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Mitochondrial DNA and the Peopling of South America

CECIL M. LEWIS JR.,1 BEATRIZ LIZRRAGA,2 RAL Y. TITO,2,3 PAUL W. LPEZ,2 GIAN CARLO IANNACONE,2 ANGEL MEDINA,2 ROLANDO MARTNEZ,2 SUSAN I. POLO,2 AUGUSTO F. DE LA CRUZ,2 ANGELA M. CCERES,2 AND ANNE C. STONE3

Abstract The initial peopling of South America is largely unresolved, in part because of the unique distribution of genetic diversity in native South Americans. On average, genetic diversity estimated within Andean populations is higher than that estimated within Amazonian populations. Yet there is less genetic differentiation estimated among Andean populations than estimated among Amazonian populations. One hypothesis is that this pattern is a product of independent migrations of genetically differentiated people into South America. A competing hypothesis is that there was a single migration followed by regional isolation. In this study we address these hypotheses using mtDNA hypervariable region 1 sequences representing 21 South American groups and include new data sets for four native Peruvian communities from Tupe, Yungay, and Puno. An analysis of variance that compared the combined data from western South America to the combined data from eastern South America determined that these two regional data sets are not significantly different. As a result, a migration from a single source population into South America serves as the simplest explanation of the data.

Critical to understanding the distribution and magnitude of human genetic variation in the Americas is deciphering how the American continents were initially colonized. The understanding of the peopling of North America has progressed signicantly from molecular anthropological studies. Unfortunately, the understanding of the peopling of South America is not as well understood. In North America the most parsimonious model for most genetic data is a single initial migration of relatively few individuals about 25,00015,000 years ago along a Pacic coastal route (Bonatto and Salzano 1997; Hey 2005; Merriwether et al. 1995b; Silva et al. 2002; Stone and Stoneking 1998) and a potentially smaller and later migration between the Laurentine and Cordilleran
1Department of Human Genetics, 4909 Buhl Building, University of Michigan Medical School, Ann Arbor, MI 48109. 2Centro de Investigacin de Bioqumica y Nutricion, Universidad Nacional Mayor de San Marcos, Lima, Peru. 3School of Human Evolution and Social Change, Arizona State University, Tempe, AZ 85287.

Human Biology, April 2007, v. 79, no. 2, pp. 159178. Copyright 2007 Wayne State University Press, Detroit, Michigan 482011309 KEY WORDS: MTDNA, HYPERVARIABLE REGION 1, AMERINDIANS, NATIVE AMERICANS, AMOVA, ANDEAN POPULATIONS, AMAZONIAN POPULATIONS, PERU, PEOPLING OF SOUTH AMERICA.

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160 / lewis et al. glaciers through the ice-free corridor about 12,550 years ago (Schurr and Sherry 2004). In South America, although there is compelling archeological and genetic evidence to suggest that the continent was peopled by 14,000 years ago (Dillehay 1999; Dixon 2001; Fuselli et al. 2003), there is no agreement regarding the number of initial migrations (Fox 1996; Lalueza et al. 1997; Luiselli et al. 2000; Moraga et al. 2000; Rothhammer et al. 2001; Rothhammer and Silva 1989, 1992; TarazonaSantos et al. 2001). In part, this lack of consensus is attributed to a unique pattern of genetic diversity in South America, resulting in a signicant challenge to understanding the initial colonization. On average, western (typically Andean) populations have higher levels of within-population genetic diversity and shorter genetic distances than eastern (typically Amazonian) populations (Fuselli et al. 2003; Lewis et al. 2005; Luiselli et al. 2000; Tarazona-Santos et al. 2001). This pattern may have originated during the initial peopling of South America. For instance, Andean and Amazonian populations may have originated from separate migration events, with Andean populations having a greater time depth (Tarazona-Santos et al. 2001) and/or less effects from bottlenecks than Amazonian populations. Alternatively, this pattern could have formed after the peopling of South America through evolutionary processes that include different population sizes and gene ow levels in western and eastern regions of the continent, all while maintaining relatively little gene ow between these regions (Fuselli et al. 2003; Lewis et al. 2005; Luiselli et al. 2000; Tarazona-Santos et al. 2001). These two explanations of the genetic pattern in South America are not mutually exclusive; however, both are dependent on how well a migration from a single source population into South America accounts for the genetic variation observed. If a migration from a single source population is responsible for the peopling of both western and eastern South America, then the variation found within these regions should be similar. Alternatively, if these regions were peopled by genetically diverged source populations, then variation found in the western region should be different from that in the eastern region. To test the null hypothesis of no genetic divergence between western and eastern regions, we combine the data for the Andean groups and the Amazonian groups into two separate regional data sets and assess the percentage of variation between these macro data sets. The present analysis uses mtDNA hypervariable region 1 (HV1) sequences from 21 South American groups (Figure 1). These groups include four new population samples from three Peruvian communities from Tupe, Yungay, and Puno.

