Supplementary material 2
Animal dental calculus from Sima del Elefante
The Sima del Elefante sedimentary sequence is divided into 16 lithostratigraphic units, which range from TE7 to TE21 (from bottom to top) (Rosas et al., 2006). Unit TE9 has been described as consisting of brown mud with subangular blocks in beds that markedly slope to the north (Parés et al., 2006). Unit TE9 comprises, from bottom to top, three subunits: TE9c, TE9b, and TE9a. The subunit TE9c—which is the focus of the current research—comprises weathered limestone blocks whose decomposition sometimes affects the fossil record in contact with them (Huguet et al., in press).
A great diversity of faunal taxa—including amphibians, reptiles, fish, birds, and large and small mammals—has been documented in subunit TE9c (Table 1). Large-mammal specimens are less abundant than small-mammal specimens; however, the taxonomic diversity therein (in terms of both carnivores and ungulates) is high.
Table 1. List of vertebrates identified in level TE9c (modified from Huguet et al., in press).
Primates / Homo sp., CercopithecidaeUngulates/Subungulates / Mammuthus sp.; Sus sp.; Bison menneri; Dama vallonnetensis, Eucladoceros giulii, Stephanorinus sp., Equus altidens
Carnivores / Panthera gombaszoegensis, Lynx sp. Pannonictis cf. nestii, cf. Baranogale antiqua, Mustela cf. palerminea/praenivalis, Canis cf.mosbachensis/arnensis,Vulpes cf. alopecoides, Ursus cf. dolinensis
Osteichthyes / Salmo sp., Leuciscus sp.
Anura / Alytes obstetricans, Pelobates cultripes, Pelodytes punctatus, Bufo bufo, Bufo calamita, Hyla arborea, Rana sp., cf. Pelophylax
Caudata / Salamandra salamandra, Triturus cf. marmoratus
Squamata / Anguis fragilis, Natrix cf. natrix, Natrix cf. maura, Coronella cf. girondica, Vipera sp., Lacerta genus incertae sedis
Aves / Galliformes indet., Corvus antecorax, Corvus frugileus, Columba livia/olea, Haliaeetus albicilla, Falco cf.tinnunculus, Carduelis chloris,Remiz sp., Certhia sp., Turdus sp., Pica pica, Perdix paleoperdix, Passeriformes indet.
Chiroptera / Myotis gr. myotis/blythii, Miniopterus schreibersii, Rhinolophus ferrumequinum, Rhinolophus gr. euryale/mehelyi, Pipistrellus sp., Chiroptera indet.
Eulipotyphla / Sorex gr. runtonensis-subaraneus, Sorex margaritodon, Asoriculus gibberodon, Beremendia fissidens, Crocidura kornfeldi, Galemys cf. kormosi,Talpa cf. europaea, Erinaceus cf. praeglacialis
Rodentia / Sciurus sp., Castillomys rivas, Apodemus sp., Eliomys sp., Allophaiomys lavocati, A. burgondiae, A. nutiensis, Arvicola jacobaeus, Ungaromys nanus,Pliomys cf. simplicior, Castor sp.
Lagomorpha / Lepus terraerubrae, Oryctolagus lacosti
Samples of animal dental calculus, and sediment,were extracted to use as controls in the analysis of a sample of hominin dental calculus from TE9. In addition, animal dental calculus is a potentially useful tool to complement paleoenvironmental studies at the site, including analyses of tooth meso and micro-wear patterns, since the calculus can reveal accumulative components of animal diet, and therefore provide valuable data used to conduct environmental analyses.
In our search for dental calculus, we examined all the mandibles, maxillae, and isolated teeth of large and medium-sized ungulates from the subunit TE9c. In total, 159 specimens were examined, and eight were selected for sampling, ATA08 TE9c E31 24, ATA08TE9cH287, ATA09 TE9c K29 29, ATA10 TE9c K28 5, ATA10 TE9c L30 2, ATA10TE9cI2829, ATA11TE9cG3038, and ATA12 TE9c H27 20. These specimens,all equid and cervid individuals, were selected on the basis of their preservation condition and amount of calculus deposition (see some examples in Figure 1). All come from the subunit TE9c from which the hominin mandible was retrieved.
