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Ancient DNA from South-East Europe Reveals Different Events during Early and Middle Neolithic Influencing the European Genetic Heritage

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The importance of the process of Neolithization for the genetic make-up of European populations has been hotly debated, with shifting hypotheses from a demic diffusion (DD) to a cultural diffusion (CD) model. In this regard, ancient DNA data from the Balkan Peninsula, which is an important source of information to assess the process of Neolithization in Eu-rope, is however missing. In the present study we show genetic information on ancient populations of the SouthEast of Europe. We assessed mtDNA from ten sites from the current territory of Romania, spanning a time-period from the Early Neolithic to the Late Bronze Age. mtDNA data from Early Neolithic farmers of the Starčevo Criş culture in Romania (Câr-cea, Gura Baciului and Negrileşti sites), confirm their genetic relationship with those of the LBK culture (Linienbandkeramik Kultur) in Central Europe, and they show little genetic continuity with modern European populations. On the other hand, populations of the Middle-Late Neolithic (Boian, Zau and Gumelniţa cultures), supposedly a second wave of Neolithic migration from Anatolia, had a much stronger effect on the genetic heritage of the European populations. In contrast, we find a smaller contribution of Late Bronze Age migrations to the genetic composition of Europeans. Based on these findings, we propose that permeation of mtDNA lineages from a second wave of Middle-Late Neolithic migration from NorthWest Anatolia into the Balkan Peninsula and Central Europe represent an important contribution to the genetic shift between Early and Late Neolithic populations in Europe, and consequently to the genetic make-up of modern European populations.
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RESEARCH ARTICLE
Ancient DNA from South-East Europe Reveals
Different Events during Early and Middle
Neolithic Influencing the European Genetic
Heritage
Montserrat Hervella
1
, Mihai Rotea
2
, Neskuts Izagirre
1
, Mihai Constantinescu
3
,
Santos Alonso
1
, Mihai Ioana
4¤
,Cătălin Lazăr
5
, Florin Ridiche
6
, Andrei Dorian Soficaru
3
,
Mihai G. Netea
4,7
*, Concepcion de-la-Rua
1
*
1Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country
UPV/EHU, Bizkaia, Spain, 2National History Museum of Transylvania, Cluj-Napoca, Romania, 3Francisc
I. Rainer" Institute of Anthropology, Romanian Academy, Bucharest, Romania, 4Department of Medicine,
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 5National History Museum of
Romania, Bucharest, Romania, 6Oltenia Museum Craiova, Craiova, Romania, 7Radboud Center for
Infectious Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
¤Current address: University of Medicine and Pharmacy Craiova, Craiova, Romania
These authors share senior authorship.
*conchi.delarua@ehu.es (CR); Mihai.Netea@radboudumc.nl (MN)
Abstract
The importance of the process of Neolithization for the genetic make-up of European popu-
lations has been hotly debated, with shifting hypotheses from a demic diffusion (DD) to a
cultural diffusion (CD) model. In this regard, ancient DNA data from the Balkan Peninsula,
which is an important source of information to assess the process of Neolithization in Eu-
rope, is however missing. In the present study we show genetic information on ancient pop-
ulations of the South-East of Europe. We assessed mtDNA from ten sites from the current
territory of Romania, spanning a time-period from the Early Neolithic to the Late Bronze
Age. mtDNA data from Early Neolithic farmers of the Starčevo Crişculture in Romania (Câr-
cea, Gura Baciului and Negrileşti sites), confirm their genetic relationship with those of the
LBK culture (Linienbandkeramik Kultur) in Central Europe, and they show little genetic con-
tinuity with modern European populations. On the other hand, populations of the Middle-
Late Neolithic (Boian, Zau and Gumelniţa cultures), supposedly a second wave of Neolithic
migration from Anatolia, had a much stronger effect on the genetic heritage of the European
populations. In contrast, we find a smaller contribution of Late Bronze Age migrations to the
genetic composition of Europeans. Based on these findings, we propose that permeation of
mtDNA lineages from a second wave of Middle-Late Neolithic migration from North-West
Anatolia into the Balkan Peninsula and Central Europe represent an important contribution
to the genetic shift between Early and Late Neolithic populations in Europe, and conse-
quently to the genetic make-up of modern European populations.
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 1/20
a11111
OPEN ACCESS
Citation: Hervella M, Rotea M, Izagirre N,
Constantinescu M, Alonso S, Ioana M, et al. (2015)
Ancient DNA from South-East Europe Reveals
Different Events during Early and Middle Neolithic
Influencing the European Genetic Heritage. PLoS
ONE 10(6): e0128810. doi:10.1371/journal.
pone.0128810
Academic Editor: Luísa Maria Sousa Mesquita
Pereira, IPATIMUP (Institute of Molecular Pathology
and Immunology of the University of Porto),
PORTUGAL
Received: December 10, 2014
Accepted: April 30, 2015
Published: June 8, 2015
Copyright: © 2015 Hervella 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 Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the Spanish
Ministry of Science and Innovation, GCL2011-29057/
BOS and grant IT542-10 from the Basque
Government to Research Groups of the Basque
University System, and (UFI 11/09) from the
University of the Basque Country, UPV/EHU. MGN
was supported by a Vici Grant of the Netherlands
Introduction
The fundamental question of the relative contribution of Palaeolithic hunter-gatherers and
Neolithic farmers regarding the genetic heritage of present-day Europeans has been hotly de-
bated. Three events are believed to have had a major impact in the present-day genetic variabil-
ity of Europeans: the expansion of modern humans from Africa through the Middle-East some
46.000 years ago, the repopulation of Europe after the Last Glacial Maximum between 27.000
and 16.000 years ago, and the arrival of the Neolithic culture from Anatolia between 9.000 and
5.000 years ago [1].
The studies by Menozzi, Piazza and Cavalli-Sforza on classical genetic markers, more than
three decades ago, described a South-East to North-West PC1 component that was interpreted
as a demic diffusion of Neolithic farmers from the Middle East into Europe [23]. These data
were however challenged by DNA analysis from present-day populations ([47] among others)
and more recently by ancient DNA (aDNA) studies based on mitochondrial DNA (mtDNA)
[823]. aDNA studies of hunter-gatherers revealed a high genetic homogeneity in the pre-Neo-
lithic groups throughout Europe, whether from Scandinavia [810], Central Europe [11] or the
Iberian Peninsula [1213]. The analysis of aDNA from Early European farmer groups of the
Linear Pottery Culture (LPC, also known as Linienbandkeramik Kultur or LBK) in Central Eu-
rope suggested a genetic discontinuity in Central Europe and favored instead of a process of
Neolithic transition through a demic diffusion model (DD) [1415]: this view was based on a
high frequency of the N1a haplogroup (about 15%) in the LBK farmers [15], absent in hunter-
gatherers in this same region [11] and almost nonexistent (0.2%) in the present-day European
populations [15]. On the other hand, these first farmers shared an affinity with the modern-
day populations from the Near East and Anatolia, supporting a major genetic input from this
area during the advent of farming in Europe [15]. Studies of other Neolithic sites in the North
of France, Hungary and the Northeast of Iberian Peninsula also supported this view [1618].
