Introgression of mitochondrial DNA among Myodes voles: consequences for energetics?

This is an elec onic ep in o he o iginal a icle. This ep in may di e om he o iginal in pagina ion and ypog aphic de ail. Au ho (s): Ti le: Yea : Ve sion: Please ci e he o iginal e sion: All ma e ial supplied ia JYX is p o ec ed by copy igh and o he in ellec ual p ope y igh s, and duplica ion o sale o all o pa o any o he eposi o y collec ions is no pe mi ed, excep ha ma e ial may be duplica ed by you o you esea ch use o educa ional pu poses in elec onic o p in o m. You mus ob ain pe mission o any o he use. Elec onic o p in copies may no be o e ed, whe he o sale o o he wise o anyone who is no an au ho ised use . In og ession o mi ochond ial DNA among Myodes oles: consequences o ene ge ics? Bo a yński, Zbyszek; Al es, Paulo C; Be o, S e ano; Koskela, Esa; Mappes, Tapio; Melo-Fe ei a, José Bo a ynski, Z., Al es, P., Be o, S., Koskela, E., Mappes, T., & Melo-Fe ei a, J. (2011). In og ession o mi ochond ial DNA among Myodes oles: consequences o ene ge ics?. BMC E olu iona y Biology, 11:355. Re ie ed om h p://www.biomedcen al.com/1471-2148/11/355 2011 In og ession o mi ochond ial DNA among Myodes oles: consequences o ene ge ics? Bo a yński e al. Bo a yński e al.BMC E olu iona y Biology 2011, 11:355 h p://www.biomedcen al.com/1471-2148/11/355 (9 Decembe 2011) RESEARCH ARTICLE Open Access In og ession o mi ochond ial DNA among Myodes oles: consequences o ene ge ics? Zbyszek Bo a yński 1* , Paulo Célio Al es 2,3,4 , S e ano Be o 1 , Esa Koskela 5 , Tapio Mappes 1 and José Melo-Fe ei a 2 Abs ac Backg ound: In og ession o mi ochond ial DNA (m DNA) is among he mos equen ly desc ibed cases o e icula e e olu ion. The endency o m DNA o c oss in e speci ic ba ie s is somewha coun e -in ui i e conside ing he key unc ion o enzymes ha i encodes in he oxida i e-phospho yla ion p ocess, which could gi e ise o hyb id dys unc ion. How m DNA e icula ion a ec s he e olu ion o me abolic unc ions is, howe e , unce ain. He e we in es iga ed how mo pho-physiological ai s a y in na u al popula ions o a common oden ( he bank ole, Myodes gla eolus) and whe he his a ia ion could be associa ed wi h m DNA in og ession. Fi s , we con i med ha M. gla eolus ha bou m DNA in og essed om M. u ilus by analyzing m DNA (cy och ome b, 954 bp) and nuclea DNA ( ou ma ke s; 2333 bp in o al) sequence a ia ion and econs uc ing loci phylogenies among six na u al popula ions in Finland. We hen s udied geog aphic a ia ion in body size and basal me abolic a e (BMR) among he popula ions o M. gla eolus and es ed i s ela ionship wi h m DNA ype. Resul s: Myodes gla eolus and i s a c ic neighbou , M. u ilus, a e ecip ocally monophyle ic a he analyzed nuclea DNA loci. In con as , he wo no he nmos popula ions o M. gla eolus ha e a ixed mi o ype ha is sha ed wi h M. u ilus, likely due o in og essi e hyb idiza ion. The analyses o pheno ypic ai s e ealed ha he body mass and whole-body, bu no mass co ec ed, BMR a e signi ican ly educed in M. gla eolus emales om no he n Finland ha also ha e he in og essed mi o ype. Res ic ing he analysis o he single popula ion whe e he mi o ypes coexis , he associa ion o m DNA ype wi h whole-body BMR emained bu hose wi h mass co ec ed BMR and body mass did no . Mi ochond ial sequence a ia ion in he in og essed haplo ypes is compa ible wi h demog aphic g ow h o he popula ions, bu may also be a esul o posi i e selec ion. Conclusion: Ou esul s show ha he pheno ypic ai s a y ma kedly along he no h-sou h axis o popula ions o M. gla eolus. This a ia ion may be ela ed o adap a ion o local en i onmen s and coincides wi h he g adien o genome e icula ion be ween M. gla eolus and M. u ilus, which was assessed by m DNA in og ession. In og ession o m DNA may ha e a ec ed mo pho-physiological ai s bu do no show s ong e ec s on ei he body mass o basal me abolic a e alone. We discuss he causes and biological meaning o ou esul s and he means o cla i y hese ques ions in u u e esea ch. Backg ound Widely dis ibu ed species ace di e en selec ion p es- su es along clima ic and ecological g adien s. In his espec , basal me abolic a e (BMR) is pa icula ly p one o e ol e adap