PLOS ONERESEARCH ARTICLE

Kazak mitochondrial genomes provide
insights into the human population history ofCentral Eurasia
Ayken Askapuli ID1,2,3,4*, Miguel Vilar5,6, Humberto Garcia-Ortiz7, Maxat Zhabagin1,2,3,
Zhaxylyk Sabitov8, Ainur Akilzhanova3, Erlan Ramanculov1,2, Uli Schamiloglu1,
Angelica Martinez-Hernandez7, Cecilia Contreras-Cubas7, Francisco Barajas-Olmos ID7,
Theodore G. Schurr9, Zhaxybay Zhumadilov3,10, Marlen Flores-Huacuja7, Lorena Orozco7,
John Hawks4,11, Naruya Saitou12,13,14*a1111111111
a1111111111
a1111111111
a1111111111
a1111111111

OPEN ACCESS
Citation: Askapuli A, Vilar M, Garcia-Ortiz H,
Zhabagin M, Sabitov Z, Akilzhanova A, et al. (2022)
Kazak mitochondrial genomes provide insights into
the human population history of Central Eurasia.
PLoS ONE 17(11): e0277771. https://doi.org/
10.1371/journal.pone.0277771
Editor: Francesc Calafell, Universitat Pompeu
Fabra, SPAIN
Received: January 27, 2022
Accepted: November 3, 2022
Published: November 29, 2022
Copyright: © 2022 Askapuli et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: Mitochondrial
genome sequences of 120 Kazak individuals were
submitted to Genbank, with accession numbers:
OM194220 - OM194339 The mitochondrial
microarray SNP data used for generating pseudomitochondrial genomes (N = 80) are available at
GEO (Accession Number: GSE208402).

1 School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan, 2 National Center for
Biotechnology, Astana, Kazakhstan, 3 National Laboratory Astana, Nazarbayev University, Astana,
Kazakhstan, 4 Department of Integrative Biology, University of Wisconsin-Madison, Madison, Wisconsin,
United States of America, 5 The Genographic Project, National Geographic Society, Washington, DC, UnitedStates of America, 6 Department of Anthropology, University of Maryland, College Park, Maryland, UnitedStates of America, 7 Immunogenomics and Metabolic Diseases Laboratory, National Institute of Genomic
Medicine, Mexico City, Mexico, 8 L.N. Gumilyov Eurasian National University, Astana, Kazakhstan,
9 Department of Anthropology, University of Pennsylvania, Philadelphia, Pennsylvania, United States ofAmerica, 10 School of Medicine, Nazarbayev University, Astana, Kazakhstan, 11 Department of
Anthropology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America,
12 Population Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka, Japan,
13 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan,
14 Advanced Medical Research Center, Faculty of Medicine, University of the Ryukyus, Okinawa Ken, Japan* ayken.askapuli@wisc.edu (AA); saitounr@nig.ac.jp (NS)

Abstract
As a historical nomadic group in Central Asia, Kazaks have mainly inhabited the steppezone from the Altay Mountains in the East to the Caspian Sea in the West. Fine scale characterization of the genetic profile and population structure of Kazaks would be invaluable forunderstanding their population history and modeling prehistoric human expansions acrossthe Eurasian steppes. With this mind, we characterized the maternal lineages of 200 Kazaks
from Jetisuu at mitochondrial genome level. Our results reveal that Jetisuu Kazaks haveunique mtDNA haplotypes including those belonging to the basal branches of both West
Eurasian (R0, H, HV) and East Eurasian (A, B, C, D) lineages. The great diversity observedin their maternal lineages may reflect pivotal geographic location of Kazaks in Eurasia andimplies a complex history for this population. Comparative analyses of mitochondrial
genomes of human populations in Central Eurasia reveal a common maternal genetic
ancestry for Turko-Mongolian speakers and their expansion being responsible for the presence of East Eurasian maternal lineages in Central Eurasia. Our analyses further indicate
maternal genetic affinity between the Sherpas from the Tibetan Plateau with the Turko-Mongolian speakers.

Funding: Fieldwork and DNA preparation parts ofthe project were supported by a Targeted Funding
Program for 2014-2017 from the Science
Committee of the Ministry of Education and

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1 / 32PLOS ONE

Science of the Republic of Kazakhstan (Grant No.
not available). Center for Life Sciences, National
Laboratory Astana, Nazarbayev University received
the grant. https://www.gov.kz/memleket/entities/
edu?lang=en Part of the data analysis was fundedby the Science Committee of the Ministry of
Education and Science of the Republic of
Kazakhstan (Grant No. AP08855823), that wasreceived by the National Center for Biotechnology,
Kazakhstan. https://www.gov.kz/memleket/entities/
edu?lang=en Part of the data analysis was
supported by grants from Nazarbayev University,
Kazakhstan (Grant Award Numbers:
090118FD5332 and 091019CRP2119). US
received the grants. https://nu.edu.kz The funders
had no role in study design, data collection andanalysis, decision to publish, or preparation of themanuscript.
Competing interests: The authors have declaredthat no competing interests exist.

Maternal lineages of Kazaks from Jetisuu

Introduction
Central Asia is a region that has witnessed important events in modern human population history,
including the westward migration of nomadic pastoralists [1, 2], the eastward expansion of IndoEuropean speakers [3–5], and admixture between anatomically modern humans and archaic
humans [6–9]. Notwithstanding the historical significance of the region, in-depth, comprehensive
genetic studies of human populations in Central Asia are generally sparse. While the distributionof paternal lineages in Central Asia and Siberia is associated with language and ethnic identity [10,11], maternal lineages do not exhibit any associations with language and ethnic group, except for
an increase of West Eurasian mtDNA haplogroups in an east-to-west direction [12].
Previous studies have showed that human populations in Central Asia, including Kazaks,
harbor elevated frequencies of both East and West Eurasian maternal lineages [12–14]. To
date, there are several studies of maternal lineages in Kazak populations from the Altay Republic (Russia) [12, 15], from Xinjiang (China) [16, 17], and from Kazakhstan [13, 14, 18, 19].
While most of these studies sequenced the mitochondrial DNA (mtDNA) control region andgenotyped coding region diagnostic SNPs, few studies analyzed the mitochondrial genomes(mtGenomes) of Kazaks (N = 19) [15, 20–22]. These studies again revealed the presence ofboth East and West Eurasian lineages in Kazak populations. Ingman and Gyllensten (2007)
sequenced mitochondrial genomes (haplogroups A, D, and T) from six Kazaks. Derenko et al.
(2012, 2014) presented whole mitochondrial genome sequences for twelve Kazaks, which
belong to haplogroups M, H, HV, and U. Besides, Sahakyan and colleagues published a single
Kazak mtGenome of haplogroup U in 2017 [21].
Historically speaking, there are three major divisions of Kazak populations. They include
the Great Jüz, Middle Jüz, and Junior Jüz. The three Jüz occupy different, albeit overlapping,regions of Kazakhstan and bordering countries. While the Junior Jüz resides in the west, theMiddle Jüz inhabits central, north, and east Kazakhstan, and the Great Jüz lives in southern
regions of the country.
To evaluate the maternal lineages of Kazaks at a finer scale, and to better understand theorigins and dispersal of maternal lineages in Central Eurasia, we analyzed the mitochondrial
genome data of 200 Kazak individuals, including full mtGenomes from 120 individuals andmicroarray data based pseudo-mtGenomes for 80 individuals, all living in the Jetisuu region ofsouthern Kazakhstan (Fig 1).
These individuals belong to four clans of the Great Jüz, including Jalayir, Alban, Shapirashti, and Suan. Our results show that while a diverse array of haplogroups of Eastern andWestern Eurasian origins comprise Kazak maternal lineages, Eastern Eurasian haplogroups,
including A, B, C, D, F and G, account for about 50% of maternal genetic diversity of Jetisuu
Kazaks. These findings indicate that mtDNA can help delineate historical processes of population formation in communities that are patrilineal in their social organization. We also performed comparative analyses of mtDNA variation in Jetisuu Kazaks and previously published
mtGenomes (N = 2074) from 21 ethnic groups from across Central Eurasia. Our analyses indicate that mtDNA haplogroups are associated with language background at the language familylevel, while the presence of East Eurasian lineages in Central Eurasia is associated with the
expansion of Turko-Mongolian speakers.

