Genomic Insights Into the Population History of Circumpolar Arctic Dogs

Home , Dog breed, Sled dog, Therkel Mathiassen, Wolfdog

UNIVERSITY OF COPENHAGEN

STOCKHOLM UNIVERSITY

PhD Thesis Tatiana Richtman Feuerborn

Genomic Insights into the Population History of Circumpolar Arctic Dogs

Supervisors: Anders J. Hansen Love Dalen Mikkel-Holger Strander Sinding Kerstin Liden

Submitted: 29th February 2020 UNIVERSITY OF COPENHAGEN

STOCKHOLM UNIVERSITY

PhD Thesis

Tatiana Richtman Feuerborn

Genomic insights into the population history of circumpolar Arctic dogs

Supervisors: Anders J. Hansen Love Dalén Mikkel-Holger Strander Sinding Kerstin Lidén

Submitted: 29th February 2020

Cover Image: Tatiana Richtman Feuerborn

“In point of fact they [Canadian Inuit Dogs] are probably the purest bred dogs in the world, being so securely segregated from the rest of the canine world.” Lindsay 1935

/ Table of Contents

List of papers 5

Candidate’s Contributions 6

English Summary 7

Dansk abstract 8

Svensk sammanfattning 9

Introduction 10 Domestication of the Dog 10 The Role of Dogs in the Arctic 10 Human History in Siberia 11 Human History in the North American Arctic 12 Dogs in the Siberian/Eurasian Arctic 14 Dogs in the North American Arctic 15 Implications of Recent Contact Between the West and the Arctic on Arctic Dogs 16 Epidemics in Arctic Dogs 18 Relationship Between Wolves and Sled Dogs 19 Objectives 21

Methods and Materials 22 Materials 22 Palaeogenetics 22 DNA Extraction 25 Next Generation Sequencing (NGS) 25 Computational Methods 26

Results & Discussion 29 Mitochondrial Haplotype Frequency Shifts Associated Human Cultures 29 Identification and Dietary Stable Isotope Analysis of Mammal Fur in Arctic Clothing 30 Population Structure in Arctic Dogs 31 Gene Flow From Non-Arctic Dogs into Arctic Dogs 33 Introgression From Wolves into Arctic Dogs 35 Mitigation Against Human Contamination in Sequenced Faunal Libraries 36

Future Directions 38

References 39

Acknowledgements 48

/ List of papers

I. Ameen, C.*, Feuerborn, T. R. *, Brown, S.K.*, Linderholm, A.*, et al. (2019) ‘Specialised sledge dogs accompanied the Inuit dispersal across the North American Arctic’, Proceeding of the Royal Society B., 286, h ttps://doi.org/10.1098/rspb.2019.1929

II. Harris, A.* Feuerborn, T. R.*, Sinding, M.-H. S., Nottingham, J., Knudsen, R., Rey-Iglesia, A., Schmidt, A.-L., Appelt, M., Gronnøw, B., Alexander, M., Eriksson, G., Dalén, L., Hansen, A.J., and Lidén, K. (Submitted) ‘Archives of human-dog relationships: Genetic and stable isotope analysis of Arctic fur clothing’

III. Feuerborn, T. R. , Carmagnini, A. , Gopalakrishnan, S., Losey, R., Appelt, M., Grønnow, B., Schmidt, A.-L., Gilbert, M.T.P., Meldgaard, M., Larson, G., Dalen, L., Hansen, A.J.*, Sinding, M.-H.S*, Frantz, L.A* (Manuscript) ‘Geno mic insight into the population history of Siberian dogs’

IV. Feuerborn, T. R. , Gopalakrishnan, S., Fernández Díaz-Maroto, P., Appelt, M., Grønnow, B., Schmidt, A.-L., Rankin, L., Gilbert, M.T.P., Dalen, L., Meldgaard, M., Sinding, M.-H.S.*, Hansen, A.J.* (M anuscript) ‘Pre-contact Inuit dog genomes show a lost wealth of dog diversity in the North American Arctic’

V. Feuerborn, T. R, Pečnerová, P., Ersmark, E., Dehasque, M., Krzewinska, M., Lagerholm, V.K., Munters, A., Rodriguez, R., Ureña, I., von Seth, J., van der Valk, T., Götherström, A., Dalen, L., Díez-del-Molino, D. (Manuscript) ‘Competitive mapping allows to identify and exclude human DNA contamination in ancient faunal genomic datasets’

* These authors contributed equally to this work.

The articles are reprinted with permission from the respective publishers.

The following paper which I contributed to is included as an appendix:

Sinding, M-H.S., Gopalakrishnan, S., Ramos-Madrigal, J., de Manuel Monter, M., Pitulko, V.V., Kuderna, L., Feuerborn, T.R., Frantz, L.A.F., Vieira, F.G., Niemann, J., Samaniego Castruita, J.A., Carøe, C., Andersen-Ranberg, E.U., Skoglund, P., Jordan, P.D., Pavlova, E.Y., Nikolskiy, P.A., Kasparov, A.K., Ivanova, V.V., Willerslev, E., Fredholm, M., Wennerberg, S.E., Heide-Jørgensen, M.P., Dietz, R., Sonne, C., Meldgaard, M., Dalén, L., Larson, G., Petersen, B., Sicheritz-Pontén, T., Bachmann, L., Wiig, Ø., Marques-Bonet, T., Hansen, A.J., and Gilbert, M.T.P. (Submitted) ‘Emergence of Arctic-Adapted Dogs at Pleistocene-Holocene Transition’

I am an author on the following papers that were published during my PhD but are not included in this thesis:

Pečnerová P., Díez-del-Molino D., Dussex N., Feuerborn T., von Seth J., van der Plicht J., Nikolskiy P., Tikhonov A., Vartanyan S., Dalén L. (2017) Genome-based sexing provides clues about behavior and social structure in the woolly mammoth. Current Biology 27(22): 3505-3510.

Rowan, B.A., Heavens, D., Feuerborn, T.R., et al. (2019) ‘An Ultra High-Density Arabidopsis thaliana Crossover Map That Refines the Influences of Structural Variation and Epigenetic Features’, GENETICS; https://doi.org/10.1534/genetics.119.302406

Candidate’s Contributions

Candidate contributions to thesis articles*

I II III IV V Conceived the study Significant Significant Substantial Substantial Significant Designed the study Significant Substantial Substantial Substantial Significant Collected the data Significant Substantial Substantial Substantial Significant Analysed the data Substantial Substantial Substantial Substantial Significant Manuscript preparation Significant Significant Substantial Substantial Significant

*Contribution Explanation: Minor: contributed in some way, but contribution was limited. Significant: provided a significant contribution to the work. Substantial: took the lead role and performed the majority of the work.

/ English Summary

The Siberian and North American Arctic have both borne witness to numerous migrations of humans and with them their dogs. This PhD thesis is based on whole genome data from 22 Siberian dogs and 72 North American Arctic dogs, in addition to 186 mitochondrial genomes Siberian and North American Arctic dogs. Mitochondrial genome data allowed for the identification of migration events that introduced distinct dog populations to North America, associated with different cultural complexes arriving to the region. A novel mitochondrial clade was also identified in dogs from eastern Siberia and Alaska. Genetic analysis was performed to confirm the macroscopic identification of fur used to make clothing in the Arctic in conjunction with stable isotope analyses to explore dietary differences of dog populations across the circumpolar region. The whole genome data generated for this PhD also detected and explored evidence for several gene flow events from West Eurasian dogs into the dogs of Siberia starting 10,900 BP. There was an additional gene flow event that introduced Near East related ancestry to the dogs of the Siberian Steppe before the Late Bronze Age. Dogs carrying this West Eurasian ancestry spread throughout Siberia, reaching northwestern Siberia by the Iron Age, by 2,000 BP. Further gene flow was detected later in Siberia from West Eurasia a thousand years later. North American Arctic dogs universally carry the Near East related ancestry that is seen in Siberian dogs starting in the Bronze Age, showing it had reached the Bering Strait before the ancestors of the Inuit departed Siberia for Alaska. Once in North America Inuit dogs experienced several other gene flow events from pre-contact subarctic dogs, modern European dogs, and wolves. The population structure seen in North American Arctic dogs reflects geography and the subsequent isolation as well as population turnover events associated with catastrophic epidemics in the dog populations. Finally, a simple method was developed to evaluate and remove human contamination from ancient DNA datasets originating from faunal taxa. All together this thesis has compiled genomic information from 94 Arctic dogs to shed light upon the genetic history of these dogs from the early Holocene through to the present day. This dataset has been able to provide insight not only into past dynamics of Arctic dogs but also a much needed resource for understanding and preserving the indigenous dog populations still present in the Arctic that face continued challenges of globalisation and climate change.

/ Dansk abstract

Det Sibiriske og Nordamerikanske Arktis har være vidne til talrige folkevandringer af historiske kulture og med disse vandringer fulgte folks hunde. Denne afhandling bygger på fuld-genom-data fra 22 Arktisk Sibiriske og 72 Arktisk Nordamerikanske hunde, samt 186 mitokondrielle genomer fra Arktiske hunde fra Sibirien og Nordamerika. Det mitokondrielle genom data tydeliggøre de individuelle folkevandringer, hvormed ny hunde populationer indførtes til Nordamerika i forbindelse med indvandring af nye kulture. Yderligere viser data en hidtil ukendt mitokondriel klade i hunde fra Østsibirien og Alaska. Genetiske undersøgelser blev brugt til at bekræfte makroskopiske artsbestemmelser af pels, brugt i klædedragter af Arktiske folk. Pels bestemt som hund, blev brugt til stabilisotopiske undersøgelser, der viser forskellige i diæt hos forskelig hunde populationer cirkumpolært i Arktis. Data fra fulde genomer, tydeliggøre adskillige bølger af genetisk diversitet fra Vesteurasiske hunde der bliver indblandet i Sibiriske hunde, allerede fra for 10,900 år siden. Der ses opblanding med Mellemøstlig hundediversitet i hunde fra den Sibiriske steppe før sen bronzealder. Denne opblandede diversitet spredes op igennem Sibirien og når Nordvestsibirien i jernalderen, hvorefter yderligere Vesteurasiske hundediversitet optræder i Sibirien efter jernalderen. Alle hunde i det Nordamerikanske Arktis, nedstammer delvist fra de Sibiriske bronzealderhunde med Mellemøstlig opblanding, hvilket bevidner at denne genetiske diversitet nåede til Beringstrædet før Inuitterns forfædre spredtes til Alaska fra Sibirien. Efter ankomsten til Nordamerika, bliver Inuithundene påvirket af andre kilder af genetisk diversitet, fra Indianerhunde, ulve og tilslut moderne Europæiske hunde. Den populationsstruktur der opstår blandt de Nordamerikanske Arktiske hunde, afspejler isolation i specifikke geografiske områder, samt kollaps af lokale hunde populationer, som følge af katastrofale epidemier. Sidst i afhandlingen præsenteres en metode til at opfange og fjerne genetisk forurening af moderne menneske DNA fra nedbrudt forhistorisk-DNA fra dyr. Som helhed samler denne afhandling fuld-genom-data fra 94 Arktiske hunde, for at belyse deres genetiske historie fra tidlig Holocæn frem til i dag. Denne data giver ikke bare indsigt i Arktiske hundes historie og dynamik gennem tid, men er også en tiltrængt resurse til at forstå og bevare de oprindelige hundepopulationer, der stadig findes i Arktis men som er pressede af globalisering og klimaændringer.

/ Svensk sammanfattning

Där har varit många migrationer av människor, och med dem deras hundar, i de sibiriska och nordamerikanska delarna av Arktis. Denna doktorsavhandling är baserad på hela genomdata från 22 sibiriska hundar och 72 nordamerikanska arktiska hundar, samt 186 mitokondriella genom från arktiska hundar från Sibirien och Nordamerika. De mitokondriella genomen möjliggjorde identifiering av migrationer av hundar som introducerade distinkta populationer till Nordamerika, associerade med olika kulturella komplex som anlände till regionen. En ny mitokondriell klad identifierades också hos hundar från östra Sibirien och Alaska. Genetiska analyser utfördes för att bekräfta den makroskopiska identifieringen av päls som användes för att göra kläder i Arktis tillsammans med stabila isotopanalyser för att undersöka dietskillnaderna hos hundpopulationer i den cirkumpolära regionen. Hela genomdata som genererats i denna avhandling upptäckte och undersökte bevis för flera genflöden från västra -eurasiska hundar till sibiriska hundar med början för 10 900 B.P. Det fanns ytterligare ett genflöde före den sena bronsåldern som introducerade börd från Främre Orienten till hundar från den sibiriska stäppen. Hundar som bär denna väst-eurasiska härkomst spred sig över Sibirien och hade nått de nordvästra delarna vid tiden för järnålderns början. Ytterligare genflöde upptäcktes senare i Sibirien från Västeurasien med början för 1 000 år sedan. De arktiska hundarna från Nordamerika har samma börd från Främre Orienten som ses i de sibiriska hundarna i början av bronsåldern, vilket visar att detta genflöde hade nått Berings sund innan förfäderna till inuiterna lämnade Sibirien för Alaska. När de hade nått Nordamerika upplevde de inuitiska hundarna flera andra genflöden från för-kontakt subarktiska hundar, moderna europeiska hundar och vargar. Befolkningsstrukturen i de arktiska hundarna från Nordamerika återspeglar geografi och den efterföljande isoleringen, samt befolkningsomsättningar förknippade med katastrofala epidemier i hundpopulationerna. Slutligen utvecklades en enkel metod för att utvärdera och ta bort mänskliga kontamineringar från gammalt DNA som härrör från fauna. Sammantaget har denna avhandling sammanställt genomisk information från 94 arktiska hundar för att belysa den genetiska historien för dessa hundar från tidigt Holocen till nutid. Dessa data har kunnat ge insikter i den historiska dynamiken hos arktiska hundar, samt också tjäna som en välbehövlig resurs för att förstå och bevara de inhemska hundpopulationer som fortfarande finns i Arktis och som står inför fortsatta utmaningar av globalisering och klimatförändringar.

/ Introduction

Domestication of the Dog

For millennia, dogs (C anis lupus familiaris) have played a central role in human societies following their domestication in the Upper Palaeolithic, sometime between 15,000 and 30,000 years ago (1–5). Dogs were the first animal species to be transformed from a wild animal to a tame working animal or pet. However, it is not clear where, when, or how and potentially how many times dogs have been domesticated, although Eurasia is the most agreed upon region for the initial domestication (3,5–7). It is often hypothesised that the process started as a mutually beneficial collaboration between humans and a grey wolf-like canid (C anis lupus) . The most widely accepted theory about the nature of the earliest close association between dog ancestors and humans involves a process called ‘self-domestication’. This theory suggests that wolves scavenging refuse at human camps and settlements, slowly began showing ‘tame’ behaviour towards humans, eventually manifesting in full domestication. Subsequent to their domestication, the role of the early dogs in human society is unknown. During this period, humans were hunter-gatherers relying on the exploitation of natural resources and living in a world with diverse megafauna - including several carnivores that could be a threat or competition to humans. In this context, it is tempting to imagine the benefits of having dog-like canids in the vicinity of humans to provide protection, assistance in hunting, and perhaps a source of food. Utilising dog-like canids while hunting could have improved the success rates and thereby increased the stability of the communities hosting the dogs. Since then, the collaboration between humans and dogs has expanded and evolved. Human selection for specific traits, behaviour, and skills has resulted in the wide array of phenotypic diversity and specialised dog types that can be seen today.

