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Population genetics and DNA preservation in ancient human remains from Eastern Spain
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Forensic Science International: Genetics Supplement Series 1 (2008) 462–464 www.elsevier.com/locate/FSIGSS
Research article
Population genetics and DNA preservation in ancient human remains from Eastern Spain C. Gamba a, E. Ferna´ndez a, A. Oliver b, M. Tirado a, C. Baeza a, A.M. Lo´pez-Parra a, E. Arroyo-Pardo a,* a
Depto. Toxicologı´a y Legislacio´n Sanitaria, Facultad de Medicina, Universidad Complutense, Madrid 28040, Spain b Seccio´n de Arqueologı´a. Museo de Bellas Artes de Castello´n, Castello´n, Spain Received 16 August 2007; accepted 7 October 2007
Abstract This work aims to establish the genetic relationship between the different settlers of Eastern Spain and also to determine the conditions of DNA preservation. We studied two overlapping sequences (16,126–16,251 and 16,256–16,369) from mitochondrial HVR-I in 37 bone and teeth samples from 17 archaeological sites of Spanish Levant. Consistence of the results was established by repeated replication of amplifications. Approximately, 50% of the samples yielded reproducible results. The high efficiency in DNA recovery indicates that sample preservation mainly depends on the depositional environment rather than on sample age. Haplogroup V, an alleged marker of Paleolithic newcomers in Europe, has been found in an unusual elevated frequency (1 Calcolithic and 2 Iberian samples). This result could suggest a more southern distribution of Palaeolithic ice refugia. Moreover, we found Haplogroup L in Calcolithic samples. This may suggest the presence of a prehistoric African genetic background in eastern Iberia. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Ancient DNA; Mitochondrial DNA; Eastern Iberia
1. Introduction The East of Spain has been populated at least since Palaeolithic times [1]. Due to its strategic location, opened to the Mediterranean shore, it has been prone to have received genetic influences from other populations. Present work tries to evaluate this hypothesis through mtDNA Hypervariable region I (HVR-I) characterization of ancient samples from Castello´n and Valencia provinces, dated back to Roman, Iberian, Bronze and Chalcolithic ages. Obtained results will be interpreted in terms of genetic continuity/discontinuity between these periods. The effect of the environment and the age of the sample over DNA preservation will be evaluated in this study.
Chalcolithic, 2 Bronze Age, 6 Iberian Culture and 3 Roman) located in Eastern Spain (see Fig. 1 and Table 1). 2.2. Extraction The external surface of the samples was removed first with a Sand-blaster, and afterwards they were irradiated with UV light and crushed in a freezer mill filled with liquid Nitrogen. Samples were then washed several times with EDTA 0.5 M and incubated in a lysis solution (EDTA 5 mM, Tris–HCl 10 mM, SDS 0.5%, proteinase K 50 mg/ml) overnight at 36 8C. DNA was extracted using a modified Phenol–Chloroform protocol [2] and concentrated with microcons (Centriplus 30000, Millipore).
2. Material and methods 2.3. PCR amplification and sequencing 2.1. Samples 36 samples were studied (21 bones and 15 teeth) belonging to 36 different individuals from 17 archaeological sites (6 * Corresponding author. Tel.: +34 91 3941576; fax: +34 91 3941606. E-mail address: [email protected] (E. Arroyo-Pardo). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.037
A 244-bp fragment was studied from mitochondrial HVRI by the independent amplification of two short overlapping sequences (positions 16,126–16,369) [2]. Qiagen Multiplex PCR was implemented for ancient DNA. Amplification products were sequenced using a Applied Biosystems 310 sequencer.
C. Gamba et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 462–464
463
Fig. 1. Location of sample sites.