Results
mtDNA haplogroups in Jetisuu Kazaks
As shown in Table 1, Jetisuu Kazaks harbor at least 56 different haplogroups. Seven of them
(C4, D4, F1, G2, H2, T2, and U5) occur at an average frequency of 3.5% or higher and

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2 / 32PLOS ONEMaternal lineages of Kazaks from Jetisuu

Fig 1. Map of Jetisuu and the sampling sites. 1. Engbekshi Kazak; 2. Talghar; 3. Ile; 4. Almati; 5. Karasay; 6. Jambil; 7. Balkash; 8. Karatal; 9. Köksuu; 10.
Taldikorghan; 11. Eskeldi; 12. Kerbulak; 13. Panfilov; 14. Kegen; 15. Rayimbek.
https://doi.org/10.1371/journal.pone.0277771.g001

appear in at least three of the clans surveyed in this study. Another six haplogroups (A5, B4,
C5, D5, N9, and U2) appeared at a frequency of 2.0–3.0% and occurred in at least two of theclans. The remaining 43 haplogroups were represented by just one to three individuals(0.5–1.5%). Only B4, D4, G2, H2, and U5 mtDNAs were found in all four Jetisuu Kazak
populations. In addition, haplogroup and genetic diversity was associated with the sample
size in the Jetisuu Kazak populations (Tables 1 and 2), with Jalayirs (N = 76) having thehighest number of haplogroups and Suans (N = 26) having the lowest haplogroup diversity(Tables 1 and 2).

mtDNA control region based PCA analysis
We compared mtDNA profiles of the Jetisuu Kazaks with geographically adjacent populations based on haplogroup frequencies determined from the mtDNA control region
sequences, since only control region data were available for most of the populations analyzed. As shown in the resulting PCA (Principal Component Analysis) plot, three groupingsof populations appear in this analysis: (1) Turko-Mongolians clustered around indigenous
Altaians; (2) Sino-Tibetan speaking groups; and (3) Uyghurs-Uzbeks (Fig 2). The TurkoMongolian cluster was comprised of Turkic speaking Altaians at the center, surrounded by
Turkic speakers (Kazak populations from Jetisuu (Kazakhstan), from Xinjiang (China), andfrom the Altay Republic (Russia), and Kirghiz from Kyrgyzstan) and Mongolic speakers(Mongols from Inner Mongolia and Xinjiang (China), and Barghuts from Inner Mongolia).
Notwithstanding the vast geographic distance between them, Mongols and Barghuts fromPLOS ONE | https://doi.org/10.1371/journal.pone.0277771 November 29, 2022

3 / 32PLOS ONEMaternal lineages of Kazaks from Jetisuu

Table 1. Mitochondrial DNA haplogroup frequencies in Kazak populations from Jetisuu, Kazakhstan.
Hg
A

Alban
1

2.86

A12

Jalayir

Shapirashti

Shapirashti

Suan

JetiKaz

2

I1

1

1.69

1

0.5

1

1.32

1

1.69

2

1

I2

1

1.69

1

0.5

1

1.69

1

0.5

I4

1

0.5

1.69

1

3.85

1

3.85

1

2.86

1

1.32

2.86

1

1.32

1
2

3.39

1

2.86

6

7.89

4

6.78

B5
C5

3

5.08

C7

1

1.69

14.29

Jalayir

4

1

5

Alban

1.69

A5

D4

Hg

1

B4

D1

JetiKaz

2.63

A14

C4

Suan

2

1

1.32

10

13.16

11

18.64

1

3.85

2

1

J1

4

2

K1

3

1.5

M10

11

5.5

M5

1
1

1

1.32

1

1.32

1

1.32

2.86

1

1.69

1

3.85

2.86

3

1.5

1

0.5

1

0.5

1

0.5

1

0.5

2

1

5

2.5

M65

1

1.69

1

0.5

M7

1

1.69

1

0.5

N1

1

2.86

1

1.32

16

N9

2

5.71

1

1.32

1

2.86

1

0.5

1

1.32

1

0.5

5

19.23

32
6

3

R0

11.54

11

5.5

R1

1
1

3.85
3.85

2

1

5

2.5

D5

3

3.95

3

5.08

F1

6

7.89

2

3.39

3
1

3.85

1

0.5

R2

1

2.86

1

0.5

7

9.21

5

8.47

1

3.85

15

7.5

R9

1

2.86

1

1.32

2

1

1

1.32

1

0.5

T1

1

2.86

1

1.32

2

1

6

3

T2

4

5.26

11

5.5

3

1.5

U2

1

2.86

3

3.95

1

0.5

U4

1

2.86

1

1.32

2

2.63

F2
G2

2

5.71

G3
H

4

11.43

H1

1

2.86

H10

1

H13
H2

2

5.71

H3

1

2.86

1

1.69

2

3.39

1

3.85

1.32

2

2.63

1

1.69

3

1.5

U5

2

5.71

8

10.53

3

5.08

13

6.5

U7

1

2.86

1

0.5

V14

5

8.47

2

3.39

1

1.69

2

7.69

1

3.85

1

3.85

1

3.85

4

2

3

1.5

7

3.5

2

1

1

0.5
0.5

H44

1

3.85

1

0.5

W3

1

1.32

1

H5

1

3.85

1

0.5

W6

1

1.32

1

0.5

1

0.5

X2i

1

0.5

2

1

Z1

2

1

1

0.5

Z3

1

1.32

1

0.5

Z4

1

1.32

1

0.5

H6
H8
H9

1

1

1.32

1

1.32

1

1.69

2.86

1

1.69
2

7.69

HV

1

1.69

1

0.5

HV1

1

1.69

1

0.5

1

0.5

Hg

24

33

26

18

59

3

1.5

N

35

76

59

26

200�

HV14
HV2

1

2.86
2

2.63

Notes: Haplogroups in this table are based on the haplogroup calls obtained via Haplogrep 2.0 [23] and mtPhyl 5.003 [24];�

The total number of mtDNA analyzed is N = 200, including four samples with ambiguous clan identity, but are from the four clans (their haplogroups are D4, HV2,

C5, and N9, respectively) and 196 samples with unambiguous clan identities; Frequencies are shown in italics (%); Hg: Haplogroups; N: Number of samples; JetiKaz: all
200 Kazak samples from Jetisuu; Total number of haplogroups: 59.
https://doi.org/10.1371/journal.pone.0277771.t001

Inner Mongolia clustered close to Mongols, Kazaks, and Kirghiz in Central Asia. By contrast, Uyghurs and Uzbeks are genetically distant from their geographic, religious, and linguistic kin in Central Asia.
With regard to Kazak populations, they did not form a unique (single) cluster, but instead
were scattered around the PCA plot (Fig 2). This distribution reveals an elevated maternal
genetic diversity in Kazaks, as noted previously [12]. Mitochondrial haplogroup frequencies in
Kazaks also varied depending on sampling sites and sample sizes. The most extreme disparities
were observed between Kazak populations from Xinjiang (XJ_Kaz_a and XJ_Kaz_b), while ananalogous situation was also observed in Altaian Kazaks (Alt_Kaz_a and Alt_Kaz_b), indicating the pronounced effect of sampling (size, coverage, location etc.).

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Table 2. Results from Tajima’s Neutrality Test [33].
Populations

Seq. Type

N

S

Ps

Θ

π

D

Alban

Pseudo15

160

0.0096566

0.0029698

0.0016957-1.8932561

Jalayir

Shapirashti

Suan

Jetisuu Kaz

Genuine20192

0.0115879

0.0032663

0.0018621-1.7843685

Combined

35308

0.0185889

0.0045138

0.0019007-2.2005190

Pseudo25

208

0.0125536

0.0033246

0.0019530-1.6451853

Genuine51443

0.0267367

0.0059425

0.0021242-2.3341813

Combined

76526

0.0317460

0.0064770

0.0021572-2.3233549

Pseudo27

221

0.0133382

0.0034605

0.0019516-1.7178648

Genuine32276

0.0166576

0.0041362

0.0020340-1.9549262

Combined

59383

0.0231155

0.0049751

0.0021047-2.0590438

Pseudo13

142

0.0085702

0.0027617

0.0018354-1.5346953

Genuine13153

0.0092341

0.0029757

0.0020451-1.4321028

Combined

26244

0.0147263

0.0038591

0.0020544-1.8558244

Pseudo80412

0.0248657

0.0050204

0.0019058-2.1460627

Genuine120689

0.0415837

0.0077574

0.0020337-2.4686793

Combined

200854

0.0515420

0.0087761

0.0020823-2.4548640

Notes: This analysis involved 200 mitochondrial sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for eachsequence pair (pairwise deletion option). There were 16569 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [34].
Abbreviations: N = number of sequences, S = Number of segregating sites, Ps = S/n, n = total number of sites, Θ = Ps/a, π = nucleotide diversity, and D is the Tajima
test statistic [35].
https://doi.org/10.1371/journal.pone.0277771.t002

