Distribution of Y-chromosomal haplotypes in the Sherpas of the Khumbu Valley (Nepal) Pamela Tozzo 1 , Luciana Caenazzo 1 , Silvano Presciuttini 2 , Elena Ponzano 1 , Irene Amoruso 3 , Gianumberto Caravello 3 1 Department of Environmental Medicine and Public Health, Legal Medicine Unit, University of Padua, (Italy) 2 Center of Statistical Genetics, University of Pisa, (Italy) 3 Department of Environmental Medicine and Public Health, Hygiene Section, University of Padua, (Italy) MATERIALS AND METHODS Population Buccal swab samples were collected in 2008, during a meeting of the Sherpa Khumbu Community held in Namche Bazaar, the main village of Khumbu, from 25 male Khumbu Sherpas. Samples were processed in the Forensic Genetics Laboratory of the Department of Environmental Medicine and Public Health, Legal Medicine Unit, University of Padua, (Italy). DNA extraction DNA extraction from samples was performed with QIAamp® DNA Microkit following the procedures described by the manufacturer (QIAGEN). About 30-50 of DNA were recovered in 30 of final solution. DNA amplification Multiplex PCR was performed on 0.5 to 1.0 ng of target DNA for each sample using AmpFlSTR YfilerTM PCR Amplification Kit (Applied Biosystems), in accordance with the protocols described in the kit User Manual. PCR reaction was executed in a GeneAmpRPCR System 9700 (Applied Biosystems) following the manufacturer's instruction except for minimal modifications of the final reaction volume that was of 12.5 L (9.5 L of master mix + 3.0 L of DNA solution) instead of 25.0 L. Also, 6 extra amplification cycles were performed for some samples. PCR thermocycling parameters were the following: initial hot-start incubation step at 95°C for 11min, 30 amplification cycles (denaturing at 94°C for 1 min, annealing at 61°C for 1 min and extension at 72°C for 1 min) and final extension at 60°C for 80 min. Electrophoresis Amplified STRs were separated by capillary electrophoresis using an ABI PrismR 3130 genetic analyzer (Applied Biosystems). GeneScan-500 Internal Lane Size Standard (LIZ-500) was used as the internal standard. The size of the PCR products was classified with Gene Map V2 software, applying Yfiler Allelic Ladder (Applied Biosystems) as a comparison. Statistical analysis Mutational distance was calculated for each pair of haplotypes as the absolute difference between the number of repeats observed at each locus, summed across loci. Each haplotype was paired with all others. Median-joining network analysis was computed by the program Network 4.5.1.6 , using the phylogenetic median-joining network algorithm (7,8). In the present work the locus DYS385 was excluded, as it is composed of two loci whose alleles are indistinguishable. From the matrix of all possible D m values, a Neighbour Joining (NJ) tree was obtained using the Clustering Calculator tool (9) from which the corresponding phenogram was drawn using the Phylodendron tool (10). ? LUKPA (RECENT SUB CLAN) TAKTOK (MINYAKPA) GOLE (MINYAKPA) GOLE (MINYAKPA) TAKTOK (MINYAKPA) TAKTOK (MINYAKPA) TAKTOK (MINYAKPA) TAKTOK (MINYAKPA) TAKTOK (MINYAKPA) GOPARMA (THIMI) TIBETAN LAMA (LAMA) PINASA (MINYAKPA) PINASA (MINYAKPA) SALAKA (THIMI) SALAKA (THIMI) CHUSHERWA (RECENT SUB CLAN) SHANGKCU (RECENT SUB CLAN) CHUSHERWA (RECENT SUB CLAN) GARJA (MINYAKPA) GARJA (MINYAKPA) CHUSHERWA (RECENT SUB CLAN) PINASA (MINYAKPA) CHAHWA Tab. 1: Twenty-five Y-chromosome haplotypes -STR loci. The DYS385 locus is ignored. RESULTS AND DISCUSSION A total population sample of 25 specimens was investigated in this study and 17 different haplotypes were detected. Three haplotypes occurred three times, two occurred two times, and 12 were unique (Table 2). For calculating the inter-haplotype mutational distances we excluded locus DYS385, since its dual product cannot be resolved into alleles of different loci. The distribution of the number of mutational steps was included between 0 (identical haplotypes) and 17. The sample was composed of three clusters of haplotypes, all connected to a number of related haplotypes (small m), but separated one from the other by a number of steps, indicating their probable derivation from the same ancestor haplotype. The table 3 shows the NJ phenogram of the 25 haplotypes based on the matrix of the D m values, together with declared Ru affiliation. In addition, it reports the origin of the Ru genealogies from the ancestral groups that Sherpa people identify as their founding predecessors. Three main clusters of haplotypes are clearly identified. It is noteworthy that two clusters include individuals