i ely, since i has been shown o be he i- able and co ela es wi h i ness componen s in endo he ms [1-8]. Se e al compa a i e s udies ha e p o- posed ha pa e ns o a ia ion in me abolic a e ma ch ce ain clima ic a iables in widely dis ibu ed g oups o species [9,10], sugges ing ha he le el o BMR esponds o di e en selec ion egimes gene a ed by local condi- ions. BMR is gene a ed mainly in physiologically impo - an in e nal o gans including he kidneys, b ain and li e [11,12] wi h mos o he physiological ac i i y loca- lized in ce ain compa men s o cells in hese o gans. Mi ochond ia a e he main cellula ene gy “ ac o ies”, supplying o ganisms wi h ene gy s o ed in ATP mole- cules. The a e o ene gy p oduc ion by mi ochond ia is c ucial o an indi idual’s i ness and is hough o be de e mined by na u al selec ion [13-15]. Mi ochond ia * Co espondence: [email p o ec ed] 1 Cen e o Excellence in E olu iona y Resea ch, Depa men o Biological and En i onmen al Science, Uni e si y o Jy äskylä, P.O. Box 35 YAC, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle Bo a yński e al.BMC E olu iona y Biology 2011, 11:355 h p://www.biomedcen al.com/1471-2148/11/355 © 2011 Bo a yńński e al; licensee BioMed Cen al L d. 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 (h p://c ea i ecommons.o g/licenses/by/2.0), 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 wo k is p ope ly ci ed. a e cha ac e ized by hei own genome and ha bou enzyma ic elemen s o ae obic me abolic pa hways. Howe e , oxida i e phospho yla ion (OXPHOS), he p ocess esponsible o ene gy p oduc ion, depends no only on he enzymes encoded in he in e nal o ganelle genome (mi ochond ial DNA [m DNA]), bu also on nuclea -encoded polypep ides which in e ac o enable he enzyma ic eac ions. The e o e, selec ion may ac on and cons ain he co-e olu ion o mi ochond ial and nuclea genes [16]. In e es ingly, be ween-species ans- e o m DNA has been equen ly obse ed among many g oup o animals and plan s [17-22]. Such ans e has been adi ionally conside ed neu al, howe e , i may be maladap i e, i in og ession b eaks he coe- ol ed mi o-nuclea complexes [16], o adap i e, as m DNA unc ion may ep esen impo an selec i e alue [23-26]. In e speci ic m DNA ans e obse ed among he b oadly dis ibu ed oden genus Myodes [27-30] o e s an ideal si ua ion o es 1) i e icula e e olu ion has an impo an and ecognizable pheno ypic e ec and 2) whe he na u al selec ion may in luence m DNA in o- g ession. The bank ole, Myodes gla eolus,isdispe sed ac oss di e en clima ic and geog aphic zones, om he Medi e anean o beyond he A c ic Ci cle [29,31] (Fig- u e 1). The dis ibu ion o i s close ela i e, he no he n ed-backed ole (o ed ole), M. u ilus, is es ic ed o no he n pa s o he ci cum-bo eal zone, ex ending o he A c ic zone [29,32]. The ed ole occu s om no h- e n Fennoscandia and no he n Eu asia, whe e i is pa ly sympa ic wi h he bank ole, o he no he n la i- udes in No h Ame ica. In og essi e hyb idiza ion be ween he bank ole and he ed ole has been docu- men ed ac oss a as geog aphic a ea, om Sweden o cen al and sou he n Russia [33,34] including no he n Finland [27]. All hese cases showed in og ession o m DNA alone and no in og ession o he nuclea gen- ome has been epo ed [27,28]. In his wo k we e isi ed a geog aphic egion whe e m DNA in og ession be ween M. gla eolus and M. u i- lus has been p e iously desc ibed [27]. Fi s , using sequence da a bo h om m DNA and nuclea DNA, we con i m ha m DNA in og ession om M. u ilus in o M. gla eolus occu s na u ally in Finland, wi h he in o- g essed haplo ype being ixed in he no he nmos popula ions. Since he mi ochond ia house he ene gy machine y o he cells, we analyzed a ia ion in wo mo pho-physiological ai s (body size and basal me a- bolic a e) among M. gla eolus popula ions, and ound di e ences be ween emales om popula ions wi h di - e en m DNA ypes, al hough he e ec o geog aphy could no be excluded. The unde pinnings and conse- quences o his a ia ion a e discussed. Resul s Gene ic a ia ion In o al, 201 oles apped du ing summe 2008 in 6 locali ies in Finland (Figu e 1) we e analyzed o a ia- ion in mi ochond ial and nuclea ma ke s (Addi ional ile 1: Table S1, Addi