Amphibia-Reptilia 28 (2007): 97-121 - iucn-tftsg.org · Amphibia-Reptilia 28 (2007): 97-121 Phenotypic plasticity leads to incongruence between morphology-based taxonomy and genetic

Amphibia-Reptilia 28 (2007): 97-121

Phenotypic plasticity leads to incongruence betweenmorphology-based taxonomy and genetic differentiation in

western Palaearctic tortoises (Testudo graeca complex;Testudines, Testudinidae)

Uwe Fritz1, Anna K. Hundsdörfer1, Pavel Široký2, Markus Auer1, Hajigholi Kami3, Jan Lehmann4,

Lyudmila F. Mazanaeva5, Oguz Türkozan6, Michael Wink7

Abstract. Tortoises of the Testudo graeca complex inhabit a patchy range that covers part of three continents (Africa, Europe,Asia). It extends approximately 6500 km in an east-west direction from eastern Iran to the Moroccan Atlantic coast andabout 1600 km in a north-south direction from the Danube Delta to the Libyan Cyrenaica Peninsula. Recent years haveseen a rapid increase of recognized taxa. Based on morphological investigations, it was suggested that this group consistsof as many as 20 distinct species and is paraphyletic with respect to T. kleinmanni sensu lato and T. marginata. Based onsamples from representative localities of the entire range, we sequenced the mitochondrial cytochrome b gene and conductednuclear genomic fingerprinting with ISSR PCR. The T. graeca complex is monophyletic and sister to a taxon consistingof T. kleinmanni sensu lato and T. marginata. The T. graeca complex comprises six well-supported mtDNA clades (A-F).Highest diversity is found in the Caucasian Region, where four clades occur in close neighbourhood. This suggests, inagreement with the fossil record, the Caucasian Region as a radiation centre. Clade A corresponds to haplotypes from theEast Caucasus. It is the sister group of another clade (B) from North Africa and western Mediterranean islands. Clade Cincludes haplotypes from western Asia Minor, the southeastern Balkans and the western and central Caucasus Region. Itssister group is a fourth, widely distributed clade (D) from southern and eastern Asia Minor and the Levantine Region (NearEast). Two further clades are distributed in Iran (E, northwestern and central Iran; F, eastern Iran). Distinctness of these sixclades and sister group relationships of (A + B) and (C + D) are well-supported; however, the phylogeny of the resultingfour clades (A + B), (C + D), E and F is poorly resolved. While in a previous study (Fritz et al., 2005a) all traditionallyrecognized Testudo species were highly distinct using mtDNA sequences and ISSR fingerprints, we detected within the T.graeca complex no nuclear genomic differentiation paralleling mtDNA clades. We conclude that all studied populations ofthe T. graeca complex are conspecific under the Biological Species Concept. There is major incongruence between mtDNAclades and morphologically defined taxa. Morphologically well-defined taxa, like T. g. armeniaca or T. g. floweri, nest withinclades comprising also geographically neighbouring, but morphologically distinctive populations of other taxa (clade A:T. g. armeniaca, T. g. ibera, T. g. pallasi; clade D: T. g. anamurensis, T. g. antakyensis, T. g. floweri, T. g. ibera, T. g.terrestris), while sequences of morphologically similar tortoises of the same subspecies (T. g. ibera sensu stricto or T. g. iberasensu lato) scatter over two or three genetically distinct clades (A, C or A, C, D, respectively). This implies that pronouncedmorphological plasticity, resulting in phenotypes shaped by environmental pressure, masks genetic differentiation. To achievea more realistic taxonomic arrangement reflecting mtDNA clades, we propose reducing the number of T. graeca subspeciesconsiderably and regard in the eastern part of the range five subspecies as valid (T. g. armeniaca, T. g. buxtoni, T. g. ibera, T.g. terrestris, T. g. zarudnyi). As not all North African taxa were included in the present study, we refrain from synonymizingNorth African taxa with T. g. graeca (mtDNA clade B) that represents a further valid subspecies.

1 - Museum of Zoology (Museum für Tierkunde), Nat-ural History State Collections Dresden, KönigsbrückerLandstr. 159, D-01109 Dresden, Germanye-mail: [email protected]

2 - Department of Biology and Wildlife Diseases, Facultyof Veterinary Hygiene and Ecology, University of Vet-erinary and Pharmaceutical Sciences, Palackého 1-3,CZ-612 42 Brno, Czech Republic

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

4 - Haspekrogen 10, DK-2880 Bagsværd, Denmark

Introduction

Tortoises of the genus Testudo Linnaeus, 1758are distributed over most of the southwestern

5 - Department of Zoology, Dagestan State University, 37aM. Gadyeva st., 367025 Makhachkala, Russia

6 - Department of Biology, Faculty of Science and Arts,Adnan Menderes University, 09010 Aydın, Turkey

7 - Institute of Pharmacy and Molecular Biotechnology(IPMB), Heidelberg University, INF 364, D-69120 Hei-delberg, Germany

© Koninklijke Brill NV, Leiden, 2007. Also available online - www.brill.nl/amre

98 U. Fritz et al.

Palaearctic Region (Ernst et al., 2000; Fritz andCheylan, 2001). The greatest portion of thisarea is inhabited by representatives of the Tes-tudo graeca complex. These chelonians are alsoknown as spur-thighed tortoises, a name refer-ring to two prominent horny thigh tuberclespresent in most individuals. The patchy rangeof spur-thighed tortoises covers part of threecontinents (Africa, Europe, Asia) and extendsapproximately 6500 km in an east-west direc-tion from eastern Iran to the Moroccan Atlanticcoast and about 1600 km in a north-south direc-tion from the Danube Delta to the Libyan Cyre-naica Peninsula (fig. 1). Spur-thighed tortoisesoccur under very different climatic and envi-ronmental conditions, ranging from a Mediter-ranean climate with mild, frost-free winters toan extreme continental steppe climate with se-vere winter frost.

While T. graeca Linnaeus, 1758 was longaccepted as single polytypic species with fourto seven subspecies (Mertens, 1946; Wermuth,1958; Wermuth and Mertens, 1961, 1977;Anderson, 1979; Ernst and Barbour, 1989;Gasperetti et al., 1993; Fritz et al., 1996; Ernst etal., 2000; Buskirk et al., 2001; Fritz and Chey-lan, 2001), its taxonomy has fluctuated greatlyin recent years. Based on morphology, manyspecies and subspecies were described or resur-rected, in part in grey literature (Chkhikvadzeand Tuniyev, 1986; Weissinger, 1987; High-field and Martin, 1989a, b, c; Highfield, 1990;Chkhikvadze and Bakradze, 1991, 2002; Perälä,1996, 2002a; Pieh, 2001; Pieh and Perälä, 2002,2004; van der Kuyl et al., 2002; table 1), andsome authors suggested that as many as 20 dis-tinct species were traditionally lumped underT. graeca (Bour, 1989; Highfield and Martin,1989a, b, c; Highfield, 1990; Gmira, 1993a, b,1995; David, 1994; Pieh, 2001; Perälä, 2002a,b; Pieh and Perälä, 2002, 2004; see also Guyot-Jackson, 2004). In contrast, Fritz et al. (1996)argued that most taxa within the T. graeca com-plex are badly defined and that two distinct lin-eages exist, one including the populations ofsmall to moderately sized tortoises with light

coloration occurring in Spain, North Africa andalong the Levantine coast, and a second lin-eage consisting of the populations with largerand darker coloured tortoises from the rest ofthe range. Gmira (1993a, b, 1995) and Perälä(2002b) proposed that species of the T. graecacomplex are paraphyletic with respect to twoother traditional Testudo species that were neverconsidered part of the T. graeca complex (T.kleinmanni Lortet, 1883 sensu lato, T. mar-ginata Schoepff, 1792). As a consequence, sys-tematics and taxonomy in these tortoises be-came a much debated field (Gasperetti et al.,1993; Ernst et al., 2000; Buskirk et al., 2001;Fritz and Cheylan, 2001; van der Kuyl et al.,2002, 2005; Harris et al., 2003; Guyot-Jackson,2004; Perälä, 2004a; Semyenova et al., 2004;Carretero et al., 2005; Korsunenko et al., 2005).In this study we treat all taxa within the T.graeca complex provisionally as subspecies ofT. graeca.

Some recent studies on mtDNA sequencevariation of T. graeca, mainly in the westernMediterranean (Álvarez et al., 2000; van derKuyl et al., 2002, 2005; Harris et al., 2003), usedthe slowly evolving mitochondrial 12S rRNAgene and in part 426 bp of the cytochrome b

gene (Álvarez et al., 2000) and 411 bp of the D-loop (van der Kuyl et al., 2005). These studiessuggested distinctly less taxonomic differentia-tion than morphological investigations. In con-trast, Semyenova et al. (2004) and Korsunenkoet al. (2005) found four eastern subspecies (T.g. ibera, T. g. nikolskii, T. g. pallasi, T. g.terrestris) distinct using nuclear genomic fin-gerprinting (Randomly Amplified PolymorphicDNA = RAPDs). If it is considered that RAPDsmay reflect population-specific, and not taxon-specific, differentiation (e.g. Haig et al., 1994,1997; Prior et al., 1997; Tassanakajon et al.,1997; Gomes et al., 1998; Vanlerberghe-Masuttiand Chavigny, 1998; Palkovacs et al., 2003),these contradictory findings are not surprising.

Here we present for the first time a range-wide phylogeography for the T. graeca com-plex that includes most nominal taxa (table 1).

Testudo graeca complex 99

Figure 1. Range of the Testudo graeca complex (shaded) and collection sites of samples. Neighbouring localities combined;for exact localities see table 2. Symbols correspond to distinct mtDNA clades. Range according to Gasperetti et al. (1993)and Buskirk et al. (2001); some records of introduced tortoises omitted.

We used the mitochondrial cytochrome b genethat in testudinids is phylogenetically more in-formative than the 12S rRNA gene and a power-ful tool for revealing their matrilineal differen-tiation (Caccone et al., 1999; Palkovacs et al.,2002; Austin et al., 2003; Fritz et al., 2005a,2006). To find out whether gene flow takes placebetween populations harbouring different ma-trilineages, we applied ISSR (Inter-Simple Se-quence Repeat) nuclear genomic fingerprinting,a technique that has been shown to be useful forTestudo species (Fritz et al., 2005a) and otherchelonians as well (Wink et al., 2001; Guick-ing et al., 2002; Schilde et al., 2004; Fritz et al.,2005b). ISSR PCR produces species-specificnuclear fingerprints in a wide range of organ-isms (e.g. Gupta et al., 1994; Zietkiewicz et al.,1994; Wink et al., 1998; Bornet and Branchard,2001; Fritz et al., 2005a, b; Hundsdörfer andWink, 2005), allowing identification of inter-specific hybrids (Wink et al., 2001; Schilde etal., 2004), and of gene flow and introgression(Wolfe et al., 1998; Nagy et al., 2003; Fritz etal., 2005b).

Materials and methods

Sampling

We studied 94 samples from localities covering most of thedistribution range of the Testudo graeca complex (fig. 1;table 2). Blood samples were obtained by coccygeal veinpuncture of wild or captive tortoises. Alternatively, muscletissue was extracted from thighs of frozen carcasses priorto alcohol preservation. Samples were either preserved inan EDTA buffer (0.1 M Tris, pH 7.4, 10% EDTA, 1%NaF, 0.1% thymol) or in ethanol, and stored at −20◦C untilprocessing. Remaining blood, tissue and DNA samples arepermanently kept at −80◦C in the blood and tissue samplecollection of the Museum of Zoology, Dresden.

