After writing my three little essays and finally consolidating them into one essay I was left with one question unanswered. The anthropologists who had done the studies were quite vague about where specifically in Cantral America the roots and fruit trees were brought from. My search focused upon their words, the Caribbean Coastal part of Central America. My search results landed me in Belize and Guatemala which is north of Belize. Both places once belonged to the ancient Mayans. If the Guanahatabey were speaking a derivative of the Mayan language, then it makes a lot of sense that the Lucayan interpreter would not understand what was being said by the Guanahatabey that had been encountered because that Lucayan spoke a derivative language of Arawak. The Caribs spoke a language that also differed from that of the Taino and the Guanahatabey. It was specific to the Caribbean islands. My search also led me to an old scientific paper that Charles Lalueza Fox et al had written based upon mtDNA studies that had been done on ancient Taino assemblages - bones. What irked me was that the title had the word extinct applied to the Taino People. (Rouse) used that word and was worked over verbally by the descendants of the Taino people for using it at a conference. The mapping out of the supposed immigration route that had been taken by the ancient Taino people is pretty linear in its nature and is also derived from Rouse. Despite that the paper is important because it shares the actual numbers within the ancient Taino sequences. If you look closely with pen and paper in hand when you get to that part of the paper, write them down. Then go back to my first essay to compare the numbers to the numerical sequences of my ancestors the Guanahatabey. You will see that both numbers almost begin the same but eventually you will also see that they differ from each other quite distinctively. In any case, here is the paper as published by Charles Lalueza Fox. Ann. Hum. Genet. (2001), 65, 137±151 Printed in Great Britain 137 MtDNA from extinct Tainos and the peopling of the Caribbean C. LALUEZA-FOX, F. LUNA CALDERO!N#, F. CALAFELL$, B MORERA$ and J. BERTRANPETIT$ Seccion Antropologia, Dept. Biologia Animal, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain # Departamento de Antropologia FõUsica, Museo del Hombre Dominicano, Santo Domingo, Republica Dominicana; Universidad Nacional Pedro Heniquez Urenh a, Republica Dominicana Unitat de Biologia Evolutiva, Facultat de Cie[ ncies de la Salut i de la Vida, Universitat Pompeu Fabra, Barcelona, Spain (Received 10.7.00. Accepted 30.11.00) summary Tainos and Caribs were the inhabitants of the Caribbean when Columbus reached the Americas; both human groups became extinct soon after contact, decimated by the Spaniards and the diseases they brought. Samples belonging to pre-Columbian Taino Indians from the La Caleta site (Dominican Republic) have been analyzed, in order to ascertain the genetic affinities of these groups in relation to present-day Amerinds, and to reconstruct the genetic and demographic events that took place during the peopling of the Caribbean. Twenty-seven bone samples were extracted and analyzed for mtDNA variation. The four major Amerindian mtDNA lineages were screened through ampli®cation of the speci®c marker regions and restriction enzymatic digestion, when needed. The HVRI of the control region was ampli®ed with four sets of overlapping primers and sequenced in 19 of the samples. Both restriction enzyme and sequencing results suggest that only two (C and D) of the major mtDNA lineages were present in the sample: 18 individuals (75%) belonged to the C haplogroup, and 6 (25%) to the D haplogroup. Sequences display speci®c substitutions that are known to correlate with each haplogroup, a fact that helped to reject the possibility of European DNA contamination. A low rate of Taq misincorporations due to template damage was estimated from the cloning and sequencing of different PCR products of one of the samples. High frequencies of C and D haplogroups are more common in South American populations, a fact that points to that sub-continent as the homeland of the Taino ancestors, as previously suggested by linguistic and archaeological evidence. Sequence and haplogroup data show that the Tainos had a substantially reduced mtDNA diversity, which is indicative of an important founder effect during the colonization of the Caribbean Islands, assumed to have been a linear migratory movement from mainland South America following the chain con®guration of the Antilles. introduction When Christopher Colombus reached two of the Greater Antilles (Bahamas and Hispaniola) Correspondence: Jaume Bertranpetit, Unitat de Biologia Evolutiva, Facultat de Cie ncies de la Salut i de la Vida, Universitat Pompeu Fabra, C. Dr. Aiguader 80, 08003 Barcelona, Spain. Tel: (3493) 542 28 40; Fax (3493) 542 28 02. E-mail: jaume.bertranpetit!cexs.upf.es during his ®rst discovery voyage, in 1492, he was greeted by indigenous people who called themselves Tainos. At that time, Columbus was convinced of having arrived in either Japan or China; later he changed his mind, and, believing he had reached India, called the aborigines `Indians, a misleading name for the