Molecular Archaeological Study Of Ancient Equine Unearthed From The Miaoliang Site In Jingbian,shaanxi Province

As the only extant member of the Equidae family,the genus Equus has a long history of development,going through a long evolutionary process from the appearance of the ancestor of all equine,the Hyracotherium,to the different species of domestic horses that people see today.The present-day genus Equus consists mainly of horses,donkeys and zebras,which can be broadly divided into caballine and non-caballine clades,but some unknown equine still existed in the past.With the application of molecular biotechnology in the field of zooarchaeology,unique and extinct equine such as Equus(Sussemionus)ovodovi have been discovered.E.ovodovi is a member of the subgenus Equus(Sussemionus),which was first divided for its unique dental characters,and subsequently more morphological identification work and molecular biology studies on the subgenus Equus(Sussemionus)were carried out,finally identifying E.ovodovi as a separate species under the subgenus Equus(Sussemionus).Written records of "horses" in northern China date back to the Xia and Shang dynasties,and many horse bones have been unearthed in the northern Shaanxi region.Many scholars have suggested that domestic horses may have appeared in the Longshan period,but there is a lack of samples and data to support whether the horse bones unearthed in this period can be identified as domestic horses.Archaeological data show that horse bones are often found in Yin and Shang period,but relatively few Neolithic horse bones have been unearthed,which is an important point in the study of the origin of the domestic horse.The Miaoliang site in Jingbian,Shaanxi province,is a Neolithic site in the northern Shaanxi region,covering both the Yangshao and Longshan periods.A large number of ancient horse bones were excavated from the Miaoliang site.These bones could not be accurately determined as domestic horses by morphological analysis,to further determine their species,we selected 17 of these samples for molecular archaeological studies.These samples' ancient DNA informations can provide useful information for studying the origin and spread of early domestic horses.We successfully extracted mitochondrial genomes from 17 samples from the Miaoliang site and combined them with mitochondrial sequences from samples excavated at three sites(Honghe site,Muzhuzhuliang site,Beiyuan site)in northern China around 4,000 years ago,and performed phylogenetic analysis,principal component analysis,and mediated network graph analysis using reference genomes of horse and donkey.These analyses showed that these equine excavated from the Miaoliang site are not domestic horses but belong to the extinct wild horse E.ovodovi.The E.ovodovi group is more distantly related to horses and more closely related to donkeys and zebras,and diverged earlier than the donkey and zebra clades,possessing an ancient,independent maternal ancestor.We also found that the E.ovodovi group is divided into two main branches,one of which is dominated by the samples in Shaanxi region(EOV1)and the other by the samples in Heilongjiang and Siberian regions(EOV2).In the EOV2 branch,the Honghe site group(EOV2a)and the earlier Pleistocene group(EOV2b)are also diverging.There is a lack of E.ovodovi sample sequences in Inner Mongolia,and issues related to gene exchange and dispersal pathways within the E.ovodovi population need to be further investigated.Through the molecular archaeological study of horse bone samples excavated from the Miaoliang site,combined with the sequence of equine faunal samples excavated from the Honghe site,the Muzhuzhuliang site,and the Shatangbeiyuan site,which are about 4,000 years old,we found that they are not domestic horses but undomesticated wild horses,which indicates the necessity to carry out morphological analysis and molecular archaeological research on the remains of ancient equine excavated in the Longshan period,in order to provide more clues to the origin of the Chinese domestic horses.