However, an ancient mtDNA study of a Neolithic site in the Mediterranean region of Europe,
namely in the Iberian Peninsula, led to the proposal of a dual model for explaining the Neolith-
ic dispersion process in Europe: DD in Mediterranean area and CD in Central Europe [19].
On the other hand, it has also been proposed that the mtDNA variability in the Cantabrian
Fringe (nine archaeological sites of both Hunter-Gatherers and Farmers) is best explained by a
model of randomrather than clinal dispersal of Neolithic farmers in Europe, with different genet-
ic influence in different geographical regions and in different periods of time [12]. In regard to
Central Europe, a comprehensive study on mtDNA from archaeological sites spanning from the
Early Neolithic to the Early Bronze Age identified four marked genetic shifts during the Neolithic
period. This diachronic study reported a marked genetic shift between the Early/Middle and Late
Neolithic populations, with a key role for Late Neolithic cultures in shaping the genetic diversity
of modern central Europe genetic diversity [21]. How did this marked genetic shift between
Early/Middle and Late Neolithic could occur in a relatively limited period of time is unclear.
Additionally, a recent mtDNA study on a sample of 15 Near Eastern farmers has revealed
genetic affinities between these earlier farmer communities and modern populations from Cy-
prus and Crete, suggesting that the Neolithic was first introduced into Europe through pioneer
seafaring colonization [22].
Finally, the study of the genomes of a 7,000-year-old farmer from Germany and eight ~8,000-
year-old hunter-gatherers from Luxembourg and Sweden have shown that most present-day Eu-
ropeans derive from at least three highly differentiated populations. Besides, authors have pro-
posed that early European farmers had a ~44% ancestry from a basal Eurasianpopulation [23].
While much has been learned by the aforementioned studies, two crucial aspects have not
been taken into consideration. Firstly, archaeological data show that the Neolithic expansion
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 2/20
Organization for Scientific Research and an ERC
Consolidator Grant (310372). The archeological work
was supported by two grants of the Romanian
National Authority for Scientific Research, CNCS
UEFISCDI, project numbers PNII-ID-PCCE-2011-2-
0013 and PNII-ID-PCE-2011-3-1015. The funders
had no role in study design, data collection and
analysis, decision to publish, or manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
from Anatolia was not a single event but was represented by several waves of migrants [24]. In
this respect the Proto-Sesklo culture in Greece, from which directly Starčevo-Crişin the North
Balkans and indirectly LBK in Central Europe originate [2526] represents only the first great
wave of Neolithisation of Europe [27]. A later great wave of migration from North-West Ana-
tolia led to important cultures of South-Eastern Europe such as Vinča and Boian cultures [28].
Secondly, there is a total absence of aDNA data from South-East Europe in the current models.
In the present study we have assessed the mtDNA variability from 63 individuals recovered
from 10 archaeological sites in Romania spanning a period of five and a half millennia (c.
63001100 cal BC) between the Early Neolithic to the Late Bronze Age in Romania (Table 1,
Fig 1). This is a strategic area of South-East Europe, from which different prehistoric human
groups have passed and later spread throughout Europe. These sites encompass several major
cultural events: i. the first Neolithic complex of the Gura Baciului- Cârcea group (also called
Precrişculture) of Starčevo-Crişculture, which has the same origin in the Proto-Sesklo culture
and it is partially contemporary with LBK culture in Central Europe; ii. the Boian, Zau and
Gumelniţa cultures, that represent a continuum of a second migration in the Middle/Late Neo-
lithic and Eneolithic, which has its origin in North-West Anatolia (Demircihoyuk) through
East Bulgaria [2829]; iii. the Eneolithic complex of Decea Mureşului, that represents a possible
eastern migration [3032]; and iv. the Early and Late Bronze Age complex of Floreşti-Polus,
that represents new migratory movements most likely originating in the North steppes of the
Black Sea [29]. The aim of the study is to shed light on the genetics of the different waves of mi-
gration of Neolithic and Bronze Age populations penetrating Europe from Anatolia and the
steppes north of the Black Sea. We also assess the genetic impact of prehistoric events in the ge-
netic composition of the present-day European populations.
Results
The mtDNA variability of prehistoric groups from Romania
Ancient DNA analysis was performed from 80 teeth remains belonging to 63 individuals recov-
ered from ten prehistoric sites (Table 1,Fig 1 and S1 Table). We have performed
14
C dating for
eleven human remains from six Romanian sites (S8 Fig). One of the samples was discarded
Table 1. Prehistoric samples from Romania analysed in the present study: Chronology, Cultural stages (also in Supporting Information S1 Table),
Archaeological sites and Sample size (I.D.: Identification name; N analysed: Number of individuals analysed; N rep: number of individuals with re-
producibility results).
Chronology and culture Site I.D. N
analysed
N
rep
Early Neolithic (E_NEO) (65005500 BC) (Cârcea/Gura Baciului/PrecrişCulture) Gura Baciului GB 2 2
Negrileşti NE 1 1
Cârcea CA 2 2
Middle/Late Neolithic and Eneolithic (M_NEO) (55004500 BC) (Boian-Zau and Gumelniţa
cultures)
Iclod I 3 3
Vărăşti Va/
BV
14 14
Curăteşti Cu 2 2
Sultana-Valea
Orbului
Su 16 12
Sultana-Malu Roşu SMR 10 10
Eneolithic (Eneol) (45003800 BC) (Decea Mureşului culture) Decea Mureşului DM 2 2
Early Bronze Age (E_BA) (26002100 BC) (Copăceni culture) Floreşti-Polus P 2 2
Late Bronze Age (L_BA) (15001100 BC) (Noua culture) Floreşti-Polus P 9 9
doi:10.1371/journal.pone.0128810.t001
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 3/20
because it provided inconsistent dating, while the others were consistent with the archeological
dating. Fifty nine informative mtDNA sequences were obtained from a total of 63 individuals,
accounting for an overall efficiency of 93% (4 individuals were discarded due to inconsistent re-
sults) (S2 Table, Supporting Information). A number of individuals (17%) has been replicated
independently, which consisted in performing the extraction, amplification and sequencing of
two samples from the same individual by different researchers at different periods of time. The
number of molecular targets was quantified for each extract by means of RT-qPCR. The results
showed that the number of molecules/μl in the extracts ranged between 20066000 (S2 Table),
values falling within the limits proposed for reliable aDNA studies [33].
In order to identify any possible contamination that might have occurred in the different
stages of the laboratory work, at least two extraction controls and several PCR negative controls
were included in each amplification reaction. The rate of contamination for this analysis was
1.3%.
In addition, a total of 192 PCR products from 26 individuals were cloned, of which a mini-
mum of ten clones per PCR product were selected and sequenced (S6 Table). The results were
used to determine the degree of coincidence between the consensus sequence of the clones and
the sequence obtained by direct sequencing. A mean of 8.20 mutations per fragment cloned
(~100 pb) were rejected as these mutations were found uniquely in different clones. These mu-
tations have been considered as artefacts resulting from post-mortem damage to aDNA.
