Detecting Sex and Selection in Ancient Cattle Remains Using - DocsLib

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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Svensson, E.M., Axelsson, E., Vretemark, M., Makowiecki, D., Gilbert, M.T.P., Willerslev, E., Götherström, A. Insights into Y chromosomal genetic variation and effective population size in the extinct European aurochs Bos primigenius. Manuscript II Svensson, E., Götherström, A., (2008) Temporal fluctuations of Y-chromosomal variation in Bos taurus. Biology Letters, 4(6):752-754 III Svensson, E.M., Götherström, A., Vretemark, M. (2008) A DNA test for sex identification in cattle confirms osteometric results. Journal of Archaeological Science 35(4):942-946 IV Telldahl, Y., Svensson, E.M., Götherström, A., Storå, J. Typing late prehistoric cows and bulls-osteology and genetics of cattle at the Eketorp fortification and settlement on Öland, Sweden. Submitted V Svensson, E.M., Anderung, C., Baubliene, J., Persson, P., Malmström, H., Smith, C., Vretemark, M., Daugnora, L., Götherström, A. (2007) Tracing genetic change over time using nuclear SNPs in ancient and modern cattle. Animal Genetics, 38(4):378-383 VI Svensson, E.M., Jakobsson, M., Vretemark, M., Telldahl, Y., Persson, P., Malmborg, G., Götherström, A. Signs of contrasting selection in medieval and modern North European cattle. Manuscript

Reprints were made with permission from the respective publisher.

Additional papers not included in the thesis

I Ala-Poikela, M., Svensson, E., Rojas, A., Horko, T., Paulin, L., Valkonen, J., Kvarnheden, A. (2005) Genetic diversity and mixed infections of begomoviruses infecting tomato, pepper and cucurbit crops in Nicaragua. Plant pathology, 54(4):448– 459 II Malmstrom, H., Svensson, E.M., Gilbert, M.T., Willerslev, E., Gotherstrom, A., Holmlund, G. (2007) More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA. Molecular Biology and Evolution, 24(4):998-1004 III Gilbert, M.T.P., Jenkins, D.L., Higham, T.F.G., Rasmussen, M., Malmström, H., Svensson, E.M., Sanchez, J.J., Cummings, L.S., Yohe, I.I. R.M., Hofreiter, M., Götherström, A., Willer- slev, E., (2009) Response to comment by Poinar et al. on "DNA from pre-clovis" human coprolites in Oregon, North America. Science, 325:148 IV Eklund, D.M., Svensson, E., Kost, B. (2010) Physcomitrella patens: a model to investigate the role of RAC/ROP GTPase signaling in tip growth. Journal of Experimental Botany, 2010 In press

Contents

Introduction ...... 11 Domestication...... 11 Why study domesticates ...... 12 Domestic animals...... 12 Farming throughout history ...... 14 The spread of farming ...... 14 Prehistoric animal husbandry ...... 15 The history of breeding ...... 16 Modern breeds and effects of breeding...... 18 Genetic effects from domestication and breeding ...... 19 Signatures of selection in domesticates ...... 19 The cattle genome under selection ...... 20 Methods ...... 22 Ancient DNA as a tool to access temporal data ...... 22 Properties of ancient DNA ...... 22 Genetic markers ...... 26 Single nucleotide polymorphisms ...... 27 Mitochondrial DNA ...... 27 Y chromosomal DNA ...... 28 Sequencing and Genotyping ...... 28 Pyrosequencing ...... 29 SNPstream ...... 31 454/FLX ...... 33 Analysing temporal data ...... 35 Ascertainment bias...... 36 Research aims ...... 38 General aims ...... 38 Specific aims ...... 38 Summaries of papers ...... 39 Paper I: Insights into Y chromosomal genetic variation and effective population size in the extinct European aurochs Bos primigenius...... 39 Paper II: Temporal fluctuations of Y-chromosomal variation in Bos taurus...... 40

Paper III: A DNA test for sex identification in cattle confirms osteometric results...... 40 Paper IV: Typing Late Prehistoric cows and bulls-osteology and genetics of cattle at the Eketorp fortification and settlement on Öland, Sweden...... 42 Paper V: Tracing genetic change over time using nuclear SNPs in ancient and modern cattle...... 43 Paper VI: Signs of contrasting selection in medieval and modern North European cattle ...... 44 Conclusions and future prospects ...... 46 Svensk sammanfattning ...... 48 Acknowledgements ...... 51 References ...... 54

Abbreviations

aDNA Ancient DNA bp Base pair(s) BP/BC Before present/Christ DNA Deoxyribonucleic acid dN Non-synonymous substitution rate dS Synonymous substitution rate kb Kilobase (103 base pairs) LD Linkage disequilibrium MRCA Most recent common ancestor mtDNA Mitochondrial DNA Ne Effective population size PCR Polymerase chain reaction SNP Single nucleotide polymorphism TRB Trichtbächer kultur

Introduction

Domestication of animals like goat, sheep, pig, horse and cow laid the foundation for the modern sedentary society (Diamond, 2002). Going from a hunter-gatherer lifestyle to a farming lifestyle is perhaps the most revolution- ising process in our species history. Throughout the time since domestication major changes have occurred in all of the major farm animals. Humans have selected for traits that were beneficial for us, resulting in the multitude of breeds seen today. Already at the time of Moses, key concepts in cattle breeding were recognized.

Ye shall keep my statutes. Thou shalt not let thy cattle gender with a diverse kind: thou shalt not sow thy field with two kinds of seed: neither shall there come upon thee a garment of two kinds of stuff mingled together. (Holy Bi- ble, Leviticus 19:19)

In this thesis I have investigated genetic signs of breeding in ancient remains of domestic cattle (Bos Taurus), and the Y chromosomal genetic diversity in extinct European aurochs (Bos primigenius).

Domestication Domestication can be seen as the adaptation of a wild animal to human society (Price, 2002), this process is likely to confer phenotypic changes to the animals. It is likely that wild animals were selected for traits like tameness and docility, selection for these traits have also been shown to confer morphological changes to the animal (Price, 1999; Trut, 1999; Vonholdt BM, 2010). This is also seen in the archaeological record, where remains from domestic animals are smaller than their wild ancestors. However, since this change might take several generations, it is not easy to distinguish a domestication event, as it happens, from the archaeological record. Often kill off patterns, the sex and age profile of when animals were slaughtered, can offer a more obvious distinction between wild and managed animals (Zeder & Hesse, 2000).

11 Why study domesticates Ever since they were first domesticated, the major farm animals have been selected for traits appreciated by or beneficial to humans. This has resulted in an enormous phenotypic diversity, rarely observed in wild populations of mammals. This makes domestic animals ideal for studies of the genetic basis for phenotypic variation (Andersson, 2001). Because selection for particular traits has been a lot stronger in many breeds than in for example humans domestic animals can serve as a models for mapping of disease genes, see for example Wilbe et al. (2010). The rapid genetic change that occurs when breeding also makes domestic animals suitable for evolutionary studies. Charles Darwin used the artificial selection in domestic species as a template for natural selection in the process of evolution (Darwin, 1868). Later on Sewall Wright’s studies of cattle and guinea pigs lead to the development of some of the most important theories in genetics and evolutionary biology, the inbreeding coefficient and the shifting balance theory (Wright, 1922, 1931, 1978, 1980). Domestic animals are of utter importance for the food production in the world, as well as providers of companionship. In all domesticates many breeds suffer from low diversity and the problems associated with that. Also breeding for specific traits can cause problems. For example selection for hairlessness in dogs is associated to no development of teeth (Drogemuller et al., 2008), and in Ridgeback dogs the selection for hair ridge also leads to predisposition to dermoid sinus (Hillbertz et al., 2007). The decreased fertility in dairy cattle associated with high production is yet another example (Boichard et al., 2003). Interesting questions arise in light of this loss of genetic diversity: For how long have domesticates sustained this? Is it a result of the domestication process, later selection, or both? Last but not the least; livestock are also of interest for the studies of past human societies since they can be used as a proxy for human movement and culture.

Domestic animals The dog was the first animal to be domesticated, although under different circumstances compared to all other domesticates. Dogs were domesticated in hunter-gatherer societies, before the transition to a farming lifestyle. There is still contradicting views on exactly where this happened, but it can be dated to at least 5000 years before humans started to take control over their nutritional sources (Vila et al., 1997; Leonard et al., 2002; Savolainen et al., 2002; Pang et al., 2009). Although animal domestication is not restricted to one geographic area, it is clear that the Fertile Crescent (that is parts of present day Iran, Iraq, Syria and Turkey) is a hotspot. For example, the area where the first indications of

12 goats herding appear, archaeozoological assemblages from Ganj Dahre, in western Iran shows signs of managed herds of goats at 10000 BP (Zeder & Hesse, 2000). Molecular evidence shows that 90% of all goats carry the mitochondrial haplogroup A (Naderi et al., 2008), based on haplogroup diversity and distribution, eastern Anatolia seems to have been one of the most important centres for domestication. However, the archaeological data from Iran are also supported by the diversity in the mitochondrial haplogroup C, indicating that a second domestication took place in Iran, in southern Zagros (Naderi et al., 2008). Sheep were domesticated in mountainous regions in Southeast Europe and central/southwest Asia, some 11,000-15,000 years ago. It is thus one of the earliest livestock animals to be domesticated, five different mitochondrial lineages have been found, but so far the wild ancestor to sheep is unknown (Meadows et al., 2007). Domestic pigs have a more diverse history. Several large scale studies of mitochondrial DNA (mtDNA) from both modern and ancient pigs, show that they were domesticated in several places in Asia and Europe, and that these events were separated both in time and space (Larson et al., 2005; Larson et al., 2007a; Larson et al., 2007b; Larson, 2010). The domestication of the horse is perhaps, from a genetic perspective, the most complex of all. Modern and ancient mtDNA suggests that many female lineages were involved in the process (Vila et al., 2001; Jansen et al., 2002), and occasionally parts of the variation have been traced back to wild horses (Lira et al., 2010). The Y chromosome, however, shows a complete lack of variation in domestic horses (Lindgren et al., 2004) suggesting that very few males were used, alternatively, breeding on stallions has been intense.

