Gene 498 (2012) 5967

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Gene
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High levels of Paleolithic Y-chromosome lineages characterize Serbia

Maria Regueiro a, Luis Rivera a, Tatjana Damnjanovic b, Ljiljana Lukovic b, Jelena Milasin b, Rene J. Herrera a,
a
b

Department of Molecular and Human Genetics, College of Medicine, Florida International University, Miami, FL, USA
Institute of Biology and Human Genetics, School of Medicine, University of Belgrade, Visegradska 26, 11000 Belgrade, Serbia

a r t i c l e

i n f o

Article history:
Accepted 19 January 2012
Available online 31 January 2012
Keywords:
Y-chromosome
Neolithic transition
Agricultural revolution
Serbia
Y-STRs
Y-SNPs

a b s t r a c t
Whether present-day European genetic variation and its distribution patterns can be attributed primarily to the
initial peopling of Europe by anatomically modern humans during the Paleolithic, or to latter Near Eastern Neolithic input is still the subject of debate. Southeastern Europe has been a crossroads for several cultures since Paleolithic times and the Balkans, specically, would have been part of the route used by Neolithic farmers to enter
Europe. Given its geographic location in the heart of the Balkan Peninsula at the intersection of Central and
Southeastern Europe, Serbia represents a key geographical location that may provide insight to elucidate the interactions between indigenous Paleolithic people and agricultural colonists from the Fertile Crescent. In this
study, we examine, for the rst time, the Y-chromosome constitution of the general Serbian population. A
total of 103 individuals were sampled and their DNA analyzed for 104 Y-chromosome bi-allelic markers and
17 associated STR loci. Our results indicate that approximately 58% of Serbian Y-chromosomes (I1-M253, I2aP37.2 and R1a1a-M198) belong to lineages believed to be pre-Neolithic. On the other hand, the signature of putative Near Eastern Neolithic lineages, including E1b1b1a1-M78, G2a-P15, J1-M267, J2-M172 and R1b1a2-M269
accounts for 39% of the Y-chromosome. Haplogroup frequency distributions in Western and Eastern Europe reveal a spotted landscape of paleolithic Y chromosomes, undermining continental-wide generalizations. Furthermore, an examination of the distribution of Y-chromosome liations in Europe indicates extreme levels of
Paleolithic lineages in a region encompassing Serbia, Bosnia-Herzegovina and Croatia, possibly the result of Neolithic migrations encroaching on Paleolithic populations against the Adriatic Sea.
2012 Elsevier B.V. All rights reserved.

1. Introduction
The transition from huntergatherer subsistence to more efcient,
productive and dependable agricultural practices during the Neolithic
in Europe, roughly 10,000 years ago, was one of the most important
demographic events since anatomically modern humans (AMH)
rst entered the continent in the Upper Paleolithic (Ammerman and
Cavalli-Sforza, 1984). Two primary scenarios have been proposed to
describe the spread of farming and domestication across Europe,
namely the Demic Diffusion Model, which supposes large-scale
expansions and replacement of indigenous Paleolithic populations
by agriculturalists from the Near East (Ammerman and Cavalli-Sforza,
1984), and the alternative Cultural Diffusion Model which suggests

Abbreviation: STR, Short Tandem Repeat; SNP, Single nucleotide polymorphism;

KYA, Thousand years ago; aDNA, ancient DNA; mtDNA, mitochondrial DNA; LBK, Linearbandkeramik culture; AMH, Anatomically modern humans; PCR, Polymerase chain
reaction; RFLP, Restriction-fragment length polymorphism; ISOGG, International
Society of Genetic Genealogy; MDS, multidimensional scaling; SPSS, Statistical Package
for the Social Sciences; MJ, Median joining; HD, Haplotype diversity; BC, Before Christ;
LGM, Last Glacial Maximum; TMRCA, Time to most recent common ancestor.
Corresponding author at: Department of Molecular and Human Genetics, College
of Medicine, Florida International University, Modesto Madique Campus, OE 304,
Miami, FL 33199, USA. Tel.: + 1 305 348 1258; fax: + 1 305 348 1259.
E-mail address: herrerar@u.edu (R.J. Herrera).
0378-1119/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2012.01.030

