Silvia Guimaraes
email: biguimaraes@hotmail.com

Benjamin S Arbuckle

Joris Peters
email: joris.peters@palaeo.vetmed.uni-muenchen.de.

Sarah E Adcock
email: adcock@uchicago.edu

Hijlke Buitenhuis
email: hijlkebuitenhuis@gmail.com

Hannah Chazin
email: h.chazin@columbia.edu

Ninna Manaseryan
email: ninna_man@yahoo.com

Hans-Peter Uerpmann
email: hans-peter.uerpmann@uni-tuebingen.de

Thierry Grange
email: thierry.grange@ijm.fr

Eva-Maria Geigl
email: eva-maria.geigl@ijm.fr

©ifce-L Gerard

Ancient DNA shows domestic horses were introduced in the southern Caucasus and Anatolia during the Bronze Age

Keywords: Equus, horse, phylogeography, paleogenetics, ancient DNA, domestication

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Introduction

The domestication of the horse ca. 5,500 years ago represents one of the most important technological innovations in the ancient world [START_REF] Drews | Early Riders: The beginnings of mounted warfare in Asia and Europe[END_REF][START_REF] Levine | Late Prehistoric Exploitation of the Eurasian Steppe[END_REF]. With the harnessing of horsepower, political, economic and social relationships throughout the ancient world were transformed as horses revolutionized transportation and impacted patterns of trade, warfare and migration (e.g., [START_REF] Drews | Early Riders: The beginnings of mounted warfare in Asia and Europe[END_REF][START_REF] Levine | Late Prehistoric Exploitation of the Eurasian Steppe[END_REF][START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF]). Archaeological, organic residue and genetic analyses suggest that the domestic horse originated in the Central Asian steppes, then spread into eastern Europe and later into Southwest Asia (SWA) (e.g., [START_REF] Levine | Late Prehistoric Exploitation of the Eurasian Steppe[END_REF][START_REF] Bökönyi | The earliest waves of domestic horses in East Europe[END_REF][START_REF] Benecke | Der Mensch und seine Haustiere[END_REF][START_REF] Uerpmann | Die Domestikation des Pferdes im Chalkolithikum West-und Mitteleuropas[END_REF][START_REF] Benecke | Equids in Time and Space: Papers in Honour of Véra Eisenmann[END_REF][START_REF] Greenfield | Horses and humans: The evolution of human-equine relationships[END_REF][START_REF] Outram | The earliest horse harnessing and milking[END_REF][START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF]). In particular, data from the Botai hunter-gatherer culture in Kazahkstan suggest that by the mid to late fourth millennium BCE horses were bitted, milked, selected for the TRPM1 coat-color locus, as well as kept in enclosures and were therefore under intensive management [START_REF] Outram | The earliest horse harnessing and milking[END_REF][START_REF] Anthony | Bit wear, horseback riding, and the Botai site in Kazakstan[END_REF][START_REF] Olsen | Prehistoric steppe adaptation and the horse[END_REF][START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF]. A recent study of ancient horse genomes, however, challenged the view that modern domestic horses derived from Central Asia, since "Botai-like" horses were shown to be the ancestors of NE Asian Przewalski's horses but not the main source of ancient or modern domestic horses [START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF][START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF]. Another recent study ruled out a second potential horse domestication center, the Iberian Peninsula, showing that Iberian wild horses went extinct without leaving significant traces in the genomes of modern horses [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF]. There are, however, two more areas that have been proposed as domestication centers for modern horses: the Pontic-Caspian steppe [START_REF] Anthony | The Horse, the Wheel, and Language: How Bronze-Age Riders from the Eurasian Steppes Shaped the Modern World[END_REF], and Anatolia [START_REF] Benecke | Equids in Time and Space: Papers in Honour of Véra Eisenmann[END_REF][START_REF] Arbuckle | Animals and inequality in Chalcolithic Anatolia[END_REF]. Although the former has been long hypothesized as the likely source of domestic horses [START_REF] Anthony | Bit wear, horseback riding, and the Botai site in Kazakstan[END_REF][START_REF] Olsen | Prehistoric steppe adaptation and the horse[END_REF][START_REF] Shaler | Domesticated animals. Their relation to man and to his advancement in civilization[END_REF], the latter region has been poorly explored regarding its role in horse domestication processes [START_REF] Martin | Horses and humans: the evolution of human-equine relationships[END_REF][START_REF] Ünal | 'da At Hititçe Kikkuli at eğitimi metinleri ve "Tavlaya çekmek"le ilgili teknik bir ayrıntı[END_REF][START_REF] Arbuckle | Archaeozoology of the Near East XII[END_REF], despite its long history of wild horse exploitation and its reputation for breeding valuable horses in Classical Antiquity [START_REF] Toynbee | Tierwelt der Antike -Bestiarium romanum[END_REF]. The origin of the domestic horse in Anatolia, and more generally in SWA, continues to represent a complex archaeological puzzle. A combination of textual, iconographic and archaeozoological data suggest that by the mid to late third millennium BCE, domestic horses were introduced from neighboring mountain regions into Mesopotamia (modern Iraq and NE Syria), where they were often referred to in cuneiform texts as ANŠE-KURRA ("donkey of the mountain") [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF][START_REF] Littauer | Wheeled Vehicles and Ridden Animals in the Ancient Near East[END_REF][START_REF] Owen | The first equestrian: An Ur III glyptic scene[END_REF][START_REF] Oates | Prehistoric Steppe Adaptation and the Horse[END_REF][START_REF] Zarins | The Domestication of Equidae in Third-Millennium BCE Mesopotamia[END_REF]. Initially kept only in small numbers, horses rose to prominence across SWA within a few centuries in association with the spread of chariots, a technological innovation of the second millennium BCE [START_REF] Drews | Early Riders: The beginnings of mounted warfare in Asia and Europe[END_REF][START_REF] Clutton-Brock | Horse Power. A History of the Horse and the Donkey in Human Societies[END_REF]. Since horse domestication in the Eurasian steppes, a region historically known for its "horse cultures", likely began in the fourth or perhaps even fifth millennium BCE [START_REF] Anthony | The Horse, the Wheel, and Language: How Bronze-Age Riders from the Eurasian Steppes Shaped the Modern World[END_REF][START_REF] Bökönyi | Akten Internationalen Interdisziplinären Kolloquiums Freie Universität Bernfried Schlerath[END_REF][START_REF] Zeuner | A History of Domesticated Animals[END_REF][START_REF] Uerpmann | Domestication of the horse: when[END_REF], it has long been argued that SW Asian horses are the descendants of these early domesticates, which arrived in the region via poorly understood participation in Pontic-Caspian-Transcaucasian interaction spheres or population movements [START_REF] Sherratt | Prehistoric steppe adaptation and the horse[END_REF][START_REF] Anthony | The horse, the wheel, and language: How Bronze-Age riders from the Eurasian steppes shaped the modern world[END_REF].

Another hypothesis argues that Anatolia played a central role in the transmission of domestic horses into Syro-Mesopotamia [START_REF] Sherratt | Prehistoric steppe adaptation and the horse[END_REF][START_REF] Becker | Akten Internationalen Interdisziplinären Kolloquiums Freie Universität[END_REF][START_REF] Uerpmann | The Ancient Distribution of Ungulate Mammals in the Middle East. Beihefte zum Tübinger Atlas des Vorderen Orients Reihe A[END_REF], and an Anatolian contribution to early domestic populations has been suggested [START_REF] Bökönyi | Equids in the Ancient World[END_REF][START_REF] Benecke | Archaeozoology of the Near East III. Proceedings of the third international symposium on the archaeozoology of southwestern Asia and adjacent areas[END_REF][START_REF] Vila | Equids in time and space: Papers in honour of Vera Eisenmann[END_REF][START_REF] Arbuckle | Zooarchaeology at Acemhöyük[END_REF]. Indeed, archaeological data indicate the widespread presence of wild horses (Equus ferus) and also of so-called hydruntines (a subspecies of the Asiatic wild ass named E.hemionus hydruntinus [START_REF] Bennett | Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA[END_REF]) in early and middle Holocene Anatolia that were regularly exploited [START_REF] Martin | Horses and humans: the evolution of human-equine relationships[END_REF][START_REF] Arbuckle | Archaeozoology of the Near East XII[END_REF][START_REF] Arbuckle | Zooarchaeology at Acemhöyük[END_REF][START_REF] Buitenhuis | The Early Settlement at Aşıklı Höyük; Essays in Honor of Ufuk Esin[END_REF]. The continuity of human-horse interactions from the ninth millennium through the second millennium BCE, when domesticates are known from archaeological contexts, led to the hypothesis that Anatolian wild horses may have been a potential source population for domestic horses. The lack of reliable osteomorphological criteria for differentiating the skeletal remains of wild and domestic horses, however, has hampered attempts to address the hypothesis of a local domestication. Therefore, the cultural processes and mechanisms triggering the widespread appearance of domesticates in the late third millennium are still elusive. In this study, we take advantage of the abundance of archaeological horse remains from the central Anatolian plateau [START_REF] Martin | Horses and humans: the evolution of human-equine relationships[END_REF][START_REF] Buitenhuis | The Early Settlement at Aşıklı Höyük; Essays in Honor of Ufuk Esin[END_REF][START_REF] Payne | Equids in the Ancient World[END_REF][START_REF] Uerpmann | The salvage excavations at Orman Fidanlıgı: A Chalcolithic site in inland northwestern Anatolia[END_REF][START_REF] Arbuckle | Zooarchaeology at Kösk Höyük[END_REF][START_REF] Arbuckle | Chalcolithic caprines, Dark Age dairy and Byzantine beef[END_REF] to provide the first rigorous test of the hypothesis of Anatolian horse domestication applying paleogenetics.

Complete present-day mitochondrial genomes have revealed 18 major haplogroups (A-R), the radiation times of which date mostly to the Neolithic and later periods [START_REF] Achilli | Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication[END_REF]. In contrast, studies of the mitochondrial hypervariable region in ancient horses have shown that domestic horses exhibit a much higher amount of genetic variation in mitochondrial lineages compared to cattle, sheep, and pigs [START_REF] Vila | Widespread origins of domestic horse lineages[END_REF][START_REF] Jansen | Mitochondrial DNA and the origins of the domestic horse[END_REF][START_REF] Cieslak | Origin and history of mitochondrial DNA lineages in domestic horses[END_REF][START_REF] Lira | Ancient DNA reveals traces of Iberian Neolithic and Bronze Age lineages in modern Iberian horses[END_REF]. Furthermore, most mitochondrial lineages observed in domestic horses already existed prior to domestication [START_REF] Lippold | Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication[END_REF]. These analyses of ancient horses did not yield a clear phylogeographic structure that would allow the spatio-temporal origin(s) of horse domestication to be identified. This was interpreted as suggesting that the mobility of wild horses in northern Eurasia allowed constant population reshuffling and repeated recruitment of wild local mares, precluding the establishment of a phylogeographic structure [START_REF] Lippold | Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication[END_REF][START_REF] Warmuth | Reconstructing the origin and spread of horse domestication in the Eurasian steppe[END_REF].

