Ancient DNA Reveals the Chronology of Walrus Ivory Trade from Norse Greenland

bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Title:

Ancient DNA reveals the chronology of walrus ivory trade from Norse Greenland

Short title:

Tracing the medieval walrus ivory trade

Bastiaan Star1*†, James H. Barrett2*†, Agata T. Gondek1, Sanne Boessenkool1*

* Corresponding author † These authors contributed equally

1 Centre for Ecological and Evoluionary Synthesis, Department of Biosciences, University of Oslo, PO Box 1066, Blindern, N-0316 Oslo, Norway. 2 McDonald Institute for Archaeological Research, Department of Archaeology, University of Cambridge, Downing Street, Cambridge CB2 3ER, United Kingdom.

Keywords:

High-throughput sequencing | Viking Age | Middle Ages | aDNA | Odobenus rosmarus rosmarus bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Abstract

The search for walruses as a source of ivory –a popular material for making luxury art objects

in medieval Europe– played a key role in the historic Scandinavian expansion throughout the

Arctic region. Most notably, the colonization, peak and collapse of the medieval Norse colony

of Greenland have all been attributed to the proto-globalization of ivory trade. Nevertheless, no

studies have directly traced European ivory back to distinct populations of walrus in the Arctic.

This limits our understanding of how ivory trade impacted the sustainability of northern

societies and the ecology of the species they relied on. Here, we compare the mitogenomes of

27 archaeological walrus specimens from Europe and Greenland (most dated between 900 and

1400 CE) and 10 specimens from Svalbard (dated to the 18th and 19th centuries CE) to partial

mitochondrial (MT) data of over 300 modern walruses. We discover two monophyletic

mitochondrial clades, one of which is exclusively found in walrus populations of western

Greenland and the Canadian Arctic. Investigating the chronology of these clades in our

European archaeological remains, we identify a significant shift in resource use from

predominantly eastern sources towards a near exclusive representation of walruses from

western Greenland. These results provide empirical evidence for the economic importance of

walrus for the Norse Greenland settlements and the integration of this remote, western Arctic

resource into a medieval pan-European trade network.

Introduction

Atlantic walrus (Odobenus rosmarus rosmarus) ivory was a popular material for the

manufacture of luxury art objects in medieval Europe. With isolated earlier and later exceptions,

its use started in the 10th century, peaked with the Romanesque art style of the 12th century, and

subsequently declined (1-4). This European demand for ivory has been considered a major

economic incentive for exploration of the North Atlantic region and the Arctic (5-9). While the

Atlantic walrus is widely distributed –with populations being found from Siberia to Canada

(10)–, of all potential sources it is Norse Greenland for which ivory trade has been considered

particularly important. First, the initial exploration and settlement of Greenland c.980-990 CE

has been attributed to the hunt for ivory (7). Second, the 13th- and early 14th-century peak in

transAtlantic trade to Greenland, and architectural (particularly church) investments there, have

been similarly connected with the walrus (6, 9, 11-14). Finally, the abandonment of the Norse

colony – its Western Settlement in the 14th century and its (more southern) Eastern Settlement

in the 15th century – has been blamed on the declining popularity of walrus ivory in Europe

and/or on a switch to alternative sources such as Svalbard or Russia (7, 15). To evaluate these

hypotheses, we identify the Arctic sources of walrus imports to Europe between the 10th and

15th centuries using ancient DNA (aDNA).

The hunting of walruses for ivory by Norse Greenlanders is testified by archaeological

finds of maxillae, maxillae fragments, tusk offcuts, postcanine teeth and carved objects (9, 16,

17). Post-cranial walrus bones (that represent hunting for meat rather than ivory) are rare from

Norse sites in Greenland, particularly from the Eastern Settlement. Based on historical sources,

most hunting took place further north along the west coast, mainly around Disko Bay (9, 18-

21). The actual transport of walrus products –ivory, hide ropes and even a decorated walrus

skull– from Greenland to Europe as gifts, tithes and trade goods is also described in historical

sources. Although these sources vary in their historicity and are dated to the 13th century CE or bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

later, they describe practices thought (by their medieval authors and modern scholars) to have

had an earlier origin (6, 7, 13, 21). Yet another major source of ivory was the Barents Sea region

of Arctic Fennoscandia and Russia. This eastern source was documented as early as the late 9th

century CE, when the Arctic Norwegian chieftain Ohthere visited the court of King Alfred of

Wessex in England (22, 23). The continued importance of this source is implied by the great

abundance of walrus ivory known from medieval Novgorod –an important trading town with

an extensive network into Arctic Fennoscandia and Russia (24, 25)– and by the hunting of the

Arctic European walrus well into the 20th century (26). Finally, walruses were also initially

hunted in Iceland during its colonization in the late 9th and 10th centuries (7, 9, 27). By the 12th-

13th centuries, when the island’s earliest laws and narrative texts were first recorded, Icelandic

walruses were reduced to isolated visitors only (9, 28). Icelandic ivory finds were probably

from local hunting during the Viking Age (7), but there exists no empirical evidence on the

geographic origin of the ivory imported to European trading centers such as Trondheim,

Bergen, Oslo, Dublin, London, Sigtuna and Schleswig during the chronology of the Norse

Greenland settlements. Here we fill this gap in present knowledge.

Genetic analyses of modern walruses reveal significant population structure based on

microsatellite variation (29-32), mitochondrial (MT) restriction fragment length polymorphism

(RFLP) (29, 30) and partial MT sequence variation (33), which agrees with high levels of

observed site fidelity (34, 35). Of particular relevance for this study, is the observation of a

unique set of MT haplotypes that occur solely in western Greenland and the Canadian Arctic

(29, 30, 33, 36, 37). We therefore presumed it possible to use mitogenomic data to identify the

origin of archaeological walrus specimens traded from or via Greenland. Although aDNA can

trace such specimens towards their biological source (38-40), it is difficult to apply this

destructive technique to objects of fine art such as worked ivory. Nonetheless, partial walrus

skulls (rostrums) with in-situ tusks were also transported to Europe (41). In rare instances these bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

‘ivory packages’ remain intact, while in others they were broken up to extract the ivory (41-

46). These rostrums and rostrum fragments serve as proxies for the ivory they carried and can

be sampled for biomolecular analyses without the need to damage ivory artefacts.

