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Pig Domestication and Human-Mediated Dispersal in Western
Eurasia Revealed through Ancient DNA and Geometric
Morphometrics
Claudio Ottoni,
y,1,2,3
Linus Girdland Flink,
y,4,5,6
Allowen Evin,
7,8
Christina Geo¨rg,
9,10
BeaDeCupere,
11
Wim Van Neer,
1,11
La
´
szlo
´
Bartosiewicz,
12
Anna Linderholm,
4
Ross Barnett,
4
Joris Peters,
13
Ronny Decorte,
2,3
Marc Waelkens,
14
Nancy Vanderheyden,
2
Franc¸ois-Xavier Ricaut,
15
Canan C¸akırlar,
11,16
O
¨
zlem C¸evik,
17
A. Rus Hoelzel,
5
Marjan Mashkour,
8
Azadeh Fatemeh Mohaseb Karimlu,
8
Shiva Sheikhi
Seno,
8
Julie Daujat,
7,8
Fiona Brock,
18
Ron Pinhasi,
19
Hitomi Hongo,
20
Miguel Perez-Enciso,
21
Morten Rasmussen,
22
Laurent Frantz,
23
Hendrik-Jan Megens,
23
Richard Crooijmans,
23
Martien Groenen,
23
Benjamin Arbuckle,
24
Nobert Benecke,
10
Una Strand Vidarsdottir,
25
Joachim Burger,
9
Thomas Cucchi,
7,8
Keith Dobney,
7
and Greger Larson*
,4
1
Center for Archaeological Sciences, Department of Earth and Environmental Sciences, University of Leuven, Leuven, Belgium
2
Laboratory of Forensic Genetics and Molecular Archaeology, Department of Forensic Medicine, UZ Leuven, Belgium
3
Department of Imaging & Pathology, University of Leuven, Leuven, Belgium
4
Durham Evolution and Ancient DNA, Department of Archaeology, Durham University, Durham, United Kingdom
5
School of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom
6
Earth Sciences Department, Natural History Museum, London, United Kingdom
7
Department of Archaeology, University of Aberdeen, Aberdeen, Scotland, United Kingdom
8
UMR 7209 CNRS/Muse
´
um National d’Histoire Naturelle, Paris, France
9
Institute of Anthropology, Johannes Gutenberg University Mainz, Mainz, Germany
10
German Archaeological Institute, Eurasian Division, Berlin, Germany
11
Royal Belgian Institute of Natural Sciences, Brussels, Belgium
12
Institute of Archaeological Sciences, Department of Archaeometry and Archaeological Method, Lora
´
nd Eo¨tvo¨s University,
Budapest, Hungary
13
Department of Veterinary Sciences, Institute of Palaeoanatomy, Domestication Research and the History of Veterinary Medicine,
Ludwig Maximilian University Munich, Munich, Germany
14
Sagalassos ArchaeologicalProject,UniversityofLeuven,Leuven,Belgium
15
CNRS FRE 2960, Laboratoire AMIS d’Anthropologie Mole
´
culaireetImageriedeSynthe
`
se, Universite
´
de Toulouse, Toulouse, France
16
Institute of Archaeology, University of Groningen, Groningen, The Netherlands
17
Faculty of Letters, Department of Archaeology,UniversityofThrace,Edirne,Turkey
18
Oxford Radiocarbon Accelerator Unit, Research Laboratory for Archaeology, University of Oxford, Oxford, United Kingdom
19
Molecular Population Genetics, Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland
20
Department of Evolutionary Studies of Biosystems, Graduate University for Advanced Studies, Hayama, Kanagawa, Japan
21
Institucio
´
Catalana de Recerca i Estudis Avanc¸ats (ICREA), Centre for Research in Agricultural Genomics (CRAG) and Facultat de
Veterinaria, Department of Animal Science, Universitat Autonoma Barcelona, Bellaterra, Spain
22
Centre for GeoGenetics, Copenhagen, Denmark
23
Wageningen University, Animal Breeding and Genomics Centre, Wageningen, The Netherlands
24
Department of Anthropology, Baylor University
25
Department of Anthropology, Durham University, Durham, United Kingdom
y
These authors contributed equally to this work.
*Corresponding author: E-mail: greger.larson@durham.ac.uk.
