**3. Results and discussion**

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 extrac-

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

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

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

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

TOPO TA Cloning® Kits (Invitrogen), following the supplier's instructions.

tion, since it was the only anthropological material available.

some modifications [20–22, 43].

116 Mitochondrial DNA - New Insights

polymorphisms (RFLPs) [43, 46].

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 produced by the postmortem damage of aDNA.

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 evidence of kinship among these individuals.

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


and the Near East (9.91%) (**Figure 1**). Haplogroup J shows similar frequencies in El Aramo and in the extant population of the Near East (13%), and lower frequencies in the present-day populations of Asturias and Europe (10.5 and 8.6%, respectively). Haplogroup U5b in El Aramo Mine has a frequency of 7%, which is close to that observed in the extant population of Europe (6%) and considerably higher than that detected in the current populations of Asturias and the Near East (0.7 and 0.05%, respectively) (**Figure 1**). Lastly, haplogroup I3 shows in El Aramo a value (7%) that is considerably higher than that observed in the current populations, among which the population of the Near East (3%) shows the highest frequency value for this haplogroup, which is absent in the current population of Asturias and very infrequent in Europe (1.8%) (**Figure 1**). To sum up, the main differences in the frequencies of the mitochondrial lineages of El Aramo Mine and the present-day populations lie in haplogroups H, U5b, and I3. The frequency distribution of the mitochondrial haplogroups of El Aramo Mine was compared with that of other Chalcolithic groups of the Iberian Peninsula and Central Europe [9, 18]. For this analysis, five population groups were defined: (1) El Aramo Mine (Late Chalcolithic-Early Bronze Age) without BBC associated (ARAMO), (2) Chalcolithic from the Cantabrian fringe of the Iberian Peninsula without BBC (CA\_Cantabrian), (3) Chalcolithic from Iberia without BBC (CA\_Iberian), (4) Chalcolithic from Iberia associated with BBC (BBC\_Iberian), and (5) Chalcolithic from Central Europe associated with BBC (BBC\_CE) (**Figure 2**). All the populations included in this analysis share the presence of haplogroups H and T, although the distribution of their frequencies is different. The group of El Aramo shows the highest frequency value for haplogroup H (60%), followed by the two Chalcolithic groups of the Iberian Peninsula without BBC, CA\_Cantabrian and CA\_Iberian (39 and 31.2%, respectively); the lowest frequencies of haplogroup H were found in the BBC-associated groups of the Iberian Peninsula (BBC\_Iberian, with 22%) and Central Europe (BBC\_CE, with 29%). This frequency distribution of haplogroup H does not support the hypothesis of Brandt et al. [16], who suggest that the BBC was spread from the Iberian Peninsula toward Central Europe by haplogroup H carriers, since the highest frequency values were not found in population associated with the BB culture; moreover, the group of the Iberian Peninsula (BBC\_Iberian) has

**Figure 1.** Frequency distribution of mitochondrial haplogroups H, T, J, U5b, and I3 in the sample of El Aramo Mine

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(Asturias, present study) and the modern populations of Asturias, Europe, and the Near East.

the lowest value (22%) (**Figure 2**).

The number of molecules of endogenous mtDNA (molecules/μl), the polymorphisms of HVS-I, HVS-II, those of the coding region (RFLPs), and the mitochondrial haplogroup to which each of the samples belong are indicated.dIndicates that this sample has been duplicated.

\* The positions of the polymorphisms of HVS-I must have 16,000 added since it is HVS-I (16,000–16,400 pb).

