Supporting InformationLindqvist et al. 10.1073/pnas.0914266107

Fig. S1. mtDNA sequence coverage per site over the entire mtDNA genome from the 454 shotgun fragment runs of the Poolepynten specimen.

Lindqvist et al. www.pnas.org/cgi/content/short/0914266107 1 of 8

Poolepynten specimen

14KB U. arctosBaranof Island

12WH U. arctosA9105 U. arctosAdmiralty Island

76824 U. arctosKodiak Island

2495 U. maritimusDiomedeLittle Diomede Island

2629 U. maritimusSavoongaSt. Lawrence Island

Poolepynten coastal cliffs(78°27′N, 11°44′E)Prins Karls ForlandSvalbard

Arct

ic C

ircle

NorthPole

Fig. S2. Map of the Arctic showing sampling sites of specimens analyzed in this study.

Lindqvist et al. www.pnas.org/cgi/content/short/0914266107 2 of 8

U. maritimus SAVO2629U. maritimus DIOM2495U. maritimus AJ428577U. maritimus NC_003428

Poolepynten specimen

U. arctos AdmA91-05U. arctos Adm12WH

U. arctos Baranof14KBU. arctos EU497665

U. arctos Kodiak76824U. arctos NC_003427

U. spelaeus EU327344U. spelaeus NC_011112

U. americanus NC_003426U. thibetanus NC_009971U. thibetanus NC_008753

U. malayanus NC_009968Ursus ursinus NC_009970

Arctodus simus NC_011116Tremarctos ornatus NC_009969

Ailuropoda melanoleuca FM177761

100

100

57

87100

100

100

100

100

100 100

97

10080

10099

Fig. S3. Molecular phylogeny of the Ursidae based on maximum parsimony (MP) analysis of complete mtDNA genome sequences [excluding the variable-number tandem repeats (VNTR) repeat region of the D loop]. One most-parsimonious tree of 8715 steps resulted from an analysis using a comprehensive MPapproach with a variety of tree space exploration techniques as implemented in TNT (1). Bootstrap support values are shown below branches.

1. Goloboff PA, Farris JS, Nixon KC (2008) TNT, a free program for phylogenetic analysis. Cladistics 24:774–786.

giant panda

spetacled bearAmerican giantshort-faced bearsloth bear

sun bear

American black bear

Asian black bear

Asian black bear

cave bear

cave bear

Kodiak bearbrown bear (east)

brown bear (west)

brown bear (Baranof)

brown bear (Admiralty)

brown bear (Admiralty)

Poolepynten specimenpolar bear

polar bear (E Greenland)

polar bear (Alaska)

polar bear (Alaska)

100

100

100

100100

100

100

100

100

100

100

100

100

93

100

92

90

99

Fig. S4. Molecular phylogeny of the Ursidae based on maximum likelihood (ML) analyses of complete mtDNA genome sequences (excluding the VNTR repeatregion of the D loop). The phylogram with bootstrap support values shown below branches resulted from ML analysis using a rapid bootstrap algorithm asimplemented in RAxML (1). The mtDNA genome sequences (previously published and this study) are identical to the ones shown in Fig. S3. The previouslypublished brown bear sequences represent “eastern” (east) and “western” (west) lineages (2).

1. Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol 75:758–771.2. Bon C, et al. (2008) Deciphering the complete mitochondrial genome and phylogeny of the extinct cave bear in the Paleolithic painted cave of Chauvet. Proc Natl Acad Sci USA 105:

17447–17452.

Lindqvist et al. www.pnas.org/cgi/content/short/0914266107 3 of 8

Umar_2495, Alaska

Umar_IDNR23479, Barents Sea/Svalbard

Umar_IDNR23530, Barents Sea/Svalbard

Umar_IDNR23395, Barents Sea/Svalbard

Umar_IDNR7944, Barents Sea/Svalbard

Poolepynten specimen

Umar_AF303111

Umar_2629, Alaska

brown bear ‘eastern lineage’

modern polar bear

brown bear Alaska/Yukon

Fig. S5. Maximum clade probability tree from a BEAST analysis of short mtDNA control-region sequences. The analysis is based on 197 base-pair sequences fromcarbon-datedbrownbears (inblackbold) (1), theancientPoolepynten specimen(in red),andmodernbrownbearsandpolarbears, including four sequencesofmodernpolar bear from the Barents Sea/Svalbard (in blue). Numbers at nodes indicatemean ages inmillion years. BEAST parameters used: the General Time Reversible (GTR)substitutionmodel, Invariant Sites (I), andGamma4 (Γ4) siteheterogeneitymodelwitha relaxeduncorrelated log-normal clock, a coalescentprior, andaMarkov chainMonte Carlo (MCMC) chain length of 50,000,000. Note that the Svalbard subfossil is considerably older in origin than any modern Svalbard polar bear sampled.

