This study presents DNA typing of bone remains belonging to 7 male skeletons anthropologically individualized and carbon dated to the period 776-991 year AD. When DNA typing was successful, morphological and genetic sex assessments were concordant. All individuals were male, 5 were adults aged between 30 and 70 years and 2 were around 17 years old.
In spite of the fact that the skeletons were buried under similar conditions, different degrees of preservation were observed during the anthropological examination, generally consistent with different DNA yields. Accordingly, different samples showed varying degrees of genetic information. No fatherhood or other paternal relationships were demonstrated among them.
Confidence in authenticity is always a concern in human ancient DNA studies, and strict measures have been suggested to increase the reliability of the study (15
), as well as to perform a self-critical and rational analysis of the data (28
). The following criteria speak in favor of the authenticity of the results presented here: a) careful procedures were observed for specimen handling and preparation, b) clean negative controls and reagent blanks were used, c) reproducible and unambiguous genetic profiles were obtained in different laboratories (no mixtures were found), d) the same profiles were obtained from different samples of the same skeleton, e) a different genetic profile was observed in every skeleton typed, e) there was a concordance of morphological and genetic sexing, and f) the greatest amplification efficiency of shorter STRs was observed.
It is not surprising that the samples that gave the greatest DNA quantity (femur 1 and 3) also presented complete profiles with Identifiler® Plus. However, when amplified with the NGMTM kit, only femur 1 yielded a complete profile, whereas femur 3 yielded results only for the shortest STR markers. Seven of the 10 non-amplified markers were shared with the Identifiler® Plus results. Given that they have similar sizes in both multiplexes, a possible explanation could be that different extracts had DNA with different degrees of degradation. Additionally, it can be suggested that Identifiler® Plus may provide better amplification efficiency than NGMTM when dealing with difficult samples. It is striking that femur 5, in spite of having had a low DNA yield, produced a partial profile with MiniFilerTM and Identifiler® Plus.
Two mtDNA haplogroups were observed based on HVS1 sequences in our samples, H and U5a. The haplogroup composition of the studied individuals fit well in the European pattern. The haplogroup H, which is the most common in our sample (5 out of 6 typeable individuals), is widely present in European populations with frequencies above 30% (30
), including the contemporary population of Spain (35
). When evaluating haplogroup H sub-haplogroups, it was observed that frequency distributions of sub-haplogroups H1 and H3 demonstrated frequency peaks both centered in Iberia and the surrounding areas (33
). Thus, further mtDNA analysis by HVS2 sequencing and the study of coding region SNPs would be valuable in order to resolve deeper lineages within the haplogroup H.
Femur 3 was assigned to mtDNA haplogroup U5a. The most ancient mitochondrial haplogroup U5 is found in northern and southern Europe, whereas U5a is mainly restricted to southern Europe, with some diverged individuals present in the northwest (22
). It has been argued that expansions of U5-subclusters, particularly U5a and U5b, occurred after the last glacial maximum period, during re-occupation of large areas of northern Europe by refugees from the Pyrenees region (37
). The frequency of haplogroup U5a in Spanish populations is approximately 8% (35
It should be noted that there was a match between femur 1 and 5, and femur 4 and rib 1, indicating that these two pairs of individuals may be maternally related.
HVS1 data do have a limited power of discrimination in a forensic (or anthropological) context and thus many mtDNA haplogroups are poorly defined by the control region. Since only the HVS1 region was analyzed, sharing the same HVS1 profile does not necessarily imply that two mtDNAs absolutely belong to the same haplogroup (38
The Y chromosome haplogroup R determined for femur 1 and 3 is in agreement with the geographical origin of the studied human remains. The majority of European Y chromosomes belong to this clade (39
). Particularly, the sub-haplogroup R1b1b2, determined in femur 1, occurs at a high frequency throughout western Europe (according to www.YHRD.org
This genetic study is part of an interdisciplinary research project that integrates the investigations of historians, archeologists, and anthropologists. There is of yet no historical or archeological evidence to infer the origin of this human group, so integration of different approaches may allow a better understanding of the origin of these people and possibly of the circumstances of their death and burial.
In summary, the case presented here shows that despite the antiquity of the samples, the combined use of current common forensic genetic systems allows the retrieval of valuable and interesting genetic information. With greater sensitivity of detection methods and more human identity genetic markers that are amenable to analysis of degraded samples, more challenging anthropological samples may disclose the secrets of their genetic make-up.