Materials and Methods

Cheek swabs were collected from 14 Aymara speakers and 30 Quechua speakers from Puno, 16 Jaqaru speakers from Tupe, and 36 Quechua speakers from Yungay. The Human Subjects Committee at the University of New Mexico and Arizona State University approved all sampling protocols. Extraction of DNA followed a protocol similar to that described by Richards et al. (1993). The 9-bp dele-

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mtDNA and the Peopling of South America / 161

Figure 1. Location of Central and South American populations analyzed.

tion and three diagnostic restriction enzymes (HaeII, AluI, and HincII) were used to categorize each individual into one of the four major native American haplogroups for mtDNA, as described previously (Stone and Stoneking 1998). Primers L15926 (5-AAACTAATACACCAGTCTTGTAAACC-3) to H16410 (Handt et al. 1996) were used to sequence the mtDNA HV1 region by PCR. Sequencing in both directions was performed on an ABI 377 DNA sequencer in the Arizona State University core DNA laboratory using the BigDye protocol from Applied Biosystems (Foster City, California). In addition to the four new data sets, HV1 sequences from 43 native American communities were collected from previously published sources (Bailliet et al. 1994; Batista et al. 1995; Bert et al. 2001, 2004; Bonatto and Salzano 1997; Budowle et al. 2002; Demarchi et al. 2001; Dipierri et al. 1998; Fuselli et al. 2003; Ginther et al. 1993; Goicoechea et al. 2001; Kittles et al. 1999; Kolman and Bermingham 1997; Kolman et al. 1995; Lalueza et al. 1997; Lewis et al. 2005;

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162 / lewis et al. Malhi et al. 2001; Merriwether et al. 1995a; Mesa et al. 2000; Moraga et al. 2000; Rickards et al. 1999; Rubicz et al. 2003; Saillard et al. 2000; Santos et al. 1994; Schmitt et al. 2004; Schurr et al. 1990; Shields et al. 1993; Starikovskaya et al. 1998; Stone and Stoneking 1998; Torroni et al. 1992, 1993; Vona et al. 2005; Ward et al. 1991, 1993, 1996). This database includes 21 data sets from North America, 5 data sets from Central America, and 21 data sets from South America. Criteria for selection were based on having a sample size of 10 or greater with known linguistic heritage and with representative sequences from position 16,040 to 16,362 (Anderson et al. 1981; Andrews et al. 1999). The North and Central American data sets were used to compare the level of genetic differentiation between regions of South America to the level of genetic differentiation among regions of the Americas as a whole. Haplotype diversity (h) and nucleotide diversity () within groups were calculated using Neis (1987) formulas
2 h = n 1 pi n 1 i

(1)

and pi pj dij L
k

i=j j<i

(2)

for all 47 communities from North, Central, and South America. Nucleotide diversity was calculated using Arlequin 2.0 (Schneider et al. 2000). Groups were classied as Andean if they were currently living on or west of the Andean mountains. The Yaghan were classied as Andean because they appear to be related to Pehuenche and other Chilean tribes (Moraga et al. 2000). Although this classication could be contested, the inclusion or exclusion of the Yaghan has no signicant effect on these analyses. Groups were classied as Amazonian if they were currently living in or adjacent to the Amazon rainforest and savannas. The populations from the department of Beni (Ignaciano, Movima, Trinitario, and Yuracare) are located in a region that includes the Andean foothills and the Amazon (Bert et al. 2004). For this reason three different strategies were used to group these data sets. In the rst strategy the Beni data sets were included with the Andean data sets. In the second strategy the Beni data sets were included with the Amazonian data sets. In the nal strategy the Beni data sets were excluded from all analyses. Statistica 6.0 (StatSoft 2001) was used to perform Mann-Whitney U tests on mean haplotype and nucleotide diversity. Mann-Whitney U tests were used to compare Andean means to Amazonian means and to all non-Andean means. Because these multiple comparisons largely lacked independence, repeated mea-

