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A comprehensive exploration of the genetic legacy and forensic features of Afghanistan and Pakistan Mongolian-descent Hazara

Authors:
Guanglin He 何光林 at Sichuan University
Guanglin He 何光林
Atif Adnan at Xiamen University
Atif Adnan
Allah Rakha at University of Health Sciences Lahore
Allah Rakha
Ivy Hui-Yuan Yeh 葉惠媛 at Nanyang Technological University
Ivy Hui-Yuan Yeh 葉惠媛
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Abstract and Figures

Afghanistan and Pakistan are rich with a complex landscape of culture, linguistics, ethnicity and genetic legacy at the crossroads between Indian-Subcontinent and Central Asia. Hazara people have historically been suggested to be Mongolian decedents but seldom been genetically studied. To dissect the genetic structure and explore the forensic characteristics of Hazara people, we first genotyped 30 Insertion/deletion (Indel) markers in 468 samples from 2 aboriginal Hazara populations from Afghanistan and Pakistan, and 100 East Asian comparative Bouyei samples using the Investigator ® DIPplex kit. Subsequently, we carried out a comprehensive population genetic analysis from four different datasets: 8,895 30-Indel genotype data from 51 populations, 15,895 30-Indel allele frequency data from 98 populations, 1,048 genotypes of 993 STRs and Indels from 53 HGDP populations and whole-genomes (621,799 single nucleotide polymorphisms) from 165 worldwide Human origin reference populations, to further unravel the genetic complexity between Hazara and worldwide human populations using various statistical analysis. We find that 30 Indels are in accordance with HWE, and informative and polymorphic in both Central Asians Hazara and East Asian Bouyei populations. The forensic combined probability of exclusion is larger than 0.9943 and the cumulative power of discrimination is larger than 0.99999999999936. These forensic parameters show the high level of diversity, which makes the Indel amplification system suitable for forensic routine work and may be used as a supplementary assay for routine forensic investigation. The results from pairwise genetic distances, MDS, PCA, and phylogenetic relationship reconstruction demonstrate that present-day Hazaras are genetically closer to the Turkic-speaking populations (Uyghur, Kazakh, and Kyrgyz) residing in northwest China than with other Central/South Asian populations and Mongolian. Outgroup and admixture f3, f4, f4-ratio, qpWave, and qpAdm results further demonstrate that Hazara shares more alleles with East Asians than with other Central Asians and carries 57.8% Mongolian-related ancestry. Overall, our findings suggest that Hazaras have experienced genetic admixture with the local or neighboring populations and formed the current East-West Eurasian admixed genetic profile after their separation from the Mongolians.
Genetic relationships among 8895 unrelated individuals from 51 worldwide human populations based on raw genotype data of 30 Indels included in the Investigator® DIPplex kit. (A). Geographic positions and population size of Qiannan Bouyei Pakistan and Afghanistan Hazara populations and all reference populations used in the Indel-based population comparisons. (B). Principal component analysis among 8895 individuals from four continental regions. (C). Multidimensional scaling plots display the genetic affinity between three investigated populations and other 48 reference populations. (D). The neighbor-joining tree showing the phylogenetic relationships between Hazara, Bouyei populations and reference populations on the basis of the pairwise Fst standard genetic distance.
Genetic relationships among 8895 unrelated individuals from 51 worldwide human populations based on raw genotype data of 30 Indels included in the Investigator® DIPplex kit. (A). Geographic positions and population size of Qiannan Bouyei Pakistan and Afghanistan Hazara populations and all reference populations used in the Indel-based population comparisons. (B). Principal component analysis among 8895 individuals from four continental regions. (C). Multidimensional scaling plots display the genetic affinity between three investigated populations and other 48 reference populations. (D). The neighbor-joining tree showing the phylogenetic relationships between Hazara, Bouyei populations and reference populations on the basis of the pairwise Fst standard genetic distance.
… 
Individual genetic ancestry components inferred from the Structure results among 8895 individuals from 51 populations based on the genetic variations of 30 Indels with a predefined population ranging from 2 to 9 (k = 2˜9). The optimized k value is 5 using the Structure Harvester.
Individual genetic ancestry components inferred from the Structure results among 8895 individuals from 51 populations based on the genetic variations of 30 Indels with a predefined population ranging from 2 to 9 (k = 2˜9). The optimized k value is 5 using the Structure Harvester.
… 
The pairwise Nei's genetic distances between Pakistan Hazara (A), Afghanistan Hazara (B), Qiannan Bouyei (C) and worldwide reference populations calculated based on 30 Indel variants.
The pairwise Nei's genetic distances between Pakistan Hazara (A), Afghanistan Hazara (B), Qiannan Bouyei (C) and worldwide reference populations calculated based on 30 Indel variants.
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Genetic heterogeneity and homogeneity between Hazara and other 52 populations from HEPH-HGDP based on the combined genetic variations of 783 microsatellites and 210 insertion/deletion polymorphisms. (A). The genetic affinity among 56 worldwide populations. (B). The phylogenetic relationship constructed using the neighbor-joining algorithm based on the pairwise Fst genetic distance. (C). Genetic ancestry among 1048 individuals from 53 populations inferred from the results of structure using 993 informative and polymorphic markers.
Genetic heterogeneity and homogeneity between Hazara and other 52 populations from HEPH-HGDP based on the combined genetic variations of 783 microsatellites and 210 insertion/deletion polymorphisms. (A). The genetic affinity among 56 worldwide populations. (B). The phylogenetic relationship constructed using the neighbor-joining algorithm based on the pairwise Fst genetic distance. (C). Genetic ancestry among 1048 individuals from 53 populations inferred from the results of structure using 993 informative and polymorphic markers.
… 
Outgroup-f ⁠ 3 results based on the genetic variations of 621,799 single nucleotide polymorphisms. (A). Outgroup-f ⁠ 3 statistics values of form f ⁠ 3 (Hazara, X; Yoruba). (B) Outgroup-f ⁠ 3
+1
Outgroup-f ⁠ 3 results based on the genetic variations of 621,799 single nucleotide polymorphisms. (A). Outgroup-f ⁠ 3 statistics values of form f ⁠ 3 (Hazara, X; Yoruba). (B) Outgroup-f ⁠ 3
… 
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UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
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2. Materials and methods
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UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
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J?ED 56
3. Results
 080>4. /4@0<=4>C :9:?6,>498 2080>4. 10,>?<0= ,8/ 19<08=4.
.3,<,.>0<4D,>498 -,=0/ 98 >30  8/06=
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:;B C7HA;HI ?D +?7DD7D EKO;? <=>7D?IJ7D 7D:
UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
*7A?IJ7D "7P7H7 FEFKB7J?EDI I I>EMD ?D .78B;I - DE :;L?7J?ED
<HEC "1 ?I E8I;HL;: ?D J>H;; IJK:?;: FEFKB7J?EDI 7<J;H J>; ED<;H
HED? EHH;9 J?ED F   ;F7HJKH; <HEC & ?I ?:;DJ?U;: ?D J>;
EDBO F7?H E< "& 7D: "& F  ?D *7A?IJ7D "7P7H7 FEF
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KB7J?EDI =;D;J?9 7WD?JO ;N?IJI 8;JM;;D <=>7D?IJ7D "7P7H7 7D: .KH
A?9IF;7A?D= FEFKB7J?EDI /O=>KH %OH=OP 7D: %7P7A> 7D: =;D;J?9
:?L;H=;D9; M?J> C;H?97D 7D: <H?97D FEFKB7J?EDI +?7DD7D
EKO;? >7I J>; 9BEI;IJ =;D;J?9 H;B7J?EDI>?F M?J> !K?P>EK EKO;?
 7D: '?7E  .EF J>H;; 9ECFED;DJI E< ?D:?L?:K7BB;L;B
* ;NFB7?D  L7H?7D9;I <HEC J>; JEJ7B L7H?7J?EDI  ?=  "7P
7H7 FEFKB7J?EDI 7H; FB79;: 9BEI;H JE 7IJ I?7D FEFKB7J?EDI 7D: BE97J;:
7FFHEN?C7J;BO ?DJ;HC;:?7J; FEI?J?ED 7CED= I?7D <H?97D 7D: C;H
?97D FEFKB7J?EDI ?D J>; JME:?C;DI?ED7B * FBEJI +?7DD7D EKO;? ?I
=;D;H7BBO EL;HB7FF;: M?J> 7IJ I?7DI
.E <KHJ>;H >7L; 7D ?DI?=>J 7J J>; ?DJH7 7D: ?DJ;HFEFKB7J?ED L7H?7
J?ED 7CED= "7P7H7 7D: 7J7I;JY H;<;H;D9; FEFKB7J?EDI JME:?C;DI?ED7B
I97B?D= FBEJI 87I;: ED J>; F7?HM?I; IJ =;D;J?9 :?IJ7D9; C7JH?N M;H;
9EDIJHK9J;:  ?=  <H?97D (?=;H?7D ?I ?IEB7J;: M?J> EJ>;HI 7D: I?J
K7J;: M?J>?D J>; KFF;H FEI?J?ED ?L; C;H?97DI 7H; FB79;: ?D J>; B;<J
BEM;H FEI?J?ED KHEF;7DI M?J> -EKJ> C;H?97D /HK=K7O7D 7H; BE97B
?P;: ?D J>; 9;DJH7B FEI?J?ED 7D: 7IJ I?7DI ;N9;FJ <EH .KHA?9IF;7A?D=
FEFKB7J?EDI 7H; BE97J;: 7J J>; H?=>J BEM;H FEI?J?ED .ME "7P7H7 FEF
KB7J?EDI 7H; 9BKIJ;H;: 9BEI;BO 7D: =HEKF;: M?J> %7P7A> /O=>KH 7D:
%OH=OP FEFKB7J?EDI M>?9> 7H; FB79;: ?D J>; ?DJ;HC;:?7J; FEI?J?ED 8;
JM;;D KHEF;7D 7D: 7IJ I?7D EKO;? ?I =HEKF;: 9BEI;BO M?J> ->7D=
>7? "7D 7D: 3KDD7D 3? *>OBE=;D;J?9 9BKIJ;H;: H;IKBJI 9EHH;IFED: JE
J>; 9EDJ?D;DJ7B =;E=H7F>?97B EH?=?D  ?=  *7A?IJ7D "7P7H7 ?I UHIJ
9BKIJ;H;: M?J> %KG7 /O=>KH 7D: J>;D 9BKIJ;H;: M?J> #B? %7P7A> 7D:
<=>7D?IJ7D "7P7H7 +?7DD7D EKO;? M7I UHIJ 9BKIJ;H;: M?J> 3KDD7D 3?