In contrast, extant domestic horses exhibit remarkably little variation in the male Y chromosome line, with only one haplotype so far identified in modern domesticates, which led to the early claim of a single domestication event for horses [START_REF] Lindgren | Limited number of patrilines in horse domestication[END_REF][START_REF] Kavar | Domestication of the horse: Genetic relationships between domestic and wild horses[END_REF]. Paleogenomic analyses of ancient specimens, however, observed additional male lineages in prehistoric populations prior to domestication and revealed that genetically diverse male founders were involved in early domestication [START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF][START_REF] Lippold | Discovery of lost diversity of paternal horse lineages using ancient DNA[END_REF]. This diversity was subsequently reduced, likely as a result of more directed human selection likely starting in the Iron Age and continuing during Roman times [START_REF] Wutke | Decline of genetic diversity in ancient domestic stallions in Europe[END_REF], and again during the Islamic conquest and the Byzantine-Sassanid war after 7 th -9 th c. CE [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF].

Paleogenetic evidence from genetic loci associated with coat color in horses argues for a diversification of coat color starting in the Bronze Age and is considered associated with an early stage of the domestication process [START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF][START_REF] Pruvost | Genotypes of predomestic horses match phenotypes painted in Paleolithic works of cave art[END_REF]. Since the appearance of new coat colors is common in domestic taxa compared to their wild counterparts, they provide a useful marker for identifying domestic horses in archaeological assemblages.

Up to now, the origins of domestic horses in Anatolia remained elusive, but careful recovery of horse remains from well-stratified archaeological contexts in Anatolia and in the neighboring Caucasus together with progress in paleogenetic approaches now make it possible to specifically address the processes responsible for the origins of domestic horses in this part of western Asia. For this project, we combined morphological classification of equid remains, which can be hampered by a lack of diagnostic anatomical and/or biometrical criteria [START_REF] Bennett | Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA[END_REF][START_REF] Geigl | Eurasian wild asses in time and space: Morphological versus genetic diversity[END_REF], with paleogenetic analysis of mitochondrial, Y chromosome DNA, and autosomal DNA markers related to coat color to trace the spatio-temporal dynamics of the emergence of domestic horses in Anatolia. We analyzed over 100 equid remains from 14 prehistoric sites in central Anatolia and the Caucasus covering most of the Holocene (9,000 BCE -1,000 CE) to gain insights into the origins of domestic horses in Anatolia, a pivotal issue in Near Eastern history.

Results

Based on our extensive experience with ancient animal remains from SWA including Anatolia, we expected DNA to be highly degraded in most samples that we had collected, of which only a few were petrous bones. Indeed, DNA in osseous remains from SWA is notoriously poorly preserved, petrous bones being an exception, although not all of them contain preserved endogenous DNA. For this reason, we decided to rely on a highly optimized metabarcoding approach combining the sensitivity of PCR and the efficiency of next-generation sequencing (NGS) specifically tailored to highly degraded ancient DNA [START_REF] Guimaraes | A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa[END_REF]. This approach has been shown to recover DNA molecules that escape shotgun sequencing [START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF] or sequence capture [START_REF] Massilani | Past climate changes, population dynamics and the origin of Bison in Europe[END_REF] and, if primers are optimized in silico and in vitro, is highly locus-specific [START_REF] Guimaraes | A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa[END_REF]. Used in combination with methods minimizing contamination [START_REF] Champlot | An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications[END_REF], as well as statistically sufficient replications [START_REF] Guimaraes | A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa[END_REF] (see also Materials & Methods section as well as Supplementary Materials), it at least equals or can occasionally be superior to DNA capture methods, which are plagued by biases (e.g., [START_REF] Albrechtsen | Ascertainment biases in SNP chips affect measures of population divergence[END_REF]). We have employed it for the study of various species, including horses [START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF][START_REF] Guimaraes | A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa[END_REF][START_REF] Coté | A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites[END_REF][START_REF] Ottoni | The palaeogenetics of cat dispersal in the ancient world[END_REF].

Here, we analyzed 111 equid remains from eight sites in Central Anatolia as well as six sites in the Caucasus dating from the Early Neolithic to the Iron Age (ca. 9,000 -500 BCE, see TabS1), with a few samples dating to later, historic periods. This approach had been developed and optimized previously to produce reliable data from highly degraded samples [START_REF] Guimaraes | A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa[END_REF][START_REF] Coté | A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites[END_REF][START_REF] Ottoni | The palaeogenetics of cat dispersal in the ancient world[END_REF], and it has been used successfully in situations where shotgun sequencing was not effective enough to genotype a large proportion of phenotype-associated SNPs [START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF]. We targeted the mitochondrial hypervariable region as well as 18 specific single nucleotide polymorphism (SNP) regions diagnostic for the 18 major mitochondrial haplogroups considered diagnostic in earlier studies ((47) and TabS2). These SNPs are sufficiently diagnostic to recapitulate the essential features of the mitogenome phylogeny (Fig. 1). Moreover, we analyzed six regions of the Y chromosome, four anonymous Y-linked fragments and two fragments of the amelogenin gene to evaluate male inheritance ((54, 56, 57) and TabS3). Finally, we chose a set of eight diagnostic SNPs in seven genes associated with the coat color in horses, including basic colors (bay, black, chestnut and grey), diluted phenotypes (silver and cream), spotted or painted phenotypes (overo, tobiano and sabino) and leopard spotting ((11); TabS4).

Genotyping versus osteological determination

From the 111 analyzed equid remains, 77 (70%) yielded ancient DNA results and could be genotyped in independent triplicate PCR experiments (TabS1). We obtained 14 different caballine (E. caballus) mitochondrial haplogroups previously defined in present-day horses [START_REF] Achilli | Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication[END_REF], as well as a new haplogroup, herein termed X, that belongs to the OPQ subtree (Fig 1, Tab S2). Moreover, from ten specimens we obtained haplotypes characteristic of donkeys (E. asinus). Finally, seven specimens yielded haplotypes belonging to E. hemionus clade H1, which Bennett et al. assigned to E. hemionus hydruntinus [START_REF] Bennett | Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA[END_REF] and are therefore referred to as 'hydruntine' below.

Genotyping and osteological determination agreed in 48 of the 57 remains that were assigned osteologically, with various degrees of certainty, to one of the equid species (84 %) (TabS1). In particular, 38 of the 40 remains assigned osteologically to wild or domestic horses showed the corresponding mitochondrial DNA (95%). Agreement was also obtained for three out of six hydruntine and seven out of eleven donkey remains. Of the twenty remains that could not be assigned osteologically to one of the aforementioned species, we identified sixteen horses, two donkeys, and two hydruntines through mitochondrial DNA typing. Genotyping and osteological determination disagreed in only four osteologically unambiguously assigned cases: two bone specimens determined osteologically as hydruntines yielded horse mitochondrial DNA, whereas two others classified osteologically as horses carried a donkey and a hydruntine mitochondrial DNA. Finally, one unassigned equid was determined genetically as a hybrid, more precisely a mule, since it carried horse mitochondrial and donkey Y chromosomal DNA (TabS1: specimen CD6189).

Diachronic pattern of maternal lineages

The 12 Anatolian horse remains predating ~4500 BCE carried either the mitochondrial haplogroup P or a new mitotype, termed herein X, not previously identified in modern or ancient horses (Fig. 1-2; TabS1-S2). Neither the P nor the X haplogroup have been documented so far beyond Anatolia in contemporaneous or older samples [START_REF] Vila | Widespread origins of domestic horse lineages[END_REF][START_REF] Cieslak | Origin and history of mitochondrial DNA lineages in domestic horses[END_REF][START_REF] Weinstock | Evolution, systematics, and phylogeography of Pleistocene horses in the new world: a molecular perspective[END_REF][START_REF] Lorenzen | Species-specific responses of Late Quaternary megafauna to climate and humans[END_REF][START_REF] Orlando | Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse[END_REF][START_REF] Elsner | Ancient mtDNA diversity reveals specific population development of wild horses in Switzerland after the Last Glacial Maximum[END_REF]. This strongly suggests that these two haplogroups represent the unique signature of a local wild horses native to the Anatolia plateau. After 2,200 BCE, this pattern changed profoundly, with 13 new mitochondrial haplogroups appearing in faunal assemblages from the Bronze and Iron Ages (Fig. 2; TabS1). Among the Bronze and Iron Age specimens, the pre-Bronze Age haplogroup P represents only 6% of the obtained haplotype spectrum (2 out of 33 remains), with both specimens dating to the earliest phase of this period (c. 2,000 BCE). Moreover, in post-3,300 BCE specimens, haplotype X is no longer detected. The novel haplotypes detected in our archaeological sample correspond mainly to haplogroups Q (11 remains), G (5 remains), and N (5 remains), while haplogroups A, B, D, E, H, I, L, Q account for the remaining 20 specimens (TabS1). These results indicate a nearly complete population turnover from the late third millennium BCE onward and corresponds well with iconographic and textual evidence for the appearance and dispersal of horse management in Anatolia and Mesopotamia [START_REF] Owen | The first equestrian: An Ur III glyptic scene[END_REF][START_REF] Zarins | The Domestication of Equidae in Third-Millennium BCE Mesopotamia[END_REF].

In the Caucasus, the earliest specimen that yielded a genetic result dates to the third millennium BCE and corresponds to haplogroup Q. Of the remaining 13 specimens, all excavated from archaeological contexts dating to the second millennium BCE, 11 represent a diverse array of haplogroups including A, B, C, E, FG, G, and Q. Taken together, this change in haplotypes in both Anatolia and the Caucasus is statistically highly significant (Fisher's test p-value 5.7x10 -6 ). The remaining two samples were identified as haplogroup P, presumably representing a continuation of the native Anatolian matriline into the Late Bronze Age.

Paternal lineages and hybrids

Paternal lineages were genotyped through six different loci on the Y chromosome (TabS3). Y chromosomal DNA data are less numerous and none were obtained from remains older than the Bronze Age, which must be due to poor DNA preservation. In 19 specimens dating to the Bronze Age or subsequent periods, however, the Y chromosome haplotype could be determined. Of these, 12 belong to E. caballus and six to E. asinus, and one specimen that was identified more generally as asinine (TabS1 and S3).

