Mitochondrial phylogeography of European pond turtles(Emys orbicularis, Emys trinacris) – an update∗

Uwe Fritz1, Daniela Guicking2,3, Hajigholi Kami4, Marine Arakelyan5, Markus Auer1,

Dinçer Ayaz6, César Ayres Fernández7, Andrey G. Bakiev8, Antonia Celani9, Georg Džukic10,

Soumia Fahd11, Peter Havaš12, Ulrich Joger13, Viner F. Khabibullin14, Lyudmila F. Mazanaeva15,

Pavel Široký16, Sandro Tripepi9, Aitor Valdeón Vélez17, Guillermo Velo Antón7, Michael Wink2

Abstract. Based on more than 1100 samples of Emys orbicularis and E. trinacris, data on mtDNA diversity and distributionof haplotypes are provided, including for the first time data for Armenia, Georgia, Iran, and the Volga, Ural and Turgay RiverBasins of Russia and Kazakhstan. Eight mitochondrial lineages comprising 51 individual haplotypes occur in E. orbicularis,a ninth lineage with five haplotypes corresponds to E. trinacris. A high diversity of distinct mtDNA lineages and haplotypesoccurs in the south, in the regions where putative glacial refuges were located. More northerly parts of Europe and adjacentAsia, which were recolonized by E. orbicularis in the Holocene, display distinctly less variation; most refuges did notcontribute to northern recolonizations. Also in certain southern European lineages a decrease of haplotype diversity isobserved with increasing latitude, suggestive of Holocene range expansions on a smaller scale.

1 - Museum of Zoology (Museum für Tierkunde), NaturalHistory State Collections Dresden, A. B. Meyer Build-ing, D-01109 Dresden, Germanye-mail: uwe.fritz@snsd.smwk.sachsen.de

2 - Institute of Pharmacy and Molecular Biotechnology,Heidelberg University, INF 364, D-69120 Heidelberg,Germany

3 - Current address: University of Kassel, FB1, Systematikund Morphologie der Pflanzen, Heinrich-Plett-Str. 40,D-34132 Kassel, Germany

4 - Department of Biology, Faculty of Sciences, GorganUniversity of Agricultural Sciences and Natural Re-sources, Golestan Province, Iran

5 - Department of Biology, Yerevan State University, AlekMaukyan 1, Yerevan, 375025, Armenia

6 - Ege University, Faculty of Science, Department ofBiology, Zoology Section, TR-35100 Bornova-Izmir,Turkey

7 - Grupo de Ecoloxía Evolutiva, Departamento deEcoloxía e Bioloxía Animal, Universidade de Vigo,E.U.E.T. Forestal, Campus Universitario, E-36005Pontevedra, Spain

8 - Institute of Ecology of the Volga River Basin, RussianAcademy of Sciences, Komzina 10, Togliatti, 445003,Russia

9 - Dipartimento di Ecologia, Università degli Studi dellaCalabria, I-87030 Arcavacata di Rende, Italy

10 - Institute for Biological Research “Siniša Stankovic”,Bulevar Despota Stefana 142, 11060 Belgrade, Serbia

11 - Département de Biologie, Faculté des Sciences,Université Abdelmalek Essaâdi, BP. 2121, Tétouan,Morocco

12 - Fauna Carpatica, Titogradská 18, SK-040 23 Košice,

The mitochondrial cytochrome b gene (cyt b)became a frequently used marker for infer-ring phylogeography in reptiles (e.g. Brown andPestano, 1998; Burbrink et al., 2000; Carranzaet al., 2000, 2002; Surget-Groba et al., 2001;Harris et al., 2002; Austin et al., 2003; Pod-nar et al., 2005; Poulakakis et al., 2005; Fritzet al., 2006a) and several studies on Euro-pean pond turtles (Emys orbicularis, Emys tri-nacris) were based on this marker gene. WhileLenk et al. (1999) provided a nearly range-wide phylogeography, their study suffered froma patchy sampling for many parts of the range

Slovakia13 - Staatliches Naturhistorisches Museum, Pockelsstr. 10,

D-38106 Braunschweig, Germany14 - Faculty of Biology, Bashkir State University, Frunze

