Native American Mitochondrial DNA Analysis Indicates That the Amerind and the Nadene Populations Were Founded Bytwo Independent Migrations

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Native American Mitochondrial DNA Analysis Indicates That the Amerind and the Nadene Populations Were Founded byTwo Independent Migrations

Antonio Torroni,* TheodoreG. Schurr,* Chi-Chuan Yang,* EmokeJ. E. Szathmary? Robert C. Williams? Moses S. Schanfield? Gary A. Troup,** WilliamC. Knowler,w Dale N. Lawrence,*$ KennethM. Weisss and Douglas C. Wallace*

*Centerfor Genetics and Molecular Medicine and Departments of Biochemistry and Anthropology, Emory University School of Medicine, Atlanta, Georgia 30322, +Officeof the Dean, Faculty of Social Science, University of Western Ontario, London, Ontario, Canada N6A 5C2,*Department of Anthropology, Arizona State University, Tempe, Arizona 85281, @AnalyticalGenetic Testing Center, Inc., Denver, Colorado80231, **Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131, -Diabetes and Arthritis Epidemiology Section, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Phoenix, Arizona 85014, "Division of Host Factors, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, and BDepartment of Anthropology and Graduate Program in Genetics, Pennsylvania State University, University Park, Pennsylvania 16802 Manuscript received April 19, 1991 Accepted for publication September 13, 1991

ABSTRACT Mitochondrial DNAs (mtDNAs) from 167 American Indians including 87 Amerind-speakers (Amerinds) and 80 Nadene-speakers (Nadene) were surveyed for sequence variation by detailed restriction analysis. All Native American mtDNAs clustered into oneof four distinct lineages, defined by the restriction site variants: HincII site loss at np 13,259, AluI site loss at np 5,176, 9-base pair (9- bp) COII-tRNALYsintergenic deletion and HaeIII site gain at np 663. The HincII np 13,259 and AluI np 5,176 lineages were observed exclusively in Amerinds and were shared by all such tribal groups analyzed, thus demonstrating that North, Central and South American Amerinds originated from a common ancestral genetic stock. The 9-bp deletion and HaeIII np 663 lineages were found in both the Amerinds and Nadene but the Nadene HaeIII np 663 lineage had a unique sublineage defined by an RsaI site loss at np 16,329. The amount of sequence variation accumulated in the Amerind HincII np 13,259 and AluI np 5,176 lineages and that in the Amerind portion of the HaeIII np 663 lineage all gave divergence times in the order of 20,000 years before present. The divergence time for the Nadene portion of the HaeIII np 663 lineage was about 6,000-10,000 years. Hence, the ancestral Nadene migrated from Asia independently and considerably more recently than the progenitors of the Amerinds. The divergence times of both the Amerind and Nadene branches of the COII-tRNALY"deletion lineage were intermediate between the Amerind and Nadene specific lineages, raising the possibility of a third source of mtDNA in American Indians.

HEconsiderable cultural diversity, linguistic tongues is disputed (DIAMOND1990; LEWIN 1990). T complexity (SPENCERet al. 1977) andbiological Estimates of the time of arrival of the first migrants variation (NEEL1978; NEEL and THOMPSON1978; have also varied widely (MARSHALL 1990; MORELL SZATHMARY1984) of Native Americans has been the 1990). Dating of skeletal remains and Clovis lithic subject of much speculation. The observed diversity artifacts yield values of 13-14,000 years before pres- has at times been attributed to multiple migrations of ent (YBP) (TAYLORet al. 1985; NELSONet al. 1986) Asiatic peoples to the Americas, a few migrations of while archaeological studies have reported North and ethnically distinct peoples, or in situ differentiation of South American sites which date to more than 33,000 ancestral Native Americans occurring after theinitial YBP (GUIDONand DELIBRIAS1986; DILLEHAY and colonization of the New World(LAUGHLIN 1988; COLLINS1988), with the best North American sites SZATHMARY1991). A recenthypothesis proposes that startingat 20,000-25,000 YBP(ADOVASIO et al. three waves of migrationpopulated the Americas 1983). (WILLIAMSet al. 1985; GREENBERG,TURNER and ZE- To further investigate the populating of the Amer- GURA 1986), corresponding to the tripartite division icas, we have examined the mtDNA variation of Na- of Native American languages [Amerind, Nadeneand tive American populations. Becauseof its maternal Eskaleut] (GREENBERG 1987). However,the common inheritance (GILESet al. 1980), the mtDNA accumu- origin of the numerous Amerind languages and the lates sequential mutations along radiating female lin- time depthrequired to develop this plurality of eages. The high mutation rate of the mtDNA