DNA extraction, marker gene amplification and sequencing

Total genomic DNA was isolated by an overnight incubationat 37◦C in lysis buffer (10 mM Tris, pH 7.5, 25 mMEDTA, 75 mM NaCl, 1% SDS) including 1 mg of proteinaseK (Roth or Merck), followed by purification with eitherthe DTAB method (Gustincich et al., 1991) or a phenol-chloroform protocol (Sambrook et al., 1989). The primersmt-A1 5′-CCC CCT ACC AAC ATC TCA GCA TGA TGAAAC TTC G-3′ or mt-a-neu2 5′-CTC CCA GCC CCA TCCAAC ATC TCA GCA TGA TGA AAC-3′ and mtf-na 5′-AGG GTG GAG TCT TCA GTT TTT GGT TTA CAAGAC CAA TG-3′ or mt-Fr 5′-CTA AGA AGG GTG GAGTCT TCA GTT TTT GGT TTA CAA-3′ were used foramplification of an approximately 1150 bp long mtDNA

100 U. Fritz et al.Ta

ble

1.Ta

xaof

the

Test

udo

grae

caco

mpl

ex.S

ever

alau

thor

sbe

lieve

that

furt

her

taxa

exis

tbut

assi

gnno

nam

es(t

wo

addi

tiona

lLev

antin

eta

xaw

ithin

T.g.

anta

kyen

sis:

Perä

lä,2

002a

,b;

furt

her

taxa

with

inT.

g.ib

era:

Perä

lä,2

002b

;Pie

het

al.,

2002

;afu

rthe

rta

xon

inT

urkm

enia

:Pie

han

dPe

rälä

,200

1).I

tis

deba

ted

whe

ther

the

nam

eTe

stud

ow

hite

iBen

nett

inW

hite

,183

6is

appl

icab

leto

any

Nor

thA

fric

anpo

pula

tion

orno

t(Pi

ehan

dPe

rälä

,200

2,20

04;v

ande

rK

uyle

tal.,

2002

,200

5;Pe

rälä

,200

4a).

Pieh

and

Perä

lä(2

002,

2004

)tr

eatT

.whi

teia

sdo

ubtf

ulna

me

that

cann

otbe

allo

cate

dto

ace

rtai

npo

pula

tion,

whi

leva

nde

rK

uyl

etal

.(20

02,

2005

)us

eT.

g.w

hite

ifo

rse

vera

lN

orth

Afr

ican

popu

latio

nsfr

omw

ithin

the

rang

eof

othe

rta

xa.

Mor

eove

r,va

nde

rK

uyle

tal.

(200

2)pr

opos

edth

atSa

rdin

ian

T.gr

aeca

mig

htre

pres

enta

nad

ditio

nals

ubsp

ecie

sal

thou

ghth

eydi

dno

trep

ortm

orph

olog

ical

orge

netic

dist

inct

ion.

Val

idity

ofTe

stud

ofla

vom

inim

aral

isH

ighfi

eld

and

Mar

tin,1

989

isge

nera

llyno

tac

cept

ed(E

rnst

etal

.,20

00;

Bus

kirk

etal

.,20

01;

Pieh

and

Perä

lä,2

002,

2004

;va

nde

rK

uyl

etal

.,20

02,2

005)

.(+

)Sa

mpl

esof

resp

ectiv

eta

xon

stud

ied

for

pres

ent

pape

r,(−

)no

tst

udie

d,(?

)un

clea

r,se

eab

ove.

MSL

=m

axim

umsh

ell

leng

th.M

SLfo

rTe

stud

ogr

aeca

grae

cafr

omPi

ehan

dPe

rälä

(200

4),f

orT.

g.ib

era

sens

ula

tofr

omB

uski

rket

al.(

2001

),fo

rT.

g.la

mbe

rti

and

T.g.

mar

okke

nsis

from

Pieh

and

Perä

lä(2

004)

,and

for

T.g.

whi

tei

from

Ern

stet

al.(

2000

);ot

her

data

from

Guy

ot-J

acks

on(2

004)

.Dis

trib

utio

nra

nges

asde

fined

inor

igin

alde

scri

ptio

ns,i

fno

toth

erw

ise

stat

ed.

Taxo

nM

SL(m

m)

MtD

NA

clad

eTy

pelo

calit

yA

ppro

xim

ate

rang

e

+Te

stud

ogr

aeca

anam

uren

sis

Wei

ssin

ger,

1987

267

DA

nam

urum

,sou

ther

nT

urke

yC

entr

alM

edite

rran

ean

coas

tof

Tur

key

+Te

stud

ogr

aeca

anta

kyen

sis

Perä

lä,1

996

250

DA

ntak

ya,s

outh

east

ern

Tur

key

Lev

ant

with

out

cent

ral

and

sout

hern

coas

tal

part

(Per

älä,

2002

a)+

Test

udo

grae

caar

men

iaca

Chk

hikv

adze

and

Bak

radz

e,19

9125

8A

Meg

ri,s

outh

east

ern

Arm

enia

Ara

xes

Val

ley

and

adja

cent

regi

ons

inT

urke

y,A

rmen

ia,

Aze

rbai

jan,

and

Iran

(Pie

het

al.,

2002

)+

Test

udo

grae

cabu

xton

iBou

leng

er,1

921

267

EM

anjil

,bet

wee

nR

esht

and

Kas

win

,Ira

nSo

uthw

este

rnco

rner

ofC

aspi

anSe

a,Ir

an(P

eräl

ä,20

04b)

+Te

stud

ogr

aeca

cyre

naic

aPi

ehan

dPe

rälä

,200

220

5B

Dar

nah,

east

ern

Lib

yaC

yren

aica

Peni

nsul

a,L

ibya

+Te

stud

ogr

aeca

flow

eriB

oden

heim

er,1

935

154

DN

egev

,pro

babl

yvi

cini

tyof

Gaz

a,Pa

lest

ine

(res

tric

ted

byB

our,

1989

,but

see

Perä

läan

dSh

acha

m,2

004)

Coa

stal

plai

nsof

Gaz

a,Is

rael

and

Leb

anon

(Per

älä,

2002

a;Pe

rälä

and

Shac

ham

,200

4)

+Te

stud

ogr

aeca

grae

caL

inna

eus,

1758

belo

w21

4B

Sant

aC

ruz

near

Ora

n,A

lger

ia(r

estr

icte

dby

Mer

tens

and

Mül

ler,

1928

)N

orth

east

ern

Mor

occo

(eas

tern

part

ofM

edite

rran

ean

coas

tan

dad

jace

ntin

land

regi

ons)

,A

lger

ia(P

ieh

and

Perä

lä,

2004

)+

Test

udo

grae

caib

era

Palla

s,18

14se

nsu

stri

cto:

260

sens

ula

to:

358

sens

ust

rict

o:A

,Cse

nsu

lato

:A

,C,D

Tbi

lisi,

Geo

rgia

(des

igna

ted

byB

our,

1987

)Se

nsu

stri

cto:

Kur

aR

iver

Bas

in,

Cau

casu

s(P

eräl

ä,20

02a;

Dan

ilov

and

Milt

o,20

04);

iber

ase

nsu

lato

incl

udes

also

sout

heas

tern

Eur

ope

and

vast

part

sof

Asi

aM

inor

(Bus

kirk

etal

.,20

01)

−Te

stud

ogr

aeca

lam

bert

iPie

han

dPe

rälä

,200

421

4?

22km

nort

hof

Tét

ouan

,Mor

occo

Nor

ther

nM

oroc

co(w

este

rnpa

rtof

Med

iterr

anea

nco

ast)

−Te

stud

ogr

aeca

mar

okke

nsis

Pieh

and

Perä

lä,

2004

237

?Ta

rmile

te,M

oroc

coN

orth

wes

tern

Mor

occo

(Atla

ntic

coas

tan

dad

jace

ntin

land

regi

on)


Tabl

e1.

(Con

tinue

d).

Taxo

nM

SL(m

m)

MtD

NA

clad

eTy

pelo

calit

yA

ppro

xim

ate

rang

e

+Te

stud

ogr

aeca

nabe

ulen

sis

(Hig

hfiel

d,19

90)

belo

w18

0B

Fore

st7-

8km

nort

hwes

tof

Nab

eul,

indi

-re

ctio

nof

Gro

mba

lia,T

unis

ia(r

estr

icte

dby

Pieh

and

Perä

lä,2

004)

Tun

isia

,adj

acen

tLib

ya(P

ieh

and

Perä

lä,2

002)

+Te

stud

ogr

aeca

niko

lski

iC

hkhi

kvad

zean

dT

u-ni

yev,

1986

297

CN

ebug

Settl

emen

t,T

uaps

eC

ount

y,K

rasn

odar

Dis

tric

t,R

ussi

aN

orth

ern

Bla

ckSe

aco

ast

ofR

ussi

aan

dG

eorg

ia(B

uski

rket

al.,

2001

)+

Test

udo

grae

capa

llas

iC

hkhi

kvad

zean

dB

akra

dze,

2002

247

AG

ilyar

y-D

agSe

ttlem

ent,

Dag

esta

n,R

ussi

aC

aspi

anSe

aco

asta

lare

ain

Dag

esta

nan

dad

jace

ntA

zerb

ai-

jan

(Dan

ilov

etal

.,20

04)

+Te

stud

ogr

aeca

pers

esPe

rälä

,200

224

0E

,F3

mile

sW

Lal

abad

Vill

age,

25m

iles

NW

Ker

man

shah

,Ker

man

shah

anPr

ovin

ce,

wes

tern

cent

ralI

ran

Zag

ros

Mou

ntai

ns(I

ran)

and

adja

cent

mou

ntai

nch

ains

−Te

stud

ogr

aeca

sous

sens

isPi

eh,2

001

249

?A

gadi

r,M

oroc

coSo

uthw

este

rnM

oroc

co,

regi

onof

Mar

rake

chan

dre

gion

sout

hwes

tof

Hig

hA

tlas

Mou

ntai

ns(P

ieh

and

Perä

lä,2

004)

+Te

stud

ogr

aeca

terr

estr

isFo

rssk

ål,1

775

254

DA

lepp

o,Sy

ria

(by

neot

ype

desi

gnat

ion;

Perä

läan

dB

our,

2004

)N

orth

ern

Mes

opot

amia

(Bou

ran

dPe

rälä

,200

4;Pe

rälä

and

Bou

r,20

04)

?Te

stud

ogr

aeca

whi

teiB

enne

ttin

Whi

te,1

836

292

?A

lgie

rsan

dits

envi

rons

,A

lger

ia(d

esig

-na

ted

byH

ighfi

eld

and

Mar

tin,

1989

b,bu

tse

ere

mar

ks)

See

rem

arks

+Te

stud

ogr

aeca

zaru

dnyi

Nik

olsk

y,18

9627

5F

Bır

jand

,K

hora

san

Prov

ince

,no

rthe

aste

rnIr

an(r

estr

icte

dby

Perä

lä,2

002a

)E

aste

rnIr

an(P

eräl

ä,20

02a)

102 U. Fritz et al.Ta

ble

2.St

udie

dsa

mpl

esof

the

Test

udo

grae

caco

mpl

exan

dou

tgro

ups.

Que

stio

nm

arks

deno

teun

cert

ain

taxo

nom

ical

loca

tions

due

toco

llect

ion

site

sal

ong

rang

ebo

rder

s(f

orA

diya

man

,T

urke

y,se

eT

ürko

zan

etal

.,20

03).

MT

DD

num

bers

refe

rto

com

plet

evo

uche

rsp

ecim

ens,

MT

DT

num

bers

tobl

ood,

tissu

eor

DN

Asa

mpl

esin

the

colle

ctio

nof

the

Mus

eum

ofZ

oolo

gy,

Dre

sden

.Pro

visi

onal

HD

num

bers

ofsa

mpl

esst

udie

dby

Fritz

etal

.(20

05a)

repl

aced

bype

rman

entM

TD

num

bers

.Acc

essi

onnu

mbe

rsre

fer

tom

tDN

Ase

quen

ces.