Figure 1. Examples of deer mandibles showing dental calculus from subunit TE9c: (A) ATA08 TE9c H28 7; (B) ATA09 TE9c K29 29.
The dental calculus sampling took place in a direct manner, through the use of dental instruments (probe with three angles); in no case was there any damage to any tooth surface. Several studies have demonstrated that calculus sampling does not macroscopically nor microscopicallydamage the tooth (Henry and Piperno, 2008). Some of the specimens showed relatively large areas with dental calculus in the enamel folds; this was especially the case with deer premolars and molars. In these cases, part of the calculus was left in situ for further analysis. Generally, 2–4 mm2 of calculus was extracted per specimen. The teeth were photographed before and after extraction. Extraction took place onto aluminium foil, which was folded and inserted individually into Eppendorf tubes. The samples remained in these tubes until their extraction for analysis at the Archaeobotany Laboratory of the Universitat Autònoma de Barcelona (UAB) in Spain. Each sample of animal dental calculus was split in two for various analyses to be conducted on each piece. In the case of the largest samples (i.e. ATA08 TE9c H28 7 and ATA11 TE9c G30 38), one part of the calculus was selected for chemical analysis that used sequential thermal desorption–gas chromatography–mass spectrometry (TD–GC–MS) and pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) (see S1). Samples were degraded for microscopic microfossil analysis in the Archaeobotany Laboratory of the UAB, where they were also weighed with a microbalance (with samples ranging from 0.0018 g to 0.0133 g); they were then mounted on glass slides and analysed for their surviving content. Some studies have highlighted the risks of laboratory contamination from modern plant microremains (e.g. Crowther et al., 2014). However, the UAB Archaeobotany Laboratory follows a weekly laboratory cleaning regimen; in addition, all work surfaces were wiped with hot water and caustic soda (5% Sodium hydroxide [NaOH]) prior to sample processing. We processed and analysed the animal calculus on the same day, to mitigate even further the possibilities of environmental contamination or of sample destruction. Additionally, two contamination control samples were placed in the laboratory and examined at regular intervals.
The samples were dissolved in a 10% solution of HCl for 50min. After visual inspection, two of the samples (ATA10 TE9c L30 2 and ATA11 TE9c G30 38) were centrifuged at room temperature at 13,000 rpm for 15 min. The remaining samples (ATA08 TE9c E31 24, ATA08TE9c H28 7, ATA09 TE9c K29 29, ATA10 TE9c K28 5, ATA10 TE9c I28 29, and ATA12TE9cH27 20) were left for an additional 15 min (i.e. 65 min in total). After this time, the remaining samples were subjected to the same procedure, and then rinsed twice in distilled water. All pellet material was mounted on microscope slides, in 50% glycerol and water. The slides were sealed and left for 2 hours. Microscopic analysis was carried out with an Olympus IX 71 inverted microscope, using magnifications between 50× and 200×; images were captured with a Colour View camera and Cell D imaging system. All visible material was counted and photographed, to be subsequently compared to a reference collection of microfossils from modern plants. Some minute portions of calculus were not completely dissolved (see Figure 2), and so a second microscopic analysis was later conducted. Following the methodology of Hardy et al. (2016), part of the sealant was removed and a 5% solution of HCl was injected into the slide, with the objective of dissolving the calculus matrix under controlled conditions. This process generated some small bubbles, but they were not an impediment to analysis.
We retrieved microremains from animal dental calculus, albeit in very low quantities. It is worth mentioning the presence of bimodal starches (most probably plants in the grass family Poaceae) in ATA08 TE9c H28 7, and a small number of micro-charcoal fragments in samples ATA08 TE9c H28 7, ATA10 TE9c I28 29, ATA12 TE9c H27 20, and ATA10TE9cL302.
Supplementary references
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Hardy, K., Radini, A., Buckley, S., Sarig, R., Copeland, L., Gopher, A., Barkai, R., 2016. Dental calculus reveals inhaled environmental contamination and ingestion ofessential plant-based nutrients at Lower Palaeolithic Qesem Cave Israel. Quat. Int. 398, 129-135
Henry, A.G., Piperno, D.R., 2008. Using plant microfossils from dental calculus to recover human diet: a case study from Tell al-Raqa’i, Syria. J. Archaeol. Sci. 35, 1943-1950.
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