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Resolving the matrilineal relationship of seven Late Bronze Age individuals from Stillfried, Austria
Forensic Science International: Genetics 36 (2018) 148–151
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsigen
Research paper
Resolving the matrilineal relationship of seven Late Bronze Age individuals from Stillfried, Austria
T
⁎
Walther Parsona,b, , Mayra Eduardoffa, Catarina Xaviera, Barbara Bertoglioc,d,e, Maria Teschler-Nicolaf,g a
Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria Forensic Science Program, The Pennsylvania State University, University Park, PA, USA c Dipartimento di Sanità Pubblica, Medicina Sperimentale e Forense, Unità di Medicina Legale e Scienze Forensi, Università di Pavia, Pavia, Italy d LABANOF (Laboratorio di Antropologia e Odontologia Forense), Dipartimento di Scienze Biomediche per la Salute, Sezione di Medicina Legale, Università degli studi di Milano, Milan, Italy e Molecular and Cellular Biology, PhD Program of the University of Pavia, Italy f Department of Anthropology, Natural History Museum, Vienna, Austria g Department of Anthropology, University of Vienna, Austria b
A B S T R A C T
In 1976 human remains of seven individuals were discovered in a storage pit located within the Late Bronze Age (9th century B.C.) settlement Stillfried an der March, Austria. In contrast to the common funeral rite of cremation typical for the Urnfield culture (1300–800 B.C.) the individuals’ skeletal remains were found outstandingly preserved (Figure S1). As a result, the burial was subject to various investigations, including two conflicting genealogical pedigree reconstructions, one of which was favoured by later geological fingerprinting. We performed mitochondrial (mt)DNA testing in order to genetically characterize the remains and shed light into the matrilineal relationship of the seven individuals that were earlier anthropologically identified as three adults (two women and a man) and four subadults (one female and three males). MtDNA was analysed using Primer Extension Capture and Massively Parallel Sequencing. The results were by and large in conflict with both pedigree models but confirmed some of the details that were elaborated in previous studies. Whereas both pedigree models suggested that all children were related to one or both females, mtDNA analyses revealed that only one subadult male resulted in the same mitotype as one adult female. All other children yielded different mitotypes indicating that they were maternally unrelated to the two females and between each other.
1. Introduction Stillfried (an der March) is a small village located in eastern Lower Austria in close vicinity to the Slovakian border. It is internationally known for its archaeological excavations that have been performed systematically between 1969 and 1989 and include discoveries from the Palaeolithic to the Roman Era [1–4]. In 1976, the remains of seven individuals, three adults and four subadults (age-at-death: 3, 5–6, 7–8 and 9 years, Figures S1 and S2), were discovered in a Late Bronze Age (9th century B.C.) storage pit (V1141) beneath the rampart of the fortification of Stillfried [5]. The individuals were - for reasons yet unknown - not cremated, which is in contrast to the normative burial treatment exercised in that epoch (Urnfield culture, 1300 - 800 BCE). These unusual findings motivated research to shed more light into the societal and cultural life of the North Alpine/ Middle Danube groups of the Late Bronze Age. In 1976, the remains were measured, documented and recovered en bloc in order to protect and preserve this rare archaeothanatological
⁎
testimonial [6]. Among other investigations, two bioanthropological analyses including an interpretation of the genealogical relationship between the seven individuals were conducted. The first analysis was performed by Emil Breitinger in 1980 and involved age-at-death estimation, morphological sexing, description of pathological and traumatic alterations, and functional adaptations [5]. Breitinger meticulously collected numerous individual details in an attempt to provide the reader with the possibility of a ‘self-contained judgement’ of the discoveries, perhaps driven by his inability to unambiguously interpret the findings [5]. However, based on his observations he excluded a ritual inhumation of the individuals but suggested a rather quick disposal of the corpses. Breitinger postulated that the remains belonged to a ca. 30-year-old man (St. 1), two ca. 40- and ca. 45-year-old women (St. 3 and St. 5) and four children, of which three were male (St. 2, 3 years; St. 4, 7–8 years; and St. 6, 5–6 years) and a 9-year-old female (St. 7, Fig. 1). He concluded a genealogical relationship based on a variety of different morphological characteristics that identified the two female adults as mothers and the male adult as father of the four children
Corresponding author at: Institute of Legal Medicine, Medical University of Innsbruck, Müllerstrasse 44, 6020 Innsbruck, Austria. E-mail address: [email protected] (W. Parson).
https://doi.org/10.1016/j.fsigen.2018.07.005 Received 23 February 2018; Received in revised form 2 July 2018; Accepted 4 July 2018 Available online 10 July 2018 1872-4973/ © 2018 Elsevier B.V. All rights reserved.