We analyze the mitogenomes of 24 archaeological walrus rostrums and three tusk

offcuts. Most specimens have been dated by archaeological context, associated artefacts or (in

one case) a runic inscription to the period of the Norse occupation of Greenland. Two late

outliers that post-date this period are included for comparison, one from a context dating 1500-

1532 CE and one post-dating 1600 CE. Two other samples are undated, being from old

excavations, but are probably medieval in origin. The specimens were originally obtained from

excavations and subsequently archived in museum collections, although in Le Mans a rostrum

with a 13th-14th century runic inscription on one of its tusks has come down to posterity intact

(41, 47). Four of our medieval rostrums serve as controls, having been excavated at the site of

Igaliku (Gardar) in Greenland’s former Eastern Settlement (48). In addition, we analyze 10

control samples from 18th and 19th-century Svalbard. We compare our data to 306 modern and

19th-century walrus specimens from the Barents Sea region, Greenland and the Canadian Arctic

(32, 33, 36, 37) to infer the geographical origin of European walrus imports during the

chronology of the Norse occupation of Greenland. bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Results

We obtained 520 million paired sequencing reads for 37 samples that passed downstream

filtering (Fig. 1A, Table S1). The libraries of these samples contained 0.01 to 71% endogenous

DNA and yielded 4.8 to 438-fold MT coverage (Table S2). The reads show the typical patterns

of fragmentation and deamination expected from post-mortem degradation (Fig. S1). Including

a de novo Pacific walrus MT sequence as outgroup (Supporting Information), a Bayesian

phylogenetic analysis of 346 SNPs reveals two fully supported monophyletic clades in the

archaeological Atlantic walrus data (Fig. 1B). The medieval walruses belong to either clade,

whereas all the 18th and 19th century CE specimens from Svalbard fall into one clade. We

tentatively define the clade containing the Svalbard samples as eastern, and the other clade as

western (Fig. 1B). Of the four Greenland samples, two specimens fall into the western and two

into the eastern clade (Fig. 1B). The two major lineages form distinct haplotype genealogies,

with each clade separated by 21 or 18 substitutions from the most recent common ancestor

(MRCA; Fig. S2). Using two different substitution rates, the time to the MRCA was estimated

to have been 23,400 (95% HPD: 14,539-34,522) to 251,120 (95% HPD: 163,819-355,131)

years ago.

A Principle Component Analysis (PCA) shows two significantly differentiated clusters

supported by clade-specific SNPs that are located throughout the entire mitogenome (Fig. S3).

We investigated if these clade-specific SNPs can be associated with RFLP (29, 30) and control

region (CR) population data (32, 33, 36, 37). First, the RFLP studies focused on ND1, ND2,

and ND3/4 MT gene regions (29, 30). We find 5 clade-specific SNPs in ND1, 3 in ND2 and 9

in ND3/4 (Supporting Information, Fig. S3C). At least one of these SNPs alters a restriction

enzyme sequence motive depending on clade membership in each of these ND genes

(Supporting Information). The combination of enzymes (29, 30) can therefore separate the

western and eastern clades we identify in our mitogenomes –on multiple restriction sites and bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

within each ND gene region. While these RFLP analyses report four MT clades, the data show

a pronounced bimodal divergence, with the majority of distinctive restriction sites and highest

statistical support observed between clade 1 and any combination of clades 2-4 (29, 30).

Following Born et al. (2001), clades 2-4 comprise 94% of the Northwest Greenland and 52%

of the West Greenland population (Fig. 1C). Crucially, these clade 2-4 haplotypes are

exclusively identified in western Greenland, and are absent in the Northeast Atlantic (29, 30).

Clade 1 is found in every individual in the Northeast Atlantic and in various proportions in

western Greenland (Fig. 1C).

Second, we investigate a 499 base pair (bp) section (between 15328 and 15827 bp) of

an extensive CR population dataset (32, 33, 36, 37). We observe three SNPs in this section, for

which 12 out of 15 archaeological western clade specimens share a distinct A15564 C15760 C15779

haplotype (Supporting Information, Fig. S3C). Reanalyzing the CR data for this ACC haplotype

(32, 33, 36, 37) we identify 38 out of 306 Atlantic walruses with the same haplotype

(Supporting Information, Table S3). The ACC haplotype is fixed in the Northwest Greenland

population, co-occurs mixed with other haplotypes in Canada, yet is absent from the Northeast

Atlantic (Fig. 1C). The distribution of this CR haplotype is therefore analogous to the RFLP

distribution. We derive that the RFLP and CR analyses detect variation explained by the

monophyletic lineages of our medieval and 18th and 19th century CE specimens. Moreover,

these analyses reveal a consistent distribution, whereby a subset of haplotypes is geographically

restricted to western Greenland and Canada. Since the ACC haplotype of our western clade is

only found in western Greenland and Canada, we conclude that the European archaeological

specimens belonging to this clade came from Norse Greenland, either because they were hunted

by the Norse or because they were traded from further north and west via contact with

indigenous Dorset or Thule peoples (18, 19, 21, 49, 50). In contrast, haplotypes of the eastern

clade may originate from either side of the Atlantic Ocean (Fig. 1C). bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

The dates of the archaeological walrus specimens cover the entire chronology of the

Norse Greenland occupation, with two outliers post-dating its abandonment (Fig. 2A, Table

S3). Before the founding of Greenland’s bishopric in the 1120s (probably by 1125) CE (51) –

during the settlement phase and the early period of Norse occupation– we identify one western

and six eastern clade specimens in Europe (Fig. 2A). In contrast, during the later occupation,

between the 1120s and the end of the 14th century, we observe 10 western and two eastern clade

specimens. The timing of this significant increase (p-value = 0.0063, Fisher's exact test) in

western clade specimens thus coincides with new ecclesiastical infrastructure in Greenland (51)

and the height of Romanesque ivory carving in Europe (3). We have not yet discovered

European walrus rostrums that date specifically to the century of Norse Greenland’s

abandonment (the 1400s). By this date walrus ivory had long gone out of fashion, with the

Gothic style using different materials such as elephant ivory (4). Of the two later outliers in our

dataset (both from Trondheim), one from the archbishop’s palace is of the western clade, dated

1500-1532. It may represent redeposition of an earlier find, or the latest known export of a

walrus rostrum from Greenland. The second outlier postdates 1600 CE and is of the eastern

clade.

Based on RFLP data, eastern clade specimens make up nearly half (48%) of the modern

West Greenland population –which is closest to the Disko Bay hunting grounds of the

Greenland Norse (Fig. 1C). Albeit a small sample size, the medieval Greenland specimens from

Gardar also have such a 50/50 distribution, suggesting long-term temporal stability of these

haplotype frequencies (Fig. 1B) and showing that eastern clade trade specimens could also have

originated from Norse Greenland. We calculated the binomial probability of the observed ratio

of western and eastern clade archaeological samples in the period before and after the founding

of the Greenlandic bishopric c.1125 CE (excluding the two outliers from Trondheim), given

variable contributions from western Greenlandic/Canadian and Northeast Atlantic/European bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Arctic sources. This probability is calculated assuming that populations in the Northeast

Atlantic are fixed for the eastern clade, whereas those in western Greenland and Canada

comprised a mixture of clades, following the RFLP frequencies (48% eastern clade) of the West

Greenland population. The probability distribution of the samples dated before the bishopric's

founding shows evidence of geographical admixture, with a most likely contribution of a

western source between 20 to 30% (Fig. 2B). In contrast, the samples dated after the founding

and before c.1400 CE show evidence for a near 100% western clade source (Fig. 2B). In other

words, in its early period, it is statistically unlikely that Norse Greenland was an exclusive

source of walrus for Europe. Arctic Europe (the Barents Sea region) is the most likely

alternative based on the Ohthere account of the late 9th century. Iceland and (less likely, due to

difficult summer ice conditions) Northeast Greenland are other possibilities (7, 21). In the later

period between c.1125 and c.1400 CE, however, the number of observed eastern clade samples

can –with high statistical probability– have come entirely from Norse Greenland together with

the western clade specimens.

