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
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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
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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
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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.
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