(;enetics 130: 153-162 uanuary, 1992) 154 A. Torroni et al. (BROWN,GEORGE and WILSON1979; WALLACEet al. cell enzyme genes (SZATHMARY, FERRELLand GERSHOWITZ 1987)permits discrimination between even closely 1983). related populations. In previous studies of Amerind TLe Navajo are southern Athapaskans who are closely related genetically (SZATHMARY 1983) andlinguistically to mtDNAs, we reported that North, Central and South the northern Athapaskan Indians living in Alaska and Can- American populations exhibited high frequencies of ada. Their ancestors were hunting-gathering people from the same set of rare Asian variants which suggested northwestern Canada who migrated to the southwest US that they derived from a common ancestral populationafter 1000 AD (HASKELL1987). Having increased to the (WALLACE,GARRISON and KNOWLER1985; SCHURRet present 150,000 from a total of no more than 15,000 people in 1868 (SPENCERet al. 1977), theNavajo are now dispersed al. 1990). Because these mtDNAs represented only a throughout the 30,000 square mile reservation in relatively small subset of all haplotypes observed in Asia (JOHN- isolated family units (WILLIAMSet al. 1981; TROUPet al. SON et al. 1983; HORAI, GOJOBORIand MATSUNAGA 1982). The bloodsanalyzed in this study represent two 1984; HORAI and MATSUNAGA1986; HARTHARA, segments of the population, a northern one centeredat the Public Health Service Indian Hospital inKeams Canyon, HIRAI andOMOTO 1986; CANN,STONEKING and WIL- Arizona, inwhich Caucasian admixture was estimated at SON 1987; HARIHARAet al.. 1988; STONEKINGet al. 1.1% (WILLIAMSet al. 1981; 1985), and a southern one 1990; BALLINGERet al. 1992), we hypothesized that based in Albuquerque, New Mexico, where individuals from all Amerinds derivedfrom an Asiatic population a wider region of the reservation were sampled and Cauca- which experienced a dramatic reduction of its effec- sian admixture was estimated at 5.0% (TROUPet al. 1982). The Pima of southwestern Arizona are descendants of tive size and genetic variation before or during its the Hohokam who migrated north from northwestern Mex- migration to the Americas. We now report a quanti- ico around 300 B.C., and belong to the Uto-Aztecan lan- tative analysis of mtDNA sequencevariation for some guage family which includes the Papago, Hopi and several Nadene and Amerind tribeswhich indicates that these northern Mexican tribes (MATSONet al. 1968; HAURY1976). two groups derived from distinct populations which Upon entering and settling the area, they are believed not to have intermingled with the Puebloan peoples who already originated at very different times. occupied the region, but may have mixed considerablywith other Southwest Indians (MATSONet al. 1968). In 1977, the MATERIALS AND METHODS Pima numbered some 8,000 people (SPENCERet al. 1977). Gm allotype studies indicated that Caucasian admixture in Samples: Blood cells inthe form of lymphoblasts,platelets the Pima was 1.0% (WILLIAMSet al. 1985, 1986). Bloods or buffy coats were obtained from 167 subjects including were collected atthe Gila River Indian Community in 80 Nadene and 87 Amerinds. * The Nadene were comprised Sacaton, Arizona, by the NIDDK as part of a diabetes study of 30 Dogrib (NW Canada), 48 Navajo (Arizona and New (KNOWLERet al. 1978). Mexico) and 2 Tlingits (Alaska), and their bloods represent The lowland Maya occupy parts of Mexico, Guatemala a random sampling of individuals within each respective and Honduras, and are known to have inhabited the area tribal group. The Amerinds were comprised of 30 Pima continuously for at least 5,000 years (MACNEISH 1983). (Arizona), 1 Hopi (Arizona), 1 Pomo (California), 27 Maya Bloodsamples were drawn from individualsliving in an (Mexico) and 28 Ticuna (Brazil). The Pima and Ticuna isolated villagein the central Yucatan Peninsula. Becauseof individuals from whom blood was taken were known to be the small size and remoteness of the village, most of its unrelated for at least three generations, whereas the Maya inhabitants were related at least at the second cousin level, represent a random sample of that tribal group. however, only one member per family cluster was analyzed The Dogrib, speakers of anorthern Athapaskan lan- in this study. These people speak Yucatec, one ofmany guage, are the largest Indian group in the Canadian North- Maya languages belonging tothe Mexican Penutian west Territories. Their traditional subsistence economywas subgroup (GREENBERG1987). Ananalysis of blood types based on hunting and fishing in the boreal forest and adja- and serum proteins in nearby villages revealed approxi- cent barrenlands. Aboriginally they were subdivided into mately 10% European admixture inthis population (our several regional bands, ie., socioterritorial groupsthat unpublished data). moved withinparticular regions for much ofthe year (HELM The Ticuna are a linguisticallydistinct and geographically 1981). The bloods used in this study were obtained from isolated tribe living inthe Amazonian rain forest of western adult members of the Rae Band (SZATHMARY,RITENBAUGH Brazil (MESTRINER,SIMOES and SALZANO 1980;NEEL et al. and GOODBY 1987). In 1979 about 1600people (75% of all 1980). A study of blood group markers indicated only 2.2% Dogrib) were members of this band. Although traditionally non-native admixture in this tribe (NEELet al. 1980). Since nomadic, by 1960 themajority of the Rae Band had settled 1942 the Brazilian Ticuna population has increased from into permanent communities located within regional band 2,000 to 1,0001 persons (SALZANO,CALLEGARI~ACQUES and areas. Consanguineal and affinal tiescontinue to link people NEEL 1980), and this recent rapid population growth has in different settlements. Accordingly the Dogrib form a been accompanied by migrations from jungle to river settle- recognizable genetic unit among other Athapaskan-speak- ments (LAWRENCE,BODMER and BODMER1980; NEEL et al. ers, asshown by genetic distance analyses (SZATHMARY 1980;SALZANO, CALLEGARI-JACQUES and NEEL 1980). 1983). The maximum European admixture in the group Blood samples were taken from individuals living in three was 8.7%, based on blood group, serum protein and red villages located along the Rio Solimoes(LAWRENCE, BODMER and BODMER1980). ’ The Native American tribal groups termed “Amerinds”are the descend- For convenience, in someof our analyses we have ants of the original migrants (ie., Paleoindianderived),not the migrant grouped populations linguistically. Thus, the Amerinds in- group itself; this term is borrowed from the linguistic classificatory term clude the Pima, Maya, Ticuna, Pomo and Hopi, and the “Amerind”(GREENBERG 1987). to permit us to group the non-Nadenetribes. By using “Amerind we do not imply any agreement or disagreement with Nadene include the Dogrib, Tlingit and Navajo. linguistic issues surrounding the term. Methods: mtDNAs were extracted from blood cells using Mitochondrial DNA Analysis 155 procedures in SCHURRet al. 1990 or a modification of those. 000 ooo-o- In the modified method, buffy coats or platelet pellets were incubated in a 50O-pl digestion solution containing 100 mM 000 ooo-o- NaCI, 10 mM Tris-CI, pH 7.4, 5 mM NaZEDTA, pH 8.0, 000 00000000 0.5% sodium dodecyl sulfate, and 500 pg/ml proteinase K, 000 oooocvmc at 55" for 10-1 6 hr, mixed with 250 pl cold 5 M KOAc 000 oooo-r* with vigorous shaking for 5 min, then incubated at 0" for 000 0000-- 10 min, and thelysed cell debris pelleted through microcen- 000 oo-o*m trifugation. The supernatant was then twice extracted with phenol-chloroform, and genomic DNA within the aqueous 000 ooomom phase ethanol precipitated. 000 oooomm All mtDNAs were polymerase chain reaction (PCR) am- 000 oooomm plified (SAIKIet al. 1985) in nine overlapping segments which 000 00000000 encompassed the entiremtDNA genome (APPENDIX A). Each PCR segment was independently digested with 14 restric- 000 o-ooo- tion endonucleases (AM,AvaII, DdeI, HaeIII, HhaI, HinfI, 000 o-ooo- HpaI, HpaII, MboI, RsaI, TaqI, BamHI, HaeII, HincII), and 000 00000000 c; the resulting fragments resolved by electrophoresis in 1 .O- 000 0000-- 2.5% NuSieve + 1.0% SeaKem agarose (FMC BioProducts) 000 omooom gels and detected by ethidium bromide fluorescence. The 000 o-ooo- fragments observed for each PCR segment were restriction 000 o-ooo- mapped by the sequence comparison method (JOHNSONet 000 o-ooo- al. 1983; CANN,BROWN and WIJSON1984), the composite restriction maps of these sets of segments representing in- 000 ooo-o- dividual haplotypes is given in APPENDIX B. The distribution 000 ooo-o- and frequency of haplotypes among tribal groups is shown oom ooooom in Table 1. Sequence divergences: Mean intra- and intergroup se- 000 ooo-o- quence divergences were estimated with NEI and TAJIMA'S 000 -0000- (1983) maximum likelihood procedure using the computer 000 00000m program DREST (graciously provided by L. JIN). This procedure considers the ratioof shared sites to the total number 000 o-ooo- of sites between two haplotypes, and the mean length of the 000 o-ooo- restriction enzyme recognition sequences to calculate an 000 omooom initial estimate of x (the probability that the two mtDNAs 000 o-ooo- have different nucleotides at a given nucleotide position). 000 ooo-o- Using this initialestimate, A is solved iteratively using equa- 000 o-ooo-m tion 28 and the sequence divergence (6) is estimated by 000 o-ooo- equation 21 (NEI and TAJIMA1983). 00- 00000- Sequence divergence values within and among the tribal 00- 00000- groups are presented in Table 2. Divergence values shown in standard type represent interpopulational comparisons, 00- ooooom those underlined represent intrapopulational comparisons, 00- 00000- and those inbold type represent interpopulation diver- oom ooooom oo= omomo- gences corrected for intrapopulational variation, 6 = 6, - mI 0.5(6, + 6J, where 6, is the mean pairwise divergence between individuals within a single population (X), 6, is the corresponding value for a second population (Y),and 6, is 000 ooo-o- the mean pairwise divergence between individuals belong- 000 ooo-o- ing to the two different populations (X and Y) (NEI and 000 ooo-o- TAJIMA1983). The Tlingit, Hopi and Pomo samples were coo* ooomm- too small in number to be used as separate units for diver- - gence calculations among tribal groups, but were included 000 0000-- in estimates of overall Nadene and Amerind divergences. 000 oooomm Phylogeneticanalysis: The evolutionary relationships -00 00000-* among the 50 Native American haplotypes were inferred p"m 00000- - m using parsimony analysis (PAUP 3.0m; SWOFFORD1989). All but two haplotypes(28 and29) segregated into fourwell 000 ooomom defined clusters, A, B, C andD, each of whichwas delineated 000 ooomom by a distinctive genetic marker (Table 1; Figure 1). The 000 omooom dendrogram presented in Figure 1 is rootedfrom HYP- 2" - ooomomm ANC, "hypothetical ancestor a"(CANN, STONEKINGand WILSON1987), and is 77 mutational steps in length with a consistency index of 0.833. When mtDNA haplotypes from modern Africans (our unpublished data) were substituted from HYPANC and used as outgroup, the branching order did not change. This dendrogram represents a tree gener- ated by the subtree pruning and regrafting (SPR) branch 156 A. Torroni et al.