1. Barnes I, Matheus P, Shapiro B, Jensen D, Cooper A (2002) Dynamics of Pleistocene population extinction in Beringian brown bears. Science 295:2267–2270.

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Fig. S6. (Continued)

Lindqvist et al. www.pnas.org/cgi/content/short/0914266107 5 of 8

Fig. S6. Correct length of the VNTR for each mtDNA genome as determined by an Agilent 2100 bioanalyzer with the DNA 7500 kit.

Lindqvist et al. www.pnas.org/cgi/content/short/0914266107 6 of 8

Table S1. Sample information and summary of bear 454 sequencing runs

MID*Sample

IDMSM archive

no.† Species‡ Tissue Location454reads

No. mtreads

Complete(non-VNTR)§ Coverage

GenBankaccessionnumbers

1 503950 NK157104 U. arctos Muscle Alaska (Chilkoot Lake) 83,842 2 No NA NA2 14KB NK157109 U. arctos Muscle Alaska (Baranof Island) 82,790 3,425 Yes 50 GU5734893 12WH NK157111 U. arctos Muscle Alaska (Admiralty Island) 108,532 2,571 Yes 36 GU5734864 A91-05 NK157120 U. arctos Muscle Alaska (Admiralty Island) 10,428 4,866 Yes 70 GU5734875 84688 NK157123 U.arctos Muscle Alaska (Chichagof Island) 44,266 5 No NA NA6 76946 NK157129 U. arctos Muscle Alaska (Kodiak Island) 146 18 No NA NA7 76824 NK157130 U. arctos Muscle Alaska (Kodiak Island) 78,018 891 Yes 12 GU5734918 542 NK157137 U. maritimus Homogenate Alaska (Barrow) 120,195 261 No 3.5 NA9 2495 NK157142 U. maritimus Muscle Alaska (Little Diomede Island) 70,862 4,685 Yes 68 GU57349010 2629 NK157143 U. maritimus Muscle Alaska (St. Lawrence Island) 25,560 1,855 Yes 23 GU573485NA NA NA U. maritimus Tooth Svalbard (Poolepynten) 482,364 1,290 Yes 14 GU573488

*MID, multiplex identifiers (Methods); NA, not applicable.†Aliquots of the modern bear tissues are archived at the Museum of Southwestern Biology (MSM).‡The modern polar bear samples were harvested in 1998 (542), 2001 (2495), and 2002 (2629), that is, before listing of the species in the Endangered Species Act,by the U.S. Fish and Wildlife Marine Region 7 Mammal Management Office.§Four samples provided too low percentages of mitochondrial reads to assemble the mt genome.

Lindqvist et al. www.pnas.org/cgi/content/short/0914266107 7 of 8

Table

S2.

Summaryofresu

ltsfrom

differentBEA

STan

alyses

based

ondifferentdatasets(this

study)

andco

mparisonofdivergen

cetimes

foundin

other

releva

nt,previously

published

studies.

Amea

nag

eof12

0thousand(k)ye

arsforthePo

olepyn

tensu

bfossilpolarbea

rwas

usedin

additionto

datafrom

twocarbon-dated

cave

bea

rmtgen

ome

sequen

ces(1,2)

Time-MRCA*

Yuet

al.,20

07(3)

Krause

etal.,20

08(2)

Bonet

al.,20

08(1)

Brown-A

BC-polaronly

excludingrepea

t

Sun-black-cav

e-brown-

ABC-polarex

cluding

repea

t(rep

orted

inFig.1)

Sloth-sun-black-cav

e-

brown-A

BC-polar

includingrepea

t

Sloth

+sun/black/

brown/cav

e/polar

6.34

(5.95–

6.73

)5.39

(4.2–6.86

)3.0(2.2–3.8)

NA

NA

2.59

(1.32–

4.05

)

Sun/black

+cave

/polar/

brown

6.13

(5.54–

6.72

)5.05

(3.9–6.48

)2.8(2.1–3.5)

NA

2.60

(1.85–

3.42

)2.49

(1.26–

3.86

)

Cav

e+brown/polar

NA

2.75

(2.1–3.57

)1.6(1–2.1)

NA

1.43

(1.03–

1.88

)1.39

(0.67–

2.16

)Su

n+black

5.67

3(5.09–

6.26

)4.58

(3.51–

5.89

)2.4(1.7–3)

NA

2.35

(1.58–

3.16

)2.12

(0.88–

3.43

)Asiatic+American

black

5.19

(4.6–5.78

)4.08

(3.11–

5.27

)2.1(1.4–2.7)