Forensic Science International: Genetics 36 (2018) 148–151
W. Parson et al.
Fig. 1. Different kinship reconstructions according to a) Breitinger [5] and Szilvássy et al [7]. Gray-shaded individuals were assumed locals according to geological fingerprinting by [8].
using the IonTorrent Server analysis pipeline including filtering and alignment. MtDNA sequence variants were manually reported relative to the rCRS [13] based on phylogenetic alignment considerations according to [14,15]. Polynucleotide stretches were not analysed. Haplogroups were determined based on Phylotree, build 17 (www. phylotree.org; [16]) using the haplogrouping function in EMPOP (www.empop.online; [17]).
(Fig. 1a, [5]). Based on Breitingers description of the remains and additional investigations Szilvássy and co-workers [7] proposed a different interpretation of kinship eight years later suggesting the 45-year-old woman and the adult man as parents of the four children (Fig. 1b). The two different kinship reconstructions were later evaluated by Strontium (Sr) isotope analyses (geological fingerprinting), which favoured Breitingers model [8]. In order to characterize the mtDNA in the seven individuals and shed further light on the possible relations, we analysed mitochondrial DNA (mtDNA) of the remains of the seven individuals. Due to the age and the degradation state of the mtDNA extracted from tooth samples we employed Primer Extension Capture (PEC) analysis [9], which resulted in interpretable mitochondrial haplotypes (mitotypes) to test the conflicting pedigree models.
3. Results and discussion DNA extracted from teeth and/or tooth samples of the seven individuals uncovered at the Late Bronze Age storage pit V1141 in Stillfried, Austria was subjected to PEC mtDNA analysis in order to evaluate earlier postulated relationships between these individuals. This analysis resulted in full control region mitotypes in all samples except for individual St. 4, where a 27 bp gap did not yield a readable sequence in hypervariable segment I (HVS-I, 16101–16128; Table 1, Figure S3). Additional segments of the mtDNA coding region could be successfully sequenced in all seven samples providing additional discrimination power and haplogroup-informative sites. Negative controls were clean; the positive control runs yielded the expected haplogroup (hg) A10 sequence (data not shown). The mitotypes from the tooth samples showed the expected post-mortem damage patterns due to increased C-T misincorporations (data not shown), thus corroborating that the genuine mtDNA was analysed and not modern contamination (for further detail see [9]). PEC analysis resulted in unambiguously different mitotypes in individuals St. 1 (hg U5a2), St. 2 (hg J1c2), St. 3 and 4 (both hg H*), and St. 7 (hg I2), whereas St. 5 and St. 6 shared an identical hg H5 mitotype in the overlapping ranges (Table 1). The observed mitotypes were all belonging to haplogroups typical of the west Eurasian phylogeny [18] and some of them also observed in other contemporaneous locations, such as the Lichtenstein Cave (Germany, 1000 BCE) by Schilz and co-authors [19] who found hgs H, J, T2, and U in their remains. The Stillfried mitotypes were not observed in a total of 26,127 control region (16024-576) sequences in EMPOP (v3/R11; [17]), except for the shared hg H5 mitotype in St. 5 and St. 6 that was observed once (Table 1). Laboratory staff handling the specimens and other study participants were excluded as donors of the mitotypes observed in St.1–7 (data not shown). The two pedigree models proposed by Breitinger [5] and Szilvássy and co-authors [7] mainly differ by the adult females giving birth to the four children. Breitinger observed skeletal modifications at the sacral and hip bones of both females, based on which he attested motherhood to both of them [5], while Szilvássy and co-authors [7] postulated that only the 45-year-old female (St. 5) had given birth to the four children (Fig. 1). The two pedigree models were evaluated using geological fingerprinting in 2016. Teschler-Nicola and co-authors [8] investigated the 87Sr/86Sr isotope ratios in dentine and enamel of the individual’s teeth and the soil background in Stillfried and concluded that individuals St. 2, St. 3 and St. 4 could be considered locals, whereas the
2. Materials and methods 2.1. Samples, DNA extraction and mtDNA quantification Teeth (permanent or deciduous molars) were carefully extracted from the lower jaw of the seven individuals recovered from the Stillfried Late Bronze Age storage pit V1141 (Table 1). They were kindly provided by the stakeholder of the findings, the Natural History Museum, Vienna, Austria for mtDNA analysis. The teeth stemming from individuals 1 (St. 1) and 4 (St. 4) used for a pilot study were grinded using a Retsch grinding mill MM400 (Retsch GmbH, Haan, Germany). For the other five individuals solely powder of the teeth was obtained using a medical dentist drill. DNA was extracted following the protocols described in [10] for samples St. 1 and St. 4 and [11] for the remaining samples (Table 1). Estimation of mtDNA copy number was performed using a 51bp target SYBR green assay (Power SYBR, Thermo Fisher Scientific (TFS), Waltham, MA, USA) with primers and probe target regions published in [12]. DNA of the positive control was extracted from whole blood independently in another laboratory. 2.2. Library preparation, primer extension capture (PEC) and sequencing The PEC method applied here targeted the mtDNA control region. A detailed protocol is provided in [9]. Positive control DNA was sheared for 40 min using the Ion Shear Kit (TFS) and the sequencing library was prepared using the IonXpress Fragment Library Kit (TFS) according to the manufacturer’s protocol in an independent laboratory. DNA extracted from the tooth samples was directly subjected to library preparation as described above. The libraries were amplified using the IonXpress Fragment Library Kit (TFS) and 10 PCR cycles according to manufacturer’s protocol. The PEC reactions were performed as described in [9]. Two libraries (St. 1 and St. 4) were sequenced using the Ion PGM (TFS) with manual template preparation (Ion OT2 (TFS)) and subsequent template enrichment according to manufacturer’s protocol [9]. The remaining five samples were sequenced on the Ion S5 (TFS) with automated template preparation using the IonChef pipeline (TFS) according to manufacturer’s protocol. Raw data analysis was performed 149