TABLE 2 mtDNA divergences of Amerinds and Nadene

Population Dogrib Navajo Pima AmerindsMaya Nadene Ticuna Dogrib 1.6 -C 1.5 5.2 f 3.5 13.3 -t 6.5 8.0 f 4.7 17.4 f 7.4 Navajo 1.3 f 0.9 6.2 f 3.8 13.2 -t 6.3 9.0 f 5.0 17.9 2 7.3

Pima 6.0 f3.6 2.8 f 1.4 13.0 -t 6.0 14.9 f 6.8 16.8 f 7.3 Maya 2.0 k 1.4 0.7 f 3.20.3 k 1.3 10.4 f 5.1 17.9 f 7.5 Ticuna 9.7 2 5.83.6 f3.4 2.3 2 1.2 5.8 f 1.9 13.8 -t 6.2

Nadene 5.0 f 3.3 13.3 f 6.3 Amerinds 3.3 +: 1.3 15.1 f 6.7 Intrapopulation divergences are along the diagonal (underlined), interpopulation divergence above the diagonal in standard type, and interpopulation divergences corrected for intrapopulation variation (NEI and TAJIMA1983) below the diagonal in bold type. Divergence values and standard errors are presented X lo4. The Nadene include 2 Tlingit, and theAmerinds include 1 Hopi and 1 Pomo.

