Mi ochond ial Genomes Re eal Slow Ra es o Molecula
E olu ion and he Timing o Specia ion in Bea e s
(
Cas o
), One o he La ges Roden Species
Susanne Ho n
1
*, Wal e Du ka
2
, Ronny Wol
3
, Aslak E mala
4
, Anneg e S ubbe
5
, Michael S ubbe
5
,
Michael Ho ei e
1¤
1Max Planck Ins i u e o E olu iona y An h opology, Leipzig, Ge many, 2Depa men o Communi y Ecology, Helmhol z-Cen e o En i onmen al Resea ch-UFZ, Halle,
Ge many, 3Depa men o Molecula E olu ion and Animal Sys ema ics, Uni e si y o Leipzig, Leipzig, Ge many, 4Finnish Game and Fishe ies Resea ch Ins i u e, Helsinki,
Finland, 5Ins i u e o Zoology, Uni e si y o Halle-Wi enbe g, Wi enbe g, Ge many
Abs ac
Backg ound:
Bea e s a e one o he la ges and ecologically mos dis inc oden species. Li le is known abou hei
e olu ion and e en hei closes phylogene ic ela i es ha e no ye been iden i ied wi h ce ain y. Simila ly, li le is known
abou he iming o di e gence e en s wi hin he genus Cas o .
Me hodology/P incipal Findings:
We sequenced comple e mi ochond ial genomes om bo h ex an bea e species and
used hese sequences o place bea e s in he phylogene ic ee o oden s and da e hei di e gence om o he oden s as
well as he di e gence e en s wi hin he genus Cas o . Ou analyses suppo he phylogene ic posi ion o bea e s as a sis e
lineage o he scaly ailed squi el Anomalu us wi hin he mouse ela ed clade. Molecula da ing places he di e gence ime
o he lineages leading o bea e s and Anomalu us as ea ly as a ound 54 million yea s ago (mya). The li ing bea e species,
Cas o canadensis om No h Ame ica and Cas o ibe om Eu asia, al hough simila in appea ance, appea o ha e
di e ged om a common ances o mo e han se en mya. This esul is consis en wi h he hypo hesis ha a mig a ion o
Cas o om Eu asia o No h Ame ica as ea ly as 7.5 mya could ha e ini ia ed hei specia ion. We da e he common
ances o o he ex an Eu asian bea e elic popula ions o a ound 210,000 yea s ago, much ea lie han p e iously
hough . Finally, he subs i u ion a e o Cas o mi ochond ial DNA is conside ably lowe han ha o o he oden s. We
ound e idence ha his is co ela ed wi h he longe li e span o bea e s compa ed o o he oden s.
Conclusions/Signi icance:
A phylogene ic analysis o mi ochond ial genome sequences sugges s a sis e -g oup ela ionship
be ween Cas o and Anomalu us, and allows molecula da ing o species di e gence in cong uence wi h paleon ological
da a. The implemen a ion o a elaxed molecula clock enabled us o es ima e mi ochond ial subs i u ion a es and o
e alua e he e ec o li e his o y ai s on i .
Ci a ion: Ho n S, Du ka W, Wol R, E mala A, S ubbe A, e al. (2011) Mi ochond ial Genomes Re eal Slow Ra es o Molecula E olu ion and he Timing o
Specia ion in Bea e s (Cas o ), One o he La ges Roden Species. PLoS ONE 6(1): e14622. doi:10.1371/jou nal.pone.0014622
Edi o : William J. Mu phy, Texas A&M Uni e si y, Uni ed S a es o Ame ica
Recei ed July 13, 2010; Accep ed No embe 10, 2010; Published Janua y 28, 2011
Copy igh : ß2011 Ho n e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: This wo k was suppo ed by he Volkswagen Founda ion and he Max Planck Socie y. The unde s had no ole in s udy design, da a collec ion and
analysis, decision o publish, o p epa a ion o he manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: [email p o ec ed]
¤ Cu en add ess: Depa men o Biology, Uni e si y o Yo k, Yo k, Uni ed Kingdom
In oduc ion
Da ing back o app oxima ely 40 million yea s, oday he amily
o bea e s, Cas o idae, is ep esen ed by only wo ex an species,
Cas o canadensis in No h Ame ica and Cas o ibe in Eu asia. Bo h
species a e cha ac e ized by hei la ge body size, being he second
la ges oden , and hei semi-aqua ic li es yle [1,2]. Howe e ,
se e al aspec s o he ea ly his o y and e olu ion o bea e s emain
unclea . Ea lie a emp s o hei phylogene ic placemen ela i e
o o he oden s we e di icul because o a lack o ixed
mo phological di e ences, poo axon sampling in many gene ic
s udies, limi ed sequence da a in p e ious s udies, and con empo-
aneous adia ions o mul iple oden lineages. Mo e ecen
molecula da a s ongly suppo he placemen o Cas o wi hin a
‘‘mouse- ela ed clade,’’ con aining se e al amilies including
Pede idae, Anomalu idae, Mu idae, Dipodidae, Geomyidae, and
He e omyidae [3,4,5,6]. P e ious mul igene s udies ha e sugges ed
Geomyoidea o be he closes ela i es o bea e s [5,6]. Howe e ,
he b anches leading o bo h g oups di e ged e y ea ly in oden
e olu ion and ansposon inse ion analyses a e inconclusi e wi h
ega d o hei monophyly [7,8].
No only he phylogene ic placemen o bea e s wi hin oden s
is no comple ely unde s ood, li le is also known abou he iming
o he specia ion e en leading o he wo ex an bea e species.
Bo h ha e a s ikingly simila pheno ype, making hem almos
indis inguishable in he ield; he e o e molecula me hods a e
applied o di e en ia e hem [1,9]. Despi e he mo phological
simila i ies, sub le mo phological and biochemical ea u es and
PLoS ONE | www.plosone.o g 1 Janua y 2011 | Volume 6 | Issue 1 | e14622
di e en ch omosome numbe s suppo hei dis inc ion as
di e en species [10]. The ossil eco d p o ides u he in o ma-
ion on he iming o Cas o specia ion. The appea ance o
he genus Cas o in Eu asia and No h Ame ica was es ima ed
o he la e Miocene and he Pliocene, be ween 9–4.9 mya
[11,12,13,14,15,16]. Cas o is assumed o ha e eme ged in Eu asia
as a close ela i e o S eneo ibe [11,12,14,15,16], and o ha e
subsequen ly dispe sed o No h Ame ica ia he Be ing land-
b idge [12,13,15,16,17]. This dispe sal e en was es ima ed o 4.9–
6.6 mya [13,15]. Howe e , since he es ima es o he ea lies
appea ance o Cas o on bo h con inen s o e lap, i is no en i ely
clea in which di ec ion he dispe sal o Cas o ook place.
