Liao valley is transroute for Koreanic and Japonic O3a from Central Plains, and C3e from the northern steppes

C3e first appeared in Dashanqian and O3a entered the latter from the Yellow River Valley area

Yinqiu Cui, Hongjie Li, Chao Ning, Ye Zhang, Lu Chen, Xin Zhao, Erika Hagelberg and Hui Zhou Y Chromosome analysis of prehistoric human populations in the West Liao River Valley, Northeast China BMC Evolutionary Biology201313:216© Cui et al.; licensee BioMed Central Ltd. : 30 September 2013

The West Liao River valley in Northeast China is an ecologically diverse region, populated in prehistory by human populations with a wide range of cultures and modes of subsistence. To help understand the human evolutionary history of this region, we performed Y chromosome analyses on ancient human remains from archaeological sites ranging in age from 6500 to 2700 BP.

47 of the 70 individuals provided reproducible results. They were assigned into five different Y sub-haplogroups using diagnostic single nucleotide polymorphisms, namely N1 (xN1a, N1c), N1c, C/C3e, O3a (O3a3) and O3a3c. We also used 17 Y short tandem repeat loci in the non-recombining portion of the Y chromosome. There appears to be significant genetic differences between populations of the West Liao River valley and adjacent cultural complexes in the prehistoric period, and these prehistoric populations were shown to carry similar haplotypes as present-day Northeast Asians, but at markedly different frequencies.

The most ancient populations of the West Liao River valley exhibited a high frequency (71%) of haplogroup N1-M231. Because of the short amplicons needed for the ancient samples, it was not possible to type the diagnostic site P43 of sub-haplogroup N1b, so samples that yielded negative M128 and TAT mutations were defined as N1 (xN1A, N1c). Besides being the only haplogroup in the Halahaigou site, N1 (x N1a, N1c) was also predominant in the Niuheliang and Dadianzi sites. In the Dashanqian site, there were two subtypes of N1-M231: N1 (xN1a, N1c) and N1c-TAT. One of the nine Dashaqian samples was N1 (xN1a, N1c), and three were N1c (Table 1). N1 is particularly widespread in northern Eurasia, from the Far East to Eastern Europe. Its subtype, N1c, is found at low frequency but has high STR variability in northern China, suggesting that this region was N1c’s centre of expansion [11].

A single instance of O3a (xO3a3) was observed in the Neolithic Hongshan and Xiaoheyan sites, although this haplogroup was observed in just under half of the Bronze Age individuals. The Upper Xiajiadian individuals of the late Bronze Age had different subtypes of O3a-M324, O3a3c-M117. O3a-M324 is found today in most East Asian populations, and its subtype O3a3c-M117 occurs at the highest frequency in modern Sino-Tibetan populations [1213].

C3-M217 is the most widespread haplogroup in Central Asia, South Asia, Southeast Asia, East Asia, Siberia and the Americas, but is absent in Oceania. Its sub-branch C3e-P53.1 is found only in Northeast Asia with low STR diversity, suggesting a recent origin in this region [6]. All individuals with the haplogroup C3-M217 in the ancient populations of the West Liao River valley belonged to the sub-branch C3e, except one from the Niuheliang site, who had an unidentified subtype. One instance of C3e-P53.1 was found in the Dashanqian site, while all 12 individuals of the Jinggouzi site belonged to this subtype. The Jinggouzi people originated in the North China steppe, and our findings support the view that C3e originated in the north.

Y chromosome STR analysis

All ancient samples were analyzed at 17 Y chromosome STR loci. Due to DNA damage, only 21 of the 47 individuals yielded results for at least three loci in two independent extractions. Consensus data are reported in Additional file 1: Table S1. The DYS389II, DYS438 and DYS635 loci frequently failed to amplify, probably because of their longer length. The inverse relationship between amplification efficiency and PCR fragment is further support for the authenticity of the extracted DNA, as ancient DNA is presumably degraded while modern DNA contamination would exhibit longer fragment lengths.

There are only two Y-chromosome haplotypes in the Jinggouzi site suggesting that individuals are paternally closely related, despite being buried in separate tombs. In the other sites in our study, we detected no potential paternal relatives among ancient individuals of the same haplogroup.


