North-south differentiation and a region of high diversity in European wolves (Canis lupus)

No h-Sou h Di e en ia ion and a Region o High Di e si y in Eu opean Wol es ( Canis lupus ) As id V. S onen 1 *, Bogumiła Je˛d zejewska 1 , Cino Pe oldi 2,3,4 , Di e Demon is 5 , E o e Randi 3,6 , Magdalena Niedziałkowska 1 , Małgo za a Pilo 7 , Vadim E. Sido o ich 8 , Iho Dykyy 9 , Josip Kusak 10 , Elena Tsinga ska 11 , Ilpo Kojola 12 , Alexand os A. Ka amanlidis 13,14 , Ai a s O nicans 15 , Vladimi A. Lobko 16 , Vi alii Dumenko 17 , Sylwia D. Cza nomska 1 1Mammal Resea ch Ins i u e, Polish Academy o Sciences, Białowiez_a, Poland, 2Depa men o Biosciences, Aa hus Uni e si y, Aa hus, Denma k, 3Aalbo g Uni e si y, Depa men 18/Sec ion o En i onmen al Enginee ing, Aalbo g, Denma k, 4Aalbo g Zoo, Aalbo g, Denma k, 5Depa men o Biomedicine, Aa hus Uni e si y, Aa hus, Denma k, 6Labo a o io di Gene ica, Is i u o Supe io e pe la P o ezione e la Rice ca Ambien ale, Ozzano Emilia (BO), I aly, 7Museum and Ins i u e o Zoology, Polish Academy o Sciences, Wa szawa, Poland, 8Ins i u e o Zoology, Na ional Academy o Sciences o Bela us, Minsk, Bela us, 9Depa men o Zoology, Biological Facul y, I an F anko Na ional Uni e si y o L i , L i , Uk aine, 10 Depa men o Biology, Facul y o Ve e ina y Medicine, Uni e si y o Zag eb, Zag eb, C oa ia, 11 BALKANI Wildli e Socie y, So ia, Bulga ia, 12 Finnish Game and Fishe ies Resea ch Ins i u e, Oulu, Finland, 13 ARCTUROS, Ci il Socie y o he P o ec ion and Managemen o Wildli e and he Na u al En i onmen , Thessaloniki, G eece, 14 Depa men o Ecology and Na u al Resou ces Managemen , No wegian Uni e si y o Li e Sciences, A ˚s, No way, 15 La ian S a e Fo es Resea ch Ins i u e ‘‘Sila a’’, Salaspils, La ia, 16 Zoological museum o Odessa, Na ional I.I. Mechniko Uni e si y, Odessa, Uk aine, 17 Biosphe e Rese e Askania No a, Askania-No a, Chaplynka Dis ic , Khe son Region, Uk aine Abs ac Eu opean wol es (Canis lupus) show popula ion gene ic s uc u e in he absence o geog aphic ba ie s, and ac oss ela i ely sho dis ances o his highly mobile species. Addi ional in o ma ion on he loca ion o and di e gence be ween popula ion clus e s is equi ed, pa icula ly because wol es a e cu en ly ecolonizing pa s o Eu ope. We e alua ed gene ic s uc u e in 177 wol es om 11 coun ies using o e 67K single nucleo ide polymo phism (SNP) loci. The esul s suppo ed p e ious indings o an isola ed I alian popula ion wi h lowe gene ic di e si y han ha obse ed ac oss o he a eas o Eu ope. Wol es om he emaining coun ies we e p ima ily s uc u ed in a no h-sou h axis, wi h C oa ia, Bulga ia, and G eece (Dina ic-Balkan) di e en ia ed om no hcen al wol es ha included indi iduals om Finland, La ia, Bela us, Poland and Russia. Ca pa hian Moun ain wol es in cen al Eu ope had geno ypes in e media e be ween hose iden i ied in no hcen al Eu ope and he Dina ic-Balkan clus e . O e all, indi idual geno ypes om no hcen al Eu ope sugges ed high le els o admix u e. We obse ed high di e si y wi hin Bela us, wi h wol es om wes e n and no he n Bela us ep esen ing he wo mos di e en ia ed g oups wi hin no hcen al Eu ope. Ou esul s suppo he p esence o a leas h ee majo clus e s (I aly, Ca pa hians, Dina ic-Balkan) in sou he n and cen al Eu ope. Indi iduals om C oa ia also appea ed di e en ia ed om wol es in G eece and Bulga ia. Expansion om glacial e ugia, adap a ion o local en i onmen s, and human- ela ed ac o s such as landscape agmen a ion and equen killing o wol es in some a eas may ha e con ibu ed o he obse ed pa e ns. Ou indings can help in o m conse a ion managemen o hese apex p eda o s and he ecosys ems o which hey a e pa . Ci a ion: S onen AV, Je˛d zejewska B, Pe oldi C, Demon is D, Randi E, e al. (2013) No h-Sou h Di e en ia ion and a Region o High Di e si y in Eu opean Wol es (Canis lupus). PLoS ONE 8(10): e76454. doi:10.1371/jou nal.pone.0076454 Edi o : Michael Ho ei e , Uni e si y o Yo k, Uni ed Kingdom Recei ed Janua y 26, 2013; Accep ed Augus 23, 2013; Published Oc obe 11, 2013 Copy igh : ß2013 S onen 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 s udy was unded by BIOCONSUS (Resea ch Po en ial in Conse a ion and Sus ainable Managemen o Biodi e si y, 7 h F amewo k P og amme con ac no. 245737), BIOGEAST (Biodi e si y o Eas -Eu opean and Sibe ian la ge mammals on he le el o gene ic a ia ion o popula ions, 7 h F amewo k P og amme con ac no. 247652), The Mammal Resea ch Ins i u e a he Polish Academy o Sciences, he Polish Minis y o Science and Highe Educa ion (g an s no. 6P04F 09421 and NN 303 418437), EURONATUR (Ge many), Ma ie Cu ie T ans e o Knowledge Fellowships o CP and JK (p ojec BIORESC in he 6 h F amewo k P og amme, con ac no. MTKD-CT-2005-029957), g an s o CP om he Danish Na u al Science Resea ch Council (g an numbe : #11-103926, #09- 065999 and 95095995) and he Ca lsbe g Founda ion (g an numbe 2011-01-0059), ISPRA (Ins i u o Supe io e pe la P o ezione e la Rice ca Ambien ale) and he I alian Minis y o En i onmen . 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: as id.s [email protected] In oduc ion Popula ion gene ic s uc u e can occu ac oss ela i ely sho dis ances in he absence o geog aphic ba ie s in highly mobile species, such as lynx (Lynx canadensis) [1], coyo es (Canis la ans) [2], and wol es (C. lupus) [3]. Wol es a e now ecolonizing se e al a eas o Eu ope, including wes e n Poland and eas e n Ge many, F ance, and Swi ze land (e.g. [4,5]). Coloniza ion p ocesses a e s ill poo ly unde s ood and, despi e legal p o ec ion in mos Eu opean coun ies, illegal killing and acciden al mo ali y emain widesp ead h ea s o wol su i al [6,7]. P e ious s udies using mi ochond ial DNA (m DNA) and mic osa elli e ma ke s sugges - ed a highly di e gen I alian popula ion wi h ela i ely low gene ic di e si y ollowing long- e m isola ion and an ex ensi e bo leneck [8,9]. Mo e in o ma ion on Eu opean wol popula ion s uc u e and he loca ion o and di e gence be ween popula ion clus e s is needed o unde s and e olu iona y his o y and in o m conse a- ion managemen . PLOS ONE | www.plosone.o g 1 Oc obe 2013 | Volume 8 | Issue 10 | e76454 P e ious indings based on 14 mic osa elli e loci [3] sugges ed ha sou he n and no hcen al Eu opean wol es may comp ise one popula ion, bisec ed by a second popula ion ex ending om eas e n Poland in o Bela us, Uk aine, and Russia. I was ne