Forensic Science International: Genetics 36 (2018) 148–151
W. Parson et al.
Table 1 Summary of studied remains from seven individuals placed in a Late Bronze Age storage pit in Stillfried, Austria. Sample designation and description of the individuals according to Breitinger, [5]. DNA extraction according to Loreille et al. [10] and Dabney et al. [11]. MtDNA specific quantitation values are indicated as mtDNA Genome equivalents per microliter. The control region (16024-576) of the mitotypes was searched in the EMPOP database (empop.online; v3/R11). Haplogrouping was performed using the EMPOP haplogrouping function on the basis of Phylotree (phylotree.org, build 17 [16],). Mitotypes are reported relative to the rCRS [13]. Mutations in brackets indicate low coverage reads (< 3). N.A. … not available. Sample
St. 1
St. 2
St. 3
St. 4
St. 5
St. 6
St. 7
Extr. blank 1
Extr. blank 2
Description Tooth Extraction Quantitation [51bp mtG/μl] EMPOP CR search (v3 R11, N = 26,127 CR) Haplogroup Mitotype
male 30 y. 36 [10] 225.00 0 U5a2 73G 263G 750G 16192T 16256T 16270T 16526A
male 3 y. 48 [11] 2,277.61 0 J1c2 73G 185A 188G 228A 263G 295T 462T 489C 750G (2706 G) 4216C 8860G 11251G 11719 A 12612G 13708A 14766T 14798C 15326G 15452A 16069T 16126C
female 40 y. 36 [11] 488.85 0 H* 263G 750G 16114T 16362C 16519C
male 7-8 y. 37 [10] 378.00 0 H* 263G 750 G (1719 A) 16261T
female 45 y. 47 [11] 2,364.13 1 H5 263G 456T 750G 16304C
male 5-6 y. 85 [11] 2,386.73 1 H5 263G 456T 750G 15326G 16304C
female 9 y. 36 [11] 5,468.84 0 I2 73G 152C 199C 204C 207A 250C 263G 573.1C 750G 1719 A 2706 G 4769G 8251A 8860G (10034C) (11719 A) 12501A (12705 T) 13780G 14766T 15043A 15326G 15758G 15924G 16129A 16223T 16390A 16391A 16519C
NA NA [10] 0.00 NA NA NA
NA NA [11] 3.47 NA NA NA
() polymorphisms observed with 1 or 2x coverage but phylogenetically plausible
Sequencing ranges (more than2x coverage): St. 1: 1–911 4935–4951 8200–8514 8591-8711 8715–8734 14076-14152.15716–16569. St. 2: 1–982 1590–1787 2155–2444 3346–3649 3681–3865 3959–4289 4420–4515 4656–4696 4980–5503 6331-6657 7376–7497 7673–7692 7694–7728 7730–7888 8130–9023 9026-9028 9033–9079 9206–9210 9212–9241 9244–9247 9249–9263 9265–9288 9308–9663 9866–9892 10050–10158 10175–10294 10296–10312 10515–10648 10743–11370 11474–11558 11565–11619 11656–11780 11915–12635 12889–14853 14904–15040 15278–16,569. St. 3: 1–872 2174–2226 2228–2241 3483–3596 5300–5371 8324–8543 8610–8755 8757–8762 11260–11297 12253-12283 12310–12414 12416-12417 12419–12433 13802–13837 15366–15567 15785–16,569. St. 4: 1–862 2140–2151 4021–4023 4932–4947 5153–5162 6224–6236 6491–6499 8070-8531 8704–8714 8788-8798 9418–9424 11039–11050 11057–11231 11294–11305 11987–11989 11991-12005 12007–12024 13078–13088 13617–13624 14038–14128 14130–14149 14670–14681 15450–15461 15637–15742 15779–16101 16,128–16,569. St. 5: 1–899 8265-8493 8593–8660 10878–10930 13749–13753 13756–13817.15826–16569. St. 6: 1–909 911-920 1752–1806 1864-1947 2183–2275 2280-2300 3512–3532 4997-5023 5264–5333 5335-5359 7338–7342 7344-7348 7350–7364 7366-7368 7370–7375 7377-7396 7398–7443 8132-8742 8935–8977 9208-9221 9266–9267 9269-9278 9505–9520 9522-9605 10803–10985 11098-11380 11981–12129 12138-12142 12304–12406 12909-12922 13209–13215 13217-13313 13938–14394 14602-14706.15302–1559015716–16569. St. 7: 1–945 947-955 957–972 1035-1075 1609–1976 2107-2484 2682–2770 3114-3912 3923–4307 4336-4526 4594–4809 4844-5696 5706–5722 5725-5755 5757–5815 5856-5885 6383–6617 6753-6864 6900–6910 7376-7496 7632–8024 8068-9190 9339–9798 10242-10328 10477–11406 11441-11716 11720–11748 11750-11751 11895–11901 11922-12196 12212–12223 12234-12252 12254–12644 12761-12795 12804–15129 15257-16,569.