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mtDNA and the Peopling of South America / 163 sure correction was not performed. P values were required to be less than 0.05 to be signicant. An analysis of molecular variance (AMOVA) was performed using Arlequin 2.0 (Schneider et al. 2000) following Excofer et al.s (1992) method of estimating indexes. AMOVAs were conducted on interregional comparisons of North, Central, and South America as well as on intraregional comparisons of the Andes and Amazon. Percentages of variation, estimates of xation indexes, and signicance of these estimates were considered. Arlequin applies a nonparametric permutation approach to calculate the signicance of estimated indexes. Ten thousand permutations were used to calculate the p values. Of particular interest are the estimates of CT, because they assess whether the combined data from the Andean region and the combined data from the Amazonian region are signicantly different from the total data for the South American continent. Arlequin 2.0 was used to estimate a genetic distance for all possible pairs of the populations. The genetic distance is the average number of nucleotide substitutions per site, assuming the substitution model described by Tamura and Nei (1993) with a gamma shape parameter of 0.26, as suggested by Meyer et al. (1999). The distance matrix was visualized using a multidimensional scaling plot and Statistica 6.0 (StatSoft 2001). The multidimensional scaling plot displays a distance matrix in a number of dimensions designated by the researcher. Goodness of t for the plot was calculated through a stress value. Typically, stress values of 0.10 or less are considered to have low distortion (Kruskal 1964).

Results
The sequence and haplogroup data are provided in Tables 14 for the Yungay, Tupe, Quechua Puno, and Aymara Puno, respectively. In these samples haplogroup B was most frequent, especially in the Aymara speakers from Puno (71.4%) and the Jaqaru speakers from Tupe (68%). Although all four haplogroups were present in the Quechua speakers from Puno and Yungay, the Aymara and Tupe samples lacked haplogroup A. These new Andean data sets have high to moderate levels of diversity. The haplotype diversities were 0.954, 0.875, 0.974, and 0.967 for the Yungay, Tupe, Quechua Puno, and Aymara Puno, respectively. The nucleotide diversities were 0.018, 0.025, 0.018, and 0.017, respectively, for these same communities. These diversity estimates are congruent with those from other sampled Andean populations (Table 5). The Mann-Whitney U test results indicate that Andean populations have signicantly higher diversity estimates than non-Andean populations (Table 6). With one exception, all tests were signicant at the 0.05 level, regardless of whether the comparisons were between the Andean and the Amazonian groups or between the Andes and all non-Andean groups studied. The exception was when the average nucleotide diversities from the Andean groups were compared to average nucleotide diversities combined for the Amazonian and Beni groups (0.097). A plot

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Table 1. MtDNA Sequence Data Amplified from Positions 16,040 to 16,362 (Anderson et al. 1981; Andrews et al. 1999) from the Yungay Sample

Haplogroup

Haplotype

16093

16094

16111

16129

16140

16168

16174

16189

16209

16217

16223

16260

16261

16270

16271

16278

16290

16292

16293

16298

16311

16319

16325

16327

16357

16360

16362

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A B B B B B B B B B B C C C C C D D D D

Anderson YU001 YU002 YU003 YU004 YU005 YU006 YU007 YU008 YU009 YU010 YU011 YU012 YU013 YU014 YU015 YU016 YU017 YU018 YU019 YU020

1 4 2 1 1 2 1 1 1 3 1 5 4 1 2 1 2 1 1 1

T . . . . . . C . . . . . . . . . . . . .

T . . . . . . . . . . . . . C . . . . . .

C T . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . A . . A .

T . . . . . . C . . . . . . . . . . . . .

C . . . T T . . T T T . . . . . . . . . .

C . . . . . . . . . . . . . . . . . . . T

T . C C C C C C C C C C . . . C C . . . .

T . . . . . . . . . . . . . . . . . C . .

T . C C C C . C C C C C . . . . . . . . .

C T . . . . T . . . . . T T T T T T . T T

C T . . . . . . . . . . . . . . . . . . .

C . . . . T . . . . . . . . . . . . . . .

C . . . T . . . . . . . . . . . . . . . .

T C . . . . . . . . . . . . . . . . . . .

C T . . T . . . . . . . . . . . . . . . .

C T . . . . . . . . . . . . . . . . . . .

C . . . . . . . . . . . T . . . . . . . .

A . . . . . . . . . . G . . . . . . . . .

T . . . . . . . . . . . C C C C C . . . .

T . . . . . . . . . . . . C . . . . . . .

G A . . . . . . . . . . . . . . . . . . .

T . . . . . C . . . . . C C C C C C C C C

C . . . . . . . . . . . . T T T T . . . .