Street 32, Ufa, 450074, Bashkortostan, Russia15 - Department of Zoology, Dagestan State University,

37a M. Gadyeva st., Makhachkala, 367025, Dagestan,Russia

16 - Department of Biology and Wildlife Diseases, Facultyof Veterinary Hygiene and Ecology, University ofVeterinary and Pharmaceutical Sciences, Palackého1-3, CZ-612 42 Brno, Czech Republic

17 - Departamento de Vertebrados, Aranzadi Society ofSciences, Zorroagagaina 11, E-20014 Donostia-SanSebastián, Gipuzkoa, Spain

*This study is dedicated to the late Peter Lenk

© Koninklijke Brill NV, Leiden, 2007. Amphibia-Reptilia 28 (2007): 418-426Also available online - www.brill.nl/amre

Short Notes 419

(North Africa, Iberian and Apennine Peninsu-las, France, East Europe, Kazakhstan, Turkey,Caucasus, Iran and Turkmenistan). Subsequentpapers used a much denser sampling but co-vered only small regions (Fritz et al., 2004,2005a, b, 2006b; Kotenko et al., 2005). Here weprovide a range-wide update on haplotype di-versity and distribution, and present for the firsttime data on pond turtles from Armenia, Geor-gia, Iran, and the Volga, Ural and Turgay RiverBasins of Russia and Kazakhstan. This note isbased on more than 1100 cyt b sequences ofknown-locality samples and specimens of un-known geographic origin and is likely to repre-sent the largest data set ever published for west-ern Palaearctic vertebrates. Our paper is aimedas a starting-point for further research, indicat-ing still badly-sampled regions and suggestingfuture research directions.

Sampling techniques, PCR and sequencingare described in Lenk et al. (1999) and Fritzet al. (2005a). Besides samples from nativewild-caught turtles or known-locality speci-mens from captive breeding projects, samples ofconfiscated, pet trade, or wild-caught allochtho-nous turtles were studied. Sequences were ap-proximately 970-1125 bp long and aligned man-ually for haplotype determination. Haplotypenomenclature follows Lenk et al. (1999) andFritz et al. (2004, 2005a, b, 2006b): Roman nu-merals designate major clades of haplotypes asrevealed by phylogenetic analyses (=mtDNAlineages); within each lineage individual haplo-types are distinguished by consecutive letters.

In addition to the previously identified 48haplotypes in nine lineages (I-IX; Lenk et al.,1999; Fritz et al., 2004, 2005a, b, 2006b) wefound eight new haplotypes, belonging to al-ready known lineages. For many haplotypes de-scribed in earlier studies, distributional data areprovided here for the first time. The geographicorigin of lineage IX (with haplotype IXa only),discovered in a pet trade turtle (Fritz et al.,2004), is still unknown however (table 1).

Based on an alignment of 1031 bp, rela-tionships of haplotypes are illustrated using a

TCS 1.21 parsimony network (Clement et al.,2000) and a MP strict consensus tree rootedwith the closely related Nearctic taxa Actinemysmarmorata and Emydoidea blandingii (PAUP*4.0b10, TBR branch-swapping algorithm, allcharacters with equal weight, stepwise randomaddition of 10 sequences; Swofford, 2002). Forthe ingroup taxa 957 characters were constant,30 were variable but parsimony-uninformativeand 44 were parsimony-informative; for all taxa877 characters were constant, 33 variable char-acters were parsimony-uninformative and 121were parsimony-informative. Maximum likeli-hood estimates for genetic differences of allnine lineages and most haplotypes were de-picted in a recently published ML phylogram(Fritz et al., 2005a); uncorrected p distanceswere reported in the same study.