Native Americans that has remained in use to this day. Thus, the wrong and biased perceptions 138 C. Lalueza-Fox and others of Westerners about Caribbean aborigines date back to the very ®rst moment both cultures collided. However, we dont really know what the Tainos thought about the Spaniards, since they were extinguished in just one or two generations after this ®rst contact, decimated by the harsh treatment of the Spaniards and the diseases they brought with them. It is difficult to know how many people were killed during this process of extinction; according to different authors they could have numbered between 2 and 7 million throughout the Caribbean (Ubelaker, 1992; Crawford, 1992). At the beginning of the 16th century, to replace the decreasing Tainos as agricultural and mining labour, the Spaniards brought African slaves (Kiple, 1984), who came to constitute the major present-day human substratum in the Caribbean. Despite claims of Taino heritage survival in some rural communities in the east of Cuba, it must be concluded that, after 500 years of cultural and genetic disruption, the original Caribbean people have disappeared forever as a distinct human group. The study of the so-called Black Caribs from Belize (Monsalve & Hagelberg, 1997), a population which is presumed to derive from the admixture of Island Caribs with West African slaves, illustrates the limitations of working with the highly admixtured modern Caribbean populations, since at least 16 of the 17 sequences found were clearly of African origin. Therefore, we need to rely on ancient DNA analysis if we want to know the genetic affinities of these groups in relation to the other peoples of the Americas. By the time of Columbus, and according to the Spanish chroniclers, there were two main human groups in the Caribbean, the Tainos, and the Caribs (whose name is the source of the regions name). The Tainos inhabited la Hispaniola, Puerto Rico, the east of Cuba, and probably Jamaica, the Bahamas, and the Turks and Caicos Islands, while the Caribs inhabited the Windward Islands and Guadeloupe (Rouse, 1986, 1993). The latter group ± sometimes called Island Caribs ± was culturally related to some mainland American groups (called Mainland Caribs), that were established mainly in Venezuela. The Tainos consisted of hierarchical societies organized into chiefdoms; they had advanced agricultural techniques that allowed them to establish some settlements of thousands of inhabitants, with ceremonial squares and ball game courts. In contrast, the Caribs were ferocious nomadic hunters that raided the Taino villages, expanding from the South through the Lesser Antilles. In addition to Tainos and Caribs, there were other groups at Columbus times: the so-called Arawaks, inhabitants of Trinidad and the Guianas, and the Guanajuatabeys, inhabitants of West Cuba. The names of the Caribbean groups and the languages they spoke are a source of debate among scholars; it seems that both Tainos and Island Caribs spoke Arawakan languages that belong to the Equatorial sub-family, in the Equatorial-Tucanoan family (Ruhlen, 1991). In contrast, the Mainland Caribs spoke Caribbean languages, which are classi®ed into the Macro- Carib subfamily, within the Ge-Pano-Carib family (Greenberg, 1987; Ruhlen, 1991). The existence of some words with clear Caribbean origin in the language of the Island Caribs points to a close relationship with the Mainland Caribs. The original homeland of the Taino groups in mainland South America is more controversial. Archaeological evidence shows that the Caribbean area was already settled by 5000 b.c. ; however, it has been suggested that the direct ancestors of the Tainos might have come from populations that migrated from the Lower Orinoco Valley, the Guianas or Trinidad and Tobago, around 1000 b.c. Thereafter, they undertook a long series of voyages, from one island to another, progressing from the mainland to the Lesser Antilles and from there to the Greater Antilles, eventually mixing with or pushing west the pre-existing populations, like the Guanajuatabeys. The islands are so close to one another that, with three exceptions, it is possible to see the next island in the migratory chain. If this hypothesis is correct, the peopling of the Caribbean had to take place as a linear migratory movement from South East to North mtDNA from extinct Caribbean Indians 139 West, following the chain con®guration of the Antilles Islands. Therefore, whether or not the Caribbean was peopled from South-America is a hypothesis that can be reliably explored with ancient DNA analysis. The vast majority of ancient DNA studies have been based on the analysis of mitochondrial DNA (mtDNA). This cytoplasmic genome has a better chance of recovery, since a cell with a single copy of the nuclear genome can contain several thousand copies of the mtDNA genome. MtDNA has been widely used as a molecular tool for reconstructing the history of present-day human populations, by virtue of its special evolutionary properties, such as a rapid mutation rate relative to nuclear DNA, lack of recombination and maternal inheritance (Avise, 1986; Stoneking, 1993). In the Americas, many studies have shown that most of the mtDNA of Amerindian populations