e heless no ed ha hese clus e s may comp ise u he subs uc u e, because o he addi ional clus e s indica ed by he m DNA esul s. Al hough sou he n and no hcen al Eu opean wol es we e g ouped in o one popula ion, ou indi iduals om sou he n Eu ope (G eece and Bulga ia) included in an analysis o 48K single nucleo ide polymo phism (SNP) ma ke s appea ed o be di e gen om wol es in no hcen al Eu opean coun ies including Poland and Li huania [10]. Mo eo e , ecen in es iga- ions o mo phology [11] and ecology [12] in a ious pa s o Eu ope sugges ha p e iously de ined popula ion clus e s migh be u he esol ed. Ea lie analyses ypically examined ,50 ma ke s, whe eas new genomic ools such as SNP ma ke s pe mi yping o se e al housand loci and, wi h an adequa e sample o ep esen a i e indi iduals, imp o ed esolu ion o popula ion gene ic s uc u e and e olu iona y p ocesses (e.g. [13]). Mic osa elli es ypically ha e apid mu a ion a es, and a bias owa d highly polymo phic loci migh esul in o e es ima es o gene ic di e si y [14]. Consequen ly, ampli ica ion o e en a ew hund ed SNPs should imp o e e alua ion o gene ic p o iles compa ed wi h a smalle panel o mic osa elli e ma ke s. We examined spa ial gene ic pa e ns in Eu opean wol es o de e mine whe he esul s based on SNP analyses 1) appea consis en wi h p e ious indings om m DNA and mic osa elli es, and 2) imp o e esolu ion o popula ion gene ic s uc u e ac oss he con inen . Al hough ou s udy ocused on wol es, he esul s may help unde s and pa e ns o gene ic a ia ion, popula ion s uc u e, and gene low in o he highly mobile species ha occu a low densi ies. Ma e ials and Me hods DNA Ex ac ion and Geno yping All samples we e collec ed om animals ound dead o om wol es legally ha es ed o pu poses o he han esea ch. The p ojec was ca ied ou unde con ac (no. 4184/B/P01/2009/ 37) wi h he Polish Minis y o Science and Highe Educa ion in compliance wi h all equi emen s. No e hics pe mi was equi ed as he p ojec did no in ol e collec ion o samples om li e animals. Samples om Finland, La ia, Russia, Bela us, Uk aine, Slo akia, C oa ia, Bulga ia, and G eece we e ob ained om collabo a o s and used wi h hei pe mission. We ex ac ed genomic DNA om issues o n = 272 Eu opean canids sampled 1995–2010, using a DNeasy Tissue Ki (Qiagen) acco ding o he manu ac u e ’s p o ocol. We pe o med DNA quan i y and pu i y con ol using he spec opho ome e Nano- D op ND-1000 (NanoD op Technologies, Inc., Wilming on, Delawa e, USA) and examined DNA quali y using elec opho esis wi h a 1% aga ose gel. Samples we e geno yped a AROS Applied Bio echnology A/S in Aa hus, Denma k, o 170 000 loci using he CanineHD BeadChip mic oa ay om IlluminaH(Illumina, Inc., San Diego, Cali o nia, USA) acco ding o hei In inium HD Assay Ul a assay p o ocol. Samples included n = 20 I alian dogs and h ee known i s -gene a ion cap i e wol -dog hyb ids o help iden i y and emo e indi iduals suspec ed o ha e dog ances y. The dogs we e o unknown b eed/ances y sampled in illages close o wol dis ibu ions. Fou wol samples ( om Bela us, G eece and Uk aine) we e p ocessed and geno yped in duplica es o e i y geno yping eliabili y and showed consis en indi idual p o iles. We used GenomeS udio TM and accompanying guidelines om Illumina [15] o call geno ypes o analyses o wol gene ic s uc u e (Table S1). I alian canids may ha e highe le els o ela edness due o hei isola ed s a us [8,9]. Acco dingly, we pe o med a sepa a e e alua ion o I alian wol es (Table S1). We de e mined pai s o wol es wi h an iden i y-by-descen sco e o .0.5 (equi alen o pa en -o sp ing o sibling ela ions) using PLINK [16] and emo ed one indi idual pe pai o educe he in luence o ela edness among indi iduals on popula ion gene ic s uc u e. The sc eening esul ed in a sample o n = 177 Eu opean wol es (Fig. 1). We es ima ed gene ic a ia ion, including obse ed and expec ed he e ozygosi y, and he pe cen ages o polymo phic loci, missing alleles, and loci no in Ha dy-Weinbe g equilib ium (HWE) in PLINK o he I alian (n = 50) and o he Eu opean wol es (n = 127) based on 79 536 au osomal SNPs p io o applying il e s o geno yping and mino allele equency. Subsequen ly, we pe o med quali y con ol o a me ged ile o 79 462 SNPs o he 177 wol es (Table S2). This esul ed in a inal da a se o 67 784 (67K) high-quali y au osomal SNPs o u he analyses. S a is ical Analyses o Gene ic S uc u e We pe o med p incipal componen analyses (PCA) on a subse o ma ke s p uned o linkage disequilib ium in PLINK (we emo ed SNPs wi h pai wise geno ypic associa ions ( 2 ).0.8 wi hin a window o 50 SNPs) using he adegene -package [17] in R 2.14.2 [18]. Subsequen ly, we e alua ed popula ion gene ic s uc u e using a Bayesian in e ence model in he p og am STRUCTURE 2.3.3 [19]. The STRUCTURE app oach has become a s anda d me hod o e alua ing he numbe o gene ic clus e s in he da a se while assuming equilib ium gene ic condi ions (Ha dy-Weinbe g and linkage equilib ium). These condi ions may none heless no be ul illed in all si ua ions, including ha o wol es sampled ac oss he Eu opean con inen . Thus i could be in o ma i e o also e alua e da a wi h PCA me hods ha a e 1) wi hou such equilib ium assump ions, and 2) be e able a iden i ying ansi ions in gene ic p o iles mo e accu a ely desc ibed as clines, which may be mo e di icul o de ec han clus e s [20]. We used 10 000 bu n-in uns ollowed by 10 000 Ma ko chain Mon e Ca lo epe i ions in STRUCTURE and e alua ed K = 1– 10 possible popula ion clus e s. Each pa ame e se ing was epea ed h ee imes. We used he admix u e model and allowed allele equencies o be co ela ed among popula ions. Ini ial assessmen s con i med p e ious epo s o an isola ed I alian wol popula ion [8,9], and he sepa a ion was su icien ly s ong ha i was necessa y o emo e he I alian wol es o esol e he emaining samples in o biologically meaning ul clus e s (da a no p esen ed). We he e o e di ided he da a se and in es iga ed s uc u e wi hin I aly and he emainde o Eu ope sepa a ely using K = 1–10. We used STRUCTURE Ha es e .06.92 [21] and CLUMPP 1.1.2 [22] o summa ize he ou pu , which included es ima es o Del a K [23], and plo ed indi idual assignmen s wi h Dis uc 1.1 [24]. We es ima ed he obse ed and expec ed he e ozygosi y, and he pe cen age o loci no in HWE, o he majo popula ion clus e s in PLINK [16]. Finally, we calcula ed F ST be ween all pai s o popula ion clus e s iden i ied by PCA and STRUCTURE using GENEPOP . 