Cattle The oldest remains from domestic taurine cattle come from the archaeological site Shillourokambos on Cyprus. As the island did not hold an aurochs population, the early cattle remains must be from domestic animals. The site, including the remains has been dated to 8200-7500 BC (Vigne, 2000). Other evidence point to Çatalhöyük as a centre for domestication, archaeological evidence from this Turkish site shows that cattle were of great cultural importance (Clutton-Brock, 1999). But if all archaeozoological evidence from the region is examined, it seems more likely that smaller domestic cattle from neighbouring regions were transported into Anatolia. At the same time that domestic cattle appear in the region, large wild cattle are also present, and it cannot be excluded that the breeding practice included recruitment of local aurochs into the domestic heard. A combination of zooarchaeological data and radiocarbon dating of the site of Erbaba, in central Anatolia, suggest that domestic cattle were present in Anatolia at 6500 BC (Arbuckle & Makarewicz, 2009).

13 Genetic evidence from both modern and ancient DNA points to the major domestication event of taurine cattle taking place in the Near East (Loftus et al., 1994; Bailey et al., 1996; Bradley et al., 1996; Troy et al., 2001; Bol- longino et al., 2006; Achilli et al., 2009). There is no conclusive evidence from ancient DNA or modern cattle that points to local domestication in Europe. But a few rare “aurochs” haplotypes have been found in modern cattle (Achilli et al., 2008). However it is hard to tell if these haplotypes were present at low frequency in Near Eastern aurochs, or if indeed a hand- ful of European aurochs chromosomes have survived until today. The introduction of cattle to Europe took at least three different routes, the Mediterranean and the Danubian route (Beja-Pereira et al., 2006) and from Africa to Spain across the strait of Gibraltar (Anderung et al., 2005). The investigation of nucleotide diversity in Neolithic European cattle also shows that it was higher in areas close to these entry points (C. Anderung manuscript), consistent with what is expected after a domestication event. A similar pattern has also been found in modern breeds (Troy et al., 2001; Me- dugorac et al., 2009).

Farming throughout history The importance of farming for human society cannot be over-estimated. Here, I start by outlining the spread of farming, focusing on Europe, continuing with details of what is known of animal husbandry and breeding, with the focus on cattle.

The spread of farming Around 10,000 years ago the first signs of farming emerged in the Near East. Crops like wheat and barley started to be cultivated, and wild animals were domesticated, as described above. These changes conferred an enormous shift in human culture and the spread of the new ideas was fast. Using radiocarbon data from archaeological sites the diffusion rate of Neolithic farmers has been estimated to an average of 1km per year (Cavalli Sforza, 1997). A number of questions relates to the rise of farming. Why it happened is a long standing debate. It might have been a by-product of social changes (see for example Brian Hayden in Models of Domestication in Transitions to Agriculture in Prehistory ,1992), or a means to increase the carrying capac- ity of the land (see for example Lewis Binford in Post-Pleistocene Adapta- tions in New Perspectives in Archaeology, 1968). However, the question suitable to study with genetic tools is how farming spread, through the diffusion of ideas and knowledge, or through the spread of people. Raising domestic animals rather than depending on hunting changed people’s use of animals (use of wool, traction, and milk). This more intense use

14 of animals, led Andrew Sherrat to propose the idea of a secondary products revolution. This is a disputed theory, which nevertheless serves to highlight the profound changes that animal husbandry made on archaeological com- munities and their management of land and resources. Dairying is seen as one of a number of interlinked innovations that transformed the economy of the Late Neolithic of South East Europe and the Copper/Bronze Age of Cen- tral Europe (Sherratt, 1981). There is evidence from lipid analysis placing the use of milk in north western Anatolia as far back in time as 6500 BC (Evershed et al., 2008), and by 4000 BC there is evidence of milk being used in Britain (Copley et al., 2003). These finds rather point to a development of farming in different ways in different areas. The high allelic richness found in milk genes in modern North European cattle, combined with human lactase persistence and the distribution of Neolithic farmers, lead Beja-Pereira and colleagues to suggest a gene-culture co evolution between cattle milk proteins and human lactase persistence (Beja-Pereira et al., 2003; Itan et al., 2009). Farming was introduced to Northern Europe and Scandinavia during the Neolithic Stone Age and continued to increase in importance during the beginning of the Bronze Age. The domesticated animals that were reared com- prised a mixture of cattle, pigs and sheep/goats, the earliest cattle remains are from around 4000 BC. While only a small percentage of wild animals are found in the archaeological record of the Trichtbächer kultur (TRB) sites (the earliest farming group in Scandinavia,) it is clear that some traditional exploitation of wild resources such as hunting of game, fishing and fowling prevailed alongside farming (Midgley, 1992).

Prehistoric animal husbandry During the Scandinavian Iron Age (500 BC-1050 AD), cattle had a central role in the farming system. Archaeological and zooarchaeological assemblages indicate that domesticates were important at this stage, for example the occurrences of large farm units throughout Northern Europe (Myrdal, 1996). But it is the medieval period that stands out as a time of new innovations in farming (Thoen, 1997; Davis & Beckett, 1999) and it is likely that it also affected the cattle. Osteological analysis of a large number of bones shows that cattle were significantly smaller during medieval times (Sten, 1994; Myrdal, 1999). The reasons for this size reduction is not know, but it is likely to be a combination of the way that cattle were kept, and possibly breeding for small cattle that did not require much energy to survive the long winters. Cattle often make up more than 50% of the bone assemblages (Vre- temark, 1997), and were thus the most important livestock in many places. Kill off patterns show that cows were often kept until old age, while males

15 were slaughtered when they reached their full size (Vretemark, 1994; Vre- temark, 1997), this pattern is consistent with a milk production economy. During medieval times there was an increased demand for butter in Swe- den, as is evident from the taxes being paid in butter (Myrdal, 1996, 1999). The many mythological creatures and superstitions connected to milk also show how important it was (Myrdal, 1999). Although it has been estimated that a medieval cow produced significantly less than a modern cow, on average 300-400 kg/year (Björnhag, 1994) compared to modern milk breeds producing more than 15 times as much (Björnhag, 1994; Flori, 2009), it is likely that increasing specified demands fuelled specialisation. There was also a constant demand for beef (Poulsen 1997) during medieval time, which increased during the later medieval time. This made ox- trades economically important during the 16th century. Oxen were transported over large distances, and in Scandinavia this is perhaps best illus- trated by the demands from the mining industry. Massive amounts of oxen were required, not only to be used as draught power and food resource, but also for rope production from their hides. This engaged large scale ox breeders in various parts of Sweden (Myrdal, 1999), and is probably the first time that a commercial need for larger animals occurred in Northern Europe. Tens of thousands of oxen were transported every year. In Europe this trade had started a bit earlier and involved hundreds of thousands of animals. For example, in 1485 duty was paid on 13 020 oxen being transported from Den- mark to the Dutch market. This number had grown to 35 000 oxen in the 1540s (Poulsen, 1997). Oxen were also used as draught animals, at least in most parts o Sweden, in northern Sweden horses were preferred. Compared to horses oxen are relatively cheap, they need less food and the food can be of poorer quality. By the 19th century, horses had replaced oxen in many parts of Sweden. Some differences in status can also be seen, larger farms that could keep both horses and oxen continued to use oxen as draught labour, while smaller farms kept only a horse, for all types of tasks (Wiking-Faria, 1986; Söder- berg, 2002).

The history of breeding The concept of breed is quite fluid. To begin with it is a cultural distinction that is followed by reproductive isolation. A breed can be defined as a group of animals that share similar special characteristics of inheritance. Following this definition it is clear that the history of breeding extends much further back in time than just the last 200 years. If reading the bible as a historic source, one can find evidence pointing to breeding of sheep. Sheep with different colours should be kept separate (for example in Genesis 30-31), and the colour of the wool also changed over time into different varieties. Alexander the great is said to have selected Indian cattle for improvement of

16 the Macedonian breed (discussed in Darwin 1868). There are further indications indicating that the concept was known in the Hellenic/Roman eras as more than 150 horse breeds have been described by written roman and Greek sources (Hyland, 1990). Actually, an early form of purposeful selection may well have been practiced already during the domestication process. It has for example been noted that pigmentation in horses was important already during, or right after domestication (Ludwig et al., 2009). As noted above, the first written records of farming in Europe come from authors active in the Roman Empire. Columella in his De Re Rustica provided a detailed description of cattle diseases and the number of injuries oxen suffered when they were used for work. He also discussed the problems of using cows for work, as this decreased their fertility. Furthermore, detailed instructions on how to breed fowl based on colour, and other phenotypic characteristics were provided. Virgil, in his Georgicas, suggests that when selecting animals for breeding to note the tribe, the lineage and the sire. This shows that already some 2000 years ago people were managing their livestock in a non random way. In his extensive work Animals and Plants under Domestication Charles Darwin recognized three types of selection in livestock, namely methodical, unconscious and natural selection. The last is independent of human, and often even works opposite to selection strategies practiced by humans (Dar- win, 1868). The methodical selection is exemplified as that practiced by several fa- mous breeders, Bakewell, Bates and Lord Leicester just to mention a few, in the 18th/19th century England, following a predetermined standard the breed is modified. Unconscious selection is in principle not different from methodical selection, renders the most valued animals to be preserved and the less valued destroyed. To separate it from methodical can be hard, but it is likely that methodical selection would result in faster change.

We have also reason to believe that selection, carried on so far unconsciously that there was at no one time any distinct intention to improve or change the breed, has in the course of time modified most of our cattle; for by this process, aided by more abundant food, all the lowland British breeds have increased greatly in size and in early maturity since the reign of Henry VII. (Darwin, 1868).

Before modern veterinary medicine, there were limits for how far selection and breeding could go. Cattle that were selected for size to a degree that they had problems giving birth could simply not be bred on any further. This can be compared to Belgian blue today, a breed that often needs surgery in order to give birth. Once breed books were established, selection for breed uniformity happened over a few generations, eroding breeds of their natural

17 variation (Pitt, 1920). In a summary of European breeds from 1860 (Moll), engravings of fifty-five different breeds are given, illustrating how special- ized and diversified cattle breeds had become in the 19th century. But Charles Darwin (1868) made the following reflexion; It is, however, probable that several of these differ very little from each other, or are merely synonyms.

