AMERICAN JOURNAL OF HUMAN BIOLOGY 00:0000 (2016)

Original Research Article

Genetic Heritage of Croatians in the Southeastern European Gene PoolY

Chromosome Analysis of the Croatian Continental and Island Population
1
1


,1 DUBRAVKA HAVAS
 AUGUSTIN,

,2 MATE MUSTAC
,3
JELENA SARAC,
* TENA SARIC
NATALIJA NOVOKMET,1 NENAD VEKARIC
,5 BRANIMIR NEVAJDA,6 ANTON GLASNOVIC
,6 SASA
 MISSONI,1,7

BLAZENKA
GRAHOVAC,4 MILJENKO KAPOVIC
SIIRI ROOTSI,8 AND PAVAO RUDAN1,9
1
Institute for Anthropological Research, 10000 Zagreb, Croatia
2
Institute for Historical Sciences, Croatian Academy of Sciences and Arts, 20000 Dubrovnik, Croatia
3
Occupational Health Clinic, 23000 Zadar, Croatia
4
Department of Pathology and Pathological Anatomy, School of Medicine, University of Rijeka, 51000 Rijeka, Croatia
5
Department of Biology and Medical Genetics, School of Medicine, University of Rijeka, 51000 Rijeka, Croatia
6
Dubrava University Hospital, 10000 Zagreb, Croatia
7
Josip Juraj Strossmayer University of Osijek, School of Medicine, Osijek, Croatia
8
Estonian Biocentre and Institute for Molecular and Cell Biology, Department of Evolutionary Biology, , University of Tartu,
51010 Tartu, Estonia
9
Anthropological Center of the Croatian Academy of Sciences and Arts, 10000 Zagreb, Croatia

Objectives: The research objective of this study is to enlarge and deepen the Y chromosome research on the Croatian population and enable additional insights into the population diversity and historic events that shaped the current genetic landscape of Croatia and Southeastern Europe (SEE).
Materials and Methods: A high-resolution phylogenetic and phylogeographic analysis of 66 biallelic (SNPs) and 17
microsatellite (STRs) markers of the Y chromosome was performed using 720 Croatian samples. The obtained results
were placed in a wider European context by comparison with 4450 samples from a number of other European
populations.
Results: A high diversity of haplogroups was observed in the overall Croatian sample, and all typical European Y
chromosome haplogroups with corresponding clinal patterns were observed. Three distinct genetic signals were identifiable in the Croatian paternal gene pool - I2a1b-M423, R1a1a1b1a*-M558, and E1b1b1a1b1a-V13 haplogroups.
Discussion: The analyses of the dominant and autochthonous I2a1b-M423 lineage (>30%) suggest that SEE had a
significant role in the Upper Paleolithic, the R1a1a1b1a*-M558 lineage (19%) represents a signal from present day
Slavic populations of Central Europe in the Croatian population, and the phylogeography of the E1b1b1a1b1a-V13
clade (around 9%) implies cultural diffusion of agriculture into Europe via the Balkan Peninsula. Am. J. Hum. Biol.
C 2016 Wiley Periodicals, Inc.
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00:000000, 2016.