We could attribute the horse Y chromosomal sequences to two of a total of four horse haplotypes that have been described previously [START_REF] Wutke | Decline of genetic diversity in ancient domestic stallions in Europe[END_REF]: five remains were carriers of haplotype Y-HT-1, which is the major haplogroup present in modern horses, while four carried the extinct haplotype Y-HT-3 and three could not be determined due to SNPs that did not yield sufficient sequence coverage.

One specimen originating from Çadır Höyük yielded Y chromosomal SNPs corresponding to a jack ass, whereas the mitochondrial DNA corresponded to a horse (TabS1 and S3), thus reflecting the presence of a hybrid (mule) dating to the Iron Age. Interestingly, the mitotype of this individual was L, a mitotype not encountered in SWA before the Bronze Age.

Coat color

We genotyped SNPs associated with coat color variations [START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF][START_REF] Pruvost | Genotypes of predomestic horses match phenotypes painted in Paleolithic works of cave art[END_REF]. As discussed above, retrieval of nuclear DNA data in addition to mtDNA requires better ancient DNA preservation. Therefore, as for the Y chromosome, nuclear SNPs could not be genotyped in a reliable manner in samples predating the Early Bronze Age (TabS1 and S4). Altogether, we obtained SNPs from 43 specimens, allowing us to infer the coat color for 33 individuals including, 25 horses, six donkeys, one hydruntine, and one mule. In our data set, we identified the mutant allele for all but two out of the eight interrogated SNPs. In particular, only the mutant alleles for overo and cream were missing while the other six genetic variants were present. Consequently, a large part of the diversity of mutations affecting the coat color already observed in ancient northern Eurasia [START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF] proved present in Bronze Age horses in SWA (TabS4). Our results allowed us to attribute a coat-color to 25 horses from the Bronze Age and later periods. We identified seven horses with a wild type bay-colored coat, one with a bay-sabino, eight with a chestnut-colored coat, two each with the colors chestnut-tobiano, chestnut-silver, leopard, and black, one with a Bay-tobiano coat-color and one specimen whose DNA preservation was not good enough to discriminate between chestnut and bay (TabS1 and S4). This diversification in the coat color distribution is statistically significant (Fisher's test p-value 1.25x10 -3 ).

As expected, the six donkeys and the hydruntine did not harbor any of the mutant SNPs that humans selected for in domestic horses. The sample that was identified as a mule carried one mutant allele in both the ASIP and MC1R genes, most likely originating from its horse mother, which are associated with a baytobiano coat in horses (TabS1 and S4). Importantly, in specimen AC8811 from Early Bronze Age Acemhöyük, a chestnut coat color is combined with mitotype P, representing the local Anatolian wild horse matriline. This combination indicates that local Anatolian mares were incorporated into domestic herds in the Early Bronze Age.

Discussion

Wild and domesticated horses in Anatolia

Our genotyping of 60 ancient horses was designed to elucidate the long-standing question of a local domestication of horses in Anatolia. Our results allow us to conclude that domestic horses were introduced into the Caucasus and Anatolia by at least 2,000 BCE, presumably from the Eurasian steppes. This conclusion is based on the fact that in Anatolia, local horse populations prior to ca. 4,500 cal BCE carried only two mitochondrial haplogroups, P and X, the latter being a previously unrecorded haplotype which belongs to the O-P-Q subtree (Fig. 1). So far, these haplogroups have not been encountered elsewhere in Eurasia in contemporaneous or earlier contexts. Furthermore, haplotype X, likely had a limited temporal occurrence in Holocene Anatolia possibly disappearing after 5500 BCE. The foregoing supports our conclusion that these two haplogroups reflect the local mitochondrial signature of wild horses hunted in Anatolia in the early and middle Holocene (Fig. 2). We propose that the P and X haplogroups evolved independently in Anatolia during the late Pleistocene and early Holocene with little or no gene flow from neighboring wild horse populations due to geographic barriers separating Anatolia from northern Eurasia, namely the Bosporus, as well as the Caucasus and the Zagros mountain ranges. Thus, our study provides the first evidence showing that Anatolia was home to a genetically distinct population of wild horses, which based on archaeozoological findings were widely exploited during the Neolithic and Chalcolithic periods [START_REF] Martin | Horses and humans: the evolution of human-equine relationships[END_REF][START_REF] Arbuckle | Archaeozoology of the Near East XII[END_REF][START_REF] Buitenhuis | The Early Settlement at Aşıklı Höyük; Essays in Honor of Ufuk Esin[END_REF]. Equid remains from early Neolithic Aşıklı Höyük, Neolithic/Chalcolithic Köşk Höyük, and Late Chalcolithic/Early Bronze Age Çadır Höyük are representative of these wild Anatolian horses.

Around 2,000 BCE, we observe a statistically significant decline in the frequency of this local wild horse mitochondrial signature as the P haplogroup becomes rare and the X haplotype disappears completely. Presumably the low frequency of haplotype X in pre-Bronze Age horse assemblages (2 out of 11 in our dataset) offers an explanation as to why it did not survive in Anatolia into historic times.

Parallel to this, the diversity of maternal lineages in archaeological horse remains from the Caucasus and Anatolia increased drastically from two to fourteen (Fig. 2), all of which identified previously in present-day horses [START_REF] Achilli | Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication[END_REF] as well as in Eneolithic, Chalcolithic and Early Bronze Age horses from SE Europe and Kazakhstan [START_REF] Cieslak | Origin and history of mitochondrial DNA lineages in domestic horses[END_REF]. In these studies, mitochondrial haplogroups display no phylogeographic structure in Eurasia, which is consistent with the absence of significant physical barriers across the vast Eurasian steppes. This suggests that horse populations in the Eurasian steppes were panmictic likely explaining the high diversity of present-day domestic horse populations. It would also account for the rapid diversification observed in our dataset upon introduction in the Caucasus and Anatolia. This sudden appearance of allochthonous lineages coincides with the emergence of iconographic and epigraphic evidence for horses and horse riding at the end of the third millennium BCE and argues for substantial imports of domestic forms and hence against an independent local domestication process [START_REF] Owen | The first equestrian: An Ur III glyptic scene[END_REF][START_REF] Oates | Prehistoric Steppe Adaptation and the Horse[END_REF][START_REF] Zarins | The Domestication of Equidae in Third-Millennium BCE Mesopotamia[END_REF].

Our results obtained from genotyping of alleles associated with variations in coat color reinforces this conclusion, since Bronze Age horses in Anatolia and the Caucasus show mutations corresponding to coat color variants thought to have been selected during domestication in the Eurasian steppes, such as chestnut, black, and silver [START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF]. Mutations associated with coat color dilutions or spotting appeared later in our data set (after 1,200 BCE). Thus, we conclude from our genetic data that the domestic horses introduced to Anatolia in the Bronze Age carried mutations found earlier in northern Eurasia [START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF] and therefore predominantly derived from stocks imported from this vast region. Although the ultimate geographic origins of this allochthonous population cannot be defined with the data at hand, the Eurasian steppes north of the Black Sea seems a most plausible candidate.

Interestingly, we found that the local Anatolian P haplotype persisted in Bronze Age domestic horses in Anatolia and the Caucasus at low frequency (8% in our dataset) as well as in present-day horses, suggesting that wild Anatolian mares were incorporated into domestic herds probably very soon after the introduction of domestic horses in the region and prior to their local extinction in the wild. This is in agreement with our observation that at least two out of the four Bronze Age horses carry the P haplogroup. Since these individuals also carry a mutant coat color allele (AC8811 and TS2, Supp. Table 1), it is most likely that they represent domestic horses whose maternal ancestors were recruited from local wild Anatolian populations, which follows a pattern observed elsewhere whereby local mares were proposed to be recruited into domestic herds resulting in high mitogenomic diversity [START_REF] Vila | Widespread origins of domestic horse lineages[END_REF][START_REF] Cieslak | Origin and history of mitochondrial DNA lineages in domestic horses[END_REF]. Our results suggest a rapid shift from hunting to herding following the introduction of domestic horses in the Early Bronze Age, which likely correlates with a terminal decline in the wild horse population in Anatolia and the Caucasus indicated by the low frequencies of their remains in faunal assemblages [START_REF] Boessneck | Tierknochen und Molluskenfunde aus Munbāqa[END_REF][START_REF] Uerpmann | The Ancient Distribution of Ungulate Mammals in the Middle East., Beihefte zum Tübinger Atlas des Vorderen Orients[END_REF].

The diachronic pattern in Anatolia and the Caucasus of the two Y chromosomal haplotypes, Y-HT-1 and Y-HT-3, hints at the possibility of a population dynamics similar to that documented for northern Eurasia. In this latter region, Y-HT-1 constituting the predominant Y haplotype in present-day horses, drastically increased after the onset of domestication reaching fixation in the gene pool of the domestic horse by the Middle Ages, while Y-HT-3 declined over time until its disappearance [START_REF] Wutke | Decline of genetic diversity in ancient domestic stallions in Europe[END_REF]. In our Anatolian dataset, both haplotypes occurred at comparable frequencies around 2,000 BCE, but here too, Y-HT-3 declined over time with the most recent horse that carried Y-HT-3 originating from the Caucasus at around 1,300 BCE. This reduction of Y chromosome diversity is presumably the result of strong selection of stallions [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF].

Since northern Eurasia, and in particular the Pontic-Caspian steppe, is currently the most likely origin for the domestic horses brought into Anatolia, there are two possible introductory routes, one via southeastern Europe and one via the Caucasus. The route across the Bosporus has been postulated on the basis of the earliest zooarchaeological evidence for domestic horses in the southern Balkans at the Early Bronze Age site of Kanligeçit around 2,600-2,300 BCE [START_REF] Benecke | Equids in Time and Space: Papers in Honour of Véra Eisenmann[END_REF]. The coat colors of ten horses from this site were genotyped and revealed a highly biased distribution of coat color mutations with six out of ten homozygous black horses (a/a) four of which also show the leopard spotting (LP), plus two bay-colored horses with leopard spotting; no chestnut mutation was detected [START_REF] Ludwig | Twenty-five thousand years of fluctuating selection on leopard complex spotting and congenital night blindness in horses[END_REF]. This pattern strongly contrasts with our results in Anatolia and the Caucasus, where chestnut (e/e) is the earliest coat color variant, while black and leopard mutants remain very rare (only 2/25). These differences argue against the introduction of a domestic horse population similar to that found at Kanligeçit. Moreover, there is no archaeological evidence for horse management in western Anatolia in the third millennium BCE, providing little additional support for the hypothesis of an early introductory route across the Bosporus [START_REF] Uerpmann | Troia and the Troad: Scientific approaches[END_REF][START_REF] Çakırlar | Patterns of animal exploitation in western Turkey: from Paleolithic molluscs to Byzantine elephants[END_REF].