I espec i e o dispe sal di ec ion, since he wo ex an species o
Cas o a e na i e o ei he Eu asia o No h Ame ica, i seems
easonable o assume ha a mig a ion ac oss he Be ing s ai
could ha e ini ia ed he specia ion o C. canadensis and C. ibe
[10,14,15] (Fig. 1). Howe e , no s udy o da e a emp ed ob aining
a sequence based molecula da e o he iming o di e gence
be ween he wo species. As mi ochond ial genomes a e a ailable
o a numbe o oden axa [18], he addi ion o Cas o
mi ochond ial genomes could shed ligh no only on he deep
phylogeny o bea e s, bu also on he iming o di e gence o o he
oden s and wi hin he genus i sel . Finally, mi ochond ial genome
da a should allow o de e mina ion o he o e all subs i u ion a e
in bea e mi ochond ial DNA, which allows e ospec i ely
in es iga ing and da ing popula ion gene ic p ocesses.
He e we p esen he i s comple e mi ochond ial genomes o
bea e s. We use hese DNA sequences o in es iga e he
phylogene ic posi ion o bea e s wi hin oden s, da e e olu iona y
e en s wi hin he ex an membe s o he amily Cas o idae and
explo e he subs i u ion a e o hei mi ochond ial DNA.
Resul s
Phylogene ics
We sequenced mi ochond ial genomes o one C. canadensis and
i e C. ibe u ilizing long ange PCR, DNA hyb idiza ion cap u e
and 454 sequencing (Table S1 and and Suppo ing Re e ences
S1). Assemblies de i ed om hyb idiza ion cap u e showed a
conside ably smoo he ead dis ibu ion han hose de i ed om
long ange PCR (Fig. 2). The consensus sequences ob ained we e
aligned wi h a a ie y o oden and ou g oup mi ochond ial
genomes o phylogene ic analyses and molecula da ing (Table
S2). We employed se e al di e en me hods o in es iga ing he
phylogene ic ela ionships in ou da ase . All me hods, Bayesian
app oaches, maximum likelihood (ML), neighbo joining (NJ) and
maximum pa simony (MP) eco e ed a well suppo ed, mono-
phyle ic clade o he Cas o indi iduals. In Fig. 3 we p esen a
maximum clade c edibili y ee om BEAST wi h suppo alues
om all phylogene ic me hods. Highes suppo was ob ained o
he sis e g oup ela ionship o C. canadensis and C. ibe , he la e
ep esen ed by a monophyle ic clade o i e subspecies (C. ibe ssp.
albicus,belo ussicus/o ien oeu opaeus,bi ulai, u inicus, and pohlei). A
posi ion o Cas o wi hin he mouse ela ed clade o oden s, as
sis e o he scaly ailed squi el Anomalu us, was suppo ed wi h
e y high and maximum suppo , espec i ely, om Bayesian
analyses as well as wi h boo s ap alues highe han 70 in ML
analyses. The squi el ela ed clade and Hys icogna hi we e
eco e ed wi h high suppo alues in all phylogene ic me hods.
Al hough he monophyly o oden s was no suppo ed in NJ and
MP, i ecei ed maximum suppo om Bayesian and a boo s ap
alue o 70 in ML analyses. Howe e , he ela ionship be ween he
h ee majo oden clades emains enigma ic, since hei
b anching o de could no be esol ed. Since di icul ies in
esol ing he oo o he oden ee ha e been a ibu ed o he
e ec o long b anch a ac ion, causing oden sequences o
clus e wi h ou g oups [18,19], we conduc ed addi ional ML
analyses wi h a mo e ep esen a i e sample o ou g oup sequences
(Table S2). These addi ional ou g oup sequences could no
imp o e he esolu ion o he ea ly b anchings, ins ead, he ML
boo s ap suppo alue o oden monophyly dec eased om 70
o 52 (Table S3). Howe e , since he monophyly o oden s was
suppo ed in ou Bayesian and ML ees based on he smalle se
o axa as well as o he s udies on nuclea DNA sequences and
ansposon inse ions [7,8], we ne e heless a emp ed es ima ing
he ime elapsed since a monophyle ic o igin o all oden s using a
molecula da ing app oach implemen ed in BEAST [20].
Figu e 1. Dis ibu ion o bea e s and sampling si es. Bea e s li e in No h Ame ica (C. canadensis, s iped a eas) and Eu asia (C. ibe , g ey
a eas). Black a eas ma k C. ibe elic popula ions om which he cu en popula ions de eloped. C. canadensis was in oduced in Eu ope and Asia
(s iped a eas, dis ibu ion no exac ly known o Kamcha ka). Numbe s indica e bea e popula ions sampled o mi ochond ial genome sequencing.
C. ibe was sampled in he a eas o he elic popula ions (1,3,4,5) o close o hem (2) [31]. C. canadensis was sampled in a Eu opean in oduced
popula ion (6). A ow: mig a ion ia he Be ing land b idge is sugges ed o ha e ini ia ed Cas o specia ion a ound 8–7.6 million yea s ago (mya). Map
ed awn om [1], [31] and [55].
doi:10.1371/jou nal.pone.0014622.g001
Cas o Mi ochond ial Genomes
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Di e gence es ima es
We es ima ed ha all oden s sha ed a common ances o a ound
67 mya (CI: 57–76 mya) (Table S4). A ound 54 mya (CI: 44–64 mya),
he b anch leading o bea e s di e ged om he common ances o
wi h he scaly ailed squi el Anomalu us. The mi ochond ial DNA o
he wo bea e species C. ibe and C. canadensis sha ed a mos ecen
common ances o be ween 8 mya (mean) and 7.6 mya (median) (CI:
3.7–13 mya; Table S4). Wi hin Eu asian bea e s, he indi iduals
coming om di e en elic popula ions sha ed a common ances o
a ound 210,000 yea s ago (CI: 110,000-340,000 ya).