Modern breeds and effects of breeding Depending on which species are included there are, on a worldwide basis, around 7600-3000 different breeds of the most common domestic species (Ruane, 1999; Groeneveld et al., 2010). During the first 6 years of this century 62 breeds have gone extinct (Groeneveld et al., 2010), and between 20- 30% of all farm animals are at risk of extinction (Ruane, 1999; Simianer et al., 2003; Groeneveld et al., 2010). For example in Sweden all five native cattle breeds are threatened by extinction. Often genetic distance between breeds is measured using microsatellite loci, because many breeds are selected for different purposes mainly manifested in the coding parts of the genome, this approach may underestimate the distance between breeds (Ru- ane, 1999). Li et al. (2006) estimated the degree of population differentiation using SNPs in three genes under selection, and compared it to microsatellite data. They found that the differentiation was larger between breeds for the three coding SNPs compared to the microsatellites. The last 50 years of industrialised farming has driven many old breeds close to extinction. This is indeed a genetic catastrophe, because these old breeds contain important genetic variation. They are often well adapted to their environment, and often produce more per energy put into the animal (although not totally) compared to breeds selected for high production (Taberlet et al., 2008; Medugorac et al., 2009). When investigating the genetic diversity for North European cattle breeds Kantanen et al. (2000) estimated divergence times between different breeds to range from some 5000 years, for the most diverged (Icelandic cattle and Danish Jersey), to 1100 years (Icelandic and Nordland cattle). Many of these breeds are on the verge of extinction, but they are an important genetic resource, with a long population history in the area, and are thus an important resource for conservation. The divergence times also indicate that cattle from the same geographic areas were kept separated already as far back in time as several thousand years (Kantanen et al., 2000), although more recent population replacements cannot be ruled out.

18 Genetic effects from domestication and breeding It is important to make a distinction between artificial selection and natural selection. Artificial selection brings about changes a lot faster than does natural selection. Artificial selection is goal-oriented, with the prioritisations from humans for a desired phenotype as the goal. Thus, often has nothing to do with the actual fitness of the animal.

Signatures of selection in domesticates The intensive selection in cattle today makes it possible to identify chromosomal regions that have changed due to artificial selection. It is also possible to separate this pattern from that of natural selection or genetic drift (Gomez- Raya et al., 2002). When genomic scans for signs of selection are performed in cattle, regions under selection linked either to traits involved in production or domestication have been found (Elsik et al., 2009; Flori, 2009; Gibbs et al., 2009; Hayes et al., 2009; MacEachern et al., 2009). Signatures of selection can also be seen when comparing the genome of a domestic animal to that of its wild relative. For example, dogs have been found to have accumulated slightly deleterious mutations since the time of domestication. The dN/dS ratio was found to be 50% higher in dogs than in wolfs (Cruz et al., 2008). The authors explain this as a result of relaxation of selective constraint, this ultimately leads to an increase in functional genetic variation (Cruz et al., 2008). The same pattern has been observed in pigs and wild boar when investigated for a coat colour gene (Fang et al., 2009), equal number of substitutions were found in both domestic pig and wild boar. All but one of the changes in pigs were non-synonymous leading to shifts in coat colour. In wild boar it is not beneficial to have a conspicuous coloration, thus there is purifying selection against non-synonymous changes (Fang et al., 2009). This type of comparisons also has the potential to find domestication genes, such as the teosinte branched1 gene, which carries a mutation respon- sible for the morphological differences between maize and teosinte (Zeder et al., 2006). In one of the most elegant domestication studies using aDNA, tb1 was sequenced in ancient maize cobs from Mexico. This showed that maize genetically similar to modern maize was present in this area at 4400 years ago (Jaenicke-Despres et al., 2003). The recent re-sequencing of the complete genomes from eight different populations of domestic chicken along with the genome of the wild ancestor, Red Jungle fowl, revealed a candidate for a domestication locus in chicken (Rubin et al., 2010). These studies show how powerful the joint analysis of the genome from a domestic species and its wild ancestor can be; unfortunately in cattle the wild ancestor is extinct.

19 The cattle genome under selection The cattle genome consists of 58 autosomes and two sex chromosomes (Melander, 1959), the size is almost 3,000,000,000bp, and when the complete genome sequence was released in 2009 several interesting properties were uncovered (Elsik et al., 2009). There are at least 22,000 protein coding genes in the cattle genome, of which 80% are shared with humans. Genes associated with; reproduction, immunity, lactation, and digestion were over represented when it comes to changes in number and organization (Elsik et al., 2009). There is extensive linkage disequilibrium (LD) in many cattle breeds (Farnir et al., 2000; Qanbari, 2010). LD extends further than in humans, but is hard to detect at distances over 200 kb (Gautier et al., 2007; Gibbs et al., 2009). Two hundred years after the introduction of breed books all commercial cattle breeds form very tight clusters that are genetically separate (Gibbs et al., 2009). This is consistent with the fact that contemporary breeding for alternate agricultural purposes has led to a genome wide divergence among beef and dairy breeds (McKay et al., 2008; Hayes et al., 2009). It also fits well with what is known about the early breed formation, where animals of similar phenotypes were crossed, and the breeders strived for uniformity in visible characteristics (Pitt, 1920). Using Bayesian demographic estimates, both Ho et al. (2008)and Finlay et al. (2007) found that a population expansion was likely to have occurred in cattle close to the time for domestication, the estimates were based on mitochondrial DNA in both studies. The large scale data currently available for cattle allows the estimation of effective population size and demographic history. Using estimates of effec- 2 tive population size (Ne) from the level of LD, as measured by r , a bottle- neck was found and dated to 1500 generations ago. Assuming a generation time of 5 years, this corresponds to the spread of cattle out of the centre of domestication (Gautier et al., 2007). At around this point in time the effective populations size was estimated to 3 000 (Hayes et al., 2008), another significant decline in Ne occurred 50-100 generations ago, placing it some- time during medieval-historic times and predating the start of breed books (Gautier et al., 2007; Villa-Angulo et al., 2009). During the medieval time there was an accelerating development in farming; cattle were also reared differently than before. The low estimates of Ne, between 500-1000 at this time, are most likely an effect of intensified selection of some sort, although not in the modern sense. Consistent with the last 50 years extensive artificial selection an Ne as low as 50 (Qanbari, 2010) has been observed in some breeds. It is well known among animal breeders that there has been an erosion of genetic diversity over the last two decades (Meuwissen, 2009). The selection for high production has in many cases led to health problems in cattle, as breeds selected for

20 high production also suffer from lower fertility among other health issues. Milk production often leads to a negative energy balance, which is harmful for the animal (Breukink & Wensing, 1996). This has lead to a continuous decline of fertility and reproductive efficiency in Holstein cows (Lucy, 2001; Boichard et al., 2003; Pryce, 2004). Compared to another domestic species with a similar number of breeds, (e.g. the dog), cattle must have had a large ancestral population size. The reason for this assumption is that selection has been very intensive in both dogs and cattle during the last few hundred years. But the cattle genome still contains surprisingly high variation. There is even a substantial amount of polymorphism still shared between bison and cattle, two species that diverged more than 2 million years ago (de Roos et al., 2008). These findings are also supported by whole genome SNP analysis. Given the small Ne and the low level of linkage equilibrium, LD, noted by the Bo- vine HapMap Consortium indicates that effective population sizes were much larger in the recent past (Gibbs et al., 2009). However, how recent, is hard to tell. Interestingly enough, Ne has declined for all cattle breeds examined by the Bovine HapMap Consortium, but in humans, during the same time span, the opposite have occurred, an exponential expansion of Ne (Gibbs et al., 2009).

21 Methods

Ancient DNA as a tool to access temporal data The first sequence from an extinct animal, the quagga, was reported in 1984 (Higuchi et al., 1984). With the invention of PCR (Mullis, 1987) it became possible to amplify DNA from degraded material, and ancient genetic data from bone tissue now accessible (Hagelberg et al., 1989) . Although the amplification process produced problems with contamination in ancient material (Zischler et al., 1995; Austin et al., 1997) the field soon expanded, mainly focusing on mitochondrial DNA from extinct species (Bailey et al., 1996; Ovchinnikov et al., 2000; Bunce et al., 2003; Lister et al., 2005). Soon the usefulness of ancient DNA to understand past population histories not accessible with just modern DNA became evident (Leonard et al., 2000; Shapiro et al., 2004; Anderung et al., 2005; Larson et al., 2005; Malmström et al., 2008; Valdiosera et al., 2008).

Properties of ancient DNA There are three major properties of ancient DNA that makes it more difficult to work with compared to DNA extracted from fresh samples. Contamina- tion, fragmentation and degradation all lead to damages in the DNA (Pääbo, 1989; Hofreiter et al., 2001; Malmström et al., 2005; Briggs et al., 2007).

Contamination Problems of contamination of authentic aDNA with extraneous DNA has been widely reported (Richards, 1995; Kolman & Tuross, 2000; Malmström et al., 2005; Sampietro et al., 2006; Linderholm et al., 2008; Green et al., 2009). The problem has mainly been reported from studies of ancient humans, since it is highly challenging to separate authentic sequences from the contaminants. However, when proper protocols are followed (Malmström et al., 2007; Dissing et al., 2008), one can eliminate much of the contamination. Also new analytical tools have been developed to separate authentic aDNA from contamination (Helgason et al., 2007). Contaminating human DNA is also a problem in studies of animal remains (Malmström et al., 2007), but if this is taken into consideration it is possible to design the primers so that they do not target human DNA. In fact, the ratio of authentic animal DNA to human DNA can be used as a measure of the quality of the

22 samples (Malmström et al., 2007). Samples with a ratio of less than 70% cow to 30 % human mtDNA, have for example lower success rate for autosomal single nucleotide polymorphisms, SNPs, (E, Svensson unpublished data). Reports on contamination from modern animal DNA are rare, but do occasionally occur. Leonard et al. (2007) did for instance observe contamination from domestic animal mtDNA in common PCR reagents, and Bol- longino et al. (2008) found cattle bones from caves to be contaminated by goat DNA. These results are disturbing as ancient DNA has been an important tool in the investigations of domestic animals (Anderung et al., 2005; Bollongino et al., 2006; Edwards et al., 2007; Larson et al., 2007a; Larson et al., 2007b; Storey et al., 2007; Scheu et al., 2008; Edwards et al., 2010). Cow, pig and chicken were the most abundant sources for the contamination (Leonard et al., 2007). This is in concordance with some of my own unpublished data, where pig sequences appears in cattle remains. In this particular case, the sequencing feature of the pyrosequencing software was used, so only short fragments of about 50 bp were retrieved (Mashayekhi & Ronaghi, 2007) and it was thus not possible to assign the pig sequence to a particular haplogroup. In order to quantify the level of contamination from domestic animals in ancient DNA extracts and also the risk for cross contamination between samples we typed ancient cattle (n = 9) and sheep (n = 12) remains for three mitochondrial SNPs separating the two species in an experimental set up similar to that in (Malmström et al., 2007). Each sample was extracted twice and for every four DNA extracts two blanks were included. We use the allele quantification feature of the Pyrosequencing software, Figure 1A to measure the amount of cow and sheep DNA in the 21 samples and negative controls. We also quantified the human contamination in the samples. Our data show that the majority of both cattle and sheep samples were contaminated by human DNA. No contamination of sheep DNA was detected. We found a few PCRs (12 out of the 216 PCRs performed) where minute amounts of cattle DNA were found in sheep samples, but not at a level that would affect the actual scoring of a genotype Figure 1B (E. Svens- son, unpublished data). This shows the robustness of pyrosequencing genotyping system to low levels of contamination or sequence damage. Since none of the negative extraction controls indicate any sign of animal contamination we argue that the contamination most likely originates from pre- laboratory handling of the samples. We also conclude that special precau- tions should be taken when only few and fragmented mtDNA sequences are retrieved from poorly preserved samples, since a contamination level of 5%, as detected by Leonard et al. (2007) could give rise to false results. How- ever, large-scale population genetic studies, where large numbers of samples are successfully amplified and numerous replications for each marker are

23 performed are not likely to suffer from misinterpretations of results due to contamination.