Early Neolithic: Starčevo Crişculture
The mtDNA variability observed in the samples from Early Neolithic in Romania (E_NEO)
(n = 5, Gura Baciului, Negrileşti, and Cârcea sites), showed five haplotypes (haplotype diversi-
ty = 0.99±0.0395) that were assorted into four European haplogroups (H, HV, J and T1a)
(Table 2). The haplogroup H is the most frequent in the present-day European populations
and the haplogroups J and T1 are suggested to be as markers of the Neolithic diffusion from
Near East [5].
Fig 1. Geographic location of ten Romanian sites analyzed in the present study. (The figure has been
provided by M. Rotea and T. Károly).
doi:10.1371/journal.pone.0128810.g001
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 4/20
Table 2. Haplotype (ht) and haplogroup (hg) mtDNA distribution resulting of the analysis of 62 ancient individuals from Romania.
Chronology Sample ht % hg %
Early Neolithic (E_NEO) GB2 ht1 20 J 20
GB3 ht2 20 HV 20
NE-1 ht 42 20 H 40
Ca1 ht16 20 H
Ca2 ht17 20 T1a 20
Middle/Late Neolithic and Eneolithic (M_NEO) BV1; Va4; Va8; Su7; Su12; Su16; Su9; SMR-1; SMR-3; SMR-6; SMR-8 ht16 27 H 58.5
BV2 ht18 2.4 H
Va3 ht21 2.4 H
Va6 ht23 2.4 H
Va11 ht27 2.4 H
Va12 ht28 2.4 H
Su11; SMR-5 ht33 4.8 H
Su14 ht35 2.4 H
Su15 ht36 2.4 H
SMR-4 ht38 2.4 H
SMR-7 ht39 2.4 H
SMR-9 ht40 2.4 H
SMR-10 ht41 2.4 H2
Cu1 ht12 2.4 U5 12.2
Su3 ht13 2.4 U5
Su13 ht34 2.4 U
Su1 ht30 2.4 U4
Su8 ht32 2.4 U5b
I8; I9 ht4 4.8 J 12.2
Va2 ht20 2.4 J
Va5 ht22 2.4 J
Va9 ht25 2.4 J
Cu2 ht29 2.4 K 4.8
Su4 ht31 2.4 K
Va1 ht19 2.4 T1 4.8
I6 ht3 2.4 T1a
Va7 ht24 2.4 W 2.4
Va10 ht26 2.4 HV0 2.4
SMR-2 ht37 2.4 R 2.4
Eneolithic (Eneol) DM3 ht5 50 K 100
DM4 ht6 50 K
Early Bronze Age (E_BA) P11 ht7 50 K 100
P12A ht7 50 K
Late Bronze Age (L_BA) P24 ht9 12.5 H1 37.5
P25 ht10 12.5 H
P30 ht15 12.5 H
P26 ht11 12.5 HV 25
P29 ht14 12.5 HV
P27 ht12 12.5 U5 25
P28 ht13 12.5 U5
P22; P23*ht8 12.5 W 12.5
(*only considered one sample).
doi:10.1371/journal.pone.0128810.t002
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 5/20
Middle/Late Neolithic and Eneolithic: Boian, Zau and Gumelniţa cultures
The sample of this chronological sequence from Romania is represented by 41 individuals
from five different sites. Boian culture (c. 53004500 cal BC) can be framed in the Middle Neo-
lithic period, while Gumelniţa (c. 45004000 cal BC) corresponds to the final stage of the Neo-
lithic in Romania, called Eneolithic (also known as Chalcolithic or Copper Age) [28].
Gumelniţa and Boian are two related cultures, having the same area, same type of settlements,
economy and burials, being only different in their chronology. Most archeologists believe that
these two cultures represent a continuum [28,34]. The samples from the Iclod site belong to
the Zau culture (who is contemporary with both Boian and Gumelniţa). Therefore, we decided
to analyse the samples belonging to Boian, Gumelniţa and Zau cultures together: for the sake
of simplicity we will call them M_NEO during the population genetic analysis. In addition, no
statitically significance differences were found between these sites, supporting the decision to
analyse them together. The analysis of their mtDNA variability showed 29 mitochondrial hap-
lotypes (haplotype diversity = 0.8095±0.0052), which were assorted into eight different hap-
logroups (H, HV, R, J, K, T, U, W) (Table 2). The most frequent is haplogroup H (58.5%),
which showed a high diversity including 13 different haplotypes, while the next most frequent
haplogroups were U (12.2%) and J (12.2%). Within haplogroup U five different haplotypes can
be seen, with four of them corresponding to the subhaplogroups that were frequent in the Eu-
ropean hunter-gatherers (U5 and U4). The haplogroups J and T (T1), which have been pro-
posed as genetic markers of the Neolithic demic diffusion from the Near East [5], showed a
frequency of 12.2% and 4.8% respectively. These values are similar to those found in modern
European populations, and the same was true for the rest of the haplogroups (K, W, HV, R).
Eneolithic: Decea Mureşului non-indigenous culture
The samples of Eneolithic in Romania were obtained from two individuals recovered from the
Decea Mureşului site (samples identified as Eneol) and belonging to a cultural phenomenon
known under the same name. Generally, archaeologists consider that the Decea Mureşului cul-
ture is the result of a migration of non-indigenous populations coming from the North Pontic
steppes [31]. The material culture of these intrusive communities differs fundamentally from
that of the local Eneolithic cultures (e.g. Boian, Gumelniţa, Petresti, Cucuteni, Tiazapolgar,
etc.) [28]. This is the reason why the samples of the Decea Mureşului culture were analysed
separately of other cultures from the same chronological sequence (e.g. the local Gumelniţa
culture).
The two different mitochondrial haplotypes obtained in two individuals recovered from the
Decea Mureşului cemetery correspond to haplogroup K. These haplotypes are unique, not
found in any prehistoric sample, either Romanian or European (Table 2). These mitochondrial
DNA haplotypes have only been found, albeit with a low frequency, in the present-day Middle
East populations (1%).
Early and Late Bronze Age: Copăceni and Noua cultures
The two individuals from the Copăceni group, an Early Bronze Age site (E_BA), showed two
different haplotypes, which are included in haplogroup K. These haplotypes are common in
present-day and ancient European populations. On the other hand, the mtDNA data obtained
from Noua Culture, a Late Bronze Age site (L_BA) in Romania, correspond to eight different
haplotypes (haplotype diversity = 0.8889±0.0074), assorted into four European haplogroups
(H, HV, U5 and W) (haplogroup diversity = 0.8214±0.1007). It should be highlighted that the
haplotypes ht12 and ht13 in the L_BA site, belonging to subhaplogroup U5 (one of the most
ancient in Europe) were also found in the Middle-Late Neolithic (M_NEO) groups from
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 6/20
Romania (Table 2). One of the haplotypes (ht8 corresponding to haplogroup W) was found in
two different individuals in the L_BA site (P22 and P23) (Table 2). As archaeological and an-
thropological context suggested a possible kinship relation between these two individuals, the
analysis of five autosomic STRs in the samples was performed (AMG, D13S317, D2S1338,
D18S51, D16S5399 AmpFlSTR MiniFiler PCR amplication Kit, Life Technologies); this genetic
analysis confirmed that they likely were sister and brother (initially called Romeo and Juliet
as they were thought to be a young couple of lovers [35]) (S2 Table). For this reason, only one
of these two individuals has been included in the diversity and statistical analysis.