Table 1 Analysed samples Archaeological site
Location
Malpaso cave Jovellus sepulchre Al Cingle de Cova Negra Ferrero cave Assud de Villarreal, 2nd sepulchre Costa Lloguera sepulchre Mas d’Abad cave Mac¸anet cave Castellet de Bernabe´ Los Villares Puig de la Nau Torrelo´ Bonerot Sant Josep Tossal de Sant Miquel Mas d’Arago´ L’Hostalot
Castellnovo (Castello´n) Benica`ssim (Castello´n) Borriol (Castello´n) Artana (Castello´n) Almassora (Castello´n) Castello´n Les Coves de Vinroma` (Castello´n) Llucena (Castello´n) Lı´ria (Valencia) Caudete de las Fuentes (Castello´n) Benicarlo´ (Castello´n) Castello´n La Vall d’Uixo´ (Castello´n) Lı´ria (Valencia) Cervera del Maestre (Castello´n) Villanova d’Alcolea (Castello´n)
Total
2.4. Criteria of authenticity Some criteria of authenticity were followed in this study: analysis in an exclusive ancient DNA lab, physical separation of pre- and post-PCR procedures, equipment exclusive for aDNA, removal of the external surface of the samples analysis by a single researcher, use of ‘‘blanks’’ in extraction and amplification, repeated amplifications from the same extract, phylogenetic sense (comparison with lab staff and archaeologists/anthropologists), DNA quantification. 3. Results Approximately, 50% of the samples yielded reproducible results: 17 samples for the two fragments and 3 samples only for one of them (See Tables 1 and 2). By comparing the obtained haploypes with public databases it was possible to assign an haplogroup to most of the sequences. Haplogroup H, the most common nowadays in Europe, was found at all age classes. Haplogroup V, an alleged
No.
Sample
Age
4 1 1 1 1 6 1 1 2 1 9 2 1 3 1 1
Teeth Tooth Tooth Bone Bone Teeth Bone Tooth Bones Bone Bones Bones Bone Bones Tooth Tooth
Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Bronze Age Bronze Age Iberian Iberian Iberian Iberian Iberian Iberian Roman Roman
36
marker for human post-glacial expansion from southwestern refugia [3–4] has been found in an unusual elevated frequency (1 Chalcolithic and 3 Iberian samples: 12.5% and 33.3%, respectively). Moreover, the Chalcolithic period is characterized by the possible presence of haplogroup L3, a typical subsaharian haplogroup [5]. 4. Discussion The high efficiency in DNA recovery obtained indicates that sample preservation mainly depends on the depositional environment rather than on sample age. Moreover, cave environment seems to aid DNA preservation as it maintains temperature in a constant interval over years. Thermal history has been proved to play an important role in the preservation of other biomolecules as amino acids [6]. The unique haplotype shared by individuals from different chronologies is CRS. However, as this is the more frequent haplotype nowadays, its presence alone does not allow us to infer the existence of genetic continuity through those periods. Despite the reduced
464
C. Gamba et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 462–464
Table 2 Obtained results Sample
Arch. site
Period
Haplotype
Haplogroup
1MP1 4MP12 2MP4 1JO1 1AV1 COST1 COST3 COST4 1MN1 CB2 LV1 PB14 PB18 PB19 PB20 TORR1 TORR2 TSM1 TSM2 1MAR2
Malpaso cave Malpaso cave Malpaso cave Jovellu´s sepulchre Assud de Villareal, 2nd sepulchre Costa Lloguera sepulchre Costa Lloguera sepulchre Costa Lloguera sepulchre Mac¸anet cave Castellet de Bernabe´ Los Villares Puig de la Nau Puig de la Nau Puig de la Nau Puig de la Nau Torrelo´ Bonerot Torrelo´ Bonerot Tossal de Sant Miquel Tossal de Sant Miquel Mas d’Arago´
Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Bronze Age Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Roman
16223T, 16325C, 16362C 16298C* 16218T, 16223T 16218Y, 16223Y 16218Y, 16223Y 16293R* 16223Y CRS CRS 16163G, 16186T, 16189C, 16250Y, 16294T, 16301Y 16298C 16293T, 16298C CRS CRS 16293T, 16298C 16223T, 16292Y, 16295Y, 16304Y, 16311Y 16223T, 16292Y, 16295Y, 16304Y CRS* 16224Y, 16234Y, 16311Y CRS
D V L3? L3? L3? H (60%) L3a? H H T1 V V H H V W W H? K H
*
Indicates the failure of the recovery of the first fragment.
