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Received: 7 November 2016 | Revised: 31 October 2017 | Accepted: 9 December 2017

DOI: 10.1002/ajpa.23380

RESEARCH ARTICLE

Detection of mitochondrial haplogroups in a small avar-slavic


population from the eigth–ninth century AD

Lukas Sebest 1 | s1 | Csaba Bogn
Marian Baldovič1 | Adam Frtu ar1 |
Klaudia Kyselicov
a2,4 | Ľudevít K us4
adasi1,3 | Radoslav Ben

1
Department of Molecular Biology, Faculty
of Natural Sciences, Comenius University,
Abstract
Mlynska Dolina, Ilkovicova 6, Bratislava 842
Objectives: In the sixth century AD, Avars came to Central Europe from middle Eurasian steppes
15, Slovak Republic
2
and founded a strong Empire called the Avar Khagante (568–799/803 AD) in the Pannonian basin.
Faculty of Medicine, Institute of Physiology,
Comenius University, Sasinkova 2, Bratislava During the existence of this empire, they undertook many military and pugnacious campaigns. In
813 72, Slovak Republic the seventh century, they conquered the northern territory inhabited by Slavs, who were further
3
Biomedical Research Center Slovak recruited in Avar military and were commissioned with obtaining food supplies. During almost 200
Academy of Sciences, Slovak Academy of years of Avar domination, a significant influence by the Avar culture (especially on the burial rite)
Sciences, Dubravska cesta 9, Bratislava 845
and assimilation with indigenous population (occurrence of “East Asian”cranial features) could be
05, Slovak Republic
4
noticed in this mixed area, which is supported by achaeological and anthropologcal research.
Department of Anthropology, Faculty of
Natural Sciences, Comenius University, Therefore we expected higher incidence of east Eurasian haplogroups (introduced by Avars) than
Mlynska Dolina, Ilkovicova 6, Bratislava 842 the frequencies detected in present-day central European populations.
15, Slovak Republic
Materials and methods: Mitochondrial DNA from 62 human skeletal remains excavated from the
Correspondence Avar-Slavic burial site Cífer-P
ac (Slovakia) dated to the eighth and ninth century was analyzed by
Marian Baldovič, Department of Molecular the sequencing of hypervariable region I and selected parts of coding region. Obtained haplotypes
Biology, Faculty of Natural Sciences,
were compared with other present-day and historical populations and genetic distances were cal-
Comenius University, Mlynska Dolina,
Ilkovicova 6, 842 15 Bratislava, Slovak culated using standard statistical method.
Republic.
Results and discussion: In total, the detection of mitochondrial haplogroups was possible in 46
Email: baldovic@fns.uniba.sk
individuals. Our results prooved a higher frequency of east Eurasian haplogroups in our analyzed
Funding information population (6.52%) than in present-day central European populations. However, it is almost three
Grant of Comenius University Grant/Award
times lower than the frequency of east Eurasian haplogroups detected in other medieval Avar pop-
Number: UK/293/2016; Diagnostics of
socially important disorders in Slovakia, ulations. The statistical analysis showed a greater similarity and the lowest genetic distances
based on modern biotechnologies, Grant/ between the Avar-Slavic burial site Cifer-Pac and medieval European populations than the South
Award Number: ITMS 26240220058; Siberian, East and Central Asian populations.
Creating Competitive Centre for research
and development in the field of molecular Conclusion: Our results indicate that the transfer of Avar genetic variation through their mtDNA
medicine Grant/Award Number: ITMS was rather weak in the analyzed mixed population.
26240220071; Research & Developmental
Operational Programme, funded by the
KEYWORDS
ERDF.
ancient DNA, haplotype, medieval population, PCA, SNP

1 | INTRODUCTION Khaganate (568 AD) with its center on the territory of present-day
Hungary (see Figure 1). The northern edge of the newly acquired Avar
Nomadic Avar tribes came to Europe from middle Eurasian steppes in territory adjoined the predominantly lowland areas densely inhabited

the second half of the sixth century AD (Cilinska, 1992). After the by Slavs. At the beginning of the seventh century, the Avar Khaganate
defeat of the Gepids and the departure of Lombards, the Avars settled began to expand throughout this territory and part of the local Slavic
in the Pannonian basin and founded an empire called the Avar tribes was conquered. The primarily agricultural Slavic population was

Am J Phys Anthropol. 2018;1–18. wileyonlinelibrary.com/journal/ajpa V


C 2018 Wiley Periodicals, Inc. | 1

2 | SEBEST ET AL.

FIGURE 1 Territorial expansion of the Avar Khaganate in the territory of Central Europe (Zabojník, 2004). (a) Boundaries of the early Avar
Khaganate; (b) boundaries of the late Avar Khaganate; arrow—location of the burial site Cífer-Pac (Slovakia)

used to obtain food supplies and Slavic men as a vanguard in Avars are dated to the eighth century and Graves 39–119 are dated from the
military campaigns (Bokes, 1946). In the second half of the seventh eighth to the first half of the ninth century. No skeletal remains were
century, the boundaries of the Avar Khaganate ranged into the terri- preserved in 25 graves due to complete decomposition of bones. The

tory of present-day Slovakia (cca 15% of its territory) (Cilinska, 1992; remaining 94 graves contained skeleteal remains of 101 individuals.
Zabojník, 1999) (see Figure 1). This set included 61 adults (20 males, 29 females, and 12 individuals
Thus the life of Slavic tribes was associated with Avars for almost with undeterminable sex), 7 adolescents (4 males, 2 females, and 1 indi-
two centuries until the downfall of the Avar Khaganate (799/803 AD). vidual with undeterminable sex) and 33 children (Baldovič, 2010). No
During this period, it is possible to observe the influence of the Avars cremation burials occured on this burial site and the inventory of
on the Slavic culture, nowadays manifested mainly in the change of the graves was relatively diverse. Most of the graves contained only pot-
burial rite. The Slavic burial custom of the cremation was gradually tery and objects of everyday use like needles, spindle whorls (female
replaced by inhumation typical for the Avars. This argument is sup- graves), iron knives and buckles (male graves), just a few individuals
ported by the lack of skeletal burials of Slavic people from the sixth to were burried with more valuable things or weapons: gilded bronze
eighth century (Bottyan, 1975) and their increased incidence on the forging (CP33), necklace with metal and glass beads (CP22), two rings
burial sites of later period of the Avar Khaganate on the territory of and lance (CP28), axe and lance (CP85), casted belt decorations with
present-day Slovakia (Frankenberger, 1935; Stloukal & Hanakova, animal or plant ornaments (CP15, CP24, CP28, CP33). Furthermore,
1974; Thurzo & Korbačkov
a, 1983; Vondrakova, 2002). Avars also ten horse rider graves (CP9, CP12, CP15, CP16, CP17, CP24, CP28,
inhumated their dead with horses, weapons (male graves) and jewellery CP33, CP34, and CP109) were excavated. The walls of these graves
(female graves), which frequently occurred in mixed Avar-Slavic burial were fortified with wooden posts and they contained skeletal remains
sites as well. Hence, the intermix of these two ethnic groups on the of riders and probably their horses. In addition, the skull analysis of 22
occupied territory (the majority of inter-ethnic marriages consisted individuals from this burial site revealed 6 cases of individuals with typ-
from Avar males and Slavic females) cannot be excluded, which is sup- ical “East Asian” cranial features (CP2, CP3, CP61, CP63, CP72, CP105)
ported by the incidence of “East Asian “cranial features (originally char- and 9 cases of mixomorphic individuals with both “East Asian” and
acteristic for Avars and relatively common at the mixed burial sites “European” cranial features including 3 individuals with a higher per-
with the presence of Avar ethnicity) (Barford, 2001; Belcastro & Fac- centage of “East Asian” features (CP55, CP66, CP69) (Baldovič, 2003).