Comparison of ancient and present-day populations from Romania
A pairwise Fst test based on the mitochondrial haplotype variability showed significant differ-
ences between ancient (present study) and modern Romanian populations [36](S3 Table). No
conclusions can be drawn for Eneol and E_BA populations due to the small sample size of
those groups (n = 2). When the analysis was performed on the mitochondrial haplogroup vari-
ability, the M_NEO and present-day Romania (ROM) populations did not show statistical dif-
ferences. Analysis of Median Joining Network within prehistoric Romanian populations
(presented in the S1 Fig), showed that the most frequent haplotype was rCRS (the central node
in the Network, ht16 in Table 2), that was shared by individuals from the Early Neolithic
(E_NEO), Middle/Late Neolithic and Eneolithic (M_NEO) and present-day (ROM) groups.
Two other shared haplotypes in this network were the 16270 (ht13, U5 in Table 2) and 16192
16270 (ht12, U5 in Table 2), polymorphisms that appeared in M_NEO and L_BA groups. The
rest of the haplotypes are specific to each archeological/cultural group.
As it can be seen in the network (S1 Fig), the higher haplotype diversity corresponded to
mtDNA lineages from Middle/Late Neolithic and Eneolithic (M_NEO), where haplogroup H
presented a high frequency and diversity values (S1 Fig). Therefore, a network including the
haplotypes of both the M_NEO and the present-day Romanian [36] populations was built in
order to analyze the mtDNA variability shared by these two populations (S2 Fig). It can be ob-
served that most of the shared polymorphisms belong to haplogroup H.
Comparison of ancient populations from Romania with other ancient
populations from Europe
Early Neolithic from Romania. The first Neolithic inhabitants of Europe are described
archeologically as belonging to the Aegean Early Neolithic cultures [27], from which the bear-
ers of both the Starčevo-Criş-Körös complex in Serbia, Romania and Hungary [28,37] and the
Linear Pottery culture in Central Europe (LBK) [21] emerged. No statistical significant differ-
ences were found between mtDNA frequency distribution of these two cultures which is in line
with the archaeological evidence of a common origin in the Sesklo cultural complex. It is note-
worthy to observe that the haplogroup N1a found in the individuals of LBK culture and which
is considered a hallmark of the Early Neolithic populations in Central Europe was absent in the
Starčevo-Crişculture groups; however, a bias due to the low number of Early Neolithic samples
from Romania cannot be excluded as a cause for this difference (S3 Fig).
Middle/Late Neolithic and Eneolithic from Romania. The population corresponding to
the Boian, Zau and Gumelniţa cultures from Romania studied here (n = 41) was compared
with populations of Central Europe represented by the Baalberge, Salzmünde and Bernburg
cultures [21], because of their chronological proximity. The S4 Fig shows that both groups
share similar frequencies for haplogroups J, R, U and W, whereas important differences were
found for haplogroups H (58.5% in Romania and 22% in Central Europe), K (4.8% and 17% re-
spectively) and T (4.8% and 14.8% respectively). Haplogroups N and X were absent in the
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 7/20
Middle/Late Neolithic and Eneolithic (M_NEO) Romanian population. This led to statistically
significant differences between Romanian and Central European Neolithic populations for
both mtDNA haplogroups and haplotypes (p = 0.00000±0.0000). Median Joining Network
analysis of the mtDNA haplotypes of M_NEO groups from Romania and Central Europe dis-
played differences in their haplotypes distributions (S5 Fig). The only shared polymorphisms
are those corresponding to the rCRS (central node of the Network) and polymorphisms 16069
(haplogroup J) and 16298 (haplogroup HV).
The mitochondrial haplotypes obtained in two individuals recovered from the Decea Mure-
şului site belonged to haplogroup K (Table 2). Therefore, we have performed a Median Joining
Network for this haplogroup (S6 Fig), which includes all haplotypes corresponding to the an-
cient populations of Romania (present study), Czech Republic (Vedrovice) [38], Near Eastern
[22], as well as present-day populations (Romania, Near Eastern and Eastern Europe). The net-
work showed that the only shared polymorphisms between Decea Mureşului samples and the
rest are those of the central node and two other polymorphisms shared with ancient and mod-
ern Near Eastern populations.
Early and Late Bronze Age from Romania. Important population shifts due to migratory
events coming especially from the East occurred in the Bronze Age on the present territory of
Romania. The Early Bronze Age II phase of Floreşti-Polus site is represented by a novel culture
(Copăceni group) characterized by the presence of tumuli and megaliths, and associated with
the Yamnaya culture from the Crimea/Volga basin [29,35]. From this stage, only two individu-
als were available, who showed the same haplogroup K. In contrast, the late phase of Floreşti-
Polus site represents a new migration event related to the Noua-Sabatinovka culture [29,35].
Therefore we compared the mtDNA haplogroup frequency of L_BA individuals from Polus
with a Bronze Age group from Ukraine [39](S7 Fig). These two Bronze Age populations
shared haplogroups H, U and W, with the largest differences referred to the frequency of hap-
logroup W. The Bronze Age Ukraine population presented the highest mtDNA haplogroup di-
versity, due most likely to its large sample size. Significant statistical differences between these
groups have not been detected.
Multivariate analysis: Ancient populations from Romania in the context
of past and present-day populations
We have analyzed the variability of mtDNA haplogroups of ancient Romania groups in the
context of other ancient and present-day populations from Europe and Middle East (S4 Table)
through two different multivariate analyses: PCA and MDS, Figs 2and 3. Eneolithic (Eneol)
and Early Bronze Age (E_BA) samples from Romania were excluded due to their small sample
size. In Figs 2and 3, the Principal Component Analysis (PCA) and Multidimensional Scaling
Analysis (MDS) are shown.
The two first components of the PCA explained 47% of the variance. PC1, representing 30%
of the total variance, was related to the haplogroups D, C, M and N (0.962, 0.952, 0.942 and
0.717 respectively). Present-day European populations lay at one end of this axis, the opposite
end being associated to the Middle East populations. Prehistoric populations are distributed
following a heterogeneous pattern between these two extremes (Fig 2). Early Neolithic
(E_NEO) populations from Romania and Central Europe clustered together, while the Middle/
Late Neolithic and Eneolithic (M_NEO) population from Romania is not clustered with the
Middle Neolithic from Central Europe, but with the modern European populations instead.