**Table 1.** Results of the analysis of mtDNA in 15 individuals of El Aramo mine.

by nine different mitochondrial haplotypes, five of which correspond to haplogroup H-rCRS (33%), which is the most frequent one in the present-day European population (15–30%) and in the current population of Asturias ( 23.5%). The rest of the haplogroups detected in El ̴ Aramo mine have low frequencies in the extant populations of Europe and the Near East [54, 55, 61]. Haplogroup T has a frequency of 13% in El Aramo, which is close to that of the presentday population of Asturias (11.2%) and higher than that of the populations of Europe (6.8%)

**Figure 1.** Frequency distribution of mitochondrial haplogroups H, T, J, U5b, and I3 in the sample of El Aramo Mine (Asturias, present study) and the modern populations of Asturias, Europe, and the Near East.

and the Near East (9.91%) (**Figure 1**). Haplogroup J shows similar frequencies in El Aramo and in the extant population of the Near East (13%), and lower frequencies in the present-day populations of Asturias and Europe (10.5 and 8.6%, respectively). Haplogroup U5b in El Aramo Mine has a frequency of 7%, which is close to that observed in the extant population of Europe (6%) and considerably higher than that detected in the current populations of Asturias and the Near East (0.7 and 0.05%, respectively) (**Figure 1**). Lastly, haplogroup I3 shows in El Aramo a value (7%) that is considerably higher than that observed in the current populations, among which the population of the Near East (3%) shows the highest frequency value for this haplogroup, which is absent in the current population of Asturias and very infrequent in Europe (1.8%) (**Figure 1**). To sum up, the main differences in the frequencies of the mitochondrial lineages of El Aramo Mine and the present-day populations lie in haplogroups H, U5b, and I3.

The frequency distribution of the mitochondrial haplogroups of El Aramo Mine was compared with that of other Chalcolithic groups of the Iberian Peninsula and Central Europe [9, 18]. For this analysis, five population groups were defined: (1) El Aramo Mine (Late Chalcolithic-Early Bronze Age) without BBC associated (ARAMO), (2) Chalcolithic from the Cantabrian fringe of the Iberian Peninsula without BBC (CA\_Cantabrian), (3) Chalcolithic from Iberia without BBC (CA\_Iberian), (4) Chalcolithic from Iberia associated with BBC (BBC\_Iberian), and (5) Chalcolithic from Central Europe associated with BBC (BBC\_CE) (**Figure 2**). All the populations included in this analysis share the presence of haplogroups H and T, although the distribution of their frequencies is different. The group of El Aramo shows the highest frequency value for haplogroup H (60%), followed by the two Chalcolithic groups of the Iberian Peninsula without BBC, CA\_Cantabrian and CA\_Iberian (39 and 31.2%, respectively); the lowest frequencies of haplogroup H were found in the BBC-associated groups of the Iberian Peninsula (BBC\_Iberian, with 22%) and Central Europe (BBC\_CE, with 29%). This frequency distribution of haplogroup H does not support the hypothesis of Brandt et al. [16], who suggest that the BBC was spread from the Iberian Peninsula toward Central Europe by haplogroup H carriers, since the highest frequency values were not found in population associated with the BB culture; moreover, the group of the Iberian Peninsula (BBC\_Iberian) has the lowest value (22%) (**Figure 2**).

by nine different mitochondrial haplotypes, five of which correspond to haplogroup H-rCRS (33%), which is the most frequent one in the present-day European population (15–30%) and in the current population of Asturias ( 23.5%). The rest of the haplogroups detected in El ̴ Aramo mine have low frequencies in the extant populations of Europe and the Near East [54, 55, 61]. Haplogroup T has a frequency of 13% in El Aramo, which is close to that of the presentday population of Asturias (11.2%) and higher than that of the populations of Europe (6.8%)