ional ile 2: Table S2, Addi ional ile 3: Table S3, Addi ional ile 4: Table S4 and Addi- ional File 5; Accession numbe s: [GenBank: JF929975- JF930131]). A e allelic phase de e mina ion (numbe o emo ed haplo ypes pe gene, wi h a lowe p obabili y o 0.95, o LCAT,G6pd,BRCA1 and GHR we e 1, 0, 0 and 5, espec i ely) he numbe o alleles p esen in M. gla eolus nuclea genes a ied be ween 7 and 10 (Table 1). The phylogenies econs uc ed o each o he nuclea genes we e consis en ac oss me hods and coin- cided wi h he expec ed assignmen o indi iduals o he species (Figu e 2), since bo h M. gla eolus and M. u i- lus (see geog aphic dis ibu ion in Figu e 1) we e eco - e ed as monophyle ic wi h high s a is ical suppo (Figu e 2). The cy b sequences ob ained we e mos likely o mi ochond ial o igin and no o nuclea in e- g a ed copies, as he eading ame was unb oken (no s op codons we e ound) and hecomposi iono he hi d codon posi ion was ypical (A 40.5%, C 39.4%, G 2.2% and T 17.8%) compa ed o he a e age in mam- mals (A 39%, C 36%, G 3% and T 21%; [35]). 5 12 3 56 4 Myodes u ilus M. gla eolus T apped popula ions In og essed, no -in . and mixed pop. Myodes u ilus M. gla eolus T apped popula ions In og essed, no -in . and mixed pop. Figu e 1 Dis ibu ion o bank ole Myodes gla eolus (g ay) and ed ole M. u ilus (s iped a ea). Numbe s in whi e ci cles on he enla ged map o Finland e e o he localiza ions o apped popula ions: 1 - Tammela (SW), 2 - Vi olah i (SE), 3 - Kannus (CW), 4 - So kamo (CE), 5 - Kola i (NW) and 6 - Sa ukoski (NE). Species dis ibu ions a e : Amo i e al. 2009 and Linzey e al. 2009. Bo a yński e al.BMC E olu iona y Biology 2011, 11:355 h p://www.biomedcen al.com/1471-2148/11/355 Page 2 o 12 Polymo phism was ound in 124 si es (137 when includ- ing Swedish popula ion and indi iduals apped in 2009 in Cen al Eas Finland) which de ined 81 haplo ypes (99 when including Swedish and 2009 indi iduals) wi hin M. gla eolus (Table 2). Con a y o in e ences based on he sequences o he ou nuclea genes, he cy b haplo ypes sampled in M. gla eolus did no o m a monophyle ic clade, and was ins ead spli in o wo clades, only pa ially o e lapping wi h species assign- men . One clade, he M. u ilus- ype m DNA (RUT), g ouped 57 (65 when including Swedish and specimens sampled 2009) indi iduals o M. gla eolus oge he wi h M. u ilus specimens, sugges ing m DNA in og ession. The second clade, M. gla eolus- ype m DNA (GLA), only g ouped haplo ypes speci ic o M. gla eolus (Figu e 3). The geog aphic dis ibu ion o each o hese hap- log oups was pa ly disjoin , wi h Sou he n and Cen al Finland (1-4 on Figu e 1) being occupied by he GLA ype m DNA, whe eas he No h and Cen al-Eas (4-6) popula ions had he RUT ype. One popula ion loca ed in Cen al-Eas e n Finland exhibi ed bo h mi o ypes (popula ion 4: Figu e 1). Nuclea DNA sequence a ia ion wi hin M. gla eolus was also de ec ed (Table 1). The equency spec um o mu a ions did no signi ican ly di e om he neu al mu a ion-d i expec a ions o mos genes, as de e - mined by Tajima’sDandFu’s Fs (Table 1). Fo cy b, sequence di e si y o indi iduals wi h he GLA and RUT mi o ype was compa able and high (Table 2). GLA and RUT ype cy b sequences di e ed by 0.098 (0.014) and 0.102 (0.014) subs i u ions pe si e, espec i ely, om M. u ocanus sequences, while he di e gence be ween GLA and RUT mi o ypes was 0.070 (0.011) (ne be ween g oup a e age dis ances and i s s anda d e o s calcu- la ed by boo s apping 1000 imes). The misma ch ana- lysis o cy b sequences om M. gla eolus showed a bimodal dis ibu ion o he numbe o pai wise di e - ences (Figu e 4a), which illus a es he exis ence o wo di e gen lineages. Simila esul s we e ob ained o he popula ion om Eas -Cen al Finland (popula ion 4, So kamo; Addi ional ile 6: Figu e S1d), whe e he m DNA lineages coexis . The misma ch dis ibu ions analyzed sepa a ely o each o he m DNA ype (Figu es 4b and 4c) and popula ion we e unimodal (Addi ional ile 6: Figu e S1). The goodness o i es o de ia ion om he expec a ion unde he Sudden Expansion Model ejec ed he model o he da a se including all bank oles wi h hei own m DNA (Figu e 3b; p = 0.019). All signi ican Tajima’sDand Fu’sF s alues we e nega