4.1.4 [25]. Eu opean Wol Popula ion Gene ic S uc u e PLOS ONE | www.plosone.o g 2 Oc obe 2013 | Volume 8 | Issue 10 | e76454 Resul s Gene ic Va ia ion Obse ed and expec ed he e ozygosi y was lowe in I alian wol es han in he es o Eu ope (Table 1). The pe cen age o missing loci was highe o he I alian popula ion, whe eas ewe loci we e polymo phic. Howe e , he I alian popula ion showed a smalle pe cen age o loci no in Ha dy-Weinbe g equilib ium. Popula ion Gene ic S uc u e o Eu opean Wol es The PCA e ealed a highly isola ed I alian popula ion (Fig. 2a), which is isible on he i s PC axis. The second PC axis e lec s s uc u ing in he es o he Eu opean sample, and e ealed no ob ious di e en ia ion wi hin he I alian wol es. We subsequen ly excluded I alian wol es o esol e s uc u ing o o he Eu opean samples. He e we obse ed ou ma kedly di e gen indi iduals om sou he n Uk aine (Fig. S1). A e emo al o he ou ou lying sou he n Uk aine indi iduals, we ound ha wol es om C oa ia, Figu e 1. Map o Eu opean wol dis ibu ion showing wol samples (n = 177) and popula ion clus e s. Map o Eu opean wol dis ibu ion showing wol samples (n = 177) and popula ion clus e s de ec ed using 67K single nucleo ide polymo phism (SNP) ma ke s. Dis ibu ion map p epa ed by he La ge Ca ni o e Ini ia i e o Eu ope (lcie.o g) based on Linnell e al. 2008. Wol es also occu in a eas ma ked ‘wi hou da a’, bu hei dis ibu ion is unce ain. doi:10.1371/jou nal.pone.0076454.g001 Table 1. Basic gene ic measu emen s o da a om I alian and o he Eu opean wol es analysed o 79,536 au osomal single nucleo ide polymo phism (SNP) ma ke s. Sample (sample size) H obs (SE, 95% CI) H exp (SE, 95% CI) Pe cen polymo phic loci Pe cen missing loci Pe cen loci no in HWE { I aly (n = 50) 0.1673 (0.0006, 0.1661– 0.1685) 0.1761 (0.0006, 0.1749–0.1773) 83.79 2.201 0.35 Eu ope o he * (n = 127) 0.2589 (0.0006, 0.2577– 0.2601) 0.2800 (0.0006, 0.2788–0.2812) 99.95 0.294 0.98 Obse ed and expec ed he e ozygosi y (H obs and H exp ) a e shown wi h s anda d e o (SE) and 95% con idence in e als (CI). *Bela us, Bulga ia, C oa ia, Finland, G eece, La ia, Poland, Russia, Slo akia, Uk aine. { Pe cen loci no in Ha dy-Weinbe g equilib ium a e Bon e oni co ec ion o mul iple es s. doi:10.1371/jou nal.pone.0076454. 001 Eu opean Wol Popula ion Gene ic S uc u e PLOS ONE | www.plosone.o g 3 Oc obe 2013 | Volume 8 | Issue 10 | e76454 G eece and Bulga ia (hence o h he Dina ic-Balkan popula ion, see [26]) o med a sepa a e clus e (Fig. 2b). Wi hin his clus e , C oa ian wol es appea o cons i u e a dis inc g oup on he hi d PC axis (Fig. 2c). Wol es om he Ca pa hian Moun ains in cen al Eu ope (Slo akia and wes e n Uk aine) occupy an in e media e posi ion on he i s axis (Fig. 2b). Ano he clus e comp ising indi iduals (excep he ou ou lie s ha we e emo ed) om sou he n and eas e n Uk aine (he ea e he Uk aine S eppe) is in e media e be ween Ca pa hian and no he n Eu opean wol es (Fig. 2b). A g adien in gene ic p o iles wi hin no hcen al Eu ope is isible on he second axis, bu in con as o he i s PC axis his g adien wi hin no hcen al Eu ope does no appea o co e- spond wi h geog aphic dis ance (Fig. 2b,c). The highes and lowes alues e lec wol es om no he n Bela us (and one Russian indi idual sampled nea he bo de o Bela us and La ia), and wes e n Bela us, espec i ely. The a ia ion in p o iles wi hin Bela us he e o e exceeds ha o all o he wol es wi hin no h- cen al Eu ope, including indi iduals om he no he nmos sampling egion o Finland. The emainde o he samples om Russia, La ia, Poland, and sou he n Bela us showed high o e lap among wol p o iles (Fig. 2b,c). The esul s o I aly (Fig. 3, Fig. S2) iden i ied ce ain ou lie s ha had been sampled in he No he n and Cen al Apennines ( egions 1 and 2), bu sugges ed no ob ious popula ion clus e s. The STRUCTURE esul s o all Eu opean wol es we e in acco d wi h he PCA in showing a highly di e gen I alian popula ion (Fig. 4). STRUCTURE esul s o Eu opean samples wi hou I aly concu ed wi h he PCA (Fig. 5), and he e was highes suppo o K = 2 and subsequen ly K = 4 popula ion clus e s (Table S3). K = 2 showed di e en ia ion be ween no h- cen al and sou he n Eu ope. K = 3 iden i ied di e gen p o iles in Uk aine (p ima ily), whe eas K = 4–5 sugges ed u he di e en- ia ion be ween geno ypes om he Ca pa hian Moun ains and he Uk ainian S eppe. Ce ain indi iduals in no he n Bela us and Russia appea o ha e a ypical p o iles (K = 4–5), whe eas K = 7 iden i ied he ou sou he n Uk ainian ou lie s (Fig. S1) as a sepa a e clus e . STRUCTURE esul s o I alian wol es (Fig. 6) we e in acco d wi h he indings om he PCA, and K = 2 popula ion clus e s ecei ed he highes suppo (Table S4). Al hough some indi iduals had di e gen p o iles he e was no ob ious geog aphic s uc u e wi hin he coun y. Obse ed and expec ed he e ozygosi y alues we e ma kedly lowe o I alian wol es han o he ou o he majo popula ion clus e s (Table 2) and wi hin-clus e analyses o o he Eu opean wol es educed he pe cen age o loci no in HWE. F ST alues be ween pai s o popula ion clus e s indica ed he p esence o a highly di e en ia ed (i.e., F ST .0.15, [27]) wol popula ion in I aly (Table 3). We pe o med addi ional analyses in STRUCTURE o e alua e whe he ou lie indi iduals had p o iles simila o ha o dogs and known wol -dog hyb ids om I aly (n = 16), including h ee i s -gene a ion wol -dog hyb ids. The esul s (da a no p esen ed) based on 21K SNPs indica ed ha he ou ou lie s om sou he n Uk aine (Fig. S1) had dog ances y o q dog $0.10 ( ange 0.16–0.34). Six I alian indi iduals showed dog ances y q dog $0.10 ( ange 0.10–0.35). Th ee o he Eu opean ou lie s ha had been emo ed p io o PCA and STRUCTURE analyses showed appa en dog ances y. These we e om no he n Poland (q dog = 0.22), wes e n Uk aine (q dog = 0.38), and G eece (q dog = 0.76). None o he wol es om Bela us showed dog ances y. Figu e 2. P incipal componen analysis o Eu opean wol es using 67K single nucleo ide polymo phism ma ke s. P incipal componen analysis o Eu opean wol es using 67K single nucleo ide polymo phism (SNP) ma ke s. a) Colou plo o all wol es (n = 177) whe e gene ic simila i y is ep esen ed by simila colou s and spa ial p oximi y. b) Indi iduals om Eu ope (excluding I aly and ou ou lie s om sou he n Uk aine, n = 123) wi h he 1 s and 2 nd