Figure 1. Pyrograms from the genotyping of mitochondrial sheep DNA. The puta- tive SNP is variable between sheep (C) and cow (T). In (A) the allele quantification analysis option is used and one can see that there is 9.3 % cattle contamination in this particular PCR. However when the genotype analysis option is used (B) the correct genotype is scored. In this particular case the SNP is a C to T transition, which is also the most common type of DNA damage seen in aDNA, so a low level of damaged could also explain the results. The height of the peaks (relative intensity on the y-axis) is proportional to the number of nucleotides incorporated.

Fragmentation The average fragment size of aDNA is considerably smaller than for modern DNA (Pääbo, 1989; Noonan et al., 2006). Although longer DNA fragments may be present in the material, spontaneous base-loss events, creating non- coding abasic sites, and certain base modifications can block the PCR amplification of aDNA templates (Hoss et al., 1996). The reason for fragmentation is oxidative damages that affects the sugar-phospahte backbone of the DNA and alters it (Hoss et al., 1996). Briggs et al. (2007).showed that purines are overrepresented at positions adjacent to breaks in aDNA Figure 2 shows that even for a relatively well preserved material, 800 year old bones from Scandinavia, fragments longer than 130 bp are rare. A similar pattern is also observed in Cave bears (Pääbo et al., 2004). The problems of fragmentation in degraded material can be overcome by targeting very short fragments (Valdiosera et al., 2006). The fragementation level can actually be used as authentication. Among the guidelines for how to authenticate aDNA is that of appropriate molecular

24 behaviour (Cooper & Poinar, 2000). It states that “PCR amplification strength should be inversely related to product size”. Fragment length is now often used a criterion for the authentication of aDNA (Malmström et al., 2005; Malmström et al., 2007; Linderholm et al., 2008; Green et al., 2009; Malmström et al., 2009).

Figure 2. Frequency (in %) of extractions from cattle samples yielding specific proportion of authentic ancient DNA, n = 117 for 70 and 124 bp, and n = 113 for 178 and 180 bp. More samples provide short authentic aDNA fragments compared with contaminating DNA fragments, whereas less samples provide long authentic aDNA fragments. Figure from Malmström et al. (2007), reproduced with permission from the publisher.

Damages Aside from being fragmented and prone to contamination, aDNA also suf- fers from damaged nucleotides. It is now well established that the most common type of damage is deamination, through hydrolysis, of cytosine to uracil, or possibly to hydroxyuracil. This leads to the C->T or G->A substitutions so often observed in aDNA sequences, the so called type II damage (Pääbo et al., 2004; Briggs et al., 2007). If damaged bases are not taken into account when analysing sequence data retrieved from aDNA, this can lead to overestimates of sequence divergence and misinterpretations of demographic processes (Ho et al., 2007a; Ho et al., 2007b; Axelsson et al., 2008; Rambaut et al., 2009). Note the propor- tionally lower number of damages and errors in a sequence sequenced to low coverage compared to one with high coverage Figure 3.

25

Figure 3. The proportion and type of damages or sequencing errors in relation to the base in the reference sequence. Sequence damages from a sequence sequenced with high coverage (A) and in one with lower coverage (B). Figure from paper (VI).

Genetic markers Over the years a variety of molecular markers have been used including Al- lozymes, AFLPs, and RFLPs (Schlotterer, 2004). Especially microsatellites

26 have proven useful in population genetic studies, and since they are highly variable, only few markers needs to be analysed in order to find structure among populations (Ellegren, 2004; Schlotterer, 2004). While they have been widely used in diversity studies of wild and domestic species using modern DNA (Tapio et al., 2005; Vali et al., 2008; Medugorac et al., 2009; Li & Kantanen, 2010), they have also been used in a few aDNA studies (Edwards et al., 2003; Ricaut et al., 2005; Ludwig et al., 2008; Cappellini et al., 2010; Hawass et al., 2010). However, microsatellites in ancient DNA have yet not become a wider success. The reason for this probably lies in the properties of microsatellites, since they are based on differences in fragment size (Ellegren, 2004), they are not suitable for aDNA since, as we just saw in Figure 2, the success rate of aDNA is highly correlated to the length of the fragment (Wandeler et al., 2003). Also, the instability of these markers, which is also why they contain high levels of variation, also increases chi- mera results (such as Stutter) in degraded or low copy number DNA (Gill et al., 2000).

Single nucleotide polymorphisms SNPs are biallelic markers with a relatively low variability (the mutation rate is estimated to 10-8-10-9 mutations per site per generation) (Brumfield et al., 2003). SNPs are abundant in the human genome, there are approximately 1 SNP per 1000 bp (Shastry, 2002) and cattle have a similar rate of variation. Since they are dispersed throughout the whole genome, SNPs are present both in coding and in non coding regions, and they can be either synonymous or non-synonymous, making them suitable markers for the study of selection. One disadvantage of SNPs is their low information content (Schlotterer, 2004) making large scale studies necessary in order to detect population structure or differentiation. Methods for large scale SNP analysis have been developed, there is for example an 800K SNP chip available for cattle, and thus genotyping modern material is an easy and cheap alternative to retrieve data on genomic variation. Allelic dropout of one allele is a problem when analysing DNA of low quality, but repeating the PCR a sufficient number of times can resolve this (Gagneux et al., 1997). Due to the short fragment length targeted in analysis of SNPs they are excellent markers for aDNA studies (Rompler, 2006; Gilbert et al., 2008b; Ludwig et al., 2009; Malmström, 2010).

Mitochondrial DNA Mitochondrial DNA (mtDNA) is present in multiple copies in almost all cells. It is inherited through the female line and thus not passed on by sperm. There is no recombination in mtDNA. Therefore the only source of variation

27 is mutations. Add to this the high mutational rate and it is easy to see why it has been the preferred marker in many evolutionary studies (Pakendorf & Stoneking, 2005). For example studies on cattle have shown its suitability for tracing population history (Loftus et al., 1994; Bradley et al., 1996; Troy et al., 2001; Edwards et al., 2007). And with the possibility to relatively easy retrieve complete mitochondrial sequences, the amount of information provided by the system has yet increased (Ho, 2010). However, the system also harbours some serious drawbacks, making it unsuitable for certain types of studies. Most important, it is just one locus. Thus, whatever information retrieved from mitochondrial DNA will only reflect this particular systems history, not necessarily the populations, or the species. And if history is yet inferred from the system, it can only be the maternal history (Vila et al., 2001; Lindgren et al., 2004). Although the system has been useful in providing a general picture of the history of a population, it reveals little about selection or breeding. The mitochondrial DNA only contains a few genes of which none, as far as I know, is important in animal breeding. The selective forces working on the mitochondria may be quite different from those acting on most of the nuclear genes (Björnerfeldt et al., 2006).

Y chromosomal DNA The Y chromosome is a paternally inherited marker, and is therefore a good complement to mtDNA for studies of population history (Vila et al., 2001; Lindgren et al., 2004; Pérez-Pardal, 2010). This chromosome is gene poor and with low level of diversity in many species (Hellborg & Ellegren, 2004). Since it does not recombine the only differences observed are those that occur through the accumulation of mutations. The Ne of the Y chromosome is 1:4 of that of autosomes this and the 5-6 times more cell divisions in the male germline can explain the relatively high mutation rate on Y, as compared to autosomes (Charlesworth, 2003; Underhill & Kivisild, 2007).

Sequencing and Genotyping Although recent years technological development in sequencing; the most common approach to retrieve aDNA is still Sanger sequencing, although this is changing rapidly. When sequencing aDNA the full potential of Sanger sequencing is not taken advantage of, since the maximum read length widely exceeds that of the aDNA fragments. Alternative methods, described below, may be more suitable for aDNA.

28 Pyrosequencing This sequencing by synthesis method takes advantage of the properties of several enzymes to detect the incorporation of nucleotides by the generation of light (Ronaghi et al., 1998). The pyrosequencing reaction is performed in real time and the time from preparation to results is short (Langaee & Rona- ghi, 2005). The principles for the method can be seen in Figure 4. It is based on a 4 enzyme system; one nucleotide is added at a time according to a dis- pensation order specific for each SNP assay. If a nucleotide is incorporated by the DNA polymerase inorganic pyrophosphate (PPi) is released, this in turn is converted to ATP by ATP sulfurylase. The ATP then drives the con- version of luciferin to oxyluciferin, by the firefly enzyme luciferase. In this process light is generated and measured by a CCD camera. One initial weak- ness of the method was how to dispose of excess ATP and unincorporated nucleotides. Apyrase, a nucleotide degrading enzyme from potatoes, was thus incorporated into the reaction. It allows the nucleotides to be added sequentially without any washing steps in between. The dATP added to the pyrosequencign reaction is not regular dATP since that produces false signals, acting as a substrate for luciferase, but -thio-dATP (Ronaghi et al., 1998; Ronaghi, 2001; Fakhrai-Rad et al., 2002). An example of the result output from the software, a pyrogram, can be seen in Figure 1. The intensity of the light is proportional to the number of nucleotides incorporated, as can also be seen in Figure 7. G. Pyrosequencing has been found to have a high concordance rate as well as a high proportion of successful genotyping calls (Ronaghi, 2001; Chen et al., 2003). Aside from being used for genotyping, pyrosequencing can also be used for sequencing, as it can routinely amplify around 60 bases (Mashayekhi & Ronaghi, 2007). The technique has been adapted to large scale sequencing (Ronaghi et al., 2007). The main advantages of the pyrosequencing technique for ancient DNA are the short fragments needed, the robustness of genotyping, the allele quantification feature implemented in the software, the speed of the analyses, and that nucleotides adjacent to the SNP are also sequenced. The last feature allows for the identification of er- roneous DNA (especially alternative loci) that may have the same sequence at the SNP site but differ at other positions. Pyrosequencing has been used successfully in a number of high profile aDNA studies (Götherström et al., 2005; Gilbert et al., 2008b; Ludwig et al., 2009; Malmström, 2010). Al- though multiplexing is possible, such systems are complicated to design and most assays, especially for aDNA, are run as single plexes. As larger data- sets, both of SNPs and in sample numbers are becoming more the standard in aDNA pyrosequencing may become more of a secondary method, used for replication of results from e.g. the large scale SNP chips or for analysis of extremely degraded material.

