AMERICAN JOURNAL OF HUMAN BIOLOGY 26:142–150 (2014)

Original Research Article

Brazilian Quilombos: A Repository of Amerindian Alleles CAROLINA CARVALHO GONTIJO,1 CARLOS EDUARDO GUERRA AMORIM,1 NEIDE MARIA OLIVEIRA GODINHO,1,2 RAFAELA CESARE PARMEZAN TOLEDO,1 ADRIANA NUNES,3 WELLINGTON SILVA,4 MARIA MANUELA DA FONSECA MOURA,5 1 ~  CARLOS COUTINHO DE OLIVEIRA,6 RUBIANI C. PAGOTTO,5 MARIA DE NAZARE  KLAUTAU-GUIMARAES, JOSE AND SILVIENE FABIANA DE OLIVEIRA1,7* 1 Laboratorio de Genetica, Departamento de Genetica e Morfologia, Instituto de Ci^encias Biol ogicas, Universidade de Brasılia, 70910-900 Brasılia, DF, Brazil 2 Instituto de Criminalıstica Leonardo Rodrigues, 74425-030 Goi^ ania, GO, Brazil 3 Departamento de Arqueologia, Universidade Federal de Rond^ onia, 76801-059 Porto Velho, RO, Brazil 4 Faculdade Adventista da Bahia, Caixa Postal 18, 44300-000 Cachoeira, BA, Brazil 5 Departamento de Biologia, Universidade Federal de Rond^ onia, 76801-059 Porto Velho, RO, Brazil 6 Departamento de Medicina, Universidade Federal de Rond^ onia, 76801-059 Porto Velho, RO, Brazil 7 Jackson Laboratory for Genomic Medicine, University of Connecticut Health Center, 06032 Farmington, CT, USA

ABSTRACT: Objectives: As a consequence of colonization of the Americas and decimation of the native population, an important portion of autochthonous genetic variation has been lost. However, some alleles have been incorporated into the growing populations of admixed mestizos. In this study, we evaluated the potential of African-derived communities in Brazil to be repositories of Amerindian alleles and, by extension, a source of information on American prehistory. Methods: In this study, we describe the genetic variation of 15 ancestry informative markers (AIMs) of autosomal origin in two quilombos, Brazilian populations mainly of African descent, Santo Ant^onio do Guapore (SAG; N 5 31), and Santiago do Iguape (STI; N 5 37). We compared the AIMs from these populations to those of other African–Brazilian populations, and to the Distrito Federal (N 5 168), an urban population representative of Brazilian genetic diversity. Results: By admixture analysis, we found that the SAG and STI communities have a much higher proportion (over 40%) of Amerindian contribution to their gene pools than other admixed Brazilian populations, in addition to marked African contributions. Conclusions: These results identify two living African–Brazilian populations that carry unique and important genetic information regarding Amerindian history. These populations will be extremely valuable in future investigations into American pre-history and Native American evolutionary dynamics. Am. J. Hum. Biol. 26:142–150, C 2014 Wiley Periodicals, Inc. V 2014. The migration and admixture of three main parental groups—namely Amerindians, Europeans, and SubSaharan Africans—gave rise to the current Brazilian population. In the year 1500, when the first Portuguese caravels reached the Brazilian shore, they met an Amerindian population estimated at 1–10 million people (Fundac¸~ ao Nacional do Indio - FUNAI: www.funai.gov.br; Rosenblat, 1954; Steward, 1954; Cunha, 2013). Contact with the Portuguese led to admixture and a drastic reduction of the autochthonous population, including a disappearance of many Amerindian ethnic groups (Santos et al., 1995). This autochthonous population is now estimated, after recent significant growth, at 817,000 people (FUNAIwww.funai.gov.br), comprising approximately 0.4% of the Brazilian population. Nevertheless, their genetic contribution to the Brazilian gene pool is significant, as shown by several population genetics studies (see for instance Godinho et al., 2008; Lins et al., 2010; Pena et al., 2011). It is estimated that of all African slaves taken to the Americas, a total number ranging from 3.6–10 million people, about 40% ended up at Brazilian ports (see review by Amorim et al., 2012). According to Mello e Souza (2006), during the centuries when African slaves were taken to the Americas, they were captured and boarded in different regions of Sub-Saharan Africa. The State of Bahia received mainly slaves from the Gulf of Benin region, while other Brazilian regions received most of their slaves from Congo and Angola (Verger, 1968). In 1850, the Slave Trade was officially made illegal in Brazil, C 2014 Wiley Periodicals, Inc. V

but the trafficking continued and African slaves were brought in mainly from West Africa until long after that (Nishida, 1993). Once on American soil, Africans and their descendants resisted slavery in many ways, such as committing suicide, displaying aggression to their owners, and through other forms of rebellion. However, the main form of resistance was through escape and hiding in remote sites, where African slaves formed isolated communities known in Brazil as quilombos (Neme and Andrade, 1987). Quilombos have been discovered all over the Brazilian territory, except in the northern states of Acre and Roraima (Fundac¸~ ao Cultural Palmares- http://palmares.gov.br). Despite variable degrees of isolation, people of different ancestries inhabited most quilombos (Karasch, 1996; Reis and Gomes, 1996; Ribeiro, 2006), making their genetic constitution quite heterogeneous and complex

Contract grant sponsors: Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico (CNPq) and Coordenac¸~ ao para Aperfeic¸oamento de Pessoal de Nıvel Superior, Brazil (CAPES). *Correspondence to: S.F. de Oliveira, Universidade de Brasılia—Campus Darcy Ribeiro, Instituto de Ci^ encias Biol ogicas, Departamento de Gen etica e Morfologia, Laborat orio de Gen etica, 70910-900 Brasılia, DF, Brazil. E-mail: [email protected] Received 30 August 2013; Revision received 5 December 2013; Accepted 15 December 2013 DOI: 10.1002/ajhb.22501 Published online 4 February 2014 in Wiley Online Library (wileyonlinelibrary.com).