Discussion

We reconstruct a chronology of long-distance ivory trade during the medieval period by

investigating complete mitogenomes of archaeological Atlantic walrus specimens from

Greenland, Svalbard and Europe. Specifically, we distinguish whether individual walruses

were obtained from a western Greenland source and our work has evolutionary, ecological and

archaeological implications.

We discover that Atlantic walrus comprises two major, monophyletic MT lineages.

Several observations support a hypothesis that these lineages have evolved in glacial refugia on

either side of the Atlantic Ocean. First, we estimate a divergence date between 23400 and

251120 years ago, well before or during the last glacial maximum (LGM). Second, (sub)fossil

walrus bones dated over 10k years BP have been found on both the European and the North

American continent at lower latitudes compared to the walrus’s modern distribution (52, 53).

Third, glacial refugia on either side of the Atlantic have also been proposed to explain (partial)

MT divergence in marine mammals with a similar coastal ecology like walrus, such as harbor

seal (Phoca vitulina) (54), harbor porpoise (Phocoena phocoena) (55) and grey seal

(Halichoerus grypus) (56). Based on these observations, we conclude that the two MT lineages

have evolved in spatial separation on either side of the Atlantic Ocean.

Such a scenario further explains the distinct geographical distribution of RFLP and CR

population data, which show that haplotypes associated with the western clade occur solely in

western Greenland and Canada (29-32, 37). The lack of such haplotypes in the Northeast

Atlantic, despite the genetic analysis of hundreds of specimens covering multiple decades (29-

32, 37), indicates that gene flow has been asymmetrical, with eastern clade females dispersing

to the western Atlantic but not vice versa. This dispersal follows the direction of the East

Greenland Current (57), suggesting that ocean currents influence the tendency of walrus

dispersal. Historically, a similar asymmetrical pattern has been observed on a regional scale in bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Baffin Bay –with walruses migrating counter-clock-wise, following the direction of the coastal

currents and the breakup of sea ice (31, 58). Our data suggest that dispersal has been persistent

in its direction since these two MT lineages came into secondary contact. As a result, eastern

clade walruses are now found in the entire range of Atlantic walrus, yet western clade walruses

have not yet been observed in the Northeast Atlantic.

With our discovery of two MT lineages we provide quantitative evidence on the

geographic origin of walrus imports to medieval Europe, and with that re-evaluate past

hypotheses on the role of walrus ivory in the origins, efflorescence and collapse of Norse

Greenlandic society. Only one of our seven samples predating the 1120s CE is of the western

clade. The other six eastern clade samples of this period could have come from a variety of

sources, including Greenland, but it is probable that most derived from the Northeast Atlantic.

Thus the theory that walrus ivory was a primary motive for the initial exploration and settlement

of Greenland may need reconsideration (7). Conversely, 10 of 12 specimens dating between

the 1120s and c.1400 CE are of the western clade and arrived in Europe via Norse Greenland.

Moreover, the observed ratio of eastern and western clade specimens has a high probablility of

deriving exclusively from western Greenland. Thus, the documented heyday of Norse

Greenlandic settlement, trade and architectural elaboration (particularly evident in churches and

their accoutrements) – between the 12th and 14th centuries – did coincide with exports of walrus

ivory. In fact, our data suggest that the Greenland trade of this commodity may have held a near

monopology in western Europe. The historically and/or archaeologically attested walrus hunts

of the 9th-10th centuries in the Barents Sea region and Iceland may have declined or ended by

the early 12th century, either because of rising Greenlandic exports or local

reduction/extirpation of populations.

The reasons for the signficant shift in trade cannot be inferred from aDNA alone and

historically contingent local factors, as well as broader socioeconomic and environmental bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

developments can be invoked as potential contributors. In 13th-14th century Iceland it was

believed that the Greenlanders used walrus products as gifts to influence the policies of

Scandinavian monarchs, for example in the story of Einar Sokkason (Grænlendinga tháttr) (2).

Although the historicity of Einar’s use of walrus ivory to secure a Greenland bishop and

episcopal see in the early 12th century cannot be confirmed, tithes (including papal dues) were

paid in this material during the 13th and 14th centuries (1, 6). Thus both local initiative and the

reach of panEuropean church infrastructure potentially played a role in the naissance and

maintenance of the Greenland ivory trade. Moreover, the 11th to 13th centuries represent a period

of major demographic and economic growth in Europe, in part due to environmental conditions

favourable to cereal agriculture (59). A growing urban and elite demand was served by transport

from increasingly distant sources in a process of proto-globalization. Our discovery shows that

Greenland was well integrated into this network.

Less can be said about the end of the Greenland colony based on the evidence here. Is

the absence of sampled (or known) European finds of walrus rostrums from the 15th century

evidence for the end of trade? Is the single, 16th-century western clade specimen an example of

the last, isolated, Greenland export, or is it a redeposition of an earlier find? These are classic

challenges of archaeological interpretation. Nonetheless, it is a conspicuous observation that

Greenland may have been the exclusive supplier of walrus ivory to Europe between the 1120s

and the 14th century. The demise of Norse Greenland would therefore have reduced European

supplies of this raw material, whereas a decline in demand would have undermined Greenland’s

social and economic organization. Whatever other factors have been influential –from the Little

Ice Age (60-63), to gradual out-migration (62, 63), to the impact of the Black Death (1346-

1353) on European markets (2, 59)– the cessation of trade in walrus ivory must have been

significant for the end of Greenland’s Eastern and Western settlements. bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Conclusion

Here, we show that the Atlantic walrus comprises two monophyletic MT lineages of which one

is exclusively found in western Greenland and Canada. By analyzing archaeological walrus

remains in Europe, this discovery allows us to infer an increasing trade of walrus ivory from

Norse Greenland. Greenland is often discussed as a general example of both human resilience

and vulnerability in the face of environmental and economic change (61, 64-67). Thus, the

implications of this study –that the influence of ecological globalization for the Greenlandic

Norse started small yet became paramount– extend far beyond medieval Europe.