The network (fig. 1) is flock-like and hap-lotypes cluster into eight major branches. Onebranch splits into lineages I and II (E. o. orbic-ularis and related subspecies); the other eightbranches correspond with lineages III (E. tri-nacris) and lineages IV to IX (remaining E. or-bicularis subspecies). Lineages I and II are in-terconnected over two loops; a further loop oc-curs within lineage IV. Under MP phylogeneticanalysis (fig. 2), monophyly of haplotypes oflineages II, III, IV, V and VII is well-supported,while monophyly for lineage I and VI haplo-types is only weakly supported. Lineages VIIIand IX are represented by only one haplotypeeach that is located outside of all other lineages.Lineage III (E. trinacris) constitutes the sistergroup of a weakly supported clade containinglineages I-II and IV-IX (E. orbicularis); a well-supported clade of lineages I and II is the sistergroup of the remaining lineages IV-IX within E.orbicularis. Bootstrap support for monophylyof the clade comprising lineages IV-IX is below50% however. Within lineage VI, North Africanhaplotypes (VIc, VIf) are paraphyletic with re-spect to European haplotypes (VIa, VIb, VId,VIe).

Regarding geographic distribution and phylo-geography, our new data confirm the findings of

420 Short Notes

Tabl

e1.

Freq

uenc

y,ge

ogra

phic

dist

ribu

tion

and

acce

ssio

nnu

mbe

rsof

mito

chon

dria

lha

plot

ypes

ofE

mys

orbi

cula

ris

(lin

eage

sI

and

II,I

Vto

IX)

and

Em

ystr

inac

ris

(lin

eage

III)

.Onl

yna

tive

turt

les

wer

eco

nsid

ered

for

dist

ribu

tion

whi

leal

lse

quen

ced

sam

ples

wer

eco

nsid

ered

for

freq

uenc

yof

hapl

otyp

es(b

rack

eted

,nu

mbe

rof

allo

chth

onou

san

dpe

ttr

ade

turt

les

ofun

know

nge

ogra

phic

orig

in).

Ast

eris

ked

dist

ribu

tion

data

prev

ious

lyun

publ

ishe

d.

Hap

loty

peFr

eque

ncy

Geo

grap

hic

Dis

trib

utio

nA

cces

sion

Ref

eren

ceN

umbe

r

Ia15

9(6

1)C

entr

alan

dea

ster

nPo

land

,L

ithua

nia,

Ukr

aine

(not

Cri

mea

),D

on,

*Vol

ga,

*Ura

lan

d*T

urga

yR

iver

Bas

ins

inR

ussi

aan

dK

azak

hsta

n,B

lack

Sea

regi

ons

ofR

oman

ia,B

ulga

ria,

Tur

key

and

*Geo

rgia

,*re

gion

sof

Gor

iand

Tbi

lisi(

Geo

rgia

),*K

lado

vo(S

erbi

a)

AJ1

3140

7L

enk

etal

.(19

99),

Fritz

etal

.(20

04)

Ib36

(16)

Eas

tern

Gre

ece,

Bul

gari

aA

J131

408

Len

ket

al.(

1999

),Fr

itzet

al.(

2004

)Ic

39(3

)C

entr

alA

nato

lia,

Tur

kish

Bla

ckSe

are

gion

,so

uthe

rnC

rim

ea(U

krai

ne),

Dag

esta

nan

dK

alm

ykia

(Rus

sia)

AJ1

3140

9L

enk

etal

.(19

99),

Fritz

etal

.(20

04),

Kot

enko

etal

.(20

05)

Id2

(1)

Cen

tral

Ana

tolia

AJ1

3141

0L

enk

etal

.(19

99),

Fritz

etal

.(20

04)

Ie1

Nor

ther

nC

rim

ea(U

krai

ne)

AY

6528

65Fr

itzet

al.(

2005

a),

Kot

enko

etal

.(20

05)

If3

*Izm

irPr

ovin

ce(T

urke

y)A

Y65

2866

Fritz

etal

.(20

05a)

Ig10

*Cen

tral

Ana

tolia

,*w

este

rnT

urki

shB

lack

Sea

regi

onA

Y65

2867

Fritz

etal

.(20

05a)

Ih1

Nor

th-e

aste

rnU

krai

neA

Y65

2868

Fritz

etal

.(20

05a)

,K

oten

koet

al.(

2005

)Ii

3So

uth-

east

ern

Cri

mea

(Ukr

aine

)A

Y65

2879

Fritz

etal

.(20

05a)

,K

oten

koet

al.(

2005

)Ij

1(1

)*U

nkno

wn

AY

6528

80Fr

itzet

al.(

2005

a)II

a27

0(1

08)