Sam

ple

Taxo

nL

ocal

ityM

TD

Acc

essi

onnu

mbe

rIS

SR(G

AA

) 5

ISSR

(GA

CA

) 4

ISSR

L18

AN

AM

UR

EN

SIS

1Te

stud

ogr

aeca

anam

uren

sis

Tur

key:

Ana

mur

um;3

6◦03

′ N32

◦ 51′

ET

257

AJ8

8834

7A

NA

MU

RE

NSI

S2

Test

udo

grae

caan

amur

ensi

sT

urke

y:G

azip

asa;

36◦ 1

7′N

32◦ 1

8′E

T25

9A

J888

348

AN

AM

UR

EN

SIS

3Te

stud

ogr

aeca

anam

uren

sis

Tur

key:

Gaz

ipas

a;36

◦ 17′

N32

◦ 18′

ET

258

AM

2309

46A

NA

MU

RE

NSI

S4

Test

udo

grae

caan

amur

ensi

sT

urke

y:Sa

hayi

Sitig

eS

Seri

kan

dA

spen

dos;

36◦ 5

0′02

′′ N31

◦ 09′

01′′ E

T15

1A

J888

356

++

+A

NA

MU

RE

NSI

S5

Test

udo

grae

caan

amur

ensi

sT

urke

y:Si

de;3

6◦46

′ 52′′

N31

◦ 23′

56′′ E

T61

0A

M23

1002

++

+A

NA

MU

RE

NSI

S6

Test

udo

grae

caan

amur

ensi

sT

urke

y:Si

de;3

6◦47

′ 18′′

N31

◦ 24′

25′′ E

T60

9A

M23

0948

++

+A

NA

MU

RE

NSI

S7

Test

udo

grae

caan

amur

ensi

sT

urke

y:W

Side

;36◦

49′ N

31◦ 2

1′E

T25

3A

M23

1003

++

AN

AM

UR

EN

SIS

8Te

stud

ogr

aeca

anam

uren

sis

Tur

key:

WSi

de;3

6◦49

′ N31

◦ 21′

ET

254

AM

2310

04+

++

?A

NA

MU

RE

NSI

S9

Test

udo

grae

caan

amur

ensi

s?T

urke

y:M

ersi

n;36

◦ 49′

N34

◦ 39′

ET

260

AM

2310

05A

NTA

KY

EN

SIS

1Te

stud

ogr

aeca

anta

kyen

sis

Isra

el:T

iber

ias;

32◦ 4

8′N

35◦ 3

1′E

T16

44A

M23

0947

AN

TAK

YE

NSI

S2

Test

udo

grae

caan

taky

ensi

sIs

rael

:Tib

eria

s;32

◦ 48′

N35

◦ 31′

ET

1376

AJ8

8834

4A

NTA

KY

EN

SIS

3Te

stud

ogr

aeca

anta

kyen

sis

Isra

el:T

iber

ias;

32◦ 4

8′N

35◦ 3

1′E

T13

77A

J888

345

AN

TAK

YE

NSI

S4

Test

udo

grae

caan

taky

ensi

sJo

rdan

:Jar

ash;

32◦ 1

1′N

35◦ 5

1′E

T13

80A

J888

346

AN

TAK

YE

NSI

S5

Test

udo

grae

caan

taky

ensi

sSy

ria:

Ant

iLeb

anon

Mts

.(Ja

bale

shSh

arqi

):M

a’lu

la;3

3◦51

′ N36

◦ 33′

ET

2179

AM

2309

49A

NTA

KY

EN

SIS

6Te

stud

ogr

aeca

anta

kyen

sis

Syri

a:A

ntiL

eban

onM

ts.(

Jaba

lesh

Shar

qi):

Sayd

naya

;33◦

42′ N

36◦ 2

2′E

T21

74A

M23

0950

AN

TAK

YE

NSI

S7

Test

udo

grae

caan

taky

ensi

sSy

ria:

Jaba

lalN

usay

rıya

h:A

ynal

Bay

dah;

34◦ 5

9′N

36◦ 2

0′E

T21

83A

M23

0951

++

+A

NTA

KY

EN

SIS

8Te

stud

ogr

aeca

anta

kyen

sis

Syri

a:Ja

bala

lNus

ayrı

yah:

Jour

ine

(atQ

alat

Mer

za);

35◦ 3

9′N

36◦ 1

6′E

T21

88A

M23

0952

AN

TAK

YE

NSI

S9

Test

udo

grae

caan

taky

ensi

sSy

ria:

Jaba

lalN

usay

rıya

h:M

asya

f;35

◦ 04′

N36

◦ 21′

ET

2181

AM

2309

53+

++

AN

TAK

YE

NSI

S10

Test

udo

grae

caan

taky

ensi

sSy

ria:

Jaba

lDur

uz:A

lKaf

r;32

◦ 39′

N36

◦ 38′

ET

2169

AM

2309

54A

NTA

KY

EN

SIS

11Te

stud

ogr

aeca

anta

kyen

sis

Syri

a:Ja

balD

uruz

:As

Suw

ayda

’;32

◦ 43′

N36

◦ 35′

ET

2162

AM

2309

55A

NTA

KY

EN

SIS

12Te

stud

ogr

aeca

anta

kyen

sis

Syri

a:Ja

balD

uruz

:hal

f-w

ayA

sSu

way

da’

toSa

leh;

32◦ 4

1′N

36◦ 4

1′E

T21

63A

M23

0956

AR

ME

NIA

CA

Test

udo

grae

caar

men

iaca

Tur

key:

Ara

xes

Riv

erV

alle

y:V

ilaye

tIgd

ır:M

elek

li;39

◦ 57N

’44

◦ 06′

ET

2095

AM

2309

57+

++

BU

XTO

NI

1Te

stud

ogr

aeca

buxt

oni

Iran

:Now

shar

near

Man

jil;3

6◦44

′ 03′′

N49

◦ 25′

12′′ E

T22

60A

M23

0958

+B

UX

TON

I2

Test

udo

grae

cabu

xton

iIr

an:S

Res

ht:b

etw

een

Sara

van

and

Ros

tam

abad

;36◦

57′ N

49◦ 3

3′E

T22

65A

M23

0959

++

+B

UX

TON

I3

Test

udo

grae

cabu

xton

iIr

an:S

Res

ht:b

etw

een

Sara

van

and

Ros

tam

abad

;36◦

57′ N

49◦ 3

3′E

T22

67A

M23

0960

BU

XTO

NI

4Te

stud

ogr

aeca

buxt

oni

Iran

:SR

esht

:bet

wee

nSa

rava

nan

dR

osta

mab

ad;3

6◦57

′ N49

◦ 33′

ET

2268

AM

2309

61+

++

BU

XTO

NI

5Te

stud

ogr

aeca

buxt

oni

Iran

:Sefi

dR

ud;3

7◦22

′ 20′′

N48

◦ 08′

10′′ E

T19

99A

M23

0962

++

+C

YR

EN

AIC

ATe

stud

ogr

aeca

cyre

naic

aL

ibya

:Cyr

enai

ca,a

ppro

x.32

◦ 25′

N21

◦ 20′

ED

4281

9A

J888

341

++

+

Testudo graeca complex 103Ta

ble

2.(C

ontin

ued)

.

Sam

ple

Taxo

nL

ocal

ityM

TD

Acc

essi

onnu

mbe

rIS

SR(G

AA

) 5

ISSR

(GA

CA

) 4

ISSR

L18

FL

OW

ER

I1

Test

udo

grae

caflo

wer

iIs

rael

:Bet

Zev

i,K

arm

elM

ts.;

32◦ 4

3′N

34◦ 5

8′E

T13

74A

M23

0963

FL

OW

ER

I2

Test

udo

grae

caflo

wer

iIs

rael

:Bet

Zev

i,K

arm

elM

ts.;

32◦ 4

3′N

34◦ 5

8′E

T13

75A

M23

1006

FL

OW

ER

I3

Test

udo

grae

caflo

wer

iIs

rael

:Ros

hH

a’A

yin;

32◦ 0

7′N

34◦ 5

8′E

T13

78A

M23

0964

++

+F

LO

WE

RI

4Te

stud

ogr

aeca

flow

eri

Isra

el:R

osh

Ha’

Ayi

n;32

◦ 07′

N34

◦ 58′

ET

1379

AM

2310

07+

+G

RA

EC

ATe

stud

ogr

aeca

grae

caM

oroc

co:N

EO

tatO

ulad

elH

ajj;

33◦ 3

7′20

′′ N03

◦ 06′

39′′ W

T22

94A

M23

0965

++

+M

AL

LO

RC

ATe

stud

ogr

aeca

grae

cas.

l.Sp

ain:

Mal

lorc

a:N

Cal

vià;

39◦ 3

6′N

02◦ 3

1′E

D42

822

AJ8

8834

2SA

RD

INIA

Test

udo

grae

cagr

aeca

s.l.

Ital

y:Sa

rdin

ia:S

inis

Peni

nsul

a;ap

prox

.40◦

00′ N

08◦ 2

5′E

T11

13A

J888

343

SIC

ILY

1Te

stud

ogr

aeca

grae

cas.

l.It

aly:

Sici

ly:M

arsa

la;3

7◦48

′ N12

◦ 27′

ET

2230

AM

2309

66IB

ER

A1

Test

udo

grae

caib

era

Aze

rbai

jan:

Kat

ekh;

41◦ 3

9′N

46◦ 3

4′E

T14

61A

M23

1008

++

IBE

RA

2Te

stud

ogr

aeca

iber

aA

zerb

aija

n:K

atek

h;41

◦ 39′

N46

◦ 34′

ET

1462

AM

2310

09+

++

IBE

RA

3Te

stud

ogr

aeca

iber

aA

zerb

aija

n:SW

Bey

laqa

n,ne

arD

ashb

urun

;39◦

43′ N

47◦ 3

4′E

T14

46A

M23

0969

++

+IB

ER

A4

Test

udo

grae

caib

era

Aze

rbai

jan:

SWB

eyla

qan,

near

Das

hbur

un;3

9◦43

′ N47

◦ 34′

ET

1447

AM

2309

67+

+IB

ER

A5

Test

udo

grae

caib

era

Aze

rbai

jan:

SWB

eyla

qan,

near

Das

hbur

un;3

9◦43

′ N47

◦ 34′

ET

1448

AM

2309

68+

++

IBE

RA

6Te

stud

ogr

aeca

iber

aB

ulga

ria:

Alb

ena;

43◦ 2

2′27

′′ N28

◦ 05′

00′′ E

T72

4A

M23

1010

IBE

RA

7Te

stud

ogr

aeca

iber

aB

ulga

ria:

Alb

ena;

43◦ 2

2′53

′′ N28

◦ 04′

97′′ E

T72

5A

J888

349

IBE

RA

8Te

stud

ogr

aeca

iber

aB

ulga

ria:

Alb

ena;

43◦ 2

2′37

′′ N28

◦ 05′

14′′ E

T72

7A

J888

350

IBE

RA

9Te

stud

ogr

aeca

iber

aB

ulga

ria:

Piri

n;41

◦ 33′

N23

◦ 34′

ET

1365

AM

2310

11IB

ER

A10

Test

udo

grae

caib

era

Bul

gari

a:Ž

elez

ino;

41◦ 2

8′N

25◦ 5

6′E

T23

32A

M23

1012

IBE

RA

11Te

stud

ogr

aeca

iber

aG

eorg

ia:M

tskh

eta;

41◦ 5

0′N

44◦ 4

3′E

D40

654

AM

2310

13+

+IB

ER

A12

Test

udo

grae

caib

era

Gre

ece:

15km

EA

lexa

ndro

úpol

is;4

0◦50

′ N26

◦ 00′

ET

1615

AM

2310

14+

++

IBE

RA

13Te

stud

ogr

aeca

iber

aG

reec

e:be

twee

nD

rám

aan

dX

anth

i;41

◦ 11′

N24

◦ 37′

ET

1614

AM

2310

15+

++

IBE

RA

14Te

stud

ogr

aeca

iber

aG

reec

e:E

vros

Del

ta,a

ppro

x.25

kmE

Ale

xand

roúp

olis

;40◦

46′ N

26◦ 0

5′E

T16

16A

M23

1016

IBE

RA

15Te

stud

ogr

aeca

iber

aG

reec

e:K

osIs

land

;36◦

52′ 3

7′′N

27◦ 1

7′76

′′ ET

821

AJ8

8835

1IB

ER

A16

Test

udo

grae

caib

era

Gre

ece:

Kos

Isla

nd;3

6◦53

′ 36′′

N27

◦ 17′

49′′ E

T82

3A

J888

352

IBE

RA

17Te

stud

ogr

aeca

iber

aR

epub

licof

Mac

edon

ia:S

kopj

e;42

◦ 00′

N21

◦ 28′

ET

1975

AM

2310

17IB

ER

A18

Test

udo

grae

caib

era

Rom

ania

:His

tria

;44◦

35′ N

28◦ 4

2′E

T13

62A

M23

1018

++

+IB

ER

A19

Test

udo

grae

caib

era

Rom

ania

:His

tria

;44◦

35′ N

28◦ 4

2′E

T13

99A

M23

1019

++

+IB

ER

A20

Test

udo

grae

caib

era

Rom

ania

:His

tria

;44◦

35′ N

28◦ 4

2′E

T16

34A

M23

1020

IBE

RA

21Te

stud

ogr

aeca

iber

aT

urke

y:M

enem

en;3

8◦37

′ N27

◦ 04′

ET

268

AM

2310

21IB

ER

A22

Test

udo

grae

caib

era

Tur

key:

Seyd

iseh

ir;3

7◦25

′ 19′′

N31

◦ 50′

05′′ E

T15

0A

J888

355

++

+

104 U. Fritz et al.

Tabl

e2.