Associate editor: Sarah Tishkoff
Article
ß The Author 2012. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Open Access
824 Mol. Biol. Evol. 30(4):824–832 doi:10.1093/molbev/mss261 Advance Access publication November 22, 2012
at Durham University on March 26, 2013http://mbe.oxfordjournals.org/Downloaded from
Abstract
Zooarcheological evidence suggests that pigs were domesticated in Southwest Asia 8,500 BC. They then spread across
the Middle and Near East and westward into Europe alongside early agriculturalists. European pigs were either domes-
ticated independently or more likely appeared so as a result of admixture between introduced pigs and European wild
boar. As a result, European wild boar mtDNA lineages replaced Near Eastern/Anatolian mtDNA signatures in Europe and
subsequently replaced indigenous domestic pig lineages in Anatolia. The specific details of these processes, however,
remain unknown. To address questions related to early pig domestication, dispersal, and turnover in the Near East, we
analyzed ancient mitochondrial DNA and dental geometric morphometric variation in 393 ancient pig specimens rep-
resenting 48 archeological sites (from the Pre-Pottery Neolithic to the Medieval period) from Armenia, Cyprus, Georgia,
Iran, Syria, and Turkey. Our results reveal the first genetic signatures of early domestic pigs in the Near Eastern Neolithic
core zone. We also demonstrate that these early pigs differed genetically from those in western Anatolia that were
introduced to Europe during the Neolithic expansion. In addition, we present a significantly more refined chronology for
the introduction of European domestic pigs into Asia Minor that took place during the Bronze Age, at least 900 years
earlier than previously detected. By the 5th century AD, European signatures completely replaced the endemic lineages
possibly coinciding with the widespread demographic and societal changes that occurred during the Anatolian Bronze
andIronAges.
Key words: pig domestication, wild boar, Neolithic, phylogeography.
Introduction
The transition from hunting and gathering to agriculture is
one of the most important biocultural processes in human
history (Diamond and Bellwood 2003). Though this transition
took place in numerous locations across the globe
(Purugganan and Fuller 2009), the earliest stages of animal
domestication in western Eurasia are recorded in the north-
ern Fertile Crescent in the 9th millennium BC (Zeder 2008,
2011). Recent evidence suggests that the establishment of
food production was followed by rapid population growth
(Bocquet-Appel 2011) and agropastoral economies often
spread through demic diffusion (Gignoux et al. 2011). This
was certainly the case for Southwest Asia where, following the
development of agricultural economies, farmers migrated
into Europe during the Neolithic bringing with them domes-
tic crops and livestock (Bramanti et al. 2009).
The increased resolving power of new genetic and morpho-
metric techniques has allowed for the identification of
fine-scale population differences across wide temporal and
geographic contexts and the capability of tracking these dif-
ferences through time and space. For example, DNA derived
from modern animal (Naderi et al. 2008; Chessa et al. 2009)and
plant (Myles et al. 2011; van Heerwaarden et al. 2011)domes-
ticates have been used to unravel geographic origins and dis-
persal patterns. The use of modern data alone, however, can be
problematic. Past domestic populations often underwent
dramatic bottlenecks, demographic fluctuations (including
complete replacement), and admixture with wild relatives,
thus obscuring the genetic signatures of earlier populations
(Larson, Albarella, et al. 2007; Larson et al. 2012).
Analyses of ancient DNA (aDNA) have overcome this issue
by typing (pre)historic populations and allowing for the direct
observation of genetic signatures through time. This
approach has generated new insights related to past genetic
diversity (Fernandez et al. 2006), wild–domestic hybridization
(Bollongino et al. 2008), and human migration (Larson,
Albarella, et al. 2007; Larson, Cucchi, et al. 2007). Similarly,
novel morphometric methods, including geometric
morphometrics (GMM), have been successfully applied to
document changes between wild and domestic animals
(Larson, Cucchi, et al. 2007) and plants (Terral et al. 2010)
and to track the phenotypic evolution of past populations
(Cucchi et al. 2009).
Zooarcheological evidence demonstrates that wild boar
were domesticated independently in the Near East by at
least 8,500 BC (Conolly et al. 2011; Ervynck et al. 2001). By
examining pig bones recovered from the Pre-Pottery
Neolithic layers at Cayonu Tepesi (10,000–6,300 BC, Erim-
O
¨
zdog
ˇ
an 2011) in southeastern Anatolia, Ervynck et al.
