Journal of Genetics and Genomics xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of Genetics and Genomics

Journal homepage: www.journals.elsevier.com/journal-of-genetics-
and-genomics/

Original research

Maternal genetic history of ancient Tibetans over the past

4,000 years
Ganyu Zhang a, b, 1, Can Cui a, b, 1, Shargan Wangdue c, 1, Hongliang Lu d, Honghai Chen e,
Lin Xi c, Wei He c, Haibing Yuan d, Tinley Tsring c, Zujun Chen c, Feng Yang d,
Tashi Tsering c, Shuai Li d, Norbu Tashi c, Tsho Yang c, Yan Tong c, Xiaohong Wu f,
Linhui Li c, Yuanhong He d, Peng Cao a, Qingyan Dai a, Feng Liu a, Xiaotian Feng a,
Tianyi Wang a, b, Ruowei Yang a, Wanjing Ping a, Ming Zhang e, Xing Gao a, Yichen Liu a, *,
Wenjun Wang a, g, *, Qiaomei Fu a, b, h, *
a
Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences,
Beijing 100044, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
Tibet Institute for Conservation and Research of Cultural Relics, Lhasa 850000, China
d
Center for Archaeological Science, School of Archaeology and Museology, Sichuan University, Chengdu, Sichuan 610064, China
e
School of Cultural Heritage, Northwest University, Xi’an, Shaanxi 710069, China
f
School of Archaeology and Museology, Peking University, Beijing 100871, China
g
Science and Technology Archaeology, National Centre for Archaeology, Beijing 100013, China
h
Shanghai Qi Zhi Institute, Shanghai 200232, China

A R T I C L E I N F O A B S T R A C T

Article history: The settlement of the Tibetan Plateau epitomizes human adaptation to a high-altitude environment that
Received 17 January 2023 poses great challenges to human activity. Here, we reconstruct a 4,000-year maternal genetic history of
Received in revised form Tibetans using 128 ancient mitochondrial genome data from 37 sites in Tibet. The phylogeny of haplotypes
14 March 2023
M9a1a, M9a1b, D4g2, G2a’c, and D4i show that ancient Tibetans shared the most recent common
Accepted 14 March 2023
Available online xxx
ancestor (TMRCA) with ancient Middle and Upper Yellow River populations around the Early and Middle
Holocene. In addition, the connections between Tibetans and Northeastern Asians varied over the past
4,000 years, with a stronger matrilineal connection between the two during 4,000e3,000 BP, and a
Keywords:
Ancient DNA
weakened connection after 3,000 BP, that were coincident with climate change, followed by a reinforced
Tibetans connection after the Tubo period (1,400e1,100 BP). Besides, an over 4,000-year matrilineal continuity was
Population history observed in some of the maternal lineages. We also found the maternal genetic structure of ancient Ti-
Mitochondrial genome betans was correlated to the geography and interactions between ancient Tibetans and ancient Nepal and
Pakistan populations. Overall, the maternal genetic history of Tibetans can be characterized as a long-term
matrilineal continuity with frequent internal and external population interactions that were dynamically
shaped by geography, climate changes, as well as historical events.
Copyright © 2023, The Authors. Institute of Genetics and Developmental Biology, Chinese Academy of
Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Tibet Autonomous Region (also called “Xizang”) is located at the