Current research on dog domestication is confounded by the fact that the earliest domestic dogs and their wolf ancestors are, as of yet, unidentified. The absence of these pieces presents a challenge in reconstruction of the evolutionary history of dogs. However, indisputable domesticated dog remains from as early as 14,500 years ago have been discovered in Germany at the Bonn Oberkassel site (8–10). Additional ancient dog remains found in the Siberian High Arctic have been dated to 9,500 years ago, which shows that dogs had already spread into regions with extreme environments ( 11).

The first ancient dog genome to be published came from a 4,800 year old Irish Neolithic dog which indicated that dogs may have been domesticated in multiple events (5). Additional Neolithic dog genomes from Germany dated to 7,000 and 4,700 years ago disputed a dual origin of dogs and instead supported a single origin of dogs in Eurasia between 20,000 and 40,000 years ago with continuity of dogs during the European Neolithic (4). Genomic sequencing of an ancient wolf from the Pleistocene Siberia, dated to 35,000 years ago, showed that Arctic dogs derive part of their ancestry from Pleistocene wolves (12). While these ancient remains are unable to tell the complete story of dogs from the point of domestication to today, they have provided a great deal of insight into the origin of dogs.

The Role of Dogs in the Arctic

Following their domestication, dogs settled into human society and played a variety of roles, these roles varied culturally, geographically, and temporarily, becoming invaluable in their ability to herd, guard, assist with hunting, and provide companionship among others (9). Historically, dogs have been used in a variety of

/ ways to aid human transportation, including acting as pack animals for carrying materials and draft animals for pulling sleds and travois (13–15). The use of dogs as draft animals is seen as a defining feature for life in the Arctic. The combined power of a team, whether only two dogs or twenty, confers a much greater strength and endurance than a person alone, enabling humans to travel further, faster, and the potential to pull many more kilograms than a human alone could (16). They also possess the potential to find their way home when lost, for example during blizzards and whiteouts (15,17). This use of dogs is particularly remarkable evidenced by findings of remains and sled materials dated back to at least 9,500 years old on the New Siberian Islands (11,18). This in turn indicates that dog sledding has occurred in Siberia for millennia. Simil ar to Siberia, dog sledding has been an important technology for transportation in the North American Arctic where dog sledding is a hallmark of Inuit culture ( 19).

In the Arctic, dogs have frequently spent two lifetimes with their human companions. In the first one, their living-lifetime, they work as hunters, herders as well as pack and draft animals. In their second, their post-living-lifetime, they serve new roles such as food or protection from the elements as clothing. Across the Arctic, dog fur has been utilised as a material for making garments. Dogs are equipped for survival in the Arctic in part by their insulating fur which can also be utilised by humans post mortem. Most mammals have tripled layered fur, comprised of inner down hair, intermediate awn hair, and the outer guard hair, which together create a thick, well-insulated coat especially during winter. The guard hairs work to repel water while the inner layers primarily function for thermoregulation. The properties of dog fur when included in clothing provide much needed protection from the elements. Historically dog fur has predominantly been used as trim for the sleeves and hoods to insulate and prevent frost buildup. Dog fur is particularly valuable as a result of its resistance to repeated freeze-thaw cycles.

Archaeological sites in both Siberia and North America contain the telltale signs of butchery on the remains of dogs (13,20–22). At sites like Ust-Polui, signs of butchery can be seen on many of the dog bones, indicating that Arctic dogs served numerous purposes in the Iron Age society including sledding, consumption, and likely ritual purposes ( 20,21).

Human History in Siberia

Arctic Siberia was occupied by humans potentially as early as 45,000 cal. BP (23–25), however the occupation was discontinuous due to the extreme climatic conditions (24,26). At least three major prehistoric influxes of people have led to the settlement of Northeastern Siberia since the Late Pleistocene (25). The earliest group, Ancient North Siberians, was a population of distant relation to the early hunter-gatherers in West Eurasia (25). The second major wave brought people who were related to populations in East Asia which was ancestral to ‘Ancient Palaeo-Siberians’ and related to present-day groups in Northeastern Siberia and Native Americans (25). The final prehistoric wave of human migration into Northeastern Siberia occurred during the Holocene bringing a population known as ‘Neo-Siberians’ who were related to the people of East Asia (25). In succession, each of these major waves of people entering Northern Siberia resulted in the near replacement of the previous population.

The Bronze Age in Siberia and the Eurasian Steppe, 6,000 to 3,000 BP, marked a period that witnessed an era of striking cultural changes. After the Early Bronze Age, the Ancient Palaeo-Siberian ancestry which had previously been widespread across Siberia became confined to Northeastern Siberia (25). The remarkable cultural changes and the disappearance of ancestry which was once widespread can be linked to the arrival of

/ numerous groups to the region (27,28). These large-scale migrations have been detected through material culture changes in the archaeological record in addition to signals of admixture between distinct human groups and population replacements (27,29). The Bronze Age migrations in the Steppe and Siberia brought groups from Western Eurasia into these eastern regions of Eurasia (30). The Andronovo cultural complex, a collection of closely related cultural groups, arrived to the West Siberian Plain from the region around present-day central Kazakhstan approximately 4,000 BP (28). The largest migration in the Late Bronze Age also brought humans from present day Kazakhstan to the West Siberian Steppe ( 28).

Similar to the Bronze Age, several waves of human migration occurred during the Iron Age, 3,000-1,500 BP, in Siberia and the Steppe (31). Starting around 3,000 BP, groups related to the Scythian cultural complex spread across huge stretches of the Eurasian Steppe (30,31). Migrations from northern Siberia to the south, also around 3,000 BP, has been postulated to be connected to a period of cooling and in turn making the northern regions less habitable ( 32).

Currently, there are over 40 groups with distinct languages and cultures in Northwestern Russia and Siberia (29). Genetic studies of modern groups in Siberia have revealed different levels of ancestry from non-Siberian populations regionally (29). Over the last few centuries there have been new migrations into Siberia from outside of the region. According to oral tradition in the Yamal region, the Nenets arrived at the Lower Ob and Yamal Peninsula after the 17th century AD when they brought their nomadic reindeer herding with them (33). Archaeological investigations and oral traditions both show that the earlier inhabitants of the region lived in houses underground and used dogs instead of reindeer as transportation (33). Reindeer herding in Kamchatka, on the other hand, was introduced as late as the early 20th century AD (34). The onset of the Russian exploration and occupation of Kamchatka in the 17th and 18th centuries AD brought the presence of people from the west along with their diseases to the region and a gradual change in lifeways was initiated ( 34,35).

Human History in the North American Arctic

The general consensus in archaeological research supports a scenario where the first humans to settle North America arrived from Siberia at the Pleistocene-Holocene transition or at the end of the Late Pleistocene (36–39). This initial migration of people from Siberia to North America brought the ancestor to the native groups of both North and South America ( 40–42).

Fig. 1. Distinct migrations of people into the Americas.

/ A second migration arriving from Siberia to North America during the middle of the Holocene, around 5,000 BP, stayed concentrated in the Arctic region. This second wave brought cultures related to the Arctic Small Tool Tradition of the western Bering Strait in a cultural complex known as the Pre-Inuit, Paleo-Eskimo or Paleo-Inuit (43). Paleo-Inuit cultures are thought to have arrived in the North American Arctic around 5,000 years ago and include cultures such as the Ipiutak, Norton, Kachemak, Pre-Dorset, Dorset, Saqqaq, and Independence (44,45). Humans first entered Greenland around 4,500 BP when the Saqqaq, a subset of the Paleo-Inuit cultural complex, arrived. The Saqqaq occupation lasted until approximately 2,800 BP, making it the longest-lasting culture in Greenland to date (46). Coinciding with the Saqqaq occupation in West Greenland a culture known as Independence I occupied North Greenland between 4,500 and 3,800 BC (46,47). Two more Paleo-Inuit cultures occupied Greenland Following the Saqqaq and Independence I eras of Greenland’s history, these are known as the Greenlandic Dorset (2,800-2,000 BP) in South Greenland (48) and the Late Dorset (1,250 - 700 BP) in Northwestern Greenland ( 47,49).

The ‘Thule culture’ was first recognised by Therkel Mathiassen in Greenland, who named the archaeological culture after the district where the first remains were found and the expedition which found the remains in 1925, the Thule District and the Second Thule Expedition respectively (50). The district and expeditions in Greenland were named after the most northerly trading station in the world, which was called such by Knud Rasmussen as inspired by the “Ultima Thule” in classics (51,52). In keeping with the resolution of the Inuit Circumpolar Council (53), academics transitioned away from the use of the term Eskimo to Inuit which they themselves have adopted (43). A further shift has also been recommended to transition to the use of ‘Inuit culture’ rather than ‘Thule culture’ for the same rationale (51). The Inuit culture, which spread from the western shores of Alaska through to the eastern shores of Greenland, originated in the Bering Sea region, not from any of the Paleo-Inuit cultures which already occupied the North American Arctic (54). The Old Bering Sea, Birnirk, and Punuk cultures of eastern Siberia and the Bering Strait preceded the Thule culture, from one of these cultures it is thought the Inuit culture arose by 1,000 BP, although it remains debated as to where in Beringia the culture first arose ( 45,54).

The Inuit culture rapidly spread across the North American Arctic (45,51). The swift migration from Alaska to Greenland was enabled by the universal use of dog sleds, in addition to their specialised maritime technology - umiaks and kayaks (45,50). Eastern migration of the Inuit culture began from Alaska travelling via the Canadian Arctic. The Inuit arrived in the Eastern Arctic, eastern Canada and Greenland, around 800 years ago, with many suggesting that the migration occurred within a single generation (45,51,55,56). In Greenland, archaeological and genetic evidence suggests the Inuit colonisation of the country consisted of multidirectional waves of migration originating from Ellesmere Island, Canada, and arriving in the Thule District in Northwest Greenland (45,51,56). From the Thule District, the Inuit travelled in two directions east and south. The eastern migration travelled across northern Greenland, reaching Northeast Greenland shortly after arriving in the Thule District. The southern migration travelled south from the Thule District down the west coast, reaching the Sermilik and Ammassalik Fjords around 600 years ago (57,58). In the past it has also been suggested that East Greenland was populated at least in part from Northeast Greenland, although the general accepted theory is that populations from West Greenland settled East Greenland ( 58). FIGURE

Studies of the genetic ancestry of people in North America and Chukotka determined that Na-Dene and Eskimo-Aleut speaking people, such as Athabaskans and Eskimo-Aleut speakers, universally possess ancestry attributed to a Paleo-Inuit related ancestor (59). Furthermore, current day Inuit, Yup’ik, and Aleutian

/ Island populations in the North American Arctic, in addition to Na-Dene speakers, share Siberian Paleo-Inuit related ancestry ( 59).

A few centuries before the arrival of the Inuit, the Norse settled Greenland. In the 10th century CE the Norse arrived in Greenland, initiating the first contact between Europe and Greenland (60). Archaeological evidence from Norse and Thule archaeological sites suggests that there was contact between the two cultures, although the extent and nature of this contact is unknown (60). While the Inuit spread across much of Greenland’s coast the Norse occupied only three settlements, referred to as the East, middle and West Settlements. On their settlements the Norse are thought to have maintained their agricultural lifestyle, they had even brought with them their livestock (60,61). The Norse occupation of Greenland lasted for approximately 450 years, ending around 500 years ago (60,61). The disappearance of the Norse from Greenland has been attributed to many causes, including climate change, decline in contact with Europe, failure to adapt their lifestyle to the environment, and conflict with local Thule populations ( 60).

Several centuries after the disappearance of the Norse from Greenland, European contact resumed with the arrival of Dutch, English, Norwegian and Danish whalers visiting Greenland during the seventeenth and eighteenth centuries CE (55,62). Later, Denmark and Norway renewed their connection with Greenland during their eighteenth century AD colonisation of the country (55). The arrival of Hans Egede at the behest of King Frederik IV of Denmark-Norway marked the beginning of the later only Danish colonisation of Greenland in 1721 (55). The founding of permanent settlements or villages in west Greenland by missionaries led to the transition from seasonal occupations to sedentary settlement establishment on the West coast during the eighteenth century. Contact between Europeans and the Inuit on the East coast and Thule District occurred during the nineteenth century ( 55).

As a result of the interconnected migration histories of humans and dogs, patterns of ancestry connecting the dogs of North America to dogs of Siberian origin will likely be revealed through genomic study of ancient Arctic dogs similar to those seen in humans.

Dogs in the Siberian/Eurasian Arctic

Wolves have occupied Siberia for longer than humans, dogs on the other hand have only been in Siberia since the early Holocene. Debate about the dog/wolf status of some of the earliest ‘dogs’ in Siberia remains a contentious issue (63–65). Dogs arrived early to the High Arctic alongside humans, evidence of this can be seen at the Zhokhov site on the New Siberian Islands when the islands were still attached to the Siberian mainland (11). Direct radiocarbon dating of a dog from Zhokhov revealed that these dogs represent the earliest known dogs in the High Arctic starting by 9,500 cal. BP (7). In addition to dog remains, excavations at the Zhokhov site also recovered organic materials associated with sleds such as sled runners (11,18). The combination of both dogs and sleds being present on the site contemporaneously has been suggested to represent the earliest evidence of not only dogs in the High Arctic but also dogs being used for the pulling of sleds (11). Morphological studies of the shape and size of skulls from the dogs at Zhokhov showed that these skulls belonged to fully domesticated dogs and not dogs at a midway point during the domestication process, which has been corroborated by genetic studies (7,11,66). Excavations at other Siberian sites, such as Afontova Gora, Cape Vhodnoi, and Ust-Polui materials related to dog sledding have also been found, including toggles for harnessing dogs to sleds ( 11,67).

/ In the Trans-Baikal region of Siberia, various sites dated to the Siberian Early Neolithic around 8,000 years ago, have been excavated that contained the remains of dogs (13). The dogs in this region appear to have been used for hunting and potentially burden carrying. During this time dogs have also been found in articulated burials (13). Later during the Iron Age, a shift appears to have occurred when dogs began to be consumed (13). In the neighbouring Cis-Baikal, dog remains have been recovered from around 7,300 years ago in an articulated burial which is the oldest known dog burial in Cis-Baikal (68). Articulated burials of animals are often associated with ritual and personal or societal significance as a result of the nature of the burials with ‘grave goods’ and the deliberate exertion needed for interments ( 9,68).

On the Yamal Peninsula and around the River Ob, there has been widespread and varied usage of dogs (20,33,69). The Iron Age site, Ust-Polui, was occupied from 2,200 to 1,800 BP, during this period of settlement numerous dog remains were deposited on the site (20). Sled materials were also recovered from the site indicating that the dogs at Ust-Polui were likely being used as draft animals for pulling the sleds (20,21). Cutmarks on numerous dog bones suggests that pulling sleds was not their only use at Ust-Polui during the Iron Age (20). Later in the same region arose the settlement of Tiutei-Sale-I around 1,000 BP (33). The limited number of reindeer bones and equipment associated with reindeer herding has been interpreted as the occupants of the site not being reindeer herders like the later groups in the area, however similar to the earlier settlements in the area dogs were present on the site ( 33).

