European Journal of Human Genetics (2009) 17, 820 – 830
& 2009 Macmillan Publishers Limited All rights reserved 1018-4813/09 $32.00
www.nature.com/ejhg
ARTICLE
Y-chromosomal evidence of the cultural diffusion of
agriculture in southeast Europe
Vincenza Battaglia1, Simona Fornarino1,12, Nadia Al-Zahery1, Anna Olivieri1, Maria Pala1,
Natalie M Myres2, Roy J King3, Siiri Rootsi4, Damir Marjanovic5,6, Dragan Primorac7,8,
Rifat Hadziselimovic5, Stojko Vidovic9, Katia Drobnic10, Naser Durmishi11, Antonio Torroni1,
A Silvana Santachiara-Benerecetti1, Peter A Underhill3 and Ornella Semino*,1
1
Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia, Italy; 2Sorenson Molecular Genealogy
Foundation, Salt Lake City, UT, USA; 3Department of Psychiatry and Behavioral Sciences, School of Medicine, Stanford
University, Stanford, CA, USA; 4Department of Evolutionary Biology, University of Tartu and Estonian Biocentre, Tartu,
Estonia; 5Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Sarajevo, Bosnia and
Herzegovina; 6Genos doo, Zagreb, Croatia; 7Medical School, Split University, Split, Croatia; 8Medical School, Osijek
University, Osijek, Croatia; 9Faculty of Medicine, University of Banja Luka, Banja Luka, Bosnia and Herzegovina;
10
Forensic Laboratory and Research Center, Ministry of the Interior, Ljubljana, Slovenia; 11Medical School, University of
Tetovo, Tetovo, Former Yugoslavia Republic of Macedonia
The debate concerning the mechanisms underlying the prehistoric spread of farming to Southeast Europe is
framed around the opposing roles of population movement and cultural diffusion. To investigate the possible
involvement of local people during the transition of agriculture in the Balkans, we analysed patterns of
Y-chromosome diversity in 1206 subjects from 17 population samples, mainly from Southeast Europe. Evidence
from three Y-chromosome lineages, I-M423, E-V13 and J-M241, make it possible to distinguish between
Holocene Mesolithic forager and subsequent Neolithic range expansions from the eastern Sahara and the Near
East, respectively. In particular, whereas the Balkan microsatellite variation associated to J-M241 correlates with
the Neolithic period, those related to E-V13 and I-M423 Balkan Y chromosomes are consistent with a late
Mesolithic time frame. In addition, the low frequency and variance associated to I-M423 and E-V13 in Anatolia
and the Middle East, support an European Mesolithic origin of these two clades. Thus, these Balkan Mesolithic
foragers with their own autochthonous genetic signatures, were destined to become the earliest to adopt
farming, when it was subsequently introduced by a cadre of migrating farmers from the Near East. These initial
local converted farmers became the principal agents spreading this economy using maritime leapfrog
colonization strategies in the Adriatic and transmitting the Neolithic cultural package to other adjacent
Mesolithic populations. The ensuing range expansions of E-V13 and I-M423 parallel in space and time the
diffusion of Neolithic Impressed Ware, thereby supporting a case of cultural diffusion using genetic evidence.
European Journal of Human Genetics (2009) 17, 820–830; doi:10.1038/ejhg.2008.249; published online 24 December 2008
Keywords: Balkan Neolithic; farming transition; peopling of Europe; Y-chromosome haplogroups
*Correspondence: Dr O Semino, Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia 27100, Italy.
Tel: þ 39 0382 985543; Fax: þ 39 0382 528496;
E-mail: semino@ipvgen.unipv.it
12
Present address: Human Evolutionary Genetics, CNRS URA 3012,
Institut Pasteur, Paris, France.
