Methods used for Identification

With the discovery of the first royal cachette, scientific investigations also began. The latest technology such as X-rays was used very early on. Over the course of time, there were various approaches by Egyptology, medicine, anthropology and molecular genetics. The dates of reign used in this meta-analysis are an approximation, following generally used chronological models [83].

Inscriptions

These results are entirely independent from the other investigation methods, using archaeological methods only: Some mummies carried restoring inscriptions on the secondary non-royal coffins, on wrapping and shrouds.

The identification dockets can be classified the following:

• Only the name (Type 1)
• Name, titles and sometimes information on the date when the restoration was made in the 3rd Intermediate Period (Type 2)

This archaeological identification is considered to be generally correct, especially the labels directly on the mummy bandages. It must be emphasized that there has been not one mummy from the royal cachette which had been proven wrongly identified by the ancient identification dockets [17]. For this reason, Herbert Winlock has taken the position valid since 1932 that, quote: “…the docket written by the ancient officials must be accepted unless there is very strong evidence against it.” [9,84].

Historical monuments of the Amarna period with inscriptions are also used to identify persons, especially mummies without labels. Tutankhamun is confirmed as the son of Akhenaten and Nefertiti by a relief now in Ashmunein [35]. The inscription in room γ of the royal tomb in El-Amarna undoubtedly names Nefertiti as the mother of the baby, who with good arguments can actually only be identified as Tutankhamun [26,28].

Other theories assume that the relief is meant to represent an abstract concept of rebirth [85,86]. However, such theological interpretations remain uncertain and speculative. Ultimately, it is the scientific-historical facts that count: Maketaton died far too young (about 10 years) to be seriously considered as Tutankhamun's mother; therefore Dodson describes Nefertiti as the perfectly plausible candidate for Tutankhamun's mother [87].

Some historical background knowledge is mandatory to understand the feasibility of presented identifications, thus brief historical notes on the respective royals are given. [83,88–93].

Anthropology

The first serial investigation was made by Grafton E. Smith (1871-1937) and was published in 1912 as monograph [94]. He measured the mummies to determine similarities between the cranium and the face. The ancient designations of the mummies were generally accepted. The age estimate was based on macroscopic examination. Douglas Derry examined the newly discovered mummies of Tutankhamun, the mummy from KV 55, King Psusennes I and other mummies a few decades later using similar methods [95–97].

Derry used similar cranio-facial measurements and analyses were used to strengthen the close morphological relationship between Tutankhamun and the mysterious male in tomb KV55 [97].

The Ramesside kings of the 19th and the Sethnakhtide kings of the 20th Dynasty were first autopsied by G. E. Smith after their discovery in the two royal cachettes DB 320 and KV 35 [94]. Smith measured the mummies to establish cranio-facial resemblances. Several mummies carried nametags and inscriptions on the mummy wrapping as part of their restauration during the later 21st and 22rd Dynasties. Some other mummies are anonymous but can be dated within the dynasty based on observation of the used mummification technique.

The cranio-facial study presented in 1992 focussed on the age and identity of several kings of the 18th Dynasty and the possibility of misinterpreting some of the ancient designations was discussed [98–101]. Recently further studies on the body height and cranial data of the Pharaohs have been undertaken [102,103]. Height and body proportions are hereditable to a high degree, thus being an indicator of bloodline connections [104–106].

Facial Resemblance

Similarity in appearance and morphology have repeatedly been used to insert the royal mummies into genealogical sequences: The cranio-facial-study presented in 1992 raised further questions about the age and identity of some kings of the 18th Dynasty, and the possibility of misinterpretation of some ancient name docket was discussed [98–101].

Radiology

The serial X-ray investigation of the kings by Harris and Weeks revealed first insights [107,108]. The new technologies provided some additional information, but also caused further controversy in some cases [98]. In some cases, the X-rays suggested a much lower age at death than had been assumed because of the years of reign, e.g., in the case of Thutmosis III. CT scans confirmed certain morphological similarities, indicating a close relationship between kings and presumed causes of death, but neither proving nor disproving identity [109].

