Queen Nefertari, the favourite Royal Consort of Pharaoh Ramses II (Ancient Egypt, New Kingdom, 19th Dynasty c. 1250 BC) is famous for her beautifully decorated tomb in the Valley of the Queens. Her burial was plundered in ancient times yet still many objects were found broken in the debris when the tomb was excavated. Amongst the found objects was a pair of mummified legs. They came to the Egyptian Museum in Turin and are henceforth regarded as the remains of this famous Queen, although they were never scientifically investigated. The following multidisciplinary investigation is the first ever performed on those remains. The results (radiocarbon dating, anthropology, paleopathology, genetics, chemistry and Egyptology) all strongly speak in favour of an identification of the remains as Nefertari’s, although different explanations—albeit less likely—are considered and discussed. The legs probably belong to a lady, a fully adult individual, of about 40 years of age. The materials used for embalming are consistent with Ramesside mummification traditions and indeed all objects within the tomb robustly support the burial as of Queen Nefertari.

The aim of the research is to answer—via a multidisciplinary approach to a long historical debate–a complex question. Do the human remains found in the tomb belong to Queen Nefertari’s original burial? ( Fig 3 ). The remains of Nefertari are considered as highly important for History and Egyptology since Nefertari is one of the most famous Queens of Ancient Egypt.

Nefertari was the most beloved wife of King Ramses II and played an active role in foreign politics. Her ancestry is unknown. Based on the legible/decipherable inscriptions on a fragment of a faience knob head or pommel found in her tomb, speculations were raised [ 4 , 5 ]. The item carries the throne name ‘Kheper-Kheperu-Ra’ and, is, therefore, connected with King Ay [ 6 ], who ruled Egypt for a few years after Tutankhamun (Turin Mus. Egizio Inv. Suppl. 5162) [ 2 , 7 ]. However, Nefertari did not carry the title ‘Daughter of a King’, which suggests that she was probably not from the main royal line. Because of the chronology, it seems quite unlikely that she was King Ay’s daughter, perhaps she was Ay’s grand-daughter [ 6 , 8 ] ( Fig 1 ). Other scholars emphasize that both Ramses II’s royal wives, Isisnofret [ 9 ] and Nefertari, had a non-royal background [ 10 ]. Nefertari married Ramses when he was crown prince during the reign of his father Sety I. The age at which Ramses II succeeded to the throne of Egypt is uncertain, possibly around his 25 th year [ 10 ]. Nefertari was then presumably the same age as her husband or slightly younger (ca. 20–25 years). She gave birth to four sons (Amun-hir-khepeshef, Pa-Ra-wenem-ef, Mery-Ra and Mery-Atum) and four daughters (Baketmut, Nefertari, Merytamun and Henuttaui). Within the succession line, Nefertari’s sons were always preferred to Queen Isisnofret’s although, in the end, the crown went to Merenptah, a son of Queen Isisnofret. Queen Nefertari, as attested by reliefs, attended the opening ceremony of the rock-cut temples of Abu Simbel in the year 24 of Ramses II’s reign (ca. 1255 BC) ( Fig 2 ) [ 11 ]. After that event, she disappeared. She was absent at the Sed-festival of Ramses II’s 30 th regal year. She probably died around his 25 th year of reign [ 10 ]. As reconstructed from historical records, Nefertari probably reached an age of about 40 to 50 years (minimum 16 + 24 years or maximum 25 + 25 years) whereas Queen Isisnofret I died later, in year 34. Subsequently Ramses II married three of his daughters: Bint-Anat, Merytamun and Nebettaui [ 10 ].

The tomb of Queen Nefertari (QV 66), the second Great Royal Wife of King Ramses II (lifetime ca. 1303–1213 BC), was discovered by Ernesto Schiaparelli (1856–1928) in the Valley of the Queens in 1904. Her burial had been looted in antiquity, so no trace of the original entrance had been preserved. Besides the famous wall paintings, a series of broken remains (e.g. a damaged pink granite sarcophagus, broken furniture, jars, shabtis, other various small items), a pair of sandals and two fragmented mummified legs (parts of tibiae and femora) are preserved. All these items and the human remains are currently housed in the (Museo Egizio Turin, Suppl. 5154 RCGE 14467) [ 1 – 3 ]. (Table A in S1 File ).

