Two factors suggested the possibility of successful isolation of authentic DNA sequences prior to the procedure, indicating that the remains were deposited in favourable conditions. Firstly, the slightly alkaline soil at a pH of 8.1 in samples from the New Assyrian and the Islamic levels contained 16.3% and 18.9% of CaCO 3 respectively. Secondly, the depth at which remains were deposited (1.5–2.0 m below the surface) suggested rather moderate than high temperature of the remains’ surroundings. Both chemical and physical parameters resulted probably in preservation of collagen content amounting to 3.41% [18] in the tooth of specimen MK 11G107.

The present study was undertaken mostly as an attempt to confirm the origin of individual MK 11G107 living 1.3–1.5 Kyrs ago whose remains were found in Tell Masaikh. Anthropological examination of the skeleton revealed features that might be recognized as signs of anaemia, and probably thalassemia [19], which in turn might suggest him having been a newcomer, e.g. from the neighbouring Mediterranean region.

mtDNA Sequence

Changes found within the HVR-I sequence of specimen MK 11G107 indicated the presence of the M4b1 haplogroup according to HaploGrep [20] (M65a by the earlier nomenclature and PhyloTree). Details are given in figure 1A and 2. The haplogroup has likely arisen in the region of the Tibetan plateau [21], thus suggesting that the origin of the individual could have been rather an Asian than European one.

The relatively high degree of DNA preservation in the skeleton MK 11G107 inspired a further attempt to isolate DNA from three other specimens, one additional from Tell Masaikh (MK) and two others from Tell Ashara (ancient Terqa, TQ) sites. In contrast to the earlier published haplogroup K found in individual from Terqa [3], an analysis of the HVR-I revealed that individuals TQ 28F 112 and MK 13G 117 belonged to M49 with different haplotypes, and TQ 28F 256 to M49 or M61 (Fig. 3). The identified clades have probably arisen between 25 and 58 Kyrs ago also on the Indian subcontinent, where they still occur [21], [22]. Having sufficiently high access to skeletal material of specimen MK 11G107, a more advanced molecular analysis was undertaken, which showed changes also within HVR-II and the coding region, confirming haplogroup M4b1, presumed earlier only on the basis of the HVR-I sequence (Fig. 1A and 3). An analysis of commonly available databases revealed several haplotypes almost identical with that of MK 11G107, present in people living today in Tibet [23], the Himalayas (Ledakh) [24] and Pakistan [25] (Fig. 1B), and having a reverse mutation at the hot spot 16311 [23] in common with the fossil haplotype [26], [27]. Using median joining network [28] we attempted to draw the four observed haplotypes from those of the extant neighbouring populations (Tab.S3) which confirmed their restriction to the South, East and Southeast Asia regions (Fig. 4). They belong mostly to subclades of South Asian-Tibet specific haplogroups absent today in Mesopotamia [26], [27] (Fig. 3). Only complete sequencing will help to narrow down the geography and indicate precise origin of the studied individuals. However, at this step of the analysis a continuity between Trans-Himalaya and Mesopotamia regions in ancient time is likely which has been broken down as a result of recent population movements. Probably, significant depopulation resulting from the Mongolian invasions of the late 13th century AD [29], followed by repopulation by Bedouin tribes in the 17th century [30] and farmers from southern Anatolia and western Syria during the 19th and 20th century [31] are among the possible factors which may have shifted the gene pool profile of the region.

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larger image TIFF original image Download: Figure 3. Identified haplotypes of studied individuals as seen from direct sequencing (one of four runs each performed on different portions of pulverized different teeth). HVR-I, HVR-II and coding region of mtDNA for individual MK 11G 107; HVR-I for individuals MK 13G 117, TQ 28F 112 and TQ 28F 256. Obtained consensus sequences have been submitted to GenBank and assigned accession numbers KF462390–KF462396. https://doi.org/10.1371/journal.pone.0073682.g003

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larger image TIFF original image Download: Figure 4. Median joining network Median joining network [28] of four individuals living in the middle Euphrates valley between 0.2 Kyrs AD and 2.5 Kyrs BC. M–J network of three observed haplogroups (M49, M61 and M4b) shows the distribution of haplotypes among populations from different geographical regions. Circle sizes are proportional to the number of mtDNAs with that haplotype. The data has been taken from published and unpublished sources given in supplementary (Table S3). https://doi.org/10.1371/journal.pone.0073682.g004

Assuming the mentioned origin of the identified haplogroups and a result of median joining network analysis, it may be claimed that the studied individuals represent genetic association with the Indian subcontinent. The fact that the studied individuals comprised both males and a female, each living in a different period and representing different haplotypes, suggests that the nature of their presence in Mesopotamia was rather long-lasting than incidental. The close ancestors of specimens TQ 28F 112 and TQ 28F 256 could fall within the population founding Terqa, the historical site constructed probably in the early Bronze Age [1], at time only slightly preceding the dating of the skeletons. All the studied remains could have been also left by descendants of much earlier migration waves spreading clades of macrohaplogroup M from the nearby subcontinent. It cannot be excluded that among them were people involved in the founding of the Mesopotamian civilizations. For instance, it is commonly accepted that the founders of Sumerian civilization came from the outside of the region, their exact origin is, however, still a matter of debate. It is suggested that migrants of Iranian, Indian [32], [33] or even Tibetan affinity [34] founded the Sumerian civilization, which suggestion can be supported by comparing the Tibeto-Burman and Sumerian languages [35]. The migrants could have entered Mesopotamia earlier than 45 centuries ago, during the lifetime of the oldest studied individual, as the Tibetan Plateau was peopled more than 20 Kyrs ago [21], [36]. However, one also should consider the possibility that studied individuals belonged to the groups of itinerant merchants moving along a trade route passing near or through the region, since a recent comparative study of strontium, oxygen, and carbon isotopes content in enamel indicates that people from Indus Valley were present in southern Mesopotamia 3 Kyrs BC [37]. We believe that the identification of mtDNA sequences itself should be acknowledged as significant, leaving its detailed interpretation for further research involving a larger number of specimens, representing other Mesopotamian regions and various periods.