7D: J>;D =HEKF;: M?J> >?D;I; .7?%7:7? FEFKB7J?EDI 1; IK8I;GK;DJBO
:?II;9J;: ?D:?L?:K7B 7D9;IJHO 9ECFED;DJI 7CED=  ?D:?L?:K7BI KI
?D= J>; CE:;B87I;: 7B=EH?J>C 7D: ?D<;HH;: J>; :;J7?B;: 7D9;IJHO 9EC
FEI?J?EDI KD:;H J>; FH;:;UD;: 7D9;IJHO IEKH9; FEFKB7J?EDI <HEC  JE 
% T I I>EMD ?D ?=  M?J> % ?D9H;7I?D= <HEC JE  C;H?
97D <H?97D KHEF;7D .?8;JEKHC7D :EC?D7DJ 7D: 7IJ I?7D :EC
?D7DJ 7D9;IJH?;I IK8I;GK;DJBO 7FF;7H ?D J>; ?D:?L?:K7B 7D9;IJHO L;HJ?
97B B?D; 1>;D J>; H;<;HH;: 7D9;IJHO IEKH9; FEFKB7J?EDI 9EDJ?DK; JE ?D
9H;7I; JE  :?<<;H;DJ FEFKB7J?EDI :;H?L;: J>;?H 7D9;IJHO <HEC 7J B;7IJ
JME FH;:;UD;: 7D9;IJHO FEFKB7J?EDI 1; 97D IJ?BB E8I;HL; J>; I?C?
B7H FHEFEHJ?ED7B 7D9;IJHO E< ;79> ?D:?L?:K7B <HEC J>; I7C; 9EDJ?D;D
J7B =HEKFI EH B7D=K7=; <7C?B?;I #D J>; EFJ?C?P;: UL; FH;:;UD;: 7D9;I
JHO IEKH9;I ?D<;HH;: <HEC J>; -JHK9JKH; "7HL;IJ;H <=>7D?IJ7D "7P7H7
?I 9ECFEI;: E<  7D9;IJHO <HEC KHEF;7D 7D:  <HEC 7IJ
I?7D .>;  .?8;JEKHC7D :EC?D7DJ 7D9;IJHO  <H?97D 7D
9;IJHO 7D:  C;H?97D 7D9;IJHO M;H; 7BIE ?:;DJ?U;: ?D <=>7D?IJ7D
"7P7H7 *7A?IJ7D "7P7H7 >7H8EHI  7D9;IJHO <HEC KHEF;7D 7D:
 <HEC 7IJ I?7D 7I M;BB 7I  <HEC .?8;JEKHC7D :EC
?D7DJ 7D9;IJHO  <HEC <H?97D 7D9;IJHO 7D:  C;H?97D 7D
9;IJHO +?7DD7D EKO;? >7H8EHI  7D9;IJHO 9ECFED;DJ <HEC 7IJ
I?7D
 080>4. 30>0<920804>C ,8/ :3C692080>4. <06,>498=34:= -0>A008 ,D,<,
9?C04 2<9?:= ,8/  A9<6/A4/0 :9:?6,>498= -,=0/ 98 >30 ,66060 1<0;?08.C
/4=><4-?>498 91  8/06=
#D 7::?J?ED JE ;NFBEH?D= J>; =;D;J?9 I?C?B7H?J?;I 7D: :?<<;H;D9;I KD
:;H J>; =;D;J?9 L7H?7J?EDI E< J>; ;DJ?H; FEFKB7J?EDI 8;?D= =;DEJOF;:
L?7 J>; #DL;IJ?=7JEH]R #*FB;N A?J :7J7I;JZ 9EDI?IJ?D= E<  ?D
:?L?:K7BI <HEC  MEHB:M?:; >KC7D FEFKB7J?EDI  ?=  M7I ;C
FBEO;: JE 9ED:K9J;: J>; I;9ED: FEFKB7J?ED =;D;J?9 7D7BOI?I KI?D= F7?H
M?I; (;? IJ7D:7H: =;D;J?9 :?IJ7D9; CKBJ?:?H;9J?ED7B I97B
UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
Fig. 2. #D:?L?:K7B =;D;J?9 7D9;IJHO 9ECFED;DJI ?D<;HH;: <HEC J>; -JHK9JKH; H;IKBJI 7CED=  ?D:?L?:K7BI <HEC  FEFKB7J?EDI 87I;: ED J>; =;D;J?9 L7H?7J?EDI E<  #D:;BI M?J> 7 FH;:;
UD;: FEFKB7J?ED H7D=?D= <HEC  JE  A T .>; EFJ?C?P;: A L7BK; ?I  KI?D= J>; -JHK9JKH; "7HL;IJ;H
?D= FBEJI FEFKB7J?EDB;L;B FH?D9?F7B 9ECFED;DJI 7D7BOI?I 7D: F>OBE
=;D;J?9 H;B7J?EDI>?F H;9EDIJHK9J?ED !;D;J?9 :?IJ7D9; C;7IKH;I E< (;?
8;JM;;D J>; J>H;; ?DL;IJ?=7J;: =HEKFI 7D: EJ>;H  H;<;H;D9; FEFK
B7J?EDI M;H; 7II;II;: 7D: B?IJ;: ?D ?=  7D: .78B; - -JHED= =;
D;J?9 7WD?JO ?I E8I;HL;: 7CED= JME "7P7H7I (;?   "7P
7H7 FEFKB7J?EDI I>EM J>; 9BEI;IJ 7D: B7H=; I?C?B7H =;D;J?9 :?IJ7D9;I
M?J> %7P7A> FEFKB7J?EDI <EBBEM;: 8O J>; /O=>KH %OH=OP ED=N?
7D= 7D: "K? H;I?:?D= ?D J>; DEHJ>M;IJ;HD H;=?ED E<
>?D7  ?= – .7?%7:7?IF;7A?D= EKO;? ?I =;D;J?97BBO 9BEI;IJ JE
J>; =;E=H7F>?97BBO 9BEI; =HEKFI IK8I;GK;DJBO <EBBEM;: 8O .?8;JEKH
C7D 7D: .KHA?9IF;7A?D= FEFKB7J?EDI  ?=  *7JJ;HDI E< =;D;J?9
I?C?B7H?JO ?D<;HH;: <HEC >;7JC7F E< (;? =;D;J?9 :?IJ7D9; 7H; 9EDI?I
J;DJ M?J> J>; H;IKBJI H;L;7B;: 8O J>; F7?HM?I; IJ UN7J?ED ?D:;N  ?=
KH; - *EFKB7J?EDB;L;B * M7I UHIJ 97HH?;: EKJ 87I;: ED J>; 7B
B;B?9 <H;GK;D9O 9EHH;B7J?ED  ?=KH; - JE 7II;II J>; =;D;J?9 H;B7J?ED
I>?F .EF UL; 9ECFED;DJI >7L; ;NJH79J;:
Fig. 3. .>; F7?HM?I; (;?’I =;D;J?9 :?IJ7D9;I 8;JM;;D *7A?IJ7D "7P7H7  <=>7D?IJ7D "7P7H7  +?7DD7D EKO;?  7D: MEHB:M?:; H;<;H;D9; FEFKB7J?EDI 97B9KB7J;: 87I;: ED  #D:;B
L7H?7DJI
UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
 L7H?7J?EDI <HEC J>; JEJ7B L7H?7D9; .>; UHIJ FH?D9?F7B 9EC
FED;DJ ;NFB7?DI   E< J>; JEF UL; I;F7H7J?D= J>;
C;H?97D 7D: 7IJ I?7D FEFKB7J?EDI <HEC EJ>;H =HEKFI .>; I;9ED:
FH?D9?F7B 9ECFED;DJ 799EKDJI <EH  L7H?7D9;  E< J>;
JEF UL; :?IJ?D=K?I>?D= <H?97D 7D: C;H?97D FEFKB7J?EDI <HEC EJ>
;HI 7IJ I?7D FEFKB7J?EDI <HEC "CED='?;D .7?%7:7? $7F7D;I;
%EH;7D 'ED=EB?9 -?D?J?9 .KD=KI?9 7D: .?8;JEKHC7DIF;7A?D= B7D
=K7=; =HEKFI 7H; 9BKIJ;H;: JE=;J>;H 9BEI;BO 7D: BE97B?P;: 7J J>; B;<J
CEIJ ;D: E< J>; N7N?I KHEF;7D FEFKB7J?EDI 7H; =HEKF;: JE=;J>;H
M?J> J>; -EKJ> 7D: 1;IJ I?7D #D:EKHEF;7D IF;7A;HI 7D: J>H;;
-EKJ> C;H?97D FEFKB7J?EDI 7H; BE97J;: ?D J>; ?DJ;HC;:?7J; FEI?J?ED
8;JM;;D (EHJ> C;H?97D 7D: <H?97D FEFKB7J?EDI #J ?I ?DJ;H;IJ?D= JE
UD: J>7J JME "7P7H7 =HEKFI 7H; 9BKIJ;H;: 9BEI;BO M?J> .KHA?9IF;7A
?D= FEFKB7J?EDI <HEC DEHJ>M;IJ >?D7 H7J>;H J>7D BE97B EH D;?=>8EH
?D= #D:EKHEF;7D IF;7A;HI ?D:?97J?D= J>; I?C?B7H =;D;J?9 FHEUB; 8;
JM;;D "7P7H7 /O=>KH %7P7A> 7D: %OH=OP :K; JE J>; =;D;J?9 7I
I?C?B7J?ED ?D J>; F7IJ JME >KD:H;: O;7HI +?7DD7D EKO;? ?I 9BEI; JE
!K?P>EK '?7E  ?=KH; - .E <KHJ>;H ?BBKC?D7J; J>; F7JJ;HDI E< =;
D;J?9 H;B7J?EDI>?F 8;JM;;D "7P7H7 EKO;? 7D: EJ>;HI 87I;: ED J>;
=;D;J?9 :?IJ7D9; L7H?7D9; M; 9EDIJHK9J;: 7 JME:?C;DI?ED7B I97B?D=
FBEJ  ?=KH; - ?C;DI?ED J7A;I ?DJE 799EKDJ J>; 7IJ I?7D ;D
JH7B I?7D KHEF;7D 7D: (EHJ> C;H?97D =;D;J?9 :?<<;H;D
J?7J?ED 9B?D; ?C;DI?ED H;V;9JI J>; <H?97D KH7I?7D 7D: (EHJ> C;H
?97D =;D;J?9 :?<<;H;DJ?7J?ED 9B?D; *>OBE=;D;J?9 H;B7J?EDI>?F H;9EDIJHK9
