Brief communication Molecular analysis of the Kwday Dn Ts'finchi ancient remains found in a glacier in Canada.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 119:288 –291 (2002) Brief Communication: Molecular Analysis of the Kwäday Dän Ts’ı̀nchi Ancient Remains Found in a Glacier in Canada M. Victoria Monsalve,1* Anne C. Stone,2 Cecil M. Lewis,2 Allan Rempel,1 Michael Richards,3 Dan Straathof,1 and Dana V. Devine1,4 1 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada 2 Department of Anthropology, University of New Mexico, Albuquerque, New Mexico 87131-1086 3 Department of Archaeological Sciences, University of Bradford, Bradford, UK BD7 1DP 4 Canadian Blood Services, Vancouver, British Columbia V6T 2B5, Canada KEY WORDS ancient DNA; Native American; mtDNA haplogroups; control region; biochemical analysis ABSTRACT DNA was extracted from the frozen remains of a man found in the northwest corner of British Columbia, Canada, in 1999. His clothing was radiocarbondated at ca. 550 years old. Nitrogen and carbon content in whole bone and collagen-type residue extracted from both bone and muscle indicated good preservation of proteinaceous macromolecules. Restriction enzyme analysis of mitochondrial DNA (mtDNA) determined that the remains belong to haplogroup A, one of the four major Native American mtDNA haplogroups. Data obtained by PCR direct sequencing of the mtDNA control region, and by sequencing the clones from overlapping PCR products, were duplicated by an independent laboratory. Comparison of these mtDNA sequences with those of North American, Central American, South American, East Siberian, Greenlandic, and Northeast Asian populations indicates that the remains share an mtDNA type with North American, Central American, and South American populations. Am J Phys Anthropol 119:288 –291, 2002. In August 1999, the frozen remains of a man were found on a glacier in Canada’s Tatshenshini-Alsek Park near the Pacific coast border with Alaska. His clothing was radiocarbon-dated at 500 ⫾ 30 BP (Beattie et al., 2000). The local Champagne-Aishihik people named the remains “Kwäday Dän Ts’ı̀nchi” (KDT), i.e., Long-Ago Person Found. With the consent of a committee comprised of First Nations representatives and British Columbia’s Archaeology Branch, we extracted mitochondrial DNA (mtDNA) from KDT hard and soft tissues and determined that his mtDNA belongs to haplogroup A, one of the four common Native American haplogroups. It is generally accepted that the first people to move into North America passed over the Bering land bridge at the end of the Pleistocene, ⬃12,000 – 31,000 years ago (Marshall, 2001). The descendants of the first inhabitants eventually formed the Pacific Northwest populations and fall into at least three linguistic groups (Greenberg, 1987). The Tlinglit and Athabaskan are Na-Dene, while the Haida speak an isolated language, although they have genetic affinities with Na-Dene speakers (Shields et al., 1993). The Tsimshian, Nuu-Chah-Nulth, and Bella Coola, also inhabitants of the Northwest Pacific coast, speak Amerindian languages. The Champagne-Aishihik First Nations aboriginal language is Tutchone, a member of the Athabaskan language family. MATERIALS AND METHODS © 2002 WILEY-LISS, INC. © 2002 Wiley-Liss, Inc. The protocol and consent form for the study of ancient human remains were reviewed by the Clinical Research Ethics Board Committee at the University of British Columbia. To minimize risk of contamination by contemporary DNA, we were provided first access to the KDT remains to take tissue samples for DNA extraction. Pieces of the upper left humerus and overlying muscle were selected for their excellent preservation, and removed under sterile conditions. The bone sample was split into two pieces of 100 mg and 200 mg to provide for DNA extraction in separate laboratories. Grant sponsor: British Columbia Heritage Trust. *Correspondence to: Maria Victoria Monsalve, Ph.D., Department of Pathology and Laboratory Medicine, 2211 Wesbrook Mall, University of British Columbia, Vancouver BC V6T 2B5, Canada. E-mail: email@example.com Received 15 August 