Conflict of interest
remaining individuals must have originated from someplace else (Fig. 1). Geological fingerprinting thus somehow supported Breitingers model that suggested St. 3 as mother of St. 2 and St. 4 (Fig. 1a). Interestingly, mtDNA analyses performed in this study do not corroborate any of these models, instead suggesting a different relation. According to the established mitotypes the 40-year-old female St. 3 is excluded as mother from any of the four children, whereas the 45-yearold female St. 5 can be considered mother or another maternally related individual of the 6-year-old boy St. 6 (Table 1, Fig. 2), confirming the interpretation of the female protecting her son [7] (or a close maternal relative).
The authors declare no conflict of interest. Acknowledgements The authors would like to thank Ronald Mühl and Bernd Bernegger (both Vienna) for technical assistance with the extraction of the teeth and Cordula Berger, Anna König, Daniel Entstrasser, and Manuel Nitz (Innsbruck) for DNA extraction of the teeth from individuals St. 1 and St. 4. 150
Forensic Science International: Genetics 36 (2018) 148–151
W. Parson et al.
[6]
[7]
[8]
Fig. 2. Matrilineal pedigree reconstruction based on mtDNA analysis (this study). The mitotypes further exclude any other maternal relationship between the three adults and the four children. The role of the 30-year-old male (St. 1) and a potential relationship to the four children remains unclear pending further nuclear DNA analysis.
[9]
[10]
[11]
Appendix A. Supplementary data [12]
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigen.2018.07.005.
[13]
References [14] [1] C. Eibner, Die Mehrfachbestattung aus einer Grube unter dem urnenfelderzeitlichen Wall in Stillfried an der March, NÖ. Forschungen in Stillfried 4, Veröffentlichungen der Österreichischen Arbeitsgemeinschaft für Ur- und Frühgeschichte 13/14, 1980, pp. 107–142. [2] F. Felgenhauer, Geschichte der prähistorisch-archäologischen Erforschung von Stillfried. Forschungen in Stillfried 1, Veröffentlichungen der Österreichischen Arbeitsgemeinschaft für Ur- und Frühgeschichte 6, 1974, pp. 7–20. [3] F.Stillfried Felgenhauer, Lebensraum des Menschen seit 30.000 Jahren: Archäologischer Fundplatz von internationaler Bedeutung; Objekt interdisziplinärer Forschung von bedeutendem Rang: Ergebnisse der Ausgrabungen und Forschungen 1996-1989. Forschungen in Stillfried 9-10, (1996), pp. 9–29. [4] I. Hellerschmid, Die urnenfelder-/hallstattzeitliche Wallanlage von Stillfried an der March: Ergebnisse der Ausgrabungen 1969-1989 unter besonderer Berücksichtigung des Kulturwandels an der Epochengrenze Urnenfelder-/ Hallstattkultur, Mitteilungen der Prähistorischen Kommission, Wien, 2006. [5] E. Breitinger, Skelette aus einer späturnenfelderzeitlichen Speichergrube in der
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Wallburg von Stillfried an der March, NÖ. Forschungen in Stillfried 4, Veröffentlichungen der Österreichischen Arbeitsgemeinschaft für Ur- und Frühgeschichte 13/14, 1980, pp. 45–106. J. Szilvassy, Kritscher H. Präparation, Rekonstruktion und Interpretation von sieben menschlichen Skeletten aus einer urnenfelderzeitlichen Speichergrube in Stillfried an der March, Niederösterreich. Annalen Naturhistorisches Museum Wien 91 (1990), pp. 125–143. J. Szilvassy, H. Kritscher, G. Hauser, Eine urnenfelderzeitliche Mehrfachbestattung in Stillfried an der March, NÖ, in: F. Felgenhauer, J.S.H. Kritscher, G. Hauser (Eds.), Stillfried Archäologie – Anthropologie, Veröffentlichungen des Museums für Urund Frühgeschichte Stillfried, 1988, pp. 9–76. M. Teschler-Nicola, J. Irrgeher, T. Prohaska, Wohnsitz und Genealogie der sieben Menschen aus der späturnenfelderzeitlichen Vorratsgrube V1141 von Stillfried an der March – eine archäometrische Ergänzung anlässlich eines “Schauplatzwechsels”, Mitteilungen der Anthropologischen Gesellschaft in Wien CXLVI (2016), pp. 159–168. M. Eduardoff, C. Xavier, C. Strobl, A. Casas-Vargas, W. Parson, Optimized mtDNA control region primer extension capture analysis for forensically relevant samples and highly compromised mtDNA of different age and origin, Genes 8 (2017). O.M. Loreille, T.M. Diegoli, J.A. Irwin, M.D. Coble, T.J. Parsons, High efficiency DNA extraction from bone by total demineralization, Forensic Sci. Int. Genet. 