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An unexpected case in the prehistory of the Iberian Peninsula: Biogeographical origin analysis through mitochondrial DNA

Published:September 16, 2017DOI:https://doi.org/10.1016/j.fsigss.2017.09.080

      Abstract

      We have determined the mitochondrial haplotype (A73G T16172C C16223T G16257A C16261T) on a Bronze Age Individual from an archaeological site located in Alaejos (Valladolid, Central Spain). This haplotype is typical from N9a2′4′5 or N9a2′4′5′11 mitochondrial haplogroups, which are nowadays widely distributed in the Asian region. Here, we compare and discuss the frequency of this haplogroup in nowadays and in ancient human populations, especially in contemporaries of the individual under study.
      The biogeographical analysis has been carried out by amplifying two overlapping fragments of the Hipervariable Region I (HRVI) and other two of the Hipervariable Region II (HRVII) of the mitochondrial DNA (mtDNA). Moreover the mtDNA haplogroup was assigned by the analysis of mtSNPs in coding regions. Furthermore, the experimental process was performed by duplicate to obtain a reliable haplogroup. Along the all process, ancient DNA authenticity criteria were observed.

      Keywords

      1. Introduction

      The Archaeological site called “El Tablón” (Alaejos, Valladolid, Central Spain) is located on the south side of Duero River, and it was discovered during the Valladolid-Salamanca motorway construction [
      • Domínguez Bolaños A.
      Primera campaña de excavación arqueológica en el yacimiento de El Tablón (Alaejos, Valladolid), Autovía de Castilla N-620, Tramo Alaejos-Cañizal. 1997–1998.
      ]. “El Tablón” is a typical pits field with many ProtoCogotas I Archaeological Culture materials (Medium Bronze Age). In two of the excavated pits of this site (concretely number 31 and 44) there were exhumed human remains from two different individuals (both in individual burials).
      The present work is focused on the analysis of the exhumed individual from the burial number 31. This pit, dated by radiocarbon about 3220 ± 35 YBP (Years Before Present) [
      • Domínguez Bolaños A.
      Primera campaña de excavación arqueológica en el yacimiento de El Tablón (Alaejos, Valladolid), Autovía de Castilla N-620, Tramo Alaejos-Cañizal. 1997–1998.
      ], seems to be a secondary burial, judging by the lack of anatomical connections, the absence of some skeletal remains and its deterioration. The bioanthropological analysis determined that the individual was probably a young man, between 16 and 19 years old.
      Mitochondrial DNA (mtDNA) is the most used genetic marker in the analysis of ancient/critical samples, mainly because the samples have more quantity of mtDNA and usually it yields most successful results than nuclear DNA (nuDNA) [
      • Palomo-Díez S.
      Caracterización Genética de las Poblaciones de las Edades del Cobre y del Bronce en la Submeseta Norte de la Península Ibérica. pHD Thesis.
      ]. Though mtDNA cannot provide the same kind of information than nuDNA, – and therefore being less informative for the identification and also in order to stablish close kinship relationships-, it can be very useful to know other kind of information about the individuals, like their probable biogeographical origin, or to study the maternal mitochondrial lineage [
      • Palomo-Díez S.
      Caracterización Genética de las Poblaciones de las Edades del Cobre y del Bronce en la Submeseta Norte de la Península Ibérica. pHD Thesis.
      ].
      In any mtDNA analysis we must know the population of origin of the studied individual, but when we are facing up to ancient samples, it is not possible to know with certainty whether the population remained unchanged or not, migratory movements, bottleneck phenomena, or which was the genetic population composition of the historic period when our individual lived [
      • Palomo-Díez S.
      Caracterización Genética de las Poblaciones de las Edades del Cobre y del Bronce en la Submeseta Norte de la Península Ibérica. pHD Thesis.
      ,
      • Hervella M.
      • Izaguirre N.
      • Alonso S.
      • Fregel R.
      • Alonso A.
      • Cabrera V.M.
      • de la Rua C.
      Ancient DNA from hunter-Gatherer and farmer groups from northern Spain supports a random dispersion model for the neolithic expansion into europe.
      ]. The only way to analyze our results is to compare with nowadays databases and studies [
      • van Oven M.
      PhyloTree Build 17: growing the human mitochondrial DNA tree.
      ,
      • Röck A.W.
      • Dür A.
      • Van Oven M.
      • Parson W.
      Cocept for estimatingmitochondrial DNA haplogroups using a maximum likelihood approach (EMMA).
      ,
      • Barral-Arca R.
      • Pischedda S.
      • Gómez-Carballa A.
      • Pastoriza A.
      • Mosquera-Miguel A.
      • López-Soto M.
      • Martinón-Torres F.
      • Álvarez-Iglesias V.
      • Salas A.
      Meta-Analysis of MItochondrial DNA variation in the Iberian peninsula.
      ] or to compare with the few ancient DNA studies performed until today [
      • Palomo-Díez S.
      Caracterización Genética de las Poblaciones de las Edades del Cobre y del Bronce en la Submeseta Norte de la Península Ibérica. pHD Thesis.
      ,
      • Hervella M.
      • Izaguirre N.
      • Alonso S.
      • Fregel R.
      • Alonso A.
      • Cabrera V.M.
      • de la Rua C.
      Ancient DNA from hunter-Gatherer and farmer groups from northern Spain supports a random dispersion model for the neolithic expansion into europe.
      ].
      Along this work we have analyzed the mtDNA of the archaeological individual exhumed from the 31 pit of El Tablón archaeological site, with the aim of establishing his mtDNA haplotype and haplogroup, in order to discuss about his possible biogeographical origin.