To the easternmost extreme of Siberia, the Bering Strait the oldest dog and dog sled materials are found associated with the Old Bering Sea and Punuk Cultures from the Bering Sea regions starting around 2,000 years ago (70). Humans along with their dogs departed Siberia from the western coast of the Bering Strait and eventually arrived in Alaska.

Dogs in the North American Arctic

The first port of entry for dogs entering and staying in the North American Arctic from Siberia was Alaska with the Paleo-Inuit. From Alaska, they spread with people throughout the North American Arctic. Alaska’s shores and islands are scattered with archaeological sites left by these early arrivals. Some of these sites contain an abundance of dog remains like the Uyak site on Kodiak Island where hundreds of remains were found (71,72). Cape Krusenstern, also in Alaska, has an archaeological record stretching over thousands of years, from early Paleo-Inuit occupations to the present day, containing a rich assortment of archaeological materials including dogs ( 73). Other sites have more limited preservation or evidence for dogs.

The earliest culture to arrive in Greenland was the Saqqaq who arrived together with the ir dogs to northwestern Greenland from Ellesmere Island in eastern Canada. Dog remains have been recovered during archaeological excavations of Saqqaq sites in West Greenland - such as Qeqertasussuk, Qajaa, and Nipisat I (16). Many of the dog bones from Qeqertasussuk and Qajaa were discovered with cut marks consistent with butchery, suggesting that dogs were used as a food source during times of need (16). Based on the low number of individuals (two or three dogs) per Saqqaq site, it has been concluded that dogs smaller populations during the Saqqaq era (16) and thus have played a different role than the sled dog of the later Inuit occupation ( 74).

Throughout the geographical range of the Dorset culture in the Eastern Arctic, there is a remarkable near absence of dog remains (16). Dorset sites across Canada, such as Tikilik, Lagoon, and Mill Island, have all

/ yielded dog bones, showing that dogs were not completely absent during the Dorset era (75–77). Archaeological excavations of Dorset sites in Greenland have not revealed the presence of dogs through skeletal material. However, DNA extracted from sediments in Greenland have contained dog DNA, suggesting that, while the skeletal material has not been preserved, dogs were present in Greenland on Dorset sites (78). Unfortunately, due to the absence of physical remains and the limited amount of dog DNA in the sediment, little is known about the dogs of the Dorset people. A Dorset age site in the Disko Bay region yielded organic materials that have been interpreted as being the remains of a dog sled, currently the only known Dorset dog sled materials (79). The limited representation of dogs in Dorset and Independence sites is likely due to a combination of poor preservation, small populations of dogs, and possible confusion between wolf and dog remains (16). For the first three thousand years of human history in Greenland, there appears to have been sporadic use of dogs in small numbers possibly for hunting, individual pack animals, and occasional food sources ( 16).

While Paleo-Inuit dogs may have been used as pack animals, the absence of sled materials and small population sizes indicates that their use with sleds was not likely to have been prevalent (16). This contrasts with the Inuit culture, which is the first North American Arctic culture to have possessed large groups of dogs along with the first known appearances of sleds in North America (9). In Greenland, the Inuit culture spread from the Thule District east to the north of Greenland and south to West Greenland and further on to East Greenland. The success of the Thule settlement of the North American Arctic and Greenland has been connected to the technology and the specialised dogs that came with them ( 16,74,80).

Implications of Recent Contact Between the West and the Arctic on Arctic Dogs

Indigenous dogs cross the Arctic share a common origin in Siberia, supported by archaeological and modern genetic data. From there, they travelled across the Bering Strait into Alaska. There has been varied studies of the mixture of Arctic dogs in Siberia and North America with non-Arctic dogs as a result of Industrialisation and Globalisation in the last three centuries. The same stands true for the genetic impact of industrialisation and colonisation on Arctic dogs and the changes they have undergone over the last three hundred years.

Genetic studies of Arctic dogs have generally focused on Arctic breeds, dogs officially recognised by kennel clubs, such as Siberian Huskies, Samoyeds, Siberian Laikas, Kamchatskaya ezdovaya, Alaskan Malamute, Canadian Inuit/Eskimo Dog, and Greenland Sled Dog. Despite their official recognition, not all of these breeds have been studied and there have been few studies specifically centred around the genetic study of Arctic dogs (81–83). Recent genetic studies on these breeds have revealed a mosaic of European ancestry. Nevertheless, not much in known about current or past populations of these indigenous dogs as finding have focused on populations of the Arctic breeds living outside of the Arctic. Some of this European ancestry likely occurred during the Gold Rush in Alaska and Canada when many European dogs were brought to the North American Arctic for the first time (82). The Alaskan Husky, a racing sled dog, can also be found in Alaska and beyond, although they are not a cohesive population representative of a ‘breed’. Alaskan huskies have a more diverse ancestry compared to other Arctic dogs. During the Gold Rush, local dogs from Alaska were mixed with European breeds and Siberian Huskies to create a new variety of dogs for pulling sleds and racing, which is reflected in their genetic composition. Genetic studies of partial mitochondrial and nuclear genomes have confirmed the shared heritage of modern Arctic Dogs rooted in Siberia, while simultaneously highlighting some differences between populations as is the case with the Alaskan huskies (3,7,82,84).

/ Conversely, partial mitochondrial genomes have been used to suggest that Greenland Sled Dogs and the Canadian Inuit Dog are a single breed, reflecting their shared origin and ancestry ( 81,85).

In North America, Greenland was the earliest to witness contact between Europeans and the local Inuit populations. The first dogs of European origin to arrive in Greenland were brought by the Norse. Although the Norse disappeared from Greenland, it remains unknown if there has been a lasting legacy of the Norse occupation left through the contact between the Norse and Inuit dogs. Later, when the whalers began visiting Greenland, starting around 300 years ago, ships brought their dogs ashore to Greenland where they reportedly mixed with the local dogs (86). In the late 19th century, the explorer Robert Peary attempted to crossbreed Greenland Sled Dogs with Newfoundland Dogs in an attempt to produce ‘a better breed’ (86). While these dogs are said to have been large and strong, they lacked the resistance to cold and starvation necessary for survival in winter and lasted only three to four generations (86). In Greenland, the more recent contact between Arctic dogs and non-Arctic dogs has been more limited compared to other regions of the North American Arctic in part as a result of legislation ( 87).

Admixture between Arctic dogs and non-Arctic dogs has not been the only consequence of the contact between the Arctic and the West. Colonisation and industrialisation have resulted in changes to settlement patterns and by default dog movements, pressures on population size, and the redistribution of dogs on top of health implications of the comingling of Arctic dogs and non-Arctic dogs.

Early 20th century Arctic explorations in the East Arctic (encompassing the Arctic Archipelago, the neighbouring Canadian mainland, and Greenland) embarked upon archaeological and ethnographic studies of the communities of the past and present (88). These exploration expeditions utilised local dogs to pull sleds with the teams and their equipment (86). During the Danish Literary Expedition, Knud Rasmussen brought dogs from West Greenland to Cape York to ‘introduce new blood’ to the dog population in Northwestern Greenland (86). Later the Second Thule Expedition brought dogs from Upernavik north to Cape York during their journey, facilitating long distance movement of dogs in Greenland (86). Degerbøl reported that in the Cape York region, it was not customary for dog owners to trade or sell dogs with each other until the ‘baptised’ Greenlanders in West Greenland began communicating with people in Cape York (86). Knud Rasmussen’s expedition by dog sled across the breadth of the North American Arctic as part of the Fifth Thule Expedition was powered by dogs from the Eastern Arctic, making it the first European expedition to traverse the Northwest Passage in its entirety by dog ( 89).

Before the introduction of reindeer (Rangifer tarandus) to Kamchatka, and later mechanical transportation methods, the Kamchatskaya ezdovaya dog breed, registered in 1991, were the main means of transportation in the region by the indigenous Kamchadal, Itelmen, and Koryaks (34,35). In the eighteenth century, Steller noted the already apparent changes to the mobility patterns in Kamchatka resulting from the Russian occupation and the demanding fishing quotas they placed on the local populations (35). The dog population in Kamchatka dramatically increased at the turn of the 20th century to keep up with the fish quotas (34). Conversely, during and following World War II, the dog population declined in Kamchatka (34). The Beringia Dog Race was founded in 1990 which later contributed to the dog sledding tourism industry. As a result of the race new dogs, Siberian Huskies, were imported in 2014 to “revitalise” the local populations (34).

Records also show the large scale movements of dogs according to the regional demand for them in Canada for use by the Canadian Royal Mounted Police (RCMP). For example, in 1899 during the Yukon Gold Rush,

/ 140 dogs were brought from the coast of Labrador (90). Although, the RMP are reported to have regionally favoured the use of Siberian Huskies over the local Canadian Inuit Dogs (17,91). Community or family relocations were also responsible for the movement of large numbers of dogs. For example, approximately one hundred dogs were moved hundreds of kilometres from Mittimatalik (Pond Inlet) and Qausuittuq (Resolute Bay) when seven families relocated in 1953 (15). There was also said to be ‘frequent’ exchange of dogs between Canada and Greenland by Arctic expedition teams and by the local populations during at least the nineteenth and twentieth centuries, although this was most likely on a small scale.

European/colonial contact with dogs in the Canadian Arctic is frequently discussed in connection to the police slaughter of dogs in the 1950s and 1960s in the East Canadian Arctic (17,90–92). The killing of dogs was in relation to the Ordinance Respecting Dogs 1929, officially to stop the spread of disease. However, it is widely held to have been “a direct attempt to force the Inuit population of Eastern Canada to assimilate by destroying Inuit dog sociality and thus removing their mode of transportation” (90). Until the early 2000s the slaughter was widely denied, a changing tide arrived in the form of research and independent commission in the last decade (93). Regardless of the rational thousands dogs are thought to have been killed during the mid twentieth century (91,92). Between the nineteenth and twenty-first centuries the number of Canadian Inuit Dogs are thought to have dropped from around twenty thousand to only a few hundred individuals ( 91).

Over the last hundred years, there has been a transition away from having free roaming dogs to picketting them, tethering them to a set location with a chain. Picketing eliminates the dogs’ ability to move freely through the community. The motivation for this transition has been in large part to improve safety by reducing conflict between dogs and people, principally children and the elderly (91). The health implications of this transition have been both advantageous and adverse. Dogs that are picketted cannot run throughout the settlement spreading contagion to the rest of the dog population. Furthermore, picketting dogs has led to a shift away from a free breeding dog population to more explicit mate selection for the dogs by people. However, picketing can result in unhygienic settings for the dogs to live in, repeatedly exposing dogs to the same pathogen as can be the case with the canine distemper virus.

Following outbreaks of disease in Greenland, initially thought to have been brought by European dogs, the official veterinarian of Greenland at the time, S.R Hjortlund, advised restricting the import of non-indigenous dogs to the sledding regions of Greenland in 1904 (87). Decades later, in 1998, legislation was introduced officially prohibiting the import of dogs to the sled dog district of Greenland in order to preserve the health and genetic purity of the dogs (Act. No 18).

Epidemics in Arctic Dogs

Danish colonists in Greenland brought their European dogs with them. European dogs were thought to have carried Eurasian pathogens after the occurrence of disease outbreaks amongst local Greenland Sled Dog populations (91). The apparent increased virulence of these diseases in Greenland was linked to the isolation of dogs in the Arctic, thereby reducing their resistance to diseases to which they had not previously been exposed (87). Rabies-like epidemics in Greenland have been recorded for over 150 years, the earliest recorded occurred in 1859 (94). Particularly in Cape York, Thule District, there were reports of epidemics that had a heavy toll on the dog population. An epidemic in 1898, thought to be rabies, resulted in the survival of only three dogs (86). Later investigation indicated that local Arctic fox populations were the cause of the rabies epidemics in northern Greenland, rather than European dogs (94). A distemper virus

/ outbreak spread across the North American Arctic in the 1980s, reaching Greenland in 1988 with devastating results. In Qaanaaq alone, 80% of the dogs died as a result of the disease (85,95). Epidemics such as these resulted in large population turnover and have undoubtedly left a lasting mark on the present-day populations of dogs in these locations, thereby making the modern populations distinct from the populations in the same location in earlier decades and centuries. The Qaanaaq area was repopulated with dogs from various settlements in West Greenland and even imported Canadian Inuit Dogs from North Baffin.

Parallelling many of the outbreaks of disease in Greenland were contemporaneous epidemics in Canada. As previously discussed, the health status of the dogs in Eastern Canada, particularly Nunavut, has been at the centre of their dwindled population size during the twentieth century. The distemper epidemic in the 1980s was first reported in Canada before making its way to Greenland. The first reported connection between arctic foxes and sled dogs in Canada occurred in 1916 (94). The hamlet of Naujaat in Nunavut, also known as Repulse Bay, lost up to 90% of their dogs in 1930 to the distemper virus ( 85).

Similarly, in Kamchatka, an epidemic in 1889 reduced the population of dogs on the peninsula by nearly half, from approximately 11,000 to 6,500 (34). The spread of disease during the 1889 epidemic in Kamchatka has been attributed to the Russian colonisation (34). The picketing of dogs has also been suggested to be a contributing factor to the decline in population size and health of the dogs due to an increased exposure to mosquitoes in the summer ( 34).

In the Arctic, where dogs live in comparatively high density and undertake long journeys, the spread of disease can take hold and spread rapidly through a population and into others. Given the record of periodic contagions that have decimated the dog populations, strong genetic effects have undoubtedly been observed in these populations. For example, significant decrease of genetic diversity. In cases where population replacements took place, a new genetic signature should be present in the locality where the replacement occurred. On the other hand, in cases where a near replacement took place, the population while carrying this new genetic signature may inadvertently benefit from the influx of ‘new blood’ and thus increased diversity. Despite the potential for heavy inbreeding, the general health and low levels of genetic diseases in Arctic dogs have been attributed to the intense survival and performance-based pressure placed on the dogs ( 85).

Relationship Between Wolves and Sled Dogs

“For times immemorial the Eskimos have wanted to get wolf blood into their dogs, the idea being to impart the ferocity of the wolf to the bear-hound.” - Peter Freuchen ( 96)

Arctic explorers and anthropologists have reported that Arctic groups encouraged the hybridisation of their sled dogs with local wolf populations to strengthen their dog teams (96–100). On the other hand, some reports state that hybrids often make poor sled dogs and as such wolf traits are selected against (96,101). Hybridisations of dogs and wolves are genetically and physically possible as the result of their close genetic relationship. Despite the widespread reports of hybridisation between dogs and wolves, there is very limited evidence in the offspring of these events outside of the context of breeds such as the Saarloos Wolfhound and Czechoslovakian Wolfdog. Deliberate hybridisation of dogs and wolves typically involved picketing, or tying-up, a female dog in oestrous in an area visited by wolves in to attract male wolves ( 100,102).

A bias has been observed in the wild showing evidence for female wolf-male dog hybridisation being more dominant than male wolf-female dog hybrids (102,103). The appearance of hybrids with a wolf father and dog mother has rarely been observed in populations studied in Europe (102). The reason for the limited

/ evidence for wolf-dog hybridisation remains unclear, although this has been suggested to be linked to biological and behavioural constraints, such as male wolf aggression towards dogs, social compatibility, and differences in fertility cycles ( 102,103).

Fig. 2. Global distribution grey wolves (Canis lupus). The present-day distribution (as of 2003) is depicted in the darkest shade of grey and historical distribution is indicated in a lighter grey. The map was downloaded on 26 February 2020 from Wikimedia commons where it is freely available. The original colour scheme was converted into greyscale.

