Mitochondrial DNA diversity in 17thЦ18th century remains from Tenerife (Canary Islands).код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 127:418–426 (2005) Mitochondrial DNA Diversity in 17th–18th Century Remains From Tenerife (Canary Islands) Nicole Maca-Meyer,1 Vicente M. Cabrera,1 Matilde Arnay,2 Carlos Flores,3 Rosa Fregel,1 Ana M. González,1 and José M. Larruga1* 1 Departamento de Genética, Universidad de La Laguna, 38271 La Laguna, Tenerife, Spain Departamento de Prehistoria e Historia Antigua, Universidad de La Laguna, 38271 La Laguna, Tenerife, Spain 3 Unidad de Investigación, Hospital Universitario Nuestra Sen̄ora de Candelaria, 38010 Tenerife, Spain 2 KEY WORDS Canary Islands; aDNA; mtDNA; haplotypes; admixture ABSTRACT Mitochondrial DNA sequences and restriction fragment length polymorphisms were retrieved (with >80% efﬁciency) from a 17th–18th century sample of 213 teeth from Tenerife. The genetic composition of this population reveals an important ethnic heterogeneity. Although the majority of detected haplotypes are of European origin, the high frequency of sub-Saharan African haplotypes (15.63%), compared to that of the present-day population (6.6%), conﬁrms the importance of the Canary Islands in the black slave trade of that epoch. The aboriginal substrate, inferred from the U6b1 haplotypes (8.59%), has also decreased due to European input. Finally, the presence of Amerindian lineages (1.5%) reveals that the Canary Islands have also received genetic ﬂow from America. Am J Phys Anthropol 127: 418–426, 2005. ' 2005 Wiley-Liss, Inc. Due to their geographic position off the northwest African coast (latitudes 278370 and 298240 ), the Canary Islands played a crucial role in the discovery and colonization of the Americas, as they were the last harbor before the transoceanic voyages (Fig 1). Moreover, a large number of Canarians were forced to embark, contributing to the settlement of the New World. At that time, these Canary Islanders were a very heterogeneous ethnic population, formed by Europeans from very different countries, Berber and sub-Saharan African slaves, and aborigines, all of them contributing in different degrees (Flores et al., 2001). Today, the presence of speciﬁc sub-Saharan and North African mitochondrial DNA (mtDNA) and Y-chromosome haplotypes attest to the impact of these African contributions in the Islands (Rando et al., 1999; Flores et al., 2001, 2003). Furthermore, the detection of some Canarian-speciﬁc mtDNA haplotypes, belonging to the U6 sub-haplogroup, in Cuban samples demonstrate that the Canarian migrations to the Americas were genetically inﬂuential (Torroni et al., 1999). The need for architectonic repairs in Concepción Church, located in Santa Cruz, the harbor and actual capital of the Tenerife Island, enabled us to obtain human remains of the population of this locality of the 17th–18th centuries. At this time, the European colonization of the New World was at its peak, and Santa Cruz was the most signiﬁcant harbor linking the Canary Islands to the Americas. The knowledge of the genetic composition of this historic population and its comparison with the present-day population could shed light on the degree of European and African impact on the indigenous inhabitants of the islands, and could unravel past undetected inﬂuences with the Americas. Besides immigration, the aboriginal population suffered important changes, such as drastic diminution in size due to epidemics and emigrations forced by imposition or famine. In order to determine the genetic composition of this historic population at a molecular level, the remains were analyzed using mtDNA sequences from the ﬁrst segment of the hypervariable region (HVRI) and diagnostic restriction-fragment length polymorphisms (RFLP). Since the excavated graves are from under a sacred ﬂoor, there is the possibility of obtaining a biased estimate of the real population. However, it is well-documented that, in contrast to other places, all deceased people in Santa Cruz of that period, including slaves, were buried in the Concepción Church (Sanz de Magallanes, 2001). # 2005 WILEY-LISS, INC. MATERIALS AND METHODS Samples Two hundred and eight teeth, belonging to 33 grave plots, were sampled from Concepción Church Grant sponsor: Ministerio de Ciencia y Tecnologia; Grant number: BMC2001-3511; Grant sponsor: Gobierno de Canarias; Grant number: COF2002-015. *Correspondence to: José M. Larruga, Departamento de Genética, Facultad de Biologı́a, Universidad de La Laguna, 38271 Tenerife, Spain. E-mail: firstname.lastname@example.org Received 31 January 2003; accepted 15 June 2004. DOI 10.1002/ajpa.20148 Published online 3 February 2005 in Wiley InterScience (www. interscience.wiley.com). 