143

QUILOMBOS: A REPOSITORY OF AMERINDIAN ALLELES

(Bortolini et al., 1999; Luizon et al., 2008; Scliar et al., 2009; Palha et al., 2011; Ribeiro et al., 2011; Amorim et al., 2011a). The Amerindian presence in these quilombos came about in two main ways: (1) enslaved natives would sometimes insurrect alongside the slaves of African origin and thus take refuge in the quilombos; or (2) native women would be kidnapped by the quilombolas (quilombo inhabitants) and became part of quilombo communities, as it was often easier for men of African descent to escape from captivity than it was for women (Karasch, 1996). The European presence was mostly a consequence of forced reproduction on farms as well as exchanges with urban areas, including the trading of goods and information (Reis and Gomes, 1996; Ribeiro et al., 2011). Population genetics has been used as a tool to understand the quilombos’ history, owing to the genetic diversity present in these communities and the lack of consistent or comprehensive documentation on their existence (Karash, 1996; Reis and Gomes, 1996; Bortolini et al., 1999; Luizon et al., 2008; Scliar et al., 2009; Amorim et al., 2011a; Palha et al., 2011; Ribeiro et al., 2011; Amorim et al., 2012). Genetic studies have shown that the quilombos have a high African contribution and variable contributions from Amerindians and Europeans (Bortolini et al., 1999; Ribeiro-dos-Santos et al., 2002; Oliveira et al., 2006; Barbosa et al., 2006; Carvalho et al., 2008; Santos et al., 2010; Assis et al., 2011; Palha et al., 2011; Amorim et al., 2011a; 2012), and some of these communities, such as Mocambo (Amorim et al., 2011a), contain a particularly high proportion of Amerindian genes compared with other populations. In stark contrast, the general Brazilian population presents a different genetic pattern and history, in which the European contribution predominates in all regions. As such, there is a much lower and more variable proportion of African content, and very low to negligible contributions from native Amerindian population (Godinho et al., 2012; Lins et al., 2010; Pena et al., 2011; Giolo et al., 2012). Despite the interest in Amerindian genetic history of Brazil (Callegari-Jacques et al., 2011; Reich et al., 2012), sample availability and access is rather limited compared with populations of other ethnic backgrounds. Over the last few decades, sample collection in autochthonous communities has become much more complicated to conduct, as major ethical issues have been raised and the discussion has gained momentum, being later amplified by the Yanomam€o scandal that came to public attention by the year 2000 (Hurtado and Salzano, 2004). In Brazil, the debate culminated in the publication of the RE CNS 196/ 96 (Conselho Nacional de Sa ude, - CNS - http://conselho. saude.gov.br/), that guides bioethics in research in Brazil (centered in the protection of the researcher and most importantly of the participant), and the RE CNS 304/00 (CNS - http://conselho.saude.gov.br/), that establishes some further parameters specific to the research with Amerindian groups. Practical consequences for researchers include the need for informed consent and new criteria regarding storage of and access to the samples and information associated. As an outcome, most native samples currently under analysis were collected long before legislation changes. Even if sample collection were straightforward, the drastic decline in Amerindian population size has very likely resulted in loss of genetic variability

within these populations (FUNAI- www.funai.gov.br). Therefore, the ability to access Amerindian genetic variability without having to directly analyze Amerindian samples might be of great strategic importance. The research presented herein aims to describe the genetic structure of two Brazilian populations of marked African ancestry and to evaluate their potential to be repositories of Amerindian alleles. Because quilombos might prove an useful source of biological material and genetic data, this work can be a first step to use these kind of population for learning more about Amerindian prehistory. SUBJECTS AND METHODS Populations Santo Ant^onio do Guapore (SAG) is a quilombo located in the Guapore River Valley in the state of Rond^onia in Northern Brazil (12 300 3700 S; 63 320 4300 W). It is 140 km from the closest urban area and 800 km from the state capital (Porto Velho). By the beginning of the eighteenth century, the expansion of agriculture and mining to the Guapore Valley led to an influx of African and Africandescendant slaves to the region. Most of the African slaves were men, and thus the quilombos that formed among those who managed to escape captivity were mostly men (Teixeira, 2010). The quilombos tended to acquire women from indigenous Amerindian communities by kidnapping, who were then incorporated into the growing quilombola populations (Rocha Pombo, 1935). In 1997, the population of SAG was estimated at 100 people (Teixeira, 1997). The quilombo Santiago do Iguape (STI) is located 44 km from Cachoeira, a village in Rec^oncavo Baiano (12 370 0400 S; 38 570 2100 W). STI was founded in the sixteenth century by Jesuits in an area that belonged to the Engenho S~ ao Domingos da Ponta, one of the most important Brazilian sugar producers at the time and the oldest sugar exporter in Brazil (Barickman, 1999, 2003). Tapuia and Guarani Amerindians previously occupied this area, the former taken to the region as slaves (Alencastro, 2000). Currently, its 3000 inhabitants live off the proceeds from small-scale agriculture and small businesses. As a candidate Brazilian population with which to compare to the above quilombos, we selected the Distrito Federal (DF), a large urban Brazilian population estimated at over 2.5 million people. DF was formed in Central Brazil during the 1960s transition to make Brasilia Brazil’s capital city, an event that resulted in the rapid influx of people from all five regions of the country where admixture had been occurring over the previous centuries. These settlement dynamics and history make it reasonable to assume that the genetic variation of the DF population is representative of the country as a whole. As some our previous work has suggested (data not published), DF presents a high European genetic contribution to its gene pool, followed by Africans and Amerindians. The sample analyzed is compound by individuals born in DF, mostly students. Sampling and DNA extraction Blood was collected from 31 volunteers in SAG, 37 in STI, and 168 in DF. The volunteer donors from SAG were chosen during visits to the community by a research group from the Universidade Federal de Rond^ onia. Those from STI were collected in community health programs run by the Faculdade Adventista da Bahia in partnership American Journal of Human Biology

144

C.C. GONTIJO ET AL.

with the Fourth Regional Health Division of the State of Bahia and local health boards. Biological material was processed and stocked under 220 C. DNA was extracted from the buffy coat using the blood genomic Prep Mini Spin Kit (GE Healthcare) for SAG and DF samples, and GFXTM Genomic Blood DNA Purification Kit (Amersham Pharmacia Biotech) for STI samples, both following the manufacturer’s instructions. Ethical aspects Sample collection and analyses were approved by the ethics committees of the institutions where each project was initiated (STI: CEP/UnB protocol number 021.0.000.012-04; SAG: CEP/NUSAU 018; DF: CEP/UnB — Universidade de Brasilia —oˆ protocol number 25000.102003/2001-05/CONEP – 2932). All donors signed an informed consent form prior to sample collection, as per the official Brazilian guidelines for ethics in research. Genetic markers Ancestry informative markers (AIMs) are molecular markers that show a great allelic frequency differential (d) among populations (Shriver et al., 1997). While the mean d found for biallelic loci between the main ethnic groups is approximately 15%–20% (Dean et al., 1994), AIMs are defined by a high d, often assumed at 50% (Shriver et al., 1997). Because the d value directly influences the precision of admixture estimates, AIMs are ideal markers for this type of analysis. We tested a set of autosomal 15 markers that are commonly used for ancestry inference and for which we had information available for all parental populations. These were: TPA25 and PV92 (Novick et al., 1995); APO (Batzer et al., 1996); ECA (Nakai et al., 1994); FXIIIB (Kass et al., 1994); D1 (Batzer et al., 1995); Sb19.3 and GC (Parra et al., 1998); AT3-I/D (Liu et al., 1995); LPL (Gotoda et al., 1992); OCA2 (Lee et al., 1995); RB2300 (Bookstein et al., 1990); DRD2A, DRD2B, and DRD2C (Gelernter et al., 1998). The methods for amplification and subsequent analysis can be found in the cited literature. Statistics We analyzed our data within and between populations using standard methods in population genetics. In addition to studying the genetic variability between the STI, SAG, and DF populations, we compared our results to those of other populations previously described in the literature, including the main parental groups to the Brazilian population (Africans, Europeans, and Amerindians), as well as other quilombos—Kalunga (KAL), Rio das R~ as (RRS), Riacho de Sacutiaba (SAC), and Mocambo (MOC). Those populations, similar to STI and SAG, are also Afroderived and their demographics, history, and genetics have been studied in detail (Amorim et al., 2011a, 2012). Hardy–Weinberg equilibrium (HWE), heterozygote excess and deficiency, and linkage disequilibrium between the pairs of loci (DL) were tested using exact tests in GENEPOP 3.4 (Raymond and Rousset, 1995). HWE was tested using the Guo and Thompson (1992) exact test, while the other two parameters were tested using Fisher’s exact test. SAG and STI were compared with each other and to DF by Fisher’s exact test for genic and genotypic differentiation (Goudet et al., 1996) using the same software. For all of analyses, we assumed that a 5 0.05. F staAmerican Journal of Human Biology