Gene flow between wolves and dogs over the thousands of years since domestication has been seen very infrequently (104,105). In the context of dog sledding, there are conflicting reports and opinions of deposition of hybrids for sledding. During the Fifth Thule Expedition, a female Greenland Sled Dog was bred with a male wolf. They reported that while the offspring were “large, powerful, and ferocious” their long legs were a hindrance when pulling sleds over uneven ice and problematic on bear hunts (96). Similarly, Knud Rasmussen embarked upon a similar attempt by bringing a female Greenland Sled Dog back to Denmark with him to the Zoological Gardens in Copenhagen to breed with a captive wolf (96). The attempt was unsuccessful, but he was able to acquire two hybrids from the Stockholm Zoological Gardens, one (a female) survived the journey to Cape York, Greenland in 1916 (96). She was uninterested in pulling sleds despite enticement or punishment. Furthermore, she was unsuccessful in her attempts to attract the male dogs (96). These ethnographic accounts demonstrate the feasibility (or infeasibility) of crossing Greenland Sled Dogs and wolves. However, they also highlight the frequent inaptitude of these hybrids with regard to sledding. Nevertheless, anecdotal reports of wolf-dog hybrids continue to be widespread in Greenland today.

Due to the bias in wolf-dog hybridisation towards female wolf-male dog events these offspring are most likely to be raised by the mother in the wild and the mitochondrial genome of the wolf mother will be passed to the offspring. These events are difficult to detect genetically in past populations as a result of the reduced likelihood of finding one of these individuals in a domestic context. If the reports of Knud Rasmussen and the Fifth Thule Expedition represent the general outcome of dog-wolf hybrids then these admixture events do not often become widespread within the population. Whole genome studies of Greenland Sled Dogs, specifically from regions of Greenland where overlap can be seen between wolf and dog/human territories are needed to address the question of whether introgression from local wolf populations can be detected in the Greenland Sled Dog after their arrival to Greenland from Canada.

/ Objectives

The aim of this thesis was to investigate the origin and ancestry of dogs from across the Arctic through time using biomolecular methods. The migrations of dogs alongside people in the Arctic have been studied through their genetic signatures.

The specific aims of this PhD are as follows:

● Test whether mitochondrial DNA and geometric morphometrics support a scenario in which multiple prehistoric migration events brought distinct dogs to Arctic North America (Paper I). ● Explore the use of shallow shotgun sequencing to genetically identify animal source of fur used to make clothing in the Arctic and yield new insights on regional variation in the diets of domestic dogs and wild species through stable isotopic analysis (P aper II) . ● Evaluate the continuity of dogs in Siberia during the Holocene and test if there have been periodic migration events bringing dogs to Siberia through admixture between Siberian dogs and other Eurasian dog populations (P aper III) . ● Investigate the population structure of dogs in the North American Arctic through time and the genomic impact of contact between Arctic North America and Europe on dogs (P aper IV) . ● Examine evidence for localised and widespread detectable introgression events between wolves and Arctic dogs and if present determine whether this originates from Pleistocene or Holocene wolves (P aper III and IV) . ● Develop a method to computationally evaluate and remove human contamination in faunal ancient DNA libraries and the implications downstream (P aper V) .

/ Methods and Materials

Materials

The dog remains from archaeological and ethnographic collections were processed at the Swedish Museum of Natural History in Stockholm, Sweden, and the Lundbeck Centre for Centre for GeoGenetics in Copenhagen, Denmark, within the Ancient and Historical DNA facilities. Samples from 97 dogs were recovered and sequenced from sites from the Eurasian and North American Arctic. An additional 135 samples were acquired from ethnographic collections from the late nineteenth and early twentieth centuries. 28 saliva samples were collected with buccal swabs from living dogs across Greenland between 2017 and 2019. The samples included in this thesis were derived from the bone, tooth, skin/hide, or saliva of dogs or putative dogs.

Bone and Tooth

Samples obtained from bones and teeth were cleaned of surface contaminants prior to sampling to aid in the reduction of contaminant DNA. Surface cleaning was conducted through superficial drilling of the outer surface with a Dremel drill. The samples which were obtained from teeth both the outer surface and the enamel layer of the tooth were removed by drilling. Between 30 and 100 mg of bone powder was drilled for extraction from the bone sample. For bone samples that were derived from the petrous bone, see Table xx. For all other bone samples, the densest part of the available bone was drilled. For tooth samples, 15 to 30 mg of powder was drilled from the cementum layer of the tooth.

Ethnographic Skin

Macroscopic identification of hides used as materials for clothing identified garments which possessed dog fur stored in the ethnographic collections of the National Museum of Denmark. 63 samples were taken from clothing which were believed to be dog hides. Samples were taken from the hide approximately the size of a grain of rice for DNA extraction. Sterile scalpels were used to remove hair from the hides, the hair was retained for later analyses.

Buccal Swab

Buccal swab (Isohelix) were used to collect saliva samples from Greenland Sled Dogs living. Sampling involved rubbing a swab on the inner surface of the cheek/lip of a dog for 15 to 60 seconds, dependent upon the temperament of the dog. After the dog was sampled, the swab was placed in a tube and within two hours an Isohelix Dri-Capsule was added to the tube to dehydrate the sample for storage at room temperature for up to three years.

Palaeogenetics

The 1980s witnessed the advent of a new field, palaeogenetics, when DNA was first sequenced from extinct species and ancient humans (106,107). Palaeogenetics involves the study of ancient DNA (aDNA), or DNA from ancient or degraded sources such as archaeological materials or museum collections. Over the last four decades, vast technological improvements and methodological developments have enhanced the accuracy, capacity, and affordability of DNA sequencing. These advances have been particularly beneficial to

/ palaeogenetics which is plagued by samples with poor preservation resulting in low level of endogenous DNA and exceedingly short fragment lengths.The polymerase chain reaction (PCR) was developed during this period which benefited the studies using aDNA through the ability to efficiently replicating DNA fragments (108). The advent of Next Generation Sequencing (NGS) further enabled the sequencing capacity and facilitated the increase in data generation (109). In combination, these developments have led to the formation of palaeogenetic studies, enabling the study of organisms in the past, including extinct species. Palaeogenetic studies have identified ways to investigate topics such as species formation, domestication processes, migration patterns, among many other topics.

Precautions Against Contamination

Ancient DNA studies have faced a monumental challenge in the form of contamination. Early aDNA studies were initially unknowingly impacted by the incorporation of contaminant DNA which only came to light after the publication of results which lead to doubts about the future potential of the field when results could not be replicated (110–112). Resulting from the discovery of the risk of contamination, strict guidelines were put into place to avoid these complications in future studies ( 113–115).

Precautions against contamination in aDNA studies include the physical separation of laboratories working on aDNA and modern DNA as well as the prohibition of performing PCR reactions in aDNA cleanlab during the sample preparations (113,116). Furthermore, clean labs are equipped with PCR and fume hoods with positive airflow and ultraviolet (UV) lighting to aid in the reduction of contamination. Frequent and thorough bleach cleaning is also used to sterilise all surfaces, equipment, and materials in cleanlabs to decrease the risk of cross-contamination.

Clean labs enforce strict dress codes in order to limit the introduction and exposure of DNA to the cleanlab and samples. Typically lab users are required to wear a full body suit, two layers of latex/nitrile gloves, disposable sleeves, a hairnet, shoe/sock covers, cleanlab specific shoes, and a facemask ( 115).

Samples may become contaminated during handling at excavations and/or curation as well as environmental contamination from their deposition and/or storage prior to entering aDNA facilities. To curtail contamination which was introduced to samples prior to their entering aDNA facilities, specific protocols are used prior to extraction. In general, prior to drilling for bone/tooth powder or crushing bone/tooth to obtain powder, the outer surface of the bone/tooth is removed through superficial drilling of the top millimetre of the surface (115). Short UV treatment to the outer surface of bones and teeth can be used to crosslink the DNA on the surfaces in combination with the removal of the surface (115). Other treatments include pre-digestion of the bone/dentine/cementum powder or skin for 15 to 60 minutes in an extraction buffer to remove surface contaminant DNA, or bleach washing of the material which has been shown to improve the endogenous DNA content of the specimens ( 117).

This study attempted to limit contamination by conducting all extraction and library building activities for the ancient and historical samples in purpose-built clean labs at the Lundbeck Centre for GeoGenetics in Copenhagen and the Swedish Museum of Natural History in Stockholm, neither of which have processed modern DNA. These labs are located in separate buildings from any modern DNA laboratories and outfitted with the recommended facilities and abide by standard aDNA cleaning.

/ Post-Mortem DNA Damage

Subsequent to the death of a cell or organism, the repair processes in cells used to repair and maintain DNA ceases. In addition to the cessation of repair, DNA also undergoes several forms of post-mortem damage (118). DNA fragmentation and deamination are two prominent forms of degradation which afflicts aDNA samples ( 119).

The double stranded DNA breaks into smaller fragments as a result of processes such as depurination and cleavages (118). Depurination occurs through hydrolysis which results in the cleavage of the phosphodiester backbone in purines, adenine and guanine nucleotides, causing DNA fragmentation (120). DNA decays exponentially in relation to the pH level, humidity, and temperature of the environment and time since deposition (121). Therefore, DNA is typically preserved better in permafrost environments such as those in the Arctic, whereas tropical environments where the setting is hot and humid generally result in limited DNA preservation (122,123). NGS technologies require relatively short fragment size making aDNA samples well suited for sequencing on these platforms, although frequently the fragment size is too short to efficiently utilise the sequencing capacity.

Deamination is also a process which impacts the preservation of DNA and downstream analyses. Cytosines are deaminated to uracil through hydrolysis. Polymerases misincorporates thymine in the place of uracils during library building and amplification leading to an increase in thymines where cytosines were originally. Deamination primarily occurs on single stranded DNA typically found at the ends of fragments, resulting in cytosine to thymine misincorporations at the 5’ ends and guanine to adenine misincorporations at the 3’ ends (116,124).

Fig. 3 Deamination patterns as seen with mapDamage at the ends of sequenced reads. The left plot shows C to T deamination and the right plot shows the G to A deamination.

Post-mortem damage can be accounted for during lab preparation and downstream analyses. Uracil-DNA-glycosylase (UDG) treatment can be done following DNA extraction to limit the uracils in the DNA fragments by removing uracils and introducing strand breaks where uracils were previously through cleavages with enzymes such as USER Enzyme (NEB, USA). Bioinformatic methods can also be used to remove deaminated sites computationally. One such method for mitigating against the misincorporation of thymine and adenine is to computationally trim the bases at the end of fragments. Another method to reduce the impact of deamination is to remove transitions, point mutations of a purine mutating to a purine or a pyrimidine to a pyrimidine, from the dataset. Bioinformatic tools such as mapDamage can be utilised to quantify and assess the presence of deamination in samples after sequencing ( 125).

/ DNA Extraction

DNA was extracted from 243 samples during the course of the PhD. A pre-digestion step was performed prior to the extraction in order to improve endogenous content of the samples. A 45 minute incubation of the bone/tooth powder and hide sample with 315 μL of 0.5M EDTA and 7.5 μL of 10 ng/μL proteinase K was utilised for the pre-digestion procedure. After the incubation, the samples were centrifuged for 5 minutes at 13,000 rpm and the supernatant was removed. The undigested bone/tooth powder underwent an overnight digestion with 630 μL of 0.5M EDTA, 70 μL of 1M UREA, and 15 μL of 10 μg/μL proteinase K, following (126). The hide samples were incubated overnight with a lysis buffer according to (127). Following the overnight incubation, the samples were concentrated with VivaSpin Columns and purified using MinElute purification kit (QIAGEN) as per the protocol used in (126).

Following the DNA extraction, screening libraries were built for all extracts using the blunt ending single-tube library preparation (BEST) protocol, which was designed specifically for working with degraded DNA (128). The DNA fragments in the extract underwent blunt end repair followed by a denaturing step at 65°C. Following the blunt ending, Illumina adapters were ligated to the ends of the DNA fragments and a ‘fill in’ step in which the nicks in the DNA strands are filled in. A single purification step was included in the library build after the ‘fill in’ step with MinElute purification kit (QIAGEN). For screening, a single reaction of the library was amplified per sample. Samples with successful amplifications were sequenced on lanes containing 40 samples.

For samples which underwent deep sequencing, the BEST protocol was also used with adjustment depending on the platform on which the samples were being sequenced. For samples sequenced on Illumina platforms, the same protocol as used in screening was maintained for some samples while others underwent USER (Uracil-Specific Excision Reagent) treatment, from New England BioLabs, to remove cytosines which had denatured to uracils. The USER treatment added three steps before the BEST protocol: 1) a three hour incubation at 37C, 2) a ten minute incubation at 65C, and 3) a MinElute purification. After the USER treatment, the protocol followed BEST as before.

The libraries which underwent sequencing on the BGISeq platform also followed the BEST protocol with the only alteration being the adapters and indexes, which were utilised due to the differences in the sequencing platform requirements.

The modern saliva samples from Greenland were extracted with an adapted protocol from the DNeasy Blood & Tissue Kit (Qiagen). The buccal swabs were incubated at 37C for one hour on a rotating plate, after which the supernatant was removed and purified using the DNeasy Blood & Tissue Kit. Libraries were built from these samples using the BEST protocol for sequencing on Illumina platforms.

Next Generation Sequencing (NGS)

A total of 210 libraries were shotgun sequenced to low depth (1,160 to 35,709,413 reads) for screening to determine endogenous DNA content, which varied between 0% and 76%, at the SciLifeLab Sequencing Facilities and Danish High-Throughput DNA Sequencing Centre. Ancient/historical samples (n=81) were selected for deeper sequencing based upon the results of the screening. Subsequent sequencing for deeper coverage was performed at the SciLifeLab Sequencing Facilities, Danish High-Throughput DNA Sequencing

/ Centre, and BGI Sequencing Facilities to obtain more comprehensive genome coverage (336,361 to 369,312,025 reads). A subset of the samples that underwent deeper sequencing had additional libraries built to incorporate a uracil-treatment to compensate for the deamination which affects DNA-post mortem. Additional sequencing for some samples to improve coverage was performed on BGI-Seq platforms, see Table xx. An additional 28 modern samples were sequenced without screening with between 19.4% and 71.9% endogenous DNA to obtain between 1 and 11x coverage of the nuclear genome.

Illumina Shotgun Sequencing

Three Illumina sequencing platforms were used within the scope of this study for either shallow shotgun sequencing for the purposes of screening or for deeper shotgun sequencing to obtain nuclear genome coverage. For the purpose of screening, the Illumina HiSeq2500 was used with paired-end sequencing for 125bp at the SciLifeLab Sequencing facilities in Stockholm, Sweden and with single-end sequencing for 80bp at the National High-throughput DNA Sequencing Centre in Copenhagen, Denmark, Paper I, II, III, IV, and V. The USER treated libraries were deep sequenced on the Illumina HiSeqX at SciLifeLab, Paper III and IV. Deep sequencing of non-USER treated libraries was also performed using the HiSeqX, Paper III and IV. The modern genomes from saliva samples were also sequenced on an Illumina platform: NovaSeq, P aper IV.

