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RESULTS
On the basis of 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. 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 polymerase chain reaction (PCR) and the efficiency of next-generation sequencing (NGS) specifically tailored to highly degraded ancient DNA (60). This approach has been shown to recover DNA molecules that escape shotgun sequencing (15) or sequence capture (61) and, if primers are optimized in silico and in vitro, is highly locus specific (60). Used in combination with methods minimizing contamination (62), as well as statistically sufficient replications (60) (see also Materials and Methods and the Supplementary Materials), it at least equals or can occasionally be superior to DNA capture methods, which are plagued by biases [e.g., (63)]. We have used this approach for the study of various species, including horses (15, 60, 64, 65).
Here, we analyzed 111 equid remains from eight sites in central Anatolia and six sites in the Caucasus dating from the Early Neolithic to the Iron Age (ca. 9000 to 500 BCE; see table S1), 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 (60, 64, 65), and it has been used successfully in situations where shotgun sequencing was not effective enough to genotype a large proportion of phenotype-associated single-nucleotide polymorphisms (SNPs) (15). We targeted the mitochondrial hypervariable region and 18 specific SNP regions diagnostic for the 18 major mitochondrial haplogroups considered diagnostic in earlier studies [(47) and table S2]. 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 table S3]. Last, 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 gray), diluted phenotypes (silver and cream), spotted or painted phenotypes (overo, tobiano, and sabino), and leopard spotting (table S4) (11).
Fig. 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. (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 additionally reveals the position of the X sequence found in two ancient Anatolian remains. The numbers by the nodes indicate their corresponding bootstrap values.
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 (table S1). We obtained 14 different caballine (Equus caballus) mitochondrial haplogroups previously defined in present-day horses (47) and a previously unidentified haplogroup, here termed X, that belongs to the subtree of the O, P, and Q haplotypes (Fig. 1 and table S2). Moreover, from 10 specimens, we obtained haplotypes characteristic of donkeys (Equus asinus). Last, seven specimens yielded haplotypes belonging to E. hemionus clade H1, which Bennett et al. (41) assigned to E. hemionus hydruntinus 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%) (table S1). In particular, 38 of the 40 remains assigned osteologically to wild or domestic horses showed the corresponding mitochondrial DNA (mtDNA) (95%). Agreement was also obtained for 3 of 6 hydruntine and 7 of 11 donkey remains. Of the 20 remains that could not be assigned osteologically to one of the aforementioned species, we identified 16 horses, 2 donkeys, and 2 hydruntines through mtDNA typing. Genotyping and osteological determination disagreed in only four osteologically unambiguously assigned cases: Two bone specimens determined osteologically as hydruntines yielded horse mtDNA, whereas two others classified osteologically as horses carried a donkey and a hydruntine mtDNA. Last, one unassigned equid was determined genetically as a hybrid, more precisely a mule, because it carried horse mitochondrial and donkey Y chromosomal DNA (table S1: specimen CD6189).
Diachronic pattern of maternal lineages
The 12 Anatolian horse remains predating ~4500 BCE carried either the mitochondrial haplogroup P or a previously unidentified mitotype, termed here X, not previously identified in modern or ancient horses (Figs. 1 and 2 and tables S1 and S2). Neither the P nor the X haplogroup has been documented so far beyond Anatolia in contemporaneous or older samples (48, 50, 66–69). This strongly suggests that these two haplogroups represent the unique signature of a local wild horses native to the Anatolia plateau. After 2200 BCE, this pattern changed profoundly, with 13 new mitochondrial haplogroups appearing in faunal assemblages from the Bronze and Iron Ages (Fig. 2 and table S1). Among the Bronze and Iron Age specimens, the pre-Bronze Age haplogroup P represents only 6% of the obtained haplotype spectrum (2 of the 33 remains), with both specimens dating to the earliest phase of this period (c. 2000 BCE). Moreover, in post-3300 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, and Q account for the remaining 20 specimens (table S1). These results indicate a nearly complete population turnover from the late third millennium BCE onward and correspond well with iconographic and textual evidence for the appearance and dispersal of horse management in Anatolia and Mesopotamia (26, 28).
Fig. 2 Mitochondrial and coat color diversity before (top) and after (bottom) 2000 BCE.
(Left) Evolution of mitochondrial haplotype diversity of horses in Anatolia and the southern Caucasus. (Right) 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.
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, and 11 represent a diverse array of haplogroups including A, B, C, E, FG, G, and Q. Together, this change in haplotypes in both Anatolia and the Caucasus is statistically highly significant (P = 5.7 × 10 −6, Fisher’s test). 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 (table S3). 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 6 to E. asinus, and one specimen that was identified more generally as asinine (tables S1 and S3).
We could attribute the horse Y chromosomal sequences to two of a total of four horse haplotypes that have been described previously (57): 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 jackass, whereas the mtDNA corresponded to a horse (tables S1 and S3), thus reflecting the presence of a hybrid (mule) dating to the Iron Age. 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 (11, 58). 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 (tables S1 and S4). Together, we obtained SNPs from 43 specimens, allowing us to infer the coat color for 33 individuals, including 25 horses, 6 donkeys, 1 hydruntine, and 1 mule. In our dataset, we identified the mutant allele for all but two 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 (11) proved present in Bronze Age horses in SWA (table S4). 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 (tables S1 and S4). This diversification in the coat color distribution is statistically significant (P = 1.25 × 10−3, Fisher’s test).
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 bay tobiano coat in horses (tables S1 and S4). 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.