affiliated to Ru deriving from the proto-Ru Minyakpa. This suggests that the Minyakpa proto-Ru included ancestors with different Y-chromosome haplogroups. According to this view, the only Ru (Taktok) that is common to both these high-order haplotypes, would still be heterogeneous in terms of Y-chromosome lineages. On the other hand, an association emerges between Y-chromosome haplotypes and Ru affiliation. As previously highlighted we have to consider that isonymous individuals are expected to carry the same Y-chromosome, and observed inconsistencies could be due also to genetic mutations, illegitimacy, adoptions, or surname polyphyletic origin. We conclude that the oral tradition of the Sherpas has generated a family identification system similar to that being used in societies with a formalized patrilineal transmission of surnames. Overall, from our preliminary results we're strongly encouraged to carry on the discussed study further. We can thus draw some research guidelines for future investigations to contribute to a better understanding of the dynamics of the human populations in this area of the world. The first step would be that of enlarging the population sample of male Khumbu Sherpas, so that we'll be able to determine the haplotype of at least one member for each existing clan. We also look forward to performing a new sampling campaign in the eastern Tibetan region of the Kham, in the places from which Sherpa migration supposely started at the end of the 15 th century. ABSTRACT Due to their high degree of polymorphism Y chromosome STRs (Y-STRs) are an highly informative tool both in the forensic field and in population genetics studies. The Y chromosome is paternally inherited and since the non-recombining region of the Y (NRY) lacks of homologous recombination during meiosis, it is passed down from father to son virtually unchanged, except for new mutational events. Our preliminary work is part of a wider research aimed at defining the Rue kinship and possibly the ancient migration history of the Sherpa population dwelling nowadays in the Himalayan region of the Khumbu, in Nepal. Our first approach has been that of establishing the actual correspondence between each Ru, the traditional Sherpa patrilinear inherited clan, and Y- STR haplotypes, identified through the analysis of 17 STRs in a population sample of 25 male Khumbu Sherpas, using the AmpFlSTRR YfilerTM PCR amplification kit. As in other societies which use patrilinear family denominations, the Sherpa society is organised in patrilinear clans, or Ru: inheritance of Ru and Y chromosome transmission thus follow a common paternal lineage. On the basis of the Y-haplotypes obtained a Ru genealogical tree was made. INTRODUCTION Y-STRs are highly polymorphic markers and are used to discover further variation within haplogroups. The recent characterization of many new Y chromosomal markers in populations worldwide has contributed to increase their usefulness for studies of genetic anthropology, which until recently lagged far behind mitochondrial DNA and maternal lineage studies (1). In societies that use patrilineal family denominations, surnames and Y-chromosome follow a common pattern of inheritance. As a son inherits the Y chromosome of his father, it follows that males sharing the same surname might also share the same haplotype in the nonrecombining segment of the Y chromosome (2). Therefore, isonymous individuals are expected to carry the same Y-chromosome, and observed inconsistencies can be ascribed to genetic mutations, illegitimacy, adoptions, or surname polyphyletic origin. The word Sherpa is a combination of two Tibetan words: "Shyar" (East) and "Pa" (people), or "People of the East". Several sources report that they migrated from the Kham area of Eastern Tibet to the present area in Eastern Nepal after crossing the Nang-pa-la pass (5780 m) around 1600 c.e . (3,4). The land of the thus lies in the northern side of the Solukhumbu district along the Dudh Koshi River. The Sherpa are Mongoloid in physical aspect and speak the 'Sino Tibetan language'; their literature and philosophy came from old Tibetan religious books. At the 2001 census, the Nepalese Sherpa population consisted of 154,622 individuals. The official surname of most Sherpa people is : the main reason is that when the Nepali-speaking government officers went into the Sherpa-speaking districts in the 1960's to register Sherpas as citizens of Nepal, they didn't ask anyone's surname; people that were obviously Sherpa were simply registered as . The only exception to this rule