Original sample of mummified tissue taken from the interior compartment of femura and tibiae contained 108 mg of material. A sample of 79.1 mg was taken for analysis and treated in a soxhlet system. A sequence of solvents (chloroform, hexane, acetone and ethanol) was used to remove resins and waxes [ 20 ]. The remaining sample with a mass of 61.7 mg underwent modified acid base treatment [ 21 ]. The short treatment in room temperature (instead of 60C) was applied because the material underwent rapid dissolution. Only 20% (i.e. 13.5 mg) of the sample remained after ABA. The remaining material was weight into tin cups for a combustion Elemental Analyser and subsequent graphitization [ 22 ] two targets were prepared from the material: one contained mainly powder of the tissue and the second the remaining of the sample. These were then analysed using MICADAS, which is a dedicated 14 C AMS instrument at the AMS facility, ETH Zurich [ 23 ]. The measured 14 C content (F14C) was normalised to the standard Oxalic Acid 2 corrected for blank values and isotopic fractionation using delta 13 C measured on graphite see Hadjas 2008 [ 21 ]. Radiocarbon ages were calculated following the convention of Stuiver and Polach 1977 [ 24 ]. OxCal program [ 25 ] and INTCAL13 [ 26 ] data set were used to calibrate to calendar ages.

The analyses were performed at the dedicated ancient DNA laboratory at the Institute of Evolutionary Medicine of the University of Zurich. For genetic analysis the samples were cleaned using a 1% bleach solution to remove contaminating DNA from modern individuals that had handled the mummified remains and samples before air drying and crushed in a SPEX freezer mill (6770) to form a fine powder. The DNA was released from the bone powder by decalcification for 48 hours (12 hours 55°C and 36 hours at room temperature) in a 0.45M EDTA (Ethylenediaminetetraacetic acid) solution with 100mg Proteinase K added to remove excess proteins and inhibit enzymatic activity. The DNA was released from the soft tissue using the extraction buffer (10mM Tris-HCl, 10mM NaCl, 5mM CaCl2, 2,5mM EDTA, 2% SDS, 40mM DTT and 100mg/ml Proteinase K) for 18 hours at 55°C. The supernatant, containing released DNA, was then subjected to a Phenol-Chloroform extraction to remove any further proteins (mixed twice with 25:24:1, phenol:chloroform:isoamyl alcohol, and the DNA containing supernatants removed, final wash with chloroform), before being concentrated with a modified QiaQuick PCR purification method (final elution incubation at 37°C for 5 minutes to maximise DNA yield). DNA extracts were subjected to conventional PCR amplification of the HVRI of the mtDNA D-loop with four overlapping primer sets and to a sexing assay using real-time PCR as previously described [ 19 ] Each extract was analyzed for mtDNA data twice and for sexing data three times, and three non-template extraction controls and reagent blanks were processed in parallel with each PCR.

The amorphous organic residues impregnating the textile samples taken from the knee assemblage were chemically characterized and identified using gas chromatography (GC-MS). After initially grinding samples to a fine powder, a weighed amount of these ground samples (from 10–90 mg) was taken. These samples were then extracted with an appropriate volume (0.5–2 mL) of chloroform-methanol solution (2:1 v/v; 3x60 min sonication). After centrifugation (20 min, 1000 rpm) the supernatant solvent was removed from the residue and placed in a vial. The three extracts were combined and the solvent reduced by rotary evaporation. Following transfer of the combined extracts to a screw-capped vial, the remaining solvent was removed by evaporation under a gentle stream of nitrogen at 40°C. The residue was reweighed to give total lipid extracts (TLE). The TLEs were trimethylsilylated using N,O-bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich Chemical Co., St Louis, MO, USA) containing 1% of trimethylchlorosilane (50 μl, 70°C, 1 hour). Excess BSTFA was then removed under a gentle stream of nitrogen and the derivatized sample redissolved in dichloromethane and analyzed by GC-MS. GC-MS analysis of the total lipid extract of each sample was performed on a a Hewlett-Packard 5890 Series II gas chromatograph fitted with a split injector (325°C) interfaced to a Trio 1000 mass spectrometer (electron voltage 70eV, filament current 200 uA, source temperature 170°C, interface temperature 325°C). The acquisition was controlled by Windows based MasSpecII32 Data System, in full scan mode (35–650 amu). Separation was performed on a fused silica capillary column (30 m x 0.25 mm i.d) coated with 0.25 um 5% phenyl methyl polysiloxane (DB-5). Initially the GC was held at 40°C for 5 minutes and then temperature programmed from 40°C-350°C at 8°C min and held at final temperature for 20 minutes (total time 63.75 minutes), with helium as the carrier gas (constant flow 1 ml/min, initial pressure of 45 kPa, splitless injection 1 min). Identification of compounds was achieved on the basis of both their mass spectra (NIST Mass Spectral Database and reference compounds) and relative retention times (relative retention indices (RRIs)).

