**2. Material and methods**

the variability of the mtDNA of past populations, it is possible to infer population movements

One of the most studied population movements is Neolithization, the transition from a nomadic hunter-gatherer to an agro-pastoralist lifestyle. The debate on the mechanisms of the Neolithic transition has been framed within a dichotomy based on either a demic (DD) or a cultural diffusion (CD). According to the DD model, the migrating people bringing new knowledge experienced some gene flow with the local hunter-gatherer groups. On the other hand, the CD model postulates that the Neolithic transition was mediated mainly through the transmission of the agro-pastoralist system without substantial movement of

However, several DNA studies on different ancient European populations indicated a more complex pattern for the Neolithic transition. Unlike the initial proposal based on classical genetic markers that suggested a major migration wave [3], further studies have shown that the Neolithization process varied in different regions, occurring along several different routes into and across Europe, and having a different genetic impact on the various regions and at

The mtDNA frequency distribution observed in hunter-gatherers and farmers from Europe provides support for a random dispersion model for Neolithic farmers, with different impacts on the various geographic regions (Central Europe, Mediterranean Europe, and the

The transition from the Neolithic to the Chalcolithic period in Europe has been debated. Previous mitochondrial DNA analyses on ancient Europeans have suggested that the current distribution of haplogroup H was modeled by the expansion of the Bell Beaker culture (BBC) out of Iberia during the Chalcolithic period. In addition, it has been suggested that these groups with Bell Beaker (BB) culture in Central Europe represented a population movement from the Iberian Peninsula [16]. However, according to the mtDNA variability in Chalcolithic groups from the Cantabrian fringe of Iberia, no genetic relationships have been detected between these Iberian and Central European groups [17]. This suggestion has been confirmed

by the recent study [18] about the Beaker phenomenon and genomic data of Europe.

analysis allows discriminating endogenous sequences from exogenous sequences.

*Paleogenetics* consists of the recovery and analysis of DNA obtained from the remains of individuals from the past, through polymerase chain reaction (PCR) and Sanger sequencing (ancient DNA—aDNA). These techniques are mainly applied in the analysis of mtDNA and fragments of nuclear DNA [9, 10, 19–22]. Since 2005, with the development of next-generation sequencing (NGS) technologies, it has been possible to retrieve also genomic data (*Paleogenomics*) from prehistoric European humans [23, 24]. This technology has allowed overcoming the apparently insurmountable difficulties associated with the deficient preservation of genetic material and the contamination of ancient DNA samples by modern DNA. NGS allows sequencing all those molecules that are present in DNA extracts (intact, contaminant, and damaged molecules, DNA from other organisms, etc.). The subsequent bioinformatic

that shaped the current genetic variability of our species.

people [2].

various times [4–17].

114 Mitochondrial DNA - New Insights

Cantabrian fringe) [9].

**1.2. Paleogenetics and paleogenomics**

In this chapter, we have analyzed the human remains from El Aramo Mine discovered in 1888, a mine located in the Asturias region in the Cantabrian fringe of the Iberian Peninsula [41]. The direct 14C analysis of the human remains from this mine indicated a dating between the Late Chalcolithic period and the Early Bronze Age. The anthropological remains from El Aramo Mine consists of 9 skulls and 12 skeletal remains. We have isolated DNA mainly from dental pieces (since it is the material that offers the greatest guarantees when recovering DNA). However, in some cases, we had to pulverize bone remains in order to perform DNA extraction, since it was the only anthropological material available.

Romania [10, 53, 54]. The present-day populations database corresponds to that described in [10], to which the present-day population of Asturias, where El Aramo Mine is located, has

Paleogenetics of Northern Iberian from Neolithic to Chalcolithic Time

http://dx.doi.org/10.5772/intechopen.76438

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The FST distance matrix between present and ancient populations was calculated from the mtDNA haplogroup frequencies using Arlequin 3.11 [57]. Relationships between populations were studied through Multidimensional Scaling analysis (MDS), based on the FST distance matrix, using SPSS 20 Software. Median Joining Network (MJN) for certain haplogroups was generated to infer phylogenetic relationships between the mitochondrial lineages from the Paleolithic period to the present day using Network software v4.5.0.0 (available at http://www. fluxus-engineering.com). Different mutation weights were applied in accordance with previous papers [58–60], and the point insertions and deletions were excluded from the analysis.

Mitochondrial DNA variability was analyzed in 21 skeletal remains recovered from El Aramo Mine, in the Asturias region (Cantabrian fringe of the Iberian Peninsula). The quantification of the template mtDNA number of each of the samples showed values above 1000 molecules/μl in all the DNA extracts from teeth and values below 1000 molecules/μl in all the DNA extracts from bones (**Table 1**). These results indicate the greater efficiency of the DNA extraction from teeth when compared to that from bones. Furthermore, in order to authenticate the results, 5.26% of the samples analyzed were duplicated, and these results were consistent in all the samples (**Table 1**). Moreover, 24 PCR products were cloned, estimating an average of 7.46 mutations per cloned fragment (~100 bp). These mutations have been interpreted as artifacts