(2001) identified a disproportionate decrease in molar
tooth size over two millennia. They interpreted this pattern
to be the result of a long-term in
situ domestication process
that led to the emergence of morphologically domestic pigs
by 6,800 BC (early Pottery Neolithic). Similar, though conten-
tious, claims for human controlled pig breeding between
8,200 and 7,500 BC have been made at Cafer Ho¨yu
¨
k
(Helmer 2008)andNevaliC¸ori (Peters et al. 2005)insouth-
eastern Anatolia. The introduction of wild boar to Cyprus by
at least 9,700–9,400 BC, however, indicates that humans were
actively manipulating wild boar populations for millennia
before the emergence of domestic pigs (Vigne et al. 2011;
Vigne et al. 2009).
Though the zooarcheological evidence demonstrates that
pigs were first domesticated in Southwest Asia, virtually all
modern domestic pigs from western Eurasia possess mito-
chondrial signatures similar (or identical) to European wild
boar (Larson et al. 2005). Ancient DNA extracted from early
Neolithic domestic pigs in Europe resolved this paradox by
demonstrating that early domestic pigs in the Balkans and
central Europe shared haplotypes with modern Near Eastern
wild boar (Larson, Albarella, et al. 2007). The absence of Near
Eastern haplotypes in pre-Neolithic European wild boar sug-
gested that early domestic pigs in Europe must have been
introduced from Anatolia by the mid 6th millennium BC
before spreading to the Paris basin by the early 4th millen-
nium BC (Larson, Albarella, et al. 2007).
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By 3,900 BC, however, virtually all domestic pigs in Europe
possessed haplotypes originally only found in European wild
boar. This genetic turnover may have resulted from the accu-
mulated introgression of local female wild boar into imported
domestic stocks or from an indigenous European domestica-
tion process (Larson, Albarella, et al. 2007). After the genetic
turnover had taken place in Europe, aDNA from Armenian
pigs indicated that European domestic pigs were present in
the Near East by the 7th century BC at the end of the Iron Age
where they replaced indigenous Near Eastern domestic
mtDNA lineages (Larson, Albarella, et al. 2007). Crucially,
the archeological record attests to rapid demographic and
societal changes during the Late Bronze Age (1,600–1,200
BC) and Iron Age (1,200–600 BC), including large-scale mi-
grations and the expansion of trade and exchange networks
across the Mediterranean and the Black Sea region (Sagona
and Zimansky 2009).
To establish a more precise geographic and temporal
framework of mitochondrial Sus haplotypes in Anatolia and
to address questions related to the mitochondrial turnover in
Armenia at the end of the Iron Age, we obtained mitochon-
drial sequences from 39 modern wild boar and 393 archeo-
logical wild and domestic pigs from 48 Near Eastern sites
spanning the Pottery Neolithic (7,000 BC) to the 15th
century AD from western Turkey to southwestern Iran
(fig. 1, supplementary fig. S1a and table S1, Supplementary
Material online). We analyzed our novel data alongside pre-
viously published ancient and modern sequences (supple-
mentary table S2, Supplementary Material online). In
addition, we performed a dental morphological assessment
of 46 archeological specimens (with known genetic haplo-
types) using traditional osteometric and GMM methods to
assess the correlation between genetic and morphometric
variation (fig. 2).
Results and Discussion
Genetic Signatures of Early Anatolian Domestic Pigs
Remains of the earliest domestic livestock are found in
Southwest Asia 9,000–8,000 BC (Zeder 2008). Unlike
sheep, goats, and cattle that likely became domesticated
through a prey pathway, pigs (like dogs and cats) probably
followed a commensal pathway that began with an initial
habituation phase before proceeding to a partnership that
ended in controlled breeding (Ervynck et al. 2001; Zeder
2012). The protracted time over which pig domestication
took place likely included a predomestic management
phase that may have been widespread across the region
(Vigne 2011).