that the indigenous Paleolithic huntergatherer groups adopted new

lifestyle strategies by acculturation with relatively little genetic inuence from groups originating in the Near East (Whittle, 1996).
Investigations of the gene pools of modern populations have generated conicting results regarding the level of Neolithic contribution
to the European present day gene pool. Analysis of the mitochondrial
DNA control region, for example, indicates that the major extant lineages throughout Europe predate the Neolithic expansion and that
the spread of agriculture was a cultural inltration accompanied by
only a relatively minor genetic contribution from Near Eastern agriculturalists (Richards et al., 1996). Similarly, early Y-chromosome
studies reported that more than 80% of European men have inherited
their Y chromosomes from Paleolithic ancestors that dispersed into
Europe starting 40,000 years ago (40 KYA). Only 20% of the paternal
genetic component of Europeans was found to be from Neolithic
farmers that migrated into Europe starting 10 KYA (Semino et al.,
2000). However, reinterpretation of the previous data through application of an admixture model performed by Chikhi et al. (2002) suggested that the contribution of genes from the Near East to Europe
was 50% and 65% when using Basques and Sardinians, respectively,
as proxy Paleolithic parental populations in the admixture analyses.
Further resolution of the Y-chromosomal lineages, which has provided
the ability to distinguish European Mesolithic Y lineages (E1b1b1a2V13 and I2a2-M423) from subsequent Neolithic range expansions

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M. Regueiro et al. / Gene 498 (2012) 5967

from the Near East (J2b2-M241), has led support to the cultural
diffusion model in Southeast Europe (Balkans) (Battaglia et al., 2009),
as well as in Sardinia (Morelli et al., 2010). However, using the microsatellite diversity within R1b1a2-M269 (the most prevalent Y haplogroup in Western Europe), Balaresque et al. (2010) have recently
suggested that most European Y lineages arrived with Near Eastern
farmers across Anatolia during the Neolithic.
In addition to the analyses of modern DNA, paleogenetic studies
of ancient DNA (aDNA) have allowed for the direct comparison of
the early inhabitants of Europe with their contemporary counterparts. Mitochondrial DNA (mtDNA) testing of early Neolithic sites in
France (Deguilloux et al., 2011), as well as Germany, Austria and
Hungary (Linear Pottery Culture, Linearbandkeramik culture or LBK,
7.07.5 KYA) (Haak et al., 2005) has revealed a discontinuity between
the gene pools of early Neolithic farmers and modern day European
matrilineages. As a result, Haak et al. (2005) propose that the
more likely explanation is the Paleolithic survival theory in which
early female Neolithic farmers were genetically diluted by resident
native huntergatherers. However, additional analyses have provided
evidence that European huntergatherers also are genetically distinct
from modern Europeans (Bramanti et al., 2009). Interestingly, the
most common modern European Y-haplogroups (e.g., R1b, R1a, I
and E1b1) were not found in the Early Neolithic samples from
Central Europe (LBK). Instead, these ancient specimens were found
to share substantial genetic afnities with present day Near East
and Anatolia (Hgs F* and G2a3) (Haak et al., 2010). By contrast,
aDNA data from Neolithic communities in the Iberian Peninsula
(Catalonia) (Sampietro et al., 2007) display genetic similarities with
modern populations in the area, suggesting that the spread of
the Neolithic was neither genetically nor geographically a uniform
process in Europe and may reect regional differences.
Therefore, whether present-day European genetic variation and its
distribution patterns largely reect the initial peopling of Europe by
AMH in the Paleolithic times, or the Neolithic migrations from the Near
East is still a subject of debate. In an attempt to shed light upon the complexities surrounding the introduction of farming in Southeastern Europe, we analyze here the Y-chromosomal composition of the general
Serbian population. Serbia is especially important given its geographic
location in the heart of the Balkan Peninsula at the intersection of Central
and Southeastern Europe and its proximity to the Near East.
2. Materials and methods
2.1. Sample collection
A total of 103 male individuals belonging to the general population of Serbia were analyzed. Genealogical information from each
donor was recorded for at least two generations in order to establish
regional ancestry. All samples were procured with informed consent
following the ethical guidelines stipulated by the research institutions
involved in this project. DNA extractions from blood were performed
using the salting-out method (Miller et al., 1988).
2.2. Y-chromosome haplotyping
A total of 104 Y-chromosome bi-allelic markers (Karafet et al.,
2008; Myres et al., 2011; Underhill et al., 2010) were hierarchically
genotyped by standard methods, including PCR/AFLPs, PCR/RFLP
and allele-specic PCR (Gayden et al., 2008). The phylogenetic relationship of polymorphic markers and the haplogroups that they dene are included in Fig. 1. Haplogroup designation is in accordance
with the International Society of Genetic Genealogy (ISOGG, 2011).
DNA samples under the background of Y-haplogroups E-M96,
I-M258, J2-M172 and R1-M173 were also typed for 17 Y-STR loci
(DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392,
DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458,

