Among the most pivotal events in human history was the domestication of wild animals. Once animals could be utilized as livestock, they provided daily labor as well as necessities in the form, for example, of clothing and food, all for the maintenance of human civilization [1–3]. The horse has a unique position in history, as it had perhaps the most profound impact on human society, having decisively influenced transportation, trade, and human life itself [4]. The advent of interregional communication, the expansion of trade, the enhancement of agricultural productivity, and even the spread of diseases - all were accelerated using by the use of domesticated horses [5].

The horse, despite its relatively low population density, has shown the widest geographic distribution among livestock [3]. During the Pleistocene, large herds of Equus ferus (wild horses) roamed the plains of Asia, Africa, Europe, and North America, though their habitats had become somewhat reduced by the end of the Ice Age [6]. The earliest domestication of the horse seems to have occurred on the Kazakhstan steppes between the fifth and fourth millennia BCE [7]. The overall genetic and archaeological evidence obtained to date shows evidence of several horse domestications in a variety of regions [4,8–15].

Over the past several decades, genetic analyses on mito chondrial DNA (mtDNA) have been performed for different horse breeds. The first-ever report of full-sequence horse mtDNA (16,660 bp) was that of Xu and Arnason [16]. From then on, many researchers have continued to conduct mtDNA research on the following subjects: Japanese domestic horse [17], Jeju Island horse [18], African ass, plains zebra and horse [19], equid remains from archaeological sites of late-Pleistocene deposits [8], Oriental and European breeds along with American mustangs [9], German heavy draught horse breeds [20], Iberian horses [21], indigenous ancient and modern Irish horse populations [10], ancient Chinese domestic horse [22], domestic horses in modern China [11], ancient Iberian horse remains [12], whole mitochondrial genomes of domestic and E. przewalskii horses [4], as well as mitogenomes of modern horses in Asia, Europe, and the Americas [13]. Recently, Cieslak et al [23], Achilli et al [13], and Yang et al [15] all reported that horses could be genetically classified into several major mitochondrial lineages (A to G) and 19 clusters.

Such genetic information is crucial to any comprehensive understanding of the domestication of horses and their subsequent geographical spread in history. However, those studies notwithstanding, the accumulated mitogenetic data on ancient or medieval horses remains insufficient. This is true especially for Korean horses. Our current study therefore was undertaken to uncover genetic information on skeletal horse remains collected at a 15th century archaeological site located in the capital (Old Seoul City) of the Joseon Kingdom (1392–1910 CE). We endeavored to obtain insights into the genetic traits of the medieval Korean horse that had prevailed in Korea prior to the introduction of Western horses in the 20th century.

The PCR products of the specimens under electrophoresis were shown as specific bands (11 amplicons) for the cytochrome b, tRNA, and D-loop region of E. caballus mtDNA, while no amplified bands were observed in the extractions of the negative controls (Figure 3). The cloned PCR products (n = 5 for each amplicon) were then sequenced and aligned to determine the consensus sequence (total length: 989 bp) (Supplementary Table S3). In NCBI/BLAST search, we could not find any taxa 100% identical to the Gongpyeongdong horse mtDNA sequence. Nevertheless, some sequences in GenBank showed remarkably high similarity (above 99%), as follows (Table 2): E. caballus reported from South Korea (KF038159.1, 99.49%; KF038162.1, 97.57%), Iran (JN398455.1, 99.49%), China (MG001421.1, 99.39%; MG001418.1, 99.39%; MG001419.1, 99.29%), and Central Asia (JN398452.1, 99.39%; JN398450.1, 99.09%) (Figure 4).

As seen in Table 2, we also found that the Gongpyeong dong mitotype shares 97.97% identity with E. przewalskii, which is not much different from other E. caballus horses (ranging approximately from 97% to 99% identity). On the other hand, the Gongpyeongdong mitotype is distinct from those of the donkey (E. asinus; 95.94%) and imperial zebra (E. grevyi; 90.86%). This pattern was further confirmed by pairwise distance analysis. As seen in Figure 5, the outgroup breeds (E. asinus and E. grevyi) were genetically remote (0.076 and 0.082, respectively) from the Gongpyeongdong case, whereas the pairwise distances remarkably close to the latter were those of E. caballus taxa reported from South Korea (0.005), Iran (0.005), China (0.006 to 0.008), Central Asia (0.008), and other countries (Figure 5). Using mtDNA cytochrome b, tRNA, and D-loop sequences of the Gongpyeongdong case and other E. caballus taxa from GenBank, the phylogenetic tree could be reconstructed (Figure 6).