falls into four major lineages (named `A, `B, `C, `D), primarily de®ned by speci®c mtDNA markers (Schurr et al. 1990; Torroni et al. 1992, 1993b, 1994; Horai et al. 1993). Haplogroup A is de®ned by an HaeIII site at np 663, haplogroup B by a COII}tRNALys intergenic 9bp deletion, haplogroup C by an AluI site at np 13262 and haplogroup D by the absence of the AluI site at np 5176. Sequence data show a correlation between these lineages and particular mutations in the Control Region I of the mtDNA genome (Torroni et al. 1993a). An additional residual ®fth founding haplogroup, named `X, has been recently described (Bandelt et al. 1995). This lineage, ancestrally related to the lineage X found in some European populations, is characterized, at its basal level, by some RFLP and control region markers, such as ± 1715 DdeI, 16517 HaeIII, and the 16223T-16278T substitutions; in the Americas, it has only been found in populations from North America. Greenberg et al. (1986) postulated that three different migrations (Amerind, Na-Dene and Eskimo-Aleut speakers) from Asia across the Bering Straits peopled the Americas. However, the ®rst sequence data (Ward et al. 1991) showed a rather high mtDNA diversity in one single tribe, suggesting a much more complex scenario than that expected from the three-migration model. Subsequent genetic studies (Horai et al. 1993; Torroni et al. 1993a, 1993b) demonstrated that the Native American mtDNAs clustered in few, but relatively deep, lineages that were widespread along the continent and not restricted to any particular ethnic group or linguistic family. The ubiquity of the Native American mtDNAs in Asia suggested that a single initial migration into America, instead of successive migration waves, was a more plausible scenario (Merriwether et al. 1995; Merriwether & Ferrell, 1996). From that common mitochondrial founding pool, different demographic events would have produced the differences observed among present-day Native American populations, thus complicating the interpretation of both genetic and ethnohistorical data (Forster et al. 1996). The purpose of this study is to recover mtDNA from pre-Columbian Taino remains from Hispaniola (Dominican Republic) to ascertain the genetic affinities of these groups in relation to present-day mainland Amerinds and to reconstruct the process of peopling of the Caribbean Islands, along with the possible existence of demographic events during that process, such as genetic drift or bottlenecks. The future aim of this project is to analyse the genetic composition of the pre-Columbian remains from other Caribbean Islands, to provide a clear picture of the whole migration process; if successful, this can constitute a case study on ancient human migrations similar to that of Polynesia, although on a smaller scale. materials and methods DNA extraction and ampli®cation Twenty-seven bone samples from the pre- Columbian site of La Caleta (Repu!blica Dominicana) were analyzed. The site is located 25 km east of Santo Domingo city, and is one of the most important Taino necropolises in the island; the bodies are buried with Boca Chica style ceramics, ornaments and tools (unpublished 140 C. Lalueza-Fox and others data). Samples were chosen from well preserved post-cranial bones, and belong to different individuals; the radiocarbon dating of several individuals has yielded dates from 670³70 a.d. to 1680³100 a.d. ; however, most of the dates are pre-Columbian. Extraction was undertaken with strict procedures to minimize the potential for contamination, in a positive-air pressure room separated from the main laboratory. Sterile gloves, face masks, sterile reagents, pipette ®lter tips and frequent bleaching of the working surfaces were some of the precautions adopted during the process. In addition, the laboratory where the analysis was done is totally new, and no extraction of DNA from Amerindians had ever been performed there. The external surface of the bone samples (³1 mm) was removed with a sterile surgical blade. Between 1 and 2 g of the bone samples were powdered in a coffee grinder; between each extraction, the grinder was washed with bleach. The powder was washed overnight, with shaking, in 10 ml of 0.5 M EDTA pH 8.0 at 37 °C; after centrifugation, the supernatant was removed, and the remaining sample was incubated overnight at 37 °C with 8.5 ml of water, 1 ml 5% SDS, 0.5 ml 1 m Tris-HCl pH 8.0 and 50 ll of 1mg}ml proteinase K. After incubation, the digests were extracted three times, ®rst with phenol, second with phenol-chloroform and third with chloroform, and the aqueous phase was concentrated by dialysis centrifugation using Centricon-30 microconcentrators (Amicon) to a 100±200 ll volume. One microlitre of template was subjected to 35 cycles of ampli®cation in 25 ll-reaction volume containing 1 unit of Taq polymerase (Ecogen, Madrid, Spain), 10¬ reaction buffer, 2.5 mm MgCl#, 0.2 mm dNTPs, 12 mg}ml of BSA and 20 pmoles of each pair of primer. Each cycle consisted of 1 min steps, with denaturation at 94 °C, annealing at 55 °C and extension at 72 °C. Negative controls (extraction blanks and PCR blanks) were undertaken along with the ancient samples, to monitor against contamination; no positive controls were