BGISeq Shotgun Sequencing

Sequencing was also performed on 58 libraries at Beijing Genomics Institute (BGI) sequencing facilities. The BGISeq-500RS platform and the MGISEQ-2000 platform were utilised when sequencing at the BGI facilities in their Shenzhen and Latvian facilities, respectively. The libraries sequenced on BGI platforms were constructed using the same protocol as those sequenced on Illumina platforms with the only differences being the adapters and primers which were platform specific, Paper II, III, IV and V (128). The libraries sequenced on BGI sequencing platforms did not undergo USER treatment, P aper III and IV.

Computational Methods

Data Processing

In Paper I, IV and V SeqPrep (v.1.1) was used to remove adapters and merge the paired-end reads following sequencing (h ttps://github.com/jstjohn/SeqPrep) . For the samples included in Paper III and II AdapterRemoval (v.2.2.4) was utilised for removing adapters and collapsing the paired-end reads ( 129).

For each dataset in this PhD thesis, a custom pipeline was used to process the sequencing data. The sequenced reads were mapped to the dog reference genome, CanFam3.1 (130), using BWA (version) (131). Filtering and alignment processing was conducted with SAMtools (132) and PCR duplicates were removed with picardTOOLS (h ttp://broadinstitute.github.io/picard/) . For Paper I, II, III, and IV the mitochondrial genome of the dog was extracted from the whole genome alignment with SAMtools, to remove NUMT, nuclear mitochondrial DNA, reads.

Data Analyses

In Paper I & II, consensus sequences were called for each mitochondrial genome using htsbox (h ttps://github.com/lh3/htsbox) . For Paper I a maximum likelihood phylogeny was constructed from the

/ mitochondrial genomes with at least 3x mean coverage for 186 samples generated in the study and 221 published mitochondrial genomes using RAxML (133,134). A tip dated phylogeny was also constructed in BEAST (v.2.4) (135,136). The effective population size through time was estimated using Bayesian Skyline analyses generated with BEAST in P aper I.

For Paper II, the raw sequences were mapped with BWA to a panel of mitochondrial reference genomes from a series of taxa in order to identify the best potential source for the fur from which the sample was derived. The raw sequencing reads were also analysed with the DIAMOND protein aligner (137) to include a wider panel of taxa and to act as a second source of confirmation for the taxa identifications.

Introgression of non-Arctic dog lineage into Arctic dogs was tested with the D-statistic in qpDstat from AdmixTools (138) for Paper III & IV. The outgroup was set as the black-backed jackal, the source population was set as various non-Arctic dog populations, the target population was set as the Arctic dog of interest, and the Zhokhov dog was set as the sister population for Paper III & IV. D-statistic tests for additional gene flow from European dogs into North American Arctic dogs was also performed with other North American Arctic dogs as the sister population in Paper IV. The same procedure was followed to test for wolf introgression into Arctic dogs using qpDstat. To test for wolf introgression the outgroup was again set as the black-backed jackal, the source population was set as various modern and Pleistocene wolf populations, the target population was set as the Arctic dog of interest, and the sister population was set as all other dogs Paper III & IV. To test whether any North American Arctic dog had more wolf gene flow than other North American Arctic dogs the sister population was also restricted to only other North American Arctic dogs P aper IV.

Maximum likelihood tree construction with simulated migration events to account for lack of fit in the maximum likelihood tree using allele frequency data (139). In Paper III & IV TreeMix was run on various subsets of the datasets to include and exclude samples based on coverage and relation to the other samples/populations. In accordance with the TreeMix software recommendations all sites with missing data were excluded, bootstrapping was performed in 50 replicates per run with window sizes of 500 SNPs to account for linkage disequilibrium, and up to eight migrations were simulated. The bootstrap support was calculated and visualised with the BITE software package for R ( 140).

For simple phylogenetic reconstruction neighbour joining trees were built based on identity-by-state matrices calculated by PLINK for samples >2x mean coverage of the genome and low coverage samples >0.1x coverage of the genome for Paper III & IV. The trees were all built with an outgroup, the black-backed jackal and with 100 bootstrap replicates using 1,000,000 random SNPs for each replicate.

To test for shared drift between populations f3-outgroup statistics were calculated with qp3Pop from AdmixTools (138) in Paper III & IV. Similar to the D-stats the black-backed jackal was used as the outgroup while the Arctic dog of interest was set as the target and all other modern and ancient dogs were set as the sister population.

In Paper V shallow shotgun sequencing data from mammoths and dogs were mapped to three reference genomes - the dog, mammoth, and human - to evaluate the amount of human contamination in ancient faunal libraries. These reads were quantified, characterised by their read length, and the post-mortem damage was characterised. The raw data was also mapped to a concatenated reference panel containing the target reference (the CanFam3.1 genome for dog samples or the LoxAfr4 genome for the mammoth) and the

/ human reference genome (Hg19). The reads mapping to the concatenated reference genome were extracted according to the species and filtered to remove reads with low mapping qualities. The that aligned to each species in the concatenated reference were then characterised by the volume, read length, and post-mortem damage.

/ Results & Discussion

Mitochondrial Haplotype Frequency Shifts Associated Human Cultures

Archaeological evidence and human genetic research has identified multiple waves of human migration from Siberia into North America, each of these spread beyond the shores of Alaska (36,39,44,45). In the North American Arctic skeletal remains of dogs have been recovered from both Paleo-Inuit and Inuit sites starting around 5,000 BP; although, the relationship between the dogs of these two cultural complexes remains unexamined. It remains unknown whether Paleo-Inuit dogs are the ancestors of the later Inuit dogs or if the Inuit were accompanied to North America by their own dogs directly from Siberia.

Fig. 4 Phylogenetic topology and geographic distribution of haplotypes through time. (a) The A-clade mitochondrial haplotypes of dogs inferred by maximum-likelihood analyses depicting the four subclades discussed in the text with their respective bootstrap support (for the whole tree see electronic supplementary material). (b) Geographical origin of North American dog samples and cultural affiliation. Pie charts indicate the abundance of subclades. Sites with more than one sample are shown in boxes with representation of sample number and haplotype. Modern samples outside of the North American Arctic were excluded from the map and pie chart. Culture dates represent the earliest and latest appearance of each group in the North American Arctic within this dataset.

In Paper I, complete (n=186) and partial (n=40) mitochondrial genomes were sequenced to explore the phylogenetic history of dogs in the North American Arctic. Previous studies have shown that dogs can be found in one of six mitochondrial clades, A-F (1,2,7,63). This study identified four main subclades within the A clade: A1a, A1b, A2a, and A2b (Fig. 4a). One of these subclades, A2b, had previously only been identified in pre-contact dogs (PCDs) of the subarctic Americas. PCDs are exclusively found in the A2b subclade and disappeared after the introduction of European dogs (7). This study found that some dogs in the North American Arctic also fall into the A2b subclade. The cultural complex that first exclusively settled the North American Arctic - the Paleo-Inuit - brought dogs with them from Siberia, this can be seen by the arrival of dogs in North America falling into a second distinct subclade of A, A2a (Fig. 4b). The next major wave of people moving into the North American Arctic, the Inuit cultural complex, also brought new dogs

/ with them, as seen through additional haplotypes associated with mitochondrial clades being introduced to the region, namely haplotypes from the A1a and A1b subclades (Fig. 4b).

The introduction of haplotypes associated with the distinct subclades of the mitochondrial phylogeny demonstrated the introduction of new dogs to the North American Arctic with the arrival of each new cultural group to the continent. Morphological study of North American dogs using geometric morphometrics also showed that not only did these dogs carry with them novel mitochondrial haplotypes they also possessed different morphologies. Paleo-Inuit dogs possessed proportionally wider craniums, more elevated braincases, narrower lower M1 and a less developed mandibular ascending ramus than Inuit dogs.

Changes in haplotype frequency were observed after European contact with the Arctic. The most notable of these changes was seen in the increased frequency of the A1a haplotypes in the Arctic. These haplotypes were only found in two dogs before the arrival of Europeans but are the most prevalent haplotypes found in modern dogs outside of the Arctic. The increase in dogs being imported to the Arctic following the colonisation and regionally increasing settlement during eras such as the Gold Rush may have been one of the factors in this frequency shift. Other factors may also have played into this such as changes in settlement patterning as a result of colonisation and industrialisation when new permanent settlements were established.

A completely novel mitochondrial clade was also identified in Paper I, the X clade. Haplotypes from this clade were discovered in seven dogs ranging between 6,660 and 75 BP. Despite not previously being discovered dogs with these haplotypes were not restricted to only one particular region. X clade mitochondrial genomes were found in dogs in Chukotka, Kamchatka, Khasansky, and Alaska.

Identification and Dietary Stable Isotope Analysis of Mammal Fur in Arctic Clothing

Paper II assesses and explores the macroscopic identification of dog fur on garments curated by the National Museum of Denmark using mitochondrial DNA and stable isotope data. Fur macroscopically identified as dog fur was sampled from clothing items collected in the Arctic in the nineteenth and twentieth centuries CE.

Shallow shotgun sequenced libraries were aligned to a panel of mitochondrial reference genomes to identify the species of origin, this was used complimentary to DIAMOND for taxonomic assignment. Of the 68 specimens samples 47 had sufficiently concentrated DNA extracts for sequencing. The methods implemented in this study proved to be insufficient for samples that received very shallow sequencing, < 50,000 reads, which was the case for 8 samples allowing for identification of 36 specimens as well as the 11 previously published specimens (Fig. 5). The study was able to identify 19 samples as having originated from dogs/wolves, samples with mitochondrial coverage of ≥3x (n=17) were used for phylogenetic tree construction. Three specimens were identified as foxes and for one sample mitochondrial coverage was sufficient for phylogenetic tree construction with red and arctic foxes, supporting a species identification of red fox. A further 8 samples were identified as wolverine, the second most abundant species in the samples studied, highlighting the macroscopic similarity of dog and wolverine fur. Additional taxa were identified in the dataset such as lynx, reindeer, bear, and rodent (Fig. 5).

/ Fig. 5. Genetically identified taxa from fur samples. A) Counts taxa identified genetically from this study and the published samples from the collection (141) . B) Counts of dog/wolf haplotypes carried by dogs/wolves in the collection. C) Genetically identified fur samples mapped to the region of collection.

Stable isotopes extracted from the hairs of the fur samples showed that the marine sourced diet of dogs from Canada and Greenland clearly distinguished the dogs from wolves. As a result of the consistently strong marine dietary input fur samples from Canada and Greenland that could not be identified genetically were able to be identified as dogs rather than wolves through their stable isotope values. Unlike the dogs of North America the isotopic distinction between dogs and wolves in Siberia was less categorical. Dogs from Kamchatka surprisingly did not have isotopic levels consistent with a diet based on salmon as reported in several ethnographic accounts (34,35,142). Dogs from Nenets and Khanti communities in the Northern and Polar Urals generally had dietary stable isotope values consistent with a diet of mixed terrestrial and anadromous fish sources.

Population Structure in Arctic Dogs

PCA and phylogenetic analyses identified population structure in the dogs of the Arctic. In Paper III, clustering was seen in Siberian dogs depending on their temporal or regional context in the PCAs. Phylogenetic construction from Siberian dog genomes showed that not surprisingly the clustering seen in the PCA is likewise seen in phylogenetic trees. The topology seen in the neighbour joining tree reiterates the tree structure seen in earlier studies (5,7) with three major clades of dogs largely reflective of geography showing clades of: East Asian, Arctic, and West Eurasian/African dogs (Fig. 6a). Siberian dogs from sites such as the early Holocene site of Veretye and the Bronze Age Sites of Ishkinino and Krasnosamarskoe are found clustered with West Eurasian dogs (Fig. 6). Regions with serial sampling through time show phylogenetic separation of samples, dependent upon the age of the site, this is most apparent in northwestern Siberia around the Yamal Peninsula.

/ Fig. 6, Neighbour joining tree and Principal component analysis (PCA). A) Neighbour joining tree of dogs with at least 2x coverage, bootstrapped with 100 replicates. Branches coloured by geographical origin and icons at the branch tip indicate sample age. C) PCA of Siberian dogs with publically available reference panel of dogs, including Greenland Sled Dogs (GSD), Alaskan Malamutes (AM), Siberian Huskies (SH), a Samoyed (SM), pre-contact North American dogs (PCD), and Bronze Age Steppe dogs (BA Steppe).

Paper IV found that the structure seen in the Siberian Arctic is maintained when North American Arctic dogs are added to the mix. Unlike Siberia that shows population structure relating to time or associated culture and location, the structure seen in the dogs of the North American Arctic relates primarily to geography, Fig. 7. The difference between the factors driving the population structures in Siberia and North America most likely reflect the relatively short window of time that Inuit dogs have occupied the North American Arctic as well as their shared cultural and Siberian origin. Reflective of geography Alaskan dogs fall between Siberian and East Arctic dogs while dogs from eastern Canada and Greenland are situated more closely together in both PCAs and phylogenies.

Within Greenland there is clear regional population structure echoing migration events and subsequent isolation, Fig. 7. The population structure in Greenland attests to a northward migration from Northwest to Northeast Greenland. However, interaction between Northeast and East Greenland does appear to have occurred. Changes in population structure can be seen regionally through time. An example of this can be found in Northeast Greenland where humans and their dogs disappeared in the nineteenth century until a permanent settlement was re-established in 1925 and populated by people and dogs from Tasiilaq (143). Similarly, periodic epidemics in Northwest Greenland can be seen in the discontinuous population structure of Northwest Greenland dogs through time.

Fig. 7 A) Neighbour joining tree rooted with the black-backed jackal as the outgroup including only samples with at least 2x mean coverage of the nuclear genome. Only dogs in the ‘Arctic’ dog clade are shown. The branch colour reflects the dog’s region of origin, icons at the branch tip indicates the sample age. Bootstrap support of nodes less than 75% are indicated.

/ Gene Flow From Non-Arctic Dogs into Arctic Dogs

In Paper III, 21 Siberian dog genomes were sequenced from ancient and historical dogs, between 11,000 and 60 years old and for Paper IV 95 North American dog genomes were used to examine interactions between Arctic dogs and dogs outside of the Arctic.

In the oldest samples, directly dated to 10,900 BP, in the dataset two dogs from the Veretye site in Karelia, western Russia, mixed ancient European and Arctic could be seen. A second, independent gene flow event was detected in Bronze Age dogs from the Siberian Steppe bringing West Eurasian, specifically from the Near East, ancestry into the Steppe, Fig. 8. This Near East related ancestry spread reaching the extremes of Siberia by 2,000 BP. The combination of Arctic and West Eurasian ancestry can be observed at the Iron Age site of Ust’-Polui on the Yamal Peninsula around 2,000 BP. Additional gene flow can be seen in the Yamal region by 1,000 years later at the sites of Tiutei-Sale 1, Yarte 6 , and Ust’-Boikar where a stronger affinity can be seen between dogs from these sites and West Eurasian dogs, again the signal for this gene flow is strongest from Near East and Siberian Steppe related sources.