17TH–18TH CENTURY MTDNA IN TENERIFE 419 Fig. 1. Geographic distribution of Canarian Archipelago, indicating sampled localities. C, Concepción Church; SB, Ermita de San Blas. Arrows indicate Moorish and black slave trade routes. Fig. 2. Distribution of grave plots analyzed in this study. (Fig. 2), and ﬁve additional teeth were from the Ermita de San Blas in Candelaria (approximately 15 km from Santa Cruz). Both samples are dated to the 17th–18th centuries, and were therefore considered a sole population. Care was taken to choose teeth that did not show fractures. Furthermore, in order to avoid sampling repetitions, whenever possible, only one type of tooth was chosen by grave, preferentially upper or lower left canines, depending on the availability in each grave. When diverse types of teeth had to be used per grave, differences in sex and sequence of the samples were established, in order to make use of the maximum number of them. Jaw fragments were available in 15 cases, so that independent extractions from single individuals were prepared in separate areas and by different researchers in consecutive years. Extraction Prior to extraction, the surface of the tooth was thoroughly washed with 15% HCl, rinsed with ultraviolet (UV)-treated ddH2O, and dried under a UV lamp for 5 min on each side. Each tooth was then placed between two sterilized metal plates and crushed with a hammer, and the pieces were introduced into sterile 15-ml tubes (Costar). DNA was extracted according to a modiﬁed silica-based protocol (Höss and Pääbo, 1993). Brieﬂy, 1–2 ml of a commercial guanidine thiocyanate solution (DNAzol1; Chomczynski et al., 1997) were added to each tube and incubated, at room temperature, for 3–4 days. After this incubation, the supernatant was passed through commercial silica columns (QIAquick1, Qiagen; Yang et al., 1998), according to the manufacturer’s recommendations. Ampliﬁcation For mtDNA HVRI analyses, seven primer pairs were designed in order to amplify overlapping fragments, with sizes ranging from 82–124 bp (Table 1). HVRI fragments were PCR-ampliﬁed in 50-ml reactions using 7–9 ml of DNA extract in 1 PCR buffer (16.6 mM (NH4)SO4, and 67 mM Tris-HCl, pH 8.8), 2.5–4 mM MgCl2, 0.2 mM dNTPs, 5 pmol of each primer, and 1.75 U of Taq DNA polymerase (Ecogen/Bioline). Reactions were submitted to 35 ampliﬁcation cycles with each one consisting of 10-sec steps, with denaturation at 948C, annealing at the corresponding temperature (Table 1), and extension at 728C. Three negative controls were included in each ampliﬁcation in order to increase the probability of detecting contamination. PCR products were separated in 6% polyacrylamide gels (PAGE) and visualized with ethidium bromide staining. Positive ampliﬁcations were puriﬁed using ammonium acetate (Maniatis et al., 1982). Four additional primer pairs were designed to enable restriction fragment length analysis (RFLP) of the four most common African (Chen et al., 1995) and European (Torroni et al., 1996) haplogroupspeciﬁc sites (Table 1). RFLP fragments were PCR-ampliﬁed in 10-ml reactions using 2 ml of DNA extract. Reactions were submitted to 35 ampliﬁcation cycles with each one consisting of 10-sec steps, with denaturation at 948C, annealing at the corresponding temperature (Table 1), and extension at 728C. Ampliﬁcations were directly digested in 15-ml reaction volumes with the restriction enzyme according to the manufacturer’s recommendations. These digestions were completely loaded in 6% PAGE, stained with ethidium bromide, and visualized under UV. For sex determination, primers were designed to amplify a small region in intron 1 of the amelogenin gene, giving products of 60 and 66 bp for the X and Y chromosomes, respectively (Table 1). PCR was carried out in 