*Nav

arra

,H

uesc

aan

dno

rthe

rnM

edite

rran

ean

coas

tof

Spai

n,w

este

rn,

sout

hern

and

cent

ralF

ranc

e,D

anub

eB

asin

,sou

th-e

aste

rnB

alka

nPe

nins

ula

AJ1

3141

1L

enk

etal

.(19

99),

Fritz

etal

.(20

05b)

IIb

31(1

)N

orth

-eas

tern

Ger

man

y,w

este

rnPo

land

(mai

nly

Ode

rB

asin

)A

J131

412

Len

ket

al.(

1999

),Fr

itzet

al.(

2005

b)II

c3

*Bal

aton

and

Vel

ence

Lak

es(H

unga

ry)

AJ1

3141

3L

enk

etal

.(19

99)

IId

1(1

)*U

nkno

wn

AJ1

3141

4L

enk

etal

.(19

99)

IIe

1(1

)*U

nkno

wn

AY

6528

69Fr

itzet

al.(

2005

a)II

f2

(1)

*Pla

tam

onas

(Mac

edon

ia,G

reec

e)A

Y65

2881

Fritz

etal

.(20

05a)

IIg

1(1

?)D

épar

tem

entA

ïn(F

ranc

e)?

AY

6528

70Fr

itzet

al.(

2005

a,b)

IIh

2D

épar

tem

entP

yrén

ées-

Atla

ntiq

ues

(Fra

nce)

AY

6528

88Fr

itzet

al.(

2005

a,b)

IIi

1D

épar

tem

entG

iron

de(F

ranc

e)A

Y65

2882

Fritz

etal

.(20

05a,

b)II

j1

(1)

*Unk

now

nA

Y65

2889

Fritz

etal

.(20

05a)

IIk

1(1

)*U

nkno

wn

AY

6528

83Fr

itzet

al.(

2005

a)

Short Notes 421Ta

ble

1.(C

ontin

ued)

.

Hap

loty

peFr

eque

ncy

Geo

grap

hic

Dis

trib

utio

nA

cces

sion

Ref

eren

ceN

umbe

r

IIIa

12(1

or2)

Sici

ly,p

erha

psC

alab

ria

(Ita

ly)

AJ1

3141

5L

enk

etal

.(19

99),

Fritz

etal

.(20

05a)

IIIb

1(1

)U

nkno

wn

AJ1

3141

6L

enk

etal

.(19

99)

IIIc

34(2

)Si

cily

AY

6528

90Fr

itzet

al.(

2005

a)II

Id1

Sici

lyA

M23

0632

Fritz

etal

.(20

06b)

IIIe

1Si

cily

AM

2306

33Fr

itzet

al.(

2006

b)IV

a14

9(8

8)A

pulia

and

Adr

iatic

coas

tof

Ital

y,w

est

coas

tof

Bal

kan

Peni

nsul

a(n

otC

epha

loni

aan

dPe

lopo

nnes

us),

Cor

fu,E

vvia

(Gre

ece)

AJ1

3141

7L

enk

etal

.(19

99),

Fritz

etal

.(20

05a)

IVb

3C

epha

loni

a(G

reec

e)A

J131

418

Len

ket

al.(

1999

)IV

c7

(2)

Pelo

ponn

esus

(Gre

ece)

AJ1

3141

9L

enk

etal

.(19

99)

IVd

22(8

)So

uthe

rnA

pulia

(Ita

ly),

*Tiv

at(M

onte

negr

o)A

Y65

2871

Fritz

etal

.(20

05a)

IVe

1(1

)U

nkno

wn

AY

6528

84Fr

itzet

al.(

2005

a)IV

f1

(1)

Unk

now

nA

Y65

2872

Fritz

etal

.(20

05a)

IVg

1Pe

lopo

nnes

us(G

reec

e)A

Y65

2873

Fritz

etal

.(20

05a)

IVh

8C

alab

ria,

sout

hern

Apu

lia(I

taly

)A

Y65

2874

Fritz

etal

.(20

05a)

IVi

2So

uthe

rnA

pulia

(Ita

ly)