Hawass and Salem have recently published additional CT-scans of some kings from the 19th and 20th Dynasties [20].

Serological Studies

During this decade of the first systematic X-ray investigation of the 1960s, a detailed anatomical examination of the mummy from KV 55 and serological tests of mummies from the Amarna period also provided important scientific information for identification. [110–113].

The blood groups amongst the Amarna rulers were similar, but this could not resolve their exact relationships. Tutankhamun and the KV 55 male have the same blood group A2 and MN. Thus, they could be brothers or – if their mother shares the same groups – father and son. The solution father-son is supported by genetics [25].

Hair Identification

Hair analyses have so far played a very minor role and have only been used for identification in one case: In Tutankhamun's tomb, Howard Carter (1874-1939) found a series of miniature coffins with the inscription of Queen Tiye (Cairo Nat. Mus. JE 60697-60700, Carter No 320a-d). They contained a lock of hair (Cairo Nat. Mus. JE 60701, Carter No 320). This was given in Tutankhamun's funeral as a memento of his ancestor, Queen Tiye [114]. The hair was compared with the wavy hair of the Elder Lady in KV 35, leading to a proposed identification as Tjye [100,115,116] which was later confirmed by genetic testing in 2010 [25].

Genetics

Scott Woodward made the first attempt at genetic analyses of some of these mummies, but the results remained unpublished; however, parts of the data was shown in a documentary movie [37,117,118]. He discovered the historically known incest also in the genetic data sets, especially at the beginning and at the end of the 18th Dynasty and proposed that the mummy CG 61074 is Amenhotep III. After the millennium, molecular genetics opened a new door into the past, but the feasibility of acquiring authentic ancient DNA from Egyptian mummies is still debated. Another project to obtain genetic profiles of the Pharaohs was suggested by Sakuji Yoshimura, but declined in 2000 [37].

Zink and Nerlich postulated that DNA analysis was feasible based on the lower temperature in the tombs, the beneficial fact that mummies are dry and that natron increases the pH-value [119]. The success rate of amplifying DNA by PCR from ancient samples is generally 50-60% [120].

Other studies based on the rate of decay of DNA in papyrus rejected the concept that authentic DNA from ancient Egypt could survive at all [121]. Factors such as humidity, chemicals, temperature and modern contamination pose a challenge to molecular genetic studies [122]. A study on the extent of modern contamination was presented by Malmström, who examined animal remains for human DNA: all 29 samples contained human DNA due to modern contamination, but in 25 cases authentic animal DNA was also found [123]. To rule out contamination, procedures have been developed to ensure the collection of authentic ancient DNA [124–126]. In principle, all studies on ancient DNA published before 2010 must be questioned and should be carried out again. To ensure authentic and credible data they suggest:

• Extraction of clean samples in new excavations, the traditional cleaning of bones must be avoided: no washing, no chemicals and separate storage from modern samples; and investigators should handle them only with gloves and wear forensic suits.
• For old material, which is possibly contaminated, a decontamination strategy should be used first.
• Search for criteria of authenticity (short DNA strands of less than 300 base pairs and authentic aDNA should contain modified bases).
• UV irradiation and removal of the surface material, extraction from the bone cortex or dentine.

For the Tutankhamun Family project, such safety protocols were applied [32,42,127]. Several facts speak clearly in favour of authentic aDNA [7]:

• All female genetic profiles were negative for Y-Chromosome markers.

• All male mummies showed homozygous (i.e. hemizygous) Y-chromosomal profiles.
• The profiles and haplotypes of all mummies showed individual differences and therefore could not have originated from the same source of putative contaminant DNA.
• The combination of nuclear data (Y- and autosomal chromosome–related markers) complemented each other.
• Different biopsies and extractions on each mummy resulted in...