The mitochondrial variability obtained from the 21 human remains from El Aramo Mine showed 15 different haplotypes (genetic diversity: 0.9608 ± 0.0394). The nine skulls studied presented nine different mitochondrial haplotypes, which allow us to rule out the existence of maternal kinship among these individuals. The 12 postcranial remains analyzed showed 7 different mitochondrial haplotypes, which, compared with the haplotypes of the skulls, lead to reject possible coincidences, since the postcranial remains were not associated with the skulls. Finally, the minimum estimated number of individuals was 15, with 15 different mitochondrial haplotypes described (**Table 1**). The high genetic diversity obtained in El Aramo site allows us to indicate that it is a representative sample of the original population, with no

The 15 mitochondrial haplotypes obtained from El Aramo Mine were classified into 5 different mitochondrial haplogroups (H, T, J, U5b, and I3), with a genetic diversity of 0.6381 ± 0.1288 and a heterogeneous distribution of their frequency values (60, 13, 13, 7, and 7%, respectively). Haplogroup H is the most frequent one in the population of El Aramo (60%), whose value is close to that shown by the current population of the Asturias region (56%), where El Aramo Mine is located, and much higher than the average value found in European (45%) and Near Eastern (16%) populations [54, 55, 61] (**Figure 1**). In El Aramo, haplogroup H is represented

been added [55, 56].

**3. Results and discussion**

produced by the postmortem damage of aDNA.

evidence of kinship among these individuals.

In the case of teeth, we have selected those without caries or deep fissures that might extend into the dental pulp. The surface of the teeth was thoroughly cleaned with acids and ultraviolet (UV) irradiation to eliminate any possible DNA contaminants [42]. In the case of bones, the surface was thoroughly cleaned by abrasion and pulverized using a Freezer miller. Then we extracted DNA from bone and dental tissue by means of the phenol/chloroform method with some modifications [20–22, 43].

The sequencing of a 399 bp (nps 16,000–16,399) segment of HVS-I and 394 bp (nps1–394) of HVS-II of the mtDNA as per [44] was conducted by amplifying 6 overlapping fragments of 93–133 bp in length. The protocol followed and the primers used are described in [9, 45]. Likewise, in order to verify the obtained mtDNA haplogroups, the nucleotide position of the coding region of mtDNA was determined by means of PCR-restriction fragment length polymorphisms (RFLPs) [43, 46].

The extraction of DNA and the preparation of samples for PCR were performed in a sterile chamber with positive pressure, free of modern DNA, in which no post-PCR process had ever been carried out. Ancient DNA results were validated through the application of the following criteria [47, 48]: (1) suitable clothing was used (disposable cap, gloves, mask and laboratory coat), (2) controls were applied to detect contamination during the extraction process and in each one of the amplifications, (3) Real-time PCR quantification of amplifiable DNA to quantify one mtDNA fragment of 113 bp was conducted [9, 49], (4) a duplicate analysis was performed on the greatest possible number of individuals, and (5) Cloning of PCR products was performed with subsequent sequencing of the clones. The cloning was carried out using TOPO TA Cloning® Kits (Invitrogen), following the supplier's instructions.

The mitochondrial variability resulted from El Aramo Mine was compared with other ancient and present-day populations. With respect to hunter-gatherers, three groups were considered: one from Scandinavia, one from Central Europe [13, 14, 50, 51], and one from the Cantabrian fringe of the Iberian Peninsula [9, 17, 33, 52]. Regarding the Neolithic DNA, 14 populations were selected: 3 from the Near East [15], 4 from Central and Eastern Europe [16, 45], 5 from the Mediterranean area of Europe (Hungary, Romania, Catalonia and France) [6, 7, 10, 12], and 2 from northern Iberia [9, 11]. With regard to the Chalcolithic groups, we considered one from Central Europe with BB artifacts associated [16, 18], one from the Cantabrian fringe of Iberia without BC culture (Longar and SJAPL sites) [9] and another two from Iberia, one with BB culture, and another one without BB culture [BBC: Arroyal (Burgos), Camino de las Yeseras (Madrid), Humanajes (Madrid), La Magdalena (Madrid), and Paris Street (Barcelona). Without BBC: Camino del Molino (Murcia), Bolares (Extremadura), el Sotillo, chabola de la Hechicera (Alava), el Mirador (Burgos), La Mina, Trocs (Huesca), and El Portalón (Burgos)] [18]. The Bronze Age period is represented by three groups from Siberia, Kazakhstan, and Romania [10, 53, 54]. The present-day populations database corresponds to that described in [10], to which the present-day population of Asturias, where El Aramo Mine is located, has been added [55, 56].

The FST distance matrix between present and ancient populations was calculated from the mtDNA haplogroup frequencies using Arlequin 3.11 [57]. Relationships between populations were studied through Multidimensional Scaling analysis (MDS), based on the FST distance matrix, using SPSS 20 Software. Median Joining Network (MJN) for certain haplogroups was generated to infer phylogenetic relationships between the mitochondrial lineages from the Paleolithic period to the present day using Network software v4.5.0.0 (available at http://www. fluxus-engineering.com). Different mutation weights were applied in accordance with previous papers [58–60], and the point insertions and deletions were excluded from the analysis.