AY

6528

85Fr

itzet

al.(

2005

a)IV

j2

Sout

hern

Apu

lia(I

taly

)A

Y65

2886

Fritz

etal

.(20

05a)

IVk

1(1

)*U

nkno

wn

AM

1179

37T

his

stud

yV

a18

1(5

2)N

orth

ern

Med

iterr

anea

nco

asto

fSp

ain,

sout

hern

Fran

ce,w

estc

oast

ofA

penn

ine

Peni

n-su

la,C

alab

ria,

Bas

ilica

ta,s

outh

ern

Apu

lia(I

taly

),C

orsi

ca,S

ardi

nia

AJ1

3142

0L

enk

etal

.(19

99),

Fritz

etal

.(20

05a)

Vb

3C

alab

ria,

sout

hern

Apu

lia(I

taly

)A

Y65

2875

Fritz

etal

.(20

05a)

Vc

7*C

alab

ria,

Bas

ilica

ta(I

taly

)A

Y65

2876

Fritz

etal

.(20

05a)

Vd

5*C

alab

ria

(Ita

ly)

AM

2698

87T

his

stud

yV

Ia46

(1or

2)*A

ltoT

rás-

os-M

onte

s(P

ortu

gal)

,*G

alic

ia,

Hue

lva,

*Mad

rid

and

Ciu

dad

Rea

l(S

pain

),M

edite

rran

ean

coas

tof

Spai

n,pe

rhap

sD

épar

tem

entP

yrén

ées-

Atla

ntiq

ues

(Fra

nce)

AJ1

3142

1L

enk

etal

.(19

99),

Fritz

etal

.(20

05b)

VIb

1A

lgar

ve(P

ortu

gal)

AJ1

3142

2L

enk

etal

.(19

99)

VIc

1M

iddl

eA

tlas

(Mor

occo

)A

J131

423

Len

ket

al.(

1999

)V

Id5

Ale

ntej

o(P

ortu

gal)

,*L

eón,

Hue

sca

and

Ebr

oD

elta

(Spa

in)

AJ1

3142

4L

enk

etal

.(19

99)

VIe

2*G

alic

ia(S

pain

)A

Y65

2877

Fritz

etal

.(20

05a)

VIf

3*R

ifM

ts.(

Mor

occo

)A

M26

9888

Thi

sst

udy

422 Short Notes

Tabl

e1.

(Con

tinue

d).

Hap

loty

peFr

eque

ncy

Geo

grap

hic

Dis

trib

utio

nA

cces

sion

Ref

eren

ceN

umbe

r

VII

a27

(2or

3)*A

rmen

ia(A

raxe

sR

iver

),A

zerb

aija

n,*G

ilan,

*Maz

anda

ran

and

*Gol

esta

n(I

ran)

;a

doub

tful

reco

rdex

ists

for

the

regi

onbe

twee

nV

olga

and

Ura

lR

iver

s(n

orth

ern

Cas

pian

Sea,

Kaz

akhs

tan)

,see

text

AJ1

3142

5L

enk

etal

.(19

99)

VII

b1

Unk

now

nA

J131

426

Len

ket

al.(

1999

)V

IIc

2*A

zerb

aija

n,*M

azan

dara

n(I

ran)

AM

2698

89T

his

stud

yV

IId

1*M

azan

dara

n(I

ran)

AM

2698

90T

his

stud

yV

IIe

1*A

zerb

aija

nA

M26

9891

Thi

sst

udy

VII

f2

*Aze

rbai

jan,

*Gol

esta

n(I

ran)

AM

2698

92T

his

stud

yV

IIg

1*G

ilan

(Ira

n)A

M26

9893

Thi

sst

udy

VII

Ia2

Ana

mur

(sou

ther

nT

urke

y)A

Y65

2878

Fritz

etal

.(20

04,2

005a

)IX

a1

(1)

Unk

now

nA

Y65

2887

Fritz

etal

.(20

04,2

005a

)

Tota

l11

07(3

57to

361)

Short Notes 423

Figure 1. Parsimony network (TCS, spring tree) of all 56known mtDNA haplotypes of Emys orbicularis (haplotypesof lineages I-II, IV-IX) and Emys trinacris (haplotypes oflineage III). Branch lengths correspond to inferred numberof nucleotide changes along each branch; open circles anddots, identified or hypothesized haplotypes, respectively.Each line between circles or dots indicates one substitution;circle size corresponds to approximate frequency of haplo-types (table 1); size classes: 1, 2-9, 10-25, 26-50, 51-150,151-200, >200 recorded haplotypes.