chini, 2001; Cilinska, 1992; Fo
 thi, 2000; Rubini & Zaio, 2011). As it was previously mentioned, Avars came to the Pannonian
The evidence of the coexistence of Avars and Slavs in the common basin from middle Eurasian steppes in the sixth century AD. But their
area provides various archeological sites like Cífer-P
ac (South-western exact origin has not been yet fully specified within this geographical
Slovakia). Excavation in this area during 1971–1983 revealed a relatively area. It is assumed that the Avars (as they are known in Europe) were a
large Avar-Slavic burial site dated to the eighth to ninth century part of the tribe Jou-Jan, which came from Central Asia around 552

 & Fusek, 1984; Cilinsk
(Chropovsky a, 1976; Kolník, 1975). The burial site  ski & Dąbrowska, 1979). According to another theory, the
AD (Szyman
includes 119 inhumation graves with varying degrees of preservation. European Avars were Pseudoavars originating from Var and Chun
Based on the accompanying inventory of the graves, Graves 1–38ab tribes inhabited Volga-Ural region (Avenarius, 1990). Avars are also

SEBEST ET AL. | 3

associated with Hephthalites, which were a confederation of peoples washed, dried and irradiated by UV-light (254 nm) for 30 min. The
in Central Asia (Pohl, 2002). According to the present data of mtDNA crowns were removed by a rotatory saw (Dremel 542). Subsequently
haplogroups distribution, Slavic populations are characterized in com- the remaining roots were irradiated by UV-light (254 nm) for 30 min
mon by the west Eurasian haplogroups and the present-day popula- and mechanically milled to fine powder.
tions of Volga-Ural region and Central Asia (where Avars presumably Prior bone samples collection, a tiny layer (1–2 mm) of the surface
originated from) are typical by the mixture of west and east Eurasian was removed from the part with the narrowest circumference (the
haplogroups (Bermisheva, Tambets, Villems, & Khusnutdinova, 2002; thickest substantia compacta). Then a small bone fragment from this
Comas et al., 2004; Kivisild et al., 1999; Malyarchuk, Derenko, Deni- part was cut out, irradiated by UV-light (254 nm) for 30 min and sepa-
sova, & Kravtsova, 2010b). Therefore we assumed that the portion of rately mechanically milled to fine powder.
detected mitochondrial haplogroups in this analyzed historical popula- Every tooth or bone sample was prepared separately using sterile
tion will be predominantly of west Eurasian origin (HV, JT, UK, I, W, tools to avoid the risk of contamination with modern DNA or cross-
and N) but a smaller proportion may consist of east Eurasian hap- contamination between the samples.
logroups (A, B, C, D, F, G, Y, and M). Our expectation is supported by In our study, we used the DNA extraction method from bones and
the detected incidence of east Eurasian haplogroups (specifically A, B, teeth described by Rohland and Hofreiter (2007).
C, D, G, M*, N9a, and Y) in present-day European populations, where
premised that these haplogroups were introduced to the European 2.3 | PCR amplification
mitochondrial gene pool by Asian nomadic populations (Huns, Avars,
Five pairs of primers were designed for the overlapping amplification of
and Mongols) during their migrations or invasions in Middle Ages
the hypervariable region I (HVR I) of mitochondrial DNA control region
(Brandstätter, Niederstätter, Pavlic, Grubwieser, & Parson, 2007b; Her-
between positions 15,985–16,398 and three pairs of primers were
vella et al., 2014; Malyarchuk, Vanecek, Perkova, Derenko, & Sip,
designed for amplification of the selected sequences of mitochondrial
2006; Mielnik-Sikorska et al., 2013) as well as by the detection of east
DNA coding region between positions 4,142–4,262, 6,957–7,081, and
Eurasian haplogroups (15.3% of all haplogroups) in the other medieval
12,250–12,377. The PCR conditions of each amplificon were optimal-
€sz et al., 2016).
Avar populations (Cso
ized (see Table 1). PCR amplification was carried out using 1 ml of
isolated aDNA in a 30 ml reaction volume containing a final concentra-
2 | MATERIALS AND METHODS tions of 13 HOT FIREPol 103 Buffer B1 (Solis BioDyne), 0.7 mM each
primer, 0.7 mM dNTPs, 4 mM MgCl2, 0.1 U HOT FIREPol DNA Poly-
2.1 | Samples
merase (Solis BioDyne), and ddH2O. Thermal cycling started with 12
The DNA analysis was performed on a set of 62 human skeletal min at 958C, followed by 35 cycles of 30 s at 958C, 30 s at 55.5–588C,
r-Pac (Slovakia). The set
remains form the Avar-Slavic burial site Cife and 35 s at 728C, followed by 7 min at 728C, finishing in a 48C hold.
consisted of 20 adult males, 29 adult females, 3 adolescent males, 2 The yield of amplification was evaluated by electrophoretic separa-
adolescent females and 8 children. Age was assessed according to the tion of 10 mL of PCR product on 2% agarose gel (Sea Kem LE agarose,
status of tooth eruption (Ubelaker, 1987), dental abrasion (Lovejoy, Lonza). The samples which had amplified each of five amplicons and
Meindl, Pryzbeck, & Mensforth, 1985), sutture obliteration (Meindl & without detected contamination in the negative extraction and nega-
Lovejoy, 1985), and morphological changes in the appearance of facies tive PCR control were further analyzed.
symphysialis and facies auricularis (Hanihara & Suzuki, 1978; Lovejoy
et al., 1985). Sex was assesssed according to morphological features of 2.4 | mtDNA sequencing and haplogroups
the cranium, pelvis, and long bones (Acs ri, 1970; Bruzek,
adi & Nemeske determination
2002; Ferembach et al., 1979; Hunger & Leopold, 1978; Işcan &
Sequencing reactions were carried out using the Big Dye 1.1 Termina-
 et al., 1993; Phenice,
Derrick, 1984; Loth & Henneberg, 1996; Novotny
tor sequencing kit (Applied Biosystems) and 8 pairs of previously
1969; Ubelaker, 1984), listed in the morphometric chart. Body height
described primers (Table 1). Sequencing products were analyzed on an
was assesed with regression formulas developed by Sjøvold (1990).
ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). The
The determination of “East Asian” cranial features was performed using
nucleotide sequences of all previously mentioned amplicons (encom-
the method of face horizontal profiling (Debec, 1961) and the method
passing positions 15,985–16,398 in HVR I, positions 4,142–4,262,
of six facial proportion measurements (Bass, 1995). Due to the lack of
6,957–7,081, and 12,250–12,377 in coding region) were compared
sufficiently preserved skulls, these analyses were performed only on
with the revised Cambridge Reference Sequence (Andrews et al.,
the set of 22 individuals. Teeth and long bones were sampled for DNA
1999). Haplogroups were determined based on the incidence of single
isolation (at least two samples per individual).
nucleotide polymorphisms in the HVR I and selected sequences of the
coding region of mtDNA. The database PhyloTree build 17, accessed
2.2 | Sample preparation and DNA extraction
18 February 2016 (Van Oven & Kayser, 2009) was used for identifica-
The teeth were decontaminated by their submersion in a bleach solu- tion of mtDNA halogroups. For construction of phylogenetic trees, the
tion (sodium hypochlorite) for one minute. After that, the teeth were software netViz 7.0 was used.

4 | SEBEST ET AL.