10-ml reactions using 6 ml DNA extract. Reactions were submitted to 40 ampliﬁcation cycles with each one consisting of 10-sec steps, with denaturation at 948C, annealing at 458C, and extension at 728C. PCR products were completely loaded in 8% PAGE, stained with ethidium bromide, and visualized under UV. 420 N. MACA-MEYER ET AL. TABLE 1. List of primers used in this study, annealing temperature, and product size Primer sequence (50 ?30 ) HVRI L1F CTCCACCATTAGCACCCAAAGC H1R AGCGGTTGTTGATGGGTGAGTC L2F GGAAGCAGATTTGGGTACCAC H2R TGGTGGCTGGCAGTAATGTACG L3F CACCCATCAACAACCGCTAT H3R TGATGTGGATTGGGTTTTTATGTA L4FN GGTACCATAAATACTTGACCACCTG H4RN TTTGGAGTTGCAGTTGATGTG L5F CAAGCAAGTACAGCAATCAACC H5R CTGTTAAGGGTGGGTAGGTTTG L6FN CTCCAAAGCCACCCCTCAC H6RN GGGACGAGAAGGGATTTGAC L7F AGCCATTTACCGTACATAGCACA H7R TGATTTCACGGAGGATGGTG RFLP L3557 GCTCTCACCATCGCTCTTCT H3623 GGCTAGGCTAGAGGTGGCTA L42101 CCACTCACCTAGCATTACTTA H42271 ATGCTGGAGATTGTAATGGGT L6977 GGCCTGACTGGCATTGTATTA H7052 TGATGGCAAATACAGCTCCT L12253 ATGCCCCCATGTCTAACAAC T92 ATTACTTTTATTTGGAGTTGCACCAAGATT Amelogenin Amel-A3 CCCTGGGCTCTGTAAAGAATAGTG Amel-C AATRYGGACCACTTGAGAAAC R ¼ A/G; Y ¼ C/T 1 2 3 Temperature (8C) Product size (bp) 50 112 50 82 46 112 50 117 46 103 50 108 46 103 45 105 (57 þ 48) 52 59 (31 þ 28) 45 115 (100 þ 1570 þ 30 þ 15) 48 105 (75 þ 30) 45 60/66 Maca-Meyer et al., 2001. Torroni et al., 1996. Sullivan et al., 1993. Cloning and sequencing PCR products were ligated into pGEM-T vectors (Promega). Colonies were plated on selective IPTG/ X-gal agar plates, and white colonies were selected. Several clones (at least three) were sequenced for each fragment until an unambiguous sequence was obtained. PCR fragments were directly sequenced using the same primer pairs as for the ampliﬁcation, and clones were sequenced using M13 universal primers. All primers were labeled with g32-ATP, and the fmol1 DNA Cycle Sequencing System (Promega) was used for sequencing. Prevention of contamination All DNA extractions and PCR setups were performed in a dedicated laboratory physically separated from the main Department of Genetics. This laboratory was constantly irradiated with UV lamps and frequently cleaned with bleach. All sample manipulations were performed in a laminar ﬂow cabinet, with dedicated pipettes and sterile ﬁlter tips (Tip One, Star Lab). Solutions were commercially acquired when possible; otherwise, they were autoclaved twice and UV-treated. Laboratory coats, face shields, hats, and sterile gloves were used at all times. All metallic material was sterilized in an oven at 2008C for at least 2 hr. In the ﬁrst stages of the study, the effectiveness of the decontamination process before the DNA extraction was veriﬁed in the following way: a tooth was immersed in a solution containing chicken DNA and then submitted to the decontamination protocol. Another tooth was processed in the same way but without decontaminating the surface. Both cases were submitted to PCR ampliﬁcations, using speciﬁc primers that amplify chicken mtDNA cyt b. In the ﬁrst case, no ampliﬁcation products were obtained, while in the second, a 400-bp product was observed after the ampliﬁcation. To monitor contamination during the extraction process, an extraction blank was processed together with each tooth. PCR contamination was monitored by means of three negative controls in each reaction. Statistical analysis Sequences were sorted into haplogroups according to Richards et al. (2000). Haplotype diversity was calculated as k/n, where k is the number of haplotypes and n the sample size. Population differentiation tests (Raymond and Rousset, 1995) were computed by means of the Arlequin 2000 computer program (Schneider et al., 2000). Admixture estimates were calculated with four different estimators. ADMIX.PAS (kindly provided by Dr. Long) was used to estimate mL (Long, 1991). ADMIX 2.0 (Dupanloup and Bertorelle, 2001), was used to calculate mY (Bertorelle and Excofﬁer, 1998). Both mL and mY were based on haplogroup frequencies, considering each haplogroup as an 17TH–18TH CENTURY MTDNA IN TENERIFE allele of the same locus. The third estimate is based on the geographic origin (G.O.) of haplogroups. We considered the lineages belonging to haplogroups L1, L2, and L3 as containing