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Ancient DNA Reveals Matrilineal Continuity in Present-
Day Poland over the Last Two Millennia
Anna Juras
1
*, Miroslawa Dabert
2
, Alena Kushniarevich
3
, Helena Malmstro
¨m
4,5
, Maanasa Raghavan
4
,
Jakub Z. Kosicki
6
, Ene Metspalu
3,7
, Eske Willerslev
4
, Janusz Piontek
1
1Department of Human Evolutionary Biology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poznan, Poland, 2Molecular Biology Techniques Laboratory,
Faculty of Biology, Adam Mickiewicz University in Poznan, Poznan, Poland, 3Evolutionary Biology Group, Estonian Biocentre, Tartu, Estonia, 4Centre for GeoGenetics,
Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark, 5Department of Evolutionary Biology, Uppsala University, Uppsala, Sweden,
6Institute of Environmental Biology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poznan, Poland, 7Department of Evolutionary Biology, University of Tartu,
Tartu, Estonia
Abstract
While numerous ancient human DNA datasets from across Europe have been published till date, modern-day Poland in
particular, remains uninvestigated. Besides application in the reconstruction of continent-wide human history, data from
this region would also contribute towards our understanding of the history of the Slavs, whose origin is hypothesized to be
in East or Central Europe. Here, we present the first population-scale ancient human DNA study from the region of modern-
day Poland by establishing mitochondrial DNA profiles for 23 samples dated to 200 BC – 500 AD (Roman Iron Age) and for
20 samples dated to 1000–1400 AD (Medieval Age). Our results show that mitochondrial DNA sequences from both periods
belong to haplogroups that are characteristic of contemporary West Eurasia. Haplotype sharing analysis indicates that
majority of the ancient haplotypes are widespread in some modern Europeans, including Poles. Notably, the Roman Iron
Age samples share more rare haplotypes with Central and Northeast Europeans, whereas the Medieval Age samples share
more rare haplotypes with East-Central and South-East Europeans, primarily Slavic populations. Our data demonstrates
genetic continuity of certain matrilineages (H5a1 and N1a1a2) in the area of present-day Poland from at least the Roman
Iron Age until present. As such, the maternal gene pool of present-day Poles, Czechs and Slovaks, categorized as Western
Slavs, is likely to have descended from inhabitants of East-Central Europe during the Roman Iron Age.
Citation: Juras A, Dabert M, Kushniarevich A, Malmstro
¨m H, Raghavan M, et al. (2014) Ancient DNA Reveals Matrilineal Continuity in Present-Day Poland over the
Last Two Millennia. PLoS ONE 9(10): e110839. doi:10.1371/journal.pone.0110839
Editor: David Caramelli, University of Florence, Italy
Received May 20, 2014; Accepted September 20, 2014; Published October 22, 2014
Copyright: ß2014 Juras et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: Funding provided by Polish Ministry of Science and Higher Education; grant N N303 406836, Estonian Research Council IUT24-1 and the European
Regional Development Fund (European Union) through the Centre of Excellence in Genomics to Estonian Biocentre and University of Tartu. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: ajuras.anthropology@gmail.com
Introduction
Continuity of human occupation in the territory of Central
Europe, modern-day Poland in particular, and its relation to the
origins of the Slavs have been widely discussed in the archaeo-
logical, linguistic and historical literature; however, these questions
still remain contentious [1–5]. At present, vast territories of East-
Central and South-East Europe are inhabited by Slavic popula-
tions [5]. Three groups of present-day Slavs are identified on the
basis of their linguistic affinities: Western Slavs (Poles, Czechs and
Slovaks), Eastern Slavs (Ukrainians, Belarusians and Russians) and
Southern Slavs (Croatians, Bulgarians, Slovenians, Bosnians,
Macedonians, Montenegrins and Serbians) [6]. It is supposed that
all Slavs, besides their linguistic affinity, also share a common
place of origin, although the latter is still inconclusive [5].
Several hypotheses have been advanced regarding the origin
and early migrations of Slavs, of which two - autochthonous and
allochthonous - have predominated. According to the autochtho-
nous hypothesis, territories around Oder and Vistula rivers (in
present-day Poland) were continuously inhabited by ancestors of
Slavs from the Roman Iron Age (0–400 AD), or perhaps even
further back in time from the Bronze Age (3200–600 BC) [7] until
the Medieval Age (500–1500 AD) [8]. In contrast, the allochtho-
nous theory suggests the discontinuity of settlements between
Roman Iron Age and Medieval Age in the territory of present-day
Poland. Allochthonists hypothesize that the Slavs originated in the
Pripet and Middle Dnieper River basins in modern-day Ukraine,
from where they migrated to the west and south of Europe in the
beginning of 5
th
century AD and inhabited the lands of present-
day Poland, which was previously occupied by Germanic tribes
during the Roman Iron Age [9]. However, morphological analyses
of skeletal materials from present-day Poland have suggested a
continuity between Roman Iron Age (represented by Przeworsk
and Wielbark cultures) and Medieval Age populations [10,11] thus
providing less support to the allochthonous model.