Although living in proximity to each other, the four Kazak populations from Jetisuu exhibited considerable diversity in mtDNA lineages (Figs 2 and 3 and Table 1). While Albans had
the highest number of West Eurasian lineages, Suans had the fewest. Jalayir and Shapirashti
were positioned between Albans and Suans in the PCA plot. As indicated before, mtDNA haplogroup frequency distribution in Jetisuu Kazaks might have been influenced by sample size.
Overall, no pronounced differences were observed in the phylogenetic affiliation of the four
populations (SF-1).
As shown in Fig 3, East Eurasian haplogroups A, B, C, D, F, and G occurred in almost all
populations in Central Asia, with D being the most frequent one. West Eurasian haplogroups(H, HV, V, J, T, U, W, and X) including several subbranches of R were also found in TurkoMongolian speakers, albeit at relatively low frequencies. Haplogroups H and U were the mostcommon West Eurasian haplogroup in Central Asia, and also occurred in the Jetisuu Kazaks
at appreciable frequencies. In contrast, the West Eurasian haplogroups were almost absent in
Sino-Tibetan speaking groups, but they harbored high frequencies of East Eurasian haplogroups M7 and M8.
In the PCA plot, Han Chinese and Dungan (Hui) from Xinjiang clustered together. Yet, thefrequencies of the maternal lineages in Dungans considerably differed from those in Han Chinese. This result suggested that the maternal gene pool of Dungans did not exclusively derivefrom Han Chinese, even though Dungans supposedly shared a common maternal ancestry
with them and speak Chinese. In Central Asia, intermarriages between Dungans and Turkic
speakers are not uncommon because of shared religious beliefs. As an example, Dungans harbored West Eurasian maternal lineages such as U2, X2, T1, and HV [29], which are virtuallyabsent in Han Chinese. The Han Chinese from Xinjiang also exhibited different maternal lineages compared to other Turko-Mongolian groups in Central Asia, indicating their recent
arrival in Xinjiang. Despite the overall similarity of mtDNA diversity in Han Chinese andTibetans, the latter clustered closer to Mongols and Kazaks.

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Fig 2. Geographic distribution of the human populations and the mtDNA control region haplogroup frequency based PCA.
Notes on Fig 2: Jeti_AL [Alban (N = 35)], Jeti_JA [Jalayir (N = 76)], Jeti_SH [Shapirashti (N = 59)], and Jeti_SU [Suan (N = 26] are
the four Kazak populations from Jetisuu, Kazakhstan; Jeti_KZ [Four populations from Jetisuu combined, plus 4 individuals with
ambiguous clan identity (N = 200)]; Altaian [Altaians (N = 490) from the Altay Republic, Russia] [25]; AltKaz_a [Kazaks (N = 237)
from the Altay Republic, Russia] [12]; AltKaz_b [Kazaks (N = 98) from the Altay Republic, Russia] [15]; Barghut [Barghuts (N = 149)

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from Hulunbuir, Inner Mongolia, China][15]; IM_Mon [Mongols (N = 48) from Inner Mongolia, China] [26]; KZ_Uyg [Uyghurs(N = 55) from Penjim, Panfilov, Almati, Kazakhstan], KG_KirT [Kirghiz (N = 48) from Talas, Kirghizstan], KG_KirS [Kirghiz(N = 47) from Sari-Tash, Kirghizstan], KZ_Kaz [Kazaks (N = 55) from Kegen, Almati, Kazakhstan] [13]; Tibetan [Tibetans (N = 6109)
from across Tibet, China] [27]; XJ_Hui [Dungans (Hui) (N = 45)], XJ_Mon_a [Mongols (N = 49)], XJ_Kaz_a [Kazaks (N = 53)],
XJ_Uzb [Uzbeks (N = 58)], XJ_Uyg [Uyghurs (N = 47)], and XJ_Han [Han Chinese (N = 47)] [All six populations from Xinjinag,
China] [17]; XJ_Kaz_b [Kazaks (N = 151) from several locations in Xinjiang, China, including b.1: Altay, b.2: Ile (Kulja), b.3: Buratala,and b.4: Urumqi, Sanji, and Kumul] [16]; XJ_Mon_b [Mongols (N = 106) from Xinjiang, China, but exact sampling site is unknown][28].
https://doi.org/10.1371/journal.pone.0277771.g002

Mongols from Xinjiang (XJ_Mon_a and XJ_Mon_b) and Inner Mongolia (IM_Mon) cluster together with Barghuts who speak a Mongolian language. Although characterized predominantly by East Eurasian maternal lineages, Mongolian speakers also harbored certain West
Eurasian haplotypes (e.g., U, H, and HV, as shown in Fig 3) [15] at minimal frequencies, most
likely due to their links with Turkic speakers in Central Asia. Xinjiang Mongols further sharedmultiple maternal lineages with Kazaks from Xinjiang and Kazakhstan. This finding might be
explained by their common Altaic ancestry, geographic proximity, similarity in subsistence
patterns, and intermarriage during the periods of Jungar, Kalmak, and Kazak Khanates.
By contrast, Mongols from Inner Mongolia shared a certain number of maternal haplotypes
with Kazaks. This finding may imply their descent from a common ancestral group at the timeof Genghis Khan or even before. Among Turkic speakers from Central Asia, Uyghurs andUzbeks exhibited elevated frequencies of West Eurasian lineages, indicating that a substantial
number of maternal lineages may have been contributed by Indo-European speaking peoples
to the gene pools of Uyghurs and Uzbeks, even though they are Turkic speakers.

Analysis of mtDNA variation
We subjected the mtDNA sequences to statistical analysis to evaluate patterns of diversity in
Kazaks. Values of Tajima’s D are significantly deviated from neutrality, implying that population expansion and/or natural selection might have played a role in shaping genetic diversity
in Kazaks (Table 2). The disparity between the number of segregating sites and the average
number of nucleotide differences was more pronounced when the number of sequences washigher. However, it seems that negative Tajima’s D characterizes mitochondrial sequences(both control region and whole genome sequences) from many human populations [12, 30–
32].

Comparative phylogenetic analysis of mtGenomes from Central Eurasia
Here we discuss the genetic features of maternal lineages present in Kazaks from Jetisuu in thecontext of central Eurasia, including Central Asia, Siberia, and Eastern Europe. We retrieved
over 2000 full mtGenome sequences from GenBank [36], and used them to conduct a comparative analysis of the maternal genetic diversity in the four language families in Eurasia, namely,
Altaic, Uralic, Sino-Tibetan, and Indo-European (Table 3).
Altaic speakers were the main focus of the analyses in the current study, since we aimed tounderstand their origins from the maternal perspective and evaluate the maternal gene pool of
Kazaks in relation to those of other Altaic speakers. The mtDNA sequences of Slavs [37–39]
and Tajiks [32] were included in the analysis in an effort to trace the origins of the West Eurasian maternal lineages (e.g., H, V, and U) found in Kazaks. We further included Hungariansequences [40] to investigate whether Hungarians had any maternal genetic affinity with
Kazaks and other Altaic speakers, since Hungarians have historical connections with Central
Asia. Lastly, we analyzed mtGenomes of Tibetans [41] and Sherpas [42], because Tibetan

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7 / 32PLOS ONEMaternal lineages of Kazaks from Jetisuu

Fig 3. A heatmap for the mtDNA haplogroups and their relative frequencies. Notes on Fig 3: the heatmap was
generated for the haplogroup frequency data that was used for PCA analysis in Fig 2.
https://doi.org/10.1371/journal.pone.0277771.g003

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8 / 32PLOS ONEMaternal lineages of Kazaks from Jetisuu

speakers have been neighbors of Altaic speakers for millennia and have interacted with themduring various time periods of history.
In the current study, we were able to build high resolution of phylogenetic trees from the
mtGenome sequences of the Jetisuu Kazak populations and evaluate their maternal genetic
affinities with populations from Central Asia and other regions. Comparative analyses werecarried out using 2074 full mtGenome sequences from 21 ethnic groups (Tables 3 and S4). The
analyses were performed in three rounds: (1) all sequences (N = 2274), including those from
Jetisuu Kazaks and other Central Eurasian populations, were analyzed together to identify
their affinities and haplogroup affiliations; (2) Central Eurasian and Jetisuu Kazak sequencesthat belonged to the haplogroups A, B, C, D, F, G, H, T, and U, were selected and aligned
together with sequences from the Phylotree Build 17.0 [46] (N = 1155; Tables 4 and S5 andS6). Subsequent analyses were separately performed for each of the haplogroups. Below, we
present the results of the final round of phylogenetic analyses, focusing on each of the major
haplogroups which characterize maternal genetic composition of the Jetisuu Kazaks.
In order to evaluate genetic distance among populations, we estimated pairwise FST values
and performed PCA analysis with them (Fig 4). In the PCA plot shown in Fig 4, Kirghiz, Hungarians, and Tibetans were positioned at the tips of a triangular pattern, indicating they were
genetically quite distant from each other, while other populations were scattered largely
around the edges of the imaginary triangle. Altaic speakers clustered together in the top left
quarter of the PCA plot (Fig 4), with Tubas and Tatars being exceptions. While Tubas werepositioned close to Sherpas, Tatars separated from the other Turkic speaking populations andclustered together with Tajiks. An unexpected observation was the separation of Sherpas from