Material and Methods

We sampled 37, morphologically identified walrus bone and ivory specimens from Western

Europe, Greenland and Svalbard (Fig. 1A, Table S1). The Svalbard specimens were dated based

on the documented use of the hunting stations from which they were collected. The Le Mans

specimen was dated based on the characteristics of a runic inscription on an in situ tusk. The

other specimens were dated by archaeological context and/or associated artefacts. Direct

radiocarbon dating was not used because precise marine reservoir correction requires a ΔR

value which cannot be predicted without catch location (68).

All DNA extraction and pre-PCR protocols were performed in a dedicated laboratory at

the University of Oslo following strict aDNA precautions (69, 70). Samples were exposed to

UV (Supporting Information) before milling using a custom designed stainless-steel mortar

(71) or a Retsch MM400 mixer mill. Extraction used a combined bleach and pre-digestion

protocol (72), apart from the Greenland samples for which only pre-digestion was used (73).

Blunt-end Illumina libraries were built (39, 74) amplifying ligated DNA with sample-specific

seven bp indexes in the P7 primer (Supporting Information). Libraries were sequenced (125 bp

paired-end) on an Illumina HiSeq 2500 and demultiplexed allowing zero mismatches in the

index tag.

Sequencing reads were processed using PALEOMIX (75) (collapsing forward and

reverse reads using AdapterRemoval v1.5 (76)) and aligned to the Pacific walrus (O. r.

divergens) nuclear genome (77) and the Atlantic walrus mitogenome (78) with BWA aln

v.0.7.5a-r405 (79). Read data from the Pacific walrus genome project (77) were aligned to the

Atlantic walrus mitogenome to obtain a Pacific walrus MT sequence (Supporting Information).

Alignments with a quality score (MapQ) of <25 were removed and aDNA damage patterns

were investigated using mapDamage v.2.0.6 (80). Genotypes were obtained using GATK v.

3.4.46 (81) Haplotypecaller with ploidy set to 1, after duplicate removal (Picard Tools v.1.96) bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

and indel realignment (GATKs IndelRealigner). Genotypes were jointly called using default

settings (GATKs Genotypecaller), and filtered with BCFTOOLS v. 1.6 (82) using filters -i

'FS<60.0 && SOR<4 && MQ>30.0 && QD > 2.0' and --SnpGap 10. Indels were excluded

using VCFTOOLS v.0.1.14 (83) and genotypes with a quality below 15 and a read depth below

3 were set as missing.

Phylogenetic analyses were performed using BEAST 2.4.7 (84) with Pacific walrus as

outgroup. Following jModeltest 2 (v0.1.10) (85), the HKY model was implemented with the

Yule tree prior and a strict clock model. Timing to the most recent common ancestor (MRCA)

was estimated using a faster rate of 0.75 x 10-7 substitutions/site/year from southern elephant

seal CR data (86) and a slower rate of 0.7 x 10-8 estimated from baleen whale cytochrome b

data (87, 88), because the CR mutates relatively fast compared to other parts of the mitogenome

(89). The MCMC (10 million gen, pre-burnin 1 million gen) was sampled every 1000 trees and

ESS values above 200 confirmed convergence (Tracer v.1.6.0 (90)). The maximum clade

credibility tree was drawn after a 10% burnin using TreeAnnotator and the tree was visualised

in FigTree (v.1.4.3). A haplotype genealogy was drawn using Fitchi (91). Differentiation among

lineages was assessed using smartPCA, EIGENSOFT v.6.1.4 (92) obtaining clade supporting

SNPs using snpweightoutname (Fig. S3). Clade specific modification of RFLP motivs (29, 30)

was obtained using Find Motif, IGV (93) (Supporting Information). CR population data (32,

33, 36, 37) were aligned to the Atlantic walrus MT genome and genotypes at position 15564,

15760 and 15779 were scored in IGV (94) (Supporting Information, Table S3). Finally, the

binomial probability of observing different western and eastern clade ratios before and after

c.1125 CE was calculated by simulating variable contributions of a western

Greenlandic/Canadian and Northeast Atlantic source, assuming 100% eastern clade specimens

in the Northeast Atlantic populations and 48% in western Greenland/Canada. Probabilities for

source admixture were scaled to one for each period. bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Acknowledgements

This work was supported by the Nansenfondet, the Research Council of Norway projects

262777 and 230821 and the Leverhulme Trust project “Northern Journeys” MRF-2013-065.

We thank M Skage, S Kollias, A Tooming-Klunderud and H Rydbeck from the Norwegian

Sequencing Centre. Samples (and information regarding their chronology) were kindly

provided by: National Museum of Denmark (J Arneborg), Natural History Museum of Denmark

(KM Gregersen, KH Kjær), NTNU Museum of Natural History and Archaeology (A

Christophersen, JA Risvaag, J Rosvold), University Museum, Bergen (G Hansen, AK

Hufthammer, S Nordeide), Museum of Cultural History, University of Oslo (M Vedeler), NIKU

(LM Fuglevik), National Museum of Ireland (M Sikora, A Halpin), MOLA (L Blackmore, A

Pipe), Sigtuna Museum (A Söderberg), Schleswig-Holsteinische Landesmuseen Schloss

Gottorf (V Hilberg, U Schmölcke) and Le musée Vert, Le Mans (N Morel). AH Pálsdóttír and

S Wickler also helped obtain samples.

Data availability

All ancient read data are available at the European Nucleotide Archive (ENA,

www.ebi.ac.uk/ena) under study accession number PRJEB25536.

Author contributions

BS, JHB and SB designed the study. AG performed the laboratory work. BS performed the

genetic analyses. SB performed the Bayesian analyses. BS, JHB & SB interpreted the results.

JHB identified and selected ancient samples. BS, JHB & SB wrote the paper.