TABLE 1. Allele frequency of 13 AIMs in African, Amerindian, and

European parental stocks Parental Allele or haplotype APO*1 D1*1 ECA*1 FXIIIB*1 PV-92*1 SB19.3*1 TPA-25*1 RB2300/BamHI*1 GC*1S GC*1F DRD2/TaqI (111) DRD2/TaqI (121) DRD2/TaqI (112) DRD2/TaqI (122) DRD2/TaqI (211) DRD2/TaqI (221) DRD2/TaqI (212) OCA2*1 LPL*1 AT3*1

Africa a

0.500 0.000b 0.273a 0.083a 0.225f 0.410d 0.409a 0.920c 0.069d 0.841d 0.500i 0.064i 0.023i 0.005i 0.194i 0.077i 0.135i 0.122c,j 0.970c,f 0.850c,j

Europe b

0.989 0.455a 0.469a 0.420a 0.152f 0.910d 0.557b 0.3333 0.607c 0.156c 0.183i 0.618i 0.011i 0.000i 0.013i 0.015i 0.153i 0.769c,j 0.489c,f 0.2633,g,j

Americas 1.000a 0.553e 0.700a 1.000i 0.875a 0.649h 0.675a 0.186h 0.542g 0.339g 0.343i 0.034i 0.047i 0.005i 0.018i 0.032i 0.511i 0.488g 0.492c,h 0.051g,h

For the markers OCA2, LPL and AT3, the values shown are a weighted mean of the frequencies found in the literature. References: aBatzer et al., 1994; bBatzer et al., 1996; cParra et al., 1998; dParra et al., 2001; eBattilana et al., 2002; fShriver et al., 2003; gBonilla et al., 2004; h Luizon et al., 2008; iRajeevan et al., 2005; jTom as et al. 2002.

tistics were calculated by the software Genetic Data Analysis (GDA) (Lewis and Zaykin, 1997), according to Weir and Cockerham (1984). Confidence intervals of 95% and 99% were obtained by bootstrapping with 10,000 replications. The three DRD2 polymorphisms investigated here were analyzed as one haplotype. For resolving phases, we chose the EM-zipper algorithm implemented with Arlequin 3.5 (Excoffier and Lischer, 2010) with default settings. The Allele Frequency Database (ALFRED Rajeevan et al., 2005) nomenclature was used, which consists of a plus signal (1) for the presence of a TaqI restriction site and a minus signal (2) for its absence, for the three polymorphic sites A, D, and B in that order. Genetic admixture was estimated by the gene-identity method described by Chakraborty (1985) using the ADMIX3 software. A trihybrid model consistent with Brazilian history was assumed, using Amerindians (Brazilians and Peruvian Quechuas), Europeans (Iberians and Italians), and several Sub-Saharan Africans (mainly Bantu and Yoruba) as parental groups. The parental frequencies are listed in Table 1. The results presented were obtained with the weighted approach. Allelic and haplotypic frequencies were used to generate two Nei’s geneticdistance matrices using different sets of populations and markers as follows: one set took into account the data available for SAG, STI, and DF with a subset of 10 markers (GC, APO, AT3, D1, LPL, OCA2, PV92, RB2300, Sb19.3, and TPA25), while the other considered the largest available number of loci (TPA25, PV92, APO, D1, AT3, LPL, and OCA2) for the populations included in the first set and the quilombos KAL, RRS, SAC, and MOC. The matrices were obtained using the GENDIST software from the PHYLIP package (Felsenstein, 2004) and were used to generate principal coordinate graphics by the NTSYS software (Rohlf, 2001). PCO graphics were used to

145

QUILOMBOS: A REPOSITORY OF AMERINDIAN ALLELES

TABLE 2. AIM data—sample size (n), observed allelic frequencies, haplotypic frequencies (DRD2Taq), and expected (He) and observed (Ho) het-

erozygosities for Santo Ant^ onio do Guapore, Santiago do Iguape, and Distrito Federal populations Santo Ant^ onio do Guapor e

Santiago do Iguape

Allele

Distrito Federal

Allele

Allele

Marker

n

1

2

n

1

2

n

1

2

TPA25 APO PV92 ECA FXIIIB D1 Sb19.3 AT3 LPL RB2300 OCA2 GC DRD2A DRD2B DRD2D DRD2Taq*(111) DRD2Taq*(1–1) DRD2Taq*(11–) DRD2Taq*(1– –) DRD2Taq*(–11) DRD2Taq*(– –1) DRD2Taq*(–1–) DRD2Taq*(– – –) mean n He

31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31 31

0.371 0.887 0.500 0.500 0.468 0.387 0.500 0.500 0.613 0.468 0.468 0.226 0.403 0.242 0.210 0.000 0.098 0.206 0.099 0.011 0.101 0.025 0.460

0.629 0.113 0.500 0.500 0.532 0.613 0.500 0.500 0.387 0.532 0.532 0.516 0.597 0.758 0.790 – – – – – – – –

37 34 37 – – 36 37 33 33 36 31 37 36 36 33 36 36 36 36 36 36 36 36 35.43

0.378 0.765 0.568 – – 0.236 0.581 0.742 0.803 0.514 0.210 0.270 0.319 0.167 0.303 0.020 0.074 0.100 0.108 0.016 0.193 0.000 0.488

0.622 0.235 0.432 – – 0.764 0.419 0.258 0.197 0.486 0.790 0.635 0.681 0.833 0.697 – – – – – – – –

168 168 167 169 166 166 167 169 159 167 164 161 167 – – – – – – – – – – 166

0.512 0.905 0.267 0.444 0.398 0.380 0.787 0.364 0.553 0.509 0.662 0.478 0.392 – – – – – – – – – –

0.488 0.095 0.733 0.556 0.602 0.620 0.213 0.636 0.447 0.491 0.338 0.308 0.608 – – – – – – – – – –

0.417

0.443

0.391

0.439

0.468 Ho 0.469

TABLE 3. Admixture estimates (percentage) and standard error (SE) for two Brazilian quilombo communities—Santo Ant^ onio do Guapore and

Santiago do Iguape—and Distrito Federal Santo Ant^ onio do Guapor e Parental population African European Amerindian R-square

Santiago do Iguape

Distrito Federal

%

SE

%

SE

%

SE

37.60 20.40 42.00

0.024 0.071 0.076

56.80 02.50 40.70

0.003 0.009 0.010

22.9 61.4 15.7

0.008 0.019 0.018

0.964

make it simpler to interpret the genetic distances observed among all populations analyzed, and among those populations and their putative parental groups. PCO derives from Principal Component Analysis (PCA). Both methods simplify the analysis of multivariate data (Cavalli-Sforza et al., 1996), and hence are more straightforward means to present genetic distance data. RESULTS All analyzed markers were polymorphic in the three populations. Table 2 shows observed allelic frequencies (and haplotypic frequencies for the DRD2 polymorphisms), sample size, and observed and expected heterozygosity. In SAG, all markers behaved as expected by the HWE model, and showed no excess or deficiency of heterozygotes. In STI, three loci presented deviations from the HWE model (P < 0.05), namely TPA25, PV92, and GC. The tests also indicated a deficiency of heterozygotes for the first two markers and an excess of the latter. Seven out of eight possible DRD2 haplotypes were observed in