Overall, a similar conclusion can be inferred from PC2 (17% of the total variance). In this case,
the variation is explained by haplogroup H, which had the highest correlation value with this
component (0.691). The M_NEO group from Romania showed a high frequency for
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 8/20
haplogroup H (58.5%), basically similar to modern Europeans, but different from the Early
Neolithic groups from Romania.
Finally, a MDS providing a two-dimensional view of a F
ST
distances matrix was performed.
F
ST
values were calculated according to the frequency of the mitochondrial haplogroups. The
results of this analysis are shown in Fig 3, with a reliable graphic representation of the genetic
distances (RSQ of 0.99071 and Stress of 0.07553). As previously shown, hunter-gatherer popu-
lations in Scandinavia [810] and Central Europe [11,21] (HG_SCA and HG_CE) are clearly
different from all other populations in the analysis. The Early Neolithic groups from Romania
and Central Europe [1415,21] (E_NEO_Romania and E_NEO_CE) are close despite differ-
ences in haplogroup distribution (S3 Fig). In contrast, the Middle Neolithic groups from Ro-
mania and Central Europe [21] (M_NEO_Romania and M_NEO_CE) are separated. In
the case of Romania, the M_NEO group had a higher genetic distance from the Early
Neolithic (E_NEO_Romania) than with the present-day Romanian population. On the con-
trary, Early and Middle Neolithic populations in Central Europe [21] lay closer to each
other than any of them with the present population of the same area. Lastly, the Late Bronze
Age Romanian group is closer to Bronze Age from Ukraine than to the M_NEO_Romania
(Fig 3).
Fig 2. Principal Component Analysis (47% of the total variance) performed considering mtDNA haplogroup frequencies of the ancient and
present-day European and Near East populations. In green Neolithic populations, in pink Hunter-Gatherer groups (HG), in yellow ancient and present-day
Romania groups, present-day European population in blue and present-day Near East population in orange. Interpretation based on the haplogroup
frequency has been written on both PC (Absence of haplogroups D, M, C and N on one side of the first component and absence of haplogroup H on the top of
the second component). PC1 represents 30% of variance and PC2 represents 17% of variance.
doi:10.1371/journal.pone.0128810.g002
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 9/20
Discussion
In the present study we analysed mtDNA from 59 Neolithic, Eneolithic and Bronze Age indi-
viduals recovered from ten archaeological sites in Romania (Table 1), in order to evaluate the
potential genetic impact of the different ancient populations in South-East Europe spanning
from Early Neolithic to the Late Bronze Age (6300 BC to 1100 BC) on the genetic composition
of present-day European populations.
The Early Neolithic farmers in Europe
One of the most hotly debated aspects concerning the origin of Europeans is represented by the
relative contribution of Palaeolithic/Mesolithic hunter-gatherers versus the Neolithic farmers for
the genetic heritage of modern populations. Two major models for the role of Neolithic farmers
and the spread of agriculture have been proposed: a demic diffusion (DD) model and a cultural
diffusion (CD) model. In the DD model the Neolithic farmers have a much bigger genetic impact
on the make-up of modern Europeans than in de CD model. Although early analyses considered
only two models, a number of mtDNA studies in Neolithic populations have indicated a more
complex pattern for Neolithic transition. Thus, the random dispersion model proposes that Neo-
lithic farmers had a different impact on the various geographic regions (central Europe, Mediter-
ranean Europe and Cantabrian fringe), at different periods of time [12,17,2023].
Studies from Central and West Europe, especially the analysis of mitochondrial diversity of
LBK culture groups, showed no continuity between the first farmers of Europe and the modern
Fig 3. Multidimensional Scaling Analysis performed by haplogroup frequencies of the ancient and present-day European and Near East
populations. In green Neolithic populations, in pink hunter-gatherer groups and in yellow ancient and present-day Romanian groups, present-day European
population in blue and present-day Near East population in orange. Stress: 0.07553 and RSQ: 0.99071.
doi:10.1371/journal.pone.0128810.g003
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 10 / 20
Europeans, thus proposing that these Neolithic pioneers had little genetic impact on the cur-
rent European population [11,1415,21]. This hypothesis is supported by our data, which
show a close genetic proximity of Early Neolithic group from Romania (Starčevo-Crişculture)
with Early Neolithic populations such as LBK but no genetic continuity with modern Roma-
nian populations (Figs 2and 3,S3 Table). These data are in line with the idea of a common ori-
gin of the LBK and Starčevo-Crişcultures from the Aegean Neolithic cultures of Northern
Greece/Thessaly, the first Neolithic complex in Europe [24]. The differential distribution of the
mtDNA haplogroup in both Early Neolithic groups (S3 Fig)highlighting the absence of N1a
lineage in E_NEO_Romania, a Neolithic marker in Central Europemay reflect a differential
genetic impact of the Neolithic pioneers in these areas.
The genetic shift between Early and Middle Neolithic in Europe
A comprehensive study of mtDNA spanning a period from the Early Neolithic to the Bronze
Age in Central European populations has been recently completed [21]. In this study, by com-
paring different Neolithic populations of Central Europe with a Central European metapopu-
lation, the authors proposed four major demographic events. Their analysis supported a
model of continuity between Late Neolithic and modern European populations, while Early
and Middle Neolithic populations showed a limited genetic impact in this region. A similar
genetic shift has been identified by an exhaustive analysis based on haplogroup H [40], show-
ing a minimal genetic continuity between Early Neolithic and Middle/Late Neolithic groups
in Central Europe, which the authors consider a previously unrecognised major genetic tran-
sition[40].
Several scenarios have been proposed to account for this genetic shift between Early/Middle
and Late Neolithic in Central Europe, suggesting an influence of the CWC (Corded Ware cul-
ture) from the East and of the BBC (Bell Beaker culture) from the West in the Late Neolithic.
The impact of people of the CWC culture, in turn massively influenced by a possible influx of
populations from the East from the Yamnaya culture, has been proposed to be especially im-
portant [41]. While this idea is certainly possible, none of the models studied to date have
taken into consideration another possible and obvious explanation, namely a new wave of Neo-
lithic migration into Europe through the traditional routeof the Balkan Peninsula. This new
wave of Neolithic migrations are represented by Vinča and Dudeşti cultures (55005000 BC),
that trace their origin in North-West Anatolia on the basis of ceramics features [28]. The
Boian, Zau and Gumelniţa cultures from Middle-Late Neolithic (M_NEO) from Romania are
the direct continuation of this cultural complex; the M_NEO group from Romania displayed
differences in haplotype (S5 Fig) and haplogroup distributions (S4 Fig) with the Middle Neo-
lithic from Central Europe.
Interestingly, the genetic analysis of a relatively large number of samples of Boian, Zau and
Gumelniţa cultures in Romania (n = 41) (M_NEO) identified a close genetic proximity be-
tween this Neolithic group and the Eastern and Central extant European populations. This was
shown in the multivariate analysis, where M_NEO and modern populations from Romania are
very close, in contrast with Middle Neolithic and modern populations from Central Europe
(Figs 2and 3). Whereas the genetic analysis of modern populations from Central Europe
showed a limited genetic impact of the E_NEO_CE and M_NEO_CE groups in this region
[21], the mtDNA data of the M_NEO groups from Romania suggest a high genetic impact on
modern population in this region (see S2 Fig for shared polymorphisms). The above men-
tioned data allow us to suggest that the populations of this putative second wave of Neolithic
migration from Anatolia caused a much stronger impact on the genetic make-up of the Euro-
pean populations than the earlier farmers of the Starčevo-Crişand LBK cultures.