world’s highest and largest plateau, the Tibetan Plateau, with an
average altitude of more than 4,000 m. Extreme aridity, low tem-
perature, severe hypoxia, and lack of animal and plant resources in
* Corresponding authors.
E-mail addresses: yichen.liu@ivpp.ac.cn (Y. Liu), wangwenjun@ivpp.ac.cn
the Tibetan Plateau, pose a great challenge to human survival (Zhang
(W. Wang), fuqiaomei@ivpp.ac.cn (Q. Fu). et al., 2021; Chen et al., 2022). Due to this harsh and relatively
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.jgg.2023.03.007
1673-8527/Copyright © 2023, The Authors. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by
Elsevier Limited and Science Press. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: G. Zhang, C. Cui, S. Wangdue et al., Maternal genetic history of ancient Tibetans over the past 4,000 years, Journal of
Genetics and Genomics, https://doi.org/10.1016/j.jgg.2023.03.007
G. Zhang, C. Cui, S. Wangdue et al.
isolated environment, cultures with features specific to Tibetans were
developed (Huo, 2019). However, a large number of archaeological
materials unearthed in the Tibetan Plateau suggest this region is not
completely isolated from the outside (Tong, 1985; Huo, 2019). Cul-
tures in the Tibetan Plateau were widely influenced by and interacted
with the civilizations from surrounding regions, including East Asia,
South Asia, and Central Asia since the prehistory period (Huo, 2019;
Zhang et al., 2021; Chen et al., 2022).
The archaeological evidence of human activities in the Tibetan
Plateau was dated before the last glacial maximum (LGM, 22e18
thousand years ago, kya) (Madsen et al., 2006; Zhang and Li, 2002).
Humans populated most regions of the Tibetan Plateau in the Early
and Middle Holocene (11.6e6 kya) (Shargan and Pu, 2005; Rhode
et al., 2014; Zhang et al., 2016; Huo, 2019). Interestingly, the
manufacturing technologies of early stone tools found in the Tibetan
Plateau are mainly associated with Northern China during this period
(Tong, 1985; Liu et al., 1986; Qian et al., 1988; Lu, 2015). In addition,
the metal materials (such as copper and iron remains) were widely
discovered after 3,000 BP in Tibetan archaeological sites (Tong,
1985; Shargan and Pu, 2005; Huo, 2014). Many archaeological dis-
coveries with regional cultural characteristics, such as the petro-
glyphs and large stone relics, may reflect Tibet-specific cultural
features (Huo and Li, 1991; Shargan and Pu, 2005). However, evi-
dence also suggests extensive interactions between Tibetans and
cultures of other regions. For instance, the bronze mirrors with han-
dles from the Qugong site may reflect the communications with
South Asia or Central Asia (Huo, 1994; Zhao, 1994; Shargan and Pu,
2005), and the golden materials discovered in southern Tibet are
characteristic of the Steppe nomads (Huo, 2001). Tubo was a period
of the unified regime in Tibet, which rose in the seventh century and
ended in the ninth century (1,400e1,100 BP) (Beckwith, 1993; Yang
et al., 2021). During the prosperity of Tubo, the expansion of the
Tubo Dynasty widely affected neighboring areas (Huo, 1995, 2009)
and was accompanied by cultural interactions (especially with the
Tang Dynasty), including the exchange of commodities and tech-
nologies, etc. (Zhang, 1995; Li, 2009). In summary, although Tibetan
culture has strong local characteristics, the interactions with cultures
from other regions could be observed since the Stone Age. However,
it is unclear whether the cultural interactions were accompanied by
gene interactions, as the two are not necessarily concurrent in human
history.
Genetic studies on present-day Tibetans can shed light on the
evolution process at high altitudes. The mitochondrial DNA studies
reveal that the Tibetan Plateau-related haplotype M62 in present-day
Tibetans might be carried by the humans who first arrived in the Ti-
betan Plateau before the LGM (Zhao et al., 2009; Qin et al., 2010).
Similar results could be found in the Y chromosome studies, which
indicates the local ancient genetic components may have been
maintained in present-day Tibetan populations (Shi et al., 2008;
Wang et al., 2018). Moreover, several studies revealed that Northern
Chinese populations have interacted with and genetically contributed
to Tibetans (Qin et al., 2010; Qi et al., 2013; Lu et al., 2016; Yang et al.,
2017; Li et al., 2019). It is worth noting that the genetic connections
with neighboring regions, including Southeast Asia (Kang et al., 2013)
and South Asia (Cole et al., 2017), have been observed. Despite
genetic events that can be inferred using genomes of present-day
Tibetans, ancient DNA obtained from sub-fossils offer time-
stamped molecular data for reconstructing a finer genetic history.
Ding et al. sequenced ancient mitochondrial genomes on individuals
with chronological data to explore the genetic structure, covering the
lowland Gansu-Qinghai and several highland regions in the Tibetan
Plateau. This study revealed that the lower altitude populations in
Northern China spread to the highlands around 4,750e2,775 BP. In
addition, the plateau populations, at least partly, genetically
contributed to present-day Tibetans (Ding et al., 2020). Even so, the
2
Journal of Genetics and Genomics xxx (xxxx) xxx
population genetic structures and spatiotemporal differentiation in
the hinterland of the Tibetan Plateau, and the genetic interactions
with the neighbors remain to be studied in depth.
In this study, we obtained 128 complete mitochondrial genomes
of 178 archaeological and historical samples from 37 sites covering
western Tibet (Ngari), southwestern Tibet (western Shigatse),
southern Tibet (eastern Shigatse, Lhasa, and Shannan), northern
Tibet (Naqu), and northeastern Tibet (Nyingchi and Chamdo), with the
dates of the samples spanning from 4,000 BP to 27 BP. Utilizing the
large temporal and geographical scale mitogenomic data, this study
explores the maternal genetic structure of Tibetans and the changes
over time, as well as the population interactions within and sur-
rounding Tibetan regions.
Results
Samples and database preparation
We captured mitochondrial DNA (mtDNA) of 178 ancient samples
from 37 archaeological sites in Tibet (Table S1), ranging from 4,000 to
27 BP. Removing 26 individuals with relatively high contamination
(>4%), 6 individuals with a high proportion of ambiguous bases “N”,
11 individuals with low mitochondrial coverage, and 7 individuals with
potential kinship (the individuals with identical mtDNA sequences in
the same site), resulted in a final dataset of 128 individuals with
coverage between 2.21- and 730.90- fold (Table S1). Besides, we
collected seven ancient Tibet samples (Ding et al., 2020) and one
ancient Nepal sample (Jeong et al., 2016) from the Tibetan Plateau in
previous studies. They are grouped with our new samples according
to spatiotemporal information, archaeological background, and ge-
netic similarity, the details are shown in Table S1 and Supplementary
data.
In total, 136 individuals after 4,000 BP were used in downstream
analyses, including 128 new samples and 8 previous published data
(Table S1), and we divided them into four periods following their
archaeological dates (Fig. 1): (1) 4-3 K (~4,000e3,000 BP, n ¼ 7); (2)
3-1.4 K (~3,000e1,400 BP, n ¼ 93); (3) 1.4-1.1 K (~1,400e1,100 BP,
the Tubo period, n ¼ 11); (4) post1.1 K (~1,10027 BP, n ¼ 25). In each
period, the individuals were grouped by different regions: Ngari in
western Tibet (WXZ); western Shigatse in southwestern Tibet
(SWXZ); Lhasa, Shannan, and eastern Shigatse in southern Tibet
(SXZ); Naqu in northern Tibet (NXZ), Nyingchi and Chamdo in
northeastern Tibet (NEXZ). Finally, the 136 individuals were com-
bined into 15 groups (WXZ_4-3 K, n ¼ 5; SXZ_4-3 K, n ¼ 1; NEXZ_4-
3 K, n ¼ 1; WXZ1_3-1.4 K, n ¼ 23; WXZ2_3-1.4 K, n ¼ 21; WXZ3_3-
1.4 K, n ¼ 23; SWXZ_3-1.4 K, n ¼ 6; SXZ_3-1.4 K, n ¼ 7; NEXZ_3-
1.4 K, n ¼ 10; NXZ_3-1.4 K, n ¼ 3; NXZ_1.4-1.1 K, n ¼ 5; SXZ_1.4-
1.1 K, n ¼ 6; SXZ_post1.1 K, n ¼ 21; NEXZ_post1.1 K, n ¼ 2;
WXZ_post1.1 K, n ¼ 2) for further studies, according to spatiotem-
poral information, archaeological background, and genetic analysis.
The details are shown in Table S1 and Supplementary data.
We collected 816 previously published ancient mtDNA data from
East and West Eurasia, ranging from the Early Neolithic to the His-
toric Era (Table S2). We grouped them as NEA (Northeastern Asian),
SEA (Southeastern Asian), and CWE (Central-west Eurasian). We
also collected 4,679 present-day whole mitochondrial genomes of
individuals from Tibetans of different regions, NEA, SEA, Nepal, and
Pakistan. Their references and detailed group information were
shown in Table S3. Firstly, we explored the maternal genetic profiles
of ancient and present-day Tibetans. In the discriminant analysis of
principle components (DAPC) plot, the subgroups of present-day
Tibetans did not show obvious genetic differentiation, and had a
large overlap with ancient Tibetans (Fig. S1). Besides, we noticed
China_TibetSherpa (shown as blue stars) clustered with ancient and
present-day Tibetans, while Nepal Sherpas separated from ancient
G. Zhang, C. Cui, S. Wangdue et al. Journal of Genetics and Genomics xxx (xxxx) xxx

WXZ SWXZ SXZ NXZ NEXZ

4000 2

6
2
3000
16 2 1
8 1
1
1
5
37 3 7
2 1
15
12 1
WXZ 1 1

2
NXZ 1400 2 6
3
SWXZ 2
SXZ
1
NEXZ 3
1100 2
Elevation (m)
high:8305 1 2
14
5
4
2 1
1 1
low:-264
Calibrated years before present (BP)

Fig. 1. Geographic and temporal distributions of archaeological sites on the Tibetan Plateau. The map on the left shows the locations of the archaeological sites of the involved ancient
Tibetan individuals for this study. Each symbol represents one site and different shapes represent different periods. The right part is timeline of archaeological sites, dated back to 4,000
BP. The numbers of samples are at the right of the site points.