Lenk et al. (1999) and Fritz et al. (2005a) in thata high diversity of distinct mtDNA lineages andhaplotypes occurs in southern regions where pu-tative glacial refuges were located (fig. 3, ta-ble 1). With the advent of Holocene warming,more northerly parts of Europe and adjacentAsia were rapidly recolonized by E. orbicu-laris from few refuges in the Balkans and thenorthern Black Sea/northern Caucasus region,resulting in the respective mtDNA lineages indecreasing haplotype diversity with increasingdistance to the refuge (long distance dispersalmodel of Hewitt, 1996); most refuges did notcontribute to northern recolonizations.

With exception of a doubtful record of haplo-type VIIa, the northernmost parts of the range ofE. orbicularis are occupied only by three hap-lotypes (Ia, IIa and IIb), while lineages I andII exhibit a much higher diversity in the south.The same pattern, suggestive of Holocene range

Figure 2. Strict consensus of six equally parsimonioustrees of all Emys haplotypes using sequences of Actinemysmarmorata (accession numbers AJ131430, U81344) andEmydoidea blandingii (AF258869, AJ131432) as outgroup(CI = 0.7585, RI = 0.9120, RC = 0.6917; 207 steps). Num-bers above nodes are bootstrap values (1000 resamplings)greater than 50. The six equally parsimonious trees differonly in the branching pattern of lineage IV haplotypes.

expansions on a smaller scale, is observed inlineages IV and V with several endemic haplo-types in the southernmost parts of their ranges;northern portions harbour only haplotypes IVaor Va. A previous record of haplotype VIIa forthe northern Caspian Sea region (Lenk et al.,1999) should be treated with care. It was basedon a turtle kept in the zoological garden of Al-maty (Kazakhstan), and we cannot exclude lo-

424 Short Notes

Figure 3. Geographic distribution of mtDNA lineages in Emys orbicularis (I-II, IV-IX) and Emys trinacris (III). Neighbouringlocalities combined; overlapping symbols indicate syntopic occurrence of distinct lineages; question marks, doubtful recordsfor lineage III in Calabria (southern Italy) and lineage VII in the northern Caspian region. Evidently allochthonous specimensnot considered.

cality confusion. Otherwise, lineage VII occursfar away, in the eastern central Caucasus andalong the south coast of the Caspian Sea (fig. 3,table 1).

The wide distribution of lineage I haplotypesaround the Black Sea suggests that the entireBlack Sea region served as glacial refuge. Onthe other hand, the localized distribution of en-demic lineage I haplotypes (table 1) indicatesthat distinct microrefuges existed there. An un-expected finding was haplotype Ia in easternGeorgia (regions of Gori and Tbilisi: one recordeach from Kareli, Gldanula River and Udabno).These sites are situated in the Kura River sys-tem that drains into the Caspian Sea. In the sameriver system occurs another mitochondrial lin-eage (VII) farther east, suggesting that lineagesI and VII meet somewhere in the central Cauca-sus. This situation is echoed in the phylogeogra-phy of Testudo graeca. Also in this species twodistinct mitochondrial lineages meet or occur in

closest neighbourhood in the central Caucasus(Fritz et al., 2007).

Besides the wide-spread haplotype IIa, themost differentiated haplotype of lineage II(IIf) occurs on the south-eastern Balkan Penin-sula (table 1), suggesting the refuge for lin-eage II was located there. From the south-eastern Balkans, lineage II most probably spreadalong the valleys of the Vardar and JužnaMorava Rivers northward to the Danube catch-ment basin from where central and western Eu-rope (Fritz et al., 2005b) and, via the MoravianGate, the Oder River Basin (Germany, Poland)were reached.