Subs i u ion a es
As bea e s a e he la ges oden s o which a mi ochond ial
genome sequence is cu en ly a ailable, we explo ed he
Figu e 2. Sequencing co e age plo s o bea e mi ochond ial genomes. Plo s o C. . ssp. bi ulai ampli ied by long ange PCR (g ey line) and
C. . ssp. pohlei en iched by hyb idiza ion cap u e (black line). Co e age was mo e e en when hyb idiza ion cap u e was used ins ead o long ange
PCR. Peaks o sequencing co e age a e isible o C. . ssp. bi ulai (g ey) in p oximi y o p iming si es (indica ed on op o he plo s).
doi:10.1371/jou nal.pone.0014622.g002
Figu e 3. Time ee o oden s and ou g oups. Bea e s sha e a common ances o wi h Anomalu omo pha a ound 54 mya (CI: 44–64 mya); he
only ex an bea e genus Cas o sepa a ed in o wo species om 8–7.6 mya onwa ds (CI: 3.7–13 mya). The common ances o o he Eu asian elic
popula ions was es ima ed o ha e li ed a ound 210,000 ya (CI: 0.11–0.34 mya). The ee depic ed is a maximum clade c edibili y ee om BEAST
analyses based on a 16,352 bp alignmen (including gaps). Bayesian pos e io p obabili y (.0.6) and boo s ap suppo alues (.50) a e shown a
he b anches and sepa a ed by slashes o M Bayes, maximum likelihood, neighbo joining and maximum pa simony, espec i ely. Diamonds indica e
ossil calib a ions (Table S4). Paleoc.: Paleocene; Oligoc.: Oligocene; Pl: Pliocene, PH: Pleis ocene and Holocene. Cas o ibe po, u, bi, in and al indica e
he subspecies sampled (see Table S1).
doi:10.1371/jou nal.pone.0014622.g003
Cas o Mi ochond ial Genomes
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mi ochond ial subs i u ion a e in compa ison o o he oden s.
The e is some e idence ha he subs i u ion a e is linked o li e
expec ancy and body mass [21] e en hough his connec ion is
con o e sial [22]. Animals like he bea e , wi h highe li e
expec ancy and la ge body mass a e he e o e expec ed o show
dec eased subs i u ion a es. Molecula subs i u ion a es o he
axa in ou da ase we e es ima ed in he BEAST analysis and a e
depic ed on each b anch in Fig. 4 (see also Table S5). The only
oden s wi h a lowe mi ochond ial a e we e Sciu us and Myoxus in
he squi el- ela ed clade. Wi hin he mouse- ela ed clade he
mi ochond ial ip a es o bea e s we e signi ican ly lowe han
hose o he o he membe s o his clade (p- alue = 0.005). Also,
when he dis ibu ion o a es o bea e s was compa ed o he
dis ibu ion o a es o all o he oden s in he da ase , he a es o
bea e s we e signi ican ly lowe (p- alue = 0.014, Mann-Whi ney
U- es , Fig.4, Table 1). This di e ence became e en mo e e iden
when ins ead o ip a es a e age a es leading o he indi idual
axa we e used (see me hods and Table S1). I es ed ac oss all
gli es ( oden s and lagomo phs), he ip and a e age a es o
bea e s did no di e signi ican ly anymo e (Table 1).
We ela ed he in e ed subs i u ion a es o body mass and
li e his o y ai s o oden s as had been done ea lie [21] (Fig. 5,
Table S6). In linea eg essions, mo e han 30 pe cen o he
a ia ion in ip a es (R
2
= 0.34, p- alue = 0.0225) and a e age
a es (R
2
= 0.40, p- alue = 0.0113) could be explained by maxi-
mum li espan. Thus conside ably mo e a ia ion in li espan was
a ibu able o mi ochond ial a e a ia ion han had been
es ima ed p e iously (below 20 pe cen in [21]). Howe e , o
body mass and age a sexual ma u i y, no signi ican co ela-
ions wi h subs i u ion a es could be iden i ied (p- alues.0.05,
Table S6).
Discussion
The phylogene ic ela ionships o ex an oden amilies ha e
been di icul o esol e. Howe e , se e al p e ious molecula
s udies ecognized h ee majo phylogene ic clades o oden s: he
mouse- ela ed clade, C enohys ica ( ela i es o he guinea pig)
and he squi el- ela ed clade (Fig. 3). This b anching was
suppo ed by nuclea DNA analyses [3,5,7,8] as well as some
s udies including mi ochond ial DNA [6,23]. We ind his
phylogeny o be also suppo ed by comple e mi ochond ial
nucleo ide sequences. In acco dance wi h s udies on nuclea
DNA [3,5,6], Anomalu us is eco e ed as an ea ly membe o he
mouse- ela ed clade. In con as , an ea lie analysis o comple e
mi ochond ial genome sequences did no eco e Anomalu us
wi hin he mouse- ela ed clade [18], possibly due o educing
he sequences o he coding egions o some o he phylogene ic
in e ences.
The oo o he oden ee is much mo e di icul o esol e.
Al hough we ob ain high suppo o oden s as a monophyle ic
g oup, con i ming ea lie s udies [3,5,8,24,25], as in p e ious
Figu e 4. Compa ison o mi ochond ial subs i u ion a es among gli es. Phylogene ic ee o gli es ( oden s and lagomo phs) wi h b anch
leng hs om he BEAST analysis. Mi ochond ial subs i u ion a es a e shown on he b anches o nex o each axon in uni s o subs i u ions pe
million yea s. Bea e s exhibi sho b anches and subs i u ion a es o bea e s a e he lowes wi hin he mouse- ela ed clade, signi ican ly lowe han
hose o mos o he oden s (see also Table 1).
doi:10.1371/jou nal.pone.0014622.g004
Cas o Mi ochond ial Genomes
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s udies, we could no de e mine he b anching o de o he h ee
majo clades [3,5,18]. Howe e , ou analyses a e based on
mi ochond ial DNA and he e o e ep esen a single genomic
locus only. Also, since many oden s exhibi high subs i u ion a es,
pa o he lowe esolu ion o he deepe nodes migh esul om
mu a ional sa u a ion o he sequences. Analyses o a la ge
numbe o nuclea loci should imp o e he esolu ion o he deepe
phylogene ic nodes wi hin oden s in he u u e.