T AB LE 1 Oligonucleotide primers used for the amplification of HVR I and selected sequences in coding region of mitochondrial DNA

Primer pairs Sequence Lenght of product Annealing

Amp1_F 50 -AGCACCCAAAGCTAAGATTCTA-30 132 bp 55.58C

Amp1_R 50 -TGGTGGCTGGCAGTAATGTA-30

Amp2_F 50 -TCAACAACCGCTATGTATTTCG-30 115 bp 55.78C

Amp2_R 50 -AGGGGGTTTTGATGTGGATT-30

Amp3_F 50 -ACCATAAATACTTGACCACCTGTAG-30 133 bp 55.78C

Amp3_R 50 -GAGGGGTGGCTTTGGAGT-30

Amp4_F 50 -ACAGCAATCAACCCTCAACT-30 120 bp 56.38C

Amp4_R 50 -CGGTAAATGGCTTTATGTACTATG-30

Amp5_F 50 -CAAACCTACCCACCCTTAACA-30 117 bp 55.78C


0 0
Amp5_R 5 -CAAGGGACCCCTATCTGAGG-3

Codreg_Amp1_F 50 -GATTCCGCTACGACCAACTC-30 121 bp 588C


0 0
Codreg_Amp1_R 5 -TAGGTTTGAGGGGGAATGCT-3

Codreg_Amp2_F 50 -GGCCTGACTGGCATTGTATT-30 125 bp 588C


0 0
Codreg_Amp2_R 5 -AAGCCTCCTATGATGGCAAA-3

Codreg_Amp3_F 50 -CAACATGGCTTTCTCAACTTTT-30 128 bp 588C


0 0
Codreg_Amp3_R 5 -GAAGTCAGGGTTAGGGTGGTT-3

2.5 | Population genetic analyses Only intact teeth without caries and well preserved long bones
were used for the isolation of DNA because intact teeth and compact
In order to infer population affinities a principal component analysis
bone tissues significantly reduce the impact of environmental factors
(PCA) as well as F-statistics (FST) distances were carried out based on
on the quality of DNA and increase the time of its usability.
data of mtDNA haplogroups frequencies. PCA was preformed by use
To prove the authenticity of the historical mtDNA haplotypes, two
of the prcomp function for categorical PCA, implemented in R3.1.3 (R
independent isolations per individual (from tooth and bone) were
Foundation of Statistical Computing, 2015) and plotted in a 2D space,
made, and the sequencing data (all amplicons were sequenced with for-
displaying the first two principal components. Arlequin 3.5 software
ward and reverse primer) from both isolations had to be coincident.
(Excoffier & Lischer, 2010) was used to calculate the pairwise FST
The haplotypes of researchers directly involved in the process of bone
genetic distances. In total, 33 mtDNA haplogroups in our analyzed
or teeth specimen collection and processing, and DNA isolation were
ancient population and other 21 historical populations (11 European
determined and compared with the mtDNA haplotypes from skeletal
and 10 Asian populations) were considered in this analysis. For more
samples (see Table 3). An aditional control method to the detection of
detailed information see Table 2.
modern DNA contamination were the internal parallel controls where
the DNA was isolated from animal bones from the same burial site as
2.6 | Precautions and DNA autheticity testing the analyzed remains. The PCR amplification of human mtDNA was
All steps of DNA isolation were carried out in accordance with the pre- also carried out from these animal DNA isolates as a paralel negative
cautions and conditions required for the work with aDNA (Cooper & control.
Poinar, 2000).
During the bones and teeth sampling, sample preparation and 3 | RESULTS
DNA extraction, the appropriate protective clothing and equipment
(gloves, face masks, laboratory coats) was used in order to eliminate We performed DNA isolation from tooth and bone samples of skeletal
the risk of contamination with recent DNA. All laboratory tools (pip- remains from 62 individuals excavated from the Avar-Slavic burial site
ettes, tweezers, etc.) and workspaces were cleaned with bleach, DNA- Cífer-Pac (Slovakia) dated to the eighth to ninth century. The complete
remover or irradiated under UV-light (254 nm) for 60 min before use. sequence of HVR I of mitochondrial DNA was amplified and sequenced
All used solutions were prepared using ultrapure H2O (Milipore) and from 46 individuals. No data were obtained from nine individuals, due
subsequently this solutions were autoclaved or UV-irradiated (254 nm) to the bad preservation of their skeletal remains. Data from six individ-
for a few minutes. The procedures of bones and teeth sampling, sample uals were discarded for giving incomplete sequence data for determina-
preparation, DNA extraction and PCR amplification were carried out in tion of haplogroup and the results of one individual were also
separate premises dedicated for work with ancient DNA. discarded for haplotype match with the person handling the samples.


T AB LE 2 Frequencies of mtDNA haplogrous of 22 historical Eurasian populations used for PCA


SEBEST

SVK_AvSl HUN_C HUN_AV CB_CZ HUN_L ITA_L ITA_m SPA_m POL_m ICE_m DAN_V NOR_V MON_X CHIN_X CHIN_WZ RUS_Y MIN_BR KAZ_BRIA ALT_Sc SIB_K SIB_B SIB_A

A 2.17 6.93 0 0 0 0 0 0 0 0 0 1.79 17.39 6.25 12.12 3.51 5 8.82 4.55 15.38 5.26 12.5
ET AL.

B 0 1.98 0 0 0 0 0 0 0 0 0 0 2.17 12.5 6.06 5.26 0 0 0 0 0 0

C 0 2.97 3.85 0 0 0 0 0 0 0 0 0 10.87 31.25 3.03 36.84 5 0 9.09 34.62 10.53 41.67

D 0 5.94 3.85 0 0 0 3.7 0 0 0 0 0 43.48 43.75 24.24 38.6 5 0 27.27 3.85 5.26 0

G 0 1.98 0 0 0 0 0 0 0 0 0 0 2.17 6.25 0 0 0 2.94 4.55 3.85 0 0

F 0 0.99 3.85 0 0 0 0 0 0 0 0 0 8.7 0 0 3.51 10 0 4.55 0 0 0

H 36.96 21.78 38.46 21.74 32.14 50 66.67 60.71 40 32.88 34.48 39.29 0 0 3.03 1.75 10 20.59 0 0 10.53 0

HV 4.35 1.98 3.85 0 7.14 0 0 0 10 1.37 0 7.14 0 0 0 0 0 5.88 13.64 0 0 0

I 0 1.98 0 0 7.14 3.57 0 0 0 6.85 13.79 3.57 0 0 0 0 5 5.88 0 0 0 0

J 21.74 4.95 0 17.39 14.29 17.86 3.7 14.29 10 15.07 13.79 17.86 2.17 0 0 3.51 0 8.82 4.55 0 5.26 0

K 4.35 1.98 3.85 0 7.14 0 3.7 1.79 10 13.7 6.9 7.14 0 0 0 1.75 5 0 13.64 0 15.79 0

M 4.35 1.98 3.85 0 0 0 3.7 3.57 0 0 0 0 4.35 0 27.27 1.75 0 8.82 0 0 0 0

N 0 0 0 0 0 0 0 0 0 0 0 0 0 0 18.18 0 0 0 0 0 0 0

N1a 0 2.97 0 0 3.57 0 0 0 0 0 3.45 0 0 0 0 0 0 0 0 0 0 0

N1b 0 0 0 4.35 7.14 0 0 0 5 0 0 0 0 0 0 0 0 2.94 0 0 0 0

R/R0 0 1.98 0 4.35 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0

T 0 3.96 3.85 0 0 3.57 0 1.79 0 2.74 3.45 0 0 0 0 0 0 5.88 0 7.69 0 12.5

T1 0 1.98 3.85 13.04 0 0 0 0 5 0 0 0 0 0 0 0 5 5.88 4.55 0 5.26 0

T2 4.35 5.94 11.54 13.04 7.14 7.14 14.81 3.57 0 6.85 6.9 3.57 0 0 0 0 5 0 0 3.85 0 0

U 0 1.98 0 0 3.57 0 0 5.36 0 0 0 0 0 0 0 0 0 8.82 0 0 5.26 0

U1 2.17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

U2 0 1.98 3.85 0 0 3.57 0 7.14 0 0 0 0 4.35 0 0 0 5 5.88 0 7.69 0 8.33

U3 0 0 3.85 0 0 0 0 0 0 1.37 0 1.79 0 0 0 0 0 0 0 0 5.26 0

U4 6.52 4.95 3.85 4.35 10.71 3.57 0 0 0 4.11 0 1.79 0 0 0 0 15 0 0 0 15.76 8.33

U5 2.17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

U5a 2.17 7.92 3.85 8.7 0 10.71 0 0 0 4.11 6.9 7.14 4.35 0 0 0 20 2.94 13.64 19.23 5.26 16.67

U5b 0 0.99 0 0 0 0 0 0 0 4.11 3.45 7.14 0 0 0 0 0 2.94 0 0 5.26 0

U7 2.17 0 0 4.35 0 0 3.7 0 0 0 3.45 0 0 0 0 0 0 0 0 0 0 0


(Continues)
|
5

6 | SEBEST ET AL.