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 11 / 20
This hypothesis is supported by the larger number of archaeological sites for the Middle/
Late Neolithic and Eneolithic cultures compared with Early Neolithic cultures in South-East
Europe, which indicates higher population numbers [2829]. It is reasonable to hypothesize an
interaction of the Vinča-Dudeşti and Zau-Boian-Gumelniţa cultures with the Late Neolithic
cultures of Central Europe. This would have led to gene flow and permeation in Central Europe
cultures of mtDNA lineages from the second great Neolithic migrations of South-East Europe,
and may have had an important contribution to the genetic shift between Early and Late Neo-
lithic populations in Europe. The hypothesized contribution of Middle Neolithic migrations
from North-West Anatolia into the Balkan Peninsula and Central Europe may explain the po-
sition of the BBC (Late Neolithic in Central Europe), close to the M_NEO groups from Roma-
nia in the multivariate analysis (Figs 2and 3).
One last aspect concerns the presence of U haplogroups in four individuals from two of the
Middle/Late Neolithic sites: Curatesti and Sultana-Valea Orbului. While it could be argued
that these individuals share a genetic background with European hunter-gatherers [42] that in-
teracted with and adopted farmer lifestyles, more genetic studies to include local hunter-gath-
erer populations and nuclear DNA are needed to discern such a possibility. On the other hand,
it should be pointed out that no statistical differences of mtDNA between the Curatesti and
Sultana-Valea Orbului sites and the other Middle/Late Neolithic populations from Romania
were detected.
Two Eneolithic (Eneol) individuals from Romania have been analyzed, showing the same
mitochondrial haplotype (haplogroup K) (Table 2). These haplotypes are unique, not found in
any mtDNA database of ancient populations. The network performed with the haplotypes cor-
responding to haplogroup K (S6 Fig) showed that the two individuals from the Decea Mureşu-
lui site shared polymorphisms with the ancient and present-day populations from the Near
East. Although the two individuals from Decea Mureşului are associated to the Suvorovo cul-
ture from the North-Pontic steppes [2932], and this has been suggested to represent the first
contact between Transylvania and North-Pontic steppes, we have not found genetic evidence
in the present study to support this hypothesis.
Bronze Age and the influence of migrations from the East
The archeological data from the Bronze Age in the central Transylvanian plateau of Romania
describe at least three major cultures, two of them probably originating and being related to
cultures from the East: 1) the Early Bronze Age represented by Copăceni group in the Floreşti-
Polus site, which is related to the Yamnaya culture [29]; and 2) the Late Bronze Age complex
from Floreşti-Polus site which is related to the Noua-Sabatinovka culture from the North of
Black Sea [29]. The most representative number of samples (n = 9) corresponded to the Late
Bronze Age (L_BA_Romania). This sample showed a closer genetic similarity with the Bronze
Age population from Ukraine than to any other ancient population from Romania. Both F
ST
distance (S3 Table) and multidimensional scaling analysis (Figs 2and 3) showed significant dif-
ferences between Late Bronze Age and Middle Neolithic from Romania, although both popula-
tions shared two haplotypes corresponding to haplogroup U5 (ht12 and 13) (Table 2). These
results could reflect the influence of migrations from the East into the Bronze Age population
of Romania. On the other hand, the unusual mtDNA haplogroup distribution [(H (37.5%), U
(25%), HV (25%), W (12.5%)], described in the L_BA_Romania group and the genetic distance
to the modern Romania population (Fig 3), suggest that the contribution of L_BA_Romania to
the present-day Romanians was relatively limited. Nevertheless, studies on more individuals
are necessary to draw definitive conclusions. Also, the impact of the early Bronze Age
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 12 / 20
migrations on the modern South-East Europeans cannot be assessed in our study, due to the
low number of samples.
Finally, in this study we report genetic information on the Neolithic and Bronze Age popu-
lations of the Balkan Peninsula, a crucial piece of the puzzle integrating the major demographic
and cultural changes that took place from the Neolitic period onwards in South-East Europe.
Based on aDNA studies from sites of the Starčevo-Crişculture (Cârcea/Gura Baciului/Negri-
leşti sites), we confirm their genetic relationship with the LBK culture, both originating in the
Proto-Sesklo cultures of Northern Greece. In addition, our data support the strong genetic dif-
ferences between these first European farmers and the later Neolithic farmers. In addition, we
provide for the first time a glimpse to the genetic make-up of the farmers from a later Neolithic
migration from Anatolia Vinča and Dudeşti cultures that later evolved in the Boian, Zau and
Gumelniţa cultures in South-East Europe. The strong genetic resemblance of individuals from
these cultures with the modern populations leads us to propose the hypothesis that they had an
important contribution to the genetic heritage of Eastern and Central Europeans. In contrast,
no such influence could be demonstrated for Late Bronze Age migrations.
All in all, these data leads to the hypothesis that the Early to Middle/Late Neolithic genetic
transition in South-East Europe was strongly influenced by a second migration of farmers
from Anatolia during the Middle Neolithic. This scenario may thus lead to a model in which a
cultural diffusion process initially brought into Central Europe by small numbers of farmers of
the Starčevo-Crişand LBK cultures was later accompanied by a demic expansion of larger
numbers of immigrant farmers of the Vinča-Dudeşti and Boian-Gumelniţa cultures. Addition-
al studies are needed in order to define in detail the Neolithic processes of migration in South-
East Europe, including an assessment of the local Mesolithic populations, and a more extensive
study assessment of Neolithic and Bronze Age Balkan cultures.
Materials and Methods
Populations
A mtDNA analysis of a total of 63 individuals recovered from ten sites located in Northern and
Southern Romania was carried out; the chronology of these sites ranges from Early Neolithic to
the Late Bronze Age. The Early Neolithic (65005500 cal BC) sites of Cârcea (Dolj county),
Negrileşti (Galaţi county) and Gura Baciului (Cluj county) are associated to the Starčevo-Criş
culture (VI millennium BC). Another five sites correspond to Middle/Late Neolithic and Eneo-
lithic period (55003800 cal BC): Iclod (Cluj county), Vărăşti (Călăraşi county), Curăteşti
(Călăraşi county), Sultana-Malu Roşu(Călăraşi county) and Sultana-Valea Orbului (Călăraşi
county). The Late Eneolithic period (45003800 BC) is represented by Decea Mureşului site
(Alba county), and finally the Bronze Age period by the site of Floresti-Polus (Cluj county).
These ten sites were put into several cultural and chronological groups, in order to characterize
changes in the mtDNA variability from Early Neolithic (E_NEO) to Late Bronze Age (L_BA)
(Fig 1 and Table 1,S7 Fig).