We first tested the geographic correspondence between
archeological and genetic evidence for pig domestication by
mapping the geographic distribution of genetic signatures
derived from modern wild boar in Anatolia and the Near
East (fig. 2). A phylogenetic tree, based on 661 bp (base
pairs) of the mitochondrial DNA (mtDNA) control region,
revealed a previously observed topology (Larson, Albarella,
et al. 2007) that included three well-supported phylogeo-
graphic clades: two clades with pigs found exclusively in the
Near East (NE1 and NE2) and a European clade. Of the 192
novel ancient sequences (supplementary table S4,
Supplementary Material online), all those that possessed
one of three Anatolian/Near Eastern mtDNA lineages
(Arm1T, Y1, or Y2) (Larson, Albarella, et al. 2007)belonged
to the NE2 clade (supplementary fig. S2a, Supplementary
Material online). In modern animals, the NE1 clade has
been identified only in Near Eastern wild boar (supplemen-
tary table S2, Supplementary Material online) and is yet to be
found in any modern or ancient domestic pigs. The geo-
graphic distributions of the NE1 and NE2 clades overlap
only in Iran, Iraq, and in the Caucasus (fig. 2 and supplemen-
tary fig. S2b, Supplementary Material online). Given the ab-
senceofNE1boarinAnatolia,andthecompletelackofNE1
signatures in modern or ancient domestic pigs, it is plausible
that the first domestic pigs in Anatolia belonged to the NE2
clade.
To establish the specific mtDNA lineage of one of the
earliest domestic pig populations, we successfully extracted
and sequenced DNA from one specimen excavated from an
early Pottery Neolithic layer (6,800–6,500 BC) at C¸ayo¨nu
¨
Tepesi, representing the final stages of the proposed in
situ domestication process (Hongo and Meadow 1998;
Ervynck et al. 2001). This specimen possessed the Arm1T
haplotype that (along with the Y1 haplotype) is the dominant
signature in other Neolithic and Bronze Age Anatolian Sus
remains (fig. 1).
We then contrasted the frequencies of NE2 lineages across
Southwest Asia (fig. 1). Like numerous other pig clades dis-
tributed across the Old World (Larson et al. 2005), the distri-
butions of Y1 and Arm1T are geographically partitioned. Y1 is
significantly more frequent in western Anatolia (Fisher’s exact
test; P <
0.001), whereas Arm1T has a much wider distribu-
tion
and dominates in southeastern Anatolia, Armenia, Syria,
Georgia, and Iran (Fisher’s exact test; P < 0.001) (fig. 1).
Despite the limited sample size, the combined zooarcheo-
logical and genetic data suggest that at least the Arm1T lin-
eage was present in the first domestic pigs in western Eurasia.
Anatolian Origins of European Neolithic Pigs
A previous DNA study of modern and ancient, wild and do-
mestic pigs demonstrated that the earliest domestic pigs in
Europe possessed one of two NE2 clade haplotypes: Y1 or Y2.
Because both of these haplotypes clustered with others found
in modern Anatolian and Near Eastern wild boar, the authors
concluded that Y1 and Y2 lineages were indigenous to
Anatolia and were later transported into Europe by migrating
farmers at the onset of the European Neolithic. The lack of
ancient Anatolian samples, however, precluded a direct dem-
onstration of that assertion (Larson, Albarella, et al. 2007).
The ancient Anatolian data presented here reveal that
both morphologically wild and domestic Neolithic pigs (dis-
tinguished using logarithmically indexed linear osteometrics)
possessed Y1 haplotypes (fig. 1, supplementary fig. S3a and
table S5, Supplementary Material online) and were present at
three archeological sites in western Anatolia: Bademag
˘
acı
(6,400–6,100 BC) (De Cupere et al. 2008), Ulucak Ho¨yu
¨
k
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FIG.1. A spatiotemporal depiction of ancient pig haplotypes. Rows represent eight chronological periods, and columns pertain to sites organized along a longitudinal axis from west to east. Approximate locations
of the archeological sites from which the samples are derived are shown as numbered circles on maps beneath the horizontal axis. Asterisks indicate directly AMS-dated samples. The question mark signifies not
enough material was available for AMS dating. Slashed boxes indicate samples on which GMM analyses were performed. Pie charts to the right of each row summarize the haplotype frequencies for each
chronological period across all sites. Columns pertain to one or two sites except for two columns that consist of several sites: Armenia (Sevkar-4, Areni-1, Khatunarkh, Shengevit, Lchashen, Tmbatir, Pilorpat,
Beniamin, and Tsakaektsi) and Iran (Qaleh Rostam, Qare Doyub, Qelı
¯
ch Qo
¯
ı
¯
neq, Dasht Qal’eh, Doshan Tepe, Malyan, Mehr Ali, Chogha Gavaneh, and Gohar Tepe).