Table 3. Information on whole mitochondrial genome sequences which used for comparison.
Nr.
1

Language
AltaicTurkic2Ethnicity

N

Reference

Altaian35

Derenko et al. 2012; Derenko et al. 2010; Derenko et al. 2007;

AltKaz11

Derenko et al. 2014;

Kazak

7

Sahakyan et al. 2017; Ingman and Gyllensten 2007;

3

Kirghiz

125

Peng et al. 2018

4

Tatar

73

Malyarchuk et al. 2010

5

Tuba

33

Sukernik et al. 2012; Zhao et al. 2009; Volodko et al. 2008;

6

Uyghur734

Peng et al. 2018; Zheng et al. 2017 [43]; Zhao et al. 2009

7

Uzbek

5

Sahakyan et al. 2017; Zhao et al. 2009; Ingman et al. 2000;

Barghut

14

Derenko et al. 2014; Derenko et al. 2012;9

Buryat231

Derenko et al. 2007; Derenko et al. 2010; Derenko et al. 2012; Derenko et al. 2014; Derenko et al. 2017/2018;10

Kalmak3

Palanichamy et al. 2015 [44]; Derenko et al. 2014; Derenko et al. 2010;

11

Mongol7

Ingman & Gyllensten 2007; Sahakyan et al. 2017; Kong et al. 2003; Protasova et al. 2016 [45];8

Mongolic

12

Uralic

Ugric

Hungarian95

Malyarchuk et al. 201813

Sino-Tibetan

Tibetic

Tibetan231

Zhao et al. 2009

Sherpa76

Kang et al. 2013

Persian

Tajik

223

Peng et al. 2018

SlavicBelrussian7

Mielnik-Sikorska et al. 2013; Malyarchuk et al. 2008;

17

Czech

13

Mielnik-Sikorska et al. 2013; Malyarchuk et al. 2008;

18

Polish30

Mielnik-Sikorska et al. 2013; Malyarchuk et al. 2008;

14
15
16

Indo-European

19

Slovak22

Mielnik-Sikorska et al. 2013; Malyarchuk et al. 2008;

20

Russian98

Mielnik-Sikorska et al. 2013; Malyarchuk et al. 2008; FTDNA;

21

Ukranian

1

Malyarchuk et al. 2008

Total number of mtG sequences (N):2074

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Table 4. Central Eurasian mtGenomes analyzed together with phylotree mtGenomes.
Count

Haplogroup

Jetisuu

Central Eurasia

Phylotree

Total N

1

A

9

53

26

88

2

B

4

13

7

24

3

C

17

68

22

107

4

D

37

112

57

206

5

F

12

42

22

76

6

G

17

54

28

99

7

H

28

126

116

270

8

T

13

60

38

111

9

U

15

123

36

174

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Tibetans, with the former being closer to Turkic speakers, especially Tubas. Interestingly, Hungarians were clustered together with Russians in the bottom right quarter of the PCA plot.

Major East Eurasian Lineages
Haplogroup A. Haplogroup A is one of the founding haplogroups for Asia and the Americas. Haplogroup A was first identified by Schurr et al. [47] and further described by Torroni
et al. [48] as one of the major maternal lineages of Native Americans. In our sample set from
Jetisuu, we observed three major subbranches of A, namely A5, A12, and A14. The diverse
branching pattern of haplogroup A suggested that it was one of the founding maternal lineagesof Kazaks.
Haplogroup A5 contained mtDNA sequences from Buryats, Kazaks, Uyghurs, and Japanese(Fig 5), with the Japanese sequences AP010701 and AP008874 being identical to those in
Uyghurs and Buryats. This result suggested that Japanese have some maternal genetic affinities
with Turkic speakers (Kazaks and Uyghurs) and Mongolic speakers (Buryats), as suggested bysome models of Altaic language family evolution, e.g., Trans-Eurasian language model [49].

Fig 4. Geographic distribution of the human populations included in the analysis of mitochondrial genomes from Central Eurasia and the mtGenome pairwise FST
value based PCA. Notes on Fig 4: Populations having more than 30 sequences were included in the PCA analysis, and thus the dataset contained 1954 sequences fromprevious studies (Table 3) and 120 Kazak mtGenome sequences from Jetisuu. Sample IDs of the sequences were given in S7 Table. The pseudo-mtGenomes (N = 80) were
excluded from the PCA analysis. Kazak [Kazaks from Jetisuu, Kazakhstan, i.e., Jeti_Kaz on the map]; Alt_Kaz [Kazaks from the Altay Republic, Russia]; Buryat [Buryats
from the Buryat Republic, Russia]; Hungarian [Hungarians from Hungary]; Kirghiz [Kirghiz from Xinjiang, China (TJ_Kir) and Kirghiz from Tajikistan (TJ_Kir)];
Russian [Russians from Russia]; Sherpa [Sherpas from Zhangmu, Tibet, China]; Tajik [Tajiks from Xinjiang, China (XJ_Taj) and Tajiks from Tajikistan (TJ_Taj)]; Tatar[Tatars from the Tatar Republic, Russia]; Tibetan [Ethnic Tibetans and other ethnic groups from Tibet, China (Tib_1–5)]; Tuba [Tubas from the Tuba/Tuva Republic,
Russia]; Uyghur [Uyghurs from Xinjiang, China (Uyg_1–2)].
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Fig 5. Schematic phylogenetic tree for the haplogroup A mitochondrial genomes. Note: Alt: Altaian; Uyg: Uyghur; Kir: Kirghiz; Hu: Hungarian.
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PLOS ONEMaternal lineages of Kazaks from Jetisuu

Kazaks, Uyghurs, and Japanese had additional mtDNA lineages representing multiple
branches of A5, implying that the possibility of recent admixture was highly unlikely.
In the Phylotree Build 17.0 [46], A14 does not have any sub-branches. It contains only a single Altaian sequence (EF153775), which is included in our dataset (Alt178.MD07). Our phylogenetic analysis identified new subbranches of A14, which were formed by sequences from
Turkic speaking Altaians (A14a), Kazaks (A14b), Kirghiz (A14c), and Uyghurs (A14d andA14e). This configuration suggested that these sequences might derive from a common Turkic
maternal ancestor.
Two sequences from Jetisuu, along with a sister branch from Hungary, formed a distinct
clade (A12a). The two Kazak sequences (JA021 and SH111) were named A12a3 and A12a4,
respectively, whereas the Hungarian sequence (HuD1.BM18) was named A12a5. One of thetwo sequences representing A12a in the Phylotree Build 17.0 [46] came from an Evenk(EF397560), and the second from a Mansi (AY519488).
Haplogroup B. Haplogroup B is ubiquitous in Asia, Oceania, and Americas [12, 15, 25,
50–53]. It may have originated in Southeast Asia [50, 51], and is estimated to be 50 kyr old[54]. In support of this view, an mtDNA with the basal sequence motive of haplogroup B was
found in a 40-kyr-old ancient human remain excavated from Tianyuan Cave near Beijing,
China [55].
Two of its sub-branches, B4 and B5, were found in Jetisuu Kazak populations. In Fig 6, B4j
contains three sequences, one each from a Kazak (B4j1), a Uyghur (B4j3), and a Buryat (B4j2),
indicating that the haplogroup is shared among Turko-Mongolian speakers. With these data,
we confirm the motif of B4j [3548 4080 5300 6122 11893 11941 13911 14248 15172 16223
16362] that was given a preliminary status in the Phylotree Build 17.0 [46].
A subclade of B4, B4b1a3, which was first described and named by Derenko and colleagues[15], was found in Altaians, Buryats, and Uyghurs (S4 Table). Found in Turko-Mongolian
speakers, this haplotype is phylogenetically closest to the Native American B2 branch.
B4c1b2b, a rare branch of B4 previously found in Chuvash people [15], has its sister branch(B4c1b2a) in Uyghurs (S4 Table), indicating the possible existence of the lineage in the ProtoTurkic stock population.
In our sample set, B5b was represented predominantly by sequences from Turko-Mongolian speakers (N = 13), including Turkic speaking Altaians, Kazaks, Kirghiz, and Uyghurs, andMongolic speaking Buryats, except for a single sequence in a Tibetan. Two Kazak and one
Uyghur sequences also did not belong to any previously known branches in the clade of B5b.
Therefore, the Kazak sequences, SH017 and SH020, were named B5b7a and B5b7b, respectively, and the Uyghur sequence was named B5b6.
In the Phylotree Build 17.0 [46], we noted Japanese sequences which represented several
sub-branches of B5b. For instance, Jetisuu Kazak sequence SU112 showed similarities withJapanese sequence AP008546 as shown in Fig 6. Although it is evident that Japanese mtDNAs
show similarities to those in Turko-Mongolian speakers from Central Asia and Siberia, B5b
seems to have a wide geographic distribution. B5b5 [KF540694] comes from Hakka, a Sinitic
speaking native group from Taiwan [56], while B5b4 [KF849981] is (mostly likely) from Han
Chinese [57].
Haplogroup C. Haplogroup C mtDNAs are found in human populations throughout
Asia and the Americas [12, 15, 25, 51, 58]. In Jetisuu Kazak populations, seventeen individuals
harbored haplogroup C mtDNAs, which represented 8.5% of the total sample size (Table 1).
Except for C1, which is an American branch [59], all major sub-branches of haplogroup C
appeared in the Jetisuu Kazak populations, including C4, C5, and C7. Most of the Jetisuu
Kazak C4 sequences belonged to C4a, with one C4b and one C4d mtDNA also being present.

