Ancient DNA As a Means to Investigate the European Neolithic

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 323

Ancient DNA as a Means to Investigate the European Neolithic

HELENA MALMSTRÖM

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 UPPSALA ISBN 978-91-554-6940-5 2007 urn:nbn:se:uu:diva-8162 !!" #$!! % % % & ' ( ) * '

+, -' !!"' . . + / * ' .

' # #' "! ' ' / 0 1"231344351!34'

( % 3 % % % '' ' 6 % ) 7 ) % %% % ) %' ( ) % ' * % % 7 % ' . 7 8 9 ) ) % % % %: ' + : % ) . % . ' - / % ) . . % ' / 3 : % : ;3 : ' / % : % % ' ( ) 3 % ) ' / -< / : % #! 3 % % ) % ) % %: % 7 3' / % ) % '

. .

! " ! # ! " $ ! % &' ()! ! "*+,-./ !

= - +, !!"

/ 5435 / 0 1"231344351!34 $ $$$ 325 8 $>> '7'> ? @ $ $$$ 325 9 List of papers

I Malmström H, Storå J, Dalén L, Holmlund G and Götherström, A (2005) Extensive human DNA contamination in extracts from ancient dog bones and teeth Mol Biol Evol 22:2040-2047

II Malmström H, Svensson E, Gilbert MTP, Willeslev E, Göther- ström A, Holmlund G (2007) More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA Mol Biol Evol 24:998-1004

III Malmström H, Vilà C, Gilbert MTP, Storå J, Willerslev E, Holmlund G, Götherström A Barking up the wrong tree: modern northern European dogs fail to explain their origin Submitted

IV Malmström H, Götherström A, Gilbert MTP, Brandström M, Storå J, Molnar P, Bendixen C, Holmlund G, Willerslev E An- cient human DNA: authentication through FLX-sequenced PCR products in conjunction with bleach pre-treatment, quantitative PCR, assessment of asymmetric molecular behaviour and negative controls Manuscript

V Malmström H, Linderholm A, Dalén L, Lidén K, Evershed R, Storå J, Molnar P, Gilbert MTP, Willerslev E, Holmlund G, Götherström A Different allele frequencies in the lactase gene in Scandinavian Neolithic populations and the development of dairy product consumption Manuscript

Paper I and II are reproduced with permission from the publisher.

Contents

Introduction...... 9 Tracing human prehistory through genetics...... 11 Human genetics...... 12 Early humans and Neandertals ...... 12 Hunter-gatherers and farmers ...... 14 The secondary product revolution ...... 17 Animal genetics...... 18 Methodological aspects of ancient DNA anlayses...... 21 The nature of ancient DNA ...... 21 DNA survival...... 21 DNA damage ...... 23 Contamination ...... 24 Authenticity ...... 25 Technical aspects...... 28 DNA quantification ...... 28 DNA sequencing...... 30 Pyrosequencing...... 30 Large scale sequencing...... 31 Research aims ...... 35 Investigations ...... 36 Paper I: Extensive human DNA contamination in extracts from ancient dog bones and teeth...... 36 Materials and methods...... 36 Results and discussion ...... 37 Paper II: More on contamination: the use of asymmetric molecular behaviour to identify authentic ancient human DNA...... 38 Materials and methods...... 39 Results and discussion ...... 39 Paper III: Barking up the wrong tree: modern northern European dogs fail to explain their origin ...... 39 Materials and methods...... 40

Results and discussion ...... 40 Paper IV: Ancient human DNA: authentication through FLX-sequenced PCR products in conjunction with bleach pre-treatment, quantitative PCR, assessment of asymmetric molecular behaviour and negative controls ...41 Materials and methods...... 41 Results and discussion ...... 42 Paper V: Different allele frequencies in the lactase gene in Scandinavian Neolithic populations and the development of dairy product consumption ...... 43 Materials and methods...... 43 Results and discussion ...... 43 Concluding remarks...... 45 Svensk sammanfattning ...... 47 Bakgrund ...... 47 Artikel I: Human DNA kontamination är vanligt förekommande i extrakt baserade på ben och tänder från förhistoriska hundar...... 48 Artikel II: Mer om kontamination: att använda asymetriskt molekylärt beteende för att identifiera autentiskt DNA från förhistoriska humanprov ...... 49 Artikel III: Att spåra Nordeuropeiska hundars ursprung genom analys av moderna hundar kan ge missvisande resultat ...... 50 Artikel IV: DNA från förhistoriska människor: verifiering av resultat genom FLX-sekvenserade PCR produkter samt förbehandling med klorin, kvantitativ PCR, analys av molekylär nedbrytningsgrad och negativa kontroller ...... 51 Artikel V: Olika allelfrekvenser i laktasgenen i Neolitiska populationer från Skandinavien och utvecklingen av konsumtionen av mjölkprodukter ...... 52 Acknowledgements...... 53 References...... 55

Front page by Lotta Tomasson

Abbreviations

aDNA Ancient DNA bp Base pair DNA Deoxyribo nucleic acid hg Haplogroup ht Haplotype HV Hypervariable region LCT Lactase gene LD Linkage disequilibrium LGM Last Glacial Maximum LP Lactase persistence MRCA Most recent common ancestor mtDNA Mitochondrial DNA nt nucleotide PC Principal Component analysis PCR Polymerase chain reaction SNP Single nucleotide polymorphism UNG Uracil-N-glycosylase

Introduction

The traces of past human activity are all around us. Some are deliberate like the megalithic structures in Stonehenge or the pyramids of Egypt. In grave deposits we find traces of human and animal fossils, often together with for example weapons and household utensils. In close proximity we may also find remnants of house structures, food remains and tools. The evidences in the archaeological record supply clues about the daily life of prehistoric societies. Further, by using for instance site stratigraphy, typology and radio- carbon dating, the prehistoric remains may be placed in a chronological con- text (Renfrew 1991). Fossil records imply that the first modern humans reached Europe about 45 000 years ago (Gamble 1999). For a while they lived side by side with indigenous Neandertal populations. With the peak of the laste Ice Age (the last glacial maximum, LGM) the hunter-gatherers were forced to retreat southwards. At the end of the Ice Age people in the refugias started re- colonising Europe (Dennell 1985). The archaeological records display major changes in human societies starting 10 000 years ago in the Fertile Cresent of the Near East (Childe 1925). Here, we find the first signs of domesticated animals and plants (Harris 1996; Clutton-Brock 1999). This transition from a hunter-gatherer society towards a farmig society is usually referred to as the “Neolithisation process”. With it came major changes such as more permanent settlements and later also villages, the development of more defined social structures, technological advances and also organized warfare and more diseases (Diamond 2002). This new lifestyle rapidly spread across Europe and reached Scandinavia about 6 200 years ago (Renfrew 1987; Malmer 2002). Even though the archaeological record provides us with clues about prehistoric events, some major questions still remain. From where came the humans that initially colonized Europe? Did these humans and Neandertals ever interact? How did the Neolithisation process reach Europe? Here, molecular genetic analyses may provide a further tool in helping us understand the past. Studies on genetic diversity in modern populations do, for instance, indicate an African ancestry of all modern humans (Cann et al. 1987; Ing- man et al. 2000) and studies on modern domesticates give us an idea about the antiquity and number of domestication events that occurred (Vilà et al.

9 1997; Luikart et al. 2001; Savolainen et al. 2002). However, when using data from modern populations to reconstruct historical events, there is always a risk that more recent demographic events obscure earlier ones (Leonard et al. 2002; Barnes et al. 2007). This problem may be overcome by directly studying DNA from ancient samples. The relationship between modern humans and Neandertals has been addressed this way (Krings et al. 1997; Ovchin- nikov et al. 2000). Also, using this method the question that has been central within archaeology for the last 80 years, whether agriculture was spread via migrating farmers or via the transmission of ideas, may be addressed (Childe 1925; Dennell 1985). The former case would be manifested through a genetic discontinuity between Mesolithic hunter-gatherers and Neolithic farmers, while in the latter case we would see a genetic continuity between the two groups. Ancient DNA provides numerous possibilities to investigate ancient populations, their movements, diseases and much more (Brown 1992; Hagelberg et al. 1994; Handt et al. 1994). However, the nature of ancient DNA causes several problems, especially when working with ancient human samples that may share haplotypes with modern contamination (Gilbert et al. 2005c; Sampietro et al. 2006). To mimimise the risk of retrieving erroneous results due to the degraded state of ancient DNA, the low copy number and the risk of contamination with modern DNA, several standard precautions are usually adopted (Cooper and Poinar 2000). However, even when these are followed (Caramelli et al. 2003), ancient human results are questioned (Abbott 2003). Although some researchers are trying to develop more reliable techniques for ancient human DNA analyses (Kemp and Smith 2005; Sampietro et al. 2006; Helgason et al. 2007), many focuses on plant (Allaby et al. 1999; Jaenicke-Despres et al. 2003) and animal remains (Vilà et al. 2001; Leonard et al. 2002; Anderung et al. 2005; Svensson et al. 2007) and use them as a proxy for prehistoric human activities. Here I will describe how a molecular genetic approach can be used to trace the prehistory of humans in Europe. I will also describe what we know about the nature of ancient DNA and highlight some of the new techniques that may help us retrieve data of better quality and quantity.

10 Tracing human prehistory through genetics

This section aim at compiling archaeological data as well as genetic data from modern and ancient humans and from domestic animals in order to review the processes of colonisation, migration and domestication in Europe from between approximately 40 000 to 4 500 years ago. I will however, start with a breaf introduction to the concept of genetic diversity and how it is studied. The molecular diversity in a population is caused by mutations and gene flow followed by drift and selection and is seen as nucletide substitutions, insertions and deletions. The amount and pattern of variation is influenced by evolutionary processes and the demographic history of the population. Due to different modes of inheritance mitochondrial, Y-chomosomal and nuclear DNA markers display the lineage history of females, males and of both sexes respectively. Both mitochondrial DNA and Y-chromosomal DNA are haploid systems where a particular combination of genetic characteristics is defined as a haplotype (ht) and where a monophyletic group of haplotypes are defined as a haplogroup (hg). The mitochondrial DNA is particularly well suited for ancient DNA analyses and for investigating phylogeographic patterns in recent times (the last tens of thousands of years) due to its high abundance per cell, its high mutation rate and non-recombining mode of inheritance. Y-chromosomal single nucleotide polymorphisms (SNPs) are also non-recombining and suitable for ancient DNA analyses since short sequences may be investigated. One advantage of the Y-chromosome compared to mtDNA is that substitutions are largely unique event polymorphisms while the mtDNA often display parallel and back mutations (Stumpf and Goldstein 2001). The diploid nuclear DNA is often investigated through microsatellite markers (short repetitive elements with varying number of repeats between alleles) to detect the history and evolution of populations, species etcetera (Ellegren 2004). The alleles of microsatellite markers do, however, often contain too many bases and they are usually too unstable to be suitable for analyses of degraded ancient DNA. Networks or phylogenetic trees are constructed to visualise the genetic variation and sometimes also the geographic distribution of the variation. The approach when the history of a gene sample is reconstructed from the present and back in time to the most recent common ancestor (MRCA) is known as coalescence. By estimating mutation rates and assuming specific models for mutations the divergence time between lineages may be calcu-

11 lated. There are, however, many uncertainties when applying molecular clock estimations (Ho and Larson 2006). Several characteristics in nucleotide diversity are known to be shaped by different demographic events. For example, center of origin of a population is usually assumed to be the region in which individuals display the highest genetic diversity. The antiquity of that particular population has allowed mutations to accumulate over time. When populations goes through bottle-necks, for instance when a group of people migrate away from the initial source population or when a certain part of an animal group is selected for domestication purposes, the genetic diversity decrease (Nei 1975). If such a group (deriving from a single source population) expands quickly, like often seen in domesticates, the shape of networks usually display a star-like structure (Slatkin and Hudson 1991) and excess of low frequency alleles increases the level of heterozygosity and absence of linkage disequilibrium (LD) (Watterson 1986). Or if such a small population does not experience much admixture, genetic drift causes alleles to be lost and genetic diversity to decrease (Kimura 1955). When specific non-synonymous regions display reduced genetic diversity it can often be deduced to one of two forms of selection. Either purifying selection in which mutations are eliminated through their deleterious effect on the function of the protein, or positive selection where mutations are maintained in elevated levels in populations due to their positive effect on the function of the protein. A typical signal for strong positive selection is that tightly linked loci may hitchhike with the allele under selection so that all become fixed in a so-called selective sweep (Zeder et al. 2006). The best historical view of a population would of course be achieved through studying several genetic markers (in coding and non-coding regions) with different mode of inheritance in both modern and ancient populations (Ramakrishnan et al. 2005).

Human genetics Early humans and Neandertals The earliest fossils of Homo sapiens first appear in Africa and the Near East more than 100 000 years ago (Valladas 1988; Schwarcz 1992), in mainland Asia around 67 000 years ago (Swisher et al. 1996) and in Europe and West- ern Asia around 40-59 000 years ago (Stringer and Grun 1991; Wolpoff and Frayer 1992; Hublin et al. 1996). These fossils differ morphologically from their predecessors by, for example, displaying a more gracile skeleton, a more voluminous cranium (except for Neandertals) and smaller teeth (Stringer 1985; Tattersall 1986). Today there is a general agreement between molecular anthropologists and paleontologists that the ancestors of anatomically modern humans developed in Africa. When they dispersed throughout

12 the world and how they developed into modern humans are, however, debated issues. The “multiregional hypothesis” advocates that the transforma- tion into Homo sapiens occurred approximately at the same time in different parts of the world (Wolpoff et al. 1988). It is proposed that fossil evidence show cultural, morphological and temporal continuity between archaic and modern humans in regions outside of Africa, for instance between Neander- tals and modern Europeans and between Homo erectus and modern Asians (Thorne and Wolpoff 1981). Another theory is the “single African origin” (Stringer and Andrews 1988). Here, a single more recent center of origin with subsequent migration of morphologically modern humans in all direc- tions replacing other archaic hominids is proposed. This theory is favoured by several studies on genetic variation in modern human populations. For instance, Cann and collegues (1987) investigated restriction fragment length polymorphism (RFLP) among the mtDNA in five different populations. By assembling the haplotypes (ht) found into a phylogenetic tree using maximum parsimony they found that the oldest clade derived from African populations and that the most recent common ancestor (MRCA) of all the haplotypes was approximately 200 000 years old. Inves- tigations of the sequence divergence between complete mtDNA sequences from humans of diverse origins, gave similar time estimates for the MRCA, and further suggested a divergence between African and non-African populations around 52 28 000 years ago and a following population expansion in the people leaving Africa (Ingman et al. 2000). Phylogenetic trees based on polymorphisms in binary markers of the non-recombining region (NRY) of the human Y chromosome suggest that the expansion out of Africa took place a bit earlier, between 35-89 000 years ago (Underhill et al. 2000), while polymorphisms in autosomal microsatellite markers suggests a dispersal around 150 000 years ago (Goldstein et al. 1995). When analysing the diversity in nuclear sequences reported dates for MRCA are approximately three to four times older, as the population size is also larger (Harding et al. 1997; Kaessmann et al. 1999). It has also been argued that the out of Africa model may be more complex than previously thought (Zhao et al. 2000). According to the fossil record the predecessor to Neandertals, Homo hei- delbergensis appeared in Europe and Western Asia over 400 000 years ago and had fully developed into Neandertals about 170 000 years ago. A few thousands of years before they vansihed (around 27 000 years ago), anatomically modern humans appear in the area, and it seems as if they coex- isted for some time (Smith et al. 1999). The question of how these groups relate to each other has been widely adressed using ancient DNA (aDNA) technology. During the last ten years, mtDNA sequences from the hypervariable region (HV) have been retrieved from over ten different Neandertal specimens (Krings et al. 1997; Krings et al. 1999; Krings et al. 2000; Ovchinnikov et al. 2000; Schmitz et al. 2002; Caramelli et al. 2003; Serre et al. 2004b; Lalueza-Fox et al. 2005; Orlando et al. 2006). All of these show

13 that the Neandertal sequences constitutes a monophylethic group, and of similar distance to any randomly selected modern human mtDNA sequences, implying that they were a separate species. The split between Neandertals and the ancestors of modern humans have beed estimated to approximately 500 000 years ago. According to mtDNA data and simulation data there has been little or no admixture (0.1-25%) between Neandertals and modern humans in Europe (Krings et al. 1997; Currat and Excoffier 2004; Serre et al. 2004b). The mtDNA however, only represents one maternally inherited locus and it may have been affected by drift or sexual bias in reproduction. Recently, two independent research groups presented vast amounts of Nean- dertal nuclear data by using the Genome Sequencer GS20 platform for mas- sive parallel pyrosequencing (see also section Large scale sequencing) (Green et al. 2006; Noonan et al. 2006). Their estimates imply a divergence time between Neandertals and the ancestors of modern humans around 700 000 ago while the actual population split was later, some 370 000 years ago (Noonan et al. 2006). Green and collegues observed a high level of shared SNP alleles between the Neandertal sequences and modern humans, especially on the X chromosome, and suggest that it may be due to geneflow from modern human males into the Neandertals population. Noonan and collegues investigated the presence of a modest level of admixture of Nean- dertal autosomal DNA into the modern gene pool, as proposed through modern data and population modelling (Plagnol and Wall 2006). However, they did not find any matches of low-frequency derived autsomal alleles between modern Europeans and the Neandertal, and thus, concluded that an admixture of Neandertals and anatomically modern humans was unlikely (Noonan et al. 2006). Additional Neandertal genomic studies may, however, elucidate several of these questions in the future.