29

Figure 4. Principles of the pyrosequencing reaction. Top panel shows PCR and subsequent clean up. In this example, biotin (represented as a circle) is used for separation of the strands during denaturation and washing. After single stranded DNA is formed a sequence primer is annealed in preparation for pyrosequencing. The example shows the genotyping of a C/T SNP. Since the SNP is sequenced with a reverse sequencing primer, the nucleotides detected in the pyrosequencign software will be G (in this example) or A. If another nucleotide is added to this particular site no light is formed and the nucleotide is immediately degraded by Apyrase. Figure adapted from (http://www.pyrosequencing.com/)

30 SNPstream The SNPstream system is a single nucleotide primer extension (mini- sequencing) method (Syvanen, 2001) based on DNA polymerase assisted allele specific extension of primers located immediately upstream of the polymorphism to type (Bell et al., 2002). Paper IV is, to my knowledge, the first report on this method being applied to ancient DNA. It involves a multiplex PCR reaction with 12 SNP-specific PCR assays, (48 plex is also available), followed by a clean-up step using standard ExoSAP protocol. The multiplex PCR and subsequent extension reactions are organised into different panels, since only two types of fluorescently labelled terminators can be used in each multiplex, for example C and T. The individual SNPs within the multiplex are identified according to the position of the arrayed oligonucleotides within each well, using the comple- mentary 5´tag sequence of the extension primer Figure 5. The captured fluorescent primers are scanned and a CCD camera is used to photograph the intensity. Individual sample genotype data is then generated based on the relative fluorescent intensities for each spot, and then computer-processed (Bell et al., 2002; Syvanen, 2005). An example of the graphical display can be seen in Figure 6. For modern DNA the success rate is high (96-98%) and the accurate call rate is >99% (Backström et al., 2006; Ragoussis, 2009). Our ancient samples had much lower success rate, ranging from 30-80% depending on the quality of the DNA. Due to allelic dropout the accuracy for call rate was also lower than for modern DNA, but not different from what is seen when pyrosequencing is used (paper IV).

31 .

Figure 5. The principles of the SNPstream method. A primer anneals immediately upstream to the SNP, ddNTPs labelled with two different dyes (TAMRA (T), Fluo- rescein (F) are added and a single elongation step is performed. The locus specific oligonucleotide is used for hybridization. Figure adapted from http://www.medsci.uu.se/molmed/snpgenotyping/methods.htm

32

Figure 6. Allelic SNP discrimination scatter plot. It shows the clustering of genotypes for some of the ancient samples from paper (IV) in the SNPstream analysis. The three genotype groups form three clusters, TT in the blue cluster, CT in the orange and CC in the green cluster. A) Example of allelic dropout/deamination leading to C-T, the black dot in the circle is a duplicate sample that is genotyped as CT once while the majority of genotypes are CC. B) The same sample as in A genotyped for another SNP, also this time the majority of replicates fall in the CC cluster, but one is typed as CT. Also note all the samples that don’t reach the quality cut off, pink dots under the horizontal line.

454/FLX The 454 (or as it is called now, FLX) was the first of the so called second generation sequencing platforms to be introduced (Margulies et al., 2005). It is based on emulsion based library construction and then sequencing by synthesis in picolitre-sized wells Figure 7. Unless specific regions are targeted by PCR or capturing, all the second generation sequencing techniques relies on shot gun sequencing. The 454 sequencing chemistry is the same as the one used in pyrosequencing (Ronaghi et al., 1998), but the solid support chemistry allows the extension of read length (Margulies et al., 2005) be- yond that of regular pyrosequencing (Mashayekhi & Ronaghi, 2007). The 454/FLX technology was adapted to aDNA shortly after its introduction (Noonan et al., 2006; Poinar et al., 2006), the low sequencing cost per base and the for aDNA so suitable fragment length of 100-300 bp (Pääbo, 1989) has made it the preferred technique in many aDNA studies (Gilbert et al., 2007; Gilbert et al., 2008a; Green et al., 2008; Hofreiter, 2008; Meyer et al., 2008; Millar et al., 2008; Malmström et al., 2009; Miller et al., 2009; Willerslev et al., 2009; Edwards et al., 2010). Whole mitochondrial genomes have become standard, and the first whole complete ancient genome retrieved using 454 technology has also been published (Miller et al., 2008).

33

Figure 7. The principles of FLX sequencing. (A) Genomic DNA is isolated and fragmented. (B) Adaptors A and B are ligated onto the fragmented DNA, and the DNA is made single stranded. (C) Fragments are bound to beads, favouring one sequence per bead. (D) Emulsion PCR of one bead per PCR reaction droplet is run. The result is beads carrying millions of copies of a unique DNA template. (E) Beads carrying single-stranded DNA are deposited into wells of a fiber-optic slide, one bead per well. Smaller beads carting the enzymes needed for sequencing are also deposited into each well (not shown). (F) The clonal fragments are sequenced using pyrosequencing. G) The flow order for a particular sequence. The intensity of the light is proportional to the number of nucleotides incorporated. Figure courtesy of 454 Sequencing © 2010 Roche Diagnostics.

Other next generation sequencing techniques as Solexa has seen an improvement in read length, and earlier this year the first whole ancient human genome retrieved with Solexa sequencing was published (Rasmussen et al., 2010). Since Solexa sequencing generates a lot more reads per run and is cheaper per base (Shendure & Ji, 2008), it will probably become more common in aDNA studies (Allentoft et al., 2009; Krause et al., 2010). The development of true single nucleic acid sequencing may be the next step forward in the retrieval of ancient DNA. There are several techniques available for this, of which HELISCOPE is one. Other advertised platforms are based on similar pretreatment as the 454, but new concepts for the sequencing technique, for example Ion Torrent. It remains to be seen which ones that survive the competition.

34 Analysing temporal data In many cases aDNA have proven to be crucial in order to fully understand the genetic history of a species (Shapiro et al., 2004; Dalen et al., 2007; Valdiosera et al., 2007; Malmström et al., 2008; Valdiosera et al., 2008; Campos et al., 2010). However, Depaulis et al. (2009) showed that the ne- glect of acknowledging heterochrony in a dataset can result in substantial misinterpretations of past population history. Especially two programs have been developed for the analysis of sequence data from multiple time points: Serial Simcoal (SSC) (Anderson et al., 2005) and BEAST (Drummond et al., 2005; Drummond & Rambaut, 2007). SSC is a program used to simulate population genetic data from multiple time points, it is possible to model complex demographic scenarios, and it has been used in a number of aDNA studies (Valdiosera et al., 2008; Malm- ström et al., 2009; Campos et al., 2010). Although temporal data may provide for more power in hypothesis testing, there are also specific problems relating to the interpretation of such data. Navascues & Emerson (2009) show that coalescence-based Bayesian inferences using heterochronous mtDNA are susceptible to an upward bias in the estimation of substitution rates. This is caused by misspecifications of the model used when data comes from populations with complex demographic histories. If post mortem damages are not properly accounted for in phylogenetic studies using ancient DNA, they will mimic variation in the most recent parts of a phylogeny, and thereby provide for overestimations of variation, mutational rates, and eventually the rate of the clock in the tree (Axelsson et al., 2008). BEAST uses a Bayesian framework to estimate mutation rate and effective population size in data sampled from multiple time points (Drum- mond et al., 2002; Shapiro et al., 2004; Ho et al., 2008; Campos et al., 2010). None of the above mentioned programs use the infinite sites model, appropriate when simulating or analyzing SNP data. However, SNPs can be a useful tool in aDNA as estimates of allele frequency data from multiple time points can be a powerful approach to detect for example selection. Changes of allele frequencies are often related to the strength of selection (Bollback, 2008) thus, the frequencies will change over time. Such changes may also be explained by demographic events, or by genetic drift, even in reasonably large populations (Ramakrishnan & Hadly, 2009). The newly developed program COMPASS (Jakobsson, 2009) allows the investigation of this, and is suitable to use with most nuclear molecular markers as it uses the infinite sites model. In a recent study Malmström et al. (2010) used COMPASS to show that the frequency observed in an ancient population could only be explained by population replacement or very strong selection.

35 COMPASS was also used in paper IV in this thesis in order to separate selection from drift in several autosomal SNPs. COMPASS and BEAST suffer from not allowing multiple populations, but when analysing DNA from temporal data, one should keep in mind that it is rarely possible to work with the concept of populations, mainly due to the lack of exact dating and origin of the samples. Of course populations could be inferred using a structuring algorithm, for example STRUCTURE (Pritchard et al., 2000), or similar programs. However, such analysis requires a fair number of individuals, and much genetic data from each of them, which is not easily obtained from ancient samples. Another way to explore past demographic events is the level of heterozygosity. This is a quite simple but effective tool to investigate signs of selection, since variation in genetic diversity, especially lower individual heterozygosity, can be taken as a sign of selection. Genetic drift can also lower the level of heterozygosity, but it is expected to affect every marker in the genome. By including neutral markers in the study the effect of genetic drift can be assessed, thereby allowing us to single out genes that demonstrate extremely low levels of heterozygosity, which in turn is a sign of selection. One can thus compare changes in individual heterozygosity from different time points to explore past changes in selection pressure.

Ascertainment bias The systematic bias introduced by the criteria used to select the individuals/loci in which genetic variation is assayed, is a major problem in many population genetic studies (Brumfield et al., 2003; Morin et al., 2004; Schlotterer, 2004). In SNP studies this mainly occurs due to the selection of the most variable loci from just a small panel of individuals used for SNP discovery, and then typing those SNPs for populations other than the one used to ascertain the SNPs. The method used to ascertain the SNPs can have a large effect (Nielsen & Signorovitch, 2003). The problem has been ac- knowledge for quite some time, see for example Rogers and Jorde (1996). A recently published striking example is that by Schuster et al. (2010), when African individuals were genotyped on a large scale SNP chip developed mainly from European SNP data, the Africans expressed lower heterozygosity than the Europeans, however genomic data shows that there is a high proportion of heterozygous SNPs in these individuals. One of the major effects of ascertainment bias is that rare SNPs are miss- ing since loci with alleles at low frequency are not selected. This selection of high frequency SNPs can make the time to the MRCA longer (Clark et al., 2005). The lack or rare alleles increases average heterozygosity of polymorphic sites, while the average heterozygosity of all sites is underestimated since SNPs with rare alleles are excluded (Clark et al., 2005).