0.999

0.997

SAG and STI, although DRD2*(111) was absent in SAG and DRD2*(–1–) in STI. In both populations, the modal haplotype was DRD2T*(– – –). In DF, two loci (TPA25 and APO) presented deviations from the HWE. Paired genic and genotypic comparisons showed significant differences between the SAG/STI, SAG/DF, and STI/ DF pairs (P < 1023). Global and pair-wise Fst(SAG/STI: 0.031; SAG/DF: 0.04; STI/DF: 0.128) were statistically significant. In spite of the small sample and population sizes, FIS estimates were not significant in either STI or SAG. These results reveal that, aside from being different from each other, the genetic profiles of the SAG and STI are significantly different from the DF urban population to which they were compared. Seven pairs of loci presented linkage disequilibrium (LD) in SAG: FXIIIB/AT3 (P 5 0.001), FXIIIB/OCA2 (P 5 0.005), D1/GC (P 5 0.048), Sb19.3/AT3 (P 5 0.025), DRD2/Sb19.3 (P 5 0.011), DRD2/APO (P 5 0.013), and DRD2/LPL (P 5 0.009). In STI, only two pairs were found to be in LD: AT3/TPA25 (P 5 0.043) and AT3/RB2300 (P 5 0.038). In DF, six pairs were found to be in LD: AT3/LPL (P 5 0.0047), American Journal of Human Biology

146

C.C. GONTIJO ET AL.

Fig. 1. Principal coordinate analysis generated from a Nei’s genetic distance matrix including Santo Ant^ onio do Guapor e, Santiago do Iguape, Distrito Federal, and (a) the three parental groups (Amerindians, Africans, and Europeans), and (b) the three parental groups and the quilombos Kalunga, Rio das R~ as, Riacho de Sacutiaba, and Mocambo.

AT3/PV92 (P 5 0.0019), D1/GC (P 5 0.0238), OCA2/GC (P 5 0.0159), PV92/D1 (P 5 0.0415), and Rb2300/LPL (P 5 0.0103). Weighted admixture estimates (Table 3) reveal that both Quilombo populations contain substantial genetic representation from both Amerindian and African lineages, with the European contribution being much smaller. In contrast, the genetic profiles of the DF revealed substantial genetic contributions of European origin followed by African origin, with the smallest contribution coming from Amerindians. The PCO graphs obtained from the genetic distance matrices are shown in Figures 1a and 1b. As shown in Figure 1a, SAG and STI are in an intermediate position between Amerindians and Africans, and relatively further away from Europeans. DF, on the other hand, is closer to the latter. In Figure 1b, all hybrid populations are located between the three parental groups, which were set apart by the first component. The second component set STI, SAG, and MOC between Africans and Amerindians and all other populations between Africans and Europeans. To have a better picture of the African descendants in Brazil, other quilombos (listed in Fig. 1a, b) were included in this analysis: they were used, as does DF, as comparison groups that allow us to understand SAG and STI in relation to other populations. DISCUSSION As a result of distinct African migration patterns throughout Brazil’s history of slavery, together with varying degrees of mixing with indigenous Amerindian populations, quilombos became important repositories of both Amerindian and African genetic ancestry. In the present study, we found differences in genetic composition between the SAG, STI, and DF. Both the SAG and STI populations carried significantly higher proportions of alleles that were of Amerindian and African origin than did the urban DF, which carried alleles largely European in origin. However, we observed distinct genetic ancestry American Journal of Human Biology

patterns even between the two quilombo populations we studied. Among the SAG, a quilombo settled in an area also co-occupied by Amerindians, we found that the parental admixture estimate was higher for the Amerindian than for the African component, unlike what we expected given the examples from other quilombos found in the literature (Bortolini et al., 1999; Luizon et al., 2008; Scliar et al., 2009; Amorim et al., 2011a), and yet consistent with its history and geographic location. On the other hand, we found that the admixture estimate in STI shows a higher African contribution than an Amerindian one. The smaller European presence—and distinct proportions of Amerindian ancestry—in these groups might reflect geographic isolation and other cultural and evolutionary factors, but nevertheless uncover important genetic patterns that are likely to reflect their admixture histories. Even though an Amerindian genetic component has been described repeatedly in quilombos, mainly due to the relationship between these groups and native tribes (Bortolini et al., 1999; Luizon et al., 2008; Scliar et al., 2009; Amorim et al., 2011a, 2012), we found that Amerindian contributions observed in SAG and STI are much higher than those described in other Brazilian quilombos previously studied. Specifically, we estimated Amerindian contributions for SAG (42%) and STI (40.7%) significantly higher than the highest Amerindian contributions previously described (20%) (Amorim et al., 2011a, b; Palha et al., 2011). Such admixture pattern, uncommon in quilombos, reflect their heterogenic history and makes them likely repositories for Amerindian genetic variability, as stated before. A point worthy of note is the relatively low R-square and high standard error (SE) obtained for SAG. Those values indicate the need for the inclusion of more AIMs in order to clarify the Amerindian and African contributions which, considering the SE, overlap. Compared to urban Brazilian populations, quilombos usually carry a higher proportion of African genes (Godinho et al., 2008; Lins et al., 2010; Pena et al., 2011). Our data indicate a high European contribution to DF’s gene pool, followed by the African and Amerindian

QUILOMBOS: A REPOSITORY OF AMERINDIAN ALLELES

contributions, in that order. Likewise, autosomal Short Tandem Repeats (STRs) show a European contribution of at least 60% for each of the five Brazilian regions considering urban populations (Godinho et al., 2008), with variable contributions by Africans and Amerindians. The analysis of indels in northern (Santos et al., 2010; Francez et al., 2012) and southern Brazilian populations (Santos et al., 2010) show similar results—a high European contribution with variable contributions by Africans and Amerindians, although this set of markers detected a higher Amerindian contribution. Uniparental markers indicate a higher European contribution in all Brazilian regions in male lineages, followed by Africans and, in a considerably smaller proportion, Amerindians (CarvalhoSilva et al., 2001; Grattapaglia et al., 2005). In contrast, the female contribution of each parental group was quite balanced (Alves-Silva et al., 2000). These data, along with the data on the quilombos, help to portray the ancestry of the current Brazilian population, where Europeans were the main contributors, followed by Africans and Amerindians. When an admixed population is formed, the frequency of any given locus is expected to reach an intermediate level relative to what is observed in the parental populations and proportional to the contribution of each parental population. This can be directly visualized by studying AIMs, given their ability to detect allelic frequency differentials (d) among populations (Shriver et al., 1997). Through AIM analysis we were able to visualize allelic frequency patterns for most of the analyzed markers, with the exception of some DRD2 haplotypes in SAG and STI, and the GC*1F allele in STI. Such discrepancies might reflect population structuring and genetic drift, and in the case of markers assumed to be neutral, selection is unlikely. Furthermore, in small human groups, there is a tendency toward homozygosity due to drift and endogamy, what could be the cause for the departure from HWE observed for TPA25 and PV92 in STI. However, no excess homozygosity was observed in SAG, despite its small size. Possible explanations are recent migration into SAG and drift intense enough to counter balance the effects of endogamy. Despite shared aspects of their history of origin, SAG and STI presented differences in allelic and genotypic frequencies. The great geographic distance between SAG and STI, along with the different demographic dynamics that they were subjected to, including gene flow to and from neighboring populations, are plausible explanations for these differences. The founder African populations that contributed to the formation of these quilombos could also play a role, given that these quilombos were founded at different times and were made up of people captured and enslaved in different regions of Africa (Schwartz, 1988; Mello e Souza, 2006). Studies with other genetic markers showed that about 70% of the African slaves brought to Brazil were from Angola, Congo, and Mozambique (Zago et al., 1992; Figueiredo et al., 1994). However, the population from Northeastern Brazilian states, where STI is located, present an African component of heterogeneous origin (Silva et al., 2010). LD can result from physical proximity between genes in the genome or from evolutionary factors such as drift, natural selection, and gene flow. LD can therefore indicate past demographic events such as expansions and retractions in population effective size, the existence of popula-