Genetic studies on present-day Slavic-speaking populations
have also addressed the complex genetic history of the Slavs [12–
15]. The comparison of the complete mitochondrial genome
sequences revealed a number of lineages that seem specific for
Central and Eastern Europe. Moreover, based on age estimations,
PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e110839
the authors suggest a genetic continuity of some Slavic mitochon-
drial lineages from at least the Bronze Age [15].
Ancient DNA (aDNA) provides direct genetic evidence for past
demographic events. Mitochondrial DNA (mtDNA) from skeletal
remains has been particularly successful in reconstructing the
evolutionary history of European populations (e.g. [16–21]).
However, no large-scale aDNA study on putative ancestral
populations of modern-day Slavs have been reported thus far.
Ancient DNA datasets from regions geographically adjacent to
present-day Poland are limited to Iron and Middle Age samples
from Denmark [22–25], Neolithic samples e.g. from the Linear-
bandkeramik culture from Germany [26] and Bronze Age samples
from Ukraine, Bulgaria and Moldova [27].
Therefore, to provide fresh perspectives on the debate of genetic
continuity in Central Europe during the last two millennia, and to
contribute to the resolution of the complex origin of the Slavs, we
present the first population-level ancient mtDNA analysis on
samples originating from six archaeological sites in Poland. The
studied samples date to the Roman Iron Age, represented by
Wielbark and Przeworsk cultures and to the Medieval Age. Our
aim is to determine matrilineal genetic structure of ancient
populations in the area comprising contemporary Poland, their
relationships to one other and to other ancient and modern
human populations from Europe, and to investigate potential
genetic continuity between populations spanning two millennia.
Materials and Methods
Archaeological sites and samples
The skeletal material studied here originated from burial sites
located in present-day Poland dating to the Roman Iron Age and
the Medieval Age (n = 72) (Figure 1). The Roman Iron Age
samples (RoIA) comprised 38 human remains from cemeteries in
Kowalewko (n = 11) and Rogowo (n = 13) assigned to the Wielbark
culture, and two burial sites in Karczyn (n = 12) and Ga˛ski (n = 2)
assigned to the Przeworsk culture. The Wielbark culture extended
in the north-eastern territories of contemporary Poland during the
1
st
to the 4
th
century AD. The Przeworsk culture was present in
the Western, Central and Southern Poland from the 3
rd
century
BC to the 5
th
century AD. Both Wielbark and Przeworsk cultures
were dated to the Roman Iron Age based on the archaeological
context. Medieval Age (ME) samples comprised 34 human
remains recovered from cemeteries in Cedynia (n = 18) and
Ostro´w Lednicki (n = 16). Detailed information about each
sample, the archaeological context of the burial sites and their
geographic origins is presented in Table 1 and Table S1. The
handing history of samples is not well recognized; however seems
to be minimal due to obtained results since aDNA was obtained
only from intact teeth that are thought to be less prone to modern
human DNA contaminations than other skeletal parts [28]. The
permission for collecting samples for aDNA studies from all
mentioned above archaeological sites was provided by the
supervisor of the skeletal materials, Head of the Institute of
Anthropology and Department of Human Evolutionary Biology at
Adam Mickiewicz University in Poznan.
Extraction of ancient DNA
Sampling was performed using disposable gloves, facemasks and
body suits to minimize the risk of contamination from modern
humans. Two teeth were collected from each individual. All pre-
PCR work was conducted in laboratories dedicated exclusively to
the analysis of low copy number DNA (Centre for GeoGenetics at
the University of Copenhagen, Denmark and Ancient DNA
Laboratory at the Adam Mickiewicz University in Poznan,
Poland). The outer surface of the teeth was decontaminated using
0.1 M HCl, followed by drilling of the teeth and treatment of the
powder with 0.5% NaOCl [29]. DNA was extracted using a silica-
column based method [29,30] which was modified by the addition
of urea to the extraction buffer [31]. DNA extractions from the
two teeth from each individual were performed at separate times.