Fig. 8: Steppe and West Siberian dogs. Ai-iii.) Outgroup-f3 statistics for samples TRF.04.04, TRF.05.05, and TRF.05.14 with qp3Pop. The outgroup was set as the black-backed jackal and the sister population was set as modern and ancient dogs from across the world. F3 values for specific individuals listed on map: GSD (Greenland Sled Dog, Tasiilaq_51602), A.EU (Ancient European, HXH), A.NE (Ancient Near East, ASHQ01), A.SS (Ancient Siberian Steppe, TRF.04.04), and the individual with the highest f3 value when compared to the sample. Bi-iii.) D-statistic test of West Eurasian ancestry into West Siberian dogs from qpDstat. The outgroup was set as the black-backed jackal, and the sister population was set as the 9,500BP Zhokhov dog in red or set as a modern Greenland Sled Dog (GSD Tasiilaq 51602) in blue in the formula: (D(Jackal, SOURCE; sample, sister population)).

The West Eurasian signal is also seen in all North American Arctic dogs in Paper IV, demonstrating that dogs with West Eurasian ancestry had reached the Bering Strait before the ancestors of Inuit dogs had left Siberia on their migration to North America. As expected all North American Arctic dogs, prior to European contact, show no additional signal for Eurasian gene flow as a result of the geographical isolation. However, over the last few centuries non-Arctic dogs have been imported to North America and there has subsequently

/ been admixture between European and Inuit dogs. A similar pattern can be seen in historical post-contact dogs from Siberia receiving additional gene flow from European dogs in P aper III.

Some of the North American Arctic dogs also appear to have experienced additional gene flow from dogs other than Inuit dogs once in North America. Pre-contact dogs from Eastern Eastern Canada in this dataset unanimously show gene flow from an earlier North American dog population as seen through an increased allele sharing between Pre-contact Arctic Candian dogs and a PCD at the 4,000 BP site of Port au Choix, Fig. 9. However, given the limited sample size for comparison, a single individual with >1x coverage, and absence of genomic information from Paleo-Inuit dogs, such as the Dorset, a precise cultural origin cannot currently be identified. This signal for PCD related ancestry can also be seen in most post-contact historical Canadian dogs and two individuals outside of Canada, one in Alaska and another in Greenland. The Alaskan individual presenting this signal may be evidence for a separate admixture event between Inuit and Paleo-Inuit dogs. As this PCD signal is not universal across the North American Arctic it does not appear that this is the result of an early admixture event that occurred in Alaska before the spread into the rest of the North American Arctic. The sole individual from pre-contact East Greenland is from Misigtoq, a site that is thought to have been occupied between 400 to 200 BP (9), the introgression event most likely transpired in Canada. Modern Greenland Sled Dogs generally show strong signals for modern European gene flow when compared to the pre-contact and historical post-contact dogs from Greenland.

Fig. 9 D-statistic calculations from AdmixTools utility qpDstat using 1,685,511 SNPs to test for relative gene flow from precontact (PCD) subarctic dogs into North American Arctic dogs in comparison to other North American Arctic dogs. Z score from the D-statistic calculation incorporating the standard error is plotted per sample coloured by sample origin and dashed red lines plotted indicating statistical significance at -3.3 and 3.3. For all calculations only other North American Arctic dogs were used as sister populations, the source population was tested as the PCD dog from Port au Choix, and the black-backed jackal was used as an outgroup, D(Jackal, Port au Choix; sample, sister population). A) Pre-contact and post-contact North American Arctic dogs, excluding Greenland Sled Dogs. B) Modern Greenland Sled Dogs.

Together these results show that despite their relative isolation, dogs of the Arctic have encountered other dog populations in Siberia paralleling human migrations prehistorically and again in the modern era reflecting colonisation and globalisation.

/ Introgression From Wolves into Arctic Dogs

In Paper III, Siberian dogs were assessed for introgression from Pleistocene and modern wolf. When compared to dogs from across Eurasia and Africa, Siberian dogs share more alleles with wolves than other dogs across Asia share with wolves. This signal is strongest when a 33,000 BP wolf is tested as the source of this gene flow from wolves into Siberian dogs, Fig. 10.

Fig. 10: D-statistics as calculated by qpDstat to test for gene flow from A.) Pleistocene wolf and B) modern wolf into Siberian and Arctic dogs. The plotted results testing different wolves as source populations are coloured by the dog sample being tested. The red dashed line marks -3.3 and +3.3 corresponding to the statistically significant Z-score. The sister population, Y, was tested as modern village and breed dogs, ancient West Eurasian dogs, and all Siberian dogs in the dataset, in D(Jackal, wolf; X, Y).

Modern wolf gene flow into Siberian dogs was also detected in several dogs. One of the 10,900 BP dogs from Veretye, a dog from Lake Baikal, and a dog sample collected in the twentieth century from the Amur Delta had statistically significantly signal for gene flow from modern wolves. Interestingly, each of these dogs is from a site or region where multiple dog genomes were sequenced but modern wolf gene flow was detected in only a single individual from each region.

Alternatively, North American Arctic dogs almost all have statistically significant signals for gene flow from wolves into dogs when compared to other dog populations, this is found in pre- and post-contact dogs alike, including modern Greenland Sled Dogs, as seen in Paper IV, Fig. 11. This universally detected gene flow from wolves into North American Arctic dogs supports a scenario in which the ancestor of North American Arctic dogs in Siberia admixed with wolves before departing Siberia but this did not become pervasive in Siberia or, alternatively, this introgression event occurred upon arrival to North America where an early Inuit dog, most likely in Alaska, admixed with wolves and this spread east to Canada and Greenland.

Nevertheless, when gene flow from wolves into North American Arctic dogs is examined relative to other North American Arctic dogs, to determine if there have been additional population or individual specific admixture events, very few samples showed significant signals for wolf introgression. A pre-contact Canadian dog showed signal for wolf introgression supporting a local introgression event occurring in the East Arctic. Modern Greenland Sled Dogs do not show consistently significant signals for wolf introgression when compared to other North American Arctic dogs.

/ Fig. 11: D-statistic calculations with qpDstat (AdmixTools) using 564,419 SNPs testing for gene flow from Wolves into North American Arctic dogs. Z score from the D-statistic calculation incorporating the standard error is plotted per sample and coloured by sample origin. For all calculations the sister population was set as dogs from a global distribution and the black-backed jackal was set as the outgroup, D(Jackal, WOLF; sample, sister population). Ai-iii) Pre-contact and post-contact North American dogs with at least 1x mean coverage of the genome, excluding Greenland Sled Dogs. Ai) Alaskan wolf, Aii) Greenland wolf, Aiii) Chukotka wolf tested as source. Bi-iii) Modern Greenland Sled Dogs, Bi) Alaskan wolf, Bii) Greenland wolf, and Chukotka wolf tested as source.

These results suggest that while there have been admixture events between North American Arctic dogs and wolves they appear to be infrequent. In the current dataset there is no evidence for introgression from wolves in the last two centuries. However, the wolf ancestry North American Arctic dogs do possess sets them apart from their counterparts in Siberia.

Mitigation Against Human Contamination in Sequenced Faunal Libraries

Ancient DNA studies have been plagued by complications of contamination since the early days of the field (113,144–148). The introduction of NGS technologies has lead to a rapid increase accumulation of genomic data from ancient sources as the cost of sequencing has become increasingly more affordable. However, challenges remain in the authentication of the DNA sequenced. Numerous steps in the preparation of genomic data can introduce contaminant human DNA to ancient samples, imposing challenges on cost-effective sequencing and accurate interpretation of data. Standard procedure when working with ancient DNA in a laboratory setting typically follows one or more protocols for reducing modern contamination while sampling or prior to extraction beyond the standard clean lab setting, ie. UV or bleach treatment

/ (117,149). We developed a simple method for removing contaminant human DNA from sequenced ancient DNA libraries to be used in conjunction with field and anti-contamination methods.

Initial mapping of faunal data from ancient DNA libraries to the human reference genome showed us that in this dataset most (>95%) samples contained less than 0.071% of reads mapped. Nevertheless, there were some samples that showed up to 38.9% of the reads mapping to the human reference. After realigning reads mapped to the target reference genome a variable but generally low level of human contamination was identified with up to 1.3% of reads the BAM file being remapped to the human reference.

Fig. 12: Characterization of endogenous and human contaminant reads in faunal BAM files. A) Comparisons of PMDR and mRL for all mammoth samples. B) mRL for mammoth sequences mapping to the elephant or the human parts of the concatenated reference. C) PMDR for mammoth sequences mapping to the elephant or the human parts of the concatenated reference. D) Comparisons of PMDR and mRL for all ancient dog samples. E) mRL for dog sequences mapping to the dog or the human parts of the concatenated reference. F) PMDR for dog sequences mapping to the dog or the human parts of the concatenated reference. In all cases, **: p-value < 0.01 and ****: p-value < 0.0001.

The presence of contaminant human sequences in ancient faunal BAM files represents a challenge for downstream analyses based in evolutionary conserved parts of the genome, such as coding regions. The mapped contaminant sequences are concentrated in these regions as a result of the conserved nature of the sequences. Our method uses competitive mapping to identify and remove human contamination from faunal datasets through alignment to concatenated reference genomes. Subsequent to alignment reads that are unmapped, mapped to the human reference genome, and have low mapping quality are removed after mapping to the concatenated reference, retaining only the reads mapping to the target reference genome of the species of interest. Our results indicate the loss of data is on average 1.35%, although it varies between 0.6% and 4.1%. The reads mapped to the human reference are characterised by short read lengths and low post-mortem damage scores (Fig. 12).

/ Future Directions

Overall, this PhD explored the circumpolar movement of dogs and their interactions with dogs and wolves within and outside of the Arctic in both Siberia and North America using nuclear and mitochondrial genome data. The dataset of 94 Arctic dog nuclear genomes and 186 mitochondrial genomes generated for this PhD represent a massive increase of ancient DNA data for dogs, including the first nuclear genomes of pre-contact North American Arctic dogs.

Future directions to be pursued with this dataset include investigation of heterozygosity levels, patterns of selection, the potential selection of genes as the result of introgression events, and modelling of demographic patterns. Future studies looking at the evolution of dogs in Siberia could use this dataset with the addition of archaeological samples in eastern Siberia to dog deeper into the timing of the arrival of West Eurasian gene flow to the region, older samples from northwest Siberia and the Steppe would also be aid this investigation. Additional study of dogs of the North American Arctic would benefit from the addition of further sampling of pre-contact dogs from Canada and Alaska. In particular Paleo-Inuit dogs from Alaska, Canada, and Greenland would allow for the investigation of the signal for PCD ancestry in dogs from Canada and Greenland.

/ References

1. Savolainen P, Zhang Y-P, Luo J, Lundeberg J, Leitner T. Genetic evidence for an East Asian origin of domestic dogs. Science. 2002 Nov 22;298(5598):1610–3.

2. Pang J-F, Kluetsch C, Zou X-J, Zhang A-B, Luo L-Y, Angleby H, et al. mtDNA data indicate a single origin for dogs south of Yangtze River, less than 16,300 years ago, from numerous wolves. Mol Biol Evol. 2009 Dec;26(12):2849–64.

3. Wang G-D, Zhai W, Yang H-C, Wang L, Zhong L, Liu Y-H, et al. Out of southern East Asia: the natural history of domestic dogs across the world. Cell Res. 2016 Jan;26(1):21–33.

4. Botigué LR, Song S, Scheu A, Gopalan S, Pendleton AL, Oetjens M, et al. Ancient European dog genomes reveal continuity since the Early Neolithic. Nat Commun. 2017 Jul 18;8:16082.

5. Frantz LAF, Mullin VE, Pionnier-Capitan M, Lebrasseur O, Ollivier M, Perri A, et al. Genomic and archaeological evidence suggest a dual origin of domestic dogs. Science. 2016 Jun 3;352(6290):1228–31.

6. Fan Z, Silva P, Gronau I, Wang S, Armero AS, Schweizer RM, et al. Worldwide patterns of genomic variation and admixture in gray wolves. Genome Res. 2016 Feb;26(2):163–73.

7. Ní Leathlobhair M, Perri AR, Irving-Pease EK, Witt KE, Linderholm A, Haile J, et al. The evolutionary history of dogs in the Americas. Science. 2018 Jul 6;361(6397):81–5.

8. Morey DF. In search of Paleolithic dogs: a quest with mixed results. J Archaeol Sci. 2014 Dec 1;52:300–7.

9. Morey DF. Dogs: Domestication and the Development of a Social Bond. Cambridge University Press; 2010. 385 p.

10. Janssens L, Giemsch L, Schmitz R, Street M, Van Dongen S, Crombé P. A new look at an old dog: Bonn-Oberkassel reconsidered. J Archaeol Sci. 2018 Apr 1;92:126–38.

11. Pitul’ko VV, Kasparov AK. Archaeological dogs from the Early Holocene Zhokhov site in the Eastern Siberian Arctic. Journal of Archaeological Science: Reports. 2017 Jun 1;13:491–515.

12. Skoglund P, Ersmark E, Palkopoulou E, Dalén L. Ancient wolf genome reveals an early divergence of domestic dog ancestors and admixture into high-latitude breeds. Curr Biol. 2015 Jun 1;25(11):1515–9.

13. Losey RJ, Nomokonova T, Fleming LS, Kharinskii AV, Kovychev EV, Konstantinov MV, et al. Buried, eaten, sacrificed: Archaeological dog remains from Trans-Baikal, Siberia. Archaeological Research in Asia [Internet]. 2018 Mar 8; Available from: http://www.sciencedirect.com/science/article/pii/S2352226718300060

14. Henderson N. REPLICATING DOG TRAVOIS TRAVEL ON THE NORTHERN PLAINS. Plains Anthropol. 1994;39(148):145–59.

15. Jobie Showmik Weetaluktuk SCC, editor. The cultural magazine of the Nunavik Inuit: special Issue Qimmiit Eskimo Dogs. Vol. 12. Culture and Linguistics Program of the Departmen t of Indian and Northern Affairs, Canada; 2000.

16. Morey DF, Aaris-Sørensen K. Paleoeskimo Dogs of the Eastern Arctic. Arctic. 2002;55(1):44–56.

/ 17. Montcombroux G. The Inuit Dog of the Polar North. Whippoorwill Press; 2015.

18. Pitul’ko VV, Kasparov AK. Ancient Arctic Hunters: Material Culture and Survival Strategy. Arctic Anthropol. 1996;33(1):1–36.

19. Mathiassen T. Archaeology of the central Eskimos: II. The Thule culture and its position within the Eskimo culture. Copenhagen: Gyldendalske; 1927. (Report of the fifth Thule expedition 1921-24; vol. IV).

20. Losey RJ, Nomokonova T, Gusev AV, Bachura OP, Fedorova NV, Kosintsev PA, et al. Dogs were domesticated in the Arctic: Culling practices and dog sledding at Ust’-Polui. Journal of Anthropological Archaeology. 2018 Sep 1;51:113–26.

21. Losey RJ, Fleming LS, Nomokonova T, Gusev AV, Fedorova NV, Garvie-Lok S, et al. Human and Dog Consumption of Fish on the Lower Ob River of Siberia: Evidence for a Major Freshwater Reservoir Effect at the Ust’-Polui Site. Radiocarbon. 2018 Feb;60(1):239–60.

22. Gotfredsen AB. The faunal material of the Saqqaq site Nipisat 1, Sisimiut District, west Greenland. Man, Culture and Environment in Ancient Greenland Report on a Research Programe. 1998;124–32.

23. Pitulko VV, Tikhonov AN, Pavlova EY, Nikolskiy PA, Kuper KE, Polozov RN. Paleoanthropology. Early human presence in the Arctic: Evidence from 45,000-year-old mammoth remains. Science. 2016 Jan 15;351(6270):260–3.