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Fig 6. Schematic phylogenetic tree for the haplogroup B mitochondrial genomes. Note: Alt: Altaian; Bur: Buryat; Uyg: Uyghur; Kir: Kirghiz; Tib:
Tibetan.
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A refined nomenclature was used for the subbranches of C4 after constructing a phylogenetic
network for the mtGenome sequences.
C4a1a encompassed sequences from mainly Turko-Mongolian speakers, but also contained
sequences from Indo-European speakers, such as Russians (Rus32), Poles (Pol422), and Tajiks(Taj126 and others), Uralic speaking Hungarians (HuS22), and Sino-Tibetan speakers(Tib034), indicating much deeper temporal origin and wide dispersal of the clade (S4 Table
and Fig 7). Similarly, C4a1a4 contained four Jetisuu Kazak sequences in addition to multiple
sequences from various ethnic groups, including Altaians, Buryats, Kirghiz, Tibetans, andUyghurs. The five sub-branches of C4a1a4 were named as C4a1a4a through C4a1a4e, of which
C4a1a4e was represented by a Jetisuu Kazak sequence (Fig 7). The tree topology suggested that
C4a1a4 could also be one of the founding maternal lineages for Turko-Mongolians. In the
Phylotree Build 17.0 [46], C4a1a4 is represented by just two sequences from Siberia, one fromthe Shors and another from the Buryats, the latter being included in our dataset.
Haplogroup C5 has four sub-branches, including C5a, C5b, C5c, and C5d, each of which
also having their own subclades. There are both C5a and C5b sequences in our Jetisuu Kazak
populations (Fig 8). C5a1 is represented by a single branch in the Phylotree Build 17.0 [46].
Two of our Kazak sequences, SU013 and KZ103, clustered within this clade, and were named
C5a1c1 and C5a1c2, respectively. Altaian, Buryat, and Uyghur sequences also grouped within
this cluster, with the Altaian sequence (Alt121) being defined as C5a1b in the Phylotree Build
17.0 [46].
C5b1 was represented by sequences from Kazaks, Uyghurs, Buryats, and Yakuts (Fig 8).
C5b1a included one Kazak sequence (SH026), and several other sequences from Altaians, Buryats, and Kirghiz, while Kazak sequence (SH026) belonged to C5b1a3. Two Jetisuu Kazak
sequences, SH108 and SH011, belonged to subclade C5b1b1, which has a single branch represented by two Yakut sequences (FJ951438 and KF148480) in the Phylotree Build 17.0 [46]. Its
sister branch C5b1b2 appeared in Uyghurs. The Kazak and Uyghur sequences were morederived compared to the ones found in Yakuts and Buryats, as shown in Fig 8. The diagnostic
mutation for C5b, an insertion at np 1719 (1719.1G), should be reconsidered, since all 15
mtGenomes in our data set possessed a back mutation at this position (@1719.1G).
In light of this evidence, haplogroup C5 can be considered as one of the founder lineages of
Turko-Mongolian speakers, or Altaic speakers more generally. All but one of the sequences inC5b belong to Turko-Mongolian speakers, including Altaians, Buryats, Kazaks, Kirghiz,
Uyghurs, and Yakuts. The remaining sequence (AY615359) appeared in Nganasans, who
speak a Samoyedic language of the Uralic language family but have mixed with Tungusic
speaking and other groups in the past [60].
Haplogroup D. Haplogroup D, first discovered in Native Americans [47, 48, 61], is amajor East Eurasian lineage, one that occurs in Northeast and Southeast Asia, and Siberia but
also the Americas [15, 26, 27, 31, 32, 41, 48, 50–52, 58]. In Jetisuu Kazaks, we found threebranches of haplogroup D, namely D1, D4, and D5. These lineages account for 19.5% of thematernal genetic diversity in Jetisuu Kazaks, making it the most common haplogroup in thispopulation.
Within D4m (Fig 9), we observed one Jetisuu Kazak sequence. Together with close relatives
in Uyghurs and Kirghiz, the Kazak sequence formed a new subbranch (D4m3). As shown in
Fig 9, D4m was represented by sequences from Turkic (Kazak, Kirghiz, and Uyghur), Mongolian (Buryat) and Tungusic (Even, KF148265) speakers, along with Japanese (AP008432),
implying a common Altaic origin for them.
Most of the D5 sequences analyzed in this study belonged to D5a2a1 (Fig 10). The branching patterns of the D5 clades were complex, and the branches are formed by the sequencesfrom Turko-Mongolian speakers, Tibetans, and Japanese.

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Fig 7. Schematic phylogenetic tree for the haplogroup C4a1 mitochondrial genomes. Note: Alt: Altaian; Bur: Buryat; Uyg: Uyghur; Kir:
Kirghiz; Tib: Tibetan.
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PLOS ONEMaternal lineages of Kazaks from Jetisuu

Fig 8. Schematic phylogenetic tree for the haplogroup C5 mitochondrial genomes. Note: Alt: Altaian; Bur: Buryat; Uyg: Uyghur; Kir: Kirghiz.
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PLOS ONEMaternal lineages of Kazaks from Jetisuu

Fig 9. Schematic phylogenetic tree for the haplogroup D4m mitochondrial genomes. Note: Alt: Altaian; Bur: Buryat; Uyg:
Uyghur; Kir: Kirghiz; Tib: Tibetan; Tub: Tuba/Tuva;. KF148265 (Even) and AP008432 (Japanese) were from the Phylotree
Build 17.0 [46].
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Fig 10. Schematic phylogenetic tree for the haplotype D5a2a1. Note: Bur: Buryat; Kaz: Kazak; Kir: Kirghiz; Tib: Tibetan; Uyg: Uyghur; The following
sequences were obtained from the Phylotree Build 17.0 [46]: AP013256, AP008854, AP013197, AP010743, AP011023, AP008536, AP013256, and AP009424
are Japanese sequences; JF824956 is from China, but unknown ethnic origin; FJ383195 is from India; AY255162 is a Han Chinese sequence.
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The D5a2a1+@16172 lineage contained sequences from Buryats, Kazaks, Tibetans, Tuba,
and Uyghurs. This clade had four subbranches, which included three new ones (D5a2a1c,
D5a2a1d, and D5a2a1e), in addition to one (D5a2a1a) already recorded in the Phylotree Build
17.0 [46]. We also propose the new branch of D5a2a1 defined by a back mutation at 16092, but
not the back mutation at 16172, which occurs in D5a2a1f (Fig 10). The newly named clade has
three subbranches formed by Buryat, Kazak, and Tibetan sequences, respectively, and implying that it has a wide geographic area of distribution.