Southeastern Europe (SEE), has been inhabited since

the Middle Paleolithic according to prehistoric archaeological findings of Proto-Aurignacian culture in the Bulgarian cave Bacho Kiro 3743 kya (Kozlowski et al., 1982;
Kozlowski and Otte, 2000; Teyssandier et al., 2006; Hoffecker, 2009). A considerable number of mtDNA and Y
chromosome population-genetic studies of the SEE region
reflect the turbulent and complex demographic history of
this region, influenced by gene flow from various parts of
Eurasia and a long history of intermixing. It has been suggested that SEE has played a key role in the Upper Paleolithic recolonization of the wider European area, as well
as in the Neolithic spread of agriculture from the Near
East, serving as a gateway between the Middle East and
the rest of Europe (Battaglia et al., 2009; Forenbaher and
Miracle, 2005; Kovacevic et al., 2014; Marjanovic et al.,
2005; Mellars, 2004; Primorac et al., 2011; Semino et al.,
2000, 2004).
Croatia, as a SEE country, has been subject to extensive
multidisciplinary anthropological researches for over four
decades (Rudan et al., 2004 and the references therein)
and is situated in a key region between the rest of Europe
and the Near East (Kovacevic et al., 2014). The research
objective of this particular study is to better characterize
the Y chromosome diversity of the Croatian population
and enable additional insights into the genetic specifics
and historic events that shaped the current genetic landscape of SEE and Croatia. The specific aims of this study
C 2016 Wiley Periodicals, Inc.
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are to: (i) assess the genetic structure and variability of

Croatian paternal genetic heritage; (ii) depict the phylogenetic and phylogeographic relationships inside the three
dominant Y chromosome haplogroups (hgs) in Croatia,
and (iii) place the Croatian paternal genetic heritage into
a wider European context.
MATERIALS AND METHODS
Sample
The sample consists of 720 randomly chosen male individuals; 336 coming from the continental part of Croatia
Contract grant sponsor: the Ministry of Science, Education and Sports
of the Republic of Croatia; Contract grant number: 196-1962766-2751;
Contract grant sponsor: the European Union European Regional Development Fund through the Centre of Excellence in Genomics, Estonian Basic
Research; Contract grant number: SF 0270177s08; Contract grant sponsor: Estonian Science Foundation; Contract grant number: 7445; Contract
grant sponsor: Institutional Research Funding from the Estonian
Research Council; Contract grant number: IUT24-1.

*Correspondence to: Jelena Sarac;
Institute for Anthropological
Research, Gajeva Street 32, 10000 Zagreb, Croatia. E-mail: jelena.sarac@
inantro.hr
Additional Supporting Information may be found in the online version
of this article.
Conflict of interest: None to declare.
Received 24 August 2015; Revision received 22 March 2016; Accepted 10
May 2016
DOI: 10.1002/ajhb.22876
Published online 00 Month 2016 in Wiley Online Library
(wileyonlinelibrary.com).


J. SARAC
ET AL.

Fig. 1. Map of Croatia with exact location of all the sampled subpopulations within the sample.

TABLE 1. Genetic diversity, mean number of pairwise differences and average gene diversity over loci
Haplogroup
I2a1b-M423
R1a1a1b1aa-M558
E1b1b1a1b1a-V13
TOTAL (STRs)
TOTAL (SNPs)a
a

No. of haplotypes

Haplotype diversity 6 SD

MNPD

Average GD over loci

57
48
21
126
720

56
44
17
114

0,999 6 0,003
0,995 6 0,005
0,981 6 2,580
0,998 6 0,001
0,993

5,514 6 2,690
5,895 6 2,864
5,109 6 1,627
10,833 6 4,961

0,324 6 0,175
0,310 6 0,167
0,268 6 0,151
0,637 6 0,323

Nei genetic diversity measure

and 384 from eight Croatian islands (Fig. 1 and Supporting Information Table 1). Samples were taken from the
DNA bank of the Institute for Anthropological Research,
Zagreb. The blood for DNA analyses was collected during
fieldwork carried out by the Institute for Anthropological
Research over a 12-year span (19972009), following the
approval of the ethical committee of the Institute for
Anthropological Research and School of Medicine, University of Zagreb, Croatia. Participants gave their
informed consent to participate in the research. Siblings
and male relatives were avoided in the selection of the
samples for DNA analysis based on obtained genealogical data, as well as examinees without at least two generations of ancestors living in the sampled subpopulation.
All of the subsequent laboratory and molecular analyses
were performed at the Institute for Anthropological
Research in Zagreb and at the Estonian Biocentre Tartu,
Estonia.
American Journal of Human Biology