DYS635, GATA H4) using the AmpFlSTR Yler kit (Applied Biosystems, Foster City, CA). PCR was performed as described by
the manufacturer (Applied Biosystems, 2006) and the resulting
amplicons were separated in an ABI Prism 3130xl Genetic Analyzer.
The GeneMapper software v3.2 was employed to determine fragment sizes and alleles were designated through comparisons to the
allelic ladder supplied by the manufacturer.
2.3. Data analyses
A list of previously published collections utilized for the phylogenetic analyses performed in this study is provided in Supplementary
Table 1. Pairwise genetic distances (Fst) based on haplogroup
frequencies, as well as Nei's gene diversity (Nei, 1987) estimates
based on 15 loci were calculated with the Arlequin v3.5 program
(Schneider et al., 2000). Multi-dimensional Scaling (MDS) analysis,
utilizing pair-wise Fst distances, was performed with the statistical
software package SPSS ver. 16.0 (SPSS, 2001). Contour maps based
on the frequencies of haplogroups E1b1b, G2a, I, J, R1a, and R1b
were generated using the Kriging procedure with the aid of the Surfer
Software (version 9, Golden Software Inc., Golden, CO, USA www.
goldensoftware.com). Median-joining (MJ) networks (Bandelt et al.,
1999) based on the Y-STR proles of individuals possessing haplogroup E1b1b1a1-M78, I2a2-M423 and R1a1a-M198 derivatives
were constructed with the NETWORK 4.5.1.6 software package
available at www.uxus-engineering.com. Networks were generated
using the MJ algorithm, with the microsatellite loci weighted proportionally to the inverse of the repeat variance observed in each Yhaplogroup lineage (Martinez et al., 2007). All phylogenetic analyses
were performed utilizing the 7 Y-STR loci (DYS19, DYS389I, DYS389II,
DYS390, DYS391, DYS392 and DYS393) in common among the
published collections. Haplotype diversity (HD) indices, based on
15 Y-STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392,
DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458,
DYS635, GATA H4), were computed utilizing Arlequin ver. 3.11
(Schneider et al., 2000). The age based on microsatellite variation
within haplogroups E, I and R was estimated as reported earlier by
Zhivotovsky et al. (2004) and modied according to Sengupta et al.
(2006). Genealogical mutation rates were also utilized as estimated
in Goedbloed et al. (2009). However, due to the contentions associated with the current calibrations of the Y-STR mutation rates
(Burgarella and Navascus, 2011; Goedbloed et al., 2009; Ravid-Amir
and Rosset, 2010; Zhivotovsky et al., 2004), as well as the limitations
of the assumptions made for time estimations, the absolute dates
generated in this study should only be taken as relative estimates.
Furthermore, as the estimates assume that the accumulated microsatellite variation depends not only on the mutation rate, but also on the
time since the haplogroup was initially founded by a single male, it is
possible that superimposed migration waves know to have occurred
in the Balkans could have inated these parameters.
Because the diversity within a particular lineage should reect
its relative age, intra-haplogroup diversity (mean microsatellite variance: Vp) (Kayser et al., 2001) was also estimated across the 15 loci.
The DYS385a/b loci were not considered in network, time estimation,
haplotype diversity and mean variance calculations given the duplicative nature of this locus. In addition, the size of the DYS389I allele
was subtracted from the DYS389II for all analyses. See in Supplementary Table 2 the 17 Y-STR haplotypes for the samples derived for the
above mentioned haplogroups.
3. Results
3.1. Haplogroup diversity
Fig. 1 illustrates the phylogenetic relationships of Y-chromosome
haplogroups and their frequencies in the general Serbian population.

M. Regueiro et al. / Gene 498 (2012) 5967

61

Fig. 1. Phylogenetic relationships of the Y-chromosome haplogroups and frequencies (%) for the population analyzed. Markers shown in italics were not genotyped and are included
to provide context. Haplogroup designation is in accordance with the International Society of Genetic Genealogy (ISOGG, 2011).

The most frequently observed haplogroups in Serbs are I-M258 (38%),

E1b1b1-M35 (18%) and R1a1a-M198 (20%), each exhibiting a high
level of haplotype diversity (0.9973, 0.9942 and 0.9905, respectively)
(Table 1). Haplogroup I is the only major clade of the Y phylogeny
that is widespread throughout Europe but is practically absent elsewhere (Rootsi et al., 2004). The M423 mutation (Underhill et al.,
2007), which characterizes the previously unresolved paragroup
I2a*-P37.2, accounts for the majority (77.2%) of East European I chromosomes (Battaglia et al., 2009) and is particularly frequent in Serbia,
where it represents 29.1% of this population's total paternal gene
pool. With the exception of one I2b1c-P78 derived individual,
the remaining I chromosomes in Serbia are characterized either by
I1-M253 (2.9%) or its downstream derivative I1-P109 (4.9%).