Hunter-gatherers and farmers The earliest modern humans often referred to as Cro-Magnons, appear to have arrived in Europe from an eastward direction. Several cultural phases may be destinguished in the Franco-Cantabrian region, in central Europe and in Russia throughout the Upper Paleolithic (40-10 000 years ago) (Gamble 1999). These hunter-gatherer produced clay figurines and cave arts depicting large wild animals and they display technological differences in stone tools production. With the advent of the last Ice Age (20 000-15 000 years ago) Northern Europe and most of the British Isles became covered by ice and the humans were forced to retract southward to areas free of ice. During the following deglaciation, human poplations appear to have migrated both in north and soutward direction following expanding game populations (Dennell 1985). During the Mesolithic (starting around 10 000 years ago) people spread futher north than before and southern Scandinavia becomes populated. The archaeological record indicate a habitation preference to-

14 wards open areas close to water and new developments in tool technology are seen (Bogucki 1988). The transition towards the Neolithic starts in the Fertile Crescent of the Middle East around 10 000 years ago. The changes in human society brought about with the introduction of agriculture are so enormous that Childe termed it the “Neolithic revolution” (today we know that it was not a single event, but rather a recurrent process). The initial domestication can be traced through preserved bones of domestic animals and through remains of seeds and pollen for instance. It is also seen through more permanent settlement arrangemets, increased amounts of food storage items (some of which even have grain impressions), and in traces of forest clearance and field systems. Some argue that population growth, a lower abundance of game animals or an increased need to buffer against environmental uncertainties are the reasons for this shift, and others believe the reasons are more in line with a reli- gious or ideological shift. The Neolithisation had a major impact on the society. Small villages were formed, social structures became more diverse and many technological advances proceeded. However, growing populations also led to increased amounts of epidemics, and fortification structures show the increased need for protection from enemies (Harris 1996; Diamond 2002). This new lifestyle expanded raplidly towards Europe, and reached the Southeastern parts around 8 500 years ago, Northern Europe around 7 000 years ago and, with a delay of about one thousand years, Scandinavia around 6 200 years ago. For nearly a century, the major question within archaelogy has been how this Neolithic package spread. Although many archaeologists argue for a diffusion of ideas from Neolithic areas to adjacent Mesolithic societies (Barker 1985; Dennell 1985; Tilley 1994), others argue that agriculture was brought to Europe with immigrating farmers that replaced the indigenous Mesolithic hunter-gatherers (Childe 1925; Renfrew 1987). Ac- cording to some, the process was much more complex, and that a mixture of the two above hypotheses are probably more accurate (Zvelebil 1986). The question of the settlement of Europe has further been adressed through mtDNA analyses on modern European and Near East populations. Initially, principal component (PC) values of allele frequency variation from high-resolution restriction fragment length polymorphism (RFLP) markers were used to create synthetic maps (Cavalli-Sforza 1994). The first PC was estimated to account for about 25% of the total variation seen and it showed a gradient from southwest (the Near East) to northeast (Europe). This was interpreted as a support for the spread of farming with immigration through demic diffusion. In recent years, mtDNA haplogroups have been more cler- aly defined and phylogeographically resolved through a combination of RFLPs, control region sequences and coding region polymorphisms (Torroni et al. 1996; Richards et al. 1998; Macaulay et al. 1999; Richards et al. 2000; Pereira et al. 2005). The haplogroups that are most common in Europe, such as H, K, T, U and V, are also present in the Near East and probably evolved

15 and migrated towards Europe from there. The variation seen in basal haplogroups have been used to provide timeframes for their dispersal and estimates of the relative genetic contribution of differerent prehistoric migration events. It has been shown that only 20% of the modern European gene pool account for migration events during the Neolithic and an even smaller fraction (<10%) seem to derive from the initial colonisation of Europe by anatomically modern humans (Richards et al. 2000; Richards et al. 2002). In- stead the major part of European lineages (about 75%) seems to have arrived to Europe prior to the late glacial maximum (LGM). There are signs of reduction in diversity due to population contraction at the onset of the LGM and also of re-expansions from ice-free refugias afterwards (Torroni et al. 1998; Richards et al. 2000; Torroni et al. 2001; Tambets et al. 2004; Pereira et al. 2005). Analyses of the non-recombining part of the Y chromosome generally show similar patterns to the mtDNA results. There are several haplogroups that are common in Europe, like I, J, K, N, R1a, R1b, and E3b (Zerjal et al. 1997; Hurles et al. 1999; Underhill et al. 2000). Many of these display a southwestern to northeastern cline and they seem to have arisen in Europe before the LGM, become isolated in refugias followed by an expansion phase after the Ice Age (Semino et al. 2000; Rootsi et al. 2004). A smaller fraction of the haplogroups, again approximately 20%, seems to derive from immigrating Neolithic farmers. The phylogeographic patterns displayed by Y chromosome SNPs are, however, much stronger compared to that of the mtDNA. This may, at least partly, be explained by differences in mobility between sexes, where females might migrate more frequently compared to males in patrilocal societies (Seielstad et al. 1998). Although the allele frequency clines shown by nuclear DNA polymorphisms are similar to those displayed by the maternal and paternal markers, they indicate a substantially higher (over 50%) Neolithic demic component (Chikhi et al. 1998; Dupan- loup et al. 2004). It has been suggested that part of this clinal pattern may be due to assertainment bias when using highly variable biallelic markers (Currat and Excoffier 2005). The few ancient DNA studies that have targeted the question of the spread of agriculture during the Neolithic have come to somewhat different results. Maternal lineagens have mainly been investigated by amplification of the HVS1 region, additional coding region polymorphisms and subsequent cloning. Haak and collegues (Haak et al. 2005) investigated 24 samples from central Europe that belonged to the Neolithic Linear pottery culture (7 000-7 500 BP). Although eighteen of the samples belonged to haplogroups of typical western Eurasian origin 25% showed different haplotypes of the N1a lineage that is extremely rare today (0.2%). They discarded the possibility that genetic drift had affected the N1a lineage by means of a demographic model and suggested that their results may indicate that although a small group of people (belonging to N1a) carried the farming practices into Europe

16 the larger indigenous Mesolithic societies in surrounding areas adopted that knowledge and with time outnumbered the initial immigrating farmers. In contrast, Sampietro and collegues (Sampietro et al. 2007) conducted phylogeographic analyses of 11 Impressed Ware pottery samples from Spain (5500 BP) and found haplogroups of similar frequencies as modern samples from the region. They suggest that the Neolithic spread of farming included different maternal processes in central Europe compared to the Mediterra- nean region where cultural diffusion had a major impact in the former and demic diffusion in the latter region. Other studies have reported sequence results from few Neolithic samples (Handt et al. 1994; De Benedetto et al. 2000; Rollo et al. 2006) and from even fewer Paleolithic samples (Caramelli et al. 2003). By investigating more samples from Neolithic, Mesolithic and Paleolithic times we may achieve further understanding about the introduction of farming practices in Europe.

The secondary product revolution There has been a debate regarding how the initial farming practices were conducted. According to Sherrat (Sherratt 1981) the archaeological and archaeozoological record indicate that domesticated livestock was first used mainly for the meat, and only later for secondary products such as milk, wool and transport. This critical phase of change around 6 000 years ago was termed “the secondary product revolution”. However, more recent analyses of archaeozoological data and of preserved dairy fat residues from vessels indicate an earlier onset (8 000 to 6 000 years ago) of dairying practices (Bogucki 1984; Copley 2005; Craig 2005). The benefit of milk consumtion as an alternative source of nutrition is evident. However, not all humans have the ability to digest non-processed milk sugar (lactose) during adulthood. When and how the genetic trait of lactase persistence (LP) spread are debated issues. The cultural-historical hypotesis advocate that lactase persistence alleles were initially rare and rose rapidly in frequency due to cultural- driven selection associated with the introduction of darying practices during the early Neolithic (Simoons 1970; McCracken 1971; Kretchmer 1972; Beja-Pereira et al. 2003). An opposing theory, the reverse cause hypothesis, suggests that dairying was adopted in certain populations that already pos- sessed the ability to digest raw milk (Bayless 1971). The ability to digest milk sugar depends on the activity of the lactase- phlorizin hydrolase enzyme in adulthood. The activity is positively corre- lated with mutations that are inherited in a dominant fashion and that are associated with the lactase gene (LCT) (Enattah et al. 2002; Poulter et al. 2003). This trait varies remarkably throughout the world and is present in high frequencies in Northern Europeans (up to 90%) and in African and Middle Eastern pastoralist populations while virtually absent in East Asia (Bersaglieri et al. 2004). Recent studies have shown that the causative SNPs

17 vary between European and African populations, and thereby suggest a con- gruent evloution of the trait in Africa and Eruope (Tishkoff et al. 2007). The European lactase persistence allele seems to have arisen between 5 000 and 10 000 years ago and extensive linkage disequilibrium (LD) surrounding the allele suggest it has been subjected to recent positive selection (Bersaglieri et al. 2004). Ancient DNA analysis has the potential to tell us when and among which populations the lactase persistence alleles arose. In an attempt to answer these questions, Burger and collegues (Burger et al. 2007) investigated lactase persistence alleles in eight Neolithic, one Mesolithic and one Medieval individual from five different localities dispersed over Europe. Only in the medieval sample did they observe the lactase persistence allele. Since lactase persistence seems to have been rare in early European farmers they conclude that the high frequencies of LP seen in the region today must have arisen through cultural driven selection after the introduction of dairying practices. Combining studies on several additional ancient human samples from Meso- lithic and Neolithic localities with analyses of milk residues on pottery and with studies on milk selection in cattle might help to clarify the picture in in the future.

Animal genetics An alternative way to trace human history, at least during the last 10 000 years, is by studying the domesticates. The domestication was an anthropo- genic process that brought animals into human societies. Therefore these animals provide as much information on human prehistory as they do on their own. When humans took control over the reproduction, nutritional supply and living space of these wild animals and plants they ultimately altered their physiology and behaviour (Clutton-Brock 1999). Eventually morphological changes became measurable between the domesticates and their pro- genitors, but the process demanded some time and it is difficult to distinguish the earliest domesticates in the archaeological record from their wild ancestors (Bökönyi 1974). For example, body size reduction, shortening of facial bones and size reduction of teeth and brain are common features among domesticates (Clutton-Brock 1999). Many of the wild species that became successfully domesticated had behavioural characteristics that made them suitable to work with (like being capable to breed in captivity and hav- ing dietary habits that could be controlled and supplied by humans) (Diamond 2002). The archaeological record indicates that Paleolithic hunter-gatherer first took control over wolves, and in time modified them into domestic dogs. Recent genetic studies further suggest that this initial domestication occurred in East Asia (see below). However, the major change in human society in

18 our part of the world started around 10 000 years ago in the Fertile Crescent or the Near East. Here, remains from the majority of domesticates that later arrived to Europe are found (Clutton-Brock 1999; Lev-Yadun et al. 2000). MtDNA variation in modern cattle, pigs, sheep and goats generally indicate that domestication of these species were geographically restricted within or close to the Fertile Crescent (although usually occurred in more than one area) (Luikart et al. 2001; Troy et al. 2001; Larson et al. 2005; Pedrosa et al. 2005). Other genetic markers often indicate a more complex picture (Vilà et al. 2005). Ancient DNA studies on Y-chromosomal markers in cattle do, for instance, indicate male introgression and backcrossing with local wild pro- genitors (which is not indicated by mtDNA analyses on ancient cattle and auroxen) (Götherström et al. 2005; Edwards et al. 2007). It has also been shown that horses differ from other domesticates. Both modern and ancient horses display a high level of mitochondrial variation indicating that several domestication events took place over a long period of time (Vilà et al. 2001; Jansen et al. 2002). A strong sex bias in the process of domestication has further been shown in horses as displayed by low genetic diversity on the Y- chromosome (Lindgren et al. 2004). Several attempts have also been made to identify the trading or migration routes humans followed when domesticates were dispersed. A Mediteranean trading route has for instance been identified for sheep, and Bronze Age cattle display haplotypes indicating trad- ingbetween Africa and Iberia (Anderung et al. 2005; Pereira et al. 2006). Here I use the first animal to become domesticated, the dog (Canis familiaris), as a more detailed example on how genetic analyses combined with archaeological data may reveal when, where and how domestication occurred. Some of the earliest morphologically distinct domestic dogs are 13 to 17 000 year old finds from Russia (Sablin 2002) and 10 000 and 9 000 year old remains from Central Europe and Scandinavia respectively (Benecke 1987). The close bond between humans and dogs is manifested in 10 000-12 000 year old human burials containing dogs (Davis 1978) and even in separate dog burials in Scandinavia (Jonsson 1988). Whether the initial domestication was an active process where humans started taming young wolves from the wild or a more passive process where the initial contact consisted of wolves coming in close proximity to human settlements when scavenging for food is not clear. It is, however, probable that domestic dogs at quite an early stage were selected for different purposes such as hearding, hunting or guarding (Clutton-Brock 1999). Genetic studies on modern dogs and wolves from around the world have recently given several clues that complement the archaeological record regarding when, where, how and from what ancestor the domestic dog derives. It has, for instance, been shown that all dogs derive from grey wolfs (Canis lupus) (Vilà et al. 1997; Savolainen et al. 2002; Angleby and Savolainen 2005) and not from a variety of wolfs or jackals as previously hypothesized. Phylogenetic trees based on substitutions and deletions in the mitochondrial

19 control region in a variety of modern dogs and wolves display between four and six distinct clades. These clades may represent separate domestication events involving at least four founding females. After the initial domestication there has probably been recurrent admixture with wild wolves, as indicated by different wolf haplotypes dispersed among the dog sequences. The male lineage seems quite similar to that of females and (using only 10 dog samples) five domestication events have been recognized with Y- chromosomal biallelic single nucleotide polymorphism (SNP) markers (Natanaelsson et al. 2006). The high genetic diversity of MHC, resulting from balancing selection acting to maintain a well functioning immune system, also indicate that multiple (probably over 20) domestication events have taken place (Vilà et al. 2005). It has further been suggested that the domestication started in East Asia (Savolainen et al. 2002). Modern dogs in this area show a significantly higher genetic diversity in the mitochondrial control region compared to dogs in other regions. They also display a large number of unique sequences, indicating that enough time has passed to accumulate unique genetic signatures, and that this population therefore is of ancient origin. At least the three largest of the previously described mitochondrial clades fall into this group. Depending on whether a monophyletic or a poly- phyletic origin is assumed for the major maternal clade MRCA calculation results suggests a domestication time of 10 0000 to 15 000 years ago (Vilà et al. 1997; Savolainen et al. 2002). The general agreement is that domestication at least predates the earliest fossils of domestic dogs. Ancient DNA analyses also indicate that domestic dogs existed already before 12 000-14 000 years ago (Leonard et al. 2002). Mitochondrial control region sequences from 37 American and 11 Alaskan dogs predating Euro- pean arrival to the New World fall into clade I or IV and do not show any similarities with native wolf sequences. Thus, humans brought domestic dogs with them when they colonised the Americas. The logic of this argu- ment suggests that domestic dogs must be at least as old as the colonization of the New World, and as this occurred some 12-14 000 years ago, domestic dogs are at least that old. Domestic linages have also been found in 14 600 to 3 000 year old Italian remains (Verginelli et al. 2005).

20 Methodological aspects of ancient DNA anlayses

Ancient DNA differs from modern DNA in several aspects. Only small amounts of DNA (if any) do survive over thousands of years and, over time, bases become altered due to molecular damage. The small DNA quantities may further be hard to separate from more abundant, modern sources of DNA. Traditionally extracted DNA is PCR amplified and cloned into plasmid vectors before sequenced. The process of retrieving ancient DNA has been both time-consuming and expensive. Today, science offers new techniques that not only help us retrieve more data but also help us charachterise our samples in more detail. An understanding of the nature of ancient DNA and, of the possibilities offered by different analythical techniques, will help us to determine how to best analyse precious ancient remains in order to retrieve reliable results.

The nature of ancient DNA

DNA survival The most common type of samples that are preserved in archaeological records, and that may contain ancient DNA, is bones and teeth. Generally, the protection of teeth by the enamel surface, renders them the better option for ancient DNA retrieval. Compact bones such as femur and tibia might also be quite well preserved although preservation/DNA abundance can vary locally in a bone sample. Other sources for ancient DNA might be materials such as coprolites, hair and leather (Poinar et al. 2003; Vuissoz 2004; Gilbert et al. 2004b), or even charred or dessicated plant remains (Allaby et al. 1999). Although ancient DNA analyses have been conducted on mummified human remains (Pääbo 1985; Zink and Nerlich 2003), there are still some controversy regarding the credibility of such results especially since they have been found in hot environments (Marota et al. 2002). The majority of pre-historic sites, does however, not contain any of the above-mentioned types of remains. Even when this is the case, plant and animal DNA have

21 been retrieved from permafrost and temperate sediments (Willerslev et al. 2003). How well DNA survives in these types of materials does not necessarily correlate with the age of the sample (Pääbo 1989; Höss et al. 1996; Poinar et al. 1996). Instead, the surrounding environment seems to be an important factor, as shown by correlations between archaeological sites and long-term DNA survival. Within-site variability due to different micro-environments is also seen (Hagelberg et al. 1991). One of the initial environmental factors affecting DNA survival post mortem is the extent of microbial activity (Pääbo 1989; Burger et al. 1999) and rapid desiccation is usually favourable for DNA survival. Favorable soil properties are high salt concentrations, neutral or slightly alkaline pH, low levels of humic and fulvic acids, and a low water content (Hagelberg et al. 1991; Lindahl 1993; Tuross 1994). Correlations between DNA survival and environmental factors such as high aridity and a low level of exposure to radiation have also been seen. One of the key parameters, however, is the thermal history of the sample and it was early recognized that low mean annual temperatures are favorable (Smith et al. 2001; Smith et al. 2003). The oldest authenticated ancient DNA results are mainly from permafrost or cave environments. Cores from Greenland have yielded genetic data from plants and insects that are 450 000 to 800 000 years old (Willerslev et al. 2007), and bear fossils that are at least 400 000 years old from the Atapuerca cave system in Spain have yielded genetic data as well (Valdiosera et al. 2006). These results exceed by far the predicted maximal DNA survival age of about 10 000 years in temperate regions and 100 000 years in colder environments. The quantity and quality of DNA in ancient specimens is also influenced by post-excavation treatments (Burger et al. 1999; Pruvost et al. 2007). Samples stored for many years in museum collections, usually in room temperature, show a decrease in amplification success rate compared to freshly excavated samples. This may be due to the elevated temperatures during storage and the washing procedures that reduce the pH and salt content of the sample. Long-term storage of ancient specimens would benefit from storage in colder environments, preferably in freezers. There are mainly two approaches used for pushing the limit of retrieving ancient DNA. It is very common to target multi-copy DNA, such as mitochondrial and chloroplast DNA, since they are more likely to be preserved compared to nuclear DNA. The other approach takes in to account the fact that the average fragment size of ancient DNA is small (Pääbo 1989; Noo- nan et al. 2006). Therefore, by targeting small fragments it is possible to extend the temporal range further (Valdiosera et al. 2006).