Received 2 September 2008; revised 17 November 2008; accepted 20
November 2008; published online 24 December 2008
Introduction
Europe was colonized by modern humans about 40 000
years ago and underwent a second colonization wave
during the Neolithic, with the spread of farming.1,2 The
relative Palaeolithic and Neolithic contribution to the
current European gene pool has been widely debated and is
still under discussion.3 – 6 Two opposing models have been
Transition to farming in the Balkans
V Battaglia et al
821
cited to account for the spread of farming in Europe: the
demic diffusion model, which implies a movement of
people and therefore a significant Near Eastern genetic
input,7 and the cultural diffusion model, which, on the
contrary, considers the transition to agriculture as a
cultural phenomenon, and therefore without major
changes at the genetic level.8 Archaeological evidences
suggest, however, that the spread of agriculture has been a
complex process characterized by migrations and local
admixture.9,10
Genetic studies have described allele frequency clines for
different markers along the European colonization
routes.11 – 13 These have been interpreted in favour of
demic diffusion and considered a strong indication of a
Neolithic contribution to the modern European gene
pool.14,15 However, a number of simulation studies have
demonstrated that allele frequency clines can also occur in
range expansions where admixture is not present.16 – 19 The
impact of the Neolithic in the Balkans, as archaeological
records show, is regarded to be considerable,20 although
Mesolithic hunter-gatherers were present in this region just
before the first appearance of Neolithic pottery.21 Thus, the
question to address is the following: can we detect presentday signals of interactions between indigenous Mesolithic
people and agricultural colonists in the southeast European
gene pool? In this regard, particularly useful are
Y-chromosome markers, whose distribution is often highly
correlated with their phylogeny.22
Interestingly, the Y-chromosome gene pool of southeast
European populations is characterized both by ‘autochthonous’ markers, such as haplogroup (Hg) I, present in the
Balkans in pre-Neolithic times,23,24 and by markers mainly
belonging to Hgs E and J, which originated outside Europe,
in Africa and the Middle East, respectively.12,13,25 – 28
In addition, new Y-chromosome polymorphisms have
added further sophistication to phylogenetic relationships,
29 – 32
especially within Hgs E, J and I,
providing the
opportunity to more fully evaluate the above issue. This
possibility induced us to carry on a deeper genetic structure
characterization of the Balkan area through the analysis of
80 Y-chromosome bi-allelic markers and 12 linked STR loci
in 1206 subjects from 17 population samples mainly from
southeast Europe.
Materials and methods
Samples
The sample consists of 1206 unrelated male individuals
from 17 population samples (Figure 1). Two-hundred and
thirty-five of these, namely 64 Albanians from Former
Yugoslavia Republic of Macedonia, 29 Croatians from
Osijek, 75 Slovenians and 67 northeast Italians (from the
province of Trento), are reported here for the first time. The
remaining include samples reported earlier,23,27,33 and
consist of 104 Caucasians (38 Balkarians and 66
Georgians), 149 Greeks (92 from Athens and 57 from
Macedonia), 55 Albanians (collected in Tirana), 89 Croatians, 99 Polish, 75 Czechs, 92 Ukrainians, 53 Hungarians
and 255 Bosnia-Herzegovinians (84 Bosniacs, 90 Croats
and 81 Serbs). Blood samples were collected from healthy
unrelated adults after obtaining informed consent. DNA
was extracted from whole blood according to the
standard phenol/chloroform procedure, followed by ethanol
precipitation.
In addition, P37.2* samples identified from a screening
of the Sorenson Molecular Genealogy Foundation collection (over 14 000 Y chromosomes from more than 100
countries) were also included.
Examined bi-allelic markers and microsatellites
Eighty Y-specific unique event polymorphisms (Figure 2)
were examined in hierarchical order. Two new mutations
Figure 1 Geographic location of the studied samples: 1, Greeks; 2, Macedonian Greeks; 3, Albanians; 4, Albanians from Former Yugoslavia
Republic of Macedonia (FYROM); 5, Bosniacs; 6, Bosnia-Croats; 7, Bosnia-Serbs; 8, Croats; 9, Croats from Osijek; 10, Slovenians; 11, northeast Italians;
12, Hungarians; 13, Czechs; 14, Poles; 15, Ukrainians; 16, Georgians; and 17, Balkarians.
European Journal of Human Genetics
Transition to farming in the Balkans
V Battaglia et al
822
were discovered: M507 (AC006376.2: g.148697 T-G) and
M521 (AC012068.5: g.5219 C-T). The first is associated
with I-M253 and was discovered while typing M227, the
second is associated with E-M78* and was ascertained while
typing V12.
Bi-allelic markers were analysed by PCR/AFLPs (YAP34
and 12f213), by PCR/RFLPs (P37,35 M9, M269,36 V12, V13,
V22, V27, V32 and V6528,30) or by PCR/DHPLC (M241,
M253, M267, M285, M287, M304, M343, M365, M367 –
M369,29 M410,37 M423, M429, M43831 and M406,38 and
those reported in the YCC39).
Nomenclature used for Hg labelling is in agreement with
YCC39 conventions and recent updating.31,32,38 Subsets of
samples belonging to E-V13, J-M241 and I-M423 Hgs were
also analysed at 12 STR loci: DYS19, YCAIIa/b, DYS388,
DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS439
and DYS460 by using multiplex reactions according to
STRBase information (http://www.cstl.nist.gov/biotech/
strbase/y20prim.htm) and ABI PRISMs 3100 DNA Sequencer, internal size standard and GeneScan fragment analysis
software. DYS44538 was analysed separately in J-M410
samples.