**Molec/μl HVS-I\* HVS-II RFLPs HG**

73G *DdeI10394-;* 

73G *DdeI10394-;* 

73G *DdeI10394-;* 

73G *DdeI10394-;* 

*HaeII9052+; HinfI12308+*

*NlaIII4216+*

73A *Alu7025-* H

*NlaIII4216+*

73G *DdeI1715-; Alu10032+* I3

*NlaIII4216+*

*NlaIII4216+*

*Alu7025-* H

*Alu7025-* H

*Alu7025-* H

*Alu7025-* H

U5b1b

T2c

J1c

J1c

T1

315.1C

263-309.1C-315.1C-320C

263G-315.1C

315.1C

Skull 1 (bone) AR13 854.6 rCRS 73A *Alu7025-* H

270T

Skull 4 (tooth) AR21 33668.7 189C 73A *Alu7025-* H

Skull 6 (tooth) AR32 18706.4 51G 73A *Alu7025-* H1

294T-296T

362C

223T

AR12 133777.7 69T-126C 73G *DdeI10394-;* 

320T-360T

187T-189T-294T

The number of molecules of endogenous mtDNA (molecules/μl), the polymorphisms of HVS-I, HVS-II, those of the coding region (RFLPs), and the mitochondrial haplogroup to which each of the samples belong are indicated.dIndicates

Fibule (bone) ARH13 312.6 086C-129A — *Alu7025-* H

The positions of the polymorphisms of HVS-I must have 16,000 added since it is HVS-I (16,000–16,400 pb).

**SAMPLE SAMPLE** 

118 Mitochondrial DNA - New Insights

Hemimandible (tooth)

(tooth)

(tooth)

\*

Right Hemimandible

Left Hemimandible

that this sample has been duplicated.

**ID**

Skull 2 (bone) AR15 551.4 rCRS 73A-263G-

Skull 5 (tooth) AR31 17194.3 rCRS 73A-150T-

Skull 7 (tooth)d AR42 2078.3 rCRS 73A-153T-

Skull 9 (tooth) AR5 178331.7 rCRS 73A-263G-

AR7-9 153133.6 86C-129A-

AR6 141554.8 69T-126C-

Skull 8 (tooth)d AR41 5847.2 126C-292T-

Mandible (tooth) AR11 37374.7 126C-355T-

Right femur (bone) ARH10 372.5 126C-163G-

**Table 1.** Results of the analysis of mtDNA in 15 individuals of El Aramo mine.

Skull 3 (tooth)d AR22 89190.2 183C-189C-

**Figure 2.** Frequency distribution of mitochondrial haplogroups H, T, J, U5b, and I3 in the sample of El Aramo Mine (Asturias, present study), and in other Chalcolithic groups, both without BB culture (CA\_Cantabrian and CA\_Iberian) and with BBC associated (BBC\_Iberian and BBC\_Central Europe).

With respect to haplogroup T, there is also a heterogeneous distribution among the five population groups analyzed. The group of El Aramo shows the highest value for this haplogroup (13%); the Chalcolithic groups associated with BBC (BBC\_Iberian and BBC\_CE) have frequencies of 5.5 and 7.8%, respectively, which are lower than that of CA\_Cantabrian (9.5%) and higher than that of CA\_Iberian (2.08%). Given the fact that haplogroup T has been proposed as a marker of the diffusion of the Neolithic culture, the heterogeneity of the frequencies of this haplogroup in the Chalcolithic populations supports the model of random Neolithic cultural diffusion (Hervella et al. [9]). This behavior has also been observed in the MJN (**Figure 3**), where T haplotypes of prehistoric and present-day populations were shown. In MJN main nodes are shared by all the populations compared, both prehistoric and modern. The polymorphisms that define the two T haplotypes of El Aramo Mine are shared with Neolithic and Chalcolithic groups, indicating their relation. Furthermore, the polymorphisms of one of the lineages found in El Aramo Mine are shared with current samples of Europe and the Near East, showing its prevalence to the present time (**Figure 3**).