amount of samples, some interesting conclusions regarding to the ancient Iberian genetic background could be drawn. Haplogroup V is considered to have originated at the Cantabrian fringe during the last Ice Age, from which it is thought to have expanded northwards after the ice retreat 10,000–15,000 YBP [3–4]. However, ancient DNA analysis failed to detect this haplogroup in ancient Basque population [7]. Its presence in Eastern Spain in Chalcolithic and Iberian periods could be explained as a result of prehistoric migrations from the north, but also this fact could be indicating a more southern distribution of Palaeolithic ice refugia. The present work also suggests the presence of a prehistoric African genetic background in eastern Iberia. The existence of genetic African lineages in prehistory has been ascertained in other North Iberian populations also in Calcolithic period [2–8]. Together these results point out at a pre-Calcolithic migration from Africa into Iberia with subsequent genetic replacement. Acknowledgments This work has been funded by the Research Project CGL2006-07828/BOS of the Spanish Ministry of Education and by the Da´valos Fletcher Foundation—Fine Arts Museum of Castello´n.
Conflict of interest None.
References [1] I. Barandiara´n et al., Prehistoria de la Penı´nsula Ibe´rica, Ariel Prehistoria, 4th edition, 2004. [2] E. Ferna´ndez, Polimorfismos de DNA mitocondrial en poblaciones antiguas de la Cuenca Mediterra´nea, Ph.D. Thesis, University of Barcelona, 2005. [3] A. Torroni, et al., MtDNA analysis reveals a major late Paleolithic population expansion from south-western to north-eastern Europe, Am. J. Hum. Genet. 62 (1998) 1137–1152. [4] A. Torroni, et al., A signal, from human mtDNA, of postglacial recolonization in Europe, Am. J. Hum. Genet. 69 (4) (2001) 844–852. [5] A. Salas, et al., The making of the African mtDNA landscape, Am. J. Hum. Genet. 71 (5) (2002) 1082–1111. [6] T. Torres, et al., Bear dentine aspartic acid racemization analysis: a proxy for the dating of Pleistocene cave infills, Archaeometry 44 (3) (2002) 417– 426. [7] N. Izaguirre, C. de la Rua, A mtDNA analysis in ancient Basque populations: implications for haplogroup V as a marker for a major paleolithic expansion from southwestern Europe, Am. J. Hum. Genet. 65 (1) (1999) 199–207. [8] A. Alzualde, et al., Insights into the ‘‘isolation’’ of the Basques: Mtdna, Am. J. Phys. Anthropol. 130 (3) (2006) 394–404.
Forensic Science International: Genetics Supplement Series 1 (2008) 462–464 www.elsevier.com/locate/FSIGSS
Research article
Population genetics and DNA preservation in ancient human remains from Eastern Spain C. Gamba a, E. Ferna´ndez a, A. Oliver b, M. Tirado a, C. Baeza a, A.M. Lo´pez-Parra a, E. Arroyo-Pardo a,* a
Depto. Toxicologı´a y Legislacio´n Sanitaria, Facultad de Medicina, Universidad Complutense, Madrid 28040, Spain b Seccio´n de Arqueologı´a. Museo de Bellas Artes de Castello´n, Castello´n, Spain Received 16 August 2007; accepted 7 October 2007
Abstract This work aims to establish the genetic relationship between the different settlers of Eastern Spain and also to determine the conditions of DNA preservation. We studied two overlapping sequences (16,126–16,251 and 16,256–16,369) from mitochondrial HVR-I in 37 bone and teeth samples from 17 archaeological sites of Spanish Levant. Consistence of the results was established by repeated replication of amplifications. Approximately, 50% of the samples yielded reproducible results. The high efficiency in DNA recovery indicates that sample preservation mainly depends on the depositional environment rather than on sample age. Haplogroup V, an alleged marker of Paleolithic newcomers in Europe, has been found in an unusual elevated frequency (1 Calcolithic and 2 Iberian samples). This result could suggest a more southern distribution of Palaeolithic ice refugia. Moreover, we found Haplogroup L in Calcolithic samples. This may suggest the presence of a prehistoric African genetic background in eastern Iberia. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Ancient DNA; Mitochondrial DNA; Eastern Iberia
1. Introduction The East of Spain has been populated at least since Palaeolithic times [1]. Due to its strategic location, opened to the Mediterranean shore, it has been prone to have received genetic influences from other populations. Present work tries to evaluate this hypothesis through mtDNA Hypervariable region I (HVR-I) characterization of ancient samples from Castello´n and Valencia provinces, dated back to Roman, Iberian, Bronze and Chalcolithic ages. Obtained results will be interpreted in terms of genetic continuity/discontinuity between these periods. The effect of the environment and the age of the sample over DNA preservation will be evaluated in this study.