Statistical analysis
Hg diversity (H) was computed using the standard method
of Nei.40 Principal components (PC) analysis was performed on Hg frequencies using Excel software implemented by Xlstat. The relative amount of accumulated
diversity, as a function of geography, was evaluated
through the mean microsatellite variance estimated for
each population with a sample size of at least five
individuals. Hg frequency and variance maps were generated by using Surfer Golden software following the Kriging
procedure.41 Within specific Hgs, Median-Joining (MJ)
networks42 were constructed using Network 4.5.0.0
program (Fluxus Engineering, http://www.fluxus-technology.
com). Networks were calculated by the MJ method, where
e ¼ 0 and microsatellite loci were weighted proportionally to
the inverse of the repeat variance observed in each Hg and
after having processed the data with the reduced-median
method. The age of microsatellite variation within Hgs
was evaluated employing the methodology described by
Zhivotovsky et al43 as modified according to Sengupta et al.37
A microsatellite evolutionary effective mutation rate of
6.9 104 was chosen as it is suitable for use in situations
where the elapsed time frame is Z1000 years or B40
generations, clearly appropriate given the prehistoric time
depths being explored in this study.44
Figure 2
It is worth mentioning that ambiguities related to past
episodes of population history (eg, size fluctuations,
bottlenecks, etc.) create inherent uncertainties in the
calibration of the YSTR molecular clock; thus the estimated
ages of microsatellite variation should be considered with
caution.
Results
Figure 2 illustrates the phylogenetic relationships of
Y-chromosome Hgs and their distribution in the examined
southeast European populations. The main Hgs observed
in Europe45 (E, I, J, R1a and R1b) contribute differently
to the gene pool of the various East European areas, Hg I
and Hg R being the most represented always and Hg E and
Hg J being mainly frequent in the southern Balkan
populations.
Hg I is restricted to western Eurasia23 and is particularly
frequent in the Balkans where it characterizes 36.3% of the
total Y chromosomes. Two of its branches, I-M223 and
I-M253, are scarcely represented in southeast Europe, the
first being only sporadically observed and the second
showing frequencies around 5%, with higher values
(around 9%) in Macedonian Greeks and Croats.
Differently, the recently described M423 SNP,31 which
characterizes the previously paraphyletic P37 clade,
accounts for the majority (77.2%) of the East European Hg
I chromosomes. Its diffusion seems not to have affected
the neighbouring North Italian populations, where low
incidences (0–2%) are observed. The I-M423 sub-clade is
characterized by a frequency distribution with high Central
Balkan values (470% in Bosnia-Herzegovina) and decreasing
frequencies moving from the southern Dinaric Alps to
northern Croatia. Although I-M423 comprises virtually all
the I-P37.2 Balkan-related chromosomes reported earlier,31
we have also detected one I-P37.2* Albanian subject
and, from a screening of previously identified P37.2
chromosomes (Rootsi et al23 and SMGF collection), 30
further P37.2* subjects, two from Moldavia23 and 28 of
either documented or presumed western European ancestry,
were identified.
Hg R1 is common throughout western Eurasia22 and
accounts for more than 30% of the Balkan Y-chromosome
pool. With the exception of one R1a*-SRY10831.2 and five
R1b1-M343* individuals, all the remaining R1 lineages
belong to R1a1-M17* and R1b1b2-M269. These two subclades, which show in Europe opposite-frequency gradients
with maximum incidences in eastern and western regions,
Phylogeny of Y-chromosome haplogroups and their frequencies (%) in the examined populations. Nomenclature and haplogroup
labelling according to the Y Chromosome Consortium (http://ycc.biosci.arizona.edu/) updated according to Karafet et al.32 *Paragroups:
Y chromosomes not defined by any phylogenetic downstream-reported and -examined mutation. aIntrapopulation haplogroup diversity. The terminal
markers of haplogroups E-V12 and E-V13 (V32 and V27, respectively) were typed but did not show any variation.