36 In order to avoid ascertainment bias in ancient DNA studies, one would need to do large scale sequencing to find polymorphisms present in the ancient population. Previously this was an impossible task given the high fragmentation of ancient DNA and poor preservation of DNA. However with the introduction of high-throughput sequencing into the field of ancient DNA (Noonan et al., 2006; Poinar et al., 2006) it is now feasible (Allentoft et al., 2009). SNPs ascertained from ancient material are still quite rare but recently an ancient human genome was published (Rasmussen et al., 2010), where SNPs previously not reported were found. This shows the potential of ascertaining SNPs in ancient samples, however it should be stressed that the preservation conditions for this specimen was excellent (Hoss et al., 1996; Smith et al., 2003), which is not the case for most ancient samples.

37 Research aims

General aims The main aim of this thesis was to investigate the pattern of genetic variation in cattle populations not yet exposed to modern breeding strategies, as well as in the extinct aurochs. This was achieved through both large-scale sequencing and genotyping of autosomal SNPs of ancient, modern and historical samples.

Specific aims I Explore the variation in Y chromosomal introns in the extinct Euro- pean Aurochs. II Evaluate the use of a Y chromosomal SNP as a phylogeographic marker for ancient cattle. III Evaluate morphological sexing methods used in archaeozoology by developing a method for molecular sex identification. IV Investigate changes in farming practice with a combined genetic, osteological, and archaeological approach V Investigate the potential of autosomal SNPs to trace genetic changes in cattle over time. VI Use coding and neutral SNPs typed in ancient and modern cattle to detect genes under selection in ancient cattle.

38 Summaries of papers

Paper I: Insights into Y chromosomal genetic variation and effective population size in the extinct European aurochs Bos primigenius. The now extinct aurochs is the progenitor of all domestic cattle (Loftus et al., 1994; Bradley et al., 1998; Troy et al., 2001). The last female, died in 1627 in the Jaktorów Forest, Poland from natural causes (Rokosz, 1995), the information about population size and structure is scarce if not non existing. We used PCR targeted FLX sequencing of six Y chromosomal introns previously characterized in cattle (Hellborg & Ellegren, 2003; Götherström et al., 2005), to investigate the genetic diversity in aurochs. We sampled ten aurochs from England, Hungary, Germany and Poland, dating from Bronze Age to early medieval time. DNA was extracted according to (Svensson et al., 2007) and PCR amplified using a multiplex approach to cover all the introns. Fragments from these individual were MID tagged and sequenced using the 454 platform. We retrieved a total of 1124 bp from six Y chromosomal introns, in 8 of the aurochs. The previously reported UTY19 A/C SNP was detected in all eight aurochs, placing them in haplogroup Y2. The ancient distribution of Y1 and Y2 haplotypes as identified from the UTY19 SNP suggests it is not possible to discriminate between Near Eastern and European Y- chromosomal lineages. A total of four polymorphic sites remained in the 1124 bases after correcting for damages and sequencing errors. We used Bayesian simulations to analyse the likely background for this pattern. By varying the mutational rate and effective population size within reasonable limits and under a constant population size, we estimated the probability for finding exactly four polymorphic sites, and also for finding four or less polymorphic sites in our em- piric data. We also estimated the possibility of finding exactly one polymorphic site in modern domesticates under similar conditions and compared it to a previously published dataset (Götherström et al., 2005). Assuming a mutational rate in between what have been suggested for cattle compared to bison and water buffalo (Nijman et al., 2008), the male effective population size is likely to have been somewhere between 20.000 and 80.000 individuals.

39 The European aurochs population is likely to have retained an effective population size large enough to maintain much variation. This could have effect on future investigations on possible hybridization between domestic cattle and European aurochs. That is, if it occurred, it should have brought in notable amounts of variation into domestic cattle in the areas where it occurred.

Paper II: Temporal fluctuations of Y-chromosomal variation in Bos taurus. Phylogeographic patterns observed in modern species often look very clear and give strong signals for population genetic subdivision based on geographic locations (Taberlet et al., 1998). However on more than one occa- sion patterns seen in modern data do not hold up when temporal data is analysed (Valdiosera et al., 2007; Valdiosera et al., 2008), this is true not only for wild species but has been found in humans (Krause et al., 2007) as well as in dogs (Malmström et al., 2008). In modern taurine cattle there is a strong pattern for a north south gradient seen when looking at the Y chromosome. Taurine cattle have been found to belong to either haplogroup Y1 or Y2, where Y1 is more common in western/ northern European breeds and Y2 is more common in southern Europe (Götherström et al., 2005). Hap- logroup Y1 has also been found to be common in aurochs and Neolithic cattle. In this study we typed 38 historic cattle remains mainly from Scandi- navia and 3 aurochs, for a SNP in the UTY19 intron that has been shown to separate the two haplogroups (Götherström et al., 2005). We did not find any geographic structure in the ancient samples, instead we found fluctuations over time, and for example medieval Swedish bulls had allele frequencies similar to modern south European bulls, while 18th century Swedish bulls were more similar to modern breeds from Britain and Holland than to Swed- ish cattle. Thus, this Y chromosomal SNP is not a good marker for aurochs introgression into domestic cattle, but the pattern observed is more likely an effect of recent cattle keeping. This marker thus has the potential to uncover past changes in the male lineages of cattle both over time and space.

Paper III: A DNA test for sex identification in cattle confirms osteometric results. The study of animal bones from archaeological sites, zoo archaeology, can provide important insight into the relationship between humans and animals in pre-history. One of the most important and commonly performed analyses of the bones is measurements for sex identification. This is often crucial in

40 order to understand different strategies of animal exploitation. For example an economy mainly based on milk production will produce a kill off pattern with a lot of young cattle males and a lot of older female animals, while the natural attrition pattern expected in case of disease outbreak or poor husbandry makes it equally likely for males and females to die. In cattle (Bos taurus) one of the most commonly used fragments for sexing of skeletal elements is metapodials (Armitage&Clutton-Brock 1976, Vretemark 1994, 1997, Mennerich 1968, Thomas 1988). It has been shown that there is a sexual bimodality in the breadth of the distal trochlea of the metapodials of cattle due to the impact of heavier weight load on the fore limbs (Griegson, 1982, Jewell 1963, Vretemark, 1997). In order to evaluate the morphological system a genetic assay for sex identification in cattle was developed. It is based on the two homologous genes ZFX and ZFY located on the X and Y chromosome respectively (Aasen & Medrano, 1990). A SNP differentiates between males and females. In position 243 both males and females have a T on the ZFX gene, but males have a C instead of T on ZFY (Werner et al., 2004). The use of a SNP for sex identification is especially suitable for ancient DNA since only a short fragment needs to be analysed, as fragment size is correlated to success rate in work with ancient DNA (Noonan et al., 2005; Noonan et al., 2006; Malmström et al., 2007). Twenty six metacarpals from the Swedish medieval town Skara were randomly selected. Measurements were taken according to Bartosiewicz et al. (1997). DNA was extracted as in (Malmström et al., 2007). Pyrosequenced, in both directions, was used for genotyping (Ronaghi et al., 1998; Ronaghi, 2001; Svensson et al., 2007). When working with ancient DNA allelic dropout is a factor that needs to be taken into account, we therefore estimated the occurrence of allelic dropout and the minimum number of genotypes needed in order to confirm a female. The dropout rate was 26%, but if 4 PCRs per sample are performed the probability of mis- typing a male as a female is p<0.001. The molecular sex could be identified from 22 of the bones; in all of the bones the molecular sex was confirmed. When logistic regression was used to test for correlation between genetic status and morphology a strong sig- nificance was found (Chi2=28, 75,625, p=<0.001). Our results thus support the reliability of traditional osteological sex identification. Traditionally ancient husbandry systems have been analysed via the construction of demographic profiles that integrate the sample distribution of age at time of death with sex ratio. This type of analysis has been hampered by an inability to attribute age and sex to a single specimen. Thus the sample of individuals represented by the mortality profile may not be the same individuals as those represented in male/female ratios, and the distribution of the sexes across age classes, an essential parameter in husbandry systems, cannot be directly estimated just from morphological data.

41 Although the DNA based sex identification is costly both in time and resources it can be especially useful in cases when osteolgical analysis cannot be performed, for example on young animals or if the material is very fragmented.

Paper IV: Typing Late Prehistoric cows and bulls- osteology and genetics of cattle at the Eketorp fortification and settlement on Öland, Sweden. Our knowledge on strategies for cattle breeding in Scandinavia mainly relies on osteological studies of bones recovered in archaeological settings. Body size has been used to a wide extent to discuss prehistoric breeding and changes in body size have often been seen as a sign of improvement of livestock e(Davis & Beckett, 1999; Albarella et al., 2008; Davis, 2008). The presence of cattle breeds is difficult to evaluate since the observations at Eketorp are based on a limited number of skeletal elements. To rely solely on osteometric data may lead to over interpretations of the data since differences in animal husbandry also affect the size and robustness of cattle. Fur- thermore, all skeletal elements are not equally suitable for comparisons. Analyses of aDNA offer possibilities to identify breeds and may complement with types of information that osteolology cannot provide. Here we combine an osteological and molecular approach in order to investigate breeding strategies and animal usage at the Eketorp fortification on the Isl- and of Öland in Sweden. Eketorp is one of a few large and well excavated late Iron Age sites in southern Scandinavia, and may be used as a model site for the rest of the area. The fortification was inhabited during three different periods; here we compare the use of cattle in period II (400-700AD) to that during period III (1000-1300AD). More than 600 metapodials were selected for osteological analysis; bones were selected to represent the full morphological range as well as samples displaying pathologies. Also 28 calves from both period II and III were selected for molecular sexing to investigate the slaughter patterns of male and female calves. One hundred and thirty three of the elements were selected for molecular sexing, and part of the samples were also genotyped for three SNPs involved in coat color, disease resistance and growth. All males were also genotyped for the SNP differing between Y1 and Y2. Using SNPs to detect the process of breeding is easier than to detect specific breeds, as breed identification requires a large number of genetic markers (McKay et al., 2008). We expect that breeding would cause changes in single genetic systems over time to a much stronger degree than genetic drift.

