Analysis of Paternal Lineages in the Chinese Populations of Macau and Shanghai

Analysis of Paternal Lineages in the Chinese Populations of Macau and Shanghai José Guilherme Moreira Alexandre Mestrado em Genética Forense Departamento de Biologia 2013 Orientador Professora Doutora Maria João Prata Martins Ribeiro, Professora Associada c/ Agregação na Faculdade de Ciências da Universidade do Porto e Investigadora no Instituto de Patologia e Imunologia da Universidade do Porto Coorientador Doutor Luis Alvarez Fernandez, PhD, Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP) Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/_________ José Guilherme Moreira Alexandre Analysis of Paternal Lineages in the Chinese Populations of Macau and Shanghai Faculdade de Ciências da Universidade do Porto Mestrado em Genética Forense 2013 “If I have seen further, it is by standing on the shoulders of Giants” Isaac Newton Dissertação de candidatura ao grau de Mestre em Genética Forense submetida à Faculdade de Ciências da Universidade do Porto. O presente trabalho foi desenvolvido no Instituto de Patologia e Imunologia da Universidade do Porto e sob orientação da Professora Doutora Maria João Prata Martins Ribeiro. Dissertation for applying to a Master’s Degree in Forensic Genetics submitted to the Faculty of Sciences of the University of Porto. The present work was developed at the Institute of Molecular Pathology and Immunology of the University of Porto and under the scientific supervision of Professor Maria João Prata Martins Ribeiro. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai i ! ! AGRADECIMENTOS À Professora Maria João Prata, minha orientadora, gostaria de agradecer em primeiro lugar, por toda a ajuda e incentivos que me deu ao longo do trabalho, com preciosas sugestões, e pela oportunidade que me deu de participar neste projecto. Ao Luís, meu co-orientador, pela disponibilidade e boa disposição com que sempre me ajudou quando necessário, e pela contribuição que deu para a realização da tese. Ao Professor António Amorim, pela oportunidade que me concedeu de frequenter este Mestrado e por tudo o que me ensinou nestes últimos dois anos. Ao grupo de Genética Populacional da Universidade Complutense de Madrid, pela forma como me receberam e por todo o apoio que me prestaram, nomeadamente a Ana Lopez, o Eduardo Arroyo e o Qi Huang. A todo o grupo de Genética Populacional, pela forma como me recebeu e por estar sempre disponível quando era necessário. À Cíntia, à Sofia Marques e à Sofia Quental. A todos os meus colegas de Genética Forense, grupo com uma grande disponibilidade e entre ajuda, com quem foi um prazer partilhar estes dois últimos anos. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ii ! ABSTRACT East Asia, the geographical region bordered by the Plateau in the southeast, by the Bering strait in the northeast, and extending into island southeast Asia is still a scarcely studied, with uncertainty surrounding the when and how the first settlements of humans reached the are, and especially the region now known as China. Currently, 56 ethnic groups are recognized in China, though the concept of ethnicity is not necessarily correlated with genetic ancestry, since this often failed to recognize pre-existing anthropological and demographic realities. In our work, we aimed to explore the genetic background of the Shanghai and Macau populations, in the broader context of population diversity in China, as well as to enrich the knowledge of the region, combining genetics with ethnic patterns, linguistic affiliation and historical records. Furthermore, we compared the results obtained in Shanghai and Macau, in order to evaluate whether signs were retained of the different geographical, historical and cultural factors that might have shaped the Y chromosome diversity in both populations, as well as China in general. In order to do so, we have sampled genetic material from 135 unrelated males, 85 from Shanghai and 50 from Macau. Samples were tested for 25 Y-SNPs using two multiplex systems – Multiplex O and Multiplex 1 and in addition for the Yap element. They were also genotyped for 17 different Y-STR markers using the AmpFℓSTR® Yfiler® PCR Amplification Kit (Applied Biosystems). As expected, the large majority of the Y lineages in this study belonged to the O haplogroup (M175): 89.5% in Macau and 81.5% in Shanghai. Furthermore, high haplogroup diversity was shown to be high for the studied populations. The levels of haplotype diversity were similarly very high in Macau and Shanghai, a feature which was also shared by other Chinese populations used to perform comparisons. Among those, the Muslim and Tibetan populations presented, however, the lowest levels of diversity, probably due to the peculiarities of their history and religious beliefs that led to a certain isolation of these populations. Although populations from China were shown to be clearly substructured, no correlation was found between geographical distance was and degree of genetic relatedness between populations. Concerning the population from Macau, despite the long shared history with Portugal, no evidence was found testifying a male-mediated genetic influence of the Portuguese. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ix ! FIGURES Figure 1 – Progressive X-Y differentiation 3 Figure 2 – Y chromosome structure 4 Figure 3 – Inheritance patterns of autosomes, Y chromosome and mtDNA 6 Figure 4 – Relative position of the 17 Y-STR loci commonly used in Y Chromosome testing 8 Figure 5 – Stepwise Mutation Model (SMM). 9 Figure 6 – Y chromosome haplogroups spatial distributions 10 Figure 7 – Global distribution of Y haplogroups 12 Figure 8 – Phylogenetic tree of the Y chromosome haplogroups studied. 30 Figure 9 – Schematic representation of the three step strategy followed in this study 32 Figure 10 – Example of an electropherogram obtained from the O specific multiplex 34 Figure 11 – Discrimination of the two YAP alleles in a polyacrylamide gel 36 Figure 12 – Map of China showing the location of the studied populations 38 Figure 13 – Frequency of the O-M175 lineages in Shanghai and Macau. 43 Figure 14 – Y-SNP gene diversity values 47 Figure 15 – Rst Genetic Distance MDS representation 54 Figure 16 – Correlation map between genetic distance and geographic distance 55 Figure 17 – Graphic representation of the FCT, FSC and FST 57 Figure 18 – Barrier representation of the RST genetic distances. 60 FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai x ! ! ABREVIATIONS AMOVA Analysis of Molecular VAriance bp base pair BP Before Present ddNTP dideoxyNucleotide-TriPhosphate DNA DeoxyriboNucleic Acid Mb Megabases MDS MultiDimensional Scaling MNPD Mean Number of Pairwise Differences MSY Male Specific region of the Y chromosome NRY NonRecombining region of the Y chromosome PAGE PolyAcrylamide Gel Electrophoresis PCR Polymerase Chain Reaction RFLP Restriction Fragment Length Polymorphism SBE Single Base Extension SMM Stepwise Mutation Model SNP Single Nucleotide Polymorphism SRY Sex determining gene STR Short Tandem Repeat YAP Y Alu Polymorphic element YBP Years Before Present YCC Y Chromosome Consortium YHRD Y chromosome Haplotype Reference Database Yp Y chromosome short arm Yq Y chromosome long arm FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 1 INTRODUCTION FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 2 FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 3 Y-Chromosome “The properties of the Y chromosome read like a list of violations of the rulebook of human genetics. It is not essential for the life of an individual, males have it but females do well without it. One half consists of tandemly repeated satellite DNA and the rest carries few genes, and most of it does not recombine. However, it is because of this disregard for the rules that the Y chromosome is such a superb tool for investigating recent human evolution from a male perspective and has specialized, but important, roles in medical and forensic genetics.” [1] Origin/Structure Nowadays, the two human sex chromosomes, X and Y, look very different from each other. Their origin, however, is believed to have been the same, evolving from a pair of homologous autosomes that followed two different evolutionary routes, around 240-320 million years before present. [1-3] Evidence indicates that the X-Y differentiation occurred only after X-Y recombination ceased, as a consequence of successive chromosomal inversions. This was likely a stepwise process that started with the acquisition of the sexdetermining gene (SRY) by one of the autosome pair. [1, 4]. This successive repression of recombination between the two chromosomes led to an expansion of the nonrecombining region (NRY), which now represents 95% of the chromosome. Figure 1 – Progressive X-Y differentiation, leading to loss in recombination from stratum 1 to strata 2, 3 and 4, four inversions are postulated. Each inversion reduced the size of the pseudoautosomal (X-Y recombining) region. [Adapted from Bruce T. Lahn and David C. Page, 1999] FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 4 The lack of recombination along with the action of other evolutionary forces led the