2001; accepted 15 March 2002. DOI 10.1002/ajpa.10116 Published online in Wiley InterScience (www.interscience.wiley. com). MTDNA OF 530-YEAR-OLD HUMAN REMAINS Macromolecules analysis To determine the presence of proteinaceous material in the samples, the percentage of nitrogen (%N) and percentage of carbon (%C) contents of two aliquots of the bone and one aliquot of the muscle tissue were measured. The preservation of macromolecules was assessed by measuring the nitrogen and carbon content of whole bone and extracted collagen-type residue, using an elemental analyzer (Ovchinnikov et al., 2000). Collagen was extracted from the two bone samples and muscle sample, using methods outlined elsewhere (Richards et al., 2000), with the addition of a chloroform:methanol lipid extraction for one bone sample and the muscle sample. The percentage of carbon and nitrogen in the extracted collagen was also assessed (Ambrose, 1990). DNA extraction, amplification, and sequencing Standard precautions were taken to minimize contamination from contemporary sources. After removal of superficial tissue, the bone was ground in a freezer mill and the muscle in a mortar refrigerated with liquid nitrogen. As a control for extraction of the bone DNA, parallel grinding and extraction of DNA from the antler bone from a moose found near the remains were carried out. An extraction without any sample was also done as a control on each occasion. Two extractions of DNA from the bone and one from the muscle were done separately. The DNA extraction methods used for the humerus and muscle were described elsewhere (Hagelberg and Clegg, 1991; Guhl et al., 1999; Yang et al., 1998). The aqueous phase was concentrated and purified with sequential additions to Centricon-30 microconcentrators (Amicon). To screen for the markers that define the four major haplogroups in Native Americans (Torroni et al., 1992), the DNA samples were subjected to PCR amplification and subsequent restriction enzyme analysis, as previously described (Monsalve et al., 1994). Double-stranded PCR fragments of the mtDNA control region were amplified, using the primer pairs L15996 (5⬘CTCCACCATTAGCACCCAAAG) (Ward et al., 1991) and H16218 (GATTGCTGTACTTGCTTGTA) (Handt et al., 1996), L16192 (CCATGCTTACAAGCAAGT) and H16401 (5⬘TGATTTCACGGAGGATGGTG) (Higuchi et al., 1988), and L16131 (5⬘CACCATGAATATTGTACGGT) and H16303 (TGGCTTTATGTACTATGTAC) (Handt et al., 1996). PCR amplification of 1 L of DNA was performed in a 25-L reaction volume containing 200 M of each dNTP (Pharmacia), 2.0 mM MgCl2, 2 mg/mL BSA (Boehringer Mannheim), 20 pmol of each primer, and 1.0 unit of Taq DNA polymerase (BRL). Amplification consisted of 40 cycles of 94°C (40 sec), 55°C (20 sec), and 72°C (1 min); the denaturation step of the first cycle was lengthened to 5 min to ensure complete denaturation of the template, and the final extension step was 10 min. Amplification products were electrophoresed on 2% agarose gels to separate nonspecific 289 amplification products and excess primers; excised bands were purified using QIAquick Gel Extraction Kit (Qiagen). Sequencing reactions were done in a thermocycler DNA with 90 ng of DNA template and 3.2 pmol primer, using the ABI BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems). Dyebased DNA sequencing was done in an Applied Biosystem ABI PRISM 378 DNA Sequencer with a long read protocol. DNA sequence data obtained from the three samples on separate occasions were unambiguous. At the University of New Mexico, DNA was extracted as described (Hoss and Pääbo, 1993) followed by PCR using 5 sets of primers: L16055 and H16142, L16131 and H16218, L16209 and H16303, L16287 and H16356, and L16347 and H16410 (Handt et al., 1996; Stone and Stoneking, 1998). Each 30-L PCR contained 5 L of DNA extract, 1 unit of AmpliTaq Gold polymerase (Roche Molecular Systems), 10 mM Tris HCL (pH 8.3), 50 mM KCL, 0.1 mM each of the four deoxyribonucleotide triphosphates (Pharmacia), 2.5 mM MgCl2, 2 mg/mL BSA (Boehringer Mannheim), and 0.2 mM of each primer. Forty cycles of PCR were carried out (30 sec at 94°C, 30 sec at 54°C, and 30 sec at 72°C) in an