Sandals (also exhibited in the Turin museum) were found among other objects, such as the fragments of a stone sarcophagus of Nefertari bearing an inscription, 34 wooden shabtis bearing her name, two lids of coffers, fabric, broken pottery and fragments of wooden statues in Turin. Mus. Egizio (List B in S1 File ). Almost all objects are either inscribed with the name of Nefertari or, at least, their styles link them to the 19 th Dynasty. The objects were philologically and epigraphically tested for consistency with the hypothesized time period and foot length can be used for forensic reconstruction of body height [ 17 , 18 ], using the regression equation for female [ 18 ] (87.906 + 3.165 x 24.5 = 165 cm).

Proportionality rule was applied to compare the ancient samples with the knees from QV 66. Testing of z-score (165 cm– 158 cm) / SD 7 = +1 SD (the less favourable data of 3 rd Intermediate Period) was implemented. By comparing the condyle’s width to Cape Coloured data, sex dimorphism characteristics were tested (studies from ancient Egyptian samples being wanted). The size of the QV 66 knees was then assessed for sex determination via a measurement of both condyles on scaled X-ray pictures (Table A in S1 File ).

To look for knee sizes, the condyle’s width was compared to modern samples (young females Cape Coloured) and to ancient samples from Metapontum (Italy 700–300 BC) [ 12 , 13 ] (Table A in S1 File ). Different body height formulae (for modern and for prehistoric remains) [ 14 – 16 ] were used to investigate the individual’s stature. Comparison of the dimensions of QV 66 knees with those of modern poor Sub-Saharan African females was implemented [ 12 ].

Results

Anthropometric reconstruction Both knee condyles show a ca. 83–85 mm width if mummified soft tissues are included and ca. 79–80 mm if only the bone is considered. A condyle width of ca 83–84 mm indicates that QV 66 knees were slightly slimmer than those of the younger and poorest women from Sub-Saharan Africa. There is no formula to re-calculate knee width from living to dead, only an estimate the greatest difference would be ca. 1.5 mm in knee width between living and dead persons [12,27,28]. Moreover, it was also possible to determine—acknowledging a certain degree of uncertainty—that the bones found in QV66 belonged to an individual whose stature ranged between 165 cm and 168 cm (Table A in S1 File).

Comparing knees with ancient and modern samples Assessment of the size of the QV 66 knees revealed them to be female with a 90% likelihood. With a single exception, the knees from QV 66 belong to an individual taller than e.g. the average ancient Greek women’s range (Table A in S1 File). From the size and proportion of the knees, the most likely body height of QV 66 female was determined to be 165 cm (+/- 2.5 cm). Compared with data about women from the New Kingdom (average 156 cm) and 3rd Intermediate Period (average 158 cm), she was taller than the average Egyptian woman [29]. The QV 66 female was approximately one Standard Deviation taller than average (or taller than 84% of the women of her time). The estimated height of ca. 165 cm is confirmed independently by the calculation of foot size and body height reconstruction obtained from the sandals found in the tomb, which, indeed, belonged to an individual of ca. 165 cm (see below). Compared to e.g. ancient Greek females QV 66 female is 95% above the ancient Greek female range and close to the average male (she is equal in height to ancient Greek and Egyptian men) [13,29].