European Journal of Human Genetics
Transition to farming in the Balkans
V Battaglia et al
C
Macedonian-Greeks
Albanians
FYROM-Albanians
Bosnia-Serbs
Bosniacs
Bosnia-Croats
Osijek-Croats
Croats
Slovenians
North-East-Italians
Hungarians
Czechs
Poles
Ukrainians
Georgians
Balkarians
N
RPS4Y711
Greeks
823
92
57
55
64
81
84
90
29
89
75
67
53
75
99
92
66
38
1.1
M174
M33
YAP
M75
M81
E1b1b1a1
V13
E1b1b1a2
16.3 19.3 23.6 34.4 19.8 13.1
V22
V65
M521
E1b1b1a3
1.1
E1b1b1a5
2.2
E1b1b1c
2.2
G2a*
2.2
G2a3c
1.1
*
P15
M287
1.6
E1b1b1a*
M123
M285
M69/M52/M370/M82
1.8
*
M78
M201
1.5
2.5
V12
M35
M406
2.6
8.9
10.3
1.8
6.7
2.7
1.5
7.5
1.5
1.9
1.3
11.9
1.9
4.0
M253
1.5
1.8
3.1
1.8
1.6
1.1
1.2
3.6
1.1
13.8
1.1
1.8
4.0
1.1
1.5
3.3
30.3 26.3
1.3
1.5
1.1
2.2
I1*
*
M21
M227
7.6
1.1
H1a11.8
M168
M170
M258
E1a
E*1b1b1
*
7.0
3.6
4.7
2.5
1.8
I1b*
3.6
7.9
6.7
9.0
3.8
9.3
4.0
1.2
1.1
2.6
2.6
1.5
1.1
M72
I1e
M507
1.1
I2*
*
1.1
3.5
2.5
I2a*
*
M423
1.3
2.6
1.6
6.5
I2a1*
17.5 14.5
9.4
34.6 42.9 73.3 27.6 32.6 20.0
9.4
14.1 17.4
1.5
1.3
P37.2
M438
M359
M429
M223
I2b1
M267
M365
J1b
*
J2a*
M367
M368
M369
M67
M102
2.7
1.3
2.4
1.3
1.0
3.0
10.6 15.8
1.5
J2a1k
1.1
1.8
2.5
J2b*
2.2
3.5
3.7
1.2
M241
J2b2
4.3
1.8
2.5
2.4
J2b3
2.2
L2
3.5
3.6
1.6
4.8
1.1
1.8
3.4
6.0
1.2
14.5 14.1
1.3
1.1
1.1
1.1
1.9
1.5
3.0
3.4
2.2
1.3
3.4
1.1
4.0
M343/P25
1.1
1.5
1.0
M56
*
M64.2
*
5.3
2.6
2.2
5.3
1.5
6.2
2.2
2.7
1.0
6.5
1.1
R1a*
M269
M124
3.0
1.1
Q
M207
15.2
1.1
4.5
N1
M17
1.1
L3
*
PK3
*
3.0
1.5
*
SRY10831.2
M70
1.1
2.4
DYS445=6
M242
M173
1.2
3.3
M214/LLY22g
M74
M45
3.5
1.2
6.3
4.3
M357
M9
3.3
3.6
J2a1b*
M92
M280
M76
M317
M11
M20
1.8
J2a1b1
*
M68
M12
2.2
J2a1a
M47
M410
M172
M89
3.6
J1*
*
1.1
1.6
I2a2
M26
12f2
M304
2.4
I2a1a
1.8
R1a1*
16.3 10.5
R1b1*
2.2
9.1
1.6
R1b1b2
17.4 14.0 18.2 18.8
13.6 15.5 12.2 37.9 27.0 38.7 10.4 56.6 41.3
56.6 50.0 10.6 13.2
1.3
6.2
3.6
2.2
12.4 21.3 41.8 15.1 33.3
5.3
18.2
2.2
R2
4.3
T
a
H
1.8
3.3
1.9
1.3
1.1
9.1
7.9
4.5
7.9
1.5
0.910 0.904 0.870 0.822 0.821 0.776 0.443 0.773 0.802 0.768 0.795 0.652 0.714 0.630 0.714 0.862 0.888
European Journal of Human Genetics
Transition to farming in the Balkans
V Battaglia et al
824
Both its main branches, J-M410 and J-M12/M102*, were
observed; although the first is scattered in different
sub-clades (J-M67, J-M92 and J-DYS445-6) with distinct
local patterns, the second is most represented by
J-M241.