used. PCR products were electrophoresed in 0.8% low-melting agarose gels in TA buffer and visualized with ethidium bromide staining. Positive ampli®cation bands were excised from the gels, melted at 65 °C for 20 min and eluted in 100±200 ll of sterile water, depending on the intensity of the band. The samples were subjected to a further 15 cycles of PCR, with limiting primers, annealing at 57 °C, one initial step at 94 °C for 5 min and one ®nal step at 72 °C for 5 min. The PCR products were puri®ed with the silica binding method (modi®ed from Ho$ ss & Pa$ a$ bo, 1993); 20 ll of reaction volume was mixed with 100 ll of 8.2 m NaI and 40 ll of silica suspension, and left for 5 min at room temperature. After a spin, the supernatant was removed and the silica pellet washed twice with 250 ll of 70% ethanol. The nucleic acids were eluted in 20±30 ll of water; 2±6 ll of these samples was used as the template for direct sequencing on an ABI 377TM automated DNA sequencer (Applied Biosystems, Foster City, CA, USA), according to the suppliers instructions. Four sets of overlapping primers (L16055- H16142, L16131-H16218, L16209-H16356, and L16347-H16410) published elsewere (Handt et al. 1996; Stone & Stoneking, 1998), were used to amplify 354 bp of the mtDNA control region I, between positions 16056 and 16409 (Anderson et al. 1981). All samples that yielded positive ampli®cations and sequences were extracted twice; additional sequences were randomly generated from the second extracts, using the shorter primer sets. To obtain additional support for the attribution of the four primary mtDNA Amerindian lineages, small fragments of mtDNA containing the speci®c marker of each lineage were ampli®ed in the same, previously sequenced, samples. Four sets of primers were used. For the haplogroup A, L635 and H709 (Handt et al. 1996); haplogroup B, L8215 and H8297 (Wrischnik et al. 1987); haplogroup C, L13257 and H13393 (Handt et al. 1996;Ward et al. 1991, respectively); haplogroup D, L5054 and H5190 (Stone & Stoneking, 1998; Handt et al. 1996, respectively). The PCR products were digested overnight at 37 °C with 0.5 ll of the appropriate restriction enzyme, and subsequently electrophoresed in 3%agarose gels, mtDNA from extinct Caribbean Indians 141 Fig. 1. Map of Central, South America and the Caribbean, with the populations included in this study. Abbreviations are: AMA, Amazonas, CAY, Cayapa, EMB, Embera, GAV, Gavia4 o, HUE, Huetar, KUN, Kuna, NGO, Ngo$be!, QUI, Quiche, TAI, Tainos, WOU, Wounan, XAV, Xavante, YAN, Yanomami, ZOR, Zoro. except for the haplogroup B ampli®cations that were directly electrophoresed. Cloning of PCR products To estimate the rate of misincorporations due to the template damage or Taq errors in our sample, two different PCR ampli®cations (L16209-H16410 and L16209-H16356) from the same sample (u163) were cloned and sequenced. Twelve microliters of the PCR product were treated with T4 polynucleotide kinase, puri®ed by phenol-chloroform extraction and MicroSpin Column centrifugation and then ligated into a SmaI pUC18 plasmid vector, for 2 h at 16 °C, following the suppliers instructions (SureClone Ligation Kit-Pharmacia, Upssala, Sweden). Five microliters of the ligation product was transformed into 100 ll of competent cells and grown in 200 ll of LB medium for 1 h before plating on IPTG}X-gal agar plates. Colonies were left to grow overnight at 37 °C; white colonies were added to 50 ll PCR reactions for 25 cycles; inserts that yielded the expected size in a electrophoresed gel were excised, puri®ed with silica and sequenced following the procedures described. Statistical analysis Intrapopulation mtDNA variation in the Tainos was measured by two parameters. Nucleotide diversity (p) was computed as p¯ (n}n®1) Rl i= (1-xi #), where n is the sample size, l the sequence length and xi the frequency of each nucleotide at position i (Nei, 1987). Sequence diversity (hs) was estimated as hs ¯ (n} n®1)Rk i= (1®pi #), where k is the number of different sequences and pi the frequency of each sequence (Nei, 1987). The pairwise difference distribution (mismatch distribution) (Rogers & Harpending, 1992; Harpending et al. 1993) was also computed. To provide a populational framework for testing the peopling of the Caribbean, all Meso and South American groups published, with ethnic attribution and large sample sizes, have been considered (Fig. 1). The populations used 142 C. Lalueza-Fox and others Table 1. mtDNA haplogroup attribution from the ampli®cation and enzymatic restriction of the speci®c markers in the Taino samples Samples (Sequenced) nt663 HaeIII COII}tRNALys 9 bp deletion nt13262 AluI nt(®)5176 AluI HAPLOGROUP 166 ® ® D 196 ® D* 189 ® ® ® D 167 ® ® ® D 70 ® ® ® C 45 ® ® ® C 187 ® ® ® C 154 ® ® C 182 ® C 71 ® ® ® C* 48 ® ® ® C 191 ® ® C 162 ® C* 53 ® ® C 170 ® ® C 54 ® ® ® C 51 ® ® C* 58 ® C 163 ® ® ® C (Not sequenced) 50 ® ? ? ? 175 ® ® C 164 ® ® C 197 ® C 72 ® ? ? ? 