      2. Material and methods

      We selected two samples from the exhumed individual, with the aim of doing all the experimental procedure by twice, according with the authenticity ancient DNA criteria [
      • Pääbo S.
      • Poinar H.
      • Serre D.
      • Jaenicke-Despres V.
      • Hebler J.
      • Rohland N.
      • Kuch M.
      • Drause J.
      • Vigilant L.
      • Hofreiter M.
      Genetic analyses from ancient Dna.
      ].
      Firstly, each sample surface was cleaned with a sandblaster DentalFarm Model Base 1 Plus using pressurized aluminum-oxid and radiated with UV light. Then, one of the samples was pulverized, using a mill cooled with liquid nitrogen, obtaining a quantity of powder enough for DNA extraction, according to silica and Thiocyanate Guanidine based extraction method [
      • Rohland N.
      • Hofreiter M.
      Ancient DNA extraction from bones an teeth.
      ]. The other sample was not powdered, because it was analyzed according to a nondestructive extraction DNA protocol, according to Gomes and Palomo-Díez [
      • Gomes C.
      • Palomo-Díez S.
      • Roig J.
      • López-Parra A.M.
      • Baeza-Richer C.
      • Esparza-Arroyo A.
      • Gibaja J.
      • Arroyo-Pardo E.
      Nondestructive extraction DNa method from bones or teeth: true of false?.
      ].
      We amplified mtDNA HVI and HVII regions by four different primer pairs [
      • Fernández E.
      Polimorfismos de ADN mitocondrial en poblacione santiguas de la Cuenca Mediteránea. Tesis doctoral.
      ,
      • Martínez-Labarga C.
      • Rickards O.
      La utilización del ADN antiguo en la investigación de la historia evolutiva humana.
      ], two pairs to amplify HVI by short overlapping fragments and other two to HVII. We performed at least two amplifications of each marker and sample to replicate all the results. The mitochondrial DNA sequences were analyzed with Mutation Surveyor® V5.0.1 software.
      The full procedure was performed in an exclusive isolated ancient DNA laboratory satisfying all the ancient DNA authenticity criteria [
      • Pääbo S.
      • Poinar H.
      • Serre D.
      • Jaenicke-Despres V.
      • Hebler J.
      • Rohland N.
      • Kuch M.
      • Drause J.
      • Vigilant L.
      • Hofreiter M.
      Genetic analyses from ancient Dna.
      ].