Therefore we can note that our initial extraction efficiency was 83.9%

€ry et al., 2007); HUN_AV, medieval

ska et al., 2015); MON_X, nomadic Xiongnu population (Mongolia) (Key-


SVK_AvSl HUN_C HUN_AV CB_CZ HUN_L ITA_L ITA_m SPA_m POL_m ICE_m DAN_V NOR_V MON_X CHIN_X CHIN_WZ RUS_Y MIN_BR KAZ_BRIA ALT_Sc SIB_K SIB_B SIB_A

Republic, Russia/Mongolia) (Allentoft et al., 2015; Gonzalez-Ruiz et al., 2012; Pilipenko et al., 2010; Ricaut et al., 2004); SIB_K, Bronze Age population of Late Krotovo culture from western Siberia (Russia)
(Molodin et al., 2012); SIB_B, Bronze Age population from western Siberia (Baraba steppe) (Rusiia) (Molodin et al., 2012); SIB_A Bronze Age population of Andronovo culture from western Siberia (Russia)
SPA_m, medieval Spaniard population (Spain) (Alzualde et al., 2006 ); POL_m, medieval Slavic population (Poland) (Jura set al., 2015); ICE_m, medieval Icelandic population (Iceland) (Helgason et al., 2001);

2015; Keyser et al., 2009); KAZ_BRIA, Bronze and Iron Age populations from central Asia (Kazakhstan) (Allentoft et al., 2015; Lalueza-Fox et al., 2004); ALT_Sc, Iron Age Scytho-Siberian population (Altai
zy et al., 2010); MIN_BR, Bronze Age populations from south Siberia/central Asia (Russia) (Allentoft et al.,
ser-Tracqui et al., 2003; Kim et al., 2010); CHIN_X, Xianbei population (inner Mongolia, China) (Changchun et al., 2006); CHIN_WZ, populations from Wanggu and Zhenzishan (inner Mongolia, China) (Fu
but the final efficiency was 74.2%.

€ sz et al., 2016); HUN_L, medieval Lombard


0

0
The set of 46 individuals with completely sequenced HVR I of

€ lad (Hungary) (Alt et al., 2014); ITA_L, medieval Lombards from Piedmont region (Italy) (Vai et al., 2015); ITA_m, medieval population from Tuscany (Italy) (Guimaraes et al., 2009);
5.26
mitochondrial DNA contained five individuals from horse rider graves,
0

0
three individuals with “East Asian” cranial features and three mixomor-

3.85
phic individuals with the prevalence of “East Asian” cranial features.
0

0 We determined a total of 33 various haplotypes and classified 24 dif-


ferent haplogroups or their sub-groups. The frequency of west Eurasian
0

0
haplogroups was 93.48% and the frequency of east Eurasian hap-
€ mo
logroups was 6.52%.
2.94

€ sz et al., 2016; To

For more detailed information see Table 4.


0

The PCA of our analyzed historical population and 21 historical


akyova et al., 2016; Cso

European and Asian populations was computed based on mtDNA hap-


0

logroup frequencies (Table 2). PCA of all historical populations showed


clustering of analyzed Cífer-Pac population with other medieval Euro-
1.75

1.75

SVK_AvSl, medieval Avar-Slavic population from Cífer-Pac (Slovakia) (this study); HUN_C, population from Hungarian conquest-period (Hungary) (Cso
0

pean populations and also revealed significant difference between his-


torical European (incluing Cífer-Pac population) and Asian populations
(see Figure 2).
6.06

€ sz et al., 2016) ; CB_CZ, two medieval Hungarian-Slavic contact zone populations (Slovakia, Croatia) (Cs
0

Pairwise FST genetic distances were calculated based on 810


DAN_V, medieval population of Danes and Vikings (Denmark); NOR_V, medieval population of Norwegian Vikings (Norway) (Krzewin

ancient HVR I sequences of 22 historical populations from Eurasia.


Similarly as the PCA results, pairwise FST values of analyzed Cífer-P
ac
0

population indicated non-significant differences with all historical Euro-


pean populations. For more detailed information see Table 5.
0

4 | DISCUSSION
1.79
0

The anthropological and archeological findings indicate that the Slavic


3.45

ethnic group constituted the majority of the analyzed population from


0

et al., 2007, 2009); RUS_Y, Yakut population from eastern Siberia (Sakha republic, Russia) (Crube

the burial site Cífer-Pac, and therefore we compared our results with
2.74

2.74

1.37

the mitochondrial haplogroup frequencies of the present-day Slavic


0

populations (mostly Slovaks, Czechs, and Poles). In case of the hap-


10

logroups with relatively wide geographical distribution (west and cen-


0

tral Eurasia) or haplogroups that are relatively rare in present-day Slavic


1.79

population, we compared the disribution and origin of their haplotypes


0

with the haplotypes of relevant present-day or historical populations (if


possible).
0

In addition, we also performed the population genetic comparison


of obtained mtDNA haplogroup frequencies in our analyzed historical
0

population with available historical Eurasian mtDNA data in order to


evaluate its genetic distance from other populations.
0

Haplogroup H was the most frequent haplogroup in the analyzed


(Allentoft et al., 2015; Molodin et al., 2012).

historical population from the burial site Cífer-Pac. It reached fre-


8.7

quency 43.5%, which correlate with the frequencies of H haplogroup


in present-day Slavic populations (40.1–47.9%) (Achilli et al., 2004;
3.85

Avar population (Hungary) (Cso

Malyarchuk et al., 2003). Our result is also comparable with the H fre-
0

quencies among Slovaks (45.5%), Czechs (44.13%), and Poles (45.18%)


(Continued)

1.98

1.98

1.98

3.96

(Malyarchuk et al., 2002, 2006, 2008a). The claim about the major
0

population from Szo

Slavic part of the analyzed historical population from Cífer-Pac could


be further supported by the significantly lower frequency of hap-
2.17

2.17

2.17

logroup H in present-day populations (11–14%) as well as in historical


T AB LE 2

populations (25.92%) of Central Asia (Kivisild et al., 1999; Lalueza-Fox


U8

YZ
W
V

et al., 2004; Loogväli et al., 2004).



SEBEST ET AL. | 7

T AB LE 3 Haplogroups of researchers directly involved in the process of specimen collection and processing, and DNA isolation