Genotyping
The samples were surveyed for a total of 66 different
biallelic markers using either restriction fragment length
polymorphism (RFLP) analysis, in/del assays or direct
Sanger sequencing (Fig. 2). The nomenclature used in
this study is adopted from the International Society of
Genetic Genealogy (2015). Y-DNA Haplogroup Tree 2015,
Version: 10.102, Date: 9 December 2015, http://www.
isogg.org/tree/[Date of access: December 9th, 2015].
The order of marker genotyping was based on the known
haplogroup hierarchy (www.isogg.org/tree; Karafet et al.,
2008) and previously observed haplogroup composition and
frequencies in this part of Europe. Each sample was tested
for SNPs after the initial amplification using polymerase
chain reaction and primer sets designed for each marker.
List of sequences and primers for each marker are listed in
Supporting Information Tables 2 and 3.

CROATIAN PATERNAL GENETIC HERITAGE IN SEE

Fig. 2. Phylogenetic tree with all the tested markers and haplogroup frequencies.

A subset of 126 samples designated as haplogroup

I2a1b-M423 (N 5 57), R1a1a1b1a*-M558 (N 5 48) or
E1b1b1a1b-V13 (N 5 21) was typed for 17 short tandem
repeats (Y-STRs) using the Applied Biosystems
AmpFlSTR Yfiler Kit and according to manufacturer recommendations (Supporting Information Table 4). The
named three haplogroups were chosen for further STR
analysis, because they harbored > 60% of the total paternal genetic varation in Croatia. For the multicopy STR
DYS389I,II the DYS389b value was DYS389I subtracted
from DYS389II (Cooper et al., 1996). The fragments were
run on a ABI PRISM 3130xl Genetic Analyzer (Applied
Biosystems) and the results were analyzed using the GeneMapper 4.0 program (Applied Biosystems).
Phylogenetic analyses
Y-STR haplotypes were used to construct phylogenetic
networks for haplogroups I2a1b-M423, R1a1a1b1a*-M558
and E1b1b1a1b-V13, using the program Network 4.6.0.0
and Network Publisher program (Fluxus-Engineering,
Suffolk, England, UK) and applying the median-joining
algorithm (Bandelt et al., 1999). STR loci were weighted
according to Helgason et al. (2000) and the sizes of the
nodes are proportional to the number of individuals. The
networks were performed utilizing eight STR loci to enable comparisons with a significant number of published
data collections. After reviewing the currently published
literature, we found that the Battaglia et al. (2009) paper
was still the most relevant source of comparable Southeastern European datasets (eight STR loci) for I2a1 and
E-V13 clade. Although there are studies with a higher
number of STRs available, there are often problems with
the compatibility of STR sets used across studies or with a

lack of haplogroup defining SNPs, which makes comparisons difficult.

Statistical analyses
A pairwise difference method based on molecular differences between the haplotypes was used for calculating
pairwise Fst genetic distances between populations. They
were obtained from Y-STR haplotypes using Arlequin 3.5
software (Excoffier and Lischer, 2010). Principal Component Analysis (PCA) and Multidimensional Scaling
(MDS) were used for visual representation of genetic distances and differences in haplogroup diversity and frequency between the populations. PCA plots were
constructed using the free software POPSTR (http//harpending.huamnevo.utah.edu/popstr/) based on biallelic
markers and MDS plots using the program XLSTAT 2013.
5.06 software (http://www.xlstat.com/en/). Spatial frequency maps were obtained by using the hg frequencies
reported in Figure 2. The frequency data were converted
into isofrequency maps using the Surfer software (version
8, Golden Software, Golden, CO), following the kriging
algorithm.
RESULTS
NRY haplogroup composition and frequencies
A high level of haplogroup diversity was observed in the
sample (Fig. 2) and the Croatian genetic profile corresponds to its geographic position and to the previously
reported patterns of Y chromosome diversity in SEE (Battaglia et al., 2009; Kovacevic et al., 2014; Marjanovic
et al., 2005; Pericic et al., 2005; Regueiro et al., 2012).
Some 36 different subhaplogroups were present in the
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J. SARAC
ET AL.

