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FICHATÉCNICA

TÍTULO

EDITORES:

DESIGN GRÁFICO:

EDIÇÃO:

IMPRESSÃO E ACABAMENTOS:

DATA DE EDIÇÃO:

TIRAGEM:

DEPÓSITO LEGAL:

ISBN:

ArchaeoAnalyticsChromatography and DNA analysis in archaeology

César Oliveira, Rui Morais e Ángel Morillo Cerdán

João Lobarinhas – Município de Esposende

Município de Esposende

NPRINT

Novembro 2015

300 exemplares

401486/15

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.

© Todos os direitos reservados. All rights reserved.

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.

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ArchaeoAnalytics Chromatography and DNA analysis in archaeology

Editors

CÉSAR OLIVEIRARUI MORAISÁNGEL MORILLO CERDÁN

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TRACING THE HISTORY OF THE HORSE IN IBERIA AND NORTH AFRICA THROUGH ANCIENT DNA

RAQUEL MATOSO SILVAcE3c, FCUL, Universidade de Lisboa, Portugal

MIM BOWERMcDonald Institute, University of Cambridge, United Kingdom

CLEIA DETRYUNIARQ, FLUL, Universidade de Lisboa, Portugal

SILVIA VALENZUELAUniversity of Sheffield, United Kingdom

CARLOS FERNÁNDEZ-RODRÍGUEZDep. Historia, FFL, Universidad de León, Spain

SIMON DAVISIGESPAR, Lab. Arqueociências, Lisboa, Portugal

JOSÉ ANTÓNIO RIQUELME CANTALDep. Geografía y Ciencias del Territorio, Universidad de Córdoba, Spain

DIEGO ÁLVAREZ LAODep. Geología, Universidad de Oviedo, Spain

ANA MARGARIDA ARRUDAUNIARQ, FLUL, Universidade de Lisboa, Portugal

CATARINA VIEGASUNIARQ, FLUL, Universidade de Lisboa, Portugal

CRISTINA LUÍSCIUHCT, MUHNAC, Universidade de Lisboa, Portugal.

Email: raquel.smas@gmail.com

Ancient DNA analysis has been an emergent area

of research during the past few decades. With the

development of new molecular biology techniques it

has become easier to retrieve genetic information from

archaeological samples than was previously thought

possible. This is of great importance as it helps us

to clarify species phylogenies and understand the

evolution of animals and plants.

This area has been important in the study of

domestication, since it sheds light on the processes

through which animals became part of human societies.

Here we address the domestication of the horse. Despite

their significance in shaping societies through prehistoric

and historic times, the nature and timing of horse

domestication has been hard to document. We initiated

ABSTRACT

the first comprehensive study of horse domestication

in Iberia using an archaeogenetics and osteometrics

approach. Gene flow across the Mediterranean has

been shown to be a significant factor in other domestic

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.

We present preliminary data resulting from the

ancient DNA analysis of archaeological populations

from Portugal, Spain, Morocco, Algeria and Tunisia, and

provide new insights into potential founder populations

of extant domestic horses. We explore the relationship

of archaeological and present day genetic types and

shed light on the history of horse and its domestication.

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

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INTRODUCTION

Among domesticated animals, the horse differs from others because, some time in the

past, horses were no longer bred solely as a food resource. Horses attained a unique role

as animals of transportation and especially warfare, changing societies on a continent-wide

scale (Levine, 2005). The horse became valuable and prestigious in many cultures, even being

gifted from one ruler to another as a sign of nobility (Levine, 1999; Hyland, 2003).

Despite the pivotal role horses have played in the history of human societies, the process

and timing of their domestication is not yet well understood. Recent research (Outram et al.,

2009) shows the earliest domestic horses present in Botai, Kazakhstan, around 5000 years

ago. It is unarguable that the central Asian steppe was significant for horse domestication

(Anthony, 2007) and both genetics and archaeology point to this geographic region contributing

significantly to the domestic horse gene pool (e.g. Levine, 2005; Warmuth et al., 2011; Achilli

et al., 2012). However, previous genetic research (Vilà et al., 2001; Jansen et al., 2002; Cieslak

et al., 2010) has shown that horses were domesticated a number of times and in a number of

locations. The central Asian steppe was undoubtedly one of these locations, but how many

other geographic regions also contributed to the domestic horse gene pool, and which ones?