Y chromosome to become one of the smallest chromosomes in size (60 million base pairs) and also in gene content, since the absence of recombination incapacitates the Y chromosome to eliminate deleterious mutations, a characteristic known as Muller’s Ratchet. [2] Although, as mentioned before, 95% of the chromosome does not recombine during male meiosis, X and Y chromosomes still undergo recombination in two specific regions called pseudoautosomal regions, regions that still behave as do autosomes. These two regions, located in each tip of the Y chromosome, are named PAR 1 - located on the short arm spanning approximately 2.6 Mb - and PAR 2 - located on the long arm and with approximately 0.32 Mb - and are fundamental in male meiosis, since they have a role in chromosome alignment (Figure 2). Figure 2 – Y chromosome structure. The Y chromosome is divided into a short (Yp) and a long arm (Yq), divided by a centromeric region (cen). Inside the MSY region can be distinguished the heterochromatic region and the eurochromatic region, further divided in X-degenerated, X-transposed and ampliconic regions. (Adapted from Hughes et al., 2010) Recently, the NRY has been renamed to male specific region (MSY). This happened because, even though X-Y recombination doesn’t occur, abundant intrachromosomal recombination has been reported. The MSY contains two different regions, the heterochromatic and the euchromatic region. [5] The heterochromatic region is composed mainly by long repetitive sequences that can extend for around 40 Mb. The euchromatic region includes all the genes identified in the Y-chromosome and ranges for about 23Mb, divided in both arms of the chromosome. This region includes the X-transposed sequence, originated from an X to Y transposition and therefore possesses great homology with the X chromosome, the X-degenerated region, a remnant of the ancient autosomes from FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 5 which the X and Y chromosomes evolved, and ampliconic sequences that allow intrachromosomal recombination due to their great similarity to other sequences in the MSY. [5, 6] FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 6 Uniparental Markers The vast majority of the human genome is inherited from both parents and undergoes recombination. It suffers, therefore, a complex reshuffling process of the genetic information in each generation. There are, however, two exceptions, the Male Specific region of the Y-chromosome (MSY) and the mtDNA. These types of genetic systems, unlike the autosomes, are transmitted from generation to generation without variation, unless when mutation occurs (Figure 3). [7] Figure 3 – Inheritance patterns of autosomes, Y chromosome and mtDNA. The mtDNA and MSY pass through generations escaping meiotic recombination, being transmitted as a haploid single block. Autosomes pass through generations suffering recombination. Thus, since the Y chromosome is paternally inherited and the mtDNA is maternally inherited, uniparental markers are not appropriated for individual discrimination. They are however capable of defining male and female lineages, a characteristic that represents a noticeable advantage to population ancestry studies. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 7 Y Chromosome: Genetic Markers Different types of polymorphisms can be found in the Y chromosome, each one with particular characteristics. The two main classes are the bi-allelic markers, which define haplogroups, and the multi-allelic markers, that define haplotypes. One of their main differences is the mutation rate, which results in distinct characteristics that permits a wide-range of different analysis. Bi-allelic markers (Y-SNPs) Bi-allelic markers on the Y chromosome (Y-SNPs), include Single Nucleotide Polymorphisms (Y-SNPs) and an Alu element insertion (Y Alu Polymorphism – YAP). The Alu element was the first Y bi-allelic marker to be discovered, and since then a large amount of new polymorphisms has been described, given that SNPs are thereafter the most frequent class of polymorphisms in the human genome. [8] SNPs present a low frequency of mutation, around 10-8 per generation, and therefore most usually arise by unique events: when two alleles are present, it is accepted that the derived allele arose, as a unique event, from an ancestral form. [9] In the forensic field SNPs have a number of characteristics that make them very appropriate for forensic studies, as the very low mutation rate and together with the short of DNA segments necessary to amply for their detection afford them desirable characteristic not only in the analysis of degraded samples, but also in general in the field of population genetics, due to their power to differentiate major human populations. Furthermore, Y chromosome haplogroups present a very well studied geographic distribution, revealing high population specificity, allowing to infer about the demographic events and evolutionary history of a population. [6, 10] On the other hand, SNPs have some limitations. In order for their power of discrimination meet that of the nowadays-used STRs in the new multiplexes, 60 SNPs would be necessary. [6, 11, 12] More than 600 bi allelic Y-chromosome markers have now been characterized and integrated in the phylogenetic tree of Y-haplogroups [13], defining 311 haplogroups. Nonetheless, new mutations are constantly being discovered and FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 8 await to be included in the YCC tree, which is being regularly updated. [14] Multiallelic Polymorphisms (Y-STRs) The human genome is overflowing with repetitive sequences, scattered all over the autosomes and sexual chromosomes. [9] Among these sequences, the Short Tandem Repeats (STRs) or Microsatellites are the most commonly used in Population and Forensic Genetics. STRs consist of 1 to 6 bp repetitions units, whose number varies between individuals. Repetitions from different STRs can be dealt together, since they define haplotypes, increasing this way the informative power. These markers present higher mutation rates when compared to the SNPs, which underlies their common multiallelism. This characteristic gives the STRs a higher discrimination power between individuals, and therefore, can be used to differentiate Y-chromosome haplotypes with fairly high resolution due to their higher mutation rates. Furthermore, STRs typing usually requires simpler techniques than SNPs and can be simultaneous tested PCR using multiplex systems (Figure 4). Although the power of discrimination of each Y-SNP marker is lower than that of YSTR markers, a broad combination of both types of markers contributes highly to increase the discrimination capacity. Y-SNP markers provide, however, an additional advantage over Y-STRs because they have more capacity for distinguishing human population groups and individuals within specific populations. [9, 15] Figure 4 – Relative position of the 17 Y-STR loci commonly used in Y Chromosome testing, such as the commercial AmpFℓSTR® Yfiler® PCR Amplification Kit (Applied Biosystems). FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 15 would have arise from gene flow among the archaic populations, and from natural selection for favourable genes and characteristics. [29, 30] Nowadays, however, the “Out of Africa” theory is the most consensually accepted, when it comes to explain the origin of the modern human being. According to this model the transition from Homo sapiens occurred less than 200,000 years BP, in Africa, and only after this transition the dispersal towards the rest of the world occurred, replacing the archaic human forms present in those areas. [31] East Asia Framework Once it became generally accepted that modern humans evolved recently in Africa, the times and routes of migration to East Asia – the geographical region bordered by the Himalayan Plateau in the southeast, by the Bering strait in the northeast, and extending into island southeast Asia - remained controversial. Different researchers insisted upon three main scenarios. One postulated that northern populations of East Asia migrated to the south, mixing then with the Australian ancestors who had settled in Southeast Asia. Contrarily, another model suggested that the northern populations of East Asia evolved from the southern settlers. However, an explanation also exists holding that northern and southern East Asia populations evolved independently since the late Pleistocene, more than 10,000 years ago. [31-33] To obtain insights into this question, Y chromosome diversity has been widely explored. There are four dominant Y chromosome haplogroups in East Asia – OM175, C-M130, D-M174 and N-M231 – accounting for about 93% of the East Asia Y chromosomes. Other haplogroups, such as E-SRY4064, G-M201, H-M69, I-M170, JP209, L-M20, Q-M242, R-M207 and T-M70, account for around 7% of the males in East Asia. [32] Haplogroup O-M175 is the largest haplogroup in East Asia, comprehending around 75% of the Chinese population. O-M175 gave rise to three downstream haplogroups, O1a-M119, O2-M268 and O3-M122, totaling around 60% of the males in East Asia. [33, 34] While the current Y chromosome diversity