ArchaeoAnalytics - Chromatography and DNA analysis in archaeology 01 - Chromatography and DNA analysis in archaeology ArchaeoAnalytics

FICHA TÉCNICA

TÍTULO ArchaeoAnalytics Chromatography and DNA analysis in archaeology

EDITORES: César Oliveira, Rui Morais e Ángel Morillo Cerdán

DESIGN GRÁFICO: João Lobarinhas – Município de Esposende

EDIÇÃO: Município de Esposende

IMPRESSÃO E ACABAMENTOS: NPRINT

DATA DE EDIÇÃO: Novembro 2015

TIRAGEM: 300 exemplares

DEPÓSITO LEGAL: 401486/15

ISBN: 978-989-99468-1-1

The Esposende City Council supported both this book and the International Symposium “Archaeoanalytics 2014 - Chromatography and DNA Analysis in Archaeology”, which took place in Esposende on September 12th 2014 under the Celebrations of Augustus Bimillennium. The Portuguese Foundation for Science and Technology (FCT) partially supported this initiative and the research results under the research project “Dialogue among sciences - Multidisciplinary analysis of navigability and anchoring during the Roman period (Esposende)” (PTDC/EPH-ARQ/5204/2012). César Oliveira acknowledges FCT for his research contract under Programa Ciência 2008.

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A reprodução total ou parcial deste livro, sob qualquer forma, carece de aprovação prévia e expressa dos respetivos autores e do Município de Esposende. The total or partial reproduction of this book, in any form, requires previous written permission of the respective authors and the Esposende Municipality.

02 ArchaeoAnalytics - Chromatography and DNA analysis in archaeology 03

Editors CÉSAR OLIVEIRA RUI MORAIS ÁNGEL MORILLO CERDÁN ArchaeoAnalytics Chromatography and DNA analysis in archaeology - Chromatography and DNA analysis in archaeology ArchaeoAnalytics

04 ArchaeoAnalytics - Chromatography and DNA analysis in archaeology 217 We present preliminary data resulting from the present preliminary data resulting from the We provide new insights into potential founder populations provide new insights into potential founder populations explore the relationship of extant domestic horses. We and types genetic day present and archaeological of shed light on the history of horse and its domestication. the the first comprehensive study of horse domestication in Iberia using an archaeogenetics and osteometrics approach. Gene flow across thebeen shown to be a significant factor in other domestic Mediterranean has species and also frequently reported for horses in historic documents. Since no recent studies on horse domestication have included samples from North Africa, this study is pioneering in this respect. ancient DNA analysis of archaeological populations from Portugal, Spain, Morocco, Algeria and Tunisia, and ABSTRACT MIM BOWER CLEIA DETRY SIMON DAVIS CRISTINA LUÍS CATARINA VIEGAS CATARINA SILVIA VALENZUELA SILVIA DIEGO ÁLVAREZ LAO DIEGO ÁLVAREZ RAQUEL MATOSO SILVA RAQUEL MATOSO ANA MARGARIDA ARRUDA University of Sheffield, United Kingdom CARLOS FERNÁNDEZ-RODRÍGUEZ JOSÉ ANTÓNIO RIQUELME CANTAL Dep. Geología, Universidad de Oviedo, Spain cE3c, FCUL, Universidade de Lisboa, Portugal Dep. Historia, FFL, Universidad de León, Spain IGESPAR, Lab. Arqueociências, Lisboa, Portugal UNIARQ, FLUL, UniversidadeUNIARQ, de Lisboa, FLUL, UniversidadePortugal de Lisboa, Portugal UNIARQ, FLUL, Universidade de Lisboa, Portugal CIUHCT, MUHNAC, Universidade de Lisboa, Portugal. McDonald Institute, University of Cambridge, United Kingdom Dep. Geografía y Ciencias del Territorio, Universidad de Córdoba, Spain TRACING THE HISTORY OF THE HORSE IN IBERIA AND NORTH AFRICA THROUGH ANCIENT DNA ANCIENT AFRICA THROUGH IBERIA AND NORTH THE HORSE IN THE HISTORY OF TRACING This area has been important in the study of study the in important been has area This Ancient DNA analysis has been an emergent area Email: [email protected] their significance in shaping societies through prehistoric through societies shaping in significance their and historic times, the nature and timing of horse initiated We document. to hard been has domestication domestication, since it sheds light on the processes the on light sheds it since domestication, through which animals became part of human societies. Here we address the domestication of the horse. Despite evolution of animals and plants. evolution of animals and plants. archaeological samples than was previously thought archaeological samples than was previously thought possible. This is of great importance as it helps us to clarify species phylogenies and understand the of research during the past few decades. With the it techniques biology molecular new of development from information genetic retrieve to easier become has - Chromatography and DNA analysis in archaeology ArchaeoAnalytics