PC axes showing ou main gene ic clus e s: Dina ic-Balkan – g een: C oa ia (n = 10), da k ed: Bulga ia (n = 10), da k blue: G eece (n = 9); Ca pa hian Moun ains – black: Slo akia (n = 1), u quoise: Wes e n Uk aine (n = 10), o ange: Polish Ca pa hian Moun ains (n = 1); Uk ainian S eppe – yellow: Eas e n Uk aine (n = 7), da k g ey: Sou he n Uk aine (n = 5); No hcen al Eu ope – g ay: Finland (n = 8), ligh g een: La ia (n = 10), blue: Russia (n = 15), ed: No he n Bela us (n = 8), o ange: Poland (excep Polish Ca pa hian Moun ains, n = 15), iole : Wes e n Bela us (n = 6), da k g een: Sou he n Bela us (n = 8). c) Indi iduals om Eu ope (excluding I aly and ou ou lie s om sou he n Uk aine, n = 123) showing he 2 nd and 3 d PC axes. Sampling and clus e s as in b), excep he Dina ic-Balkan clus e o which C oa ia and Bulga ia/G eece o med sepa a e g oups. doi:10.1371/jou nal.pone.0076454.g002 Eu opean Wol Popula ion Gene ic S uc u e PLOS ONE | www.plosone.o g 4 Oc obe 2013 | Volume 8 | Issue 10 | e76454 Discussion Ou esul s indica e clea gene ic di e gence be ween I alian wol es and indi iduals om o he Eu opean coun ies. We ound di e en ia ion be ween p o iles om no he n and sou he n Eu ope, wi h indi iduals om he Ca pa hian Moun ains in cen al Eu ope displaying in e media e geno ypes. Ou esul s also e eal high gene ic di e si y wi hin Bela us ha exceeded he a ia ion obse ed in neighbou ing coun ies. The p esence o a dis inc I alian popula ion wi h compa a i ely low alues o he e ozygosi y acco ds wi h ea lie epo s o long- e m isola ion and ela i ely low gene ic di e si y [8,9]. Ou indings o I aly seem compa able wi h ea lie esul s based on analyses o 48K SNP loci [10]. A dis inc sub-popula ion, o igina ing om a small numbe o wol es dispe sing om he Apennines, has also been epo ed in he I alian Alps [9]. We only had one sample om he Alps, and we e hus unable o e alua e he exis ence o a sepa a e clus e in his egion. The highe pe cen age o missing loci o he I alian wol es may be explained by lowe quali y samples, as hese we e no esh issues bu ob ained om animals ha we e ound dead. Reduced da a quali y may ha e augmen ed homozygosi y alues, al hough low a iabili y in I alian wol es has also been epo ed om s udies based on mic osa elli e [8] and m DNA ma ke s [28]. Ou esul s suppo he p esence o dis inc wol popula ions in Eu ope [3] and de ec ed addi ional gene ic s uc u e. We iden i ied one clus e in he Ca pa hian Moun ains, which seems consis en wi h m DNA and mic osa elli e esul s om Cza - nomska e al. [29]. They ound wol es om he Polish Ca pa hians o be di e gen om indi iduals sampled in he no he n lowlands, al hough he wo egions lie well wi hin wol dispe sal dis ance [30,31]. Al hough ou Ca pa hian samples o igina ed om wes e n Uk aine and Slo akia, wol es om he Polish pa o his moun ain ange may ha e simila p o iles. Ca pa hian indi iduals we e dis inc om Dina ic-Balkan wol es, which in ou sample comp ised he mos isola ed g oup ou side I aly acco ding o PCA and S uc u e esul s. Impo an ly, howe e , we did no ha e samples om Romania, and an impo an esea ch p io i y will be o de e mine whe he a g adien in wol p o iles migh be p esen and ex end om he Ca pa hian Moun ains in o he Dina ic-Balkan popula ion. Ca pa hian indi iduals we e mo e simila o wol es om he eas (i.e. he Uk ainian S eppe) han hey we e o wol es om no hcen al Eu ope, al hough wol es in no he n Poland (pa o he no hcen al popula ion clus e ) a e ne e heless geog aph- ically close o he Ca pa hian Moun ains han o he Uk ainian S eppe. Fac o s o he han geog aphic dis ance appea he e o e o be impo an in shaping popula ion s uc u e. The Ca pa hian Moun ains a e a mee ing poin o di e en wol haplog oups and subpopula ions based on m DNA analyses [3,32]. Cza nomska e al. [29] no ed he appa en p esence o a sepa a e clus e in eas e n Poland, be ween he Ca pa hians and he no he n lowlands. This Figu e 3. P incipal componen analysis o I alian wol es (n = 50) using 67K single nucleo ide polymo phism ma ke s. P incipal componen analysis o I alian wol es (n = 50) using 67K single nucleo ide polymo phism (SNP) ma ke s. Red = No he n Apennines (n = 14), blue = Cen al Apennines (n = 21), g een = Sou he n Apennines (n = 14), black = a single sample om he Alps. doi:10.1371/jou nal.pone.0076454.g003 Figu e 4. STRUCTURE esul s o Eu opean wol es (n = 177) using 67K single nucleo ide polymo phism ma ke s. STRUCTURE esul s o Eu opean wol es (n = 177) using 67K single nucleo ide polymo phism (SNP) ma ke s and K2 popula ion clus e s. doi:10.1371/jou nal.pone.0076454.g004 Figu e 5. STRUCTURE esul s o Eu opean wol es (n = 127, wi hou I aly and ou lie s). STRUCTURE esul s o Eu opean wol es (n = 127, wi hou I aly and ou lie s) using 67K single nucleo ide polymo phism (SNP) ma ke s and K = 2–10 popula ion clus e s. doi:10.1371/jou nal.pone.0076454.g005 Eu opean Wol Popula ion Gene ic S uc u e PLOS ONE | www.plosone.o g 5 Oc obe 2013 | Volume 8 | Issue 10 | e76454 acco ds wi h Pilo e al. [3]’s obse a ion o a popula ion ex ending om eas e n Poland in o sou he n Bela us, no he n Uk aine and Russia. Gu sky [33] epo ed a ‘wol - ee bel ’ be ween Ca - pa hian and lowland wol es in Uk aine, and he di e gence be ween he Ca pa hian and Uk ainian S eppe clus e sugges s ha (e ec i e) dispe sal be ween hese a eas may be limi ed. Fu he mo e, da a on mo phology and popula ion his o y indica e ha wol es ecolonized sou he n Uk aine om he eas [34,35]. The PCA and STRUCTURE esul s a e gene ally in ag eemen , al hough PCA appea s be e able o iden i y clines and ecognize clus e s ep esen ed by only a ew indi iduals. Al hough we used a la ge numbe o ma ke s, depa u e om he expec ed equilib ium condi ions, such as unde lying gene ic s uc u e, may ha e a ec ed he STRUCTURE esul s [19]. Simila ly, F ST alues should be in e p e ed wi h cau ion conside ing ha some clus e s a e based on ,20 samples. Ge en e al. [36] ound eas -wes en i onmen al g adien s o be s ongly associa ed wi h popula ion s uc u e in No h Ame ican wol es, and no h-sou h s uc u e has also been epo ed (e.g. [37]). Fine-scale di e en ia ion is documen ed in ce ain a eas wi h ab up en i onmen al ansi ions, such as he Paci ic Coas o Canada and sou heas e n Alaska [38,39]. Isola ion and expansion om di e en glacial e ugia [8,9,40] and adap a ion o local en i onmen s and ecological