1 (2007) 191–195. J. Dabney, M. Knapp, I. Glocke, M.T. Gansauge, A. Weihmann, B. Nickel, et al., Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 15758–15763. H. Oberacher, H. Niederstätter, C.G. Huber, W. Parson, Accurate determination of allelic frequencies in mitochondrial DNA mixtures by electrospray ionization timeof-flight mass spectrometry, Anal. Bioanal. Chem. 384 (2006) 1155–1163. R.M. Andrews, I. Kubacka, P.F. Chinnery, R.N. Lightowlers, D.M. Turnbull, N. Howell, Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA, Nat. Genet. 23 (1999) 147. H.J. Bandelt, W. Parson, Consistent treatment of length variants in the human mtDNA control region: a reappraisal, Int. J. Legal Med. 122 (2008) 11–21. W. Parson, L. Gusmao, D.R. Hares, J.A. Irwin, W.R. Mayr, N. Morling, et al., DNA Commission of the International Society for Forensic Genetics: revised and extended guidelines for mitochondrial DNA typing, Forensic Sci. Int. Genet. 13 (2014) 134–142. M. van Oven, M. Kayser, Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation, Hum. Mutat. 30 (2009) E386–94. W. Parson, A. Dür, EMPOP–a forensic mtDNA database, Forensic Sci. Int. Genet. 1 (2007) 88–92. A. Torroni, A. Achilli, V. Macaulay, M. Richards, H.J. Bandelt, Harvesting the fruit of the human mtDNA tree, Trends Genet. 22 (2006) 339–345. F. Schilz, Molekulargenetische Verwandtschaftsanalysen am prähistorischen Skelettkollektiv der Lichtensteinhöhle, Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-AugustUniversität zu Göttingen, 2006.
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsigen
Research paper
Resolving the matrilineal relationship of seven Late Bronze Age individuals from Stillfried, Austria
T
⁎
Walther Parsona,b, , Mayra Eduardoffa, Catarina Xaviera, Barbara Bertoglioc,d,e, Maria Teschler-Nicolaf,g a
Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria Forensic Science Program, The Pennsylvania State University, University Park, PA, USA c Dipartimento di Sanità Pubblica, Medicina Sperimentale e Forense, Unità di Medicina Legale e Scienze Forensi, Università di Pavia, Pavia, Italy d LABANOF (Laboratorio di Antropologia e Odontologia Forense), Dipartimento di Scienze Biomediche per la Salute, Sezione di Medicina Legale, Università degli studi di Milano, Milan, Italy e Molecular and Cellular Biology, PhD Program of the University of Pavia, Italy f Department of Anthropology, Natural History Museum, Vienna, Austria g Department of Anthropology, University of Vienna, Austria b
A B S T R A C T
In 1976 human remains of seven individuals were discovered in a storage pit located within the Late Bronze Age (9th century B.C.) settlement Stillfried an der March, Austria. In contrast to the common funeral rite of cremation typical for the Urnfield culture (1300–800 B.C.) the individuals’ skeletal remains were found outstandingly preserved (Figure S1). As a result, the burial was subject to various investigations, including two conflicting genealogical pedigree reconstructions, one of which was favoured by later geological fingerprinting. We performed mitochondrial (mt)DNA testing in order to genetically characterize the remains and shed light into the matrilineal relationship of the seven individuals that were earlier anthropologically identified as three adults (two women and a man) and four subadults (one female and three males). MtDNA was analysed using Primer Extension Capture and Massively Parallel Sequencing. The results were by and large in conflict with both pedigree models but confirmed some of the details that were elaborated in previous studies. Whereas both pedigree models suggested that all children were related to one or both females, mtDNA analyses revealed that only one subadult male resulted in the same mitotype as one adult female. All other children yielded different mitotypes indicating that they were maternally unrelated to the two females and between each other.