E. przewalskii horse, which inhabits the Mongolian grasslands, is morphologically distinct from modern horses, and thus has been thought to be the direct descendant of wild horse E. ferus [13]. However, Ishida et al [17] revealed by phylogenetic analysis that E. przewalskii is within the genetic variation of the Japanese domestic horse. They conjectured that the chromosome number change of E. przewalskii might have occurred rather recently. Another recent study revealed that E. przewalskii is likely a horse that was domesticated once and then returned to the wild, like the mustang of North America [14].

In Korean history, descriptions of domesticated horses are seen as early as the 2nd century BCE [3]. In the 12th century CE, Koreans determined their horses to be comprised of at least six breeds, as dependent on their body shapes [3]. However, those classifications were based only on appearance, not on any scientific evidence. In this regard, the Jeju Island horse (Jorangmal in Korean) is important to any understanding of the genetic traits of ancient or medieval Korean horses because Western horses were actively introduced starting in the 20th century, with the result that indigenous Korean horses became much rarer. Actually, many scholars consider the Jeju Island horse to represent the indigenous Korean horse populations [30].

Nevertheless, as historical and genetic research on horses proceeded, it turned out that the reality was not so simple. For instance, when Mongolians invaded the Korean Kingdom in 1276, they established ranches on Jeju Island to raise their horses. Therefore, the genetic traits of Jeju Island horses might have been seriously influenced by introgression of Mongolian breeds [3]. In fact, this conjecture was further confirmed by an mtDNA D-loop analysis exhibiting multiple origins (possibly representing Mongolian and non-Mongolian breeds) in Jeju Island horses’ maternal lineage [18]. Simply put, indigenous Korean horses have not remained the same but have been constantly changed by introgression of foreign horses in history [3,30]. This hypothesis also was supported by our phylogenetic analysis (ML method) findings. Based on the previous reports of mtDNA haplogroup using cytb and D-loop sequences [13,14,18,28,29,31–36] (Supplementary Table S4), the Gongpyeongdong horse was clustered together with the E. caballus taxa of haplogroup Q. According to Achilli et al [13], a horse haplogroup such as Q shows frequency peaks in Asia (13.80%) and a decline to the Middle East (10.42%) and Europe (3.85%). Ning et al [29] also confirmed that the genetic origin of haplogroup Q is likely East Asia [29]. In this sense, the present Gongpyeongdong case, the 15th century horse bones found on the mainland of Korea, can be regarded as genetically representative of the horses long established in East Asia. In the phylogenetic tree, we also noted that Jeju Island horses belong to the different clades of B, D, E, I, J, O, and Q (Figure 6). Among them, haplogroups O and Q are mostly localized to East Asia [13,29]; the ancestral haplotypes of haplogroup D, moreover, also have been localized to Asia [29]. On the other hand, the Jeju Island horse of haplogroup I looks somewhat different from those of haplogroups D, O, and Q, because the former is known to be common among Western and Southern European horses [29]. Taken together, modern Jeju Island horses include different maternal haplogroups with various genetic backgrounds. We can conjecture that various horse breeds with different genetic origins left their genetic traits among modern Korean horses.

By obtaining a consensus mtDNA sequence from a horse bone collected at a 15th century archaeological site (Gongpyeongdong) in Old Seoul City, we confirm that the horse belongs to haplogroup Q, the genetic origin of which could be traced back to East Asia. Our data reported on the maternal lineage of Joseon period horse bones are meaningful for understanding the domestication history in East Asia, which has yet to be comprehensively revealed. We look forward to further relevant aDNA reports on ancient or medieval horse bones found at South Korean archaeological sites.