Am. J. Hum. Genet. 65:199±207, 1999 An mtDNA Analysis in Ancient Basque Populations: Implications for Haplogroup V as a Marker for a Major Paleolithic Expansion from Southwestern Europe N. Izagirre and C. de la RuÂa Euskal Herriko Unibertsitatea, Animali Biologia eta Genetika Saila, Zientzi Fakultatea, Bilbao, Spain Summary Introduction mtDNA sequence variation was studied in 121 dental Molecular analyses have shown that most mtDNA mu- samples from four Basque prehistoric sites, by high-res- tations have accumulated sequentially along radiating olution RFLP analysis. The results of this study are cor- maternal lineages that have diverged as human popu- roborated by (1) parallel analysis of 92 bone samples, lations have colonized different geographic regions of (2) the use of controls during extraction and ampli®- the world. Studies based on sequence data from hyper- cation, and (3) typing by both positive and negative re- variable segments of mtDNA, in conjunction with RFLP striction of the linked sites that characterize each hap- haplotype data from the entire mtDNA, have revealed logroup. The absence of haplogroup V in the prehistoric the existence of European-speci®c haplogroups (H±K, T, samples analyzed con¯icts with the hypothesis proposed V, and W), whereas other haplogroups (such as U and by Torroni et al., in which haplogroup V is considered X) are shared between Europeans and Africans and be- tween Europeans and modern Amerindians, respectively as an mtDNA marker for a major Paleolithic population (Torroni et al. 1996, 1998). expansion from southwestern Europe, occurring In a recent paper, Torroni et al. (1998) analyzed the »10,000-15,000 years before the present (YBP). Our distribution and frequency of haplogroups V and H in samples from the Basque Country provide a valuable a wide range of modern populations from Eurasia. On tool for checking the previous hypothesis, which is based the basis of analysis of the frequency and extent of var- on genetic data from present-day populations. In light iation accumulated within haplogroup V, these authors of the available data, the most realistic scenario to ex- suggested that this haplogroup is most likely to have plain the origin and distribution of haplogroup V sug- originated 10,000±15,000 years before the present gests that the mutation de®ning that haplogroup (4577 (YBP) in a region covering the northern part of the Ibe- NlaIII) appeared at a time when the effective population rian Peninsula and the southwestern part of France. Sim- size was small enough to allow genetic drift to actÐand ilarly, they proposed that haplogroup V might constitute that such drift is responsible for the heterogeneity ob- a marker of a major Paleolithic expansion from south- served in Basques, with regard to the frequency of hap- western Europe, which took place after the Last Glacial logroup V (0%±20%). This is compatible with the at- Maximum. Thus, the Paleolithic population of south- tributed date for the origin of that mutation western Europe would have contributed extensively to (10,000±15,000 YBP), because during the postglacial the mitochondrial gene pool of all central and northern period (the Mesolithic, »11,000 YBP) there was a major European populations, including the Saami and the demographic change in the Basque Country, which min- Finns. imized the effect of genetic drift. This interpretation does Interdisciplinary studies involving mtDNA sequences, not rely on migratory movements to explain the distri- RFLP data, and archeological ®ndings can provide a bution of haplogroup V in present-day Indo-European better insight into population history (Bertranpetit et al. populations. 1995; Richards et al. 1996). Still, several points remain to be resolved satisfactorily. At the genetic level, one of Received September 10, 1998; accepted for publication April 23, the main dif®culties is the estimation of mutation rates, 1999; electronically published June 3, 1999. which vary considerably in different regions of the mi- Address for correspondence and reprints: Dr. C. de la Ru a, Euskal tochondrial genome (Howell et al. 1996; Mumm et al. Herriko Unibertsitatea (UPV/EHU), Animali Biologia eta Genetika 1997; Parsons et al. 1997). Archaeologically, hypotheses Saila, Zientzi Fakultatea, P.O. Box 644, 48080 Bilbao, Spain. E-mail: based on migrational events (Otte 1990) are losing [email protected] q 1999 by The American Society of Human Genetics. All rights reserved. ground, since it has become dif®cult to sustain the idea 0002-9297/99/6501-0026$02.00 that the presence of similar artifacts in distant popula- 199 200 Am. J. Hum. Genet. 