MJ Research PTC 200 cycler. Eight microliters were electrophoresed in 2.8% NuSieve (FMC) agarose gels. PCR product bands were excised from the gel and used for reamplification, as described by Stone and Stoneking (1998). The reamplified DNA was purified with Qiagen QIAquick-spin columns. The purified products were then sequenced with the ABI Prism Big Dye Sequencing Kit (PE Applied Biosystems) and a model 377 DNA sequencer. The sequence reported in this paper has been deposited in the GenBank database (accession no. AF 502945). RESULTS AND DISCUSSION Modern bone has a bulk %N value of 3– 4% due to the presence of proteinaceous material. Measurement of the KDT sample provided bulk %N values of 1.5% and 1.6% in the two pieces of bone and 0.8% in the muscle tissue, which indicated the presence of relatively large amounts of proteinaceous material. The %C was 51.1% and 52.6% in the pieces of bone, and 25.3% in the muscle. Determination of macromolecules in the collagen alone can also indicate preservation of the samples. Collagen extracted from modern bovine tendon (sigma collagen) was 15.2–16.7%N and 47.0 – 47.6%C (Richards, 1998). Collagen extracted from the two bone samples had %C values of 47% and 42%, and %N values of 17 % and 16%, and collagen extracted from the muscle sample had 42%C and 14 %N. These are all similar to the modern bovine samples, and therefore indicate the presence of good-quality collagen (Richards et al., 2000; Richards, 1998). Modern bone is approximately 20% collagen by mass, and the two bone samples yielded 18.7% and 19.2% collagen-type material, while the muscle sample yielded 3.7% collagen-type material. The ⫹663HaeIII, ⫹5176AluI, and ⫹13259HincII sites and the absence of the 9-bp deletion catego- 290 M.V. MONSALVE ET AL. rized the DNA of these human remains as belonging to haplogroup A, one of the major mtDNA haplogroups identified in Native Americans (Torroni et al., 1992). All Native American Natives have been categorized as belonging to mtDNA haplogroups A, B, C, or D (Torroni et al., 1993), or X (Brown et al., 1998). Haplogroup A is the most common in the North America Natives. It is found in the highest frequencies in the Dogrib Continental Na-Dene (Torroni et al., 1993) and in the Haida from the Queen Charlotte Islands (Torroni et al., 1993); both groups are inhabitants of the Northwest Pacific coast. This haplogroup is also found in high frequencies in Central American Natives (Torroni et al., 1994). The frequency is lower in South America and is not found in some groups from the Amazon (Torroni et al., 1993) and the Andes (Lalueza-Fox, 1996). This haplogroup has also been found in ancient remains from the Norris Farms Oneota in North America (Stone and Stoneking, 1998) and in ancient remains from South America (Monsalve et al., 1996). This haplogroup is not restricted to any linguistic or ethnic group. Thus, there is no reason to conclude that the remains are not of an ancestor of the contemporary people of the region. The presence of haplogroup A in the KDT remains suggests that he is one of the descendants of the first inhabitants who arrived from Asia. Haplogroups A–D have been found in Asian populations (Torroni et al., 1993). Haplotype X, on the other hand, has not been found in Asian populations, and it has been postulated that this mtDNA was brought to Beringia/ America by the eastward migration of people who were also ancestral to Europeans (Brown et al., 1998). A total of 405 bp of the hypervariable region I (HVRI) on the mtDNA was amplified with three overlapping polymerase chain reaction (PCR) fragments with lengths of 222 bp, 209 bp, and 172 bp. Products from independent PCR amplifications from the 222-bp and 209-bp fragments from the humerus were also cloned into pBluescript vector and sequenced (Fig. 1). Comparison of a 362-bp fragment of the control region HVRI with the reference sequence (Anderson et al., 1981) revealed 16111T, 16189C, 16223T, 16290T, 16319A, and 16362C polymorphisms. These six substitutions were found in 1 of 41 Haida analyzed (Ward et al., 1993). An extension of the survey of the HVRI control