(Con

tinue

d).

Sam

ple

Taxo

nL

ocal

ityM

TD

Acc

essi

onnu

mbe

rIS

SR(G

AA

) 5

ISSR

(GA

CA

) 4

ISSR

L18

IBE

RA

23Te

stud

ogr

aeca

iber

aT

urke

y:L

ake

Van

:10

kmN

Van

;38◦

34′ N

43◦ 2

4′E

T38

2A

J888

354

++

IBE

RA

24Te

stud

ogr

aeca

iber

aT

urke

y:L

ake

Van

:Bud

akli;

38◦ 2

3′56

′′ N42

◦ 37′

56′′ E

T38

1A

M23

1022

+IB

ER

A25

Test

udo

grae

caib

era

Tur

key:

Lak

eV

an:K

üçük

su;3

8◦27

′ N42

◦ 20′

ET

380

AM

2310

23+

+IB

ER

A26

Test

udo

grae

caib

era

Tur

key:

Lak

eV

an:E

rcis

;39◦

02′ N

43◦ 2

3′E

T27

21A

J888

353

++

+SI

CIL

Y2

Test

udo

grae

caib

era

Ital

y:Si

cily

:Cam

pobe

llodi

Maz

ara;

37◦ 3

8′N

12◦ 4

5′E

T21

42A

M23

0970

NA

BE

UL

EN

SIS

1Te

stud

ogr

aeca

nabe

ulen

sis

Tun

isia

:Sou

sse

(Sus

ah);

35◦ 5

0′N

10◦ 3

9′E

T14

9A

M23

0971

NA

BE

UL

EN

SIS

2Te

stud

ogr

aeca

nabe

ulen

sis

Tun

isia

D42

893

AM

2309

72N

IKO

LSK

II1

Test

udo

grae

cani

kols

kii

Rus

sia:

NT

uaps

e;44

◦ 11′

N39

◦ 06′

ET

1808

AM

2309

73+

+N

IKO

LSK

II2

Test

udo

grae

cani

kols

kii

Rus

sia:

Sukk

o,SE

Ana

pa;4

4◦47

′ N37

◦ 23′

ET

1806

AM

2309

74+

PAL

LA

SI1

Test

udo

grae

capa

llas

iA

zerb

aija

n:K

olan

i;41

◦ 11′

N49

◦ 08′

ET

1450

AM

2309

75+

++

PAL

LA

SI2

Test

udo

grae

capa

llas

iA

zerb

aija

n:K

olan

i;41

◦ 11′

N49

◦ 08′

ET

1451

AM

2309

76+

+PA

LL

ASI

3Te

stud

ogr

aeca

pall

asi

Aze

rbai

jan:

Kol

ani;

41◦ 1

1′N

49◦ 0

8′E

T14

52A

M23

0977

++

PAL

LA

SI4

Test

udo

grae

capa

llas

iR

ussi

a:D

ages

tan:

Dag

esta

nski

eO

gni;

42◦ 0

7′N

48◦ 1

1′E

T23

88A

M23

0978

++

PAL

LA

SI5

Test

udo

grae

capa

llas

iR

ussi

a:D

ages

tan:

Dag

esta

nski

eO

gni;

42◦ 0

7′N

48◦ 1

1′E

T23

89A

M23

0979

++

+PA

LL

ASI

6Te

stud

ogr

aeca

pall

asi

Rus

sia:

Dag

esta

n:D

ages

tans

kie

Ogn

i;42

◦ 07′

N48

◦ 11′

ET

2390

AM

2309

80+

+PA

LL

ASI

7Te

stud

ogr

aeca

pall

asi

Rus

sia:

Dag

esta

n:Z

elen

omor

sk;4

2◦45

′ N47

◦ 42′

ET

873

AM

2310

00+

++

PAL

LA

SI8

Test

udo

grae

capa

llas

iR

ussi

a:D

ages

tan:

Zel

enom

orsk

;42◦

45′ N

47◦ 4

2′E

T87

4A

M23

1001

++

+?

PAL

LA

SI9

Test

udo

grae

capa

llas

i?A

zerb

aija

n:A

bshe

ron

Peni

nsul

a:N

ovkh

ani;

40◦ 3

2′N

49◦ 4

7′E

T14

53A

M23

0981

++

+?

PAL

LA

SI10

Test

udo

grae

capa

llas

i?A

zerb

aija

n:A

bshe

ron

Peni

nsul

a:N

ovkh

ani;

40◦ 3

2′N

49◦ 4

7′E

T14

54A

M23

0982

++

+P

ER

SES

1Te

stud

ogr

aeca

pers

esIr

an:A

njır

Ava

nd;3

2◦30

′ N54

◦ 26′

ET

2283

AM

2309

83P

ER

SES

2Te

stud

ogr

aeca

pers

esIr

an:6

kmN

EM

eshg

ınSh

ahr;

38◦ 2

6′N

47◦ 4

2′E

T14

27A

M23

0984

++

PE

RSE

S3

Test

udo

grae

cape

rses

Iran

:6km

NE

Mes

hgın

Shah

r;38

◦ 26′

N47

◦ 42′

ET

1428

AM

2309

85+

+P

ER

SES

4Te

stud

ogr

aeca

pers

esIr

an:N

eyrı

z;29

◦ 12′

N54

◦ 19′

ET

2282

AM

2309

86+

++

PE

RSE

S5

Test

udo

grae

cape

rses

Iran

:Sha

hr-e

Bab

ak,M

aym

and;

appr

ox.3

0◦07

′ N55

◦ 07′

ET

2284

AM

2309

87+

++

PE

RSE

S6

Test

udo

grae

cape

rses

Iran

:Sha

hr-e

Bab

ak,M

aym

and;

appr

ox.3

0◦07

′ N55

◦ 07′

ET

2285

AM

2309

88+

++

PE

RSE

S7

Test

udo

grae

cape

rses

Iran

:EE

sfah

an:K

uhpa

yeh;

32◦ 4

3′N

52◦ 2

6′E

T22

69A

M23

0989

++

+P

ER

SES

8Te

stud

ogr

aeca

pers

esIr

an:N

ır;3

1◦30

′ N54

◦ 08′

ET

2272

AM

2309

90+

+?

PE

RSE

S9

Test

udo

grae

cape

rses

?Ir

an:b

etw

een

Ger

mıa

ndR

azay

-Am

ırA

bad

(Am

ırK

andı

);38

◦ 53′

N48

◦ 00′

ET

2286

AM

2309

91+

++


Tabl

e2.

(Con

tinue

d).

Sam

ple

Taxo

nL

ocal

ityM

TD

Acc

essi

onnu

mbe

rIS

SR(G

AA

) 5

ISSR

(GA

CA

) 4

ISSR

L18

TE

RR

EST

RIS

1Te

stud

ogr

aeca

terr

estr

isSy

ria:

hills

NW

Ale

ppo:

Dar

Ta’i

zzah

;36◦

17′ N

36◦ 5

1′E

T21

93A

M23

0992

++

+T

ER

RE

STR

IS2

Test

udo

grae

cate

rres

tris

Syri

a:hi

llsN

WA

lepp

o:Q

alat

Sam

an;3

6◦22

′ N36

◦ 51′

ET

2194

AM

2309

93T

ER

RE

STR

IS3

Test

udo

grae

cate

rres

tris

Tur

key:

Mar

din;

37◦ 1

9′N

40◦ 4

7′N

T26

7A

M23

0994

TE

RR

EST

RIS

4Te

stud

ogr

aeca

terr

estr

isT

urke

y:St

reet

E-9

9be

twee

nH

ilvan

and

Çay

larb

asi;

37◦ 3

9′N

T36

4A

M23

0995

39◦ 0

6′E

?T

ER

RE

STR

IS5

Test

udo

grae

cate

rres

tris

?T

urke

y:A

diya

man

;37◦

46′ 0

9′′N

38◦ 2

1′35

′′ ET

378

AM

2309

96?

TE

RR

EST

RIS

6Te

stud

ogr

aeca

terr

estr

is?

Tur

key:

Adi

yam

an;3

7◦46

′ N38

◦ 16′

NT

264

AM

2309

97Z

AR

UD

NY

I1

Test

udo

grae

caza

rudn

yiIr

an:S

agha

nd,s

outh

ern

bord

erof

Kav

ırD

eser

t;32

◦ 33′

N55

◦ 13′

ET

2280

AM

2309

98+

++

ZA

RU

DN

YI

2Te

stud

ogr

aeca

zaru

dnyi

Iran

:Tab

as,s

outh

ern

bord

erof

Kav

ırD

eser

t;33

◦ 35′

N56

◦ 56′

ET

2273

AM

2309

99+

++

OU

TG

RO

UPS

:H

ER

MA

NN

IH

ER

MA

NN

I1

Test

udo

herm

anni

herm

anni

Ital

y:R

occa

tede

righ

i;43

◦ 02′

N11

◦ 05′

ED

4159

0A

J888

362

HE

RM

AN

NI

HE

RM

AN

NI

2Te

stud

ohe

rman

nihe

rman

niSo

uth

Ital

yD

4297

2A

J888

364

HO

RSF

IEL

DII

KA

ZA

CH

STA

NIC

ATe

stud

oho

rsfie

ldii

kaza

chst

anic

aK

azak

hsta

nD

4420

1A

J888

365

HO

RSF

IEL

DII

RU

STA

MO

VI

Test

udo

hors

field

iiru

stam

ovi

Iran

:Gor

gan;

36◦ 5

0′N

54◦ 2

6′E

T14

19A

J888

366

KL

EIN

MA

NN

I1

Test

udo

klei

nman

niU

nkno

wn

D44

284

AJ8

8837

0K

LE

INM

AN

NI

2Te

stud

okl

einm

anni

Unk

now

nD

4428

5A

J888

371

MA

RG

INA

TA1

Test

udo

mar

gina

taG

reec

e:Pe

lopo

nnes

e:D

ídim

i;37

◦ 28′

N23

◦ 07′

ET

388

AJ8

8831

8M

AR

GIN

ATA

2Te

stud

om

argi

nata

Ital

y:Sa

rdin

ia:P

ittul

ongo

;40◦

59′ N

09◦ 3

5′E

T11

17A

J888

332

106 U. Fritz et al.

fragment (cytochrome b gene and adjacent portion of tRNA-Thr gene). PCR was performed in a 50 µl volume (BioronPCR buffer or 50 mM KCl, 1.5 mM MgCl2, and 10 mMTris-HCl, 0.5% Triton X-100, pH 8.5) containing 1 unit ofTaq DNA polymerase (Bioron), 10 pmol dNTPs and 5 or10 pmol of each primer. The PCR program consisted of a5 min initial denaturation at 94◦C, followed by 35 cyclesof 45 s at 94◦C, 52 s at 55-60◦C and 80 s at 72◦C witha final elongation step of 10 min at 72◦C, or alternatively,a 4 min initial denaturation at 94◦C, then 30 cycles of45 s at 94◦C, 60 s at 52◦C and 120 s at 72◦C with a10 min final elongation step at 72◦C. PCR products weresequenced directly on both strands on ABI or MegaBace1000 (Amersham Biosciences) sequencers using the internalprimers mt-c2 5′-TGA GGA CAA ATA TCA TTC TGAGG 3′ or mt-c-For2 5′-TGA GGV CAR ATA TCA TTYTGA G-3′ and mt-E-Rev 5′-GCA AAT AGG AAG TATCAT TCT GG-3′ or mt-E-Rev2 5′-GCR AAT ARR AAGTAT CAT TCT GG-3′.