Chalcolithic, 2 Bronze Age, 6 Iberian Culture and 3 Roman) located in Eastern Spain (see Fig. 1 and Table 1). 2.2. Extraction The external surface of the samples was removed first with a Sand-blaster, and afterwards they were irradiated with UV light and crushed in a freezer mill filled with liquid Nitrogen. Samples were then washed several times with EDTA 0.5 M and incubated in a lysis solution (EDTA 5 mM, Tris–HCl 10 mM, SDS 0.5%, proteinase K 50 mg/ml) overnight at 36 8C. DNA was extracted using a modified Phenol–Chloroform protocol [2] and concentrated with microcons (Centriplus 30000, Millipore).
2. Material and methods 2.3. PCR amplification and sequencing 2.1. Samples 36 samples were studied (21 bones and 15 teeth) belonging to 36 different individuals from 17 archaeological sites (6 * Corresponding author. Tel.: +34 91 3941576; fax: +34 91 3941606. E-mail address: [email protected] (E. Arroyo-Pardo). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.037
A 244-bp fragment was studied from mitochondrial HVRI by the independent amplification of two short overlapping sequences (positions 16,126–16,369) [2]. Qiagen Multiplex PCR was implemented for ancient DNA. Amplification products were sequenced using a Applied Biosystems 310 sequencer.
C. Gamba et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 462–464
463
Fig. 1. Location of sample sites.
Table 1 Analysed samples Archaeological site
Location
Malpaso cave Jovellus sepulchre Al Cingle de Cova Negra Ferrero cave Assud de Villarreal, 2nd sepulchre Costa Lloguera sepulchre Mas d’Abad cave Mac¸anet cave Castellet de Bernabe´ Los Villares Puig de la Nau Torrelo´ Bonerot Sant Josep Tossal de Sant Miquel Mas d’Arago´ L’Hostalot
Castellnovo (Castello´n) Benica`ssim (Castello´n) Borriol (Castello´n) Artana (Castello´n) Almassora (Castello´n) Castello´n Les Coves de Vinroma` (Castello´n) Llucena (Castello´n) Lı´ria (Valencia) Caudete de las Fuentes (Castello´n) Benicarlo´ (Castello´n) Castello´n La Vall d’Uixo´ (Castello´n) Lı´ria (Valencia) Cervera del Maestre (Castello´n) Villanova d’Alcolea (Castello´n)
Total
2.4. Criteria of authenticity Some criteria of authenticity were followed in this study: analysis in an exclusive ancient DNA lab, physical separation of pre- and post-PCR procedures, equipment exclusive for aDNA, removal of the external surface of the samples analysis by a single researcher, use of ‘‘blanks’’ in extraction and amplification, repeated amplifications from the same extract, phylogenetic sense (comparison with lab staff and archaeologists/anthropologists), DNA quantification. 3. Results Approximately, 50% of the samples yielded reproducible results: 17 samples for the two fragments and 3 samples only for one of them (See Tables 1 and 2). By comparing the obtained haploypes with public databases it was possible to assign an haplogroup to most of the sequences. Haplogroup H, the most common nowadays in Europe, was found at all age classes. Haplogroup V, an alleged
No.