condi ions [12,41] may ha e in luenced he ex en and di ec ion o gene low in Eu opean wol es. Human- ela ed ac o s such as landscape agmen a ion and de elopmen [8,42–44], high hun ing p essu e [45–47] including poaching [6,7], may also ha e in luenced pa e ns o dispe sal. Mo eo e , wa s and up isings o e he pas 150 yea s seem o ha e exe ed a s ong in luence on wol dynamics in pa s o he s udy a ea [48], and may hus ha e in luenced gene low. Al hough wol es migh ha e had an ex ensi e dis ibu ion in no he n Eu asia du ing he la e Pleis ocene [49,50], expansion om a ious e ugia and eplacemen o di e en lineages appea o ha e played an impo an ole in s uc u ing wol gene ic a ia ion in Eu ope [32]. Subsequen admix u e would none he- less be expec ed o limi di e gence ac oss well-connec ed popula ions wi h equen gene low. Ecological and beha iou al ac o s such as p ey selec ion could he e o e play a mo e impo an ole han geog aphic dis ance in shaping wol gene ic s uc u e, as epo ed in no hcen al Eu ope [12,51]. The p esence and abundance o wild ungula es in Eu ope, wi h la ge species gene ally occu ing in he no h, may in luence he spa ial o ganiza ion o wol popula ions in he absence o (majo ) ba ie s o dispe sal. Moose (Alces alces) and also wild o es eindee (Rangi e a andus ennicus) a e impo an wol p ey in a eas o no he n Eu ope [45,47,52] whe eas sou he n Eu opean wol es o en ely on smalle species including li es ock [53,54]. Dina ic- Balkan wol es we e epo ed o ha e smalle and di e en ly shaped skulls han indi iduals om he Se bian po ion o he Ca pa hian Moun ains [11]. No h Ame ican esea ch has sugges ed ha wol size, in pa icula ha o males, may in luence he abili y o cap u e and handle la ge p ey, whe eas smalle wol es may be ad an aged in cap u ing smalle and swi e species [55]. Di e en ial selec ion associa ed wi h p ey de ence mecha- nisms and he ai s equi ed o o e come hese (e.g. size e sus speed) migh in luence he di e en ia ion obse ed be ween no he n and sou he n Eu ope. The possibili y o na al habi a - biased dispe sal, including he p esence o asymme ical dispe sal be ween highland and lowland a eas [2,56], also me i s u he a en ion. Wol es in Bela us exhibi ed unexpec ed di e si y and s uc u e, and wes e n and no he n Bela us wol es showed he mos di e gen geno ypes wi hin no hcen al Eu ope. The coun y is loca ed nea he cen e o ou sampling a ea and he e a e no majo landscape ba ie s o dispe sal whe eas wol ha es is high [45–47]. We would he e o e ha e p edic ed Bela us wol geno ypes o be simila o hose obse ed in neighbou ing Figu e 6. STRUCTURE esul s o I alian wol es (n = 50). STRUCTURE esul s o I alian wol es (n = 50) using 67K single nucleo ide polymo phism (SNP) ma ke s showing esul s o K = 2, 6, and 10. Regions wi hin I aly a e he No he n Apennines (wi h a single Alpine sample in he inal posi ion, n = 15), he Cen al Apennines (n = 21), and he Sou he n Apennines (n = 14). doi:10.1371/jou nal.pone.0076454.g006 Table 2. Basic gene ic measu es o majo popula ion clus e s o Eu opean wol es, iden i ied by PCA and STRUCTURE analyses, based on da a om 67,784 SNP ma ke s. Popula ion clus e Sample size H obs (SE, 95% CI) H exp (SE, 95% CI) Pe cen loci no in HWE { No hcen al Eu ope { 60 0.2648 (0.0006, 0.2636–0.2660) 0.2744 (0.0007, 0.2730–0.2758) 0.37 Uk ainian S eppe (sou h and eas ) 12 0.2922 (0.0007, 0.2908–0.2936) 0.2920 (0.0006, 0.2908–0.2932) None Ca pa hian Moun ains (Uk aine wes , Slo akia) 12 0.2419 (0.0008, 0.2403–0.2435) 0.2505 (0.0007, 0.2491–0.2519) None Dina ic-Balkan (G eece, Bulga ia, C oa ia) 29 0.2550 (0.0007, 0.2536–0.2564) 0.2639 (0.0007, 0.2625–0.2653) 0.22 I aly 50 0.1649 (0.0007, 0.1635–0.1663) 0.1742 (0.0007, 0.1728–0.1756) 0.39 Obse ed and expec ed he e ozygosi y (H obs and H exp ) a e shown wi h s anda d e o (SE) and 95% con idence in e als (CI). { Finland, La ia, Russia, Bela us (sou h egion), and Poland. Excluding ou lie s om wes e n and no he n Bela us, and Russia. doi:10.1371/jou nal.pone.0076454. 002 Eu opean Wol Popula ion Gene ic S uc u e PLOS ONE | www.plosone.o g 6 Oc obe 2013 | Volume 8 | Issue 10 | e76454 coun ies. Ea lie analyses iden i ied a dis inc m DNA subpopu- la ion in his egion [3]. Al hough i o e lapped wi h he sampling a ea o ou di e gen indi iduals, he la e did no ha e he haplo ype (H7) ypical o his (small) subpopula ion, bu a haplo ype (H1) common h oughou no heas e n Eu ope [3]. The o igin o he high di e si y wi hin Bela us is unclea , and me i s u he in es iga ion. We did no obse e dog ances y in Bela ussian wol es, bu dogs and wol es can in e b eed wi h golden jackals (C. au eus) [57] and his migh ha e occu ed in Bulga ia (A.E. Mou a, unpubl. da a). We we e unable o e alua e his possible sou ce o in og ession, bu golden jackals a e no known o occu in Bela us a p esen [58]. The high le els o wol ha es epo ed o Bela us [45–47] could h ea en he long- e m conse a ion o local gene ic a ia ion. Hun ing mo ali y may ha e augmen ed immig a ion in o Bela us, and he di e gen indi iduals migh ep esen long-dis ance mig an s. Sampling o wol es a he o he eas could help cla i y he high di e si y obse ed wi hin his coun y. Wol es in Finland appea ed well-connec ed o popula ions in Russia and sou hwa d, despi e he geog aphic dis ance. Jansson e al. [59] none heless epo ed signs o isola ion and inb eeding in Finish wol es analysed wi h a se o 17 mic osa elli e ma ke s, and an ea lie analysis wi h 10 mic osa elli e ma ke s sugges ed ma ked bu ecen di e en ia ion among wol es in Finland and hose o he Ka elia and A khangelsk egions o Russia [60]. Such disc epancies migh , a leas in pa , be explained by he use o di e en gene ic ma ke s [14], al hough ac o s such as he lowe sample size in ou s udy may also ha e played a ole. A s udy o a c ic wol es in No h Ame ica based on 14 mic osa elli es [37] obse ed a la ge numbe o popula ion clus e s han a s udy o he same a ea (wi h ewe bu mo e e enly dis ibu ed samples) using .26K SNP ma ke s [61]. The highe mu a ion a e and a iabili y in mic osa elli es may pe mi mo e apid de ec ion o popula ion s uc u e a e y ecen di e gence imes, al hough his could be balanced by employing a la ge sui e o SNP ma ke s [62]. We iden i ied ce ain canids wi h appa en dog ances y, including ou indi iduals om sou he n Uk aine whe e mul iple ins ances o wol -dog hyb idiza ion ha e