The PC analysis, from the perspective of population Hg
frequencies (Figure 3), reveals a tight cluster of populations
not comprising southern Balkan and Caucasian
groups. Common to this cluster are lower frequencies
of Hgs, G-M201 and J-M410, and higher frequencies of Hgs,
I-M423, E-V13 and J-M241. Whereas the first two are
primarily Middle Eastern Hgs and have been shown to
be associated with the early Neolithic colonization of
Crete,38,46 Italy,47,48 and southern Caucasus, I-M423, E-V13
and J-M241, in spite of parallel Balkan patterns of
distribution, have clearly different origins.30,31,38
Their comparison can therefore provide insights into the
complex interaction between the European Mesolithic
foragers and the Middle Eastern Neolithic farmers
respectively, still display high values in northern Balkans
and sensibly decrease southward. R-M269 chromosomes
are common in the Balkans and Anatolia and, according to
the observed internal divergence of their 49a,f branches,29
most likely predated the origin of agriculture. However, the
current lack of informative Hg sub-division within these
populations (Figure 2) does not allow, at this time, to
evaluate the role of R-M269 chromosomes during the
transition to agriculture.
E-M35 is the only branch of Hg E observed in this survey.
It is prevalently represented by E-M78 chromosomes,
almost completely (490%) belonging to the recently
described30 E-V13 sub-clade. Only four E-M78*, which do
not belong to any already described sub-clade, have been
observed in the southern Balkans. Two of them (from
Greece) turned out to be characterized by the mutation
M521 and therefore represent a new M78 lineage.
The majority of the Balkan Hg J Y chromosomes
belong to the J-M172 sub-Hg and range from 2% to 20%.
Gr
Alb-F
Alb-A
Sr-B
Mac-Gr
Cr-H
Bs-B
Cz
Slo
Pl
Uk Hun
Cr-B
Cr-O
NE-I
Bk
Geo
Figure 3 PC analysis performed using haplogroup frequencies in the populations of this study. Gr, Greeks; Mac-Gr, Macedonian Greeks; Alb-A,
Albanians; Alb-F, Albanians from FYROM; Sr-B, Bosnia-Serbs; Bs-B, Bosniacs; Cr-B, Bosnia-Croats; Cr-O, Croats from Osijek; Cr-H, Croats of Croatia; Slo,
Slovenians; NE-I, northeast Italians; Hun, Hungarians; Cz, Czechs; Pl, Poles; Uk, Ukrainians; Geo, Georgians; Bk, Balkarians. Thirty-four percent of the
total variance is represented. Insert illustrates the contribution of each haplogroup.
European Journal of Human Genetics
Transition to farming in the Balkans
V Battaglia et al
825
Discussion
during the transition to farming society in the Balkans.
These Hgs, although characterized by different distribution
patterns of frequency and variance (Figure 4),
display networks of microsatellite haplotypic variation
(Supplementary Figures S1 – S3), all consistent with a
Balkan expansion.
Various episodes of population movement have affected
southeast Europe, and the role of the Balkans as a longstanding gateway to Europe from the Near East is
illustrated by the phylogenetic unification of Hgs I and J
by the basal M429 mutation.31 This evidence of common
75
70
0.30
65
60
55
0.25
50
45
0.20
40
35
0.15
30
25
20
0.10
15
10
I-M423
0.05
5
0
0.00
35
0.35
30
0.30
25
0.25
20
0.20
15
0.15
10
0.10
5
0.05
0
0.00
E-V13
0.25
15
0.20
10
0.15
0.10
5
0.05
J-M241
0
0.00
Figure 4 Frequency (left) and variance (right) distributions of the main Y-chromosome haplogroups, I-M423, E-V13 and J-M241, observed in this
survey. Frequency data are reported in Figure 2, variance data are relative to the examined microsatellite reported in the Supplementary Table S2. We
acknowledge that interpolated spatial frequency surfaces should be viewed with caution because of sample size.41 K Data from this study. Frequency
and variance values were assigned to sample-collection places (dots). Population samples (geographically close) with less than five observations were
pooled and the corresponding variance assigned to a middle position of the pooled sample locations. þ Data from the literature.13,23,27,28,36,45,49 – 54
European Journal of Human Genetics
Transition to farming in the Balkans
V Battaglia et al
826
ancestry suggests that ancestral IJ-M429* Y chromosomes
probably entered Europe through the Balkan route sometime before the Last Glacial Maximum. They subsequently
evolved into Hg J in the Middle East and Hg I in Europe in a
typical disjunctive phylogeographic pattern. Such a geographic corridor is likely to have experienced additional
subsequent gene flows, including the migration of agricultural colonists from the Middle East. Pottery is a useful
proxy for the spread of farming both spatially and
temporally. The first appearance of pottery in the Adriatic
region was in Corfu at 6500 BC and reached the northern
most Adriatic B1000 years later.21 Its dispersal provides a
comparative template for spatial and temporal patterns of
Y chromosome Hg diversity observed in this area.