J?ED H;IKBJI 7H; FH;I;DJ;: ?D ?=  ?L; 7FF7H;DJ 9B7:;I 7H; ?:;DJ?U;:
(EHJ> C;H?97D KHEF;7D <H?97D .KHA?9IF;7A?D= 7D: .?8;JEKH
C7DIF;7A?D= FEFKB7J?EDI .ME 7:C?N;: 9B7:;I 7H; I?CKBJ7D;EKIBO :;
J;9J;: ED; 9EDI?IJ?D= E< -EKJ>1;IJ I?7DI 7D: -EKJ> C;H?97D 7D:
J>; EJ>;H 9ECFEI?D= E< 7IJ I?7D FEFKB7J?EDI M?J> J>; ;N9;FJ?ED E<
.KHA?9 7D: .?8;JEKHC7D IF;7A;HI )KH H;IKBJI H;L;7B J>; IJHED= 9EHH;
B7J?ED 8;JM;;D =;D;J?9 7WD?JO 7D: =;E=H7F>O 7D: B?D=K?IJ?9 7<UB?7J?ED
 080>4. ,F84>C 91 ,D,<, ?8/0< >30 2080>4. -,.52<9?8/ 91 "
" :9:?6,>498 2080>4. @,<4,>498= 91  %&$=8/06=
!;D;J?9 H;B7J?EDI>?FI 8;JM;;D "7P7H7 /O=>KH 7D: 'ED=EB?7D ?D
J>; 9EDJ;NJ E< MEHB:M?:; >KC7D FEFKB7J?EDI M;H; UD7BBO :?II;9J;: ;C
FBEO?D= J>; =;DEJOF; :7J7 E<  -.,I#D:;BI <HEC  FEFKB7J?EDI
?D9BK:;: ?D J>; "**" ';7IKH;I E< =;D;J?9 :?IJ7D9;I 7H; FH;
I;DJ;: ?D .78B; - M>?9> I>EMI J>; "7P7H7’I 9BEI;IJ 7WD?JO ?I JE
/O=>KH  IJ  7D: J>; I;9ED: 7D: J>?H: 9BEI;IJ 7WD?J?;I 7H;
JE BE97B *7J>7D  IJ 7D: 'ED=EB?7D  IJ <EBBEM;:
8O ;DJH7B-EKJ> KHKI>E 7D: 7IJ
Fig. 4. *>OBE=;D;J?9 H;B7J?EDI>?F I>EMI J>; =;D;J?9 I?C?B7H?J?;I 7D: :?<<;H;D9;I 8;JM;;D JME "7P7H7 FEFKB7J?EDI ED; EKO;? 7D: EJ>;H  MEHB:M?:; >KC7D FEFKB7J?EDI <HEC <H?97
KHEF; 1;IJ-EKJ> I?7 7IJ I?7 7D: C;H?97 87I;: ED J>; 7BB;B; <H;GK;D9O 9EHH;B7J?ED E<  #D:;BI
UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
I?7D 2?8; 7D: 7KH .>; =;D;J?9 C7A;KF E< "7P7H7 ?I :?IJ?D9J JE C;H
?97D -KHK?  IJ  <EBBEM;: 8O C;H?97D %7H?J?7D7 *?C7 7D:
EBEC8?7D 'ED=EB?7D >7I J>; 9BEI;IJ =;D;J?9 H;B7J?EDI>?F M?J> .K@?7
<EBBEM;: 8O EJ>;H DEHJ>;HD 7IJ I?7D FEFKB7J?EDI E< 7KH ";P>;D
2?8; 7D: DEHJ>;HD "7D .>; F7JJ;HDI E< =;D;J?9 I?C?B7H?JO 7D: :?<<;H
;D9; 8;JM;;D /O=>KH 7D: J>;?H MEHB:M?:; H;<;H;D9; FEFKB7J?EDI 7H;
I?C?B7H JE J>; UD:?D=I E< J>; "7P7H7 FEFKB7J?ED .ME:?C;DI?ED7B '-
FBEJI FKJ "7P7H7 7D: /O=>KH ?D J>; ?DJ;HC;:?7J; FEI?J?ED 8;JM;;D 7IJ
I?7D 7D: J>; '?::B; 7IJ EH KHEF;7D =HEKFI 8KJ 9BEI;H JE J>; ;DJH7B
-EKJ> I?7D J>7D JE 7IJ I?7D FEFKB7J?EDI  ?=  <H?97D C;H?
97D 7D: J>; ?IEB7J;: %7B7I> FEFKB7J?ED >7L; 7 :?IJ?D9J H;B7J?EDI>?F M?J>
EJ>;HI 7I H;FEHJ;: 8O J>; H;9;DJ M>EB;=;DEC; >?=>:;DI?JO =;D;J?9
L7H?7J?ED IJK:O 56 1; J>;D H;9EDIJHK9J;: 7 F>OBE=;D;J?9 H;B7J?EDI>?F
JH;; 87I;: ED J>; D;?=>8EH@E?D?D= 7B=EH?J>C KI?D= J>; F7?HM?I; IJ :?I
J7D9; C7JH?N 97B9KB7J;: <HEC J>; =;D;J?9 L7H?7J?EDI <HEC  FEBOCEH
F>?9 C7HA;HI  ?=  BB  MEHB:M?:; FEFKB7J?EDI 7H; =;D;H7BBO 9BKI
J;H;: ?DJE I?N =;D;J?9 7WD?JO 9B7:;I C;H?97D 7IJ I?7D )9;7D?7D
<H?97D ;DJH7B-EKJ> I?7D 7D: KHEF;7D 7D: J>; '?::B; 7IJ FEF
KB7J?EDI "7P7H7 ?I UHIJ =HEKF;: M?J> /O=>KH 7D: J>;D =HEKF;: M?J>
EJ>;H 7IJ I?7D FEFKB7J?EDI 8KJ DEJ 9BKIJ;H;: M?J> =;E=H7F>?97B D;?=>
8EH?D= -EKJ> I?7D FEFKB7J?EDI .E :?II;9J J>; "7P7H7 7D9;IJHO 9EC
FED;DJ KD:;H J>; =;D;J?9 L7H?7J?ED ?D J>; 7J7I;J[ M; 9ED:K9J;: J>;
-JHK9JKH; 7D7BOI?I M?J> FH;:;UD;: 7D9;IJHO FEFKB7J?EDI <HEC  ?D9H;7I
?D= JE   ?=  J %  C;H?97D 7D: 7IJ I?7D FEFKB7J?EDI 7H;
I;F7H7J;: <HEC J>; EJ>;H FEFKB7J?EDI 8O I>7H?D= J>; I7C; 9EBEH /D?GK;
<H?97D KHEF;7D 7IJ I?7D 7D: )9;7D?7D :EC?D7DJ 7D9;IJHO 9EC
Fig. 5. !;D;J?9 >;J;HE=;D;?JO 7D: >ECE=;D;?JO 8;JM;;D "7P7H7 7D: EJ>;H  FEFKB7J?EDI <HEC "*""!* 87I;: ED J>; 9EC8?D;: =;D;J?9 L7H?7J?EDI E<  C?9HEI7J;BB?J;I 7D: 
?DI;HJ?ED:;B;J?ED FEBOCEHF>?ICI  .>; =;D;J?9 7WD?JO 7CED=  MEHB:M?:; FEFKB7J?EDI  .>; F>OBE=;D;J?9 H;B7J?EDI>?F 9EDIJHK9J;: KI?D= J>; D;?=>8EH@E?D?D= 7B=EH?J>C 87I;:
ED J>; F7?HM?I; IJ =;D;J?9 :?IJ7D9;  !;D;J?9 7D9;IJHO 7CED=  ?D:?L?:K7BI <HEC  FEFKB7J?EDI ?D<;HH;: <HEC J>; H;IKBJI E< IJHK9JKH; KI?D=  ?D<EHC7J?L; 7D: FEBOCEHF>?9
C7HA;HI
UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
FED;DJI IK8I;GK;DJBO ;C;H=; <HEC %   JE %   1; ;IJ?C7J;: J>;
A   7I J>; 8;IJ % L7BK; 1; IJ7HJ JE E8I;HL; M?J>?DFEFKB7J?ED IK8
IJHK9JKH;I ?D 7D9;IJHO 9ECFED;DJ 7II?=D?D= M>;D J>; ?D<;HH;: 7D9;I
JHO FEFKB7J?EDI B7H=;H J>7D  % <H?97D FEFKB7J?EDI A;;F >ECE
=;D;EKI M>;D % 8KJ FEFKB7J?ED IK8IJHK9JKH;I 7H; ?:;DJ?U;: M?J>
% L7BK;I H7D=?D= <HEC  JE  IK9> 7I *O=CO FEFKB7J?EDI ?7A7 7D:
'8KJ? I>EM?D= J>;?H KD?GK; 9ECFED;DJ 7J %  )9;7D?7D 7D: 7IJ
I?7D F;HI?IJ;DJBO A;;F >ECE=;DEKI ;L;D M?J> B7H=;H % L7BK;I ;D
JH7B-EKJ> I?7D 7D: '?::B; 7IJ FEFKB7J?EDI >7L; L7H?78B; 7D9;IJHO
9ECFED;DJI C7?DBO :;H?L?D= <HEC =;E=H7F>?97B 7:@79;DJ 7IJ I?7D 7D:
KHEF;7D FEFKB7J?EDI 1; E8I;HL;  KHEF;7D H;B7J;: 7D9;IJHO
 7IJ I?7D H;B7J;: 7D9;IJHO  <H?97D H;B7J;: 7D9;IJHO
 )9;7D?7D H;B7J;: 7D9;IJHO 7D:  C;H?97D H;B7J;: 7D9;I
JHO ?D "7P7H7 FEFKB7J?ED 1; DEJ; J>7J J>; <H?97D 7D: )9;7D?7D H;
B7J;: 7D9;IJHO C7O DEJ 8; H;B?78B; :K; JE J>; L;HO BEM F;H9;DJ7=;I -?C
?B7H 7D9;IJHO 9ECFEI?J?ED ?I E8I;HL;: ?D J>; /O=>KH FEFKB7J?ED :;H?L
?D=  7D9;IJHO <HEC KHEF;7D 7D:  <HEC 7IJ I?7D FEF
KB7J?EDI .>; 'ED=EB?7D FEFKB7J?ED :;H?L;I  7D9;IJHO <HEC 7IJ
I?7DI  <HEC KHEF;7DI 7D:  <HEC EJ>;HI 1; UD: J>7J
"7P7H7 I>EMI 7 CEH; I?C?B7H =;D;J?9 FHEUB; M?J> 7IJ1;IJ KH7I?7D
7:C?N;: /O=>KH FEFKB7J?ED J>7D M?J> 'ED=EB?7DI .>;I; E8I;HL;: H;