been epo ed [63,64]. Howe e , he p esence o hese indi iduals did no al e he o e all esul s. The pu a i e hyb ids appea o sugges he p esence o back-c ossed indi iduals (a i s gene a ion wol -dog hyb id b eeding back in o he wol popula ion) in se e al Eu opean coun ies. Hyb idiza ion equi es u he in es iga ion ac oss Eu ope o de e mine he occu ence and ex en o dog, and possible golden jackal, in og ession, and how such p ocesses may a ec wol gene ic s uc u e, beha iou , ecology, and in e ac ions wi h humans ([6,65,66] and A.E. Mou a, unpubl. da a). Wi hin he Dina ic-Balkan clus e , we obse ed di e gence be ween wol es om C oa ia and indi iduals om G eece and Bulga ia. A ecen e alua ion o he Eu opean wol dis ibu ion sugges ela i ely good landscape connec i i y om he Ca - pa hians and sou hwa d (Fig. 1), al hough he la ge Dina ic- Balkan wol popula ion likely exhibi s subs uc u ing [26]. Despi e a bo leneck in he ea ly 1990s, he p esen C oa ian popula ion appea s o demons a e high le els o gene ic a ia ion ([67] and e e ences he ein), and connec i i y wi hin C oa ia has been well- p ese ed despi e ecen landscape de elopmen [68]. C oa ian haplo ypes ha e ea lie been ound o clus e wi h Bulga ia and he Alps [67]. Gene low is expec ed o occu be ween he C oa ian pa o he Dina ic popula ion and wol es in Slo enia, Bosnia & He zego ina, and u he sou heas in Mon eneg o, Se bia and he o me Yugosla Republic o Macedonia [26], as well as wi h wol es in Bulga ia and G eece [67]. P e ious in es iga ions in Bulga ia and G eece also sugges ed ela i ely high haplo ype di e si y [28,32,69], and wol es om his pa o he con inen may encompass a signi ican po ion o he di e si y p e iously ound in he la ge and con inuous Eu opean popula ion [28,67]. Fu he sampling is needed o esol e he gene ic s uc u e in his pa o Eu ope, and should aim o include samples om he a ea ex ending om C oa ia sou hwa d o Bulga ia and G eece. The de ini ion o managemen uni s om popula ion gene ic da a should conside he ex en o which popula ions a e demog aphically independen [70]. Such independence migh be shaped, a leas in pa , by en i onmen al and ecological in luences on dispe sal. Imp o ed esolu ion o dispe sal p e e ences could he e o e in o m conse a ion managemen in exis ing Eu opean popula ions and in a eas p esen ly being ecolonized by wol es and o he wide- anging species. Suppo ing In o ma ion Figu e S1 P incipal componen analysis o Eu opean wol es (n = 127) using 67K single nucleo ide polymo - phism (SNP) ma ke s. Gene ic di e si y is ep esen ed by dis ance and colou ; indi iduals u he away and wi h mo e di e en colou s ha e mo e di e gen geno ypes. The i s axis ep esen s 3.6% o he a ia ion, he second axis 2.4%. (DOC) Figu e S2 P incipal componen analysis o I alian wol es (n = 50) using 67K single nucleo ide polymo - phism (SNP) ma ke s. Gene ic di e si y is ep esen ed by dis ance and colou ; indi iduals u he away and wi h mo e di e en colou s ha e mo e di e gen geno ypes. The i s axis ep esen s 5.7% o he a ia ion, he second axis 4.3%. (DOC) Table 3. F ST alues be ween pai s o majo Eu opean wol popula ion clus e s iden i ied by PCA and STRUCTURE analyses. Clus e (n) No hcen al Eu ope { (n = 60) Uk ainian S eppe (n = 12) Dina ic-Balkan (n = 29) Ca pa hian Moun ains (n = 12) Uk ainian S eppe (n = 12) 0.030 – – – Dina ic-Balkan (n = 29) 0.046 0.053 – – Ca pa hian Moun ains (n = 12) 0.046 0.056 0.056 – I aly (n = 50) 0.197 0.236 0.218 0.250 Popula ion in o ma ion is p o ided in Table 2. Pai wise compa isons showing high (F ST .0.15) di e en ia ion a e shown in bold; n – numbe o samples. { Finland, La ia, Russia, Bela us (sou h egion), and Poland. Excluding ou lie s om wes e n and no he n Bela us, and Russia. doi:10.1371/jou nal.pone.0076454. 003 Eu opean Wol Popula ion Gene ic S uc u e PLOS ONE | www.plosone.o g 7 Oc obe 2013 | Volume 8 | Issue 10 | e76454 Table S1 Quali y con ol o single nucleo ide polymo - phism (SNP) da a om n = 272 canids (n = 96 I alian and n = 176 om o he a eas o Eu ope) o e alua ion o wol popula ion s uc u e. The esul ing da a se had n = 177 samples (n = 50 om I aly and n = 127 om o he a eas o Eu ope). (DOC) Table S2 Quali y con ol o 79 462 single nucleo ide polymo phism (SNP) loci in Eu opean wol samples, esul ing in a da a se o 67 784 SNP loci. (DOC) Table S3 Summa y o STRUCTURE esul s o Eu ope minus I aly and ou lie s (n = 127, 67K SNPs) o 3 epe i ions o each K- alue. These sugges highes Del a K suppo o K2, hen K4. (The e y high alue o K9 is no eliable as he uns o K10 did no con e ge). (DOC) Table S4 Summa y o STRUCTURE esul s o I alian wol es (n = 50, 67K SNPs) o 3 epe i ions o each K- alue. The esul s sugges highes Del a K suppo o K2. (DOC) Acknowledgmen s We hank Z. Ande sone-Lilley, A.N. Bune ich, R. Caniglia, E. Fabb i, L. Geo giadis, J. Goszczyn ´ski, M. Kolesniko , L. K ambokoukis, R. W. Mysłajek, S. Nowak, J. Ozolins, K. Pe zanowski, and o he pe sons and o ganisa ions ac oss many coun ies ha con ibu edsamplesand in o ma ion o make his s udy possible. Special hanks o W. Je˛d zejewski o his help in he ea ly s ages o his p ojec , including he ini ia i e o s a ing he sample collec ion. The La ge Ca ni o e Ini ia i e o Eu ope gene ously sha ed da a on he cu en wol dis ibu ion, and M. Go´ ny p epa ed he map. Da a accessibili y. Single nucleo ide polymo phism da a and lis o loci no in Ha dy-Weinbe g equilib ium o each popula ion clus e a chi ed in D yad en y doi:10.5061/d yad.9s1 9. Au ho Con ibu ions Concei ed and designed he expe imen s: SDC BJ MN CP MP ER AVS. Analyzed he da a: AVS. W o e he pape : AVS. Funding acquisi ion: SDC BJ MP ER. P ojec coo dina ion: SDC BJ. Sample collec ion: SDC ID VD BJ AAK IK JK VAL MN AO MP ER VES ET. Guidance du ing analyses: DD CP ER. Manusc ip e ision: AVS BJ CP DD ER MN MP VES ID JK ET IK AAK AO VAL VD SDC. Re e ences 1. Rueness EK, S ense h NC, O’Donoghue M, Bou in S, Elleg en H, e al. (2003) Ecological and gene ic spa ial s uc u ing in he Canadian lynx. Na u e 425: 69– 72. 2. Sacks BN, B own SK, E nes HB (2004) Popula ion s uc u e o Cali o nia coyo es co esponds o habi a -speci ic b eaks and illumina es species his o y. Mol Ecol 13: 1265–1275. 3. Pilo M, Jed zejewski W, B anicki W, Sido o ich VE, Jed zejewska B, e al. (2006) Ecological ac o s in luence popula ion gene ic s uc u e o Eu opean g ey wol es. Mol Ecol 15: 4533–4553. 