In contrast, our identification of several allochthonous mitochondrial lineages as well as coat color mutations appearing broadly contemporaneously in the Southern Caucasus and in central Anatolia argues in favor of a dispersal route via the Caucasus. Indeed, the abundance of horse bones and images of horses in Maikop culture settlements and burials of c. 3300 BCE in the northern Caucasus led to the suggestion that horseback riding began in the Maikop period [START_REF] Anthony | The Horse, the Wheel and Language. How Bronze-Age Riders from the Eurasian Steppes Shaped the Modern World[END_REF]. In addition, recent studies of ancient human genomes showed continuous gene flow between Copper Age steppes and Caucasus peoples [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF][START_REF] Lazaridis | Genomic insights into the origin of farming in the ancient Near East[END_REF][START_REF] De Barros Damgaard | The first horse herders and the impact of early Bronze Age steppe expansions into Asia[END_REF], and later, during the Bronze Age, between Mesopotamia, Anatolia, the southern and northern Caucasus and the steppes [START_REF] Wang | Ancient human genome-wide data from a 3000-year interval in the Caucasus corresponds with eco-geographic regions[END_REF]. This exchange between human groups appears to intensify during a century long period of cooling and desertification, known as the 4.2 ky event (e.g., [START_REF] Szczęsny | Was the 4.2 ka Event an Anthropogenic Disaster[END_REF] but see [START_REF] Voosen | New geological age comes under fire: Massive drought or myth? Scientists spar over an ancient climate event behind our new geological age[END_REF]) which may have impacted subsistence strategies and social networks in the steppe zone (e.g., [START_REF] Wang | Ancient human genome-wide data from a 3000-year interval in the Caucasus corresponds with eco-geographic regions[END_REF][START_REF] Shishlina | Counterpoint: Essays in Archaeology and Heritage Studies in Honour of Professor[END_REF]). On present evidence, this climatic event seems to be broadly contemporaneous with the arrival of non-local horse mitochondrial haplogroups and coat colors in the Caucasus and Anatolia and linked to the expansion of horse husbandry and possibly Indo-European languages (e.g., [START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF][START_REF] Anthony | The Horse, the Wheel and Language. How Bronze-Age Riders from the Eurasian Steppes Shaped the Modern World[END_REF], but see [START_REF] Démoule | Mais où sont passés les Indo-Européens ? Le mythe d'origine de l'Occident[END_REF]). Although the cultural processes initiating the dispersal of horse husbandry south of the Caucasus are currently difficult to address, it may relate to human population movements into the Caucasus and subsequently into Anatolia beginning in the late third millennium BCE.

Other equids

The present study identified a mule, offspring of a female horse and a male donkey, from an Early Iron Age context (dated to 1,100 -800 BCE) at central Anatolian Çadır Höyük. Mules have been identified based on osteological criteria and more recently on genomic data in the European Iron Age and Roman periods [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF][START_REF] Armitage | Roman mules[END_REF][START_REF] Peters | Römische Tierhaltung und Tierzucht. Eine Synthese aus archäozoologischer Untersuchung und schriftlich-bildlicher Überlieferung[END_REF][START_REF] Johnstone | [END_REF]. Although several specimens have been tentatively identified as mules from Bronze and Iron Age contexts in SWA [START_REF] Peters | Madaba Plains Project: the 1994 season at Tall al-Umayri and subsequent studies[END_REF][START_REF] Becker | Maison de l'Orient et de la Mediterranee[END_REF][START_REF] Mohaseb | Elamica: on history and culture of Elam and its neighboring regions[END_REF], this is the most ancient genomic evidence for a mule in SWA. Since wild donkeys are not native to Anatolia, the jack ass involved must have been a domestic animal [START_REF] Clutton-Brock | Horse Power. A History of the Horse and the Donkey in Human Societies[END_REF][START_REF] Uerpmann | The Ancient Distribution of Ungulate Mammals in the Middle East., Beihefte zum Tübinger Atlas des Vorderen Orients[END_REF]. The parent mare of the mule from Çadır Höyük was also a domestic animal as it carried an allochthonous mitochondrial haplogroup and contributed two coat color mutant alleles to its offspring. Although not a surprise, the presence of a mule in the Anatolian Iron Age, reflects a new role for domestic horses that emerged as they moved into SWA where domestic donkeys had been utilized since the fourth millennium BCE [START_REF] Boessneck | Tierknochenfunde der Ausgrabungen des Deutschen Archäologischen Instituts Baghdad in Uruk-Warka, Iraq[END_REF] and where a tradition of equid (donkey x hemione) hybridization emerged in the third millennium BC [START_REF] Zarins | The Domestication of Equidae in Third-Millennium BCE Mesopotamia[END_REF]. This situation reflects a true integration of horses into SW Asian equid economies and is an early example of intentional livestock engineering. Being the most expensive species mentioned on a price list for livestock dating to the Hittite period (~1,600 -~1,178 BCE (90), mules were obviously highly valued.

In addition to horses, our results provide unambiguous evidence that Neolithic, Chalcolithic and Bronze Age people in Anatolia also hunted hydruntines, the hemione subspecies E. hemionus hydruntinus, once inhabiting large parts of Anatolia [START_REF] Bennett | Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA[END_REF]. This is not a trivial result since the osteological identification of the hydruntine is not obvious [START_REF] Bennett | Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA[END_REF][START_REF] Geigl | Eurasian wild asses in time and space: Morphological versus genetic diversity[END_REF], an observation confirmed in our study illustrating disagreement between genotyping and osteological determination in six cases. At the same time it also confirmed the results of another genetic study presented by us illustrating that no other subspecies of hemione populated the Anatolian plateau [START_REF] Bennett | Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA[END_REF]. As such, the most recent evidence in our data set for an Anatolian hydruntine dates to ~2,200 BCE, which is in agreement with results reported earlier [START_REF] Bennett | Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA[END_REF]. The combined data thus suggest that hydruntines went extinct in Anatolia during the Late Bronze Age more or less at the same time as local wild horses perhaps in response to the same combination of factors including increased aridity associated with the 4.2 ky event, competition for pasture resources with growing numbers of livestock, and hunting pressure perhaps related to practices of elites hunting [START_REF] Zarins | The Domestication of Equidae in Third-Millennium BCE Mesopotamia[END_REF].

Conclusions

Our study of ancient equid remains from Anatolia and the southern Caucasus covering ~9,000 years of the Holocene analyzed the dynamics over time of mitochondrial lineages and tested the hypothesis of Anatolia as a center of horse domestication. We were able to identify two new mitotypes characteristic of local Anatolian wild horses, which were regularly exploited in the early and middle Holocene. However, we identified a pattern of genetic change that does not reflect a gradual process involving the local population but rather a sudden appearance ~2,000 BCE of non-local lineages that are still present in domestic horses. We also show that these imported horses exhibited coat colors that are absent in local wild horses prior to domestication. Moreover, continuation of Anatolian maternal lineage P into the Bronze Age implies some limited incorporation of local mares into domestic herds. These patterns of change indicate that domestic horses were introduced into Anatolia perhaps via the Caucasus region during the Bronze Age and provide, for the first time, a date for the beginning of the exploitation of domestic horses in Anatolia and Transcaucasia. They also argue against local independent domestication of the horse in this region. Indeed, our results strongly suggest that Anatolia was not a primary source for domestic horse lineages but, as observed in other regions, local matrilines were incorporated into herds of imported domestic horses, which were also hybridized with local donkeys to create mules. The ultimate geographic origins of the imported domestic herds remain to be determined but by eliminating Anatolia directs further attention to the adjacent regions of the Black Sea.

Material and Methods

Archaeological bones

The provenance of the analyzed horse specimens is described in the Supplementary Materials paragraph 1 and table S1.

Genetic analysis

Sample preparation, DNA extraction, DNA purification and following pre-PCR procedures were carried out in the high-containment facility of the Jacques Monod Institute in Paris physically separated from areas where modern samples are analyzed and dedicated exclusively to ancient DNA analysis using the strict procedures for contamination prevention previously described [START_REF] Champlot | An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications[END_REF][START_REF] Bennett | Library construction for ancient genomics: single strand or double strand?[END_REF]. Bone specimens were cleaned and ground in a freezer mill, and DNA extracted and purified as previously described [START_REF] Massilani | Past climate changes, population dynamics and the origin of Bison in Europe[END_REF][START_REF] Gorgé | Analysis of Ancient DNA in Microbial Ecology[END_REF] (also see Supplementary Materials paragraph 2.1.). Genetic analyses were performed using aMPlex-Torrent, a multiplex PCR assay coupled to NGS optimized to genotype reliably highly damaged ancient DNA at high throughput [START_REF] Guimaraes | A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa[END_REF][START_REF] Coté | A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites[END_REF][START_REF] Ottoni | The palaeogenetics of cat dispersal in the ancient world[END_REF]. The approach relies on careful design of primers that were optimized in silico and in vitro and on the systematic replication in triplicates of each PCR ((60) and Supplementary Materials). Moreover, we used it in combination with rigorous methods of elimination of DNA molecules contaminating commercially purchased reagents [START_REF] Champlot | An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications[END_REF], and of previous PCR amplification products with UDG treatment [START_REF] Pruvost | Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies[END_REF], which also removes cytosine deamination products [START_REF] Pruvost | DNA diagenesis and palaeogenetic analysis: critical assessment and methodological progress[END_REF]. This approach proved extremely useful to retrieve selected markers more efficiently and at much lower costs than with shotgun sequencing [START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF]. Compared to DNA capture approaches, it provides a higher yield since capture approaches tend to leave gaps in the genomic regions that are targeted, which then have to be filled via PCR approaches ( (61); supplementary information).