Sequence alignment, phylogenetic analyses and geneticdistances

The corresponding sequences from both strands were com-bined with the software DNAsis 7.00 (copyright HitachiSoftware Engineering Company, 1991) for every samplewith the resulting consensus sequences being aligned man-ually using BioEdit 7.0.5.2 (Hall, 1999). Sixteen sequences(AJ888341-56) were already published in a previous study(Fritz et al., 2005a); sequences of all other Testudo speciesfrom this paper served as outgroups for our phylogeneticanalyses (table 2). Data were analyzed using Bayesian in-ference of phylogeny as implemented in MrBayes 3.1 (Ron-quist and Huelsenbeck, 2003), maximum parsimony (MP;equal weighting) and neighbour joining (NJ; with model-corrected maximum likelihood distances) as implementedin PAUP* 4.0b10 (Swofford, 2002). Bayesian analysis wasperformed using four chains of 1 000 000 generations sam-pling every 100 generations and with the first 1000 gen-erations discarded as burn-in (with which only the plateauof the most likely trees was sampled). The best evolution-ary model for the data (also included in the analysis) wasestablished by hierarchical likelihood testing using Mod-eltest 3.06 (Posada and Crandall, 1998). For the ingroupspecies, 934 of 1134 aligned sites were constant, 74 char-acters were variable but parsimony-uninformative, and 126variable characters were parsimony-informative. Due to theprotracted computation times for MP analyses, the num-ber of equally most parsimonious solutions that were savedneeded to be limited using the maxtrees option. However,to determine whether or not this constraint was limitingthe search unduly, maxtrees was set to 10 000, 50 000 and100 000 in three separate analyses and the resolution andtopologies of the resulting strict consensus trees were com-pared. All calculations resulted in MP trees of 603 steps(CI = 0.6385, RI = 0.9135). To test the robustness of ob-tained branching patterns, bootstrap permutations (Felsen-stein, 1985) were run under both MP (setting maxtrees =1000, nreps = 1000) and NJ (ML-distances; nreps = 1000).Genetic distances (uncorrected p distances) were calculated

with PAUP* 4.0b10; previously published sequences (Fritzet al., 2005a) were used for comparison with other Testudospecies.

Nuclear genomic fingerprinting and analysis

ISSR employs a single PCR primer, binding to di- or trin-ucleotide repeat motifs (microsatellites), which are abun-dant in eukaryotic genomes (Tautz and Renz, 1984; Con-dit and Hubbell, 1991). Since sequences of microsatellitesare conserved over a wide range of organisms, universalprimers can be applied. Amplified regions correspond to thenucleotide sequence between two inverted simple sequencerepeat (SSR) priming sites (Wolfe et al., 1998; Bornet andBranchard, 2001). SSR regions appear to be scattered evenlythroughout the genome (Tautz and Renz, 1984; Condit andHubbell, 1991), resulting in a large number of polymorphicbands. ISSR markers are inherited in a dominant or codom-inant Mendelian fashion (Gupta et al., 1994; Tsumura et al.,1996). They are interpreted as dominant markers scored asdiallelic with ‘band present’ or ‘band absent’. The absenceof a band is interpreted as primer divergence or loss of alocus through the deletion of the SSR site or chromosomalrearrangement (Wolfe and Liston, 1998; Wolfe et al., 1998).ISSR fingerprints are usually diagnostic for species-leveltaxa (e.g. Gupta et al., 1994; Zietkiewicz et al., 1994; Winket al., 1998; Bornet and Branchard, 2001; Fritz et al., 2005a,b; Hundsdörfer and Wink, 2005). Individuals from contactzones of distinct subspecies as well as interspecific hybridsshare with both parental taxa diagnostic bands. Thus, lim-ited or non-existing gene flow is reflected by distinct band-ing patterns for reproductively isolated taxa, while a highpercentage of shared bands is indicative of gene flow orincomplete differentiation (Wolfe et al., 1998; Nagy et al.,2003; Fritz et al., 2005b).

We used two non-anchored primers that yielded species-diagnostic banding patterns for Testudo in a previous study(Fritz et al., 2005a), (GAA)5, annealing temperature 40◦C,and (GACA)4, annealing temperature 55◦C. In addition, weapplied the short anchored primer L18 (CTC GGG AAGGGA), annealing temperature 45◦C, that was useful in an-other chelonian genus, Emys (Fritz et al., 2005b). Each PCRwas performed with approximately 60 ng template DNA ina 25 µl volume [10 pmol of the primer and 0.625 nmol ofeach dNTP, except dATP: 0.28 nmol cold dATP plus 0.1µl radioactive α-33P-dATP solution (370 MBq/ml, Amer-sham Biosciences), 0.75 units of Taq polymerase (SIGMA)and water, buffered with 10 mM Tris-HCl, 50 mM KCl,0.5% Triton X-100, 1.5 mM MgCl2] covered by two dropsof mineral oil. Thermo-cycling was performed with a TrioThermo block TB1 (Biometra, Göttingen). Following an ini-tial 5 min denaturation at 94◦C, the program consisted of 30cycles of 45 s at 94◦C, 60 s at the respective annealing tem-perature, 120 s at 72◦C and 5 min at 72◦C for final elonga-tion. DNA fragments were separated by PAGE in a verticalapparatus (Base Acer Sequencer, Stratagene) for 2.5-4 h at65 W. The denaturing gel [6 M Urea, 100 ml Long RangerSolution, Biozym (PA), 100 ml TBE buffer (10x: 1 M Tris,0.83 M Boric Acid, 10 mM EDTA, pH 8.6)] had a size of45 × 30 cm and a thickness of 0.25 mm. After drying, an


X-ray film (Hyperfilm-MP, Amersham) was exposed to thegel for at least 12 h and developed.

Fragment patterns were analyzed manually. Unmis-takably identifiable bands were transferred into a pres-ence/absence matrix scoring each particular fragment.(GAA)5 yielded 33 fragments for 50 samples, (GACA)4 29fragments for 45 samples, and L18 resulted in 89 fragmentsfor 46 samples. To enable comparison of variation withinT. graeca and other organisms, we report average Dice andJaccard distance values (Jaccard, 1901, 1908; Dice, 1945;Nei and Li, 1979), calculated with RAPDistance 1.04 (Arm-strong et al., 1996), following the appeal for standardizationin ISSR PCR analyses by Hundsdörfer and Wink (2005).Based on the presence/absence matrix, cluster analyses (NJtrees) as implemented in PAUP* 4.0b10 (Swofford, 2002)were calculated for banding patterns of each primer and fora combined dataset for all three primers (151 characters for36 samples). Robustness of trees was tested by bootstrap-ping (2000 replicates) under the 50% consensus criterion.

Results

MtDNA sequence variation and phylogeny

Monophyly of the Testudo graeca complex wasconfirmed by high posterior probability andbootstrap values under all tree building meth-ods; its sister taxa are T. kleinmanni and T. mar-ginata (fig. 2). All phylogenetic analyses re-sulted in six well-supported major clades (A-F)within the T. graeca complex, and the MP topol-ogy obtained was corroborated by all maxtreessettings.

Average uncorrected p distances between theT. graeca complex and other Testudo speciesrange between 8.870% and 12.662%. Withinthe T. graeca complex, a mean of 3.346% wasobserved. This value clearly exceeds sequencedivergences within other Testudo species, andwithin-divergences of clades B and E corre-spond approximately to that within the poly-typic T. hermanni (table 3).

Within the T. graeca complex, clade A com-prises haplotypes distributed in the East Cauca-sus. It is sister to another clade (B) that corre-sponds to haplotypes from the western Mediter-ranean islands of Mallorca, Sardinia and Sicily(which are thought to be inhabited by allochtho-nous tortoise populations; Buskirk et al., 2001)and from North Africa. Clade C includes haplo-

types from western Asia Minor, the southeast-ern Balkans and the western and central Cauca-sus Region. Its sister group is a fourth, widelydistributed clade (D) from southern and east-ern Asia Minor and the Levantine Region (NearEast). Two further clades (E, F) are distributedin Iran (one clade in northwestern and centralIran; the other in eastern Iran).

While the distinctness of each of these sixclades and the sister group relationships be-tween (A + B) and between (C + D) were un-equivocally supported, the phylogeny of the re-sulting four clades (A + B), (C + D), E and Fwas poorly resolved (small cladograms in fig.2). Under Bayesian analysis, there was weaksupport for a sister group relationship of the twomajor western clades, i.e. ((A + B) + (C + D)),whereas MP and NJ resulted in a sister group re-lation between a weakly supported clade ((A +B) + E, F) and (C + D).

Nuclear genomic fingerprinting

In contrast to obvious geographical sequencedifferentiation of the mitochondrial genome, wefound a quite homogenous pattern in nuclear ge-

Figure 2. Bayesian phylogram of mtDNA haplotypes forspur-thighed tortoises (Testudo graeca complex), rootedwith all other Testudo species. Taxon names within T.graeca complex are assigned according to morphologyand geographic provenance and follow Chkhikvadze andTuniyev (1986), Weissinger (1987), Chkhikvadze andBakradze (1991, 2002), Perälä (2002a, b, 2004b), Pieh etal. (2002) and Pieh and Perälä (2002, 2004). Questionmarks denote uncertain taxonomic allocations due to local-ities along range borders. Locality names (Mallorca, Sar-dinia, Sicily) instead of taxon names indicate introducedor allochthonous tortoises from the western Mediterranean.Numbers following taxon or locality names refer to table 2,where exact localities are given. Symbols correspond to dis-tinct mtDNA lineages and to figure 1. Branch lengths areBayesian estimates and proportional to the scale bar withthe unit being a mean number of nucleotide changes persite. Numbers at crucial nodes represent posterior proba-bilities and MP and NJ bootstrap values (1000 replicates)greater than 50; NJ using ML distances. Dashes indicatethat the respective branch was not supported. Small clado-grams show alternative topologies for major groups withinT. graeca complex. For clade D from southern and easternAsia Minor and the Levant, see also figure 7.

108 U. Fritz et al.


Tabl

e3.

Unc

orre

cted

pdi

stan

ces

(per

cent

ages

)w

ithin

and

betw

een

clad

esof

the

Test

udo

grae

caco

mpl

exan

dot

her

Test

udo

spec

ies,

base

don

ada

tase

tof

1000

bp(m

tDN

A,c

ytoc

hrom

eb

gene

).M

ean

dist

ance

sbe

twee

nsp

ecie

sor

clad

esar

egi

ven

belo

w,r

ange

s(i

nita

lics)

abov

eth

edi

agon

al.T

hew

ithin

-tax

onse

quen

cedi

verg

ence

isgi

ven

inbo

ldon

the

diag

onal

(mea

nan

dra

nge)

.Pol

ytyp

icsp

ecie

sas

teri

sked

.Seq

uenc

esfr

omFr

itzet

al.(

2005

a):T

.her

man

nibo

ettg

eriA

J888

357-

60,T

.her

man

nihe

rman

niA

J888

361-

64,T

.hor

sfiel

diik

azac

hsta

nica

AJ8

8836

5,T.

hors

field

iiru

stam

oviA

J888

366,

T.kl

einm

anni

AJ8

8837

0-71

,T.m

argi

nata

AJ8

8830

8-40

.

nTe

stud

ogr

aeca

*–

all

Cla

deA

Cla

deB

Cla

deC

Cla

deD

Cla

deE

Cla

deF

Test

udo

herm

anni

*Te

stud

oho

rsfie

ldii

*Te

stud

okl

einm

anni

Test

udo

mar

gina

ta

Test

udo

grae

ca*

–al

l

943.

346

(0.0

00-7

.953

)–

––

––

–10

.562

-15.

087

9.84

5-14

.609

8.05

7-10

.928

7.25

6-10

.665

Cla

deA

14–

0.30

4(0

.000

-1.1

48)

3.90

8-6.

149

4.20

8-5.

423

4.00

8-5.

834

3.70

7-5.

732

3.30

7-4.

020

12.0

94-1

4.05

011

.186

-13.

060

10.0

20-1

0.55

18.

868-

10.2

56

Cla

deB

7–

4.66

41.

515

(0.0

00-2

.440

)5.

206-

7.95

34.

868-

7.42

94.

709-

7.44

73.

707-

4.99

011

.208

-13.

314

11.0

25-1

3.60

88.

994-

10.4

548.

870-

10.3

82

Cla

deC

20–

4.67

75.

990

0.24

3(0

.000

-0.9

21)

2.09

2-4.

081

3.97

4-6.

212

3.70

7-4.

509

12.0

20-1

5.08

710

.587

-14.

609

9.19

7-10

.928

7.65

8-9.

844

Cla

deD

37–

4.48

65.

591

2.61

20.

789

(0.0

00-2

.509

)3.

607-

6.93

93.

009-

4.88

311

.049

-13.

692

9.84

5-12

.544

8.05

7-9.

703

7.28

8-9.

514

Cla

deE

9–

4.27

45.

419

4.85

84.

409

1.34

3(0

.000

-3.0

46)

2.50

5-4.

309

11.2

28-1

4.02

810

.374

-13.

916

8.91

8-10

.922

7.89

4-10

.665

Cla

deF

7–

3.54

54.