40 ® ® ® ? D 185 ® ® ® D 150 ? ? ? ? accounts for unresolved enzymatic digestion due to low ampli®cation efficiency. * attributions were con®rmed by sequencing of the control region. are the Cayapas (Rickards et al. 1999), Embera, Gavia4o (Ward et al. 1996), Huetar (Santos et al. 1994), Kuna (Batista et al. 1995), Mapuches (Ginther et al. 1993), Ngo$be! (Kolman et al. 1995), Quiche (Boles et al. 1995), Wounan (Kolman & Bermingham, 1997), Xavante (Ward et al. 1996), Yanomami (Torroni et al. 1993a) and Zoro (Ward et al. 1996), as well as some individuals from related tribes (Yanomama, Wayampi, Kayapo, Arara, Katuena, Portujara, Awa-Guaja, and Tiriyo) grouped into`Amazonas (Santos et al. 1996). Analyses including these samples were made considering sequences between positions 16024 and 16383. A distance matrix between populations was generated using the mismatch-intermatch distance. Principal coordinates analysis was performed on the distance matrix with the NTSYS programme, version 1.70 (Applied Biostatistics, Inc, Setanket, NY, USA). In order to understand the history of the sequences found in the Tainos, we have also performed a median network analysis (Bandelt et al. 1995) with 218 sequences from South American populations. To simplify the phylogeny obtained, we have repeated the network only with the C haplogroup sequences, which includes most of the Taino sequences. Analysis of Molecular Variance (AMOVA) (Excoffier et al. 1992) was carried out with the Arlequin 2000 package (Schneider et al. 2000). results The ampli®cation results of the speci®c haplogroup markers are shown in Table 1, along with the putative haplogroup assignment. The ampli- ®cation efficiency varied from one haplogroup to another, probably due to differences in the primer design and the length of the ampli®ed mtDNA from extinct Caribbean Indians 143 Table 2. Polymorphic sites of the sequences found in the Tainos 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 1 1 1 2 2 2 2 2 2 3 3 3 3 4 2 2 8 2 4 5 6 6 9 1 2 2 6 0 Sample HAPLOGROUP 6 9 9 3 2 4 3 5 8 1 5 7 2 0 166 D [ [ [ T [ [ [ [ [ [ C [ C [ 196 D [ [ [ T [ [ [ [ [ [ C [ C [ 189 D [ A [ T [ [ [ [ [ [ C [ C [ 167 D [ [ [ T T [ [ [ [ C C [ C T 70 C [ [ [ T [ [ [ [ C C C T [ [ 45 C [ [ [ T [ [ [ [ C C C T [ [ 187 C [ [ [ T [ [ [ [ C C C T [ [ 154 C [ [ [ T [ [ [ [ C C C T [ [ 182 C [ [ [ T [ [ [ [ C [ C T [ [ 71 C [ [ [ T [ [ [ [ C [ C T [ [ 48 C [ [ [ T [ [ [ [ C [ C T [ [ 191 C [ [ [ T [ [ [ [ C [ C T [ [ 162 C [ [ C T [ [ [ [ C [ C T C [ 53 C [ [ C T [ [ [ [ C [ C T C [ 170 C [ [ [ T [ [ [ [ C [ [ T C [ 54 C [ [ [ T [ [ C [ C [ [ T [ [ 51 C [ [ [ T [ [ [ T C [ C T [ [ 58 C [ [ [ [ [ G [ [ C [ C T C [ 163 C C [ [ [ [ [ [ [ C [ C T [ [ Base positions are compared to the Cambridge reference sequence (Anderson et al. 1981). product; for instance, it was possible to amplify the A haplogroup region in almost all the samples (25 out of 27); in contrast, only 16 samples yield PCR products for the D haplogroup. In some of the C and D haplogroup ampli®cations, the bands were so faint that the ®nal result remained unclear. It should be noted that ampli®cation is independent of the results of the enzymatic digestion and, therefore, the ampli®cation efficiency does not bias the haplogroup attribution. The ampli®cation efficiency was higher for the control region, maybe due to a better primer design (Table 2). In the best preserved specimens (ten samples), it was possible to obtain the 354 bp region with only two overlapping fragments (L16055-H16218 and L16209-H16410). The widely described degradation of ancient DNAinto fragments, usually smaller than 200 bp (Pa$a$ bo, 1989; Lalueza-Fox, 1996a), made necessary the ampli®cation of nine samples (71, 182, 53, 154, 187, 196, 58, 51, 170) in four fragments. A partial sequence, with a G in np 16212 (L16131-H16218 fragment), was obtained for the no. 197 sample; however, since it was not possible to extend the sequence or reproduce it subsequently, this has not been included in Table 2. In addition, seven samples failed to yield ampli®able DNA. The low ampli®cation efficiency and low reproducibility of some samples most likely re¯ects severe DNA degradation and a small number of template molecules. The sequence markers obtained, corresponding to the mtDNA lineages (C in np 16325 and C in np 16362 for the haplogroup D, and C in np 16298, C in np 16325 and T in np 16327 for the haplogroup C), con®rmed the haplogroup attribution (Forster et al. 1996). Taking into account the consensus haplogroup assignation, inferred from both the haplogroup markers and the sequences, it can be summarized that 75%of the Tainos studied belonged to the C haplogroup (N¯18) and 25% (N¯6) to the D haplogroup. No presence of the two other major Amerindian haplogroups (A and B) was detected. The highest limit for a 95% con®dence interval for no observation in a sample size (N) of 24 individuals can be estimated through the Poisson distribution 1®e−FN¯0.95, were F is the frequency of an unobserved haplogroup; therefore, other haplogroups could be present in the Taino 144 C. Lalueza-Fox and others Table 3. MtDNA lineage frequencies in Amerindian populations Linguistic classi®cation mtDNA lineages frequencies (%) and Population n A B C D Others References ESKIMO-ALEUT Old Harbor, Eskimos 115 61.7 3.5 0.0 34.8 0.0 Merriwether et al. 1995b Ouzinbie, Eskimos 41 73.2 0.0 4.9 14.6 7.3 Merriwether et al. 1995b Gambell, Eskimos 50 58.0 0.0 14.0 26.0 2.0 Merriwether et al. 1995b Savoonga, Eskimos 49 93.9 0.0 0.0 2.0 4.1 Merriwether et al. 1995b St. Paul, Aleuts 