1. Introduction Stillfried (an der March) is a small village located in eastern Lower Austria in close vicinity to the Slovakian border. It is internationally known for its archaeological excavations that have been performed systematically between 1969 and 1989 and include discoveries from the Palaeolithic to the Roman Era [1–4]. In 1976, the remains of seven individuals, three adults and four subadults (age-at-death: 3, 5–6, 7–8 and 9 years, Figures S1 and S2), were discovered in a Late Bronze Age (9th century B.C.) storage pit (V1141) beneath the rampart of the fortification of Stillfried [5]. The individuals were - for reasons yet unknown - not cremated, which is in contrast to the normative burial treatment exercised in that epoch (Urnfield culture, 1300 - 800 BCE). These unusual findings motivated research to shed more light into the societal and cultural life of the North Alpine/ Middle Danube groups of the Late Bronze Age. In 1976, the remains were measured, documented and recovered en bloc in order to protect and preserve this rare archaeothanatological
⁎
testimonial [6]. Among other investigations, two bioanthropological analyses including an interpretation of the genealogical relationship between the seven individuals were conducted. The first analysis was performed by Emil Breitinger in 1980 and involved age-at-death estimation, morphological sexing, description of pathological and traumatic alterations, and functional adaptations [5]. Breitinger meticulously collected numerous individual details in an attempt to provide the reader with the possibility of a ‘self-contained judgement’ of the discoveries, perhaps driven by his inability to unambiguously interpret the findings [5]. However, based on his observations he excluded a ritual inhumation of the individuals but suggested a rather quick disposal of the corpses. Breitinger postulated that the remains belonged to a ca. 30-year-old man (St. 1), two ca. 40- and ca. 45-year-old women (St. 3 and St. 5) and four children, of which three were male (St. 2, 3 years; St. 4, 7–8 years; and St. 6, 5–6 years) and a 9-year-old female (St. 7, Fig. 1). He concluded a genealogical relationship based on a variety of different morphological characteristics that identified the two female adults as mothers and the male adult as father of the four children
Corresponding author at: Institute of Legal Medicine, Medical University of Innsbruck, Müllerstrasse 44, 6020 Innsbruck, Austria. E-mail address: [email protected] (W. Parson).
https://doi.org/10.1016/j.fsigen.2018.07.005 Received 23 February 2018; Received in revised form 2 July 2018; Accepted 4 July 2018 Available online 10 July 2018 1872-4973/ © 2018 Elsevier B.V. All rights reserved.
Forensic Science International: Genetics 36 (2018) 148–151
W. Parson et al.
Fig. 1. Different kinship reconstructions according to a) Breitinger [5] and Szilvássy et al [7]. Gray-shaded individuals were assumed locals according to geological fingerprinting by [8].
using the IonTorrent Server analysis pipeline including filtering and alignment. MtDNA sequence variants were manually reported relative to the rCRS [13] based on phylogenetic alignment considerations according to [14,15]. Polynucleotide stretches were not analysed. Haplogroups were determined based on Phylotree, build 17 (www. phylotree.org; [16]) using the haplogrouping function in EMPOP (www.empop.online; [17]).
(Fig. 1a, [5]). Based on Breitingers description of the remains and additional investigations Szilvássy and co-workers [7] proposed a different interpretation of kinship eight years later suggesting the 45-year-old woman and the adult man as parents of the four children (Fig. 1b). The two different kinship reconstructions were later evaluated by Strontium (Sr) isotope analyses (geological fingerprinting), which favoured Breitingers model [8]. In order to characterize the mtDNA in the seven individuals and shed further light on the possible relations, we analysed mitochondrial DNA (mtDNA) of the remains of the seven individuals. Due to the age and the degradation state of the mtDNA extracted from tooth samples we employed Primer Extension Capture (PEC) analysis [9], which resulted in interpretable mitochondrial haplotypes (mitotypes) to test the conflicting pedigree models.