Table 1
Haplotype diversity and mean variance based on 15 loci.
Haplogroup

Final marker

Haplotype diversity SD

Mean variance

M35
V13
M258
M423
M258(xM423)
M173
M198
M458
M198(xM458)
M269
M269(xM412)
M304
M172

19
16
39
30
9
29
21
7
14
8
5
8
7

0.9942 0.0193
0.9917 0.0254
0.9973 0.0066
0.9977 0.0094
0.9722 0.0640
0.9951 0.0106
0.9905 0.0178
0.9524 0.0955
0.9327 0.0017
1.0000 0.0625
1.0000 0.1265
1.0000 0.0625
1.0000 0.0764

0.315
0.306
0.871
0.732
0.063
0.602
0.384
0.448
0.306
0.380
0.367
0.496
0.355

Haplogroup R-M207, which constitutes greater than 30% of the

Balkan Y-chromosome pool (Battaglia et al., 2009), was detected
at a similar frequency in Serbia (28.2%). Of this haplogroup's subclades, R1a1a*-M198 and its downstream mutation, R1a1a7-M458,
are the most prominent, constituting 14.6% and 5.8% of Serbian patrilineages, respectively. It should be noted that the paragroup R1a1a*M198, which has been associated with the Indo-Aryan expansions
(Keyser et al., 2009; Underhill et al., 2010), was observed at relatively
high frequencies (14.6%). R1b1a2-M269 chromosomes, on the other
hand, are observed in Serbia (8%) at low levels comparable to those
reported for the Balkan Peninsula (>5%) (Battaglia et al., 2009).
Downstream from the latter sub-haplogroup, two Serbian samples
are characterized by the M405 mutation and one by the U152 derivative, while the remaining chromosomes (~ 5%) belong to paragroups
R1b1a2a-L23*(xM412) (~3%) and R1b1a2-M269*(xL23) (~2%).
All E-M96 derived chromosomes belong to sub-haplogroup
E1b1b1-M35, with a single downstream mutation (E1b1b1a2-V13)
within this branch constituting the majority of Serbian M35 lineages
(15.5% total). With respect to haplogroup J, only one Serbian sample
belongs to the J1-M267 sub-clade while the remaining Y chromosomes possess J2-M172 derivatives. Both branches of J2, i.e., J2aM410 and J2b-M102, were observed in the general Serbian collection
at relatively low frequencies (3% and 2.9%, respectively). Although
several J2a sub-clades (J2a*-M410, J2a4b*-M67 and J2a4b1-M92)
were observed in the present study, haplogroup J2b-M102 is represented exclusively by the M205 mutation (2.9%). It is also worth
noting that the lack of J2b2-M241 derivatives observed in the Serbian
population is consistent with its low diversity in the Balkans (mean
variance of 0.121 within J2b2-M241, Battaglia et al., 2009) and all
Serbian J2b-M102 chromosomes tested are entirely represented by
its J2b1-M205 sub-lineage. Only one Serbian sample was found to

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M. Regueiro et al. / Gene 498 (2012) 5967

extensive haplotype sharing, within E1b1b1a1-M78, I-M258 and

R1a1a-M198, suggests substantial genetic ow within the Balkans.
3.3. Distribution of Paleolithic and Neolithic Y-chromosome lineages in
Serbia and through Europe

Fig. 2. MDS plot of pairwise Fst distances estimated based on haplogroup frequencies
at the resolution of the major branches (AT) of the Y chromosome phylogram
(Stress= 0.13900). Ser (Serbia, Present study), Bos (Bosnia-Herzegovina Bosniacs), Croa
(Croatia), KoAl (Kosovar Albanians), Mac (Macedonia), Alb (Albania), Cre (Crete), Gre
(Greece), SLOVE (Slovenia), LIT (Lithuania), LAT (Latvia), SLOK (Slovakia), BEL (Belarusian),EST (Estonia), UKR (Ukrainian), POL (Poland), LEB (Lebanon), SYR (Syria), ANA (Anatolia), IRN (North Iran), IRS (South Iran), UZB (Uzbekistan), TUR (Turkmenistan), TAJ
(Tajikistan), IND (India), PAK (Pakistan). See Supplementary Table 1 for the complete list
of the reference populations.