22 DNA damage DNA molecules from ancient specimens are usually damaged. There are several processes causing the damage and ultimately they lead to reduction in the amount of retrievable DNA, reduction in the size of retrievable sequences as well as modification of bases. Understanding these processes and their effects on ancient DNA helps us to use proper assays (such as amplification of short overlapping fragments and cloning and sequencing of multiple clones) in order to retrieve (any) DNA and to avoid erroneous interpretation of sequence data. When an organism dies the enzymatic repair mechanism of the cell ceases to function. Initially, the DNA becomes exposed to nucleases in the cell and to degradation caused by bacteria, fungi and insects. This leads to a reduction of the total DNA and to DNA strand breaks that will accumulate over time. Chemical processes such as hydrolysis and oxidation further damage the DNA molecules (Pääbo 1989). Hydrolytic cleavage of phosphodiester and glycosidic bonds causes single stranded nicks. The latter form is also known as depurination, which upon occurrence form baseless or apurinic sites (AP sites). Ancient DNA has also been shown to contain oxidative residues that are induced by free radicals that are created for instance by ioniz- ing radiation (Poinar et al. 1996). These residues are mainly caused by hy- dantoin derivates of pyrimidines and they probably (block) hinder strand elongation during PCR. Together, DNA fragmentation and amplification blocking limit the maximum length of ancient DNA sequences that can be retrieved, sizes usually ranging from less than one hundred to a few hundred base pairs. These problems can, however, be overcome by targeting short amplicons and by targeting overlapping fragments. Chemical degradation also produces altered bases that can lead to nucleotide misincorporation during amplification. This form of damage is, in a way more severe, since it may result in errors in the DNA sequence. Such miscoding lesions may be due to hydrolytic deamination of cytosine to uracil, 5- metyl-cytosine to tymine, adenine to hypoxantine and guanine to xanthine. The most common form of lesions observed in ancient DNA datasets is transitions (substitution of a purine for a purine or a pyrimidine for a pyrimidine). These have been referred to as Type 1 (A G/ T C) and Type 2 (C T/ G A) damages (Hansen et al. 2001). There have been reports suggesting different pattern among these damage derived miscoding lesions, where some have argued for an almost complete dominance of Type 2 damages and others for a substantial presence of Type 1 substitutions in addition to the Type 2 damages. The most recent consensus is, however, that Type 2 substitutions are predominant, with the majority caused by deamination of C to T and with a smaller fraction of G to A substitutions caused by an unknown lesion (perhaps deamination) (Stiller et al. 2006; Gilbert et al. 2007). There are still debates regarding the interpretation of the observed Type 1

23 substitutions. Some argue that they may be caused by template switching during amplification (“juping PCR”), by modern DNA contamination or by DNA polymerase enzyme errors during amplification (Pääbo 04). Others hypothesize that fragmentation during the preparational steps at the positions where Type 1 substitutions occur, or true damage that varies in individual ancient samples may be causative. It is important to deal with possible nucleotide misincorporation regard- less of if it is cause by DNA polymerase errors or by damage. Modified bases are usually observed when PCR products from ancient specimens are cloned and sequenced. If an erroneous base is amplified during late stages in the PCR, the cloned sequences will show singleton substitutions, which are usually easy to distinguish from the over all consensus sequence. If, however, it happens during early stages of the PCR, a majority of the cloned sequences from one amplicon may show consistent substitutions. This type of error can be reduced by repeated amplifications followed by cloning and sequencing (Hofreiter et al. 2001a). If a substitution truly stems from the ancient template DNA, and not from erroneous bases, it would be consis- tently seen in a majority of the clones and in clones from different amplifications. Errors caused by the deaminated form of cytosine (uracil) can also be minimized by adding uracil-N-glycosylase (an enzyme that removes uracil) to the ancient DNA extract prior to amplification (Pääbo 1989; Hofreiter et al. 2001a). Another reason, making it extremely important to interpret the pattern of nucleotide misincorporation in several different amplicons, is that some (often phylogenetically informative) nucleotide positions seem to be specifically error-prone. Some even argue that ancient DNA extracts may contain some unknown mutagen that induces mutations in modern DNA (Pusch and Bachmann 2004).

Contamination Only a fraction of prehistoric specimens actually contain ancient template molecules (Höss et al. 1996) and it is usually only possible to amplify small quantities of quite damaged DNA from these samples. Therefore, even minute amounts of DNA contamination may yield in false positive results. The enormous severity of contamination was recognized quite early (Handt et al. 1994). There are some classical examples of early results from millions of years old dinosaurs, amber entombed insects and magnolia leafs that are now believed to derive from contamination, either due to human DNA contamination or due to the fact that it has been impossible to replicate the results (Pääbo and Hofreiter 2004). A contaminant sequence may be introduced at several stages. A human specimen can be contaminated during deposition or burial, during the excavation, during storage at museums or during handling of the specimen when conducting morphological investigations. Exogenous

24 DNA can further be introduced during the extraction or amplification procedures (Brown 1992). Contamination occurring during the laboratory procedures may be detected when negative extraction and amplification controls (test tubes without bone powder) yield amplifiable sequences. This is commonly seen when analyzing both human (Izagirre and de la Rua 1999; Yang et al. 2003) and non-human samples (Leonard 2006). Contamination in negative controls is often derived from few template molecules and is therefore usually seen sporadically. Sometimes zero, and often about a few up to approximately 10% of the negative controls are usually reported as contaminated. It has also been proposed that contamination may be abundant even when no sequences are obtained from the negative controls. This is explained by a “carrier effect” in which low concentrations of contaminant DNA is thought to be PCR unamplifiable through adherence to plastic ware and equipment (Cooper 1992; Handt et al. 1994; Leonard 2006). If this is the case, the contaminant will become amplified in the presence of DNA extracts containing the actual samples, without being detected in the controls. This phenomenon can also be seen if the contamination derives from the specimen and it has been observed at several occasions in both human (Handt et al. 1996; Krings et al. 1997; Kolman and Tuross 2000; Vernesi et al. 2004) and non-human specimens (Handt et al. 1994; Richards 1995). Direct handling and washing seem to contaminate both sample surfaces and to some extent, depending on preservation and porosity, also the interior of bones and teeth (Salamon et al. 2005; Gilbert 2005b; Sampietro et al. 2006). The risk of getting erroneous data through contamination varies depending on the abundance of the contaminant species in the environment surrounding the sample or the reagents used (Gilbert et al. 2005c). Analysing ancient human specimens pose the greatest threat. Not only because it is humans that excavate the samples, perform the DNA extractions and produce the laboratory reagents used, but also because modern contaminants may share haplotypes or alleles with the ancient material, making it difficult to separate the contamination from the authentic sequences (Richards 1995; Abbott 2003; Sampietro et al. 2006). Over the years several standard precautions (discussed in the Authenticity section) have been proposed and utilized to authenticaticate ancient DNA results (Cooper and Poinar 2000). If these precautions suffice for producing reliable data from ancient human samples is a matter still under debate (Barbujani and Bertorelle 2003).

Authenticity Errors in ancient DNA sequences can be caused by a variety of factors such as nucleotide misincorporation due to damage and DNA polymerase errors, jumping PCR and by nuclear insertions of mitochondrial sequences (so called numts). Due to the low amount of template molecules in ancient

25 DNA extracts (Pääbo 1989; Handt et al. 1996) modern DNA contamination poses the biggest threat, especially when working with prehistoric human samples. There are several strategies used to autheticate ancient DNA results (Handt et al. 1996; Poinar et al. 1996; Krings et al. 1997; Cooper and Poinar 2000). In this section, I will start by reviewing the strategies employed to avoid erranous results and continue with strategies used to detect erroneous results.

Avoiding erronoeous results To avoid laboratory based contamination and back contamination from previous PCR products ancient DNA research is usually conducted in a pre- PCR laboratory that is physically isolated from areas in which modern DNA or PCR products are analysed (Lindahl 1993). It is also common to use a workflow in which personnel only move from ancient to modern laboratories (Willerslev et al. 2004). The workstations and disposable labware are regu- larly cleaned with oxidants such as sodium hypoclorite and/or UV- irradiation and laboratory reagents (at least inorganic ones) may be decontaminated with UV (Pääbo and Hofreiter 2004). Disposable protective cloth- ing, facial masks, disposable gloves and shoe covers are used to protect the sample from the laboratory staff. The laboratory may further be epuipped with high preassure or whith hepa-filtered hoods to make sure no DNA enter the room. There are several methods applied to remove or avoid sample contamination. The most common methods focus on decontaminating the outer layer of a sample by UV-irradiation, surface removal or surface cleaning (with oxidants or acids) followed by powderisation only of the interior of the sample (Richards 1995; Kolman and Tuross 2000; Caramelli et al. 2003; Vernesi et al. 2004). Recent studies have further shown that bleach pretreatment of powderised samples is highly efficient for removal of contaminant DNA (Kemp and Smith 2005; Salamon et al. 2005). The advantage of the latter method is that contaminant DNA that has permeated into the sample may be removed. Another option is to avoid sample contamination by removing samples from the archaeological site when they are still embedded in soil and to excavate them in a controlled laboratory environment. PCR carry-over contamination may be controlled with the UNG-system, where DNA is amplified with uracil instead of thymin, and PCR reactions are pre-treated with uracil before amplification. This way all PCR products that may contaminate a reaction are destroyed, while the targeted DNA is left intact (Kwok and Higuchi 1989; Hofreiter et al. 2001a).

Detecting erroneous results As mentioned earlier, negative controls (samples to which no material is added) are extracted and amplified in paralell with extracts from ancient samples to monitor contamination that may appear during the laboratory

26 procedure from reagents and solutions (Pääbo 1989). Another option, although not as commonly employed, is to use faunal remains associated with the human samples under investigation as negative controls (Cooper and Poinar 2000). In this way false negative results due to a possible carrier effect may be avoided. The elevated risk for contamination in ancient material has also lead to a complex replication system. Reproducibility is often tested by conducting several independent extractions (preferably from different parts of the same specimen) and amplifications. It has also been suggested that at least 10% of the investigated samples or all key results should be subjected to reproduction at a second laboratory to control for intra-laboratory contamination (Willerslev et al. 2004). There are no set rules as to how many replications are needed to produce reliable data although the International Society for Forensic Genetics recommend analysis from human mtDNA from degraded samples to be analysed twice on separate occasions and to reject a sample if a negative control yields the same sequence as the sample (Bär et al. 2000). When investigating SNPs Taberlets (Taberlet et al. 1996) mathematical model for reliable genotyping of autosomal loci from extracts with few template molecules provides one strategy. Here it is suggested that two replications for heterozygous loci and seven replications for homozygous loci should be employed to achieve a confidence level of 99% of the result. The probability is based on the assumtion that the allelic dropout is 100%. An alternative would be to actually calculate the allelic dropout frequency, as suggested by Gagneux and colleques (Gagneux et al. 1997). This approach rarely demands more than four replications to reach 95% probability of not missing any heterozygotes (Svensson et al. 2007; Svensson in press). Ancient DNA results should preferably be based on extracts containing at least 1 000 template molecules to minimise the effect of damaged templates (Handt et al. 1994; Krings et al. 1997). The template number may be investigated through competitive PCR or through real-time PCR (see DNA quantification section for more details). A large number of templates do, however, not necessarily imply that the sequences are endogenous to the sample. Indi- rect evidence that endogenous DNA have survived may be provided through quantifying animal DNA in associated animal remains (Cooper and Poinar 2000) and by investigating the biochemical preservation of other macro- molecules such as amino acids (Poinar et al. 1996). Further support may be given if PCR products show an inverse relationship between amplicon length and amplification yield (Cooper and Poinar 2000). This test of “appropriate molecular behaviour” is based on the assumption that DNA degrades as a loose function of temperature and time (Smith et al. 2001). Since the true endogenous DNA is much older than contaminant DNA the relative ratio between short and long amplicons should be higher for the endogenous DNA.

27 Through molecular cloning it may be possible to determine the extent of contamination, post mortem damage and PCR artefacts that is present within a sample (Krings et al. 1997). Amplicons displaying several highly abundant sequence types are usually discarded as the sample may have been contaminated by several sources. If one sequence type is in majority and remaining sequence variation display shattered singleton substitutions that may be the result of degradation of the original sequence then it looks more authentic. Some even suggest that an exess of C to T and G to A transitions in cloned sequences from an ancient amplicon may be a sign that ancient template DNA is present (Rudbeck et al. 2005; Topf et al. 2006). A statistical approach for the identification of endogenous template DNA exploiting the damage pattern has further been proposed (Helgason et al. 2007). However, caution is needed since contaminant sequences older than ten years may display damage levels similar to those of truly ancient sequences (Sampietro et al. 2006). It is also recommended to amplify several overlapping fragments to confirm the authenticity of sequence variation and to be able to detect numts. The variation in the sequences should also make phylogenetic sense and should not consist of artificial combinations of several phylogenetically unrelated polymorphisms (Bandelt 2004). Since there are many factors influencing the reliability of ancient DNA results Gilbert and collegues (Gilbert et al. 2005c) advocate that thorough information about the samples and the processes they are exposed to ought to be presented in more detail in future research papers to enable the reader to form an opinion regarding the authenticity of the results presented.

Technical aspects DNA quantification To know the amount of DNA present in an ancient extract helps us to determine the reliability of upcoming sequence results. If too few DNA templates have survived there is, for instance, a risk that damaged templates may take over the amplification reaction and become in majority by the last PCR cycle. This is also true for polymerase errors occurring at early cycles of amplification. The inverse relationship between amplification efficiency and DNA fragment length, as often used to authenticate ancient DNA results, may further be investigated using DNA quantification. Mainly two methods have been applied in the ancient DNA field; competitive PCR or real-time PCR. The earliest technique for quantification of template molecules was competitive PCR (Handt et al. 1994). This is a relative quantification technique in which the unknown target PCR product is compared with competitor DNA of known amounts. The competitor is usually constructed by insertion

28 of a modified target sequence into vector and the concentration is measured by optical density. A dilution series of the competitor is amplified together with set amount of the unknown extract and the intensity between the different types of bands are compared on an ethidium bromide stained agarose gel. This technique has been used to investigate mitochondrial DNA template amounts in pre-historic human samples (Handt et al. 1994; Handt et al. 1996; De Benedetto et al. 2000), in Neandertal specimens (Krings et al. 1997) and in extinct moa specimens (Cooper et al. 2001). However, measurements based on end-point values are quite unreliable since inhibitors and reagent limitations may affect the final amount of the product. The final amounts are also affected by differences in PCR efficiency as the two types of target molecules (actual sample and competitor) differ in length. Real-time PCR is a simultaneous amplification and quantification reaction where, instead of an end-point measure, the initial copy number of a target sequence is determined (Arya et al. 2005). Fluorescent PCR products are measured with a CCD camera as they accumulate during each amplification cycle. Since detection take place during the exponential growth phase of the reaction, the amounts of fluorescence from the PCR product is directly proportional to the amounts of starting templates. The DNA-extracts with unknown template numbers are amplified in parallel with a serial dilution of the target sequence with known amounts of templates. This standard DNA is usually constructed from a synthetic single stranded oligo or from a plasmid containing the template sequence. A standard curve is created by plotting the log of initial copy numbers against the cycle threshold value (CT, the point at where the curve passes a set threshold). The standard curve is then used to determine the initial copy number of the unknown samples (Higuchi et al. 1993). There are various chemistries used for real-time amplicon detection (Araya et al. 2005) although two are more commonly used in ancient DNA research. TaqMan/ is a sequence specific probe based chemistry that quan- tifies templates of approximately 100 bp length. This dually labeled probe hybridizes to the single stranded target after denaturation. The 5´ reporter dye (FAM, TET or VIC) is suppressed by the 3´ quencher (TAMRA or non- flourescent labels). The 5´-3´exonuclease activity of the Taq DNA polymerase cleaves the probe during elongation. As the quencher is separated from the reporter a fluorescent signal is released. This process occurs at each cycle and fluorescence measured is proportional to the amount of accumu- lated PCR products. A cheaper alternative to TaqMan is the SYBR Green chemistry. SYBR Green I is a DNA-binding dye that binds into the minor groove of non-specific double stranded DNA at the end of the elongation phase of each amplification cycle (Morrison et al. 1998). A strong fluorescent signal is emitted upon binding and the signal decrease when DNA is denatured. Since the dye is not primer-specific it binds to any double stranded molecules in the reaction, including primer dimers and non-specific

29 products. Therefore this chemistry has a stricter requirement on primer de- sign. It is possible to control for non-specific products and dimers by plotting the fluorescence as a function of temperature (Ririe et al. 1997). If more than one melting peak is present it is an indication that more than one amplicon have been measured. In recent years, real-time quantification has been applied to ancient DNA studies in order to determine the reliability of sequence results. It is highly suitable for these types of studies due to the high sensitivity where templates may be detected down to single molecules. The main advantages of using SYBR Green to quantify ancient samples are that short fragments can be analysed (a probe does not have to fit between the primers) and that the ex- act sequence between the primers does not need to be known before the analysis. It is not possible, however, to quantify several templates in a multi- plex reaction. This method has been used to quantify Pleistocene and Holo- cene bovine DNA (Pruvost 2004). The probe based chemistry have, for instance, been used to quantify mitochondrial DNA from late Pleistocene ground sloth coprolites (Poinar et al. 2003), from human mitochondrial or nuclear DNA in forensic casework (Andreasson et al. 2002b; Andréasson et al. 2006b) and for simultaneous quantification and sex determination in ancient or degraded human samples (Andréasson and Allen 2003; Alonso et al. 2004; Niederstätter 2007).

