1. Sanxo Llop, a site on the banks of the Serpis River

1L’Alqueria de Sant Andreu - La Vital - Sanxo Llop is a large archaeological site located on the banks of the river Serpis, near the sea. The first settlement of the site occurred in the 5th millennium BCE, although its development peaked at the end of the 3rd millennium BCE. It continued to be inhabited with varying degrees of occupation in the following millennia until the present day. The archaeological remains found in the area during the intervention correspond to negative structures whose primary function must have been storage of food, most likely as silos for cereal. In later stages, when the storage structures decayed they were turned into pits to dump and dispose materials, some even becoming burial spaces. However, a number of them were built ex profeso to act as sepulchers. Some of them had abundant animal offerings: suids, ovicaprids, dogs and even cetaceans.

2Within the perimeter of the prehistoric site, an area of later occupation used for inhumations was discovered. The age of this newfound necropolis is between 660 CE and 760 CE according to the radiocarbon dating, which corresponds to the Visigothic period. In addition, it was noted that the four burials with human remains display typological variety. The sepulcher Number 50, which is the subject of this article, was a double inhumation. The burial had been made inside a silo with of cylindrical shape, and consisted of an adult individual and a child. Both bodies displayed contracted anatomical positions due to the fact that both skeletons adapted to the dimensions and shape of the silo. The unusual nature of the burial raised a number of questions. One of them was the possibility of both individuals being related. The aim of this work was to elucidate the question of the relationship between the two individuals buried in the silo using ancient DNA recovered from their skeletal remains (fig. 1).

Fig. 1. Location of the Sanxo Llop Necropolis, with respect to the rest of sampled necropolis.

2. Anthropological and funerary analysis

3Burial number 6 is a collective burial in a grave delimited by boulders that contained a collection of three individuals: an adult between 40-60 years old and two children; one between 10-14 years old and another around 7-8 years of age.

4Tomb 41 consisted of an individual burial located in a pit delimited by ashlars that contains the female skeleton of 20-30 years of age lying in supine position, elbows bent and forearms crossed over the abdomen. Such position of the forearms resting on the abdomen and not with the arms parallel to the thorax with the elbows extended is characteristic of later periods, especially from the 7th century CE onwards (Sastre et al. 2020).

5Burial 65 consisted of a grave dug in the ground whose dimensions were adapted to the size of the body of the deceased. The pit contained the skeletal remains of an adult between 40 and 60 years of age, placed in supine position with the arms parallel to the thorax and the articulations of both elbows extended, with the hands dorsal on the pelvis.

6The double inhumation in pit number 50 where the two individuals analyzed were found, had a flat base and a frustoconical shape. Its diameter was 1.26 metres at the base and 0.94 metres at the mouth; and a depth of 0.89 metres. It is an older silo reused as a burial pit. Inside the silo there was a double, primary burial, made up of a male adult and a female subadult. The adult individual had an age between 20-25 years and an estimated height of 1.68 metres. The body was deposited in supine position, adapting to the shape and dimensions of the silo. It was oriented S-N, with the head to the South and the feet to the North. The subadult (8-10 years old) subject was placed in left lateral decubitus position, oriented SE-NW with the head to the SE and the feet to the NW. The position is conditioned by the dimensions and frustoconical shape of the silo.

7From the stratigraphic record and the osteological observations of the general position of the skeleton and of each individual bone, we can reconstruct the original position of the bodies. The funerary container necessarily conditioned the disposition of the corpses and had a direct influence on the evolution of the decomposition of the body and on taphonomic processes. Both skeletons were covered by the base of a large dolium, which would serve as a covering for the grave. However, between the base of the dolium and the skeletons there is a sandy-clay layer. The displacement of some bones outside their own anatomical position and the natural volume of the body (fibula and left radius and ulna of the adult individual) indicates that the decomposition of the body occurred in an empty environment. Therefore, the sediment located between the dolium and the skeletons is a layer formed by infiltration that progressively filled the empty space. So when the dolium that covered the tomb collapsed, it rested on the infiltrated sediment that had already filled the empty space in which both corpses were deposited (fig. 2).

Fig. 2. Burial no. 50.