swapping method. Although no shorter trees were found, they could exist and the numberof trees equal in length to Figure 1 is probably large. Bootstrap analysis of the branching order of the dendrogram was also performed. However, most American Native haplotypes are separated by a few site differences. Hence, the resampling of haplotypes for tree-building did not create enough definition in the branching order ofall trees to permit a statistically significant test of their relationships and the analysis could not be completed unless the MUL- PARS (multiple parsimony) option was turned off. This meant that branch swapping was carried out on only one randomly chosen tree of minimum length at each resampling of the data. Nevertheless, given this reduced level of robustness of testing, 100 bootstrap replications produced a consensus tree nearly identical to Figure 1 with a consistency index of 0.844 and a length of 77 mutational steps. The major internal nodes distinguishing each cluster were present in 63-78% of the treesgenerated. Hence, even - I OM though no statistically significant levelof association of 31 haplotypes could be obtained, the bootstrap analysis sup- 033 0% ported the segregation of mtDNAs into the observed clus- 0 35 ters. C 0% Intragroup sequence divergence for each cluster were --04.3 032 1.37 calculated according to NEIand TAJIMA(1983). Divergence I .a 0 39 times for each group were estimated using a mtDNA evo- om lutionary rate of 2.0-4.0% per million years (MYR), a range I 0 41 J 042 which encompasses most estimates of the average mtDNA evolution rate2 (BROWN 1980; MIYATAet al. 1982; STONE- D 0 45 ’ KING, BHATIAand WILSON 1986; CANN, STONEKINCand 044 ,046 0 47 WILSON1987; WALLACEet al. 1987; STONEKINCet al. 1990) 048 I 1.49 (Table 3). OM HVPANC RESULTS AMERiND 0 NADENE 8 SHAREDBETWEEN AMERINDS AND NADENE “HYPANC HYPOTHETICAL ANCESTOR Restriction analysis: A total of 67 polymorphic A = +Haell1 np 663 B = 9-BPDeletion C = -H/ncilnp 13259 D = -AM np 5176 Haplotypes 5 and 6 have the Rsal np 16239 siteloss characteristic of the Nadene sites and the 9-base pair (9-bp) COII/tRNALYs inter- genic deletion (CANNand WILSON1983; HORAIand FIGURE1 .-A phylogenetic tree of 50 Native American mtDNA MATSUNACA1986; WRISCHNIKet al. 1987; HERTZ- haplotypes, A, B, C and D indicate the four haplotype clusters or BERG et al. 1989; SCHURRet al. 1990) were observed lineages, with the key below the dendrogram indicating the characteristic mutation of each one. The numbers at the end of each in this analysis. These defined 50 haplotypes (Table branch refer to distinct mtDNA haplotypes, with black circles indicating those present in Amerinds, white circles those present in For themtDNA coding regions, WALLACEel al. (1987) estimateda replacement mutation rateof about 1.8 X lo-’ substitutions/site/year (SSY), Nadene, grey circles those present in both groups, and“HYPANC” or 0.18%/myr, and a synonymous mutation rate of about 32.3 X lO-’SSY, the hypothetical ancestor. Individual branch lengths are propor- or 3.2%/myr. The non-coding D-loop sequence, which represents approxi- tional to the number of mutational steps between haplotypes. mately 7.0% of the total mitochondrial genome, evolves much faster, about 8.4%/myr (GREENBERG,NEWFJORD and SUGINO1983; VIGILANTet al. 1989; HORAIand HAYASAKA1990). Mitochondrial DNA Analysis 157 TABLE 3 be closer to the Nadene (0.080-0.090%) than they Sequence divergence (SD) and divergence time (DT) of Native are to the Pima (0.149%)and Ticuna (0.179%).The American mtDNA haplotype clusters reduced divergence between the Maya and the Na- dene groups is primarily due to their sharing haplo- ClusterPopulation n N SD (XlO‘) DT (YBP) type 1. This haplotype was present in 38.8% of the A Amerinds 10 21 7.1 k4.2 17,750-35,500 Nadene and 18.5%of the Maya but is not found in Nadene 4 60 2.1 f 2.05,250-10,500 either the Pima or the Ticuna. This result could be B Amerinds 10 223.7 f 3.09,250-18,500 explained by either gene flow from the Nadene to Nadene (Navajo) 6 18 3.2 k 2.58,000-16,000 some Amerind populations, the loss of haplotype 1 in C Amerinds 14 10.226 f 25,500-51,0005.2 the Pima and Ticuna or admixture with a third mi- D Amerinds 7 17 7.3 f18,250-36,500 4.2 gratory group. Evolutionary relationships: Analysis of the phylo- For each population, n = the number of haplotypes in each cluster; N = the total number of mtDNAs observed in each cluster; genetic relationships among Native American haplo- SD = sequence divergence presented X104; and DT = divergence types revealed four well-defined mtDNA lineages,’ time in YBP calculated by using the evolutionary rate of 2-4%/ designated clusters A-D (Figure 1). myr. If all cluster B haplotypes are considered to have derivedfrom a single migration and are pooledtogether, the corresponding The haplotypes in cluster A are associated with the values are: n = 15; N = 40; SD = 3.8 k 3.0; and DT = 9,500- Hue111 np 663 site. This site has previously been 19,000 YBP. observed at low frequencies in mtDNAs of East Asians [Han Chinese and Koreans (BALLINGERet al. 1992)] 1). The 14 restriction endonucleases used in this sur- and Chicanos4 (CANN,STONEKINC and WILSON1987). vey screened an average of 373 sites/genome or over ClusterA is divided into two subclusters radiating 10% of the mtDNA sequence per individual. from haplotypes 1 and 9. Haplotype 1 was found in Genetic variation within Native American popu- 38.8% of the Nadene, 18.5%of the Maya and 5.0% lations: Intra- and intergroup sequence divergences of Taiwanese Han (haplotype 56 in the Asian phylo- divide the populations into two groups that parallel geny of BALLINCERet al. 1992). Haplotype 9 