24. Pitulko VV, Nikolsky PA, Girya EY, Basilyan AE, Tumskoy VE, Koulakov SA, et al. The Yana RHS site: humans in the Arctic before the last glacial maximum. Science. 2004 Jan 2;303(5654):52–6.

25. Sikora M, Pitulko VV, Sousa VC, Allentoft ME, Vinner L, Rasmussen S, et al. The population history of northeastern Siberia since the Pleistocene. Nature. 2019 Jun;570(7760):182–8.

26. Goebel T. The “Microblade Adaptation” and Recolonization of Siberia during the Late Upper Pleistocene. In: Robert G. Elston and Steven L. Kuhn, editor. Thinking Small: Global Perspectives on Microlithization. 2002. p. 117–31. (12).

27. Allentoft ME, Sikora M, Sjögren K-G, Rasmussen S, Rasmussen M, Stenderup J, et al. Population genomics of Bronze Age Eurasia. Nature. 2015 Jun 11;522(7555):167–72.

28. Molodin VI, Pilipenko AS, Romaschenko AG. Human migrations in the southern region of the West Siberian Plain during the Bronze Age: archaeological, palaeogenetic and anthropological data. in prehistory and early … [Internet]. 2012; Available from: https://www.degruyter.com/downloadpdf/books/9783110266306/9783110266306.93/9783110266306.93 .xml

29. Wong EHM, Khrunin A, Nichols L, Pushkarev D, Khokhrin D, Verbenko D, et al. Reconstructing genetic history of Siberian and Northeastern European populations. Genome Res. 2017 Jan;27(1):1–14.

30. Unterländer M, Palstra F, Lazaridis I, Pilipenko A, Hofmanová Z, Groß M, et al. Ancestry and demography and descendants of Iron Age nomads of the Eurasian Steppe. Nat Commun. 2017 Mar 3;8:14615.

31. Damgaard P de B, Marchi N, Rasmussen S, Peyrot M, Renaud G, Korneliussen T, et al. 137 ancient human genomes from across the Eurasian steppes. Nature. 2018 May;557(7705):369–74.

32. Vaskul IO, Zherebtsov IL, Murygin AM, Klenov MV. Arctic and subarctic zones of the European North-East of Russia in the Iron Age and the Middle Ages. IOP Conf Ser: Earth Environ Sci. 2019 Aug 6;302(1):012005.

/ 33. Fitzhugh WW. Searching for the Grail: Virtual Archaeology in Yamal and Circumpolar Theory. In: R Gilberg And Hans, editor. Fifty Years of Arctic Research Anthropological Studies From Greenland to Siberia. The National Museum of Denmark; 1997. p. 102–18. ( Publications of the National Museum Ethnographical Series).

34. Strecker L. Northern relations : People, sled dogs and salmon in Kamchatka (Russian Far East). In: Dogs in the North. Routledge; 2018. p. 61–86.

35. Steller GW. History of Kamchatka: Collected Information Concerning the History of Kamchatka, Its Peoples, Their Manners, Names, Lifestyles, and Various Customary Practices. Fairbanks: University of Alaska Press; 1774.

36. Moreno-Mayar JV, Potter BA, Vinner L, Steinrücken M, Rasmussen S, Terhorst J, et al. Terminal Pleistocene Alaskan genome reveals first founding population of Native Americans. Nature. 2018 Jan 11;553(7687):203–7.

37. Pedersen MW, Ruter A, Schweger C, Friebe H, Staff RA, Kjeldsen KK, et al. Postglacial viability and colonization in North America’s ice-free corridor. Nature. 2016 Sep 1;537(7618):45–9.

38. Potter BA, Baichtal JF, Beaudoin AB, Fehren-Schmitz L, Haynes CV, Holliday VT, et al. Current evidence allows multiple models for the peopling of the Americas. Sci Adv. 2018 Aug;4(8):eaat5473.

39. Goebel T, Waters MR, O’Rourke DH. The late Pleistocene dispersal of modern humans in the Americas. Science. 2008 Mar 14;319(5869):1497–502.

40. Posth C, Nakatsuka N, Lazaridis I, Skoglund P, Mallick S, Lamnidis TC, et al. Reconstructing the Deep Population History of Central and South America. Cell [Internet]. 2018 Nov 8 [cited 2018 Nov 9];0(0). Available from: http://www.cell.com/cell/retrieve/pii/S0092867418313801?_returnURL=https%3A%2F%2Flinkinghub. elsevier.com%2Fretrieve%2Fpii%2FS0092867418313801%3Fshowall%3Dtrue

41. Raghavan M, Steinrücken M, Harris K, Schiffels S, Rasmussen S, DeGiorgio M, et al. POPULATION GENETICS. Genomic evidence for the Pleistocene and recent population history of Native Americans. Science. 2015 Aug 21;349(6250):aab3884.

42. Raghavan M, Skoglund P, Graf KE, Metspalu M, Albrechtsen A, Moltke I, et al. Upper Palaeolithic Siberian genome reveals dual ancestry of Native Americans. Nature. 2014 Jan 2;505(7481):87–91.

43. Friesen TM. On the naming of Arctic archaeological traditions: The case for Paleo-Inuit. Arctic [Internet]. 2015;68(3). Available from: https://journalhosting.ucalgary.ca/index.php/arctic/article/download/67546/51449

44. Raghavan M, DeGiorgio M, Albrechtsen A, Moltke I, Skoglund P, Korneliussen TS, et al. The genetic prehistory of the New World Arctic. Science. 2014 Aug 29;345(6200):1255832.

45. Friesen M. Pan-Arctic Population Movements. In: Max Friesen And, editor. The Oxford Handbook of the Prehistoric Arctic. Oxford University Press; 2016.

46. Grønnow B. Independence I and Saqqaq. In: Max Friesen And, editor. The Oxford Handbook of the Prehistoric Arctic. Oxford University Press; 2016.

47. Bjarne Grønnow And. Palaeo-Eskimo Migrations into Greenland: The Canadian Connection. In: Jette Arneborg And, editor. In Dynamics of Northern Societies: Proceedings of the SILA/ NABO Conference on Arctic and North Atlantic Archaeology. National Museum, Copenhagen; 2006. p. 59–74.

48. Jensen JF. Greenlandic Dorset. In: Max Friesen And, editor. The Oxford Handbook of the Prehistoric

/ Arctic. Oxford University Press; 2016.

49. Appelt M, Damkjar E, Friesen M. Late Dorset. In: Max Friesen And, editor. The Oxford Handbook of the Prehistoric Arctic. Oxford University Press; 2016.

50. Mathiassen T. Archæology of the Central Eskimos: I. Descriptive Part. Copenhagen: Gyldendal; 1927. (Report of the fifth Thule expedition, 1921-24; vol. IV).

51. Whitridge P. Classic Thule [Classic Precontact Inuit]. In: The Oxford handbook of the prehistoric Arctic. Oxford University Press New York; 2016. p. 827–50.

52. Rasmussen K. The Fifth Thule Expedition, 1921-24. The Danish Ethnographical and Geographical Expedition from Greenland to the Pacific. Geogr J. 1926;67(2):123–38.

53. Inuit Circumpolar Council Resolution 2010 – 01 [Internet]. [cited 2020 Jan 18]. Available from: https://www.inuitcircumpolar.com/wp-content/uploads/2019/01/iccexcouncilresolutiononterminuit.pdf

54. Mason O. Thule Origins in the Old Bering Sea Culture. In: Max Friesen And, editor. The Oxford Handbook of the Prehistoric Arctic. Oxford University Press; 2016.

55. Gulløv H-C. Inuit-European Interactions in Greenland. In: Max Friesen And, editor. The Oxford Handbook of the Prehistoric Arctic. Oxford University Press; 2016.

56. Mc Ghee R. The Timing of the Thule Migration. Polarjorschung. 1984;54(1):1–7.

57. Gilberg R. Thule. Arctic. 1976;29(2):83–6.

58. Mathiassen T. Prehistory of the Angmagssalik Eskimos. CA Reitzel; 1933.

59. Flegontov P, Altınışık NE, Changmai P, Rohland N, Mallick S, Adamski N, et al. Palaeo-Eskimo genetic ancestry and the peopling of Chukotka and North America. Nature. 2019 Jun;570(7760):236–40.

60. McGovern TH. The Archaeology of the Norse North Atlantic. Annu Rev Anthropol. 1990;19:331–51.

61. McGovern TH. Cows, Harp Seals, and Churchbells: Adaptation and Extinction in Norse Greenland. Hum Ecol. 1980;8(3):245–75.

62. Allen RC, Keay I. Bowhead Whales in the Eastern Arctic, 1611-1911: Population Reconstruction with Historical Whaling Records. Environ Hist Camb. 2006;12(1):89–113.

63. Thalmann O, Shapiro B, Cui P, Schuenemann VJ, Sawyer SK, Greenfield DL, et al. Complete mitochondrial genomes of ancient canids suggest a European origin of domestic dogs. Science. 2013 Nov 15;342(6160):871–4.

64. Germonpré M, Jimenez E-L, Sablin M. Palaeolithic and prehistoric dogs and Pleistocene wolves from Yakutia: Identification of isolated skulls. J Archaeol Sci. 2016 Nov 27;78(1-19):1–19.

65. Perri A. A wolf in dog’s clothing: Initial dog domestication and Pleistocene wolf variation. J Archaeol Sci. 2016 Apr 1;68:1–4.

66. Lee EJ, Merriwether DA, Kasparov AK, Nikolskiy PA, Sotnikova MV, Pavlova EY, et al. Ancient DNA analysis of the oldest canid species from the Siberian Arctic and genetic contribution to the domestic dog. PLoS One. 2015 May 27;10(5):e0125759.

67. Astakhov SN. Paleolithic of the Yenisei: Paleolithic sites on the Afontova Gora in the Krasnoyarsk.

/ St.-Petersburg: European House (in Russian); 1999.

68. Losey RJ, Bazaliiskii VI, Garvie-Lok S, Germonpré M, Leonard JA, Allen AL, et al. Canids as persons: Early Neolithic dog and wolf burials, Cis-Baikal, Siberia. Journal of Anthropological Archaeology. 2011 Jun 1;30(2):174–89.

69. Drake AG, Coquerelle M, Kosintsev PA, Bachura OP, Sablin M, Gusev AV, et al. Three-Dimensional Geometric Morphometric Analysis of Fossil Canid Mandibles and Skulls. Sci Rep. 2017 Aug 25;7(1):9508.

70. Mason OK. The Contest between the Ipiutak, Old Bering Sea, and Birnirk Polities and the Origin of Whaling during the First Millennium A.D. along Bering Strait. Journal of Anthropological Archaeology. 1998 Sep 1;17(3):240–325.

71. Steffian AF. Fifty years after Hrdlicka: Further excavation of the Uyak site, Kodiak Island, Alaska. Contributions to the anthropology of southcentral and southwestern Alaska. 1992;141–64.

72. Heizer RF. Archaeology of the Uyak Site, Kodiak Island, Alaska. Vol. 17. University of California Press; 1956.

73. Giddings JL, Anderson DD. Beach Ridge Archaeology of Cape Krusenstern: Eskimo and Pre-Eskimo Settlements around Kotzebue Sound Alaska. Publications in Archaeology 20 National Park Service. 1986;U.S. Department of the Interior, Washington, D.C.

74. Park RW. Dog remains from Devon Island, NWT: archaeological and osteological evidence for domestic dog use in the Thule culture. Arctic. 1987;184–90.

75. Murray MS. Economic change in the Palaeoeskimo prehistory of the Foxe Basin, NWT [Internet]. macsphere.mcmaster.ca; 1996. Available from: h ttps://macsphere.mcmaster.ca/handle/11375/12973

76. Arnold CD. Possible Evidence of Domestic Dog in a Paleoeskimo Context. Arctic. 1979;32(3):263–5.

77. Park RW. The Dorset-Thule Succession in Arctic North America: Assessing Claims for Culture Contact. Am Antiq. 1993;58(2):203–34.

78. Seersholm FV, Pedersen MW, Søe MJ, Shokry H, Mak SST, Ruter A, et al. DNA evidence of bowhead whale exploitation by Greenlandic Paleo-Inuit 4,000 years ago. Nat Commun. 2016 Nov 8;7:13389.

79. Jensen JF. Stone Age of Qeqertarsuup Tunua (Disko Bugt): A Regional Analysis of the Saqqaq and Dorest Cultures of Central West Greenland. Museum Tusculanum Press; 2009. 272 p.

80. Jensen JF. Thule Culture Settlement Patterns in Peary Land. In: Grønnow B, editor. On the Track of the Thule Culture from Bering Strait to East Greenland. National Museum of Denmark; (15).

81. Brown SK, Darwent CM, Sacks BN. Ancient DNA evidence for genetic continuity in arctic dogs. J Archaeol Sci. 2013 Feb 1;40(2):1279–88.

82. Brown SK, Darwent CM, Wictum EJ, Sacks BN. Using multiple markers to elucidate the ancient, historical and modern relationships among North American Arctic dog breeds. Heredity . 2015 Dec;115(6):488–95.

83. Huson HJ, Parker HG, Runstadler J, Ostrander EA. A genetic dissection of breed composition and performance enhancement in the Alaskan sled dog. BMC Genet. 2010 Jul 22;11:71.

84. Shannon LM, Boyko RH, Castelhano M, Corey E, Hayward JJ, McLean C, et al. Genetic structure in village dogs reveals a Central Asian domestication origin. Proc Natl Acad Sci U S A. 2015 Nov

/ 3;112(44):13639–44.

85. MacRury IK. The Inuit Dog: Its Provenance, Environment, and History. Inuit Sled Dog International; 1991.

86. P DMF. Report of the fifth Thule Expedition 1921-24. the Danish expedition to Arctic North America in charge of Knud Rasmussen Vol. 2, no. 4-5. Vol. 2. Copenhagen: Gyldendal; 1935.

87. Hjortlund S. Om de smitsomme Hundesygdomme i Nordgrønland og deres Bekæmpelse. Meddelelser fra Direktoratet for den kongelige grønlandske handel for aarene. 1907;1907(4):57–81.

88. T. Max Friesen and Owen. K. Mason. Introduction: Archaeology of the North American Arctic. In: T. Max Friesen and Owen. K. Mason, editor. The Oxford Handbook of the Prehistoric Arctic. Oxford: Oxford University Press; 2016. p. 1–26.

89. Rasmussen K. Across Arctic America: narrative of the fifth Thule expedition. GP Putnam’s Sons, New York; 1927.

90. Wishart RP. The police and dogs during the early patrol years in the Western Canadian Subarctic : An inter-species colonial cooperation? In: Dogs in the North. Routledge; 2018. p. 152–71.

91. McHugh S. “A flash point in Inuit memories”: Endangered Knowledges in the Mountie Sled Dog Massacre. ESC: English Studies in Canada. 2013;39(1):149–75.

92. Kingston L. The Destruction of Identity: Cultural Genocide and Indigenous Peoples. N Y Law Sch J Hum Rights. 2015 Jan 2;14(1):63–83.

93. Lévesque F. Threatening the fantasy of an Arctic welfare state : Canada, Quebec and Inuit dogs in Qikiqtaaluk and Nunavik between 1957 and 1968. In: Dogs in the North. Routledge; 2018. p. 172–90.