827
Ancient DNA and GMM Data from Near Eastern Pigs
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(6,400–5,900 BC) (C¸akırlar 2012), and Mentes¸e Ho¨yu
¨
k
(6,000 BC). The presence of these lineages corroborates
the supposition that the earliest domestic pigs in Europe
originated from populations originally domesticated in the
Near East, conclusively linking the Neolithization of Europe
with Neolithic cultures of western Anatolia (Larson, Albarella,
et al. 2007; O
¨
zdog
ˇ
an 2005).
The Y1 haplotype does not appear to be associated with
either wild boar or early domestic pigs in eastern parts of
Anatolia, and it is completely absent in Iranian and
Caucasian pigs where the Arm1T lineage dominates.
Intriguingly, though Arm1T is present in early domestic pigs
in eastern Turkey, this lineage has yet to be identified in either
ancient or modern European pigs. This temporal and geo-
graphic pattern (fig. 1) could be the result of two different
processes. First, it is possible that genetically differentiated
wild boar populations in eastern and western Anatolia were
domesticated independently. More likely, however, is a
(a)
(b)
FIG.2. Panel (a) depicts a schematic phylogenetic tree derived from an alignment of 267 modern wild boar from western Eurasia. Red, green, and gray
triangles refer to the well-supported European, Near Eastern 1 (NE1), and Near Eastern 2 (NE2) clades, respectively. Branches supported by P > 0.99 are
indicated by a black circle. A more detailed representation of the tree including support values is presented in supplementary figure S2a, Supplementary
Material online. The NE2 clade includes all ancient Near Eastern haplotypes depicted in figure 1. Panel (a) also shows the approximate geographic
distribution of modern wild boar belonging to these clades. Areas with overlapping distributions are represented in dark. A more detailed depiction is
presented in supplementary figure S2b, Supplementary Material online. Panel (b) presents molar size (M
2
) and shape (M
2
and M
3
) differences between
ancient pigs assigned to European (red) and Near Eastern (gray) mtDNA clades. Differences in shape calculated along linear discriminant analysis (LDA)
axes are displayed in overlapping shapes in the upper right. The arrow indicates a statistically significant size reduction in the M
2
between European
and Near Eastern pigs. Numbers following “N=” represent sample sizes, and single and triple asterisks represent significance to the P < 0.05 and P < 0.01
levels.
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scenario in which southeastern Anatolian wild boar were ini-
tially domesticated and subsequently transported west out of
the Neolithic “core zone” (O
¨
zdog
˘
an 2011). Then, following
admixture with female wild boar indigenous to western
Turkey, they acquired the local Y1 lineage that prevailed
over the Arm1T lineage in this area.
The route along which domestic pigs traveled to arrive in
western Anatolia remains unknown. The presence of mor-
phologically domestic pig remains by the 7th millennium BC
(Pottery Neolithic layers) at the site of Yumuktepe, in
south-central Turkey (Buitenhuis and Caneva 1998), and at
the early 7th millennium BC layers of Ulucak (C¸akırlar 2012;
C¸ilingirog
˘
lu 2012) near the eastern Aegean coast, contrasted
with the general dearth of pigs during the same period in
central Anatolia (Conolly et al. 2011), however, suggest that
one of the possible routes was along the Mediterranean coast.
Timing and Nature of the Anatolian Pig Turnover
A previous study (Larson, Albarella, et al. 2007)demonstrated
that domestic pigs with mitochondrial haplotypes predomi-
nantly found in Europe replaced mitochondrial lineages in
Armenia that possibly originated from the early domestic
swine herds in the Neolithic core zone by 700 BC. Because
that study did not include ancient pigs from central or west-
ern Anatolia, the scale and timing of this proposed eastward
dispersal and replacement by European domestic pigs re-
mained unresolved.
The temporal and geographic distribution of genetic
haplotypes presented in our study demonstrates that the
first AMS radiocarbon-dated pig with European ancestry
(haplotype A) appeared almost 1,000 years earlier than the
Armenian samples, in a Late Bronze Age context (1,600–
1,440 BC) at Lidar Ho¨yu
¨
k(fig. 1). An apparently even earlier
MiddleBronzeAgespecimenfromthesamesitealsopos-
sessed a European signature, but a direct radiocarbon date for
this specimen could not be obtained.
Our data also show that European pigs are unlikely to have
arrivedinAnatoliabefore2,000BCsincetheEarlyBronzeAge
layers at Bademag
˘
acı and Lidar Ho¨yu
¨
k (in southwestern and
southeastern Anatolia, respectively) only possess indigenous
Near Eastern pig lineages. The frequency of pigs with
European ancestry increased rapidly from the 12th century
BC onwards, and by the 5th century AD, domestic pigs pos-
sessing a Near Eastern genetic signature had all but dis-
appeared across Anatolia and the southern Caucasus.