Preparation of reagents and solutions was conducted under sterile
conditions and appropriate precautions (e.g. UV irradiation) were
taken to avoid modern DNA contamination. Negative controls
were set up during extractions (one control for every four samples)
and amplifications (one control for every eight PCR reactions).
Three faunal samples, contemporary with the human skeletal
samples, were retrieved from one of the locations (Kowalewko)
and were used as controls to screen for contamination from
modern human sources both during and post-excavation.
Mitochondrial DNA analysis
Seven sets of overlapping primer pairs (Table S2) were used to
amplify 360 base pairs (bp) of the first hypervariable region
(HVRI) of the mtDNA control region between nucleotide
positions (nps) 16043-16403, according to the revised Cambridge
Reference Sequence (rCRS) (NC_012920.1) [32]. Haplogroup-
diagnostic nps in HVRII and mtDNA coding region were
amplified using eighteen primer pairs, respectively, and HVRI
regions between nps 16043-16132 and 16307-16403 were
amplified using M13-tailed primers (Table S2). PCR reactions
were set up as follows: 2 ml DNA extract, 0.5 U Platinum Taq
DNA Polymerase High Fidelity (Invitrogen), 1X High-Fidelity
PCR Buffer, 2 mM MgSO
4
, 0.8 mg/ml RSA (Calbiochem),
200 mM each of dNTPs (Invitrogen), 500 nM of each primer and
ddH
2
Oupto25ml. The thermocycling conditions were as follows:
initial denaturation at 94uC for 4 minutes; 42 cycles of 94uC for 30
seconds, 52uC–60uC depending on the primer pair, (Table S2) for
20 seconds, 68uC for 20 seconds; and final extension at 72uC for
10 minutes. PCR products were visualized through electrophoresis
on 2% agarose gel.
Almost all analyzed fragments, including HVRI, HVR-II and
coding region fragments containing diagnostic SNPs were cloned
and sequenced. Only fragments comprising nps 16048-16132 and
16307-16403 were amplified with M13-tailed primers and directly
sequenced without cloning. Amplicons were cloned into pCR2.1-
TOPO vector and transformed into competent E. coli cells (One
Shot E. coli) using TOPO TA cloning kit (Invitrogen), following
the supplier’s instructions. More than twelve bacterial colonies
from each cloning experiment were screened for inserts using M13
universal primers. At least four positive clones were sequenced for
each case. PCR products were purified with exonuclease I and
Fast alkaline phosphatase (Fermentas) and sequenced using
BigDye Terminator v3.1 kit and ABI Prism 3130xl Genetic
Analyzer (Applied Biosystems), following manufacturer’s instruc-
tions. Amplicons generated with M13-tailed primers were
sequenced in both directions using M13 universal primers.
In order to determine consensus sequence and detect post-
mortem damages or/and possible contaminations, alignment of
mtDNA sequences was performed using BioEdit v.7.0.5.3 (http://
www.mbio.ncsu.edu/BioEdit/bioedit.html). Polymorphic posi-
tions in HVRI, HVRII and coding region sequences were scored
against the rCRS sequence. Haplogroups were assigned following
the established hierarchy of mtDNA phylogeny [33].
Populations used in comparative analyses
In order to compare mtDNA profiles of RoIA and ME samples
to modern populations, we compiled a database of published
mtDNA diversity in contemporary Poles, Czechs, Slovaks,
Ancient DNA Reveals Matrilineal Continuity in Present-Day Poland
PLOS ONE | www.plosone.org 2 October 2014 | Volume 9 | Issue 10 | e110839
Belarusians, Ukrainians, Russians (from the European part),
Slovenians, Bosnians, Croatians, Bulgarians, Serbs, Germans,
Finns, Estonians, Latvians, Lithuanians, Macedonians and Swedes
(Table S3). To determine genetic distances between ancient
populations, we also compiled a dataset of published mtDNA
diversity in populations from Iron Age, Early Christian period and
Middle Age from present-day Denmark (Germanic tribes), and
Neolithic population from Linear Pottery Culture (LBK) from
Germany (Table S3).
For haplotype sharing analysis we considered sequences
bounded by np 16043 to 16403 of the HVRI for both aDNA
samples in this study and those from modern populations of our
database. All matches between haplotypes were scored taking into
account their haplogroup affiliation. Because different studies have
used different phylogenetic resolution of mtDNA, for haplotype
sharing analysis and for MDS we considered the phylogenetic
resolution depth reached in our aDNA samples.