On the basis of 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. 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 polymerase chain reaction (PCR) and the efficiency of next-generation sequencing (NGS) specifically tailored to highly degraded ancient DNA (60). This approach has been shown to recover DNA molecules that escape shotgun sequencing (15) or sequence capture (61) and, if primers are optimized in silico and in vitro, is highly locus specific (60). Used in combination with methods minimizing contamination (62), as well as statistically sufficient replications (60) (see also Materials and Methods and the Supplementary Materials), it at least equals or can occasionally be superior to DNA capture methods, which are plagued by biases [e.g., (63)]. We have used this approach for the study of various species, including horses (15, 60, 64, 65).
Here, we analyzed 111 equid remains from eight sites in central Anatolia and six sites in the Caucasus dating from the Early Neolithic to the Iron Age (ca. 9000 to 500 BCE; see table S1), 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 (60, 64, 65), and it has been used successfully in situations where shotgun sequencing was not effective enough to genotype a large proportion of phenotype-associated single-nucleotide polymorphisms (SNPs) (15). We targeted the mitochondrial hypervariable region and 18 specific SNP regions diagnostic for the 18 major mitochondrial haplogroups considered diagnostic in earlier studies [(47) and table S2]. 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 table S3]. Last, 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 gray), diluted phenotypes (silver and cream), spotted or painted phenotypes (overo, tobiano, and sabino), and leopard spotting (table S4) (11).
Fig. 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. (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 additionally reveals the position of the X sequence found in two ancient Anatolian remains. The numbers by the nodes indicate their corresponding bootstrap values.
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 (table S1). We obtained 14 different caballine (Equus caballus) mitochondrial haplogroups previously defined in present-day horses (47) and a previously unidentified haplogroup, here termed X, that belongs to the subtree of the O, P, and Q haplotypes (Fig. 1 and table S2). Moreover, from 10 specimens, we obtained haplotypes characteristic of donkeys (Equus asinus). Last, seven specimens yielded haplotypes belonging to E. hemionus clade H1, which Bennett et al. (41) assigned to E. hemionus hydruntinus 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%) (table S1). In particular, 38 of the 40 remains assigned osteologically to wild or domestic horses showed the corresponding mitochondrial DNA (mtDNA) (95%). Agreement was also obtained for 3 of 6 hydruntine and 7 of 11 donkey remains. Of the 20 remains that could not be assigned osteologically to one of the aforementioned species, we identified 16 horses, 2 donkeys, and 2 hydruntines through mtDNA typing. Genotyping and osteological determination disagreed in only four osteologically unambiguously assigned cases: Two bone specimens determined osteologically as hydruntines yielded horse mtDNA, whereas two others classified osteologically as horses carried a donkey and a hydruntine mtDNA. Last, one unassigned equid was determined genetically as a hybrid, more precisely a mule, because it carried horse mitochondrial and donkey Y chromosomal DNA (table S1: specimen CD6189).
Diachronic pattern of maternal lineages
The 12 Anatolian horse remains predating ~4500 BCE carried either the mitochondrial haplogroup P or a previously unidentified mitotype, termed here X, not previously identified in modern or ancient horses (Figs. 1 and 2 and tables S1 and S2). Neither the P nor the X haplogroup has been documented so far beyond Anatolia in contemporaneous or older samples (48, 50, 66–69). This strongly suggests that these two haplogroups represent the unique signature of a local wild horses native to the Anatolia plateau. After 2200 BCE, this pattern changed profoundly, with 13 new mitochondrial haplogroups appearing in faunal assemblages from the Bronze and Iron Ages (Fig. 2 and table S1). Among the Bronze and Iron Age specimens, the pre-Bronze Age haplogroup P represents only 6% of the obtained haplotype spectrum (2 of the 33 remains), with both specimens dating to the earliest phase of this period (c. 2000 BCE). Moreover, in post-3300 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, and Q account for the remaining 20 specimens (table S1). These results indicate a nearly complete population turnover from the late third millennium BCE onward and correspond well with iconographic and textual evidence for the appearance and dispersal of horse management in Anatolia and Mesopotamia (26, 28).
Fig. 2 Mitochondrial and coat color diversity before (top) and after (bottom) 2000 BCE.
(Left) Evolution of mitochondrial haplotype diversity of horses in Anatolia and the southern Caucasus. (Right) 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.
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, and 11 represent a diverse array of haplogroups including A, B, C, E, FG, G, and Q. Together, this change in haplotypes in both Anatolia and the Caucasus is statistically highly significant (P = 5.7 × 10 −6, Fisher’s test). 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 (table S3). 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 6 to E. asinus, and one specimen that was identified more generally as asinine (tables S1 and S3).
We could attribute the horse Y chromosomal sequences to two of a total of four horse haplotypes that have been described previously (57): 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 jackass, whereas the mtDNA corresponded to a horse (tables S1 and S3), thus reflecting the presence of a hybrid (mule) dating to the Iron Age. 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 (11, 58). 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 (tables S1 and S4). Together, we obtained SNPs from 43 specimens, allowing us to infer the coat color for 33 individuals, including 25 horses, 6 donkeys, 1 hydruntine, and 1 mule. In our dataset, we identified the mutant allele for all but two 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 (11) proved present in Bronze Age horses in SWA (table S4). 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 (tables S1 and S4). This diversification in the coat color distribution is statistically significant (P = 1.25 × 10−3, Fisher’s test).
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 bay tobiano coat in horses (tables S1 and S4). 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.