Tibetans and clustered with several present-day Tibetans. In the
Haplogroup Sharing analysis (Fig. S2C), present-day Tibetans of
different regions shared similar genetic patterns sharing about 50%
of present-day Tibetan related haplotypes, and about 30%e40% of
haplotypes with ancient Tibetans. The frequency of shared haplo-
types with ancient Tibetans did not show evident genetic differences
across groups of present-day Tibetans. Noticeably, Tibetan Sherpas
share a higher level of haplotypes (9.58%) with Nepal Sherpas
compared to other present-day Tibetan groups (0.33%e1.94%).
Similar results can be observed in FST results, with a low FST value
observed between China_TibetSherpa and Nepal_Sherpa (0.093)
(Table S4).
The maternal genetic connections between Tibetans and NEA
populations varied during different periods in the past 4,000
years
Complete mitochondrial genome results showed that the ancient
Tibetan individuals can be unambiguously allocated into hap-
logroups M*, M8, M9, G, D, N1, A, and R, which can be further
assigned into 54 haplotypes (Fig. S2A; Table S1). We did the Hap-
logroup Sharing analysis using the 4,679 published data among
different regions to explore the maternal genetic pattern of the
different ancient Tibetan groups (Fig. S2C; Table S3). The result
shows the ancient Tibetan groups shared relatively high-level hap-
lotypes associated with NEA populations (1.43%e11.33%). To un-
derstand the maternal genetic structure of ancient Tibetans, we
performed DAPC analysis of the newly sequenced Tibetan in-
dividuals with other representative ancient populations (Fig. 2A). The
results show that all individuals were mainly divided into four clusters:
NEA, SEA, CWE, and Tibetan populations, suggesting a unique
maternal genetic structure of ancient Tibetans (Fig. 2A). Also, the
ancient Tibetans cluster closer with NEA populations compared with
other involved populations, which is consistent with the result of
Haplotype Sharing analysis (Fig. S2C). In addition, we observed Ti-
betans and NEA populations formed a cluster in the FST heatmap
(Fig. 2B), which further indicate that ancient Tibetans had close
maternal genetic connections with NEA populations.
To understand the genetic connections of Tibetans with NEA
populations, we further analyzed the shared haplotypes among
them. We observed Tibetans shared several haplotypes (such as
Aþ152, B4, D4g2, D4h, D4i, D4j1, D5a, F1a’c’f, F1þ16189, G2a’c,
G2b, M9a, and Zþ152) with NEA populations, especially those in the
middle Yellow River (Middle YR) region, which are related to Han-
related populations (Qingtai, Shimao, and Taosi population) (Miao
et al., 2021; Xue et al., 2022), (Figs. S3 and S4; Tables S1 and S2).
In the network analysis, we observed several ancient Tibetans and
YR populations shared a common ancestor and separated into
different branches (such as M9a1a, M9a1b, D4g2, G2a’c, and D4i)
(Figs. 3B, 3E, S3). The phylogeny of some haplotypes (M9a1a,
M9a1b, D4g2, and G2a’c) show ancient Tibetans and Middle YR
Han-related populations (Shimao, Qingtai, and Taosi) form two
distinct clades and shared a common ancestor around
12,517e9,385 BP, based on a molecular clock-based method
(BEAST) (Figs. 3A and S5). It can be linked to the Han-Tibetan split
during early Holocene, as suggested before (Lu et al., 2016; Zhang
et al., 2019, 2021). Particularly, the observed phylogeny and the
TMRCA of the M9a1a1c1b1a clade also fits the hypothesis that a
Neolithic expansion of this lineage lead to the enrichment of this
haplogroup M9a1ac1b in Tibet, as suggested in Peng et al. (2011).
Moreover, we observed that the haplotypes D4i and D4j1 are shared
between ancient Tibetans and Zongri individuals, suggesting a
connection between highland Tibetans and the populations in the
upper Yellow River (Upper YR) region. The Phylogenetic trees show
that they share several variants, such as 16294, 1734, and 7055
(Figs. 3F and S6A). Interestingly, the network of haplotype D4j1
shows a star-like pattern, the Upper YR individual (4,750 BP) located
in the center of the D4j1 network, and connected to several ancient
Tibetans (WXZ1_3-1.4 K, SXZ_3-1.4 K, SWXZ_3-1.4 K, and
NEXZ_post1.1 K) (Fig. S3I), indicating a Tibetan-wide (southern,
western, southwestern, and northeastern Tibetan individuals)
connection between these Upper YR individuals and ancient

3
G. Zhang, C. Cui, S. Wangdue et al. Journal of Genetics and Genomics xxx (xxxx) xxx

A B
WXZ_4-3K Nepal_2.4-1.2K
NEXZ_4-3K Pakistan_3.2-2.8K China_TibetSherpa(82)
SChina_TibetChamdo(5)
SXZ_4-3K GQ_Pukagongma_3-2.8K SChina_TibetLhasaShannan(16)
WXZ1_3-1.4K GQMajiaY_4.8-3.8K Present-day Tibet SChina_TibetNagqu(17)
SChina_TibetShigatse(18)
WXZ2_3-1.4K GQZongri_5-4K SChina_TibetShigatseT(86)
WXZ3_3-1.4K Shimao_4.2-3.2K SChina_NyingchiMonpa(22)
SChina_NyingchiLhopa(91)
SWXZ_3-1.4K Taosi_4.2-3.7K SChina_NyingchiD(91)
SXZ_3-1.4K CSteppe_4.5-3.7K WXZ_4-3K(5)
NXZ_3-1.4K CSteppe_3.8-1.7K WXZ1_3-1.4K(23)
WXZ2_3-1.4K(21)
NEXZ_3-1.4K FJ_4.6-4.2K Tibet Ancient Tibet WXZ3_3-1.4K(23)
NXZ_1.4-1.1K YN_3.5-3.2K NEXZ_3-1.4K(10)
SXZ_3-1.4K(7)
SXZ_1.4-1.1K YNHC_post3K SWXZ_3-1.4K(6)
WXZ_post1.1K GX_post1.7K SXZ_1.4-1.1K(7)
SXZ_post1.1K(21)
SXZ_post1.1K Nepal Nepal_2.4-1.2K(7)
NEXZ_post1.1K preShimao_4.8-4.6K(21)
Shimao_4.2-3.2K(101)
Middle YR Taosi_4.2-3.7K(40)
QT_5.3-5.1K(52)
GQMajiaY_4.8-3.8K(10)
GQQijia_4.3-3.5K(16)
Upper YR GQKayue_3.2-2.6K(8)
CWE Nepal SD_7-4.6K(33)
SD_post4.6K(49)
Other NEA NXJ_2.9-2K(15)
EXJ_2.3-2.1K(27)
NEA EXJ_3.2-2.7K(25)
NEA SWXJ_2.7-2.3K(12)
FJ_4.6-4.2K(11)
SEA YN_3.5-3.2K(5)
YNHC_post3K(11)
GX_post1.7K(32)
Tagar_2.7K(8)
Sarmatian_2.4-2K(9)
CenSaka_2.6-2.4K(8)
Pakistan Nomad_2.5-1.7K(6)
TianSaka_2.3-2K(9)
WuKang_2.3-1.7K(10)
TianHun_1.9-1.5K(23)
CWE Pakistan_3.2-2.8K(84)
Pakistan_post2.2K(22)
SXJ_2.5-1.9K(19)
CSteppe_4.5-3.7K(8)
CSteppe_3.8-1.7K(45)
Turkmen_5.5-4.9K(14)
Turkmen_4.6-3.2K(14)
Uzbek_3.9-3K(25)
Iran_5.5-4.6K(5)
Iran_4.9-4.2K(9)
NChina_Han(138)
Han SChina_Han(168)
SEA Nepal Sherpas Nepal_Sherpa(83)

Nepal_Sherpa
SChina_Han
NChina_Han
Iran_4.9-4.2K
Iran_5.5-4.6K
Uzbek_3.9-3K
Turkmen_4.6-3.2K
Turkmen_5.5-4.9K
CSteppe_3.8-1.7K
CSteppe_4.5-3.7K
SXJ_2.5-1.9K
Pakistan_post2.2K
Pakistan_3.2-2.8K
TianHun_1.9-1.5K
WuKang_2.3-1.7K
TianSaka_2.3-2K
Nomad_2.5-1.7K
CenSaka_2.6-2.4K
Sarmatian_2.4-2K
Tagar_2.7K
GX_post1.7K
YNHC_post3K
YN_3.5-3.2K
FJ_4.6-4.2K
SWXJ_2.7-2.3K
EXJ_3.2-2.7K
EXJ_2.3-2.1K
NXJ_2.9-2K
SD_post4.6K
SD_7-4.6K
GQKayue_3.2-2.6K
GQQijia_4.3-3.5K
GQMajiaY_4.8-3.8K
QT_5.3-5.1K
Taosi_4.2-3.7K
Shimao_4.2-3.2K
preShimao_4.8-4.6K
Nepal_2.4-1.2K
SXZ_post1.1K
SXZ_1.4-1.1K
SWXZ_3-1.4K
SXZ_3-1.4K
NEXZ_3-1.4K
WXZ3_3-1.4K
WXZ2_3-1.4K
WXZ1_3-1.4K
WXZ_4-3K
SChina_NyingchiD
SChina_NyingchiLhopa
SChina_NyingchiMonpa
SChina_TibetShigatseT
SChina_TibetShigatse
SChina_TibetNagqu
SChina_TibetLhasaShannan
SChina_TibetChamdo
China_TibetSherpa
0 0.15 0.2 0.35