061

3.95

43.

510

3.08

10.

150

(0.0

00-0

.401

)10

.562

-12.

224

10.0

90-1

2.31

48.

460-

9.21

87.

256-

8.71

7

Test

udo

herm

anni

*8

12.6

6213

.203

12.4

0413

.154

12.4

5312

.605

11.6

151.

183

(0.0

00-2

.752

)9.

931-

13.0

5710

.673

-11.

907

9.87

1-11

.864

Test

udo

hors

field

ii*

211

.737

11.9

9412

.328

12.3

0811

.243

11.9

3011

.420

11.6

222.

461

(–)

9.99

0-11

.333

10.0

72-1

1.78

8

Test

udo

klei

nman

ni2

9.41

010

.184

9.60

19.

872

8.82

89.

746

8.99

511

.454

10.6

620.

000

(–)

6.45

2-7.

301

Test

udo

mar

gina

ta33

8.87

09.

433

9.70

39.

207

8.48

18.

973

7.88

411

.086

10.9

496.

847

0.22

0(0

.000

-1.1

01)

110 U. Fritz et al.

nomic fingerprints. All studied samples shared ahigh percentage of bands. The primer (GACA)4

resulted in average distances of 0.30 (Dice; Neiand Li) and 0.44 (Jaccard), (GAA)5 in 0.35(Dice; Nei and Li) and 0.50 (Jaccard), and L18,which produced the most variable fingerprints,in average distances of 0.23 (Dice; Nei and Li)and 0.37 (Jaccard).

Using NJ cluster analyses, the datasets forthe three single primers resulted in unresolvedpolytomies for most samples under the 50%bootstrap consensus criterion, and those sam-ples that clustered with weak to high bootstrapsupport often belong to different mtDNA lin-eages and different taxa. The NJ tree of the(GAA)5 dataset serves as example (fig. 3, left).The combined dataset of 151 characters fromthe three primers resulted in fewer polytomies(fig. 3, right); however, also in this tree none ofthe major clusters corresponds with taxon limitsor mtDNA lineages.

Systematics, zoogeography and discussion

Incongruence of morphology, taxa and geneticdata

Populations of spur-thighed tortoises comprisein some regions (Spain, North Africa, Levant)small to medium-sized, mainly yellow-colouredtortoises, while in other parts of the range largersized, dark individuals occur. However, onto-genetic variation of shell coloration and pro-portions is considerable. Different age classesof Bulgarian Testudo graeca display nearly theentire range of shell coloration, with young,small adults being light-coloured and aged,large adults being dark-coloured. Also shellproportions are known to change much duringgrowth (Fritz et al., 1996).

Nevertheless, such characters (shell shapeand proportions, colour pattern, in part alsoscutellation characters) were repeatedly usedfor taxon delineation, leading to recent de-scriptions or resurrections of several morpho-logically diagnosable taxa (Pieh, 2001; Perälä,

2002a; Pieh and Perälä, 2002, 2004). For Mo-roccan T. graeca it was demonstrated, how-ever, that such variation corresponds rather topopulation-specific differentiation than to tax-onomic distinctness (Harris et al., 2003; Car-retero et al., 2005).

Using ISSR fingerprinting we found no sup-port for any taxonomic differentiation and noneof our six mtDNA clades within the T. graecacomplex is congruent with any morphologi-cally defined taxon. Also a distinction betweena North African-Levantine lineage, comprisingmainly yellow-coloured tortoises of small tomoderate size and a second lineage of larger anddarker tortoises, occurring in Southeast Europeand the Near and Middle East, as suggested byFritz et al. (1996), is not corroborated by ourdata (figs 1-2). While all studied North Africantortoises fall into one clade (B), the sister groupof this clade is not formed by Levantine haplo-types but by haplotypes from the East Caucasus(clade A), representing populations with largeand dark tortoises.

According to our mtDNA data, this East Cau-casian clade A is composed of haplotypes fromtortoises that belong to T. g. armeniaca, T. g. ib-era and T. g. pallasi. The North African cladeB contains haplotypes of the nominal taxa T.g. graeca, T. g. cyrenaica and T. g. nabeulen-sis as well as most specimens from the westernMediterranean islands. Clade C, found in theBalkans, in western Asia Minor, and in the west-ern and central Caucasus, comprises sequencesrepresenting T. g. ibera and T. g. nikolskii. CladeD from southern and eastern Asia Minor and theLevant is composed of haplotypes of T. g. ana-murensis, T. g. antakyensis, T. g. floweri, T. g.ibera, T. g. terrestris and one (allochthonous)tortoise from Sicily. Clade E from northwest-ern and central Iran consists of haplotypes fromT. g. buxtoni and T. g. perses, and the easternIranian clade F contains haplotypes assigned toT. g. perses and T. g. zarudnyi.

From a morphological point of view, cladesA, C, E and F contain populations with dark-coloured tortoises of medium to large size.


Figure 3. Unrooted NJ bootstrap 50% majority-rule consensus trees of ISSR fingerprints for samples of the Testudograeca complex. Left, primer (GAA)5, based on 50 samples and 33 fragments. Right, combined dataset of primers(GAA)5, (GACA)4 and L18, based on 36 samples and 151 fragments. Numbers along branches represent bootstrap values(2000 replicates) greater than 50. For further explanations see figure 2.

Taxa in these clades differ mainly in shellshape and in part in colour pattern. Tortoisesof clade B are generally lighter coloured andsmall- to moderately-sized (table 1). Most vari-ation is found in clade D that comprises small-to medium-sized, mainly yellow-coloured tor-toises (T. g. antakyensis, T. g. floweri, T. g.terrestris) as well as huge, dark brownishindividuals, either with flat, elongated shells (T.g. anamurensis) or distinctly domed shells (T. g.ibera from Lake Van Region, Turkey).

However, even within populations attributedto T. g. antakyensis by Perälä (2002a) consider-able morphological differences are known to oc-cur, reflecting the same general pattern. WithinSyria, morphological variation was shown tofollow Gloger’s Rule in that tortoises fromarid regions are distinctly lighter coloured (andsmaller) than tortoises from more humid re-

gions. Further, tortoises living on dark basalticsoil, as in the Jabal Duruz Region, are signifi-cantly darker coloured than tortoises from otherregions with light substrate (figs 4-6; Fritz etal., 1996). We observed the same pattern also inother regions of the Near East during field work.This suggests stabilizing selection by environ-mental pressure as a source of morphologicalvariation. Similarity of general body and sub-strate coloration is a well-known phenomenonalso in other animal species (‘substrate races’;Mayr, 1963).

Haplotypes within clade D, that containsamong others all T. g. antakyensis sequences,group into three weakly to well-supported sub-clades. These subclades rather correspond tosequences from neighbouring regions than totaxonomic segregation or morphological sim-ilarity (fig. 7). This is most obvious in the

112 U. Fritz et al.

well-supported subclade from Israel, Jordan and

southern Syria, which consists of sequences

from the nominal taxa T. g. antakyensis and T.

g. floweri. While the sequences of the four T. g.

floweri and a T. g. antakyensis from Jordan (an-

takyensis 4) represent small-sized, mainly yel-

low tortoises from arid regions, other sequences

from T. g. antakyensis originate from larger and

darker tortoises that live in moister environ-

ments (antakyensis 1-3: Tiberias, Israel). The

three Syrian T. g. antakyensis sequences in this

subclade (antakyensis 10-12) are from dark-

coloured tortoises from the basaltic Jabal Duruz

Region.

Testudo graeca armeniaca is a further promi-

nent example for incongruence of morpholo-

gical and genetic differentiation. This taxon is

characterized by a depressed shell and flat-

tened forearms. In general appearance it resem-

bles T. horsfieldii Gray, 1844 (Chkhikvadze and

Bakradze, 1991; Pieh et al., 2002), a species that

is not closely related to the T. graeca complex

and allocated to the genus Agrionemys by some

authors (Fritz and Cheylan, 2001; Parham et al.,

2006). The mtDNA haplotype of a T. g. arme-

niaca is nested within the East Caucasian clade

A together with other, morphologically distinc-

tive tortoises with domed shell from the same

region (fig. 2; table 2). Both T. g. armeniaca

and T. horsfieldii occur in regions with strictly

continental climate with severe, cold winters.

Farther, both are steppicolous tortoises that dig

and inhabit deep burrows, suggesting that envi-

ronmental pressure and the similar mode of life

results via selection in similar phenotypes and

masks phylogenetic relationships.

Also in another Testudo species, T. mar-

ginata, environmental pressure was shown to re-

sult in considerable phenotypic variation (size

reduction, persisting juvenile characteristics in

a poor region of the range; Fritz et al., 2005a),

suggestive of considerable phenotypic plasticity

in Testudo species.

How many species?

Based on morphology and zoogeographic con-siderations, it was repeatedly suggested thatTestudo graeca is composed of several distinctspecies (Bour, 1989; Gmira, 1993a, b, 1995;David, 1994; Pieh, 2001; Perälä, 2002a, b; Piehand Perälä, 2002, 2004). In contrast to Gmira(1993a, b, 1995) and Perälä (2002b), whosemorphological datasets argued for the para-phyly of the T. graeca complex with respectto other Testudo species (T. kleinmanni sensulato, T. marginata), our mtDNA data confirmeda monophyletic T. graeca complex and its sis-ter group relationship to T. kleinmanni and T.marginata. This agrees with other recent stud-ies employing genetic markers (mtDNA data:van der Kuyl et al., 2002; Fritz et al., 2005a;Parham et al., 2006; nuclear genomic finger-printing: Fritz et al., 2005a).

Obviously, species delineation depends onthe adopted species concept, and there is an on-going debate about the benefits and shortcom-ings of different competing species concepts(e.g. Ereshefsky, 1992; Wheeler and Meier,2000; Agapow et al., 2004; Coyne and Orr,2004). There is no doubt that the six mtDNAlineages within the T. graeca complex couldbe recognized as representing full species un-der the Evolutionary (e.g. Wiley and May-den, 2000) or Phylogenetic Species Concepts(e.g. Cracraft, 1983, 1987; Mishler and The-riot, 2000; Wheeler and Platnick, 2000; seealso the review in Coyne and Orr, 2004), al-though morphological characterization of thesespecies would be difficult (see below). How-ever, the most restrictive species concept forbisexual animals, the Biological Species Con-cept of Mayr (e.g. 1942, 1963, 2000), de-fines species as reproductively (and thus ge-netically) isolated groups of populations. Un-der this species concept, mitochondrial genomicmarkers alone are not conclusive, mainly dueto their matrilineal inheritance and the differ-ent effective population sizes of mitochondrialand nuclear genomes, resulting in different pat-terns for incomplete lineage sorting and intro-


gression (Funk and Omland, 2003; Ballard andWhitlock, 2004).

ISSR PCR provides an ideal tool for obtain-ing information about nuclear genomic differ-entiation and gene flow. In distinct ‘biologicalspecies’, differentiation of nuclear and mito-chondrial genomes is typically congruent andISSR profiles differ clearly (e.g. for cheloni-ans: Wink et al., 2001; Guicking et al., 2002;Schilde et al., 2004; Fritz et al., 2005a, b). Insuch cases, introgression and hybridization areindicated by shared bands in otherwise distinctbanding patterns. In contrast, if populations stillform a genetic continuum, ISSR profiles do notdiffer markedly. ISSR also allows identificationof taxa in which introgression of mtDNA mightmask nuclear genomic differentiation (Gupta etal., 1994; Zietkiewicz et al., 1994; Wink et al.,1998, 2001; Wolfe and Liston, 1998; Wolfe etal., 1998; Guicking et al., 2002; Nagy et al.,2003; Schilde et al., 2004; Fritz et al., 2005a, b).

In a previous study, ISSR profiles of all fivetraditionally recognized Testudo species wereshown to be significantly distinct (in NJ clus-ter analysis bootstrap support values of 100 foreach species; Fritz et al., 2005a). Using the sameprimers, (GAA)5 and (GACA)4, plus one ad-ditional primer (L18) shown to be useful inother chelonians (Emys; Fritz et al., 2005b), wefound little differentiation within the T. graecacomplex, despite extensive mtDNA differenti-ation. As we can exclude homoplasy due tothe excellent discrimination of the other Testudospecies, the similarity of fingerprints within theT. graeca complex indicates either a high levelof gene flow or a low level of differentiation(including incomplete lineage sorting). We con-clude that T. graeca represents a single poly-typic species under the Biological Species Con-cept.