DNA sequencing The main DNA sequence determination method has been based on Sanger sequencing (Sanger et al. 1977). Although robust, there are limitations that are especially evident when the method is used for ancient DNA research. First, the read quality is usually low at least for the 20 first nucleotides read. Second, the method is optimized for long read lengths and sequencing fragments below 100 bp are not recommended. It may therefore be difficult to apply the technique on the short amplicons (usually deriving from direct amplification or from cloned PCR products) in ancient DNA sequences. In recent years, new sequencing techniques have been developed that are highly applicable in ancient DNA research.

Pyrosequencing Pyrosequencing/ is a sequencing-by-synthesis technology where DNA synthesis is monitored through a cascade of enzymatic reactions that generates visible light that is proportional to the number of incorporated nucleotides (Ronaghi et al. 1996; Ronaghi 2001). This technique is highly suitable for ancient DNA sequences since it allows short (up to 100 bp) sequences to be read and since high quality reads are obtained even from the nucleotides situated directly after the sequencing primer.

30 Single stranded DNA is prepared for pyrosequencing through amplification of the template using one unmodified and one biotinylated primer. The biotinylated strand is selected through immobilization on to streptavidin coated beads followed by denaturation and washing. A sequencing primer is hybridized on to the single stranded amplicon. The sequencing templates are placed in the pyrosequencing machine together with the deoxynucleotide triphosphates (dNTPs), enzymes (DNA polymerase, ATP sulfurylase, luciferase and apyrase) and substrates (adenosine 5´ phosphosulfate and luciferin) required for the reaction. The nucleotides are added to the reaction one at a time. When a nucleotide is complementary to the single stranded template it is incorporated by the DNA polymerase. The polymerase release pyrophosphate and ATP is generated and further converted to visible light through enzymatic reactions. The light is captured with a CCD camera and the signal will generate a peak in a pyrogram that is proportional to the number of incorporated nucleotides. Before the addition of the next of the four nucleotides unincorporated nucleotides and excessive ATP is degraded by apyrase. Pyrosequencing has been used for SNP typing of nuclear DNA from medieval cattle (Svensson et al. 2007), mtDNA from Bronze Age cattle (Anderung et al. 2005) and of Neolithic aurochsen Y chromosomal DNA (Götherström et al. 2005). In the forensic field Pyrosequencing is used for SNP detection in human identification cases (Andréasson et al. 2002a) and in molecular autopsies (Jin et al. 2005). Further, pyrosequencing is used to determine species affiliations in unknown samples (Balitzki-Korte et al. 2005; Karlsson in press). There is also an allele quantification mode within the pyrosequencing software that is highly suitable for determining relative amounts of alleles that may be caused by sample mixture or contamination (Andréasson et al. 2006a).

Large scale sequencing With the recent arrival of the Genome Sequence GS201 and FLX1 DNA Sequencing Systems (Roche/454 Life Sciences), the pyrosequencing technology was taken a step further. It is now possible to sequence up to 25 million base pairs in one four-hour run (Margulies et al. 2005). This technology is highly suitable for ancient DNA analyses since the read length of approximately 100-300 base pairs are within the expected range of preserved DNA from most fossil remains (Pääbo 1989). The amount of sequences generated also allows the recovery of endogenous DNA from the fossils even when a large proportion of the generated sequences are of exogenous origin (Höss et al. 1996; Noonan et al. 2005).

Figure 1. Genome Sequencer GS20 and FLX A) Genomic DNA is isolated, fragmented, ligated to adaptors and separated into single strands. B) and C) The fragments are bound to beads under conditions that favour one fragment per bead. An emulsion-based amplification occurs within each droplet. D) The DNA strands are denatured and beads with single stranded clones are deposited into wells on a slide. E) Smaller beads with enzymes required for pyrosequencing are added. F) The clonal fragments are sequenced using pyrosequencing. Courtesy of 454 Life Sci- ences.

This technique involves the generation of libraries through random fragmentation of isolated DNA (Margulies et al. 2005). The DNA is blunt end ligated to common biotinylated primers (so called adaptors, containing region for amplification and sequencing primer and a key sequence) and exposed to limited dilution. After denaturation, the single stranded sequences are immobilized on to Streptavidin coated beads. Each bead is captured in an oil droplet containing the PCR reaction mix. This emulsion amplification is carried out in a clonal fashion since the limited dilution should produce approximately one template per bead. After breaking the emulsion, the beads are transferred on to small reaction wells (approximately one bead per well)

32 on a fibre optic slide. Even smaller beads, containing pyrosequencing enzymes, are deposited in each well and simultaneous sequencing by synthesis is carried out. The light emitted during the reaction is detected with a CCD camera. The signal intensity is further normalized and the quality of each base read is estimated when the sequences are processed. When the technique is applied in ancient DNA research, the degraded state of the DNA renders it unnecessary to randomly fragment the DNA library. Instead, libraries are often constructed from total DNA extracts or from extracts that has been cloned and amplified with vector primers prior to GS20 sequencing (Noonan et al. 2005; Green et al. 2006; Noonan et al. 2006; Poinar et al. 2006; Stiller et al. 2006). This way large amounts of genome-wide random fragments have been retrieved from single samples. There have also been several suggestions on how to use the sequencing power of the genome sequencer platforms on multiple samples that requires cloning. One way is to apply different samples on to different sections of the picotitre plate (Thomas et al. 2006). This approach is at the moment limited since there are only between eight to sixteen different sections on the plates. A larger sample set can be processed if the samples are allowed to be pooled together. By using unique coded primers for initial amplification, multiple homologous amplicons can be identified after sequencing through this unique code (Binladen et al. 2007). Since the genome sequencing technology enables the generation of vast amounts of nuclear as well as mitochondrial sequence data it has been read- ily adopted by the ancient DNA field. By analyzing genome-wide sequences, the divergence between extinct species such as the wolly mammoth (Poinar et al. 2006) or Neandertals (Green et al. 2006; Noonan et al. 2006), have been estimated through comparison with modern relatives. It has also been possible to investigate the process of damage derived nucleotide misincorporation in more detail since the single stranded template can be deduced (Stiller et al. 2006). The major draw back of the method is the inability to replicate a result. The aim should therefore be to get as high sequence cover- age as possible to minimize the risk of interpreting sequence errors as authentic results. Another next-generation solution for large-scale sequencing is based on the Solexa/ Sequencing Technology and the Illumina Genome Analyzer (www.solexa.com). As in the GS20 and FLX technology, single DNA molecules are prepared by random fragmentation and ligation to adaptors, followed by clonal amplification and high throughput sequencing by synthesis. There are, however, several differences between the systems. For instance, the number of base pairs generated in one Solexa run is higher (one billion bp) although the sequence reads are shorter (up to 50 bp). A solid-phase amplification is employed when the sequencing templates have been immobilized on to a flow cell surface. The fluorescently labeled modified nucleotides (A, C, T and G, with reverse termination property) are added simulta-

33 neously in each cycle of the sequencing and the base calling is given confidence scores by the software. Although several Solexa-based ancient DNA studies are underway, none have been published to date.

34 Research aims

1. To investigate the nature and extent of human DNA contamination in bones and teeth from museum specimens. 2. To find methods that will yield reliable DNA data from ancient human remains. 3. To use these methods to investigate genetic relationship between different populations living in Neolithic Scandinavia.

35 Investigations

Paper I: Extensive human DNA contamination in extracts from ancient dog bones and teeth Ancient DNA (aDNA) sequences, especially those of human origin, are no- toriously difficult to analyse due to molecular damage and exogenous DNA contamination (Richards 1995; Handt et al. 1996; Kolman and Tuross 2000). Because of this several guidelines have been developed for authentication of aDNA results (Cooper and Poinar 2000). These include for example the use of isolated aDNA facilities, parallel processing of multiple negative controls with actual samples, quantification of target DNA and sequencing of cloned amplicons. However, results authenticated using these guidelines are still questioned especially since modern contaminants may share haplotypes with the ancient material (Abbott 2003). It has further been shown that ancient samples may be contaminated even when accompanying negative controls are not. Proposed explanations for this is either that a carrier effect where low concentrations of contaminant DNA in negative controls are rendered PCR unamplifiable through adherence to plastic tube walls (Cooper 1992; Handt et al. 1994; Kolman and Tuross 2000) or that the contamination derives from the bone samples rather than from laboratory reagents (Richards 1995; Kolman and Tuross 2000). We examine the nature of contamination by real-time PCR quantification, cloning and sequencing of human mitochondrial DNA (mtDNA) in non- human museum specimens and in negative controls.

Materials and methods To separate between authentic aDNA and modern human contamination we sampled and extracted DNA from 28 ancient and one modern dog (Canis familiaris) from 10 different museum collections. Standard precautions for preventing contamination were followed. Prior to extraction samples were decontaminated through UV-irradiation and only the interior of each sample was powderised. The number of contaminating and authentic sequences in the ancient dog extracts, extraction controls and PCR controls were quantified using real-time PCR. Two distinct assays were created to quantify similar fragment lengths from each control region sequence of either human or dog mtDNA (148 bp and 152 bp respectively). Nine of the contaminant hu-

36 man amplicons were cloned and these, as well as the amplicons from the dog extracts and from extraction and PCR blanks, were sequenced. A statistical approach was used to assess the differences in the amount of human and dog DNA in the dog samples and negative controls. The diversity in the clone- derived contaminant human sequences was compared to a recently published data set of aDNA sequences from human remains (Vernesi et al. 2004). We also tested the possible presence of a carrier effect by extracting and quantifying human and dog standard DNA in different concentrations where dog DNA was used to simulate contamination and human DNA to simulate potential carrier molecules. Analysis of covariance was used to test if the retrieved number of dog molecules was affected by different input amounts of human DNA.

Results and discussion By quantifying human and dog mtDNA we were able to investigate the amount of contamination and authentic ancient mtDNA sequences that were present in ancient dog specimens and negative controls. We found preserved dog sequences based on multiple extractions and amplifications from a majority of the dog samples and in similar amounts as has been accounted for in other quantification studies (Handt et al. 1996; Krings et al. 1997; Stone and Stoneking 1998). However, we also found contaminating human sequences in all DNA extracts from the ancient dog samples as well as in the majority of the negative controls. The proportion of human DNA contamination in our ancient dog specimens greatly exceeded the amount of authentic dog DNA. Further, the amounts of human DNA was significantly higher in the ancient dog extracts than in the negative controls. This suggests that the major source of the contamination derives from pre-laboratory handling of the specimens. This is strongly supported by several studies in which human DNA contamination has been reported in ancient human (Handt et al. 1996; Stone and Stoneking 1998; Kolman and Tuross 2000; Gilbert 2005b) and non-human specimens (Handt et al. 1994; Richards 1995; Hofreiter et al. 2001b) but not in accompanying negative controls. The more extensive contamination in negative controls in this study probably exceeds the more commonly reported low-level contamination in other studies (Izagirre and de la Rua 1999; Yang et al. 2003) due to the high sensitivity of the methods employed. Other methods than the ones we used may be more efficient in removing contamination from samples although sample surface removal, sample encasement and bleach treatment have failed in providing samples free from human contamination (Richards 1995; Kolman and Tuross 2000; Gilbert 2005b). When cloned human amplicons from the ancient dog extracts were sequenced we found between one and six haplotypes per amplicon. The fact that some cloned amplicons yielded only one type of sequence and since the

37 proportion of contamination greatly exceeded the amount of authentic DNA suggests that the consensus of sequenced clones from amplicons would not always yield an authentic sequence. This is further corroborated by the fact that we found no significant differences in nucleotide diversity or number of haplotypes when our data set was compared to a set of data deriving from ancient human samples (Vernesi et al. 2004). Since all dog samples from ten different collections contained human DNA we believe that pre-laboratory sample contamination is a problem of a general nature. When specimens are contaminated replications and reproduc- tions as well as cloning of amplicons may yield reproducible although false results. The present application of the authentication criteria may not suffice for aDNA studies on human remains.

Paper II: More on contamination: the use of asymmetric molecular behaviour to identify authentic ancient human DNA Authenticating DNA results from ancient humans is extremely difficult. As contamination mainly derives from exogenous DNA in the specimens (Richards 1995; Hofreiter et al. 2001b; Paper I) replication in an independent laboratory may fail to identify contaminant sequences. Contaminant sequences may further behave like ancient DNA (aDNA) in amplicon cloning (Sampietro et al. 2006; Paper I). Thus, authentication procedures designed to avoid and detect contaminating DNA are of little help in studies on ancient human remains. It has been suggested that the degree of degradation vary between authentic and contaminant sources of DNA and therefore might be used to authenticate aDNA results (Cooper and Poinar 2000). This has been referred to as “appropriate molecular behaviour”. Since DNA degrades as a loose function of temperature and time (Smith et al. 2001) and contaminant DNA is younger in age than the true endogenous DNA it is to be expected that contaminant DNA would display lower relative levels of short to long DNA fragments compared to authentic endogenous DNA. Here we quantify mitochondrial DNA (mtDNA) fragments of different sizes derived from contaminant human and authentic dog and cattle DNA to measure the quantitative relation between short and long fragments of the two sequence types (contamination vs authentic aDNA). We also treat a subsection of the powderised samples with bleach (Kemp and Smith 2005; Salamon et al. 2005) prior to extraction and investigate the decontamination efficiency in comparison with previously generated data from the same specimens without bleach pre-treatment.

38 Materials and methods DNA was extracted, amplified and real-time quantified (Paper I) from twenty-five ancient dog (Canis familiaris) samples (the majority were from the same specimens as in Paper I) and from extraction and PCR blanks. Prior to extraction the samples were polished with sandpaper and the bone powder was incubated in bleach. Two different sizes of mtDNA control region fragments (approximately 110 bp and 150 bp) were targeted for both contaminant human and authentic ancient dog DNA. In addition, DNA was extracted from 34 medieval cattle (Bos taurus) remains. Conserved primers of a 16S mtDNA region with substitutions specific to either human or cattle was designed in four differently sized versions (70, 124, 178,180 bp) and after amplification the proportion of human and cattle DNA was quantified using pyrosequencing. Statistical measurements were used to compare the amount of contamination in the bleach pre-treated samples with the previous data on untreated samples and to compare the amount of contamination in sample extracts and negative controls. The proportion of short to long fragments in contaminant human and authentic dog or cattle samples was calculated.

Results and discussion Bleach pre-treatment of samples were shown to significantly reduce the amount of contamination. It also reduced the amount of authentic DNA but to a lesser degree. The contamination from the ancient dog extracts were typically below one hundred templates while a large portion of the authentic dog DNA contained several hundreds to a few thousands of templates. Even though heavily reduced the level of contamination in the ancient dog extracts was still significantly higher than in the negative controls. The increase in DNA yield with decreased fragment size was significantly higher for authentic dog or cattle DNA than for contaminant human DNA. This pattern could be detected with two different quantification methods and in two different sets of data. We therefore conclude that that the degradation pattern in DNA extracts from ancient specimens provides a quantitative difference between authentic aDNA and modern contamination.

Paper III: Barking up the wrong tree: modern northern European dogs fail to explain their origin Geographic distribution of genetic diversity in modern domestic animals has often been used to infer centres of domestication. Analyses of mtDNA diversity in modern dogs have revealed four to six mitochondrial haplogroups (hg) and recurrent domestication or backcrosses between domestic dogs and wild wolfs (Vilà et al. 1997). While three hgs are distributed throughout the

39 world one (D) is restricted mainly to Europe and is especially abundant in breeds originating from Scandinavia (Vilà et al. 1997; Savolainen et al. 2002; Angleby and Savolainen 2005; Pires et al. 2006). Similar patterns of fragmented genetic diversity have been used to argue for local domestication in other species (Jansen et al. 2002). However, applying phylogeography to domestic species is more complicated since human activities may alter the genetic composition (Notter 1999) or geographic position (Leonard et al. 2002) of domesticates. We have typed mtDNA from ancient Scandinavian dogs to investigate whether the high frequency of a rare dog haplogroup (D) in northern Europe is evidence for a local domestication event.