72 25.0 0.0 1.4 66.7 6.9 Merriwether et al. 1995 b CONTINENTAL NA-DENE Dogrib 30 100.0 0.0 0.0 0.0 0.0 Torroni et al., 1993a Dogrib 124 88.7 0.0 2.4 0.0 8.9 Merriwether et al. 1995b Navajo 48 58.3 37.5 0.0 0.0 4.2 Torroni et al. 1993a Apache 25 64.0 16.0 12.0 8.0 0.0 Torroni et al. 1993a HAIDA NA-DENE Haida 38 92.1 0.0 7.9 0.0 0.0 Ward et al. 1993 Haida 25 96.0 0.0 0.0 4.0 0.0 Torroni et al. 1993a NORTHERN AMERIND Bella Coola 25 60.0 8.0 8.0 20.0 4.0 Torroni et al. 1993a Bella Coola 32 78.1 6.25 9.4 6.25 1.6 Ward et al. 1993 Nuu-Chah-Nulth 63 44.5 3.1 19.1 26.7 13.3 Ward et al. 1991 Nuu-Chah-Nulth 15 40.0 6.7 13.3 26.7 13.3 Torroni et al., 1993a Ojibwa 28 64.3 3.6 7.1 0.0 25.0 Torroni et al. 1993a Mohawk 18 46.4 10.5 13.8 0.6 28.7 Merriwether et al. 1995b Maya 27 51.9 22.2 14.8 7.4 3.7 Torroni et al. 1993a Mixe 16 62.5 31.3 6.2 0.0 0.0 Torroni et al. 1994 Muskoke 71 36.6 15.5 9.9 38.0 0.0 Merriwether et al. 1995b CENTRAL AMERIND Mixtec Alta 15 73.4 13.3 13.3 0.0 0.0 Torroni et al. 1994 Mixtec Baja 14 92.9 7.1 0.0 0.0 0.0 Torroni et al. 1994 Zapotec 15 33.3 33.3 33.3 0.0 0.0 Torroni et al. 1994 Pima 30 6.7 50.0 43.3 0.0 0.0 Torroni et al. 1993a CHIBCHA-PAEZAN Teribe 20 80.0 20.0 0.0 0.0 0.0 Torroni et al. 1994 Guatuso 20 85.0 15.0 0.0 0.0 0.0 Torroni et al. 1994 Boruca 14 21.4 71.5 0.0 7.1 0.0 Torroni et al. 1993a Kuna 16 100.0 0.0 0.0 0.0 0.0 Torroni et al. 1993a Kuna 63 71.4 28.6 0.0 0.0 0.0 Batista et al. 1995 Guaymi 16 68.8 31.2 0.0 0.0 0.0 Torroni et al. 1993a Bribi}Cabecar 24 54.2 45.8 0.0 0.0 0.0 Torroni et al. 1993a Huetar 27 70.4 3.7 0.0 25.9 0.0 Santos et al. 1994 Ngo$be! 46 67.4 32.6 0.0 0.0 0.0 Kolman et al. 1995 Cayapa 120 29.1 40.0 9.2 0.0 21.7 Rickards et al. 1999 Atacama 13 23.1 69.2 7.7 0.0 0.0 Bailliet et al. 1994 Atacamen4 o 50 12.0 72.0 10.0 6.0 0.0 Merriwether et al. 1995b Yanomama 24 0.0 16.7 54.2 29.2 0.0 Torroni et al. 1993a ANDEAN Quechua 19 26.3 36.8 5.3 31.6 0.0 Merriwether et al. 1995b Aymara 172 6.4 67.4 12.2 14.0 0.0 Merriwether et al. 1995b Mapuche 39 15.4 38.5 20.5 25.6 0.0 Ginther et al. 1993 Mapuche 58 5.3 32.7 20.6 31.1 10.3 Bailliet et al. 1994 Huilliche 38 5.3 28.9 18.4 47.4 0.0 Bailliet et al. 1994 Huilliche 80 3.75 28.75 18.75 48.75 0.0 Merriwether et al. 1995b Pehuenche 100 2.0 9.0 37.0 52.0 0.0 Merriwether et al. 1995b Aonikenk 15 0.0 0.0 26.7 73.3 0.0 Lalueza-Fox 1996 Kawe!skar 19 0.0 0.0 15.8 84.2 0.0 Lalueza-Fox 1996 Ya!mana 11 0.0 0.0 90 10 0.0 Lalueza-Fox 1996 Selknam 13 0.0 0.0 46.2 46.2 7.7 Lalueza-Fox 1996 EQUATORIAN TUCANOAN Piaroa 10 50.0 0.0 10.0 40.0 0.0 Torroni et al. 1993a Wapishana 12 0.0 25.0 8.3 66.7 0.0 Torroni et al. 1993a Ticuna 28 17.9 0.0 32.1 50.0 0.0 Torroni et al. 1993a Zoro 30 20.0 6.7 13.3 60.0 0.0 Ward et al. 1996 mtDNA from extinct Caribbean Indians 145 Table 3. (cont.) Linguistic classi®cation mtDNA lineages frequencies (%) and Population n A B C D Others References Gavia#o 27 14.8 14.8 0.0 70.4 0.0 Ward et al. 1996 Tainos 24 0.0 0.0 75.0 25.0 0.0 Present study GE-PANO-CARIB Makiritare 10 20.0 0.0 70.0 10.0 0.0 Torroni et al. 1993a Macushi 10 10.0 20.0 30.0 40.0 0.0 Torroni et al. 1993a Kraho 14 28.6 57.1 14.3 0.0 0.0 Torroni et al. 1993a Marubo 10 10.0 0.0 60.0 30.0 0.0 Torroni et al. 1993a Mataco 28 10.7 35.7 0.0 53.6 0.0 Torroni et al. 1993a Xavante 25 16.0 84.0 0.0 0.0 0.0 Ward et al. 1996 LINGUISTIC CLASSIFICATION NO-SPECIFIED Colombians 20 50.0 20.0 25.0 5.0 0.0 Horai et al. 1993 Chileans 45 4.5 22.2 40.0 33.3 0.0 Horai et al. 1993 ANCIENT GROUPS Norris Farm-Oneota 108 31.5 12.0 42.6 8.3 5.6 Stone and Stoneking 1998 Great Salt Lake Fremont 32 0.0 73.0 13.0 7.0 7.0 Parr et al. 1996 Fig. 2. MtDNA lineage frequencies in Amerindian populations grouped by broad geographic regions (North, Central and South America). X haplogroup has only been found in North America, the black bar in South America correspond to a lineage described in the Capayas. population with frequencies up to 12.5% and would not have been detected with the present sample size of 24 individuals. Data on the haplogroup frequencies for other Amerindian populations have been compiled from previously published papers, and have been grouped according to linguistic and geographic criteria (Table 3). The most obvious pattern of variation in these frequencies is still geographical, as some authors have suggested (Merriwether et al. 1995; Lalueza-Fox, 1996b). When grouped in the three main geographic entities of the continent (North American, Central American and South American Amerinds) marked differences in the distribution of the mtDNA lineages can be observed (Fig. 2). Cloning results The sequence of the clones obtained for one sample (no. 163) consistently shows the substitutions found in the direct sequencing of the sample (16298 [C] 16325 [C] and 16327 [T]), as well as some singletons (Fig. 3). Since none of the singletons are shared in two or more clones, these most probably correspond to cloning artifacts and not to template damage and Taq misincorporations; the latter would yield multiple clones sharing the substitution (Krings et al. 1997). Thus, the DNA from the no. 163 sample is remarkably well preserved (only 5 singleton substitutions in 2864 nucleotides sequenced, error rate}1000 bp of 1.75). Since there did not seem to be