be derived for the J1c3-P58 mutation that is distinctive of the Arabicspeaking populations (Chiaroni et al., 2010).
3.2. Population relationships
To assess the phylogenetic relationships between the general
population of Serbia and 27 other geographically targeted populations
obtained from the literature, an MDS plot and MJ networks were constructed (Fig. 2 and Supplementary Fig. 1A, B, C). The MDS plot (Fig. 2)
reveals a loosely associated cluster of Southeastern European populations that occupies the middle to upper right half of the graph, while
the Northeast European collections cluster on the lower right portion
of the plot. The clustering of the Balkan collections is most likely
the result of high frequencies of I-M258, E-M96 and R-M207.
The network projection based on the Y-STR proles of individuals
harboring the E1b1b1a1-M78 mutation is provided in Supplementary
Fig. 1A. Ten different Serbian haplotypes are found, a scattered distribution congruent with the high haplotype diversity (0.9917
0.0254) (Table 1) present in Serbia and in the Balkan Peninsula.
The phylogenetic relationships of haplotypes within I-M258 lineages
are displayed in Supplementary Fig. 1B. As in the previous network,
the high diversity of the Serbian collection is mirrored by the widespread distribution of haplotype clusters, which exhibits 20 distinct
haplotypes. The distribution of haplotypes within the R1a1a-M198
lineage, illustrated in the network projection provided in Supplementary Fig. 1C, similarly correlates with the high Serbian diversity
observed within this haplogroup (0.9905 0.0178) (Table 1). The

In order to compared the proportions of Y-chromosome markers

that may signal Neolithic migrations from the Near East as part of
the Agricultural Revolution, and the paternal heritages from autochthonous Paleolithic populations, the Neolithic and post-Neolithic
E1b1b, G, J1, J2 (Battaglia et al., 2009) and R1b (Balaresque et al.,
2010), and Paleolithic R1a (Perii et al., 2005; Semino et al., 2000;
Wells et al., 2001) I1 and I2a (Battaglia et al., 2009; Rootsi et al.,
2004) lineages were examined in the general population of Serbian
as well as in different regions of Europe (see Supplementary Table 1
for populations references). Fig. 3 illustrates the levels of the aforementioned haplogroups in Europe, exhibiting a focal point of extreme
frequencies (low or high) in the northwestern corner of the Balkan
Peninsula, encroached at the coastal edge of the Adriatic Sea. The cumulative frequencies of Paleolithic and Neolithic lineages, as presented in the contour maps (Figs. 3G and H, respectively), illustrate,
region by region, an inverse relationship.
4. Discussion
The primary objective of the current study was to characterize, for
the rst time, the Y-chromosome lineages in the general population
of Serbia and phylogenetically compare them with those from
previously reported geographically targeted reference populations,
in an effort to provide insights into the relative contribution of
the Neolithic patrilineages from the Near East to Europe during
the Agricultural Revolution. For the purpose of the ensuing
discussion, the end of the Paleolithic and the beginning of the
Mesolithic began in Europe with the Holocene warm period around
11.6 KYA and ended with the introduction of farming during the
Neolithic which in the Balkan Peninsula started around 8.57.2 KYA
(Price, 2000).
4.1. Gene ow from the Eurasian steppes and in situ European
diversication
Haplogroup R1a1a-M198, which is detected at levels greater than
10% along a geographic range that extends from South Asia to Central
East Europe (Underhill et al., 2010), occurs at a frequency of 16%
in Southeastern Europe (Perii et al., 2005), while exhibiting particularly high haplotype diversity in Serbia (0.9905 0.0178). Three
major episodes of gene ow have been described to explain the
high R1a haplotype diversity in the Balkans: (1) re-colonization
from the refugium in the Ukraine (early post-LGM, ~2012 KYA)
(Passarino et al., 2001; Semino et al., 2000); (2) migrations from the
Pontic steppe associated with the Indo-European Kurgan culture
(30001000 B.C.) (Rosser et al., 2000; Semino et al., 2000); and,
more recently, (3) the massive Slavic migration (5th7th centuries)
(Bara et al., 2003; Perii et al., 2005). As postulated by Gimbutas