Materials and methods Twenty-four ancient dog (Canis familiaris) remains from eight Scandinavian localities (four Medieval and four Neolitihic) were analysed and seven were also replicated at an independent laboratory. Genetic data generated in this study was combined with data generated in previous studies (Paper I; Paper II). Two mtDNA control region fragments were amplified and sequenced to generate 219 bp in total. The polymorphisms were compared with previously published sequences (Savolainen et al. 2002) using a reduced median network (Bandelt et al. 2000) to determine haplogroup affiliations. The haplogroup frequencies within the ancient data set were compared to those in modern dogs (Angleby and Savolainen 2005).

Results and discussion Reproducible sequence data for the full 219 bp sequence was recovered from 18 samples. Seven of these belonged to haplogroup A that also encompass more than 70% of modern dogs around the world (Savolainen et al. 2002) and eleven samples belonged to the now rare haplogroup C. Although six samples only yielded reproducible results from a 107 bp sequence they could still be characterised as belonging to the same haplogroups as the other samples. There was a significant difference in the frequency of haplogroup D between the ancient (0%) and modern Scandinavian (33%) dogs. Our results indicate that hg frequencies have been altered in Scandinavian dogs since their first arrival. We find no evidence for prehistoric canid domestication in Scandinavia. Our data further calls for caution when using modern sequences of domestic animals to interpret the history of domestication. Ge- netic diversity may change rapidly for instance due to intense selective breeding, backcrossing with wild ancestors (Götherström et al. 2005; Vilà et al. 2005; Svensson et al. 2007) and migration of human groups with geneti- cally different domesticates (Leonard et al. 2002). While diversity and branching patterns of mtDNA may be used to study the number and possible

40 antiquity of domestication events, phylogeographical patterns in domestic species may indeed not be suitable to base discussion on their geographical origin.

Paper IV: Ancient human DNA: authentication through FLX-sequenced PCR products in conjunction with bleach pre-treatment, quantitative PCR, assessment of asymmetric molecular behaviour and negative controls Ancient human DNA is an area with a high profile, generating much interest (Handt et al. 1994; Caramelli et al. 2003; Haak et al. 2005; Burger et al. 2007; Sampietro et al. 2007), but it is also problematic and plagued with contamination from modern human DNA with similar genetic appearance as the ancient samples, making the results difficult to authenticate (Abbott 2003; Gilbert et al. 2005c; Sampietro et al. 2006). Several screening methods have been proposed to discriminate between modern DNA contamination and authentic ancient DNA. For instance, samples with relatively high levels of endogenous DNA may be more resilient to contamination (Gilbert 2005b). Such samples can be identified using quantitative PCR (qPCR) (Handt et al. 1996). Further, as contamination is younger than authentic DNA it is likely to be better preserved and less fragmented, and can therefore be identified by examining the degradation ratio (Noonan et al. 2006; Paper II). Sample-based contamination may also be removed already prior to DNA extraction through bleaching of powdered bone (Kemp and Smith 2005; Salamon et al. 2005; Paper II). Finally, the use of molecular cloning to identify the number of DNA sources present within PCR amplicons may be helpful (Handt et al. 1996), although to retrieve reliable statistical support, increased number of clones needs to be examined (Bower et al. 2005). Re- cent publications have demonstrated that a suitable way to retrieve large amounts of clone sequences from amplicons is to use genomic sequencing platforms to sequence pooled PCR products (Margulies et al. 2005; Binladen et al. 2007). We investigate the efficacy on combining bleach pretreatment, quantification of DNA in human samples and in non-human controls and examination of degradation ratios with the use of the FLX platform to generate reliable DNA sequences from ancient humans.

Materials and methods DNA was extracted from 71 Neolithic human samples from Sweden, 3 Me- dieval samples from Argentina, 35 Neolithic non-human samples (mostly harpseals) and from 53 extraction blanks. Powderised bone samples had

41 been treated with bleach prior to extraction. Real-time quantitative PCR was used to assess the total human DNA content of a short (80 bp) and a long (136 bp) coding region mtDNA fragment and to calculate degradation ratios. Real-time PCR was also used to quantitate the amount of coding region seal DNA, from the seal samples, to compare if the amount of human DNA in human samples was similar to the amount of seal DNA in seal samples. The human-specific mtDNA HVS1 region was amplified in 7 overlapping fragments using primers with unique 4 bp 5´tags. The total set of amplicons were pooled and sequenced using the Roche Genome Sequencer FLX system. The sequences were then sorted into separate data sets that could be identified to individual samples (through the unique 5´tags) and to specific fragments (through the primer combinations used in each of the 7 HVS1 fragments) and to a residual set with sequences that could not be assigned to specific samples. Median joining networks was constructed to visualize the sequence variation in amplicons from one of the fragments (2a) in 3 different sample types (human > 1000 copies, human < 1000 copies and non-human samples). Networks was also used to compare sequences from 30 Neolithic human samples, with > 1000 copies, > 20 clones from each amplicon and a degradation ratio > 1, with sequences retrieved from non-human samples and with sequences from modern Scandinavians.

Results and discussion A total of 85,596 sequence reads from the FLX could be accurately attrib- uted to specific samples and HVSI fragments. We found significant differences between human samples and negative controls (non-human, extraction blanks and PCR blanks) with regard to the number of human-specific mtDNA copies and the number of HVSI “clonal” sequences. The amount of human mtDNA retrieved from Neolithic human samples was similar to the amount of seal mtDNA in the Neolithic seal samples. We were able to com- pile complete mtDNA HVSI sequences from 30 Neolithic human samples from Sweden. The sequence results are supported by > 1000 mtDNA copies per sample, > 20 clones per amplicon and a degradation ratio > 1. Network analyses show that these sequences have one major consensus while the non- human samples from human sequences with < 1000 mtDNA copies show several major haplotypes which is consistent with contamination. Further- more, the few human sequences that shared haplotypes with non-human sequences (non-human HVSI, compiled using less stringent criteria than for human samples) were all similar to CRS that is a common haplotype in present day Europe. Twenty-two of the human samples shared haplotypes with modern populations. We conclude that FLX sequencing in combination with rigorous tests for possible contamination may be useful to retrieve large amounts of authentic “clonal” sequences from ancient human samples.

42 Paper V: Different allele frequencies in the lactase gene in Scandinavian Neolithic populations and the development of dairy product consumption Even though genetics and culture may interact (Renfrew 2000), finding direct evidence of this is difficult. The genetics behind lactase persistence (Enattah et al. 2002), the ability to consume unrefined milk during adulthood, may be an exception. The frequency of a mutation (-13910 in the lactase gene) linked to this trait is strongly associated with present-day dairy cultures and the geographical areas where they have developed. For example, cattle genes related to milk shows a geographical affinity for these areas (Beja-Pereira et al. 2003), and fat residues in archaeological pottery indicating actual usage of milk have been found here (Copley et al. 2003). It has been suggested that present day geographical distribution of lactase persistence and the -13910 substitution is a result of culturally induced selection over a short period of time, especially in Europe (Kuokkanen et al. 2005; Tishkoff et al. 2007), and that this selection was due to the introduction of farming (Burger et al. 2007). Here we investigate if the ability to consume unrefined milk was present in populations with different nutritional specialisation in Scandinavia during the middle Neolithic (5 300 to 4 500 years ago).

Materials and methods The material consisted of duplicate extractions from 36 middle Neolithic humans that had previously yielded high-quality mitochondrial DNA data (Paper IV). The samples derived from four hunter-gatherer sites (n=14) and one farming site (n=4) in Sweden were extracted together with negative controls (seal samples from the hunter-gatherer sites and extraction blanks). The –13910 C/T substitution in the nuclear lactase gene was amplified and alleles were identified using pyrosequencing. The ancient lactase data was statisti- cally compared to earlier published data from modern Swedes. The previously yielded HVSI data (Paper IV) was compared to modern Swedish, Norwegian and Saami populations using pair wise Fst.

Results and discussion Even after thorough sample quality pre-selection only 50% (18 out of 36) of our samples yielded reproducible results and among these, as also noted in other ancient DNA studies (Svensson et al. 2007; Svensson in press), allelic dropout was high. A lower number of positive results were detected in negative controls (in 4/43 seal amplicons, 4/41 extraction blanks and 6/39 PCR blanks). The allele associated with lactase persistence was found in 50% of

43 the farmer samples and in 10% of the hunter-gatherer samples. The farming samples did not differ from modern Swedes whereas the hunter-gatherers did. Further, the Neolithic samples were significantly different from modern Norwegian and Saami samples when mitochondrial HVSI was compared. These results may, however, be influenced by five of the hunter-gatherer samples sharing a haplotype not found among any published modern populations. We base the authenticity of our results on the fact that the samples had been pre-screened for contamination in a previous study (Paper IV), that the success rate for retrieving alleles differ between the human samples and the negative controls, and that the allele frequencies differ between these two sample types. Our data suggests that the frequency of the allele linked to lactase persistence in the investigated farmer population was, already 5 500 years ago, closer to modern Swedish frequencies than to those seen in the contemporary hunter-gatherers. This may be caused by cultural induced selection in the farmers. An alternative explanation that would not rely on an extreme cultural induced selection pressure, but with limited archaeological support (Lidén in press), would be that only those with the genetic base for consuming unrefined milk became farmers while remaining people stayed hunter- gatherers.

44 Concluding remarks

Genetic studies on modern and ancient material may shed light on some of the classical questions in human prehistory. By investigating the genetic diversity in ancient human as well as in ancient domesticate samples, a direct window to the past is opened. The processes underlying the transition from hunter-gatherers to farmers are slowly being resolved, and genetics is one important part in the jigsaw. The field of ancient human DNA has, however, been hampered with problems relating to DNA contamination from modern humans. Guidelines and criterias developed to detect and avoid contamination have in many cases been proven inadequate. This thesis has shown that modern human DNA contamination is mainly derived from pre-laboratory handling of the museum specimens. After iden- tifying this major source, it is possible to minimise the contamination through various decontamination methods. Keeping thorough track of the nature of the DNA content by using real-time quantitative PCR, assessing the asymmetric molecular behaviour (the quantitative ratio between short and longer DNA fragments), and investigating vast amounts of clones from independent DNA extracts in both human and non-human samples may help to identify authentic sequences from contaminant DNA. In this specific case I have shown that ancient mitochondrial sequences as well as nuclear SNPs retrieved from Neolithic humans and dogs may be authenticated using criteria based on these particulars. The field of ancient DNA is moving into a new era after more than 20 years without any major technological advances. When the first mitochondrial sequences (less than 400 bp) from Neandertals were retrieved it was greated as a major breakthrough. One year ago more than a million nucleotides of nuclear Neandertal DNA were published. With the development of techniques like pyrosequencing and SNaPshots, more degraded DNA may be analysed, and thereby DNA from older material. The recent releases of genomic sequencing platforms, for instance the Genomic Sequencer GS20 and FLX or the Solexa sequencing technology, also enable us, as with the Neandertal specimens, to retrieve enormous amounts of data. This, in turn, pushes the field of ancient DNA in to the era of bioinformatics, as this is the only way to work with the amount of data produced by the new platforms. An interesting era, demanding close collaboration between molecular genetics, bioinformatics, archaeology, and paleozoology is behind

45 the corner, and we have the luxury to participate and shape the development of this era.

46 Svensk sammanfattning

Bakgrund

Med jordbrukets introduktion i området kring den bördiga halvmånen i Turkiet för cirka 10 000 år påbörjades de kanske mest samhällsomvälvande händelser som människan varit med om. I princip lades grunden till det sam- hälle vi lever i idag. Hur jordbruket spreds in i Europa är dock oklart. Blev de jägar-samlande urinvånarna i Europa inspirerade att börja med jordbruk och djurhållning genom kontakter med bönder, eller var det de tidigaste bönderna som migrerade in i Europa och slutligen ersatte jägar- samlarsamhällena? Kan övergången från ett jägar-samlarsamhälle till jord- bruksbaserad ekonomi ha påverkats av de gentiska förutsättningarna som olika människor hade? Kan det exempelvis ha varit så att människor som hade förmågan att tåla mjölk var de som började utnyttja boskap för mjölk- produktion eller var det tvärtom, att förmågan att tåla mjölk blev mer och mer vanlig i de kulturer som övergick till jordbruk? Det här är klassiska frågor som kan belysas med hjälp av DNA-analyser. Frågan kring huruvida människor med kunskap om jordbruk migrerade eller om lokala jägar-samlare själva förändrar sin livsstil handlar ur ett biologiskt perspektiv om kontinuitet i genetisk variation eller genflöde. Hur och när jordbruket spreds i olika riktingar kan undersökas med hjälp av DNA- analyser både på förhistoriska människor och på domesticerade växter och djur. Den genetiska diversiteten som en population uppvisar ger oss ledtrådar kring de bakomliggande demografiska processerna. När dylika studier utförs på ett förhistoriskt material är de ofta baserade på det maternellt ärvda mitokondrie-DNAt eftersom det finns i så mycket högre upplaga än vad vanligt kärn-DNA gör. Genom att studera punktmutationer i korta DNA-fragment (SNPs) kan man även få fram resultat som speglar paternella skeenden (via Y-kromosomen). Om korta fragment undersöks i gener, områden som är kopplade till specifika egenskaper, kan man även studera hur dessa egenskaper förändras, till exempel i samband med selektiv avel. Alla dessa typer av markörer skulle kunna bli aktuella i studier av förhistoriskt humant material. Molekylärgenetiska analyser på förhistoriska människor är dock problema- tiska. DNA-kontamination från moderna människor är så allmänt förekom- mande att det kan vara svårt att hävda att resultat baserade på förhistoriska människor är äkta. Om man till exempel lyckas påvisa en genetisk kontinui-

47 tet med förhistoriskt humant DNA flera tusen år tillbaka i tiden är det kanske så att man egentligen har analyserat modernt kontaminerande DNA, och det man ser är inte alls bevis för en lång kontinuitet. Med ökade kunskaper om vari de största problemen ligger (att oftast endast korta DNA-fragment över- lever, att kemiska skador kan uppstå inuti DNA-strukturen över tid, och hur kontaminationen ter sig i förhållande till det äkta förhistoriska DNAt) och med modern teknik (realtids-kvantifiering av DNA, pyrosekvensering och genomsekvensering) skapas nu nya förutsättningar att kontrollera DNA- kvalitet och kvantitet och för att producera större mängder data än vad som var möjligt tidigare. Målet med den här avhandlingen har varit att försöka fastställa hur mycket modern human DNA-kontamination som vanligtvis finns i benmaterial förvarade på våra museer. Nästa steg har varit att undersöka hur man meto- dologiskt bör gå tillväga för att kunna eliminera kontaminationen och identifiera de äkta förhistoriska DNAt. Detta har inneburit en hel del metodutveck- ling. I förlängningen har syftet hela tiden varit att med hjälp av trovärdiga DNA-analyser försöka spåra skeenden kopplade till introduktionen av jor- bruk i Skandinavien.

Artikel I: Human DNA kontamination är vanligt förekommande i extrakt baserade på ben och tänder från förhistoriska hundar DNA-sekvenser som kommer ifrån förhistoriska prov, speciellt de som här- rör från människor, är mycket svåra att analysera. De innehåller bland annat molekylära skador samt utifrån kommande kontaminerande DNA. På grund av detta har ett flertal riktlinjer utvecklats som är till för att verifiera DNA resultat från förhistoriska prov. Dessa innefattar bland annat att arbetet bör utföras i ett isolerat speciallaboratorium, att blankprover bör processas i an- slutning till de förhistoriska proven, att DNA sekvenserna bör baseras på flera kloner från amplifierat DNA och att resultaten bör reproduceras. Efter- som kontaminerande DNA kan dela sekvensvariant, så kallad haplotyp, med det förhistoriska provet, och eftersom det har visat sig att blankprover kan visa negativa resultat trots att proven är kontaminerade, är det fortfarande problematiskt att arbeta med DNA-analyser på förhistoriska människor. I denna artikel undersöker vi systematiskt hur omfattande problemet med modern human DNA kontamination är vid analys av förhistoriska prov. Vi gör detta genom att extrahera DNA från 28 förhistoriska hundprov och sedan realtids-kvantifiera hur mycket kontaminerande humant mitokondriellt DNA och hur mycket autentiskt mitokondriellt hund-DNA proven innehåller. Vi fann att många av de förhistoriska hundproven innehöll autentiskt för- historiskt hund-DNA. Men det visade sig också att majoriteten av DNA-

48 extrakten från hund innehöll human DNA-kontamination och att denna kontamination proportionellt översteg mängden hund-DNA. Vi fann även att blankprover ofta var kontaminerade men med betydligt färre molekyler jäm- fört med kontaminationen i hundextrakten. När kloner från kontaminerade PCR-produkter undersöktes fann vi även att dessa kan påminna om hur autentiskt förhistoriskt DNA ser ut. Det vill säga de kan innehålla en sekvens- typ i majoritet och några sekvenstyper som enbart skiljer sig från denna med enstaka substitutioner. Den stora mängd human DNA kontamination vi fann i hundextrakten visar att de metoder som oftast används för dekontamination av ben- och tand- prov är otillräckliga och att kontaminationen kan vara så hög att den över- skuggar de autentiska förhistoriska DNA-sekvenserna som finns i ett prov. Eftersom majoriteten av kontaminationen härrörde från hundprovs-extrakten slöt vi oss även till att den mesta kontaminationen uppkommer genom han- tering och tvätt av ben och tänder och inte från de efterföljande laborativa processerna. Detta innebär att falska resultat baserade på kontamination skulle kunna reproduceras och att riktlinjerna för verifiering av DNA-resultat inte alltid kan till att spåra kontamination i förhistoriska prov.