Amphora (spike) Praia de Belinho − 2014 Foto. Museu D. Diogo de Sousa

218 ArchaeoAnalytics - Chromatography and DNA analysis in archaeology 219 , et al. ., 2005), and ., 2011; Achilli , 2002; Cieslak , 2011) suggest et al et al et al. et al. , 2012; Colominas- et al. , 1996). Nevertheless, recent ., 2001; Jansen et al. , 2007; Warmuth et al et al. , 2002; Cieslack et al., 2010), a large number et al. , 2015) show that gene flow occurred in wheat and cattle from North Africa et al. 2010) has shown that horses were domesticated a number of times and in a number of 2010) has shown that horses were domesticated ., 2012). However, previous genetic research (Vilà We initiated research on an extensive collection of living and archaeological horses fromhorses archaeological and living of collection extensive an on research initiated We Furthermore, an area of research that has hitherto been overlooked, is the possible Furthermore, an area of research that has hitherto been overlooked, is the The Iberian Peninsula formed a refugium for many plant and animal species duringThe Iberian Peninsula formed a refugium Despite the pivotal role horses have played in the history of human societies, the process horses have played in the history of Despite the pivotal role Among domesticated animals, the horse differs from others because, some time in the others because, the horse differs from animals, Among domesticated ancient DNA from archaeological horses in order to facilitate accurate temporal and spatialancient DNA from archaeological horses in order to facilitate accurate temporal analyses ofmodelling of past population demographics. This paper presents the preliminary Iberia and North Africa, using an osteometric and population genetics approach. We included We Iberia and North Africa, using an osteometric and population genetics approach. carried out on the possible gene flow between the horse populations of these regions. in Research in this area could bring new insights into the history of horse domestication this region and contact between people across the Mediterranean in prehistory. particular, those from North Africa. Recent research (Oliveira Barbera has been into the Iberian Peninsula at various periods in prehistory. But no research influence of horse populations from around edges of the Mediterranean basin, in of individuals are required if population structure is to be demonstrated. Therefore, the of individuals are required if population structure is to be demonstrated. Therefore, order in increased significantly be to needs Iberia from samples horse ancient of number gene pool. to further investigate the contribution of Iberian horses to the domestic horse especially from the south, where the horses are thought to have persisted even during especially from the south, where the horses are thought to have persisted even of the high the last Ice Age (e.g. Andrade, 1954; Gonzaga, 2004; Gomes, 2010). Because genetic diversity of horses (e.g. Jansen population genetics from zooarchaeological samples, indicated a lack of evidence for population genetics from zooarchaeological horse in the Iberian Peninsula. However, these an independent domestication of the of samples from the Iberian Peninsula (n = 67), studies only included a limited number Gonzaga, 2004; Andrade, 1926; Zeuner, 1963; Uerpmann, 1995; Royo Gonzaga, 2004; Andrade, 1926; Zeuner, (e.g. Muñiz is supported by the archaeological record DNA (mtDNA) et al. (2010) and Lira et al. (2010) based on mitochondrial works by Cieslack independent focus of horse domestication, due to its latitude and isolation from the rest ofindependent focus of horse domestication, (e.g. authors by several raised been previously had theory This Pyrenees. the by Europe Pleistocene horse populations which, prior to the LGM were widely dispersed across EuropePleistocene horse populations which, prior (e.g. Bendrey, 2012). Previous works (Seco-Morais a possible have formed the LGM could survived horse populations which post glacial the that other geographic regions also contributed to the domestic horse gene pool, and which ones?other geographic regions also contributed & Lunt, 2007), and this most probably includedthe last glacial maximum (LGM) (Gómez et al., how manybut locations, one of these undoubtedly was steppe Asian locations. The central (Anthony, 2007) and both genetics and archaeology point to this geographic region contributing genetics and archaeology point to this (Anthony, 2007) and both significantly to the domestichorse gene pool (e.g. Levine, 2005; Warmuth et al and timing of their domestication is not yet well understood. Recent research (Outram is not yet well understood. and timing of their domestication years5000 around Kazakhstan, Botai, in horses present domestic earliest the shows 2009) ago. It is unarguable that the central Asian steppe was significant for horse domestication scale (Levine, 2005). The horse became valuable and prestigious in many cultures, even beingThe horse became valuable and prestigious scale (Levine, 2005). 1999; Hyland, 2003). to another as a sign of nobility (Levine, gifted from one ruler past, horses were no longer bred solely as a food resource. Horses attained a unique role longer bred solely as a food resource. past, horses were no societies on a continent-wide and especially warfare, changing as animals of transportation INTRODUCTION the genetics results obtained from archaeological horse samples collected in the Iberian Peninsula and North Africa, in order to contribute to the overall characterisation of the domestication of the horse. Our study presents the first ancient DNA data from North Africa. In this matter, this study is unique.