We eco e ed he six bea e mi ochond ial genomes in one
monophyle ic g oup wi hin he mouse- ela ed clade o oden s
(Fig. 3). This phylogene ic posi ion o bea e s was indica ed
al eady in ea lie analyses based on nuclea gene sequences and
ansposon inse ions [5,7,8]. Now, mi ochond ial genomics adds
ano he line o e idence o his ela ionship. In ou da ase , he
mi ochond ial sequence o he scaly ailed squi el Anomalu us was
mos closely ela ed o hose o he bea e s. Howe e , he e is no
mi ochond ial genome sequence a ailable o he Geomyoidea
(gophe s and kanga oo a s), which ha e been ound o be mos
closely ela ed o bea e s in mul igene s udies [5,6] and could
a ach o he e olu iona y b anch leading o bea e s e en la e
han Anomalu us.
We es ima ed di e gence imes wi hin oden s in BEAST using
six ossil calib a ion poin s and a Bayesian elaxed molecula clock
app oach [20] and ound all oden s o sha e a common ances o
a ound 67 mya (CI: 57–76 mya) (Table S4). This iming is
ema kably coinciden wi h he mass ex inc ion e en ha ma ked
he ansi ion be ween he C e aceous and Te ia y (K-T
bounda y). A ha ime dinosau s wen ex inc and a majo
aunal change ook place all o e he wo ld. The o igin o oden s
has been da ed bo h ea lie and la e in o he s udies: a ound 96
mya [26], 71–89 mya [6], a ound 72 mya [3] and a ound 62 mya
[18]. Ou es ima e lies in be ween he ange o ea lie es ima es,
close o he K-T bounda y and p eda ing he explosi e adia ion
o oden s in he ea ly Eocene (da ed o a ound 55mya) [27].
Thus, he changing bio a a he K-T bounda y may ha e been
causal o a i s oden adia ion.
A ound 54 mya (CI: 44–64 mya), a phylogene ic lineage leading
o bea e s di e ged om i s common ances o wi h Anomalu -
omo pha (Anomalu us and Pede es). Thus, bea e s p obably ha e a
e y long e olu iona y his o y, which migh explain hei
ecological and mo phological peculia i ies. In a p e ious s udy
using 5.5 kb o nuclea and mi ochond ial DNA sequences [3], he
di e gence o bea e s and Anomalu omo pha was es ima ed e en
olde , a mo e han 65 mya. Howe e , since Geomyoidea a e
po en ially e en mo e closely ela ed o bea e s, addi ional DNA
sequences o Geomyoidea will be equi ed o p o ide in o ma ion
on he beginning o bea e e olu ion.
Simila o he di e gence o he amily Cas o idae, he
di e gence ime o he wo ex an bea e species has no ye been
es ima ed wi h much p ecision. The o igin o he ex an bea e
genus Cas o has been sugges ed o lie in Eu asia a some ime
be ween 9.7 and 5.2 mya based on he ossil eco d and simila i ies
wi h S eneo ibe [14,15,16]. Howe e , since he e is o e lap wi h he
ea lies appea ance o Cas o in No h Ame ica (6.6 mya o 7.5 mya
[12,28]), he geog aphical o igin o Cas o emains unce ain.
Independen o ha , a mig a ion o ancien Cas o ia Be ingia and
he subsequen geog aphical isola ion o he wo popula ions in
Eu asia and No h Ame ica, espec i ely, mos likely led o he
di e gence o Cas o lineages ha ul ima ely ga e ise o he
mode n species C. ibe and C. canadensis. Acco ding o ou
molecula da ings, he wo bea e species sha ed a common
ances o a ound 7.6–8 (CI: 3.7–13) mya. This molecula es ima e
is in iguingly close o he ea lies ossil bea e emains in No h
Ame ica possibly da ing as ea ly as 7.5 mya [12]. The Be ingian
land b idge allowed aunal exchange be ween Eu asia and No h
Ame ica un il i s i s looding 5.4–5.5 mya and hen again se e al
imes du ing he Pliocene and Pleis ocene [29]. Thus, a mig a ion
o e he Be ingian landb idge a ound 8–7.6 mya has likely been
he s a ing poin o he specia ion be ween C. ibe and C.
canadensis, each on a di e en con inen (Fig. 1).
Despi e a success ul e olu iona y his o y ac oss he Palaea c ic o
se e al millions o yea s, bea e popula ions dec eased d ama ically
in size in mo e ecen his o y. Ex ensi e hun ing by humans and
Table 1. Mi ochond ial subs i u ion a es o Cas o di e signi ican ly om hose o o he oden s.
Median a e a ips Median a es a e aged
Cas o s. es o mouse-clade U = 36, p- alue = 0.004998 ** U = 42, p- alue = 0.002591 **
Cas o s. es o oden s U = 58, p- alue = 0.01375 * U = 60, p- alue = 0.007005 *
Cas o s. es o gli es U = 62, p- alue = 0.1075 U = 60, p- alue = 0.1457
Resul s o Mann-Whi ney U- es s es ing he null hypo hesis ha subs i u ion a es o Cas o a e iden ical o he a es o o he oden s (Wilcoxon ank sum es wi h
con inui y co ec ion as implemen ed in R).
*: signi ican , ,0.05;
**: highly signi ican , ,0.005.
doi:10.1371/jou nal.pone.0014622. 001
Figu e 5. Linea eg ession o a e age subs i u ion a es and
li espan o oden s. Roden s wi h highe mi ochond ial subs i u ion
a es exhibi a sho e li espan (R squa ed = 0.4011, p- alue = 0.0113).
