Mitochondrial genomes reveal slow rates of molecular evolution and the timing of speciation in beavers (Castor), one of the largest rodent species

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 PLoS ONE | www.plosone.o g 2 Janua y 2011 | Volume 6 | Issue 1 | e14622 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 PLoS ONE | www.plosone.o g 3 Janua y 2011 | Volume 6 | Issue 1 | e14622 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 PLoS ONE | www.plosone.o g 4 Janua y 2011 | Volume 6 | Issue 1 | e14622 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 PLoS ONE | www.plosone.o g 5 Janua y 2011 | Volume 6 | Issue 1 | e14622 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 PLoS ONE | www.plosone.o g 6 Janua y 2011 | Volume 6 | Issue 1 | e14622 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. 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Halley DJ, Rosell F (2003) Popula ion and dis ibu ion o Eu opean bea e s (Cas o ibe ). Lu a 46: 91–101. Cas o Mi ochond ial Genomes PLoS ONE | www.plosone.o g 9 Janua y 2011 | Volume 6 | Issue 1 | e14622