Fig. 3. Each contour map was obtained by converting the haplogroup frequencies from data from the literature into spatial-frequency maps. Spatial gradient map illustrate clines in
frequency for (A) haplogroup E1b1b1, (B) haplogroup G, (C) haplogroup J, (D) haplogroup R1b, (E) haplogroup I, (F) haplogroup R1a, (G) haplogroups I and R1a, (H) haplogroups
E1b1b1, G, J and R1b. Data from the literature: Anatolia, England, Scotland, Wales, Germany, Albania, Greece, Crete, Thrace, Macedonians (Skopje), Bosnia, Croatia, Spain (Castile,
Catalonia, Seville, Pyrenean, Galicia), Basque, France, Italy (Apennine), Sicily, North Netherlands (Frisians), Republic of Moldova [Gagauz (Kongaz and Etulia)], Hungaria, Portugal,
Romanians, Russia (Komi), Russia (North and Central), Sweden (Sami), Slovenians, Ukrainians. See Supplementary Table 1 for the complete list of the reference.Each contour
map was obtained by converting the haplogroup frequencies from data from the literature into spatial-frequency maps. Spatial gradient map illustrating clines in frequency for
(A) haplogroup E1b1b1, (B) haplogroup G, (C) haplogroup J, (D) haplogroup R1b, (E) haplogroup I, (F) haplogroup R1a, (G) haplogroups I and R1a, (H) haplogroups E1b1b1,
G, J and R1b. Data from the literature: Anatolia, England, Scotland, Wales, Germany, Albania, Greece, Crete, Thrace, Macedonians (Skopje), Bosnia, Croatia, Spain (Castile, Catalonia,
Seville, Pyrenean, Galicia), Basque, France, Italy (Apennine), Sicily, North Netherlands (Frisians), Republic of Moldova [Gagauz (Kongaz and Etulia)], Hungaria, Portugal, Romanians,
Russia (Komi), Russia (North and Central), Sweden (Sami), Slovenians, Ukrainians. See Supplementary Table 1 for the complete list of the references.

M. Regueiro et al. / Gene 498 (2012) 5967

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M. Regueiro et al. / Gene 498 (2012) 5967

M. Regueiro et al. / Gene 498 (2012) 5967

(1970), the Kurgan expansions would have resulted in the spread of

the Indo-European languages, and the dispersal of the R1a haplogroup believed to have been present in the majority of the Kurgan
paternal gene pool (Zerjal et al., 1999), as supported by analysis of
ancient (3.80.1 KYA) Kurgan Y chromosomes from across south
central Siberia (Keyser et al., 2009). Such analyses revealed that,
with the exception of one individual (belonging to haplogroup C),
all male samples were exclusively R1a1 (N = 9) (Keyser et al., 2009).
The relatively old expansion time (14.0 3.3 KYA) (Supplementary Table 3), associated mean variance (0.384) and high haplotype diversity (0.9905 0.0178) (Table 1), also evident in the phylogenetic
network (Supplementary Fig. 1C), among Serbian R1a1a-M198 carriers, are consistent with previous studies (Perii et al., 2005;
Semino et al., 2000; Wells et al., 2001) that suggest that the common
ancestor for all R1a1a-M198 individuals in the Balkans existed in Paleolithic times. The R1a1a7 dening marker, M458, exhibits a Central
and Eastern European compartmentalization within the R1a haplogroup (Underhill et al., 2010). While R1a1a7-M458 displays high
diversity among Slavic and Finno-Ugric peoples (coalescent time
~ 11 KYA), R1a1a*(xM458) has the most diversity among IndoAryan and Dravidian speakers (coalescent time in India ~14 KYA)
(Underhill et al., 2010). Analysis of both chromosomes in Serbia
revealed higher R1a1a7-M458 diversity than R1a1a*(xM458) and
yielded coalescent times at ~14 KYA (mean variances ~ 0.448) and
11 KYA (mean variance 0.306), respectively. Therefore, this high diversity and estimated age of microsatellite variation in Serbia, might
be inated by the aforementioned superimposed migrations involving R1a individuals that penetrated Europe. This hypothetically inated diversity and time estimates may articially place the R1a
haplogroup in the Paleolithic (Perii et al., 2005; Semino et al.,
2000) in Europe rather than during the early Halocene (Klyosov,
2009).
4.2. Genetic signature from the Near East and autochthonous European
lineages
The role of the Balkans as a gateway into Europe from the Near
East has been illustrated by IJ-M429 Y chromosomes which are
thought to have entered Europe through the Peninsula shortly before
the Last Glacial Maximum (LGM) (Battaglia et al., 2009; Semino et al.,
2000; Underhill et al., 2007). Descendent Y-chromosomes bearing
the M429 mutation then segregated to form haplogroup I-M258
somewhere in Europe close to the Near East, subsequently giving
rise to haplogroups I1-M253, I2a-P37.2 and I2b1-M223 (Underhill
et al., 2007). Haplogroup I is thought to have played a central role in
the process of human recolonization of Europe from isolated glacial
refugia after the LGM. Moreover, the expansion phase of I1-M253
and I2a-P37.2, exhibiting different phylogeographies, is thought to
have occurred later, during the early Holocene corresponding with
the beginning of the Mesolithic age (Rootsi et al., 2004). The age
based on 15 Y-STR loci for all I lineages in Serbia (N = 39) was estimated to be 20.0 4.0 KYA, a value in agreement with the population
divergence time for Europe estimated by Rootsi et al. (2004) (23.0
7.7 KYA), further supporting that the Gravettian technology/culture (archeologically dated at approximately 24 KYA in the Upper Paleolithic) spread across Europe with the migrations of people from
the Middle East, Anatolia and the Balkans carrying the I-M258 mutation ~ 28,00022,000 years ago. The high haplotype diversity of I2a2P37.2/M423 lineages in Serbia (0.9977 0.0094) also supports the
hypothesis that the P37.2 mutation has been present in the Balkans
before the LGM (Rootsi et al., 2004; Semino et al., 2000). Moreover,
the age of I2a2-M423 chromosomes in Serbs based on accumulated
Y-STR variation, is ~ 9000 years ago (Table 1) and is consistent with
previously reported time frames in the Early Holocene, during the European Mesolithic period (Battaglia et al., 2009; Perii et al., 2005;
Rootsi et al., 2004; Underhill et al., 2007). Our data corroborate the