42 We did uncover trends in various measurements that we interpret as a shift, possibly towards a farming economy where cattle gained a new role. We also discovered patterns, which are compatible with early breeding and an increasing level of specialisation. We see a varied use of cattle at Eketorp and utilization strategies that require breeding efforts and conscious deci- sions on actions such as castration. For example, draught oxen had to be trained. There was a change in kill off pattern, suggesting that meat was more important during the later phase of the settlement. All but two males, one from period II and one from period III, belong to haplogroup Y2 suggesting that the population was quite homogenous also over time. There was a clear difference in the use of males and females, as visible from the pathologies, males displayed pathologies associated with draught, and females displayed pathologies for which the cause is not fully under- stood. Despite of this we have found little evidence for the use of specific breeds for specific purposes. The usage of the bulk of the cattle seems to have been constant from period II to period III, indicating that the shift we describe was not a fast one. The use of specific (genetic) breeds seems to be a later phe- nomenon. However, taken together, the genetic result and the morphology suggest that there is a difference between the population from period II and that from period III.

Paper V: Tracing genetic change over time using nuclear SNPs in ancient and modern cattle. The knowledge of animal husbandry during medieval and historic time is scarce, and in need of further investigation (Albarella, 1999). The analyses of ancient autosomal DNA, and in particular coding DNA, holds great potential for the study of past evolutionary events (Brown, 1992). Since aDNA is highly fragmented (Pääbo, 1989), SNPs are particularly suitable markers for targeting nuclear DNA since only short fragments are needed to retrieve the genetic information. And they are also suitable for investigating breeding and breeds, as many of them are in the coding region, or linked to them. In order to investigate the possibilities of tracing genetic change through time using nuclear markers we selected three SNPs that have been associated with phenotypic traits in modern cattle. The melanocortin receptor 1 (MC1R) influences coat colour, the SNP we analyzed results in dominant black coat colour (Klungland et al., 1995). The SNP analyzed in the Leptin (LEP) gene has been associated with body fat deposition as well as milk yield and content in dairy breeds (Buchanan et al., 2002; Buchanan et al., 2003). Finally we analysed one SNP in the Toll-like receptor 4 (TLR4), this is a candidate gene for disease resistance, the ancestral allele is found in both

43 taurine and indicine cattle, while the derived allele is only found in taurine cattle (White et al., 2003). We successfully extracted (Yang et al., 1998), PCR amplified, Pyrose- quenced (Ronaghi et al., 1998), and retrieved genotypic results from 111 medieval and historic cattle bones as well as from 93 cattle from modern breeds. The DNA preservation for 64 of the bones was also assessed using collagen extraction and quantification (Collins, 1998). We analysed these data using common statistics and found a decline in individual heterozygosity for all three SNPs. We interpreted this as possible signs of selection, however since no neutral markers were analysed we could not rule out genetic drift as the cause of the decrease. We did show that the method holds great power to investigate the development of variation over time using SNPs in ancient material.

Paper VI: Signs of contrasting selection in medieval and modern North European cattle Cattle are the most important animal for humans throughout history, as it has provided food, hides and draught labour. But although selection for beneficial traits to humans is likely to have occurred, little is known prior to the formation of breeds during the 19th century (Wheaton-Smith, 1960). By targeting 8 SNPs in region likely to have been under selection; milk, meat, colour and disease resistance (Klungland et al., 1995; Lien et al., 2000; Bu- chanan et al., 2003; White et al., 2003; Nilsen et al., 2009), and 6 neutral SNPs (Werner et al., 2004), we aimed to separate signals changes caused by drift from other demographic signals, such as selection. We hypothesize that observed changes in the level of heterozgosity and allele frequencies over time in coding markers, given that no significant change is observed in neutral markers, would be a sign of selective breeding. The medieval period was a time of new innovations in farming (Thoen, 1997; Davis & Beckett, 1999) and it is likely that it affected also the cattle, therefore we focused on material from this period. We used pyrosequencing and SNP stream to genotype 426 ancient and modern cattle for the 14 SNP. Heterozygosity and allele frequencies were compared over time and the program COMPASS (Jakobsson, 2009) was used to simulate different demographic scenarios. There is a general trend for a decrease in heterozygosity in coding markers, while most of the non-coding markers either did not change in heterozygosity level or even increased over time. In particular MC1R and IGF1 stand out as showing a significantly lower heterozygosity in modern animals (p=0.00094 and p=0 respectively). If a constant population size of 1000 individual is assumed, which is a reasonable historic population size for cattle (Qanbari, 2010), the allele fre-

44 quencies in modern cattle for all SNPs except MC1R can be explained by drift. To accommodate the modern allele frequency Ne must have been as low as 100 to 200 individuals at some point in time, if the larger Ne is assumed the prehistoric Ne must have been as low as 1000 individuals. When this reduction in Ne happened is harder to pinpoint, given the data from our simulations, it is equally likely to have happened 50 years ago as during medieval times. We have shown that there was small but significant differences in allele frequencies that have been associated with coat colour and growth, between cattle populations from different localities in Iron Age and Medieval Swe- den. However none of these changes were as dramatic as the ones taking place during the last 300 to 50 years. The formation of breeds and the subsequent use of only a few bulls and artificial insemination have lead to an almost complete depletion of individual genetic variation as shown by several SNPs in coding genes, with the major changes occurring in the IGF1 and MC1R genes.

45 Conclusions and future prospects

In this thesis I have shown that analysis of nuclear markers dispersed in the genome can be used to detect signs of past selection. And, that there is much information to be gained by using aDNA as a tool when investigating past selection pressures and demographic history in a species. However, relying on individual SNPs to detect population differentiation is not feasible with the genotyping techniques used in this thesis. Since the information content in SNPs is low, a large number of SNPs is needed in order to assign individuals to populations. One alternative is to select markers that form haplotypes, since haplotypes contains more information than SNPs. This way differences in haplotype frequencies can be detected between different times and locations, even if no signal in allele frequency is detected. Another way is to take advantages of the Bovine SNP chips. Although ascertainment bias will be a major issue when using them for ancient DNA they could provide information on past population structures. Even if only a fraction of the SNPs on the chip will work for ancient DNA that amount will by far exceed that of the largest ancient SNP studies so far. In the near future it may even be possible to sequence the genome of a sample of ancient individuals, and use the genome data to design new SNP chips, and thereby overcome the ascertainment bias. With large scale shot gun sequencing combined with targeted sequencing of ancient genomes, long lost variation can be discovered and regions of specific interest for further understanding of the domestication process and early selection can be investigated. The archaeozoological record is full of interesting material that has the potential to answer questions about early cattle keeping, cattle origin and prehistoric breeding. For example sites where there are morphological differences between bones at different time points or even at the same time. There are sites where shifts in the bone assemblages can be associated to different cultures, just to mention some of the questions that wait being re- searched. Here I have shown that genetics in combination with extensive osteological investigations can be a lot more powerful than any of the two disciplines on its own to address these questions. With the new techniques applied to aDNA it should be possible to thoroughly investigate the origin of today’s breeds and to finally find an answer to the question if there was any introgression between European aurochs and domestic cattle. During my years as a PhD student I have had the opportunity to experi- ence a methodological revolution; large scale sequencing has made it possi-

46 ble to retrieve large amounts of data from ancient DNA (Rasmussen et al., 2010), and I think that we are only in the beginning of this exciting and complete shift of paradigm. So far genome scale ancient DNA have focused mainly on well preserved permafrost specimens (Gilbert et al., 2007; Gilbert et al., 2008c; Rasmussen et al., 2010) or with unusually large resources at hand (Green et al., 2008) if the preservation is poorer. Now the first complete aurochs genome is underway (Edwards et al., 2010), and an initial test of using the large scale SNP chips to genotype aDNA looks promising (Decker et al., 2009). These technological advances in combination with development of better methods for analysing the data (Anderson et al., 2005; Helgason et al., 2007; Bollback, 2008; Depaulis, 2009; Jakobsson, 2009) will truly allow for the full potential of using temporarily sampled DNA. Finally, something we tend to forget is that with the move into this era of large scale retrieval of data, the authentication criteria will need to be ad- justed.

47 Svensk sammanfattning

Steget från jägare-samlare till bofasta jordbrukare är kanske det mest revolu- tionerande i mänsklighetens historia. Våra vanligaste tamdjur, ko, gris, får och get domesticerades alla i området kring Eufrat och Tigris, för omkring 10 000 år sedan. Jordbruket spreds sedan med en hastighet om ungefär 1 km per år till närliggande områden och Europa. Den nötboskap som är vanligast i Europa i dag bär alla på mitokondrieDNA som har sitt ursprung från dessa områden, dvs. den fertila halvmånen. På så sätt har man med genetik kunnat spåra kons ursprung till den vilda uroxen. Det har även funnits uroxar i Eu- ropa men det finns inga entydiga bevis för att dessa skulle ha bidragit gene- tiskt till dagens nötboskap. Uroxen är idag utdöd, den sista oxen dog i Polen 1627, därför är det svårt att göra genetiska jämförelser mellan uroxar och moderna kor. Men det är ändå möjligt, med hjälp av gammalt DNA, dvs. DNA från arkeoosteologiska lämningar och modern storskalig sekvenseringsteknik har vi sekvenserat över 1000 baser från uroxens Y kromosom. Med simuleringar har vi upp- skattat den effektiva populationsstorleken i uroxar under de senaste 5000 åren till mellan 20000 och 80000 hanar. En sådan stor population bör ha kunnat behålla mycket av den genetiska variationen över ett långt tidsinter- vall, och även under den tid de delade Europa med tamboskapen. Genetiska skillnader på Y-kromosomen används ofta för att visa på ursprung av olika populationer, i modern nötboskap räcker det med en variabel position, så kallad SNP, för att separera djuren i två genetiska grupper, så kallade haplogrupper, Y1 och Y2. Dessa grupper är separerade geografiskt, med Y1 framför allt i nord-västeuropeiska raser, medan Y2 är vanligast i syd-europeiska och turkiska raser. Denna SNP har tidigare analyserats i uroxar och tamkor från bondestenåldern i tyskland. Det visade sig då att alla uroxar tillhörde Y1 gruppen, vilket sågs som ett tecken på att vilda tjurar hade parat sig med tamkor och på så vis fört vidare sitt DNA till nu levande individer. När vi analyserade fler uroxar, från Ungern och England, samt 38 tjurar från Sverige, fann vi inte samma tydliga geografiska mönster. Alla uroxar tillhörde Y2. Detsamma gällde för de medeltida korna, medan det i djur från 1700-talet fanns lika många från båda grupperna. Moderna Svenska djur tillhör alla Y1. Så istället för att visa på hybridisering mellan tamkor och uroxar för flera tusen år sedan, tyder det genetiska mönstret från denna markör på att djur var mobila under senmedeltid och historisk tid och att

