Magazine R629

pool [1,6]. In addition, a low previously described for other Correspondences genetic diversity was observed Neandertals. among Neandertals, similar to Genealogical analysis that observed among modern of the mtDNA Mitochondrial [5]. With the gradual sequences with a reduced accumulation of more Neandertal median network has shown two DNA of an Iberian sequences, it should be groups of sequences within the Neandertal possible to start exploring the Neandertal mtDNA variation phylogeography of Neandertals. [4]. Some haplotypes, those suggests a El Sidrón is a karstic of Mezmaiskaya and Monte system in Asturias (Northern Lessini, are clearly more diverse population ), where some Neandertal and branch near the root in the bones were accidentally genealogy. Both have an A at affinity with discovered by cavers in 1994. position 16,078 and T at position other European The site has been under 16,154 of the HVR1, while the systematic excavation since rest of the sequences are more Neandertals 2000 [9] and has yielded several similar to each other and share hundred skeletal remains, a G in position 16,078 and a C in Carles Lalueza-Fox1,*, belonging to at least eight position 16,154; these positions Johannes Krause2, David hominids. Most remains were are very stable in modern Caramelli3, Giulio Catalano3,4, found at the Galería del Osario humans and, therefore, not prone Lucio Milani3, María Lourdes (43º 23’ 01’’ N, 5º 19’ 44’’ W), to recurrent mutations. The Sampietro4, Francesc Calafell4, which consists of a small described El Sidrón sequence Cayetana Martínez- Maza5, lateral gallery leading around belongs to the latter group, Markus Bastir5, Antonio 220 meters deep into the karst along with Feldhofer 1 and 2 García-Tabernero5, Marco de la system. Previously, one tooth and Vindija 75–80. Placing these Rasilla6, Javier Fortea6, Svante was subjected to ancient DNA data within the framework for Pääbo2, Jaume Bertranpetit4 analysis and yielded a short the post-glacial recolonisation and Antonio Rosas5 47 bp long Neandertal mtDNA of Europe [11] suggests that sequence [10]. Although Central European Neandertals this suggested that the cave (including those from ) Mitochondrial DNA (mtDNA) environment was favourable to along with the Iberian Peninsula sequences have been retrieved ancient DNA preservation, the Neandertals might represent so far from twelve Neandertal level of DNA fragmentation and a relatively homogenous ( neanderthalensis) the low ratio of endogenous genetic group with a common specimens: Feldhofer 1 and 2 versus contaminant sequences in [1,2], Mezmaiskaya (around 5%) prevented the 1111111111111111111111111111111111111111 in [3], Monte Lessini retrieval of more genetic data. To 6666666666666666666666666666666666666666 0000111111111111112222222222222233333334 in [4], Vindija 75, 77 and lower the rate of contamination 3789001123455678880233445566679912466680 7863781299846982399304346823681910412940 80 in Croatia [5,6], with DNA, an adult femur b and in [6,7], fragment designated for ancient La Chapelle-aux- Saints and DNA analysis was excavated

Rochers de Villeneuve in in September 2005 under strict CRS AATTCCCCGACTGCTAATTCACTGCAC-CCCATCCGTGGC FE1 GG.CTTTTATTC.T..CCCTGT.AAGTA.T.GCT..C..T FE2 GG...... ATTC.T.CCCCTGT.AA.TA.T.GCT..C.. [6,8] and Sidron 441 in Spain [9]. clean conditions and kept frozen MEZ .C.....ATT.AT.CCCCTGT.AA.TA.T.GCTT.C. MLS G...... ATT..T.CCCCTGT.AAGTA....CT.A.AAT There are only six Neandertal at -20ºC. To our knowledge, this V75 ...... ATTC.T.CCCCTGT.AAGTA.T.GCT..C. V80 GG...... ATTC.T.CCCCTGT.AAGTA.T.GCT..C. sequences (two from Feldhofer, is the first time that a Neandertal SI2 G...... ATTC.TCCCCCTGT.AAGTA.T.GCT..C. SCL TGTCAA.TATTTGCT V77 T.AAG two from Vindija, one from sample has been excavated with SI1 T.AAGTA. ENG T.AA. Mezmaiskaya and one from precautions specifically designed RVI T.AA. Monte Lessini) that cover a to prevent contamination and CHP T.AA. significant section (>300 bp) of further DNA damage. the hypervariable region 1 (HVR1) The sequence obtained by Current Biology of the