration of modern humans into Eastern Asia during the last Ice Age.
Am. J. Hum. Genet. 65, 1718–1724, doi: 10.1086/302680 (1999).
3. Shi, H. et al. Y-chromosome evidence of southern origin of the East Asian-specic haplogroup O3-M122. Am. J. Hum. Genet.
77, 408–419, doi: 10.1086/444436 (2005).
4. Consortium, H. P.-A. S. et al. Mapping human genetic diversity in Asia. Science 326, 1541–1545, doi: 10.1126/science.1177074
(2009).
5. Su, B. et al. Polynesian origins: insights from the Y chromosome. Proc Natl Acad Sci USA 97, 8225–8228 (2000).
6. Jin, L. & Su, B. Natives or immigrants: modern human origin in east Asia. Nat. ev. Genet. 1, 126–133, doi: 10.1038/35038565
(2000).
7. Zhang, X. et al. Analysis of mitochondrial genome diversity identies new and ancient maternal lineages in Cambodian
aborigines. Nat Commun 4, 2599, doi: 10.1038/ncomms3599 (2013).
8. Lewis, M. P. Ethnologue: languages of the world. Dallas (TX): SIL International. [Internet; cited 2010 Sep],< http://www.
ethnologue.com/> (2009).
9. Chaubey, G. et al. Population genetic structure in Indian Austroasiatic speaers: the role of landscape barriers and sex-specic
admixture. Mol. Biol. Evol. 28, 1013–1024, doi: 10.1093/molbev/msq288 (2011).
10. umar, V. et al. Y-chromosome evidence suggests a common paternal heritage of Austro-Asiatic populations. BMC Evol. Biol. 7,
47, doi: 10.1186/1471-2148-7-47 (2007).
11. Sahoo, S. et al. A prehistory of Indian Y chromosomes: evaluating demic diusion scenarios. Proc Natl Acad Sci USA 103,
843–848, doi: 10.1073/pnas.0507714103 (2006).
12. Sengupta, S. et al. Polarity and temporality of high-resolution y-chromosome distributions in India identify both indigenous and
exogenous expansions and reveal minor genetic inuence of Central Asian pastoralists. Am. J. Hum. Genet. 78, 202–221, doi:
10.1086/499411 (2006).
13. ivisild, T. et al. e genetic heritage of the earliest settlers persists both in Indian tribal and caste populations. Am. J. Hum.
Genet. 72, 313–332, doi: 10.1086/346068 (2003).
14. Charavarti, A. Human genetics: Tracing India’s invisible threads. Nature 461, 487–488, doi: 10.1038/461487a (2009).
15. Basu, A. et al. Ethnic India: a genomic view, with special reference to peopling and structure. Genome es. 13, 2277–2290, doi:
10.1101/gr.1413403 (2003).
16. Metspalu, M. et al. Most of the extant mtDNA boundaries in south and southwest Asia were liely shaped during the initial
settlement of Eurasia by anatomically modern humans. BMC Genet. 5, 26, doi: 10.1186/1471-2156-5-26 (2004).
17. eddy, B. M. et al. Austro-Asiatic tribes of Northeast India provide hitherto missing genetic lin between South and Southeast
Asia. PLoS One 2, e1141, doi: 10.1371/journal.pone.0001141 (2007).
18. Zhang, X. et al. An updated phylogeny of the human Y-chromosome lineage O2a-M95 with novel SNPs. PLoS One 9, e101020,
doi: 10.1371/journal.pone.0101020 (2014).
19. Clar, P. U. et al. e Last Glacial Maximum. Science 325, 710–714, doi: 10.1126/science.1172873 (2009).
20. Cai, X. et al. Human migration through bottlenecs from Southeast Asia into East Asia during Last Glacial Maximum revealed
by Y chromosomes. PLoS One 6, e24282, doi: 10.1371/journal.pone.0024282 (2011).

www.nature.com/scientificreports/
8
SCIENTIFIC RepoRts | 5:15486 | DOI: 10.1038/srep15486
21. Li, H. et al. Paternal genetic anity between Western Austronesians and Daic populations. BMC Evol. Biol. 8, 146, doi:
10.1186/1471-2148-8-146 (2008).
22. Li, D. et al. Paternal genetic structure of Hainan aborigines isolated at the entrance to East Asia. PLoS One 3, e2168, doi: 10.1371/
journal.pone.0002168 (2008).
23. Gan, . J. et al. Pinghua population as an exception of Han Chinese’s coherent genetic structure. J. Hum. Genet. 53, 303–313,
doi: 10.1007/s10038-008-0250-x (2008).
24. angaraj, . et al. Genetic anities of the Andaman Islanders, a vanishing human population. Curr. Biol. 13, 86–93 (2003).
25. Trivedi, . et al. Molecular insights into the origins of the Shompen, a declining population of the Nicobar archipelago. J. Hum.
Genet. 51, 217–226, doi: DOI 10.1007/s10038-005-0349-2 (2006).
