Mitochondrial DNA Reveals the Trace of the Ancient Settlers of a Violently Devastated Late Bronze and Iron Ages Village

<div><p>La Hoya (Alava, Basque Country) was one of the most important villages of the Late Bronze and Iron Ages of the north of the Iberian Peninsula, until it was violently devastated around the 4th century and abandoned in the 3rd century B.C. Archaeological evidences suggest that descendants from La Hoya placed their new settlement in a nearby hill, which gave rise to the current village of Laguardia. In this study, we have traced the genetic imprints of the extinct inhabitants of La Hoya through the analysis of maternal lineages. In particular, we have analyzed the mitochondrial DNA (mtDNA) control region of 41 human remains recovered from the archaeological site for comparison with a sample of 51 individuals from the geographically close present-day population of Laguardia, as well as 56 individuals of the general population of the province of Alava, where the archaeological site and Laguardia village are located. MtDNA haplotypes were successfully obtained in 25 out of 41 ancient samples, and 14 different haplotypes were identified. The major mtDNA subhaplogroups observed in La Hoya were H1, H3, J1 and U5, which show a distinctive frequency pattern in the autochthonous populations of the north of the Iberian Peninsula. Approximate Bayesian Computation analysis was performed to test the most likely model for the local demographic history. The results did not sustain a genealogical continuity between Laguardia and La Hoya at the haplotype level, although factors such as sampling effects, recent admixture events, and genetic bottlenecks need to be considered. Likewise, the highly similar subhaplogroup composition detected between La Hoya and Laguardia and Alava populations do not allow us to reject a maternal genetic continuity in the human groups of the area since at least the Iron Age to present times. Broader analyses, based on a larger collection of samples and genetic markers, would be required to study fine-scale population events in these human groups.</p></div

Enlarged view of the Basque Country and its provinces with the location of the village of Laguardia and La Hoya archaeological site (distance 1 km).

<p>Enlarged view of the Basque Country and its provinces with the location of the village of Laguardia and La Hoya archaeological site (distance 1 km).</p

MtDNA haplogroup composition of Laguardia (LG) and Alava (AL) population sample sets.

<p>R0* includes H and HV lineages except for H1, H3, and HV0.</p

Summary of diversity parameters for 51 mtDNA CR (16024–576) sequences from Laguardia and 56 from Alava region.

<p>Insertions at 16193, 309, and 573 were ignored for all calculations.</p

Principal Component Analysis (PCA) based on haplogroup frequencies of La Hoya archaeological samples (LH) and other historic and prehistoric populations from the literature.

<p>Neolithic: Los Cascajos, Navarre (LC); Paternanbidea, Navarre (PA); Camí de Can Grau, Barcelona (C1); San Juan Ante Portam Latinam, Basque Country (SJ). Chalcolitchic: Pico Ramos, Basque Country (PR); Longar, Navarre (LO); El Mirador Cave, Atapuerca (AT). Pre-Roman: Girona, Catalonia (C2). Post-Roman: Aldaieta, Basque Country (AL).</p

Haplogroups identified in the sample set of La Hoya.

<p>N = number of individuals.</p

Brain white matter tissue obtained from epileptic patients does not show histopathological abnormalities.

<p><b>A.</b> The DTI imaging analysis of the temporal areas used in our experiments showed diffusion tensors with elongated ellipsoidal form and grouped colouring, indicating a conserved white matter ultrastructure similar to the contralateral areas. <b>B–C.</b> The anisotropy of water molecules, quantified by the fractional anisotropy mean (FA) and the apparent diffusion coefficient mean (ADC), showed no significant differences between both epileptic (black) and contralateral (grey) white matter zones (p-value: 0.155 and 0.439, respectively) <b>D.</b> No neuronal heterotopia was observed within the white matter, with only a few neurons NeuN⁺ rarely spread throughout white matter parenchyma (white arrow). The scale bar represents 20 µm. <b>E.</b> Microglial Iba-1⁺ cells showed mainly quiescent morphology, with long branching processes and small cellular bodies. The scale bar represents 100 µm. <b>F.</b> Cortical disorganisation was also discarded by NeuN immunohistochemistry, as the neuronal layer could be perfectly differentiated (indicated by the roman numerals). The scale bar represents 20 µm. <b>G–J.</b> Haematoxylin-eosin staining revealed a normal cellularity (<b>G</b>) and the Ki67 recounts showed a normal number of cells in active phases of the cell cycle in all samples (<b>H</b>). Glioma tissue was used as positive control (<b>I–J</b>)<b>.</b> The scale bars represent 50 µm.</p

SOX2⁺ cells act as a glial progenitor <i>in vivo</i>.

<p><b>A–B</b>. After the spontaneous differentiation protocol of small spheres, the number of SOX2⁺ cells decreased, accompanied by the emergence of O4⁺ cell processes, which correspond to 58.35% ±21.11% of total cells. A small number of GFAP⁺ cells could be also detected (10.01% ±3.06%), all of them SOX2⁺. The scale bar represents 25 µm. <b>C</b>. SOX2⁺ population exhibits morphology compatible with oligodendroglial differentiation after the directed differentiation protocol, which was confirmed by the detection of myelin protein MBP. The scale bars represent 50 µm. <b>D</b>. No neuronal MAP2⁺ cells were observed in the spheres after the spontaneous differentiation of sphere-forming cells. Primary cell cultures derived from glioblastoma were used as a positive control. The scale bars represent 20 µm.</p

Schematic diagram representing our hypothesis about SOX2⁺ cell evolution <i>in vitro</i>.

<p>According to our findings, we hypothesise that SOX2⁺ cells correspond to glial restricted progenitors, spontaneously differentiating to O4⁺ pre-oligodendrocytes that finally lose SOX2 expression. In addition, a small number of GFAP⁺ astrocytes is generated. Finally, after exposure to the appropriate growth factors, SOX2⁺ cells are able to generate oligodendrocytes that express mature myelinating oligodendrocyte proteins such as MBP.</p

Proliferative cells can be isolated from adult human white matter.

<p><b>A</b>. Diagram depicting the different procedures followed in the present work for the isolation of proliferative cells from human white matter. <b>B</b>. The cell isolation protocol without (w/o) the sucrose centrifugation step gives rise to a monolayer culture, which could be expanded more than seven passages. However, only 28.57% of samples generated viable cultures with this procedure (<b>B</b>). Therefore, we added a final sucrose centrifugation, which allowed the obtaining of sphere-like colonies lightly adhered to the culture plate from all samples processed (<b>D</b>). These cells generated an adherent culture that could be expanded independently for more than five passages (<b>E</b>). When the initial spheres were passaged and reseeded at a cell density of 3000 cells/cm² or higher, new spheres were obtained (<b>C</b>), which, in turn, generated a new monolayer culture. These spheres could be passaged up to tertiary spheres, similarly to other proliferative cells isolated from adult human white matter. The scale bars represent 50 µm.</p