region in Native Americans from Central and South America indicated that only 1 Amerindian Maya of 3 (Torroni et al., 1993), 1 Amerindian Quiche of 30 (Boles et al., 1995), and 3 of 247 unrelated Brazilian individuals (Alves-Silva et al., 2000) shared this sequence. The cloned PCR contained all the substitutions that were detected by direct sequencing; one additional 16113G transition was present in one clone only. The 16189C transition was present in the PCR direct sequence products and the clones analyzed. Length differences in the cytosine homopolymer at position 16184–16193 were found in association with the 16189C transition. This transition produces an interrupted string of 10 Cs that is copied with low Fig. 1. Variable sites in DNA sequences obtained from PCR amplification of overlapping fragments. P1–P8 correspond to fragments from PCR direct sequences. A1–A6 and B1–B4 are fragments from cloned PCR products. Human reference sequence is included for comparison (Anderson et al., 1981). fidelity during mtDNA replication (Bendall and Sykes, 1995). Cloning and sequencing showed that individuals with a 16189C transition had extensive length heteroplasmy in the homopolymeric tract (Bendall and Sykes, 1995). Two of 6 clones presented a string of 11 Cs, and one a string of 9 Cs, indicating the existence of heteroplasmy in the homopolymeric tract in these remains. Both template strands gave the same result. Alternatively, it could reflect errors that arose during cloning. To independently confirm the results of the DNA analysis, a piece of the bone was analyzed at the University of New Mexico. The results corroborated those collected at the University of British Columbia. In addition, the KDT sequence did not match that of any of the investigators. The control region sequence resolves the RFLPdefined haplogroup into A1 and A2 types (Forster et al., 1996). High frequencies of type A2 have been found in American Eskimos, Haida, and Na-Dene (Forster et al., 1996). Interestingly, the KDT remains present an A2-16189C sequence variant that has been found throughout the Americas. It has not yet been determined if this variant is common in the contemporary inhabitants of the area where the remains were found. The KDT remains were found in an area equidistant between two present-day aboriginal settlements: Klukwan, a Chilkat Tlingit community near Haines, Alaska, and the Klukshu, a ChampagneAishihik First Nations settlement in the Yukon. A woven hat made of plant fibers with an attached MTDNA OF 530-YEAR-OLD HUMAN REMAINS hide chinstrap, radiocarbon-dated AD 1405–1445, was found in the vicinity of the body (Beattie et al., 2000). Its brimmed conical shape is characteristic of contemporary Northwest Pacific populations living in this area (Holm, 1988). High frequencies of mtDNA haplogroup A have been found in American Eskimos, Athabaskans, Haida, and Bella Coola from the northwest coast (Forster et al., 1996). Although the KDT remains are unique and thus are not amenable to population-level analyses, these results as well as the geographic location of the find suggest a connection to modern populations in this region. CONCLUSIONS Ancient DNA authenticity was established by: 1) verifying that nitrogen and carbon percentages in the hard and soft tissues met requirements for preservation of proteinaceous material, 2) analyzing mtDNA sequences from PCR fragments and cloning products of overlapping fragments of the HVRI control region, and, 3) validating the humerus DNA results in an independent laboratory. Comparison of the KDT mtDNA sequences with those of North American, Central American, South American, East Siberian, Greenlandic, and Northeast Asian populations indicates that the remains share an mtDNA type with the Haida of North America, Maya and Quiche from Central America, and Brazilians from South America. ACKNOWLEDGMENTS We thank the Kwäday Dän Ts’ı̀nchi Committee, and in particular Al Mackie, for facilitating the execution of this project, Dr. Owen Beattie for his constant support, and the British Columbia Royal Museum. 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