differs the postulated linguistic divisions (Table 2). The mean fromhaplotype 1 by the presence of a HaeIII np intragroup divergence value forthe Nadene is 16,5 17 site and was detected in 8.8%of the Nadene, 0.050%, while that of the Amerinds is three times 7.4%of the Maya, 7.1 % of the Ticuna. Thishaplotype higher at 0.151%. This result confirms the general is only one mutationalstep away fromthe Asian genetic cohesiveness of the Nadeneobserved in other haplotypes 28 and 103 found in 7.7% of Koreans, studies(SZATHMARY and OSSENBERG1978; SZATH- 7.1% of Han Chinese and 5.0% of Taiwanese Han MARY 1979, 1983;WILLIAMS et al. 1985) and suggests (BALLINGERet al. 1992). Therefore, haplotypes 1 and thatthe Amerinds have radiatedlonger than the 9 are likely to have been the founding haplotypes of Nadene. this lineage since they are nodal within the cluster, Within the Nadene, the intragroup divergence of the most prevalent cluster A haplotypes in both the the Navajo is much higher (0.062%)than that of the Dogrib (0.016%) (Table 2). This differenceresults Nadene and Amerinds and the only ones found in from 37.5% of the Navajo having haplotypes associ- Asia. Within cluster A two haplotypes, 5 and 6, have ated with the 9-bp deletion which is not seen in those lost the RsaI np 16,329 site, a polymorphism not of the Dogrib or Tlingits. In fact, 6 out of the 10 previously observed. This mutation occurs in all Na- Navajo haplotypes (haplotypes 13-1 8) are associated dene populations examined (50.0% of the Tlingits, with the 9-bpdeletion. However, all non-deletion 26.7%of the Dogrib, and 27.1%of the Navajo), but haplotypes observed in the Navajo (1, 5 and 9) are not in Amerinds. Therefore, the RsaI np 16,329 site shared with the Dogrib and Tlingits (Table 1). loss appears to be a specific genetic marker for the Among the Amerinds, the Pima and Ticuna have Nadene. similar intragroupdivergences (0.132-0.138%) The haplotypes in Cluster B are defined by the 9- whereas the Maya are somewhat less divergent bp deletion. The Hue111 site gain at np 16,517 was (0.104%). always found in association with the deletion. Deleted The interpopulational divergence values revealed mtDNAs were observed in 24.0%of theNative Amer- intriguing relationships between tribal groups. The ican mtDNAs analyzed, with haplotype 13 represent- South American Ticuna have similarly high diver- ing 52.5% of the mtDNAs within the cluster. This gence values relative to both theNadene and the ’ When using the term “lineage” and “haplotype,” we define “lineage” as other Amerindgroups (0.168-0.179%). The Pima aset of mtDNAsrelated to eachother by sharedcommon and unique also have similar divergence values relative to all other mutations not observed in other mtDNAs.We define “haplotype”as a distinct association of restriction endonuclease site polymorphisms for 14 enzymes groups analyzed (0.132-0.168%), although they are within one mtDNA genome. less divergent from the Navajo and Dogrib than are ‘The Occurrence of the HaeIIl np 663 site in Chicano mtDNAs (CANN, STONEKING and WILSON1987) indicates Native American admixture in this the Ticuna. On the other hand, the Maya appear to population; for this reason it cannot be considered a “Caucasian” marker. 158 A. Torroni et al. haplotype is also positioned at the nodeof the cluster, DISCUSSION is the only one of the Amerindian deleted haplotypes which is found in East Asians [2.9-7.1% (haplotype Native American mtDNA markers: Our analysis 54 in BALLINCERet al. 1992)], and is the only cluster shows that all Native American mtDNAs are charac- B haplotype shared by morethan one population terized by one of the fourfollowing mutations: HaeIII (Navajo, Pima and Maya). Consequently,haplotype np 663 site gain; 9-bp deletion; HincII np 13,259 site 13 is the most likely ancestral mtDNA of this cluster. loss/AluI np 13,262 site gain; and AluI np 5,176 site The haplotypes of cluster Care characterized by an loss.All of these mutations have been observed at A to G transition at np 13,263which simultaneously much lower frequencies in Asians (HORAI,GOJOBORI eliminates a HincII site at np 13,259 and creates an and MATSUNACA1984; HARIHARA, HIRAIOMOTO and AluI site at np 13,262 (morph-6,WALLACE, GARRISON 1986; HORAI and MATSUNAGA 1986;CANN, and KNOWLER 1985; SCHURRet al. 1990). This mu- STONEKINGand WILSON1987; HARIHARAet al. 1988; tation was always found in association with a DdeI site STONEKINCet al. 1990; BALLINCERet al. 1992). The gain at np 10,394 and anAh1 site gain at np 10,397. first three of these mutations have not been observed Haplotypes within this cluster are absentfrom the in Africans and Caucasians, whereas thelatter has Nadene but are present in all three Amerind groups been observed at low frequency in Africans but not (43.3% of the Pima, 14.8% of the Maya and 32.1% in association with the DdeI np 10,394 and AZuI np of the Ticuna)at an overall frequency of 29.9%. This 10,397 site gains as observed in Amerind cluster D mutation is also found at low frequencies inAsian haplotypes (CANN,STONEKING and WILSON 1987; mtDNAs, being observed in 1.8% ofJapanese (HORAI, BROWNet al. 1992; A. TORRONI,unpublished data). GOJOBORIand MATSUNACA1984), 1.6% of Orientals Hence, these are highly informative genetic markers (BLANCet al. 1983) and 1 out of 153 East Asians (the which can be used to confirm American Indian ad- only population samples analyzed with the same set of mixture in other American ethnic groups. enzymes, BALLINCERet al. 1992). The phylogenetic Founding haplotypes:This study suggests that only analysis indicated haplotype 43 as being central to the five haplotypes (1,9, 13, 43 and 44)founded the Amerinds, whereas