Fig. 2. The genetic analysis between Tibetans and other ancient populations including the DAPC plot and FST heatmap. A: DAPC of Tibetan individuals and other ancient populations,
including all ancient Tibetan samples and representative ancient groups in our dataset. B: The genetic distance (FST) heatmap of ancient Tibetan groups and other ancient and present-
day populations. Groups with fewer than five individuals were not included. NXZ_1.4-1.1 K was removed since its single haplotype composition caused a large genetic distance
(FST > 0.10) with other populations. DAPC, discriminant analysis of principal components.

Tibetans. In the haplotype D4i network, we found ancient Tibetans
and several Upper YR individuals located at different branches
respectively (Fig. 3E). We further estimated the TMRCA to be around
7,000 BP (7,676 BP, p ¼ 1), using a molecular clock-based method
(BEAST) (Fig. 3D).
In other lineages, we observed ancient Tibetans and NEA pop-
ulations located on the same sub-branch rather than showing a clear
split (Fig. S3). The same haplotypes Aþ152 and F1a’c’f were found in
WXZ_4-3 K and NEA populations, especially with Middle YR pop-
ulations (Tables S1 and S2). The WXZ_4-3 K individual and Middle YR
individuals are closely connected in the same sub-branch F1c shown in
the F1a’c’f network (Fig. S3F). We also found several 4-3 K individuals
in the western Tibet cluster close to NEA populations, especially the
populations from Middle YR in the DAPC plot (Figs. 2A and S7). FST
results show WXZ_4-3 K was clustered together with the other Tibetan
populations and showed a closer genetic connection with NEA pop-
ulations (average FST value: 0.095) compared with SEA populations
(average FST value: 0.14), and CWE populations (average FST value:
0.12). Among the NEA populations tested in this study, the WXZ_4-3 K
was closest to the Middle YR populations (average FST value: 0.092)
(Fig. 2B; Table S4). These results indicate that 4-3 K Tibetans had close
maternal genetic connections with NEA populations.
Compared with the 4-3 K Tibetans, the 3-1.4 K Tibetans span a
larger area, suggesting a high maternal genetic diversity, while the
1.4-1.1 K Tibetans fall in the center of Tibet-related region and thus
likely reflecting relatively homogenous maternal genetics in the DAPC
plot (Fig. 2A). We further analyzed the haplotypes among ancient
populations. We identified 15 haplotypes that are observed in Ti-
betans accounting for a high proportion (>75% of ancient individuals
possess the haplotype are Tibetans) or exclusively observed in the
ancient Tibetans (e.g., M62 and M9a1b1). Therefore, these haplo-
types are likely related to ancient Tibetans, the details are shown in
Table S5. These Tibet-related haplotypes account for 60.22% of 3-
1.4 K Tibetans and increased to 100% in 1.4-1.1 K Tibetans in the
Haplotype Frequency analysis, which indicates a decrease in the
interaction with populations from surrounding regions over time
(Table S6). Similar results are observed in the Networks and Phylo-
genetic trees of several haplotypes that frequently are observed in
ancient Tibetans, including C4a2b2, D4i3, F1g, M70, M9a1a, and
Z3a1a. Specifically, we observe the 3-1.4 K and 1.4-1.1 K Tibetans
exclusively shared several sub-branches with other Tibetans (A11a,
A21, F1g, M9a1b1, and U2b1) (Fig. S4). In addition, the higher FST
values (average: 0.12 for SXZ_1.4-1.1 K, 0.29 for NXZ_1.4-1.1 K; for
comparison, 3-1.4 K Tibetans: 0.10) indicated these genetic differ-
ences between Tibetans and the ancient individuals outside Tibet
from 1,400 to 1,100 BP (Table S4). These results indicate the
decreasing maternal genetic connection with YR populations from
3,000 to 1,100 BP.
After 1,100 BP, an increased connection between Tibetans and
YR populations can be observed in our analysis. Tibet-related hap-
lotypes went down to 56%, while 40% of NEA-related haplotypes
were observed in post1.1 K Tibetans, suggesting a closer interaction
with NEA in post1.1 K individuals than 1.4-1.1 K individuals (Fig. 4A).
The Network of sub-haplogroups D4h and D4j1 indicate the Tibetans
in some regions (SXZ_post1.1 K and NEXZ_post1.1 K) had a close
relationship with YR populations (Fig. S3C, I). In the DAPC plot,
although most of the post1.1 K individuals clustered closely with
previous 1.4-1.1 K Tibetan individuals (SXZ_1.4-1.1 K and NXZ_1.4-
1.1 K), we found several SXZ_post1.1 K individuals clustered closer
to YR populations (Fig. 2A) than 1.4-1.1 K Tibetans. Besides, the FST
heatmap showed SXZ_post1.1 K was in the Tibetan cluster and had
closer genetic distance with NEA populations (FST values:
0.037e0.11) than its precursor SXZ_1.4-1.1 K (FST values:
0.071e0.18) (Fig. 2B; Table S4). Haplotype diversity analysis showed

4
G. Zhang, C. Cui, S. Wangdue et al. Journal of Genetics and Genomics xxx (xxxx) xxx

A 0.9978 Taosi_LN_M42
M9a1a1c1a B C 7142
16291
M9a1a1c
Q T_MN_L7714
14417
M9a1a1c1
Sila_M1001F
7697 7861
0.9935 @16291
M9a1a1c1a
0.9462
Nuda_M3
M9a1a1c1b
9385BP 310 3106d

Nuda_M1 M9a1a1c1a @153

5899.XC
3107C
16249

M9a1a1c1b1
Longsangquduo_1 711
M9a1a1c1b1a
Chaxiutang_J2-13
0.9982
Middle YR 3106d
3107C
3106d
3107C
@5899.XC 3106d
3107C
309
310
309
310
309
310
309
310
303d
3106d
3106d
3107C
3106d
3107C
309
310
3106d
3107C
303d
3106d
3106d
3107C
3106d
3107C
3106d
3107C
Sangdalongguo_M2_2 3866 I187T @5899.XC @5899.XC 3106d 3106d 3107C 3106d 3107C @5899.XC @5899.XC 3106d @5899.XC 3107C 3866 I187T @5899.XC @5899.XC
5474 3107C 3107C @5899.XC 3107C @5899.XC 3107C @5899.XC @5899.XC 13072
@5899.XC @5899.XC @5899.XC 16162 @5899.XC @5899.XC 13702 R456C
Gachong_M1_1 16194 5978 15526

Nepal_IA_S10

Sangdalongguo_M5_3

Sangdalongguo_M2_2
Sangdalongguo_2_1
Latuotanggu_M3_5

Longsangquduo_1

Chaxiutang_J2-13

Latuotanggu_M51
0.4719

Latuotanggu_M6

Latuotanggu_M4

Tanggalang_M1
Tanggalang_M1

Gachong_M1_1

QT_MN_L7714

Taosi_LN_M42
Nepal_IA_M63

Sila_M1001F
Ounie_B0M4

Sazha_M12
M9a1a1c1b1a

Nuda_M3

Nuda_M1
Latuotanggu_M6

Latuotanggu_M4

Latuotanggu_M51 M9a1a1c1b1a
Latuotanggu_M3_5
Tibet-related populations and Middle YR populations
Ounie_B0M4