Other haplogroups of significanceHere, we discuss the mitochondrial haplogroups F, G, H, T, and U, which are found relatively
high frequencies in Jetisuu Kazaks. Haplogroups F and G are common in populations from
Siberia and Central Asia [12, 15], but they occur at considerably higher frequencies in East
Asia [51]. While the West Eurasian haplogroups H and T are also found in Central Asia andSiberia, their frequencies decrease in the eastward direction.
Haplogroup F is distributed in central, eastern, southern regions of Asia [12, 15, 25, 50, 51,
62, 63]. F1b1f occurs in populations that belong to Turkic (Kazak and Uyghur), Mongolic(Buryat), and Tungusic (Evenks) branches of the Altaic language family. While most subbranches of F1b1 are found in Turko-Mongolian speakers, including Buryats, Kazaks, Kirghiz,and Uyghurs, some sublineages also occur in Japanese. Similarly, F1a1 mtDNAs are found in
Turko-Mongolian speakers, Japanese, and Tibetans (S4 Table), but also occur in populations
in southeast Asia (i.e. Cambodia and the Orchid Island of Taiwan) [46]. Haplogroup G, which
was defined by Schurr et al. [64], also reflects maternal genetic connection between TurkoMongolian speakers and Japanese. For instance, in the clade of G2a5, Kazak and Buryat
sequences are derived from a Japanese sequence (AP008897). In the same way, multiple
Turko-Mongolian sequences cluster together with Japanese sequences in the clades of G2b2
and G3a2 (S1 Fig).
In our dataset, haplogroup H lineages comprised about 17% of the maternal linages in Jetisuu Kazaks, with H2 being the most common haplogroup (6.5%, Table 1). While Turkic speaking Tatars shared multiple West Eurasian haplotypes with Russians and other Slavic
populations, most of the West Eurasian haplotypes found in Kazaks and other Turko-Mongolian speakers were distantly related to those occurring in the Slavic groups. In addition, someWest Eurasian haplotypes exclusively occur in Turko-Mongolian speakers (e.g., H8b1). Thesefindings strongly support the notion that the existence of West Eurasian haplotypes in modern
Turko-Mongolian speakers are not simply due to Russian expansions in the recent centuries.
For instance, in S4 Table, the subclade of H8b1 is comprised of Buryat, Kazak, Kirghiz, andTuba sequences. In addition, H8b1 also includes a sequence (KF148188) from a Tungusic
speaking Evenk.
It is interesting to note that, in the phylogeny of haplogroup T, Tatar sequences seemed to
be ancestral to subbranches appearing in Central Asia. As shown in Fig 11, Tat42.MB10 isancestral to T2b lineages found in Buryats, Kazaks, Kirghiz, Russians, and Hungarians.
Another Tatar sequence (Tat7.BM10) is ancestral to T1a1 subbranches (S1 Text). This finding
may reflect the geographic origin of haplogroup T in Central Asia and Siberia. In support ofthis view, T1a and T2b mtDNAs were discovered in archaeological samples of Yamna Culturesites [65] in Volga region, Russia, whence the Tatars (Tat7 and Tat42) were sampled [66].
Excluding U6 and U9, all subclades of U (U1-5 and U7-8) occurred in Turko-Mongolian
speakers in Central Asia (S4 Table). While U7a was found mostly in Turko-Mongolian speakers and in the Iranian speaking Tajiks, U7b, previously believed to be absent in Central Asia[21], was discovered only in Uyghurs (S4 Table). While U4a seemed to characterize Slavic

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Fig 11. Schematic phylogenetic tree for the haplotype T2b. Note: Kaz: Kazak; Hu: Hungarian; Rus: Russian; Tat: Tatar; Uyg: Uyghur; SU006 and SH105 are
Jetisuu sequences; EU007872 and JQ798066 are sequences from the Phylotree Build 17.0 [46].
https://doi.org/10.1371/journal.pone.0277771.g011

speakers, all U8 sequences in our dataset were from Turko-Mongolians, with the exception of
one Hungarian sequence (S4 Table).

Discussion
We characterized maternal lineages of Kazak populations in Jetisuu at the mtGenome level. To
our knowledge, this is the first mtGenome study of human populations in Kazakhstan. Notwithstanding the small size and uniparental inheritance of mtDNA, it has provided important
insights into the modern human evolution [67, 68]. The mtGenome data produced in thisstudy will therefore be invaluable for reconstructing human population history and can serve
as a reference for identification of pathogenic mutations in patients with mitochondrial disorders in Central Asians.
In previously published studies, the mitochondrial genetic profiles of Kazaks and other ethnic groups in Central Asia and Siberia were characterized through the analyses of mtDNA

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PLOS ONEMaternal lineages of Kazaks from Jetisuu

control region sequences and coding region SNPs [12, 14, 19, 25, 47, 61, 69, 70] and complete
mtDNA sequencing [15, 20, 63, 71]. A mtDNA study of Kazaks from the Altay-Sayan region[12] revealed that they exhibit highly diverse maternal genetic profiles, harboring both Westand East Eurasian maternal lineages. Our results confirmed the same pattern of maternal
genetic variations in Jetisuu Kazaks, with almost all 200 individuals surveyed having distinct
haplotypes (S2 and S3 Tables).
West Eurasian lineages could have become part of the maternal gene pool of Kazaks during
Turko-Mongolian expansions (i.e., Turkic Khanates and Golden Horde) and/or Slavic expansions during Russian Empire and the Soviet Union, or perhaps even deeper in time, due prehistoric expansion of human populations. The comparative analyses of mtGenomes frommultiple ethnic groups in Central Eurasia indicate that West Eurasian haplogroups (e.g. H, T,
and U) have diverse subbranches in Kazaks and other ethnic groups in Central Asia and Siberia, with most of the subbranches being distinct from those haplotypes found in Russians andother Slavic populations. This observation indicates that Slavic expansions may not fully
account for the occurrence of West Eurasian maternal lineages in Central Asia. For instance,
multiple subbranches of U, including U2, U4, U5, and U7, are found in Jetisuu Kazaks, andthe haplogroup U has the largest TMRCA (~ 40,000 years) among the haplogroups found in
Kazaks (Table 1 and Table 2 in S1 Text). The mtDNA lineage of the 24-thousand-year-old
Malta individual also belongs to basal U [72], suggesting a deep origin for this maternal lineage
in Central Eurasia. Recent ancient DNA studies also found West Eurasian lineages in Iron Age
populations or earlier inhabitants of the Altay Mountains [5]. Interestingly, Jetisuu Kazaks harbor the same set of West Eurasian lineages (HV, N1, J, T, U, K, W, I, and X) found in the Iron
Age populations of Central Asia [5].
It can be deduced from the frequencies of these ancestral lineages in various ethnic groupsin Central Asia that, the arrival of East Eurasian lineages (e.g., Haplogroups A, B, C, and D) insouthern Central Asia, i.e. the Pamir Plateau, could possibly be attributed to Turko-Mongolian
expansions. Examples of these expansions could include early movements of Turks and Mongols, then southward movement of Mughals, and later the expansion of the Jungarian Khanate[1]. The subbranches of haplogroups A, B, C, and D found in Uyghurs from Xinjiang andTajiks from the Pamir are closely related to those of Altaians, Buryats, Kirghiz, and Kazaks (S4Table). However, such a hypothesis may require further lines of evidence, given the fact that
haplogroup C was found among Tarim Basin mummies [73, 74] and haplogroup D was foundin an Iron Age population in Xinjiang [75]. These ancient populations predate the TurkoMongolian expansions. In addition, haplogroup C7 occurs in northeast India [76], most likely
due to prehistoric migrations.
East Eurasian haplogroups found in eastern Europe are associated with those found in
Turko-Mongolians as well. A previous study tried to demonstrate paternal genetic affinities
between Hungarian and Kazak populations [77], but the coalescence time of the Y-chromosome haplogroups G1 and G2 predated the historical events linking Hungarians to Central
Asians. In addition, the arrival of haplogroup G1 in the Kazak steppes may date to recent historical time periods, as the age of this lineage in Kazaks was estimated to be ~ 600 years old[78]. On the other hand, it appears that maternal lineages A12a, C4a1a, and U8b1b may link
Hungarians with Kazaks and other Turko-Mongolian speakers (Fig 5 and S4 Table).
In the FST-based PCA analysis, Sherpa population unexpectedly exhibited a closer affinity
with Turko-Mongolians than with Tibetans (Fig 4). Our analysis indicates that certain mtDNA
lineages (D4j13 and D4j1a1) and some other haplotypes clearly connect Sherpas in Tibet (andNepal) and Turko-Mongolians in Central Asia and Siberia. D4j13 occurs in Sherpas and Turkic
speaking Kirghiz, Tatars, and Uyghurs, while D4j1a1 appears in Sherpas and Tibetans, as well
as Mongolic speaking Buryats and Turkic speaking Kirghiz and Uyghurs (S4 Table). The similar

