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Native American Mitochondrial PDF
Native American Mitochondrial PDF
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.
T HEconsiderablecultural
complexity (SPENCER
diversity, linguistic
et al. 1977) andbiological
variation (NEEL1978; NEEL and THOMPSON 1978;
tongues is disputed (DIAMOND 1990; LEWIN 1990).
Estimates of the time of arrival of the first migrants
have also varied widely (MARSHALL 1990; MORELL
SZATHMARY 1984) of Native Americans has been the 1990). Dating of skeletal remains and Clovis lithic
subject of much speculation. T h e observed diversity artifacts yield values of 13-14,000 years before pres-
has at times been attributed to multiple migrations of ent (YBP) (TAYLOR et 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;DILLEHAYand
colonization of the New World(LAUGHLIN 1988; COLLINS1988), with the best North American sites
SZATHMARY 1991). A recenthypothesis proposes that startingat 20,000-25,000 YBP(ADOVASIO et al.
three waves of migrationpopulated the Americas 1983).
(WILLIAMS et 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 t o develop this plurality of eages. The high mutation rate of the mtDNA
(;enetics 130: 153-162 uanuary, 1992)
154 A. Torroni et al.
(BROWN, GEORGEand WILSON1979; WALLACE et al. cell enzyme genes(SZATHMARY, FERRELL and GERSHOWITZ
1987)permits discrimination between even closely 1983).
TLe Navajo are southern Athapaskans who are closely
related populations. In previous studies of Amerind related genetically (SZATHMARY 1983) and linguistically 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 (HASKELL 1987). Having increased to the
present 150,000 from a total of no more than 15,000 people
(WALLACE, GARRISON and KNOWLER1985; SCHURR et in 1868 (SPENCER et 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 (WILLIAMS et al. 1981; TROUP et al.
SON et al. 1983; HORAI, GOJOBORI and 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 andOMOTO1986; CANN, STONEKING and WIL- Arizona, inwhichCaucasian admixture was estimated at
SON 1987; HARIHARA et al.. 1988; STONEKING et al. 1.1% (WILLIAMS et al. 1981; 1985), and a southern one
1990; BALLINGER et al. 1992), we hypothesized that based in Albuquerque, New Mexico, where individuals from
all Amerinds derivedfroman 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% (TROUP et 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; HAURY 1976).
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 (MATSON et al. 1968). In 1977, the
MATERIALS AND METHODS Pima numbered some 8,000 people (SPENCER et al. 1977).
Gm allotype studies indicated that Caucasian admixture in
Samples: Blood cells in the form of lymphoblasts,platelets the Pima was 1.0% (WILLIAMS et 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 smallsize 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 to the Mexican Penutian
west Territories. Their traditional subsistence economywas subgroup (GREENBERG 1987). 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, SIMOESand 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 1,000
1 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-JACQUESand 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 BODMER 1980).
’ 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
+
2.5% NuSieve 1.0% SeaKem agarose (FMC BioProducts) 0 0 0 omooom
gels and detected by ethidium bromide fluorescence. The 0 0 0 o-ooo-
fragments observed for each PCR segment were restriction 0 0 0 o-ooo-
mapped by the sequence comparison method (JOHNSON et 0 0 0 o-ooo-
al. 1983; CANN,BROWN and WIJSON1984), the composite
0 0 0 ooo-o-
restriction maps of these sets of segments representing in-
0 0 0 ooo-o-
dividual haplotypes is given inAPPENDIX B. The distribution
and frequency of haplotypes among tribal groups is shown o o m ooooom
in Table 1.