Researchers HVR I region 15,985–16,398 Coding region Haplogroups

R1 16192T, 16304C 7028C H5a

R2 16126C, 16153, 16294, 16390 7028T, 4216C T2e

R3 16093C, 16224C, 16311C 7028T K1a

R4 16111T, 16129A, 16256T 7028C H3b

The most frequent haplotype matched the revised Cambridge Ref- 2010; Brandstätter et al., 2006; Malyarchuk et al., 2003, 2008a;
erence Sequence (rCRS) in the HVR I region and carried the diagnostic Mielnik-Sikorska et al., 2013) (see Figure 1). An identical H13a haplo-
for haplogroup H polymorphism in the control region of mtDNA. It is type was detected in the present-day Slovak and Finnish populations.
relatively widespread and therefore we are not able to reliably estimate (Loogväli et al., 2004; Baldovič, 2010; Raule et al., 2014). Therefore we
the phylogeographic origin of individuals which carry this haplotype. assume the European origin of H6a, H7c and H13a subclades in the
Haplotype H characterized with polymorphisms 16093C and 16311C analyzed population.
was detected in the present-day population of Russia, Poland, Slovakia The second most frequent haplogroup was haplogroup J, up to
and Macedonia (Baldovič, 2010; Brandstätter et al., 2008; Malyarchuk 21.7% of the detected mtDNA haplotypes in the analyzed ancient pop-
et al., 2002; Mielnik-Sikorska et al., 2013; Quintana-Murci et al., 2004). ulation. This haplogroup occurs in Europe quite often (10.5%). J is char-
Haplotype H defined by polymorphisms 16206T and 16265T was the acterized by decreasing occurrence eastwards, where it reaches 6.7%
most similar to haplotypes detected among present-day Austrians in Caucasus and 2.5–5.17% in Central Asia (Comas et al., 2004; Kivisild
(16265T) and Poles (16265T and 16293G) (Brandstätter et al., 2008; et al., 1999). The frequency of haplogroup J is 6.94–11.73% in Slavic
Mielnik-Sikorska et al., 2013). Therefore we assume their European ori- speaking populations (Malyarchuk et al., 2004, 2006), especially it
gin. A specific case is haplotype H defined only by 16075C polymor- reaches frequency 8.55, 11.73, and 7.8% among Slovaks, Czechs and
phism. This polymorphism was detected in the haplotypes of present- Poles (Baldovič, 2010; Malyarchuk et al., 2002, 2006). The higher fre-
day populations of Europe, Anatolian plateau and Near East but these quency of J haplogroup in the analyzed ancient population is likely a
haplotypes contain another two to three different polymorphisms random fluctuation caused either by genetic drift or sampling. All of the
(Loogväli et al., 2004). Therefore we are not able to estimate its origin detected haplotypes are commonly presented in the recent populations
in the analyzed medieval population. of Europe, Caucasus and Near East, and likely reflect the Neolithic or
Within haplogroup H, we were able to detect some of its sub- subsequent migrations of this haplogroup from Near East to the sur-
groups and subclades respectively. Sub-group H1 was represented by rounding areas (Richards et al., 2000).
its subclades H1c and H1ar. H1 is very frequent among the Slavic popu- Another haplogroup with higher incidence was haplogroup U,
lations of east Europe (about 30% of haplogroup H) (Achilli et al., 2004; which was represented by its sub-groups or subclades: U1a, U4, U4a,
Loogväli et al., 2004; Nikitin et al. 2009). Identical haplotype H1c was U5, U5a, U7, and U8 in the analyzed ancient population.
detected only once in the present-day Slovak population (Baldovič, U1a is a cubclade of sub-group U1. This sub-group is predomi-
2010).Therefore we are not able to reliably estimate their origin in the nantly Near Eastern (2.45%) and it was also found in south-east Europe
analyzed population. In the case of H1ar, an identical haplotype was (1.1%), Caucasus (3.95%), and India (2.3%) (Derenko et al., 2012;
detected among modern-day Turks (13) (Loogväli et al., 2004) and thus Fernandez et al., 2014; Kivisild et al., 1999; Malyarchuk et al., 2002;
it could be introduced to the analyzed historical population by Avars. Richards, Macaulay, Torroni, & Bandelt, 2002). The highest frequencies
H6a is considered as European subclade of the sub-group H6, of U1 were observed in Armenia and Georgia (14.4%) (Kivisild et al.,
whereas H6b is more frequent in Central Asia and Near East, which is 1999). Identical U1a haplotype was detected in modern Polish, Azer-
explained by the long-lasting separation of European and Asian H6 baijan, Adigey (Adyghe Republic, Russia), and Druze population (ethnic
mitochondrial gene pools (Loogväli et al., 2004). An identical H6a hap- group inhabited Lebanon, Syria and Israel) (Macaulay et al., 1999;
lotype was detected in present-day European population (Czechs, Mielnik-Sikorska et al., 2013; Quintana-Murci et al., 2004). Based on
Poles, Austrians) (Brandstätter et al., 2008; Malyarchuk et al., 2002; the facts mentioned above, we are not able to reliably determine the
Mielnik-Sikorska et al., 2013). origin of U1a haplotype in the analyzed population.
H7c and H13a are subclades of sub-group H7 and H13 respec- Sub-group U4 (with its subclade U4a) has a relatively wide geo-
tively. Both of these sub-groups occur infrequently in Europe, Near graphic distribution (Malyarchuk et al., 2004) and it is very rare in cen-
East and Caucasus. An interesting fact is that all of the observed H13 tral Europe (Bramanti et al., 2009). Based on the detected ancient
haplotypes from these geographical regions belonged to the H13a sub- haplotypes defining U4 and U4a we are not able to reliably determine
clade (Brandstätter et al., 2006; Mielnik-Sikorska et al., 2013; Roostalu their origin in the analyzed population, because U4 haplotype repre-
et al., 2007). Medieval H7c haplotype is sharing much more identical sents a so far unknown lineage and the same haplotype as U4a has
positions with European haplotypes than the haplotypes detected in been detected only in the present-day Slovak population (0.09%) (Bal-
the present-day Near Eastern and Caucasian populations (Baldovič, dovič, 2010) (see Figure 3).

8 | SEBEST ET AL.

T AB LE 4 Detected HVR I and coding region polymorphisms and haplogroups of 46 individuals from burial site Cífer-Pac (Slovakia)

Individuals HVR I region 15,985–16,398 Coding region Haplogroups

CP104 female 50–59 yrs rCRS 7028C H

CP106 female 20–29 yrs rCRS 7028C H

CP10a male 30–39 yrs rCRS 7028C H

CP24* male 20–29 yrs rCRS 7028C H

CP33* male ND rCRS 7028C H

CP611 female 40–49 yrs rCRS 7028C H

CP68 female 40–49 yrs rCRS 7028C H

CP12* male 40–49 yrs 16093C, 16311C 7028C H

CP109* male 30–39 yrs 16093C, 16311C 7028C H

CP46 male 30–39 yrs 16206T, 16265T 7028C H

CP47 male 40–49 yrs 16075C 7028C H

CP43 male 20–29 yrs 16189C, 16362C 7028C H1c


2
CP69 male 40–49 yrs 16302G 7028C H1ar

CP10b male 40–49 yrs 16193T, 16219G, 16362C 7028C H6a

CP6 female 20–29 yrs 16093C, 16265G 7028C H7c

CP57 male 20–29 yrs 16234T 7028C H13a

CP58 female 50–59 yrs 16234T 7028C H13a

CP18 female 20–29 yrs 16069T, 16126C 7028T, 4216C J

CP41 ND 4–12 yrs 16069T, 16126C 7028T, 4216C J


1
CP51 female 20–29 yrs 16069T, 16126C 7028T, 4216C J

CP75 female 50–59 yrs 16069T, 16126C 7028T, 4216C J

CP70 ND 4–12 yrs 16069T, 16126C 7028T, 4216C J

CP8 ND 4–12 yrs 16069T, 16126C, 16320T, 16360T 7028T, 4216C J

CP40 male 30–39 yrs 16069T, 16126C, 16229C 7028T, 4216C J

CP100 female 601 yrs 16069T, 16126C, 16311C 7028T, 4216C J

CP76 female 20–29 yrs 16069T, 16126C, 16290T 7028T, 4216C J

CP79 male 13–29 yrs 16069T, 16126C, 16221T 7028T, 4216C J

CP662 ND 4–12 yrs 16183C, 16189C, 16249C 7028T, 12308G U1a

CP28* male 13–19 yrs 16189C, 16356C, 16362C 7028T, 12308G U4

CP4 female 20–29 yrs 16189C, 16356C, 16362C 7028T, 12308G U4

CP88 female 20–29 yrs 16134T, 16311C, 16356C 7028T, 12308G U4a

CP117 ND 4–12 yrs 16183C, 16189C, 16270T 7028T, 12308G U5

CP7 male 40–49 yrs 16256T, 16270T 7028T, 12308G U5a


2
CP55 female 50–59 yrs 16309G, 16318T 7028T, 12308G U7

CP101 male 30–39 yrs 16146G, 16342C 7028T, 4216G, 12308G U8a

CP26 female 20–29 yrs 16126C, 16294T, 16296T 7028T, 4216C T2

CP91 female 40–49 yrs 16126C, 16172C, 16294T, 16304C 7028T, 4216C T2b

CP48 male 20–29 yrs 16224C, 16311C 7028T K

CP53 male 50–59 yrs 16093C, 16224C, 16278T, 16311C 7028T K1a
(Continues)

SEBEST ET AL. | 9

T AB LE 4 (Continued)

Individuals HVR I region 15,985–16,398 Coding region Haplogroups

CP52 female 20–29 yrs 16298C 7028T HV0


1
CP72 male 50–59 yrs 16217C 7028T HV2

CP97 female 30–39 yrs 16261T,16298C, 16311C 7028T V10a

CP94 female 30–39 yrs 16192T, 16223T, 16292T, 16325C 7028T W6

CP71 female 30–39 yrs 16189C, 16223T, 16290T, 16319A, 16362C 7028T A

CP19 female 30–39 yrs 16223T, 16311C 7028T M

CP107 ND 4–12 yrs 16223T, 16304C 7028T M


a
Horse riders.
b
“East Asian” cranial features.
c
Mixomorphic cranial features.
ND, not determined; rCRS, revised Cambridge reference sequence.