8The stratigraphic record indicates that both bodies were buried in a narrow time frame, since both show the same topographic location, both are deposited on the same base of the silo and both are covered by the same stratum, without there being any type of sediment between the bones of both subjects. To determine the relative chronology of the two depositions, we must pay attention to the fact that the individuals are in contact by means of particularly weak elements. Both the axial and appendicular skeletons of both individuals were well articulated, preserving both persistent and weak anatomical connections. Therefore, the corpses had all the anatomical integrity at the moment in which they came into contact and the disposition of one has not led to the alteration of the other. It could therefore be a simultaneous burial or two inhumations carried out within a short period of time. It is difficult to be more precise. However, studying the reciprocal positions the simultaneity of the deposition can be deduced. The distribution of the two bodies in the grave also supports the assumption that both bodies were buried at the same time. Both deceased seem placed respecting the space that the body volume occupies, one from the other. In this sense, the position of the lower extremities of the adult individual compressed against the wall of the silo, with the articulation of the knees fully flexed, seems to obey the need to leave enough space for the deposition of the infantile individual (fig. 3).

Fig. 3. Plan of the UE50 burial and representation of the original position in which the bodies were buried.

9The paleopathological analysis did not reveal a violent traumatic injury or an infectious disease that could have caused the death of both in a very short space of time.

10The adult individual presents several traumatic injuries such as trauma marks in the lumbar vertebrae, signs of fracture of the left clavicle and post-traumatic myositis osificans in the distal third of the right tibia, on the insertion surface of the interosseous membrane with the fibula, at the level of the opening for the perforating branch of the peroneal artery. However, all these injuries were consolidated long before death and are undoubtedly caused by hard and continuous physical activity (fig. 4).

Fig. 4. Post-traumatic myositis osificans in the distal third of the right tibia of the male adult individual.

11Regarding the pathologies detected in the subadult individual, the incisor teeth displayed banded depressions in the dental enamel, a physical indicator of hypoplasia. This occurs during dental development and remains throughout life as proof of having suffered disease or malnutrition during childhood. An indication of possible growth and biological development problems. In addition, the child had multiple holes in the eye sockets. These holes are evidence of porotic orbital sieve (fig. 1). This pathology is associated with anemia and other diseases related to a deficient assimilation of vitamins and nutrients. Anemia is the result of a lack of iron that can be genetically inherited or a consequence of a lack of iron in commonly consumed foods. The diseases observed in the infantile subject can be related to some type of epidemic or endemic infectious disease (fig. 5).

Fig. 5. Porotic cribra orbitalia in the female infantile individual.

3. Materials and methods

12Samples were collected as petrous bones and handled in the dedicated Ancient DNA Facility at the University of Huddersfield. After screening for endogenous DNA content using USER-treated libraries, the samples were considered viable for further sequencing via three additional libraries (non-USER treated) in one Illumina HiSeq4000 lane (100 cycles) (Macrogen Inc., in South Korea). Following an initial quality check and pair-end read merging using FastQC v.0.11.558 (Andrews 2010) and leeHom (Renaud et al. 2014), the raw reads were mapped against the human reference genome (hg19 version) using BWA v.0.7.5a-r40560 aln algorithm (Schubert et al. 2012), with specifications in place (-n 0.01, -o 2, -q 20). We performed quality controls="true" on the resulting BAM files with QualiMap v.2.262 (Okonechnikov et al. 2015). Duplicate reads were removed with Samtools rmdup command and then sorted also with Samtools. Finally, we added read groups, necessary for downstream step, using Picard AddOrReplaceReadGroups. All libraries were merged into a final file using Picard MERGESAM (https://github.com/broadinstitute/picard). We confirmed aDNA authenticity by checking post-mortem damage patterns (Jónsson et al. 2013), coherent mtDNA haplogroup and sex classification across libraries. Finally, to avoid post-mortem damage bias in SNP calling, we softclipped the terminal 3 base pairs of sequencing reads using the trimBam option in bamUtil package v. 1.0.1466. With GATK Haplotype caller we retrieved all variant positions in the mitochondrial region for each sample and built haplotypes. These haplotypes were in turn submitted into HaploGrep 2.067 for classification (Kloss-Brandstätter et al. 2011). For the Y chromosome we extracted all reads mapped to ChrY into a separte BAM file that was later fed into Yleaf (Ralf et al. 2018) to check mutations against the ISOGG (International Society of Genetic Genealogy) database. We also used pathPhynder (Martiniano et al. 2022) to further confirm Y chromosome haplogroup classification. To identify accurately the genetic sex of both ancient individuals, we used a method designed for low-coverage DNA sequencing and degradation patterns (Skoglund et al. 2013). To determine whether any familial relationship existed between these two individuals buried together we made use of READ (Relationship Estimation from Ancient DNA) (Monroy Kuhn et al. 2018), which is a method designed to deal with low-coverage pseudo-haploid data. Pairwise kinship coefficients were calculated using the READ method for all samples collected to cross check for possible cross contamination.