Artikel II: Mer om kontamination: att använda asymetriskt molekylärt beteende för att identifiera autentiskt DNA från förhistoriska humanprov Risken att få falska resultat baserade på modern human DNA-kontamination är mycket stor när DNA från förhistoriska människor analyseras. Det har tidigare visats att många av de riktlinjer för verifiering av resultat, såsom reproduktion och kloning av PCR-produkter, i många fall inte räcker för att spåra eventuell kontamination. Nedbrytning av DNA startar direkt efter dö- den och ju längre tid som passerar desto mer bryts DNA-molekylerna ned. Eftersom modern DNA-kontamination inte är lika gammal som autentiska förhistoriska DNA-sekvenser borde det gå att se en skillnad i graden av nedbrytning mellan dessa två typer av DNA-källor. Vi testar denna hypotes genom att extrahera DNA från förhistoriska hund- och nötboskapsprover. För att bedöma graden av nedbrytning kvanti- fierar vi mitokondriella DNA-fragment av olika längd (korta/långa fragment) med hjälp av realtids-PCR och kvantitativ pyrosekvensering. Både kontaminerande (humant DNA) och autentiskt förhistoriskt DNA (hund- eller bo- skaps-DNA) undersöks. Eftersom vi tidigare har kunnat påvisa att ben och tänder från museisamlingar ofta är mycket kontaminerade testar vi även att rengöra benpulver med klorin före DNA-extraktionen. Mängden kontamination i dessa prov jämförs med tidigare resultat från samma prov som inte klorinbehandlats.

49 Mängden kontamination minskade kraftigt i de klorinbehandlade extrakten jämfört med de obehandlade proven. Mängden kontaminerande DNA visade sig oftast bestå av färre än hundra templatmolekyler medan mängden autentiskt DNA innehöll flera tusen molekyler. Vi observerade en kraftig ökning av antalet molekyler i de korta fragmenten jämfört med det långa fragmentet av autentiskt DNA. Den moderna DNA-kontaminationen var inte lika nedbruten och proportionen mellan korta och långa fragment var därför mindre. Därför drar vi slutsatsen att det asymmetriska molekylära nedbryt- ningsmönstret, som observeras mellan modern human DNA kontamination och autentiskt förhistoriskt DNA, kan användas för att spåra kvantitativa skillnader mellan dessa två typer av DNA-källor.

Artikel III: Att spåra Nordeuropeiska hundars ursprung genom analys av moderna hundar kan ge missvisande resultat För att spåra varifrån olika domestikat ursprungligen kommer undersöker man ofta den geografiska spridningen av den genetisk diversitet som moderna populationer uppvisar. Mitokondriella DNA-analyser utförda på moderna hundar har till exempel visat att det förekommer ett flertal maternella linjer (så kallade haplogrupper), att hundar troligtvis har domesticerats vid ett flertal olika tillfällen och att viss återkorsning med vilda vargar har inträffat. De största haplogrupperna är vitt spridda över världen men det existerar också en mindre haplogrupp, D, som återfinns i ovanligt hög frekvens hos moderna hundraser som anses härstamma från Skandinavien. Detta skulle kunna tol- kas som att vissa hundar domesticerades lokalt i Skandinavien. Vi undersöker en eventuell skandinavisk domesticering genom att analysera mitokondriellt DNA från 24 förhistoriska hundar från Sverige. Polymor- fier samt haplogruppsfrekvenser jämförs med tidigare publicerade data på moderna hundar. Den genetiska variationen i tretton av de förhistoriska hundproven indike- rade att de tillhörde haplogrupp C som är mycket ovanlig i moderna hundar. Resterande prov tillhörde den idag vanligt förekommande haplogruppen A (förekommer i cirka 70 % hos moderna hundar). Således var frekvensen av haplogrupp D i förhistoriska hundprov (0 %) signifikant skild från den frekvens som moderna Skandinaviska hundraser uppvisar (33 %). Våra resultat stödjer inte hypotesen om en lokal domesticering i Skandinavien och visar även att haplogruppsfrekensen har ändrats över tid i området. Eftersom tidigare studier har visat att genetisk variation i domestikat snabbt kan ändras till följd av människans handlingar (exempelvis selektion och migration), bör man vara försiktig med att använda fylogeografiska mönster baserade på moderna domestikat vid diskussioner om geografiska ursprung.

50 Artikel IV: DNA från förhistoriska människor: verifiering av resultat genom FLX-sekvenserade PCR produkter samt förbehandling med klorin, kvantitativ PCR, analys av molekylär nedbrytningsgrad och negativa kontroller DNA-analyser på förhistoriska människor är ett prioriterat område som ge- nererar mycket intresse. Det är dock svårt att producera trovärdiga resultat. Genom att arbeta med prov som innehåller mycket endogent DNA, genom att behandla pulveriserade benprov med klorin, genom att undersöka ned- brytningsgraden hos DNA samt genom att undersöka stora mängder klonade DNA-sekvenser, ökar man möjligheten att identifiera den modern DNA- kontamination som härrör från människor och skilja den från de äkta förhi- storiska DNA-sekvenserna. Här undersöker vi möjligheten att använda dessa metoder för att generera trovärdiga DNA-resultat från förhistoriska människor och kombinerar dem med att sekvensera det amplifierade DNAt på en FLX-sekvenator. DNA extraherades från 74 förhistoriska människor och från ett stort antal negativa kontroller (35 förhistoriska djur och 53 extraktionsblankar). Mängden av ett kortare och ett längre människo-specifikt mitokonriellt DNA-fragment un- dersöks med kvantitativ PCR, liksom även mängden säl-DNA i förhistoriska sälprov. Människobenen visade sig innehålla signifikant mer människo- DNA än vad de negativa kontrollerna gjorde och dessa mängder påminner om de mängder säl-DNA som fanns i de förhistoriska sälarna. Vi amplifierade den humana mitokondriella HVSI regionen i alla våra provtyper med hjälp av sekvensmärkta primers för 7 överlappande fragment. Alla PCR- produkter poolades sedan och sekvenserades simultant på FLX-sekvenatorn. Detta resulterade i över 80 000 sekvenser som vi kunde härleda till specifika prov med hjälp av de sekvensmärkta primrarna. Genom att skapa fylogene- tiska nätverk fann vi att sekvenserna från de människoprov som, innehöll över 1000 mitokondrie-DNA kopior, hade över 20 kloner per PCR-produkt och visade ett nedbrytningsmönster över 1 verkar komma från en enda DNA-källa. De negativa kontrollerna visade på flera olika DNA-källor vilket brukar förknippas med kontamination. Sekvenserna från de förhistoriska människorna påminner till stor del om de sekvenser man finner hos moderna Skandinaver medan de människo-specifika sekvenserna från förhistoriska sälar inte gjorde det. Våra resultat visar att det är möjligt att generera trovärdiga DNA-resultat från förhistoriska människor om ett flertal kontroller genomförs samtidigt. Den nya FLX-sekvenatorn erbjuder ett effektivt sätt att snabbt generera stora mängder DNA-sekvenser från ett stort antal PCR-produkter.

51 Artikel V: Olika allelfrekvenser i laktasgenen i Neolitiska populationer från Skandinavien och utvecklingen av konsumtionen av mjölkprodukter Man har länge ansett att kulturellt betingade beteenden kan påverka den genetiska sammansättningen i en population. Förmågan att tåla icke- processad mjölk i vuxen ålder är kopplad till substitutioner i nukleära gener. I vissa geografiska regioner är det vanligare att tåla mjölk och de här regio- nerna är ofta kopplade till nutida mjölk-konsumerande kulturer. Den substitution som är kopplad till mjölktolerans i Europa verkar ha varit utsatt för stark selektion inom ett relativt kort tidsintervall. Det har föreslagits att se- lektionen för tolerans av mjölk berodde på introduktionen av jordbruk. I den här studien undersöker vi om människor som levde i Sverige för mellan 5 300 och 4 500 år sedan hade förmågan att tåla mjölk. DNA extrakt producerades i dubbel upplaga och 18 prov från jägar-samlare (n=14) och jordbrukande människor (n=4) undersöktes för den substitution i kärn-DNAt som är kopplad till mjölktolerans i Europa. Den genetiska sammansättningen fastställdes med hjälp av pyrosekvensering och resultaten jämfördes med moderna svenskar. Hälften av de prov vi undersökte gav resultat som gick att verifiera. Den substitution som är associerad med mjölktolerans återfanns i hälften av jord- bruksproven, och endast i 10 % av jägar-samlarproven. Jägar-samlarna skil- de sig därför signifikant från moderna svenskar eftersom tolerans är mycket vanlig här idag. De prov som undersöktes i den här studien hade tidigare genererat sekvensresultat från en mitokondriell region (HVSI). När de resultaten jämfördes mellan de förhistoriska proven och moderna norrmän och samer fann vi att våra förhistoriska prov urskilde sig markant. Våra resultat verifieras genom att i en tidigare studie ha kontrollerat dessa prov för kon- tamnination med modernt DNA från människa och genom att proven då uppvisade en hög kvalitetsgrad. Även om våra negativa kontroller sporadiskt uppvisade kontamination var de resultaten inte möjliga att upprepa. Dessut- om visade den moderna kontaminationen olika allelfrekvenser jämfört med de förhistoriska proven. Våra resultat tyder på att den allelvariant som är kopplad till förmågan att tåla mjölk var nästan lika vanlig hos jordbrukande människor för cirka 5 000 år sedan som den är i Sverige idag, men att den var väldigt ovanlig hos de samtida jägar-samlarna. Det mönster vi ser kan ha orsakats antingen av kul- turdriven selektion eller av att endast de som hade förmågan att tåla mjölk ändrade livsstil från jägar-samlare till jordbrukare.

52 Acknowledgements I would like to thank my supervisors Anders Götherström and Gunilla Holm- lund for all the years of fun research we have shared, for lending me their time, knowledge and support. I really enjoyed working with you and hope we find many reasons to continue working together in the future.

Hans Ellegren, thank you for letting me be a part of a great research facility, the Department of Evolutionary Biology. A special thanks goes to my clos- est colleagues Cecilia Anderung and Emma Svensson, you guys are great! I am grateful to have gotten to know everyone at the department. Nilla and Carolina, you are always so helpful. Emma, Carles, Mikael and Cia, thanks for sharing research interests that have and will (hopefully) result in nice papers.

Department of Forensic Genetics and Forensic Toxicology in Linköping and Johan Ahlner have let me use their excellent research facilities. Everyone working here are such good friends, and I am so grateful for all the support you have given me. Even though everyone here has helped me in my research I would like to especially thank those I have worked more closely with; Irene, Lisbeth, Ulla, Helena, Ann-Britt, Anna-Lena, Bertil A and An- dreas.

Center for Ancient DNA and Evolution in Copenhagen and Eske Willerslev gave me the opportunity to explore some of the latest advances in sequencing technology. Thank you Jonas, Paula, Tommy, Tina, Eske and everyone else too for making me feel very welcome during my visits! Tom and Trine especially, thanks for dinners, movies and good company and also for all the last minute help with manuscripts!

I could not have conducted my studies without the knowledge and help from Janne Storå, Per Persson, Petra Molnar, Karl-Göran Sjögren, Maria Vrete- mark, Leena Drenzel, Marietta Douglas, Marie Ohlsén, Petter Nyberg, Leif Jonsson, Anna Nyqvist Thorsson, Marita Karlsson and Magnus Elfvendahl. Thank you all for providing me with samples to analyse.

As a researcher you always need to discuss (and sometimes discard) ideas. I thank Tom Gilbert, Cecilia Anderung, Emma Svensson, Andreas Karlsson, Love Dalén, Janne Storå, Anna Linderholm, Cristina Valdiosera, Colin Smith, Kerstin Lidén and of course Anders and Gunilla for valuable thoughts and comments.

I also thank Christian Bendixen and Richard Evershed for cooperation.

53 During my years in Linköping I have had the opportunity to borrow equipment and expertise from other departments, so thanks Uno Johansson, Liselotte Lenner, Christina Valgren, Johannes Hedman and Carina Ansell.

I would also like the opportunity to thank my dear friends, for being there and for reminding me what life is all about. The “archaeology girls” Sophie, Felicia and Waleska with families; LOIG (is that our name nowadays?) Lotta, Caroline, Jens, Erik, Axel, Danny and Jonas; and also Challe and Åsa for all long walks and talks; Oya for being there for so many years; and Pe- ter. Lotta, thank you for being my “illustrator” and helping me win those poster prices!

Ingrid Ivarsson for being a fantastic “landlady”, for always being supportive and for listening to all my conference talks on before hand.

I thank my family for their love and support, you are the best! Mum, dad and Anita, Johan and Tessan, Eva, Andreas, Victor, grandma and all cousins with families. I also thank the Helsingborg-side of the family: Verica, Slo- bodan, Suzana, Jonas, Leo and Teo.

Gunilla Angerbjörn Ahlbäck and grandma, you two inspired me to walk down the road of science.

Emil, thanks for all your love, help and support. But most of all, thank you for being you, love you!

My work has been funded by the Swedish Research Council, the Sven and Lilly Lawski grant, the Marie Curie Actions MEST-CT-2004-7909 ’GENE- TIME’ grant and by the National Board of Forensic Medicine. The Royal Swedish Academy of Science, CF Liljewalch stiftelse, Center of Forensic Science, Linköping University and Wallenbergstiftelsen funded several of my travels.

54 References

Abbott A (2003) Anthropologists cast doubt on human DNA evidence. Nature 423:468 Allaby RG, Banerjee M, Brown TA (1999) Evolution of the high molecular weight glutenin loci of the A, B, D, and G genomes of wheat. Genome 42:296-307 Alonso A, Martin P, Albarran C, Garcia P, Garcia O, de Simon LF, Garcia- Hirschfeld J, Sancho M, de La Rua C, Fernandez-Piqueras J (2004) Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies. Forensic Sci Int 139:141-149 Anderung C, Bouwman A, Persson P, Carretero JM, Ortega AI, Elburg R, Smith C, Arsuaga JL, Ellegren H, Götherström A (2005) Prehistoric contacts over the Straits of Gibraltar indicated by genetic analysis of Iberian Bronze Age cattle. Proc Natl Acad Sci U S A 102:8431-8435 Andréasson H, Allen M (2003) Rapid quantification and sex determination of forensic evidence materials. J Forensic Sci 48:1280-1287 Andréasson H, Asp A, Alderborn A, Gyllensten U, Allen M (2002a) Mitochondrial sequence analysis for forensic identification using pyrosequencing technology. Biotechniques 32:124-126, 128, 130-123 Andreasson H, Gyllensten U, Allen M (2002b) Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis. Biotechniques 33:402-404, 407-411 Andréasson H, Nilsson M, Budowle B, Frisk S, Allen M (2006a) Quantification of mtDNA mixtures in forensic evidence material using pyrosequencing. Int J Le- gal Med 120:383-390 Andréasson H, Nilsson M, Budowle B, Lundberg H, Allen M (2006b) Nuclear and mitochondrial DNA quantification of various forensic materials. Forensic Sci Int 164:56-64 Angleby H, Savolainen P (2005) Forensic informativity of domestic dog mtDNA control region sequences. Forensic Sci Int 154:99-110 Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HR (2005) Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 5:209-219 Balitzki-Korte B, Anslinger K, Bartsch C, Rolf B (2005) Species identification by means of pyrosequencing the mitochondrial 12S rRNA gene. Int J Legal Med 119:291-294 Bandelt HJ (2004) Etruscan artifacts. Am J Hum Genet 75:919-920; author reply 923-917 Bandelt HJ, Macaulay V, Richards M (2000) Median networks: speedy construction and greedy reduction, one simulation, and two case studies from human mtDNA. Mol Phylogenet Evol 16:8-28 Barbujani G, Bertorelle G (2003) Were Cro-Magnons too like us for DNA to tell? Nature 424:127

55 Barker G (1985) Prehistoric farming in Europe. Cambridge University Press, Cam- bridge Barnes I, Shapiro B, Lister A, Kuznetsova T, Sher A, Guthrie D, Thomas MG (2007) Genetic structure and extinction of the woolly mammoth, Mammuthus primigenius. Curr Biol 17:1072-1075 Bayless TM (1971) Junior, why didn´t you drink your milk? Gastroenterology 60:479-480 Beja-Pereira A, Luikart G, England PR, Bradley DG, Jann OC, Bertorelle G, Cham- berlain AT, Nunes TP, Metodiev S, Ferrand N, Erhardt G (2003) Gene-culture coevolution between cattle milk protein genes and human lactase genes. Nat Genet 35:311-313 Benecke N (1987) Studies on early dog remains from Northern Europe. J Arch Sci 14:31-49 Bersaglieri T, Sabeti PC, Patterson N, Vanderploeg T, Schaffner SF, Drake JA, Rhodes M, Reich DE, Hirschhorn JN (2004) Genetic signatures of strong recent positive selection at the lactase gene. Am J Hum Genet 74:1111-1120 Binladen J, Gilbert MT, Bollback JP, Panitz F, Bendixen C, Nielsen R, Willerslev E (2007) The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS ONE 2:e197 Bogucki P (1984) Ceramic sieves of the Linear pottery culture and their economic implications. Oxford J Archaeol 3:15-30 Bogucki PI (1988) Forest farmers and stockherders: early agriculture and its consequences in north-central Europe. Cambridge University Press, Cambridge Bower MA, Spencer M, Matsumura S, Nisbet RE, Howe CJ (2005) How many clones need to be sequenced from a single forensic or ancient DNA sample in order to determine a reliable consensus sequence? Nucleic Acids Res 33:2549- 2556 Brown TA, and Brown, K.A. (1992) Ancient DNA and the archaeologist. Antiquity 66:10-23 Burger J, Hummel S, Hermann B, Henke W (1999) DNA preservation: a microsatellite-DNA study on ancient skeletal remains. Electrophoresis 20:1722-1728 Burger J, Kirchner M, Bramanti B, Haak W, Thomas MG (2007) Absence of the lactase-persistence-associated allele in early Neolithic Europeans. Proc Natl Acad Sci U S A 104:3736-3741 Bär W, Brinkmann B, Budowle B, Carracedo A, Gill P, Holland M, Lincoln PJ, Mayr W, Morling N, Olaisen B, Schneider PM, Tully G, Wilson M (2000) DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing. Int J Legal Med 113:193-196 Bökönyi S (1974) History of domestic mammals in central and eastern Europe, Budapest Cann RL, Stoneking M, Wilson AC (1987) Mitochondrial DNA and human evolution. Nature 325:31-36 Caramelli D, Lalueza-Fox C, Vernesi C, Lari M, Casoli A, Mallegni F, Chiarelli B, Dupanloup I, Bertranpetit J, Barbujani G, Bertorelle G (2003) Evidence for a genetic discontinuity between Neandertals and 24,000-year-old anatomically modern Europeans. Proc Natl Acad Sci U S A 100:6593-6597 Cavalli-Sforza LL, Menozzi, P., and Piazza, A. (1994) The history and geography of human genes. Princeton University Press, Princeton Chikhi L, Destro-Bisol G, Bertorelle G, Pascali V, Barbujani G (1998) Clines of nuclear DNA markers suggest a largely neolithic ancestry of the European gene pool. Proc Natl Acad Sci U S A 95:9053-9058