i e (Table 2). Bo h he unimodali y o he cy b misma ch dis ibu ion wi hin clades/popula ions and nega i e and signi ican alues o es s o selec i e neu- ali y a e expec ed o popula ions unde going ecen demog aphic g ow h o /and unde posi i e selec ion. Table 1 Sequence di e si y and neu ali y es s o nuclea ma ke s o bank oles. Gene n i n h n p hπ(%) Θ (S) pe si e (%) Tajima’sDFu’sF s LCAT 386 8 6 .51(.02) .15(.12) .16(.07) -.15 -1.43 G6pd 388 7 6 .08(.02) .01(.03) .16(.07) -1.72 & -11.66* BRCA1 388 8 7 .48(.02) .09(.08) .19(.08) -1.05 -3.62 GHR 376 10 11 .74(.02) .36(.22) .27(.10) .72 2.23 n i , numbe o sampled ch omosomes; n h , numbe o haplo ypes; n p , numbe o polymo phic si es; h, haplo ype di e si y; π, nucleo ide di e si y; Θ (S) , compu ed om he numbe o seg ega ing si es. S anda d de ia ions a e shown in b acke s. & and * indica e p< 0.05 and < 0.001, espec i ely. Myodes gla eolus M. u ilus M. u ocanus - gla - u - u GHR G6pd BRCA1 LCAT glaG3 glaG6 glaG4 glaG7 glaG2 glaG5 glaG1 u G1 u G1 u G2 u G3 99/100/1.0 93/95/0.6 99/100/- 0.005 Mic o us glaL1 glaL2 glaL4 glaL8 glaL5 glaL3 glaL6 glaL7 u L1 u L1 83/80/0.8 99/100/1.0 0.005 Mic o us glaGH8 glaGH10 glaGH2 glaGH1 glaGH7 glaGH4 glaGH5 glaGH6 glaGH3 glaGH9 u GH1 u GH2 u GH3 u 1 Mic o us 99/100/1.0 60/71/0.5 97/98/1.0 0.005 glaB5 glaB8 glaB2 glaB4 glaB1 glaB3 glaB6 glaB7 u B1 u B2 u B3 u B1 u B4 u B2 u B3 Mic o us 99/100/1.0 98/98/1.0 96/91/0.6 88/88/1.0 0.005 Figu e 2 Neighbou Joining (NJ) ees o ou nuclea ma ke s. Numbe s in he species nodes ep esen s pe cen age o boo s ap alues o 1000 pseudo eplica es o NJ and maximum likelihood analyses and Bayesian pos e io p obabili ies. B anch leng h is p opo ional o he numbe o subs i u ions pe si e. Myodes gla eolus (gla) and M. u ilus ( u ) haplo ype names a e unde lined wi h ho izon al ba s e e ing o he ype o he m DNA de ec ed in he pa icula haplo ype (whi e, black, and black and whi e: GLA, RUT and bo h m DNA ypes, espec i ely). T ees we e oo ed wi h sequences o Mic o us species [GenBank: GQ267517, AB086024, AY295009, AM910792]. “-” e e o un esol ed node by speci ic me hod. Bo a yński e al.BMC E olu iona y Biology 2011, 11:355 h p://www.biomedcen al.com/1471-2148/11/355 Page 3 o 12 Gi en he a e age a e o cy och ome b di e gence o oden s [36] o 0.176% pe si e pe My and he pa a- me e s de i ed om he misma ch dis ibu ion (τ=3.5; CI = 1.9-4.7) o he in og essed haplog oup, his ans- la es o an expansion ime o app oxima ely10 500 (5 500-14 000) yea s ago. The unimodal dis ibu ion and s a -like ne wo k may imply ha in og ession o m DNA happened only once in o he Finnish popula- ions o bank oles (Figu e 3). Despi e he excess o a e alleles ha we e gene ally de ec ed in M. gla eolus in he cy b and no in he nuclea genes, he mul ilocus HKA es [37]didno ejec henullneu almodelo e olu ion (p > 0.05). Pheno ypic a ia ion All ai s measu ed we e posi i ely co ela ed (N = 191, = 0.66, 0.57 and 0.43 o BMR-BM, HW-BM andBMR-HW;p<0.001)andall epea abili yes i- ma es (coe icien s o in aclass co ela ion) we e high and s a is ically signi ican (τ> 0.62, p < 0.0003, Table 3). As body mass and BMR we e signi ican ly in lu- enced by a la i ude-sex in e ac ion in he ini ial ana- lyses o a iance (logBM: F = 3.13, p = 0.046, logBMR: F=3.41,p=0.035,Addi ional ile7:Figu eS2), u he es s we e pe o med sepa a ely wi hin sexes. Whole body BMR was highe in he GLA han in he RUT mi o ype in M. gla eolus emales (p = 0.032; Table 4, Figu e 5a). Fo males, whole body and mass- co ec ed BMR dec eased owa d he No h (p = 0.002 and 0.015) bu did no di e be ween mi o ypes ( he e ec was gene a ed mainly by Sou he n popula ions, which had he highes alues o BMR, Addi ional ile 7: Figu e S2). Female M. gla eolus wi h he GLA mi o- ype we e signi ican ly hea ie han emales wi h he RUT mi o ype (p = 0.004, Figu e 5b). Longi ude, la i ude and hei in e ac ion did no a ec a ia ion in emales’body mass and we e sequen ially excluded om he model (p > 0.1). Wi hin he sympa ic popu- la ion o wo mi o ypes (Figu e 1; popula ion 4), 8 emales and 8 males o M. gla eolus we e de ec ed wi h M. u ilus m DNA (Table 4). The in og essed emales had signi ican ly lowe alues o whole-body