4. Ciucci P, Reggioni W, Maio ano L, Boi ani L (2009) Long-Dis ance Dispe sal o a Rescued Wol F om he No he n Apennines o he Wes e n Alps. J Wildli e Manage 73: 1300–1306. 5. Nowak S, Myslajek RW, Klosinska A, Gab ys G (2011) Die and p ey selec ion o wol es (Canis lupus) ecolonising Wes e n and Cen al Poland. Mamm Biol 76: 709–715. 6. Randi E (2011) Gene ics and conse a ion o wol es Canis lupus in Eu ope. Mammal Re 41: 99–111. 7. Libe g O, Chap on G, Wabakken P, Pede sen HC, Hobbs NT, e al. (2012) Shoo , sho el and shu up: c yp ic poaching slows es o a ion o a la ge ca ni o e in Eu ope. P Roy Soc B-Biol Sci 279: 910–915. 8. Lucchini V, Galo A, Randi E (2004) E idence o gene ic dis inc ion and long- e m popula ion decline in wol es (Canis lupus) in he I alian Apennines. Mol Ecol 13: 523–536. 9. Fabb i E, Miquel C, Lucchini V, San ini A, Caniglia R, e al. (2007) F om he Apennines o he Alps: coloniza ion gene ics o he na u ally expanding I alian wol (Canis lupus) popula ion. Mol Ecol 16: 1661–1671. 10. onHold BM, Pollinge JP, Ea l DA, Knowles JC, Boyko AR, e al. (2011) A genome-wide pe spec i e on he e olu iona y his o y o enigma ic wol -like canids. Genome Res 21: 1294–1305. 11. Milenko ic ´M, S ˇipe ic ´VJ, Blagoje ic ´J, Ta o ic ´S, Vujosˇe ic ´M (2010) Skull a ia ion in Dina ic–Balkan and Ca pa hian g ay wol popula ions e ealed by geome ic mo phome ic app oaches. J Mammal 91: 376–386. 12. Pilo M, Jed zejewski W, Sido o ich VE, Meie -Augens ein W, Hoelzel AR (2012) Die a y Di e en ia ion and he E olu ion o Popula ion Gene ic S uc u e in a Highly Mobile Ca ni o e. PLOS One 7: e39341. 13. Rice AM, Rudh A, Elleg en H, Q a ns om A (2011) A guide o he genomics o ecological specia ion in na u al animal popula ions. Ecol Le 14: 9–18. 14. B ands o¨m M, Elleg en H (2008) Genome-wide analysis o mic osa elli e polymo phism in chicken ci cum en ing he asce ainmen bias. Genome Res 18: 881–887. 15. Illumina (2010) GenomeS udio TM Geno yping Module 1.0 Use Guide. An in eg a ed pla o m o da a isualiza ion and analysis. Illumina, Inc. Pa # 11318815, 168 pp. 16. Pu cell S, Neale B, Todd-B own K, Thomas L, Fe ei a MAR, e al. (2007) PLINK: A Tool Se o Whole-Genome Associa ion and Popula ion-Based Linkage Analyses. Am J Hum Gene 81: 559–575. 17. Jomba T (2008) adegene : a R package o he mul i a ia e analysis o gene ic ma ke s. Bioin o ma ics 24: 1403–1405. 18. R De elopmen Co e Team (2012) R: A language and en i onmen o s a is ical compu ing. R Founda ion o S a is ical Compu ing, Vienna, Aus ia. 19. P i cha d JK, S ephens M, Donnelly P (2000) In e ence o popula ion s uc u e using mul ilocus geno ype da a. Gene ics 155: 945–959. 20. Jomba T, De illa d S, Du ou AB, Pon ie D (2008) Re ealing c yp ic spa ial pa e ns in gene ic a iabili y by a new mul i a ia e me hod. He edi y 101: 92– 103. 21. Ea l DA, Vonhold BM (2012) STRUCTURE HARVESTER: a websi e and p og am o isualizing STRUCTURE ou pu and implemen ing he E anno me hod. Conse Gene Resou 4: 359–361. 22. Jakobsson M, Rosenbe g NA (2007) CLUMPP: a clus e ma ching and pe mu a ion p og am o dealing wi h label swi ching and mul imodali y in analysis o popula ion s uc u e. Bioin o ma ics 23: 1801–1806. 23. E anno G, Regnau S, Goude J (2005) De ec ing he numbe o clus e s o indi iduals using he so wa e STRUCTURE: a simula ion s udy. Mol Ecol 14: 2611–2620. 24. Rosenbe g NA (2004) DISTRUCT: a p og am o he g aphical display o popula ion s uc u e. Mol Ecol No es 4: 137–138. 25. Rousse F (2008) genepop9007: a comple e e-implemen a ion o he genepop so wa e o Windows and Linux. Mol Ecol Res 8: 103–106. 26. Linnell J, Sal a o i V, Boi ani L (2008) Guidelines o popula ion le el managemen plans o la ge ca ni o es in Eu ope. A La ge Ca ni o e Ini ia i e o Eu ope epo p epa ed o he Eu opean Commission (con ac 070501/ 2005/424162/MAR/B2). 27. Balloux F, Lugon-Moulin N (2002) The es ima ion o popula ion di e en ia ion wi h mic osa elli e ma ke s. Mol Ecol 11: 155–165. 28. Randi E, Lucchini V, Ch is ensen MF, Mucci N, Funk SM, e al. (2000) Mi ochond ial DNA a iabili y in I alian and Eas Eu opean wol es: De ec ing he consequences o small popula ion size and hyb idiza ion. Conse Biol 14: 464–473. 29. Cza nomska SD, Je˛d zejewska B, Bo owik T, Niedziałkowska M, S onen AV, e al. (2013) Conco dan mi ochond ial and mic osa elli e DNA s uc u ing be ween Polish lowland and Ca pa hian Moun ain wol es. Conse Gene 14: 573–588. 30. Wabakken P, Sand H, Kojola I, Zimme mann B, A nemo JM, e al. (2007) Mul is age, long- ange na al dispe sal by a global posi ioning sys em-colla ed Scandina ian wol . J Wildli e Manage 71: 1631–1634. 31. Schede J-U, Schumann G, We sin-Siela A (2010) Wo¨l e in B andenbu g – eine spu ensuche im ma¨ kischen Sand. Po sdam, Ge many. [In Ge man]. Minis e - ium u¨ Umwel . Gesundhei und Ve b auche schu z des Landes B andenbu g. 32. Pilo M, B anicki W, Je˛d zejewski W, Goszczynski J, Je˛d zejewska B, e al. (2010) Phylogeog aphic his o y o g ey wol es in Eu ope. BMC E ol Biol 10: 104. 33. Gu sky IG (1985) Uk aine and Molda ia. In: Bibiko DI, edi o . The wol His o y, sys ema ics, mo phology, ecology. Moscow. [In Russian]: Nauka Publishe s. 487–493. 34. Dumenko VP (2004) The wol Canis lupus L. in he Biosphe e Rese e ‘‘Askania No a’’ and i s egion. Repo 2. Mo phological desc ip ion o he mode n popula ion. News Biosphe e Rese e ‘‘Askania No a’’ 6: 83–102. [In Russian wi h English summa y]. 35. Dumenko VP (2005) The wol Canis lupus L. in he Biosphe e Rese e ‘‘Askania No a’’ and i s egion. Repo 1. His o y o he species. News Biosphe e Rese e ‘‘Askania No a’’ 7: 102–111. [In Russian wi h English summa y]. Eu opean Wol Popula ion Gene ic S uc u e PLOS ONE | www.plosone.o g 8 Oc obe 2013 | Volume 8 | Issue 10 | e76454 36. Ge en E, Ande son MJ, Wayne RK (2004) Clima e and habi a ba ie s o dispe sal in he highly mobile g ey wol . Mol Ecol 13: 2481–2490. 37. Ca michael LE, K izan J, Nagy JA, Fuglei E, Dumond M, e al. (2007) His o ical and ecological de e minan s o gene ic s uc u e in a c ic canids. Mol Ecol 16: 3466–3483. 38. Weckwo h BV, Talbo S, Sage GK, Pe son DK, Cook J (2005) A signal o independen coas al and con inen al his o ies among No h Ame ican wol es. Mol Ecol 14: 917–931. 39. Mun˜oz-Fuen es V, Da imon CT, Wayne RK, Paque PC, Leona d JA (2009) Ecological ac o s d i e di e en ia ion in wol es om B i ish Columbia. J Biogeog 36: 1516–1531. 