is one particular Sherpa clan (Ru) called . The Lama clan is very proud; they have always called themselves "Lama" because that is an important name in Sherpa society. In fact, the Sherpa community is divided into 18-23 clans (Ru). Ru is a Sherpa word meaning bones. The male line transmits the Ru (bones) and so the lineage determines clan membership. Four main Ru originally came from eastern Tibet: the Thimi, Minyakpa, Chawa, and Lama. Each clan gave rise to several brother-clans and continuous migration has brought many new Ru in to the area (5,6). This work is part of an interdisciplinary research aimed at clarifying the biological origin and history of the Sherpa population presently living in the Khumbu Valley of Nepal, to ascertain if the Ru affiliation of Sherpa people corresponds to the family identification and denomination that has been established in most European populations in the past centuries. Our first approach has been to establish the possible correspondence between the rhu and the Y-chromosome haplotype determined at 17 Y-STR in a sample of individuals belonging to this population. A sampling campaign was conducted at a convention of the Sherpa Khumbu community. Each contributor donated saliva after signing a consent form that explained the use of their DNA in population genetics research. The form included also a self-declared Ru affiliation: all of the contributors reported that they were not close relatives and even those sharing the same Ru were actually unaware of any kind of biological relationship between them. SAMPLES M1 M3 M4 M6 M8 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 DYS456 16 16 14 14 17 15 15 16 13 16 16 15 15 15 16 15 15 15 14 15 16 14 14 16 15 DYS389-I 13 13 10 10 14 12 12 13 10 13 13 12 12 12 14 12 12 12 10 12 13 10 10 13 12 DYS390 23 25 23 23 23 23 23 23 23 25 25 23 23 23 23 23 23 27 23 23 25 23 23 23 23 DYS389-II 29 29 27 27 30 28 28 29 27 29 29 28 28 28 30 28 30 29 27 28 29 27 28 27 28 DYS458 16 16 15 15 17 17 18 16 15 16 16 17 18 18 17 17 17 15 15 18 16 15 15 16 18 DYS19 15 15 14 14 15 14 14 15 14 15 15 14 14 14 16 14 14 16 14 14 15 14 14 15 14 DYS385a/b 13,15 11,11 15,16 15,16 11,11 14,18 13,2 13,15 15,16 11,11 11,11 14,18 13,19 13,19 11,12 14,18 18,19 17,17 15,16 13,19 11,11 15,16 15,16 11,11 13,19 DYS393 13 13 14 14 13 13 12 13 14 13 13 13 12 12 13 13 12 12 14 12 13 14 14 13 13 DYS391 10 11 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 11 10 DYS439 12 12 11 12 12 13 12 12 12 12 12 12 12 12 12 12 12 11 11 12 12 11 11 12 12 DYS635 21 21 21 21 21 21 20 21 20 21 21 21 20 20 21 21 20 20 21 20 21 21 21 21 20 DYS392 11 7 11 11 7 14 14 11 11 7 7 14 14 14 7 14 14 10 11 14 7 11 11 7 14 YGATAH4 11 11 11 11 11 12 11 11 11 11 11 12 12 12 11 12 12 11 11 12 11 11 11 11 12 DYS437 14 14 14 14 14 16 15 14 14 14 14 16 14 14 14 16 15 14 14 14 14 14 14 14 15 DYS438 11 11 10 10 11 11 11 11 10 11 11 11 11 11 11 11 11 10 10 11 11 10 10 11 11 DYS448 20.2 19 19 19 18 21 20 20.2 19 19 19 21 20 20 18 21 21 18 19 20 19 19 19 19 20 Fig. 2: Unrooted NJ phenogram of 25 Sherpa Y-chromosome haplotypes based on the mutational distance matrix (Dm). This is the sum, over all loci, of the absolute difference between the number of repeats at each locus between all pairs of haplotypes. The locus DYS385 was excluded, as it is composed by two loci whose alleles are indistinguishable. Fig. 3: Median-joining network of 25 Y-STR Sherpa haplotypes, generated using Network 4.5.1.6. Yellow dots mark the observed haplotypes (larger dots include identical haplotypes with the same colours reported in Table 1). REFERENCES 1. Crawford M. H., Anthropological Genetics: Theory, methods and applications. Cambridge University Press, 6:149-151, Cambridge, 2007. 2. J. et al., Y-chromosomal Short Tandem Repeat haplotypes in southern Croatian male population defined by 17 Loci. Croat Med Journal, 49:201-206, 2008. 3. P. Ortner, Sherry B.; High Religion: A Cultural and Political History of Sherpa Buddhism. Princeton UP, Princeton, 1989. 4. C. Fürer-Haimendorf, The Sherpas of Nepal buddhist highlanders. Murray, London, 1964. 5. Ang Tshering Sherpa, Thesis on the origin of Sherpa peopole. Namche, Khumbu, Nepal, 2007. 6. J. Wangmo, Lawudo Lama. Wisdom Publications, Boston, 2005. 7. H.J. Bandelt, P. Forster, A. Rohl, Median-joining networks for inferring intraspecific phylogenies, Mol. Biol. Evol. 16:37 48, 1999. 8. http://www.fluxus-technology.com. 9. http://www2.biology.ualberta.ca/jbrzusto/cluster.php. 10. http://iubio.bio.indiana.edu/treeapp/treeprint-form.html. View publication stats View publication stats