Haplogroup J shows high-frequency values in the groups that are not associated with BBC (Aramo (13%), CA\_Cantabrian (13%), and CA\_Iberian (23%)), with the sample of CA\_Iberian showing the highest value. The BBC-associated groups have lower frequencies, with 7.8% in the sample of BBC\_CE and 0% in BBC\_Iberian, showing once again the difference between the Chalcolithic groups with BBC and without BBC. Haplogroup J has also been proposed as a marker of the Neolithic diffusion in Europe. The distribution of frequencies of this haplogroup in the populations analyzed (**Figure 2**) shows a greater Neolithic influence in the non-BBC Chalcolithic groups. This influence of Neolithic diffusion is also observed in the MJN analysis of haplogroup J (**Figure 4**), which shows that the haplotypes of the Chalcolithic samples non- BBC are included in the mitochondrial variability of the Near East. With regard to the BBC-associated populations, the J lineages correspond solely to the BBC\_CE population, and not to the BBC\_Iberian population (**Figures 2** and **4**), highlighting the differentiation between the two groups with BB culture (BBC\_CE and BBC\_Iberian). The frequency of haplogroup U5b in El Aramo (7%) indicates the persistence of Paleolithic lineages even after the Neolithic period. The prevalence of this lineage seems to be higher in the Chalcolithic groups of the Iberian Peninsula (16.6% in CA\_Iberian and 27.8% in BBC\_Iberian) when compared to

those of Central Europe (6.3%) (**Figure 2**). The absence of haplogroup U5b in the Chalcolithic sample of the Cantabrian region (CA\_Cantabrian) is due to the lack of differentiation in the U

**Figure 4.** Median Joining Network of mitochondrial haplotypes of haplogroup J. El Aramo (red), Neolithic populations

(green), Chalcolithic (blue), BBC (pink), modern Europeans (yellow), and modern Near Easterns (orange).

**Figure 3.** Median Joining Network of mitochondrial haplotypes of haplogroup T. El Aramo (red), Neolithic populations

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(green), Chalcolithic (blue), BBC (pink), modern Europeans (yellow), and modern Near Easterns (orange).

Lastly, haplogroup I3 appears only in the sample of El Aramo (7%) and in the BBC population of Central Europe (5.5%). The origin of this mitochondrial lineage has been thoroughly debated, and the consensus reached is that it began in the recent Neolithic period [1], with

lineages published in this sample [43].

**Figure 3.** Median Joining Network of mitochondrial haplotypes of haplogroup T. El Aramo (red), Neolithic populations (green), Chalcolithic (blue), BBC (pink), modern Europeans (yellow), and modern Near Easterns (orange).

With respect to haplogroup T, there is also a heterogeneous distribution among the five population groups analyzed. The group of El Aramo shows the highest value for this haplogroup (13%); the Chalcolithic groups associated with BBC (BBC\_Iberian and BBC\_CE) have frequencies of 5.5 and 7.8%, respectively, which are lower than that of CA\_Cantabrian (9.5%) and higher than that of CA\_Iberian (2.08%). Given the fact that haplogroup T has been proposed as a marker of the diffusion of the Neolithic culture, the heterogeneity of the frequencies of this haplogroup in the Chalcolithic populations supports the model of random Neolithic cultural diffusion (Hervella et al. [9]). This behavior has also been observed in the MJN (**Figure 3**), where T haplotypes of prehistoric and present-day populations were shown. In MJN main nodes are shared by all the populations compared, both prehistoric and modern. The polymorphisms that define the two T haplotypes of El Aramo Mine are shared with Neolithic and Chalcolithic groups, indicating their relation. Furthermore, the polymorphisms of one of the lineages found in El Aramo Mine are shared with current samples of Europe and the Near

**Figure 2.** Frequency distribution of mitochondrial haplogroups H, T, J, U5b, and I3 in the sample of El Aramo Mine (Asturias, present study), and in other Chalcolithic groups, both without BB culture (CA\_Cantabrian and CA\_Iberian)

