Abstract
There is a consensus that modern humans arrived in the Americas 15,000–20,000 y ago during the Late Pleistocene, most probably from northeast Asia through Beringia. However, there is still debate about the time of entry and number of migratory waves, including apparent inconsistencies between genetic and morphological data on Paleoamericans. Here we report the identification of mitochondrial sequences belonging to haplogroups characteristic of Polynesians in DNA extracted from ancient skulls of the now extinct Botocudo Indians from Brazil. The identification of these two Polynesian haplogroups was confirmed in independent replications in Brazil and Denmark, ensuring reliability of the data. Parallel analysis of 12 other Botocudo individuals yielded only the well-known Amerindian mtDNA haplogroup C1. Potential scenarios to try to help understand these results are presented and discussed. The findings of this study may be relevant for the understanding of the pre-Columbian and/or post-Columbian peopling of the Americas.
The origin of the first Americans has been a controversial topic since the beginning of the 19th century (1, 2). Three main hypotheses for the pre-Columbian peopling of America are presently being entertained. The first hypothesis, called “Two Components,” is based on morphological studies of crania from ancient and modern Native Americans. Two apparently polar morphologies have been identified: first, a generalized (or Paleoamerican) one with dolichocephalic features, and second, a specialized Mongoloid morphology with brachycephalic features (3). The foremost representatives of the Paleoamerican type in Brazil are the skeletons from Lagoa Santa, Brazil (4), and the specialized morphology is seen in the vast majority of extant Amerindians. Proponents of this model argue that two distinct populations entered the Americas by the end of the Pleistocene, and that the transition between the cranial morphology of the Paleoamericans and the morphology of later Native Americans, which occurred around 8,000–9,000 y before present, was abrupt because of total or partial population replacement (5⇓⇓⇓–9). Of interest is the observation that multivariate analyses of skull measurements of the Paleoamerican group have shown similarity to some African groups, Australians, Melanesians, and Easter Islanders (5).
In apparent contrast, molecular data, mostly obtained through the study of extant populations, have proposed a single migration wave into South America (Single Wave model), although North America may have received additional migrations on a minor scale from Beringia in the Holocene (10). Analyses of complete mitochondrial genomes have identified at least 15 founding lineages in the extant Amerindian gene pool, including the Pan-American mtDNA haplogroups A2, B2, C1b, C1c, C1d, C1d1, D1, and D4h3a, as well as some haplogroups restricted to northern North America (sub-Arctic and Arctic regions), A2a, A2b, D2a, D3, and X2a (11⇓–13), and others that have been exclusively found in populations located in the southernmost part of South America, including B2l, D1g, and C1b13 (14, 15). It has been proposed that most of these founding lineages were generated during a period of refuge of about 5,000 years in Beringia (13, 16). In contrast, genetic analyses of the nonrecombining portion of Y chromosome haplotypes have shown a significant founder effect (17). More than 80% of Amerindian Y chromosomes belong to the single haplogroup Q1a3a-M3, whose precursors could be traced back to central Siberia (18⇓–20). Although this genetic evidence is suggestive of a single founding population for all Native South Americans, we have to realize that these studies have been largely limited to extant Amerindian groups or to relatively recent skeletal remains. Thus, molecular studies have not been capable of testing appropriately the Two Components model yet.
Based on geometric morphometric studies, González-José et al. (21) have proposed that the Paleoamerican and Mongoloid cranial morphologies are not separate “types,” but simply extremes in a continuous gradient of variation. The authors postulate that the morphological pattern defining the Paleoamerican remains can be found in the range of extant Amerindian populations and that the previous separation of samples into discrete categories represented subjective assignments. González-José et al. presented a third model, called “Recurrent Gene Flow,” which is capable of integrating morphological and molecular data (21). Using 2D geometric morphometric data they propose that this model has better explanatory performance than either the Single Wave or the Two Components models.
In the context of the genetic reconstruction of the Native American history, the study of the Botocudo Indians from Brazil might provide new information. Also known as Aimores, these Amerindians inhabited adjacent areas in the present-day states of Minas Gerais, Espírito Santo and Bahia, in Southeastern Brazil (22). They were a large community, divided into many separate groups, some of them in conflict with each other. In common, they were Macro-Je speakers, had a nomadic hunter-gatherer lifestyle, and used typical ornaments in the lips and ears, called “botoques” by the Portuguese colonizers. In 1808 the Portuguese Crown declared “Just War” (Bellum iustum) against all Indian tribes that did not accept European laws (23). The fierce Botocudo were targeted in such wars and, in consequence, became virtually extinct by the end of the 19th century (24). Their importance for the history of the peopling of the Americas was revealed by studies reporting that the Botocudo had cranial features that consistently were described as intermediate between the polar Paleoamerican and Mongoloid morphologies (25, 26). Multivariate analyses of the cranial measures of different Amerindian and Paleoamerican groups from Brazil indeed concluded that the Botocudo Indians presented sufficient similarities with the Lagoa Santa Paleoamericans to be considered candidates to be their possible descendants (27).