See Table S6 and S7 o de ails.
doi:10.1371/jou nal.pone.0014622.g005
Cas o Mi ochond ial Genomes
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habi a des uc ion a ec ed bo h bea e species [1,30] and le
Eu asia wi h a ew, isola ed elic popula ions o C. ibe a he end o
he 19
h
cen u y [30]. The di e si ica ion o hese ex an bea e
popula ions was p oposed o ha e happened du ing he las glacial
pe iod, om 115,000 ya onwa ds [31]. We da ed he las common
ances o o he ex an Eu asian bea e mi ochond ial genomes o
almos wice he age han es ima ed be o e, a ound 210,000 ya (CI:
110,000–340,00 ya). Howe e , i has been poin ed ou ha due o
he p oblems in de e mining subs i u ion a es p ecisely, such da es
should be iewed wi h cau ion and co ela ing molecula di e gence
da es wi h changes in en i onmen al condi ions may be en a i e a
bes [32]. I should be no ed ha when using ossil calib a ion poin s
like in his s udy, he di e gences owa ds he ips o he ee may be
o e es ima ed [33]. Howe e , he subs i u ion a e es ima es o he
ip b anches leading o he indi idual subspecies a e e y simila o
he o e all es ima e o he b anch om he MRCA o all oden s o
he ips o he bea e b anches, sugges ing ha o his e olu iona y
lineage, ime-dependen a ia ion in subs i u ion a es is negligible.
The e o e, he di e si ica ion o he ex an Eu asian bea e lineages
s a ed mos likely subs an ially ea lie han p e iously es ima ed.
Also, geog aphically dis inc lineages o o he Eu asian, po en ially
o es dependan species showed simila di e gence es ima es, such
as ca e bea s (173,000–414,000 ya [34]) and b own bea s (174,000–
314,000 ya [35]). The gene ic di e si y o hese popula ions was
p obably shaped by clima ic luc ua ions du ing he glacial cycles
since a ound 0.9 mya [36].
The analysis o subs i u ion a es also shows ha bea e s display
a lowe subs i u ion a e han mos o he oden s, a ea u e clea ly
isible in he b anch leng h o he phylogene ic ee (Fig. 4). This
di e ence was highly signi ican when bea e s we e es ed agains
o he membe s o he mouse- ela ed clade. The a e di e ence was
s ill signi ican when Cas o was compa ed wi h all o he oden s in
he da ase , including he squi el- ela ed clade which also showed
lowe subs i u ion a es. I has been a gued be o e ha ce ain
mo phological, physiological, and li e his o y ai s in luence DNA
subs i u ion a es. Fo example, Welch e al. [21] showed ha li e
his o y ai s like body mass, li espan and age a sexual ma u i y
a e nega i ely co ela ed wi h subs i u ion a es o e a wide ange
o mammals. Bea e s a e a ypical among oden s wi h espec o
se e al li e his o y ai s as hey ha e a much la ge body mass,
longe li espan and do no each sexual ma u i y un il 1.5–3 yea s
o age (Table S7, [1,14]). Thus, a lowe subs i u ion a e can be
expec ed o bea e s compa ed o o he oden s. In ou analysis o
mi ochond ial genomes, body mass and age a sexual ma u i y did
no co ela e signi ican ly wi h mi ochond ial subs i u ion a es,
al hough body mass did so ea lie [21]. Howe e , we ound a
signi ican nega i e co ela ion be ween he subs i u ion a e and
maximum li espan as has been p e iously epo ed o mi ochon-
d ial synonymous si es ac oss mammals [21]. Despi e he smalle
sample size in ou analysis, a la ge ac ion o he a ia ion in
subs i u ion a e was explained by maximum li espan, e en hough
we included non-synonymous si es, which did no show a
signi ican co ela ion ea lie [21]. Co ela es o subs i u ion a es
wi h li espan a e no ully explained o da e [21], bu na u al
selec ion could ha e ac ed o educe he mu a ion a e in
mi ochond ia o long li ed axa such as he bea e [37,38].
Howe e , in a genome scan ac oss 25 species, genes in ol ed
in DNA eplica ion, epai o an ioxida ion did no show
signa u es o selec ion in long-li ed axa. Ins ead, he selec ed
ea u es we e connec ed o cellula memb ane and ex acellula
collagen composi ion and hei unc ional ele ance emains
puzzling [39].
In summa y, ou s udy showed ha o e he wide axonomic
ange o gli es, da ase s comp ising whole mi ochond ial genome
sequences acili a e he in e ence o subs i u ion a es, phylogene ic
analyses and di e gence es ima es.
Ma e ials and Me hods
E hics s a emen
The issue sample o Cas o canadensis was aken om a dead
bea e , which was sho du ing he open hun ing season in Finland.
The sample o Cas o ibe ssp. albicus came om a oad kill,
dissec ed wi h he app o al om he en i onmen al agency o
Ge many, adminis a i e dis ic o Leipzig. Samples Cas o ibe
ssp. belo ussicus/o ien oeu opaeus,bi ulai, u inicus and pohlei consis ed
o bea e ail skin o hai ha was ob ained om li e animals
cap u ed wi h ne s, li e aps, and a nigh wi h a sea ch ligh and
ne ing om a boa . Animals we e eleased a e wa d. Sampling
p ocedu es we e consis en wi h guidelines o he Ame ican
Socie y o Mammalogis s o he cap u e and handling o
mammals [40]. All ca ches we e pe o med in coope a ion wi h
and unde app o al by, he Na u e Rese e ‘Malaya Sos a’ in
Russia, he Zhi ko Russian Resea ch Ins i u e o Game
Managemen and Fu Fa ming in Ki o , Russia, as well as he
Na ional Uni e si y o Mongolia in Ulaan-Baa a , Mongolia.
Samples, DNA ex ac ion and long ange PCR
DNA was ex ac ed om i e issue samples o C. ibe and om
one sample o C. canadensis (Table S1) using he DNeasy blood &
issue ki (Quiagen). Fo well p ese ed DNA, he mi ochond ial
genome was ampli ied in wo o e lapping pieces a ound 11 and
6 kb in leng h, by long ange PCR using he expand dNTPack
(Roche) acco ding o he manu ac u e ’s ins uc ions. P ime
sequences a e lis ed in Table S8. Long ange PCR p oduc s we e
shea ed using a Bio up o UCD-200 (Diagenode). Ba coding
adap o s wi h sample speci ic sequences and 454 sequencing
adap o s we e liga ed o he sonica ed p oduc s as desc ibed
p e iously [41].