IKBJI IK==;IJ J>7J *7A?IJ7D "7P7H7 C?=>J >7L; >7: =;D;J?9 9BEI;UJJ?D=
9EDJ79J M?J> KHEF;7D EH 7:@79;DJ 7:C?N;: FEFKB7J?EDI <EBBEM?D= J>;?H
I;F7H7J?ED <HEC J>; 'ED=EB?7DI
 480=.,60 2080>4. =><?.>?<0 ,8/ ,/74B>?<0 34=>9<C 91 ,D,<, <010<<0/
1<97 A3960208970 % "=
1; UD7BBO :?II;9J J>; UD;I97B; =;D;J?9 IJHK9JKH; E< "7P7H7 KI?D=
 -(*I <HEC  MEHB:M?:; FEFKB7J?EDI 7J7I;J\ 7D: FHE
L?:;: <EHC7B J;IJI <EH =;D;J?9 7:C?NJKH; KI?D= '#2.))&I .>; =;
D;J?9 7WD?JO 8;JM;;D "7P7H7 7D: EJ>;H H;<;H;D9;I H;L;7B;: 8O )KJ
=HEKF 1] ) ,D,<, *9<?-, :;CEDIJH7J;I J>7J "7P7H7 I>7H;I CEH; 7BB;
B;I M?J> 7IJ KH7I?7DI J>7D 1;IJ KH7I?7DI  ?=  K; JE J>; 7<EH;
C;DJ?ED;: 9EHH;B7J?ED 8;JM;;D "7P7H7 /O=>KH 7D: 'ED=EB?7D M;
IK8I;GK;DJBO ;IJ?C7J;: J>; I>7H;: =;D;J?9 :H?<J 8;JM;;D /O=>KH 'ED
=EB?7D 7D: EJ>;HI KI?D= J>; 1] 982964,8 ) *9<?-, 7D: 1] 'C23?<
) *9<?-, ?<<;H;DJ F7JJ;HDI E< J>; I>7H;: 7BB;B;I 7H; E8I;HL;:  ?=
T -?=D?<?97DJ D;=7J?L; L7BK;I E< J>; ,/74B>?<01G   ,D,<,
IJ7J?IJ?9I 7H; E8I;HL;: 8;JM;;D J>; H;<;H;D9;I H;IF;9J?L;BO <HEC KHE
F;7D 7D: I?7DI .78B; - M>?9> <KHJ>;H IK==;IJI "7P7H7 97HH?;I 8EJ>
I?7D 7D: KHEF;7D 7D9;IJHO
BB;B;I>7H?D= 1] ) 982964,8 ,D,<, *9<?-,  ?=  M;H; 7BIE
F;H<EHC;: -?=D?<?97DJ :;F7HJKH;I E< J>; FEI?J?L; 1] L7BK;I IK==;IJ 7D
;N9;II E< 7BB;B; I>7H?D= 8;JM;;D "7P7H7 7D: J;IJ?D= FEFKB7J?ED 2 7D:
D;=7J?L; L7BK;I ?D:?97J; CEH; I>7H;: 7BB;B;I M?J> 'ED=EB?7D )KH H;
IKBJI :;CEDIJH7J; J>7J FH;I;DJ:7O "7P7H7 I>7H;I CEH; =;D;J?9 9EC
FED;DJI M?J> 'ED=EB?7DI J>7D M?J> EJ>;H MEHB:M?:; FEFKB7J?EDI ;N
9;FJ <EH <;M FEFKB7J?EDI ?D DEHJ>;7IJ I?7 7D: -?8;H?7 <EH ;N7CFB;
)HEG;D /B9>? (=7D7I7D ";P>;D 7KH %EH;7D 7D: 37AKJ 1; J>;D
KI;: 1]H7J?E ;:(,@0 7D: ;:/7 JE ;IJ?C7J; J>; 7:C?NJKH; FHEFEH
J?EDI ?D "7P7H7 .>; 1]H7J?E IJ7J?IJ?9I ?D J>; <EHCI E< 1]=5479 *9<?-,
) ?=><,64,8=5479 *9<?-, 982964,8 ?=
Fig. 6. )KJ=HEKF1G H;IKBJI 87I;: ED J>; =;D;J?9 L7H?7J?EDI E<  I?D=B; DK9B;EJ?:; FEBOCEHF>?ICI  !?>2<9?:1G IJ7J?IJ?9I L7BK;I E< <EHC 1G ,D,<, ) *9<?-,  !?>2<9?:1]
IJ7J?IJ?9I L7BK;I E< <EHC 1G 982964,8 ) *9<?-,  !?>2<9?:1] IJ7J?IJ?9I L7BK;I E< <EHC 1G 'C23?< ) *9<?-,
UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
Fig. 7.  =>,>4=>4.= H;IKBJI E< <EHC 2 'ED=EB?7D "7P7H7 3EHK87 I>EM?D= J>; I>7H;: =;D;J?9 I>?<J 8;JM;;D "7P7H7 EH 'ED=EB?7D 7D: .;IJ?D= FEFKB7J?EDI 87I;: ED  =;D;J?9
L7H?7DJI
><,64,8 7D: 1] 2,8,=,8 *9<?-, ) ",:?,8 2,8,=,8 *9<?-, 98
2964,8 ",:?,8 H;IF;9J?L;BO H;L;7B J>7J "7P7H7 >7H8EHI 
4I9EH;   7D:  'ED=EB?7DH;B7J;: 7D9;IJHO
4I9EH;   .78B;I -T 1; KI;: /O=>KH 7D: "7P7H7 7I J>;
J7H=;J;: FEFKB7J?EDI H;D9> 7D: 'ED=EB?7D 7I J>; IEKH9; =HEKFI 7D:
3EHK87 '8KJ? KIJH7B?7D 7D: *7FK7D 7I J>; EKJ=HEKFI JE F;H<EHC J>;
;:(,@0 7D: ;:/7 JE UD: J>; BEM;IJ DKC8;H E< 7D9;IJHO IJH;7CI 7D:
;NFBEH; J>; FHEFEHJ?EDI E< 7D9;IJHO )KH ;:(,@0 H;IKBJI IK==;IJ JME 7D
9;IJHO IJH;7CI ?D "7P7H7 H7DA   :  7D: /O=>KH H7DA  
:  H;=7H:?D= JE J>; 78EL; <EKH EKJ=HEKFI 1; >7L; 9ED9BK:;:
J>7J "7P7H7 97HH?;I  H;D9>H;B7J;: 7D9;IJHO 7D:  'ED=E
B?7DH;B7J;: 7D9;IJHO 7D: /O=>KH 8H?D=I  H;D9>H;B7J;: 7D9;IJHO
7D:  'ED=EB?7DH;B7J;: 7D9;IJHO
4. Discussion
"7P7H7 FEFKB7J?EDI H;I?:?D= ?D <=>7D?IJ7D 7D: *7A?IJ7D 7H; H;
=7H:;: 7I J>; :;I9;D:7DJI E< IEB:?;HI E< J>; 'ED=EB?7 CF?H; 799EH:
?D= JE J>; >?IJEH?97B H;9EH:?D=I 7D: 39>HECEIEC7B >7FBE=HEKF 7D7BO
I?I 56 ;IF?J;
C7DO =;D;J?9 IJK:?;I ED J>; FEFKB7J?ED >?IJEHO H;9EDIJHK9J?ED E< MEHB:
M?:; ;J>DEB?D=K?IJ?97BBO 7D: =;E=H7F>?97BBO :?IJ?D9J FEFKB7J?EDI
5–6 J>; =;D;J?9 >?IJEHO 7D: <EH;DI?9 9>7H79J;H?IJ?9I E< J>; "7P
7H7 FEFKB7J?ED H;C7?D KD9B;7H 1; >;H; H;FEHJ J>; =;DEJOF;I E< 
#DI;HJ?ED:;B;J?ED #D:;B C7HA;HI ?D  I7CFB;I <HEC  78EH?=?D7B
"7P7H7 FEFKB7J?EDI <HEC <=>7D?IJ7D 7D: *7A?IJ7D 7D:  I7CFB;I
<HEC 7IJ I?7DI .7?%7:7?IF;7A?D= FEFKB7J?EDI )KH IJK:O I>;:I B?=>J
ED J>; =;D;J?9 EH?=?D IJHK9JKH; 7D: H;B7J?EDI>?F E< *7A?IJ7D 7D:
<=>7D?IJ7D "7P7H7 FEFKB7J?EDI 7D: >?D;I; EKO;? KI?D= <EKH 9ECFH;
>;DI?L; =;D;J?9 L7H?7J?ED :7J7I;JI "7P7H7 FEFKB7J?EDI 7H; FB79;: 8;
JM;;D 7IJ I?7D 7D: KHEF;7D 9BKIJ;HI ?D EKH * '- F>OBE=;
D;J?9 JH;; IK==;IJ?D= J>;?H 7:C?N;: =;D;J?9 FHEUB; 8;JM;;D 1;IJ;HD
KH7I?7D 7D: 7IJ;HD KH7I?7D 1; FHEFEI; J>7J I;B<:;9B7H;: 'ED=E
B?7D:;I9;D:7DJI E< "7P7H7 F;EFB; 7H; 7D 7:C?N;: FEFKB7J?ED :;H?L
?D= 7D9;IJHO <HEC 8EJ> 1;IJ;HD KH7I?7D 7D: 7IJ;HD KH7I?7D !;
D;J?9 7D9;IJHO :?II;9J?ED E< J>; "7P7H7 FEFKB7J?ED ?D:?97J;I J>7J J>;O
>7L; H;9;?L;: CEH; =;D;J?9 ?DVK;D9;I <HEC J>; IKHHEKD:?D= FEFKB7
J?EDI 7D: ;NF;H?;D9;: J>; :?<<;H;DJ >?IJEHO E< FEFKB7J?ED 7:C?NJKH;
7D: 7II?C?B7J?ED 9ECF7H?D= M?J> 'ED=EB?7DI 7<J;H IFB?JJ?D= <HEC J>;

UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
9ECCED 7D9;IJEH .>; IKFFEHJ?D= F7JJ;HDI E8I;HL;: ?D EKH -JHK9JKH; H;
IKBJI 87I;: ED J>;  #D:;BI FEBOCEHF>?ICI ?D  MEHB:M?:; FEFKB7
J?EDI 7D:  -.,I EH #D:;BI FEBOCEHF>?ICI <HEC  "!* FEFKB7J?EDI
97D 8; ;NFB7?D;: ;?J>;H 8O J>; H;9;DJ 7:C?NJKH; <HEC :?<<;H;DJ 7D9;IJHO
IEKH9;I IK9> 7I 7:C?N;: =;D;J?9 FHEUB; E8I;HL;: ?D /O=>KH 7D: C;H?