How many subspecies?

Based on a combined data set of 393-394 bp ofthe 12S rRNA gene and 411 bp of the D-loop,van der Kuyl et al. (2005) found among 22 spur-thighed tortoises only two well-supported hap-

lotype clades, corresponding to tortoises from(i) North Africa and (ii) Turkey (one sample)and the Near East and presumed that theserepresent only two subspecies, Testudo graecagraeca and T. g. ibera, while genetic evi-dence for the existence of other subspecies wasthought to be weak. According to locality data,the ‘ibera clade’ of van der Kuyl et al. (2005)could be identical with our clade D. However,a problem of the papers by van der Kuyl etal. (2002, 2005) was that many tortoises of un-known provenance were studied. Such speci-mens cannot be used when assessing geographicvariation (Harris et al., 2003). Moreover, sub-species names assigned by van der Kuyl et al.(2002, 2005) often did not fulfil nomenclaturalrequirements and locality data in part did notmatch the ranges of the respective taxa (Perälä,2004a). For example, van der Kuyl et al. (2005)assign tortoises from Bulgaria in part to T. g.terrestris; this taxon does not occur in Bulgaria(Bour and Perälä, 2004; Perälä and Bour, 2004;see also table 1).

In accordance with the Biological SpeciesConcept, we understand subspecies as geneti-cally distinct, geographically vicariant groupsof populations between which gene flow occurs.Distinct mitochondrial matrilineages often, butnot always, mirror such subspecific differenti-ation (Avise, 2000; but see Funk and Omland,2003).

Using the mitochondrial cytochrome b gene,we discovered distinctly more variation thanprevious studies based on the 12S rRNA gene(van der Kuyl et al., 2002, 2005; Harris et al.,2003). This is in line with other investigationsthat found in testudinids the cytochrome b geneto be more variable than the 12S rRNA gene(Caccone et al., 1999; Palkovacs et al., 2002;Fritz et al., 2005a, 2006). Neither the four sub-species that were originally thought to composeT. graeca (T. g. graeca from Spain and north-western Africa; T. g. terrestris from northeast-ern Africa, southeastern Turkey and the Lev-antine Region; T. g. ibera from Southeast Eu-rope, Asia Minor, the Caucasus Region, west-

114 U. Fritz et al.

Figure 4. Light coloured spur-thighed tortoise from arid region in Syria (Saydnaya, Anti Lebanon Mts.; maximum weight990 g, n = 17). Note healed serious injury on top of carapace.

Figure 5. Dark coloured spur-thighed tortoise from more humid environment in Syria (Qalat Saman, hills NW Aleppo,maximum weight 1900 g, n = 15).


Figure 6. Dark coloured spur-thighed tortoise from basaltic region in Syria (Jabal Duruz, maximum weight 1320 g, n = 28).

ern and central Iran; T. g. zarudnyi from easternIran; Mertens, 1946; Wermuth, 1958; Wermuthand Mertens, 1961, 1977), nor the many taxathat were later described or resurrected (table1) correspond perfectly to any of our six ma-jor mtDNA clades. This could be indicative ofinaccurate taxon limits.

Several authors agree that T. g. ibera, as cur-rently understood, might be a conglomerate ofdistinct taxa, and that the name ibera shouldbe used only for tortoises from the CaucasianKura River Basin, the area around the type lo-cality (Perälä, 2002a; Pieh et al., 2002; Danilovand Milto, 2004). However, the disagreementof subspecies delineation and mtDNA cladeswould not be resolved by using a restricted sub-species concept for T. g. ibera (table 1). Evenour sequences of T. g. ibera sensu stricto fromthe Kura River Basin are scattered over twoclades (A, C). The sequences from central Cau-casian ibera localities, one from Mtskheta only15 km away from the type locality of ibera(Tbilisi, Georgia), belong to clade C, which is

otherwise found along the northeastern BlackSea coast (=T. g. nikolskii), in the Balkans andwestern Asia Minor (=T. g. ibera sensu lato),while the ibera sequences from the more east-ern Caucasian locality Dashburun (Azerbaijan)appear in the same clade (A) as T. g. pallasiand the morphologically very distinctive T. g.armeniaca, so that a taxonomic break must beassumed within the Kura River Basin.

Until now, taxa within the T. graeca complexwere defined exclusively by morphology. Ourdata imply that genetic differentiation is maskedby pronounced morphological plasticity. Thisresults in the unexpected nesting of morpholog-ically well-defined taxa, like T. g. armeniaca orT. g. floweri, within clades comprising also ge-ographically neighbouring, but morphologicallydistinctive populations of other taxa (clade A: T.g. armeniaca, T. g. ibera, T. g. pallasi; clade D:T. g. anamurensis, T. g. antakyensis, T. g. flow-eri, T. g. ibera, T. g. terrestris), or the scatteringof sequences of tortoises of the same subspecies(T. g. ibera sensu stricto or T. g. ibera sensu lato)

116 U. Fritz et al.

Figure 7. Detail of Bayesian tree of mtDNA haplotypes for spur-thighed tortoises from southern and eastern Asia Minor andthe Levant. For further explanations see figure 2.

over two or three genetically distinct clades (A,C or A, C, D, respectively; fig. 2). Future re-search is needed to find out whether any mor-phological differences exist paralleling the sixmtDNA clades.

In any case, it is obvious that the sug-gested existence of up to 20 distinct taxa withinthe T. graeca complex (Highfield and Mar-tin, 1989a, b, c; Highfield, 1990; Pieh, 2001;Perälä, 2002a, b; Pieh and Perälä, 2002, 2004)is exaggerated and does not match genetic dif-ferentiation. To achieve a more realistic taxo-nomic arrangement reflecting mtDNA clades,we propose usage of the taxonomic arrange-ment in table 4, which is also most parsimo-nious with respect to nomenclatural changesof the formerly widely accepted subspecies de-lineation of Mertens (1946), Wermuth (1958)and Wermuth and Mertens (1961, 1977). Asnot all North African taxa were included inthe present study, we refrain from synonymiz-

ing North African taxa with T. g. graeca Lin-naeus, 1758 (mtDNA clade B) that representsa further valid subspecies. We admit that oursubspecies delineation may be imprecise alongrange borders due to introgression of mitochon-drial genomes.

Historic zoogeography

The distribution of the six major mtDNA cladeswithin the Testudo graeca complex allows somezoogeographical insights. Like in many otherorganisms (Myers et al., 2000), a remarkablyhigh diversity is found in the Caucasus Region,where four of the six mtDNA clades occur (A,C, D, E; fig. 1). The high diversity of the Cau-casus Region is often explained by an admixtureof old endemics and taxa of different geographicorigin (e.g. Satunin, 1910; Darevskii, 1967; Tu-niyev, 1990, 1995), highlighting the role of thismountain range as centre of endemism, refugearea and crossroads between the Mediterranean,


Table 4. Proposed new subspecies delineation for the eastern part of the range of Testudo graeca acknowledging mtDNAclades. For most type localities see table 1. Type localities of taxa that have not been regarded as valid in past decadesare: Testudo ibera racovitzai Calinescu, 1931 – Tutrakan, Bulgaria; Testudo ibera var. bicaudalis Venzmer, 1920 – TaurusMts., Cilicia, Turkey (Buskirk et al., 2001). We refrain from assigning the doubtful name Testudo ecaudata Pallas, 1814(see Buskirk et al., 2001; Pieh et al., 2002) to clade E although its type locality (forested parts of Persia at the Caspian Sea)suggests identity with T. g. buxtoni.

MtDNAclade

Subspecies Junior synonyms Range

A Testudo graeca armeniacaChkhikvadze andBakradze, 1991

Testudo graeca pallasi Chkhikvadzeand Bakradze, 2002

Central west coast of Caspian Sea; easternCaucasus Region and parts of central Cau-casus Region, including Araks River Valley(Armenia, Turkey)

C Testudo graeca iberaPallas, 1814

Testudo ibera racovitzai Calinescu,1931Testudo graeca nikolskiiChkhikvadze and Tuniyev, 1986

Southeast Europe, western Asia Minor,Russian and Georgian Black Sea coast,central Caucasus Region (Georgia, adjacentAzerbaijan)

D Testudo graeca terrestrisForsskål, 1775

Testudo ibera var. bicaudalisVenzmer, 1920

Southern and eastern Asia Minor, Levant

Testudo floweri Bodenheimer, 1935Testudo graeca anamurensisWeissinger, 1987Testudo antakyensis Perälä, 1996

E Testudo graeca buxtoniBoulenger, 1921

Testudo perses Perälä, 2002 Northwestern and central Iran, in the east-ern Caucasus Region probably also adjacentparts of other countries

F Testudo graeca zarudnyiNikolsky, 1896

– East Iran

Europe, and Central Asia. The complicated geo-logical history of the Caucasus Region and adja-cent East Anatolia includes repeated Oligoceneand Miocene episodes of isolation during ma-rine transgressions and reconnections with Eu-ropean and Asiatic landmasses, in part open-ing corridors to Africa via Arabia (Rögl, 1998,1999), resulting in several vicariance events forbiota.

Not only the current high diversity in the Cau-casus Region, but also the fossil record suggestsa Caucasian origin for the Testudo graeca com-plex. Fossils referred to the extinct species T.burtschaki and T. eldarica from the Medial toUpper Miocene of Azerbaijan and eastern Geor-gia (Chkhikvadze, 1983, 1989; Danilov, 2005)are the oldest representatives of this group. Inthe Pliocene, tortoises resembling T. graecawere already widely distributed, with recordsin the central and northern Caucasus Region,Ukraine, Moldavia, Romania (T. cernovi, T. ku-curganica; Chkhikvadze, 1983, 1989; Danilov,2005) and Morocco (T . aff. kenitrensis; de Lap-

parent de Broin, 2000), indicating rapid disper-sal.

The fossil record also provides evidence thatthe current patchy distribution of clade C (fig.1) is a consequence of secondary range inter-ruption. Besides the above-mentioned Pliocenefossils, there are many Pleistocene tortoise find-ings from the northern Caucasus and Black SeaRegions that are in part referred to T. graecaalready (Chkhikvadze, 1983, 1989). Thus, thecurrent gaps in the range of clade C tortoises aremost probably the result of glacial extinction.

All tree building methods favour a sistergroup relationship of the East Caucasian andNorth African clades A and B, although both arefully allopatric today and separated by a largedistribution gap (figs 1-2). From Central andWest Europe there are no tortoise fossils knownresembling unambiguously the T. graeca com-plex, despite an excellent fossil record (Bailón,2001; Buskirk et al., 2001; de Lapparent deBroin, 2001). This suggests that the founder in-dividuals of the current Spanish (and Italian)

118 U. Fritz et al.

populations were introduced. If the East Cau-casian and the North African clades A and B aresister groups indeed and an invasion of NorthAfrica via West Europe is to be excluded, a col-onization route via the Levantine Region mustbe hypothesized. However, along the Levant an-other clade (D) is distributed today, so that atleast two colonization waves must have reachedthis region.

A first dispersal event from the Caucasus Re-gion to North Africa could have occurred inthe Middle to Late Miocene, when intermittentland bridges connected North Africa with theNear and Middle East due to the northeastwardmovement of the Arabian Plate, leading laterto complete closure of the Tethys Sea (Rögl,1998, 1999). An invasion of North Africa viasuch a temporary land bridge is an attractivescenario, explaining later extermination and re-placement of geographically connecting popu-lations through clade D, now distributed in theLevantine Region (fig. 1).

Our investigation confirms that most of theintroduced spur-thighed tortoise populations onwestern Mediterranean islands originated inNorth Africa, as suggested by the previous allo-cation of these populations to the subspecies T.g. graeca (Wermuth and Mertens, 1961, 1977;Buskirk et al., 2001). However, the record of aSicilian tortoise belonging to the eastern cladeD instead of the North African clade B providesevidence for multiple introductions from differ-ent regions, and that some individuals definitelydid not come from North Africa.

Acknowledgements. F. Ahmadzadeh, H. Bringsøe, M.Cheylan, S. d’Angelo, D. Frynta, E. Geffen, M. Jirku, M.Kamler, P. Mikulícek, D. Modrý, O. Paknia, E. Roitberg,W. Wegehaupt and B.H. Zarei assisted in obtaining bloodsamples or provided samples. Most lab work was done byA. Müller (Museum of Zoology, Dresden) and H. Sauer-Gürth (IPMB, Heidelberg). The work of P. Široký profitedfrom the IGA VFU grant (2/2004/FVHE) and O. Türkozanwas supported by TÜBITAK project TBAG-2206 102T104.Thanks for comments on this paper go to O. Bininda-Emonds, S.D. Busack, D.J. Harris, M. Vences and oneanonymous reviewer.