The Iberian Peninsula formed a refugium for many plant and animal species during

the last glacial maximum (LGM) (Gómez & Lunt, 2007), and this most probably included

Pleistocene horse populations which, prior to the LGM were widely dispersed across Europe

(e.g. Bendrey, 2012). Previous works (Seco-Morais et al., 2007; Warmuth et al., 2011) suggest

that the post glacial horse populations which survived the LGM could have formed a possible

independent focus of horse domestication, due to its latitude and isolation from the rest of

Europe by the Pyrenees. This theory had previously been raised by several authors (e.g.

Gonzaga, 2004; Andrade, 1926; Zeuner, 1963; Uerpmann, 1995; Royo et al., 2005), and

is supported by the archaeological record (e.g. Muñiz et al., 1996). Nevertheless, recent

works by Cieslack et al. (2010) and Lira et al. (2010) based on mitochondrial DNA (mtDNA)

population genetics from zooarchaeological samples, indicated a lack of evidence for

an independent domestication of the horse in the Iberian Peninsula. However, these

studies only included a limited number of samples from the Iberian Peninsula (n = 67),

especially from the south, where the horses are thought to have persisted even during

the last Ice Age (e.g. Andrade, 1954; Gonzaga, 2004; Gomes, 2010). Because of the high

genetic diversity of horses (e.g. Jansen et al., 2002; Cieslack et al., 2010), a large number

of individuals are required if population structure is to be demonstrated. Therefore, the

number of ancient horse samples from Iberia needs to be significantly increased in order

to further investigate the contribution of Iberian horses to the domestic horse gene pool.

Furthermore, an area of research that has hitherto been overlooked, is the possible

influence of horse populations from around edges of the Mediterranean basin, in

particular, those from North Africa. Recent research (Oliveira et al., 2012; Colominas-

Barbera et al., 2015) show that gene flow occurred in wheat and cattle from North Africa

into the Iberian Peninsula at various periods in prehistory. But no research has been

carried out on the possible gene flow between the horse populations of these regions.

Research in this area could bring new insights into the history of horse domestication in

this region and contact between people across the Mediterranean in prehistory.

We initiated research on an extensive collection of living and archaeological horses from

Iberia and North Africa, using an osteometric and population genetics approach. We included

ancient DNA from archaeological horses in order to facilitate accurate temporal and spatial

modelling of past population demographics. This paper presents the preliminary analyses of

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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.

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.

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

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Ancient DNA analysis

Sequences obtained from ancient DNA amplification were aligned with the DNA Alignment

v 1.3.3.1 software (fluxus-engineering.com) and median joining networks were constructed

using Network 4.6.1.3. (Bandelt et al., 1999; fluxus-engineering.com). Haplogroups were

defined with reference to the nomenclature used by Cieslak et al. (2010).

RESULTS AND DISCUSSION

From the total of 106 ancient samples screened, we obtained DNA from 54 samples, and

the complete sequence of 700 bp from 33 samples (Table 1), belonging to archaeological sites

in Portugal (N=2), Spain (N=20), Algeria (N=3) and Tunisia (N=8) (Figure 2). This represents a

success rate for aDNA amplification of 29%, which is typical in aDNA studies. This is due to

the difficulties associated with analysis of ancient biomolecules which are fragmented and

chemically altered due to post-mortem DNA decay (Gilbert et al., 2003). The integrity of DNA

molecules is variable and depends on several factors such as the climate, soil chemistry and

time (Pääbo et al., 2004, Campana et al., 2013).

Table 1 - Archaeological horse bones and teeth from which mitochondrial DNA has been obtained. The dating shown is the presumed age of the archaeological site from which the samples derive. Samples listed in italics are wild horses, all others are assumed to be domesticated based on archaeological context.