suggests multiple early migration FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 16 of modern humans from Africa via Southeast Asia to East Asia, and that after the initial settlements, northward migrations during the Paleolithic Age shaped the genetic structure in East Asia, many uncertainties still persist on the population history of the region. China First Settlements Uncertainty also surrounds the when and how the first settlements of humans reached the geographical region now known as China. The Y chromosome capability to infer the age of genealogies has been a support for the OOA hypothesis, as before mentioned. In combination with archaeological evidence, it is believed that the first modern man arrived in China, via Southeast Asia, around 60 to 45 thousand YBP. Evidence also indicates that very likely a second wave of migration from the north occurred 10 to 20 thousand years later. [35, 36] A major north-south genetic differentiation in the Chinese populations is being revealed, though the northern populations retain signs of the genetic origins in the south of the continent. However, a few recent studies pointed out that East Asian populations’ origin might be more complex than assumed in the previous works. According to those studies, the East Asian migrations can be explained by a multidirectional pattern, with both south and north migrations and with a great migration from central Asia, mainly into northern China, therefore suggesting a frame with multiple ancient migrations, into and within China. [35, 37-39] Important information about earlier migrations is being obtained from the analysis of Y chromosome haplogroups. As mentioned before. In East Asia the dominant haplogroup is the O, defined by the O-M175 mutation. It emerged from an earlier NO clade, in which the haplogroups O and N shared a common mutation. It is believed that soon after the arrival in East Asia, a further mutation occurred in carriers of the NO lineages, separating them into N and O (~30.000 YBP). Later on, carriers of the N haplogroup individuals would mainly travel northwards, into Siberia, FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 17 Russia and Eastern Europe (~12.000 YBP), while individuals with the O chromosomes would establish mainly in East Asia. Ethnicity concept in China – Minzu Definitions of ethnicity and ethnic identification in China have a long and complex history. The People’s Republic of China was established in 1949, after the victory of the Chinese Communist Party in the post World War II civil war. One of the early steps undertaken by the new government was to recognize the existence of non-Han ethnic minorities. Then in PRC, “ethnic group” was connoted with the term minzu, an ambiguous word, whose meaning changed over time accompanying the symbiotic development of the concepts of “nation” and “ethnicity” in China. Table 1 – Total population of each Ethnic group considered in this study (2010 Census results). The Han are the most numbered ethnic group in China, as well in the world. The Hui, Manchu, Tibetan and Salar follow them, in terms of absolute population number. Minzu Name Population (2010) National share (%) Han 1.220.844.520 91.65 Hui 10.586.087 0.79 Manchu 10.387.958 0.78 Tibetan 6.282.187 0.47 Salar 130.607 0.0098 Currently, there are 56 officially recognized ethnic groups, or minzu, within the China population of ~1.300 million, with the majority Han population forming approximately 91% of the total (Table 1). The other 55 minority populations mostly live in the peripheral and boundary regions of the country and they range widely in size from some thousand to millions. [40] The concept of ethnicity in China is not necessarily correlated with genetic ancestry, and it is probably more appropriate to consider minzu as a political construct, rather than a real ethnological identity. This happens because after the formalization of the minzu concept, many Han communities, who lived in close association with ethnic minorities, voluntarily FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 18 adopted minority ethnic status, in order to benefit from the political and personal advantages that the ethnic minorities have. Furthermore, most of the 55 minzu minorities have not been subject to the One Child Certificate programme, and as a result, their number have increased disproportionately since the inception of this programme in 1979. [41, 42] It is tempting, when conducting population genetic studies within China, to rely on the official minzu categories to define each population. This system, however, often failed to recognize pre-existing anthropological and demographic realities, which in turn meant that many minzu exhibit significant internal genetic heterogeneity. This blend of political, demographic and historical aspects implies that the concept of ethnicity in China is a complex and intricate subject, being necessary to account for all the factors that might underlie population structure, including history, when performing and interpreting the meaning of genetic analyses. Han People The term Han is used collectively to define the majority of the Chinese population. It was brought to common usage after the fall of the Eastern Han Dynasty, often referred to as the Chinese history golden age (202 BC – AD 220). According to the 2000 Census, the Han numbered approximately 1.200 million people, and, although resident throughout most of the Chinese territory, they are more numerous in the more densely populated east region of the country. Language wise, most Han people speak the Mandarin Chinese, the official language of PRC, whose written form is uniform throughout all the population. However, the spoken Mandarin language differs from province to province, with eight recognized major dialects. As stated before, the ethnic concept in China is fluid and complex. As so, there is historical evidence of ethnic self-identification between Han population, mainly due to changing political fortunes. During the middle periods of the Qing dynasty (1700-1800 AD), some Han migrants changed their family names to Man surnames, and thus changed their perceived ethnic status. Mutually, the opposite FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 19 also occurred during the final stages of the Qing dynasty, with the intent of taking profit from economic and political advantages. [42, 43] Given the large number of individuals and degree of geographical dispersal, it the effect of historical events on Han genetic diversity should be considered at least on a province-by-province basis. Chinese Muslims The arrival of Islam into China had a lasting effect. During the Tang Dynasty (618-907 AD), people of Islamic faith, mostly men, are believed to have entered China as soldiers, merchant and political emissaries from Central Asia, Arabia and Persia. Some of these men eventually settled in China and intermarried with local Han women, founding this way the present-day Chinese Muslim communities. [44] There are ten officially recognized Muslim minorities in the Peoples Republic of China – the Bonan, Dongxiang, Hui, Kazakh, Kirghiz, Salar, Tatar, Tajik, Uygur and Uzbek – with a combined population of 91 million people. Salar People The Salar people are one of the official Muslim minzu, accounting for around 130.000 individuals, according to the last Census. The population mainly inhabit in the Autonomous County of Jisishan, in the Gansu province. Their language is of Turkic origin, similar to Uzbek and Turkman. It is believed that the Salar originated from an Oghuz tribe, which travelled east from Samarkand (currently Uzbekistan), in the 14th or 15th century. Salar people ancestors are thought to have merged with Tibetans, Han people and Mongolians to form the present-day community. [45] Hui People The Hui people are other of the official Muslim minzu, present in China. Their total population, as for 2010, was of more than 10 million individuals. Despite practicing the Islam, the Hui share many ethnic affinities with the Han people. The FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 20 majority of the Hui people is also Mandarin speaker, although retaining some of the Persian words. They are mainly concentrated in the northwestern provinces and in the Central Plain, the area on the lower reaches of the Yellow River, in an Autonomous Region called Ningxia. [44, 45] The Hui Chinese have diverse origins, and many of them are direct descendants of Silk Road travellers. The Silk Road was a series of trading routes, from Xian through Northwest China and Central Asia, to Constantinople (Istanbul), and as far as Italy. Hence, it is not surprising the ethnic diversity present in the regions of the old Silk Road, and the cultural influence that this trade route brought to China, as a result of the on going migrations that occurred. [44] Tibetan People The Tibetans account for more than 6 million people in the present Chinese population. Their ethnic group that is native of Tibet, but significant minorities also live in India, Nepal and Bhutan. Tibetans speak tibetic languages, many mutually unintelligible, which belong to the Tibeto-Burman languages family. Most