0 0 0 ooo-o-
Sequence divergences: Mean intra- and intergroup se-
quence divergences were estimated with NEI and TAJIMA'S 0 0 0 - 0 0 0 0 -
(1983) maximum likelihood procedure using the computer 0 0 0 0 0 0 0 0 m
program DREST (graciously provided by L. JIN). This pro-
0 0 0 o-ooo-
cedure considers the ratioof shared sites to the total number
of sites between two haplotypes, and the mean length of the 0 0 0 o-ooo-
restriction enzyme recognition sequences to calculate an 0 0 0 omooom
initial estimate of x (the probability that the two mtDNAs 0 0 0 o-ooo-
have different nucleotides at a given nucleotide position). 0 0 0 ooo-o-
Using this initialestimate, A is solved iteratively using equa- 0 0 0 o-ooo-m
tion 28 and the sequence divergence (6) is estimated by 0 0 0 o-ooo-
equation 21 (NEI and TAJIMA 1983). 0 0 - 0 0 0 0 0 -
Sequence divergence values within and among the tribal 0 0 - 0 0 0 0 0 -
groups are presented in Table 2. Divergence values shown
0 0 - ooooom
in standard type represent interpopulational comparisons,
0 0 - 0 0 0 0 0 -
those underlined represent intrapopulational comparisons,
and those inbold type represent interpopulation diver- o o m ooooom
0 0 0 0 0 0 0 - -
gence calculations among tribal groups, but were included
in estimates of overall Nadene and Amerind divergences. 0 0 0 o o o o m m
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
ing between 21,000-42,000 YBP.Ifmost of this culturally with Puebloan Indians, borrowing both ag-
radiation occurred after entry into theAmericas, this ricultural practices and ceremonial rituals(SPENCER et
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; SCHELL and BLUMBERC 1988).
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. B haplotypes
The third alternativeis that the cluster
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, inAsia 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 (BALLINCER et 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 (HERTZBERC et 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 theNadenethrough 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
Science Foundation grant BNS8718775 awarded to D.C.W.
they could have arrived in an independent migration
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Mitochondrial DNA Analysis 161
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and genetic relationship, pp. 185-210 in The First Americans: WILLIAMS, R. C., A.G. STEINBERG, W. C. KNOWLERand D. J.
Origins, Afjnities and Adaptations, edited by W. S. LAUGHLIN PETTITT, 1986 Gm 3;5,13,14and stated admixture: inde-
and A. B. HARPER. Gustav Fischer, New York. pendent estimates of admixture in American Indians. Am. J.
SZATHMARY, E. J. E., 1983 Dogrib Indians of the Northwest Hum. Genet. 39: 409-413.
Territories, Canada: genetic diversity and genetic relationship WRISCHNIK, L. A., R. G. HIGUCHI,M. STONEKING, H. A. ERLICH,
among subarctic Indians. Ann. Hum. Biol. 1 0 147-162. N. ARNHEIM and A. C. WILSON,1987 Length mutations in
SZATHMARY, E. J. E., 1984 Peopling of northern North America: human mitochondrial DNA: direct sequencing of enzymatically
clues from genetic studies. Acta Anthropol. 8 79-109. amplified DNA. Nucleic Acids Res. 15: 529-542.
SZATHMARY, E. J. E., 1991 Modeling ancient population relation- Communicating editor: V. G. FINNERTY
ships from modern population genetics, in Method and Theory
for Investigating the Peopling to the Americas, edited by R. BON-
NICHSEN and D. G. STEELE. Texas A&M Press, College Station,
APPENDIXA
Tex. (in press). Oligonucleotide primers for PCR amplifications of
SZATHMARY, E. J. E., R. E. FERRELLand H. GERSHOWITZ,
1983 Genetic differentiation in Dogrib Indians: serumpro-
Native American mtDNAs are shown in Table 4.
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. 1 9 694-701. 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.
3007-3023,5917-5898 55 2910
TAYLOR, R. E., L.A. PAYEN,C. A. PRIOR,P. J. SLOTA,JR., R.
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. DONAHUE and R. BERGER,1985 Major
8282-8305,10107-10088 57 1825
revisions in the Pleistocene age assignments for North Ameri-
9911-9932,11873-11851 69 1962
can human skeletons by C-14 accelerator mass spectrometry:
11673-11691,13950-13932 57 2277
none older that 11,000 C-14 years B.P. Am. Antiq. 50: 136-
13914-13930,16547-16527 47 2633
140.
16453-16472.1696-1677 61 1812
TROUP, G. M., M. S. SCHANFIELD, C. H. SINGARAJU, R. L. HARVEY,
J. JAMESON, J. CAPPERand B. BAKER,1982 Study of HLA Primer pair are numbered according to ANDERSON et al. (1 98 I),
alloantigens of the Navajo Indians of North America. Tissue with the 5’ + 3’ coordinates before the comma corresponding to
Antigens 2 0 339-35 1. the forward primer and those afterthe comma to the reverse
VIGILANT, L., R. PENNINGTON, H. HARPENDING, T. D. KXHER primer. The THused for annealing was the lowest for the primer
pair as calculated from the nucleotide sequence of each primer,
and A. C. 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