147

tion structure, and admixture (Pfaff et al., 2001; Slatkin, 2008; Amorim et al., 2011b). For example, reductions in effective population size could cause decreased recombination and consequent LD. Furthermore, isolation is another aspect to be considered, as many examples of low genetic diversity and high LD have been described (Laan €bo, 1997; Varilo et al., 2000; Katoh et al., 2002; and P€ aa Amorim et al., 2011b). In this scenario, one would expect to find LD in small and isolated populations such as STI and especially SAG, which is smaller and more isolated. Nevertheless, our results indicate that LD is more important in STI, suggesting that other factors might be influencing the likelihood of LD in these groups. Gene flow might also explain the deviations in the LD observed, such as through admixture among populations with different allelic frequencies, a phenomenon known as ALD - admixture linkage disequilibrium (Hartl and Clark, 2007; Pfaff et al., 2001). Pfaff et al. (2001) analyzed two ALD models proposed by Long (1991): (1) a hybridisolation model, which describes a population that is formed by admixture in early generations but is soon isolated from the surrounding populations; and (2) a continuous gene flow model, which describes a population formed by admixture that, unlike the first model, continues to receive gene flow. The authors tested the behavior of LD along generations and the rates at which it diminishes; in both scenarios, LD tends to disappear even between physically close loci, but at different rates (i.e., faster in hybrid-isolation). Our findings suggest that both models are plausible explanations for LD in the quilombo groups we studied, and in surprising ways. For example, even though SAG’s history and location suggest that its demographic history could be better described by hybrid-isolation, our data point toward LD by continuous gene flow. According to the hybrid-isolation model, LD is supposed to have vanished within five generations. However, we still observed LD after this generational time point in later SAG populations. Furthermore, the high Amerindian contribution and the low, but still significant, European contribution suggest the existence of gene flow among SAG and neighboring populations. In contrast, a hybrid-isolation model may better explain the LD observed among the STI, even though their demographic history and location would suggest continuous gene flow. LD was found for only two pairs of markers and the overall European contribution is estimated to be low, indicating the absence of gene flow from surrounding populations. It is noteworthy that geographic isolation alone does not account for whether gene flow is present or for its intensity, and our case other factors, cultural and economic, for instance, must be acting. Like the pairwise genic and genotypic differentiation tests and the comparisons of the admixture estimates, F statistics implied important differences among these populations. Such differences were expected, given the geographic distance that separates SAG, STI, and DF, and also because of their histories, regarding where the main contributors came from and other factors. The SAG arose within the Amazon Forest, far from any urban center, in a very isolated and inaccessible area. In contrast, the STI derive from an easily accessible area in Bahia, a more densely populated state. The accessibility to other populations allows for the existence and maintenance of migration, but there seems to have been another factor influencing gene flow in the studied populations, as our American Journal of Human Biology

148

C.C. GONTIJO ET AL.

data suggests a constant gene flow into SAG, but not into STI. Overall, the groupings observed in the PCOs reflect the admixture estimates and the allelic frequencies described herein quite well. In Figure 1a, SAG and STI show a trend of grouping together and closer to Africans and Amerindians, while DF is more similar to Europeans. The first components positioned SAG, STI, and DF between Africans and the Amerindian–European group (generated by the d between the loci analyzed). The second components clearly set these two groups apart, positioning SAG and STI between Amerindians and Africans and DF between Africans and Europeans. The relative positions of the populations on both graphs reflect the admixture patterns and the allelic frequencies quite well. On the graphs comparing SAG, STI, other hybrid populations (KAL, RAS, SAC, MOC, and DF), and the parental groups (Fig. 1b), all admixed populations were set between the parental groups, as expected. SAG and STI showed a trend toward grouping together and between Africans and Amerindians, while DF was always closer to Europeans. SAG, STI, and MOC were between Amerindians and Africans, KAL, RAS, and SAC were between Africans and Europeans, and DF was closer to Europeans. These positioning reflect quite well the admixture estimates described for all populations included in our analysis. Our results point out an Amerindian contribution in SAG and STI higher than those described for other quilombos, which might make them and groups alike repositories of Amerindian variability that could otherwise be lost or not easily assessable. Furthermore, they indicate genetic differences consistent with the geographic distance among SAG, STI and DF, and with the differences in the settlement history of the regions where they are located. An interesting point derived from LD analyses is the important role played by factors such as culture and economics to the intensity of gene flow. CONCLUSIONS The study presented herein compared two Brazilian populations of marked African ancestry to one another and to other urban and Afro-derived populations. Although the DF, SAG, and STI are all trihybrid populations comprised of genes of Amerindian, African, and European ancestry, our analyses reveal striking differences in the proportion of Amerindian and African gene information carried by SAG and STI. We clearly observed that, even after generations under gene flow, SAG and STI, as most quilombos, maintain African and Amerindian contributions above the means observed in urban areas of the country. In fact, the Amerindian contributions observed in these two populations are much higher than those described for other quilombos in the literature, maybe reflecting the relationship they might have had, or still maintain, with surrounding native populations. Furthermore, the contact with the European colonizers and enslaved Africans led to a drastic reduction of the native population in the Americas. Such reduction was caused by (1) extinction of whole ethnic groups and tribes, (2) reduction in population size, and (3) incorporation into the growing admixed population. Extinction might have led to a loss of genetic variability. Reduction in population size might have caused a bottleneck and subsequent loss American Journal of Human Biology