sample, and are shown in detail in Figure 2 and Supporting Information Tables 5 and 6. The most common hgs
were I, R, E, J, and G, which encompass 96% of the total
sample and only three subclades I2a1b-M423 (33.2%),
R1a1a1b1a*-M558 (19.3%), and E1b1b1a2a-V13 (8.75%)
harbored > 60% of the total paternal genetic variation in
Croatia.
When compared to a larger population dataset comprising 4459 samples from other European populations
(Balanovsky et al., 2008; Battaglia et al., 2009; Karanachak et al., 2013; King et al., 2011; Kushniarevich
et al., 2013; Regueiro et al., 2012), it was observed in
the PCA plot (Fig. 3) that the formed clusters clearly

reflected the European geographic architecture, and

that most of Croatian samples fit into the wider SEE
region. The first principal component encompassed 20%
of the observed variance and sharply distinguished
Northern and Central European populations from the
others, based on the frequency of R1a individuals,
while the second one accounted for about 23% of the
total variance and was a clear dividing point between
Southeastern and Southern Europe and other European
populations, based on the increased frequency of I2a2M423.
It was observed from spatial gradient maps (Figs. 46
and Supporting Information Table 7) that haplogroups
I2a1b-M423 and E1b1b1a1b-V13 both exhibited high densities within SEE populations. For example, I2a1b-M423
had a frequency peak in present-day Bosnia and Herzegovina and in Southern Croatia, and E1b1b1a1b-V13
peaked in southern parts of the Balkan Penninsula,
namely in Greece and Albania, as suggested in previous
studies (Battaglia et al., 2009; Marjanovic et al., 2005;
Rootsi et al., 2004; Underhill et al., 2007). R1a1a1b1a*M558 reaches its frequency peak in the area of todays
Belarus and Ukraine and decreases to the south, but
more prominently to the west of Europe, as noted in
Underhill et al. (2015) study. Nevertheless, its percentage
in northern Croatia and Central European populations
was still considerable.
NRY haplotype diversity

Visual representation (PCA plot) of major (sub)haplogroup

frequencies in European populations and Croatian samples. Green
diamond: North and Eastern Europe, Orange diamond: Central
Europe, Purple diamond: Southern Europe, Red diamond: Southeastern Europe, Yellow diamond: Western Europe. Abbreviations: CRO,
Croatia; BOS, Bosnia and Herzegovinia; SRB, Serbia; HUN, Hungaria; UKR, Ukraine; POL, Poland; SLO, Slovenia; CZE, Czech Republic; BLRUS, Belarus; RUS, Russia; BULG, Bulgaria; MAC-G,
Macedonian Greeks; ALB, Albania; MAC-A, Macedonian Albanians;
GRE, Greece; ITAL, Italy; FRA, France.
Fig. 3.

Phylogenetic relationships within the three dominant

Croatian NRY were evaluated by analyzing eight STR loci
on 126 samples and compared to over 500 samples from
different European populations (Supporting Information
Figs. 13, Supporting Information Table 8). The diversity
of I2a1b-M423 haplotypes was high and mirrored by the
widespread distribution of haplotype clusters, most of
which originated in south/southeastern European

Fig. 4. Spatial gradient map illustrating clines in frequency for I2a1b-M423. Comparative population data has been taken from Battaglia
et al. (2009), Regueiro et al. (2012), Underhill et al. (2007), and Varzari et al. (2013).

American Journal of Human Biology

CROATIAN PATERNAL GENETIC HERITAGE IN SEE

Fig. 5. Spatial gradient map illustrating clines in frequency for R1a1a1b1a*-M558. Comparative population data has been taken from
Underhill et al. (2015).