Y chromosome characteristics of the prehistoric population

The West Liao River valley was a cradle of Chinese civilization, together with the valleys of the Yellow River and Yangtze River, and there is considerable interest among scholars in the origin and expansions of the ancestors of the present-day inhabitants. Extensive analyses of extant populations have revealed that the most common Y chromosome haplogroup today is O-M175 (58.8%, n=176), followed by C3-M217(23.8%), N-M231(8.5%), and several relatively rare haplogroups, namely D-M174, Q1a1-M120, and R-M207 [814]. Our data reveal that the main paternal lineage in the prehistoric populations was N1 (xN1a, N1c), present in about 63% of our combined sample of all cultural complexes. It was the predominant haplogroup in the Neolithic period (89%), and declined gradually over time (Table 1). Today it is only found at low frequency in northeast Asia (Table 2). There appears to be significant genetic differences between ancient and extant populations of the West Liao River valley
Previous analyses showed that there were different frequency distributions of the sub-haplogroups used in this study in both ancient and extant populations of adjacent regions. The Yellow River valley, located in the southwest region of the West Liao River valley, was one original centre of agriculture in China. O3-M122 is the most abundant haplogroup in both ancient (80%, n=5) and extant population (53%, n=304) of the region [8, 13], but the frequency of O3-M122 only began to rise in the West Liao River valley in the Bronze Age. The ancient West Liao River valley population is significantly different from both the ancient Yellow River Valley population (P<0.01), and the extant Yellow River Valley population (P<0.01). The Miaozigou site, about 500 km west of the West Liao River valley in the central/southern region of Inner Mongolia, was settled by people of the northern branch of the Yangshao culture, an important Neolithic farming culture along the Yellow River. Our analysis of three ancient Miaozigou individuals revealed that they all belong to haplogroup N1(xN1a, N1c), while the main lineage of the Yellow River valley culture is O3-M122 [9]. The existence of N1(xN1a, N1c) in the Miaozigou site could be evidence for the expansion of the Hongshan culture during its heyday, a view supported by archaeological evidence of Hongshan influences at the Miaozigou site [15]. However, the small sample size of our current ancient genetic material and the lack of data for earlier time periods means an alternate explanation [16], in which N1(xN1a, N1c) existed across the region prior to the Neolithic, is still a possibility.The main haplogroups of Northern steppe nomad population were C3 (50.7% in the Mongolian, n=285) [8, 17, 18], and N1c (94% in the Yakut, n=184) [19]. The ancient individuals from the Jinggouzi site, a Northern Steppe nomadic culture on the western fringes of the West Liao River valley, carry a single haplogroup, C3e, divided into two sub-types on the basis of Y chromosome STR analysis. Previous mtDNA data have shown that the Jinggouzi people have closely related mtDNA types [20], suggesting that the Jinggouzi site was settled by family groups migrating from the northern steppe within a short period, which is in agreement with archaeological results [21]. Therefore, the prehistoric people of the West Liao River valley carried the characteristic N1 (x N1a, N1c) lineage, and appear both culturally and genetically distinct.
Prehistoric migrations in relation to cultural transitions
The Lower Xiajiadian culture (LXC) was an early Bronze Age culture with a highly developed agricultural society, with a subsistence strategy quite different from the hunting-gathering strategy typical of the Hongshan culture. However, the LXC people retained the microliths (tips of hunting weapons) and custom of dragon worship typical of the Hongshan culture. Most archaeologists agree that during the transition from the Neolithic to the Bronze Age, migrants carried farming technology from the Yellow River valley to surrounding areas including the West Liao River valley. In the Dadianzi people of the LXC, O3a is the main haplogroup after N1(xN1a, N1c). The former was previously shown to be the characteristic lineage for ancient populations along the Yellow River and Yangtze River valleys [9]. Previous mitochondrial DNA analyses of the Dadianzi population showed that the LXC people probably included immigrants from the Central Plains [22]. The archaeological analyses showed that farming tools and ceramic techniques can be traced to cultures from the Yellow River Basin [3]. Both the ancient genetic and archaeological data suggest that immigrants from the Yellow River valley, of type O3a, may have migrated into the West Liao River valley and influenced changes to the existing culture, but genetic drift cannot be ruled out as the cause for the observed frequencies.The Upper Xiajiadian culture (UXC) of the late Bronze Age succeeded the LXC but was completely different from the LXC. The UXC people mainly practiced animal husbandry and made bronze objects decorated with animal and other natural motifs in the style of the Eurasian steppes. The UXC individuals of the Dashaqian site had higher Y chromosome haplogroup diversity, with a lower frequency of the LXC lineage. Only one individual carried N1 (×N1a, N1c), the prevalent haplogroup before the UXC period. The O3-M122 type could have been inherited from LXC, but the existence of two different sub-types of O3, O3a (xO3a3) and O3a3c, implies continuous northward gene flow from the Yellow River valley. It is worth noting that the two northern haplogroups N1c and C3e first appeared in the ancient peoples of the Dashaqian site. N1c-TAT has the greatest frequency in populations from Northern Eurasia (see Table 2), and 94% of Yakuts belong to this haplogroup [19]. 33.3% of Dashaqian samples were N1c, and the present-day distribution of the ancient haplotype based on one STR profile search is mainly Northern Asia. The presence of N1c in the UXC might suggest that there is immigration from the north Eurasian steppes during this period.The Jinggouzi site is situated northwest of the West Liao River Valley, and was occupied by northern nomadic tribes during similar time periods (3000-2500BP) as the Dashanqian site. All ancient samples of the Jinggouzi site were assigned to C3e, suggesting northern nomads might have entered the West Liao River valley from the northwest. C3e is rare in modern populations, and is only found in Northeast Asia.