56 Childe VG (1925) The dawn of European civilization. Kegan Paul, London Clutton-Brock J (1999) A natural history of domesticated animals. Cambridge Uni- versity Press, Cambridge Cooper A (1992) Removal of colourings, inhibitors of PCR, and the carrier effect of PCR contamination from ancient DNA samples. Anc DNA Newslett 1:31-32 Cooper A, Lalueza-Fox C, Anderson S, Rambaut A, Austin J, Ward R (2001) Com- plete mitochondrial genome sequences of two extinct moas clarify ratite evolution. Nature 409:704-707 Cooper A, Poinar HN (2000) Ancient DNA: do it right or not at all. Science 289:1139 Copley MS, Berstan R, Dudd SN, Docherty G, Mukherjee AJ, Straker V, Payne S, Evershed RP (2003) Direct chemical evidence for widespread dairying in prehistoric Britain. Proc Natl Acad Sci U S A 100:1524-1529 Copley MS, Berstan, R., Mukherjee, A.J., Dudd, S.N., Straker, V., Payne, S. and Evershed, R.P. (2005) Dairying in antiquity. III. Evidence from absorbed lipid residues dating to the British Neolithic. J Arch Sci 32:523-546 Craig OE, Chapman, J., Heron, C., Willis, L.H., Bartosiewicz, L., Taylor, G., Whit- tle, A. and Collins, M. (2005) Did the first farmers of central and eastern Europe produce dairy foods? Antiquity 79:882-894 Currat M, Excoffier L (2004) Modern humans did not admix with Neanderthals during their range expansion into Europe. PLoS Biol 2:e421 Currat M, Excoffier L (2005) The effect of the Neolithic expansion on European molecular diversity. Proc Biol Sci 272:679-688 Davis JM, and Walla, F.R. (1978) Evidence for domestication of the dog 12000 years ago in the Natufian of Israel. Nature 276:608-610 De Benedetto G, Nasidze IS, Stenico M, Nigro L, Krings M, Lanzinger M, Vigilant L, Stoneking M, Pääbo S, Barbujani G (2000) Mitochondrial DNA sequences in prehistoric human remains from the Alps. Eur J Hum Genet 8:669-677 Dennell R (1985) European economic prehistory: a new approach. London Aca- demic Press, London Diamond J (2002) Evolution, consequences and future of plant and animal domestication. Nature 418:700-707 Dupanloup I, Bertorelle G, Chikhi L, Barbujani G (2004) Estimating the impact of prehistoric admixture on the genome of Europeans. Mol Biol Evol 21:1361- 1372 Edwards CJ, Bollongino R, Scheu A, Chamberlain A, Tresset A, Vigne JD, Baird JF, et al. (2007) Mitochondrial DNA analysis shows a Near Eastern Neolithic origin for domestic cattle and no indication of domestication of European aurochs. Proc Biol Sci 274:1377-1385 Ellegren H (2004) Microsatellites: simple sequences with complex evolution. Nat Rev Genet 5:435-445 Enattah NS, Sahi T, Savilahti E, Terwilliger JD, Peltonen L, Jarvela I (2002) Identi- fication of a variant associated with adult-type hypolactasia. Nat Genet 30:233- 237 Gagneux P, Boesch C, Woodruff DS (1997) Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair. Mol Ecol 6:861-868 Gamble C (1999) The Paleolithic societies of Europe. Cambridge University Press, Cambridge Gilbert MT, Bandelt HJ, Hofreiter M, Barnes I (2005c) Assessing ancient DNA studies. Trends Ecol Evol 20:541-544

57 Gilbert MT, Binladen J, Miller W, Wiuf C, Willerslev E, Poinar H, Carlson JE, Leebens-Mack JH, Schuster SC (2007) Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis. Nucleic Acids Res 35:1-10 Gilbert MT, Wilson AS, Bunce M, Hansen AJ, Willerslev E, Shapiro B, Higham TF, Richards MP, O'Connell TC, Tobin DJ, Janaway RC, Cooper A (2004b) An- cient mitochondrial DNA from hair. Curr Biol 14:R463-464 Gilbert MTP, Rudbeck, L., Willerslev, E., Hansen, A.J., Smith, C., Penkman, E.H., Prangeberg, K., Nielsen-Marsh, C.M., Jans, M.E., Arthur, P., Lynnerup, N., Turner-Walker, G., Biddle, M., Kjølbye-Biddle, B., and Collins, M. (2005b) Biochemical and physical correlates of DNA contamination in archaeological bones and teeth excavated at Matera, Italy. J Archaeol Sci 32:785-793 Goldstein DB, Ruiz Linares A, Cavalli-Sforza LL, Feldman MW (1995) Genetic absolute dating based on microsatellites and the origin of modern humans. Proc Natl Acad Sci U S A 92:6723-6727 Green RE, Krause J, Ptak SE, Briggs AW, Ronan MT, Simons JF, Du L, Egholm M, Rothberg JM, Paunovic M, Pääbo S (2006) Analysis of one million base pairs of Neanderthal DNA. Nature 444:330-336 Götherström A, Anderung C, Hellborg L, Elburg R, Smith C, Bradley DG, Ellegren H (2005) Cattle domestication in the Near East was followed by hybridization with aurochs bulls in Europe. Proc Biol Sci 272:2345-2350 Haak W, Forster P, Bramanti B, Matsumura S, Brandt G, Tanzer M, Villems R, Renfrew C, Gronenborn D, Alt KW, Burger J (2005) Ancient DNA from the first European farmers in 7500-year-old Neolithic sites. Science 310:1016-1018 Hagelberg E, Bell LS, Allen T, Boyde A, Jones SJ, Clegg JB (1991) Analysis of ancient bone DNA: techniques and applications. Philos Trans R Soc Lond B Biol Sci 333:399-407 Hagelberg E, Quevedo S, Turbon D, Clegg JB (1994) DNA from ancient Easter Islanders. Nature 369:25-26 Handt O, Krings M, Ward RH, Pääbo S (1996) The retrieval of ancient human DNA sequences. Am J Hum Genet 59:368-376 Handt O, Richards M, Trommsdorff M, Kilger C, Simanainen J, Georgiev O, Bauer K, Stone A, Hedges R, Schaffner W, et al. (1994) Molecular genetic analyses of the Tyrolean Ice Man. Science 264:1775-1778 Hansen A, Willerslev E, Wiuf C, Mourier T, Arctander P (2001) Statistical evidence for miscoding lesions in ancient DNA templates. Mol Biol Evol 18:262-265 Harding RM, Fullerton SM, Griffiths RC, Bond J, Cox MJ, Schneider JA, Moulin DS, Clegg JB (1997) Archaic African and Asian lineages in the genetic ancestry of modern humans. Am J Hum Genet 60:772-789 Harris DR (1996) The origins and spread of agriculture and pastoralism in Eurasia. UCL Press, London Helgason A, Pálsson S, Lalueza-Fox C, Ghosh S, Sigurethardottir S, Baker A, Hraf- nkelsson B, Arnadottir L, Thornorsteinsdottir U, Stefansson K (2007) A Statisti- cal Approach to Identify Ancient Template DNA. J Mol Evol 65:92-102 Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real- time monitoring of DNA amplification reactions. Biotechnology (N Y) 11:1026- 1030 Ho SY, Larson G (2006) Molecular clocks: when times are a-changin'. Trends Genet 22:79-83 Hofreiter M, Jaenicke V, Serre D, Haeseler Av A, Pääbo S (2001a) DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Res 29:4793-4799

58 Hofreiter M, Serre D, Poinar HN, Kuch M, Pääbo S (2001b) Ancient DNA. Nat Rev Genet 2:353-359 Hublin JJ, Spoor F, Braun M, Zonneveld F, Condemi S (1996) A late Neanderthal associated with Upper Palaeolithic artefacts. Nature 381:224-226 Hurles ME, Veitia R, Arroyo E, Armenteros M, Bertranpetit J, Perez-Lezaun A, Bosch E, Shlumukova M, Cambon-Thomsen A, McElreavey K, Lopez De Munain A, Rohl A, Wilson IJ, Singh L, Pandya A, Santos FR, Tyler-Smith C, Jobling MA (1999) Recent male-mediated gene flow over a linguistic barrier in Iberia, suggested by analysis of a Y-chromosomal DNA polymorphism. Am J Hum Genet 65:1437-1448 Höss M, Jaruga P, Zastawny TH, Dizdaroglu M, Pääbo S (1996) DNA damage and DNA sequence retrieval from ancient tissues. Nucleic Acids Res 24:1304-1307 Ingman M, Kaessmann H, Pääbo S, Gyllensten U (2000) Mitochondrial genome variation and the origin of modern humans. Nature 408:708-713 Izagirre N, de la Rua C (1999) An 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 65:199-207 Jaenicke-Despres V, Buckler ES, Smith BD, Gilbert MT, Cooper A, Doebley J, Pääbo S (2003) Early allelic selection in maize as revealed by ancient DNA. Science 302:1206-1208 Jansen T, Forster P, Levine MA, Oelke H, Hurles M, Renfrew C, Weber J, Olek K (2002) Mitochondrial DNA and the origins of the domestic horse. Proc Natl Acad Sci U S A 99:10905-10910 Jin M, Gock SB, Jannetto PJ, Jentzen JM, Wong SH (2005) Pharmacogenomics as molecular autopsy for forensic toxicology: genotyping cytochrome P450 3A4*1B and 3A5*3 for 25 fentanyl cases. J Anal Toxicol 29:590-598 Jonsson L (1988) The vertebrate faunal remains from the Late Atlantic settlement Skateholm in Scania, South Sweden. In: Larsson L (ed) The Skateholm project I, Man and environment. Almqvist & Wiksell International, Stockholm Kaessmann H, Heissig F, von Haeseler A, Pääbo S (1999) DNA sequence variation in a non-coding region of low recombination on the human X chromosome. Nat Genet 22:78-81 Karlsson AO, and Holmlund, G. (in press) Identification of mammal species using species-specific DNA pyrosequencing. Forensic Sci Int Kemp BM, Smith DG (2005) Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Sci Int 154:53-61 Kimura M (1955) Random genetic drift in a multiallelic locus. Evolution 9:419-435 Kolman CJ, Tuross N (2000) Ancient DNA analysis of human populations. Am J Phys Anthropol 111:5-23 Kretchmer N (1972) Lactose and lactase. Sci Am 277:71-78 Krings M, Capelli C, Tschentscher F, Geisert H, Meyer S, von Haeseler A, Grosss- chmidt K, Possnert G, Paunovic M, Pääbo S (2000) A view of Neandertal genetic diversity. Nat Genet 26:144-146 Krings M, Geisert H, Schmitz RW, Krainitzki H, Pääbo S (1999) DNA sequence of the mitochondrial hypervariable region II from the neandertal type specimen. Proc Natl Acad Sci U S A 96:5581-5585 Krings M, Stone A, Schmitz RW, Krainitzki H, Stoneking M, Pääbo S (1997) Nean- dertal DNA sequences and the origin of modern humans. Cell 90:19-30 Kuokkanen M, Butzow R, Rasinpera H, Medrek K, Nilbert M, Malander S, Lubinski J, Jarvela I (2005) Lactase persistence and ovarian carcinoma risk in Finland, Poland and Sweden. Int J Cancer 117:90-94 Kwok S, Higuchi R (1989) Avoiding false positives with PCR. Nature 339:237-238

59 Lalueza-Fox C, Sampietro ML, Caramelli D, Puder Y, Lari M, Calafell F, Martinez- Maza C, Bastir M, Fortea J, de la Rasilla M, Bertranpetit J, Rosas A (2005) Ne- andertal evolutionary genetics: mitochondrial DNA data from the iberian peninsula. Mol Biol Evol 22:1077-1081 Larson G, Dobney K, Albarella U, Fang M, Matisoo-Smith E, Robins J, Lowden S, Finlayson H, Brand T, Willerslev E, Rowley-Conwy P, Andersson L, Cooper A (2005) Worldwide phylogeography of wild boar reveals multiple centers of pig domestication. Science 307:1618-1621 Leonard JA, Shanks, O., Hofreiter, M., Kreuz, E., Hodges, L., Ream, W., Wayne, R.K., and Fleischer, R.C (2006) Animal DNA in PCR reagents plagues ancient DNA research. J Archaeol Sci 34:1361-1366 Leonard JA, Wayne RK, Wheeler J, Valadez R, Guillen S, Vilà C (2002) Ancient DNA evidence for Old World origin of New World dogs. Science 298:1613- 1616 Lev-Yadun S, Gopher A, Abbo S (2000) Archaeology. The cradle of agriculture. Science 288:1602-1603 Lidén K, and Eriksson, G. (in press) Walking on the Wild side. In: Parker Pearson M, and Larsson, M. (ed) British Archaeological review Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709-715 Lindgren G, Bäckström N, Swinburne J, Hellborg L, Einarsson A, Sandberg K, Cothran G, Vila C, Binns M, Ellegren H (2004) Limited number of patrilines in horse domestication. Nat Genet 36:335-336 Luikart G, Gielly L, Excoffier L, Vigne JD, Bouvet J, Taberlet P (2001) Multiple maternal origins and weak phylogeographic structure in domestic goats. Proc Natl Acad Sci U S A 98:5927-5932 Macaulay V, Richards M, Hickey E, Vega E, Cruciani F, Guida V, Scozzari R, Bonne-Tamir B, Sykes B, Torroni A (1999) The emerging tree of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLPs. Am J Hum Genet 64:232-249 Malmer MP (2002) The Neolithic of South Sweden. TRB, GRK and STR. Almqvist & Wiksell International, Stockholm Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, et al. (2005) Genome sequencing in microfabricated high-density picolitre reac- tors. Nature 437:376-380 Marota I, Basile C, Ubaldi M, Rollo F (2002) DNA decay rate in papyri and human remains from Egyptian archaeological sites. Am J Phys Anthropol 117:310-318 McCracken RD (1971) Lactase deficiency: An example of dietary evolution. Curr Anthropol 12:479-717 Morrison TB, Weis JJ, Wittwer CT (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954-958, 960, 962 Natanaelsson C, Oskarsson MC, Angleby H, Lundeberg J, Kirkness E, Savolainen P (2006) Dog Y chromosomal DNA sequence: identification, sequencing and SNP discovery. BMC Genet 7:45 Nei M, Maruyama, T. and Chakraborty, R. (1975) The bottleneck effect and genetic variability in populations. Evolution 29:1-10 Niederstätter H, Höchl, S., Grubweiser, P., Pavlic, M., Steinlechner, M., and Parson, W. (2007) A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA. Forensic Sci Int: Genetics 1:29-34