Haplotype sharing analysis
Haplotype sharing analysis was conducted in order to detect
mtDNA haplotypes shared between RoIA and ME samples and
modern Europeans. To this end, eighteen contemporary popula-
tions representing East-Central, Southeast and North Europe were
used in the analysis (Table S3). Several populations (Slovaks/
Czechs, Macedonians/Serbs, Bosnians/Slovenians/Croatians,
Finns/Estonians and Lithuanians/Latvians) were pooled together
so that the total sample size (N) for each population amounted to
Figure 1. Locations of the Roman Iron Age (RoIA) and Medieval Age (ME) burial sites in the territory of present-day Poland. RoIA
(stars): Kowalewko (K), Karczyn (KA), Ga˛ski (G), Rogowo (R). ME (triangles): Cedynia (C), Ostro
´w Lednicki (OL).
doi:10.1371/journal.pone.0110839.g001
Table 1. Geographical origins and archaeological contexts of ancient samples analyzed in the present study.
BURIAL SITES N SAMPLE NAMES CONTEXT
Kowalewko (K) 11 K1-K11 RoIA (100-300 AD; Wielbark culture)
Rogowo (R) 13 R1-R13 RoIA (200 AD; Wielbark culture)
Karczyn (KA) 12 KA1-KA12 RoIA (200-500 AD; Przeworsk culture)
Ga˛ski (G) 2 G1-G2 RoIA (200 BC-100 AD; Przeworsk culture)
Cedynia (C) 18 C1-C18 ME (1000-1400 AD)
Ostro
´w Lednicki (OL) 16 OL1-OL16 ME (1100-1400 AD)
N represents the number of analyzed individuals from each of the sites.
doi:10.1371/journal.pone.0110839.t001
Ancient DNA Reveals Matrilineal Continuity in Present-Day Poland
PLOS ONE | www.plosone.org 3 October 2014 | Volume 9 | Issue 10 | e110839
approximately 300 individuals (between 277 and 317). Pooling was
performed according to the linguistic affinities and geographic
location of populations. Likewise, the sample sizes for populations
with N.300 samples were decreased by sampling 300 randomly
selected individuals. Consequently, a total of twelve populations or
groups of pooled populations were used in the analysis.
The presence or absence of a particular haplotype in a given
contemporary population was marked as ’’1’’ or ’’0’’, respectively,
and the total number as well as frequency of ancient haplotypes
found in each modern population was calculated. Furthermore, all
haplotypes present in RoIA and ME samples were divided into
three classes based on their incidence in the comparative modern
populations: informative haplotypes, which were found in less than
half of the comparative populations; non-informative haplotypes,
which occurred in more than half of the comparative populations;
and unique haplotypes, which did not have exact matches in
contemporary populations. In cases where we reduced sample
sizes, we have cross-checked for informative/unique haplotypes if
they present in whole samples size.
We used the two-tailed z-test to assess statistical significance of
shared informative haplotypes between ancient and modern
populations [26]. Nonparametric bootstrapping of 1000 replicates
for each population was used to generate the confidence intervals
for the percentage of all matches, informative matches, and non-
informative matches. The analysis was performed in R (R
Development Core Team 2013) [34] using boot library [35].
Population pairwise F
ST
MtDNA haplogroup frequencies were used to determine genetic
distances between ancient and modern populations. MtDNA
haplogroups of comparative modern Slavic groups as well as Iron
Age and Middle Age populations from present-day Denmark [22–
25] and Neolithic population (LBK) from present-day Germany
[26] were used in this analysis. Slatkin’s linearized pairwise F
ST
were calculated using Arlequin v.3.5 [36]. Multidimensional
Scaling (MDS) plot was built based on population pairwise F
ST
values using Statistica v.10 StatSoft (2011).
Results
Authenticity of ancient DNA results
Reproducible mtDNA sequences were obtained from 23 out of
the 38 specimens from Roman Iron Age burial sites (Table 2), and
from 20 out of the 34 samples from the Medieval Age sites
(Table 3). The consensus nucleotide sequences were supported by
an alignment of at least 10 clones deriving from partially
overlapping amplicons obtained from at least two independent
DNA extractions (nps 16050-16130, 16119-16196, 16181-16226
and 16209-16356) (Figure S1). Therefore, if the same polymorphic
positions were detected in cloned sequences retrieved from two
teeth of each individual, the results were considered to be
authentic. In four individuals we did not obtain a fragment
16209-16356 from one of the two DNA samples extracted from
the same individual. Thus a shorter fragment of HVRI (np 16249-
16317) was amplified, cloned and sequenced. For eleven
individuals a fragment comprising nps 16050-16130 was obtained.