Early Neolithic (E_NEO_ROMANIA) (65005500 cal BC)
Five individuals from the Early Neolithic Romania period come from three sites: 1) Cârcea site
is located on the banks of the Cârcea River. Most of the human bones were found in the settle-
ment's "defense ditch" and they were among ceramic fragments and animal bones [43]. 2)
Negrileşti is a grave found at 2.90 m depth. The skeleton lying on the right side with bent legs
carried on the abdomen and the chest a deposit of snails and a stone [44]. 3) Gura Baciului
burials consisted of an inhumation and incineration pit where seven skeletons were inhumated
in a bent position [4546].
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 13 / 20
Middle/Late Neolithic and Eneolithic (M_NEO_ROMANIA) (55003800
cal BC)
Forty-five samples from individual graves have been recovered from five Middle and Late Neo-
lithic sites. From geographical point of view most of these sites are placed in southeaster area of
Romania, near Danube River (Vărăşti) or on the high terrace of Mostiştea Lake (Curăteşti, Sul-
tana-Malu Roşu, Sultana-Valea Orbului). The only exception is the Iclod cemetery that is locat-
ed in Transylvania, on the banks of the Someşul Mic River. In terms of cultural framework,
Iclod cemetery belongs to Zau culture [4748]; Curăteşti and Sultana-Valea Orbului to Boian
culture [49], and Vărăşti and Sultana-Malu Roşu are settlements belonging to Boian and
Gumelniţa communities using the same cemetery[49].
Eneolithic (Eneol_ROMANIA) (45003800 cal BC)
This period is represented by Decea Mureşului site (Alba county), dated in the end of the 5th
millennium BC. Samples for mtDNA analysis were taken from two of the discovered graves.
Exceptional grave goods and the use of ocher and stone mace-head, represent the first contact
(migration) between Transylvania and North-Pontic steppes [50].
Early Bronze Age (E_BA ROMANIA) (26002100 cal BC)
Two samples were taken from the great barrow/tumulus from Floresti-Polus (Cluj county)
[5152]. This funerary complex belongs to Copaceni group, dating from the period II of the
Early Bronze Age in Transylvania. The Yamnaya culture (Pit-Grave culture) [5354], that in-
fluence this group, appears at the end of 4th millennium BC in the north steppes of the Black
Sea [55] and, later it cover a large area to the west, including Transylvania.
Late Bronze Age (L_BA ROMANIA) (15001050 cal BC)
Nine samples for mtDNA analysis come from eight graves from Floresti-Polus (the largest ne-
cropolis of Noua culture from Transylvania) [51]. The local populations contributed to cultural
genesis of this archaeological complex (Monteoru and Komarov cultures from Moldavia and
some eastern contributionmost often attributed to the Iranian people (ancestors cimirienilor,
scythians) who, in the second millennium BC dominated a Ponto-Caspian steppes) [56].
DNA isolation and genetic studies
The processing of the ancient samples in the laboratory involved the application of a series of
strict criteria for the authentication of results, detailed in [5760]. In our case, the extraction
and preparation of the PCR was undertaken in a specific lab for aDNA, which consist in a posi-
tive-pressure sterile chamber, located in a physically separated space from the laboratory where
post-PCR processes are carried out. All the work surfaces were cleaned regularly with sodium
hypochlorite and irradiated with UV light. Suitable disposable clothing was worn (lab coat,
mask, gloves and cap). Contamination controls were applied in both the extraction and
amplification processes.
Selection of samples for performing the present study was made from teeth without caries
or deep fissures that might extend into the pulp. Whenever possible, more than one tooth was
taken from each individual for duplicate analysis, with the duplicates being analysed in various
sessions by different researchers at the University of the Basque Country (UPV/EHU).
In order to eliminate surface contamination, the teeth were subjected to a process of depuri-
nation using acids, and the entire surface was irradiated with ultraviolet light [61]. The extrac-
tion process followed the protocol described by [62]: the tissue (root of the tooth or powdered
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 14 / 20
bone) was incubated with stirring for 2 hours at 56°C in a lysis buffer (5 ml) (0.5 M EDTA pH
8.08.5; 0.5% SDS; 50 mM Tris HCl pH 8.0; 0.01 mg/ml proteinase K). The DNA was recov-
ered using phenol and chloroform and then concentrated and purified (Centricon-30, Ami-
con). Each extraction session involved two contamination controls that were applied to the
entire process, except no dental or bone tissue was added.
Analysis of mtDNA variability
Sequencing of HVR-I [nucleotide positions (nps) 15,99816,400] and HVR-II (nps 16504
429) as per [63], was undertaken in six overlapping fragments, each with a length of approxi-
mately 100 bp (base pair). Besides, the fragment between primers 8F and 8R [12] was amplified
in all samples to determine position 73 of HVR-II of the mtDNA. The amplification of each
fragment was undertaken in independent PCRs. In the case of positive amplification and the
absence of contamination, the amplifications were purified by ExoSAP-IT (USB Corporation),
with subsequent sequencing in an ABI310 automatic sequencer using chemistry based on Big-
Dye 1.1 (Life Technology).
The results obtained were edited with BioEdit software (http://www.mbio.ncsu.edu/
BioEdit/bioedit.html) and the sequences were aligned manually. The sequences obtained in the
present study are deposited in Genbank under accession numbers KR149064-KR149120.
In order to classify the mitochondrial variability of the individuals analyzed in this study, we
proceeded to amplify 11 markers, which are required for defining the 10 Caucasian hap-
logroups described [64]. The protocol and primers are described in [65]. The digestion patterns
were verified using a fragment analyzer (Bioanalyzer, Agilent Technologies).
Authentication methods
In addition to the precautions taken to avoid contamination, other authentication criteria such
as duplication, quantification, cloning and sequencing were applied.
Duplication: A duplicate analysis was performed for 10.3% of individuals at different times
and by different researchers at the University of the Basque Country (UPV/EHU).
Quantification of target DNA: Amplifiable DNA was quantified by means of the quantita-
tive PCR (qPCR) of a fragment of 113 bp length of HVR-I, using Taqman probe [66].
Cloning: In order to detect possible heterogeneities in the PCR products that may corre-
spond to either post-mortem damage and/or mixed contamination, a fragment of HVR-I was
cloned by means of the TOPO TA Cloning Kit (Invitrogen). Linkage to the vector
pCR2.1-TOPO and chemical transformation of the cells TOP10F(One Shot E. coli) were per-
formed following the suppliers instructions [12](S6 Table).
We have determined the HVR-I and HVR-II sequence of the mtDNA of the researchers
and archaeologists who handled the samples in order to discard possible contamination (S5
Table).
Confirmation of the haplogroups obtained by sequencing and cloning of the HVR I of the
mtDNA was verified by identifying the SNPs of the coding region by PCR-RFLPs.
In order to identify any possible contamination that might occur in the various stages of the
genetic analysis, at least two blanks were included in each extraction round with a control of
the PCR in each amplification reaction. If any contamination was detected, the results obtained
were discarded.