subSaharan African inﬂuence, U6 lineages as containing North African inﬂuence, and the rest as being of European origin. A derivative of this method would include the founder haplotypes, those supposedly present in the aboriginal population (Rando et al., 1999), as a North African inﬂuence. The last estimator is based on the analysis of shared lineages (L.S.) between populations. To accomplish this, the Concepción sequences were compared with published and unpublished sequences from the Iberian Peninsula, North Africa, and subSaharan Africa as the most probable parental populations. Due to the large Iberian sample size, the number of shared sequences was divided by the number of different sequences present in each parental population. RESULTS AND DISCUSSION From a total of 213 extractions, 169 sequences and/or RFLPs were obtained from 147 individuals, corresponding to an extraction efﬁciency of 80%. Of these, 19 were discarded due to the low number of ampliﬁed fragments, which made them impossible to classify into haplogroups. Six individuals were able to be classiﬁed into haplogroups because of RFLP information, but were not taken into account for haplotype studies. Contamination in the extraction blank was only detected in one case, and this extraction was discarded. In total, 122 individuals were able to be used for haplotype analysis. There were no inconsistencies in the 15 replicates, as all extractions gave the same sex, HVRI sequence, and RFLP pattern. The sexual proportion was estimated as 53.3% males and 46.7% females, based on a sample of 60 teeth. PCR contamination was sporadic and could be overridden by repeating the ampliﬁcations using new aliquots of PCR solutions. Haplotype classiﬁcation and distribution Seventy-one different haplotypes (Table 2) were found in the 122 individuals analyzed in this historical sample, which accounts for a haplotype diversity of 58%. Of these, 13 (18.3%) were considered unique to the historical sample, though some of these motifs are found, with some variants, in other populations. Most of these unique sequences belong to the African sub-haplogroup L1c, which could reﬂect an insufﬁcient number of African populations in the comparative database. The most abundant haplogroup was H, encompassing 37% of the sample (Table 3). Two sequences (1.56%) were unequivocally of American origin, belonging to haplogroups A and C, corroborating the important nexus that the Canary Islands have had in the colonization of the Americas. Lineages belonging to 421 African haplogroups L1, L2, and L3 comprised 15.63% of the sample, a much higher frequency than currently found in the Islands (Rando et al., 1999). Subgroup U6a, of North African assignation, was found at a frequency of 1.56%, while U6b1, the speciﬁc Canarian subgroup, made up a total of 8.59%. Thirty-eight percent of the lineages found in our sample (Table 2) are present in the contemporary populations of Tenerife and/or the Canary Islands (Rando et al., 1999). Another 18% of matches are speciﬁcally European, and are absent in the Canaries and North Africa. Of these, approximately 10% are found in the Iberian Peninsula, and the rest of the matches are located in other areas of the continent, predominantly on the Atlantic coast. Regarding haplotypes belonging to macro-haplogroup L, only three (4.2%) are limited to the subSaharan African region, two of them located in the Gulf of Guinea (J.M. Larruga, personal communication) and the third in Mozambique (Pereira et al., 2001), the rest are distributed along different North African and European populations. The U6 lineages, considered a North African inﬂuence, account for 5.6% of the haplotypes. However, half of them belong to the Canarian-speciﬁc subgroup U6b1, which has still not been detected in Africa (Rando et al., 1998). The reciprocal inﬂuence between the Archipelago and the Americas can also be observed with haplotype matches. Two Amerindian haplotypes were found in the historical sample, one of which is still present in the Islands. In a similar vein, Canarian-speciﬁc U6b1 haplotypes were detected in Cuba (Torroni et al., 1999). U6a lineages were observed in Brazil (Alves-Silva et al., 2000), but they do not match those found in North Africa, Portugal, Spain, or