Hg J is most common (B50%) in the Middle East and
Anatolia,27,29,47 with a spread zone spanning from northwest Africa to India.12,55 It has been related to different
Middle Eastern migrations.12,56 In addition to Hg J-M410,
Hg G-P15 chromosomes, which are also common in
Anatolia,29 have been implicated in the colonization and
subsequent expansion of early farmers in Crete, the Aegean
and Italy.38,46 – 48 Earlier studies have concluded that the
J-M410 sub-clades, J-DYS445-6 and J-M67, are linked to the
spread of farming in the Mediterranean Basin,38,47 with a
likely origin in Anatolia.29 Interestingly, J-DYS445-6 and
J-M92 (a sub-lineage of M67), both have expansion times
between 7000 and 8000 years ago (Table 1), consistent with
the dating of the arrival of the first farmers to the Balkans.
The first detection of milk residue in ceramic pottery
occurs in sites from northwest Anatolia 7000 – 8500 years
ago,58 an age that approximates the Hg-expansion times.
Regarding Hg J-M12/M102, which is discernable from
India to Europe, the M12/M102* chromosomes display a
very high YSTR diversity, whereas on the other hand, the
J-M241 sub-lineage has low diversity (Table 1) in the
Balkans, indicating different demographic histories.
Although Hg J-M241 shows high variance in India,37 its
place of origin is still uncertain. As J-M241 has older
expansion times in Sicily, Apulia and Turkey (Table 2), it
may have arrived in the Balkans from elsewhere.
On the other hand, the expansion times of Hg V13
(Table 3) are consistent with a late Mesolithic time frame.
The Greek Mesolithic, although different in its material
culture from the Natufian Mesolithic of the Levant, bears
some resemblance to the Mesolithic of southern Anatolia.60 This archaeological congruence between the Mesolithic of the Balkans and southern Anatolia may mirror the
similar E-V13 expansion times observed for Konya,
Franchthi Cave and Macedonian Greece, all approximately
9000 years ago. Moreover, E-V13 YSTR-related data from
Bulgaria and Macedonia,28 both with a variances of 0.28,
suggest an expansion time of approximately 10 000 years
ago. It is likely that the origin of V13 occurred somewhere
within the zone of these sample collections. In addition,
it is also worth noting that in the Anatolian region
European Journal of Human Genetics
Table 1 Ages of microsatellite variation and mean
variance of microsatellite loci within haplogroups
Haplogroup (N)
I-P37*-DYS388-15 (28)
I-M423 (219)
E-V13 (92)
J-M410* (23)
J-M67 (42)
J-M67* (32)
J-M92 (10)
J-DYS445-6 (10)
J-M12 (51)
J-M12/102* (15)
J-M241 (34)
Age of microsatellite
variationa ±SE b (ky)
Mean variance a
4.0±1.7
7.8±2.3
5.9±1.7
15.8±3.8
11.0±2.4
11.2±2.9
7.0±1.9
7.7±2.6
7.4±1.4
12.3±2.7
4.8±1.3
0.107
0.198
0.155
0.410
0.287
0.298
0.187
0.164
0.191
0.332
0.121
a
Values computed over the following microsatellite loci: DYS19,
DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393,
DYS439 and DYS460 (values of YCAa,b were excluded from the
calculation because of the impossibility of assigning each allele to one
or the other locus of this ambiguous system). Ages of microsatellite
variation were estimated as reported in Sengupta et al.37 Owing to the
small sample sizes, the estimates of the expansion time should be
considered indicative.
b
SE was computed over loci.43,57
Table 2 Ages of microsatellite variation and mean
variance of microsatellite loci within J-M241 haplogroup
in Turkey, the Balkans and Italy
Region (N)
Turkeyc (5)
Albaniad (8)
FYROMd (9)
Greeced,e (5)
Central Italyd (5)
Apuliad (5)
Sicilyf (11)
Age of microsatellite
variationa ±SE b (ky)
10.1±3.4
5.4±2.5
2.4±1.2
2.9±1.2
5.8±1.4
9.6±3.4
10.0±5.4
a
Values computed over the following microsatellite loci: DYS19,
DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393,
DYS439 and DYS460 (values of YCAa,b were excluded from the
calculation because of the impossibility of assigning each allele to one
or the other locus of this ambiguous system). Ages of microsatellite
variation were estimated as reported in Sengupta et al.37 Owing to the
small sample sizes, the estimates of the expansion time should be
considered indicative.
b
SE was computed over loci.43,57
c
Cinnioglu et al29 (region 5 (n ¼ 2), region 6 (n ¼ 1), region 9 (n ¼ 2));
values computed with DYS461 in place of DYS460.