As for lineage IV, displaying a classic circum-Adriatic distribution, two distinct refuges ex-isted in southern Italy and the southernmostBalkan Peninsula. The lack of the common hap-lotype IVa, distributed in Italy, Istria, Dalma-tia, Corfu and Evvia, and the occurrence of en-demic haplotypes in the Peloponnesus and on

Short Notes 425

Cephalonia (IVb, IVc, IVg) provide evidencefor the colonization of the west coast of theBalkans, Corfu and Evvia from southern Italyand not from the geographically closer southernBalkanic refuge (Fritz et al., 2005a).

Like in Mauremys leprosa (Fritz et al.,2006a), mountain chains constitute major bio-geographic barriers for mtDNA lineages in E.orbicularis (fig. 3). The Pyrenees separate theIbero-Maghrebinian lineage VI from lineages IIand V in the north, and the Apennines sepa-rate lineages IV and V in Italy. In the BalkanPeninsula, the Dinarid and Pindos Mts. are abarrier between lineage IV along the west coastand lineages I and II in the east, and south ofthe Alps occur lineages IV and V while in thenorth lineage II is distributed. In East Europethe Carpathians act as barrier between lineageII in the south and lineage I in the north; far-ther eastward the Greater Caucasus separateslineages I and VII and the Taurus Mts. (Turkey)divide lineages I and VIII. Syntopic occurrencesof distinct mtDNA lineages, most probably asresult of Holocene range expansions, are con-fined to narrow contact zones in close proximityof these mountain chains in the northern IberianPeninsula, France and the southern Apennineand Balkan Peninsulas. Obviously, marshlandsalong sea coasts and river courses were used ascolonization corridors during range expansions,enabling development of contact zones.

The basal position of the North African hap-lotypes VIc and VIf in the MP tree and inthe network branch of lineage VI (figs 1, 2)suggests that the Iberian haplotypes VIa, VIb,VId and VIe are derived from North Africanhaplotypes. This implies a complicated colo-nization history, from Europe to North Africaand back to Europe, as recently proposed forthe ribbed newt Pleurodeles waltl (Veith etal., 2004). Denser sampling is still needed inNorth Africa however, especially in Algeria andTunisia. Some Moroccan amphibians and rep-tiles that occur in habitats resembling those of E.orbicularis (Pleurodeles waltl, Mauremys l. lep-rosa) are replaced in eastern Algeria and Tunisia

by other taxa (P. nebulosus, P. poireti, M. l. sa-harica, Carranza and Arnold, 2003; Carranzaand Wade, 2004; Veith et al., 2004; Fritz et al.,2006a; distinct mitochondrial lineages of Natrixmaura, Guicking et al., 2006), suggestive of asimilar pattern in E. orbicularis. Further sam-pling is also needed in Turkey, Turkmenistan, inthe Caucasus and in the northern Black Sea re-gion to reveal haplotype diversity and potentialcontact zones there; one of these regions mustharbour lineage IX.

Besides mtDNA haplotyping, future researchshould focus on gene flow along contact zonesof distinct mitochondrial lineages. A promisingapproach will be using microsatellites as mark-ers.

Acknowledgements. Thanks for lab work and curation ofsamples go to A. Hundsdörfer, Ch. Kehlmaier, A. Müller,and H. Sauer-Gürth. Blood or tissue samples were pro-vided by B. Andreas, M. Barata, J. Budó, H. Bringsøe, X.Capalleras, R. Castilho, J.-M. Ducotterd, O. Homeier, M.Korn, M. Kuprian, M. Meeske, K.D. Milto, S. Mitrus, D.Mosimann, M. Nembrini, N.L. Orlov, J.F. Parham, R. Pod-loucky, N. Schneeweiß, J. Schwarzendrube, P. Segurado,R. Stampfer, D. Tarknishvili, and R. Wicker; M. Fischerand J.-M. Lange assisted with the production of the map.Georg Džukic’s work for this study was supported by theSerbian Ministry of Science and Environmental Protection(“Patterns of amphibian and reptile diversity on the BalkanPeninsula”, Grant 143052).

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Received: May 29, 2006. Accepted: November 17, 2006.