the Canary Islands. Matches of European haplotypes between the historical sample and American populations are restricted to the basal motifs of the most abundant haplogroups. With respect to subSaharan African lineages, an L2a sequence (223 278 294 309 390) was found in Brazil (Alves-Silva et al., 2000), and an L3b sequence (124 223 278 362) in Mexico (Green et al., 2000), that match with the historical sample. This corroborates the important role that the Canarian Archipelago played in the route of the African slave trade to the Americas. Haplogroup frequencies Haplogroup frequencies of the historical sample and present-day populations of Tenerife and the Canary Islands are shown in Table 3. Overall, the historical sample shows high frequencies for haplogroups H and J*, and African macro-haplogroup L, compared to those found in present populations. Similarly, low frequencies are found for haplogroups V, T1, T2, U5b, and K. However, an exact test of differentiation (Raymond and Rousset, 422 N. MACA-MEYER ET AL. TABLE 2. HVRI sequences and RFLP status of individuals analyzed in this study, together with populations in which these lineages were detected Haplogroup/haplotype1 N2 H CRS 126 129 145 153 168 189 248 260 290 292 311 316 362 093 311 273 311 304 311 311 357 093 263 311 Total H V* 298 335 Total V* 19 2 2 1 1 3 2 1 1 1 1 2 1 2 1 1 1 1 1 44 1 1 HVR CRS 129 311 183C 189 Total HVR* 6 1 3 2 12 RFLP3 7025AluI () 13 1 1 1 3 2 1 1 1 1 1 2 TF CAN NAf * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 1 1 1 SSA * * EU NE * * * * * * * * * * * * * * * * * AM * * * * 7025AluI (þ) 1 * 7025AluI (þ) 12308Hinf I () 4216NlaIII () 6 1 3 1 * * * * * * * A 183C 189 223 290 319 362 Total A 1 1 * C 223 298 325 327 Total C J* 069 126 069 126 311 069 126 318C 069? 126 153 285T 290 J1 069? 126 163 261 Total J T* 126 286 295 296 311 T2 126 294 296 304 T3 126 292 294 T5 126 153 292 294 Total T 1 1 5 2 1 1 1 10 4216NlaIII (þ) 3 1 1 1 4216NlaIII (þ) 1 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 1 2 2 1 6 4216NlaIII (þ) 1 4216NlaIII (þ) 1 W 223 292 223 292 295 223 292 362 Total W 1 1 1 3 X 093 189 223 278 1 183C 189 223 278? 1 7025AluI (þ) 3592HpaI () 7025AluI (þ) 12308Hinf I () 3592HpaI () 4216NlaIII () * (continued) 423 17TH–18TH CENTURY MTDNA IN TENERIFE TABLE 2. (Continued) 1 Haplogroup/haplotype 093 189 223 274 278 356 Total X 2 RFLP3 1 3 12308HinfI () N TF CAN NAf SSA EU NE * * AM I 129? 223? 391 Total I U3 189 343? 390 U5a* 192 270 294 334 U5a1 114A 192? 270 294? 129 192 256? 270 U5b 189 270 189 270 362 U6a 172 219 278? 092 172? 219? 278 U6b1 163 172 219 311 092 163 172 219 311 Total U 1 1 1 * 1 * 1 1 * 1 1 1 1 10 1 19 * 12308Hinf I (þ) 1 1 12308Hinf l (þ) 6 1 * * * * * * * * * * * * * * * * * * * * * * * * * K 093 224 311 Total K L1b 126 187 188A 189 223 264? 270? 278? 311? 093 126 187 189 223 264 270 278 293? 311? L1c 129? 187 189 223 251 294 311 129 189 223 231 274 278 311 129 187 189 223 278 286A 292 294 311 360? 129 187 189 265C 278 286A 292 294 311 360? 017 129 163 187? 189 223 278 293 294 301 311 360 L2a 223 278 294 309 390 L2b 129 213 278 390 213 223 278 390 L2c 223 278 390 L3 223 223 311 L3b 124 166 223 309 124 223 278 362 223 278 311 362? 124 223 278 311 362 L3ela 185 223 311 327 Total L 1 1 1 3592HpaI (þ) 1 1 * 1 * 3592HpaI (þ) 1 1 1 1 1 1 1 1 1 1 1 1 3592HpaI (þ) 1 3592HpaI (þ) 1 1 * * * * * * * 2 2 * * * * * 1 20 * * * * * * 1 1 1 1 1 * 3592HpaI () 1 1 * * 1 * * * Mutations were designated as their position according to Anderson et al. (1981) minus 16000. Absolute frequency. 3 Number of individuals per haplotype that share speciﬁed RFLP status. This number may differ from absolute frequency, since limited amount of DNA did not always allow RFLP typing. CRS, Cambridge Reference Sequence (Anderson et al., 1981); TF, Tenerife (Rando et al., 1999); CAN, Canary Islands (Rando et al., 1999); NAf North Africa (Rando et al., 1998; Thomas et al., 2002; Larruga, personal communication); SSA, sub-Saharan Africa (Mateu et al., 1997; Monsalve and Hagelberg, 1997; Watson et al., 1997; Rando et al., 1998; Pereira et al., 2001); EU, Europe (Crespillo et al., 2000; Richards et al., 2000; Larruga et al., 2001; Gonzátez et al., 2003); NE, Near East (Richards et al., 2000; Thomas et al., 2002); AM, America (Torroni et al., 1999; Alves-Silva et al., 2000). In bold, mutations corresponding to the basal motif. In italics, mutations not detected by sequencing, but that were added because they belong to a similar haplotype detected in neighboring populations. * Matches between historical sample and current populations from broad geographical regions. 