Fig. 6. Spatial gradient map illustrating clines in frequency for E1b1b1a1b-V13. Comparative population data has been taken from Battaglia et al. (2009), Cruciani et al. (2007), and Regueiro et al. (2012).

populations. Two dominant star-like clusters were evident

with a major portion of south/southeastern European
samples placed within them, indicating a rapid expansion
from a common founder. Unlike for hg I2a2, the prevailing
diversity of R1a1a1b1a*-M558 was among the Northern,
Eastern and Central European populations. One major
cluster was evident, with only a minor presence of South/
Southeastern Europeans. Its star-like form, relative

shortness of the branches and observed frequency pattern

in Europe were consistent with a model of recent hg R1a
diversification followed by range expansions and subsequent population growth across Europe. The dominant
portion of the E1b1b1a2a-V13 haplotype diversity
occurred among southern and Southeastern European
countries, in line with the previous findings that indicated
Southeastern/Southern Europe as the geographic source
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of this particular E-subclade (Battaglia et al., 2009;

Semino et al., 2004). One dominant cluster was evident,
with most of its representatives belonging to Balkan populations and Croatia.
It is important to stress that the proposed old age of the
I2a1b-M423 and R1a1a1b1a*-M558 lineages obtained in
previous studies (Battaglia et al., 2009; Pericic et al.,
2005; Rootsi et al., 2004; Underhill et al., 2007, 2015) has
been based on STR analysis (8 and 10 loci, respectively)
and recent studies clearly indicate that the STR-based
age calculations tend to yield overestimated dates (Batini
et al., 2015; Hallast et al., 2015; Karmin et al., 2015). For
example, the expansion time estimates of R1a lineages in
Europe have been dated much later, about 5 kya, based on
70 complete NRY sequences collected worldwide (Karmin
et al., 2015).
Some 56 different haplotypes were detected in the
I2a1b-M423 clade, congruent with its high haplotype
diversity (0.999 6 0.003) and a scattered distribution in
the phylogenetic network. The distribution of 44 different
haplotypes within the R1a1a1b1a*-M558 lineage, also
illustrated in the network projection, similarly correlated
with the high diversity observed within this haplogroup
(0.995 6 0.005). The most haplotype sharing and slightly
lower diversity values were evident within E1b1b1a1bV13 lineage (0.981 6 2.580), suggesting a somewhat
reduced degree of gene flow within the Balkan populations for this clade (Table 1).
Genetic distances obtained by the pairwise difference
method using eight STR loci (Supporting Information
Table 9) were visualized in form of multidimensional scaling plots for three major hgs (DYS19, DYS389I,
DYS389II, DYS390, DYS391, DYS392, DYS393, and
DYS439) (Supporting Information Figs. 46). As expected,
the obtained genetic distances between Croatia and other
European populations for hg I2a1b-M423 place Croatia
close to its geographic neighboring countries. The
observed peak frequency of this lineage in Bosnia (Marjanovic et al., 2005) could be responsible for its slightly outlying position in the plot. Slovenia is the only SEE
country in the plot that doesnt show affinity to this
region, which coincides with previous findings based on

mtDNA analyses (Sarac
et al., 2014). As for the
R1a1a1b1a*-M558 lineage, most SEE populations (Croatia, Bosnia, Serbia, Montenegro) are positioned relatively close to each other, indicating their genetic, as well
as geographic closeness. The only surprising result is the
E1b1b1a2-V13 plot, since the Croatian population does
not cluster tightly with the rest of the SEE ones. This
result could possibly be explained by a maritime influx
into Croatia through the Mediterranean Sea, which
brought some untypical E1b1b1a1b-V13 haplotypes into
the genetic structure of Croatia and caused its alienated
position.
DISCUSSION
Our results indicate that a high level of haplogroup and
haplotype diversity exists in the Croatian sample set, suggesting a dynamic gene flow in the SEE region throughout
history. Y chromosome haplogroup frequency distributions in the Croatian population correspond to the previously reported results for the Croatian population (Barac
et al., 2003; Pericic et al., 2005) and they fit within a
Southeast European paternal genetic landscape (BattaAmerican Journal of Human Biology

glia et al., 2009; Kovacevic et al., 2014; Marjanovic et al.,

2005; Mirabal et al., 2010; Regueiro et al., 2012). A previous high-resolution analysis of the maternal heritage of