For samples which had been confirmed by cloning as containing
aDNA free from contaminations of modern human DNA a direct
sequencing of M13-tailed amplicons comprising nps 16048-16132
and 16307-16403 was performed. Twenty nine out of 72 samples
failed during the PCR amplifications or showed inconsistent
alignment of cloned sequences and were discarded from the
analyses.
All cloned mtDNA inserts showed characteristic post-mortem
ancient DNA damages, of which 99% wereby C.T and G.A
transitions [37–39]. Sequences obtained with the same procedure
in the two independent laboratories were consistent. No evidence
of modern human DNA contamination was present in the faunal
material used as negative controls.
MtDNA haplogroup composition in ancient populations
All haplotypes identified in RoIA and ME populations belonged
to typical West Eurasian mtDNA haplogroups (hg): H, K, U, T, J,
W, HV, X, HV0, R0, and N (Table 2 and 3). There were 18
different mtDNA haplotypes found among 23 RoIA individuals
and 20 distinct mtDNA haplotypes identified among 20 ME
samples.
Haplogroup (hg) H was the most abundant hg in the RoIA
populations (60.9% of all genotyped RoIA individuals). Samples
from Rogowo comprised the highest number of individuals (10 out
of 12) assigned to hg H, of which three displayed HVRI sequence
identical to the rCRS. Three haplotypes, originating from
Kowalewko and Ga˛ski burial sites, were assigned to three sub-
branches of hg U (U5a, U5b and U3) (Table 2). Haplogroup W
was only identified in three individuals from the Kowalewko site.
Remaining hgs (T, J2a and N1a) were identified in one individual
each.
Haplogroup (hg) H was also the most frequent hg among the
ME samples (40% of all analyzed ME individuals) (Table 3).
Other frequent hgs in the ME population were K, J and X, each
occurring in two specimens. Haplogroups T, W, N1b, HV, HVO,
and R0a were only observed in one individual each.
MtDNA haplotypes shared between ancient and modern
populations
A set of 3595 modern mitochondrial haplotypes, including
majority of Slavic populations from regions geographically
adjacent to Poland, was used for comparative analysis. Haplotypes
shared between ancient and modern populations are presented in
Table S4.
Among haplotypes identified in the RoIA populations, seven
were found to be frequent in most of the modern-day populations
in our database and thus classified as non-informative. These
widespread haplotypes belong to common West Eurasian hgs H, T
and W. The 16223-16292 (hg W) haplotype represents a basal
haplotype within the phylogeny of hg W. Eight RoIA haplotypes
were infrequent in contemporary populations and were classified
as informative (Table S4). The relative frequencies of the shared
informative and non-informative RoIA haplotypes were calculated
in each of twelve present-day populations. Three modern
populations or groups of populations (Lithuanians and Latvians,
Poles, and Czechs and Slovaks) were found to contain significantly
higher percentages (p,0.05) of shared informative haplotypes with
the RoIA samples compared to other present-day populations
(Figure 2, Table S4). Notably, modern Poles shared the highest
number (nine) of informative mtDNA haplotypes with the RoIA
individuals. The remaining three haplotypes had no match in the
screened modern populations and were classified as unique (Table
S3). These unique haplotypes belonged to mtDNA hgs N1a
(16147A-16223-16248-16320-16355), with (likely) back mutation
(C.T) at the position 16172, U5a (16256-16263-16270) and W
(16192-16223-16292-16399).
Among distinct mtDNA haplotypes in the ME samples, eight
belonging to hgs H, T, K, and J were present in high frequencies
in modern-day populations and were hence classified as non-
informative (Table S4). The informative group consisted of nine
haplotypes occurring in low frequencies in six modern populations
Ancient DNA Reveals Matrilineal Continuity in Present-Day Poland
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(Table S4); however, only three out of the 12 tested modern
populations (Bulgarians, Poles and Belarusians) were found to
share significantly higher percentage (p,0.05) of informative
haplotypes with the ME samples (Figure 3). Three haplotypes,
characteristic of hg X4 (16183-16189-16223-16258-16266-16274-
16278), hg HV0 (16129-16145-16298) and hg HV (16189-16271)
did not have matches to the studied modern populations and were
thus considered as unique to the ME samples.
