Liqian people, officially recognized as Han Chinese by P. R. China, live in some small village located in Yongchang County of Gansu province, China. Many of them have light colored hair and Caucasian features, which are sharply different from Han Chinese and most ethnic minorities. Recent years, Liqian people were well known to all with controversial hypothesis of ancient Roman mercenary origin. In 1955, Homer H. Dubs proposed that some Roman soldiers captured by the Parthians after Crassus’s defeat at Carrhae in 53 B.C. were eventually hired as mercenaries by a Hun warlord in the western frontier past the boundaries of the Han Empire and were captured by the Chinese and allowed to form their own city, based on the Roman model.
The hypothesis has been adopted by some scholars. It was, however, disputed by many historians. Several decades passed, the hypothesis remains hotly debated. No direct evidence, paternal genetic contribution seems particular necessary. To test this hypothesis, we surveyed more than 12 Y chromosome binary polymorphisms by use of PCR （polymerase chain reaction, PCR-RFLP （restriction fragment length polymorphism）, DHPLC （Denaturing High Performance Liquid Chromatography） methods and 12 short tandem repeat （Y-STRs）loci by using Powerplex? Y system for 227 male individuals representing four Chinese populations: Liqians, Yugurs, Uygur and Tibetans. In comparison with worldwide populations, the following results were obtained.1.
Eleven Y-SNP haplogroup and 75 Y-STR haplotype were observed for 87 unrelated Liqian males. At the haplogroup level, the Liqians presented low genetic diversity with a single highest frequent haplogroup O3-M122 （71.3%）. When 12 fast-evolving Y-STRs were used, the genetic diversity of Liqians is high than 0.98. In present study, 77% Liqian Y chromosomes were restricted to East Asia. It is unexpected that the frequency of Haplogroup O-M175, an East Asian-specific haplogoup, is relatively higher in Liqian people than that in most populations in North China.2. Principal Component （PC） based on Y-SNPs and multidimensional scaling （MDS） analysis on basis of Y-STRs suggests that the Liqians is closely related to Chinese populations, especially Han Chinese populations, whereas greatly deviate from Central Asian and West Eurasian populations. The positions of populations within some clusters correspond well to their predefined assignments to specific regional groups.
One conclusion can also be drawn from these analyses: despite the ascertainment bias in the binary markers, PC result based on Y-SNPs is consistent with MDS result on basis of Y-STRs. In addition, further phylogenetic analysis confirmed the genetic affinity between Liqian and Han Chinese populations.3. By PC and MDS analysis, we found two old populations: Han Chinese and Mongolians, which showed close genetic relationship to Liqians. In subsequent admixture analysis, the two populations were assumed to be parental populations of Liqians, and the Liqians is regarded as hybride population. Admixture proportional analysis suggested that the genetic contribution from Han Chinese amount to 78% in Liqians.4. The Liqians and the Yugurs, regarded as kindred populations with common origins, present underlying genetic difference in Median-joining network and admixture proportional analysis.5.
Liqian population show close affinities to its geographic neighbors. This is confirmed in Mantel test （r=0.646, P =0.003）, which show a strong and highly significant partial correlation between genetics and geography among population mentioned in our study.6. Statistically, the Liqian showed non-significant genetic difference to Han Chinese in North China, and significant genetic difference to other Eurasian populations.7. When we compare Liqian minimal haplotypes （9 loci “minimal haplotype”） with worldwide data in YHRD, most Liqian haplotypes were found in East Asia and South Asia. Only two matches were found in Europe, but they belonged to East Asia-specific Haplogroup O-M122. The incompatible result probably originated from recurrent mutation of fast-evloving Y-STRs. Overall, Roman mercenary origin could not be accepted as a history truth according to paternal genetic variation, and the current Liqian population is more likely to be a subgroup of Chinese majority Han. Our studies provided genetic evidence for the origin of the Liqian people, and inriched to human genetic database. The 12 Y-STR polymorphism markers are highly discriminating in the Chinese Liqian population, and they may be powerful for paternity testing and personal identification.
In August 2013, Andronovo culture remains, including 17 tombs and 3 ritual sites, were discovered in Xinjiang Uyghur Autonomous Region, which shows that the Uyghurs are culturally and genetically linked to the Andronovo culture. The Andronovo people belonged to R1a/Z93 mixed with Q1a2a1/L54 and the R1a frequency in the Uyghurs amounts to 28.6%. On the other hand, R1a1 accounts for only 1.1% in Liquian villagers in western China and 71.3% of them belong to O3 (Zhou et al. 2007). Other West Eurasian haplogroups such as P*(xR1a) and J* were found at 8% and 1.1% respectively. The frequency of P*(xR1a) varied from 26% in the east to 45% in the south of Norway. Up to 10% of the Liquian villagers,which explains why they are born with Caucasian physical features, and they may also have the steppe ancestry linking them to the Andronovo people from Russia.