SEE (Sarac
et al., 2014) based on mitochondrial DNA
SNPs is also consistent with the results of the paternal
lineage analysis. Three NRY haplogroups that account for
the majority of Croatias paternal gene pool have been the
focus of this study, namely the I2a1b-M423, R1a1a1b1a*M558, and E1b1b1a1b1a-V13 haplogroup, which we view
as representing a Balkan, a Central-European and a Neolithic input into the Southeast European genetic
landscape.
The Balkan I2a1b-M423 haplogroup
One of the foci in this study was a I-M438 lineage with
a downstream mutation M423a marker most common
in Slovenia, Bosnia and Herzegovina, Croatia and Russia.
It is characterized as a European-specific, SEE autochthonous paternal lineage and a genetic signal of the recolonization process of Europe after LGM in the Early
Holocene (Rootsi et al., 2004). More recent studies indicate that it most likely spread following the post-Younger
Dryas recovery (Karmin et al., 2015, Fig. Suppl. 25). It is
now known that the M423 SNP accounts for the majority
of the Eastern/Southeastern European hg I chromosomes
(>75%) and virtually all I-P37.2 Y individuals in SEE
(Battaglia et al., 2009). The results of our study show a
considerable frequency of I2a1b-M423 (>30%) in Croatia
and its phylogenetic network also shows high haplotype
diversity, mirrored by the widespread distribution of haplotype clusters. The elevated frequency and high diversity
of I2a1b-M423 lineages among different SEE populations
shows a genetic signature of their common paternal history over a long period of time. The PCA plot clearly
shows a SEE cluster comprised of Bosnian and Herzegovinian, Serbian and Croatian samples based predominantly
on the I2a1b-M423 component. As shown in the I2a1bM423 spatial gradient map, a clear cline of this clade is
evident inside Europe, spreading from the area of Southern Croatia and Bosnia and Herzegovina mostly northward and eastward. Although the initial STR-based time
estimate for this clade gave support for a Upper Paleolithic origin (Pericic et al., 2005), new studies based on
whole Y chromosome sequencing suggest a somewhat
younger age of this clade, between 5 and 7.5 kya (Batini
et al., 2015; Karmin et al., 2015). In addition, the high
haplotype diversity of this lineage in Croatia reveals its
significant expansion only after the adoption of agriculture by Mesolithic hunter-gatherers in SEE (Battaglia
et al., 2009).
More evidence of its autochthonous European origin
also came recently from European aDNA studies. Lazaridis et al. (2014) sequenced nine ancient genomes (with
age estimates of cca 8 kya) and analyzed the complete
NRY sequence of five male individuals (one from Luxemburg and four from Sweden). Their results showed all five
of them belonged to the I haplogroup. The authors warned
that, at present, the limited number of ancient samples
for which Y chromosome data are available makes it difficult to assess how statistically surprising it is that this
NRY haplogroup occurs in all five of the ancient Mesolithic males but in only a quarter of present-day males
from that geographic area. They appear to argue that the
haplogroup I today is found in a wider European area at a

CROATIAN PATERNAL GENETIC HERITAGE IN SEE

much lower frequency than it occurred around 8 kya. This

haplogroup has also been found and described in a Scandinavian Neolithic hunter-gatherer from Sweden (Skoglund
et al., 2014), as well as in Neolithic remains from southern
France and northern Spain (Lacan et al., 2011a). The fact
that a significant portion of investigated Mesolithic males
belonged to haplogroup I suggests that this paternal lineage might represent a major pre-Neolithic European
clade, and the results obtained in this study support this
hypothesis.

