Molecular Genetic Analysis Of Ancient Remains From Xiaohe Cemetery

The Tarim Basin in western China, as the main crossroad contact the West and the East, positioned at a critical site on the ancient Silk Road, has played a significant role in the history of human migration, cultural developments, and communications between the East and the West. However, the questions are still obscure due to the events occurred earlier always been covered up by the latter ones. The ancient DNA can be escape from the time and recurs prehistoric demographic events, therefore, it has a unique advantages in tracing population history. Fortunately, there were many well-preserved cemeteries in Xinjiang due to the dry condition and provided an invaluable opportunity to investigate the migrations and origins of ancient populations in the region, especially the Xiaohe cemetery rediscovered in 2000 year, among which a lot of preserved-well prehistoric Bronze Age"Caucasoid"mummies were excavated, which have attracted extensive interest among scientists regarding the question:"Who these people were, and where they came from?"Xiaohe cemetery is located in the Taklamakan Desert of northwest China, at the easternmost edge of Tarim Basin, Xinjiang. The burial site comprises a total of 167 graves. It can be divided into five layers from up to down spanning several hundred years (3540 -3980BP). It is the oldest and largest archeological site with human remains thus far discovered in the Tarim Basin. Many preserved-well mummies, animal remains and plant remains were excavated and exhibits very particular and enigmatic burial features. In this study, in order to trace the origin of Xiaohe people and social relationships, we analyzed the DNA of the ancient remains, containing the human remains, cattle remains and crop remains.Firstly, we successfully gained 58 reproducible mtDNA HVRI fragments, after discarding seventeen samples due to failed amplification or irreproducible results. The 58 DNA fragments containing 26 different haplotypes can be further assigned to 17 haplogroups, which contained the East Eurasian haplogroups B, C4, D, F, the West Eurasian haplogroups H, K, U5, U7, U2e, T, R*, and the Indian peculiar lineages M3, M5, and M25 . The results showed that the genetic structure of Xiaohe population is very complex, the ancient populations originating from Europe, North Asia, East Asia, and South Asia all had genetic contribution for it. The haplogroups originated from Europe in Xiaohe people were the most complex, and occurred time was also the earliest; the haplogroups originated from North Asia in Xiaohe people had the genetic feature of high frequency and very low diversity; the haplogroups originated from East Asia and India held lower frequency in Xiaohe population, and the occurred time was later.Secondly, the entire necropolis can be divided into five layers. The fifth population (the lowest layer) carried both the East Eurasian haplogroup C4, and the West Eurasian haplogroups (H and K ), demonstrating that the Xiaohe people from the lowest layer were an admixed population originating from both the West and the East. This implied that by 4000 years ago, a West–East admixed population had already settled in the Tarim Basin. However, the haplogroups H, K, and C all are very ancient lineages, over ten thousand years old in Eurasia, whereas the civilization of the Tarim Basin, according to the archaeological materials, rose very late. The admixture therefore likely occurred elsewhere, before immigration into the Tarim Basin. Furthermore, the cultural characteristics of the Xiaohe cemetery are similar to those of the Andronovo or Afanasevo culture that appeared throughout the southern Russian steppe during the second millennium B.C. where the anthropology characteristic and genetic structure of Bronze Age people were very similar with that of Xiaohe people. Therefore, the admixed population may have close relationship with the populations settled in South Siberia during Bronze Age. It is possible that the initial admixture occurred somewhere in southern Siberia. The matrilineal ancestors of the individuals who possessed East Eurasian lineage C might have originated from south-central Siberia or northeast Siberia, begun their westward movement, met the eastward-migrating western people, and intermarriage with them in that region. In addition, the genetic diversity of population from the lowest layer is very low (0.7381), demonstrating that the individuals of the layer may have had a family relationship in matrilineal descent, among which the family with the haplotype 16298-16327 has the most members (50%). Interestingly, the female individual worshipped , buried in muddy coffin, also present this haplotype, which