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Marı´a Jose´ Casas,1* Erika Hagelberg,1 Rosa Fregel,2 Jose´ M. Larruga,2 and Ana ... - page 2 / 13

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540 CASAS

ET AL.

Muslim burial areas dating from Almohade times. Their state of preservation is very good and the archaeological documentation is excellent. From the archaeological records there is no doubt about the culture and religion of these individuals, but they are not informative about the biological background of the population. The main objectives of this study are a) to determine the genetic composition of the medieval population, b) to evaluate the impact of North African migrations, and c) to assess the evolutionary changes of the population until present. To this end, osseous and dental remains from 71 medie- val individuals and 108 saliva samples from the present population of the same geographic area were analyzed using mtDNA sequences from the first hypervariable region (HVRI) and diagnostic restriction-fragment poly- morphisms.

MATERIALS AND METHODS Medieval samples

Bone and teeth samples belonging to 71 individuals were collected from the burial sites of El Palenque (45), La Cava (8), and El Castillo (18), of the Islamic medieval town Madinat Baguh (today called Priego de Cordoba). The chronology of the human remains was determined by the archaeological records as well as by calibrated 14C dating of human bones (cal. A.D. 1218, 2r: 1025–(1218)– 1291; and cal. A.D. 1214, 2r: 1022–(1214)–1289). Dates were consistent with the Almohade epoch, and therefore the samples were considered a sole population.

In two of the sites, La Cava and El Palenque, the skel- etons were perfectly individualized and in anatomical connection. At the third site, El Castillo, samples from individualized burials were collected (4), but also teeth from isolated jaws from no individualized complete skele- tons (14).

Femur and tibia in good macroscopic preservation con- ditions, i.e., without fractures and with, at most, moder- ately porous cortices, were the preferential bones selected for sampling. Pieces of 4 cm2 were cut from the front of the shaft in minimum informative areas from an anthropological perspective, i.e., without patho- logical signs and not corresponding to muscular or liga- ment insertion areas. The sampled teeth were taken directly from their alveoli, and for those from incomplete skeletons, only inferior teeth were collected from com- plete jaws, to be sure they belonged to different individu- als. The selected teeth showed no cracks, fractures, or caries lesions.

For some individuals, two bones or teeth of the same skeleton were collected, so that independent extractions could be prepared.

Modern samples

At the ancient DNA laboratory of the University of Oslo (Norway), following the recommendations of Hagel- berg (1994), bone samples were broken into small frag- ments (<1 cm) with a hammer and grounded up in a cryogenic impact grinder (Spex Industries, Edison, NJ) in grinding vials with a capacity for 2–4 g of bone. Grinding vials and impactors of the mill were cleaned with Deconex1 (Borer Chemie AG) after use, to prevent sample carryover. After grinding, bone powder was stored in sterile tubes at 208C. Bone powder (0.8 g) was washed twice by centrifugation with 10 ml of 0.5 M EDTA (pH, 8.5), then set for lysis in 10 ml volume and finally used for a DNA phenol-chloroform extraction (Hagelberg and Clegg, 1991). The aqueous phase was desalted and concentrated using Centriplus1 Centrifugal Filter Devices (Amicon, Millipore) according to the man- ufacturer’s instructions. Several rounds of dilution of the retentate with autoclaved ddH2O and centrifugation were necessary to get a final volume of 100–200 ll with a salt concentration 1 mM. One blank control was included.

At the ancient DNA laboratory of the University of La Laguna (Tenerife, Spain) the whole surface of each bone sample was exposed to UV light for 5 min, while teeth were thoroughly washed with 15% HCl, rinsed with UV- treated ddH2O, and then exposed to UV light for 5 min. Then, the sample (bone or tooth) was placed between two sterilized metal plates and crushed with a hammer (Maca-Meyer et al., 2005). The pieces were kept in 15-ml sterile tubes (Costar). Alternatively, after exposure to UV, and instead of crushing, bone samples were cut with an electric saw, and from inside the section, bone was powdered with a dentist drill. Also alternatively, dental material from inside the crown was powdered, by prac- ticing just a small entrance from the inferior limit of the crown. Powder of bone or teeth was collected in 1.5-ml sterile eppendorf tubes. Then DNA was extracted accord- ing to a modified silica-based protocol (Ho¨ss and P¨a¨abo, 1993; Maca-Meyer et al., 2004). Briefly, 1–2 ml of a com- mercial guanidine thiocyanate solution (DNAzol1; Chomczynski et al, 1997) was added into each tube and incubated at room temperature for 2–3 days. After this incubation, the supernatant was passed through com- mercial silica columns QIAquik1 (Qiagen) (Yang et al., 1998), according to the manufacturer’s instructions. For some samples, part of the bone powder was processed as described, and another part was previously washed with 0.5 M EDTA for 10 min at room temperature, followed by centrifugation at 10,000 rpm for 1 min, before adding DNAzol. The use of alternative extraction protocols allowed us to compare the efficiency of crushing versus powdering as well as EDTA decalcification versus no decalcification. To quantify these alternative procedures, twofold serial dilutions were tested in parallel for posi- tive PCR amplification.

Saliva samples of 108 unrelated individuals who gave their informed consent were collected in FTA cards (FTA1, Whatman). All the donors and their maternal ancestors at least for two previous generations came from Priego de Co´rdoba or nearby villages within 30 km of the town.

Extraction

Prior to extraction, around 1 mm of the surface of the bone sample was removed with a dentist drill to prevent contamination.

Amplification

At the Oslo laboratory, two overlapping fragments of HVRI mtDNA with sizes of 278 and 281 bp, respectively, were amplified from the ancient extracts with primers A1(L15997)/B2(H16237) and A2(L16159)/B1(H16402), kindly provided by Eva Staalstrøm (Forensics Depart- ment, Rikshospitalet, Oslo), and used routinely in Foren- sics. A1 and A2 were as described by Wilson et al. (1995), while the sequence of B1 is TGATTTCACGGAG- GATGGT and that of B2 is CTTTGGAGTTGCAGTTGAT,

American Journal of Physical Anthropology—DOI 10.1002/ajpa

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