The first Neanderthal tooth found north of the Carpathian Mountains

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1 Author s remark: mind the fact this paper was published in the year Since that time, after several seasons of excavation and laboratory work our knowledge about the site expanded significantly. It is reflected by example by the partial redefinition of the stratigraphy and dating, which basically locate the discovered Neanderthal layers (D1, D2 and D3) inside the MIS-3 stage, not MIS-5 as it was preliminary suggested in this publication. The final publication is available at Naturwissenschaften Short Communication Mikołaj Urbanowski 1, Paweł Socha 2, Paweł Dąbrowski 3, Wioletta Nowaczewska 3, Anna Sadakierska-Chudy 4, Tadeusz Dobosz 4, Krzysztof Stefaniak 2, Adam Nadachowski 2,5 The first Neanderthal tooth found north of the Carpathian Mountains 1 Department of Archaeology, Institute of History and International Relations, Szczecin University, ul. Krakowska 71-79, Szczecin, Poland. 2 Department of Palaeozoology, Institute of Zoology, Wrocław University, ul. Sienkiewicza 21, Wrocław, Poland. 3 Department of Anthropology, Wrocław University, ul. Kuźnicza 35, Wrocław, Poland. 4 Molecular Techniques Unit, Institute of Forensic Medicine, Wrocław Medical University, ul. M. Curie-Skłodowskiej 52, Wrocław, Poland. 5 Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, ul. Sławkowska 17, Kraków, Poland. 1

2 Abstract An upper second permanent molar from a human was found alongside numerous tools of the Micoquian tradition and was excavated in Stajnia Cave, which is located over 100 km north of the Carpathian Mountains in southern Poland. The age of these finds has been established within a time-span of late Saalian to early Weichselian, most likely to OIS 5c or 5a, according to the palaeontological, geological, archaeological and absolute dating of the layer from which they were obtained. An examination of the morphology of the human molar indicates that this tooth exhibits many traits frequently occurring in Neanderthal upper molars. Although the occurrence of derived Neanderthal traits in the Stajnia molar cannot be firmly established because of degradation of its cusps, the presence of the above-mentioned features allows the assertion that this tooth belonged to a Neanderthal. The age of the Stajnia tooth and the archaeological context of this find also indicate that this molar is of Neanderthal origin. Keywords Homo neanderthalensis molar Micoquian Poland 2

3 Introduction Based on the currently known distribution of Late Middle Palaeolithic (LMP) industries, the geographical range of Neanderthals in Central Europe also includes an area north of the Carpathians. However, despite a long tradition of archaeological research in this region, there has been no direct evidence until now of the presence of Homo neanderthalensis in this part of Europe (i.e., osteological remains or mtdna). We report that excavations started in 2007 at a new LMP site in the Stajnia Cave (southern Poland, 50 36'58" N, 19 29'04" E) have yielded a single right upper second permanent human molar and a wide range of archaeological evidence of human activity, including thousands of animal bones and flint artefacts. A preliminary analysis of two other human teeth (S4300 and S4619) discovered in the LMP layers, although not as thorough as the study of the molar described here (S5000), suggests that the total number of Neanderthal remains may be increased to three individuals. Most of the items, including the molar and another presumably Neanderthal tooth, were found in a structure located in a shallow depression under the cave wall that was filled in with artefacts and bone remains during the deposition of the D1 (LMP) layer (see Electronic supplementary material (Esm) 1, The Stajnia Cave stratigraphy, and 2, The Chronology of the Stajnia Cave finds ). This layer was dated by geological and faunal analyses and was found to be in accordance with the archaeological age of the finds and the preliminary absolute dating. These analyses included AMS radiocarbon dating of a cave bear bone (Ursus spelaeus), which yielded a date of >49,000 years BP (Poz-28892, 2.6% N and 4.1% C). Other faunal evidence is typical of the early Weichselian (Vistulian) OIS 5d-a or late Middle Pleistocene cold stage (OIS 6) period, with Dicrostonyx gulielmi and Lemmus lemmus dominating. Cave bear and the other animals typical for that period, including reindeer, represent large fauna (see Esm 3, Faunal assemblage ). 3