of genetic variability, as well as an increase in the differentiation between groups. The incorporation into the admixed population might have led to the incorporation of alleles specific to native groups into that gene pool. In this case, it is reasonable to assume that admixed populations, especially those with such a high proportion of contribution from Amerindians, might represent a repository of Amerindian alleles that could have been lost or are, as a consequence of bottlenecks and drift, misrepresented in contemporary native populations. ACKNOWLEDGMENTS We would like to thank the people from Santo Ant^onio do Guapore and Santiago do Iguape for their collaboration participating in this project. We also thank the Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq) and the Coordenac¸~ ao para Aperfeic¸oamento de Pessoal de Nıvel Superior, Brazil (CAPES) for financial support. A special thanks to Anna Lisa Lucido, Scientific Writer form Jackson Laboratory for Genomic Medicine, for the manuscript review, and Daya Sisson for her comments on bioethics and its history in Brazil. All authors disclose any affiliations that have a direct interest, particularly a financial interest, in the subject matter or materials discussed here. LITERATURE CITED Alencastro LF. 2000. O trato dos viventes: a formac¸~ ao do Brasil no Atl^ antico Sul. 1ed. S~ ao Paulo: Companhia das Letras. Alves-Silva J, Santos MS, Guimara~es PEM, Ferreira ACS, Bandelt HJ, Pena SJD, Prado VF. 2000. The ancestry of Brazilian mtDNA lineages. Am J Hum Genet 67:444–461. Amorim CEG, Gontijo CC, Falc~ ao-Alencar G, Godinho NMO, Toledo RCP, Pedrosa MAF, Luizon MR, Sim~ oes AL, Klautau-Guimar~ aes MN, Oliveira SF. 2011a. Migration in Afro-Brazilian rural communities: crossing demographic and genetic data. Hum Biol 83:509–521. Amorim CEG, Wang S, Marrero AR, Salzano FM, Ruiz-Linares A, Bortolini MC. 2011b. X-Chromosomal genetic diversity and linkage disequilibrium patterns in Amerindians and Non-Amerindian populations. Am J Hum Biol 23:299–304. Amorim CEG, Gontijo CC, Oliveira SF. 2012. Migration in Afro-Brazilian rural communities: crossing historical, demographic, and genetic data. In: Crawford MH, Campbell B, editors. Causes and consequences of human migration. Cambridge: Cambridge University Press. Assis AML, Azevedo DA, Souza GRB, Santos-Filho MVC, Silva LAF. 2011. Hierarchical analysis of 15 Y-chromosome SNPs and demographic history of Afro-derived isolated communities in Alagoas Brazil. Forensic Sci Int Genet 3:e172–e173. Barbosa AAL, Sousa SMB, Ab e-Sandes K, Alonso CA, Schneider V, Costa DCC, Cavalli IJ, Azev^ edo EES. 2006. Microsatellite studies on an isolated population of African descent in the Brazilian state of Bahia. Genet Mol Biol 29:23–30. Barickman BJ. 1999. At e A V espera: O Trabalho Escravo e a Produc¸~ ao de  Ac¸ ucar nos Engenhos do Rec^ oncavo Baiano (1850–1881). Afro-Asia 2122:177–238. Barickman BJ. 2003. E se a casa n~ ao fosse t~ ao grande? Uma freguesia  ac¸ucareira no Rec^ oncavo Baiano em 1835. Afro-Asia 20-31:79–132. Battilana J, Bonatto SL, Freitas LB, Hutz MH, Weimer TA, CallegariJacques SM, Batzer MA, Hill K, Hurtado AM, Tsuneto LT, Petzl-Erler ML, Salzano FM. 2002. Alu insertions versus blood group plus protein genetic variability in four Amerindian populations. Ann Hum Biol 29: 334–347. Batzer MA, Stoneking M, Alegria-Hartman M, Bazan H, Kass DH, Shaikh TH, Novick GE, Ioannou PA, Scheer WD, Herrera RJ, Deininger PL. 1994. African origin of human-specific polymorphic Alu insertions. Proc. Natl. Acad. Sci 91:12288–12292. Batzer MA, Rubin CM, Hellmann-Blumberg U, Alegria-Hartman M, Leeflang EP, Stern JD, Bazan HA, Shaikh TH, Deininger PL, Schmid CW. 1995. Dispersion and insertion polymorphism in two small subfamilies of recently amplified human Alu repeats. J Mol Biol 247: 418–427. Batzer MA, Arcot SS, Phinney JW, Alegri-Hartman M, Kass DH, Milligan SM, Kimpton C, Gill P, Hochmeister M, Ioannou PA, Herrera RJ,

QUILOMBOS: A REPOSITORY OF AMERINDIAN ALLELES Boudreau DA, Scheer WD, Keats BJB, Deininger PL, Stoneking M. 1996. Genetic variation of recent Alu insertions in human populations. J Mol Evol 42:22–29. Bonilla C, Parra EJ, Pfaff CL, Dios S, Marshall JA, Hamman RF, Ferrell RE, Hoggart CL, McKeigue PM, Shriver MD. 2004. Admixture in the Hispanics of the San Luis Valley, Colorado, and its implications for complex trait gene mapping 68:139–153. Bookstein R, Lai CC, To H, Lee WH. 1990. PCR-based detection of a polymorphic BamHI site in intron 1 of the human retinoblastoma (RB) gene. Nucleic Acids Res 18:1666. Bortolini MC, Silva-Jr WA, Guerra DC, Remonatto G, Mirandola R, Hutz MH, Weimer TA, Silva MCBO, Zago MA, Salzano FM. 1999. Africanderived South American Populations: a history of symmetrical and asymmetrical matings according to sex revealed by bi- and uni-parental genetic markers. Am J Hum Biol 11:551–563. Callegari-Jacques SM, Tarazona-Santos EM, Gilman RH, Herrera P, Cabrera L, Santos SEB, Mor es L, Hutz MH, Salzano FM. 2011. Autosome STRs in native South America – Testing models of association with geography and language. Am J Phys Anthropol 145:371–381. Carvalho BM, Bortolini MC, Santos SEB, Ribeiro-dos-Santos AKC. 2008. Mitochondrial DNA mapping of social-biological interactions in Brazilian Amazonian African-descendant populations. Genet Mol Biol 31(1): 12–22. Carvalho-Silva DR, Santos FR, Rocha J, Pena SDJ. 2001. The phylogeography of Brazilian Y-chromosome lineages. Am J Hum Genet 68: 281–286. Cavalli-Sforza LL, Menozzi P, Piazza A. 1994. History and geography of human genes. Princeton: Princeton University Press. Chakraborty R. 1985. Gene identity in racial hybrids and estimation of admixture rates. In: Ahuja YR, Neel JV, editors. Genetic differentiation in human and others animal populations. New Delhi: Indian Anthropological Association. p 171–180. Cunha MC. 2013. Indios no Brasil — Hist oria, direitos e cidadania. S~ ao Paulo: Companhia das Letras. Dean M, Stephens JC, Winkler C, Lomb DA, Ramsburg M, Boaze R, Stewart C, Charbonneau L, Goldman D, Albaugh BJ, Goedert JJ, Beasley RP, Hwang L, Buchbinder S, Weedon M, Johnson PA, Eichelberger M, O’Brien SJ. 1994. Polymorphic admixture typing in human ethnic populations. Am J Hum Genet 55:788–808. Excoffier L, Lischer HEL. 2010. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10:564–567. Felsenstein J. 2004. PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Washington: Department of Genome Sciences University of Washington Seattle. Figueiredo MS, Olympio Silva MCB, Guerreiro JF, Pante-Souza G, Pires ACR, Zago MA. 1994. The heterogeneity of the bS cluster haplotypes in Brazil. Gene Geogr 8:7–12. Francez PAC, Ribeiro-Rodrigues EM, Santos SEB. 2012. Allelic frequencies and statistical data obtained from 48 AIM INDEL loci in an admixed population from the Brazilian Amazon. Forensic Sci Int Genet 6:132–135. Gelernter J, Kranzler H, Cubells JF, Ichinose H, Nagatsu T. 1998. DRD2 allele frequencies and linkage disequilibria including the 2141C/Del promoter polymorphism in European-American and Japanese subjects. Genomics 51:21–26. Giolo SR, Soler JMP, Greenway SC, Almeida MAA, Andrade M, Seidman JG, Krieger JE, Pereira AC. 2012. Brazilian urban population genetic structure reveals a high degree of admixture. Eur J Hum Genet 20:111– 116. Godinho NMO, Gontijo CC, Diniz MECG, Falc~ ao-Alencar G, Dalton GC, Amorim CEG, Barcelos RSS, Klautau-Guimar~ aes MN, Oliveira SF. 2008. Regional patterns of genetic admixture in South America. Forensic Sci Int Genet 1:329–330. Gotoda T, Yamada N, Murase T, Shimano H, Shimada M, Harada K, Kawamura M, Kozaki K, Yazaki Y. 1992. Detection of three separate DNA polymorphisms in the human lipoprotein lipase gene by gene amplification and restriction endonuclease digestion. J Lipid Res 33: 1006–1072. Goudet J, Raymond M, Mee€ us T, Rousset F. 1996. Testing differentiation in diploid populations. Genetics 144:1933–1940. Grattapaglia D, Kalupniek S, Guimar~ aes CS, Ribeiro MA, Diener PS, Soares CN. 2005. Y-chromosome STR haplotype diversity in Brazilian populations. Forensic Sci Int 149:99–107. Guo SW, Thompson EA. 1992. Performing the exact test of HardyWeinberg proportion for multiple alleles. Biometrics 48:361. Hartl DL, Clark AG. 2007. Principles of population genetics. Sunderland: Sinauer Associates Inc. Publishers. Hurtado AM, Salzano FM. 2004. Conclusions. In: Hurtado AM, Salzano FM, editors. Lost paradises and the ethics of research and publication. New York: Oxford University Press. p 211–228.