Sub-group U5 is of European origin and also one of the oldest sub- Haplogroup K and its subclade K1a was detected only in one indi-
groups in Europe (Soares et al., 2010). It has been suggested that its vidual from our analyzed ancient population. The frequency of this hap-
subclade U5a could be of eastern European origin (Malyarchuk et al., logroup does not exceed 10% in Europe (Peričić, Lauc, Klarić,
2010a). The U5 lineage occurs mainly in Europe but it reaches low fre- Janićijević, & Rudan, 2005), whereas it reaches 3–6% among Slavic
quencies in Near East (up to 2%) (Quintana-Murci et al., 2004). In most populations (Malyarchuk et al., 2002; Mielnik-Sikorska et al., 2013).
cases of Near Eastern U5 haplotypes, these haplotypes are derived Similarly, haplogroup K occurs also in the Near East, Caucasus, and
from European lineages, which is explained as a back migration from Central Asia (Comas et al., 1998; Macaulay et al., 1999). For detected K
Europe to Near East (Richards et al., 2000). An identical U5a haplotype lineages in ancient individuals, it is not possible to determine their ori-
was detected in the Bronze Age population from the territory of gin because identical (similar in case of K1a) haplotypes were detected
present-day Ukraine (Nikitin, 2011) and also in the Middle Neolithic independently in the populations of all geographical regions mentioned
population from the territory of present-day central Poland (Lorkiewicz above.
et al. 2015). Therefore we assume the European origin of these sub- Haplogroup T2 and its subclade T2b were also detected only in
clades in the analyzed population. one individual in our analyzed medieval population. T2 is the most fre-
Haplogroup U7 is virtually absent in Europe (Richards et al., 2000; quent in the Mediterranean area, central and western Europe (8%) and
Tambets et al., 2000). In Slavic speaking populations, it occurs at fre- it is relatively common in some parts of the Near East and Caucasus
quency 0.3% among Slovaks, 0.25% among Poles and 1.2% among (up to 10%). T2b is a predominantly European subclade because it con-
Czechs (Baldovič, 2010; Malyarchuk et al., 2008a; Mielnik-Sikorska stituted about half of T2 among Europeans and some of its branches
et al., 2013). U7 reaches frequency 2–4% in the Near East, Caucasus, (like T2b4) were detected in the Gulf area. However, this was derived
Central Asia and India, whereas it is the most frequent in some popula- from lineages of back migration from Europe as well as in case of some
tions of north-western Iran (9.7%) (Metspalu et al., 2004; Quintana- U5 subclades (Pala et al., 2012; Richards et al., 2000). In case of T2, we
Murci et al., 2004). Interestingly, the haplogroup U7 was detected inde- are not able to reliably estimate its origin because identical haplotypes
pendently in two other studies, which analyzed mtDNA from medieval were detected in present-day European, Near Eastern and Caucasian
human skeletal remains from Denmark and Norway and these studies populations (Gonzalez et al., 2003; Macaulay et al., 1999; Malyarchuk
suppose that the individuals with U7 (or their ancestors) originated et al., 2002, 2006, 2008a) and ancient Central Asian population
from the Black Sea area (Holck, 2006; Rudbeck et al., 2005). But our (Lalueza-Fox et al., 2004). The same T2b ancient haplotype has yet
detected haplotype is the most frequent haplotype in present-day pop- been detected in present-day Slovak, Czech and Portuguese population
ulations of Iran and Pakistan (Quintana-Murci et al., 2004) (see Figure (Gonzalez et al., 2003; Malyarchuk et al., 2006, 2008a), which could
3). Therefore, we suggest that this haplogroup could represent a gene indicate its European origin.
flow that was introduced by Avars. Haplogroup V (including V10a) is considered a European autoch-
U8a is very rare subclade of sub-group U8 and it is characterized thonous haplogroup and is relatively frequent in all parts of Europe. V
by dispersal but widespread distribution limited to Europe (frequency is most frequent among Saami (41.6%) and Basques (12.4%) (Tambets
0.2%), because U8a has not yet been detected in the surrounding areas et al., 2004). The distribution of haplogroup V is significantly decreasing
(Near East, Caucasus, North Africa) (Gonzalez, García, Larruga, & Cab- towards the east of Europe, where it reaches 0.5% in the Caucasus,
rera, 2006; Karachanak et al., 2012). Identical U8a haplotype was 0.2% in the Near East and is virtually absent in Central Asia (Al-Zahery
detected in recent Czech and Polish population (Malyarchuk et al., et al., 2003; Kivisild et al., 1999; Quintana-Murci et al., 2004; Tambets
2002, 2006). et al., 2004). The detected V10a haplotype is the most similar to

10 | SEBEST ET AL.

FIGURE 2 Phylogenetic networks with selected mtDNA variants of haplogroups U4, U7, HV2, A, and M. The legend shows the color code
of present day and historical populations. The number of individuals is shown by the size of circles. Polymorphisms are shown on the lines
between colored nodes (circles). References for individual populations: Adyghene republic (Russia) (Macaulay et al., 1999); ancient Bogratsky
site (Khakassia republic, Russia) (Keyser et al., 2009); ancient Cífer-Pac (Slovakia) (this study); ancient Egyin Gol (Mongolia) (Keyser-Tracqui
et al., 2003); ancient Kongemarken (Denmark) (Rudbeck et al., 2005); ancient Olon Kurin Gol (Mongolia) (Pilipenko et al., 2010); ancient
} e
Orm nykut (Hungaria) (To€ mo
€ ry et al., 2007); Bosnia and Herzegovina (Malyarchuk et al., 2003); China (Kong et al., 2003); Czech republic
(Malyarchuk et al., 2006; Mielnik-Sikorska et al., 2013); Georgia (Comas et al., 2000); India (Quintana-Murci et al., 2004); Iran (Quintana-
Murci et al., 2004); Kazakhstan (Lalueza-Fox et al., 2004); Kirghistan (Comas et al., 1999); Pakistan (Quintana-Murci et al., 2004); Poland
(Mielnik-Sikorska et al., 2013); Portugal (Gonzalez, 2003); Slovakia (Baldovič, 2010; Malyarchuk et al., 2008a, 2008b); Slovenia (Malyarchuk
et al., 2003); Tajikistan (Quintana-Murci et al., 2004); Turkmenistan (Quintana-Murci et al., 2004)

haplotypes observed in the present-day Portuguese, Slovenian, Slovak among Poles (Malyarchuk et al., 2008a, Malyarchuk, Perkova, &
and Polish population (Baldovič, 2010; Gonzalez et al., 2003; Derenko, 2008b). HV0 is also present in the Near East with an average
Malyarchuk et al., 2002, 2003) and it is probably of European origin. incidence of 0.35% (Fern
andez et al., 2014). An identical ancient HV0
Haplogroup HV0 is relatively frequent in northern, central and haplotype was detected in present-day European populations (Slovaks,
south-eastern Europe, where it reaches 4.5–11% (Ottoni et al., 2011). Czechs, Ukrainians, Austrians, Germans, Romanians) (Brandstätter
Its frequency is 39% among Slovaks, 2.9% among Czechs and 4.8% et al., 2008; Malyarchuk et al., 2008a; Mielnik-Sikorska et al., 2013) as