Sangdalongguo_2_1

10
sa
mp
les
Nepal_IA_M63

1
sa
mpl
Tibet

e
Sazha_M12

A
C
Sangdalongguo_M5_3

B
D4

E
E F

D
16294
D

G
D4i

F
Sangdalongguo_M3_28 195 195 63 195 195 195 195 195 195 195
0.9878
310 310 195 310 310 310 310 310 4936 310
0.7416 Zhangcun_M1_2
D4i3 501 501 310 501 501 501 501 501 16114 1187
1187 1187 501 1187 1187 1187 1187 1187 16318 3106d
3107C 3107C 1187 3107C 3107C 3107C 3107C 3107C D4i3 3107C
SXJ_IA_ZGLK_M83A
15884C A380P15884C A380P 3107C 15884C A380P
15884C A380P
15884C A380P
15884C A380P
15884C A380P 16092
16192 16268A 15884C A380P 16192 16192 @195 309
GQZongri_LN_L3197 3106d 310
0.4255
1
7676BP
D4i 3107C 508
0.9946 GQZongri_LN_R2111 9965 3106d

Upper YR 3107C
8292
GQZongri_LN_R2089
@16114

Sangdalongguo_M3_28
0.5755 GQZongri_LN_R2114
0.191

GQZongri _LN_R2122

GQZongri_LN_R2121

GQZongri_LN_R2114

GQZongri_LN_R2111

GQZongri_LN_R2089

SXJ_IA_ZGLK_M83A
GQZongri_LN_L3255

GQZongri_LN_L3254
GQZongri _LN_L3197
0.0753 GQZongri_LN_R2121
Tibet

Zhangcun_M1_2
0.146 GQZongri_LN_L3255 D4i Upper YR
D4i3
0.1165
GQZongri_LN_R2122
Middle YR
GQZongri_LN_L3254
CWE
700.0
Nepal Upper YR populations and Tibet-related populations
Tibet

Fig. 3. Genetic analysis of ancient Tibetans in the haplogroups M9a1a1c1 and D4i. AeC: The Bayesian phylogenetic tree (A), network (B), and phylogenetic tree (C) of haplotype
M9a1a1c1. DeF: The Bayesian phylogenetic tree (D), network (E), and phylogenetic tree (F) of haplotype D4i. Tibetans and NEA individuals were marked with red and green squares (or
circles). In Bayesian phylogenetic trees, the posterior rate is on the branch and the coalescence time is on the node.

A C
U2a/U2b/U2c Haplogroup U2
Tibet/Pakistan

U2e
CWE

Tibet-related Upper YR Middle YR Other NEA Pakistan CWE

B D Haplogroup M9a1a

M9a1a2
Tibet/Nepal

M9a1a1c1b1a
Tibet/Nepal
Tibet
Other NEA
Nepal
Pakistan
WXZ SWXZ SXZ NXZ NEXZ Nepal Pakistan
CWE
10 s

Fig. 4. The genetic analysis of ancient Tibetans between ancient Pakistan and Nepal populations. A: Haplogroup frequency of ancient Tibetans. Groups with fewer than five individuals
were not included. B: Haplogroup sharing analysis among ancient Tibet groups, Nepal and Pakistan. C: The network of sub-haplogroup U2. D: The network of haplotype M9a1a.

Tibetans during Tubo period (1.4-1.1 K) possessed lowest maternal (Fig. S2B; Table S7). These results indicate an increased maternal
genetic diversity (0.87), which had since increased after 1,100 BP genetic connection between Tibetans and populations outside Tibet
(0.94) and reached to the highest in present-day Tibetans (0.97) after 1,100 BP.