149

Karasch M. 1996. Os quilombos de ouro na capitania de Goi as. In: Reis JJ, Gomes FS, editors. Liberdade Por Um Fio. S~ ao Paulo SP Brazil: Companhia das Letras. p 240–262. Kass DH, Aleman C, Batzer MA, Deininger PL. 1994. Identification of a human specific Alu insertion in the factor XIIIB gene. Genetica 94:1–8. Katoh T, Mano S, Ikuta T, Munkhbat B, Tounai K, Ando H, Munkhtuvshin N, Imanishi T, Inoko H, Tamiya G. 2002. Genetic isolates in East Asia: a study of linkage disequilibrium in the X chromosome. Am J Hum Genet 71:395–400. €bo S. 1997. Demographic history and linkage disequilibrium Laan M, P€ aa in human populations. Nat Genet 17:435–438. Lee ST, Nicholls RD, Jong MTC, Fukai K, Spritz RA. 1995. Organization and sequence of the human P gene and identification of transport proteins. Genomics 26:354–363. Lewis PO, Zaykin D. 1997. Genetic data analysis: software for the analysis of discrete genetic data. Version 1.0. Sunderland: Sinauer Associates, Inc. Lins TC, Vieira RG, Abreu BS, Grattapaglia D, Pereira RW. 2010. Genetic composition of Brazilian population samples based on a set of twenty eight ancestry informative SNPs. Am J Hum Biol 22:187–192. Liu Y, Saha N, Low PS, Tay JS. 1995. Linkage disequilibrium between two loci (5’ untranslated exon 1 and intron 5-DdeI) of the antithrombin III gene in three ethnic groups in Singapore. Hum Hered 45:192–198. Long JC. 1991. The genetic structure of admixed populations. Genetics 127:417–428. Luizon MR, Mendes-Junior, CT, Oliveira SF, Sim~ oes AL. 2008. Ancestry informative markers in Amerindians from Brazilian Amazon. Am J Hum Biol 20:86–90. Mello e Souza M. 2006. Reis Negros no Brasil escravista: hist oria da festa de Coroac¸~ ao de Rei Congo, 1 edn. Belo Horizonte: Editora UFMG. Nakai KMD, Itoh CMD, Miura Y, Hotta K, Musha T, Itoh T, Miyakawa T, Iwasaki R, Hiramori MD. 1994. Deletion polymorphism of the angiotensin I-converging enzyme is associated with serum ACE concentration and increase risk for CAD in the Japanese. Circulation 90:2199–2202. Neme S, Andrade CO. 1987. Quilombo: forma de resist^ encia. Proposta hist orico-arqueol ogica. In: Huber G, De Souza FB, editors. Insurreic¸~ ao Negra e Justic¸a. Rio de Janeiro: OAB. Nishida M. 1993. Manumission and ethnicity in urban slavery: Salvador, Brazil, 1808–1888. Hisp Am Hist Rev 73:361–391. Novick GE, Novick CC, Yunis J, Yunis E, Martinez K, Duncan GG, Troup GM, Deininger PL, Stoneking M, Batzer MA, Herrera RJ. 1995. Polymorphic human-specific Alu insertions as markers for human identification. Electrophoresis 16:1596–1601. Oliveira SF, Ribeiro GGBL, Ferreira LB, Klautau-Guimar~ aes MN, Sim~ oes AL. 2006. History reconstruction of afro-derived isolated Brazilian populations: the contrast among female and male genetic contribution. In: Sociedad Espa~ nola de Antropologıa Fısica. Diversidad Biol ogica y Salud Humana. M urcia: Quaderna Editorial Palha TJBF, Ribeiro-Rodrigues EM, Ribeiro-dos-Santos A, Guerreiro JF, Moura LSS, Santos S. 2011. Male ancestry structure and interethnic admixture in African-descent communities from the Amazon as revealed by Y-chromosome STRs. Am J Phy Anthropol 144:471–478. Parra FC, Marcini A, Akey J, Martinson J, Batzer MA, Cooper R, Forrester T, Allison DB, Deka R, Ferrell RE, Shriver MD. 1998. Estimating African American admixture proportions by use of populationspecific alleles. Am J Hum Genet 63:1839–1851. Parra EJ, Kittles RA, Argyropoulos G, Pfaff CL, Hiester K, Bonilla C, Sylvester N, Parrish-Gause D, Garvey WT, Jin L, McKeigue PM, Kamboh MI, Ferrell RE, Pollitzer WS, Shriver MD. 2001. Ancestral proportions and admixture dynamics in geographically defined African Americans living in South Carolina. American Journal of Physical Anthropology 114:18–29. Pena SDJ, Di Pietro G, Fuchschuber-Moraes M, Genro JP, Hutz MH, Kehdy FSG, Kohlrausch F, Magno LAV, Montenegro RC, Moraes MO, Moraes MEA, Moraes MR, Ojopi EB, Perini JA, Racciopo C, Ribeiro-dosSantos AKC, Rios-Santos F, Romano-Silva MA, Sortica VA, Suarez-Kurtz G. 2011. The genomic ancestry of individuals from different geographical regions of Brazil is more uniform than expected. PLoS One 6:2. Pfaff CL, Parra EJ, Bonilla C, Hiester K, Mckeigue PM, Kamboh MI, Hutchinson RG, Ferrell RE, Boerwinkle E, Shriver MD. 2001. Population structure in admixed populations: effect of admixture dynamics on the pattern of linkage disequilibrium. Am J Hum Genet 68:198–207. Rajeevan H, Cheung KH, Gadagkar R, Stein S, Soundararajan U, Kidd JR, Pakstis AJ, Miller PL, Kidd KK. 2005. ALFRED: an allele frequency database for microevolutionary studies. Evol Bioinform Online 1:1–10. ALFRED database http://alfred.med.yale.edu. Accessed 2013 December. Raymond M, Rousset F. 1995. Genepop Version 3.3: a population genetics software for exact tests and ecumenicism. J Hered 86:248–249. Reich D, Patterson N, Campbell D, Tandon A, Mazieres S, Ray N, Parra MV, Rojas W, Duque C, Mesa N, Velez ID, Garcıa LF, Triana O, Blair S, Maestre A, Dib JC, Bravi CM, Bailliet G, Corach D, H€ unemeier T,

American Journal of Human Biology

150

C.C. GONTIJO ET AL.