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genetic affinities can be observed between Sherpas and Turko-Mongolian speakers throughhaplotypes D5a2a1, C4a2, G3a, M5, M9a1a1c1, M11, M13, and the West Eurasian lineages H10,
U2, and W. For instance, a Sherpa individual (Shr035.KL13) shares the basal W with a Uyghur(S4 Table). The existence of Y-chromosome haplogroups Q and R in Sherpas [79] may further
suggest their connections with Turkic peoples through paternal lineages as well. However, theexact nature of the genetic affinity needs to be further evaluated by Y-chromosome and wholegenome studies.
It has been noted by previous studies that Tibetans show genetic affinities with Japanesethrough their paternal [80] and maternal [51] lineages. In the current study, we identified several mitochondrial lineages that connect Kazaks and other ethnic groups from Central Asiawith both Tibetans and Japanese. Haplogroup M10 occurs in Tibetans, but it is also found in
Turkic speaking Altaians, Kazaks (e.g., AL104), and Uyghurs. Their shared maternal genetic
ancestry may explain most of the maternal genetic connections between populations from the
Tibetan Plateau and Turko-Mongolian groups.
This pattern of genetic diversity may have been partly shaped by recent genetic admixture.
Within haplogroup A15, Uyghur sequences clustered together with those from Tibetans andSherpas, revealing gene flow between Tibetans and Uyghurs despite their dissimilarities in language and religion. This genetic affinity could be attributed to geographic proximity, Tibetan
Empire, or once flourished Buddhism in Xinjiang. An analogous genetic link was observed
between Kirghiz and Tibetans in the clade of A17 (S4 Table). The directionality of gene flow,
as inferred from the branching pattern, was from Tibetans into Kirghiz.
Some clades of mtDNA (D4a3b, and D4b1, D4m, and M7) present in our dataset link Japanese with Turko-Mongolian speakers. It is known that Ryukyuans from Japan harbor a highfrequency of M7 mtDNAs [51]. All three subclades of M7, namely M7a, M7b, and M7c, arefound in Turko-Mongolian speakers (S4 Table), including Kazaks from Jetisuu (S2 and S3Tables). In addition, N9a mtDNAs are present in Turko-Mongolian speakers (e.g., N9a1,
N9a3, N9a8, and N9a9) and occur at a considerably high frequency (2.5%) in Jetisuu Kazaks,
while its sister branch N9b occurs at elevated frequencies in Ainu and Ryukyuans from Japan[51]. N9a mtDNAs are mainly found in continental Asia, including Japan [15, 31, 51, 81]. Haplogroup Y1, a subclade of N9, is also present in Buryats and Uyghurs (S4 Table), and occurs at
a high frequency in Ainu people from Japan [51]. It was hypothesized that N9a might havereached East Asia through northern route after the dispersal of macrohaplogroup N fromAfrica [51]. Kazaks and other ethnic groups harbor several basal N and R sequences (S2–S4
Tables), and this may reflect that the basal branches of macrohaplogroup N have existed inCentral Eurasia for a very long time. The discovery of a basal R mtDNA from a 45-kyr-old
individual in Siberia (Ust-Ishim) [82] and a basal N mtDNA from a 34-kyr-old individualfrom Mongolia (Salkhit) [83] further supports the notion that some subbranches of N
expanded towards East Asia through a northern route.
We found a limited number of haplogroups that exclusively characterize Turkic speakers inour dataset (e.g., A14, B4c1b2, and C4d). Interestingly, haplogroups HV1a1a, K1a17, andM13a1b1 are only found in Mongolic speaking Buryats and Barghuts. Many haplogroups,
such as B4j, B4b1a3, C4a1a4, C4a2, C4b6, C5b, D4j5b, and D4j8 to name a few, are shared
among Turkic and Mongolic speakers, i.e., Turko-Mongolians. Furthermore, a few other haplogroups, such as A5, B5b, D4b2b, D5a2a1, F1a1, F1b1, G2a5, G2b2, and G3a2, are found in
human populations from a wide geographic area spanning from the Japanese archipelago in
the East and the Kazak steppes in the West.
Some SNPs identified in certain pseudo-mtGenome sequences of Jetisuu Kazaks may
potentially lead to identification of novel mtDNA lineages in haplogroup D. These may
include coding region SNPs at nucleotide positions (nt) 3548 and 13830, and a control region

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back mutation at nt 16223 in Jetisuu Kazak mtDNA (e.g., JA005, SU009, SH012, and SH007),
although we could not rule out the possibility of incorrect SNP calls on the GenoChip microarray. In addition, SNPs at nt 2396 and nt 16519 in the pseudo-mtGenomes seem to be ambiguous, as well. Therefore, the authenticity of these SNPs needs to be evaluated and confirmed by
future DNA analysis. In addition to these variants, Haplogrep reported multiple back mutations for some mtGenome sequences (AL108, AL109, JA116, and SH119 etc.), suggesting thatsuch mtGenome sequences may require additional evaluation.
Mass migration, demographic fluctuations, and genetic drift can hinder the ability to accurately pinpointing the geographic origins and TMRCA of mitochondrial lineages. Thus, toconfirm our current findings regarding the TMRCA and demographic history of Jetisuu
Kazak maternal lineages (S1 Text), additional analyses of the West and East Eurasian lineages
found in contemporary Kazaks and other ethnic groups in Central Asia, along with an
expanded analysis of ancient DNA samples, should be undertaken.

Materials and methods
Fieldwork and DNA preparation
Blood samples were collected from ethnic Kazak individuals during several fieldwork sessions
conducted in Almati Oblast, Kazakhstan, in summer 2014 (S1 Table). All blood samples weredrawn via vacutainer (BD) by medical nurses. Written informed consent was obtained fromthe research participants. Prior to taking bloods, all of the research participants signed an
informed consent form and filled out a genealogical questionnaire. The informed consent
form and questionnaire were reviewed and approved by the Ethics Committee at the Center
for Life Sciences (CLS), National Laboratory Astana (NLA), Nazarbayev University (NU).
After fieldwork, blood samples were stored at -86˚C, and DNAs were extracted with a salt
extraction method [84] with modification, or a commercial DNA extraction kit (e.g., Qiagen)at CLS, NLA, NU.

Microarray genotyping
The genomic DNAs of 80 individuals were genotyped via the GenoChip 2.0 microarray (Illumina iSelect HD custom genotyping bead array) [85] under the framework of the of Genographic Project. The GenoChip 2.0 surveys about 750,000 SNPs from autosomes, sex
chromosomes, and the mtDNA. Accordingly, in the current study, we analyzed about 3153
mitochondrial SNPs located across mitochondrial genome, i.e., from both control and coding
regions of the mtDNA. Among the SNPs genotyped, about 400 of them were different fromthe revised Cambridge Reference Sequence (rCRS) [86], as shown in Table 2.

Pseudo-mitochondrial genome reconstruction
Pseudo-mitochondrial genomes (pseudo-mtGenomes) were reconstructed from the SNP
genotype data for 80 Kazak individuals from Jetisuu via a Python script modified from GitHub[87] (S2 Table). For the reconstruction of pseudo-mtGenomes, rCRS [86] was used as a template, and nucleotides at certain positions of rCRS were replaced with SNP calls from the
GenoChip.

Mitochondrial genome sequencing
In addition to the 80 genotyped samples, we sequenced the whole mitochondrial genomes of
120 Kazak individuals from Jetisuu. Whole mtGenome sequencing was performed on the NGS
platform of Iron Torrent at the National Institute of Genomic Medicine, Mexico City, Mexico.

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Sequence coverages of the newly generated mtGenomes were evaluated by QUALIMAP v2.2.1
after converting the raw sequence files into BAM files [88]. The average sequence coverage ofthe mtGenomes was 67.5x.

Sequence alignment
mtGenomes from Jetisuu. In the initial analysis, the pseudo-mtGenomes (N = 80) werealigned together with the genuine mitochondrial genomes (N = 120) using the online versionof MAFFT v7.0 [89, 90]. rCRS [86] and the Reconstructed Sapiens Reference Sequence (RSRS)[91] were also included in the multiple sequence alignment (MSA). The aligned mtGenomes
were further processed by the program of SNP-Sites [92] to generate an MSA VCF file. In the
final stage of data analysis, BWA [93] was used for the MSA of both pseudo and genuine
mtGenome sequences from Jetisuu (N = 200), for the sake of accuracy and consistency withthe subsequent analyses.
mtGenomes from Central Eurasia. The Jetisuu Kazak mtGenome sequences (N = 200)
were aligned together with 2074 mtGenomes from multiple ethnic groups across Central Eurasia (Tables 3 and S4) via BWA [93]. This step produced a SAM format alignment of 2274
mtGenome sequences. Subsequent file format conversions were performed through SAMtools[94], BAMtools [95], and BCFtools [96, 97].
Selection of mtGenomes for phylogenetic analysis. From the first round MSA of mtGenomes from Central Eurasia (N = 2274), subsets were selected based on haplogroup identities
and their affinity with Jetisuu Kazak sequences. Thereafter, an MSA alignment was performed
for each of the macro-haplogroups (A, B, C, D, F, G, H, T, and U) (Table 4), with sequencesfrom the Phylotree Build 17.0 [46] representing major subbranches of the haplogroups also
being added (Tables 4 and S5 and S6). All alignments were carried out via BWA [93], and file format conversions were performed through SAMtools [94], BAMtools [95], and BCFtools [96, 97].