Tibetans practice the Tibetan Buddhism, another religion present in the Chinese territory. [44] The traditional, or mythological, explanation of the Tibetan peoples’ origin is that they are descendants of the monkey Pha Trelgen Changchup Sempa. The real history, however, of the human occupation of the Tibetan plateau remains largely unknown. Archaeological evidence points toward to around 23-50 000 years ago, as the period when the peopling of the Tibetan plateau first occurred. [46] Recently, strong genetic evidence appeared of two major migrations of modern humans into the plateau, with the permanent occupation likely occurring in the early Upper Paleolithic before the Last glacial maximum, and a recent migration and population expansion beginning in the early Neolithic, coincident with the emergence of farming and yak pastoralism of the plateau. [44, 47, 48] FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 21 Manchu People Manchus are the third largest ethnic minority group in China, with over 10 million people. They are distributed throughout China, for over 31 Chinese provincial regions, making the Manchu people the largest minority group in China without an autonomous region. At least half of the population, however, lives in the Liaoning province. Most Manchu today speak Standard Chinese, while the Manchu language is only spoken by elderly people in remote northeastern China.[44] Liaoning province, the main Manchu territory, was for many centuries the border between the Chinese Empire and the ‘barbarian’ hordes. The tribes in this area absorbed, that way, a lot from the Chinese culture, through contact with the Chinese Empire, resulting in the formation of the Man (Manchu) nationality. In the time that the Chinese definitely established in Liaoning, mass migration and settlement of Han communities was recorded. Within a generation, Han immigrants outnumbered the local Manchu inhabitants. Over the times, many Manchu people changed their names to Han surnames, in order to obtain more economic and political advantages. For this reason, historically the Manchu and Han ethnicities are considered to have had a very fluid and strong connection, over the time. [43, 44] Shanghai and Macau in the context of China In the present, China, officially known as People’s Republic of China, is a sovereign state located in East Asia and also the most populated country in the world, with a population of 1.35 billion people. China is a Socialist Republic, ruled by the Chinese Communist Party, and has jurisdiction over twenty two provinces, five autonomous regions – Xinjiang, Interior Mongolia, Tibet, Ningxia and Guangxi – four municipalities – Peking, Tianjin, Shanghai and Chongqing – and two Special Administrative Regions, with a high degree of autonomy – Macau and Hong Kong. Shanghai Shanghai city is located in the Yangtze River Delta, in the Chinese east coast, being bordered by the provinces of Jiangsu and Zhejiang to the north, south and west, and bounded to the east by the East China Sea. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 22 During the Qing Dynasty (17th century), Shanghai became one of the most important seaports in the Yangtze Delta region. International attention to Shanghai, however, just grew importance in the 19th century, due to European recognition of its economic and trade potential at the Yangtze River. During the First Opium War (1839-1842), British forces occupied the city. The war ended with the 1842 Treaty of Nanjing, which allowed the British to dictate opening the treaty ports, Shanghai included, for international trade. [49] Between 1860 and 1862, rebels attacked Shanghai twice and destroyed the city’s eastern and southern suburbs, but failed to take the city. In 1862, the British settlement, to the south, and the American settlement, to the north, joined in order to form the Shanghai International Settlement. The French opted out of the Shanghai Municipal Council and maintained it’s own concession, to the southwest of Shanghai. [50] Citizens from many countries and from all continents migrated to Shanghai to live and work during the following decades, calling themselves Shanghailanders. In the 1920s and 1930s, almost 20 thousand “White Russians” and Russian Jews fled to Shanghai from the newly established Soviet Union and took shelter in Shanghai. In the 1930s, around 30 thousand Jewish refugees from Europe also arrived in the city. By 1932, Shanghai had become the world’s fifth largest city and home to 70 thousand foreigners. Shanghai is, thereupon, the most ‘occidentalized’ city in China, having a long and lasting history of different cultures and people, from all over the world. [50] Nowadays, Shanghai is, according to the last census, the house for more than 23 million people, earning this way the title for biggest Chinese city. Also according to census data, 98.8% of Shanghai’s residents are of the Han Chinese ethnicity, while 1.2% belongs to various minority groups. Macau Macau is one of the two special administrative regions of the PRC. Geographically, Macau lies on the western side of the Pearl River Delta, across Hong Kong to the east, bordered by Guangdong province to the north and facing the South China Sea to the east and south. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 23 Macau was a former Portuguese colony, having been under the administration of Portugal from the mid-16th century until 1999. Portuguese traders first settled in Macau in the 1550s, and in 1557 Macau was rented to Portugal, by the Chinese empire, as a trading port. Portugal administered the city under Chinese authority until 1887, when Macau officially became a colony of the Portuguese empire. During the 17th century, around 5 thousand slaves lived in Macau, in addition to 2 thousand Portuguese and 20 thousand Chinese. [51] Nowadays, live in Macau over 509 thousand Chinese people, among whom the Portuguese account for only around 1% of the population. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 24 Aims This study involved the characterization of the Y chromosome genetic pool in samples of two Chinese populations, from Shanghai and Macau, aiming at achieving the following main goals: • Compare the results obtained from Shanghai and Macau in order to evaluate whether signs were retained of the different factors (geographical, historical and/or cultural) that have shaped Y chromosome diversity in both populations. • Explore the genetic background of the studied populations in the broader context of population diversity in China. • Enrich the historical knowledge of the region, combining genetics with ethnic patterns, linguistic affiliation and historical records. • Assess the global pattern of population genetic substructure in China, and investigate if it had any correlation with: 1. Ethnic and linguistic affiliations 2. Geography 3. Pre-historical, historical and other recent migrations. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 31 FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 32 Genotyping Strategy The typing strategy involved three sequential steps: 1. The multiplex O was tested in all samples, given the previous knowledge that O lineages present very high incidence in East Asia. Since besides M175, the multiples also includes other 15 SNPs, it allowed to identify sublineages to which belonged O individuals. 2. Samples excluded to be O were all analysed with multiplex 1, which includes, 9 different previously mentioned SNPs. 3. Samples that did not fell into any haplogroup able to be detected with both multiplexes were lastly tested for the presence of the YAP element. Figure 9 – Schematic representation of the three step strategy followed in this study. In red are the step relying in multiplex PCR and SBE techniques while in orange that based in single PCR and PAGE. The typing strategy was determined by the known structure of the Y chromosome phylogenetic tree (Figure 8). After tested with multiplexes O and 1, a likely possibility for samples showing a derived status for SRY1532 polymorphism and ancestral status for the M213 polymorphism, was to belong to the haplogroups B*C* or DE*. This was deduced because the absence of the mutation defining the F haplogroup – M213 polymorphism– included in multiplex 1, indicated that those samples could belong to an older and more basal haplogroup in the tree, since in addition they didn’t accumulate any of the more recent mutations integrated in the YCC tree (excluding the possibility of a back mutation event). Therefore, we test the YAP polymorphism, given that it could differentiate between two likely options, haplogroup B*C* and haplogroup DE*. Since haplogroup B is found mainly in sub-Saharan Africa populations and haplogroup E is also only well represented across the African continent their presence in East Asia seemed unlikely. Multiplex!O! Multiplex!1! YAP! FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 33 Genotyping Methods SNaPshot Concerning the referred two multiplexes, a mini-sequencing reaction was performed, using the SNaPshot™ kit (Applied Biosystems). This technique involves an amplification of the surrounding region of the polymorphic site, primer annealing, adjacent