d
Present paper.
e
Greeks (n ¼ 4), Macedonian Greek (n ¼ 1).
f
Di Gaetano et al.59
of supposed Einkorn wheat origin61 (region 5 of
Cinnioglu et al29), only one V13 chromosome out of 43 is
found (PA Underhill, unpublished data). Therefore, as no
evidence at present supports the association of E-V13 Hg
with the attested origin of farming in southeast Anatolia,
the possibility of farming adoption by Balkan E-V13associated people is plausible. The low E-V13 frequency
Transition to farming in the Balkans
V Battaglia et al
827
Table 3 Ages of microsatellite variation and mean
variance of microsatellite loci within E-V13 haplogroup in
Turkey and Greece
Region (N)
Konya-Turkeyc (10)
Macedonian Greecec (8)
Greecec (8)
Nea Nikomedeia (North Greece)d (6)
Seklo and Dimini (Central Greece)d (20)
Lerna and Franchthi Cave (South Greece)d (20)
Age of
microsatellite
variationa
±SE b (ky)
9.4±2.9
8.5±4.5
4.8±1.6
8.6±4.0
4.3±1.8
9.2±4.3
a
Values computed over the following microsatellite loci: DYS19,
DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393,
DYS439 and DYS460 (values of YCAa,b were excluded from the
calculation because of the impossibility of assigning each allele to one
or the other locus of this ambiguous system). Ages of microsatellite
variation were estimated as reported in Sengupta et al.37 Owing to the
small sample sizes, the estimates of the expansion time should be
considered indicative.
b
SE was computed over loci.43,57
c
Present paper.
d
King et al.38
and STR variation observed in Crete38 indicate that if the
first Neolithic colonists came from central Anatolia, they
did not bring this Hg. The two more recent expansion
times for V13 for Greece and Sesklo and Dimini (Table 3),
dating to the Bronze Age, possibly reflect a more recent
integration of some V13 chromosomes into the populations of the first farmers represented by J-M410 and
G-M201 lineages. Both the lack of any plausible Middle
Eastern source of E-V13 during either the early Neolithic or
Bronze Age and the age of microsatellite variation observed
are consistent with E-V13 chromosomes reflecting a
Mesolithic heritage as suggested by King et al.38
As reported earlier,28 both J-M12 and E-V13 radiation
patterns overlap geographically in the Balkans (Figure 4).
Although J-M12 chromosomes were not genotyped for
M241 by Cruciani et al,28 the low YSTR diversity observed
suggests that these are predominantly M241 derivatives.
The difference between E-V13 and J-M241 (Table 1)
indicates that both E-V13 frequency and haplotype
diversity would have been greater than J-M241 components just before the episode of population growth. This
also is the case when the dating is carried out by
disregarding the mutational steps connecting the three
haplotypes that, including Turkish samples (Supplementary Figure S2), can be considered as founders.62 Whether
or not E-V13 and J-M241 participated in the same
demography remains uncertain.
The presence of E-M78* Y chromosomes in the Balkans
(two Albanians), previously described virtually only in
northeast Africa, upper Nile,28,63 gives rise to the question
of what the original source of the E-M78 may have been.
Correlations between human-occupation sites and radiocarbon-dated climatic fluctuations in the eastern Sahara
and Nile Valley during the Holocene64 provide a framework for interpreting the main southeast European centric
distribution of E-V13. A recent archaeological study reveals
that during a desiccation period in North Africa, while the
eastern Sahara was depopulated, a refugium existed on the
border of present-day Sudan and Egypt, near Lake Nubia,
until the onset of a humid phase around 8500 BC
(radiocarbon-calibrated date). The rapid arrival of wet
conditions during this Early Holocene period provided an
impetus for population movement into habitat that was
quickly settled afterwards.64 Hg E-M78* representatives,
although rare overall, still occur in Egypt, which is a hub
for the distribution of the various geographically localized
M78-related sub-clades.28 The northward-moving rainfall
belts during this period could have also spurred a rapid
migration of Mesolithic foragers northwards in Africa, the
Levant and ultimately onwards to Asia Minor and Europe,
where they each eventually differentiated into their
regionally distinctive branches.