2 424 N. MACA-MEYER ET AL. TABLE 3. Haplogroup frequencies for historical population compared with those of present-day population of Tenerife and Canary Islands (Rando et al., 1999) Haplogroup CRS H (-CRS) HVR V* A C J* J1 J1a J2 T* T1 T2 T3 T5 W X I U* U2 U3 U4 U5* U5a* U5a1 U5a1a U5b U6a U6b K M1 L1b L1c L2 L3 Total Historic 19 29 12 1 1 1 9 1 (14.85%) (22.68%) (9.38%) (0.78%) (0.78%) (0.78%) (7.03%) (0.78%) Tenerife Canary Islands 14 (19.17%) 8 (10.96%) 10 (13.70%) 3 (4.11) 44 (14.67%) 21 (7.00%) 48 (16.00%) 7 (2.33%) 1 (0.33%) 1 (0.33%) 15 (5.00%) 2 (0.67%) 3 (1.00%) 1 (0.33%) 2 (0.67%) 10 (3.33%) 11 (3.67%) 13 (4.33%) 1 (0.33%) 3 (1.00%) 8 (2.67%) 3 (1.00%) 8 (2.67%) 2 (0.67%) 1 (1.37%) 1 (1.37%) 2 (2.74%) 1 (1.37%) 1 (0.78%) 2 2 1 3 3 1 2 (1.56%) (1.56%) (0.78%) (2.34%) (2.34%) (0.78%) (1.56%) 3 (4.11%) 3 (4.11%) 3 (4.11%) 2 (2.74%) 1 (1.37) 1 (1.37%) 1 (0.78%) 1 (1.37%) 1 (0.78%) 2 (1.56%) 2 2 11 1 (1.56%) (1.56%) (8.59%) (0.78%) 2 5 4 9 (1.56%) (3.91%) (3.13%) (7.03%) 128 2 (2.74%) 1 (1.37%) 3 (4.11%) 4 (5.48%) 4 (5.48%) 1 2 6 3 2 7 3 39 12 1 6 (0.33%) (0.67%) (2.00%) (1.00%) (0.67%) (2.33%) (1.00%) (13.00%) (4.00%) (0.33%) (2.00%) 2 (2.74%) 3 (4.11%) 4 (1.33%) 10 (3.33%) 73 300 1995) revealed that these differences are not statistically signiﬁcant (P = 0.112 0.024). In synthesis, the historical sample presents lineages that account for the major inﬂuences recorded in the peopling and colonization of the Islands. In this way, the U6b1 North African lineages would represent the initial aborigine substrate, as it has not been detected in presentday North African populations. U6a lineages, wellrepresented in African populations, could have reached the Islands as a consequence of the Moorish slave trade after the European Conquest. The large number of European lineages reﬂect the impact of the colonization of the Islands, mainly by Spaniards and Portuguese. Finally, the high number of sub-Saharan African lineages present in the sample reveals the importance of the black slave trade in the Islands. Admixture estimates The above-mentioned continental inﬂuences detected in the historical Canarian population can be quantiﬁed by means of admixture algorithms. TABLE 4. Admixture estimates (%) in historical population of Tenerife, and present-day populations of Tenerife and Canary Islands1 Iberian Peninsula mY Historic Tenerife Canary Islands Canary Islands2 Mean mL Historic Tenerife Canary Islands Canary Islands3 Mean L.S. Historic Tenerife Canary Islands Canary Islands3 Mean Mean North Africa Sub-Saharan Africa 22.7 29.6 16.8 34.0 6.9 14.0 8.0 3.5 3.0 50.9 19.0 82.9 12.8 58.1 21.7 36.4 57.1 8.4 45.5 23.4 17.1 12.8 39.1 21.2 45.2 36.7 5.8 3.6 8.1 2.8 3.7 18.4 6.2 3.6 13.5 28.1 25.6 34.8 25.5 3.9 39.8 7.5 62.4 59.5 60.7 41.3 56.0 4.3 50.8 5.8 24.1 12.4 13.7 23.9 18.5 2.7 9.4 3.8 56.3 50.8 22.9 17.0 36.7 22.7 29.6 16.8 35.0 8.5 43.7 49.2 77.1 69.0 59.8 negative contribution. Calculations were redone using two parental populations: Iberian Peninsula and North Africa. 2 Dupanloup and Bertorelle, 2001. 3 Pinto et al., 1996. 1 The problem of estimating these proportions is that the parental populations, namely the Iberian Peninsula, North Africans, and sub-Saharan Africans, show a considerable amount of gene ﬂow between themselves. For this reason, we used several methods for calculating these proportions: those based on haplogroup frequencies, and those based on the geographical assignation of lineages (Table 4). The mY and mL estimators yield a major European contribution, followed by North African and sub-Saharan African contributions. However, the L.S. estimator gives a major North African contribution (62.4%), followed by the sub-Saharan African (24.1%) and the European contribution (13.5%). Finally, the G.O. estimator gives a minimum estimate of the North African contribution (21%), as only U6 lineages (and founder haplotypes) were taken