Extended Bayesian skyline plot (EBSP)
Jetisuu mtDNA sequences (N = 200) were first partitioned into HVS-I, HVS-II, and coding
regions to estimate the demographic history of Jetisuu Kazaks using the EBSP method (S1Text). The coding region was divided again based on codon start positions. Different mutation
rates were used to estimate the coalescence times based on the coding and control regions of
mtDNA. Two mutation rates, 31.43 x10-8 μ/site/year for the control region [98] and 1.71 x10-8μ/site/year for the coding region [54] were used (Table 1 in S1 Text). EBSP was performed on
BEAST 2.0 [99] with the Markov Chain Monte Carlo (MCMC) chain length of 30,000,000 andsampling every 3000 steps. Initial 3,000,000 iterations were considered pre-burn-in andexcluded. EBSP results were reviewed with Tracer v1.7 [100] and plotted via R [101].

Identification of mitochondrial haplogroups
Mitochondrial haplogroups were identified by analyzing either VCF or FASTA file via theonline version of Haplogrep 2.0 [23]. In parallel, mitochondrial haplogroups were also
obtained by analyzing FASTA format sequence files on mtPhyl 5.003 [24], which does not
require MSA. No discrepancy was observed between Geno 2.0 SNP based haplogroup calls
and the pseudo-mtGenome based haplogroup calls.

PCA using mtDNA control region haplogroup frequencies
PCA analysis (Fig 2) was performed using mtDNA haplogroup frequencies for several populations in Central Asia, together with the four Kazak populations from Jetisuu. OtherPLOS ONE | https://doi.org/10.1371/journal.pone.0277771 November 29, 2022

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populations included Altaians from the Altay Republic, Russia [25]; Kazaks and Uyghurs from
Kazakhstan, Kirghiz from Talas and Sari-Tash, Kyrgyzstan [13]; Mongols from Inner Mongolia, China [26]; Dungans (Hui), Mongols, Kazaks, Uyghurs, and Han Chinese from Xinjiang,
China [16, 17]; Kazaks from the Altay Republic, Russia [12, 15]; Barghuts from Inner Mongolia, China [15]. Most of the comparative studies identified mtDNA haplogroups based on control region sequences, i.e., HVS-I and HVS-II, and coding region SNPs. Therefore, mtDNA
haplogroup frequencies of Jetisuu Kazaks used in this PCA plot are mostly from the frequencies of basal branches of the mitochondrial phylogeny rather than terminal haplotypes. The
prcomp function of base R [101] was used for the PCA analysis, and the ggplot2 [102] and factoextra packages of R [101] were used to plot PCA results.
A heatmap was created using the R packages reshape [103] and ggplot2 [102] for the haplogroup frequency data used for the PCA.

mtGenome pairwise FST value based PCA
Pairwise FST values were calculated with pairwise_Gst_Nei function of the mmod package of R
[104]. PCA was performed using the prcomp function of base R [101]. PCA plot was made by
the R packages factoextra and ggplot2 [102].

Phylogenetic analysis
Phylogenetic networks were constructed from mtGenome sequences with Network 10.1.0.
[105]. Phylogenetic trees were also obtained through Haplogrep 2.0 [23] and mtPhyl 5.003[24]. New branches and twigs, which are not available in the current version of Phylotree(v17.0) [46] and cannot be identified by Haplogrep 2.0, were reconstructed and named basedon the comparative analyses of the phylogenetic trees from Haplogrep 2.0 and mtPhyl 5.003,
as well as the phylogenetic networks constructed by Network v10.1.0. Schematic phylogenetic
trees were drawn manually using the draw.io program [106]. mtGenome and pseudo-mtGenome sequences with ambiguous SNPs were excluded from the schematic phylogenetic trees.
Jetisuu Kazak sequences were highlighted in bold. Ethnicity was used as prefix and the initialsof the first author followed by the year of publication were used as suffix for the sample ID of
the sequences used for comparative analysis. The mtDNA sequences from Phylotree [46] werenamed after their GenBank accession number, as given in the Phylotree Build 17.0 [46]. If a
Phylotree sequence was also included in our initial dataset, it is shown in parenthesis in theschematic phylogenetic trees.

Coalescence time estimation
Coalescence times (TMRCA) were estimated via the method provided by Soares and colleagues [54] with a whole mtGenome mutation rate of one mutation per 3624 years (S1 Text).
Rho and sigma values were obtained on Network v10.1.0 [105].

Supporting information
S1 Text. Additional analysis of mitochondrial sequences.
(PDF)
S1 Fig. A phylogenetic tree of Jetisuu mitochondrial DNA (N = 200). The phylogenetic treeof the Jetisuu Kazak mtDNA was constructed via Haplogrep 2.0 [23].
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PLOS ONEMaternal lineages of Kazaks from Jetisuu

S1 Table. Number of individuals surveyed from each of the four clans and the samplingsites.
(XLSX)S2 Table. Information on the Jetisuu samples genotyped via the GenoChip microarray(N = 80).
(XLSX)S3 Table. Information on the Jetisuu samples subjected to whole mitochondrial genome
sequencing (N = 120).
(XLSX)S4 Table. Mitochondrial genomes (N = 2074) from the Central Eurasia for comparative
analyses.
(XLSX)S5 Table. Mitochondrial genomes from phylotree (N = 369) for comparative analyses.
(XLSX)S6 Table. Sample ID List of the mitochondrial genomes (N = 1155) used for the second
round mtGenome analysis. Ethnicity and GenBank ID information of the samples can befound in S2–S4 Tables.
(XLSX)S7 Table. Sample ID list of mitochondrial genomes from the Central Eurasia (N = 1954)
and Jetisuu (N = 120, excluding pseudo-mtGenomes) that were used for the mtGenome
FST value based PCA analysis. Ethnicity and GenBank ID information of the samples can befound in S3 and S4 Tables.
(XLSX)

Acknowledgments
We would like to express our gratitude to the research participants who donated their samples
for this study. We appreciate the Center for Alash History in Almati city for assisting the fieldwork. We are thankful to the local government of the Almati Oblast, and the county and village
level administrations, for their support for organizing the fieldwork. We also thank local hospitals in the Almati Oblast for providing nurses to collect blood samples. The GenoChip microarray analysis was carried out under the framework of the Genographic Project, National
Geographic Society, USA. The sequencing of the Kazak mtGenomes was supported by the
National Institute of Genomic Medicine, Mexico City, Mexico.

Author Contributions
Conceptualization: Ayken Askapuli, Lorena Orozco.
Data curation: Miguel Vilar, Humberto Garcia-Ortiz, Cecilia Contreras-Cubas, Francisco
Barajas-Olmos, Marlen Flores-Huacuja, Lorena Orozco.
Formal analysis: Ayken Askapuli, Humberto Garcia-Ortiz, Francisco Barajas-Olmos, Marlen
Flores-Huacuja.
Funding acquisition: Maxat Zhabagin, Ainur Akilzhanova, Erlan Ramanculov, Uli Schamiloglu, Zhaxybay Zhumadilov.

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Investigation: Ayken Askapuli, Maxat Zhabagin, Zhaxylyk Sabitov, Angelica Martinez-Hernandez, Lorena Orozco.
Methodology: Ayken Askapuli, Humberto Garcia-Ortiz, Cecilia Contreras-Cubas.
Project administration: Ayken Askapuli, Miguel Vilar, Maxat Zhabagin, Angelica MartinezHernandez, Lorena Orozco.
Resources: Ainur Akilzhanova, Erlan Ramanculov, Lorena Orozco.
Software: Lorena Orozco.
Supervision: John Hawks.
Writing – original draft: Ayken Askapuli.
Writing – review & editing: Ayken Askapuli, Miguel Vilar, Uli Schamiloglu, Theodore G.
Schurr, John Hawks, Naruya Saitou.

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