97D<H?97DI EH 8O J>; I>7H?D= 7D9;IJHO <HEC J>;?H 9ECCED 7D9;IJEH 8;
<EH; J>;?H :?L;H=;D9; KD:;H :?<<;H;DJ ;LEBKJ?ED7HO <EH9;I B?A;BO =;D;J?9
C7J;H?7BI 7CED= ,KII?7DI H;I?:?D= ?D J>; -?8;H?7 7D: DEHJ>;7IJ I?7D
37AKJI 56 EC8?D;: M?J> J>; FEFKB7J?ED C?=H7J?ED >?IJEHO >?IJEH
?97B H;9EH:?D=I 7D: FH;L?EKI KD?F7H;DJ7BBO ?D>;H?J;: C7HA;HI 56
9KHH;DJ =;D;J?9 B7D:I97F; E< <=>7D?IJ7D 7D: *7A?IJ7D "7P7H7 FEFKB7
J?EDI :;H?L;: <HEC H;9;DJ 7:C?NJKH; 8;JM;;D 7IJ I?7D 7D: KHEF;7D
EH ;DJH7B I?7D 7:C?N;: FEFKB7J?EDI )KH IK8I;GK;DJ M>EB;=;DEC;
=;D;J?9 ;L?:;D9; <KHJ>;H IKFFEHJI J>; 7IJ1;IJ KH7I?7D =;D;J?9 7:C?N
JKH; ?D "7P7H7 L?7 J>H;;FEFKB7J?ED 7D: <EKHFEFKB7J?ED J;IJ?D=
)KH FEFKB7J?ED =;D;J?9 IJK:?;I UD: J>; IJHED=;H =;D;J?9 7WD?JO 8;
JM;;D "7P7H7 7D: .KHA?9IF;7A?D= FEFKB7J?EDI ?D ;DJH7B I?7 <EH ?D
IJ7D9;I /O=>KH %OH=OP %7P7A> H7J>;H J>7D 8;JM;;D "7P7H7 7D: BE
97B <=>7D?IJ7D 7D: *7A?IJ7D FEFKB7J?EDI EH FH;I;DJ:7O 'ED=EB?7DI
.>; 9ECFB;N >?IJEH?97B FEFKB7J?ED C?=H7J?ED 7D: 7:C?NJKH; ;L;DJI >7L;
I>7F;: J>; 9KHH;DJ:7O ?DJ;H;IJ?D= =;D;J?9 B7D:I97F; ?D ;DJH7B 7D:
-EKJ> I?7 EH ;N7CFB; J>; /O=>KH ?I 7BIE 7 JOF?97B 7:C?N;: FEFK
B7J?ED >7H8EH?D= J>; M;IJ;HD 7D: ;7IJ;HD 7DJ>HEFEC;JH?9 JH7?JI 2K ;J
7B ED9; <EKD: J>7J 2?D@?7D= /O=>KH :;H?L;: 78EKJ  7D9;IJHO <HEC
1;IJ KH7I?7DI 7D:  7D9;IJHO <HEC 7IJ KH7I?7DI 87I;: ED J>;
;DJ?H; L7H?7J?EDI E< >HECEIEC;  .>;D J>;O <KHJ>;H :?II;9J;: 7:
C?N;: 7D9;IJHO FHEFEHJ?EDI 87I;: ED J>; >?=>:;DI?JO M>EB;=;DEC;
-(* L7H?7J?EDI J>7J "!* DEHJ>;HD /O=>KH >7I  :;H?L;: <HEC 7IJ
I?7D 7D:  <HEC 1;IJKH7I?7DI 7D: *7DI?7D IEKJ>;HD /O=>KH
>7I  7IJ I?7D 7D9;IJHO 7D:  1;IJKH7I?7DI 7D9;IJHO 56
.>; UD;I97B; 7D9;IJHO C7A;KF H;L;7B;: J>; CKBJ?FB;M7O 9EDJ79JI E9
9KHH?D= ?D 8HEDP; 7=; ?DJHE:K9?D= <EKH 7D9;IJHO IEKH9;I ?D FH;I;DJ:7O
/O=>KHI – <HEC -?8;H?7D – <HEC 7IJ I?7D –
<HEC -EKJ> I?7D 7D: – <HEC KHEF;7D 56 )KH IJK:O 87I;: ED
J>; 1G<,>49 J;IJ?D= IKFFEHJ?D= J>; 7<EH;C;DJ?ED;: 7D9;IJHO FHEFEHJ?ED E<
/O=>KH M>?9> 97HH?;I  H;D9>H;B7J;: 7D9;IJHO 7D:  'ED
=EB?7D 7D9;IJHO .>; 9ECFB;N =;D;J?9 FHEUB; E8I;HL;: ?D "7P7H7 F;EFB;
97D 8; ;NFB7?D;: 8O J>;?H =;D;J?9 9EDJ79JI M?J> 7:@79;DJ D;?=>8EHI "7P
7H7 =;D;J?9 9>7H79J;H?IJ?9I 7H; 9EHH;IFED:?D= M;BB JE >?IJEH?97B H;9EH:
?D=I 7D: B?D=K?IJ?9 7<UB?7J?ED IKFFEHJI J>;?H 'ED=EB?7D EH?=?D M?J> 7
BED=J;HC 7FFHEN?C7J;BO ED; C?BB;DD?KC E< 9EDJ79J 7D: ;N9>7D=;
M?J> ;DJH7B EH -EKJ> I?7D ;J>D?9 =HEKFI ,;9;DJBO ED; M>EB;=;DEC;
I;GK;D9?D= FHE@;9J E< 'ED=EB?7D FEFKB7J?EDI H;L;7B;: 7FF7H;DJ FEFKB7
J?ED IJH7J?U97J?ED 7CED= =;E=H7F>?97BBO EH 9KBJKH7BBO :?L;HI; JH?8;I 56
.>KI CEH; 9ECFH;>;DI?L; FEFKB7J?ED >?IJEHO E< =;D;J?9 :OD7C?9I 7:
C?NJKH; :?L;H=;D9; E< "7P7H7 7I M;BB 7I 9B;7H;H =;D;J?9 H;B7J?EDI>?FI
7CED= "7P7H7 /O=>KH 7D: 'ED=EB?7D 7H; D;;:;: JE 8; :?II;9J;: 7D:
H;9EDIJHK9J;: 87I;: ED J>; M>EB;=;DEC; I;GK;D9?D= EH >?=>:;DI?JO
=;DEJOF?D= :7J7 <HEC CEH; H;FH;I;DJ7J?L; CE:;HD 7D: 7D9?;DJ I7CFB;I
<HEC =;E=H7F>?97BBO9KBJKH7BBO :?L;HI; FEFKB7J?EDI
5. Conclusion
1; FHEL?:; J>; UHIJ 87J9> E< 9HK9?7B #D:;B H;IEKH9; 7D: <EH;DI?9 H;<
;H;D9; :7J7I;J ?D ;DJH7B-EKJ> I?7D "7P7H7 FEFKB7J?EDI 7D: >?D;I;
.7?%7:7?IF;7A?D= EKO;? M>?9> M?BB <79?B?J7J; J>; KD:;HIJ7D:?D= E<
<EH;DI?9 <;7JKH;I 7D: J>; M?:;BOKI?D= E< #D:;B87I;: 7CFB?U97J?ED IOI
J;C ?D J>; ;DJH7B 7D: -EKJ> I?7DI 7D: 7IJ I?7DI )KH UD:?D=I <HEC
J>; <EH;DI?9 C;7IKH;I ?D:?97J; J>7J 7BB  ?DL;IJ?=7J;: #D:;BI C7HA;HI
7H; ?D<EHC7J?L; 7D: FEBOCEHF>?9 ?D *7A?IJ7D 7D: <=>7D?IJ7D "7P7H7
FEFKB7J?EDI 7D: EKO;? =HEKF IK==;IJ?D= J>EI; C7HA;HI 97D 8; KI;: 7I
7 FEM;H<KB IKFFB;C;DJ7HO JEEB <EH <EH;DI?9 F7J;HD?JO 7D: F;HIED7B ?:;D
J?U97J?ED ?D J>; I?7DI 'ED=EB?7D:;I9;DJ "7P7H7 F;EFB; 7H; 7D 7:
C?N;: FEFKB7J?ED :;H?L?D= 78EKJ >7B< 7D9;IJHO <HEC 7IJ I?7DI 7D: 7D
EJ>;H >7B< <HEC 1;IJ KH7I?7DI ,;IKBJI <HEC J>; 9ECFH;>;DI?L; FEFKB7
J?ED =;D;J?9 IJK:?;I L?7 J>; F7?HM?I; =;D;J?9 :?IJ7D9;I '- * F>O
BE=;D;J?9 JH;; 7D: <EHC7B J;IJ?D= <EH 7:C?NJKH; ?D '#2.))&I :;CED
IJH7J; J>7J <=>7D?IJ7D 7D: *7A?IJ7D "7P7H7 FEFKB7J?ED 7H; =;D;J?97BBO
9BEI;H JE J>; .KHA?9IF;7A?D= /O=>KH %7P7A> 7D: %OH=OP J>7D JE J>;?H
BE97B EH 7:@79;DJ D;?=>8EHI
Author contribution
1 * 7D: !" :;I?=D;: J>?I IJK:O
 9EBB;9J;: J>; I7CFB;I
* 24 '1 7D: $! 9ED:K9J;: J>; ;NF;H?C;DJ
!" MHEJ; J>; C7DKI9H?FJ
!"  ', 7D:   7D7BOP;: J>; H;IKBJI
1 "3 7D: , CE:?U;: J>; C7DKI9H?FJ
BB 7KJ>EHI H;L?;M;: J>; C7DKI9H?FJ
Compliance with ethical standards
.>?I IJK:O ?I 9ED:K9J;: ?D 799EH:7D9; M?J> J>; IJ7D:7H:I E< J>;
;9B7H7J?ED E< ";BI?DA? 7D: 7FFHEL;: 8O J>; ;J>?97B H;L?;M 8E7H: E<
-?9>K7D /D?L;HI?JO 7D: 2?7C;D /D?L;HI?JO BB I7CFB;I 7H; E8J7?D;:
<HEC F7HJ?9?F7DJI M?J> ?D<EHC;: MH?JJ;D 9EDI;DJ
Competing (nancial interests
(ED;
Acknowledgments
.>?I MEHA M7I IKFFEHJ;: 8O J>; (7J?ED7B (7JKH7B -9?;D9; EKD:7
J?ED E< >?D7  (7DG?7D= )KJIJ7D:?D= 3EKD= .7B;DJI *HE
=H7C E< 2?7C;D /D?L;HI?JO 2 7D: KD:7C;DJ7B ,;I;7H9>
KD:I <EH J>; ;DJH7B /D?L;HI?J?;I 4%
Appendix A. Supplementary data
-KFFB;C;DJ7HO C7J;H?7B H;B7J;: JE J>?I 7HJ?9B; 97D 8; <EKD: ?D J>;
EDB?D; L;HI?ED 7J :E?>JJFI:E?EH=@<I?=;D

UNCORRECTED PROOF
00>,6 9<08=4.%.408.08>0<8,>498,6080>4.= BBB BBBB BBBBBB
References
56 - &EF;P '! .>EC7I & L7D EHF ( DI7H?*EKH - -J;M7HJ & $ED;I  $;
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B;=79O E< 4EHE7IJH?7D?IC ?D #H7D 7D: #D:?7 ?DI?=>JI ?DJE FEFKB7J?ED IJHK9JKH;
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... Later, ancient Turkic and Mongolic tribes spread westward and scattered throughout Central Asia, the Middle East, and Eastern Europe [21]. The descendants of the above-mentioned ancient people mixed and formed the modern populations in Central Asia over a long historical period [22][23][24]. ...