4 The cave was used for a wide range of activities, including lithic tool production. The traces of flint raw material storage are particularly interesting, as the cave infillings yielded dozens of flint nodules that must have been collected in the neighbourhood of the cave (up to 12 km). The nodules were processed into cores or tools, although some knives and Levallois products were made from high quality flint from the southern part of the Polish Jura, about km from the site (see Esm 4, Methods ; 4.1, Archaeological analyses ; 4.2, Geological analyses ). The assemblage is dominated by bifacial and unifacial asymmetrical knives of the Micoquian tradition (Keilmessergruppe), representing relatively high level of reduction. Archaeological finds suggest long periods of human occupation, while the technostylistic characteristics of the assemblage indicates an early stage (KMG-A according to Jöris 2006) of LMP Micoquian industries, dating the occupation of the site to the beginning of the last glaciation (Brörup Interstadial). Therefore, the assemblage fits well into local and regional LMP sequences (Schild 2005). Description and discussion The human tooth from Stajnia has four cusps and three roots (Fig. 1a,b). The middle and apical parts of the mesial and distal roots are broken off. The tooth was determined to be a right M 2 (see Esm 4.3, Palaeoanthropological analyses ). Despite functional abrasion of the Stajnia molar crown, two buccal cusps are visible: the paracone and a somewhat reduced metacone (primary trigon is well-defined); the largest cusp is the protocone (Fig. 1a). No Carabelli s cusp is present on the mesiolingual surface of the protocone. Although Neanderthal upper second molars typically exhibit Carabelli s cusp with a high frequency relative to contemporary and fossil Homo sapiens (Hs), this cusp was present only in 50% of the Neanderthal M 2 studied by Bailey (2006). The talon bears a large hypocone that scores a 4.0 on the Arizona State University Dental Anthropology System (ASUDAS) scale (Turner et al. 1991). It is worth highlighting that the frequency of occurrence of the large hypocone in 4

5 the M 2 is higher in European Neanderthals than in European Upper Palaeolithic (UP) Hs (Coppa et al. 2005). The mesio-distal (MD) crown diameter and buccolingual (BL) crown diameter of the tooth were determined. The MD diameter (length) of the crown on the Stajnia M 2 is 9.6 mm, and the BL crown diameter (breadth) is 11.0 mm. The original shape of the occlusal surface and the MD diameter were reconstructed and the corrected MD of the Stajnia tooth is mm (see Esm 4, Methods ; 4.3, Palaeoanthropological analyses ). The results of comparing the Stajnia MD and BL diameters with those from samples of hominins are presented in Tables 1S and 2S (see Esm 4.3), and these show that the value of the Stajnia MD diameter is nearest the mean calculated value for this feature in Neanderthals (see Table 1S Esm 4.3) but is also in the range of European Homo heidelbergenis and Palaeolithic Hs; in the case of the Stajnia BL diameter, a greater similarity is observed to the European Neanderthal mean, but its value is in the Middle UPHs range and below the Early UPHs range (see Table 1S Esm 4.3). Simple tooth measurements alone, however, are of limited use in taxonomic studies. Unfortunately, degradation of the cusps of the Stajnia molar prevented the analysis of the cusp angles and the occlusal polygon area as described by Bailey (2004). The relative cusp base areas of the Stajnia tooth were calculated according to the Bailey 2004 method. The pattern of relative cusp sizes established for the Stajnia tooth is protocone > paracone > hypocone > metacone, and this pattern frequently occurs on upper Neanderthal molars (Bailey 2004, 2006). The results of comparing the relative cusp base areas of the Stajnia tooth with samples of hominins are presented in Tables 3S and 4S (see Esm 4.3) and show that the value of the relative size of the Stajnia hypocone is nearest the Neanderthal mean for this trait (see Table 4S - Esm 4.3). The Stajnia M 2 exhibits a mesial interproximal groove, a trait that is characteristic of, but not unique to, Neanderthals (Frayer and Russell 1987; Schmitz et al. 2002). The detailed analysis of the shape and course of this 5