The Museu Nacional/Universidade Federal do Rio de Janeiro (UFRJ) in Rio de Janeiro has a collection of Botocudo skulls dating to the 19th century and we extracted DNA and partially sequenced the mtDNA from teeth obtained from 14 of them. Usually, when human groups colonize the territory of another population there is introgression of the mtDNA of the autochthonous population into the “invading” one because of sexually asymmetric reproduction (28). Thus, we reasoned that if the Botocudo Indians were indeed a product of genetic admixture between the original Paleoamericans and a second wave of Amerindians with specialized cranial morphologies, the chance of finding any DNA evidence of such event would be greater using the matrilineal mtDNA.
We describe herein the discovery of unexpected mitochondrial lineages considered as typically Polynesian in teeth obtained from two Botocudo skulls. We shall first present our molecular results, which have been reproduced independently in two different laboratories. We then present possible scenarios to explain how this Polynesian-Botocudo contact might have occurred. The first scenario, prehistoric, is related to the possibility of genetic continuity between the Paleoamericans from Lagoa Santa and Botocudo Indians. We follow with an imaginable historic pre-Columbian scenario, involving opportunities for more recent direct contact between Polynesia and South America before the European arrival. We then present two possible modern post-Columbian scenarios, both related to the slave trade. One scenario is connected with the practice of the “blackbirding” trade, which occurred in 1860s and brought ∼2,000 Polynesians to Peru. The second scenario is the possible arrival of Polynesian haplogroups to Brazil in modern times through the African slave trade from Madagascar, where 20% of the mtDNA belong to the B4a1a1a haplogroup (29).
Results
We sequenced the control region (first and second hypervariable segments: HVSI-HVSII) and typed specific mutations of the coding region of mtDNA extracted from teeth of 14 different Botocudo skulls. As reported previously (30), 12 of these skulls clearly belonged to the Amerindian haplogroup C1 and will not be further discussed here. The extracts from the remaining two skulls, MN00015 and MN00017, yielded mitochondrial sequences belonging to haplogroup B, with unexpected ancestries. Their description and analysis constitute the core of the present article.
Phylogenetic Analysis of Polynesian Sequences Found in Brazil.
The skulls identified as MN00015 and MN00017 in the Museu Nacional/UFRJ in Rio de Janeiro were both from adult male Botocudo individuals from the Rio Doce valley in the state of Minas Gerais, Brazil, as registered by annotations written directly on the outer aspect of their respective parietal bones. According to information of the log book of the museum, it is most likely that these crania arrived there on August 25, 1890. The date of death is not known with certainty, but it is almost certainly the second half of the 19th century.
We first extracted DNA from an intact premolar tooth of skull MN00015. Sequencing of mtDNA identified the following mutations: 73G, 146C, 151T, 6719C, 12239T, 15746G, 16189C, 16217C, 16247G, 16261T. The 9-bp deletion between the COII and tRNA (Lys) genes characteristic of haplogroup B (31) was also present. This set of mutations classified the haplotype as B4a1a, found mainly in Taiwan, Island Southeast Asia and populations on the Pacific Islands (32, 33). Further analysis identified the mutation 14022G, which classified the sample in haplogroup B4a1a1 (32⇓–34). The presence of the mutation 16247G (HVSI) and 6905A (ancestral allele), further characterized the sequence as belonging to haplogroup B4a1a1a (32⇓–34). This haplogroup is found at high frequency in Polynesia, Micronesia, parts of Near Oceania, and Easter Island (33⇓⇓–36).
Our next step was the replication of the results through the analysis of a second tooth from the same MN00015 skull, using identical methodology. All mutations were confirmed. A third tooth from MN00015 was then sent to the Centre for GeoGenetics in Copenhagen for independent validation. The 9-bp deletion in the intergenic COII/tRNA (Lys) region was confirmed by PCR and blind analysis of the control region confirmed the presence of 73G, 146C, 151T, 16189C, 16217C, 16247G, 16261T. Most importantly, the presence of 6719C, 12239T, 14022G, 15746G, and the ancestral allele at 6905 were also authenticated. Thus, we obtained confirmation in two laboratories that the mtDNA of the MN00015 skull belonged to haplogroup B4a1a1a, which is characteristic of Polynesians.
Mitochondrial DNA analysis from a tooth extracted from the second skull, MN00017, also revealed the presence of the 9-bp deletion of haplogroup B and the presence of the following mutations: 73G, 146C, 151T, 16126C, 16189C, 16217C, 16261T, and 14022G, thus classifying it as haplogroup B4a1a1 (32). These results were independently confirmed through the extraction of DNA from a second tooth of the same skull.