Because the farming LXC was replaced by the nomadic UXC and no transitional type has yet been found, it had been suggested that there might have been large-scale immigration or even population replacement by northern Asian nomads [23]. Y chromosome data show immigration components from both northern steppe tribes and farmers from the Yellow River valley. However, because all original LXC lineages in this investigation were retained in the UXC gene pool, we tend to believe that while immigrant nomads from the north played an important part in the cultural transitions in this region, they probably did not replace the preceding populations in the West Liao River valley. Instead, the cultural transitions were more likely the result of adaptations to a new lifestyle caused by climate change.
Temporal continuity of paternal lineages in the West Liao River valley
The origin and development of the prehistoric populations of the West Liao River valley, a cross road of continuous migration events, is expected to involve complex processes and population admixture. Our prehistoric population data show that the principal lineages in the region remained relatively constant from the Neolithic to the Bronze Age. In the historic period, the region was controlled mainly by nomads, including the Nüzhen, Mongolians and Manchu. The genetic structure of this period can be deduced from data of Xibe, an extant minority in Xinjiang, from the northwestern region of China. The Xinjiang Xibe originated in Northeast China and were sent to Xinjiang in 1764 by the Qing emperor to defend the frontier [24]. This population carries the original Y chromosome lineages of the prehistoric population of the West Liao River valley, with a high frequency of C3e (Table 2), whose genetic structure is similar to that of the Upper Xiajiadian.

In modern times, especially the last century, a massive number of immigrants from the south poured into this region. To investigate the extent of continuity in the paternal lineages, we examined the present-day patterns of distribution of the Y chromosome lineages observed in our ancient populations (Table 2). Except for O3a, the lineages of the prehistoric people are present today at low frequencies in the West Liao River valley. O3a continued to enter the West Liao River valley during the expansion of the Yellow River valley culture, displacing or replacing the original lineages. Today, N1 (xN1a, N1c) and C3e are mostly found in the northern Han and the northeast minority populations such as the Mongolians, Manchu, Oroqen, Xibe and Hezhe, although at low frequencies. Yi is the only population which has a relatively high frequency of N1 (xN1a,xN1c) in southern China. According to the archaeological record, one of the original branches of the ancestral Yi population was the Diqian, a nomadic ethnic group who lived in the northern steppes from 5000 to 3000 BP [25], which may explain the origin of N1(xN1a,xN1c) in the Yi people.

Our results suggest that the prehistoric cultural transitions were associated with immigration from the Yellow River valley and the northern steppe into the West Liao River valley. They reveal the temporal continuity of Y chromosome lineages in populations of the West Liao River valley over 5000 years, with a concurrent increase in lineage diversity caused by an influx of immigrants from other populations. During the cultural transitions occurring in this region, the immigration had an effect on the genetic structure of populations in this region, but no population replacement was found