65

previous assertion by Battaglia et al. (2009) that the autochthonous

haplogroup I2a2-M423 is indicative of the adoption of farming by
Mesolithic huntergatherers in the Balkans.
The sister clade to haplogroup I, haplogroup J is thought to
have emerged in the Levant just prior to the Neolithic Agricultural
Revolution (Cinniolu et al., 2004; Di Giacomo et al., 2004; Semino
et al., 2004) and thus its frequency and distribution in Serbia may
provide some evidence of the sources and relative contribution of
any Neolithic gene ow into this Balkan population. Although both
Near Eastern specic haplogroups J1-M267 and J2-M172 are found
in Serbia, haplogroup J1-M267 is represented by only one individual
that is derived for the J1c3-P58 mutation distinctive of Arabicspeaking populations (Chiaroni et al., 2010). The remaining 7 J Ychromosomes are characterize by the M172 mutation (J2) and its
downstream derivatives, J2a-M67/M92 and J2b-M102, which signal
more recent Bronze Age expansions (post-Neolithic) westward from
Turkey (Cinniolu et al., 2004; Cruciani et al., 2007; Di Giacomo
et al., 2004). In Serbia the most prevalent J haplogroup is J2-M102
(3%), consistent with previous studies of Southern Balkan Peninsula
populations (Marjanovic et al., 2005; Perii et al., 2005). Limited
inuence from Anatolia in the Serbian collection is reected in the
relatively low occurrence of the J2a4b-M67(xM92) and the J2a4b1M92 lineages (present at 1% each). Minimal genetic input from
this region is also supported by the relatively low level (5.8%) of
G2a-P15 chromosomes, the most frequent G sub-clade in Turkey
(Cinniolu et al., 2004). Considering the low levels of J1, J2 and G, it
is interesting that 5 centuries of occupation by the Ottoman empire
did not impacted substantially the patrilineage component of Serbia.
Furthermore, although only present at nominal frequencies, Serbian
J chromosomes display a very high haplotype diversity likely reecting the different Middle Eastern migrations that have been associated
with this haplogroup (Mitchell and Hammer, 1996; Semino et al.,
1996). In addition, this high diversity within J2 lineages from Serbia,
as well as other Balkan populations relative to other European groups
is consistent with the diffusion of these lineages into Europe from the
southern Balkans (Di Giacomo et al., 2004; Semino et al., 2004). Haplogroups G, J1 and J2 have been associated with the successful colonization and subsequent expansions of Neolithic pioneers (Battaglia et
al., 2009).
Haplogroup E1b1b1a-M78 is thought to have arisen in Northeastern Africa (Cruciani et al., 2004; Semino et al., 2004) with the V13
mutation later emerging somewhere in the Near East/Anatolia or
in the Balkans (Battaglia et al., 2009; Cruciani et al., 2007).
E1b1b1a2-V13 is the only branch of this sub-clade that reaches
high frequencies outside of continental Africa, representing about
85% of the European M78 derived chromosomes (Cruciani et al.,
2007). Although early studies suggested that the E1b1b1a2-V13
mutation was brought to the Balkans together with early farming
technologies (Semino et al., 2004), Battaglia et al. (2009) propose
an earlier arrival of this marker in Europe, i.e., during the late Mesolithic period, follow by a Neolithic dispersion into Europe with the
spread of farming, since there is no E1b1b1a2-V13 chromosomes in
the Fertile Crescent where farming originated. More recently, King et al.
(2011) suggested that E1b1b1a2-V13 may trace the demographic and
socio-cultural impact of Greek colonization. The diversity of E1b1b1a2V13 in the collection from Serbia, with a variance of 0.30 and an expansion time of 11.73.5 KYA supports a plausible origin of V13 chromosomes in the Balkans during late Mesolithic times (Battaglia et al., 2009;
Cruciani et al., 2007).
Early studies suggested a Paleolithic origin for R1b1a2-M269, the
major western European lineage (Perii et al., 2005; Rosser et al.,
2000; Semino et al., 2000). Although Morelli et al. (2010) suggest that
modern Western European populations do not contain the genetic signals that would imply that the R1b1a2-M269 lineage in these groups arrived from the East following a Neolithic demic diffusion, additional
analyses have led to the conclusion that R1b-M269 in Europe is young