40. Da ison J, Ho SYW, B ay SC, Ko s en M, Tammeleh E, e al. (2011) La e- Qua e na y biogeog aphic scena ios o he b own bea (U sus a c os), a wild mammal model species. Qua e na y Sci Re 30: 418–430. 41. Nosil P, Vines TH, Funk DJ (2005) Pe spec i e: Rep oduc i e isola ion caused by na u al selec ion agains immig an s om di e gen habi a s. E olu ion 59: 705–719. 42. Riley SPD, Pollinge JP, Sau ajo RM, Yo k EC, B omley C, e al. (2006) A sou he n Cali o nia eeway is a physical and social ba ie o gene low in ca ni o es. Mol Ecol 15: 1733–1741. 43. Huck M, Je˛d zejewski W, Bo owik T, Je˛d zejewska B, Nowak S, e al. (2011) Analyses o leas cos pa hs o de e mining e ec s o habi a ypes on landscape pe meabili y: wol es in Poland. Ac a The iol 56: 91–101. 44. S aka M, Paule L, Ionescu O, S ˇ o ı ´k J, Adamec M (2012) Mic osa elli e di e si y and s uc u e o Ca pa hian b own bea s (U sus a c os): consequences o human caused agmen a ion. Conse Gene 13: 153–164. 45. Sido o ich VE, Tikhomi o a LL, Je˛d zejewska B (2003) Wol Canis lupus numbe s, die and damage o li es ock in ela ion o hun ing and ungula e abundance in no heas e n Bela us du ing 1990–2000. Wildli e Biol 9: 103–111. 46. Sido o ich VE, S olya o VP, Vo obei NN, I ano a NV, Je˛d zejewska B (2007) Li e size, sex a io, and age s uc u e o g ay wol es, Canis lupus, in ela ion o popula ion luc ua ions in no he n Bela us. Can J Zool 85: 295–300. 47. Je˛d zejewski W, Je˛d zejewska B, Ande sone-Lilley Z, Balc ˇiauskas L, Ma¨nnil P, e al. (2010) Syn hesizing wol ecology and managemen in eas e n Eu ope: simila i ies and con as s wi h No h Ame ica. In: Musiani M, Boi ani L, Paque PC, edi o s. The wo ld o wol es: New pe spec i es on ecology, beha iou and managemen . Calga y, Canada: Uni e si y o Calga y P ess. 207–233. 48. Je˛d zejewska B, Je˛d zejewski W, Bune ich AN, Miłkowski L, Oka ma H (1996) Popula ion dynamics o wol es Canis lupus in Białowiez_a p ime al o es (Poland and Bela us) in ela ion o hun ing by humans, 1847–1993. Mammal Re 26: 103–126. 49. Kahlke R-D (1999) The his o y o he o igin, e olu ion and dispe sal o he la e Pleis ocene Mammu hus-Coelodon a aunal complex in Eu asia (La ge Mammals). Rapid Ci y, Sou h Dako a: Fenske Companies. 50. Ho ei e M (2007) Pleis ocene ex inc ions: Haun ing he su i o s. Cu Biol 17: R609–R611. 51. Je˛d zejewski W, Niedziałkowska M, Haywa d MW, Goszczyn ´ski J, Je˛d zejewska B, e al. (2012) P ey choice and die o wol es ela ed o ungula e communi ies and wol subpopula ions in Poland. J Mammal 93: 1480–1492. 52. Kojola I, Hui u O, Toppinen K, Heiku a K, Heikkinen S, e al. (2004) P eda ion on Eu opean wild o es eindee (Rangi e a andus) by wol es (Canis lupus)in Finland. J Zool 263: 229–235. 53. Papageo giou N, Vlachos C, S ouga is A, Tsachalidis E (1994) S a us and Die o Wol es in G eece. Ac a The iol 39: 411–416. 54. Kusak J, Majic ´Sk binsˇek A, Hube D (2005) Home anges, mo emen s, and ac i i y o wol es (Canis lupus) in he Dalma ian pa o Dina ids, C oa ia. Eu J Wildl Res 51: 254–262. 55. MacNul y DR, Smi h DW, Mech LD, Ebe ly LE (2009) Body size and p eda o y pe o mance in wol es: is bigge be e ? J Anim Ecol 78: 532–539. 56. Sacks BN, Mi chell BR, Williams CL, E nes HB (2005) Coyo e mo emen s and social s uc u e along a c yp ic popula ion gene ic subdi ision. Mol Ecol 14: 1241–1249. 57. Lindblad-Toh K, Wade CM, Mikkelsen TS, Ka lsson EK, Ja e DB, e al. (2005) Genome sequence, compa a i e analysis and haplo ype s uc u e o he domes ic dog. Na u e 438: 803–819. 58. A nold J, Hume A, Hel ai M, Mu a iu D, Spasso N, e al. (2012) Cu en s a us and dis ibu ion o golden jackals Canis au eus in Eu ope. Mammal Re 42: 1–11. 59. Jansson E, Ruokonen M, Kojola I, Aspi J (2012) Rise and all o a wol popula ion: gene ic di e si y and s uc u e du ing eco e y, apid expansion and d as ic decline. Mol Ecol 21: 5178–5193. 60. Aspi J, Roininen E, Kiiskila J, Ruokonen M, Kojola I, e al. (2009) Gene ic s uc u e o he no hwes e n Russian wol popula ions and gene low be ween Russia and Finland. Conse Gene 10: 815–826. 61. Knowles JC (2010) Popula ion genomics o No h Ame ican g ey wol es (Canis lupus) [MSc hesis]. Edmon on, Albe a, Canada: Uni e si y o Albe a. 62. Haasl RJ, Payseu BA (2011) Mul i-locus in e ence o popula ion s uc u e: a compa ison be ween single nucleo ide polymo phisms and mic osa elli es. He edi y 106: 158–171. 63. Gu sky IG (1975) Hyb idiza ion be ween wol es and dogs in na u e. Byulle en Mosko skogo Obshches a Ispy a elei P i ody, O delenie Biologii 80: 131–136. [In Russian wi h English summa y]. 64. Dumenko VP (2001) Nakhodka y odka olko-sobachikh gib ido bios e nom zapo ednike ‘‘Askaniya-No a’’ [The inding o a li le o wol -dog hyb ids in Biosphe e Rese e ‘‘Askania No a’’]. T ansac ions o Zoological Museum o Odessa Na ional Uni e si y: Ma e ials o he knowledge o animals ( auna, mo phology, me hods o in es iga ions) 4: 170–171. As op in , Odessa, Uk aine [In Russian]. 65. Godinho R, Llaneza L, Blanco JC, Lopes S, Al a es F, e al. (2011) Gene ic e idence o mul iple e en s o hyb idiza ion be ween wol es and domes ic dogs in he Ibe ian Peninsula. Mol Ecol 20: 5154–5166. 66. Hind ikson M, Mannil P, Ozolins J, K zywinski A, Saa ma U (2012) Bucking he T end in Wol -Dog Hyb idiza ion: Fi s E idence om Eu ope o Hyb idiza ion be ween Female Dogs and Male Wol es. PLOS One 7: e46465. 67. Gome c ˇic ´T, Sindic ˇic ´M, Galo A, A banasic ´H, Kusak J, e al. (2010) High gene ic a iabili y o g ey wol (Canis lupus L.) popula ion om C oa ia as e ealed by mi ochond ial DNA con ol egion sequences. Zool S ud 49: 816– 823. 68. Kusak J, P ohaska A, Hube D, Vuksˇic ´I (2009) Highway Cons uc ions and Main enance o La ge Mammals Habi a Con inui y – a C oa ian Expe ience. In: Je˛d zejewski W, Ław eszuk D, edi o s; 2009 No embe 20–22nd 2008. [In Polish wi h English summa y]; Białowiez_a, Poland. Mammal Resea ch Ins i u e. 51–59. 69. Vila` C, Amo im IR, Leona d JA, Posada D, Cas o iejo J, e al. (1999) Mi ochond ial DNA phylogeog aphy and popula ion his o y o he g ey wol Canis lupus. Mol Ecol 8: 2089–2103. 70. Palsbøll PJ, Be ube M, Allendo FW (2007) Iden i ica ion o managemen uni s using popula ion gene ic da a. T ends Ecol E ol 22: 11–16. Eu opean Wol Popula ion Gene ic S uc u e PLOS ONE | www.plosone.o g 9 Oc obe 2013 | Volume 8 | Issue 10 | e76454