      3. Results

      A total of 16 PCRs, 8 of each sample, 4 of the two overlapping fragments of HVI and other 4 of the two overlapping fragments of HVI were performed. The results were considered valid when all the replications were consistent with the same mtDNA profile. According to these conditions it was possible to establish a mtDNA haplotype into the range (16105–16399) for HVI, and (50–140) for HVII. The obtained haplotype was T16172C C16223T G16257A C16261T A73G. From this haplotype we have established the most probable haplogroup of the individual, according to the Phylotree (mtDNA tree Build 17 (18 Feb 2016)) [
      • van Oven M.
      PhyloTree Build 17: growing the human mitochondrial DNA tree.
      ] and using the Haplogrep tool [
      • Kloss-Brandstätter A.
      • Pacher D.
      • Schönherr S.
      • Weissensteiner H.
      • Binna R.
      • Specht G.
      • Kronenberg F.
      HaploGrep: a fast and reliable algorithm for automatic classification of mitochondrial DNA haplogroups.
      ] and EMPOP Query tool and database [
      • Röck A.W.
      • Dür A.
      • Van Oven M.
      • Parson W.
      Cocept for estimatingmitochondrial DNA haplogroups using a maximum likelihood approach (EMMA).
      ]. The Haplogrep assignation of the haplogroup was N9a2‘4‘5‘11, with an estimated quality of 100%, and the EMPOP assignation was N9a2′4′5.

      4. Discussion

      According to the obtained results the studied individual belongs to the mitochondrial haplogroup N9a2‘4‘5 or N9a2‘4‘5‘11. In any case, it is a typical haplogroup from Asiatic present-day human populations [
      • van Oven M.
      PhyloTree Build 17: growing the human mitochondrial DNA tree.
      ], and it is possible to observe its distribution around the world on Fig. 1, where we also can see that the haplogroup N9a2‘4‘5 in the studied archaeological site location is practically absent nowadays.
      Fig. 1
      Fig. 1Distribution of N9a2‘4‘5 mitochondrial haplogroup around the world according to EMPOP database, and the location of Alaejos (Valladolid, Central Spain) where the archaeological human remains were exhumed. (Source: EMPOP web).
      In regard to actual Spanish populations, according to Barral-Arca et al. [
      • Röck A.W.
      • Dür A.
      • Van Oven M.
      • Parson W.
      Cocept for estimatingmitochondrial DNA haplogroups using a maximum likelihood approach (EMMA).
      ] the N mtDNA haplogroup is nowadays present in the 1% of the Spanish Iberian Plateau (n = 1318 individuals) population [
      • Röck A.W.
      • Dür A.
      • Van Oven M.
      • Parson W.
      Cocept for estimatingmitochondrial DNA haplogroups using a maximum likelihood approach (EMMA).
      ]. So that it is not usual to find an N individual in today’s human population of the region. The differences between the 3220 ± 35 YBP populations and the present one can be due to unknown factors such as migrations, bottle-necks and so on. Therefore, we do not know with certainty what exactly has occurred with human populations along this time span. For a better understanding of why this individual belonged to an unusual haplogroup in this geographical region, it would be very useful to know the mtDNA composition of its contemporary populations. However, there is not enough published data about Bronze Age Spanish Iberian Plateau populations to date [
      • Palomo-Díez S.
      Caracterización Genética de las Poblaciones de las Edades del Cobre y del Bronce en la Submeseta Norte de la Península Ibérica. pHD Thesis.
      ]. The few data available shows none N haplogroup case until now, this being it is the first registered case of N9a2‘2′5 or N9á2′2′5′11 haplogroup in this geographical area [
      • Palomo-Díez S.
      Caracterización Genética de las Poblaciones de las Edades del Cobre y del Bronce en la Submeseta Norte de la Península Ibérica. pHD Thesis.
      ,
      • Barral-Arca R.
      • Pischedda S.
      • Gómez-Carballa A.
      • Pastoriza A.
      • Mosquera-Miguel A.
      • López-Soto M.
      • Martinón-Torres F.
      • Álvarez-Iglesias V.
      • Salas A.
      Meta-Analysis of MItochondrial DNA variation in the Iberian peninsula.
      ].
      In conclusion, we have not enough information to know whether this individual became from an Asiatic population (along his maternal lineage) o if he is part of a small population of N haplogroups that still exists in the current Iberian population. In any case, it is a rare finding, and the most ancient human of N9a2‘2′5 or N9á2′2′5′11 haplogroup found in this geographical region.

      Conflict of interest statement

      None.

      Acknowledgements

      This work has been possible thanks to the projects HAR2009-10105 and HAR2013-41851-P funded by the MINECO (Spanish Government) and BES2010-035322FPIz grant for Sara Palomo Díe. And by G/6401400/8000 research project (Banco Santander-Universidad Complutense de Madrid, Spain) for Cláudia Gomes.

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