SEBEST ET AL. | 11

T AB LE 5 Pairwise FST genetic distances calculated between ancient Cífer-P


ac population and other 21 historical populations

Population FST values Origin Age N

SVK_AvSl ... central Europe 8th–9th AD 46

HUN_C 0.005 central Europe 10th–12th AD 101

HUN_AV 0.006 central Europe 6th–9th AD 26

CB_CZ 0.008 central Europe 10th–11th AD 23

HUN_L 0.006 central Europe 6th AD 28

ITA_L 0.004 south-west Europe 6th–8th AD 28

ITA_m 0.005 south-west Europe 10th–15th AD 27

SPA_m 0.005 south-west Europe 6th–7th AD 56

POL_m 0.008 central Europe 11th–15th AD 20

ICE_m 0.004 north-west Europe 11th–12th AD 73

DAN_V 0.006 west Europe 8th–16th AD 29

NOR_V 0.004 north Europe 6th–11th AD 56

MON_X 0.013 central Asia 3rd BC–2nd AD 46

CHIN_X 0.017 central Asia 4th–5th AD 16

CHIN_WZ 0.022 central Asia 12th–14th AD 33

RUS_Y 0.013 north Asia 15th–19th AD 57

MIN_BR 0.010 north Asia 18th–9th BC 20

KAZ_BRIA 0.007 central Asia 23rd–4th BC 34

ALT_Sc 0.010 central Asia 5th–3rd BC 22

SIB_K 0.013 north Asia 23rd–17th BC 26

SIB_B 0.009 north Asia 13th–7th BC 19

SIB_A 0.014 north Asia 20th–13th BC 24

N, number of analyzed individuals; SVK_AvSl, medieval Avar-Slavic population from Cífer-Pac (Slovakia) (this study); HUN_C, population from Hungarian
conquest-period (Hungary) (Cso € sz et al., 2016; To
€ mo
€ry et al., 2007); HUN_AV, medieval Avar population (Hungary) (Cso € sz et al., 2016); CB_CZ, two
medieval Hungarian-Slavic contact zone populations (Slovakia, Croatia) (Csakyov a et al., 2016; Cso€ sz et al., 2016); HUN_L, medieval Lombard population
from Szo € lad (Hungary) (Alt et al., 2014); ITA_L, medieval Lombards from Piedmont region (Italy) (Vai et al., 2015); ITA_m, medieval population from Tus-
cany (Italy) (Guimaraes et al., 2009); SPA_m, medieval Spaniard population (Spain) (Alzualde et al., 2006 ); POL_m, medieval Slavic population (Poland)
(Juras et al., 2015); ICE_m, medieval Icelandic population (Iceland) (Helgason et al., 2001); DAN_V, medieval population of Danes and Vikings (Denmark);
NOR_V, medieval population of Norwegian Vikings (Norway) (Krzewin ska et al., 2015); MON_X, nomadic Xiongnu population (Mongolia) (Keyser-Trac-
qui et al., 2003; Kim et al., 2010); CHIN_X, Xianbei population (inner Mongolia, China) (Changchun, Li, Xiaolei, Hui, & Hong, 2006); CHIN_WZ, popula-
tions from Wanggu and Zhenzishan (inner Mongolia, China) (Fu et al., 2007, 2009); RUS_Y, Yakut population from eastern Siberia (Sakha republic,
Russia) (Crube zy et al., 2010); MIN_BR, Bronze Age populations from south Siberia/central Asia (Russia) (Allentoft et al., 2015; Keyser et al. 2009);
KAZ_BRIA, Bronze and Iron Age populations from central Asia (Kazakhstan) (Allentoft et al., 2015; Lalueza-Fox et al., 2004); ALT_Sc, Iron Age Scytho-
Siberian population (Altai Republic, Russia/Mongolia) (Allentoft et al., 2015; Gonz alez-Ruiz et al., 2012; Pilipenko et al., 2010; Ricaut et al., 2004);
SIB_K, Bronze Age population of Late Krotovo culture from western Siberia (Russia) (Molodin et al., 2012); SIB_B, Bronze Age population from western
Siberia (Baraba steppe) (Rusiia) (Molodin et al., 2012); SIB_A Bronze Age population of Andronovo culture from western Siberia (Russia) (Allentoft et al.,
2015; Molodin et al., 2012).

well as in the historical populations of central and eastern Europe (Lee respectively (Quintana-Murci et al., 2004). HV2 haplotype detected in
et al., 2014a, 2014b; Lorkiewicz et al. 2015; Melchior, Gilbert, Kivisild, our study was identical with present-day and ancient HV2 haplotypes
Lynnerup, & Dissing, 2008; Nikitin et al., 2017), which could indicate its detected in Central Asia and its presence in the analyzed ancient popu-
European origin. lation could be related to Avars (Comas et al., 1999; Pilipenko, Roma-
Haplogroup HV2 is very rare in Europe. The unique case of HV2 schenko, Molodin, Parzinger, & Kobzev, 2010; Quintana-Murci et al.,
incidence was observed among Slovaks and Bosnians (Malyarchuk 2004) (see Figure 2).
et al., 2003, 2008a). The major distribution area of HV2 is the Iranian Haplogroup W (including W6) belongs to the group of west Eura-
Plateau (Iran, Pakistan) and western Central Asia (Uzbekistan, Turkme- sian haplogroups with a wide geographical distribution. It is present in
nistan), where this haplogroup reaches frequencies about 10 and 2.4%, Europe, Caucasus, the Near East and also in India (Kivisild et al., 1999;

12 | SEBEST ET AL.

FIGURE 3 PCA plot of 22 historical Eurasian populations. The legend shows the color code of compared historical Eurasian populations.
Abbreviations and references for individual populations: HUN_C, population from Hungarian conquest-period (Hungary) (Cso €sz et al., 2016;
To€ mo
€ ry et al., 2007); HUN_AV, medieval Avar population (Hungary) (Cso €sz et al., 2016) ; CB_CZ, two medieval Hungarian-Slavic contact
zone populations (Slovakia, Croatia) (Csakyova et al., 2016; Cso
€ sz et al., 2016); HUN_L, medieval Lombard population from Szo € lad (Hungary)
(Alt et al., 2014); ITA_L, medieval Lombards from Piedmont region (Italy) (Vai et al., 2015); ITA_m, medieval population from Tuscany (Italy)
(Guimaraes et al., 2009); SPA_m, medieval Spaniard population (Spain) (Alzualde et al., 2006 ); POL_m, medieval Slavic population (Poland)
(Juras et al., 2015); ICE_m, medieval Icelandic population (Iceland) (Helgason et al., 2001); DAN_V, medieval population of Danes and Vikings
(Denmark); NOR_V, medieval population of Norwegian Vikings (Norway) (Krzewin ska et al., 2015); MON_X, nomadic Xiongnu population
(Mongolia) (Keyser-Tracqui et al., (2003); Kim et al., 2010); CHIN_X, Xianbei population (inner Mongolia, China) (Changchun et al., 2006);
CHIN_WZ, populations from Wanggu and Zhenzishan (inner Mongolia, China) (Fu et al., 2007, 2009); RUS_Y, Yakut population from eastern
Siberia (Sakha republic, Russia) (Crube zy et al., 2010); MIN_BR, Bronze Age populations from south Siberia/central Asia (Russia) (Allentoft
et al., 2015; Keyser et al. 2009); KAZ_BRIA, Bronze and Iron Age populations from central Asia (Kazakhstan) (Allentoft et al., 2015; Lalueza-
Fox et al., 2004); ALT_Sc, Iron Age Scytho-Siberian population (Altai Republic, Russia/Mongolia) (Allentoft et al., 2015; Gonz alez-Ruiz et al.,
2012; Pilipenko et al., 2010 Ricaut et al., 2004); SIB_K, Bronze Age population of Late Krotovo culture from western Siberia (Russia) (Molo-
din et al., 2012); SIB_B, Bronze Age population from western Siberia (Baraba steppe) (Rusiia) (Molodin et al., 2012); SIB_A Bronze Age popu-
lation of Andronovo culture from western Siberia (Russia) (Allentoft et al., 2015; Molodin et al., 2012)

Quintana-Murci et al., 2004). Based on the available mtDNA data, an (13), and Iranians (13) (Gonzalez et al., 2003; Malyarchuk et al., 2002,
identical W6 haplotype was detected in the present-day population of 2003, 2008a; Mielnik-Sikorska 2013; Quintana-Murci et al., 2004).
Slovenians (13), Poles (43), Slovaks (13), Ukrainians (13), Portuquese Therefore, it is unlikely that this haplogroup was introduced by Avars.