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M. Regueiro et al. / Gene 498 (2012) 5967

and likely associated with a Neolithic demic expansion from the Near
East through Anatolia (Arredi et al., 2010; Balaresque et al., 2010;
Myres et al., 2011). The time estimate values for Serbian R1b1a2M269(xM412) chromosomes (12.03.3 KYA, N=5) approximate the
coalescent times of the R1b1a2-M269 clade across Europe estimated by
both Myres et al. (2011) (10.21.6 KYA) and Shi et al. (2010) (median
TMRCA dates 8.512.5 KYA) based on a different inference methodology.
It is likely that our time estimate values for the Serbia population represent upper limit estimates inated by multiple migrational waves into
the area.
4.3. High pre-Neolithic signals in Serbia
Previous studies involving Paleolithic Y-chromosomes R1a (Perii
et al., 2005; Semino et al., 2000; Wells et al., 2001) and I (Battaglia
et al., 2009; Rootsi et al., 2004) report high frequencies in Balkan Peninsula. Conversely, lower levels of Neolithic and post-Neolithic Y-lineages
E1b1b, J, and G (Battaglia et al., 2009) and R1b (Balaresque et al., 2010)
have been detected in the same location. When the frequency values
are plotted on the background of a European map (see Fig. 3), it is evident that the values change clinally along the eastwest axis. Yet, it is
also apparent that superimposed on these general demic gradients,
spotted and regionalized high levels of Paleolithic and Neolithic lineages are observed. The consequences of such distribution patterns in
the assessment of whether Europeans are genetically Paleolithic or Neolithic and whether the adoption of the agriculture is the result of
acculturation or genetic ow is very region-specic and requires
the evaluation of specic areas individually.
Case in question is the extreme high levels of Paleolithic Ychromosomes observed in the general population of Serbia in this
study, as well as in the northwest Balkan Peninsula. The observed
distribution of Paleolithic and Neolithic Y-chromosome lineages is
compatible with the previously described cul-de-sac effect (Salas et al.,
1998). In population genetic terms, the bottom of the sack scenario
envisions an advancing wave of migrants pressing and encroaching
the native populations of the area against a geographical barrier (Salas
et al., 1998). In our study, the high levels of Paleolithic lineages
in Serbia and the northwest region of the Balkan Peninsula as well as
the east to west clinal frequency differences starting in Anatolia,
reaching its maximum at the coastal edge of the Adriatic Sea (Fig. 3)
are hallmarks of an expansion, possibly of farmers from the Near
East pushing on Mesolithic settlements against the bottom of a
geographical sack or obstacle, the Adriatic Sea.
5. Conclusions
Although it is difcult to discern clearly between the Mesolithic
and early Neolithic in Europe with current genetic coalescence assessment (Arredi et al., 2010; Myres et al., 2011), our data is consistent
with acculturation as the main force driving the adoption of agriculture in Serbia and in the northeast of the Balkans. The results suggest
that the majority (~58%) of Serbian Y-chromosomes belong to lineages believed to be Paleolithic (I1-M253, I2a-P37.2, R1a1a-M198),
while the putative Neolithic contributors, including E1b1b1a1-M78,
G2a-P15, J1-M267, J2-M172 and R1b1a2-M269, appear to have had
a smaller impact on the present-day Serbian gene pool, comprising
~ 39% of the observed patrilineages. Our analyses also indicate that
ne-grained regional evaluation of the gene pools may be require to
assess the relative contribution of genetic ow versus acculturation
to the adoption of agriculture and domestication in Europe.
Supplementary materials related to this article can be found online at doi:10.1016/j.jprot.2012.01.023.
Conict of interest statement
The authors declare no conict of interest.

Acknowledgments
We thank all the men who donated DNA samples used in this
study. The authors would also like to thank Tanya Simms, Tenzin
Gayden, Kristian Herrera, and Robert Lowery for their constructive
criticisms of the manuscript.
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