NA

NA

1.85

(0.72–

2.97

)Brown+polar†

1.32

(0.93–

1.71

)0.88

(0.66–

1.17

)0.6(0.3–0.8)

east

brown;

0.4(0.2–0.5)

westbrown

0.44

(0.14–

0.97

)ea

stbrown

0.49

(0.36–

0.62

)ea

stbrown;0.31

(0.24–

0.40

)westbrown

0.51

(0.29–

0.76

)ea

stbrown;0.33

(0.20–

0.49

)west

brown

ABC+polar

NA

NA

NA

0.23

(0.11–

0.50

)0.15

2(0.131

–0.17

7)0.16

7(0.132

–0.21

4)Ancien

t+modern

NA

NA

NA

0.17

(0.11–

0.32

)0.13

4(0.122

–0.14

9)0.13

8(0.122

–0.16

2)Modern

NA

NA

NA

68.6k(5.5–16

6.0k

)43

.5k(29.8–

59.0k)

46.7k(24.0–

71.2k)

Method

TREE

-PUZZ

LE:molecu

lar

clock

onco

ding

regions,onedee

pfossilcalib

rationpoint

BEA

ST:strict

clock,GTR

+Γ4

,threedee

pfossil

calib

rationpoints

BEA

ST:relaxe

dunco

rrelated

log-

norm

alclock,GTR

+I+

Γ4,twodee

pfossil

calib

rationpoints

BEA

ST:relaxe

dunco

rrelated

log-

norm

alclock,GTR

+I+Γ4

,nodee

pfossil

calib

rationpoints

BEA

ST‡:relaxe

dunco

rrelated

log-

norm

alclock,GTR

+I+Γ4

,nodee

pfossil

calib

rationpoints

BEA

ST:relaxe

dunco

rrelated

log-

norm

alclock,GTR

+I+Γ4

,nodee

pfossil

calib

rationpoints

Other

inform

ation

NA

SpeciationYule

process,

MCMCch

ainlength

=30

,000

,000

,logev

ery

1000

,Tracer

burn-in

5,00

0,00

0states

SpeciationYule

process,

MCMCch

ainlength

=10

,000

,000

,logev

ery

1000

,burn-in10

,000

states

SpeciationYule

process,

MCMCch

ainlength

=50

,000

,000

,logev

ery

1000

,Tracerburn-in

5,00

0,00

0states,a

llES

S>21

0,samplin

g50

,001

tree

s,Tree

Annotatorburn-in

1000

tree

s

SpeciationYule

process,

MCMCch

ainlength

=50

,000

,000

,logev

ery

1000

,Tracerburn-in

5,00

0,00

0states,a

llES

S>24

0,samplin

g50

,001

tree

s,Tree

Annotatorburn-

in10

00tree

s

SpeciationYule

process,

MCMCch

ainlength

=80

,000

,000

,logev

ery

1000

,Tracerburn-in

8,00

0,00

0states,a

llES

S>23

0,samplin

g80

,001

tree

s,Tree

Annotatorburn-

in10

00tree

s

*Tim

ein

Mya

(million

yearsag

o)is

indicated

forclad

esunless

labeled

otherwise.

Mea

nan

d95

%highestposteriorden

sity

(HPD

;in

paren

theses)va

lues

areshown.NA,notap

plicab

le;GTR

,Gen

eral

Time

Rev

ersible;ES

S,EffectiveSa

mple

Size

.†NC_0

0342

7,“ea

stbrownbea

rclad

e”;EU

_497

665,

“westbrownbea

rclad

e”(1).

‡Usingthesameparam

etersin

BEA

STbuttheHaseg

awa-Kishino-Yan

o(H

KY)model

instea

doftheGTR

+Iresulted

insimila

rtimeestimates

(althoughallES

S>

1100

).

1.BonC,et

al.(200

8)Decipheringtheco

mplete

mitoch

ondrial

gen

omean

dphylogen

yoftheex

tinct

cave

bea

rin

thePa

leolithic

paintedcave

ofChau

vet.ProcNatlAcadSciUSA

105:17

447–

1745

2.2.

Krause

J,et

al.(200

8)Mitoch

ondrial

gen

omes

reve

alan

explosive

radiationofex

tinct

andex

tantbea

rsnea

rtheMiocene-Plioceneboundary.

BMCEv

olBiol8:22

0.3.

YuL,

LiY-W

,Ryd

erOA,Zh

angY(200

7)Analysisofco

mplete

mitoch

ondrial

gen

omesequen

cesincrea

sesphylogen

etic

resolutionofbea

rs(U

rsidae

),amam

malianfamily

that

experiencedrapid

speciation.BMCEv

olBiol7:19

8–20

9.

Lindqvist et al. www.pnas.org/cgi/content/short/0914266107 8 of 8