to the SNP position, and primer extension of a single ddNTP (fluorescent dye-labelled dideoxynucleotide triphosphate). Since the ddNTPs are labelled with specific fluorescent dyes, it was possible to combine different polymorphisms in the same reaction. The PCR for the O haplogroup multiplex and for the multiplex 1 plus M13 were performed in a final volume of 10 µl, with 5 µl of 2x QIAGEN® Multiplex PCR Kit and 1 µl of primer mix at 2.0 µM. The volume of added DNA for each sample was of 0.5 µl, and 3.5 µl of water was added in order to complete the 5 µl volume. As for the PCR conditions, samples were submitted to a denaturation step of 15 minutes with 95ºC, followed by 30 cycles of 94ºC for 30 seconds, 60ºC for 1 minute and 30 seconds and 72º for 1 minute. Each one of the different temperatures in the cycles phase corresponds to the denaturation, annealing and elongation step, respectively. A final extension step was performed at a temperature of 72ºC for 15 minutes. Purification of the PCR products was done in order to degrade the remaining primers and nucleotides of the PCR reaction, was done using ExoSAP-IT® (USB Corp.), following the manufacturer’s specification. The SNaPshot reaction was performed in a final volume of 5 µl, with 1 µl of SNaPshot™ Multiplex Mix (Applied Biosystems), 1.5 µl of SBE (Single Base Extension) Mix and 1.5 µl of PCR product, previously purified. Finally, 1 µl of water was added to the mix to obtain the 5 µl final volumeThe samples went through 25 cycles of 96ºC for 10 seconds, 50ºC for 5 seconds and 60ºC for 30 seconds. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 34 To remove unincorporated ddNTPs from the SNaPshot reaction, a product purification was performed using SAP™ (USB Corporation, Clevelend, OH, USA), according to the manufacturer’s specifications. The separation of the mini-sequencing reaction products was done by capillary electrophoresis using the ABI 3130 Genetic Analyser (Applied Biosystems). The data obtained from the capillary electrophoresis was analysed using the GeneMapper® Software v4.0 (Applied Biosystems). Results were visualized in a single eletropherogram, corresponding each peak to a different polymorphism, with the color defining the status (Figure 10). Figure 10 – Example of an electropherogram obtained from the O specific multiplex. Each nucleotide is associated with a particular dye color, A as green, C as yellow but displayed as black, G as blue and T as red. On the top is represented the profile of an individual negative for all the polymorphisms tested with this multiplex. On the bottom is represented an individual with the O-P203 mutation. [55] FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 35 Direct Visualization For the detection of the YAP element, a simple PCR amplification was performed and then the amplified product was submitted to the Polyacrylamide Gel Electrophoresis (PAGE). The PCR for YAP was performed in a final volume of 5 µl, with 2.5 µl of 2x QIAGEN® Multiplex PCR Kit and 0.4 µl of each forward and reverse primers at 2.5 µM. The volume of added DNA for each sample was of 1.2 µl, and 0,5 µl of water was added in order to complete the 5 µl volume. Samples were submitted to an initial denaturation at the temperature of 95ºC for 15 minutes, followed by 35 cycles at 94ºC for 30 seconds, 52ºC for 90 seconds and 72ºC for 60 seconds. The final extension step was performed at a temperature of 72ºC for 10 minutes. The difference in the size of the two amplified allele products (YAP+ with 455bp and YAPwith 150bp) was clearly visualized after applying to the polyacrylamide gel with the silver staining method (Figure 11). [56] The silver staining is a sensitive method of nucleic acids detection, which permits the visualization of the electrophoresis results without any special equipment. In silver staining, polyacrylamide gels are impregnated with the soluble silver ion (Ag+) and developed by treatment with a reductant. Macromolecules in the gel promote the reduction of silver ion to metallic silver (Ag0), which is insoluble and visible, allowing bands containing nucleic acids to be seen. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 36 Figure 11 – Discrimination of the two YAP alleles in a polyacrylamide gel after application of the silver staining method. YAP positive (YAP+), Y chromosome with an inserted copy of the Alu element is represented in the image by the 455bp band and the 150bp band represents the negative YAP (YAP-). YAP+%(455bp)% % YAP,%(150bp)% FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 37 Populations Data In order to conduct comparative analyses, previously published population data were recruited from the literature for this study. Concerning STRs only, populations from China were used (Table 2 and Figure 12). Table 2 – Populations used for comparative analysis with STRs and SNPs. Fig. Code Population N Ethnia Language Reference 1 Shanghai 85 Han Shanghainese This work 2 Macau 50 Han Cantonese This work 3 Henan 276 Han Henan dialect (Dialect of Mandarin) Wu et al. (2011) 4 Shandong 131 Han Jiaoliao and Zhongyuan (Dialects of Mandarin) Yan et al. (2007) 5 Xinbin 231 Manchu Jiaoliao Mandarin and Manchu Juan He (2013) 6 Ningxia Hui 141 Hui Zhongyuan and Lanyn (Dialects of Mandarin) Hua Guo et al. (2008) 7 Shanxi 222 Han Jin and Zhongyuan (Dialects of Mandarin) Mei-Sen et al. (2011) 8 Qinghai Tibetan 167 Tibetan Khams language dialect Bofeng Zhu et al. (2008) 9 Taiwan 200 Han Mandarin Tsun-Ying et al. (2008) 10 Lhasa Tibetan 351 Tibetan Khams language Lhasa dialect Qingxia Zhang et al. (2006) 11 Qinghai Salar 133 Salar Turkic branch of the Altaic language family Bofeng Zhu et al. (2007) 12 Luzhou, Sichuan 424 Han Sichuanese Long Bing et al. (2013) a Korean 506 Korean Korean Kim et al. (2010) b Khalkh 45 Mongol Khalkha Dialect Jin et al. (2003) c Buryat 36 Mongol Buryat Dialect Kim et al. (2000) d Beijing 51 Han Mandarin Kim et al. (2000) e Xian 34 Han Jin and Zhongyuan (Dialects of Mandarin) Kim et al. (2000) f Yunnan 60 Han Linguistic diversity Jin et al. (2003) FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 38 Figure 12 – Map of China showing the location of Shanghai, Macau and other sample populations used in the comparative analysis with STR data. Correspondence between each number and population is described in Table 2. For SNPs, a sample from Korea and two from Mongolia – Buryat and Khalkh – were used besides three samples from different Chinese populations – Han from Beijing, Han from Yunnan and Xian. (Table 2 and Figure 12) Four samples from Shanghai and two from Macau did not perform well in the Y-STR typing and were not used in this analysis, since it was not possible to obtain a complete Y-STR profile. Four other different samples from Shanghai and two from Macau were not possible to type, haplogroup wise, and were not used in the Y-SNP tests. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 39 Statistical Analysis The haplogroup and haplotype frequencies, as well as the number of shared and unique haplotypes of the studied populations were calculated using Arlequin v3.5.1.3 software (Excoffier and Lischer, 2010). The Mean Number of Pairwise differences (MNPD) for the STR data and gene diversity values (Nei, 1987) for biallelic and STR loci data were estimated, as well as the matrixes of pairwise genetic distances and the corresponding p-values, by Arlequin v3.5.1.3 software. Pairwise FST distances results were computed using the Slatkin’s linearization (Slatkin, 1995) for STR data (RST) and Reynolds linearization (Reynolds et al. 1983) for bi-allelic data (FST). A statistical significance of P<0..05 was also initially considered, although a Bonferroni correction was also applied – P<0.00415 for STR data and 0.00625 for bi allelic data. Arlequin software was also used to perform analysis of molecular variance (AMOVA), considering both types of information, haplotypes and haplogroups. Such analysis allowed the estimation of the percentage of genetic variation that was observed among groups, among populations within groups and within populations. The groups for this test were chosen following geographic and ethnic criteria. For these tests SAMOVA v1.0 (Dupanloup, I., Schneider, S., Excoffier, L. 2002) was also used. SAMOVA is software which, based on a number of previously chosen groups, will then divide the studied populations following the mentioned pattern. This separation is based on three parameters of population differentiation measurement – FCT, FST and FSC. Finally, pairwise genetic distances were analysed through MultiDimensional Scale (MDS), using the SPSS v21.0 (SPSS inc.) program and the PROXCAL algorithm, where the fitness of the results was measured by the s-stress value. The pairwise genetic distances were also measured using the Barrier v2.2 (Manni et al. 2004), a software that combines genetic and geographic distances. This software computes geographic barriers by taking into account the correlation between the two types of distances, by using the Monmonier algorithm (Monmonier, 1973), assigning barriers when a high genetic differentiation between neighbouring populations exists. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 40 RESULTS AND DISCUSSION FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 47 Admitting that B and E will indeed be ruled out, the combined frequency of C, D plus N accounts in Shanghai and Macau for values somehow lower than usual in Eurasian populations. But that is compensated by the high frequency O lineages in the two samples, resulting in a combined frequency of C, D, N and O chromosomes reaching the common range in populations from Eurasia. N1c-Tat was also detected in one male from Shanghai and another from Macau. The haplogroup arose probably in China around 14 thousand years ago, experiencing afterwards a series of founder effects or strong bottlenecks in Siberia, accompanied by a secondary expansion in East Europe. Studies trace the origin of haplogroup N to Southwestern China or Southeast Asia, and the dispersion of haplogroup N has been argued as evidence for the origin of the East Asian populations. The N haplogroup emerged from an earlier NO clade, in which the haplogroups N and O shared a common mutation. It is believed that soon after the arrival in East Asia, a further mutation occurred in the NO lineages, separating them into N and O (~30.000 YBP). Later on, individuals with N chromosomes would mainly travel northwards, into Siberia, Russia and Eastern Europe (~12.000 YBP), while carriers of O haplogroup would establish in East Asia. Haplogroups O and N are, therefore, brother haplogroups. The scarce representativeness of N1c both in Macau and Shanghai fits well the low frequency of the haplogroup found across East Asia, including China where the highest frequencies were reported to be 5-7%. [62-64] One individual from Shanghai was found to belong to the R1 haplogroup, which was identified by the presence of the derived allele at M173. Two main clades within R1-M173, defined by M420 and M343-R1b, are widely spread throughout Eurasia, with the exception of the Southeast and East Asia. R1-M173 contains, however, other minor clades, still remaining incipiently characterized by known SNPs and being collectively referred to as R-M173*, which have shown to be present in the Americas, Asia and Oceania. In this study, because just a single chromosome felt in R1-M173 and given time limitation, no further SNP characterization of that lineage was performed, which would be necessary to clarify whether it could signal a possible European influence in the Han population from Shanghai. Anyway, since no other males lineages of likely European origin were detected in Shanghai, our data indicate that it was virtually null the genetic impact of the presence of European FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 48 people in what was known since the early nineteen century as “The Paris of the East”. The absence of male European lineages in Macau is still more remarkable given that for more than four centuries Macau represented the centre of Portuguese trade and culture on the South China coast. Haplogroup diversities here estimated for Shanghai and Macau are presented in Figure 14, where are also shown previously reported values for other East Asian populations. Figure 14 – Y-SNP gene diversity values. In the figure below are represented the values of Y-SNP diversity, obtained considering a set of 13 common haplogroups. Levels of haplogroup diversity are high in all populations shown in Figure 14, with the Koreans presenting the most elevated value of Y haplogroup diversity, followed by the populations from Yunnan and Shanghai that show quite similar high values 0.8073 +/- 0.0343 and 0.8025 +/- 0.0303, respectively, when compared to the other populations considered in the comparison (Figure 14). In the opposite lowest Macau 0.7595 +/- 0.0531 Yunnan 0.8073 +/- 0.0343 Xian 0.8025 +/- 0.0303 Shanghai 0.8025 +/- 0.0303 Beijing 0.7647 +/- 0.0425 Buryat 0.7270 +/- 0.0523 Khalkh 0.7121 +/- 0.0505 Korea 0.8172 +/- 0.0079 O1a* O1a1 O2* O2a O2a1 O2b* O2b1* O3* O3a* O3a3 O3a4 R N C* Other FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 49 range are the two populations from Mongolia, the Buryat and the Khalkh, presenting diversities of 0.7270 +/- 0.0523 and 0.7121 +/- 0.0505, respectively. High diversity of male lineages in the Koreans probably relies in their intricate and complex history. Although Koreans are in overall integrated in the genetic context of other Northeast Asian groups, it has been proposed a dual origin for the peopling of the Korean peninsula, with contributions from both southern and northern parts of East Asia, since the population from Korea contains lineages typical of both Southeast and Northeast Asian populations. [65] Within China, the more diverse populations are those from Shanghai, Yunnan and Xian, located in Central or Southern China. Comparatively, the population from Beijing, in the Northeast region, reveals a considerable reduced diversity. As mentioned before, Underhill et al. (2001) sustained a southeastern origin for the populations of East Asia. After the arrival of modern man in China via Southeast Asia, they began migrating toward north having made a substantial contribution to extant Northeastern Asia populations, which still retain signs not only of the southern origin but also of bottlenecks and genetic drift during the northwards migration, with consequent lose of diversity. Even though other migrations in Postglacial and Neolithic times also importantly contributed to current East Asian populations, compelling evidence suggest that the southern route shaped a south-tonorth clinal structure that is being demonstrated in East Asia (Zhong et al. 2011). Recently, a trend to diversity decrease from South to North East Asia has been clearly shown by genome-wide SNP data (Abdulla 2009), which seemingly is also supported by our Y-SNP based results notwithstanding the drawback of the small number of populations here considered. Other factors might have also contributed for the high diversities in Shanghai and Yunnan. For a long time ago, both cities are known to be important trade centres of goods. Shanghai was a well-established strategic point for trade, mainly because of the seaport. Yunnan, on the other hand, constituted the cornerstone of the Silk Road Route. The long-lasting history of migratory movements, into and out of both cities, as well as the constant renewed settlement of the cities, likely accounted for their broader spectrum of Y chromosomes. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 50 Concerning diversity in Macau, it represents indeed the lowest value compared to the Chinese populations. For that it must likely has accounted the recent, while centenary, history of human movements into this small regions that still constitutes a special administrative region in China. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 51 Haplotype Frequencies and Diversity Values In this study, a total of 127 different haplotypes (79 in Shanghai and 48 in Macau) defined by 17 Y-STR markers were identified. Only one shared haplotype was observed between two individuals from Shanghai samples. Accordingly, haplotype diversity inferred from the entire set of markers was 0.9997 +/- 0.0020 in Shanghai, while in Macau reached the maximum values 1.0000 +/- 0.0043. Both values are in range of those commonly reported for Chinese populations tested with the same set of Y-STR loci. [66-69] Table 4 – Y-STR haplotype diversity data of the 12 populations used in this study. Mean Number of Pairwsise Differences (MNPD) value on the right. Population N STR haplotype diversity MNPD Shanghai 81 0.9991 +/- 0.0021 9.537346 +/- 4.419859 Macau 48 1.0000 +/- 0.0043 9.515071 +/- 4.442024 Henan 276 0.9998 +/- 0.0003 10.268590 +/- 4.701417 Shandong 131 0.9998 +/- 0.0010 9.158191 +/- 4.239352 Xinbin Manchu 231 0.9996 +/- 0.0005 9.689253 +/- 4.455636 Ningxia Hui 141 0.9933 +/- 0.0010 10.057244 +/- 4.624071 Shanxi 222 0.9999 +/- 0.0004 10.183115 +/- 4.668170 Qinghai Tibetan 167 0.9998 +/- 0.0007 9.115576 +/- 4.215253 Taiwan 200 0.9996 +/- 0.0006 9.360804 +/- 4.317102 Lhasa Tibetan 351 0.9999 +/- 0.0002 9.074562 +/- 4.186760 Qinghai Salar 133 0.9983 +/- 0.0013 10.096605 +/- 4.642639 Luzhou (Sichuan) 424 1.0000 +/- 0.0002 9.663522 +/- 4.437385 The levels of haplotype diversity were similar and very high in all Chinese populations (Table 4) ranging from 0.9933 +/- 0.0010 in the Ningxia Hui to 1.0000 +/- 0.0002 in the Luzhou and Macau. For Y-STR lineages, no signs emerged of diversity structuring according to geography in China. Interestingly, however, the Hui and the Salar presented the lowest levels of Yhaplotype diversity. Both are Muslim populations, with a history of relative isolation from other populations, mainly due to the religious affiliation. The presence of Islamic groups in China is associated to people of Islamic faith, most of them men, who have FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 52 entered China from Central Asia, Arabia and Persia. It is believed that there are Muslim in China since around 1.400 years ago [45] and from then on the Islam expanded gradually across the entire China. Despite the multiple accounts of continuous interaction between Muslims and other people from China, the religious beliefs might have favoured some isolation, and consequently genetic drift, in certain groups, contributing to decrease levels of diversity. Whereas that can be indeed the case of the Hui and the Salar, it is noteworthy the elevated mean number of pairwise differences between haplotypes in both populations, which otherwise may reflect the assimilation of quite differentiated lineages as a consequence of interactions with other Chinese populations. Also of note that the two lowest values of MNPD are shown by the Tibetan populations from Qinghai and Lhasa, which indicated that despite the high haplotype diversity (a common feature even in other Chinese ethnic minorities [44, 59]), on average haplotypes from Tibetans and molecularly more related between each other than in other populations from China. One of the largest minority ethnic groups in China, the Manchu, presents a very high level of diversity, result that was expected from the recent extensive admixture between the Manchu with the Northern Asian and Han Chinese populations. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 53 Population Comparisons In order to investigate the affinities between the samples here studied from Shanghai and Macau and other East Asian populations, genetic distances were calculated, RST for STR data and FST for SNP data, using the data available for surrounding populations. However, since East Asia is still a scarcely studied area, data for the comparative analysis was quite limited for the markers tested, especially when it comes to SNP data. Y-STR For the comparative analysis using Y-STRs, a set of 15 different loci was used, in order to maintain the same level of resolution for all populations. The matrix of RST genetic distances obtained through AMOVA is displayed in Table 5. Table 5 – Matrix of the RST genetic distances for the 12 populations used in the STR study. All the statistical significant values are highlighted in bold. Those significant (P<0.05) are labelled with an asterisk and those values that remain significant after the Bonferroni correction (P<0.0042) with two asterisks. !! Shanghai! Macau! Shandong! Ningxia! Hui! Shanxi! Qinghai! Tibetan! Taiwan! Lhasa! Tibetan! Q.!Salar! Henan! Xinbin! Manchu! Shanghai! *% ,! ,! ,! ,! ,! ,! ,! ,! ,! ,! Macau! 0.02065*! *! ,! ,! ,! ,! ,! ,! ,! ,! ,! Shandong! 0,02410**! 0.00789% *! ,! ,! ,! ,! ,! ,! ,! ,! Ningxia!Hui! 0.06286**! 0.06732**! 0.02120**! *! ,! ,! ,! ,! ,! ,! ,! Shanxi! 0.03964**! 0.01895**! 0.00000! 0.02226**! *! ,! ,! ,! ,! ,! ,! Qinghai!Tibetan! 0.26783**! 0.26442**! 0.21807**! 0.13227**! 0.19414**! *% ,! ,! ,! ,! ,! Taiwan! 0.00331% 0.00000! 0.00000! 0.07414**! 0.04030**! 0.28784**! *! ,! ,! ,! ,! Lhasa!Tibetan! 0.15883**! 0.14741**! 0.09957**! 0.06621**! 0.11289**! 0.02337**! 0.17000**! *! ,% ,! ,! Qinghai!Salar! 0.12294**! 0.12601**! 0.09101**! 0.02084**! 0.06211**! 0.12658**! 0.14041**! 0.08575**! *! ,! ,! Henan! 0.01952**! 0.00936! 0.00000! 0.07359**! 0.03626**! 0.25675**! 0.01496**! 0.15728**! 0.13951**! *! ,! Xinbin!Manchu! 0.01969**! 0.00000! 0.00000! 0.02570**! 0.01470**! 0.19410**! 0.01261**! 0.11188**! 0.07211**! 0.02084**! *! Sichuan!Luzhou! 0.08211**! 0.00936**! 0.06625**! 0.09094**! 0.06039**! 0.28188**! 0.06956**! 0.18021**! 0.16460**! 0.07477**! 0.05989**! Between the two studied populations in this work, Macau and Shanghai, the RST value was 0.02065, with no significant statistical differences after the Bonferroni correction, attesting, therefore, for a rather high homogeneity between the two populations in which respect Y-STRs. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 54 Furthermore, among the compared samples of Han populations – which were Macau, Shanghai, Henan, Shandong, Taiwan, Shanxi, Luzhou, it was noticeable the low pairwise values of genetic distances, even though some reached statistical significance, when compared to distances involving non-Han populations, confirming previous evidence that the Han constitute a coherent genetic cluster of populations. As for the two Tibetan ethnic groups, from Lhasa and Qinghai, not surprisingly they presented very lower level of genetic differentiation, and the same happened with the two Muslim populations, the Hui from Ninxgia and the Salar ethnic populations. Historically, minorities living in China are closed populations, were the people tend to get together with other culturally and ethnic similar people, even some Muslim communities that live in close proximity have little contact or intermarriage [70], probably from the arise of the ethnic nationalism between the different minorities in the country. [44, 71]. Therefore, even though groups with identical ethnical or cultural affiliation might live geographically quite far in different provinces, such as the Tibetans Lhasa and Qinghai or the Muslims Salar and Hui, they retain firm genetic signatures of the shared history and/or ancestry. The Manchu population, from Xinbin, even though commonly categorized as a different ethnic other than the Han, genetically reveals clear affinities with the assumed Han populations. The values of genetic distances reflect the close relationship between Manchu minzu and Han throughout the Chinese history. Overall, with genetic distances ranging from 0.0000 to 0.28784, with most of them assuming statistical significance, the results provided by Y-STRs data testify that current China encompasses remarkable genetic diversity, FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 55 MDS Representation In order to obtain a visual representation of the genetic affinities between populations, the matrix of pairwise RST values was submitted to MDS and the obtained plot is presented in the Figure 15, where a color code is used to represent the different ethnic affiliations of the populations. Figure 15 – Rst Genetic Distance MDS representation. In dark red are represented the Han ethnic populations, blue the Tibetan populations, in yellow and green are the Muslim populations, Salar and Hui, respectively. In black is represented the Manchu ethnic population. Multidimensional scaling is widely used to assess and to visualize similarities or dissimilarities among a set of objects. The fitness of these results is measured by the value of S-stress, which evaluates the diagram’s stress to the dimensional fit. As described by Jorge Rocha (2000) in “A Multidimensional Scaling Stress Evaluation”, when working with twelve objects in a two dimensions Multidimensional scale, the value of S-stress should fall under 18.3%, a value high above the S-stress presented by our results, conferring them a high power of discrimination and significance. A major cluster of populations appears located in the up right side of the plot. This cluster integrates the majority of Han populations plus the Xinbin Manchu. As did indicate the analysis of values of genetic distances, the Xinbin Manchu are FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 56 positioned close to the Han populations, which is in accordance with the known historical interactions between Manchu and Han. The two Tibetan populations form a distinct cluster occupying the centre left side of the plot. Even though being from different regions – Qinghai and Tibet – the two populations evidence clear genetic ties, meaning therefore that geographical distance has not neutralized the influence of a shared culture and history. The two Muslim populations, Hui and Salar, are also clearly separated from the remaining Chinese, lying in the upper left quadrant of the plot. As for the Sichuan population, despite being aligned with other Han populations when it comes to the first dimension of the plot, the second dimension sharply discriminates this group, pushing the Sichuan alone from Hans to the lower right quadrant of the MDS. Figure 16 – Correlation map between genetic (Rst) distance and geographic distance (Km) among the 12 populations used for this study. R²%=%1.9E,05% 400% 600% 800% 1000% 1200% 1400% 1600% 1800% 2000% 2200% 0.00000% 0.00500% 0.01000% 0.01500% 0.02000% 0.02500% 0.03000% 0.03500% 0.04000% Geographic!Distance!(Km)! GenePc!Distance!