Haplogroup J shows high-frequency values in the groups that are not associated with BBC (Aramo (13%), CA\_Cantabrian (13%), and CA\_Iberian (23%)), with the sample of CA\_Iberian showing the highest value. The BBC-associated groups have lower frequencies, with 7.8% in the sample of BBC\_CE and 0% in BBC\_Iberian, showing once again the difference between the Chalcolithic groups with BBC and without BBC. Haplogroup J has also been proposed as a marker of the Neolithic diffusion in Europe. The distribution of frequencies of this haplogroup in the populations analyzed (**Figure 2**) shows a greater Neolithic influence in the non-BBC Chalcolithic groups. This influence of Neolithic diffusion is also observed in the MJN analysis of haplogroup J (**Figure 4**), which shows that the haplotypes of the Chalcolithic samples non- BBC are included in the mitochondrial variability of the Near East. With regard to the BBC-associated populations, the J lineages correspond solely to the BBC\_CE population, and not to the BBC\_Iberian population (**Figures 2** and **4**), highlighting the differentiation between the two groups with BB culture (BBC\_CE and BBC\_Iberian). The frequency of haplogroup U5b in El Aramo (7%) indicates the persistence of Paleolithic lineages even after the Neolithic period. The prevalence of this lineage seems to be higher in the Chalcolithic groups of the Iberian Peninsula (16.6% in CA\_Iberian and 27.8% in BBC\_Iberian) when compared to

East, showing its prevalence to the present time (**Figure 3**).

and with BBC associated (BBC\_Iberian and BBC\_Central Europe).

120 Mitochondrial DNA - New Insights

**Figure 4.** Median Joining Network of mitochondrial haplotypes of haplogroup J. El Aramo (red), Neolithic populations (green), Chalcolithic (blue), BBC (pink), modern Europeans (yellow), and modern Near Easterns (orange).

those of Central Europe (6.3%) (**Figure 2**). The absence of haplogroup U5b in the Chalcolithic sample of the Cantabrian region (CA\_Cantabrian) is due to the lack of differentiation in the U lineages published in this sample [43].

Lastly, haplogroup I3 appears only in the sample of El Aramo (7%) and in the BBC population of Central Europe (5.5%). The origin of this mitochondrial lineage has been thoroughly debated, and the consensus reached is that it began in the recent Neolithic period [1], with very low-frequency values found in the modern populations. In the MJN conducted for haplogroup I3, it was observed that El Aramo shares polymorphisms with Neolithic and Chalcolithic groups and with the current populations of Europe and the Near East, showing a low diversity for this lineage (**Figure 5**).

After the comparative analysis of the frequency distribution of the mitochondrial haplogroups of El Aramo and other prehistoric populations, it is possible to distinguish some general tendencies. On the one hand, there is a clear genetic differentiation between the Chalcolithic groups with BB culture (BBC\_CE and BBC\_Iberian) and those without BB culture (CA\_Iberian, CA\_ Cantabrian and El Aramo); on the other hand, there is a differentiation between the BBC groups of Central Europe and those of the Iberian Peninsula (**Figure 2**). The population of El Aramo Mine shows a distribution of mitochondrial haplotypes that is closer to that of Chalcolithic populations without BBC than to that of chalcolithic populations with BBC, highlighting the specificity of El Aramo, the high frequency of haplogroup H (60%) and the presence of haplogroup I3, whose frequency is, in addition, one of the highest ones described to the present day.

The mitochondrial variability of the population of El Aramo was analyzed in the context of other prehistoric groups and the modern populations of Europe and the Near East through a Multidimensional Scaling analysis (MDS) (**Figure 6**). The MDS was done through a matrix of FST distances, calculated by the frequencies of the mitochondrial haplogroups of hunter-gatherer,

**Figure 5.** Median Joining Network of mitochondrial haplotypes of haplogroup I3. El Aramo (red), Neolithic populations (green), Chalcolithic (blue), BBC (pink), modern Europeans (yellow), and modern Near Easterns (orange).