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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).

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

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The 27 sequences in our analysis showed 24 haplotypes which clustered into six

haplogroups. There was very little haplotype sharing, showing that the diversity in our data

was high. This is to be expected, as horse genetic diversity is high as previously referred,

however, it is significant that we captured so much genetic diversity in such a small sample

set. The analysis shows a heterogeneous mtDNA pattern within ancient horse populations

in Iberia and North Africa (Figure 3). Of the six haplogroups represented in our data, two can

be identified as being part of the three pre-domestic lineages (B, H1, J) identified by Cieslack

et al. (2010) as being confined to the Iberian Peninsula. Namely, the H1 and J lineages appear

in our Chalcolithic and Bronze Age samples from north and south of Spain, which indicates

that these lineages were widespread in Iberia during this period. In particular, Haplogroup

H1 is still found in some living Iberian breeds such as the Lusitano or Marismeño, but in

lower frequency than in ancient times (Cieslack et al., 2010). Since Haplotype H1 was at

high frequency in past populations in Iberia, it is not surprising that it is represented in

our data and at high frequency (26%). The second highest frequency haplotype in our data

is J, which occurs at 22%. Haplogroup B was not found in our data. It is significant that

Haplogroup H1 in our data is almost exclusively Bronze Age and Haplogroup J is almost

exclusively Chalcolithic. In fact, the period 3000-800 BC is represented by Haplotypes

H1 and J only. Although this might suggest low genetic diversity in horses of this period,

it is most likely that this is an artefact of low sample number. However, where sample

size is small, the highest frequency types will have a higher probability of being sampled

due to stochastic factors, therefore, we can suggest that Haplotypes H1 and J were at high

frequency in Chalcolithic and Bronze Age horse populations in Iberia.

Haplogroup X2 is the most common in living Iberian and North African samples (e.g.

Jansen et al., 2002, Royo et al., 2005, Luís et al., 2006) and appears only after the Bronze Age

in our data. This is in agreement with the findings of Cieslak et al., (2010) and Lira et al. (2010)

and suggests that wild horse genetic diversity was repeatedly sampled over time, bringing

different haplotypes into the domestic gene pool at different periods in time. This shows that

the domestication of horses was a much more complex and protracted process than that

of other animals, involving gene flow of scale far greater than the introgression from wild

populations experienced by domestic cattle, ovicaprids, or pigs (e.g. Edwards et al., 2007).

Figure 3 - Median joining network of mitochondrial D-loop from 27 ancient horses. Nodes are proportional to the frequency of individuals; colours represent ancient DNA sequences from archaeological horses according to the legend. Circled: haplogroups H1, J and X2.

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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

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

domestication process, not only in Iberia and North Africa, but also in Eurasia, and its

genetic consequences for the horse as a species.

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ACKNOWLEDGEMENTS

This study was funded by FCT (Foundation to Science and Technology), Portugal (Grant

reference: PTDC/HIS.ARQ/120183/2010) and the McDonald Institute for Archaeological

Research, University of Cambridge.

We would like to thank to the investigators and collaborators in this study: Ana

Elisabete Pires, António Matias, Catarina Ginja, Eugénia Cunha, Fethi Amani, Isabel

Fernandes, João Zilhão, Jorge Camino Mayor, Luis Berrocal Rangel, Maria Ana Aboim,

Maria do Mar Oom, Mário Varela Gomes, Miguel Ramalho, Nuno Ferreira Bicho, Rui

Mataloto, Víctor Gonçalves, Yasmina Chaïd-Saoudi, Youssef Bokbot, Museu Geológico de

Portugal and Museo Arqueológico de Astúrias. Invaluable support and advice were given

by Graeme Barker and Christopher Howe, the members of the Howe Group, Department of

Biochemistry, University of Cambridge, and the members of the Glyn-Daniel Laboratory

for Archaeogenetics, University of Cambridge.

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