Statistical Analysis
Genetic diversity [67] and genetic distances (Fst analysis) were calculated using the statistical
package Arlequin 3.11 [68].
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 15 / 20
Principal Component Analyses (PCAs) was conducted using as variables the frequencies of
the mitochondrial haplogroups obtained in this study together with the data from present-day
and prehistoric populations taken in the literature (S4 Table) (SPSS 17 Software). In addition, a
distance matrix was calculated between the populations studied and those existing in the litera-
ture by means of the Arlequin 3.11 program [68]. This distance matrix has been depicted in
two dimensions by means of a Multidimensional Scaling (MDS) analysis (SPSS 17 Software).
Furthermore, a Median-Joining Network (MJN) has been constructed using the sequences
of ancient and present-day groups from Romania and some ancient groups from Europe that
have so far been published, using the Network 4.6.0.0 program (http://www.fluxus-
engineering.com). Given the high mutation rate of HVR-I from mtDNA, we applied the substi-
tution rates obtained by Meyer et al. [6970] to establish varying mutational weights ranging
from 0 to10, for this reason some mutation remove in the networks and the reticulations
are reduced.
This manuscript involves field studies of anthropological specimens. All necessary permits
were obtained for the described study, which complied with all relevant regulations. The Na-
tional History Museum of Transylvania and Francisc I. Rainer" Institute of Anthropology, Ro-
manian Academy, gave us the permission to use these samples. We have not conducted field
work on site.
Supporting Information
S1 Fig. Median Joining Network of haplotypes distribution. Data encompass mtDNA
HVR-I (position 16024 to 16399). Haplotype distribution of the five Rumanian prehistoric
groups (present study): Early Neolithic group (green), Middle/Late Neolithic and Eneolithic
group (pink), Eneolithic group (blue), Early Bronze Age (black), Late Bronze Age (yelow).
(BMP)
S2 Fig. Median Joining Network of haplotypes distribution. Data encompass mtDNA
HVR-I (position 16024 to 16399). Haplotype distribution of the Middle/Late Neolithic and
Eneolithic group from Romania (present study, S2 Table) and present-day Romania popula-
tion (28). Middle-Late Neolithic group (pink) and present-day Romania (yellow).
(BMP)
S3 Fig. Comparison of the mtDNA haplogroup frequency between LBK (Linearbandkera-
mik, Early Neolithic culture from Central Europe) [21] and Carcea/Gura/precris (Early
Neolihic culture in Romania) (present study) populations.
(TIF)
S4 Fig. Comparison of the mtDNA haplogroup frequency between groups of Middle-Late
Neolithic from Central Europe [21] and that of Middle/Late Neolithic and Eneolithic
group from Romania (present study).
(BMP)
S5 Fig. Median Joining Network of haplotypes distribution of the Middle-Late Neolithic
groups from Romania (yellow) (present study) and those from Central Europe (blue) [21].
Data encompass mtDNA HVR-I (nps 1599916399).
(TIF)
S6 Fig. Median Joining Network of haplotypes distribution of haplogroup K. Data encom-
passes mtDNA HVR-I (position 16024 to 16399). Haplotype distribution of the three Roma-
nian prehistoric groups (present study): Middle/Late Neolithic and Eneolithic group (pink),
Eneolithic group (light blue), Early Bronze Age (dark blue). Farmers from Near Eastern (lilac)
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 16 / 20
[22] and from Czech Republic (orange) [39]. Present-day populations from: Romania (black),
East of Europe (yellow), Near Eastern (green) (S4 Table).
(BMP)
S7 Fig. Comparison of the mtDNA haplogroup frequency between: Late Bronze Age group
from Romania (present study) and Bronze Age group from Ukraine [37].
(TIF)
S8 Fig. Radiocarbon dating of human bone samples analysed in the present study.
(TIF)
S1 Table. A general chronology of sites analysed in the present study.
(DOCX)
S2 Table. mtDNA results from 63 ancient individuals from Romania, haplotype of HVR-I
and HVR-II, SNPs of the coding region, number of molecules per microlitre and haplotype
and haplogroup assignation. HVR-I: Hypervariable Region I of mtDNA (the sequence range
from position 16024 to 16399) and HVR-I: Hypervariable Region II of mtDNA (the sequence
range from position 0 to 340). rCRS: revised Cambridge Reference Sequence. The figures corre-
spond to the position in region I and II of HVR of mtDNA that changes with respect to the
rCRS.
(DOCX)
S3 Table. The F
ST
analysis. a) p-values with standard deviation (p±de) based on the haplo-
types frequencies (below the diagonal) and p-values with standard deviation (p±de) based on
haplogroup frequencies (upper the diagonal) (P<0.0027, in grey). b) F
ST
values based on the
haplotypes frequencies (below the diagonal) and F
ST
values based on haplogroup frequencies
(upper the diagonal) Ancient samples from Romania: Early Neolithic (E_NEO), Middle Neo-
lithic (M_NEO), Late Neolithic (L_NEO), Early Bronze Age (E_BA), Late Bronze Age (L_BA);
Present-day Romanian population (ROM) (Hervella et al., 2014).
(DOCX)
S4 Table. Prehistoric and present-day populations compiled from literature constituting
the database of HVR-I sequences of mtDNA for the present study.
(DOCX)
S5 Table. Mitochondrial haplotypes (HVR-I and HVR-II) of researchers and archaeolo-
gists.
(DOCX)
S6 Table. Mitochondrial DNA sequences of the clones from the samples analyzed in the
present study.
(XLSX)
Acknowledgments
We should like to thank PhD Zoia Maxim and Tiberiu Tecar for the very generous help in se-
lection of samples from the sites of northern of Romania. Finally, we would like to thank two
anonymous reviewers for giving helpful comments and thoughts on the manuscript
Author Contributions
Conceived and designed the experiments: MR MGN MH CR. Performed the experiments: MR
MGN MH CR. Analyzed the data: MR MGN MH CR. Contributed reagents/materials/analysis
Neolithic Events in South-East Europe
PLOS ONE | DOI:10.1371/journal.pone.0128810 June 8, 2015 17 / 20
tools: MR MGN MH CR SA NI MI ADS CL MC FR. Wrote the paper: MR MGN MH CR ADS
MC SA NI.
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Supplementary resources (14)

... 5000-4500 BCE), the emergence of tell settlements, the appearance of extra-muros cemeteries, the increase in density of human occupations in the landscape, the development of inter-regional trade networks, exotic items, innovations, adoption of new raw materials (i.e., copper, gold, graphite), along with different pottery styles, were recorded in the Lower Danube region (Anthony, 2010;Chapman, 2020). All these significant changes observed in the human communities from the studied area could have been due to a new wave of farmers arriving from Southwestern Anatolia, demonstrated recently by aDNA investigations (Hervella et al., 2015;Mathieson et al., 2018). The optimal climatic conditions in the region of Lower Danube region during the Early Chalcolithic had a crucial role in the thriving of these communities (Todorova and Vasjov, 2013). ...
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