Ancillary Egyptological analysis: the Sandals and Other Objects Found in Tomb QV 66 Only the faience knob with the name of King Ay found in tomb QV 66 belongs to the late 18th Dynasty and predates Ramses II and Nefertari by perhaps two generations. The poor quality of the shabtis was also a matter of speculation as they seemed ill-fitting for a burial of a great Queen [8]. A fragment of a golden object with the name of Nefertari was discovered in 1988 when the tomb was restored [8]. Other fragments of jewellery without a provenance but bearing the name of the queen are also known. They may also come from QV 66 (List B in S1 File) [8]. The sandals are made of sewn fibre and they belong to the group of type C sandals (Veldmeijer´s classification): type C variation 1; the front strap is Type 3 and back strap is type 2 [30,31]. The style is typical of the 18th– 19th Dynasties [32,33]. The sandals from QV 66 show some wear caused by the movement of the foot on the dorsal (upper) side, the ventral side could not be studied due to mounting on a display panel (neither by Veldmeijer, nor by the authors of the present study). The sandals measure 29 cm in length and 10 cm in width (Fig 11). Type C has a pointed, slightly upturned toe pointing to a modern shoe size of 39–40, if one only counts the length used by the foot, indicated by the imprints and the subtraction of the pointed end [34]. Furthermore the model clearly indicates the position of the big toe, with visible marks of the size, especially on the left sandal: It can be deduced, with a certain reservation, that the sandals’ owner had a body height ranging c. 165 cm using forensic methods [17]. Veldmeijer described the sandals as those of Queen Nefertari [30]. The fine quality manufacture and high quality of the sandals speaks in favour of royal footwear. Thus it is widely accepted, that all objects found in QV 66 seem to be part of the original burial of Queen Nefertari, broken by ancient tomb robbers [7]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 11. Sandals from tomb QV 66. Museo Egizio Turin Suppl. 5160 RCGE 14471. https://doi.org/10.1371/journal.pone.0166571.g011

Chemical analysis (data on embalming agents) The results of the chemistry of the embalming agents suggest a date earlier than the 3rd Intermediate Period, which is consistent with the evidence for the mummification materials and methods detailed below: the absence of bitumen is consistent with a New Kingdom date since it does not appear in balms from mummies until 900 BC [35,36]. The use of bitumen, and more liberal employment of tree resins in the embalming recipes, is seen in 3rd Intermediate Period mummies and later, the use and proportion of both in relation to the plant oil/animal fat base increasing over time with greatest use during the Ptolemaic and Roman Periods [35,37–39]. Bitumen was not detected in any of the samples from the knee assemblage despite selectively monitoring for the presence of hopanes (m/z 191) and steranes (m/z 217) characteristic of a true natural bitumen [40]; Constituents of coniferous and non-coniferous resins were also not detected. The biomarkers for both these natural products are highly resilient and so can be expected to survive in a burial environment such as QV66 if they were originally present (Fig 12). This is consistent with these samples being largely from the outer layers of wrappings where oils or fats are usually the main or only ‘embalming agent’ during the New Kingdom, and are used to convey religious, political and cultural identities at this time [37]. In this context, it is notable that the samples from the mummified knees all revealed a non-human animal fat as the source of the embalming agents applied liberally to their linen wrappings, with all parts of the knee ‘assemblage’ showing a very similar lipid (fat) profile suggesting a likely common origin, i.e. the same individual. The same non-human animal fat, most likely a ruminant fat, constituting the embalming agent in the outer wrappings from all three parts of the knee assemblage, combined with the absence of evidence for a natron bath being employed and other aspects of the mummification, suggest a 19th or 20th Dynasty date for the mummification. Massive sub-cutaneous stuffing, the characteristic of the 3rd Intermediate Period, is not visible. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 12. Reconstructed gas chromatography-mass spectrometry (GC-MS) total ion chromatogram (TIC) of the trimethylsilylated total lipid extract of ‘resin’/linen wrapping from left long leg fragment. Peak identities (‘n’ indicates carbon chain length; where shown, i indicates degree of unsaturation): filled triangles, C n:i indicates fatty acids. https://doi.org/10.1371/journal.pone.0166571.g012