SEBEST ET AL. | 13

Haplogroups M and A are typical east Eurasian haplogroups. They absent in Europe, whereas they reach the highest frequencies in the
reach the highest frequencies in eastern and southeastern Asia. In Near East, Caucasus, and Central Asia. The incidence of these three
Europe, M and A are very rare, and they were observed only in unique haplogroups in the analyzed historical population could be explained by
cases in central, southeastern and eastern Europe predominantly in three possible scenarios. The first, these haplogroups were the original
Slavic populations (Cvjetan et al., 2004; Malyarchuk et al., 2002, 2003, part of the Avar mtDNA gene pool. The second, these haplogroups
2006; Nikitin et al., 2009; Pliss et al., 2006). It is worth mentioning that were introduced to Avar mtDNA gene pool sometimes during their
the small population of Lemkos (inhabiting the Carpathian Highlands journey to the Pannonian basin or their numerous conquest against
territory located in south-east Poland, east Slovakia and west Ukraine) Byzantine Empire (Pohl, 2002). The third, these haplogroups were
is characterized by a high frequency (5.7%) of M haplogroup (Nikitin introduced to the historical Cífer-Pac population by intensive trade
et al., 2009). However, the haplotype data (based on the polymor- relations with the Byzantine Empire in peacetime because important
phisms in HVR I sequence) of individuals sharing M haplogroup from land and river trade routes passed through the territory of the Avar
this population is not available and therefore we can not performed Khaganate.
comparison with the haplotypes detected in medieval Cífer-Pac popu- Surprisingly, the typically east Eurasian haplogroup A and M that
lation. The detected ancient M and A haplotypes are identical or very are considered to be evidence of Avar genetic influence to the ana-
similar to the haplotypes in present-day and historical Central Asian lyzed ancient population, were detected outside of two previously
populations (Derenko et al., 2012; Keyser-Tracqui, Crubezy, & Ludes, mentioned groups of individuals from horse rider graves and individuals
2003; Kong et al., 2003; Lalueza-Fox et al., 2004; Malyarchuk et al., with “East Asian” cranial features. Haplogroup A was detected in the
€ mo
2006; Quintana-Murci et al., 2004; To € ry et al., 2007) (see Figure 2). individual CP71 and haplogroup M was detected in the individuals
Therefore, we suggested that these haplogroups were most likely intro- CP19 and CP107. Unfortunately, the analysis of cranial features of
duced to the analyzed ancient population by Avars. these three individuals could not be performed due to poor preserva-
For as much as the available archeological and anthropological tion of their skulls. The inventory of their graves was relatively poor
data indicated that the East Eurasian haplogroups might occur in the (pottery and objects of daily use). Individuals CP71 and CP19 were
groups of individuals from horse rider graves, individuals from the females. Sex of the individual CP107 cannot be reliably detected
graves with more valuable burial equipment and individuals with “East because it was juvenile. These facts suggest that these three individuals
Asian” cranial features. could probably be the descendants of Slavic men and Avar women or
In the group of individuals from horse rider graves, we were able at least mothers of these individuals could be of Avar origin.
to completely analyze the mitochondrial DNA of five individuals. Hap- The frequency of east Eurasian haplogroups in our analyzed popu-
logroup H was determined in individuals CP12, CP24, CP33, CP109, lation is 6.52%, whereas the average frequency of east Eurasian hap-
and haplogroup U4 in individual CP28. The fact that we determined no logroups in present-day central European populations is 2.15% (Bosch
typical east Eurasian haplogroup in this group is very surprising because et al., 2006; Brandstätter et al., 2007a, 2007b; Grzybowski et al., 2007;
the horse rider graves belonged to the richest equipped from the whole Handt et al., 1994; Irwin et al., 2007; , Baldovič, Kadasi, & Metspalu,
burial site and therefore their owners had to belong to a higher social 2008; Malyarchuk et al., 2002, 2003, 2006, 2008a; Mielnik-Sikorska
class. The results obtained from this specific group can be interpreted et al., 2013; Nikitin et al., 2009; Parson, Parsons, Scheithauer, & Hol-
in two different ways. First, the individuals from the horse rider graves 
land, 1998; Richards et al., 2000; Sarac et al., 2014; Vanecek, Vorel, &
were Slavs who took over the social and burial habits of Avars, what Sip, 2004; Pajnič, Balazic, & Komel, 2004). Thus we can conclude that

was a frequent phenomenom in the mixed areas (Cilinsk 
a, 1992). The the obtained results confirm our expectation about a higher frequency
second option is that these individuals were the descendants of Avar of east Eurasian haplogroups in our analyzed mixed population than in
men and Slavic women, what could ensure them a higher social status present-day central European populations. However, frequency 6.52%
in this community. However, typical Avar haplogroups were not trans- is relatively low when compared with the total frequencies of east Eur-
fered to this generation due to the matrilinear inheritance of mitochon- asian haplogroups in present-day populations of the Volga-Ural region
drial DNA. Unfortunately the analysis of “East Asian” cranial features of (cca. 26%) and Central Asia (cca. 55.8%) (Chaix et al., 2007; Comas
these individuals was not performed due to the poor preservation of et al., 1998, 2004; Derbeneva, Starikovskaya, Wallace, & Sukernik,
their skulls. 2002; Derenko et al., 2003, 2007; Devor et al., 2009; Gokcumen et al.,
Similarly in the group of individuals (CP51, CP61, and CP72) with 2008; Irwin et al., 2010; Kolman, Sambuughin, & Bermingham, 1996;
typical “East Asian” cranial features or mixomorphic individuals (CP55, Malyarchuk et al., 2010b; Phillips-Krawczak et al., 2006; Quintana-
CP66, and CP69) with the prevailing “East Asian” features only west Murci et al., 2004; Starikovskaya et al., 2005), or the historical popula-
Eurasian haplogroups were detected. The presence of cranial “East tion of Central Asia (cca. 45.5%) (Allentoft et al., 2015; Chikisheva
Asian” features without typical East Eurasian haplogroups could be et al., 2007; Gonzalez-Ruiz et al., 2012; Keyser et al., 2009; Keyser-
explained by likely asymmetric mating of Avar men with Slavic women, Tracqui et al., 2003; Kim et al., 2010; Lalueza-Fox et al., 2004; Molodin
thus increased maternally inherited mtDNA of autochthonous origin et al., 2012; Pilipenko et al., 2010; Ricaut, Keyser-Tracqui, Bourgeois,
even in the group of individuals preserving “East Asian” cranial charac- zy, & Ludes, 2004) and also historical Avar populations from the
Crube
teristics. Despite the occurrence of only west Eurasian haplogroups, 9th to 10th century AD from the territory of current south-eastern
the haplotypes H1ar, HV2, U1a, and U7 are very rare or virtually Hungary (near the city of Szeged), which is almost three fold higher

14 | SEBEST ET AL.

€ sz et al., 2016). The differences in East Eurasian hap-


(15.38%) (Cso features on skeletal remains, the transfer of Avar genetic variation
logroup frequencies of medieval Avar populations from a burial site in through their mtDNA was either weak in the analyzed mixed popula-
south-eastern Hungary mentioned earlier (which was located almost in tion or it could be almost solely mediated by Avar men and their pater-
the center of the Avar Khaganate) and the frequencies analyzed in this nal lineages. However, the presented data abouth mtDNA haplogroup
study, are notable. It could suggest a higher rate of assimilation in variations do not allow us to explore the full genetic impact of Avars
mixed populations on the borders of the Avar Khaganate than in its on this small mixed population. Therefore, the determination of
centre. However for the confirmation of this theory, it would be neces- Y-chromosomal haplogroups could bring completely different results,
sary to analyze many more such burial sites from the Avar period. requiring further analysis.
PCA and pairwise FST genetic distances used for comparison of
our analyzed historical population with other 21 historical populations ORC ID
showed that Cífer-Pac population is geneticaly more similar and closer
s 
Luka Sebest http://orcid.org/0000-0003-2439-4385
to European historical populations than the Asian historical popula-
tions. These results could have two explanations. The first, the rela-
RE FE RE NC ES
tively intensive assimilation took place in the ancient Cífer-Pac
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