important taphonomic differences among samples from the La Caleta site related to preservation, the cloning results suggest a low ratio of putative misincorporations due to template damage and Taq errors in our sample. Diversity parameters Nucleotide diversity of the Tainos was estimated at 0.0084 for the 354 bp fragment; this value is lower than that found in most of the 146 C. Lalueza-Fox and others Amerind populations studied; only the Kuna (0.0092), the Huetar (0.0097) and the Xavante (0.0083) show a similar level of reduced diversity. The sequence diversity obtained for the Tainos was 0.918; in this case, this value is higher than most of the other Amerind populations, similar to the Amazonas (0.933), Embera (0.942), Mapuche (0.912) or Wounan (0.920). In addition, the Tainos present a bell-shaped pairwise difference distribution, with a mean value of 2.96, the smallest of the Amerind populations used for comparison, but close to the values found in the Xavante (3.00), the Kuna (3.30) and the Huetar (3.50). Ayes, sic, here are the sequence numbers: Sequence sharing Some of the sequences found have been already described, especially those close to the root of the D and C haplogroups: (1) 16223 [T] 16325 [C] 16362 [C], (2) 16223 [T] 16298 [C] 16311 [C] 16325 [C] 16327 [T], and (3) 16223 [T] 16298 [C] 16325 [C] 16327 [T]. Interestingly, two previously undescribed sequences, 16223 [T] 16242 [T] 16311 [C] 16325 [C] 16362 [C] 16400 [T] and 16189 [C] 16223 [T] 16298 [C] 16325 [C] 16327 [T] 16362 [C] are very close to two Mapuche sequences already described by Ginther et al. (1993). In addition, 16263 [C] has been described in a Mongolian individual in association with some of the substitutions (16223 [T] 16298 [C] 16327 [T]) also found in our sample, while 16254 [G] and 16129 [A] substitutions have been found in Asian individuals. 16265 [T] is an unusual substitution although it has been found in some Panamanian individuals (Kolman & Bermingham, 1997), while 16126 [C] has been widely described in other populations. Taino genetic affinities The results of the principal coordinate analysis on the mismatch-intermatch genetic distance matrix are represented in Fig. 4; the ®rst two principal coordinates account for 68.5% of the total variance of the genetic distance matrix (the ®rst coordinate accounts for 42.2% and the second for 26.3%). The neighbor-joining tree (not shown) displays a topology that re¯ects a similar structure. It can be observed that most groups from Central America (the Ngo$be!, Kuna, Huetar and Quiche) are separated from the other groups, but related quite closely to one another, while the Tainos and Yanomami are opposite to them; in between them are all the other South American populations as well as the Choco! speakers from Panama!}Colombia, the Embera and the Wounan. The Xavante, a group from the south of Brazil, seem to be the most differentiated population within South America. Phylogenetic analysis of sequences In the median network of the South American sequences, the Taino individuals tend to be distributed around the central nodes of the C and D haplogroups, clustering with or close to the inferred ancestral sequences, suggesting a relative antiquity of these sequences. The C haplogroup median network is clearly star-like (Fig. 5); a visual fact supported by the low values of kurtosis (0.258³0.778) and skewness (1.026³0.398) of the networks distribution branch length (Mateu et al. 1997). This kind of star-shaped phylogeny suggests a population expansion (Forster et al. 1996). It can be observed that most of the Taino sequences are related to the founding sequence by just one, or very few, substitutions; the main inconsistencies are due to reversions in position 16362, which has already been described as a highly unstable position (Forster et al. 1996) and has a substitution rate ®ve times higher than the control region average (Meyer et al. 1999). Interestingly, the longest branches in the network correspond to sequences found in the Amazonas region, either in some Yanomami or in the tribes included within the `Amazonas group. The position of most of the Taino sequences close to the root and their lack of dispersion along the network suggests that a population expansion occurred before the formation of long branches in South American lineages, and}or before a very narrow bottleneck in the peopling mtDNA from extinct Caribbean Indians 147 ANDERSON CLONE 1A CLONE 2A CLONE 3A CLONE 4A CLONE 5A CLONE 6A CLONE 1B CLONE 2B CLONE 3B CLONE 4B CLONE 5B ANDERSON CLONE 1A CLONE 2A CLONE 3A CLONE 4A CLONE 5A CLONE 6A CLONE 1B CLONE 2B CLONE 3B CLONE 4B CLONE 5B Fig. 3. Sequences from clones generated from two different ampli®cations (L16,209-H16,410 and L16,209- H16,356) of the no. 163 Taino sample. Anderson is the Cambridge reference sequence (Anderson et al. 1981); substitutions shared in all the clones obtained are C in 16,298, C in 16,325 and T in 16,327. Fig. 4. Principal Coordinate (PC) Analysis of the distance matrix obtained from Central and South American populations. The score values have been multiplied by 100. Abbreviations are: AMA, Amazonas, CAY, Cayapa, EMB, Embera, GAV, Gavia4 o, HUE, Huetar, KUN, Kuna, NGO, Ngo$be!, QUI, Quiche, TAI, Tainos, WOU, Wounan, XAV, Xavante, YAN, Yanomami, ZOR, Zoro. of the Antilles. To test whether both putative population growths correspond to the same demographic event, we have estimated the pairwise distribution of the C sequences of the Taino and one South American population (the Yanomami), whose distribution may clearly re¯ect a past population expansion (Harpending et al. 1993). Since both mean values are quite different (2.11 for the Tainos and 1.25 for the Yanomami), this suggests we are observing the consequences of two population expansions, one associated to a founding event in the Yanomami population and another, more ancient, to the peopling of the Caribbean. 