MATERIAL AND METHODS

Research material

297 archaeological horse bones and teeth from Iberia and North Africa were collected for aDNA analysis, from a wide temporal scale (Figure 1). A network of archaeologists, researchers and institutions collaborated in this study and provided the ancient samples. - Chromatography and DNA analysis in archaeology ArchaeoAnalytics

Figure 1 - Locations and approximate time periods of horse bones and teeth samples collected for this study.

Ancient DNA extraction and amplification

We selected 106 ancient samples out of the initial 297, covering a broad range of time periods, and a wide geographical spread, including the north and south of the Iberian Peninsula, and North Africa. Ancient DNA analysis was performed in a dedicated ancient DNA laboratory at the University of Cambridge. We amplified a 700 base pair fragment of the mitochondrial D-loop in five overlapping fragments, between 15424 bp and 16107 bp with reference to the sequence published by Xu and Arnason (1994). The DNA analysis followed previously published protocols (e.g. Campana, 2011) and used a PCR based re- sequencing approach. We chose to analyse the mitochondrial D-loop in order to allow the screening of the largest number of individuals possible to enable a population genetics approach. Appropriate contamination control and authentication methods were followed. The samples will be further analysed at an independent laboratory to validate the results.

220 ArchaeoAnalytics - Chromatography and DNA analysis in archaeology 221 (2010). , 2003). The integrity of DNA et al. et al. , 1999; fluxus-engineering.com). Haplogroups were , 2013). et al. et al. 2004, Campana et al., From the total of 106 ancient samples screened, we obtained DNA from 54 samples, and ancient samples screened, we obtained From the total of 106 Sequences obtained from ancient DNA amplification were aligned with the DNA Alignment DNA the with aligned were amplification DNA ancient from obtained Sequences Table 1 - Archaeological horse bones and teeth from which mitochondrial DNA has been obtained. The dating shown 1 - Archaeological Table wild horses,is the presumed age of the archaeological site from which the samples derive. Samples listed in italics are all others are assumed to be domesticated based on archaeological context. chemically altered due to post-mortem DNA decay (Gilbert chemically altered due to post-mortem factors such as the climate, soil chemistry andmolecules is variable and depends on several time (Pääbo success rate for aDNA amplification of 29%, whichis typical in aDNA studies. This is due to the difficulties associated with analysis of ancient biomolecules which are fragmented and the complete sequence of 700 bp from 33 samples (Table 1), belonging to archaeological sites 1), belonging of 700 bp from 33 samples (Table the complete sequence a represents This 2). (Figure (N=8) Tunisia and (N=3) Algeria (N=20), Spain (N=2), Portugal in RESULTS AND DISCUSSION RESULTS v 1.3.3.1 software (fluxus-engineering.com) and median joining networks were constructed (Bandelt using Network 4.6.1.3. to the nomenclature used by Cieslak defined with reference Ancient DNA analysis - Chromatography and DNA analysis in archaeology ArchaeoAnalytics

Figure 2 - Locations and approximate time periods of horse bones and teeth, analysed for this study from which we have obtained mitochondrial DNA.