DNA hyb idiza ion cap u e and sequencing
Fo less well p ese ed samples, o which long ange PCR did
no wo k, DNA hyb idiza ion cap u e was used o en ich o
mi ochond ial genomes (Table S1). Fo his p ocess, bio inyla ed
bai molecules a e hyb idized wi h a genomic lib a y and la e
selec i ely cap u ed on s ep a idin beads [42]. Ba coded genomic
lib a ies we e p epa ed om Cas o DNA as desc ibed p e iously
[41,43], allowing he simul aneous sequencing o di e en samples
on he same 454 lane. To p oduce bai molecules, long ange
amplicons o C. ibe ssp. albicus we e used. The PCR p oduc s we e
shea ed un il hey had a leng h o a ound 300 bp, and double
s anded, bio inyla ed adap e s we e liga ed on o he ends. The
hyb idiza ion mix u e was se up as ollows: 1 mg o genomic
lib a y, 100 ng o bai and ou blocking oligos (each 2 mM) in 1x
blocking Agen (Agilen ) and 1x hyb idiza ion bu e (Agilen ).
Agilen eagen s we e om he aCGH Ki #5188-5220.
Sequences o he blocking oligos a e lis ed in Table S8. A e
dena u a ion o he mix u e a 95uC o 5 min, he hyb idiza ion
was ca ied ou in 200 ml ubes (Eppendo ), o a ing a 65uC o
48 hou s in a con en ional hyb idiza ion o en (SciGene).
A e hyb idiza ion, bio inyla ed bai molecules we e cap u ed
by incuba ion wi h 5 ml o magne ic s ep a idin co e ed beads
(Dynalbeads M270, In i ogen) o 20 min a oom empe a u e.
The mix u e was hen placed in o a magne ic ack (Beckman
Coul e #A32782) allowing he sepa a ion o magne ic beads om
he supe na an . The supe na an , con aining non- a ge mole-
cules was disca ded and he emaining beads we e washed i e
imes using 1xBWT bu e (1 M NaCl, 10 mM T is-Cl, 1 mM
Cas o Mi ochond ial Genomes
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EDTA, 0.05% Tween-20 (Sigma), pH 8.0) and once in p e-
wa med HW bu e (200 ml 10x AmpliTaq Gold bu e , 200 ml
MgCl
2
, 1.6 ml H
2
O) a 50uC o 2 min. A e one mo e wash wi h
1xBWT, he beads we e ans e ed in o a new ube wi h 100 mlo
TE-bu e (con aining 0.05% Tween-20). Finally, hyb idized
a ge molecules we e sepa a ed om he bai molecules in 30 ul
1xTE by 5 min incuba ion a 95uC in a he mocycle . The elua e
con aining he sequencing lib a y en iched o mi ochond ial
DNA, was di ec ly used o quan i ica ion and sequencing.
Sequence analyses and assembly
A e 454 lib a y p epa a ion and quan i ica ion o he lib a ies
by qPCR using emPCR p iming si es [44], sequencing was ca ied
ou on he 454 FLX pla o m. De no o assembly o 454 eads o he
mi ochond ial genomes o C. ibe ssp. albicus and C. canadensis was
done wi h unAssembly (454 Roche), sepa a ely o each o he wo
o e lapping long ange amplicons, and he o e laps joined by
hand in BioEdi [45]. Fo he emaining C. ibe specimens,
sequencing eads we e mapped on o he assembled C. ibe
mi ochond ial genome using unMapping (454 so wa e) and he
i e a i e mapping ool IMA [46]. The ou pu o he mappings was
iewed wi h cl iew (so wa e a ailable a h p://compbio.d ci.
ha a d.edu/ gi/so wa e/) and he map aligne [46]. Mapping o
he sequencing eads om one PCR de i ed sequence showed a
somewha une en co e age and peaks o ead coun s in he
p oximi y o p iming si es (Fig. 2), which could esul om
amplicons ha we e abo ed du ing he PCR sho ly a e
p iming. The gene ally obse ed di e ences in co e age be ween
bo h long ange PCR p oduc s migh e lec a ia ion in
quan i ica ion o pooling o hese p io o sequencing. Co e age
plo s om hyb idiza ion cap u e we e smoo he han hose de i ed
om long ange PCR (Fig. 2). The ob ained mappings co e ed he
comple e mi ochond ial genomes o all samples. One si e each in
bea e samples C. canadensis and C. ibe albicus had less han 3x
co e age, o which he sequence was con i med by PCR and
Sange sequencing (sample speci ic p ime s lis ed in Table S8).
Nuclea mi ochond ial inse ions (num s) could be uled ou o
long ange PCR by joining he o e laps c ea ing a ci cula
sequence, and a e no expec ed o be a p oblem o hyb idiza ion
cap u e since nuclea DNA has a much lowe copy numbe han
mi ochond ial DNA. The leng h o he epea egion be ween he
con ol egion and RNA-Phe is a minimum es ima e, since he
454 sequencing eads did no span he egion comple ely. Fo
be ween wo and 21 posi ions pe mi ochond ial genome,
co ec ions o he assembled DNA sequence we e made by hand
in BioEdi . These a ec ed mainly p o ein coding egions, whe e
he leng h o homopolyme s was co ec ed so ha he eading
ame o amino acid sequences was e ained. The ob ained
sequences we e deposi ed in GenBank wi h accession numbe s
FR691684-FR691689.
Phylogene ic analyses and di e gence es ima es
Alignmen s o DNA sequences we e done using ma 6.708b
[47] o he axa lis ed in Table S2. The comple e alignmen
including gaps had 19,419 bp in leng h. 3067 bp o he alignmen
con aining he con ol egion be ween RNA-P o and RNA-Phe
we e no well aligned due o high sequence di e gence and we e
he e o e emo ed, esul ing in an alignmen o 16,352 bp o
phylogene ic analyses.