Conflict of Interest

The authors declare no conflict of interest bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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Figures

Fig. 1. (A) Population distribution, historic trade routes and sample locations of Atlantic walrus in the northern Atlantic region. The range of modern Atlantic walrus (dark grey) and putative dispersal routes (black arrows) follow (58) and (31). Eight breeding populations are recognized (58); 1 - Foxe Basin, 2 - Hudson Bay, 3 - Hudson Strait, 4, - West Greenland, 5 - North Water, 6 - East Greenland, 7 - Svalbard/Franz Josef land, 8 - Novaya Zemlya. Historic trade routes from Greenland –including the location of Norse settlements– and northern Fennoscandia/Russia (yellow) indicate possible sources from which walrus ivory was exported to Europe during the Middle Ages. The Svalbard specimens (orange) were originally from hunting stations of the 1700s and 1800s. The other Atlantic walrus specimens (red, grey) were obtained from museum collections. (B) Bayesian phylogenetic tree obtained using BEAST (84) based on 346 mitochondrial SNPs using Pacific walrus (PAC) as an outgroup. Numbers represent the different specimens as listed in Table S1, and colors match the sampling locations as in Fig. 1A. Branches with a posterior probability of one (grey circles) are indicated. (C) Distribution of RFLP and control region (CR) haplotypes of modern Atlantic walrus populations. The RFLP clade classification follows Born, Andersen et al. (2001). The distribution of a distinct ACC CR haplotype is from 306 modern specimens (see material and methods). bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig. 2. Chronology of Atlantic walrus specimens in Europe. (A) Archaeological Atlantic walrus specimens classified as western clade (blue) or eastern clade (orange) are plotted according to their estimated age range. The start and end of the Norse Greenland occupation, the founding of the bishopric and the arrival of the Black Death (plague) in Norway are indicated (dashed red lines). For each specimen, its location (light grey) is indicated. (B) Probability of obtaining the observed sample of eastern and western clade archaeological specimens as a function of a variable contribution of a western Greenland source. The probability was calculated for those samples obtained before (dark grey) or after (light grey) the founding of the Greenland bishopric, excluding the 16th and 17th century CE specimens.

Supporting Information

Ancient DNA reveals the chronology of walrus ivory trade from Norse Greenland

Bastiaan Star, James H. Barrett, Agata T. Gondek and Sanne Boessenkool

Supplementary Notes

1. Generating a Pacific walrus mitochondrial genome sequence

2. DNA extraction and library preparation

3. Assessing Restriction Fragment Length Polymorphism in Atlantic walrus

4. Atlantic walrus control region polymorphisms

Supplementary Tables

Supplementary Figures

Supplementary Notes

1. Generating a Pacific walrus (Odobenus rosmarus divergence) mitochondrial

genome sequence

The publicly released genome sequence of the Pacific walrus (77) includes a mitogenome

assembly from Atlantic walrus (78), Genbank ID NC_004029.2). Hence, no de novo Pacific

walrus mitochondrial reference genome sequence is currently available. To create a Pacific

walrus MT genome sequence to root our phylogenetic analyses, we obtained a subset of

169,056,390 paired reads generated for the Pacific walrus genome assembly project (file

SRR575502, ENA project ID PRJNA167474, (77)) and aligned these to the Atlantic walrus

MT reference genome with PALEOMIX (75) using BWA mem v.0.7.5a-r405 (79). Both

collapsed and paired reads were used, resulting in a 13-fold coverage of the mitogenome. The

Pacific walrus alignment data and the archaeological Atlantic walrus data were further

processed simultaneously.

2. DNA extraction and library preparation

All DNA extraction and pre-PCR protocols were performed in a dedicated laboratory at the

University of Oslo following strict aDNA precautions (69, 70). Samples were exposed to UV

(10 min) on each side (total dosage of 4800 J/m2) before cutting. Dust was removed with UVed

milliQ and cut fragments were again exposed to UV (10 min) on each side (total dosage of 4800

J/m2) before milling using a custom designed stainless-steel mortar (71) or a Retsch MM400

mixer mill. Extraction used a combined bleach and pre-digestion protocol (72), apart from the

Greenland samples for which only pre-digestion was used (73). Bleach washes were done in

duplicate (150-200 mg of powder each) (72), washed with H2O and pre-digested, followed by

an overnight, second digestion (73). The two eluates were combined and concentrated bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

(Amicon-30kDA Centrifugal Filter Units), extracting DNA using Minelute (Qiagen) according

to manufacturer’s instructions. DNA was eluted in 60 µl pre-heated (60°C) EB buffer,

incubating for 15 min at 37°C (95). Negative controls were included in all extraction

experiments. Blunt-end Illumina libraries were built (39, 74) amplifying ligated DNA with

sample-specific seven bp indexes in the P7 primer. PCRs were done in triplicate, 25 µl reactions

(1.25 U PfuTurbo Cx Hotstart DNA Polymerase (Agilent), 1x buffer, 0.2 mM per dNTP, 0.2

µM P7 index primer, 0.2 µM P5 IS4 primer and 0.4 mg/ml BSA) for 13 cycles (2 min at 95°C,

13 cycles of 30s at 95°C, 30s at 60°C and 70s at 72°C, final extension of 10 min at 72°C).

Amplified products were cleaned using Agencourt® AMPure XP beads at a 1:1.7 ratio, eluted

in 30 µl of molecular grade H2O, and quantified using a Bioanalyzer 2100 (Agilent). Libraries

were sequenced (125 bp paired-end) on an Illumina Hiseq 2500 and demultiplexed allowing

zero mismatches in the index tag.

3. Assessing Restriction Fragment Length Polymorphism in Atlantic walrus

Significant population genetic structure based on mtDNA variation in Atlantic walrus has been

detected using Restriction Fragment Length Polymorphism (RFLP) analysis targeting the ND1,

ND2, and ND3/4 genes (29, 30). Specifically, several RFLP mtDNA haplotypes have been

reported that are solely found in specimens obtained from western Greenland and the Canadian

Arctic, and these haplotypes have therefore been suggested to be diagnostic markers, unique to

these western populations (29, 30). We here assess whether the RFLP haplotypes obtained in

these earlier studies are linked to the observed monophyletic divergence between the western

and eastern clade of Atlantic walrus in our ancient samples. First, we expect diagnostic SNPs

supporting the two clades found in the present study to be located in the ND1, ND2 and

ND3/ND4 region. Moreover, at least some of these diagnostic SNPs should alter the sequence bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

motive of restriction fragment binding sites specific to the restriction enzymes used in the RFLP

studies.

In our mitogenomes, we identify five diagnostic SNPs with principle component

weightings greater than 1.25 (smartPCA, EIGENSOFT v.6.1.4 (92)) in ND1 (between 2752

and 3708 bp), three SNPs in ND2 (between 3920 and 4963 bp) and nine SNPs in ND3/ND4

(between 9485 and 11568 bp). In each of these three regions, we find at least one SNP that

alters the sequence motive of a restriction enzyme according to each monophyletic clade. In

ND1 at position 2982, a G/A polymorphism distorts the GGCCC (western) binding motive of

Sau961 and HaeIII into AGCCC (eastern). In ND2 at position 4597, an A/G polymorphism

alters the CGACT (eastern) motive restricted by Hinf1 to CAACT (western). In ND3/ND4 at

position 9658, a G/A polymorphism distorts the GGCC (western) binding motive of HaeIII into

AGCC (eastern). Finally, in ND3/ND4 at position 10616, a T/C polymorphism distorts the

TTTAAA (western) binding motive of Dra1 to ATTAAA (eastern). The haplotype separation

obtained by the earlier RFLP studies using these specific restriction enzymes can therefore be

explained by diagnostic SNP differentiation that is directly linked to the two monophyletic

clades discovered in our ancient mitogenomes.