mtDNA. Here we report the amplification, cloning and seventh extensive HVR1 mtDNA sequencing of overlapping Figure 1. Genetic diversity of known Neandertals. sequence of a Neandertal. The fragments (Supplemental data) specimen stems from El Sidrón ranges from positions 16,076 Variable sites in the comparison of the Cambridge reference sequence (CRS) cave (Asturias, North of Spain) to 16,378 (Figure 1) of the and 12 Neandertal sequences, five of and was radiocarbon dated to mitochondrial genome and is them represented by short fragments. around 43,000 years of age [9]. similar, but not identical, to Abbreviations: CHP = La Chapelle aux The retrieval and the analysis previously published Neandertal Saints; ENG = Engis 2, FE1 = Feldhofer of Neandertal mtDNA sequences HVR1 sequences. Compared 1, FE2 = Feldhofer 2, MEZ = Mezmais- have allowed the exclusion of to the Cambridge reference kaya, MLS = Monti Lessini, RVI = Ro- chers de Villeneuve, SCL=Scladina, SI2= the possibility of a mitochondrial sequence for modern humans El Sidrón bone (present study), SI1 = El contribution of Neandertals (CRS), it carries one substitution Sidrón 441, V75 = Vindija 75, V77 = Vin- to the modern human gene (C in 16,178) that was not dija 77, V80 = Vindija 80. Current Biology Vol 16 No 16 R630

demographic history that is 3. Ovchinnikov, I.V., Götherström, A., Romanova, G.P., Kharitonov, V.M., A highly divergent probably different to that of other Lidén, K., and Goodwin, W. (2000). Neandertal groups placed in Molecular analysis of Neandertal DNA mtDNA sequence Eastern Europe and in the Italian from the northern Caucasus. Nature 404, 490–493. Peninsula. 4. Caramelli, D., Lalueza-Fox, C., Condemi, in a Neandertal The most recent common S., Longo, L., Milani, L., Manfredini, A., ancestor of the seven Neandertal Saint Pierre, M. de., Adoni, F., Lari, M., individual from Giunti, P., et al. (2006). A highly divergent sequences has been estimated mtDNA sequence in a Neandertal Italy to be around 250,000 ± 65,000 individual from Italy. Curr. Biol. 16, years of age (assuming an age R630–R632. 5. Krings, M., Capelli, C., Tschentscher, 1 of around 40,000 years for F., Geisert, H., Meyer, S., von Haeseler, David Caramelli , Carles these specimens) by coalescent A., Grossschmidt, K., Possnert, G., Lalueza-Fox2, Silvana Condemi3, methods (Supplemental data). Paunovic, M., and Pääbo, S. (2000). A Laura Longo4,5, Lucio Milani1, view of Neandertal genetic diversity. Nat. 1 The clade containing the Genet. 26, 144–146. Alessandro Manfredini , Michelle four most similar sequences 6. Serre, D., Langaney, A., Chech, M., de Saint Pierre2, Francesca Teschler-Nicola, M., Paunovic, M., Adoni1, Martina Lari1, Paolo (Feldhofer 1 and 2, Vindija Mennecier, P., Hofreiter, M., Possnert, 5 4 75-80 and El Sidrón, between G., and Pääbo, S. (2004). No evidence Giunti , Stefano Ricci , Antonella positions 16,076-16,378) of Neandertal mtDNA contribution to Casoli6, Francesc Calafell7, early modern humans. PLoS Biol. 2, 8 sharing the G16,078-C16,154 313–317. Francesco Mallegni , Jaume haplotype, has been estimated 7. Orlando, L., Darlu, P., Toussaint, M., Bertranpetit7, Roscoe Stanyon1, Bonjean, D., Otte, M., and Hänni, C. to be 130,000 ± 30,000 years of Giorgio Bertorelle9 and Guido (2006). Revisiting Neandertal diversity 9, age. Interestingly, this date is with a 100,000 year old mtDNA Barbujani * roughly concordant with the end sequence. Curr. Biol. 16, R400. 8. Beauval, C., Maureille, B., Lacrampe- of a dramatic glacial maximum Cuyaubere, F., Serre, D., Peressinotto, around 135,000 years ago [12]. D., Bordes, J.G., Cochard, D., Couchoud, Neandertals are documented in I., Dubrasquet, D., Laroulandie, V., Therefore, it seems that, despite et al. (2005). A late Neandertal femur Europe and Western Asia from the broad geographic range, the from Les Rochers-de-Villeneuve, about 230,000 to 29,000 years El Sidrón, Feldhofer and Vindija France. Proc. Natl. Acad. Sci. USA 102, ago. Analyses of mitochondrial 7085–7090. Neandertals may belong to the 9. Lalueza-Fox, C., Sampietro, M.L., DNA (mtDNA) from Neandertal same group of Neandertals that Caramelli, D., Puder, Y., Lari, M., samples [1,2] and other analyses Calafell, F., Martinez-Maza, C., Bastir, expanded from a hypothetical M., Fortea, J., de la Rasilla, M., et al. [3–5] appear incompatible with southern glacial refugium (2005). Neandertal evolutionary genetics; the hypothesis that Neandertals after a demographic collapse mitochondrial DNA data from the are direct ancestors of modern Iberian Peninsula. Mol. Biol. Evol. 22, associated with this glacial 1077–1081. Europeans [6,7]. However, there maximum. 