Model es was used o de e mine he op imal e olu iona y
model o he da ase [48]. The gene al ime e e sible (GTR)
model wi h a p opo ion o in a ian si es o 0.2559 and a gamma
shape pa ame e o 0.4721 was de e mined o be mos
app op ia e. Using his subs i u ion model, phylogene ic ees
we e calcula ed wi h maximum likelihood (ML). One hund ed
boo s ap eplica es we e done in Paup [49] e sion 4.0d105. The
heu is ic sea ch used ee bisec ion econnec ion (TBR) limi ed o
1000 ea angemen s pe boo s ap eplica e due o compu a ional
cons ain s.
In o de o es i he emo al o as e ol ing si es om he
sequences could imp o e phylogene ic in e ence [6], wo sho e
e sions o he o iginal alignmen we e c ea ed and also subjec ed
o ML in e ence (Table S3). One alignmen con ained all
anno a ed loci wi h 15,865 bp (coding genes, l- RNA, s- RNA,
eplica ion o igin, RNAs) and hus excluded only 487 bp o non-
anno a ed loci. The o he alignmen (con aining coding genes, l-
RNA and s- RNA) had 14,270 bp, excluding all RNAs and he
eplica ion o igin. Since hese sho ened alignmen s did no
imp o e phylogene ic in e ence wi h ML (Table S3) and he
longe alignmen wi h 16,352 bp allowed he ecogni ion o all
majo oden clades, he la e was used o he ollowing
phylogene ic in e ences and molecula da ing wi h BEAST. The
ini ial ML analyses on h ee alignmen s o di e en leng h we e
pe o med also on a la ge axon se in o de o e alua e he
opology o ou phylogene ic ee, especially he monophyly o
oden s. This la ge axon se comp ised all 39 axa lis ed in Table
S2. Due o compu a ional cons ain s, he axon se was hen
educed o 24 axa, excluding some o he ou g oup sequences o
he ollowing phylogene ic in e ences and molecula da ing wi h
BEAST.
Fo neighbo joining (NJ) he T3P model was used in Mega4
[50]. 1,000 Boo s ap eplica es we e calcula ed wi h pai wise
dele ion and gamma dis ibu ed a es among si es (gamma shape
pa ame e was 0.4729). Fo maximum pa simony (MP) 1,000
boo s ap eplica es we e done wi h close neighbo in e change
(CNI, le el 1) and ini ial ees o he CNI sea ch by andom
addi ion ees (10 eplica ions) in Mega4. T ees we e sea ched in
M Bayes .3.1.2 wi h ou pa allel sea ch chains o 10 million
gene a ions in iplica es. The sea ch chains eached simila
pos e io p obabili y le els in he iplica es as iewed in T ace
1.4.1, inal ESS we e 339. The ee iles we e combined in
logcombine and maximum clade c edibili y ees we e c ea ed
wi h T eeAnno a o implemen ed in BEAST .1.5.3 and iewed
in FigT ee .1.3.1 [51].
Molecula da ing was ca ied ou wi h BEAST .1.5.3, a
coalescence based me hod o pa ame e es ima ion [52]. Six ossil
calib a ions we e aken in o accoun as p io assump ions on
di e gence imes (diamonds in Fig. 3, Table S4) and simul a-
neously op imized wi h o he pa ame e s desc ibing he phylog-
eny. The elaxed molecula clock models implemen ed in BEAST
assume independen a es on di e en b anches o a phylogeny,
hus allowing o calib a ions in a he dis an ly ela ed axonomic
g oups. Thus, BEAST is speci ically sui ed o accommoda e he
a e a ia ion be ween lagomo phs, oden s and p ima es as well
as ha be ween oden s wi h slowe and as e a es. The sampling
p io s o ossil calib a ions we e se o be no mally dis ibu ed,
inco po a ing knowledge on di e gence es ima es, such as he
ea lies appea ance o membe s o a clade in he ossil eco d as
well as he age o an assumed monophyle ic o igin and ea ly
ela i es o he clade. Since he e a e unce ain ies o de e mining
he age o ossils and imes ha lineages ac ually di e ged, we used
a he wide anges a ound hese e en s o calib a ion (Table S4).
Fu he , we used es ima ed base equencies, an unco ela ed
logno mal elaxed molecula clock, he Yule p io o specia ion
scena ios and UPGMA s a ing ees. BEAST xml- iles we e
c ea ed in beau y (implemen ed in BEAST). Sea ch chains we e
un in BEAST o 50 million gene a ions and log iles we e w i en
e e y 5,000 gene a ions. The GTR subs i u ion model was used
Cas o Mi ochond ial Genomes
PLoS ONE | www.plosone.o g 7 Janua y 2011 | Volume 6 | Issue 1 | e14622
and he i s 10 pe cen o uns we e disca ded as bu nin.
Logcombine om he BEAST package did no emo e he
bu nin co ec ly, when combining log iles. The e o e, log iles o
iplica es o he 50 million gene a ion uns we e combined in o a
single log ile by hand and he esul s we e iewed in T ace
e sion 1.4.1 [53]. The pos e io dis ibu ions o he ossil
calib a ions we e isually inspec ed in T ace 1.4.1 and e i ied
o be symme ically bell shaped, indica ing a p ope sampling om
he p io dis ibu ion. A maximum clade c edibili y ee was
c ea ed wi h eeanno a o and iewed in FigT ee.
Compa ison o e olu iona y a es
Subs i u ion a es o he b anches leading o di e en oden
axa we e de e mined in he BEAST analyses (combined om
h ee 50 million gene a ion uns) and shown as b anch labels in
Fig ee. We es ed i bea e s di e in subs i u ion a es om o he
oden s using a Mann-Whi ney U- es , implemen ed in R 2.9.2 as
Wilcoxon ank sum es wi h con inui y co ec ion. Since his es
examines i wo dis ibu ions di e signi ican ly om each o he ,
all bea e samples we e included in he es .
We showed ha mi ochond ial subs i u ion a es o Cas o di e ed
signi ican ly om hose o he o he oden s in he da ase . An
inc ease o he obse ed a es owa ds he ips in a phylogeny (Fig. 4)
is assumed o be a esul o ansien polymo phisms p esen in he
mo e ecen imescales o a phylogeny [33]. Al hough his hypo hesis
is con o e sial [54], in ou BEAST phylogeny he es ima ed
subs i u ion a es also inc ease owa ds he ips. In o de o explo e
his possible bias due o sampling on di e en axonomic le els, we
a e aged he subs i u ion a es along all b anches leading o he
common ances o o all es ed axa (Table S5) and epea ed he
Mann-Whi ney U- es s wi h hose a e age a es (Table 1). In o de o
a e age he a es, o each o he h ee es s (Cas o s. mouse- ela ed
clade, Cas o s. es o oden s, Cas o s. es o gli es, as indica ed in
Table 1) he a i hme ic mean was calcula ed om he a es along he
b anches leading o he ances al node connec ing he es ed axa.