(Rst)!! FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 63 Final!Considerations!and!Conclusion! % % The major dispute about the peopling of East Asia among researchers is rooted in the different interpretations of the early migration history, which are based on the observed genetic divergence between northern (NEAS) and southern (SEAS) East Asian populations. [72, 73] Su et al. (2001), examining Y chromosome SNPs from Chinese populations found out that the southern populations were more diversified than the northern populations. They concluded, therefore, that the northern populations derived from the southern populations after the initial Neolithic peopling of East Asia, an interpretation that was also supported in other studied. [74, 75] However, Karafet et al. (2001) based on the lack of enough genetic divergence between SEAS and NEAS, did not sustain the hypothesis of early northward migration in East Asia. Ding et al. (2000), claiming that the genetic divergence between SEAS and NEAS could be due only to isolation by distance, maintained a northern origin as a possible explanation. The authors also argued that the southern areas are heavily populated, whereas the northern areas are parsley populated, and consequently, between regions migration accompanied by high rates of genetic drift and lineage loss in northern groups could had accounted for an asymmetry in lineage composition, implying that a northern origin could not be ruled out. More recent studies, such as that of Abdulla et al. (2009), that was based on a large scale survey of autosomal single nucleotide polymorphisms, showed that East Asian populations have a clinal structure, with haplotype diversity decreasing from south to north. The south/north cline was also demonstrated by previous mitochondrial DNA and Y chromosome studies by Kivisild et al. (2002), Shi et al. (2005) and Shi et al. (2008). The distribution of O haplogroup and subsequent clades has been interpreted as a mere reflection of the initial founder effect of the early northward effect, followed by the consequent geographic isolation. [65] Diversity in East Asia was also strongly modelled by the demographic history of the past 5000 years, during which the major population migration in the region FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 64 occurred from north to south, following the expansion of the Han culture, although, as pointed out by Hong Shi et al. (2005), more recent population movements and admixture have to be taken into account, since they might have erased signatures of the early population movements [34]. For example, Karafet et al. (2001), provided evidence that the Hui and Uygurs are recently established NEAS with various degrees of admixture among them.are recently established NEAS with various degrees of admixture among them. Viewing to extend the knowledge of Y chromosome diversity in China, in this work we have studied two Chinese populations, one from Shanghai and another from Macau. In both populations the vast majority of the Y chromosomes belonged to the O haplogroup - 81.5% in Shanghai and 89.58% in Macau, which was in fact expected since the O haplogroup is very ancient and overwhelmingly represented in East Asia. Our Y-SNP results, seemingly supported the model initially proposed by Underhill et al. (2001) of a southeastern origin for the East Asian populations, with a afterward north migration, in the sense the southern populations presented, in some way, a pattern of higher haplogroup diversity. Other factors, nevertheless, have also a key influence in the patterns of male diversity in China. Shanghai and Yunnan, two populations characterized by high levels of diversity, share the condition of having been, in the course of history, important trade points, with intense contact with other cultures and populations, which likely has played a major influence in their Y chromosome spectrum. The haplotype diversity levels were, on the other hand, very high and similar across all Chinese populations. The Muslim populations presented the lower diversities, probably due to their religious beliefs that might have instigated certain isolation around these populations. They presented, however, high MNPD, which is probably a reflection of the differentiated lineages that entered China with the Muslims, who, after settling in the territory, assimilated lineages from neighbour populations. Contrarily, Tibetan populations were characterized by low MNPD values, meaning high molecular relatedness between haplotypes, which can be a sign of a rather homogeneous set of lineages that gave rise to the nowadays Tibetan populations. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 65 The Manchu population, showed clear genetic affinities with the other Han populations, probably signalling the recent history of admixture between Manchu and neighbour Han people. Also of note, the population from Luzhou, which despite considered to be Han people, consistently appeared quite differentiated in the cluster of Han populations. The Luzhou populations, and especially the Sichuan province, have a very peculiar modern history, which might in part explain these results. In the middle of the 17th century the Sichuan province was conquered by rebels from the North – the province had already gone throw several years of war after a Manchu invasion – whom in response to the local resistance, massacred a great deal of the native population. [76] In the 18th century an earthquake resulted in the death of 100.000 people, and in the 20th century, between 1959 and 1961, the Great Chinese Famine also deeply affected this province, resulting in the death of over 9 million people (13% of the population at that time). [77, 78] Our data might be, accordingly, the result of the modern history of the region, which was shaped by warfare, famine and natural disasters. Chinese populations were found to be highly substructured, however, the role of geographical distance was shown to be not significantly correlated with the degree of genetic relatedness between populations. Distant populations did not tended to be genetically more differentiate than neighbouring populations. The results revealed, however, that level of differentiation between populations was fairly related with ethnic-cultural aspects. Populations from the same ethnic group, such as Han, Tibetans or Muslim populations, tended to be more genetically related even when living quite far from each other, than were with the neighbouring populations of different ethnic affiliation. The Macau population, with a long shared history with the Portuguese, who ruled the region for over 400 years, roused special interest. Contrarily to the reasonable expectation, no evidence was found testifying the genetic influence of the Portuguese settlers in Macau or the African slaves that were brought by them, according to the historical documentation. [51] It is remarkable that, despite being so long under the Portuguese rule, the population from Macau had not assimilated any European male lineages. This can likely be explained by the fact that the effective number of Portuguese in the territory was never much expressive. FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 66 As for the Shanghai population, the “Paris of the Orient” presented a higher Y-SNP gene diversity value, when compared to the Macau population, result that may be a consequence of the metropolitan characteristics of Shanghai, to where people were attracted to migrate, favouring population admixture and consequently increment in genetic diversity. In conclusion, China harbours remarkable genetic diversity, but also anchors equally remarkable cultural, ethnic and linguistic diversity. China is the house for over 1 billion people, with different demography, culture and history. Consequently, a very cautious approach needs to be taken when investigating the patterns of genetic diversity of Chinese populations. The gap between the official minzu categories and genetic structure is still understudied, which might introduce strong bias in the interpretation of overall patterns of genetic diversity. 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The haplotypes are presented with the following Y-STR order: DYS456, DYS389 I, DYS390, DYS389 II, DYS458, DYS19, DYS385a, DYS385b, DYS393, DYS391, DYS439, DYS635, DYS392, YGATA H4, DYS437, DYS438 and DYS448. S64 1 15 14 23 20 14 14 11 12 13 11 12 22 14 12 14 11 20 S65 1 15 14 24 16 16 16 12 13 13 10 11 23 13 12 14 11 19 S66 1 15 12 23 15 19 15 12 16 13 10 12 21 12 11 15 10 20 S67 1 15 13 23 15 19 15 11 18 13 10 12 22 11 11 14 10 21 S68 1 14 12 25 16 17 15 12 20 12 9 11 22 13 12 14 10 19 S69 1 15 15 23 17 17 16 11 16 15 10 11 21 11 12 14 10 21 S70 1 17 13 22 17 14 15 11 12 14 10 10 22 14 11 14 10 19 S71 1 15 12 21 16 18 15 11 18 12 10 13 24 11 11 14 10 20 S72 1 15 12 24 15 17 14 13 15 12 10 12 20 14 11 16 11 20 S73 1 14 12 25 19 16 15 13 21 12 10 11 21 13 13 15 10 20 S74 1 15 13 24 16 18 15 13 18 12 10 12 20 14 11 15 11 20 S75 1 16 12 23 17 16 15 12 14 13 10 13 22 14 12 14 10 18 S76 1 14 14 23 16 17 14 11 12 13 10 12 20 14 10 14 10 18 S77 1 16 12 23 17 15 15 13 14 13 10 11 20 15 12 14 10 18 S78 1 16 14 22 17 17 15 11 11 12 10 12 21 13 11 15 10 19 S79 1 17 12 23 15 15 16 12 13 13 11 12 20 14 13 14 10 18 S80 1 15 12 23 16 18 15 12 16 12 11 11 19 12 12 15 10 19 FCUP Analysis of paternal lineages in the Chinese populations of Macau and Shanghai ! 80 Table 6 – AMOVA results. All the statistical significant values are highlighted in bold (P<0.05). Y-STR (%) Significance Test (P-value) Geographic Distinction: North/South Among Groups 0.00 0.65103 +- 0.00000 Among Populations Within Groups 10.64 0.00000 +- 0.00000 Within Populations 90.31 0.00000 +- 0.00000 Geographic Distinction: Cost/Interior Among Groups 2.37 0.13196 +- 0.01129 Among Populations Within Groups 8.86 0.00000 +- 0.00000 Within Populations 88.78 0.00000 +- 0.00000 Ethnic Distinction: Muslims/Tibetans/Han Among Groups 12.50 0.00000 +- 0.00000 Among Populations Within Groups 2.64 0.00000 +- 0.00000 Within Populations 84.86 0.00000 +- 0.00000 Ethnic Distinction: Muslims/Tibetans/Han/Manchu ! ! Among Groups 9.63 0.00782 +- 0.00280 Among Populations Within Groups 3.28 0.00000 +- 0.00000 Within Populations 87.09 0.00000 +- 0.00000