3. Results and discussion DNA extracted from teeth and/or tooth samples of the seven individuals uncovered at the Late Bronze Age storage pit V1141 in Stillfried, Austria was subjected to PEC mtDNA analysis in order to evaluate earlier postulated relationships between these individuals. This analysis resulted in full control region mitotypes in all samples except for individual St. 4, where a 27 bp gap did not yield a readable sequence in hypervariable segment I (HVS-I, 16101–16128; Table 1, Figure S3). Additional segments of the mtDNA coding region could be successfully sequenced in all seven samples providing additional discrimination power and haplogroup-informative sites. Negative controls were clean; the positive control runs yielded the expected haplogroup (hg) A10 sequence (data not shown). The mitotypes from the tooth samples showed the expected post-mortem damage patterns due to increased C-T misincorporations (data not shown), thus corroborating that the genuine mtDNA was analysed and not modern contamination (for further detail see [9]). PEC analysis resulted in unambiguously different mitotypes in individuals St. 1 (hg U5a2), St. 2 (hg J1c2), St. 3 and 4 (both hg H*), and St. 7 (hg I2), whereas St. 5 and St. 6 shared an identical hg H5 mitotype in the overlapping ranges (Table 1). The observed mitotypes were all belonging to haplogroups typical of the west Eurasian phylogeny [18] and some of them also observed in other contemporaneous locations, such as the Lichtenstein Cave (Germany, 1000 BCE) by Schilz and co-authors [19] who found hgs H, J, T2, and U in their remains. The Stillfried mitotypes were not observed in a total of 26,127 control region (16024-576) sequences in EMPOP (v3/R11; [17]), except for the shared hg H5 mitotype in St. 5 and St. 6 that was observed once (Table 1). Laboratory staff handling the specimens and other study participants were excluded as donors of the mitotypes observed in St.1–7 (data not shown). The two pedigree models proposed by Breitinger [5] and Szilvássy and co-authors [7] mainly differ by the adult females giving birth to the four children. Breitinger observed skeletal modifications at the sacral and hip bones of both females, based on which he attested motherhood to both of them [5], while Szilvássy and co-authors [7] postulated that only the 45-year-old female (St. 5) had given birth to the four children (Fig. 1). The two pedigree models were evaluated using geological fingerprinting in 2016. Teschler-Nicola and co-authors [8] investigated the 87Sr/86Sr isotope ratios in dentine and enamel of the individual’s teeth and the soil background in Stillfried and concluded that individuals St. 2, St. 3 and St. 4 could be considered locals, whereas the
2. Materials and methods 2.1. Samples, DNA extraction and mtDNA quantification Teeth (permanent or deciduous molars) were carefully extracted from the lower jaw of the seven individuals recovered from the Stillfried Late Bronze Age storage pit V1141 (Table 1). They were kindly provided by the stakeholder of the findings, the Natural History Museum, Vienna, Austria for mtDNA analysis. The teeth stemming from individuals 1 (St. 1) and 4 (St. 4) used for a pilot study were grinded using a Retsch grinding mill MM400 (Retsch GmbH, Haan, Germany). For the other five individuals solely powder of the teeth was obtained using a medical dentist drill. DNA was extracted following the protocols described in [10] for samples St. 1 and St. 4 and [11] for the remaining samples (Table 1). Estimation of mtDNA copy number was performed using a 51bp target SYBR green assay (Power SYBR, Thermo Fisher Scientific (TFS), Waltham, MA, USA) with primers and probe target regions published in [12]. DNA of the positive control was extracted from whole blood independently in another laboratory. 2.2. Library preparation, primer extension capture (PEC) and sequencing The PEC method applied here targeted the mtDNA control region. A detailed protocol is provided in [9]. Positive control DNA was sheared for 40 min using the Ion Shear Kit (TFS) and the sequencing library was prepared using the IonXpress Fragment Library Kit (TFS) according to the manufacturer’s protocol in an independent laboratory. DNA extracted from the tooth samples was directly subjected to library preparation as described above. The libraries were amplified using the IonXpress Fragment Library Kit (TFS) and 10 PCR cycles according to manufacturer’s protocol. The PEC reactions were performed as described in [9]. Two libraries (St. 1 and St. 4) were sequenced using the Ion PGM (TFS) with manual template preparation (Ion OT2 (TFS)) and subsequent template enrichment according to manufacturer’s protocol [9]. The remaining five samples were sequenced on the Ion S5 (TFS) with automated template preparation using the IonChef pipeline (TFS) according to manufacturer’s protocol. Raw data analysis was performed 149