BMR han sympa ic emales wi h na i e m DNA (p = 0.049, Figu e 5a). Mass co ec ed BMR showed he same end bu was no signi ican (p = 0.262). These e ec s we e also simila o males and in he same di ec ion, bu we e also insigni ican . No signi ican di e ences be ween sympa ic mi o ypes in body mass we e de ec ed in his popula ion (Table 4; Figu e 4b). Discussion Al hough mi ochond ia se e impo an physiological unc ions in o ganisms, i s e olu ion has adi ionally been conside ed neu al. Acco dingly, m DNA a ia ion should ha e li le pheno ypic consequences in na u al popula ions [26]. Howe e , du ing he las decade e i- dence is accumula ing ha he e olu ion o m DNA is o en subjec o na u al selec ion [38,39]. O e 20 yea s ago, m DNA low be ween wo species o Myodes oles, he bank ole, Myodes gla eolus,and he ed ole,M. u ilus, was i s desc ibed [27], p o iding a po en ially good model o es whe he na u ally occu ing m DNA in og ession may in luence pheno ypic ai s. He e, in addi ion o sampling some M. u ilus specimens, we sampled M. gla eolus in 6 Finnish popula ions, including he no he n ange o he species whe e i o e laps wi h M. u ilus, whe e m DNA in og ession migh occu . We assessed m DNA in og ession by compa ing he m DNA-based phylogeny wi h ha o 4 nuclea DNA ma ke s. As expec ed, we de ec ed cong uence be ween Table 2 Sequence di e si y and neu ali y es s o cy b o bank oles wi h di e en m DNA ypes (GLA, RUT) and om di e en popula ions (see map: Figu e 1). n i n h n p hπ(%) Θ (S) pe si e (%) Tajima’sDFu’sF s All da a 201 81 124 .97(.01) 3.28(1.59) 2.11(.51) 1.50 -6.13 GLA ype m DNA 144 57 65 .95(.01) .28(.17) 1.23(.31) -2.39* -26.64* RUT ype m DNA 57 24 29 .95(.01) .37(.21) .66(.21) -1.42 + -12.51* popula ion/m DNA ype Sweden/GLA 10 6 9 .89(.07) .31(.20) .33(.17) -.37 -.88 SW/GLA 34 14 20 .87(.04) .19(.13) .51(.19) -2.12* -8.34 & SE/GLA 35 8 9 .75(.06) .14(.10) .23(.10) -1.15 -2.42 CW/GLA 43 23 27 .96(.01) .31(.19) .65(.22) -1.76* -16.49 & CE/GLA, RUT 102 30 94 .94(.01) 2.32(1.14) 1.90(.49) .73 3.94 CE/GLA 85 23 28 .92(.02) .29(.17) .59(.18) -1.54* -11.02 & CE/RUT 17 7 7 .83(.06) .26(.17) .22(.11) .72 -.90 NW/RUT 33 12 14 .89(.03) .28(.17) .36(.14) -.70 -3.37 + NE/RUT 15 9 17 .92(.04) .44(.26) .55(.23) -.77 -1.63 n i , numbe o indi iduals; n h , numbe o m DNA haplo ypes; n p , numbe o polymo phic si es; h, haplo ypes di e si y; π, nucleo ide di e si y; Θ (S) , compu ed om he numbe o seg ega ing si es. S anda d de ia ions a e shown in b acke s. + , & and * indica e p< 0.055, < 0.05 and < 0.001, espec i ely. Bo a yński e al.BMC E olu iona y Biology 2011, 11:355 h p://www.biomedcen al.com/1471-2148/11/355 Page 4 o 12 species assignmen o he indi idual oles wi h he a ia- ion in ou nuclea ma ke s (Figu e 2), and ound ha mos o he m DNA haplo ypes sampled in M. gla eolus om no he n Finland (Figu e 1) clus e wi hin he clade o M. u ilus (Figu e 3), which is a ypical conse- quence o m DNA in og ession [18-21,23]. Indeed, i is s iking o no e ha he m DNA haplo ypes om M. u ilus a e ixed in he no he n popula ions o M. gla - eolus and only in one popula ion did we ind bo h he na i e and he in og essed mi o ypes (Figu e 1). Ha ing iden i ied among he sampled specimens hose wi h na i e and in og essed m DNA, we aimed o es whe he in og ession explains di e ences in wo key pheno ypic measu es: body size and basal me abolic a e. These ai s we e s udied because size and ene - ge ic physiology a e impo an p edic o s o li e his o y ai s in animals [8,12,40]. Also, some o he mos ecen da a and heo e ical p edic ions link a ia ion in hose ai s wi h di e en i ness componen s [1,2,6]. We ound ha he epea abili y o size and me abolism was signi ican (Table 3) and ha pheno ypes a ied ma k- edly be ween dis an ly loca ed popula ions o bank oles. I has been pos ula ed ha he geog aphic a ia- ion in mo pho-physiological ai s migh be caused by clima ic di e ences among dis an locali ies [10,41]. I so, he pheno ypic di e ences a e p edic ed o be no iceable and caused by pheno ypic plas ici y o adap- a ion o di e en habi a s along en i onmen al g adi- en s, which he e could include in o ma ion inhe i ed in m DNA as a possible mechanism o physiological adap- a ion [42]. 