MtDNA haplotypes shared between RoIA and ME
populations
Comparison of mtDNA compositions of the RoIA and ME
populations revealed two shared haplotypes. The first one was
assigned to hg H with a mutation at position 16362. The second
one belonged to hg H5 with a mutation at the diagnostic position
16304. Of the analyzed individuals assigned to hg H5, three RoIA
haplotypes: 16153-16304 (sample R3), 16304 (sample K1), 16129-
16304 (sample G1) (Table 2), and one ME haplotype (16304
(sample OL1)) (Table 3), further belonged to subhaplogroup H5a1
with defining mutation at coding region position 15833.
Pairwise distances and MDS
Pairwise genetic distances were calculated in order to recon-
struct the genetic relationship between the ancient and modern
populations. Pairwise F
ST
values showed non-significant differ-
ences between the RoIA and the ME samples (p.0.01) (Table S5).
The RoIA samples differed significantly when compared to
Neolithic individuals (LBK, Germany), Ukrainians, Belarusians,
Latvians and Finns (p,0.01). The ME individuals showed no
significant genetic differences to other populations used in the
analysis (p.0.01), with the exception of the Neolithic (LBK)
population and Finns (p,0.01) (Table S5). Correspondingly, in
resulted MDS plot both ancient samples from this study (RoIA,
ME) are mostly differentiated from northeast modern Europeans
and Neolithic LBK sample, while being within the conglomerate
of the remaining populations (Figure 4).
Discussion
This is the first large-scale study presenting mtDNA profiles for
43 individuals recovered from Roman Iron Age and Medieval Age
burial sites from modern-day Poland. All mtDNA hgs identified in
RoIA and ME populations are observed in most modern Slavic
populations [40–51]. However, distinct haplotypes detected in the
ancient samples and corresponding frequencies in modern
populations have the potential to address the question of
continuity of mitochondrial lineages over time in the area of
present-day Poland.
Haplotype sharing analysis indicates that the RoIA individuals
shared the highest number of informative mtDNA haplotypes with
present day Poles (Table S4). Of particular interest are three RoIA
samples assigned to subhaplogroup H5a1, which were recovered
from the Kowalewko (sample K1), the Ga˛ski, and the Rogowo
(samples G1 and R3) burial sites (see Figure 1). Recent studies on
mtDNA hg H5 have revealed that phylogenetically older
subbranches, H5a3, H5a4 and H5e, are observed primarily in
modern populations from southern Europe, while the younger
Table 2. MtDNA haplogroups (hg) identified in Roman Iron Age populations.
SAMPLE NAMES HVRI REGION (16043-16403) CR SNPs Hg
R1 rCRS 7028C H
R2 16093C, 16129A, 16316G 7028C H
R3 16153A, 16304C 7028C, 15833T H5a1
R4 16362C 7028C H
R5 16183C, 16189C, 16356C 7028C H
R6 rCRS 7028C H
R7 rCRS 7028C H
R8 16093C 7028C H
R9 16183C, 16189C, 16356C 7028C H
R10 16129A 7028C H
R11 16069T, 16126C, 16145A, 16231C, 16261T, 16299G 10398G J2a
R12 16126C, 16294T, 16296T, 16304C T2
K1 16304C 7028C, 15833T H5a1
K2 rCRS 7028C H
K3 16223T, 16292T 8251A W
K4 16223T, 16292T 8251A W
K5 16192T, 16223T, 16292T, 16399G 8251G W
K6 16192T, 16270T 7768G U5b
K7 16343G, 16390A 14139G U3
KA1 16222T 7028C H
KA2 16147A, 16223T, 16248T, 16320T, 16355T 10238C N1a1a2
G1 16129A, 16304C 7028C, 15833T H5a1
G2 16256T, 16263C, 16270T, 16399G 15218G, 3816A U5a1
rCRS refers to the revised Cambridge Reference Sequence and CR refers to the mtDNA coding region.
doi:10.1371/journal.pone.0110839.t002
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ones, including H5a1 that was found among RoIA individuals in
our study, date to around 4.000 years ago (kya) and are found
predominantly among Slavic populations of Central and East
Europe, including contemporary Poles [15]. Notably, we also
found one ME sample belonging to subhaplogroup H5a1 (sample
OL1 in Table 3). The presence of subclusters of H5a1 in four
Table 3. MtDNA haplogroups identified in Medieval populations.