Though we did not detect European signatures in the ancient
Iranian samples (fig. 1), the eastward spread of European lin-
eages may have continued into Iran later than the Iron Age,
since European lineages have been found in wild caught
modern Iranian samples (Larson, Albarella, et al. 2007).
If European pig haplotypes were present in Anatolia at
<5% before the Middle Bronze Age, our sample size (bino-
mial distribution, n = 73, confidence interval = 95%) would
not have allowed us to detect them. To assess the possibility
that haplotypes so far found exclusively in Europe were indi-
genous to Anatolia and the Near East, we analyzed the mor-
phometric differentiation in molar size and shape between
archeological samples that possessed European and
Anatolian/Near Eastern genetic signatures. Single interbreed-
ing populations have been shown to possess deeply divergent
mitochondrial haplotypes (e.g., yaks [Guo et al. 2006])
demonstrating that maternal genetic differentiation alone is
not sufficient to infer geographic separation. Statistically sig-
nificant phenotypic differences between pigs possessing
Anatolian/Near Eastern and European haplotypes, however,
would indicate that the two populations had been evolving in
isolation from one another and that pigs with a European
genetic signature were not present in Anatolia before being
introduced by people.
A GMM analysis of 46 pigs with known genetic
signatures revealed significant differences in both molar size
(P < 0.01) and shape (P < 0.05) between European and
Anatolian/Near Eastern pigs (fig. 2 and supplementary table
S6, Supplementary Material online). European pigs possessed
overall smaller teeth and proportionally shorter and laterally
widened third molars. The concordance between genetic and
GMM signatures strongly suggests that pigs possessing
European and Anatolian/Near Eastern mtDNA lineages are
morphologically different and that European pigs were, there-
fore, introduced to Anatolia. The DNA evidence suggests that
this process may have taken place (at the latest) during the
Middle to Late Bronze Age, at least 900 years earlier than
previously inferred.
Establishingamoreprecisetemporal and geographic pat-
tern for the initial introduction and subsequent dominance of
European pigs allows for the turnover to be assessed in its
cultural context, though the limited archeological coverage of
pigs in western Anatolia precludes a definitive identification
of an entrance route. Minoans and Mycenaeans may have
initially introduced pigs during the Bronze Age when they
colonized the western Anatolian coast from the 16th to
12th centuries BC. Alternatively, pigs may have been im-
ported by the Hittites (Seeher 2011)whosekingdomex-
tended from central Anatolia to the northern Levant from
the 17th to 13th centuries BC (Bryce 2005). The lack of pigs
possessing European signatures in Bronze Age contexts from
sites in Georgia suggests that pigs did not arrive via the
Caucasus (
fig. 1).
Regardless of the exact routes of their arrival,
European domestic pigs were deliberately introduced into
Anatolia. Within two millennia, European mitochondrial lin-
eages had replaced their Near Eastern domestic counterparts
that were present, and grew in frequency in the early domes-
tic herds of the Near East over the previous 6,000 years.
Conclusions
This study addresses questions regarding the origins and dis-
persal of domestic pigs in Southwest Asia by combining gen-
etic and morphometric analyses often on the same
archeological samples. The data presented here add to the
growing body of evidence suggesting that pig domestication
was a complex, nonlinear process that took place over several
millennia and involved multiple Southwest Asian wild boar
populations (Ervynck et al. 2001; Peters et al. 2005; Vigne et al.
2009).
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More specifically, our data suggest a narrative that begins
with the domestication of pigs in Southwest Asia, at Upper
Tigris sites including C¸ayo¨nu
¨
Tepesi (Ervynck et al. 2001)and
possibly Upper Euphrates sites including Cafer Ho¨yu
¨
k
(Helmer 2008)andNevaliC¸ori (Peters et al. 2005). Early do-
mestic pigs likely possessed at least the Arm1T haplotype
(indigenous to Southwest Asia) and dispersed with humans
as the Neolithic expanded away from these centers. Once
introduced to western Anatolia, domestic swineherds
acquired a mitochondrial signature (Y1) associated with the
local wild boar most likely through admixture. The eastern
Anatolian mitochondrial lineage (Arm1T) became less fre-
quent likely as a result of this admixture process, small popu-
lation sizes, and genetic drift.