... Previous research has also focused on the formation of modern populations in the recent millennium [23,24]. A strong tribe structure has been detected among populations from this region [26]. ...
... It is thought that the expansion in Central Eurasia of C2a1a3-M504 is directly related to the activity of ancient Mongol tribes in the past millennium [26,48,49]. The high frequency of this lineage in the Hazara, Karakalpak, Kazakh, Kyrgyz, and Uyghur populations in this study is consistent with the proposed origin of these populations and previous research [24,48]. The downstream lineages of N-M231 among Kazakhs (Tables S1 and S2) unique sub-branch, L1a2a-M2398-Y236528, in these samples; this lineage separated from the lineages found in India about 1500 years ago (Tables S1 and S2, Figure S1). ...
Ancient Components and Recent Expansion in the Eurasian Heartland: Insights into the Revised Phylogeny of Y-Chromosomes from Central Asia
Article
Full-text available
  • Oct 2022
In the past two decades, studies of Y chromosomal single nucleotide polymorphisms (Y-SNPs) and short tandem repeats (Y-STRs) have shed light on the demographic history of Central Asia, the heartland of Eurasia. However, complex patterns of migration and admixture have complicated population genetic studies in Central Asia. Here, we sequenced and analyzed the Y-chromosomes of 187 male individuals from Kazakh, Kyrgyz, Uzbek, Karakalpak, Hazara, Karluk, Tajik, Uyghur, Dungan, and Turkmen populations. High diversity and admixture from peripheral areas of Eurasia were observed among the paternal gene pool of these populations. This general pattern can be largely attributed to the activities of ancient people in four periods, including the Neolithic farmers, Indo-Europeans, Turks, and Mongols. Most importantly, we detected the consistent expansion of many minor lineages over the past thousand years, which may correspond directly to the formation of modern populations in these regions. The newly discovered sub-lineages and variants provide a basis for further studies of the contributions of minor lineages to the formation of modern populations in Central Asia.
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... Due to pressure from regional countries Panjshir insurgency went underground, similarly anti IEA forces like Gen Dostam , Gen Atta Muhammad, Ahmed Masood, etc. still have considerable influence in their ethnic groups. In addition, ISKP has conducted some bold terrorist attacks in Kabul, including an attack near the Gulbadin Hikmat Yar office in the capital (He et al., 2019), Taliban lack resources to manage all. On other side, there is growing Pashtun nationalism due to PTM propaganda which is exploiting Pashtun sentiments. ...
Pakistan IEA Relations, A Game Theory Perspective
Article
Full-text available
  • Jun 2022
In ancient Greece Armies, before going to war, used to pay respect to Athena, who was the Greek goddess of wisdom, strategy, and warfare. The Athenians believed that Athena would offer them strategic guidance and protect them in battle. In contrast, Ares, who was the god of war and bloodlust was ignored. The Athenians believed that invoking Ares would bring them victory, but at the cost of more bloodshed and brutality. Therefore, they preferred wisdom from Athena because historical experience had taught them that war is won primarily with wisdom, whereas emotionalism and bloodlust only result in a Pyrrhic victory. The Khyber Pakhtunkhwa province in Pakistan has experienced a high level of terrorist violence and attacks for many years, including before and after the US withdrawal from Afghanistan. The violence in KPK is primarily linked to the ongoing conflict between the Pakistani government and various militant groups, including TTP and other extremist organizations. Although the US withdrawal from Afghanistan may have some impact on the situation in KPK, it is not the sole cause of the violence there. The conflict in KPK has deep roots, including historical grievances, ethnic and sectarian tensions, and ongoing struggles over political power and resources. In recent years, the Pakistani government has taken steps to address the violence in KPK, including increased military operations and efforts to promote peace talks with militant groups. However, the situation in the province remains volatile, and ongoing violence and instability continue to pose a significant challenge. Multipolarity in the global order is becoming visible day by day. China and Russia are coordinating policies and are working together to replace the US-dominated global order. Central and Euro Asia are strategically very important to competing powers. This has restarted a great game in the region in which Pakistan and Afghanistan are on one side victims and on the other are getting pitched against each other. Due to the surge in terrorism, the Pakistani establishment is being forced to launch /target TTP positions in Afghanistan. Taliban who are themselves under pressure due to the financial crisis coupled with ISKP attacks are not taking any action against TTP. In this essay, we will use Game Theory and the prisoner dilemma model to find out what can be the best policy for Pakistan to achieve its objectives.
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... These software packages render quite precise information, which is supported by SNPs typing results. This might be due to STRs being more polymorphic as compared to SNPs (He et al., 2019). Studies on Y-STRs are still ongoing with several new applications. ...
Editorial: Role of Y Chromosome in Molecular Anthropology, Forensics, and Genetic Genealogy
Article
Full-text available
  • Jun 2022
The Human Y chromosome has proven to be a potent tool for studying human genetic anthropology due to its haploid state and the presence of a wide variety of markers (Quintana-Murci and Fellous, 2001). The accumulations of the mutations have led to the separation of the Y lineages, which have been extensively studied (Quintana-Murci and Fellous, 2001; Knight et al., 2003; Tambets et al., 2004; Yang et al., 2014; Jobling and Tyler-Smith, 2017; Kivisild, 2017). Various markers have been utilised to study the populations and lineages over different time scales. This does make the Y chromosome more powerful than mtDNA which does not harbour the array of markers that the Y chromosome contains (Jobling and Tyler-Smith, 1995). The Y chromosome microsatellites (Y-STRs) and single nucleotide polymorphisms (SNPs) have been extensively used for forensic applications and population studies. However, Alu insertion YAP and duplications/deletions have also been useful in population studies (Hammar et al., 1981; Hammer, 1994). STRs have been extensively employed in forensic analysis and kinship testing and various commercial multiplexes are available. Specifically, Y STRs are used in the analysis of samples in sexual offence casework and various mixtures analyses. Two mega multiplexes containing several Y-STRs are Powerplex Y 23 by Promega (Thompson et al., 2013) and Y Filer Plus by Thermo Fisher Scientific (Gopinath et al., 2016). Both kits are employed by forensic laboratories for casework and are also used for kinship testing. Among the Y-STRs, a unique variety is Rapidly Mutating (RM) Y STRs which have a niche application in separating paternally related individuals (Ballantyne et al., 2010, 2012; Adnan et al., 2016; Neuhuber et al., 2022). Similarly, slowly mutating Y STRs (Baeta et al., 2018) might prove complementary to SNP markers in evolutionary studies, though the number of such markers needs to be increased for precise inferences. Y SNPs like sY81 and SRY studied initially have been followed by the detection of vast array of such markers (Underhill et al., 2000; Hinds et al., 2005; Repping et al., 2006; Karafet et al., 2008). The Y consortium published the Y chromosome phylogenetic tree topology based on 243 SNP markers (Consortium, 2002). A further 351 markers were used to develop a better-resolved tree (Karafet et al., 2008). In the current era of rapid data generation using traditional and more recently Massive Parallel Sequencing (MPS) platforms which together with the development of extremely powerful software has started to provide us with new markers and lineages. Hallast et al (Hallast and Jobling, 2017) reported 13,621 SNPs after resequencing 3.7 Mb of Male Specific Region of Y Chromosome (MSY). However, many thousands have been detected by others as well using the Massive Parallel Sequencing techniques, increasing the potential of the Y chromosome as an evolutionary and human identification tool (Francalacci et al., 2013; Poznik et al., 2013; Wei et al., 2013; Scozzari et al., 2014). These developments are helping in determining new directions in human population migrations and evolution. The topic "Role of Y Chromosome in Molecular Anthropology, Forensics, and Genetic Genealogy" was meant to generate a review of the role of various Y markers bringing to light current developments and applications within the field. Human males and females have contributed unequally to geographic expansion due to biological and behavioural differences. Dispersive genetic forces act quickly on uniparental sequences which lead to changes in sequences with one population branching off from the other. Mutations at the AZFc region or DYS448 which are regional specific or to some extent ethnic group-specific is a fine example of it (Adnan et al., 2018, 2021). These haplogroups were previously predicted using SNPs which was a conventional method but now these haplogroups can be successfully predicted from Y-STRs using different software packages like Nevgen and Whit Athey's Haplogroup Predictor Tool (Athey, 2006). These software packages render quite precise information which is supported by SNPs typing results. This might be due to STRs being more polymorphic as compared to SNPs (He et al., 2019). Studies on Y-STRs are still ongoing with several new applications. Ravasini et al have identified four phylogenetically related samples with a null allele at DYS448 and a tetrallelic pattern at DYF387S1, two Y-STRs located in the AZFc. Through MPS analysis, they found that the unusual Y-STR pattern may be due to a 1.6 Mb deletion arising concurrently or after a 3.5 Mb duplication event. Jordamovic et al., predicted the Y haplogroup using Whit Athey's Haplogroup Predictor, based on haplotype data on Powerplex Y 23 -STR markers. They found that Athey's Haplogroup Predictor offers more accurate results and a higher probability of detecting rare haplogroups as compared to SNPs results. Luo et al have characterized the genetic structure and forensic parameters of She and Hakka ethnic groups from Guangdong province of China. They ascertained that Guangdong Hakka has a close relationship with Southern Han, and the genetic pool of Guangdong Hakka was influenced by closely located, Han populations. The predominant haplogroups of the Guangdong She group were O2-M122 and O2a2a1a2-M7, while Guangdong She clustered with other Tibeto-Burman language-speaking populations. Bini et al studied the relationships between old (16-18th century remains) and the modern male population of a population isolate from Roccapelago which is a small village located in the Northern Central Apennines in Italy. 14 modern samples and 25 ancient mummies were genotyped. They utilised several techniques to ascertain relationships of the old and modern-day population from this area. Y haplogroup predictor tool (Athey, 2006) and Nevgen software (Cetkovic Gentula, 2015) were used to infer Y haplogroups from Y-STR haplotypes which showed a close relationship between the old and modern samples. Network analysis showed relationships between the two sets of samples. The combined employment of various forensic genetics and archaeogenetic techniques demonstrated the value of a multidisciplinary approach which is perhaps a way forwards for human Y chromosome analysis. Indeed Y chromosome continues to march on.