0.05 Apodemus ag a ius Mic o us ag es is Myodes gla eolus M. u ilus (whi e) and (black) 100/100/1.0 100/100/1.0 96/100/1.0 99/96/1.0 100/100/1.0 Myodes u ocanus Myodes gla eolus h6 h4 h2 h1 h3 h5 h37 h31 h30 h28 h36 h40 h68 h71 h70 h67 h65 h63 h61 h59 h57 h55 h53 h51 h47 h45 h42 h41 h38 h35 h34 h33 h29 h27 h22 h21 h19 h18 h17 h16 h14 h12 h11 h10 h9 h8 h7 h13 h15 h20 h23 h25 h39 h24 h32 h26 h49 h69 h43 h54 h56 h58 h60 h62 h64 h66 h44 h46 h48 h52 h50 h85 h83 h80 h81 h82 h84 h102 h101 h98 h90 h87 h79 h89 h97 h94 h88 h78 h76 h74 h73 h72 h95 h75 h77 h86 h93 h96 h99 h100 h91 h92 Figu e 3 Neighbou Joining (NJ) ees o cy och ome b. Numbe s in he species nodes ep esen s pe cen age o boo s ap alues o 1000 pseudo eplica es o NJ and maximum likelihood analyses and Bayesian pos e io p obabili ies. Fo simplici y he ee is collapsed in o he majo clades. B anch leng h is p opo ional o he numbe o subs i u ions pe si e. Haplo ype ne wo ks o m DNA o na i e (GLA) and in og essed Myodes gla eolus (RUT) oge he wi h M. u ilus a e p esen ed sepa a ely. O al sizes a e p opo ional o he numbe o sampled indi iduals. Poin s on he b anches indica e hypo he ical haplo ypes. Shadings o he in og essed haplo ypes e e o popula ions: ligh g ay - 4, So kamo (CE), da k g ay - 6, Sa ukoski (NE), no shading - 5, Kola i (NW). T ees we e oo ed wi h sequences downloaded om GenBank (Mic o us ag es is and Apodemus ag a ius, AY167187 and AB303226). a ) m DNA ypes: GLA and RUT b ) GLA c) RUT numbe o pai wise di e ences absolu e equency 0 1500 3000 4500 6000 01020 70 80 0 150 300 450 600 0612 Θ Θ τ 0 1 = 0.09 8 = 67.3 4 = 3.5 4 0 6 12 18 0 1500 3000 4500 6000 Figu e 4 Obse ed (black ba s) and expec ed (g ay lines) misma ch dis ibu ions. Figu es include: a) all samples o bank oles, Myodes gla eolus, b) bank oles wi h na i e m DNA ype (GLA) and c) bank oles wi h m DNA ype o ed oles M. u ilus (RUT). Values o he expansion pa ame e s a e only shown i he assump ions o he Sudden Expansion Model a e ul illed (unimodal dis ibu ion and goodness o i es : p > 0.05). Table 3 Repea abili ies o body mass, head wid h and basal me abolic a e. ANOVA ANCOVA (wi h BM) T ai N τpNτp logBM 26 .87 < .0001 26 logHW 25 .83 < .0001 25 .74 < .0001 logBMR 26 .80 < .0001 26 .62 < .0003 τ, coe icien o in aclass co ela ion based on wo epea ed measu emen s ac oss in a e age 35.5 days (min 16, max 51), based on a iance componen s om ANOVA wi h s udy indi idual as ca ego ical p edic o s, o om ANCOVA wi h body mass included as co a ia e (which p o ides epea abili y o mass- independen ai alues). Bo a yński e al.BMC E olu iona y Biology 2011, 11:355 h p://www.biomedcen al.com/1471-2148/11/355 Page 5 o 12 An in e es ing obse a ion om ou esul s is ha he no he n (and in og essed) Myodes gla eolus oles we e smalle and had lowe BMR alues han oles om he sou he n popula ions (and wi h he na i e mi o ype, Addi ional ile 7: Figu e S2). Sex speci ic es s showed ha emales wi h di e en m DNA ypes di e ed in body mass and absolu e (whole-body) me abolic a e bu no in mass-co ec ed BMR, whe eas males om no h- e n popula ions had lowe alues o absolu e and mass- co ec ed BMR, bu no body mass, and hei ai s did no a y signi ican ly be ween m DNA ypes (Table 4). Whe he he in e ed di e ences ac oss popula ions a e due o m DNA in og ession o ela ed o genome e i- cula ion is di icul o assess because bank oles om no he n popula ions a e also exclusi ely in og essed. To ci cum en his p oblem we analyzed a subsample o he da a om he single popula ion whe e bo h mi o- ypes coexis , hus elimina ing he geog aphic ac o . Al hough we did no ind a signi ican di e ence in body size be ween in og essed and non-in og essed oles (Figu e 5b), we ound ha in og essed emales had lowe absolu e BMR alues han non-in og essed ones (Table 4, Figu e 5a). Howe e , his associa ion was no signi ican when BMR was mass-co ec ed. Though, only 8 emales wi h M. u ilus m DNA ype we e sampled in his popula ion, which yields li le powe o de ec e ec s on body size and mass co ec ed BMR. The absence o a s ong ela ionship be ween m DNA ype and a ia ion in pheno ypic cha ac e s could ha e esul ed om ela i ely ecen di e gence