26. Chandrasear, A. et al. YAP insertion signature in South Asia. Ann Hum Biol 34, 582–586, doi: 10.1080/03014460701556262
(2007).
27. Zhong, H. et al. Global distribution of Y-chromosome haplogroup C reveals the prehistoric migration routes of African exodus
and early settlement in East Asia. J. Hum. Genet. 55, 428–435, doi: 10.1038/jhg.2010.40 (2010).
28. GaneshPrasad, A. et al. A late Neolithic expansion of Y chromosomal haplogroup O2a1-M95 from east to west. Journal of
Systematics and Evolution. 1–15 doi: 10.1111/jse.12147 (2015).
29. Butler, J. M. et al. A novel multiplex for simultaneous amplication of 20 Y chromosome ST marers. Forensic Sci. Int. 129,
10–24 (2002).
30. ayser, M. et al. Evaluation of Y-chromosomal STs: a multicenter study. Int J Legal Med 110, 125–133, 141–129 (1997).
31. Deln, F. et al. e Y-chromosome landscape of the Philippines: extensive heterogeneity and varying genetic anities of Negrito
and non-Negrito groups. Eur J Hum Genet 19, 224–230, doi: 10.1038/ejhg.2010.162 (2011).
32. Hammer, M. F. et al. Dual origins of the Japanese: common ground for hunter-gatherer and farmer Y chromosomes. J. Hum.
Genet. 51, 47–58, doi: 10.1007/s10038-005-0322-0 (2006).
33. He, J. D. et al. Patrilineal perspective on the Austronesian diusion in Mainland Southeast Asia. PLoS One 7, e36437, doi:
10.1371/journal.pone.0036437 (2012).
34. arafet, T. M. et al. Major east-west division underlies Y chromosome stratication across Indonesia. Mol. Biol. Evol. 27,
1833–1844, doi: 10.1093/molbev/msq063 (2010).
35. utanan, W. et al. Genetic structure of the Mon-hmer speaing groups and their anity to the neighbouring Tai populations
in Northern ailand. BMC Genet. 12, 56, doi: 10.1186/1471-2156-12-56 (2011).
36. Nonaa, I., Minaguchi, . & Taezai, N. Y-chromosomal binary haplogroups in the Japanese population and their relationship
to 16 Y-ST polymorphisms. Ann Hum Genet 71, 480–495, doi: 10.1111/j.1469-1809.2006.00343.x (2007).
37. Xue, Y. et al. Male demography in East Asia: a north-south contrast in human population expansion times. Genetics 172,
2431–2439, doi: 10.1534/genetics.105.054270 (2006).
38. Trivedi, . et al. Molecular insights into the origins of the Shompen, a declining population of the Nicobar archipelago. J. Hum.
Genet. 51, 217–226, doi: 10.1007/s10038-005-0349-2 (2006).
39. Zhivotovsy, L. A. et al. e eective mutation rate at Y chromosome short tandem repeats, with application to human
population-divergence time. Am. J. Hum. Genet. 74, 50–61, doi: Doi 10.1086/380911 (2004).
40. Excoer, L., Laval, G. & Schneider, S. Arlequin (version 3.0): an integrated soware pacage for population genetics data
analysis. Evol Bioinform Online 1, 47–50 (2005).
41. Tamura, ., Stecher, G., Peterson, D., Filipsi, A. & umar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.
Mol. Biol. Evol. 30, 2725–2729, doi: 10.1093/molbev/mst197 (2013).
42. ichards, M., Macaulay, V., Torroni, A. & Bandelt, H. J. In search of geographical patterns in European mitochondrial DNA. Am.
J. Hum. Genet. 71, 1168–1174, doi: 10.1086/342930 (2002).
43. Shi, H. et al. Genetic evidence of an East Asian origin and paleolithic northward migration of Y-chromosome haplogroup N.
PLoS One 8, e66102, doi: 10.1371/journal.pone.0066102 (2013).
Acknowledgements
We are grateful to all the volunteers for providing blood samples, and to Andrew Willden for editing
the manuscript. is study was supported by the National 973 Program of China (2012CB518202 to
X.Q.), the National Natural Science Foundation of China (31130051 and 91231203 to B.S., 31371268
and 91131001 to H.S. and 31371269 to X.Q.) and the Natural Science Foundation of Yunnan Province
(2010CI044 to H.S.).
Author Contributions
B.S. and H.S. designed the experiment; X.M.Z., X.B.Q., J.K., Z.H.Y., B.S., T.S., L.B., H.S.A., H.S., D.K.
and H.S. collected the samples; X.M.Z., S.Y.L. and X.B.Q. collected the data and conducted data analysis;
J.W.L. and H.Z. provided technical assistance in the experiments; X.M.Z., X.B.Q., H.S. and B.S. wrote
the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Zhang, X. et al. Y-chromosome diversity suggests southern origin
and Paleolithic backwave migration of Austro-Asiatic speakers from eastern Asia to the Indian
subcontinent. Sci. Rep. 5, 15486; doi: 10.1038/srep15486 (2015).
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