2014). Such recent episodes could be the proposed IndoEuropean migrations from the Pontic steppe associated
with the Yamnaya and Corded Ware cultures in Late Neolithic/Bronze Age and massive Slavic migrations in a
more recent history (3rd 6th century), together with
smaller ones that most probably occurred repeatedly since
Paleolithic times and are associated with specific historical events (Haak et al., 2015; Pericic et al., 2005; Regueiro
et al., 2012).

In situ European diversification of the

R1a1a1b1a*-M558 clade

Several theories about the origin of E1b1b1a1b1a-V13

and its spread have been proposed, and all are, in a certain way, linked to the Neolithic farmers. Early studies
suggested that the mutation was brought to the Balkans
together with early farming technologies (Semino et al.,
2004). Battaglia et al. (2009) proposed an earlier arrival of
this lineage in Europe (during the late Mesolithic period),
followed by its Neolithic dispersal into Europe with the
spread of farming. Most recently, King et al. (2011) suggested that E1b1b1a1b1a-V13 may trace the demographic
and socio-cultural impact of Greek colonization.
Researchers still debate the fate and interplay between
pioneering farmers from the Near East and local Mesolithic societies and search for the clearest signal of their
interaction. However, the phylogeography and temporal
evidence supports the idea that E1b1b1a1b1a-V13 arose
in Southern Europe, with peak frequencies among the
Albanians and Greeks (Semino et al., 2004) and with
declining patterns towards the north. The lack of any
plausible Middle Eastern source of E1b1b1a1b1a-V13 during the Early Neolithic or Bronze Age, together with the
low STR variation observed in the Middle East, additionally bolsters this view (Battaglia et al., 2009; King et al.,
2008).
We support the scenario proposed by Battaglia et al.
(2009) and additionally strengthened by two recent studies that the Mediterranean route has had a pronounced
role in the spread of farming (Lacan et al., 2011b;
Paschoua et al., 2014) and that local South European populations adopted the new technology through their contact with Near Eastern migrants and spread it further
into the European area. The high diversity of the
E1b1b1a1b1a-V13 obtained in this study supports the cultural diffusion theory that the clade originated in the
neighboring area of Southern Europe and spread only
after populations adopted farming from Near Eastern pioneers using leapfrog colonization in the Aegean/Adriatic
(Richards, 2003; Forenbaher and Miracle, 2005). Lacan
et al. (2011b) and Paschoua et al. (2014) also concluded
that the Mediterranean route had even greater influence
on the peopling of Southern Europe during the Neolithic
transition than the Central European one, based on autosomal, mitochondrial and Y chromosome DNA analysis,
giving even more importance to this lineage.