4. Atlantic walrus Control Region (CR) Polymorphism

Within 27 Atlantic walruses sampled from both sides of the Atlantic Ocean, significant

population differentiation –identifying a group of monophyletic western Greenland

haplotypes– has been detected based on the combined data from the ND1, COI and the mtDNA

control region (CR) (33). Using data from the CR region alone, however, extensive population

studies targeting hundreds of individuals show that this region lacks discriminatory power to

confidently resolve Atlantic walrus populations within the Northeast Atlantic (32) nor

distinguish between western and eastern Atlantic populations (36, 37). Nevertheless, several bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

haplotypes were found only in the western Greenland region and not in any of the studied

populations in East Greenland, Svalbard or Franz Josef land (36, 37). This observation suggests

the existence of CR haplotypes that are only found in walruses in western Greenland. We here

associate these CR haplotypes to the monophyletic divergence observed in our historic samples

and the 27 modern specimens studied by Lindqvist et al. (33).

In our mitogenome data, we identify three SNPs (A/G15564, C/T15760 and C/T15779) with

principle component weightings above 1.25 (smartPCA, EIGENSOFT v.6.1.4 (92)) that fall

within the region of the CR covered by all earlier studies, between 15328 and 15827 bp (32,

33, 36, 37). In contrast to the diagnostic SNPs in the ND1, ND2, ND3 and ND4 region (see

Supplementary Note 2), neither of these CR SNPs alone is fixed in either clade, reflecting the

lack of power of the CR to differentiate the observed divergence over the entire mitogenome.

Yet, we note that the A15564 C15760 C15779 haplotype occurs in 12 out of 15 of the archaeological

specimens from the western clade while it is not found in the eastern clade.

We investigate the occurrence of this specific ACC haplotype in 105 publicly available

CR sequences (Table S2) (32, 33, 36, 37) obtained from Genbank. These haplotypes represent

over 300 individual Atlantic walruses sampled from multiple locations east and west of

Greenland (Figure 2C). For walruses from East Greenland, Svalbard, Frans-Josef land and the

Pechora Sea we report the specimen numbers of (32) as this study includes samples analysed

by (33). Historic data from Håøya and Bjørnøya were obtained from (37) and the Northwest

Greenland samples were obtained from (33). From (36) we selected only data from those two

locations (Nottingham Island and Hoare Bay) from which more than two specimens were

sampled. All obtained sequences were aligned to the Atlantic walrus reference genome using

BWA mem v.0.7.5a-r405 (79), and their genotypes at position 15564, 15760 and 15779 were

scored in IGV (94).

We identify 38 out of 306 modern Atlantic walruses with the same CR haplotype (Table bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S2). None of the modern samples obtained from populations in the Northeast Atlantic contained

this ACC haplotype in the CR, several samples in Canada have this haplotype, all samples from

Northwest Greenland have this haplotype (Table S2, Figure 1C). This result shows that the

ACC haplotype we observe in our archaeological mitochondrial data is restricted to modern

walrus populations in western Greenland and the Canadian Arctic.

Supplementary Tables

Table S1. Sample details of archaeological Atlantic walrus specimens. The inferred genetic (e; eastern clade, w; western clade) MT lineage of each specimen is also indicated. Based on anatomical representation, animal size, archaeological context and/or MT haplotype all bones except two London specimens can be confidently interpreted as from separate animals. The two London specimens could be parts of the same walrus rostrum (representing left and right alveoli) although they were obtained from different archaeological layers. All samples were taken with museum permission and transported across international borders with the relevant CITES permits.