This same turnover pattern was evident after pigs possess-
ing domestic Y1 lineages were subsequently transported west
into Europe as far as the Paris Basin (Larson, Albarella, et al.
2007). Once domestic pigs orientating from southeastern
Anatolia but possessing the western Anatolian Y1 haplotype
arrived in Europe, they acquired European wild boar genetic
signatures and lost the Y1 haplotype through introgression of
resident wild boar mitochondria into the imported domestic
pig population. From at least the beginning of the Late Bronze
Age, and possibly several centuries before, domestic pigs of
European wild boar origin now all carrying European wild
boar mtDNA lineages were introduced to Anatolia. On this
occasion, however, swineherds did not take on the genetic
characteristics of the local populations. Instead, by the 5th
century AD, European domestic pig haplotypes had com-
pletely replaced the endemic Y1 and Arm1T lineages.
The movement of domestic pigs from western Anatolia
into Europe is consistent with recent aDNA studies of human
remains that support a demic diffusion model of the initial
Central European Neolithic (Bramanti et al. 2009). Whether
the back migration of European pigs into Anatolia reflects
human migration or trade and exchange remains unclear.
Addressing these and other questions can be accomplished
by incorporating both mitochondrial and nuclear markers in
combination with large-scale morphological analyses.
Material and Methods
Ancient Samples
We analyzed 393 ancient pig bone and tooth specimens
excavated from 48 Anatolian archeological sites (supplemen-
tary fig. S1a and table S1, Supplementary Material online). All
dates are reported in calibrated radiocarbon years BC. The
ages of the archeological remains ranged from the 10th mil-
lennium BC to the medieval era and were determined using
direct accelerator mass spectrometry (AMS) radiocarbon
dating (Beta Analytic Inc. and University of Oxford), strati-
graphic associations with AMS dates, and contextual archeo-
logical evidence. Samples dated at Oxford were treated using
standard protocols as described by Brock et al. (2010).
Genetic Analyses
Analyses were carried out in aDNA facilities in three separate
institutions: the Forensic Genetics and Molecular Archeology
department in Leuven (Belgium), the Department of
Archaeology at Durham University (United Kingdom), and
the Institute of Anthropology in Mainz (Germany) using
standard contamination precautions (Gilbert et al. 2005).
Two 120 bp fragments of the control region of the mito-
chondrial genome were amplified (Larson, Albarella, et al.
2007) and sequenced. Some fragments were cloned (supple-
mentary table S3, Supplementary Material online). Larger
control region fragments (up to 800 bp) were generated
from DNA extracts of 39 modern wild boar from the greater
geographic region (supplementary table S2, Supplementary
Material online). Modern sequences were generated at the
Department of Animal Sciences, Universitat Auto
`
noma de
Barcelona (Spain). A maximum likelihood (ML) tree was cre-
ated from an alignment of 661 bp of the control region of 267
modern wild boar using PhyML (Guindon and Gascuel 2003)
in Geneious 5.5 (Drummond et al. 2011)(supplementary text,
Supplementary Material online). Variations in substitution
model and analytical framework did not affect the topology
of the main clades. Details regarding methods, contamination
avoidance procedures, authentication, and phylogenetic ana-
lyses are described in the supplementary text, Supplementary
Material online.
Reproducible aDNA sequences were obtained from
192 of 393 specimens (48.9%, supplementary table S4,
Supplementary Material online). As expected for ancient sam-
ples (Smith et al. 2003), we observed an inverse correlation
between aDNA success frequency and sample age in nine
time bins (Spearman’s rank correlation r
2
= 0.87, P < 0.001;
supplementary fig. S1b, Supplementary Material online). All
sequences have been deposited in GenBank (JX893958–
JX894188). Variable positions of the two concatenated
ANC1 and ANC2 fragments are presented in supplementary
table S4, Supplementary Material online.AsshownintheML
tree (supplementary fig. S2a, Supplementary Material online),
the majority of the diagnostic variation is present in the ANC1
fragment (Larson, Albarella, et al. 2007). Given the greater
resolving power of this fragment, the relatively low variation
within the ANC2 fragment, and to be consistent with the
terminology developed by Larson et al. (Larson, Albarella, et al.