To amplify the mitochondrial genome, primers were designed in order to cover the complete mitochondrial genome with minimal primer dimer propensity from a multiple alignment of all the equids mitogenomes sequences present in Genbank in 2014 using the software Oligo 7 as previously described [START_REF] Guimaraes | A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa[END_REF]. The primers were then tested for efficiency and dimer formation using quantitative real-time PCR (qPCR) and optimized as described before [START_REF] Guimaraes | A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa[END_REF] and combined in three different multiplex reactions (see SI paragraphs 2.2.1., 3.1. and 3.4.1.). We proceeded in the same way to analyze the other genetic markers. To analyze the coat color, we chose a set of eight SNPs in seven genes for detecting basic colors (bay, black, chestnut and grey), diluted phenotypes (silver and cream), spotted or painted phenotypes (overo, tobiano and sabino) and leopard spotting, as described previously [START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF] (see SI paragraphs 2.2.2., 3.2. and 3.4.2.). For the analysis of the Y chromosome, we selected four anonymous Y-linked fragments, Y2B17, Y3B1, Y3B12, Y3B19 and two fragments of the amelogenin gene, AME2 and AME3, as previously described (54) (see SI paragraphs 2.2.3., 3.3. and 3.4.3.). To protect against cross-contamination between samples, we used the UNG-coupled PCR system (see Supplementary Materials paragraphs 2.2.1.-2.2.3.). For each sample, we then performed the six multiplex PCRs with different primer combinations (mtDNA, coat color and Y chromosome) (see SI paragraphs 3.4.). Each extract was amplified in triplicate and the triplicates were pooled so that, at the end, there were three pools for each extract. The PCR products were pooled in a 96-well plate and DNA libraries prepared in an automate Tecan Freedom Evo 100 ligating samplespecific Ion Torrent barcoded adaptors (see Supplementary Materials paragraph 2.3.). After amplification the size distribution and concentration of the library was assessed on the Agilent 2100 Bioanalyzer. Emulsion PCR and Ion Sphere Particle enrichment were conducted with the Ion OneTouch System (Life Technologies) using the Ion OneTouch 200 Template kit v2 DL (see Supplementary Materials paragraph 2.3.). The DNA library was sequenced on the Ion Torrent Personal Genome Machine (PGM) Sequencer using the Ion PGM 200 Sequencing Kit and Ion 314 semiconductor sequencing chips (Life Technologies) (see Supplementary Materials paragraph 2.3.). Consensus sequences were established typically from several tens to several hundreds of sequences, and only replicated sequences were considered herein.

Phylogenetic analysis of the mitochondrial DNA

A maximal likelihood phylogenetic tree was constructed from aligned complete Equus mitogenomes using RAXML (95) and a general time reversible nucleotide substitution model, with gamma categories and an estimated proportion of invariant sites. Following reduction of the mitogenomes to the fragment amplified by PCR after primer removal, a phylogenetic tree was constructed using PHYML [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF] with four gamma categories and an estimated proportion of invariant sites and a Tamura-Nei substitution model (97).

Statistical analysis

The probability (two-tailed p-values) that the changes of mitochondrial DNA haplotype and coat color diversity in Anatolia and the Caucasus after ~2000 BCE could have been observed simply by chance was estimated with the Fisher's exact test using as nominal variables time (before and after 2000 BCE) and genotype (OPQX vs other haplogroups for mitochondrial haplotypes, Bay vs other colors for coat colors).

Supplementary Materials

Ancient DNA shows domestic horses were introduced in the southern Caucasus and Anatolia during the Bronze Age.

Aşıklı Höyük

Hijlke Buitenhuis, Joris Peters

Aşıklı Höyük is an aceramic Neolithic settlement mound located next to the Melendiz River. It covers an area of 3.5-4 ha, rising 15.35 m above the Melendiz Ülain at the north and 13.16 m at the south. Under the direction of Prof. Dr. U. Esin (University of Istanbul), excavations were carried out from 1989 until 2003. Renewed excavations directed by Prof. Dr. M. Özbaşaran (University of Istanbul) started in 2009 and continued since then. Five levels, designated as 1-5 from top to bottom respectively, could be documented so far. The earliest settlement phase excavated thus far is represented by Level 5. Radiocarbon dates allow concluding that early site occupation dates to ~8,400 BCE (1, 2). The faunal remains of the site are currently analyzed by a team of zooarchaeologists including H. Buitenhuis, N. Munro, J. Peters, N. Pöllath, and M.C. Stiner. A most important finding is the fact that caprine management was already practiced by the site inhabitants [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF][START_REF] Bökönyi | The earliest waves of domestic horses in East Europe[END_REF][START_REF] Benecke | Der Mensch und seine Haustiere[END_REF] and developed further on during the occupation in the later levels to a full caprine husbandry. So far, two species of wild equids have been evidenced in the archaeofauna. Remains of wild horses (E. ferus) form a small part of this assemblage, whereas the majority belongs to hemione-like animals, likely E. hydruntinus [START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF][START_REF] Uerpmann | Die Domestikation des Pferdes im Chalkolithikum West-und Mitteleuropas[END_REF]. The faunal assemblage is curated on site at the Aşıklı Höyük dighouse, Kızılkaya village, Aksaray, Turkey. All specimens were exported with the permission of the Aksaray Museum and the Turkish Ministry of Culture and Tourism.

Acemhöyük

Benjamin Arbuckle

Acemhöyük is a large mound located at an elevation of 950 meters on the alluvial fan of the Melendiz river near the Tüz Gölü (Salt Lake) in Central Turkey. The mound is approximately 800 x 600 meters in dimension rising 20 meters above the surrounding Yesilova plain and represents the remains of a large, fortified Bronze Age urban center. The site has been excavated since 1962 by Dr. N. Özgüç, Ankara University in association with the Turkish Historical Society and the General Directorate of Antiquities and Museums and by Prof. Dr. A. Öztan of the same institution since 1989 [START_REF] Benecke | Equids in Time and Space: Papers in Honour of Véra Eisenmann[END_REF][START_REF] Greenfield | Horses and humans: The evolution of human-equine relationships[END_REF]. Acemhöyük consists of 12 levels with deposits representing Chalcolithic to Medieval occupations. The faunal assemblage at Acemhöyük is dominated by domestic sheep, goats, cattle and pigs with equids representing a tertiary resource [START_REF] Outram | The earliest horse harnessing and milking[END_REF][START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF]. The Acemhöyük faunal assemblage is curated on site at the Acemhöyük dighouse, Yeşilova, Aksaray, Turkey. All specimens were exported with the permission of the Konya Museum and the Turkish Ministry of Culture and Tourism. 24 equid specimens were submitted for ancient DNA analysis. Twenty of these samples derive from deposits stratigraphically assigned to levels XI, X, VII, V and IV representing the late Early Bronze Age (EBA) and earliest Middle Bronze Age (MBA) (associated radiocarbon dates c. 2100-1900 BCE). Many of these specimens were identified only to the genus level (Equus sp.) and could potentially represent wild horse (Equus ferus), domestic horse (Equus caballus), wild hemione/hydruntine (Equus hemionus hydruntinus), domestic donkey (Equus asinus), or one of several hybrids (horse x donkey, donkey x horse, donkey x hemione). Four of these specimens (AC14371, AC14379, AC14380, AC14427) derive from equid burials and are identified based on dental morphology and body size as donkeys or hemiones. An additional 7 of these late EBA/early MBA specimens were also identified as donkeys or hemiones rather than horses. One specimen, identified as Equus sp., derives from level III, the Assyrian Colony period (c. 1700s BC). Two specimens (Equus caballus) are from Hellenistic contexts (level I), while the last specimen (Equus caballus) is thought to come from a pit perhaps dating to the Ottoman period.

Çadır Höyük

Benjamin Arbuckle

Çadır Höyük is a 32-meter-high mound located in the Kanak Su basin situated in the Yozgat Province of north central Turkey. The modestly sized 240 x 185-meter mound boasts an occupational history spanning 6000 years (ca. 5200 BCE to the thirteenth century CE). This sequence has been explored since 1993 by the Çadır Höyük Archaeological Project led by Ron Gorny and more recently Greg McMahon and Sharon Steadman [START_REF] Anthony | Bit wear, horseback riding, and the Botai site in Kazakstan[END_REF][START_REF] Olsen | Prehistoric steppe adaptation and the horse[END_REF]. The Çadır Höyük faunal assemblage is curated on site at the Çadır Höyük dighouse, Peyniryemez, Sorgun, Turkey. Specimens were exported with the permission of the Yozgat Archaeology Museum and the Turkish Ministry of Culture and Tourism. 21 specimens derived from throughout the Çadır Höyük occupational sequence were submitted for ancient DNA analysis. These include two of specimens (CD2017, CD2015) from the Deep Sounding which were recovered from a Middle Chalcolithic context with an associated radiocarbon date of 4500 cal BCE (Beta-418460). Due to this early date, these specimens are thought to represent wild Anatolian horse (Equus ferus). Another (wild?) horse specimen (CD1875) was recovered from a Late Chalcolithic context with an associated radiocarbon date placing it in the late fourth millennium BCE (Beta418461: 3350 cal BCE). Six specimens derive from Early and Late Bronze Age contexts; ten specimens come from Iron Age deposits and include horse, donkey and Equus sp identifications. Finally, two horse specimens derive from Byzantine deposits.

Boğazköy-Hattusa

Benjamin Arbuckle

Boğazköy-Hattusa is an UNESCO World Heritage site located in north central Turkey (Çorum province) that represents the remains of a major urban center dating to the Middle and Late Bronze Age (2000-1200 BCE). Boğazköy is perhaps best known as the capital city of the Hittite empire and has been excavated by the Deutsches Archäologisches Institut under the supervision of Dr. Andreas Schachner. One specimen was provided from Hattusa for ancient DNA analysis. This specimen, an incisor tooth likely representing a domestic donkey, derives from Late Bronze Age (Hittite) deposits (excavation year 2013, context 407, bag#820).

Köşk Höyük

Benjamin Arbuckle

The site of Köşk Höyük (originally Köşk-Pınar ( 14)) represents the remains of a small agro-pastoral village located on the eastern edge of the Bor Plain in central Turkey at an elevation of 1400 meters [START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF]. The site consists of a shallow mound, about half a hectare in area, situated on the edge of a limestone ridge overlooking a permanent spring. The location of the site provides ready access to the resources of the Konya-Ereğli Plain to the west, the foothills and uplands of the Taurus Mountains to the east and south, and the most direct route from the west and south to the obsidian sources of the nearby Melendiz Mountains. Excavations at Köşk began with a series of soundings done by Dr. Nimet Özgüç (Ankara University) in 1981, followed by a more extensive excavation campaign led by Dr. U. Silistreli, in association with the Nigde Museum from 1983 to 1992, and finally by Prof Dr. Aliye Öztan from 1995 to 2012 [START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF][START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF]. The stratigraphy of Köşk has been divided into prehistoric levels V-I. Stratigraphic levels V-II have been radiocarbon dated to between 6200-5500 cal BCE and represent the Early Chalcolithic period in central Anatolia. In this period the faunal assemblage indicates that although domestic sheep and goats are the most abundant mammalian taxa, wild taxa are abundant as well. Wild cattle, boar, as well as hare and foxes were hunted and trapped at the site. However, wild equids including both wild horse (Equus ferus) and European wild ass (Equus hemionus hydruntinus) are the most abundant wild taxa representing a surprising 15-38% of the fauna from EC contexts [START_REF] Anthony | The Horse, the Wheel, and Language: How Bronze-Age Riders from the Eurasian Steppes Shaped the Modern World[END_REF]. 24 specimens from Köşk Höyük were sent for ancient DNA analysis. These specimens derive from the Early Chalcolithic levels IV and III representing the Early Chalcolithic period. 14 specimens were identified based on dental and skeletal morphology as Anatolian wild horse (Equus ferus); five specimens were identified as European wild ass (Equus hemionus hydruntinus); while the remaining five specimens were identified to genus as Equus sp (Arbuckle). All specimens were exported with the permission of the Niğde Archaeology Museum and the Turkish Ministry of Culture and Tourism.