**Figure 6.**

Multidimensional Scaling Analysis performed for haplogroup frequencies of the ancient and present-day European and Near Eastern populations. Hunter-Gatherer groups (pink), Neolithic and Chalcolithic populations (green), Bronze Age groups (purple), El Aramo Mine (red), present-day European populations (grey),

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and present-day Near Eastern populations (orange). Stress: 0.142 and RSQ: 0.94548. Abbreviations: Hunter-Gatherers (HG), Neolithic (NEO), Bronze Age (BA), presentday populations in Europe: Eastern Mediterranean (MdE), Central Mediterranean (MdC), Western Mediterranean (MdW), Northeast Europe (NE), North-Central

Europe (NC), Northwest Europe (NW), Southeast Europe (SE), and Alps (ALP).

very low-frequency values found in the modern populations. In the MJN conducted for haplogroup I3, it was observed that El Aramo shares polymorphisms with Neolithic and Chalcolithic groups and with the current populations of Europe and the Near East, showing

After the comparative analysis of the frequency distribution of the mitochondrial haplogroups of El Aramo and other prehistoric populations, it is possible to distinguish some general tendencies. On the one hand, there is a clear genetic differentiation between the Chalcolithic groups with BB culture (BBC\_CE and BBC\_Iberian) and those without BB culture (CA\_Iberian, CA\_ Cantabrian and El Aramo); on the other hand, there is a differentiation between the BBC groups of Central Europe and those of the Iberian Peninsula (**Figure 2**). The population of El Aramo Mine shows a distribution of mitochondrial haplotypes that is closer to that of Chalcolithic populations without BBC than to that of chalcolithic populations with BBC, highlighting the specificity of El Aramo, the high frequency of haplogroup H (60%) and the presence of haplogroup I3, whose frequency is, in addition, one of the highest ones described to the present day.

The mitochondrial variability of the population of El Aramo was analyzed in the context of other prehistoric groups and the modern populations of Europe and the Near East through a Multidimensional Scaling analysis (MDS) (**Figure 6**). The MDS was done through a matrix of FST distances, calculated by the frequencies of the mitochondrial haplogroups of hunter-gatherer,

**Figure 5.** Median Joining Network of mitochondrial haplotypes of haplogroup I3. El Aramo (red), Neolithic populations

(green), Chalcolithic (blue), BBC (pink), modern Europeans (yellow), and modern Near Easterns (orange).

a low diversity for this lineage (**Figure 5**).

122 Mitochondrial DNA - New Insights

farmer, Chalcolithic, Bronze Age, and present-day populations from the Cantabrian fringe, Europe, and the Near East. This analysis shows the differentiation between the two huntergatherer populations from Central Europe and Scandinavia (**Figure 6**). It has been proposed that the mtDNA variation of these groups indicates a genetic discontinuity between the huntergatherers and later populations in these two geographic regions [13, 14, 50, 51]. However, this suggested discontinuity is not so obvious in the case of the hunter-gatherers from the Cantabrian fringe who separated from those of Central Europe and Scandinavia in this analysis [9, 17] (**Figure 6**).

during which a change in the landscape and the subsistence conditions have been detected in this region [62]. El Aramo consists of males who had a strong relationship with mining, and the burials reflect a ritual related to the exploitation of the mine [63]. Therefore, it is a group with differentiated cultural characteristics within the Chalcolithic groups of the Iberian Peninsula. This cultural differentiation seems to be accompanied by a genetic differentiation, since in the MDS, El Aramo is distant from the rest of the Chalcolithic groups (**Figure 6**). These results indicate the existence of local processes in the Chalcolithic period that could be related to subsistence strategies linked to the characteristics of the environment. In the case of El Aramo (Asturias), these strategies are linked to mining activity, since the region of Asturias

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This work was supported by the Basque Government to Research Groups of the Basque University System (IT1138-16) and grant from the Spanish Ministry of Science and Innovation (GCL2016-79093/P). We are grateful to Miguel Angel de Blas Cortina, Director of the archeological intervention in El Aramo Mine, for providing archeological data and fruitful discussion.

[1] Revesz PZ. A spatio-temporal analysis of mitochondrial DNA Haplogroup I. MATEC

[2] Zvelebil M, Zvelebil KV.Agricultural transition and indo-European dispersal. Antiquity.