be ween ole species, and consequen ly, he unc ional simila i y be ween mi ochond ial and nuclea genes, as shown in ecen expe imen al ansi ions o whole genomes in D osophila species [43]. O he wise, he co-in og ession o some nuclea genes ha ha e no been included in cu en analyses, bu may be impo an o ene ge ic physiology and body g ow h a e, may ha e balanced he incompa ibili y and in luenced pa icula pheno ypes [44,45]. The obse ed sex-speci ic e ec s o m DNA on pheno ypes, isible a leas in he be ween-popula ion analyses ca ied ou he e and in ano he s udy [46], could be explained by di e en equi emen s be ween sexes connec ed o hei ep oduc i e biology [8,47]. In ac , ecen models p edic e.g. ha he le el o he a e o basal me abolism in endo he ms is a consequence o di ec ional selec ion ope a ing on ep oduc i e pe o - mance [48,49]. Acco dingly, such p ocesses a e ela ed o wo ene ge ically-cos ly emale ep oduc i e p o- cesses: p egnancy and lac a ion. I has been also expe i- men ally con i med ha di ec ional selec ion can pe o m di e en ially on size and me abolic a e be ween sexes in mammals [1,2]. Howe e , he mos impo an p edic ion o di e en ial e ec s o m DNA on pheno ype comes om he ac ha in mammals, mi ochond ia a e almos exclusi ely ansmi ed h ough emales. Consequen ly, m DNA can only ha e di ec adap i e e ec o emales and he m DNA e ec s on male pheno ype a e expec ed o esul om coun e ba- lancing e ec s o nuclea genes [50]. The p e alence o he in og essed m DNA haplo ype in he no he n popula ions o M. gla eolus in ex eme equencies - he o eign haplo ype seems ixed in hese popula ions - is s iking, and we hus aised he hypo h- esis ha m DNA in og ession could ha e been Table 4 Pheno ypic di e ences be ween bank oles wi h di e en m DNA ypes. All popula ions Sympa ic popula ion mi ochond ial DNA ype o mi ochond ial DNA ype o Myodes u ilus M. gla eolus m DNA la . Myodes u ilus M. gla eolus m DNA T ai s N mean(SD) N mean(SD) p p N mean(SD) N mean p Females BM 20 16.7(2.72) 63 19.4(3.84) .004* - 8 16.8(2.77) 29 19.7(4.56) .098 HW 13.2(.370) 13.3(.370) .163 - 13.1(.395) 13.2(.426) .317 BMR ANOVA 40.1(4.12) 43.7(6.80) .032 - 39.3(4.16) 44.4(8.47) .049 BMR ANCOVA 42.6(4.86) 42.9(4.73) - .445 -.351(1.17) .097(.928) .262 Males BM 37 21.6(4.79) 71 21.9(4.23) - .135 8 19.8(4.41) 47 21.4(4.35) .329 HW 13.4(.450) 13.4(.420) - .265 13.2(.522) 13.4(.411) .189 BMR ANOVA 48.6(8.99) 49.0(11.67) - .002* 39.4(5.68) 45.1(9.12) .374 BMR ANCOVA 48.9(8.80) 48.9(8.83) - .014* -.124(.761) .021(1.03) .705 Va ia ion in body mass (BM, g), head wid h (HW, mm) and basal me abolic a e (BMR, ml O2 min-1) is p esen ed o bank oles om “all popula ions”and om So kamo “Sympa ic popula ion”whe e mi o ypes coexis . Signi icance o he e ec s: ype o he m DNA ( u ilus s. gla eolus), la i ude (la .) and longi ude (insigni ican and excluded om able) o popula ion (6 popula ions) we e es ed in ANOVA o ANCOVA ( o BMR wi h BM as co a ia e) models on log ans o med ai s as dependen a iables. Ma ginal means (and hei SDs) o m DNA ypes o ANCOVA models we e calcula ed accoun ing o a ia ion in BM. The insigni ican e ec s (p > 0.1) we e hie a chically educed om he analyses o a iances “-”and a e no p esen ed in he able. La i ude×longi ude in e ac ions a ec ed only males log ans o med and esidual alues o HW (p < 0.045), no included in he able. * e ec s signi ican a e implemen ed Bon e oni co ec ion. Bo a yński e al.BMC E olu iona y Biology 2011, 11:355 h p://www.biomedcen al.com/1471-2148/11/355 Page 6 o 12 p omo ed by na u al selec ion. We assessed i he e- quency spec um o mu a ions o he in og essed hap- lo ypes could be biased owa ds an excess o a e alleles, a pa e n compa ible wi h pos -in og ession posi i e selec ion. Ou analysis o sequence a ia ion among he in og essed cy b haplo ypes indeed shows a pa e n ha is no compa ible wi h a model o popula ion equi- lib ium (bell-shaped misma ch dis ibu ion and nega i e and signi ican Tajima’sDand Fu’sF s alues; Table 2). This could esul om adap i e in og ession o m DNA, p omo ing he expansion o he no el haplo- ype no hwa d. Howe e , selec ion is expec ed o a ec solely he locus in ques ion,while hee ec o popula- ion