Differently from the earlier discussed Hgs, I-M423
represents the southeast European autochthonous clade
of I-P37.2. Its distribution reaches Anatolia, where, however, it is only sporadically observed (2.6%, updated from
Rootsi et al23). Also, virtually, all the I-P37.2* paragroup
members identified in this survey harbouring the peculiar
DYS388-15 trinucleotide repeat motif (not observed in any
other Hg I clade) likely represent a new rare P37.2
sub-clade. Their distribution (Supplementary Table S1)
and the associated YSTR variation age of B4000 years
(Table 1) suggest that they expanded demographically,
perhaps from central European regions during the Bronze
Age. In this scenario, the only I-P37.2* chromosome
observed in Albania, not characterized by the unusual
DYS388-15 repeat motif marker, could either represent the
consequence of a reversion event back to the ancestral
allele or be a rare representative of the ancestral P37.2 state.
The network of the STR haplotypes identified in 222 Y
chromosomes belonging to the I-M423 Hg (Supplementary
Figure S1) is characterized by a star-like shape centred on
the most frequent and diffused haplotype that is present in
all Balkan populations. The marginal positions occupied by
the three Turkish chromosomes are in agreement with a
recent gene flow. The age of accumulated microsatellite
variation associated with Hg I-M423 (Table 1) dates to
around 8000 years ago (Early Holocene). Thus, although
Hgs G and J mark the successful colonization and
subsequent demic expansions of Neolithic pioneers to
these regions, consistent with a wave of advance,19,65 the
widespread adoption of farming by Mesolithic huntergatherers in the Balkans and Central Europe is recorded in
the autochthonous Hg I-M423.
These data indicate the complex interactions between farmers and foragers rather than the large-scale
European Journal of Human Genetics
Transition to farming in the Balkans
V Battaglia et al
828
replacement of hunter-gatherers by pioneering agriculturalists during the spread from the Neolithic to the southeast
Europe. The data also indicate that I-M423 and
probably also E-V13 representatives would have been well
established in the Balkans before the arrival of a nucleus of
pioneering agriculturalists.
Thus, unlike Crete, southern and central Italy and the
southern Caucasus, the cultural transmission of the Neolithic package played an important role. Either the initial
G and J2 Hg agriculturalists who colonized the Balkans at
first flourished but later diminished in a similar manner to
that proposed regarding the Linearbandkeramik in central
Europe66 or the package was rapidly and robustly adopted
by local Mesolithic people in the southern Balkans
(plausibly characterized by E-V13), who underwent a demic
expansion and a subsequent range expansion to the eastern
Adriatic. These former foragers who had recently acquired
the Neolithic tradition participated in ‘leapfrog’ colonizations up the Adriatic, where they eventually transmitted
agricultural practices to resident Mesolithic populations
represented by I-M423 chromosomes.
Interestingly, the derived Y-chromosome scenario
strongly recalls the fourth PC synthetic map of Europe
calculated on gene frequencies at 95 nuclear loci,11 which
displays a centre in the southern Balkans and a large
surrounding area that terminates with a ‘propagule’ to the
northeast of the central Balkans. On the basis of this
observation, our assumptions could provide a possible
interpretation of the described expansion centred in the
southern Balkans.
Conclusion
This study provides a model that elevates the role of
migratory foragers with remote eastern Saharan ancestry
who, once established in Asia Minor with their own
derived genetic signature, were destined to become the
earliest converts to farming and the adherents of its further
spread into Europe. Such an interpretation finds support in
the ‘dispersal model’ of Impressed Ware in which the
‘Neolithic package’ was acquired by native groups and
subsequently diffused by interactions between farmers and
foragers.67 Although southeast Europe shows considerable
archaeological evidence of the Neolithic transition,20 our
Y-chromosome results provide biological evidence of
complexity21 in the transition to farming in terms of the
contrasting influences of pioneering agriculturalists and
Mesolithic foragers.
Acknowledgements
We are grateful to all the donors for providing the blood samples and to
all the people and institutions that contributed to their collection.
These include the Institute for Transfusion and the Institute for
Clinical Biochemistry of Sarajevo, the Cantonal Hospital and the
Primary Care Institution of Mostar, the Clinical Hospital Centre and
European Journal of Human Genetics
the Institute for Transfusion of Banja Luka, the Regional Hospitals of
Doboj and Bijeljina, from Bosnia-Herzegovina, and Professor Simun
Andjelinovic and his team from the University Hospital of Split, from
Croatia. We are also grateful to Franco Lo Curto for having provided
us with the Italian samples. We thank the three anonymous reviewers
for useful comments and suggestions. This research was supported by
the Italian Ministry of the Foreign Affaires (to OS), Compagnia di San
Paolo (to AT and OS), the Italian Ministry of the University: Progetti
Ricerca Interesse Nazionale 2007 (to AT and OS) and Estonian Science
Foundation Grant no. 7445 (to SR).
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Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)
European Journal of Human Genetics