2007), haplotype assignments for each specimen were based
on the ANC1 terminology. Additional information regarding
haplotype assignment and terminology is present in the sup-
plementary text, Supplementary Material online. The haplo-
type distribution across Southwest Asia was tested using a
Fisher’s exact test. Western Anatolia included sites from
Ulucak Ho¨yu
¨
ktoC¸amlibel Tarlası and eastern Anatolia
included sites from Sirkeli Ho¨yu
¨
ktoC¸ayo¨nu
¨
.
Morphometric Analyses
A total of 62 mandibular molars (25 M
2
and 37 M
3
)from46
ancient specimens were analyzed using traditional biometri-
cal and GMM approaches from standardized photographs
takenfromtheocclusalview(supplementary fig. S3b,
Supplementary Material online). For the traditional metrical
approach, we measured maximum length and width metrics
(two for the M
2
and three for the M
3
, supplementary fig. S3b,
830
Ottoni et al.
.
doi:10.1093/molbev/mss261 MBE
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Supplementary Material online). Two-dimensional GMM
methods (based on coordinates) were used to separately ana-
lyze size and shape variables. In total, we analyzed 7 landmarks
(homologous points) for the M
2
,8fortheM
3
, 68 sliding
semilandmarks (points along the outline of the tooth) for
the M
2
,and91fortheM
3
(Cucchi et al. 2011; Evin et al.
forthcoming)(supplementary fig. S3b, Supplementary
Material online).
Differences between ancient pigs assigned to Near Eastern
mtDNA clade NE2 and European mtDNA clades were tested
using traditional metric and GMM approaches that analyzed
both shape and log-transformed centroid size. Traditional
measurements were analyzed using a log-shape ratio (LSR)
approach (Mosimann and James 1979) that allowed a separ-
ation of shape and isometric size. Differences between clades
were tested with Kruskal–Wallis tests for size indices (centroid
and isometric) paired with boxplots and multivariate analyses
of variance for shape measures (from GMM and LSR) coupled
with linear discriminant analyses paired with leave-one-out
cross-validation percentages. Details are described in the
supplementary text, Supplementary Material online.
Supplementary Material
Supplementary text, figures S1–S3,andtables S1–S6 are avail-
able at Molecular Biology and Evolution online (http://mbe.
oxfordjournals.org/).
Acknowledgments
The authors thank Marcel Amills, Universitat Auto
`
noma de
Barcelona and Hikmet U
¨
n, Etlik Veterinary Control Central
Research Institute, Ankara (Turkey), for modern wild boar
samples, Helen Everett, AHVLA-Weybridge (UK), for DNA
extraction, and Eske Willerslev and Tom Gilbert (both at
the Center for GeoGenetics, Denmark) for assistance with
sequencing and comments on the manuscript. They also
thank Boris Gasparyan and Mary M. Voigt, College of
William and Mary (USA), for access to ancient samples.
This work was supported by grants from the Natural
Environment Research Council (NE/F003382/1), the
Leverhulme Trust (F/00 128/AX), the Belgian Program on
Inter-University Poles of Attraction (IAP 6/22), the Research
Fund of the KU Leuven (BOF-GOA 07/02), the Hercules
Foundation (AKUL/09/16), and the Deutsche Archa¨o-
logisches Institut (DAI). G.L. was supported by an RCUK Aca-
demic Fellowship, J.P. was supported by the German Science
Foundation (PE 424/10-1), M.W. received a Methusalem
grant from the Flemish Ministry for Science Policy, and C.G.
was supported by the German Archaeological Institute, Berlin
(Research Cluster 1, Project 8999539). All research within the
framework of the Sagalassos Project was carried out by the
Centre for Archeological Sciences of the KU Leuven. Arche-
ozoological research in Iran was supported by the Iranian
Cultural Heritage, Handicraft, and Tourism Organization
(ICHHTO); the UMR 7209 of the CNRS/MNHN; the British
Institute for Persian Studies; the Cultural Service of the French
Embassy in Iran; and the University of Edinburgh. Interdisci-
plinary research into the Neolithic of Southwest Iran was
supported by a joint French Japanese Cooperation project
(04.2.444) and JSPS. The archaeozoological study of Neolithic
Ulucak has been funded through an individual fieldwork
grant from the Institute for Aegean Prehistory since 2008
and C.C. was supported by the Belgian Federal Science
Policy Office (postdoctoral fellowship to non-EU researchers).
The authors also wish to thank the assistant director of
Ulucak excavation, C¸iler C¸ilingirog
˘
lu, for collaboration.
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