Troy Hans-Peter Uerpmann

The excavation of the site of Troy, located close to the Dardanelles under the hill of Issarlik in Western Turkey, has identified a succession of 9 towns and 46 occupational levels. The complex stratigraphy extends over 20 m. The bones used for the present study originate from three different levels, Troy I, Troy IV and Troy VI. Troy I is a small fortified village and corresponds to the first human occupation of the site during the Early Bronze Age. The bone samples analysed in the present study originate from Troy IV and were assigned to E. caballus. Troy IV is dated to the Middle Bronze Age and corresponds to the phase of decline of the town whose subsistence mode seems to have been based on agriculture and handcraft rather than trading. Troy VI corresponds to the golden age of the town at the end of the Bronze Age as witnessed by the construction of palaces. Only a fraction of the animal remains found during the ongoing excavations have been studied. Nevertheless, the archaeozoological data of more than 120,000 bone finds are stored in a data-base [START_REF] Arbuckle | Animals and inequality in Chalcolithic Anatolia[END_REF].

Lidar Höyük Joris Peters

Southwest of the village Lidar (SE-Anatolia, Turkey) lies the impressive settlement mound of Lidar Höyük, measuring 200 by 240 m at the base and rising 23 m above the Euphrates River terrace. Situated near a major passage across the Euphrates River, the site of Lidar Höyük was already inhabited in the Early Bronze Age. Although occupation lasted until Medieval Times, the site was not occupied permanently, as the archaeological record shows. Excavations carried out between 1979 and 1986 under the direction of Prof. Dr. H. Hauptmann † produced a rich archaeofauna totalling 62,453 vertebrate remains [START_REF] Shaler | Domesticated animals. Their relation to man and to his advancement in civilization[END_REF]. 3.8% of this assemblage pertains to equids, remains of donkeys outnumbering those of horses and hemiones. Difficulties arose to assign some larger specimens either to (small) horse, mule or Asiatic wild ass [START_REF] Shaler | Domesticated animals. Their relation to man and to his advancement in civilization[END_REF]. The 1987 excavation produced another sample of equid bone, from which specimens were selected for ancient DNA analysis, including a Bronze Age horse radius collected in Area S47.

Caucasus

Georgia

Didi Gora Hans-Peter Uerpmann

This site in the province of Kachetia is located on the plain of the Alazani in Eastern Georgia between the Black and the Caspian Seas. In the past it was probably located on a river bank. It comprises a history of three millennia, from the Early Bronze Age (c. 2800 BCE) to the Early Iron Age (c. 1,000 BCE) and is one of the largest settlement hills during the Neolithic and Bronze Age in this region. Archaeological evidence points to a semi-nomadic, seasonal lifestyle of the inhabitants, to the exploitation of ore deposits and to extensive trading contacts with other regions in the Near East. The Bronze Age layers are poor in archaeological artifacts except for obsidian flints, but very rich in faunal remains. These faunal remains, which were analyzed in the present study, are very well preserved suggesting a rapid burial. The genetically analyzed equid remains had been attributed to E. caballus based on the relative size of the bones and on their weight since the bones were heavily fragmented [START_REF] Martin | Horses and humans: the evolution of human-equine relationships[END_REF].

Tqisbolu Gora

The site of Tqisbolu Gora was occupied between 3,800 and 2,800 years ago and was thus partly contemporaneous to Didi Gora. The youngest layers of Didi Gora correspond to Iron Age layers in Tqisbolu Gora. Three equid species were identified on the two sites. The remains of domestic horses (E. caballus) are bigger than the remains of the small equids from the site. The latter could belong to E. hemionus, E. hydruntinus or to the domestic donkey E. asinus [START_REF] Martin | Horses and humans: the evolution of human-equine relationships[END_REF]. In the present study, four remains were analyzed genetically. Three of them had been attributed to E. caballus and one to E. hydruntinus based on the relative size of the bones.

Armenia

Lusakert

Nina Manaseryan

The analyzed horse remains originate from the Middle Palaeolithic Cave Lusakert 1 in the Hrazdan valley of Armenia.

Mets Sepasar

Nina Manaseryan

Mets Sepasar is a Bronze Age habitat and necropolis in Shirak. The mountain Mets Sepasar is located on the Shirak plateau in the northwest of Armenia. The archaeological studies, led by L.Yeganyan, started out from the foothills of the mountain. As a result of these excavations an Early Bronze Age settlement has been discovered. In the middle of the monument, in a volcanic cone, a sanctuary was found showing traces of festivities accompanied by sacrifices.

Tsaghkahovit and Gegharot

Hannah Chazin

Gegharot and Tsaghkahovit are multi-phase sites located in the Tsaghkahovit Plain, Armenia and have been the site of ongoing archaeological research by the joint Armenian-American Project ArAGATS [START_REF] Ünal | 'da At Hititçe Kikkuli at eğitimi metinleri ve "Tavlaya çekmek"le ilgili teknik bir ayrıntı[END_REF][START_REF] Arbuckle | Archaeozoology of the Near East XII[END_REF][START_REF] Toynbee | Tierwelt der Antike -Bestiarium romanum[END_REF]. Gegharot was occupied during both the Early and Late Bronze Age. The Late Bronze Age occupation consisted of a walled fortress and nearby cemetery. Tsaghkahovit was occupied during the Late Bronze Age, as well as during the Achaemenid period. The Late Bronze Age occupation at Tsaghkahovit consisted of a walled fortress, extra-mural room blocks, and an associated cemetery. Two of the equid specimens were from Late Bronze Age tombs (Gegharot Kurgan 1 and Tsaghkahovit BC12), and the remainder were from Late Bronze middens excavated in the Tsaghkahovit Residential Complex. Equid specimens from these sites were identified by Hannah Chazin and Belinda Monahan.

Paleogenetic Analysis

Rationale of the experimental approach

Due to the age of the archaeological samples and the climatic conditions of the area and to the fact that most of the bones that were appropriate to answer the scientific questions were not petrous bones, DNA was poorly preserved. Thus, we used a method that we have developed for poorly preserved archaeological specimens [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF]. This powerful multiplex PCR assay is highly sensitive when optimized primers are used and allows high-throughput genotyping of a large sample number since it is coupled to next-generation sequencing [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF]. We used it previously successfully to genotype various difficult ancient samples [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF][START_REF] Littauer | Wheeled Vehicles and Ridden Animals in the Ancient Near East[END_REF][START_REF] Owen | The first equestrian: An Ur III glyptic scene[END_REF][START_REF] Oates | Prehistoric Steppe Adaptation and the Horse[END_REF]. This approach is more efficient and cost-effective than sequence capture, in particular for samples with highly degraded DNA, and is informative enough for the specific questions addressed in this study. Here, we optimized the primers in silico and in vitro, i.e., tested and optimized the PCR conditions prior to the analysis of the ancient specimens. We amplified the ancient DNA extracts in the presence of dUTP and degraded products of previous PCR amplifications using UDG [START_REF] Zarins | The Domestication of Equidae in Third-Millennium BCE Mesopotamia[END_REF]. This UDG treatment eliminates in addition the products of cytosine deamination, thus increasing the accuracy of retrieved sequences [START_REF] Zarins | The Domestication of Equidae in Third-Millennium BCE Mesopotamia[END_REF]. Moreover, prior to each PCR amplification, we eliminated contaminating DNA molecules in reagents using the UVD decontamination procedure [START_REF] Clutton-Brock | Horse Power. A History of the Horse and the Donkey in Human Societies[END_REF]. This multi-level decontamination procedure enabled us to produce reliable, authentic amplicons. Finally, we performed independently each PCR amplification three times and sequenced the bulk PCR products using NGS to determine the authentic sequences.

All pre-PCR procedures were carried out in the high containment facility of the Jacques Monod Institute in Paris physically separated from areas where modern samples are analyzed and dedicated exclusively to ancient DNA analysis using the strict procedures for contamination prevention previously described [START_REF] Clutton-Brock | Horse Power. A History of the Horse and the Donkey in Human Societies[END_REF][START_REF] Bökönyi | Akten Internationalen Interdisziplinären Kolloquiums Freie Universität Bernfried Schlerath[END_REF].

DNA Extraction

The external surface of the specimens was removed with a sterile blade to minimize the environmental contamination. For each bone sample, roughly 0.2 g was ground to a fine powder in a freezer mill (Freezer Mill 6750, Spex Certiprep, Metuchen, NJ), which was then suspended in 2mL of extraction buffer containing 0.5M EDTA, 0.25M Di-sodium hydrogen phosphate (Na2HPO4), pH 8.0, 0.14M 2mercaptoethanol and 0.25 mg/mL of proteinase K and incubated under agitation at 37°C for 48 hours. Blank extractions were carried out for each extraction series. Samples were then centrifuged and the supernatant was purified with a modified protocol of the Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany) using the manifold device, as previously described [START_REF] Bökönyi | Akten Internationalen Interdisziplinären Kolloquiums Freie Universität Bernfried Schlerath[END_REF][START_REF] Zeuner | A History of Domesticated Animals[END_REF][START_REF] Uerpmann | Domestication of the horse: when[END_REF].