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Table 1 Summary of studied remains from seven individuals placed in a Late Bronze Age storage pit in Stillfried, Austria. Sample designation and description of the individuals according to Breitinger, [5]. DNA extraction according to Loreille et al. [10] and Dabney et al. [11]. MtDNA specific quantitation values are indicated as mtDNA Genome equivalents per microliter. The control region (16024-576) of the mitotypes was searched in the EMPOP database (empop.online; v3/R11). Haplogrouping was performed using the EMPOP haplogrouping function on the basis of Phylotree (phylotree.org, build 17 [16],). Mitotypes are reported relative to the rCRS [13]. Mutations in brackets indicate low coverage reads (< 3). N.A. … not available. Sample
St. 1
St. 2
St. 3
St. 4
St. 5
St. 6
St. 7
Extr. blank 1
Extr. blank 2
Description Tooth Extraction Quantitation [51bp mtG/μl] EMPOP CR search (v3 R11, N = 26,127 CR) Haplogroup Mitotype
male 30 y. 36 [10] 225.00 0 U5a2 73G 263G 750G 16192T 16256T 16270T 16526A
male 3 y. 48 [11] 2,277.61 0 J1c2 73G 185A 188G 228A 263G 295T 462T 489C 750G (2706 G) 4216C 8860G 11251G 11719 A 12612G 13708A 14766T 14798C 15326G 15452A 16069T 16126C
female 40 y. 36 [11] 488.85 0 H* 263G 750G 16114T 16362C 16519C
male 7-8 y. 37 [10] 378.00 0 H* 263G 750 G (1719 A) 16261T
female 45 y. 47 [11] 2,364.13 1 H5 263G 456T 750G 16304C
male 5-6 y. 85 [11] 2,386.73 1 H5 263G 456T 750G 15326G 16304C
female 9 y. 36 [11] 5,468.84 0 I2 73G 152C 199C 204C 207A 250C 263G 573.1C 750G 1719 A 2706 G 4769G 8251A 8860G (10034C) (11719 A) 12501A (12705 T) 13780G 14766T 15043A 15326G 15758G 15924G 16129A 16223T 16390A 16391A 16519C
NA NA [10] 0.00 NA NA NA
NA NA [11] 3.47 NA NA NA
() polymorphisms observed with 1 or 2x coverage but phylogenetically plausible
Sequencing ranges (more than2x coverage): St. 1: 1–911 4935–4951 8200–8514 8591-8711 8715–8734 14076-14152.15716–16569. St. 2: 1–982 1590–1787 2155–2444 3346–3649 3681–3865 3959–4289 4420–4515 4656–4696 4980–5503 6331-6657 7376–7497 7673–7692 7694–7728 7730–7888 8130–9023 9026-9028 9033–9079 9206–9210 9212–9241 9244–9247 9249–9263 9265–9288 9308–9663 9866–9892 10050–10158 10175–10294 10296–10312 10515–10648 10743–11370 11474–11558 11565–11619 11656–11780 11915–12635 12889–14853 14904–15040 15278–16,569. St. 3: 1–872 2174–2226 2228–2241 3483–3596 5300–5371 8324–8543 8610–8755 8757–8762 11260–11297 12253-12283 12310–12414 12416-12417 12419–12433 13802–13837 15366–15567 15785–16,569. St. 4: 1–862 2140–2151 4021–4023 4932–4947 5153–5162 6224–6236 6491–6499 8070-8531 8704–8714 8788-8798 9418–9424 11039–11050 11057–11231 11294–11305 11987–11989 11991-12005 12007–12024 13078–13088 13617–13624 14038–14128 14130–14149 14670–14681 15450–15461 15637–15742 15779–16101 16,128–16,569. St. 5: 1–899 8265-8493 8593–8660 10878–10930 13749–13753 13756–13817.15826–16569. St. 6: 1–909 911-920 1752–1806 1864-1947 2183–2275 2280-2300 3512–3532 4997-5023 5264–5333 5335-5359 7338–7342 7344-7348 7350–7364 7366-7368 7370–7375 7377-7396 7398–7443 8132-8742 8935–8977 9208-9221 9266–9267 9269-9278 9505–9520 9522-9605 10803–10985 11098-11380 11981–12129 12138-12142 12304–12406 12909-12922 13209–13215 13217-13313 13938–14394 14602-14706.15302–1559015716–16569. St. 7: 1–945 947-955 957–972 1035-1075 1609–1976 2107-2484 2682–2770 3114-3912 3923–4307 4336-4526 4594–4809 4844-5696 5706–5722 5725-5755 5757–5815 5856-5885 6383–6617 6753-6864 6900–6910 7376-7496 7632–8024 8068-9190 9339–9798 10242-10328 10477–11406 11441-11716 11720–11748 11750-11751 11895–11901 11922-12196 12212–12223 12234-12252 12254–12644 12761-12795 12804–15129 15257-16,569.
Conflict of interest
remaining individuals must have originated from someplace else (Fig. 1). Geological fingerprinting thus somehow supported Breitingers model that suggested St. 3 as mother of St. 2 and St. 4 (Fig. 1a). Interestingly, mtDNA analyses performed in this study do not corroborate any of these models, instead suggesting a different relation. According to the established mitotypes the 40-year-old female St. 3 is excluded as mother from any of the four children, whereas the 45-yearold female St. 5 can be considered mother or another maternally related individual of the 6-year-old boy St. 6 (Table 1, Fig. 2), confirming the interpretation of the female protecting her son [7] (or a close maternal relative).
The authors declare no conflict of interest. Acknowledgements The authors would like to thank Ronald Mühl and Bernd Bernegger (both Vienna) for technical assistance with the extraction of the teeth and Cordula Berger, Anna König, Daniel Entstrasser, and Manuel Nitz (Innsbruck) for DNA extraction of the teeth from individuals St. 1 and St. 4. 150
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[6]
[7]
[8]
Fig. 2. Matrilineal pedigree reconstruction based on mtDNA analysis (this study). The mitotypes further exclude any other maternal relationship between the three adults and the four children. The role of the 30-year-old male (St. 1) and a potential relationship to the four children remains unclear pending further nuclear DNA analysis.
[9]
[10]
[11]
Appendix A. Supplementary data [12]
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigen.2018.07.005.
[13]
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