only two or three of these (1, 9 radiation of the cluster, and since this haplotype is and 13) founded the Nadene. In general, these are identical to the oneobserved in East Asians(haplotype the most prevalent among the populations, are shared 65) it islikely to be the founding mtDNA for this between different tribes, are the only ones also de- lineage. tected in Asians, and are positioned at the nodes of The haplotypes of cluster D are distinguished by a the phylogenetic haplotype clusters. Except for the loss of the AluI site at np 5,176 and this mutation is nodal haplotypes and Nadene haplotype 5, all other virtually always associated with the DdeI np 10,394 mtDNA haplotypes are present at low frequencies and and AZuI np 10,397 site gains. Haplotypes within this are tribal specific (“private polymorphisms”; NEEL cluster are found exclusively amongthe Amerinds 1978), suggesting that they developed in situ in the (Pomo, Maya, Ticuna), comprising 19.5% of the sam- Americas after the initial radiation of the founding ples analyzed. This mutation has also been observed population/s in the Americas or in Siberia. in mtDNAs of 23.1% of Koreans, 14.3% of Chinese Geneticdivergence times: Calculations of intra- Han and 10.0% of Taiwanese Han (BALLINGERet al. cluster divergences yielded similarly high values for 1992). The phylogenetic analysis positioned haplotype Amerindclusters C and D (0.102% and 0.073%; 44 at the nodeof cluster D. This haplotype is the only Table 3). Likewise, the divergence value for the C~US- one shared by two tribal groups (Pomo and Ticuna) ter A haplotypes in the Amerinds was 0.071% which and is only a single mutational step away from Asian is essentially identical to that calculated for cluster D. haplotype 25 of BALLINCERet al. 1992. Hence, it is The sequence divergenceof the Nadene cluster A was probably the founding haplotype of this lineage. much lower than the Amerind specific clusters, being Caucasian admixture:Haplotypes 28 and 29of the only 0.02 1%. The overall divergence of the cluster B Maya and the Navajo, respectively, lack characteristic deletion haplotypes in Navajo, Pima and Maya, was of Native American mutations and do notcluster in any 0.038% and independent cluster B calculations for of the four Native American lineages (Figure 1). Hap- the Navajo and Amerinds gave very similar values of lotype 28 has an AluI site loss atnp 7,025 found 0.032% and 0.037%, respectively. Hence, the diver- predominantly in Caucasian mtDNAs (ANDERSONet gence of cluster B haplotypes is intermediate between al. 1981; CANN,STONEKINC and WILSON1987; SHOFF- the Amerind andNadene specific lineages. NER et al. 1990; BROWNet al. 1992), and haplotype The similarity of the sequence divergences of the 29 has a DdeI site loss at np 1,7 15 foundin about 10% Amerind clusters A, C and D suggests that these were of Caucasian mtDNAs (SHOFFNERet al. 1990; BROWN the founding lineages of the Paleoindians. Using the et al. 1992).Consequently, these haplotypes were weighted mean value of 0.084%,the Amerind probably introduced by European admixture. mtDNA lineages are estimated to have begun radiat- Mitochondrial DNA Analysis 159 ing between 21,000-42,000 YBP.If most of this culturally with Puebloan Indians, borrowing both ag- radiation occurred after entry into theAmericas, this ricultural practices and ceremonial rituals (SPENCERet timerange would suggest thatthe Americas were al. 1977). Their genetic admixture with surrounding colonized before the dates associated with the oldest peoples has also been confirmed by the presence of Paleoindian skeletal remains (14,000 YBP) and Clovis Albumin variants specific to southern Native Ameri- lithic artifacts (1 1,500YBP), and would be consistent cans (Albumin Mexico; SCHELLand BLUMBERC1988). with the estimated ages of the oldest American ar- However, this scenario does not explain how the Na- chaeological sites (35,000 YBP). vajo acquired deletion haplotypes from the Amerinds The Nadene clusters A and B (Table 3) show a without obtaining any haplotypes from Amerind clus- much more limited divergence (0.021% and 0.032%) ters C and D (Table 1, Figure Similarly, 1). the limited than the Amerind clusters A, C and D. If both these amount of divergence observed for Amerind cluster lineages observed in modern Nadene were present in B haplotypes (0.037%) can not be reconciled with the the founding Nadene, then the radiation time of the higher divergence values determined for the other Nadene would fall into the range of 5,250-16,000 Amerind clusters. YBP. The third alternative is that the clusterB haplotypes However, the only mtDNA lineage shared by all arrived in the Americas in a separate migration that Nadene tribesis cluster A. The overall Nadene cluster was intermediate between those of the Paleoindians A divergence is 0.021%, much lower thanthat of and ancestral Nadene. As this migration moved south, cluster A in the Amerinds. Since the founder cluster perhaps along the continental divide, it would have A haplotypes must have predated the Nadene radia- mixed with some Amerindtribes. The subsequent tion, this implies that the Nadene became genetically arrival and limited southward expansion by the an- distinct about 5,250-10,500 YBP. These estimates of cient Nadene would explain the preferential admix- the age of the Nadene lineages are concordant with ture of the deletion haplotype in the Nadene and the the 9,000 YBP date calculated from glottochronol- similar and intermediate divergence of cluster B hap- ogical analyses of Nadene linguistic diversification lotypes in both modern Nadene and Amerinds. Inter- (GREENBERG,TURNER and ZECURA1986). estingly enough, in