Sample Museum MT Modern Discovery Archaeological number record lineage location location site Date CE Material References WLR008 B.1950 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR009 JS366 e NHM, University of Bergen Russekeila, n.a. 18th-19th C petrous Anne Karin Hufthammer (a) Isfjorden, Svalbard WLR010 B.1947 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR011 B.2036 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR012 B.1952 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR017 B.2034 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR021 B.1877 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR022 B.1907 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR023 B.2005 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR024 B.1973 e NHM, University of Bergen Moffen, Svalbard n.a. c.1850 petrous Anne Karin Hufthammer (a) WLR029 E173:4330 e National Museum of Ireland Dublin St John's Lane c.900-1125 rostrum Maeve Sikora & Andy Halpin (a) WLR030 E173:323 w National Museum of Ireland Dublin St John's Lane c.900-1125 rostrum Maeve Sikora & Andy Halpin (a) WLR031 21273 w MOLA (Museum of London London London Guildhall late 12th- rostrum (1) Archaeology) early 13th C WLR033 21274 w MOLA (Museum of London London London Guildhall late 12th- rostrum (1) Archaeology) early 13th C WLR034 E172:4311 e National Museum of Ireland Dublin Fishamble Street II 11th C rostrum (2); Maeve Sikora & Andy Halpin (a) WLR038 N10102 e NTNU Museum of Natural Trondheim Søndre gate Felt II, 1111 or rostrum Axel Christophersen (a) History and Archaeology Felt T shortly before WLR039 BRM0/46092/1 w KHM, University of Bergen Bergen BRM0 1120-1170 rostrum (3) WLR041 N32091 w NTNU Museum of Natural Trondheim Folkebibliotekstomten 1225-1275 rostrum Axel Christophersen (a) History and Archaeology WLR042 BRM4/4176/1 w KHM, University of Bergen Bergen BRM4 late 12th C rostrum (3) to 1225 WLR043 BRM0/2709/1 e KHM, University of Bergen Bergen BRM0 1248-1332 rostrum Gitte Hansen (a) WLR044 N29494 w NTNU Museum of Natural Trondheim Folkebibliotekstomten 1150-1175 rostrum Axel Christophersen (a) History and Archaeology WLR045 BRM0/87411/1 w KHM, University of Bergen Bergen BRM0 1248-1332 rostrum Gitte Hansen (a) WLR046 N167587 w NTNU Museum of Natural Trondheim Archbishop's Palace 1500-1532 rostrum Sæbjørg Nordeide (a) History and Archaeology WLR047 N37603 e NTNU Museum of Natural Trondheim Folkebibliotekstomten post 1600 rostrum Axel Christophersen (a) History and Archaeology WLR048 N203145 w NTNU Museum of Natural Trondheim Kjøpmannsgata, n.a. rostrum Axel Christophersen (a) History and Archaeology Felt 93/2 WLR049 F51873 w NIKU, Oslo Oslo Follo D1 Vest 1150-1350 rostrum Lars Morten Fuglevik (a) WLR062 C23798 e KHM, University of Oslo Oslo Ladegårds 1903 n.a. rostrum (4); Marianne Vedeler (a) WLR063 8240a e Sigtuna Museum Sigtuna kv Urmakaren 1000-1050 tusk offcut Anders Söderberg (a) WLR064 8241a e Sigtuna Museum Sigtuna kv Urmakaren 1000-1050 tusk offcut Anders Söderberg (a) WLR065 8241b e Sigtuna Museum Sigtuna kv Urmakaren 1000-1050 tusk offcut Anders Söderberg (a) WLR067 FNR 29257 w Sigtuna Museum Sigtuna kv Trädgårdsmästaren 1200-1230 rostrum (5); Anders Söderberg (a) WLR068 n.a. e Schleswig-Holsteinische Schleswig Hafenstraße 13 12th-13th C rostrum (6); Volker Hilberg (a) Landesmuseen Schloss Gottorf WLR069 P151/2017 KMG e Natural History Museum of Igaliku (Gardar), Kirkegaarden 1926 late 10th- rostrum (7); Jette Arneborg (a); Kristian Murphy Denmark Greenland 12th C Gregersen (a) WLR070 P149/2017 KMG e Natural History Museum of Igaliku (Gardar), Kirkegaarden 1926 late 10th- rostrum (7); Jette Arneborg (a); Kristian Murphy Denmark Greenland 12th C Gregersen (a) WLR071 P153/2017 KMG w National Museum of Igaliku (Gardar), na late 10th- rostrum (7); Jette Arneborg (a); Kristian Murphy Denmark Greenland 12th C Gregersen (a) WLR072 P155/2017 KMG w National Museum of Igaliku (Gardar), na late 10th- rostrum (7); Jette Arneborg (a); Kristian Murphy Denmark Greenland 12th C Gregersen (a) WLR073 MHNLM w Musée Vert, muséum na na 13th-14th C postcanine (8, 9); Nicolas Morel (a) 2004.3.53 d'histoire naturelle du Mans tooth root (a) Personal communication. References: 1. Bowsher D, Dyson T, Holder N, & Howell I eds (2007) The London Guildhall: An Archaeological History of a Neighbourhood from Early Medieval to Modern Times (Museum of London Archaeology Service, London). 2. Caulfield D (1992) Walrus skull and tusk fragment. From Viking to Crusader: Scandinavia and Europe 800-1200, eds Roesdahl E & Wilson DW (Nordic Council, Uddevalla, Sweden), p 385. 3. Hansen G (2005) Bergen c.800-c.1170: The emergence of a town (Fagbokforlaget as, Bergen). 4. Grieg S (1933) Middelalderske Byfund fra Bergen og Oslo (A.W. Brøggers Boktrykkeri A/S, Oslo). 5. Karlsson J (2016) Spill om Djur, Hantverk och Nätverk i Mälarområdet under Vikingatid och Medeltid (Institutionen för arkeologi och antikens kultur, Stockholms universitet, Stockholm). 6. Rösch F (2015) Das Schleswiger Hafenviertel im Hochmittelalter: Entstehung – Entwicklung – Topographie. Teil II: Abbildungen, Tabellen, Katalog I. PhD (Christian- Albrechts-Universität zu Kiel, Kiel). 7. Degerbøl M (1929) Animal bones from the Norse Ruins at Gardar. Norse Ruins at Gardar: The Episcopal Seat of Medieval Greenland, eds Nørlund P & Roussell A (The Commission for Scientific Research in Greenland, Copenhagen), pp 183-192. 8. Roesdahl E & Stoklund M (2006) Un crâne de morse décoré et gravé de runes: Á propos d'une découverte récente dans un musée du Mans. Proxima Thulé 5:9-38. 9. Imer LM (2017) Peasants and Prayers: The Inscriptions of Norse Greenland (University Press of Southern Denmark, Copenhagen). bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table S2. Sequencing details of 37 archaeological Atlantic walrus samples. Estimates for library clonality and endogenous DNA content were obtained by aligning reads to the nuclear Pacific walrus reference genome (77). To obtain mitochondrial (MT) data, reads were aligned separately to the Atlantic walrus mitogenome (78). Endogenous Reads DNA MT fold Insert Sample ID (millions) Clonality (%) (fraction) coverage length(bp) WLR008 13 0.01 0.44 43 70 WLR009 16 0.01 0.09 17 80 WLR010 19 0.01 0.33 59 79 WLR011 6 0.00 0.23 8 62 WLR012 5 0.00 0.43 30 68 WLR017 20 0.01 0.68 125 78 WLR021 15 0.01 0.71 80 91 WLR022 15 0.01 0.67 89 87 WLR023 12 0.01 0.35 73 96 WLR024 16 0.01 0.69 49 81 WLR029 12 0.02 0.29 52 69 WLR030 17 0.03 0.17 74 72 WLR031 9 0.02 0.16 81 72 WLR033 17 0.03 0.01 8 94 WLR034 30 0.42 0.02 7 58 WLR038 5 0.04 0.35 16 76 WLR039 8 0.03 0.01 13 61 WLR041 1 0.01 0.48 10 70 WLR042 11 0.02 0.01 13 74 WLR043 13 0.08 0.00 9 71 WLR044 10 0.01 0.49 14 80 WLR045 12 0.02 0.11 24 75 WLR046 10 0.03 0.03 22 97 WLR047 11 0.05 0.30 9 64 WLR048 11 0.03 0.42 179 79 WLR049 12 0.02 0.02 9 79 WLR062 15 0.06 0.49 295 62 WLR063 12 0.01 0.06 46 77 WLR064 9 0.01 0.03 14 72 WLR065 12 0.02 0.37 438 73 WLR067 6 0.01 0.08 13 69 WLR068 10 0.02 0.19 104 67 WLR069 23 0.01 0.01 5 76 WLR070 27 0.01 0.02 10 72 WLR071 27 0.01 0.01 6 69 WLR072 35 0.01 0.02 10 69 WLR073 16 0.01 0.15 68 72 bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table S3. Genotypes for three control region SNPs in 105 Atlantic walrus control region sequences. These SNPs are located between 15328 and 15827 bp. For each of the sequences, we show their Genbank accession number, study from which the data was obtained, and whether the individuals with these sequences originated from western Greenland or Canada (West) or the Northeast Atlantic (East). The MT location and alleles (between brackets) for each SNP are given. The A15564 C15760C15779 haplotype that only occurs in western Greenland and the Canadian Arctic is highlighted (bold). SNP Genotype GenBank Accession Study Location 15564 (A/G) 15760 (C/T) 15779 (C/T) KJ522887.1 McLoad 2014 West A T C KJ522888.1 McLoad 2014 West A T C KJ522889.1 McLoad 2014 West A T C KJ522890.1 McLoad 2014 West G T T KJ522891.1 McLoad 2014 West A T C KJ522892.1 McLoad 2014 West A T C KJ522893.1 McLoad 2014 West G T T KJ522894.1 McLoad 2014 West A T C KJ522895.1 McLoad 2014 West G T C KJ522896.1 McLoad 2014 West A C C KJ522897.1 McLoad 2014 West A T C KJ522898.1 McLoad 2014 West A C C KJ522899.1 McLoad 2014 West G T T KJ522900.1 McLoad 2014 West G T T KJ522901.1 McLoad 2014 West G T T KJ522902.1 McLoad 2014 West A C C KJ522903.1 McLoad 2014 West A C C KJ522904.1 McLoad 2014 West A C C KJ522905.1 McLoad 2014 West A T C KJ522906.1 McLoad 2014 West G T C KJ522907.1 McLoad 2014 West A T C KJ522908.1 McLoad 2014 West A T C KJ522909.1 McLoad 2014 West A T C KJ522910.1 McLoad 2014 West G T T KJ522911.1 McLoad 2014 West A C C KJ522912.1 McLoad 2014 West G T C KJ522913.1 McLoad 2014 West A T C KJ522914.1 McLoad 2014 West A T C KJ522915.1 McLoad 2014 West A T C KJ522916.1 McLoad 2014 West A T C KJ522917.1 McLoad 2014 West A T C KJ522918.1 McLoad 2014 West A T C KJ522919.1 McLoad 2014 West A T C KJ522920.1 McLoad 2014 West A C C KJ522921.1 McLoad 2014 West A C C KJ522922.1 McLoad 2014 West A C C MF166700.1 Andersen 2017 East G T C MF166701.1 Andersen 2017 East A T T MF166702.1 Andersen 2017 East G T C MF166703.1 Andersen 2017 East G T C MF166704.1 Andersen 2017 East A T T MF166705.1 Andersen 2017 East G T T MF166706.1 Andersen 2017 East G T T MF166707.1 Andersen 2017 East A T C MF166708.1 Andersen 2017 East A C T MF166709.1 Andersen 2017 East G T C MF166710.1 Andersen 2017 East G T T MF166711.1 Andersen 2017 East G T C MF166712.1 Andersen 2017 East A C T MF166713.1 Andersen 2017 East A T C MF166714.1 Andersen 2017 East A T T MF166715.1 Andersen 2017 East G T C MF166716.1 Andersen 2017 East G T C MF166717.1 Andersen 2017 East G T T MF166718.1 Andersen 2017 East A T T MF166719.1 Andersen 2017 East G T T MF166720.1 Andersen 2017 East A T T MF166721.1 Andersen 2017 East A T T MF166722.1 Andersen 2017 East G T T MF166723.1 Andersen 2017 East A T T MF166724.1 Andersen 2017 East A T T KU710183.1 Bachman 2016 East A T T bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