T . . . . . . . C . . . . . . . . . . . .

C . . . . . . . . T . . . . . . . . . . .

T C . C . . C . . . . . . . C . . C C C C

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Table 2. MtDNA Sequence Data Amplified from Positions 16,040 to 16,362 (Anderson et al. 1981; Andrews et al. 1999) from the Tupe Sample

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Haplogroup

Haplotype

16093

16113

16129

16168

16185

16189

16192

16217

16223

16256

16260

16274

16288

16298

16304

16319

16325

16327

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B B B B B B B C C

Anderson TPE01 TPE02 TPE03 TPE04 TPE05 TPE06 TPE07 TPE08 TPE09

5 1 1 1 1 1 1 4 1

T . . . . . . . C .

A . . G . . . . . .

G . . . . . A . . .

C T . . . . . . . .

C . . T . . . . . .

T C C C C C C C . .

C . . . . . . . T .

T C C C C C C C . .

C . . . . . . . T T

C . . . . . . T . .

C . T . . . . . . .

G . . . . A . . . .

T . . C . . . . . .

T . . . . . . . C C

T . . . . . . C . .

G . A . . . . . . .

T . . . . . . . C C

C . . . . . . . T T

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Table 3. MtDNA Sequence Data Amplified from Positions 16,040 to 16,362 from the QuechuaSpeaking Puno Sample

Haplogroup

Haplotype

16051

16066

16086

16092

16093

16111

16126

16129

16145

16150

16153

16168

16169

16176

16180

16189

16217

16220

16222

16223

16256

16274

16290

16294

16298

16301

16319

16325

16326

16327

16344

16354

16357

16362

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A A B B B B B B B B B B B B B C C C C C C C D

Anderson QPNO01 QPNO02 QPNO03 QPNO04 QPNO05 QPNO06 QPNO07 QPNO08 QPNO09 QPNO10 QPNO11 QPNO12 QPNO13 QPNO14 QPNO15 QPNO16 QPNO17 QPNO18 QPNO19 QPNO20 QPNO21 QPNO22 QPNO23

A A 1 . . 1 . . 2 . . 2 . . 1 . . 1 . . 1 . . 1 . . 4 . . 1 . . 1 . . 1 . . 1 . G 1 . . 1 . . 1 . . 1 . . 1 . . 1 . . 1 . . 1 . . 1 G . 3 . .

T . . . . . . . . . . . . . . C . . . . . . . .

T . . . . . C . . . . . . . . . . . . . . . . .

T . . . . . . C . . . . . . . . . . . . . . . .

C T T . . . . . . . . . . . . . . . . . . . . .

G . . . . . A . . . A . . . . . . . . . A . . .

G C . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . .

G . . . . . . . . . . . A . . . . . . . . . . .

C . . T . . . . . . . . . T . . . . . . . . . .

C . . . . T . . . . . . . . . . . T . . . . . .

C . . . . . . T . . . . . . . . . . . . . . . .

A . . . . . C . . . . . . . . . . . . . . . . .

T . . C C . C C C . . C C . . C C . . . C C C .

T . C C C C C C C C C C C C C C . . . . . . . .

A . . . . C . . . . . . . . . G . . . . . . . .

C T . . . . . . . . . . . . . . . . . . . . . .

C T T . . . . . . . . . . . . . T T T T T T T T

C . . . . . . . . . . . . . . . . . T . . . . .

G . . . . . . A . . . . . . . . . . . . . . . .

C T T . . . . . . . . . . . . . . . . . . . . .

C T . . . . . . . . . . . . . . . . . . T . . .

T . . . . . . . . . . . . . . . C C C . C C C .

C . . . . . . . . . . . . . . . . . . . . . . .

G A A . . A . A . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . C C C C C C C C

A . . . . . . . . . . C . . . . . . . . . . . .

C . . . . . . . . . . . . . . . T T T T T T T .

C . . . . . . . . . . . . . . . T . . . . T T .

C . . . . . . . . . T . . . . . . . . . . . . .

T . . . . . . . C . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . C . . . . . . . . .

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Table 4. MtDNA Sequence Data Amplified from Positions 16,040 to 16,362 from the AymaraSpeaking Puno Sample

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Haplogroup

Haplotype

16051

16086

16111

16136

16170

16177

16189

16197

16200

16217

16223

16235

16242

16248

16258

16261

16264

16266

16298

16309

16310

16325

16327

16362

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mtDNA and the Peopling of South America / 167

B B B B B B B C C D D

Anderson APNO01 APNO02 APNO03 APNO04 APNO05 APNO06 APNO07 APNO08 APNO09 APNO10 APNO11

2 1 2 1 2 1 1 1 1 1 1

A . . . . . . . G . . .