Asia the frequency of mtDNAs The relationship of cluster B to the Nadene and characterized by the 9-bp deletion reaches its highest Amerinds is unclear. Cluster Bhaplotypes are present value in coastal populations (BALLINCERet al. 1992) in 37.5% of the Navajo, 50.0% of the Pima and 22.2% and deletion mtDNAs were virtually the only ones of the Maya but areabsent within the Dogrib and the present in the migratory groupsof Asianancestry that Ticuna. Moreover, the divergence of cluster B hap- began to colonize Polynesia around5,000 YBP lotypes is greater than the rest of the Nadene and (HERTZBERCet al. 1989). Hence this could have been lower than the rest of the Amerind lineages. a separate migration. Clearly, further clarification of The presence of cluster B haplotypes in the Navajo the origin and migrations of American Indian mt- but not in the Dogrib could have occurred in three DNAs will require characterization of the nature and ways: deletion haplotypes could have been part of the frequency of these mtDNA lineages in Asia, Siberia original Nadene genetic stock and then lostby the and the Pacific. Dogrib and the Tlingit through genetic drift; they The authors would like to thank C. RONALDSCOTT for contrib- could have beenacquired by theNadene through uting the Tlingit samples and JAM= DERRfor assistance with the admixture of their ancestors with the Amerinds; or PAUP bootstrap analysis. This workwas supported by National they could have arrived in an independent migration Science Foundation grant BNS8718775 awarded to D.C.W. and mixed with both Nadene andAmerinds. LITERATURE CITED The first explanation would require that theDogrib lost all deletion haplotypes without losing any of the ADOVASIO,J. L., J.DONAHUE, K. CUSHMAN,R. C. CARLISLE,R. nondeletion haplotypes (1, 5 and 9) which they share STUKENRATH,J. D. GUNNand W. C.JOHNSON, 1983 Evidence from Meadowcroft Rockshelter, pp. 163-189 in Early Man in with the Navajo. the New World, edited by R. SHUTLER, JR.Sage Publications, The second possibility is that the Navajo acquired Beverly Hills, Calif. the deletion haplotypes by admixture with Amerinds. ANDERSON,S., S. T. BANKIER, B.G. BARRELL, M. H. L. DE BRUIJN, This is more probable than the first hypothesis since A. R. COULSON,J. DROUIN,I. C. EPERON,D. P. NIERLICH, B. A. ROE, F. SANGER, P. SCHREIER, A. J. H. SMITH,R. STADEN the ancestors of the Navajo are believed to have and I. G. YOUNG, 1981 Sequence and organization of the separated from other northern Athapaskan peoples human mitochondrial genome. Nature 290: 457-465. and migrated south in small familial groups through BALLINGER,S. W., T. G. SCHURR,A. TORRONI,Y. Y. GAN, J. A. regions exclusively inhabited by Amerinds about 1000 HODGE, K. HASSAN, K. H. CHEN and D. C. WALLACE, A.D.(HASKELL 1987). After their movement into 1992 Southeast Asian mitochondrial DNA analysis reveals genetic continuity of ancient mongoloid migrations. 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mitochondrial DNA tRNALYsmutation. Cell 61: 931-937. mitochondrial genes sustain seventeen times more mutations. SPENCER,R. F., J. D. JENNINGS,E. JOHNSON,A. R. KING,T. STERN, Curr. Genet. 12: 81-90. K. M. STEWARTand W. J. WALLACE, 1977The Native Ameri- WALLACED. C., G. SINGH,M. T. LOTT,J. A. HODGE,T. G. SCHURR, cans. Harper & Row, New York. A. M. S. LEZZA,L. J. EISAS I1 and E. NIKOSKELAINEN, STONEKING,M., K. BHATIAand A. C. WILSON,1986 Rate of 1988 Mitochondrial DNA mutation associated with Leber’s sequence divergence estimated from restriction maps of mito- hereditary optic neuropathy. Science 242: 1427-1430. chondrial DNAs from Papua New Guinea. Cold Spring Harbor WILLIAMS,R. C., H. G. MORSE, M. D. BONNELL,R. G. RATE and Symp. Quant. Biol. 51: 433-439. T. T. KUBERSKI,1981 The HLA loci of the Hopi and Navajo. STONEKING,M., L. B. JORDE,K. BHATIAand A. C. WILSON, Am. J. Phys. Anthropol. 56 291-296. 1990 Geographic variation in human mitochondrial DNA WILLIAMS,R. C., A. G. STEINBERG,H. 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GERSHOWITZ, Native American mtDNAs are shown in Table 4. 1983 Genetic differentiation in Dogrib Indians: serumpro- tein and erythrocyte enzyme variation. Am. J. Phys. Anthropol. 62: 249-254. TABLE 4 SZATHMARY,E. J. E., and N. S. OSSENBERG,1978 Are the biolog- ical differences between North American Indians and Eskimos Oligonucleotide primers for PCR amplifications of Native truly profound? Curr. Anthropol. 19 694-701. American mtDNAs SZATHMARY,E. J- E.,C. RITENBAUGH,and C. S. GOODBY, 1987 Dietary changes and plasma glucose levels in an Amer- Primer coordinates TH(“C) Segment size (bp) indian population undergoing cultural transition. SOC.Sci. 1562-1581,3717-3701 51 2155 Med. 24: 791-804. 55 2910 TAYLOR,R. E., L.A. PAYEN,C. A. PRIOR,P. J. SLOTA,JR., R. 3007-3023,5917-5898 5317-5333,7608-7588 57 2291 GILLESPIE,J. A. J. GOWLETT,R. E. M. HEDGES,A. J. T. HULL, 7392-74 10,8921-8902 57 1529 T. H. ZABEL,D. J. DONAHUEand R. 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WILSON,1989 Mitochondrial DNA sequences in where TH= 4(C + G) + 2(T + A) - 5’. All primers were synthesized single hairs from a southern African population. Proc. Natl. at the Emory University Microchemical Facility. Acad. Sci. USA 86: 9350-9354. WALLACE,D. C., K. GARRISONand W. C. KNOWLER, 1985 Dramatic founder effects in Amerindian mitochondrial DNAs. Am. J. Phys. Anthropol. 68: 149-155. APPENDIX B WALLACE,D. C., J. YE, S. N. NECKELMANN,G. SINGH,K. A. WEBSTER and B. D. GREENBERG,1987 Sequence analysis of Figure 2 shows the polymorphicrestriction sites cDNAs for the human and bovine ATP synthase p subunit: observed in Native American mtDNA haplotypes. 162 A. Torroni et al.