5
G. Zhang, C. Cui, S. Wangdue et al.
To sum up, several ancient Tibetans shared a common ancestor
with YR populations, and separated into different branches, like the
Middle YR Han-related populations (TMRCA around 10,000 BP), and
Upper YR populations (TMRAC around 7,000 BP). In other lineages,
we observed the genetic interactions between Tibetans and NEA
populations. We observed closer maternal genetic connections with
YR populations during 4,000e3,000 BP. This connection had a trend
of decreasing during 3,000e1,400 BP and reached its trough in
1,400e1,100 BP, and the latter had the least connections with
populations outside Tibet. After 1,100 BP, the connections with YR
increased in Tibetans, and continue to the present-day Tibetans. So
the degree of the Tibetans’ connection with the outside varied during
different periods in the past 4,000 years.
The internal interaction and maternal genetic structure within
Tibetan populations in different regions
Overall, the Tibetans from different time periods clustered closer
with each other rather than the populations outside Tibet, suggesting
a unique maternal genetic structure of ancient Tibetans (Figs. 2A, S7,
S8). In addition, Tibetan individuals from different regions cluster
close to the earlier population from the same region (WXZ, SXZ, NXZ,
NEXZ) in the DAPC plot (Figs. 2A and S7), which may indicate the
local genetic connection in different regions of Tibet over the past
4,000 years. The shared branches in the Network analysis can also
suggest the genetic continuity of several haplotypes among different
regions in different periods (haplotype Aþ152 and D4j1 in WXZ, C4a
and M9a in SXZ, and F1g in NEXZ) (Figs. S3 and S4). Moreover, we
further combined the published present-day mtDNA data to explore
the relationship between our newly sequenced ancient Tibetans and
present-day populations (Table S3). The present-day populations
include present-day Tibetans of different regions, NEA, SEA, Nep-
alese and Pakistanis. In the Haplogroup Sharing analysis, present-
day Tibetans shared more haplotypes with ancient Tibetans (over
25% in every group) than the other groups (less than 10% in most
groups), indicating ancient Tibetans had the most maternal genetic
contribution to present-day Tibetans compared with other pop-
ulations. This maternal genetic contribution was also revealed by the
DAPC analysis, ancient Tibetans clustered closer with present-day
Tibetans than Han and Nepal populations (Fig. S1), which is
consistent with the view in the previous study (Ding et al., 2020).
Moreover, It is worth mentioning that we observed some deeply
diverged haplotypes (M62 and M13a) in our new sequenced data of
ancient Tibetans in different regions in our database (Table S1). Using
a molecular clock-based method (BEAST), the TMRCA of haplotypes
M62 and M13a are estimated to be around 19,422 BP and 20,986 BP,
respectively (Fig. S9). These haplotypes are among the deep lineages
maintained since the Late Pleistocene settlers in Tibet during 40e20
kya and are still present in present-day Tibetans (Qin et al., 2010; Wu
et al., 2013; Zhao et al., 2009).
Although the local genetic connection in different regions and
Tibet-specific genetic structure can be observed in a large area of
Tibet, the regional characteristics in different regions of Tibet were
revealed in our analysis. In the DAPC plot, the southwestern Tibetans
(WXZ, SXZ, and SWXZ) clustered closely with other Tibetan in-
dividuals, while northeastern Tibetans (NEXZ and NXZ) had more
overlapping areas with the NEA populations (Fig. S7). This result in-
dicates some differences in the populations in these two regions.
Moreover, the southwestern individuals have more Tibet-related
haplotypes (A11a, A21, C4a1a, C4a2b2, D4g2a1c, F1c1a1a, F1d,
M62, M9a1a1c1b1a, M9a1b1, and U2b1) and did not connect closely
with populations outside the plateau, while northeastern individuals
had more connections with NEA populations (B4, D5a, G2b, and
F1þ16189) (Figs. S3 and S4). The Network of haplotypes D5a and
F1g also revealed that the Tibetans in these two regions (southwest
6
Journal of Genetics and Genomics xxx (xxxx) xxx
and northeast) did not connect closely (Figs. S3 and S4). However,
the connections across these two regions can be observed during
specific periods. The Network of haplotype Fþ16189 shows that the
southwestern (WXZ2_3-1.4 K) and the northeastern (NEXZ_3-1.4 K)
Tibetans shared the same branch F1d in about 2,000 BP (Fig. S4).
Another connection between the populations in these two regions
was in the Tubo period, which was shown in the Network of sub-
haplogroups M9a (Fig. S4). The Tubo populations in these two re-
gions (NXZ_1.4-1.1 K and SXZ_1.4-1.1 K) shared the same haplotype
M9a1a1c1b1a. Interestingly, we did not observe this southwestern
and northeastern maternal genetic differentiation significantly in
present-day Tibetans, which may be a result of increased in-
teractions between populations from these regions. In the DAPC plot,
present-day Tibetans in these two different regions are scattered
together (Fig. S1). Less genetic distance among present-day Ti-
betans (on an average of 0.046) than ancient Tibetans (on an average:
0.089) shown in the FST values Table (Table S4). These results reveal
more closely genetic connections among Tibetans in different re-
gions in the present day.
Therefore, the local genetic continuity exists in wide areas of Tibet
from 4,000 BP to present-day reflected in some maternal lineages,
with within-regional interactions of Tibetans observed. More genetic
connections can be found among the populations in southwestern
Tibet with more maternal genetic homogeneity among Tibetans,
while the Tibetans in northeastern Tibet had more connections with
YR populations. However, the cross-region interactions between
southwestern and northeastern Tibetans can be observed in different
periods (around 2,000 BP and 1,400e1,100 BP).
The interactions between Tibetans and Nepal and Pakistan
populations since 3,000 BP
To understand the genetic relationship between Tibetans and
their neighbors, we collected the mtDNA data of ancient populations
around Tibet (Jeong et al., 2016; Narasimhan et al., 2019) (Table S2).
We found that the southwestern neighbors of Tibetans, Nepal, and
Pakistan populations, had maternal genetic connections with the
ancient Tibetans, especially with the populations in southwestern
Tibet (WXZ, SXZ, and SWXZ), but the interaction patterns among
these populations are different.
In the FST heatmap, the ancient Pakistanis had closer genetic dis-
tance with CWE populations (average FST ¼ 0.063) and showed some
affinities with the ancient Tibetans (average FST ¼ 0.078), while the
ancient Nepalese were in the Tibetan cluster (average FST ¼ 0.078) and
do not show closer connections with CWE populations (average
FST ¼ 0.18) compared with Pakistan population (Fig. 2B; Table. S4).
The DAPC plot shows that the southwestern Tibetan individuals (WXZ,
SWXZ, and SXZ) mainly scatter with other Tibetan individuals, and are
close to the CWE-related Pakistan population (Fig. S7). By contrast,
the Nepal populations clustered close to several Tibetan populations
(especially the WXZ, SWXZ, and SXZ), indicating a close maternal
genetic relationship between Tibetans and Nepal populations (Fig. 2A),
corresponding to the FST result. Moreover, the Haplogroup Sharing
analysis reveals that the sharing haplotypes between ancient Tibetans
and Pakistanis were mainly observed in the populations of western
Tibet (WXZ1_3-1.4 K and WXZ2_3-1.4 K) during 2,700e1,800 BP,
which indicates spatiotemporal connections between Tibetans and
ancient Pakistan populations (Fig. 4B) (Table S1). By contrast, haplo-
types of the Nepal population were shared with ancient Tibetans in a
wide geographical range on a long-term scale since 3,000 BP,
including all southwestern ancient Tibetans groups after 1,400 BP, and
NXZ Tibetans in 1,400e1,100 BP (Figs. 2A and S4). The proportion of
involving sharing haplotypes between ancient Tibetans and Nepalese
(14.29%e33.33%, average: 22.52%) is higher than that between Ti-
betans and Pakistanis (4.55%e11.69%, average: 10.03%), suggesting
G. Zhang, C. Cui, S. Wangdue et al.
a closer genetic relationship between ancient Tibetans and Nepalese
(Table S8). The Network and Phylogenetic tree of haplotype M70 and
Z3a1a showed the western Tibetans (WXZ1_3-1.4 K and WXZ2_3-
1.4 K) and Pakistanis connect closely and shared several variants
(such as variant 2368867 in haplotype M70, and variant 8931, 11075 in
haplotype Z3a1a), indicating the connections between them (Figs. S4,
S6D, S6E). Although we found the network of sub-haplogroup U2 and
R30 showed Tibetan and Pakistan populations shared the same clade,
they separate into different branches (Fig. S10). It is worth noticing that
U is a CWE-related haplogroup, but Tibetans had more connections
with Pakistanis in U2b rather than connecting with CWE populations
directly. Differently, the Network of sub-haplogroups M9a1a, Zþ152,