The first extensive analysis of Y variation in the Liqians was carried out in our study. This allowed us to compare our data with those previously reported in worldwide populations in order to investigate the origin and evolution。 Internal numbers are bootstrap values (%) for 1,000 replicates. Tunisian was regarded as outgroup of the Liqian people. The results based on Y-SNPs are consistent with those obtained with Y-STRs, which rein-force our findings. In the present study, it was unexpected that 71.3% Ychromosomes of the Liqians belonged to the haplogroupO3-M122, which is an East Asian-specific haplogroup (Suet al.1999; Shi et al.2005). The O3-M122 frequency in the Liqians was by far the highest one observed among pop-ulations in northern China (Xue et al.2006; Su et al.1999,2000b; Shi et al.2005). Since the Han dynasty, the Liqians have been living in northwestern China, which has been well-established by historical records (Song et al.2005).Combining with the historical records, the haplogroup distribution suggested that most of the Liqian Y chromo-somes could be traced back to northern China. The result was also reflected in PC and MDS analysis, which indi-cated that the Liqians were genetically close to Chinese populations, especially Han Chinese populations in various regions. The result was confirmed in admixture and phy-logenetic analysis (Table2, Fig.5), suggesting a strong Han Chinese paternal influence on the Liqian gene pool.The Mantel test suggests that the Liqian people, wherever they originated, must have had an extensive gene exchange with the local people.
Besides the Han Chinese people,Mongolians and Yugurs in China are genetically related to the Liqian people.Previous studies demonstrated that the male lineages of the Mongolians spread rapidly in a large part of Asia (Zerjal et al. 2002, 2003). The Hexi region in northern China, where the Liqians have settled, was controlled by the Mongols in the 13th and 14th centuries. It is reasonable that the Mongolians would have an impact on the Liqiangene pool. In our study, a small Mongolian contribution was observed in the Liqian gene pool.The Yugurs, described as a kindred population of the Liqians, were genetically close to the Liqians in PC and MDS plots, which is compatible with the admixture analysis. The paternal genetic contribution indicated that the Liqian and the Yugur populations have similar con-tribution proportions from the Han Chinese and Mongo-lian populations.
Nevertheless, the underlying genetic difference between the Liqian and Yugur populations was explored in a median-joining network and neighbor-join-ing tree (Fig.5, Fig.6). It may result from long-term isolation by distance. In addition, the Yugurs are strictly endogamous and live in autonomous regions of the central province of Gansu, while the Liqians live together withHan Chinese. Overall, the genetic difference between the Liqian and Yugur populations is statistically non-signifi-cant.In PC and MDS analysis, the Liqian population is fairly distinguished from Central Asian and West Eurasian pop-ulations. The result is incompatible with the historical hypothesis that the Liqian people derived from ancient Roman soldiers, which probably included mercenaries from West Asia, as described by Huang et al.1990. When Liqian haplotypes were compared with worldwide popu-lations in the YHRD, no matches were found only in the West Eurasian populations, and only two matches were shared by European populations and East Asian popula-tions. The two Liqian haplotypes are present in West European populations, but they belong to East Asian-spe-cific haplogroup O3. The incompatible fact probably arose from the high mutation rate of Y-STR.
It seemed that the two Y chromosomes are more likely to be Asian lineagesthan European lineages. Failure to find an apparent link between the Liqian people and ancient Roman soldiers in this study might be either because long distance migrationand intermarriage have erased earlier genetic signatures orbecause the Liqians are just a general population in north China. Moreover, it is noteworthy that a small proportion of Liqian people with mixed racial traits are not necessarily associated with ancient Roman soldiers. Along the ancient Silk Road in north China, it is common to see people with Caucasian morphological traits, which is also a classical trait of Chinese minority ethnic groups in Xinjiang (like theUygurs). Therefore, we cannot trace a Liqian origin only from morphological traits.As described above, the Liqians are closely related to Chinese populations, especially the Han Chinese in north China. In addition, the Liqian and Yugur populations arelikely to be kindred populations. No obvious signature of Roman soldier origin is observed in the Liqian paternal gene pool. A Roman mercenary origin for the Liqian people is likely to be nothing more than an interesting theory. In order to reveal genetic landscapes of the Liqians completely, complementary autosomal and mtDNA studies have to be carried out in future work.