SITES HAPLOTYPES

11111111112222222222333333333344444444445 12345678901234567890123456789012345678901234567890

29c 6630 9311 1:M 171% 3192c 3337k 3371k 3388e/3391. 3403t 38991 3981. 4310a 4546g 4769a* 5176a 5584a 5983g 6204a 7025a+ 7497e 78530 78593 8569c/8572s 87741 8858f* 9052n/9053f 9504. 9714. 9942j 10091. 10254j 10394~ 10397.. 108931 111ooa 11321. 11793c 119241 12406h/124060 13031g 132590 132590/13262a 13633e/136340 13702e* 14015a 141990* 14268g* 14304a

15073c 15172e 16049k 161450 :::::I 16329k 16389 rn/16390b 163d+g/1639Ob 16398j 16456~ 16511. Region V

FIGURE2.-Table presenting the polymorphic restriction sites observed in Native American mtDNA haplotypes. Haplotypes are

numbered according to Table 1. A “ 1 ” indicates the presence of a site and a “0” indicates the absence of a site except for region V

where ‘‘ 1 ” indicates a single copy of the 9-bp repeat (deletion) and “2”indicates two copies ofthe repeat. Sites are numbered from the first nucleotide of the recognition sequence according to the published sequence (ANDERSONet al. 198 1). Boldface numbers indicate site gains relative to the published sequence and standard type numbers indicate site losses. The 14 restriction enzymes used in the analysis are designated by the following single-letter code (after CANN,BROWN and WILSON1984): a, AluI; b, AuaII; c, DdeI; e, HaeII1; f, HhaI; g, HznfI; h, HpaI; i, HpalI; j, MboI; k, RsaI; 1, TaqI; 111, BamHI; n, HaeII; 0,HZncII. Sites separated by a diagonal line indicate either simultaneous site gains or site losses for two different enzymes or a site gain for one enzyme and a site loss for another because of a single inferred nucleotide substitution; these sites are considered to be only one restriction site polymorphism in the statistical analysis. Sites marked with an asterisk were found to be present or absent in all samples (except where polymorphic) con- trary to the published sequence, and were confirmed in mtDNAs from all major ethnic subdivisions by DNA sequencing (WALLACE et al. 1988; SHOFFNERet al. 1990; BROWNet al. 1992; A. TORRONI, unpublished data).