F1a’c’f, and F1þ16189 reveals that Nepal individuals did not form a
unique branch but scattered among WXZ and SWXZ groups and hard
to distinguish Nepal populations from the Tibetans branches since
3000 BP (Fig. 4D, S3, S4). In contrast, present-day Nepal Sherpas
(Nepal_Shepa) show fewer connections with both ancient and present-
day Tibetans. In the DAPC plot, they lie separately from ancient Ti-
betans and clustered closely with only Tingri groups (SChina_Tibet-
ShigatesT) (Fig. S1). In the heatmap (Fig. 2B), Nepal_Sherpa is far away
from Tibetans, the average FST value is 0.26 with ancient Tibetans and
0.23 with present-day Tibetans (For comparison, Nepal_2.4-1.2 K
share average FST values of 0.078 with ancient Tibetans) (Table S4).
These results could be explained by a genetic divergence between
Sherpas and Tibetans in previous studies (Bhandari et al., 2015)
Above all, we found the southwestern neighbors of Tibetans (the
Nepal and Pakistan populations) had maternal genetic connections
with the ancient Tibetans. The ancient CWE-related Pakistan pop-
ulations had connections with western Tibetans mainly in
2,700e1,800 BP, while the ancient Nepal population interacted
frequently with southwestern Tibetans since 3,000 BP, covering a
large area of Tibet (Ngari, Lhasa, Shannan, and Shigatse).
Discussion
Among 38 ancient populations outside Tibetan Plateau investi-
gated in this study (Table S2), we identify strong maternal genetic
connections between Tibetans and ancient NEA populations. Spe-
cifically, the phylogeny of haplotypes M9a1a, M9a1b, D4g2, and
G2a’c show ancient Tibetans shared a common ancestor with
ancient Middle YR Han-related populations (Shimao, Qingtai, and
Taosi) around early Holocene (12,517-9,385 BP). This observation fits
the scenario that YR populations expanded to Tibetan regions as the
climate became warmer in the Holocene, as suggested by various
archaeological, genetic, and linguistic studies (Su et al., 2000; Zhang
et al., 2016, 2019, 2021; Wang et al., 2021). In addition, a younger
divergence time around 7,000 BP is observed between Tibetans and
Upper YR Zongri population who possesses haplotype D4i. The
connection with Upper YR influenced wide areas of the Tibetan
Plateau, including western (WXZ1_3-1.4 K, Gebusailu site), south-
western (SWXZ_3-1.4 K, Nuda site), and northeastern
(NEXZ_post1.1 K, Gutong site) Tibet, as shown in the network anal-
ysis of haplotype D4j1. It supports the previous view that the popu-
lation expanded from the edge of the plateau to higher altitudes
during the Middle Holocene (Ding et al., 2020).
Overall, a long-term matrilineal continuity is shown in some
maternal lineages, and it has been maintained for over 4,000 years
and can still be observed in present-day Tibetans. It is worth
mentioning that we observed some deeply diverged haplotypes (M62
and M13a) in our newly sequenced data of ancient Tibetans in
different regions. Besides, the southern Tibetans adjacent to the
valleys of the Himalayas and Gangdise mountains carried more
deeply diverged old lineages M62 and M13a than the other Tibetans
(Table S1). It is possible that warm valleys in south Tibet serve as
refuge regions for these Late Pleistocene settlers during LGM
7
Journal of Genetics and Genomics xxx (xxxx) xxx
(Aldenderfer and Yinong, 2004; Qin et al., 2010), and therefore these
deep lineages still represent a relatively high level in these pop-
ulations. However, a better understanding of the genetic history of
these old lineages is needed to confirm this hypothesis. Moreover,
the maternal genetics of southwestern Tibetans (WXZ, SXZ, and
SWXZ groups) are closely related, while northeastern Tibetans show
more connections with NEA populations. While this observation can
be difficult to directly relate to the physical distance between the
above-mentioned populations, it can be well linked to the Tibetan
landscape. Particularly, the southwestern Tibetans were living along
the Yarlung Zangbo River and the Xiangquan River, and shared
freshwater resources have been suggested to facilitate population
interactions (Ning et al., 2020). In addition, the movements of
southwestern populations are greatly hindered by the Himalayas and
Gangdise mountains and thus their interactions are mostly restricted
to the highland populations.
Despite that the Tibetan Plateau is a relatively isolated region, the
ancient Tibetans frequently interacted with other populations (Zhang
et al., 2016; d’Alpoim Guedes and Aldenderfer, 2020; Zhang et al.,
2020). Particularly, the ancient Tibetan show strong matrilineal con-
nections with NEA populations during 4,000e3,000 BP, this connec-
tion weakened after 3,000 BP and then reinforced after the Tubo
period. The connections between Tibetans and other populations were
weakened around 3,000 BP, which is a time period when East Asia
experienced a cooling trend with a rapidly declining monsoon (Zhu,
1973; Dykoski et al., 2005; Marcott et al., 2013; Zhang et al., 2016).
It is very likely this climate change played a role in the observed
weakened interactions between Tibetans and other populations, as
Plateaus tend to be heavily influenced by climate changes and thus
pose additional stress and lead to a decrease in human activity to the
local populations (Li and Kang, 2006; Zhang et al., 2016). During the
Tubo period, all the haplotypes identified from Tubo individuals are
those well-represented in Tibetan populations, and no new haplotypes
linked to other populations are found. Also, the overall maternal ge-
netic diversity of Tubo populations is the lowest across all the ancient
Tibetan populations examined in this study. These findings are
consistent with the genetic and historical records that the Tubo pop-
ulation dynamics are mainly dominated by influence on the pop-
ulations in surrounding regions (such as Baltistan and Dulan, Qinghai)
instead of the other way around (Yang et al., 2021; Zhu et al., 2022),
and a population bottleneck occurred with climate change and
extensive wars after the Sui-Tang Warm Period (Li, 2009, 2015; Ge
et al., 2013; Yin et al., 2016; Zhang, 2021; Huo, 2022; Huang, 2002).
Besides, interactions between ancient Tibetan and ancient Nepal and
Pakistan populations are observed. Western Tibetans show connec-
tions with the geographically adjacent Pakistan populations during
2,700e1,800 BP. Southwestern Tibetans show close connections with
Nepal populations since 3,000 BP, which suggests the interactions
between these two populations occurred even before the construction
of the ancient Boni Road (Tian, 2015; Xiong, 2020). However, as only a
small number of samples representing several populations (e.g.,
WXZ_4-3 K) is collected, additional samples are required to obtain a
more comprehensive understanding of their maternal genetic structure
and history. In the future, additional efforts can be placed in obtaining
genomic data of ancient Tibetans to achieve a better understanding of
finer genetic structure as well as genetic variations that facilitated their
adaptation to this unique environment.
Material and methods
Ancient DNA extraction and library preparation
All ancient DNA work in this study was conducted in the ancient
DNA clean room at the Chinese Academy of Sciences’ Institute of
Vertebrate Paleontology and Paleoanthropology. A total of 178
G. Zhang, C. Cui, S. Wangdue et al.
ancient human individuals were collected from 37 sites in Tibet,
China. The archaeological details are provided in the Supplementary
data.
The 178 human remains were surface-cleaned and drilled for less
than 100 mg of bone powder according to the previous method
(Dabney et al., 2013). The single-stranded (denoted as “SS”) and
double-stranded (denoted as “DS”) libraries were prepared for all
samples and generated 199 libraries (Kircher et al., 2012; Meyer
et al., 2012), which were partially treated with uracil-DNA glyco-
sylase (denoted as “half UDG” in Table S1) (Rohland et al., 2015).
They were indexed according to previous protocols (Fu et al., 2013a).
We then amplified libraries for 35 cycles using the AccuPrime Pfx
polymerase to obtain enough ancient DNA for capture (Yang et al.,
2020). The P5 and P7 primers were added last to limit library
contamination rate (Kircher et al., 2012). We also used a NanoDrop
2000 spectrometer to evaluate the DNA concentration.
In-solution capture and sequencing of mtDNA
To accomplish the in-solution mtDNA, we used oligonucleotide
probes synthesized from the complete human mitochondrial
genome. Libraries were hybridized into probes that overlapped with
the mitochondrial genome as previously described (Fu et al., 2013a).
A targeted DNA sequence retrieval protocol was used to isolate
mtDNA fragments and recover complete mitochondrial genomes.
Finally, we got enriched endogenous ancient DNA.
The enriched mtDNA libraries were sequenced on Illumina MiSeq
platform with 2  76 base pair (bp) paired-end reads. Then we used
the leeHom software (Renaud et al., 2014) (https://github.com/