2.2. Multiplex PCR Amplification 2.2.1. Mitochondrial DNA Primers were designed in order to cover the complete mitochondrial genome with minimal primer dimer propensity from a multiple alignment of all the equids mitogenomes sequences present in Genbank in 2014 using the software Oligo 7 as previously described [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF]. The primers were then tested for efficiency and dimer formation using quantitative real-time PCR (qPCR) [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF]. We selected a total of 20 primer pairs (see paragraph SI 3.1.). To assess the phylogenetic consistency of our molecular assay with the selected fragments, we built a maximum likelihood tree with PHYML (33) for both the complete mitogenomes (GTR + G + I) and the concatenation of the 20 amplified fragments without primers (TN93 + G + I) [START_REF] Anthony | The horse, the wheel, and language: How Bronze-Age riders from the Eurasian steppes shaped the modern world[END_REF]. This confirmed the preservation of the salient features of the tree topology when using only the selected fragments (Fig. 1). We optimized the multiplex PCR as described before [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF] and the primer pairs were combined in three different multiplex reactions (see paragraph SI 3.4.1.). To protect against crosscontamination between samples, we used the UNG-coupled PCR system [START_REF] Arbuckle | Zooarchaeology at Acemhöyük[END_REF]. Each reaction was carried out in a 30 µl reaction volume with a final composition of 50 mM Tris/HCl, 10 mM KCl, 5 mM (NH4)2SO4, 4 mM MgCl2, pH 8.3 (@25°C), 1 mg/ml BSA (bovine serum albumin), 0.15 µM of each primer, 0.25 mM dA/G/CTPs and 0.50 mM dUTP, 2 U of Fast Start DNA polymerase, 0.5 U of uracil-N-Glycosylase (UNG) and 4 µl of DNA. The MgCl2 solution, reaction buffer and BSA were decontaminated by exposing the solutions in UV-pervious tubes (Qubit®, Life Technologies) to UV light for 10 minutes at short distance as described [START_REF] Clutton-Brock | Horse Power. A History of the Horse and the Donkey in Human Societies[END_REF]. A negative control was performed for each multiplex reaction that was processed in the same way as the samples throughout the whole experimental procedure. The cycling program consisted of 15 minutes at 37°C (carry-over contamination prevention and aDNA damage products through digestion by UNG of dUTP-labeled amplicons and of cytosine deamination products), 95°C for 10 minutes (inactivation of UNG and activation of the Fast Start DNA polymerase), followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute and a final extension step at 72°C for 4 minutes. All extracts were tested for inhibition as previously described (35).

Coat color

We chose a set of eight SNPs in seven genes for detecting basic colors (bay, black, chestnut and grey), diluted phenotypes (silver and cream), spotted or painted phenotypes (overo, tobiano and sabino) and leopard spotting, as described previously [START_REF] Uerpmann | The Ancient Distribution of Ungulate Mammals in the Middle East. Beihefte zum Tübinger Atlas des Vorderen Orients Reihe A[END_REF] (see paragraph SI 3.2.) We optimized the multiplex PCR as described before [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF] and all the primers were selected and combined in a single multiplex reaction (see paragraph SI 3.4.2.). Each reaction was carried out in a 30 µl reaction volume with a final composition of 50 mM Tris/HCl, 10 mM KCl, 5 mM (NH4)2SO4, 4 mM MgCl2, pH 8.3 (@25°C), 1 mg/ml BSA (bovine serum albumin), 0.15 µM of each primer, 0.25 mM dA/G/CTPs and 0.50 mM dUTP, 2 U of Fast Start DNA polymerase, 0.5 U of uracil-N-Glycosylase (UNG) and 4 µl of DNA. A negative control was performed for each multiplex reaction that was processed in the same way as the samples throughout the whole experimental procedure. The cycling program consisted of 15 minutes at 37°C (carry-over contamination prevention through digestion by UNG of dUTP-labeled amplicons), 95°C for 10 minutes (inactivation of UNG and activation of the Fast Start DNA polymerase), followed by 50 cycles at 95°C for 20 seconds, 55°C for 30 seconds, 72°C for 30 seconds and a final extension step at 72°C for 4 minutes.

Y chromosome

We analyzed four anonymous Y-linked fragments, Y2B17, Y3B1, Y3B12, Y3B19 and two fragments of the amelogenin gene, AME2 and AME3, as previously described (37) (see paragraph SI 3.3). We optimized the multiplex PCR as described before [START_REF] Boessneck | Pferde im 4/3. Jahrtausend v. Chr. in Ostanatolien[END_REF] and all the primers were selected and combined in two multiplex reactions (see paragraph SI 3.4.3.). Each reaction was carried out in a 30 µl reaction volume with a final composition of 50 mM Tris/HCl, 10 mM KCl, 5 mM (NH4)2SO4, 4 mM MgCl2, pH 8.3 (@25°C), 1 mg/ml BSA (bovine serum albumin), 0.15 µM of each primer, 0.25 mM dA/G/CTPs and 0.50 mM dUTP, 2 U of Fast Start DNA polymerase, 0.5 U of uracil-N-Glycosylase (UNG) and 4 µl of DNA. A negative control was performed for each multiplex reaction that was processed in the same way as the samples throughout the whole experimental procedure. The cycling program consisted of 15 minutes at 37°C (carry-over contamination prevention through digestion by UNG of dUTP-labeled amplicons), 95°C for 10 minutes (inactivation of UNG and activation of the Fast Start DNA polymerase), followed by 50 cycles at 95°C for 20 seconds, 55°C for 30 seconds, 72°C for 30 seconds and a final extension step at 72°C for 4 minutes.

For each sample, we then performed the six multiplex PCRs with different primer combinations (mtDNA, coat color and Y chromosome) that were pooled at the end in a 96-well plate. Each extract was amplified in triplicate so that, at the end, there were three pools for each extract.

ION Torrent sequencing

The following steps were performed using an automate Tecan Freedom Evo 100 equipped with a 4 channel liquid handling arm suitable for disposable tips, a gripper to move objects, a double thermoblock and an automated solution for vacuum solid phase extraction. We first performed End Repair with the NEBNext end repair module (New England Biolabs) using 20 µL of multiplex PCR (containing no more than 5 picomoles of amplicon products) in a 50 µL reaction volume with only 0.1 µL of End Repair Enzymes. The reaction was incubated for 30 minutes at 25°C and then purified using the NucleoSpin 96 PCR clean-up kit as recommended by the manufacturer (Macherey-Nagel ref. 740658). The DNA was eluted in 50 µL and 20 µL were used for the ligation of the sample-specific Ion Torrent barcoded adaptors (1 µL of annealed A+P1, 20 µM) using the NEB Next ligation module (New England Biolabs) in a 30 µL reaction volume with 1 µL of Quick Ligase. The reaction was incubated for 30 minutes at 16°C. After the ligation, 60 µL of binding buffer (i.e., NT buffer, Macherey-Nagel) was added and all samples were pooled in a tube before purification on a silica column. The pooled barcoded PCR products were size selected using either an E-Gel SizeSelect (Life technology) or the Caliper Labchip XT (Perkin Elmer) to purify the PCR products ligated to the adapters. The selected product was subjected to nick repair and amplification with the NEB OneTaq Hot Start in a 40 µL reaction volume containing Ion Torrent primers A and P1 (0.5 µM). The reaction was incubated for 20 min at 68°C (nick repair step). The DNA library was then amplified using the following program: initial denaturation at 94°C for 5 min, (94°C for 15 sec, 60°C for 15 sec, 68°C for 40 sec) for 6 cycles, final elongation at 68°C for 5 min. Products were finally purified with a Qiaquick PCR purification kit (Qiagen). The size distribution and concentration of the library was assessed on the Agilent 2100 Bioanalyzer. Emulsion

Figure 1 :

 1 Figure 1: Comparative maximum likelihood phylogenetic analyses of horse mitogenome using either the complete mitogenome sequences (left side) or the concatenated mitogenome fragments used for genotyping ancient remains (right side). The nomenclature of the horse mitochondrial haplogroups from A to R is as defined by Achilli et al., 2012 (47). Haplogroup S corresponds to an additional haplogroup obtained when adding Przewalski's horse sequences not belonging to the F haplogroup (98). The scale bar represents the number of nucleotide substitution per site as indicated. The branches separating horse, hemione and donkey sequences are not drawn to scale as indicated by the intersecting parallels. A magnified view of the O-P-Q subtree of the concatenated fragments is represented in the box on the right side. The magnified view reveals in addition the position of the X sequence found in two ancient Anatolian remains. The numbers by the nodes indicate their corresponding bootstrap values.

Figure2:

  Figure2: Mitochondrial and coat color diversity before (Upper part) and after (Lower part) 2,000 BCE. The left panel presents evolution of mitochondrial haplotype diversity of horses in Anatolia and the southern Caucasus. The right panels represent the evolution of coat color genetic diversity in these two geographic regions in the same time ranges. The area of the circles is proportional to the number of individuals present in each category.

Fig. S1 :

 S1 Fig. S1: Map showing the locations of the archaeological sites from which the analyzed specimens originate. Aşıklı Höyük (1), Acemhöyük (2), Çadır Höyük (3), Boğazköy-Hattusa (4), Köşk Höyük (5), Troy (6), Lidar Höyük (7), Didi Gora (8), Tqisbolu Gora (9), Lusakert[START_REF] Outram | The earliest horse harnessing and milking[END_REF], Mets Sepasar (11), Tsaghkahovit[START_REF] Anthony | Bit wear, horseback riding, and the Botai site in Kazakstan[END_REF], Gegharot[START_REF] Olsen | Prehistoric steppe adaptation and the horse[END_REF]

Primer name Gene region Primer seq (5'-3') Amplicon length (bp)

  Template kit v2 DL according to the manufacturer's protocol. The DNA library was sequenced on the Ion Torrent Personal Genome Machine (PM) Sequencer using the Ion PGM 200 Sequencing Kit and Ion 314 semiconductor sequencing chips (Life Technologies).3. Information to PCR primers used 3.1. PCR primers for mitochondrial DNA program : 1 cycle 37°C, 15 min, 95°C, 10 min; 50 cycles 95°C 20 sec, 55°C 30 sec, 72°C 30 sec, ; 1 cycle 72°C 4 min