60 Noonan JP, Coop G, Kudaravalli S, Smith D, Krause J, Alessi J, Chen F, Platt D, Pääbo S, Pritchard JK, Rubin EM (2006) Sequencing and analysis of Neander- thal genomic DNA. Science 314:1113-1118 Noonan JP, Hofreiter M, Smith D, Priest JR, Rohland N, Rabeder G, Krause J, Det- ter JC, Pääbo S, Rubin EM (2005) Genomic sequencing of Pleistocene cave bears. Science 309:597-599 Notter DR (1999) The importance of genetic diversity in livestock populations of the future. J Anim Sci 77:61-69 Orlando L, Darlu P, Toussaint M, Bonjean D, Otte M, Hänni C (2006) Revisiting Neandertal diversity with a 100,000 year old mtDNA sequence. Curr Biol 16:R400-402 Ovchinnikov IV, Götherström A, Romanova GP, Kharitonov VM, Lidén K, Good- win W (2000) Molecular analysis of Neanderthal DNA from the northern Cau- casus. Nature 404:490-493 Pedrosa S, Uzun M, Arranz JJ, Gutierrez-Gil B, San Primitivo F, Bayon Y (2005) Evidence of three maternal lineages in Near Eastern sheep supporting multiple domestication events. Proc Biol Sci 272:2211-2217 Pereira F, Davis SJ, Pereira L, McEvoy B, Bradley DG, Amorim A (2006) Genetic signatures of a Mediterranean influence in Iberian Peninsula sheep husbandry. Mol Biol Evol 23:1420-1426 Pereira L, Richards M, Goios A, Alonso A, Albarran C, Garcia O, Behar DM, Golge M, Hatina J, Al-Gazali L, Bradley DG, Macaulay V, Amorim A (2005) High- resolution mtDNA evidence for the late-glacial resettlement of Europe from an Iberian refugium. Genome Res 15:19-24 Pires AE, Ouragh L, Kalboussi M, Matos J, Petrucci-Fonseca F, Bruford MW (2006) Mitochondrial DNA sequence variation in Portuguese native dog breeds: diversity and phylogenetic affinities. J Hered 97:318-330 Plagnol V, Wall JD (2006) Possible ancestral structure in human populations. PLoS Genet 2:e105 Poinar H, Kuch M, McDonald G, Martin P, Pääbo S (2003) Nuclear gene sequences from a late pleistocene sloth coprolite. Curr Biol 13:1150-1152 Poinar HN, Hoss M, Bada JL, Pääbo S (1996) Amino acid racemization and the preservation of ancient DNA. Science 272:864-866 Poinar HN, Schwarz C, Qi J, Shapiro B, Macphee RD, Buigues B, Tikhonov A, Huson DH, Tomsho LP, Auch A, Rampp M, Miller W, Schuster SC (2006) Me- tagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Sci- ence 311:392-394 Poulter M, Hollox E, Harvey CB, Mulcare C, Peuhkuri K, Kajander K, Sarner M, Korpela R, Swallow DM (2003) The causal element for the lactase persistence/non-persistence polymorphism is located in a 1 Mb region of linkage disequilibrium in Europeans. Ann Hum Genet 67:298-311 Pruvost M, and Geigl, E-M. (2004) Real-time quantitative PCR to assess the authenticity of ancient DNA amplification. J Archaeol Sci 31:1191-1197 Pruvost M, Schwarz R, Correia VB, Champlot S, Braguier S, Morel N, Fernandez- Jalvo Y, Grange T, Geigl EM (2007) Freshly excavated fossil bones are best for amplification of ancient DNA. Proc Natl Acad Sci U S A 104:739-744 Pusch CM, Bachmann L (2004) Spiking of contemporary human template DNA with ancient DNA extracts induces mutations under PCR and generates nonau- thentic mitochondrial sequences. Mol Biol Evol 21:957-964 Pääbo S (1985) Molecular cloning of Ancient Egyptian mummy DNA. Nature 314:644-645

61 Pääbo S (1989) Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proc Natl Acad Sci U S A 86:1939-1943 Pääbo S, Poinar, H., Serre, D., Jaenicke-Després, V., Hebler, J., Rohland, N., Kuch, M., Krause, J., Vigilant, L., and, Hofreiter M (2004) Genetic analyses from ancient DNA. Annu Rev Genet 38:645-679 Ramakrishnan U, Hadly EA, Mountain JL (2005) Detecting past population bottle- necks using temporal genetic data. Mol Ecol 14:2915-2922 Renfrew C (1987) Archaeology and language: the puzzle of Indo-European origins. Jonathan Cape, London Renfrew C (2000) Archaeogenetics: Towards a population prehistory of Europe. In: Boyle CRK (ed) Archaeogenetics: DNA and the population Prehistory of Europe. Mc Donald Institute for Archaeological research, Cambridge Renfrew C, and Bahn, P. (1991) Archaeology. Theories, methods and practice. Thames and Hudson Ltd, London Richards M, Macaulay V, Hickey E, Vega E, Sykes B, Guida V, Rengo C, et al. (2000) Tracing European founder lineages in the Near Eastern mtDNA pool. Am J Hum Genet 67:1251-1276 Richards M, Macaulay V, Torroni A, Bandelt HJ (2002) In search of geographical patterns in European mitochondrial DNA. Am J Hum Genet 71:1168-1174 Richards MB, Macaulay VA, Bandelt HJ, Sykes BC (1998) Phylogeography of mitochondrial DNA in western Europe. Ann Hum Genet 62:241-260 Richards MB, Sykes, B.C, and Hedges, R.E.M. (1995) Authenticating DNA extracted from ancient skeletal remains. J Archaeol Sci 22:291-299 Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154-160 Rollo F, Ermini L, Luciani S, Marota I, Olivieri C, Luiselli D (2006) Fine characterization of the Iceman's mtDNA haplogroup. Am J Phys Anthropol 130:557- 564 Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3-11 Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84-89 Rootsi S, Magri C, Kivisild T, Benuzzi G, Help H, Bermisheva M, Kutuev I, et al. (2004) Phylogeography of Y-chromosome haplogroup I reveals distinct do- mains of prehistoric gene flow in europe. Am J Hum Genet 75:128-137 Rudbeck L, Gilbert MT, Willerslev E, Hansen AJ, Lynnerup N, Christensen T, Dissing J (2005) mtDNA analysis of human remains from an early Danish Christian cemetery. Am J Phys Anthropol 128:424-429 Sablin MV, and Khlopachev, G.A (2002) The earliest Ice Age dogs: evidence from Eliseevichi I. Curr Anthropol 43:795-799 Salamon M, Tuross N, Arensburg B, Weiner S (2005) Relatively well preserved DNA is present in the crystal aggregates of fossil bones. Proc Natl Acad Sci U S A 102:13783-13788 Sampietro ML, Gilbert MT, Lao O, Caramelli D, Lari M, Bertranpetit J, Lalueza- Fox C (2006) Tracking down human contamination in ancient human teeth. Mol Biol Evol 23:1801-1807 Sampietro ML, Lao O, Caramelli D, Lari M, Pou R, Marti M, Bertranpetit J, Lalu- eza-Fox C (2007) Palaeogenetic evidence supports a dual model of Neolithic spreading into Europe. Proc Biol Sci 274:2161-2167

62 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463-5467 Savolainen P, Zhang YP, Luo J, Lundeberg J, Leitner T (2002) Genetic evidence for an East Asian origin of domestic dogs. Science 298:1610-1613 Schmitz RW, Serre D, Bonani G, Feine S, Hillgruber F, Krainitzki H, Pääbo S, Smith FH (2002) The Neandertal type site revisited: interdisciplinary investigations of skeletal remains from the Neander Valley, Germany. Proc Natl Acad Sci U S A 99:13342-13347 Schwarcz HP, Grun R (1992) Electron spin resonance (ESR) dating of the origin of modern man. Philos Trans R Soc Lond B Biol Sci 337:145-148 Seielstad MT, Minch E, Cavalli-Sforza LL (1998) Genetic evidence for a higher female migration rate in humans. Nat Genet 20:278-280 Semino O, Passarino G, Oefner PJ, Lin AA, Arbuzova S, Beckman LE, De Benedic- tis G, Francalacci P, Kouvatsi A, Limborska S, Marcikiae M, Mika A, Mika B, Primorac D, Santachiara-Benerecetti AS, Cavalli-Sforza LL, Underhill PA (2000) The genetic legacy of Paleolithic Homo sapiens sapiens in extant Euro- peans: a Y chromosome perspective. Science 290:1155-1159 Serre D, Langaney A, Chech M, Teschler-Nicola M, Paunovic M, Mennecier P, Hofreiter M, Possnert G, Pääbo S (2004b) No evidence of Neandertal mtDNA contribution to early modern humans. PLoS Biol 2:E57 Sherratt A (1981) Plough and pastoralism: aspects of the secondary product revolution. In: Hodder I, Isaac, G., Hammond, N. (ed) Pattern of the past Studies in honour of David Clarke. Cambridge University Press, Cambridge, pp 261-305 Simoons FJ (1970) Primary adult lactose intolerance and the milking habit: a problem in biologic and cultural interrelations. II. A culture historical hypothesis. Am J Dig Dis 15:695-710 Slatkin M, Hudson RR (1991) Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations. Genetics 129:555-562 Smith CI, Chamberlain AT, Riley MS, Cooper A, Stringer CB, Collins MJ (2001) Neanderthal DNA. Not just old but old and cold? Nature 410:771-772 Smith CI, Chamberlain AT, Riley MS, Stringer C, Collins MJ (2003) The thermal history of human fossils and the likelihood of successful DNA amplification. J Hum Evol 45:203-217 Smith FH, Trinkaus E, Pettitt PB, Karavanic I, Paunovic M (1999) Direct radiocar- bon dates for Vindija G(1) and Velika Pecina late Pleistocene hominid remains. Proc Natl Acad Sci U S A 96:12281-12286 Stiller M, Green RE, Ronan M, Simons JF, Du L, He W, Egholm M, Rothberg JM, Keates SG, Ovodov ND, Antipina EE, Baryshnikov GF, Kuzmin YV, Va- silevski AA, Wuenschell GE, Termini J, Hofreiter M, Jaenicke-Despres V, Pääbo S (2006) Patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA. Proc Natl Acad Sci U S A 103:13578-13584 Stone AC, Stoneking M (1998) mtDNA analysis of a prehistoric Oneota population: implications for the peopling of the New World. Am J Hum Genet 62:1153- 1170 Stringer CB (1985) Middle Pleistocene hominid variability and the origin of Late Pleistocene humans. In: Delson E (ed) Ancestors: the hard evidence. Liss, New York, pp 289-295 Stringer CB, Andrews P (1988) Genetic and fossil evidence for the origin of modern humans. Science 239:1263-1268 Stringer CB, Grun R (1991) Palaeoanthropology. Time for the last Neanderthals. Nature 351:701-702

63 Stumpf MP, Goldstein DB (2001) Genealogical and evolutionary inference with the human Y chromosome. Science 291:1738-1742 Svensson EM, Anderung C, Baubliene J, Persson P, Malmström H, Smith C, Vrete- mark M, Daugnora L, Götherström A (2007) Tracing genetic change over time using nuclear SNPs in ancient and modern cattle. Anim Genet 38:378-383 Svensson EM, Götherström, A., Vretemark, M. (in press) A DNA test for sex identification in cattle confirms osteometric results. J Archaeol Sci Swisher CC, 3rd, Rink WJ, Anton SC, Schwarcz HP, Curtis GH, Suprijo A, Widi- asmoro (1996) Latest Homo erectus of Java: potential contemporaneity with Homo sapiens in southeast Asia. Science 274:1870-1874 Taberlet P, Griffin S, Goossens B, Questiau S, Manceau V, Escaravage N, Waits LP, Bouvet J (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res 24:3189-3194 Tambets K, Rootsi S, Kivisild T, Help H, Serk P, Loogvali EL, Tolk HV, et al. (2004) The western and eastern roots of the Saami--the story of genetic "out- liers" told by mitochondrial DNA and Y chromosomes. Am J Hum Genet 74:661-682 Tattersall I (1986) Species recognition in human paleontology. Journal of Human Evolution 15:165-175 Thomas RK, Nickerson E, Simons JF, Janne PA, Tengs T, Yuza Y, Garraway LA, et al. (2006) Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nat Med 12:852-855 Thorne AG, Wolpoff MH (1981) Regional continuity in Australasian Pleistocene hominid evolution. Am J Phys Anthropol 55:337-349 Tilley C (1994) A phenomenology of landscape. Berg, Oxford Tishkoff SA, Reed FA, Ranciaro A, Voight BF, Babbitt CC, Silverman JS, Powell K, Mortensen HM, Hirbo JB, Osman M, Ibrahim M, Omar SA, Lema G, Nyambo TB, Ghori J, Bumpstead S, Pritchard JK, Wray GA, Deloukas P (2007) Convergent adaptation of human lactase persistence in Africa and Europe. Nat Genet 39:31-40 Topf AL, Gilbert MT, Dumbacher JP, Hoelzel AR (2006) Tracing the phylogeography of human populations in Britain based on 4th-11th century mtDNA geno- types. Mol Biol Evol 23:152-161 Torroni A, Bandelt HJ, D'Urbano L, Lahermo P, Moral P, Sellitto D, Rengo C, Forster P, Savontaus ML, Bonne-Tamir B, Scozzari R (1998) mtDNA analysis reveals a major late Paleolithic population expansion from southwestern to northeastern Europe. Am J Hum Genet 62:1137-1152 Torroni A, Bandelt HJ, Macaulay V, Richards M, Cruciani F, Rengo C, Martinez- Cabrera V, et al. (2001) A signal, from human mtDNA, of postglacial recoloni- zation in Europe. Am J Hum Genet 69:844-852 Torroni A, Huoponen K, Francalacci P, Petrozzi M, Morelli L, Scozzari R, Obinu D, Savontaus ML, Wallace DC (1996) Classification of European mtDNAs from an analysis of three European populations. Genetics 144:1835-1850 Troy CS, MacHugh DE, Bailey JF, Magee DA, Loftus RT, Cunningham P, Cham- berlain AT, Sykes BC, Bradley DG (2001) Genetic evidence for Near-Eastern origins of European cattle. Nature 410:1088-1091 Tuross N (1994) The biochemistry of ancient DNA in bone. Experientia 50:530-535 Underhill PA, Shen P, Lin AA, Jin L, Passarino G, Yang WH, Kauffman E, Bonne- Tamir B, Bertranpetit J, Francalacci P, Ibrahim M, Jenkins T, Kidd JR, Mehdi SQ, Seielstad MT, Wells RS, Piazza A, Davis RW, Feldman MW, Cavalli- Sforza LL, Oefner PJ (2000) Y chromosome sequence variation and the history of human populations. Nat Genet 26:358-361

64 Valdiosera C, Garcia N, Dalén L, Smith C, Kahlke RD, Lidén K, Angerbjörn A, Arsuaga JL, Götherström A (2006) Typing single polymorphic nucleotides in mitochondrial DNA as a way to access Middle Pleistocene DNA. Biol Lett 2:601-603 Valladas H, Reyss, J.L., Joron, J.L., Valladas, G., Bar-Yosef, O. and Vander- meersch, B. (1988) Thermoluminescence dating of Mousterian Troto-Cro- Magnon remains from Israel and the origin of modern man. Nature 331:614-615 Watterson GA (1986) The homozygosity test after a change in population size. Ge- netics 112:899-907 Verginelli F, Capelli C, Coia V, Musiani M, Falchetti M, Ottini L, Palmirotta R, Tagliacozzo A, De Grossi Mazzorin I, Mariani-Costantini R (2005) Mitochon- drial DNA from prehistoric canids highlights relationships between dogs and South-East European wolves. Mol Biol Evol 22:2541-2551 Vernesi C, Caramelli D, Dupanloup I, Bertorelle G, Lari M, Cappellini E, Moggi- Cecchi J, Chiarelli B, Castri L, Casoli A, Mallegni F, Lalueza-Fox C, Barbujani G (2004) The Etruscans: a population-genetic study. Am J Hum Genet 74:694- 704 Vilà C, Leonard JA, Götherström A, Marklund S, Sandberg K, Lidén K, Wayne RK, Ellegren H (2001) Widespread origins of domestic horse lineages. Science 291:474-477 Vilà C, Savolainen P, Maldonado JE, Amorim IR, Rice JE, Honeycutt RL, Crandall KA, Lundeberg J, Wayne RK (1997) Multiple and ancient origins of the domestic dog. Science 276:1687-1689 Vilà C, Seddon J, Ellegren H (2005) Genes of domestic mammals augmented by backcrossing with wild ancestors. Trends Genet 21:214-218 Willerslev E, Cappellini E, Boomsma W, Nielsen R, Hebsgaard MB, Brand TB, Hofreiter M, et al. (2007) Ancient biomolecules from deep ice cores reveal a forested southern Greenland. Science 317:111-114 Willerslev E, Hansen AJ, Binladen J, Brand TB, Gilbert MT, Shapiro B, Bunce M, Wiuf C, Gilichinsky DA, Cooper A (2003) Diverse plant and animal genetic records from Holocene and Pleistocene sediments. Science 300:791-795 Willerslev E, Hansen AJ, Poinar HN (2004) Isolation of nucleic acids and cultures from fossil ice and permafrost. Trends Ecol Evol 19:141-147 Wolpoff MH, Frayer DW (1992) Neanderthal dates debated. Nature 356:200-201 Wolpoff MH, Spuhler JN, Smith FH, Radovcic J, Pope G, Frayer DW, Eckhardt R, Clark G (1988) Modern human origins. Science 241:772-774 Vuissoz A, Worobey, M., Odegaard, N., Bunce, M., Machado, C.A., Lynnerup, N., Peacock, E.E., and Gilbert, M.T.P. (2004) The survival of PCR-amplifiable DNA in cow leather. J Archaeol Sci 34:823-829 Yang DY, Eng B, Saunders SR (2003) Hypersensitive PCR, ancient human mtDNA, and contamination. Hum Biol 75:355-364 Zeder MA, Emshwiller E, Smith BD, Bradley DG (2006) Documenting domestication: the intersection of genetics and archaeology. Trends Genet 22:139-155 Zerjal T, Dashnyam B, Pandya A, Kayser M, Roewer L, Santos FR, Schiefenhovel W, Fretwell N, Jobling MA, Harihara S, Shimizu K, Semjidmaa D, Sajantila A, Salo P, Crawford MH, Ginter EK, Evgrafov OV, Tyler-Smith C (1997) Genetic relationships of Asians and Northern Europeans, revealed by Y-chromosomal DNA analysis. Am J Hum Genet 60:1174-1183 Zhao Z, Jin L, Fu YX, Ramsay M, Jenkins T, Leskinen E, Pamilo P, Trexler M, Patthy L, Jorde LB, Ramos-Onsins S, Yu N, Li WH (2000) Worldwide DNA sequence variation in a 10-kilobase noncoding region on human chromosome 22. Proc Natl Acad Sci U S A 97:11354-11358

65 Zink A, Nerlich AG (2003) Molecular analyses of the "Pharaos:" Feasibility of molecular studies in ancient Egyptian material. Am J Phys Anthropol 121:109-111 Zvelebil M (1986) Hunters in transition: Mesolithic societies of temperate Eurasia and their transition to farming. Cambridge University Press, Cambridge

Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 323

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology”.)

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-8162 2007