T . . . . . . . C . . .

C . T . . . . . . . . .

T . C . . . . . . . . .

A . . . . . . . G . . .

A . . . . . G . . . . .

T . C . . C . C . . C .

C . . . . . . . . . . .

A . . . . . . . . . . .

T C C C C C C C . . . .

C . . . . . . . T T T T

A . . . . . . . . . G .

C . . . . . . . . . . T

C . . . . . . . . . . T

A . . . C . . . . . . .

C . . . . T . . . . . .

C . T . . . . . . . . .

C . . T . . . . . . . .

T . . . . . . . C C . .

A . . . . . G . . . . .

G . . . . . . . . . A .

T . . . . . . . C C C C

C . . . . . . . T T . .

T . . . . . . . . . C C

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Table 5. Diversity Data from Contributed Populationsa

Population Ache Aleut Ancash Apache Arequipa Athabaskan Bella Coola Canadian Inuit Cayapa Central Yupik Cherokee Cheyenne Chippewa Embera Gaviao Greenland Inuit Guahibo Haida Huetar Ignaciano Inupiaq Kuna Mapuche 1 Mapuche 2 Movima Navajo Ngoebe Nuu Chah Nulth Oneonta Pehuenche Pima Puno (Aymara) Puno (Quechua) Siberian Yupik 1 Siberian Yupik 2 Sioux Surui Tayacaja Trinitario Tupe Waiwai Wounan Xavante Yaghan Yakima Yungay Yuracare Zoro n 63 163 35 183 22 40 41 46 30 25 30 39 19 44 28 82 59 41 27 15 15 63 39 34 12 164 46 59 51 24 40 14 30 52 77 16 24 65 12 16 26 31 25 15 42 36 15 28 Number of Haplotypes 3 21 23 30 17 15 10 7 8 4 12 24 6 18 7 11 11 9 7 11 5 7 11 8 8 40 7 24 23 13 20 11 23 9 12 13 4 45 10 9 10 14 4 6 20 20 11 7 Haplotype Diversity 0.204 0.618 0.948 0.777 0.965 0.853 0.894 0.479 0.837 0.650 0.881 0.965 0.725 0.930 0.862 0.746 0.854 0.684 0.709 0.933 0.800 0.592 0.895 0.834 0.894 0.906 0.763 0.946 0.900 0.902 0.918 0.967 0.974 0.651 0.719 0.975 0.370 0.971 0.955 0.875 0.837 0.912 0.677 0.848 0.893 0.954 0.952 0.741 Nucleotide Diversity 0.004 0.014 0.019 0.017 0.017 0.007 0.017 0.004 0.039 0.004 0.017 0.024 0.012 0.020 0.014 0.005 0.016 0.008 0.011 0.022 0.009 0.010 0.022 0.019 0.009 0.019 0.014 0.017 0.016 0.020 0.024 0.017 0.018 0.009 0.010 0.024 0.005 0.020 0.023 0.025 0.022 0.023 0.010 0.017 0.016 0.018 0.023 0.012 Source Schmitt et al. (2004) Rubicz et al. (2003) Lewis et al. (2005) Budowle et al. (2002) Fuselli et al. (2003) AF184627184647; Shields et al. (1993) Ward et al. (1993) AF186706186751 Rickards et al. (1999) AF011645011669 Malhi et al. (2001) Kittles et al. (1999) Malhi et al. (2001) Kolman and Bermingham (1997) Ward et al. (1996) Saillard et al. (2000) Vona et al. (2005) Ward et al. (1993) Santos et al. (1994) Bert et al. (2004) AF082222082231; Shields et al. (1993) Batista et al. (1995) Ginther et al. (1993) Moraga et al. (2000) Bert et al. (2004) AF011670011684; Budowle et al. (2002) Kolman et al. (1995) Ward et al. (1991, 1993) Stone and Stoneking (1998) Moraga et al. (2000) Kittles et al. (1999) This study This study AF013684013633 Starikovskaya et al. (1998) Malhi et al. (2001) Bonatto and Salzano (1997) Fuselli et al. (2003) Bert et al. (2004) This study Bonatto and Salzano (1997) Kolman and Bermingham (1997) Ward et al. (1996) Moraga et al. (2000) Shields et al. (1993) This study Bert et al. (2004) Ward et al. (1996)

a. Because the estimates are based on the mtDNA HV1 region from position 16,040 to 16,361 (Anderson et al. 1981; Andrews et al. 1999), they may deviate from the original published source.