94. Mørk T, Prestrud P. Arctic rabies--a review. Acta Vet Scand. 2004;45(1-2):1–9.

95. Bohm J, Blixenkrone-Møller M, Lund E. A serious outbreak of canine distemper among sled-dogs in northern Greenland. Arctic Med Res. 1989 Oct;48(4):195–203.

96. Degerbøl M, Freuchen P. Mammals. Report of the Fifth Thule Expedition 1921-24. The Danish expedition to Arctic North America in charge of Knud Rasmussen. Ph. D; 1935.

97. Frobisher SM. The second voyage of Master Martin Frobisher,. In: Hakluyt R, editor. VOYAGES: VOL V (EVERYMAN’S LIBRARY NO 338). Hakluyt Society; 1577.

98. Whitbourne R. A Discourse and Discovery of New-found-land with Many Reasons to Proove how Worthy and Beneficiall a Plantation May There be Made. 1622.

99. Allen G. Dogs of the American Aborigines. 1920. (Bulletin of the Museum of Comparative Zoology at Harvard College; vol. 63).

100. Lindsay M. Sledge: The British Trans-Greenland Expedition, 1934. London: Cassell; 1935.

101. Coppinger R, Coppinger L. Dogs: A Startling New Understanding of Canine Origin, Behavior & Evolution. Simon and Schuster; 2001. 352 p.

102. Hindrikson M, Männil P, Ozolins J, Krzywinski A, Saarma U. Bucking the trend in wolf-dog hybridization: first evidence from europe of hybridization between female dogs and male wolves. PLoS One. 2012 Oct 3;7(10):e46465.

/ 103. Vila C, Wayne RK. Hybridization between Wolves and Dogs. Conserv Biol. 1999 Feb;13(1):195–8.

104. Wang G-D, Xie H-B, Peng M-S, Irwin D, Zhang Y-P. Domestication genomics: evidence from animals. Annu Rev Anim Biosci. 2014 Feb;2:65–84.

105. Frantz LAF, Larson G. A genetic perspective on the domestication continuum. In: Hybrid Communities. Routledge; 2018. p. 41–56.

106. Higuchi R, Bowman B, Freiberger M, Ryder OA, Wilson AC. DNA sequences from the quagga, an extinct member of the horse family. Nature. 1984;312(5991):282–4.

107. Pääbo S. Molecular cloning of Ancient Egyptian mummy DNA. Nature. 1985;314(6012):644–5.

108. Pääbo S, Higuchi RG, Wilson AC. Ancient DNA and the polymerase chain reaction: the emerging field of molecular archaeology (Minireview). J Biol Chem. 1989;264(17):9709–12.

109. Poinar HN, Schwarz C, Qi J, Shapiro B, Macphee RDE, Buigues B, et al. Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Science. 2006 Jan 20;311(5759):392–4.

110. Stoneking M. Ancient DNA: how do you know when you have it and what can you do with it? Am J Hum Genet. 1995 Dec;57(6):1259–62.

111. Cooper A, Wayne R. New uses for old DNA. Curr Opin Biotechnol. 1998 Feb;9(1):49–53.

112. Wayne RK, Leonard JA, Cooper A. Full of Sound and Fury: History of Ancient DNA. Annu Rev Ecol Syst. 1999 Nov 1;30(1):457–77.

113. Cooper A, Poinar HN. Ancient DNA: do it right or not at all. Science. American Association for the Advancement of Science; 2000 Aug 18;289(5482):1139.

114. Handt O, Krings M, Ward RH, Pääbo S. The retrieval of ancient human DNA sequences. Am J Hum Genet. 1996 Aug;59(2):368–76.

115. Fulton TL. Setting Up an Ancient DNA Laboratory. In: Beth Shapiro And, editor. Ancient DNA Methods and Protocols. New York: Humana Press; 2012. p. 1–12. (Methods in Molecular Biology; vol. 840).

116. Ginolhac A, Rasmussen M, Gilbert MTP, Willerslev E, Orlando L. mapDamage: testing for damage patterns in ancient DNA sequences. Bioinformatics. 2011 Aug 1;27(15):2153–5.

117. Kemp BM, Smith DG. Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Sci Int. 2005 Nov 10;154(1):53–61.

118. Briggs AW, Stenzel U, Johnson PLF, Green RE, Kelso J, Prüfer K, et al. Patterns of damage in genomic DNA sequences from a Neandertal. Proc Natl Acad Sci U S A. 2007 Sep 11;104(37):14616–21.

119. Sawyer S, Krause J, Guschanski K, Savolainen V, Pääbo S. Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA. PLoS One. 2012 Mar 30;7(3):e34131.

120. Lindahl T, Andersson A. Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3618–23.

121. Höss M, Jaruga P, Zastawny TH, Dizdaroglu M, Pääbo S. DNA damage and DNA sequence retrieval from ancient tissues. Nucleic Acids Res. 1996 Apr 1;24(7):1304–7.

122. Smith CI, Chamberlain AT, Riley MS, Stringer C, Collins MJ. The thermal history of human fossils

/ and the likelihood of successful DNA amplification. J Hum Evol. 2003 Sep;45(3):203–17.

123. Allentoft ME, Collins M, Harker D, Haile J, Oskam CL, Hale ML, et al. The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils. Proc Biol Sci. 2012 Dec 7;279(1748):4724–33.

124. Krause J, Fu Q, Good JM, Viola B, Shunkov MV, Derevianko AP, et al. The complete mitochondrial DNA genome of an unknown hominin from southern Siberia. Nature. 2010 Apr 8;464(7290):894–7.

125. Jónsson H, Ginolhac A, Schubert M, Johnson PLF, Orlando L. mapDamage2.0: fast approximate Bayesian estimates of ancient DNA damage parameters. Bioinformatics. 2013 Jul 1;29(13):1682–4.

126. Ersmark E, Orlando L, Sandoval-Castellanos E, Barnes I, Barnett R, Stuart A, et al. Population Demography and Genetic Diversity in the Pleistocene Cave Lion [Internet]. 2015. Available from: https://www.openquaternary.com/jms/article/view/oq.aa

127. Gilbert MTP, Tomsho LP, Rendulic S, Packard M, Drautz DI, Sher A, et al. Whole-genome shotgun sequencing of mitochondria from ancient hair shafts. Science. 2007 Sep 28;317(5846):1927–30.

128. Carøe C, Gopalakrishnan S, Vinner L, Mak SST, Sinding MHS, Samaniego JA, et al. Single-tube library preparation for degraded DNA. Johnston S, editor. Methods Ecol Evol. 2018 Feb 10;9(2):410–9.

129. Schubert M, Lindgreen S, Orlando L. AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res Notes. 2016 Feb 12;9:88.

130. Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, Kamal M, et al. Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature. 2005 Dec 8;438(7069):803–19.

131. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009 Jul 15;25(14):1754–60.

132. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009 Aug 15;25(16):2078–9.

133. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014 May 1;30(9):1312–3.

134. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006 Nov 1;22(21):2688–90.

135. Bouckaert R, Heled J, Kühnert D, Vaughan T, Wu C-H, Xie D, et al. BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput Biol. 2014 Apr;10(4):e1003537.

136. Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol. 2012 Aug;29(8):1969–73.

137. Buchfink B, Xie C, Huson DH. Fast and sensitive protein alignment using DIAMOND. Nat Methods. 2015 Jan;12(1):59–60.

138. Patterson N, Moorjani P, Luo Y, Mallick S, Rohland N, Zhan Y, et al. Ancient admixture in human history. Genetics. 2012 Nov;192(3):1065–93.

139. Pickrell JK, Pritchard JK. Inference of population splits and mixtures from genome-wide allele frequency data. PLoS Genet. 2012 Nov 15;8(11):e1002967.

140. Milanesi M, Capomaccio S, Vajana E, Bomba L, Garcia JF, Ajmone-Marsan P, et al. BITE: an R package for biodiversity analyses [Internet]. bioRxiv. 2017. p. 181610. Available from:

/ https://www.biorxiv.org/content/10.1101/181610v1

141. Ameen C, Feuerborn T, Linderholm A, Brown S, Hulme-Beaman A, Lebrasseur O, et al. Specialised sledge dogs accompanied Inuit dispersal across the North American Arctic. Proceedings of the Royal Society B: Biological Sciences [Internet]. 2019; Available from: https://abdn.pure.elsevier.com/en/publications/specialised-sledge-dogs-accompanied-inuit-dispersal-acr oss-the-no

142. Krasheninnikov SP, Crownhart-Vaughan EAP. Explorations of Kamchatka, 1735--1741. Translation of the original. 1955;

143. Sandell HT, Sandell B. Archaeology and Environment in the Scoresby Sund Fjord. Museum Tusculanum Press; 1991. 152 p.

144. Green RE, Briggs AW, Krause J, Prüfer K, Burbano HA, Siebauer M, et al. The Neandertal genome and ancient DNA authenticity. EMBO J. 2009 Sep 2;28(17):2494–502.

145. Leonard JA, Shanks O, Hofreiter M, Kreuz E, Hodges L, Ream W, et al. Animal DNA in PCR reagents plagues ancient DNA research. J Archaeol Sci. 2007 Sep 1;34(9):1361–6.

146. Malmström H, Storå J, Dalén L, Holmlund G, Götherström A. Extensive human DNA contamination in extracts from ancient dog bones and teeth. Mol Biol Evol. 2005 Oct;22(10):2040–7.

147. Malmström H, Svensson EM, Gilbert MTP, Willerslev E, Götherström A, Holmlund G. More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA. Mol Biol Evol. 2007 Apr;24(4):998–1004.

148. Skoglund P, Northoff BH, Shunkov MV, Derevianko AP, Pääbo S, Krause J, et al. Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. Proc Natl Acad Sci U S A. 2014 Feb 11;111(6):2229–34.

149. Damgaard PB, Margaryan A, Schroeder H, Orlando L, Willerslev E, Allentoft ME. Improving access to endogenous DNA in ancient bones and teeth. Sci Rep. 2015 Jun 17;5:11184.

/ Acknowledgements

Firstly, I would like to thank my supervisors Anders Hansen and Love Dalen for giving me the opportunity to undertake my PhD at both the University of Copenhagen and the University of Stockholm. Together you have given me the opportunity to research a topic that I am exceedingly passionate about and given me the space to develop into an independent researcher. I couldn't appreciate more the chance to get to know both of you over the last 3.5 years whether that be on Nut Island or traipsing across Greenland.

I would also like to thank all the wonderful archaeologists and museum curators that have not only supplied samples for my projects but have invested themselves in the results and the articulation of these results and responded to my many questions about sample provenances and site information. Under this umbrella my thanks go to: Kristian Murphy Gregersen (Natural History Museum of Denmark), Christian Koch Madsen and Bo Albrechtsen (National Museum of Greenland), Martin Appelt, Bjarne Grønnow, and Ann-Lisbeth Schmidt (National Museum of Denmark), Rob Losey (University of Alberta), Greger Larson (University of Oxford), and Vladimir Pitulko (Russian Academy of Sciences). A huge thank you to Carly for coordinating our colossal project you did such an incredible job of herding the cats when it came to manuscript feedback. Also thanks to Sarah, Ben, and Chris for taking part in the experience with us it was nice to see it all come together.

I would also like to thank the numerous working groups that I had the pleasure to be a part of during the course of my PhD. My homebase with the GID group has been filled with many friendly faces with new ones arriving and old ones leaving: Carlotta, Frederik, Hussein, Ida, Lousia, Luise, Linett, Maria, Paloma, Sarah, Tobias. And a special thanks to Alba who allowed me to follow her to every office she occupied whether she wanted to or not. Also a special shoutout to Jill who translated the summary for this thesis. To my fellow Lovers, I think I regret that choice already, I can't thank you enough for immediately feeling welcome when I got to Stockholm, it is hard to imagine a group with a more collegiate feeling, but I suppose that's what happens when you play games two hours a day everyday... My thanks to you guys for the experience even those of you who I barely overlapped with: Edana, Erik, Johannes, Karin, Marianna, Nic, Nora, Patricia, and Petter (and lest we forget the addons Dani, Allison, Mos, and Eric). A special thank you to David who saved the day and pulled off an amazing feat of doing the legwork to put together a manuscript in a record amount of time, you sir are a true renegade. And a special thanks to Johanna, it was a great feeling knowing that there was someone in the same boat there with me. Also a thanks to the Queen Mary's and PalaeoBARN groups for welcoming me for my stay and making me feel welcome. Laurent, Alberto, and Greger thank you for the opportunity to spend the summer with you and collaborate, it was an incredible experience, hopefully one of many.

A big thank you to the Qimmeq Project that brought together a diverse set of people with a common interest (you'll be surprised to hear that it was dogs...). It was constantly exciting to explore Greenland together and share ideas and results about our research together. A special thank you to Morten and Pipaluk for all the extra effort that the two of you put into coordinating the project and pulling it all together.

I also have to acknowledge the support of the lifelong friends that I made through ArchSci2020. There is definitely a part of our lives now that only the 15 of us will understand. It is safe to say that our shared experiences brought us together and gave us an incredible support system of people who really understood

/ our experience. I have to give a special shout out to my fellow Copenhagen ArchSci's who without hesitation put a roof over my head when I was kindly asked to leave Sweden, Eden I don't know what I would have done without you, what could have been a miserable experience ended up being 8 of the best days of my PhD and the foundation of a lifelong friendship. And for saving the day again when I moved back to Copenhagen, I couldn't have asked for a better moving companion. To Xenia thank you for your constant support and friendship, I wouldn't trade your friendship for anything. Also, to Jonas who never hesitated to help me with my random bioinformatics questions and never turned down an opportunity to come over to play a game (or maybe just never turned down an opportunity to play with Xolo). Maddie, Stockholm wouldn't have been the same without you, I will forever look fondly on our evenings venting, eating burgers, and watching some incredible films. And to Alison, it was a great pleasure to get to know you and to collaborate with you! I enjoyed every minute working with you on our project.

I would also like to give a massive thank you to my family for their constant support and encouragement. Mom and Dad thank you for your frequent reminders of how proud you are of me and for reading my entire introduction, your feedback was invaluable. To my cousin Lena who put up with me sleeping on her floor for three months while on my fellowship in London, you are a true hero. It was wonderful getting to spend all that quality time together even if most of it was us in a perpetual state of degree related stress, but we both made it there in the end! And to my FAMILY you are the ones who literally kept me fed and clothed for the last 6 months (or let's be honest year) of my PhD. Jacquie, you are the most incredible sister, I don't know how I would have survived without your guidance with R. Well I might have survived but my plots would look very different. You are the absolute best and if I didn't already plan to love you until my dying day I certainly would now... And Xolo, of course I have to give a quick thanks to my own dog although I am not certain of how he would fair in the Arctic unlike the other dogs who made my PhD possible. To Julian, your levels of understanding and patience have far exceeded anything I could imagine possible, particularly when it came to helping me with my scripting. Thank you for being there for me, it knew I could always count on you.

Lastly, I need to give my undying thanks to Mikkel Sinding, not just supervisor but also a mentor. I don't know where this PhD or project would be without your initiative, focus, and encouragement. It was remarkable the number of days that I was feeling discouraged when you would unknowingly send a message of encouragement just when I needed it most, it was like you had a sixth sense for it. It was a constant comfort that there was always someone just as interested and invested in my project as I was. I can confidently say that you went above and beyond and that you were instrumental in making this PhD what it became.