The R1a-M420 clade arose in Western Eurasia and

today it is most frequently observed in Eastern Europe,
Western and Central Asia and southern Siberia. The
recent, highly detailed phylogeographic analysis of 2,923
R1a-M420 Y chromosomes revealed two geographically
divergent new mutations the European Z282 and the
Asian Z93 SNP, which defined major opposing clines. The
Middle East (most probably present-day Iran) has been
proposed as the plausible geographic center from which
Z282 and Z93 chromosomes spread towards Europe and
Asia, respectively (Underhill et al., 2015). This study proposed the M558 marker, which defines a sister clade to
the already defined M458 lineage, with high frequencies
in central/eastern Europe. Its prevalence drops drastically
towards Western Europe, and it occurs at lower but
informative frequencies in Balkan populations with
known Slavonic heritage. This finding raised the possibility that the wide and rapid spread of the M458 and M558
lineages was associated with an autochthonous Bronze
Age Proto-Slavic culture that arose in Central Europe
near the Vistula river, the Corded Ware culture.
The age of R1a1a1b1a*-M558 obtained by Underhill
et al. (2015) placed this lineage in the Holocene period
(about 9 kya), based on STR data analysis. However, a
more recent study conducted by Haak et al. (2015) suggests that the previous time estimates were significantly
overestimated and that the Yamnaya culture, a cultural
complex that spread across the Pontic Caspian-Ural
steppes (beginning around 3,3 kya) brought the R1a clade
into Europe in Late Neolithic. Two additional aDNA studies analyzing the Y chromosome showed that R1a individuals are absent or rare in sites dating before 5 kya (Batini
et al., 2015; Haak et al., 2008), an observation consistent
with the R1a age estimate published by Karmin et al.
(2015). In this study, the R1a1a1b*-M458 lineage is not as
frequent as its sister clade R1a1a1b1a*-M558, which is
five times more common. This finding suggests that certain Central-European migrations left a more pronounced
impact on the Croatian genetic landscape, especially the
islands where the pronounced influence of genetic drift
most probably pushed this particular R1a lineage to
higher frequencies.
Its high prevalence in our total sample (almost 20%),
together with a high haplotype diversity, speaks in favor
of several episodes of gene flow from Central Europe that
occurred during prehistory and history in SEE, which is
also evident in haplotype clusters depicted in the haplogroups phylogenetic network. These episodes were, as
seems, male-specific, rapid and relatively recent (5 kya
and younger), which makes any strong inference about
older demographic events questionable (Hellenthal et al.,

The Mesolithic E1b1b1a1b1a-V13 foragers

CONCLUSION
The genetic structure of modern SEE has been shaped
by the complexity of human movements through the Paleolithic, Neolithic, the Bronze Ages and the most recent
history of the last two millennia (involving the overlapping of different cultural and demic expansions). Molecular genetic analyses of the modern Croatian paternal
genetic pool performed in this study confirm the
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ET AL.

extraordinary heterogeneity and complexity of this population and suggest a dynamic gene flow in the Croatian
population and SEE throughout history. Our NRY analyses point to three distinct genetic signals traceable in the
Croatian paternal genetic landscape - the dominant
Balkan genetic heritage (embodied in hg I2a1b-M423),
along with a Central European input from Slavicspeaking populations (R1a1a1b*-M558) and a recognizable, but moderate Neolithic influx (E1b1b1a2-V13). However, concerning the timing of the proposed lineages,
recent studies show that most paternal lineages were
greatly affected by more recent and rapid demographic
changes that occurred in the last 5,000 years, making it
difficult to make inferences about older demographic
events. Also, one has to bear in mind that some of the haplotypes present in our sample have most probably been
brought into the SEE gene pool at different time periods
than the ones indicated in this study, some of them even
very recently, and that evolutionary processes involve
whole populations and not only certain haplogroups.
Nonetheless, this study offers the most complete picture
of Croatian paternal genetic diversity to date and offers
additional insights into the specific genetic and possible
historic events that shaped the current genetic landscape
of Croatia and the wider SEE region. Subsequent whole Y
chromosome sequencing and the advances in the aDNA
research combined with historical, archeological, linguistic, and paleoclimatic perspectives will then further
improve our understanding of the population movements
in Croatia and SEE, as well as the underlying demographic and historic processes.
Author Contributions
All authors contributed to the work presented in this
 analyzed data and wrote the paper, T. S.,
 D. H
paper. J. S.
A. and N. N. analyzed data, N. V., M. M., B. G., M. K., B.
N., A. G. and S. M. collected samples, S. R. and P. R.
supervised the project and wrote the paper.
ACKNOWLEDGMENTS
This research is a part of the project Population structure of Croatia anthropogenetic approach Laboratory
analyses were carried out at the Department of Evolutionary Biology, University of Tartu and Estonian Biocentre,
Tartu, Estonia.
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