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Table 6. Results of the Mann-Whitney U Tests
Average Haplotype Diversity 0.928 and 0.761 0.930 and 0.674 0.928 and 0.783 0.930 and 0.769 0.928 and 0.674 Average Nucleotide Diversity 0.020 and 0.015 0.020 and 0.013 0.020 and 0.015 0.020 and 0.014 0.020 and 0.013

Comparison Andean to Amazonian (with Beni) Andean (with Beni) to Amazonian Andean to all non-Andean Andean (with Beni) to all non-Andean Andean to Amazonian (no Beni included)

P Value 0.012 0.000 0.001 0.003 0.000

P Value 0.097 0.005 0.005 0.027 0.006

of these distances illustrates the high levels of haplotype and nucleotide diversity in Andean populations (Figure 2). A multidimensional scaling plot (Figure 3) shows a cluster that includes the Andean and many of the Beni populations. The raw stress value for this plot was 10.5%. The Movima are the only Beni population outside this cluster, and the

Figure 2. Scatterplot comparing haplotype and nucleotide diversity measures for the studied South American populations. Triangles, Andean populations; squares, Beni populations; circles, Amazonian populations.

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Figure 3. Multidimensional scaling of South American populations with Andean and Beni populations circled. Raw stress value was 10.5%.

Yaghan are the only Andean population outside this cluster. The Amazonian populations are not clustered with each other or with the Andean populations. The AMOVA calculations show lower percentages of variation among Andean groups than among Amazonian groups (Table 7). The percentage of variation among the Andean data sets is 6.34% when the Beni data are included and 5.52% when they are not. The percentage of variation among the Amazon data sets is 32.18% when the Beni data are included and 38.29% when they are not. However, when the Andean data sets in aggregate are compared to the Amazonian data sets in aggregate, the percentage of variation between them is near zero regardless of the placement of the Beni data. The associated CT estimates were the only nonsignicant p values from permutations. When comparing the percentage of variation between North and Central America to South America (13.26%) or when comparing the percentage of variation between Central America and South America (6.81%), we found that the associated p values for the CT estimates were all less than 0.05.

Discussion
We have found that the distribution of genetic variation within and among local South American populations is patterned differently within the western region

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Table 7. AMOVA Percentage of Variation and Fixation Indexesa
Variation Among Populations Within Groups (%) 6.34 5.52 32.18 38.29 17.72 18.73 17.94 16.83 21.42

Comparison Andean (with Beni) Andean Amazon (with Beni) Amazon Andean to Amazonian (with Beni) Andean to Amazonian (no Beni included) Andean (with Beni) to Amazonian Central America to South America North and Central America to South America

Variation Between Groups (%) NA NA NA NA 0.08 0.50 0.44 6.81 13.26

Variation Within Populations (%) 93.66 94.48 67.82 61.71 82.36 81.77 82.1 76.36 65.32

CTb
NA NA NA NA 0.001 0.005 0.004 0.068 0.132

SC
NA NA NA NA 0.177 0.186 0.179 0.180 0.248

ST
0.063 0.055 0.322 0.389 0.176 0.182 0.175 0.236 0.348

a. Fixation indexes found to be significant ( p values < 0.05) after 10,000 permutations are in bold. The p values for the Andean to Amazonian comparisons range from 0.431 to 0.482. b. The highest (closest to 1) significant p value was the Central American to South American comparison; the CT was 0.0196.

than within the eastern region, yet from the variation between these regions, we nd no evidence for differentiation. The Mann-Whitney U test and scatterplot for the haplotype and nucleotide diversity measures suggest that, on average, withinpopulation genetic diversity is signicantly higher in the Andes than in the Amazon. There was one exception to this pattern. When the estimated within-population nucleotide diversities for the Andean region were compared to those for the Amazonian and Beni region combined, we observed a nonsignicant value (0.097). This is not surprising considering that three of the four Beni communities had relatively high levels of within-population genetic diversity. The estimated ST from the AMOVA show low genetic differentiation among Andean communities (0.06), especially when compared to values among Amazonian communities (0.35). Theoretically, xation indexes of 0.00 to 0.05 suggest little differentiation, 0.05 to 0.15 suggest moderate differentiation, 0.15 to 0.25 suggest great differentiation, and >0.25 suggest very great differentiation (Wright 1978). In reality, the estimated xation indexes assume that population structures are approximated by a hierarchical model of relationships in which there is an innite number of equally sized populations, each with panmictic mating and each evolving independently (the innite island model). Moreover, the indexes assume that levels of diversity within populations are equal. However, Amazonian