148 C. Lalueza-Fox and others Fig. 5. Reduced median network of the C haplogroup mtDNA sequences from South America. Taino sequences are in black; the circles are proportional to the frequency of the sequences they represent and the substitutions involved in the Taino sequences are listed in the branches. Central node (*) includes sequences with T in 16,223, C in 16,298, C in 16,325 and T in 16,327. discussion The closest phylogenetic affinities of the Tainos are with South American populations, since high frequencies of C and D lineages are more common in South American than in Central or North American populations, in accordance with the observed clinal pattern in the geographic distribution of these lineages along the continent (Merriwether et al. 1995; Lalueza-Fox, 1996b). There is little genetic structure among the Central and South American populations; the most notable feature seems to be the clustering of most Central American populations in a distinct group from the rest. In fact, AMOVA (Analysis of Molecular Variance) revealed that, when populations (excluding the Tainos) were divided into Central and Southern American, the difference among sub-continents accounted for 10.8% of the genetic variance (signi®cantly different from zero; p¯0.00196), while 13.05% of the genetic variance could be explained by differences among the populations in each subcontinent. Geography, considered in a latitudinal sense, is probably the main differentiating factor in the genetic history of the Amerind populations, the main exception being the Tainos. Considering the position of the Tainos in the genetic analysis, it is clear that, despite being geographically close to the Central American groups, their affinities are with South American groups. In particular, the closest group to the Tainos are the Yanomami, the only South American sample available near the Orinoco Valley, a suggested area for the Taino ancestors (Rouse, 1986). The Taino sequences cluster close to the ancestral founding sequences in the median network analyses; this suggests a considerable antiquity for the origin of the mtDNA variation found in the pre-Columbian Caribbean populations and a narrow bottleneck in the founding population. In the genetic analyses, the Tainos do not seem to be particularly close to the other EquatorianmtDNA from extinct Caribbean Indians 149 Tucanoan speakers, the Zoro and the Gavia4o; instead, they cluster close to the Yanomami, who have a Chibcha-Paezan language. However, the grouping of the three Chibchan-speaker groups (Kuna, Huetar and Ngo$be!) could either be attributed to geographic or linguistic affinities. Therefore, although the three-migration hypothesis of Greenberg et al. (1986) for the Americas is not supported by genetic data, the correlation between language and mtDNA variation in particular areas, like Central America, may be noticeable. It has been suggested that some archaic archaeological horizons in the Antilles, such as the Casimiroid ¯ints, originated in Central America, which would indicate a population movement from Yucatan into Cuba and Hispaniola, maybe around 5000 bc. (Rouse, 1986). In contrast, the subsequent Ceramic traditions in the Caribbean can be traced back to the Orinoco Valley in South America, and most probably correspond to a movement of people still ancestral to the Tainos migration into the Caribbean. It is unknown whether these migrating people replaced or mixed with preexisting populations; however, the absence in the Tainos of the A and B haplogroups, that have high frequencies in Central American populations, points to an extensive replacement of the ancestral Caribbean populations. Also, some contacts between the Caribbean groups and Central American populations have been suggested in more recent times (maybe from 1000 b.c. to Columbian times), especially to explain the diffusion of ball-court structures very similar to those found in Maya cultures (Rouse, 1986). Despite the possible existence of some contacts with Central America, the genetic impact of these should have been quite small, again given the absence of the A and B lineages. The Tainos seem to be one of the Amerind groups studied so far with lowest genetic diversity. Reduced mtDNA diversity has also been described in some groups from Panama and Costa Rica, like the Huetar, Ngo$be! and Kuna (Santos et al. 1994; Kolman et al. 1995; Batista et al. 1995), and has been attributed to either postcontact demographic decline or a small founding population (Kolman et al. 1995). 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