SAMPLE NAMES HVRI REGION (16043-16403) CR SNPs Hg
C1 16111T 7028C H
C2 16362C 7028C H
C3 16354T 7028C H
C4 16080G, 16189C, 16356C 7028C H
C5 16126C 7028C H
C6 16162G 7028C H1a
C7 16224C, 16311C 1189C K1
C8 16222T, 16224T, 16270T, 16311C 146C, 152T (HVRII), 1189T K2
C9 16183C,16189C, 16223T, 16258C, 16266T, 16274A, 16278T, 16390A 146C, 195C (HVRII), 1719G X4
C10 16183C,16189C, 16223T, 16278T 146T, 195C (HVRII), 1719A X2
C11 16189C, 16271C 7028T, 14766T HV
C12 16069T, 16126C, 16145A, 16222T, 16261T J1b
C13 16126C, 16362C R0a
C14 16129A, 16145A, 16298C 7028T, 72C (HVRII) HV0
C15 16145A, 16176G, 16209C, 16223T, 16390A N1b
OL1 16304C 7028C, 15833T H5a1
OL2 16311C 7028C H
OL3 16126C,16163G, 16186T, 16189C, 16294T T1a
OL4 16069T, 16126C, 16145A, 16172C, 16222T, 16261T 10398G J1b
OL5 16223T 8251A W
rCRS refers to the revised Cambridge Reference Sequence and CR refers to the mtDNA coding region.
doi:10.1371/journal.pone.0110839.t003
Figure 2. Frequencies (%) of haplotypes shared between Roman Iron Age individuals and sampled modern Europeans. Grey bar- all
shared haplotypes; green - informative haplotypes, which not differ significantly from the average frequency (p,0.05); graphite - informative
haplotypes, which differ significantly from the average frequency (p,0.05). Abbreviations for populations: Poles (PL), Belarusians (BY), Russians
(European part) (RU), Czechs/Slovaks (CZ, SK), Bosnians/Slovenians/Croatians (BH, SLO, HR), Bulgarians (BG), Macedonians/Serbs (MA, SI), Ukrainians
(UA), Swedes (SE), Germans (DE), Lithuanians/Latvians (LT, LV), Estonians (EE), Finns (FI).
doi:10.1371/journal.pone.0110839.g002
Ancient DNA Reveals Matrilineal Continuity in Present-Day Poland
PLOS ONE | www.plosone.org 6 October 2014 | Volume 9 | Issue 10 | e110839
ancient samples belonging to both the RoIA and the ME periods,
and in contemporary Poles, indicates the genetic continuity of this
maternal lineage in the territory of modern-day Poland from at
least Roman Iron Age i.e. ,2 kya. Age estimates for other
subhaplogroups of mtDNA hg H5 (Ha5a2, H5e1a and H5u1) as
well as for U5a2b1, U5a2a and U4a are similar as for
subhaplogroup H5a1 (,4 kya) in Central and Eastern Europe
[15,42].
The evolutionary age of H5 sub-branches (,4 kya) [15] also
approximates the age of N1a1a2 subclade found in the RoIA
population (sample KA2) (Table 2). The coalescence age of
N1a1a2 is around 3.4–4 kya, making this haplotype one of the
youngest sub-branches within hg N [52]. The N1a1a2 haplotype
found in one RoIA individual was classified as unique because no
exact match was found among the twelve comparative populations
or groups of populations used in the haplotype sharing test.
Notably, a similar N1a1a2 haplotype carrying an additional
transition at position 16172 was found in a modern-day
Polish individual [53]. Taken together, the presence of
mtDNA subhaplogroups N1a1a2 and H5a1 in both the ancient
Figure 3. Frequencies (%) of haplotypes (matches) shared between Medieval Age individuals and sampled modern Europeans. Grey
bar - all shared haplotypes; green bar- informative haplotypes, which not differ significantly from the average frequency; graphite bar - informative
haplotypes, which differ significantly from the average frequency (p,0.05). For population abbreviations, see Figure 2.
doi:10.1371/journal.pone.0110839.g003
Figure 4. Multidimensional Scaling plot based on F
ST
values calculated from mitochondrial haplogroup frequencies in sampled
European populations. Modern Slavic populations and other populations adjacent to Poles (black): for populations abbreviations, see Figure 2;
ancient comparative populations (green): Danish (Iron Age) (DIA), Danish (Medieval) (DM), Neolithic (LBK, Germany) (NEO); present study populations
(red): Roman Iron Age (RoIA), Medieval Age (ME).
doi:10.1371/journal.pone.0110839.g004
Ancient DNA Reveals Matrilineal Continuity in Present-Day Poland
PLOS ONE | www.plosone.org 7 October 2014 | Volume 9 | Issue 10 | e110839
populations as well as in studied modern Poles suggests a genetic
continuity of certain matrilineages in the territory of present-day
Poland, at least from the Roman Iron Age.