65:199±207, 1999 tions can always be interpreted in migrational terms (Clark and Lindly 1991). Thus, despite the great many recent theoretical advances in population genetics (Slat- kin and Hudson 1991; Rogers and Harpending 1992; Harpending et al. 1993), the possibility of extracting and characterizing DNA from the remains of prehistoric hu- mans themselves constitutes a unique and highly valu- able contribution to the debate on the origin of the ge- netic diversity of human populations. In this study we report the frequency of mtDNA hap- logroups in four prehistoric populations of the Basque Country that are distributed over different regions of the Basque area (®g. 1). Here, we will focus on the analysis of haplogroup V in prehistoric Basque populations; this will enable us to discuss the recently proposed value of this haplogroup as a marker for a major Paleolithic ex- pansion from southwestern Europe (Torroni et al. 1998). Figure 1 Geographic map of Basque Country, showing locations and radiocarbon dates of periods of occupation of prehistoric sites. Subjects and Methods removed by a saw in a sterile environment and then were The sample considered in our work is large (121 teeth) subjected to a DNA-extraction procedure. and covers different regions of the Basque Country (®g. DNA extraction was performed according to the phe- 1). Two of the sitesÐSan Juan Ante Portam Latinam nol-chloroform method (Hagelberg and Clegg 1991). In (SJAPL) (Araba) (Etxeberria and Vegas 1988) and Lon- brief, the roots of the teeth were incubated overnight at gar (Nafarroa) (Armendariz and Irigarai 1995)Ðare lo- 377C, with agitation in a lysis buffer (0.5 M EDTA pH cated in the southern part of the Basque Country, 8.0±8.5, 200 mg proteinase K/ml, 0.5% SDS, 50 mM whereas the othersÐPico Ramos (Bizkaia) (Zapata Tris-HCl), followed by one extraction with phenol and 1995) and Urratxa (Bizkaia) (MunÄ oz and Berganza one ®nal extraction with chloroform. The DNA extract 1997)Ðlie in the Cantabrian fringe. Habitation of these obtained was concentrated and puri®ed by means of sites has been chronologically dated as having occurred Centricon-30 columns, down to a volume of 300 ml, between the Neolithic and the Bronze Age. Radiocarbon which was stored at 2207C, as three aliquots of 100 ml dates of the occupation periods, as well as the geographic each. location of the prehistoric sites analyzed in this work, In each extraction round, a control sample (a sample are presented in ®gure 1. with no tissue) was also included and was processed Before molecular analysis, all the fossil teeth recovered similarly. The DNA extraction and ampli®cation of the were classi®ed by an expert in dental anthropology, ac- ancient DNA (aDNA) samples were performed in a ded- cording to type (incisors, canines, premolars, and mo- icated laboratory in which no work with modern DNA lars), side, and jaw (upper/lower). This allowed us to had been performed. All the materials and work surfaces choose the single dental piece that maximized the sample used were routinely washed with bleach and alcohol size and thus to avoid the possibility of analyzing the (Prince and Andrus 1992) and were irradiated with UV same individual twice. Genetic analysis of this prehis- light of short wavelength (Fox et al. 1991). toric material was performed only on intact permanent We typed these prehistoric samples in order to identify teeth not showing any cracks or caries, so that the dental those haplogroups previously de®ned by Torroni et al. pulp was preserved from possible contaminants. For that (1996, table 3) in modern DNA. We typed those nucle- reason, only 121 canine teeth from a total of 1,099 were otide positions strictly necessary to correctly identify the used for analysis. The most suitable tooth was selected nine haplogroups characteristic of Caucasians: 7025 at each site, on the grounds of frequency and conser- AluI, 4577 NlaIII, 8249 AvaII, 9052 HaeII, 8994 HaeIII, vation; for instance, at site SJAPL (Araba) it was the 12308 HinfI, and 13704 BstNI. Table 1 shows the prim- right maxillary canine, and 63 intact teeth were selected ers used to amplify the fragments containing the mu- from the total of 232 right-maxillary canines. Addition- tation that de®nes each haplogroup, as well as the size ally, the selected teeth were subjected to a sterilization of both the amplicon and the restriction fragments. We process based on acids (Ginther et al. 1992), a technique designed all these primers ourselves, except the reverse with a high depurinating power capable of inactivating primer used to detect the mutation in nucleotide 12308 any potential exogenous contaminating DNA present on by HinfI. PCR ampli®cations were performed in a ®nal the surface of the teeth. The roots of the teeth were volume of 35 ml, composed of the following: 1 # ®nal Izagirre and de la Ru a: Absence of Haplogroup V in Prehistoric Basques 201 Table 1