	SNP41sh2F SNP41sh2R Y3B12-16F Y3B12-16R	GGTGGATTCTCAGTAGACAAA GGGCTGTGATGATGAAGGGTA Y3B12 GCCCCTGGTGACTCCCAGT 14683-14703 14741-14761 GGCCTTGGCGAGGAAGAAC	79	68	37 30	CytB
	PCR and Ion Sphere Particle enrichment were Ion OneTouch System (Life Technologies) using the Primer seq (5'-3') the Primer coordinates Amplicon length (bp) Sequence length (bp) Gene region GGTACAGCTTTTTAGATACAGGTTA with Ion OneTouch 200 Primers conducted SNP2F 1554-1578 81 34 16S SNP2R GGCTGCTTTTAAGCCAACTATG HVR1F ATTTCTTCCCCTAAACGACAAC 15469-15490 96 52 D-loop HVR1R ATATTGCATGTCAGGTGGGTAT Y3B19-18F Y3B19 GGCTTGATCCAAACTGCT 70 30 Y3B19-18R TCTTTTTTATTGGATTTATTGC 15543-15564 HVR5F CCGCGGGAAATCAGCAAC 15749-15775 121 73 YAme2-21F Amelogenin TCTAAGGGAGATATTCTGCACGATT 71 21 YAme2-21R TGCAATTTGTATTGGTGCTGGAGTG D-loop HVR5R GAATGGCCCTGAAGAAAGAAC 15849-15869 YAme2-23F Amelogenin CATAGGATTTATAATGTTTCTACTTTACC 71 19 YAme2-23R ACAATTATTTTGGGCATTATTGA 1613-1634 SNP4F TTCAACTCCTCTCCCTAACAA 2749-2769 78 37 3.2. Primers for alleles involved in coat color YAme2-24F Amelogenin GGAAAATGGTGGGTAGGATAGG 66 23 YAme2-24R AAGATCCCCTTCACCCTACAC ND1 SNP4R AATGCTACGGCGAGCAAGAT 2807-2826 SNP8F AATACGCAAACATCATCATGATAAA 3450-3474 91 44 Colour Primer name Gene region Primer seq (5'-3') Amplicon length (bp) Sequence length (bp) YAme3-26F Amelogenin TTGTCCCATCACACTCCTACTCG 56 10 YAme3-26R CCATGTAAGGAGATGAAACTGAG ND1 SNP8R ATTGAGTAGAGTTCTGGCAGGT 3519-3540 SNP10F GAATCGGATTTGAAATAAATCTACT Chestnut MC1R-F MC1R-E1 GCACTCACCCATGTACTACTTCA 71 29 MC1R-R GCACGTTGCTCATGCTCAC YAme3-27F Amelogenin GGAATATAAGAATAGTTGCTCAAA 72 22 YAme3-27R ATGGTAGTACAGCATTTGAAATCAAT 4025-4049 73 25 ND2 SNP10R TCTATGGTTCGGGGATTGTACTT 4075-4097 SNP11F TCGCGCATATAGGATGAATAAC Black ASIP-F ASIP AAGAAATCCAAAAAGATCAGC 77 38 ASIP-R YAme3-28F Amelogenin GAGTCTCCAGATTTGCGATTAACT 69 24 YAme3-28R TGTGAAAGAGGAATAGTTTCC ATGAGAAGTCCAAGGCCTACCT 4487-4508 120 73 ND2 SNP11R AAGAGCTGTGGATAAATAGTATGAA 4582-4606 SNP13F AATAAAATGACAATTCGAAACCAAA 4872-4896 73 25 ND2 SNP13R GAGGAGTAGGGAGGATATAACAA 4922-4944 SNP15F GAATTAGGCCAACCTGGGAC 5479-5498 75 35 COX1 SNP15R TACGAATGCATGGGCGGTTA 5534-5553 SNP17F GTATGGGCTCACCACATGTTT 6220-6240 119 76 COX1 SNP17R GTGGCTAGTCAGCTGAATACTT 6317-6338 SNP21F TAGGCCTCCCTATTGTAATTCTG 8008-8030 95 49 ATP6 SNP21R TTGAATTGAGATTAGGCGATTGT 8080-8102 SNP25F CTCAACAGCCCTTATTACGTTTA 8522-8544 76 30 Cream MATP-F MATP (SLC45A2) GCCATAACCATCACCATGATAG 65 24 MATP-R GGCCCTACAATGAAGTCAG KIT13F KIT (intron 13) 3.4. Multiplex PCR conditions CGTCATGACTCATTCGTGAGAA 63 18 KIT13R GCTCTGAAGGTAACAAGCAACTA 3.4.1. Mitochondrial DNA Tobiano KIT16F KIT (intron 16) TTTAAATGGCTTTCTTTTCTCC 59 15 KIT16R TGCCAAGTCCCTATGAATACAC Silver SILV11-F SILV Multiplex: 1 2 3 TCCTTCTTCTTCTCCCAAATCA 52 9 SILV11-R SNP2 SNP29 SNP8 GAGCTGAGCCCTGCTTCATAA Overo ENDRB-F EDNRB SNP31 SNP15 SNP21 CAGTAGTGTCCTGCCTAGTGTTC 63 17 ENDRB-R SNP11 SNP4 SNP26 TGATTCTCAGCAGTGTGGAGTTT Leopard LP-F LP SNP17 SNP32 SNP36 AGATCGAGCTGCTGAACTGG 62 22 LP-R SNP41sh2 SNP25 SNP10 TCTCCATGATCCCAAGCAAT HVR1 SNP37 SNP13 ATP6 SNP25R GGCTTGGATTATAGCTACTGCGA 8575-8597 3.3. Sequences of Y-chromosome primers HVR5 SNP41sh1
	SNP26F SNP26R PCR program: 1 cycle 37°C 15 min, 95°C 10 min; 40 cycles 95°C 15 sec, 60°C 60 sec ; 1 cycle 72°C, 4 TCCCCTTACTCAACACCTCA 9030-9049 83 40 ACGGTTTCCTTCTATTAGGCTAT 9090-9112 Sequence Length (bp) min	COX3
	SNP29F SNP29R Y2B17-1F 3.4.2. Multiplex Coat color TCCTAAACACCCACTTCACACT AGTAGGGATAATCCTAGAGCTG Y2B17 ATGTCAGGATGCCAACTGGTC 10057-10078 Y2B17-1R GTGCCCACAAGACAATGTC 10126-10147 SNP31F ATCCCCTATCAGCCCCACTT 10380-10399 SNP31R GTGTTGGCTGGCTATGAGTA Y2B17-4F Y2B17 Multiplex: 1 AGTGGCYCCCTGAAGACATT Y2B17-4R CGACRGGAAAAGGGACAGTG ASIP 10431-10450 SNP32F CTGCCACTAATACTCATAGCCAG 10421-10443 SNP32R Y2B17-5F Y2B17 GGCCACCACTGTCCCTTTT KIT13 Y2B17-5R KIT16 CAGGACAACAACCCCACTCA GCATGGTGATGTAGAGTTTTTTTC 10476-10499 SNP36F GAAAGTATGCAAGAACTGCTAAT 11654-11676 Y2B17-6F Y2B17 GGCGACATGGCAGCACTA MATP Y2B17-6R MC1R ACCTGAGCCAGTGCAGTGA SNP36R TCCTTTAAAAGTTTGAGAGAGCC 11702-11724 SNP37F TCCTCCCTCATACTAGTTTCAC Y3B1-9F Y3B1 CTCTCTCTCTAWTGGGGCAGCAG SILV11 Y3B1-9R GTCACCKGCCAGTCTCAGC LP 11795-11816 SNP37R GCATATGAGATAGTGTTTTTTACA 11887-11910 SNP41sh1F ATCAGCAATTCCCTACATCGG SNP41sh1R TAAGGGTGGCTTTGTCTACTG 14693-14713 Y3B1-12F Y3B1 Y3B1-12R TTTTGTCACTGAGGACTCTTGCA AATGTGGATTCCAGGCAGCTTCT 14637-14657 Y3B1-10F Y3B1 GCTTCAGCCTGGCTGAGACT Y3B1-10R GCACTCCCACTGGTGAAGGA EDNRB	91 71 79 71 116 74 76 67 64 58 77 73 56	47 34 31 36 32 28 25 27 16 70 35 27 16	ND4L ND4L ND4L Ser (AGY) ND5 CytB

PCR

Institut Jacques Monod, CNRS, University of Paris, Paris; biguimaraes@hotmail.com; thierry.grange@ijm.fr; evamaria.geigl@ijm.fr

[START_REF] Levine | Late Prehistoric Exploitation of the Eurasian Steppe[END_REF] Department of Anthropology, University of North Carolina at Chapel Hill, NC 27599, USA; bsarbu@email.unc.

edu[START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF] ArchaeoBioCenter and Department of Veterinary Sciences, Institute of Palaeoanatomy, Domestication and the History of Veterinary Medicine, Ludwig Maximilian University Munich, Kaulbachstr. 37/111, 80539 Munich, Germany; joris.peters@palaeo.vetmed.uni-

muenchen.de.[START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF] State Collection of Anthropology and Palaeoanatomy Munich, Bavarian Natural History Collections, Karolinenplatz 2a,

80333Munich.[START_REF] Bökönyi | The earliest waves of domestic horses in East Europe[END_REF] Department of Anthropology, University of Chicago, 1126 East 59th

Street, Chicago, Il 60637, USA; adcock@uchicago.edu[START_REF] Benecke | Der Mensch und seine Haustiere[END_REF] Groningen Institute of Archaeology, University of Groningen, 9712 ER Groningen, The Netherlands; hijlkebuitenhuis@gmail.com

Acknowledgments. We thank the directors of excavations Harald Haupmann † (Norşun Tepe), Mihriban Özbaşaran (Aşıklı Höyük) for access to samples as well as the Department of Archaeology at Bitlis Eren University, the Çorum Müzesi, Andreas Schachner and T. Emre Şerifoğlu for their support. Permission to export samples was granted by the Turkish Ministry of Culture and Tourism and the Aksaray Museum. Many thanks to Ruben Badalyan and Adam T. Smith for permission to analyze the equids from the Project ArAGATS excavations. Funding. The study was supported by the National Science Foundation BCS-1311551. The paleogenomic facility of the Institut Jacques Monod obtained support from the University Paris Diderot within the program "Actions de recherches structurantes". The sequencing facility of the Institut Jacques Monod, Paris, is supported by grants from the University Paris Diderot, the Fondation pour la Recherche Médicale (DGE20111123014), and the Région Ile-de-France (11015901). Moreover, we acknowledge support from the French national research center CNRS, the National Geographic Society, the American Research Institute in Turkey, the University of North Carolina at Chapel Hill and Baylor University, as well as the Deutsche Forschungsgemeinschaft (DFG) to JP (PE 424/10,1-3). S.E.A. was supported by a Fulbright IIE Grant and the University of Chicago.

Supplementary Material for this article is available at Supplementary Materials and Methods Supplementary Tables S1 -S4: TabS1: samples analyzed with dates and results TabS2: List of SNPs used for determination of the mitochondrial DNA haplogroups TabS3: List of diagnostic SNPs for the determination of horse mitochondrial DNA haplogroups in both the complete mitogenomes and in the reduced assay used in the present study TabS4: List of diagnostic SNPs assayed to determine horse mitochondrial DNA haplogroups TabS5: List of SNPs obtained from the Y-chromosome assay TabS6: List of genotypes obtained from the coat color assay Supplementary The authors declare no to have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

Multiplex Y chromosome