48 dagens Y-kromosomala mönster är ett resultat av bildandet av raser på 1800- talet och den avel som följde därefter. Trots den mångfald av raser som finns i världen idag är många hotade an- tingen för att det finns för få djur, detta gäller till exempel lantraserna, eller för att man med avel har drivit djuren till en mycket hård inavel. Avel för vissa önskvärda egenskaper medför en minskad genetisk variation i de regi- oner av genomet där generna för dessa egenskaper finns Trots att kor är ett av våra vanligaste husdjur, och kanske det allra viktigaste, vet man förvån- ansvärt lite om hur man har avlat före 1800–talet. Det finns bara ett fåtal källor som beskriver olika typer av djur med olika egenskaper. Om aveln var systematisk under en längre tid innan dagens moderna avel startade kan det ha konsekvenser för hur man bör bredriva modern avel. Tecken på tidig avel skulle också vara ett bevis för kons viktiga ställning i samhället, samt visa på hur komplex och utvecklad kreaturshållningen var under vikingatid och medeltid. Många studier av förhistorisk boskapsskötsel är beroende av analys av djurben och ofta har könsbestämning en central roll i tolkningen av hur man har använt djuren. Jag har med ett molekylärt test för kön testat om de mått som traditionellt används för könsbestämning är korrekta. Jag kom fram till att överensstämmelsen mellan de morfologiska måtten och det molekylä- ra testet var total. Men det är inte alla ben som går att könsbestämma baserat på mått, i sådana fall är det molekylära testet ett bra alternativ. Genom att kombinera genetik med arkeologi och undersökningar av djurben undersökte vi om det fanns några skillnader mellan de olika korna funna vid Eketorps fornborg på Öland. Denna borg har varit bebodd under flera perioder, då den fungerat som en lokal centralort, men stått tom däremellan. Borgen fungerar alltså som en tillförlitlig modell för jordbruket i Sydskandi- navien under järnålder och tidig medeltid. Jag undersökte ett stort antal ben från Järnåldern och vikingatiden, och kom fram till att det fanns små men ändå tydliga skillnader i hur man använde sig av djuren under de två perio- derna. Mjölk var viktig under både järnålder och Vikingatid, men slaktmöns- ter tyder på att kött blivit viktigare under den senare delen av borgens bo- sättning. För att undersöka om det går att använda genetiska markörer från kärn DNA (SNPar), för att hitta skillnader mellan kor från olika tidpunkter, ut- vecklade jag ett system med tre stycken SNPar, i gener som kodar för päls- färg, mjölkmängd och sjukdomsresistens. Det visade sig att dessa markörer fungerade väldigt bra och jag kunde analysera över hundra medeltida och historiska kor från Sverige, samt jämföra resultaten mot moderna djur. Jag fann att det skett en kraftig minskning i individuell genetisk variation sedan medeltiden och fram till idag. Men eftersom bara markörer i kodande regio- ner analyserats var det svårt att med säkerhet säga att detta berodde på avel, det skulle även kunna vara en slumpmässig process, så kallad genetisk drift. Eftersom metoden var så framgångsrik gick vi vidare med en större studie. Över tvåhundra djur främst från Sverige, men även från England, analysera-

49 des för 14 SNPar, 8 av dem i gener som kodar för mjölk, pälsfärg och stor- lek, samt 6 stycken så kallade neutral markörer, SNPar som inte medför nå- gon aminosyraförändring och som inte heller sitter i närheten av någon gen. Neutrala SNPar har troligtvis inte varit föremål för selektion, varken artifici- ell (dvs. avel) eller naturlig. Denna större studie visade på en förvånansvärt liten genetisk skillnad mellan medeltida kor och moderna raser när det gäller markörer i mjölk gener, men mycket stora skillnader i en gen som kodar för pälsfärg. Det visade sig att svart färg blivit betydligt vanligare i moderna djur än det var under medeltiden och historisk tid. Dessutom har den indivi- duella variationen minskat kraftigt i denna markör och allt tyder på att detta är ett resultat av kraftig avel under rasernas tillkomst på 1800-talet. Mina studier har visat på vikten av att inte enbart förlita sig på moderna data när man försöker förstå en arts genetiska historia, utan även att analysera historiska data. Jag visar också att även om en viss form av avel ägde rum under järnålder/medeltid så går dess effekter inte på långa vägar att jämföra med det som skedde med kons genom då de moderna raserna bildades.

50 Acknowledgements

First of all I want to thank my supervisor, Anders Götherström. It has been very inspiring to work with you; these years have been the most fun! I could not imagine a better PhD supervisor or collaborator. You are always so en- thusiastic (even if it’s just over a gel picture), and full of good ideas. Professor Hans Ellegren, thank you for providing an excellent research environment, being part of the Department of Evolutionary Biology is very inspiring. The first “aDNAers”, Cia Anderung and Helena Malmström for being such good support and fun company during my first years at EBC. I have learned a lot from you! The new group, Eva and Oddný, you’ve been so supportive and kind! Helena, thank you for a lot of fun and stimulating conversations about family and research, and for reading and discussing last minute things, it really helped a lot!!! Linus, Pontus, Carolin the always en- thusiastic exam workers! And Frida, it’s been a lot of fun to work together with you, let’s write some papers now! The work in this thesis could not have been done without the help from my collaborators. Erik Axelsson, Mia Vretemark, Tom Gilbert, Eske Wil- lerslev, Per Persson, Ylva Telldahl, Janne Storå, Gustav Malmborg, Colin Smith, Jurgita Baubliene, Linas Daugnora, Daniel Makowiecki, Terry O’Connor and Mattias Jakobsson. After many years in the same office, I’ve had the opportunity to share a lot of good (and bad) moments with some fantastic people. Niclas and Santi, thanks for everything but especially for “fabriken”, and the opportunity to make and listen to very funny phone calls. Anna-Karin, thanks for bringing some equality to the office, both gender wise and in messiness. The new office crew! Nicklas S, Carina S and Lucie, thank you for all sorts of distractions, R-programming, encouragement and Coke! Past and present co-workers at the department of Evolutionary Biology, this is truly a great place to work at, and it’s all thanks to the nice and smart people there. No one mentioned, no one forgotten...but I do want to thank some people in particular. Carles Vila, thank you for accepting to be my co-supervisor, and for helpful discussions. Jennifer Leonard, for science and baby talk. Nilla, Ce- cilia and Malin for always trying to help out if possible, for nice conversations and for keeping the lab up and running! Kristiina, thanks for being a friend in good and in bad, and for cheering me on! Rebecka, thanks for good

51 company during my occasional 10am coffees, for stipend discussions and for making the department a happier place. Frank, Mikael and Axel thank you for your everlasting (?) patience, and help with various computer problems. Johan and Jochen, thanks for forwarding interesting papers. Hanna, thank you for lots of fun conversations, for all your support with the teaching and for the comma! Holger, for offering to help, when I needed it the most, and for saving me from having numbered references, for that I’ll be forever grateful! Karin, for fun times with our families. But especially for leading the way this spring, it’s been so good to have you ahead of me in the process. And thank you for standing next to my computer. You have magic powers! Judith Mank, thank you for introducing me to spinning, for reading and correcting whenever needed. But most of all, thanks for being a great friend. Thanks to you I’ve kept calm (almost) and carried on! There are a lot of people I would like to thank for useful and interesting discussions: Mark Thomas, Mattias, Hanna, Mia Olsson, Judith, Greger, Tom, Helena, Cia, Pontus, Love, Anna, Kerstin, Cris, Niclas, Eva, Oddný, Robert, Matthew Collins, Simon McGrory and of course Anders. Anna Linderholm, thank you for lots of laughs, and for last minute brain- storming! A lot of my collaborations and projects could never have been initiated without the financial support from the following funding bodies: The Sven and Lilly Lawski’s fund for scientific research, The Marie Curie Actions MEST-CT-2004-7909 ‘GENETIME’ grant, The Royal Swedish Academy of Sciences, The Nilsson-Ehle Donation, P O Lundells foundation, Helge Ax:son johnssons foundation, Lars Hierta Memorial Fund, Birgit and Gad Rausing’s foundation for humanistic research, The Royal Swedish Academy of Agriculture and Forestry, Travel grant from the Wallenberg foundation, CF Liljewalchs foundation, Gertrud Thelins foundation, Sernanders foundation, The EBC Graduate school on Genomes and Phenotypes. I also want to thank Jan Ekman and Ben Hayes, for providing very interesting scientific distractions. The thought of continuing the projects gave me something to look forward to when writing felt hard. To all my friends and family! Without your support and chances for non- scientific activities these years would have been more empty and tiresome. Mia, for being a very good friend! Thank you for sushi, talks and for sharing the same passion for our “paper babies”! Lisa, my favorite sales rep! Thank you for being such a good friend! And for all the fantastic dinners and lunches on the clock’! Anders and Svante for fun company and babysiting over the years. Annika, for fun science talk about techniques and other stuff, the best fika, and for checking my facts. The Larsson family I, Kattis, Jimmy and Valle. It’s always so great hanging out with you! I’m glad that I’ll be joining you in the PhD club now. The Larsson Family II, Emma, Erik, Alva and Tuva. Thank you for very nice company over the years, fun parties and

52 for taking care of Ida. Friends from home and Uppsala, SofiaA, SofiaE, So- fiaE(N), Sara, Karin, Ingela and Anna thank you for being you! Marjo, for sharing my love for Nica, and everything associated. Allisan & Anita for the great time in Woods Hole. You are my *! The Eklund/Olsson family in Stockholm and Mjölby. Lotta Tomasson for the cow-er! I love it! Mamma, you have always told me that if I just work hard and do my best I can do what I want. I know that you worried a lot about your lazy daughter, but I finished in time. Without your constant support I would not be here today. Thank you for questioning the relevance of my projects and for always being interested! Dr Magnus Eklund, thanks for good collaboration and for all your help these last stressful months, especially with the figures! Magnus, thank you for being such a big support, and for encouraging me to work as much as needed and not feel bad about it! Love you. Ida, älskade unge, my little sunshine, when you are old enough to read this I hope that you don’t remember all the time I was working but see that if your mother can get a PhD you can do anything if you just set your mind to it. I love you so much. Nu ska mamma vara med o leka!

53 References

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