into account. Naturally, other sequences would have accompanied these lineages, but as these are shared with other populations, they were not considered of speciﬁc North African inﬂuence. For this reason, the G.O. estimator was eliminated from subsequent calculations. The demographic evolution of this population can be assessed by comparison with the admixture values obtained for the present-day population of Tenerife (Rando et al., 1999). Whereas the mY estimator does not show marked differences, mL and L.S. clearly show an important decrease of the subSaharan and North African maternal lineages in the present population, in favor of the European ones (Table 4). Proportions obtained for the whole Canary Islands sample (Rando et al., 1999) are also different, depending on the estimator used. 17TH–18TH CENTURY MTDNA IN TENERIFE Whereas mY emphasizes the North African component, mL shows a major European input, and L.S. stresses the minor sub-Saharan African ﬂow. Other mtDNA admixture results of the Canary Islands were published (Pinto et al., 1996; Dupanloup and Bertorelle, 2001), but they were based on notably smaller sample sizes than those used in this work (Table 4). If we take all these data as independent estimations of the maternal contributions to the Canarian population, we obtain coarse means giving values of 50% North African, 40% European, and 10% sub-Saharan (Table 4). In conclusion, from the mtDNA point of view, the historical population of Tenerife does not show signiﬁcant differences from the present-day population, the majority of lineages being of European origin, reﬂecting the impact of this contingent in the colonization of the Islands. The Canarian-speciﬁc subgroup U6b1, most probably of aboriginal origin, was also detected in a considerable proportion of the sample. Likewise, the presence of U6a lineages could reﬂect a minor Moorish slave inﬂuence on the Canary Islands. African lineages belonging to macrohaplogroup L are present in high frequency, attesting to the importance of the ‘‘black slave trade’’ in that historical period. Finally, the presence of Amerindian haplotypes, although in low frequency, demonstrate the importance of the Archipelago in the transoceanic voyages to the Americas. ACKNOWLEDGMENTS We thank W. Salo for his technical advice and critical reading of the manuscript. This study was supported by grants BMC2001-3511 from Ministerio de Ciencia y Technologia and COF2002-015 from Gobierno de Canarias to V.M.C. LITERATURE CITED Alves-Silva J, da Silva Santos M, Guimarães PEM, Ferreira ACS, Bandelt HJ, Pena SDJ, Ferreira Prado V. 2000. The ancestry of Brazilian lineages. Am J Hum Genet 67:444–461. Anderson S, Bankier AT, Barrell BG, Bruijn MHLDE, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, Young IG. 1981. Sequence and organization of the human mitochondrial genome. Nature 290:457–465. Bertorelle G, Excofﬁer L. 1998. Inferring admixture proportions from molecular data. Mol Biol Evol 15:1298–1311. Chen YS, Torroni A, Excofﬁer L, Santachiara-Benerecetti AS, Wallace DC. 1995. Analysis of mtDNA variation in African populations reveals the most ancient of all continent-speciﬁc haplogroups. Am J Hum Genet 57:133–149. Chomczynski P, Mackey K, Drews R, Wilﬁnger W. 1997. DNAzol1: a reagent for the rapid isolation of genomic DNA. Biotechniques 22:550–553. Crespillo M, Luque JA, Paredes M, Fernández R, Ramı́rez E, Valverde JL. 2000. Mitochondrial DNA sequences for 118 individuals from northeastern Spain. Int J Legal Med 114: 130–132. Dupanloup I, Bertorelle G. 2001. Inferring admixture proportions from molecular data: extension to any number of parental populations. Mol Biol Evol 18:672–675. Flores C, Larruga JM, González AM, Hernández M, Pinto FM, Cabrera VM. 2001. The origin of the Canary Island aborigines 425 and their contribution to the modern population: a molecular genetics perspective. Curr Anthropol 42:749–755. Flores C, Maca-Meyer N, Pérez JA, González AM, Larruga JM, Cabrera VM. 2003. A predominant European ancestry of paternal lineages from Canary Islanders. Ann Hum Genet 67:138–152. González AM, Brehm A, Pérez JA, Maca-Meyer N, Flores C, Cabrera VM. 2003. Mitochondrial DNA afﬁnities at the Atlantic fringe of Europe. Am J Phys Anthropol 120: 