Of the 28 North African samples that we studied, we obtained a total of 11 complete sequences, with a success rate of 39.3%. This is higher than that for the Iberian samples (28.2% success). This success rate is better than expected a priori when we consider previous studies (Smith et al., 2003) that demonstrate that the thermal age of the fossils – i.e. the time taken to produce a given degree of DNA degradation when temperature is held at a constant 10ºC - is important for the conservation of biomolecules in the sample, being lower temperatures better for DNA preservation. Prior to this research, very few aDNA studies of domestic animal species have included data from North Africa (Campana et al., 2013) - only cattle (e.g. (Edwards et al., 2004) and donkey (e.g. Kimura et al. 2013) have been studied - because of the poor preservation of ancient DNA from these regions and the difficulty in obtaining material for study. Interestingly, despite repeated attempts, we were not able to extract and amplify DNA from the 13 Moroccan samples we screened. This is probably due to the harsh environmental conditions that these samples were subject to. The results obtained in this study showed that two of our North African samples (LOS220 from Algeria and ALT117 from Tunisia) belonged to another equid species instead of Equus caballus, namely Equus asinus. This shows that ancient DNA analysis can be an effective tool in discerning between equid species when the analysis of bone and tooth morphology yields an unclear identification. Two further Algerian samples (FOR221 and FOR224) were radiocarbon dated and the results showed that these samples were from a recent historic period. Therefore, they were excluded from the network presented here. The samples for which we obtained the complete sequence consist of 2 horses of Late Pleistocene age, representing wild horses and 27 Holocene horses dating variously (according to the archaeological contexts from which the bones and teeth derive) to the Mesolithic (8000-7000 BC), the Middle/Late Neolithic (5000-3000 BC), the Chalcolithic (3000-1900 BC), the Bronze Age (1800-800 BC), the Iron Age (850-200 BC), the Roman period (200 BC-500 AD), and the medieval period (500-1400 AD).