Sample
Age
4 1 1 1 1 6 1 1 2 1 9 2 1 3 1 1
Teeth Tooth Tooth Bone Bone Teeth Bone Tooth Bones Bone Bones Bones Bone Bones Tooth Tooth
Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Bronze Age Bronze Age Iberian Iberian Iberian Iberian Iberian Iberian Roman Roman
36
marker for human post-glacial expansion from southwestern refugia [3–4] has been found in an unusual elevated frequency (1 Chalcolithic and 3 Iberian samples: 12.5% and 33.3%, respectively). Moreover, the Chalcolithic period is characterized by the possible presence of haplogroup L3, a typical subsaharian haplogroup [5]. 4. Discussion The high efficiency in DNA recovery obtained indicates that sample preservation mainly depends on the depositional environment rather than on sample age. Moreover, cave environment seems to aid DNA preservation as it maintains temperature in a constant interval over years. Thermal history has been proved to play an important role in the preservation of other biomolecules as amino acids [6]. The unique haplotype shared by individuals from different chronologies is CRS. However, as this is the more frequent haplotype nowadays, its presence alone does not allow us to infer the existence of genetic continuity through those periods. Despite the reduced
464
C. Gamba et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 462–464
Table 2 Obtained results Sample
Arch. site
Period
Haplotype
Haplogroup
1MP1 4MP12 2MP4 1JO1 1AV1 COST1 COST3 COST4 1MN1 CB2 LV1 PB14 PB18 PB19 PB20 TORR1 TORR2 TSM1 TSM2 1MAR2
Malpaso cave Malpaso cave Malpaso cave Jovellu´s sepulchre Assud de Villareal, 2nd sepulchre Costa Lloguera sepulchre Costa Lloguera sepulchre Costa Lloguera sepulchre Mac¸anet cave Castellet de Bernabe´ Los Villares Puig de la Nau Puig de la Nau Puig de la Nau Puig de la Nau Torrelo´ Bonerot Torrelo´ Bonerot Tossal de Sant Miquel Tossal de Sant Miquel Mas d’Arago´
Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Chalcolithic Bronze Age Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Roman
16223T, 16325C, 16362C 16298C* 16218T, 16223T 16218Y, 16223Y 16218Y, 16223Y 16293R* 16223Y CRS CRS 16163G, 16186T, 16189C, 16250Y, 16294T, 16301Y 16298C 16293T, 16298C CRS CRS 16293T, 16298C 16223T, 16292Y, 16295Y, 16304Y, 16311Y 16223T, 16292Y, 16295Y, 16304Y CRS* 16224Y, 16234Y, 16311Y CRS
D V L3? L3? L3? H (60%) L3a? H H T1 V V H H V W W H? K H
*
Indicates the failure of the recovery of the first fragment.
amount of samples, some interesting conclusions regarding to the ancient Iberian genetic background could be drawn. Haplogroup V is considered to have originated at the Cantabrian fringe during the last Ice Age, from which it is thought to have expanded northwards after the ice retreat 10,000–15,000 YBP [3–4]. However, ancient DNA analysis failed to detect this haplogroup in ancient Basque population [7]. Its presence in Eastern Spain in Chalcolithic and Iberian periods could be explained as a result of prehistoric migrations from the north, but also this fact could be indicating a more southern distribution of Palaeolithic ice refugia. The present work also suggests the presence of a prehistoric African genetic background in eastern Iberia. The existence of genetic African lineages in prehistory has been ascertained in other North Iberian populations also in Calcolithic period [2–8]. Together these results point out at a pre-Calcolithic migration from Africa into Iberia with subsequent genetic replacement. Acknowledgments This work has been funded by the Research Project CGL2006-07828/BOS of the Spanish Ministry of Education and by the Da´valos Fletcher Foundation—Fine Arts Museum of Castello´n.
Conflict of interest None.
References [1] I. Barandiara´n et al., Prehistoria de la Penı´nsula Ibe´rica, Ariel Prehistoria, 4th edition, 2004. [2] E. Ferna´ndez, Polimorfismos de DNA mitocondrial en poblaciones antiguas de la Cuenca Mediterra´nea, Ph.D. Thesis, University of Barcelona, 2005. [3] A. Torroni, et al., MtDNA analysis reveals a major late Paleolithic population expansion from south-western to north-eastern Europe, Am. J. Hum. Genet. 62 (1998) 1137–1152. [4] A. Torroni, et al., A signal, from human mtDNA, of postglacial recolonization in Europe, Am. J. Hum. Genet. 69 (4) (2001) 844–852. [5] A. Salas, et al., The making of the African mtDNA landscape, Am. J. Hum. Genet. 71 (5) (2002) 1082–1111. [6] T. Torres, et al., Bear dentine aspartic acid racemization analysis: a proxy for the dating of Pleistocene cave infills, Archaeometry 44 (3) (2002) 417– 426. [7] N. Izaguirre, C. de la Rua, A mtDNA analysis in ancient Basque populations: implications for haplogroup V as a marker for a major paleolithic expansion from southwestern Europe, Am. J. Hum. Genet. 65 (1) (1999) 199–207. [8] A. Alzualde, et al., Insights into the ‘‘isolation’’ of the Basques: Mtdna, Am. J. Phys. Anthropol. 130 (3) (2006) 394–404.