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... Recent findings based on the forensic STRs and SNPs identified the genetic link between geographically distinct Torghut Mongolians and 3000 kilometers away Jalaid Mongols . The identified shared alleles between Mongolians and Hazaras also supported the hypothesis that modern Hazaras were the descendants of ancient Mongols (He et al., 2019). Kalmyk is one of the Mongolic-speaking populations residing in Yashkul. ...
Extensive ethnolinguistic diversity at the crossroads of North China and South Siberia reflects multiple sources of genetic diversity
Article
Full-text available
  • Dec 2021
North China and South Siberia, populated by Altaic- and Sino-Tibetan-speaking populations, possess extensive ethnolinguistic diversity and serve as the crossroads for the initial peopling of America and western-eastern trans-continental communication. However, the population genetic structure and admixture history of northern East Asians remain poorly understood due to a lack of genome-wide data, especially for Mongolic-speaking people in China. We genotyped genome-wide SNPs for 510 individuals from 38 Mongolic, Tungusic, and Sinitic speaking populations. We first explored the shared alleles and haplotypes within the studied groups. We then merged with 3508 published modern and ancient Eurasian individuals to reconstruct the deep evolutionary and natural selection history of northern East Asians. We identified genetic substructures within Altaic-speaking populations: Western Turkic people harboured more western Eurasian related ancestry; Northern Mongolic people in Siberia and eastern Tungusic people in Amur River Basin (ARB) possessed a majority of Neolithic ARB related ancestry; Southern Mongolic people in China possessed apparent genetic influence from Neolithic Yellow River Basin (YRB) farmers. Additionally, we found the differentiated admixture history between western and eastern Mongolians and geographically close Northeast Hans: the former received a genetic impact from western Eurasians, and the latter retained the primary Neolithic YRB and ARB ancestry. Moreover, we demonstrated that Kalmyk people from the northern Caucasus Mountain possessed a strong genetic affinity with Neolithic Mongolian Plateau (MP) people, supporting the hypothesis of their eastern Eurasian origin and long-distance migration history. We also illuminated that historical pastoral empires in the MP contributed considerably to the gene pool of northern Mongolic people but rarely to the southern ones. We finally found natural selection signatures in Mongolians associated with alcohol metabolism. Our results demonstrated that the Neolithic ancestral sources from the MP or ARB played an important role in spreading Altaic populations and languages. The observed multi-sources of genetic diversity contributed significantly to the extensive ethnolinguistic diversity in northern East Asia.
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... Genetic characterization showed that the Uyghur and Kazakh ethnic groups were closely related to other Turkic-speaking groups while the Han and Hui populations showed their associations with other Sinitic language-speaking populations. Interestingly, the Kazakh population showed an affinity with the Mongols which suggested an ancient divergence between Kazakh and Mongols when Mongols originally migrated to present-day Xinjiang (Adnan et al., 2018a;Zhan et al., 2018;He et al., 2019). ...
The Heart of Silk Road “Xinjiang,” Its Genetic Portray, and Forensic Parameters Inferred From Autosomal STRs
Article
Full-text available
  • Dec 2021
The Xinjiang Uyghur Autonomous Region of China (XUARC) harbors almost 50 ethnic groups including the Uyghur (UGR: 45.84%), Han (HAN: 40.48%), Kazakh (KZK: 6.50%), Hui (HUI: 4.51%), Kyrgyz (KGZ: 0.86%), Mongol (MGL: 0.81%), Manchu (MCH: 0.11%), and Uzbek (UZK: 0.066%), which make it one of the most colorful regions with abundant cultural and genetic diversities. In our previous study, we established allelic frequency databases for 14 autosomal short tandem repeats (STRs) for four minority populations from XUARC (MCH, KGZ, MGL, and UZK) using the AmpFlSTR® Identifiler PCR Amplification Kit. In this study, we genotyped 2,121 samples using the GoldenEye™ 20A Kit (Beijing PeopleSpot Inc., Beijing, China) amplifying 19 autosomal STR loci for four major ethnic groups (UGR, HAN, KZK, and HUI). These groups make up 97.33% of the total XUARC population. The total number of alleles for all the 19 STRs in these populations ranged from 232 (HAN) to 224 (KZK). We did not observe any departures from the Hardy–Weinberg equilibrium (HWE) in these populations after sequential Bonferroni correction. We did find minimal departure from linkage equilibrium (LE) for a small number of pairwise combinations of loci. The match probabilities for the different populations ranged from 1 in 1.66 × 10²³ (HAN) to 6.05 × 10²⁴ (HUI), the combined power of exclusion ranged from 0.999 999 988 (HUI) to 0.999 999 993 (UGR), and the combined power of discrimination ranged from 0.999 999 999 999 999 999 999 983 (HAN) to 0.999 999 999 999 999 999 999 997 (UGR). Genetic distances, principal component analysis (PCA), STRUCTURE analysis, and the phylogenetic tree showed that genetic affinity among studied populations is consistent with linguistic, ethnic, and geographical classifications.
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Pakistani Hazara Shia victims: challenges, survival techniques, and protective needs
Article
  • Mar 2023
News reports frequently convey acts of violence against Hazara Shias in Pakistan, but there is limited empirical scholarship about lived experiences of the community. To contribute to the knowledge in this field, interviews with Pakistani Hazara Shia victims, who have also lost immediate family members to violence, were conducted. Interviews with thirteen participants took place between February and May 2021. Thematic content analysis revealed two broad areas: Challenges and fears and Coping and hope for a better future. The former showed that participants struggled with: (i) uncertainty about which factor plays a greater role in violence; (ii) facing psychological warfare and living with violence by normalising it; (iii) discrimination and exclusion; and (iv) mental trauma, drug abuse, and suicide ideation. The latter thematic area uncovered that participants persisted through: (i) religious coping and spirituality; (ii) plans for migration versus nationalism; and (iii) hope that basic human rights and special quotas would be secured in the future. The study highlights that unless it is stopped, the violence against Hazara Shias will have to be accepted as an ‘extension of genocide’ which deprives living members of the community from essentially continuing with life beyond pure survival and suffering from ‘social death’.
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Genetic admixture history and forensic characteristics of Tibeto-Burman-speaking Qiang people explored via the newly developed Y-STR panel and genome-wide SNP data
Article
Full-text available
  • Oct 2022
Fine-scale patterns of population genetic structure and diversity of ethnolinguistically diverse populations are important for biogeographical ancestry inference, kinship testing, and development and validation of new kits focused on forensic personal identification. Analyses focused on forensic markers and genome-wide single nucleotide polymorphism (SNP) data can provide new insights into the origin, admixture processes, and forensic characteristics of targeted populations. Qiang people had a large sample size among Tibeto-Burmanspeaking populations, which widely resided in the middle latitude of the Tibetan Plateau. However, their genetic structure and forensic features have remained uncharacterized because of the paucity of comprehensive genetic analyses. Here, we first developed and validated the forensic performance of the AGCU-Y30 Y-short tandem repeats (STR) panel, which contains slowly and moderately mutating Y-STRs, and then we conducted comprehensive population genetic analyses based on Y-STRs and genome-wide SNPs to explore the admixture history of Qiang people and their neighbors. The validated results of this panel showed that the new Y-STR kit was sensitive and robust enough for forensic applications. Haplotype diversity (HD) ranging from 0.9932 to 0.9996 and allelic frequencies ranging from 0.001946 to 0.8326 in 514 Qiang people demonstrated that all included markers were highly polymorphic in Tibeto-Burman people. Population genetic analyses based on Y-STRs [RST, FST, multidimensional scaling (MDS) analysis, neighboring-joining (NJ) tree, principal component analysis (PCA), and median-joining network (MJN)] revealed that the Qiang people harbored a paternally close relationship with lowland Tibetan-Yi corridor populations. Furthermore, we conducted a comprehensive population admixture analysis among modern and ancient Eurasian populations based on genome-wide shared SNPs. We found that the Qiang people were a genetically admixed population and showed closest relationship with Tibetan and Neolithic Yellow River farmers. Admixture modeling showed that Qiang people shared the primary ancestry related to Tibetan, supporting the hypothesis of common origin between Tibetan and Qiang people from North China.
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Genetic admixture history and forensic characteristics of Tibeto-Burman-speaking Qiang people explored via the newly developed Y-STR panel and genome-wide SNP data
Preprint
Full-text available
  • Apr 2022
Fine-scale patterns of population genetic structure and diversity of ethnolinguistically diverse populations are important for biogeographical ancestry inference, kinship testing and also for the development and validation of new kits focused on forensic personal identification. Analyses focused on forensic markers and genome-wide SNP data can provide new insights into the origin, admixture processes and forensic characteristics of targeted populations. Qiang people with a large sample size among Tibeto-Burman-speaking populations widely reside in the middle latitude of the Tibetan Plateau. However, their genetic structure and forensic features have remained uncharacterized due to the paucity of comprehensive genetic analyses. Here, we first developed and validated the AGCU-Y30 Y-STR panel, which contains slowly and moderately mutating Y-STRs, and then we conducted comprehensive population genetic analyses based on Y-STRs and genome-wide SNPs to explore the admixture history of Qiang people and their neighbours. The validated results of this panel showed that the new Y-STR kit was sensitive and robust enough for forensic applications. Haplotype diversity (HD) ranging from 0.9932 to 0.9996 and allelic frequencies ranging from 0.001946 to 0.8326 in 514 Qiang people demonstrated that all included markers were highly polymorphic in Tibeto-Burman people. Population genetic analyses based on Y-STRs (RST, FST, MDS, NJ, PCA and MJNs) revealed that the Qiang people harboured a paternally close relationship with lowland Tibetan-Yi corridor populations. Furthermore, we made a comprehensive population admixture analysis among Eurasian modern and ancient populations based on the shared alleles. We determined that the Qiang people were a genetically admixed population and showed the closest relationship with Tibetan and Neolithic Yellow River farmers. Admixture modelling showed that Qiang people shared the primary ancestry with Tibetan and was derived from North China, supporting the hypothesis of common origin between Tibetan and Qiang people.