10. Fortea, J., de la Rasilla, M., Martínez, are broad geographic gaps in E., Sánchez-Moral, S., Cañaveras, J.C., the sampling of Neandertal DNA Cueva, S., Rosas, A., Soler, V., Julià, R., diversity. Here, we describe Acknowledgments de Torres, T., et al. (2003). La Cueva de El Sidrón (Borines, Piloña, Asturias): the sequence of the first This study has been financed by Primeros resultados. Estud. Geol. 59, mitochondrial hypervariable region ­Consejería de Cultura del Principado 159–179. de Asturias-Universidad de Oviedo 11. Hewitt, G. (2000). The genetic legacy of the Quaternary ice ages. Nature 405, 111111111111111111111111111111111111 1 (CN-00- 184-D3; CN-01-132,133,134- B1; 907–913. 666666666666666666666666666666666666 6 000011111111111111222222222223333333 4 CN-04-152), Ficyt-Consejería de 12. Petit, J.R., Jouzel, J., Raynaud, D., 378900112345568888023345566791246668 0 Barkov, N.I., Barnola, J.-M., Basile, I., 786378129984692379930446823891041294 0 Cultura (FC- 02-PC-SPV01-27) and b Bender, M., Chapellaz, J., Davis, M., by the Min is try of Education and Delaygue, G., et al. (1999). Climate CRS AATTCCCCGACTGCAACTTCACGCAC-CATCCGTGG C Science of Spain to C.L.F. and J.B. mtDNAEve ...... A...... TC.TG.....-T.C...... and atmospheric history of the past FE1 GG.CTTTTATTC.T.C.CCTGTAAGTATGCT..C.. T (BFF2002- 10206- E and CGL2006-03987). 420,000 years from the Vostok ice core, FE2 GG...... ATTC.TCC.CCTGTAA.TATGCT..C.. ? Antarctica. Nature 399, 429–436. MEZ ?.C.....ATT.ATCC.CCTGTAA.TATGCTT.C.? ? MLS G...... ATT..TCC.CCTGTAAGTA..CT.A.AA T V75 ??...... ATTC.TCC.CCTGTAAGTATGCT..C.? ? V80 GG...... ATTC.TCC.CCTGTAAGTATGCT..C.? ? Supplemental data V77 ????????????????.????TAAG??????????? ? 1 SID ????????????????.????TAAGTA????????? ? Supplemental data are available at Secció Antropologia, Facultat de ENG ????????????????.????TAA.??????????? ? Biologia, Universitat de Barcelona, RIV ????????????????.????TAA.??????????? ? http://www.current-biology.com/cgi/ CHP ????????????????.????TAA.??????????? ? Barcelona, Spain. 2Department content/full/16/16/R629/DC1/ of Evolutionary Genetics, Max Current Biology Planck Institute for Evolutionary , , Germany. Figure 1. Neandertal diversity. References 3Dipartimento di Biologia Animale e Comparison of the Cambridge reference 1. Krings, M., Stone, A., Schmitz, R.W., Genetica, Laboratorio di Antropologia, sequence (CRS) and the putatively an- Krainitzki, H., Stoneking, M., and Pääbo, Università di Firenze, Firenze, cestral sequence to all extant human S. (1997). Neandertal DNA sequences 4 and the origin of modern humans. Cell Italy. Unitat de Biologia Evolutiva, variation (mtDNA Eve) with 11 Neandertal 90, 19–30. CEXS, Universitat Pompeu Fabra, sequences, five of them represented by 2. Schmitz, R.W., Serre, D., Bonani, G., Barcelona, Spain. 5Departamento short fragments. CHP = La Chapelle aux Feine, S., Hillgruber, F., Krainitzki, de Paleobiología, Museo Nacional Saints, CRS = Cambridge reference se- H., Pääbo, S., and Smith, F.H. (2002). de Ciencias Naturales, CSIC, quence, ENG = Engis 2, FE1 = Feldhofer The Neandertal site revisited; Madrid, Spain. 6Área de Prehistoria, 1, FE2 = Feldhofer 2, MEZ = Mezmaiskaya, interdisciplinary investigations of skeletal remains from the Neander Valley, Departamento de Historia, Universidad MLS = Monti Lessini, RIV = Rochers de Germany. Proc. Natl. Acad. Sci. USA 99, de Oviedo, Oviedo, Spain. ­Villeneuve, SID = El Sidrón 441, V75 = Vin- 13342–13347. *E-mail: [email protected] dija 75, V77 = Vindija 77, V80 = Vindija 80.