KU710184.1 Bachman 2016 East G T T KU710185.1 Bachman 2016 East G T C KU710186.1 Bachman 2016 East A T T KU710187.1 Bachman 2016 East G T C KU710189.1 Bachman 2016 East A T T KU710190.1 Bachman 2016 East A T T KU710191.1 Bachman 2016 East A T T KU710192.1 Bachman 2016 East G T T KU710193.1 Bachman 2016 East G T C KU710194.1 Bachman 2016 East G T T KU710195.1 Bachman 2016 East G T T KU710196.1 Bachman 2016 East G T T KU710197.1 Bachman 2016 East A T T KU710198.1 Bachman 2016 East A T T KU710199.1 Bachman 2016 East G T C KU710200.1 Bachman 2016 East G T T EU728544.1 Lindquist 2009 East A T T EU728545.1 Lindquist 2009 East A T T EU728546.1 Lindquist 2009 East A T T EU728547.1 Lindquist 2009 East A T C EU728548.1 Lindquist 2009 East A T T EU728549.1 Lindquist 2009 West A C C EU728550.1 Lindquist 2009 West A C C EU728551.1 Lindquist 2009 West A C C EU728552.1 Lindquist 2009 West A C C EU728553.1 Lindquist 2009 West A C C EU728554.1 Lindquist 2009 East A T T EU728555.1 Lindquist 2009 East A T T EU728556.1 Lindquist 2009 East A T T EU728557.1 Lindquist 2009 East A T T EU728558.1 Lindquist 2009 East A T T EU728559.1 Lindquist 2009 West A C C EU728560.1 Lindquist 2009 West A C C EU728561.1 Lindquist 2009 West A C C EU728565.1 Lindquist 2009 East A C T EU728566.1 Lindquist 2009 East A T T EU728567.1 Lindquist 2009 East A T T EU728568.1 Lindquist 2009 East A T C EU728569.1 Lindquist 2009 East G T T EU728570.1 Lindquist 2009 East G C T EU728571.1 Lindquist 2009 East G T C EU728572.1 Lindquist 2009 East G T C EU728573.1 Lindquist 2009 East G T C bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplementary Figures

Figure S1. aDNA fragmentation and mis-incorporation patterns of sequencing read data from 37 archaeological Atlantic walrus samples. All samples show the typical fragmentation (top four panels), elevated 5'-end C->T (bottom left panel) and elevated 3'-end G->A substitution patterns (bottom right panel) expected from sequencing authentic aDNA data. Patterns were obtained using MapDamage v. 2.0.6 after down-sampling BAM files to 1,000,000 reads if applicable. bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure S2. Haplotype genealogy graph of 37 archaeological Atlantic walruses and one Pacific walrus. Haplotypes belonging to the Western clade (blue), Eastern clade (orange) or the Pacific walrus (grey) are separated by a number of substitutions (grey edges indicated with a number for the two main Atlantic walrus branches). Circle size reflects the number of specimens with an identical haplotype, and where this is >1 the number is specified within the circle. bioRxiv preprint doi: https://doi.org/10.1101/289165; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure S3. Genetic population structure based on whole mitochondrial genome data in 37 archaeological Atlantic walruses and one Pacific walrus. a) Principle component analysis (PCA) based on 346 SNPs using smartPCA, EIGENSOFT v.6.1.4. The first principle component significantly (eigenvalue = 15.5, Tracy-Widom (TW) stat = 1.76, p-value = 0.01) differentiates the Pacific walrus (grey) from the Atlantic specimens. The second principle component significantly (eigenvalue = 7.01, TW stat = 4.26, p-value = 0.0001) differentiates the western (blue) and eastern (orange) Atlantic walrus clades. b) A bimodal distribution characterizes the SNP-weightings of the second Principle Component. SNPs with a weighting above 1.25 support the western and eastern differentiation, and those with the highest values are exclusively associated with either clade. c) SNPs supporting the western and eastern differentiation are located throughout the mitochondrial genome with a subset of SNPs (red) located in those regions (ND1, ND2, ND3/4 and the control region CR, red italic) investigated in previous studies.