6 groove, including the location of the microscopic striations, indicated that this type of defect is similar to that observed in Neanderthal teeth and also that it was probably made by thin, stiff and hard objects used as toothpicks (Frayer and Russell 1987; Ungar et al. 2001; Schmitz et al. 2002) (Fig. 2a,b) (see Esm 4.3, Palaeoanthropological analyses ). The subvertical grooves are present on the mesial and distal interproximal facets of the Stajnia tooth. These structures are present on most Neanderthal posterior teeth but are not specific only for this hominin (Villa and Giacobini 1995). The results of the scanning electron microscope (SEM) observation of the subvertical grooves morphology (see Esm 4.3 for the description of these structures and interproximal facets of the Stajnia tooth) clearly indicate the resemblance to Neanderthal subvertical grooves (see Fig. 2c,d). The degree of abrasion on the occlusal surface of the crown is 3 on Molnar s scale (Molnar 1971), as modified by Trinkaus (1995), and as Neanderthals are suggested to have a faster rate of enamel formation (Smith et al. 2007), this indicates that the individual was 20 years of age or slightly more at the time of death. The lingual root of the tooth is intermediate in form between conical and stake-shaped and is more powerfully built than the equivalent lingual root of the typical Hs M 2 (Fig. 1b) (Hillson 1996). This root is 11.8 mm long from cervix to apex. Although it s length is below the Neanderthal range for this trait ( ) and falls in the UPHs range ( ), this trait is not diagnostic in the case of upper molars, thus it cannot definitively show whether the Stajnia tooth belonged to UPHs or Neanderthal (Bailey 2005). The results of precise analysis of the radiographs of the Stajnia molar, including the recording of the data used to calculate the Taurodontism Index (TI), indicate that the Stajnia tooth exhibits a low pulp chamber (non-taurodont) (see Esm 4.3 for detailed description of the method). Taurodontism is commonly regarded as a characteristic of Neanderthals. However, the lack of this trait is also observed in some Neanderthal M 2 (e.g., the Feldhofer specimen 6

7 NN31) (Schmitz et al. 2002), and the presence of this feature is highly variable in Neanderthal maxillary teeth (Skinner and Sperber 1992). In general, the Stajnia tooth shows a combination of characteristics frequently occurring in Neanderthal upper molars, such as a hypocone ASUDAS grade more than three, a relative hypocone base area larger than the metacone, an interproximal groove, subvertical grooves, specific patterns of abrasions on the occlusal surface and an arrangement of interproximal facets (Villa and Giacobini 1995; Lebel et al. 2001; Ungar et al. 2001; Coppa et al. 2005; Bailey 2006). In spite of the fact that the state of preservation of the crown of the Stajnia tooth excludes the possibility of examining the occurrence of derived (diagnostic) Neanderthal traits, such as the Neanderthal-specific upper molar shape of the occlusal polygon and small size of the relative occlusal polygon area (Bailey 2004, 2006), the combination of morphological features (not the presence or absence of particular traits) mentioned above can diagnose this molar as Neanderthal. A sample of DNA was eluted from the Stajnia M 2, using the non-destructive technique of Rohland et al. (2004) with some modifications. The X-Y sex-specific sequence of the amelogenin gene was used to determine gender in the tested sample, as well as in comparative DNA samples obtained from humans, apes and monkeys, according to Zoledziewska and Dobosz (2003) with re-designed primers. The PCR products from each species were distinguished by length. The DNA from the tested teeth produced amplicons of 54/62 bp. This molecular analysis showed that the sample had a male genotype that was not typical of human, chimpanzee or monkey but was of an intermediate length between man and monkey. Another PCR test was performed with oligonucleotide primers designed for Neanderthalspecific mtdna, according to Krause et al. (2007). This PCR produced an 84 bp amplicon, which was directly cloned into the pcr 2.1-TOPO vector for re-sequencing. Unfortunately, 7

8 the poor state of the mtdna preservation rendered it impossible to definitively identity as Neanderthal mtdna (see Esm 4.4, Genetic analyses ). However, the tooth described here and the other two that are still under analysis do not show the most diagnostic skeletal aspects in terms of distinguishing between Neanderthal and AMH remains, but, according to the results obtained so far, they fit well into the pattern expected for Neanderthal remains. Taking into account their archaeological context and dating, the S5000 tooth is most likely the first Neanderthal tooth to be discovered north of the Carpathian Mountains. The significance of this discovery (see Esm 5, Summary ) warrants further research in the Stajnia Cave. Acknowledgements This research was supported by University of Szczecin and University of Wrocław. We thank C. Stringer for his help, R. Kruszynski, T. Compton, E. Trinkaus for their helpful comments, and S. Pääbo and J. Krause for providing the Neanderthal-specific mtdna primers and valuable suggestions. The authors declare no conflict of interest. References Bailey SE (2004) A morphometric analysis of maxillary molar crowns of Middle-Late Pleistocene hominins. J Hum Evol 47: Bailey SE (2005) Diagnostic Dental Differences between Neandertals and Upper Paleolithic Modern Humans: Getting to the Root of the Matter. In: Zadzinska E (ed) Current Trends in Dental Morphology Research. University of Lodz Press, Lodz (Poland), pp Bailey SE (2006) Beyond Shovel-Shaped Incisors: Neandertal Dental Morphology in a Comparative Context. Periodicum Biologorum 108:

9 Coppa A, Grün R, Stringer C, Eggins S, Vargiu R (2005) Newly recognized Pleistocene human teeth from Tabun Cave, Israel. J Hum Evol 49: Frayer DW, Russell MD (1987) Artificial grooves on the Krapina Neanderthal teeth. Am J Phys Anthropol 74: Hillson SW (1996) Dental Anthropology. Cambridge Univ Press, Cambridge Jöris O (2006) Bifacially backed knives (Keilmesser) in the Central European Middle Palaeolithic. In: Goren-Inbar N, Sharon G (eds) Axe Age: Acheulian Tool-making from Quarry to Discard, Equinox Publishing Ltd, London, pp Krause J, Orlando L, Serre D, Viola B, Prüfer K, Richards MP, Hublin JJ, Hänni C, Derevianko AP, Pääbo S (2007) Neanderthals in central Asia and Siberia. Nature 449: Lebel S, Trinkaus E, Faure M, Fernandez P, Guérin C, Richter D, Mercier N, Valladas H, Wagner GA (2001) Comparative morphology and paleobiology of Middle Pleistocene human remains from the Bau de l Aubesier, Vaucluse, France. Proc Natl Acad Sci USA 98: Molnar S (1971) Human tooth wear, tooth function and cultural variability. Am J Phys Anthropol 34: Rohland N, Siedel H, Hofreiter M (2004) Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens. Biotechniques 36: Schild R (2005) The killing fields of Zwoleń. A Middle Paleolithic kill-butchery-site in Central Poland. Institute of Archaeology and Ethnology, Polish Academy of Sciences, Warsaw Schmitz RW, Serre D, Bonani G, Feine S, Hillgruber F, Krainitzki H, Pääbo S, Smith FH (2002) The Neandertal type site revisited: Interdisciplinary investigations of skeletal 9

10 remains from the Neander Valley, Germany. Proc Natl Acad Sci USA 99: Skinner MF, Sperber GH (1992) Atlas of the Radiographs of Early Man. Liss, New York Smith TM, Toussaint M, Reid DJ, Olejniczak AJ, Hublin J-J (2007) Rapid dental development in a Middle Paleolithic Belgian Neanderthal. Proc Natl Acad Sci USA 104: Trinkaus E (1995) Neanderthal Mortality Patterns. J Archaeol Sci 22: Turner II CG, Nichol CR, Scott GR (1991) Scoring procedures for key morphological traits of permanent dentition: The Arizona State University Dental Anthropology System. In: Kelley MA, Larsen CS (eds) Advances In Dental Anthropology. Wiley-Liss Inc, New York, pp Ungar PS, Grine FE, Teaford MF, Pérez- Pérez A (2001) A review of interproximal wear grooves on fossil hominin teeth with new evidence from Olduvai Gorge. Arch Oral Biol 46: Villa G, Giacobini G (1995) Subvertical Grooves of Interproximal Facets in Neandertal Posterior Teeth. Am J Phys Anthropol 96:51-62 Zoledziewska M, Dobosz T (2003) Gender determination in highly degraded DNA samples. Progress in Forensic Genetics 9:

11 Figure Legends Fig. 1 The Stajnia right second maxillary molar. a Occlusal view. Abrasion is visible on the occlusal surface. (Abbreviations: Pa, paracone; Me, metacone; Pa, paracone; Hy, hypocone). b General image (mesial and occlusal views). The buccal roots were broken post-mortem. c Individual cusp base areas of the Stajnia crown Fig. 2 The interproximal groove visible on the mesial surface of the Stajnia molar. a The toothpick groove on the mesial face of the Stajnia M 2. b SEM image of the surface of the mesial wear groove on this molar. The surface exhibits parallel buccolingual striations. c SEM image of the subvertical grooves on the mesial interproximal facet of the Stajnia tooth. The grooves show a radial direction from the occlusal surface. d SEM image of the pitted and eroded surface of the subvertical groove. 11