The esul ing p- alues indica ed an e en mo e p onounced, highly
signi ican di e ence be ween he lowe a es o bea e s and he
highe a es o he o he oden s in he da ase (Table 1). Fu he , we
co ela ed he subs i u ion a es o oden s wi h li e his o y ai s, such
as body mass, li espan and age a sexual ma u i y [21] using linea
eg essions in R. In o de o a oid unbalanced da a, we collapsed all
sampled bea e s in o one da a poin o each eg ession. Measu e-
men s o subs i u ion a es and li e his o y ai s o bea e s we e
he e o e a e aged using he a i hme ic mean. Re e ences o
maximum li espan, body mass and age a sexual ma u i y a e gi en
in Table S7 and Suppo ing Re e ences S1.
Suppo ing In o ma ion
Table S1 Samples and o e iew o p ocessing o mi ochond ial
genome sequencing. Samples we e ob ained om di e en sou ce
popula ions o bea e s [S1], en iched o mi ochond ial DNA and
ba coded be o e sequencing. LR PCR: long ange PCR. Hyb:
hyb idiza ion cap u e.
Found a : doi:10.1371/jou nal.pone.0014622.s001 (0.04 MB
DOC)
Table S2 Taxa and accession numbe s o he mi ochond ial
genome sequences used. Sequences ma ked by * a e addi ional
ou g oup sequences o oden s and we e used o e alua e he
opology o ou phylogene ic ee in he ini ial ML analysis bu
we e excluded in he la e ML, NJ, MP, and Bayesian analyses due
o compu a ional cons ain s.
Found a : doi:10.1371/jou nal.pone.0014622.s002 (0.06 MB
DOC)
Table S3 Boo s ap suppo o monophyle ic clades in maxi-
mum likelihood analyses o oden mi ochond ial genome
alignmen s o di e en leng h. Suppo alues o clades eco e ed
in p e ious s udies decline when using smalle ac ions o he
sequence da a. Suppo alues a e gi en o alignmen s including
24 axa and 39 axa sepa a ed by a slash. No e ha all alignmen s
excluded a less well aligned egion con aining he con ol egion.
Found a : doi:10.1371/jou nal.pone.0014622.s003 (0.04 MB
DOC)
Table S4 Fossil calib a ions and age es ima es. P io and
pos e io alues o he ime o he mos ecen common ances o
( m ca) o monophyle ic clades de e mined by BEAST analyses a e
gi en in million yea s ago (mya). L.: la e. M.: middle. HPD:
highes pos e io densi y. S de : S anda d e o .
Found a : doi:10.1371/jou nal.pone.0014622.s004 (0.05 MB
DOC)
Table S5 Mi ochond ial subs i u ion a es o gli es. Tip a es
es ima ed by BEAST and a e aged a es gi en in subs i u ions pe
posi ion pe million yea s.
Found a : doi:10.1371/jou nal.pone.0014622.s005 (0.06 MB
DOC)
Table S6 The ela ionships o body mass, li e his o y ai s and
subs i u ion a es we e explo ed in linea eg essions. A linea mo-
del co ela ed he es ima ed a es wi h li espan and eached a
signi icance le el below 0.05 o ip as well as a e aged a es. In
con as , linea models co ela ing a es wi h body mass and age a
sexual ma u i y did no each his signi icane le el. Da a on he
body mass o he sequenced Anomalu us sp. indi idual we e
missing. Thus independen eg essions we e made o da ase s
con aining Anomalu us wi h a body mass o 700 g and 2000 g. *:
signi ican , ,0.05.
Found a : doi:10.1371/jou nal.pone.0014622.s006 (0.04 MB
DOC)
Table S7 Li e his o y ai s o oden s and e e ences. Since no
da a we e a ailable o M. kikuchii, da a o M. oeconomus was
used ins ead. Bo h axa a e closely ela ed [S19]. No e ha high
alues o li espan could esul om animals held in cap i i y.
Found a : doi:10.1371/jou nal.pone.0014622.s007 (0.07 MB
DOC)
Table S8 Oligo sequences. Long ange PCR p ime and
blocking oligos.
Found a : doi:10.1371/jou nal.pone.0014622.s008 (0.05 MB
DOC)
Suppo ing Re e ences S1 Suppo ing Re e ences
Found a : doi:10.1371/jou nal.pone.0014622.s009 (0.03 MB
DOC)
Acknowledgmen s
We hank Alexande P. Sa elje o sample collec ion, Die ich Heidecke
and Na alia Rybczynski o ui ul discussions and Ka l And eas Ni sche
o he d awing o a bea e in Fig. 1. We hank Ma hias Meye o help
wi h agging and sequencing p o ocols as well as Tomisla Ma icic o help
wi h he hyb idiza ion cap u e and wo anonymous e iewe s o help ul
commen s.
Au ho Con ibu ions
Concei ed and designed he expe imen s: SH MH. Pe o med he
expe imen s: SH. Analyzed he da a: SH. Con ibu ed eagen s/ma e i-
als/analysis ools: WD RW AE AS MS MH. W o e he pape : SH WD
MH.
Cas o Mi ochond ial Genomes
PLoS ONE | www.plosone.o g 8 Janua y 2011 | Volume 6 | Issue 1 | e14622
Re e ences
1. Bake BW, Hill EP (2003) Bea e (Cas o canadensis). In: Feldhame GA,
Thompson BC, Chapman JA, eds. Wild Mammals o No h Ame ica: biology,
managemen , and conse a ion Bal imo e, MD The Johns Hopkins Uni e si y
P ess.
2. Djoshkin WW, Sa onow WG (1972) Die Bibe de al en und neuen Wel .
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PLoS ONE | www.plosone.o g 9 Janua y 2011 | Volume 6 | Issue 1 | e14622