expansion and o he demog aphic phenomena should a ec he whole genome equally, on a e age. As he HKA es did no indica e any de ia ion om neu- al expec a ions o he mul ilocus a ios o di e gence o polymo phism (see Resul s) and bo h mi o ypes iden- i ied in M. gla eolus (GLA, and he in og essed RUT) showed simila expansion signals, ou esul s sugges ha he species i sel may ha e unde gone ecen popu- la ion expansion, sp eading he aces o m DNA in o- g ession h oughou he no he n ange o M. gla eolus. Indeed, i hyb idiza ion occu s be ween a esiden and an in ading species, in og ession is expec ed o occu in o he spa ially expanding one, in a s ochas ically neu- al phenomenon [51,52]. This scena io has been used o explain massi e m DNA in og ession among ha es [53,54] and (po en ially) in o he o ganisms [55]. In his demog aphic eplacemen p ocess, ma ke s wi h lowe in aspeci ic mig a ion a es, as is o en he case wi h m DNA, a e expec ed o in og ess mo e easily because he in lux o na i e alleles in o he in asion on is lowe [51,52]. Thus, he no he n expansion o M. gla - eolus in o he ange o M. u ilus could ha e a ou ed m DNA in og ession in a pu ely neu al manne , and could also explain he asymme y o he obse ed in o- g ession. Such asymme y could be explained by emale- biased asso a i e ma ing [56]. In a si ua ion o in e spe- ci ic con ac wi h imbalanced equencies o he in e - ac ing species, he emales o he a e species (in his case, p esumably he ou -compe ed M. u ilus) end o ma e mo e easily wi h he he e ospeci ic males, i.e., he expanding M. gla eolus. The con inuous backc ossing o hyb ids wi h he same equency-dependence o e gen- e a ions would cause in og ession o he ma e nally ansmi ed m DNA in he di ec ion o he mo e abun- dan species (M. gla eolus). This asymme y could also occu i he in asion o he ange o M. u ilus by M. gla eolus was pionee ed by males, which a e known o dispe se a he han emales in his species [57]. Conclusion This s udy unco e ed ma ked di e ences in body mass and BMR ac oss Finnish popula ions o M. gla eolus along a no h-sou h axis. These di e ences co espond wi h massi e m DNA in og ession om M. u ilus in o he popula ions o M. gla eolus, which sugges s ha genome e icula ion may p esumably be ela ed o he di e ences in pheno ype. Gi en i s ole in ene gy p o- cessing, m DNA is an ob ious candida e o unde lie physiological pheno ypic di e ences mo e di ec ly. Al hough a signal o associa ion be ween m DNA in o- g ession and whole-body BMR was de ec ed om he analysis o he popula ion whe e he na i e and o eign m DNA haplo ypes coexis , he esul s ailed o show a ela ionship be ween mass-co ec ed BMR o body mass aloneand hemi ochond ial DNA. Fu he analyses using inc eased sample sizes and mo e popula ions whe e m DNA ypes exis in sympa y will help o cla - i y his esul . Also, he co-in og ession o nuclea ele- men s may be esponsible o he al e ed pheno ype and coun e balance e en ual incompa ibili ies be ween nuclea and mi ochond ial genomes. Ob ious candida es o co-in og ession wi h m DNA a e nuclea genes in ol ed in physiological pa hways, mos no ably hose in ol ed in mi ochond ial unc ion, which a e known o co-e ol e wi h m DNA [16]. The cons uc ion o con- genic s ains in b eeding expe imen s [58,59], whe e he m DNA o m M. u ilus is ixed in a nuclea back- g ound o M. gla eolus, would allow es ing i m DNA alone al e s pheno ype, and ul ima ely, whe he he m DNA in og ession has any signi ican i ness e ec s. Me hods Sampling Voles we e apped in six popula ions along No h- Sou h and Eas -Wes g adien s in Finland, nea he owns o Tammela (SW Finland: 60°48’N:23°58’E), BMR (O ml/h; ± SE) 2 BM (g; ± SE) all popula ions sympa ic all popula ions sympa i c a) b) Figu e 5 Di e ences in pheno ype be ween mi o ypes. Di e ences in means (± SE) o a) basal me abolism (BMR) and b) body mass (BM), be ween na i e (black) and in og essed (g ay) m DNA ypes in Myodes gla eolus. Da a a e p esen ed sepa a ely o emales (ci cles) and males ( iangles) cap u ed in allopa ic ( illed) o sympa ic popula ions (open igu es). Bo a yński e al.BMC E olu iona y Biology 2011, 11:355 h p://www.biomedcen al.com/1471-2148/11/355 Page 7 o 12