391–404. Green LD, Derr JN, Knight A. 2000. mtDNA afﬁnities of the peoples of north-central Mexico. Am J Hum Genet 66:989–998. Höss M, Pääbo S. 1993. DNA extraction from Pleistocene bones by a silica-based puriﬁcation method. Nucleic Acids Res 21: 3913–3914. Larruga JM, Dı́ez F, Pinto FM, Flores C, González AM. 2001. Mitochondrial DNA characterization of European isolates: the Maragatos from Spain. Eur J Hum Genet 9: 708–716. Long JC. 1991. The genetic structure of admixed populations. Genetics 127:417–428. Maca-Meyer N, González AM, Larruga JM, Flores C, Cabrera VM. 2001. Major genomic mitochondrial lineages delineate early human expansions. BMC Genet 2:13. Maniatis T, Fritsch EF, Sambrook J. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory. Mateu E, Comas D, Calafell F, Pérez-Lezaun A, Abade A, Bertranpetit J. 1997. A tale of two islands: population history and mitochondrial DNA sequence variation of Bioko and São Tomé, Gulf of Guinea. Ann Hum Genet 61:507–518. Monsalve MV, Hagelberg E. 1997. Mitochondrial DNA polymorphisms in Carib people of Belize. Proc R Soc Lond [Biol] 264:1217–1224. Pereira L, Macaulay V, Torroni A, Scozzari R, Prata MJ, Amorim A. 2001. Prehistoric and historic traces in the mtDNA of Mozambique: insughts into the Bantu expansions and the slave trade. Ann Hum Genet 65:439–458. Pinto F, González AM, Hernández M, Larruga JM, Cabrera VM. 1996. Genetic relationship between the Canary Islanders and their African and Spanish ancestors inferred from mitochondrial DNA sequences. Ann Hum Genet 60:321–330. Rando JC, Pinto F, González AM, Hernández M, Larruga JM, Cabrera VM, Bandelt HJ. 1998. Mitochondrial DNA analysis of northwest African populations reveals genetic exchanges with European, Near Eastern, and sub-Saharan populations. Ann Hum Genet 62:531–550. Rando JC, Cabrera VM, Larruga JM, Hernández M, González AM, Pinto F, Bandelt HJ. 1999. Phylogeographic patterns of mtDNA reﬂecting the colonization of the Canary Islands. Ann Hum Genet 63:413–428. Raymond M, Rousset F. 1995. An exact test of population differentiation. Evolution 49:1280–1283. Richards M, Macaulay V, Hickey E, Vega E, Sykes B, Guida V, Rengo C, Sellito D, Cruciani F, Kivisild T, Villems R, Thomas M, Rychkov S, Rychkov O, Rychkov Y, Golge M, Dimitrov D, Hill E, Bradley D, Romano V, Cali F, Vona G, Demaine A, Papiha S, Triantaphyllidis C, Stefanescu G. 2000. Tracing European founder lineages in the Near Eastern mtDNA pool. Am J Hum Genet 67:1251–1276. Sanz de Magallanes JM. 2001. In memoriam. Enterramientos en la Parroquia Matriz de La Concepción, V Centenario (1499–1999) de la Parroquia de la Concepción de Santa Cruz de Tenerife. Canildo Insular de Tenerife. Schneider S, Roessli D, Excofﬁer L. 2000. Arlequin version 2000: a software for population genetics data analysis. Geneva: Genetics and Biometry Laboratory, University of Geneva. Sullivan KM, Mannuci A, Kimpton CP, Gill P. 1993. A rapid and quantitative DNA sex test: ﬂuorescence-based PCR analysis of X-Y homologous gene amelogenin. Biotechniques 15: 636–641. Thomas MG, Weale ME, Jones AL, Richards M, Smith A, Redhead N, Torroni A, Scozzari R, Gratrix F, Tarekegn A, Wilson JF, Capelli C, Bradman N, Goldstein D. 2002. Founding 426 N. MACA-MEYER ET AL. mothers of Jewish communities: geographically separated Jewish groups were independently founded by very few female ancestors. Am J Hum Genet 70:1411–1420. Torroni A, Huoponen K, Francalacci P, Petrozzi M, Morelli L, Scozzari R, Obinu D, Savontaus ML, Wallace DC. 1996. Classiﬁcation of European mtDNAs from an analysis of three European populations. Genetics 144:1835–1850. Torroni A, Cruciani F, Rengo C, Sellito D, López-Bigas N, Rabionet R, Govea N, López de Munain A, Sarduy M, Romero L, Villamar M, del Castillo I, Moreno F, Estivill X, Scozzari R. 1999. The A1555G mutation in the 12S rRNA gene of mtDNA: recurrent origins and founder events in families affected by sensorineural deafness. Am J Hum Genet 65:1349–1358. Watson E, Forster P, Richards M, Bandelt HJ. 1997. Mitochondrial footprints of human expansions in Africa. Am J Hum Genet 61:691–704. Yang DY, Eng B, Waye JS, Dudar JC, Saunders SR. 1998. Improved DNA extraction from ancient bones using silicabased spin columns. Am J Phys Anthropol 105:539–543.