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Genomic Insights Into the Genetic Structure and Natural Selection of Mongolians
Article
Full-text available
  • Dec 2021
Mongolians dwell at the Eastern Eurasian Steppe, where is the agriculture and pasture interlaced area, practice pastoral subsistence strategies for generations, and have their own complex genetic formation history. There is evidence that the eastward expansion of Western Steppe herders transformed the lifestyle of post-Bronze Age Mongolia Plateau populations and brought gene flow into the gene pool of Eastern Eurasians. Here, we reported genome-wide data for 42 individuals from the Inner Mongolia Autonomous Region of North China. We observed that our studied Mongolians were structured into three distinct genetic clusters possessing different genetic affinity with previous studied Inner Mongolians and Mongols and various Eastern and Western Eurasian ancestries: two subgroups harbored dominant Eastern Eurasian ancestry from Neolithic millet farmers of Yellow River Basin; another subgroup derived Eastern Eurasian ancestry primarily from Neolithic hunter-gatherers of North Asia. Besides, three-way/four-way qpAdm admixture models revealed that both north and southern Western Eurasian ancestry related to the Western Steppe herders and Iranian farmers contributed to the genetic materials into modern Mongolians. ALDER-based admixture coefficient and haplotype-based GLOBETROTTER demonstrated that the former western ancestry detected in modern Mongolian could be recently traced back to a historic period in accordance with the historical record about the westward expansion of the Mongol empire. Furthermore, the natural selection analysis of Mongolians showed that the Major Histocompatibility Complex (MHC) region underwent significantly positive selective sweeps. The functional genes, alcohol dehydrogenase (ADH) and lactase persistence (LCT), were not identified, while the higher/lower frequencies of derived mutations were strongly correlated with the genetic affinity to East Asian/Western Eurasian populations. Our attested complex population movement and admixture in the agriculture and pasture interlaced area played an important role in the formation of modern Mongolians.
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MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets
Article
Full-text available
  • Jul 2016
We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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The Genetic Ancestry of Modern Indus Valley Populations from Northwest India
Article
Full-text available
  • Dec 2018
  • AM J HUM GENET
The Indus Valley has been the backdrop for several historic and prehistoric population movements between South Asia and West Eurasia. However, the genetic structure of present-day populations from Northwest India is poorly characterized. Here we report new genome-wide genotype data for 45 modern individuals from four Northwest Indian populations, including the Ror, whose long-term occupation of the region can be traced back to the early Vedic scriptures. Our results suggest that although the genetic architecture of most Northwest Indian populations fits well on the broader North-South Indian genetic cline, culturally distinct groups such as the Ror stand out by being genetically more akin to populations living west of India; such populations include prehistorical and early historical ancient individuals from the Swat Valley near the Indus Valley. We argue that this affinity is more likely a result of genetic continuity since the Bronze Age migrations from the Steppe Belt than a result of recent admixture. The observed patterns of genetic relationships both with modern and ancient West Eurasians suggest that the Ror can be used as a proxy for a population descended from the Ancestral North Indian (ANI) population. Collectively, our results show that the Indus Valley populations are characterized by considerable genetic heterogeneity that has persisted over thousands of years.
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Whole-genome sequencing of 175 Mongolians uncovers population-specific genetic architecture and gene flow throughout North and East Asia
Article
Full-text available
  • Dec 2018
  • Nat Genet
The genetic variation in Northern Asian populations is currently undersampled. To address this, we generated a new genetic variation reference panel by whole-genome sequencing of 175 ethnic Mongolians, representing six tribes. The cataloged variation in the panel shows strong population stratification among these tribes, which correlates with the diverse demographic histories in the region. Incorporating our results with the 1000 Genomes Project panel identifies derived alleles shared between Finns and Mongolians/Siberians, suggesting that substantial gene flow between northern Eurasian populations has occurred in the past. Furthermore, we highlight that North, East, and Southeast Asian populations are more aligned with each other than these groups are with South Asian and Oceanian populations. © 2018, The Author(s), under exclusive licence to Springer Nature America, Inc.
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Genetic characterization of Y-chromosomal STRs in Hazara ethnic group of Pakistan and confirmation of DYS448 null allele
Article
Full-text available
  • May 2019
  • INT J LEGAL MED
Pakistan harbors 18 major ethnic groups and Hazara is one of the distinct but smaller groups comprising 0.090% of the total population. Hazara individuals have typical Mongolian facial features and they claim to be descendants of Genghis Khan’s army in the first quarter of the thirteenth century AD. In this study, we genotyped 153 unrelated males living in Quetta, Baluchistan, Pakistan, for a total of 26 (n=153) to 30 (n=47) Y-chromosomal STR loci. 140 unique haplotypes were developed for Hazara population using the PowerPlex Y23 loci. The Y-STR locus showed a genetic diversity ranging from 0.2384 to 0.7918, and an overall discrimination capacity (DC) of 91.5%. The Hazara population samples were profiled for three additional Y-STRs (DYS388, DYS449 and DYS460), which increased the number of unique haplotypes to 144 while the DC increased to 94.11% in Hazara Population of Pakistan. Interestingly null alleles were observed at DYS448 in 25 individuals of Hazara population. The Hazaras showed significant differences from other local populations of Pakistan as well as neighboring populations, but had considerable genetic affinities to Kazakhs and Mongols.
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Population genetic analysis of 30 insertion–deletion (INDEL) loci in a Qinghai Tibetan group using the Investigator DIPplex Kit
Article
Full-text available
  • Jul 2019
  • INT J LEGAL MED
The Qiagen Investigator® DIPplex Kit multiplexes 30 autosomal INDELs plus amelogenin for forensic use. The aim of this study was to estimate the diversity of 30 INDEL loci in a sample of 530 Tibetan individuals from Qinghai Province, China, and to evaluate the usefulness of these loci for forensic genetics. The observed heterozygosity ranged from 14.3 to 53.4%, and combined power of discrimination, matching probability, and cumulative probability of exclusion were 0.99999999999172, 8.27999 × 10⁻¹², and 0.9897, respectively, in the Qinghai Tibetan group. The results of pairwise genetic distance, principal component analysis, a multi-dimensional scaling plot, and a neighbour-joining tree between the Qinghai Tibetan and 49 reference populations revealed significant genetic differences between continental populations and a close genetic relationship between the Qinghai Tibetan and East Asian populations. This study indicates that the Investigator® DIPplex Kit can serve as an effective supplementary tool for forensic genetic tasks.
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Population Genetic Diversity and Clustering Analysis for Chinese Dongxiang Group With 30 Autosomal InDel Loci Simultaneously Analyzed
Article
Full-text available
  • Aug 2018
In comparison with the most preferred genetic marker utilized in forensic science (STR), insertion/deletion analysis possesses further benefits, like absence of stutter peak, low mutation rate, and enabling mixed stain analysis. At present, a total of 169 unrelated healthy Dongxiang individuals dwelling in Dongxiang Autonomous county of Gansu province were recruited in our study to appraise the forensic usefulness of the panel including 30 autosomal diallelic genetic markers. The insertion allele frequencies were in the range of 0.1598 at HLD 111 to 0.8550 at HLD 118. The cumulative match of probability and the combined probability of exclusion were estimated based on independence of pairwise loci, with the values of 3.96 × 10⁻¹¹ and 0.9886, respectively, which showed tremendous potential of this panel to be qualified for forensic personal identification in Chinese Dongxiang group. And it could also be used as a complementary tool for forensic parentage testing when combined with standard STR genetic markers. Furthermore, calculation of the DA distance and Fst values of pairwise populations, phylogenetic reconstruction, multidimensional scaling analysis, structure clustering analysis were also conducted to probe the genetic relationships between Dongxiang group and the other 30 reference populations. Results demonstrated that Dongxiang ethnic group might be genetically closer related with most Chinese populations involved in our study, especially Tibet groups, Xibe group, and several Han populations.
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Autosomal DIPs for population genetic structure and differentiation analyses of Chinese Xinjiang Kyrgyz ethnic group
Article
Full-text available
  • Jul 2018
In recent years, deletion and insertion polymorphisms (DIPs) were treated as a novel complementary tool with huge potential for forensic applications. In this study, we utilized 30 DIP loci to make a comprehensive research of allele frequency distribution and compute forensic parameters to evaluate the efficiency of forensic applications in the 295 unrelated healthy individuals of Kyrgyz group, and in addition, infer the genetic relationships between Kyrgyz group and 24 other previously studied groups. No significant departures from Hardy-Weinberg equilibrium and linkage disequilibrium were observed at these 30 DIP loci. The combined power of discrimination and the combined probability of exclusion for all 30 DIP loci in Kyrgyz group were 0.9999999999989 and 0.9939, respectively. Furthermore, the results of the interpopulation differentiations, phylogenetic reconstruction, population genetic structure and principal component analyses suggested that Kyrgyz group had relatively close genetic relationships with Kazakh and Uygur groups. However, it was also important to stress that 15 loci were selected out from these 30 DIP loci using the method of selecting ancestry markers, which could be utilized for further ancestry inference study relatively.
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Developmental Validation of a Novel Six-Dye Typing System with 47 A-InDels and 2 Y-InDels
Article
  • Feb 2019
  • FORENSIC SCI INT-GEN
In this study, a multiplex amplification system including 47 autosomal InDels, 2 Y-chromosome InDels, and the sex-determining marker (Amelogenin) was developed with six fluorescent dyes labeling. These InDels were selected from the previous study based on a series of criteria (0.3 < MAF < 0.5, HET > 0.4, etc). The system was designated the AGCU InDel 50 kit and was validated in a series of studies, including a degradation study; tests for sensitivity, species specificity, reproducibility, stability, applicability to case samples, balance of peak height, and PCR conditions; and a population study. The results showed that AGCU InDel 50 kit was quite sensitive, specific, stable in several PCR conditions or exposure to PCR inhibitors, especially against degradation. 74 case samples and 50 paternity cases with STR mutation events were tested using PowerPlex ® 21 System, AGCU InDel 50 kit, and Investigator DIPplex kit, and the results showed that the ratio of loci detected with the developed kit were close to Investigator @ DIPplex kit, but considerably higher than PowerPlex ® 21 System for case samples containing low amounts of degraded DNA. As for 50 paternity cases, no mutation was observed in any InDels locus, and the CPIs based on 47 autosomal InDels contained in the AGCU InDel 50 kit were all higher than those based on 30 InDels contained in Investigator® DIPplex kit, except 3 cases. In the population study, 203 unrelated individuals from the Guangdong Han population were detected using the AGCU InDel 50 kit, and the values of combined power of discrimination and combined power of exclusion were 0.999 999 999 999 999 and 0.9997, respectively. Thus, AGCU InDel 50 kit is suitable for individual identification and as a supplemental tool for paternity testing. It is reproducible, accurate and robust for forensic applications and human genetic studies.
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