Haplotype sharing test revealed that RoIA populations share
significantly higher number of haplotypes not only with present-
day Poles, and Czechs and Slovaks (Slavic populations), but also
with Lithuanians and Latvians (Table S4). Similarity in genetic
compositions among ancient populations across Central and
north-eastern Europeans may reflect their shared deep matrilineal
history being consistent with demographic history of Europe in
general [54]. Additionally, more recent events such as migration
and admixture might have also contributed to the observed genetic
similarity. For instance, archaeological evidence and historical
records show extensive cultural connections between populations
over wide areas of Europe during the Iron Age [55]. In particular,
the Roman Empire extended throughout the latitudinal breadth of
Europe in the Iron Age. During this period, trade routes crossed
the territories of modern Poland and eastern regions, inhabited by
Baltic tribes [55] thus enabling contacts between RoIA popula-
tions (in particular those belonging to Wielbark culture) and
neighboring Baltic groups, resulting not only in cultural exchange
but, possibly, also in gene flow.
The ME populations share significantly higher percentages of
haplotypes with modern Poles, Belarusians and Bulgarians (p,
0.05) (Table S4). Shared haplotypes over time might reflect genetic
continuity between populations from RoIA, ME and modern
Poles as well as a matrilineal gene flow towards east and south of
Europe at the onset of the Middle Ages, coinciding with
movements of Slavic groups [56].
Inclusion of ancient Danish samples for comparative analysis
was prompted by the fact that these groups as well as those from
the other parts of Scandinavia are thought to have descended from
Germanic tribes that occupied the territory of modern-day Poland
during the Roman Iron Age [9]. The MDS plot showed large
genetic differentiation between Iron Age individuals from Den-
mark and RoIA individuals from Poland (Figure 4), although the
F
ST
values were not statistically significant (p.0.01). Both
populations shared haplotypes belonging to subhaplogroups U3a
and T2. Notably, we did not identify mtDNA hg I, occurring at
high frequency (13%) in ancient Iron and Middle Age Danish
populations [23], among any of the 43 successfully amplified
ancient individuals from Poland.
We note that due to the uniparental inheritance of the
mitochondrial genomes and relatively small sample sizes in our
study, our observation of the various mtDNA hgs in the ancient
populations, both the presence/absence and frequencies, are likely
to be prone to stochastic events. Hence, future research including
nuclear DNA markers are necessary to further explore the genetic
connections between these ancient and modern populations,
including the question of a common origin and the extent of
admixture, if any, between them.
Conclusions
Results of our study indicate genetic continuity of mitochondrial
lineages between ancient and modern populations in the territory
of contemporary Poland. In particular, presence of sub-clades of
hg H5a1 among both RoIA and ME ancient samples and present-
day Poles, and the identification of N1a1a2 haplotype in RoIA and
contemporary Poles is consistent with the idea of continuity of
maternal lineages from at least Roman Iron Age in the region.
Our data demonstrates that present-day Western Slavs, among
analyzed Europeans, exhibit a mtDNA profile that is more similar
to one found among ancient inhabitants of Central Europe. This
observation appears to be in concordance with the autochthonous
hypothesis. Studies on the genetic profiles of other ancient Slavic
populations, especially employing nuclear markers, are necessary
for further resolution of the complex origin of the Slavs.
Supporting Information
Figure S1 Alignments of cloned aDNA sequences ana-
lyzed in this study. The first lines report the revised Cambridge
Reference Sequence (rCRS) with the numbering of the nucleotide
positions.
(PDF)
Table S1 Detailed information about all samples used
in the study involving their repository names, geograph-
ical location, context and references.
(XLS)
Table S2 PCR primers used in the present study.
(DOCX)
Table S3 Modern and ancient populations used in the
comparative analysis.
(DOCX)
Table S4 Haplotype sharing analysis.
(XLSX)
Table S5 F
ST
distances by Slatkin’s based on hap-
logroup frequencies (P
,
0.01).
(DOCX)
Acknowledgments
The authors are grateful to Anna Wrzesin
´ska from Museum of the First
Piasts at Lednica for her help in collecting samples.
Author Contributions
Conceived and designed the experiments: AJ JP EW HM MD. Performed
the experiments: AJ MR. Analyzed the data: AJ EM AK JZK. Contributed
reagents/materials/analysis tools: AJ MD EW. Wrote the paper: AJ AK
MR HM JZK.
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