grenaud/leeHom) to trim adapters and merge paired-end reads into
a single sequence (minimum overlap of 11 base pairs). Then, we
merged reads with a length of at least 30 bp and aligned them to the
mtDNA revised Cambridge Reference Sequence (rCRS) using BWA
(version: 0.5.10-evan.9-1-g44db244, https://bitbucket.org/ustenzel/
network-aware-bwa) (Li and Durbin, 2009) with arguments: n 0.01
eo 2 el 16500 to increase sensitivity. We also removed sequences
shorter than 30 base pairs and duplicates were removed using bam-
rmdup (version: 0.6.3, https://bitbucket.org/ustenzel/biohazard-
tools). After all these steps, we constructed the whole mitochon-
drial sequence.
Test for contamination
To test for contamination from present-day humans, we
compared our mtDNA fragments to 311 world-wide mtDNA se-
quences (Reich et al., 2012). Using the contamination estimator
software ContamMix (Fu et al., 2013a), we determined the fraction of
fragments matching present-day mtDNA better than the consensus
sequence. The first and last five positions of the fragments were
ignored to avoid the damage pattern detected as contamination. If
the contamination rate was > 4%, we treated this library as
contaminated (Fu et al., 2016). Libraries with a contamination level
higher than the threshold were excluded from further analyses. Of the
178 new mtDNA samples, 26 of them were excluded from our
studies. In some cases, multiple individuals were obtained from the
same tomb and shared a single mitochondrial haplotype. To avoid
potential biases introduced by matrilineal kinship, the Bioedit soft-
ware (version 7.2.5) was used for testing the kinship. Finally, seven
pairs were found with probable matrilineal kinships and the lower
coverage one was excluded in each pair (Table S1). After manually
excluding individuals with a potentially high level of contamination
(>4%), individuals with probable matrilineal kinship, and individuals
with high proportions of ambiguous base (“N”) because of a low
coverage on mtDNA, our final dataset included 128 new samples
8
Journal of Genetics and Genomics xxx (xxxx) xxx
with high coverage and low contamination rates. Detailed samples
and sequencing information are presented in Table S1.
Radiocarbon dating
Partial samples were dated with C14 dating, using the same bone
materials for aDNA extraction. The dates were calibrated with OxCal
v.4.4 using the IntCal20 (Reimer et al., 2020) curve, and presented as
95.4% confidence intervals in calibrated years before present (cal
BP). Present was defined as 1950 CE.
Haplogroup analysis
The complete sequences of the mitochondrial genomes from the
samples were aligned with rCAS using MEGA (Kumar et al., 2016).
HaploGrep2 (Weissensteiner et al., 2016), built on PhyloTree Build 17
(Oven and Kayser, 2009) were used to call the haplogroup of each
mtDNA sequence.
Discriminant analysis of principal components and
Haplogroup Sharing
We used discriminant analysis of principal components (DAPC)
(Gruenwald and Goss, 2011; Jombart et al., 2010; Pritchard et al.,
2000) which maximizes between-group differences and minimizes
within-cluster variation based on alleles to show the different
maternal genetic relationships between Tibet population and other
populations. We used the DAPC function of the “adegenet” package
in R (version 4.1.2) software to conduct a sequence-based DAPC
(Jombart et al., 2010). All Tibet samples and only partly respective
populations were used to make the distribution clearer.
The proportion of shared haplogroups and haplotypes were
calculated as a pairwise matrix, where “sharing” was defined as the
lowest denomination of counts between groups, and “not-sharing”
was the private counts not found in other groups (Ding et al., 2020).
Genetic distance analysis
Genetic distances (FST) and corresponding P-values were
calculated by Arlequin software package (version 3.5.2.2) (Excoffier
and Lischer, 2010) (Table S4). Generally, a lower FST value repre-
sents a closer maternal genetic relationship between two groups.
Heatmap was drawn with the package “pheatmap” in R to illustrate
the statistical significance of clusters based on FST .
Construction of network and phylogenetic tree
We used DNASP6 (version 6.12.03) and PopArt 1.7 to conduct a
median-joining network analysis on all the samples in the same
haplogroup (sub-haplogroup or haplotype) dataset (Bandelt et al.,
1999; Leigh and Bryant, 2013) and construct the haplotype
network. Based on network results, we grouped haplogroups as
“Tibet - related”, “Upper YR”, “Middle YR”, “Other NEA”, “Pakistan”
or “CWE” according to locations on the branches. Details could be
seen in Supplementary data. We ploted the histogram with the pro-
portion in the population. We used HaploGrep2 (Weissensteiner
et al., 2016) to construct phylogenetic tree which shows mutation
sites and reflects basic relationship between samples in same
haplogroup.
BEAST analysis
The Bayesian phylogenetic analysis was performed by BEAST
v1.10.4 (Alexei et al., 2007) with the best-fit nucleotide substitution
model HYK þ G þ I determined by Bayesian Information Criterion
G. Zhang, C. Cui, S. Wangdue et al.
(BIC) (Darriba et al., 2012) in jModeltest2 (Guindon and Gascuel,
2003). For the choice of the molecular clock (Kass and Raftery,
1995; Yang, 2007), we assumed a strict molecular clock (Fu et al.,
2013b) for phylogenetic analysis. For the tree prior (Guy et al.,
2012), we chose Bayesian Skyline and Piecewise-constant model
(Drummond et al., 2005). Using tip-calibration samples (Shapiro
et al., 2011), a total of 10 million Markov Chain Monte Carlo
(MCMC) chains were run for this model, sampling per 1,000 steps.
Effective sample size (ESS) values were checked in Tracer v1.7.1. A
total of 10% of the trees were discarded as burn-in using TreeAn-
notator v1.10.4. The phylogeny was visualized in FigTree v1.4.4.
Haplotype diversity
We also calculated Haplotype diversity to compare genetic di-
versity among different groups, which use the frequencies of two
different haplotypes randomly selected from the samples to reflect
group diversity. Haplotype diversity is an important index to measure
the degree of variation in a population. It refers to the frequency of
two different haplotypes randomly selected from a group. Generally,
a population with high haplotype diversity indicates high genetic di-
versity and rich genetic resources. If every individual in the group has
a different haplotype, the Haplotype diversity of the group is
maximum 1.
Data availability
All the mitogenome consensus sequences reported in this paper
have been deposited in the GenBase in National Genomics Data
Center (CNCB-NGDC Members and Partners, 2022), Beijing Institute
of Genomics, Chinese Academy of Sciences/China National Center
for Bioinformation, under accession number C_AA002007-
C_AA002182 that is publicly accessible at https://ngdc.cncb.ac.cn/
genbase. These sequences have also been deposited in GenBank
under accession numbers OQ707084-OQ707211 (http://www.ncbi.
nlm.nih.gov/Genbank/index.html). All data needed to evaluate the
conclusions in the paper are present in the paper and/or the Sup-
plementary data. Additional data related to this paper may be
requested from the authors.
CRediT authorship contribution statement
Ganyu Zhang, Can Cui: Formal analysis, Visualization, Writing -
Original draft, Writing - Review & Editing. Shargan Wangdue:
Archaeological materials and dating, Writing - Review & Editing.
Hongliang Lu, Honghai Chen, Lin Xi, Wei He, Haibing Yuan, Tinley
Tsring, Zujun Chen, Feng Yang, Tashi Tsering, Shuai Li, Norbu
Tashi, Tsho Yang, Yan Tong, Xiaohong Wu, Linhui Li, Yuanhong
He, Tianyi Wang, Ming Zhang, Xing Gao: Archaeological materials
and dating. Peng Cao, Qingyan Dai, Feng Liu, Xiaotian Feng,
Ruowei Yang, Wanjing Ping: Wet laboratory work. Yichen Liu:
Conceptualization, Supervision, Writing - Review & Editing. Wenjun
Wang: Conceptualization, Supervision, Formal analysis, Writing -
Original draft, Writing - Review & Editing. Qiaomei Fu: Conceptu-
alization, Project administration, Funding acquisition, Supervision,
Writing - Original draft, Writing - Review & Editing.
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgments
We would like to thank the Archaeological Teams from Tibet,
Sichuan, Shannxi and Beijing for valuable support. This work was
9
Journal of Genetics and Genomics xxx (xxxx) xxx
supported by the National Key R&D Program of China
(2021YFC1523600), the Chinese Academy of Sciences (CAS) (YSBR-
019 and XDB26000000), National Natural Science Foundation of
China (41925009), “Research on the roots of Chinese civilization” of
Zhengzhou University (XKZDJC202006), the Tencent Foundation
(through the XPLORER PRIZE), the Howard Hughes Medical Institute
(55008731). X.W. was supported by Key National Social Science
Foundation of China (16ZDA144).
Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jgg.2023.03.007.
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