4. Results

13Preservation of ancient DNA in the petrous bones (inner ear) of both individuals was relatively good given the potential for DNA preservation in the region. The adult individual had 12% of surviving endogenous DNA and for the infantile we quantified it at 18%. Subsequently, the genomes were sequenced to mean depth of coverage of around 0.26X and 0.36X respectively sharing a single Illumina lane. With the genomic data we were able to perform a test to identify genetic sex. We confirm the adult as a male in concordance with the previous forensic labeling, and the infantile as a female.

4.1. Distinct phylogeographic patterns in the mitochondrial lineages

14The genomic analysis of the individuals will be addressed in a separate publication, jointly with the rest of the samples collected from the same region. Nevertheless, we explored the phylogeography of both mitochondrial haplotypes identified in the samples. We found that the adult male was a carrier of a HV mitochondrial lineage within haplogroup HV34. This particular sub-branch of HV34 (Haplotype: 263 310 750 1438 2706 4769 7028 8860 9801 10205 10920 15326 15514 16311) is defined by four mutations (10205, 10920, 15514 and 16311). The estimation of the modern occurrence of this lineage is based on four samples, and appears to have a western Mediterranean distribution with presence both in North Africa (Morocco) and South Europe (Iberia and Italy) (fig. 2). The ancient male individual actually shares the same haplotype with another modern Moroccan individual. The sister sub-branch within HV34 occurs mainly in West Asia according to the two representatives found (Iraqi and Iranian samples). Overall the distribution of HV34 appears to be restricted to the wider Mediterranean sphere of influence. On the other hand, the mitochondrial haplogroup H2a1e1a (Haplotype: 263 310 575 750 751 951 8860 9052 15326 16124 16354) to which the infant female belonged has a different distribution. The lineage of H2a1e1a is defined by one mutation at position 16124 and has two sister branches. In addition to four daughter branches. The distribution of this clade in modern Europe is mainly northern European with representatives mostly found in Denmark and Ireland (fig. 6).

Fig. 6. Reconstructed phylogeographic trees of mitochondrial haplogroups HV34 and H2a1e1a and placement of the ancient individuals in their respective maternal trees.

4.2. A common R1b haplogroup on the paternal lineage

15The Y chromosome haplogroup recovered from the male individual is R1b1a1a2a1a (tabl. 1), with the most derived mutation being consistent with marker L11. Although due to low coverage issues, we cannot rule out the possibility of other more derived mutations being missed in this classification. This R1b-L11 haplogroup is eminently European, very common and with wide representation in the Atlantic half of western Europe. The highest frequencies of R1b-L11 occur in Ireland, Great Britain, France and Spain.