[3] Ammerman AJ, Cavalli-Sforza LL. The Neolithic Transition and the Genetics of Popu-

Web Conference. 2016;**76**:04048. DOI: 10.1051/matecconf/2016760

lations in Europe. Princeton: Princeton University Press; 1984

is one of the traditional mining areas since the Neolithic period [63].

The authors have declared that no competing interests exist.

Montserrat Hervella and Concepcion de-la-Rua\*

\*Address all correspondence to: conchi.delarua@ehu.eus

University of the Basque Country (UPV/EHU), Bizkaia, Spain

**Acknowledgements**

**Conflict of interest**

**Author details**

**References**

1988;**62**:574-583

With regard to the European Neolithic populations, the heterogeneity observed in the mtDNA haplogroup frequency variation is revealed by their position on the two-dimensional plot of the MDS analysis (**Figure 6**). On the one hand, a group of populations (Near East, Central Europe, Hungary, and Eastern Pyrenees) with high-frequency values for haplogroup N is separated from the other Neolithic populations. On the other hand, heterogeneity is also apparent within the Mediterranean area, with a Neolithic population of Southern France being closer to present-day populations in the Near East, due to its high frequency for haplogroups J and U, whereas the Neolithic populations from the Iberian Peninsula (Catalonia and the Cantabrian fringe) show lower frequency for those haplogroups (J and U) (**Figure 6**). The genetic distances observed between the European Neolithic groups suggest a different genetic impact of the Neolithic farmers from the Near East on Central Europe, Mediterranean Europe, and the Cantabrian fringe. These data support a random dispersion model for Neolithic farmers, with different impact on the various geographic regions [9].

With respect to the Chalcolithic prehistoric groups included in the analysis (BBC\_CE, BBC\_ Iberian, CA\_Iberian, CA\_Cantabrian and Aramo), those with BB culture (BBC\_CE and BBC\_ Iberian) are differentiated from the rest. On the other hand, the distance between the BBC groups of Central Europe and the Iberian Peninsula is due to both the greater persistence of Paleolithic U5 lineages in the BBC\_Iberian group and the higher frequency of Neolithic lineages T and J in the BBC\_CE group. This differentiation suggests that the relation between these two Chalcolithic groups (BBC\_CE and BBC\_Iberian) is only cultural but not genetic, supporting the study [18] about the Beaker phenomenon and genomic data from Europe, who reject the hypothesis that the genetic substrate of the BBC\_CE groups came from the BBC\_Iberian groups [16].

Regarding the Chalcolithic populations without BB culture, CA\_Iberian and CA\_Cantabrian are genetically close to one another, with El Aramo being further from them due to the high frequencies of haplogroups H and I3. These Chalcolithic groups are not distant from their Neolithic ancestor populations, although they are distant from the present-day populations of these regions (**Figure 6**), which could be attributed to a post-Neolithic population restructuring [20].

In view of the results obtained, it can be inferred that the influence of the Neolithic period on the local groups was complex, and its result could generate the existence of Chalcolithic groups in the Iberian Peninsula with genetic and cultural differences, with the latter being mainly related to the Beaker phenomenon.

The human group of El Aramo Mine, without artifacts associated with BBC, shows some peculiarities. Its chronology expands from the Chalcolithic period to the Early Bronze Age, during which a change in the landscape and the subsistence conditions have been detected in this region [62]. El Aramo consists of males who had a strong relationship with mining, and the burials reflect a ritual related to the exploitation of the mine [63]. Therefore, it is a group with differentiated cultural characteristics within the Chalcolithic groups of the Iberian Peninsula. This cultural differentiation seems to be accompanied by a genetic differentiation, since in the MDS, El Aramo is distant from the rest of the Chalcolithic groups (**Figure 6**). These results indicate the existence of local processes in the Chalcolithic period that could be related to subsistence strategies linked to the characteristics of the environment. In the case of El Aramo (Asturias), these strategies are linked to mining activity, since the region of Asturias is one of the traditional mining areas since the Neolithic period [63].