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172 / lewis et al. population samples have a wide range of diversities (see Table 1), which likely reects an amalgam of different population histories and potential sampling biases; thus for this analysis this assumption has been relaxed. Nevertheless, there is a clear pattern of little to moderate differentiation among populations in the Andes and very great differentiation among populations in the Amazon. The multidimensional scaling plot further supports this regional patterning. The arrangement of groups in the multidimensional scaling analysis illustrates the genetic similarities among Andean communities. Andean groups are clustered more closely than is expected given their geographic distances. For example, in the multidimensional scaling plot the Peruvian populations are clustered near Chilean populations, even though these populations are separated by 2,000 to 3,000 km, whereas all Amazonian populations are scattered on the periphery of the plot. One exception to this pattern is the Yaghan. The Yaghan are an island population in Tierra del Fuego, and their association with Andean populations has been debated (Moraga et al. 2000). Because of the uncertainty about how to classify them in separate analyses, the Yaghan sample was excluded from Mann-Whitney U tests and AMOVAs. We determined that the exclusion of the Yaghan had no signicant effect on the results of these analyses. In the multidimensional scaling plot Beni groups are more similar to Andean groups than to Amazonian groups. The exceptions are the Movima. Although geographically near the other Beni populations, the Movima sample is not clustered with any other population. Compared to the other Beni populations examined, the Movima are the farthest from the Andes. Because they are located predominantly east of these other Beni communities, this may suggest that they t more with Amazonian genetic patterns than with Andean genetic patterns. In addition, the Movima are members of the Macro-Tucanoan language family, a different language family than that of the other Beni communities. Consequently, linguistic isolation of the Movima, or some cultural-historical factor associated with linguistics, may also explain this divergent pattern. Although our examination supports different patterns of genetic variation in Andean versus Amazonian regions, when data for these regions are combined to form two separate macro data sets, the percentage of genetic variation between these two regions is negligible. For instance, when the data for the western and eastern regions are compared to each other, the percentages of variation are slightly negative and the estimated CT are nonsignicant. The variance components of the AMOVAs are covariances, which, by chance, can have slightly negative values when there is an absence of genetic structure. Grouping populations improperly can lead to a lack of observed genetic structure. For example, if populations are classied as Amazonian because of their current location, but historically they were associated largely with the Andes, then this could bias the statistics toward lower estimated percentages of variation between the regions. Potential classication errors were a primary concern in this analysis, especially considering the geographic location of the Beni communities. However, all AMOVAs comparing the Andes and Amazon regions were slightly

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mtDNA and the Peopling of South America / 173 negative, regardless of the placement or exclusion of the Beni data, suggesting that misclassication was not an issue for these data. Thus, even though the communities from the Andes and Amazon have separate patterns of genetic diversity, the AMOVA comparing the combined data from western South America to the combined data from eastern South America was unable to reject similarity. Because there is no evidence to suggest that these regions are genetically diverged, a migration from a single source population serves as the simplest explanation of the mtDNA HV1 data. For this single maternally inherited locus, a more complex model is not required to account for the genetic patterning. Nevertheless, if these analyses are representative, then multiple migrations could occur, given three different scenarios. Scenario 1: Genetic variation in each source population is not signicantly different. It is difcult to assess the likelihood of this scenario given the paucity of information on the distribution of genetic variation in early native Americans. However, it is interesting to note that AMOVAs comparing North and Central America to South America and comparing only Central America to South America had signicant CT estimates, with percentages of variation of 13.26 and 6.81 between the regions, respectively. Thus, if it is assumed that ancestral migrations entering South America derived from a random sampling of North or Central American variation, then from these analyses it is likely that each sample would have genetic variation that is signicantly different. Scenario 2: After the founding migrations, long-range gene ow between western and eastern South America was sufciently high enough to homogenize the variation. When the combined data sets for the Andean and Amazon groups were compared, we found slightly negative percentages of variation from the AMOVA analyses, which suggests low regional differentiation. This could be a product of long-range gene ow between these regions. On the other hand, when the data for Amazonian communities in this study were considered, we did not see evidence of recent long-range gene ow. The percentages of variation among Amazonian populations are greater than 30%. This suggests significant differentiation, which is unexpected when there is moderate long-range gene ow. Moreover, this differentiation may be underestimated because three of the six Amazonian tribes are geographically close to one another. Genetic differentiation among groups, as well as lower diversity measures within groups, is characteristic of limited gene ow and lower population sizes. The limited gene ow and reduced population sizes in the Amazon may be biogeographically related. The dense vegetation of the Amazon may limit population sizes and isolate communities. Although some archeologists have suggested that larger population sizes existed prehistorically (see, e.g., Roosevelt 1991), this theory is tenuous (Meggers 2003). Moreover, although Amazonian rivers are known to facilitate regional interactions among many Amazonian communities (Hornborg 2005), they do not appear to be a signicant channel for gene ow on this larger continental scale. Indeed, the Amazon River may have been a barrier to gene ow between the northern and southern Amazonian communities (Callegari-Jacques et