4.3. Resolving the family tree

16Samples GOG34 and GOG35 share a first degree of kinship. This observation is derived from the values obtained by measuring the average pairwise allelic differences between all pairs of samples collected in the area. Different values in the continuous scale of the normalized kinship estimator (proportion of non-matching alleles or P0) in the READ method, can identify twins/duplicates, first degree and second degree relationships, or unrelated individuals. Values of P0 >= 0.906 indicate unrelatedness, values between P0 < 0.906 and P0 >= 0.812 indicate 2nd degree, 1st degree when P0 is between 0.812 and P0 >= 0.625, and twins for values of P0 < 0.625. In the pairing of samples GOG34 and GOG35, we obtained a P0 value of 0.691, consistent with a 1st degree kinship relationship (fig. 7A). In fact, they were the only pair that had any degree of kinship among all samples analyzed. Coincidentally they were also the only ones found buried together (fig. 7C). However, a first degree of kinship can represent two scenarios: parent-offspring or full siblings. With modern and high coverage data there are varying ways of resolving this question. However, aDNA not always allows for more powerful methodologies. However, aDNA not always allows for more powerful methodologies. Nevertheless, combining the information about genetic sex and uniparental markers, we determined that these two individuals were related as parent-offspring (fig. 7B). More precisely the older male individual, although young, was the father of the younger female individual (fig. 7).

Fig. 7. A) Kinship determination based on the P0 values from READ. B) Reconstructed family tree of the individuals studied.

5. Discussion

17Late Roman burials in silos, although not frequent, have been registered in other sites (Alapont 2005, 2007, 2009; Martinez-Porral 2018). Less common are collective or double burials in these silos such as the one that occupies this work.

18Thanks to recovery of ancient DNA and the sequencing of the genomes, we have been able to reveal the existing blood ties between the two samples. We can confirm genetically that the adult male is the father of the female infantile in the double burial.

19The analyses performed on their genomes indicate that they share a first degree of kinship which allowed two possible scenarios: parent-offspring or sibling-sibling.

20The genetic sexing of the skeletons coupled with the classification of their mitochondrial DNA, established that they had to be father and daughter and allowed us to rule out the sibling-sibling scenario which was an alternative interpretation of the 1st degree kinship. The reason why they cannot be siblings is because they do not share the same mitochondrial lineage. The sibling scenario was also a serious possibility given the young age of both skeletons and the relatively small age gap between the two (approximately 10-15 years of difference).

21We can also infer that they were buried together because they were very closely related and, at some point between the years 660 and 760 CE, died together or within little time of each other for unknown reasons. The reason behind the absence of the mother at the burial can mean that she either survived them both or had died sufficiently earlier to be buried elsewhere. She could potentially be identified in another grave of the necropolis with further screenings but that is beyond the scope of this work. Based on age and proximity, we speculate that a potential candidate exists in the same necropolis if she died around the same time. Burial 41 which contained a young female skeleton, could be the mother based on the similar age to the father and the vicinity of the inhumations. Nevertheless, the lack of the remains of the mother did not prevent us from gaining some indirect information about her.

22We have gained insight into to the different phylogeographic patterns that the mitochondrial lineage of the father has, compared to that of the daughter and by extension the mother. The paternal lineage is found mainly in the western Mediterranean both in the European side and in North Africa. Furthermore, the exact same haplotype the father carries is present in modern Morocco. Whereas the maternal mitochondrial haplogroup is clearly more common in northern Europe, which in turn suggests potential diverse origins of the paternal and maternal sides of the daughter.

23This fact would be an indication that people of different geographical or familial origins were still coming together to form families exogamous in essence in the post-Roman world. This in turn also suggests that mobility for single individuals was still possible in a period as turbulent as the late antiquity during the Visigoth rule in this region of Iberia, shortly after the Byzantine invasion and Justinian Plague and just before the Islamic conquest. Alternatively, it means that the individuals forming the population of the region were still genetically heterogeneous in the 7th and 8th century CE.

24This is also backed by the genomic analysis of the two individuals. However, this will be treated in more detail in a future publication. Interestingly, both father and daughter display a non-neglectable amounts of North African genomic ancestry at a time predating the Islamic conquest. The father carries twice as much North African ancestry as the daughter. This may be taken as an indication that the unknown mother did not contribute any North African ancestry to the daughter, which as mentioned above is indicative of genetic heterogeneity in the post-Roman population of Mediterranean Iberia.

25On the paternal lineage side, the Y chromosome haplogroup offers little phylogeographic insights since it is widely spread and very common across Atlantic Europe, including Spain.

26Altogether, the picture suggests continuity of heterogeneity and mobility in Iberia even after the collapse of Roman administration.