suggested that this female had the high statue may be related to what some hypothesize was the meaning of the unusually shaped posts marking her grave, whose sexual representation perhaps indicates that the Xiaohe people placed great emphasis on reproductive symbolism.The population excavated from the fourth layer presented a more complex genetic structure. Its genetic diversity(0.9004) is obviously higher than that of population from the lowest layer, showing that following the lost of time , Xiaohe people always occurred genetic exchange with other populations , mainly containing populations from West Eurasia and India, also had a small quantity of East Asian. Moreover, the haplotype16223-16298-16309-16327 was shared by most people in this layer, as well as the female individuals worshipped.The upper three layers have same culture characteristic. Therefore, we called them as up layer. The population from the up layer had the highest genetic diversity (0.9890). It is obviously that the diversity of haplogroups originated from West Eurasia, East Eurasia and India all increased. However, the frequency of haplogroup C4 was decreased seriously. This implied that the presence of muddy coffin may be related to the people with the lineage C4.In conclusion, the later population had more complex genetic structure than that of earlier population. This suggested that after immigration into the Tarim Basin, the Xiaohe population was not isolated from other populations, but occurred frequently gene exchange. The extent of gene exchange was very high so as to the original genetic structure of Xiaohe population was changed. This may be the reason that the Xiaohe culture occurred change in the later.Thirdly, the internal transcribed spacer region (ITS region) in nuclear ribosomal DNA (rDNA) and the sequence differences between D and B genomes (IGS) were analyzed for Xiaohe wheat. The results showed that the wheat grains had similar genetic characteristic with hexaploid bread wheat which originated from Caucasia suggesting the wheat in Xiaohe cemetery is the bread wheat. The bread wheat presenting in Xiaohe cemetery implied that it had spread into Xinjiang at Bronze Age, and provided further some clues for understanding the origin of bread wheat in East Asia. Xinjiang, as the main crossroad contact the West and the East, may play a significant role in the history of eastward spread of wheat. The 5.8S gene in rDNA was analyzed for common millet excavated from Xiaohe cemetry. The result showed that common millet from Xinjiang Xiaohe cemetery have same genetic charicatristic with Chinese common millet,while different with common millet from Near East, Australia, and Europe , indicated Xinjiang common millet had same progenitor with Chinese common millet. According to the extensive and earliest presence of common millet in north china, Xinjiang common millet may original from East Asian. This indicated that the agriculture of north china have genetic contribution to Xinjiang agriculture during Bronze Age. The presence of wheat and common millet in Xiaohe cemetery indicated that the eastern agriculture culture had met the western agriculture culture in Xinjiang during Bronze Age.Fourthly, we performed mtDNA HVRI polymorphisms analysis for 17 ancient cattle samples from Xiaohe cemetery. We successfully gained14 mtDNA HVRI fragments containing 6 haplotypes, among which 10 samples were assigned to T3 lineage, 3 samples were assigned to T2 lineage, one samples attributed to T lineage. T3 both is the dominant lineage in modern European cattle and Near East cattle, and a few in East Asian cattle. T2 and T lineages mainly distributed in modern Near East cattle, a less extent in European cattle, few in East Asian cattle. In addition, the sharing sequences of Xiaohe cattle mainly distributed in Near East and Europe, some little in East Asia. This implied that the cattle was cultivated in Xiaohe may be the result of eastward spread of West Eurasian cattle, which is consistent with the origin of West Eurasian lineages in Xiaohe people.In a word, the analysis for the ancient remains from Xiaohe cemetery showed that the Tarim Basin had been occupied by a West–East admixed population since the early Bronze Age. The European, Indian and East Asian all had genetic contribution for the Xiaohe people. As the results of these population movements, various cultures were also spread into Xinjiang, such as the agriculture cultures, stockbreeding cultures, and formed the complex populations and multiply cultural coexist in Xinjiang. Our research provided molecular evidences for tracing the forming process of ancient populations of Xinjiang, and also provided insight into the population movements and culture spread in Eurasia.