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174 / lewis et al. al. 1994). Thus a common ancestry of these groups rather than more recent longrange gene ow would explain the near zero percentage of variation between the Andes and the Amazon. The larger population sizes in the Andes would retain more genetic diversity within populations than the smaller population sizes in the Amazon, which would be more subject to the loss of variation from genetic drift. That being said, the Amazon River could serve as the path of least resistance for migration(s) between the Andes and the Amazon. Additional studies evaluating communities along the Amazon River would be a valuable objective in future research, although many of these areas were affected considerably by European contact (Hornborg 2005). Scenario 3: Multiple migrations occurred with a different pattern of dispersal than that studied here. Two previously proposed models fall under this scenario. The rst model, by Lalueza and Fox (Fox 1996; Lalueza et al. 1997), is that southern populations in South America represent a separate migration from the more northern populations. In this study we have focused largely on the genetic variation in the Andes and the Amazon, and future studies of the Argentine Pampas and more southern regions are necessary. However, from our examination of the southern Andean populations, our results do not support a separate migration of southern South American populations. For example, in the multidimensional scaling plot the Pehuenche and Mapuche are placed with other Andean populations, implying a single genetic history, and, as discussed by Moraga et al. (2000), the Yaghan may be associated with the Pehuenche and other Chilean communities. A second model, by Rothhammer and colleagues (Rothhammer et al. 2001; Rothhammer and Silva 1989), proposes that one founding population entered South America from the northwest, then traveled southward through the Andes to the central highlands of Peru, then migrated eastward to occupy northwestern Argentina, eastern Brazil, and the Argentine Pampas. The other population entered South America from the north, heading eastward into Venezuela and Guyana and later occupying the central Amazon. Over time this population grew, expanded, and eventually reached the eastern slopes of the Andes to become the current inhabitants of the central Andes today. In this scenario populations from the southern Andes, such as the Mapuche and Pehuenche, could be part of the migration that occupied the Argentine Pampas. The data presented here do not support Rothhammer and colleagues model (Rothhammer et al. 2001; Rothhammer and Silva 1989). As depicted in the cluster on the multidimensional scaling plot, these Andean populations, whether central or southern, have relatively small genetic distances. In addition, nucleotide and gene diversities estimated within Andean populations are typically higher than those estimated within Amazonian populations. This is unexpected if founder effects came with the Amazonian migration. This does not reject Rothhammer et al.s (2001) hypothesis, because reduced population size in the eastern region likely reduced diversity within these populations. Nevertheless, at this time the analyses from this study do not support a founding migration from the Amazon to the Andes.

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mtDNA and the Peopling of South America / 175 In summary, the occupation of South America may have been a complex process involving multiple early migrations and subsequent population movements. However, before such complex scenarios can be supported, they have to be demonstrated to be more likely than the simpler scenarios. Using an analysis of genetic variation, we were unable to reject similarity in the genetic variation found in western compared to eastern South America. Thus these analyses of the mtDNA HV1 data are unable to reject a migration from a single source population. This suggests that later regional isolation and differential evolutionary processes led to the formation of the different patterns of diversity observed in the western and eastern regions. The greater diversity estimated within western populations and limited genetic differentiation estimated among western populations likely can be attributed to larger population sizes and more extensive gene ow within this region. The lower diversity within eastern populations and greater differentiation among eastern populations likely can be attributed to a period of reduced population sizes and more restricted gene ow within this region.
Acknowledgments We would like to thank the people who participated in this study. We would also like to thank Jeffrey Long, Keith Hunley, Graciela Cabana, and Gregory Zaro for their comments. Funding was provided by the University of New Mexico through a Latin American and Iberian Institute Field Research Grant and by the National Science Foundation (through grant BCS 0401434). Received 14 December 2005; revision received 21 September 2006.

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