222 ArchaeoAnalytics - Chromatography and DNA analysis in archaeology 223 2007). et al., , (2010) and Lira et al. (2010) et al. 2010). Since Haplotype H1 was at 2010). Since Haplotype et al., , 2005, Luís et al., 2006) and appears only after the Bronze Age et al. , 2002, Royo et al. (2010) as being confined to the Iberian Peninsula. Namely, the H1 and J lineages appear lineages J and H1 the Namely, Peninsula. Iberian the to confined being as (2010) Haplogroup X2 is the most common in living Iberian and North African samples (e.g.Haplogroup X2 is the most common in living Iberian and North African samples The 27 sequences in our analysis showed 24 haplotypes which clustered into sixhaplotypes which showed 24 in our analysis The 27 sequences populations experienced by domestic cattle, ovicaprids, or pigs (e.g. Edwards different haplotypes into the domestic gene pool at different periods in time. This shows thatdifferent haplotypes into the domestic gene pool at different periods in time. This thatthan process protracted and complex more much a was horses of domestication the of other animals, involving gene flow of scale far greater than the introgression from wild Jansen Cieslak of findings the with agreement in is This data. our in bringingand suggests that wild horse genetic diversity was repeatedly sampled over time, Figure 3 - Median joining network of mitochondrial D-loop from 27 ancient horses. Nodes are proportional to the27 ancient horses. from D-loop of mitochondrial - Median joining network Figure 3 to the legend.frequency of individuals; colours represent ancient DNA sequences from archaeological horses according Circled: haplogroups H1, J and X2. size is small, the highest frequency types will have a higher probability of being sampledsize is small, the highest frequency types can suggest that Haplotypes H1 and J were at highdue to stochastic factors, therefore, we horse populations in Iberia.frequency in Chalcolithic and Bronze Age exclusively Chalcolithic. In fact, the period 3000-800 BC is represented by Haplotypesexclusively Chalcolithic. In fact, the period low genetic diversity in horses of this period,H1 and J only. Although this might suggest of low sample number. However, where sampleit is most likely that this is an artefact is J, which occurs at 22%. Haplogroup B was not found in our data. It Bronze Age and Haplogroup J is almostHaplogroup H1 in our data is almost exclusively is significant that lower frequency than in ancient times (Cieslack lower frequency than Iberia, it is not surprising that it is represented inhigh frequency in past populations in second highest frequency haplotype in our dataour data and at high frequency (26%). The in our Chalcolithic and Bronze Age samples from north and south of Spain, which indicates Bronze Age samples from north and in our Chalcolithic and In particular, Haplogroupwere widespread in Iberia during this period. that these lineages Lusitano or Marismeño, but in living Iberian breeds such as the H1 is still found in some in Iberia and North Africa (Figure 3). Of the six haplogroups represented in our data, two can (Figure 3). Of the six haplogroups represented in Iberia and North Africa Cieslack by identified J) H1, (B, lineages pre-domestic three the of part being as identified be et al. however, it is significant that we captured so much genetic diversity in such a small sample small a such in diversity genetic much so captured we that significant is it however, ancient horse populations a heterogeneous mtDNA pattern within set. The analysis shows haplogroups. There was very little haplotype sharing, showing that the diversity in our data showing that the haplotype sharing, There was very little haplogroups. previously referred, diversity is high as as horse genetic This is to be expected, was high. Although our data include only two Pleistocene horse sequences, they fall within the network at a basal position close to the root. Furthermore, one (CRI184) is relatively closely related to our Neolithic sequence. Since previous research (e.g. Vilà et al., 2001) has placed Pleistocene horses in a separate, distant clade to other living or ancient horses, it is interesting that the Pleistocene and Neolithic horses in our data are relatively closely related. This suggests that more robust phylogenetic methods might place the Pleistocene horses within the past horse populations and not separate from them, suggesting that it might be possible to demonstrate some level of continuity of populations in Iberia. The results we have presented here are preliminary and further analyses are planned. Initially, the ancient sequences will be included into an extensive analysis of living horse populations, carried out as part of this study, and will serve to anchor the living horse phylogenies in time and space. Collation of living and ancient sequences will allow the - Chromatography and DNA analysis in archaeology use of Bayesian statistical reconstruction of population demographics over time. The data here can be combined with previously published mtDNA data to provide the basis of a large scale analysis of Old World horse genetic data. We are confident that with these further analyses we can obtain new insights into the timing and complexity of the horse ArchaeoAnalytics domestication process, not only in Iberia and North Africa, but also in Eurasia, and its genetic consequences for the horse as a species.

224 ArchaeoAnalytics - Chromatography and DNA analysis in archaeology 225 This study was funded by FCT (Foundation to Science and Technology), Portugal (Grant Portugal (Grant Technology), to Science and (Foundation was funded by FCT This study this study: Ana investigators and collaborators in would like to thank to the We LARSON, G., HO, S., HEUPINK, T., SHAPIRO, B., FREEMAN, A., THOMAS, M., ARBOGAST, R., ARNDT, B., B., ARNDT, R., ARBOGAST, M., THOMAS, A., FREEMAN, B., SHAPIRO, T., HEUPINK, S., HO, G., LARSON, E., DÖHLE, H., BARTOSIEWICZ, L., BENECKE, N., BUDJA, M., CHAIX, L., CHOYKE, A., COQUEUGNIOT, analysis of 101 cattle remains: Limits and prospects”, Journal of Archaeological Science, 31, pp. 31, pp. analysis of 101 cattle remains: Limits and prospects”, Journal of Archaeological Science, 695–710. A., VIGNE, J., BAIRD, J., CHAMBERLAIN, A., TRESSET, C., BOLLONGINO, R., SCHEU, A., EDWARDS, 15311. K. M., MARTIN, L., RUSSELL, N., HORWITZ, L. K., MCINTOSH, C. J., MACHUGH, D. 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