Bortolini MC, Salzano FM, Petzl-Erler ML, Acu~ na-Alonzo V, CanizalesQuinteros S, Aguilar-Salinas C, Tusi e-Luna T, Riba L, Rodrıguez-Cruz M, LopezAlarcon M, Coral-Vazquez R, Canto-Cetina T, Silva-Zolezzi I, Fernandez-Lopez JC, Contreras AV, Jimenez-Sanchez G, G omezV azquezMJ, Molina J, Carracedo A, Salas A, Gallo C, Poletti G, Witonsky DB, Alkorta-Aranburu G, Sukernik RI, Osipova L, Fedorova S, Vasquez R, Villena M, Moreau C, Hammer M, Barrantes R, Pauls D, Excoffier L, Bedoya G, Rothhammer F, Dugoujon JM, Larrouy G, Klitz W, Labuda D, Kidd J, Kidd K, Di Rienzo A, Freimer NB, Price AL, RuizLinares A. 2012. Reconstructing Native American population history. Nature 488:370–374. Reis JJ, Gomes FS. 1996. Introduc¸~ ao – Uma hist oria de liberdade. In: Reis JJ, Gomes FS, editors. Liberdade por um fio. S~ ao Paulo: Cia das Letras. p 9–25. Ribeiro-dos-Santos AK, Pereira JM, Lobato MRL, Carvalho BM, Guerreiro JF, Batista-dos-Santos SE. 2002. Dissimilarities in the process of formation of Curiau, a semi-isolated Afro-Brazilian population of the Amazon Region. Am J Hum Biol 14:440–447. Ribeiro D. 2006. O Povo Brasileiro: a formac¸~ ao e o sentido do Brasil. S~ ao Paulo: Companhia das Letras. Ribeiro GGBL, Abe-Sandes K, Barcelos RSS, Klautau-Guimar~ aes MN, Silva-J unior WA, Oliveira SF. 2011. Who were the male founders of rural Brazilian Afro-derived communities? A proposal based on three populations. Ann Hum Biol 38:237–240. Rocha Pombo. 1935. In: Teixeira, MAD. 2006. Quilombolas de Santo rea de Ant~onio do Guapore: conflitos nas relac¸~ oes socioambientais em a reserva ecologica. Porto Velho: Work-paper/UNIR. Rohlf J. 2001. Ntsys: numerical taxonomy and multivariate analysis system version 2.1. New York: Exeter Software Setauket. Rosenblat A. 1954. La Poblaci on Indıgena y el mestizaje en America). Buenos Aires: Editorial Nova. Santos SEB, Ribeiro-Rodrigues E, Fagundes N, Guerreiro JF. 1995. The indigenous contribution to the formation of the population of the Brazilian Amazon region. Genet Mol Biol 18:311–315. Santos NPC, Ribeiro-Rodrigues EM, Ribeiro-dos-Santos AKC, Pereira R, Gusm~ ao L, Amorim A, Guerreiro J, Zago M, Matte C, Hutz MH, Santos SEB. 2010. Assessing individual interethnic admixture and population substructure using a 48-insertion-deletion (INSEL) ancestryinformative marker (AIM) panel. Hum Mutat 31:184–190. Schwartz SB. 1988. Segredos Internos. Engenhos e escravos na sociedade colonial. S~ ao Paulo: Companhia das Letras.

American Journal of Human Biology

Scliar MO, Vaintraub MT, Vaintraub PMV, Fonseca CG. 2009. Admixture analysis with forensic microsatellites in Minas Gerais, Brazil: the ongoing evolution of the capital and of an African-derived community. Am J Phys Anthropol 139:591–595. Shriver MD, Smith MW, Jin L, Marcini A, Akey JM, Deka R, Ferrell RE. 1997. Ethnic-affiliation estimation by use of population-specific DNA markers. Am J Hum Genet 60:957–964. Silva WS, Klautau-Guimar~ aes MN, Grisolia CK. 2010. b-globin haplotypes in normal and hemoglobinopathic individuals from Reconcavo Baiano, State of Bahia, Brazil. Genet Mol Biol 33:411–417. Shriver MD, Parra EJ, Dios S, Bonilla C, Norton H, Jovel C, Pfaff C, Jones C, Massac A, Cameron N, Baron A, Jackson T, Argyropoulos G, Jin L, Hoggart CJ, McKeigue PM, Kittles RA. 2003. Skin pigmentation, biogeographical ancestry and admixture mapping. Hum Genet 112:387–399. Slatkin M. 2008. Linkage disequilibrium – understanding the evolutionary past and mapping the medical future. Nat Rev Genet 9:477–485. Steward JH. 1949. Handbook of South American Indians – The Comparative Ethnology of South American Indians. Washington: Smithsonian Institution.  cidade das ruınas grandeza e Teixeira MAD. 1997. Dos campos d’ouro a decad^ encia do colonialismo portugu^ es no Vale do Guapor e s eculos XVIII e XIX. In: Teixeira MAD, Fonseca DR, Moratto J. 2010. A presenc¸a negra em Rond^ onia: as estruturas do povoamento. Rev Eletr^ onica Afros e Amaz 2(1). Teixeira MAD, Fonseca DR, Moratto J. 2010. A presenc¸a negra em Rond^ onia: as estruturas do povoamento. Rev Eletr^ onica Afros e Amaz 2(1). Teixeira MAD. 2006. Quilombolas de Santo Antonio do Guapore: conflitos nas relac¸~ oes socioambientais em area de reserva ecologica. Porto Velho: Work-paper/UNIR. Varilo T, Laan M, Hovatta I, Wiebe V, Terwilliger JD, Peltonen L. 2000. Linkage disequilibrium in isolated populations: Finland and a young sub-population of Kuusamo. Eur J Hum Genet 8:604–612. Verger P. 1968. Flux et Reflux de la Traite des Ne`gres entre le Golfe de Benin et Baia de Todos os Santos. Paris: Mouton Press. p 720. Weir BS, Cockerham CC. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370. Tomas G, Seco L, Seixas S, Faustino P, Lavinha J. 2002. The Peopling of Sao Tome (Gulf of Guinea): Origins of Slave Settlers and Admixture with the Portuguese. Human Biology 74:397–411. Zago MA, Figueiredo MS, Ogo SH. 1992. Bantu bS cluster haplotype predominates among Brazilian blacks. Am J Phys Anthropol 88:295–298.