References

Agapow, P.-M., Bininda-Emonds, O.R.P., Crandall, K.A.,Gittleman, J.L., Mace, G.M., Marshall, J.C., Purvis, A.(2004): The impact of species concept on biodiversitystudies. Quart. Rev. Biol. 79: 161-179.

Álvarez, Y., Mateo, J.A., Andreu, A.C., Díaz-Paniagua, C.,Diez, A., Bautista, J.M. (2000): Mitochondrial DNAhaplotyping of Testudo graeca on both continental sidesof the Straits of Gibraltar. J. Heredity 91: 39-41.

Anderson, S.C. (1979): Synopsis of the turtles, crocodiles,and amphisbaenians of Iran. Proc. California Acad. Sci.,4th Ser. 41 (22): 501-528.

Armstrong, J., Gibbs, A., Peakall, R., Weiller, G. (1996):RAPDistance 1.04. Canberra, Australian National Uni-versity.

Austin, J.J., Arnold, E.N., Bour, R. (2003): Was there a sec-ond adaptive radiation of giant tortoises in the IndianOcean? Using mitochondrial DNA to investigate specia-tion and biogeography of Aldabrachelys. Mol. Ecol. 12:1415-1424.

Avise, J.C. (2000): Phylogeography. The History and For-mation of Species. Cambridge, Massachusetts, and Lon-don, Harvard University Press.

Bailón, S. (2001): Revisión de la asignación a Testudo cf.graeca del yacimiento del Pleistoceno superior de CuevaHorá (Darro, España). Rev. Esp. Herpetol. 15: 61-65.

Ballard, J.W.O., Whitlock, M.C. (2004): The incompletenatural history of mitochondria. Mol. Ecol. 13: 729-744.

Bornet, B., Branchard, M. (2001): Nonanchored inter sim-ple sequence repeat (ISSR) markers: reproducible andspecific tools for genome fingerprinting. Plant Mol. Biol.Rep. 19: 209-215.

Bour, R. (1987): L’identité des tortues terrestres eu-ropéennes: spécimens-types et localités-types. Rev. Fr.Aquariol. 13 (1986): 111-122.

Bour, R. (1989): Caractères diagnostiques offerts par lecrâne des tortues terrestres du genre Testudo. Mésogée48 (1988): 13-19.

Bour, R., Perälä, J. (2004): Testudo [graeca] terrestrisForsskål, 1775. In: Numéro spécial Testudo. Guyot-Jackson, G., Ed., Manouria 7 (22): 40-41.

Buskirk, J.R., Keller, C., Andreu, A.C. (2001): Testudograeca Linnaeus, 1758 – Maurische Landschildkröte. In:Handbuch der Reptilien und Amphibien Europas. Band3/IIIA: Schildkröten (Testudines) I, p. 125-178. Fritz,U., Ed., Wiebelsheim, Aula-Verlag.

Caccone, A., Amato, G., Gratry, O.C., Behler, J., Powell,J.R. (1999): A molecular phylogeny of four endangeredMadagascar tortoises based on mtDNA sequences. Mol.Phylogenet. Evol. 12: 1-9.

Carretero, M.A., Znari, M., Harris, D.J, Macé, J.C. (2005):Morphological divergence among populations of Tes-tudo graeca from west-central Morocco. Animal Biol.55: 259-279.

Chkhikvadze, V.M. (1983): Iskopaemye cherepakhi Kav-kaza i Severnogo Prichernomorya. Tbilisi, Metsniereba.

Chkhikvadze, V.M. (1989): Neogenovye cherepakhi SSSR.Tbilisi, Metsniereba.


Chkhikvadze, V.M., Bakradze, M.A. (1991): O sistematich-eskom polozhenii sovremennoi sukhoputnoi cherepakhiiz doliny reki Araks. Trudy Tbilissk. gosudarstven. Univ.305: 59-63.

Chkhikvadze, V.M., Bakradze, M.A. (2002): Novyi podvidsukhoputnoi cherepakhi iz Dagestana. Trudy Inst. Zool.,Akad. Nauk Gruzii 21: 276-279.

Chkhikvadze, V.M., Tuniyev, B.S. (1986): O sistemati-cheskom polozhenii sovremennoi sukhoputnoi chere-pakhi zapadnogo Zakavkazya. Soobshch. Akad. NaukGruzinsk. SSR 124: 617-620.

Condit, R., Hubbell, S.P. (1991): Abundance and DNAsequence of two-base repeat regions in tropical treegenomes. Genome 34: 66-71.

Coyne, J.A., Orr, H.A. (2004): Speciation. Sunderland,Massachusetts, Sinauer Associates.

Cracraft, J. (1983): Species concepts and speciation analy-sis. Curr. Ornithol. 1: 159-187.

Cracraft, J. (1987): Species concepts and the ontology ofevolution. Biol. Phil. 2: 329-346.

Danilov, I.G. (2005): Die fossilen Schildkröten Europas. In:Handbuch der Reptilien und Amphibien Europas. Band3/IIIB: Schildkröten (Testudines) II, p. 329-441. Fritz,U., Ed., Wiebelsheim, Aula-Verlag.

Danilov, I.G., Milto, I.G. (2004): Testudo [graeca] ib-era Pallas, 1814. In: Numéro spécial Testudo. Guyot-Jackson, G., Ed., Manouria 7 (22): 23-24.

Danilov, I.G., Milto, I.G., Mazanaeva, L.F. (2004): Tes-tudo [graeca] pallasi Chkhikvadze et Bakradze, 2002.In: Numéro spécial Testudo. Guyot-Jackson, G., Ed.,Manouria 7 (22): 34-35.

Darevskii, I.S. (1967): Skal’nye Yashcheritsy Kavkaza.Leningrad, Nauka.

David, P. (1994): Liste des reptiles actuels du monde. I.Chelonii. Dumerilia 1: 7-127.

de Lapparent de Broin, F. (2000): African chelonians fromthe Jurassic to the present: phases of development andpreliminary catalogue of the fossil record. Palaeontolo-gia Africana 36: 43-82.

de Lapparent de Broin, F. (2001): The European turtle faunafrom the Triassic to the present. Dumerilia 4: 155-217.

Dice, L.R. (1945): Measures of the amount of ecologicassociation between species. Ecology 26: 297-302.

Ereshefsky, M. (1992): The Units of Evolution. Essays onthe Nature of Species. Cambridge, Massachusetts, andLondon, Bradford Book, MIT Press.

Ernst, C.H., Barbour, R.W. (1989): Turtles of the World.Washington, DC, Smithsonian Institution Press.

Ernst, C.H., Altenburg, R.G.M., Barbour, R.W. (2000): Tur-tles of the World. World Biodiversity Database, CD-ROM Series, Windows Version 1.2. Amsterdam, Biodi-versity Center of ETI.

Felsenstein, J. (1985): Confidence limits on phylogenies: anapproach using the bootstrap. Evolution 39: 783-791.

Fritz, U., Bischoff, W., Martens, H., Schmidtler, J.F. (1996):Variabilität syrischer Landschildkröten (Testudo graeca)sowie zur Systematik und Zoogeographie im NahenOsten und in Nordafrika. Herpetofauna 18 (104): 5-14.

Fritz, U., Cheylan, M. (2001): Testudo Linnaeus, 1758 –Eigentliche Landschildkröten. In: Handbuch der Rep-tilien und Amphibien Europas. Band 3/IIIA: Schild-kröten (Testudines) I, p. 113-124. Fritz, U., Ed.,Wiebelsheim, Aula-Verlag.

Fritz, U., Široký, P., Kami, H., Wink, M. (2005a): Environ-mentally caused dwarfism or a valid species – Is Testudoweissingeri Bour, 1996 a distinct evolutionary lineage?New evidence from mitochondrial and nuclear genomicmarkers. Mol. Phylogenet. Evol. 37: 389-401.

Fritz, U., Fattizzo, T., Guicking, D., Tripepi, S., Pennisi,M.G., Lenk, P., Joger, U., Wink, M. (2005b): A newcryptic species of pond turtle from southern Italy, thehottest spot in the range of the genus Emys. Zool. Scr.34: 351-371.

Fritz, U., Auer, M., Bertolero, A., Cheylan, M., Fattizzo, T.,Hundsdörfer, A.K., Martín Sampayo, M., Pretus, J.L.,Široký, P., Wink, M. (2006): A rangewide phylogeog-raphy of Hermann’s tortoise, Testudo hermanni (Rep-tilia: Testudines: Testudinidae): implications for taxon-omy. Zool. Scr. 35: 531-543.

Funk, D.J., Omland, K.E. (2003): Species-level paraphylyand polyphyly: frequency, causes, and consequences,with insights from animal mitochondrial DNA. Annu.Rev. Ecol., Evol. Syst. 34: 397-423.

Gasperetti, J., Stimson, A.F., Miller, J.D., Ross, J.P.,Gasperetti, P.R. (1993): Turtles of Arabia. Fauna ofSaudi Arabia 13: 170-367.

Gmira, S. (1993a): Une nouvelle espèce de tortue Testu-dininei (Testudo kenitrensis n. sp.) de l’Inter Amirien-Tensiftien de Kénitra (Maroc). C.R. Acad. Sci. Paris 316(II): 701-707.

Gmira, S. (1993b): Nouvelles données sur les espècesactuelles de Testudo. Bull. Soc. Herpétol. Fr. 65/66: 49-56.

Gmira, S. (1995): Étude des chéloniens fossiles du Maroc.Paris, Cahiers de Paléontologie, CNRS Éditions.

Gomes, C., Dales, R.B.G., Oxenford, H.A. (1998): Theapplication of RAPD markers in stock discriminationof the four-wing flyingfish, Hirundichthys affinis in thecentral western Atlantic. Mol. Ecol. 7: 1029-1039.

Guicking, D., Fritz, U., Wink, M., Lehr, E. (2002): Newdata on the diversity of the Southeast Asian leaf turtlegenus Cyclemys Bell, 1834. Molecular results. Faun.Abh. Mus. Tierkd. Dresden 23: 75-86.

Gupta, M., Chyi, Y.-S., Romero-Severson, J., Owen, J.L.(1994): Amplification of DNA markers from evolution-ary diverse genomes using single primers of simple-sequence repeats. Theor. Appl. Genet. 89: 998-1006.

Gustincich, S., Manfioletti, G., del Sal, G., Schneider, C.,Carninci, C. (1991): A fast method for high-qualitygenomic DNA extraction from whole human blood. BioTechniques 11: 298-302.

Guyot-Jackson, G., Ed. (2004): Numéro spécial Testudo.Manouria 7 (22): 1-52.

Haig, S.M., Rhymer, J.M., Heckel, D.G. (1994): Populationdifferentiation in randomly amplified polymorphic DNAof red-cockaded woodpeckers Picoides borealis. Mol.Ecol. 3: 581-595.

120 U. Fritz et al.

Haig, S.M., Gratto-Trevor, C.L., Mullins, T.D., Colwell,M.A. (1997): Population identification of western hemi-sphere shorebirds throughout the annual cycle. Mol.Ecol. 6: 413-427.

Hall, T.A. (1999): BioEdit: a user-friendly biological se-quence alignment editor and analysis program for Win-dows 95/98/NT. Nucl. Acids Symp. Ser. 41: 95-98.

Harris, D.J., Znari, M., Macé, J.-C., Carretero, M.A. (2003):Genetic variation in Testudo graeca from Morocco esti-mated using 12S rRNA sequencing. Rev. Esp. Herpetol.17: 5-9.

Highfield, A.C. (1990): Tortoises of north Africa; taxonomy,nomenclature, phylogeny and evolution with notes onfield studies in Tunisia. J. Chelon. Herpetol. 1 (2): 1-56.

Highfield, A.C., Martin, J. (1989a): A revision of the Tes-tudines of North Africa, Asia and Europe. Genus: Tes-tudo. J. Chelon. Herpetol. 1 (1): 1-12.

Highfield, A.C., Martin, J. (1989b): Testudo whitei Bennett1836. New light on an old carapace – Gilbert White’sSelborne tortoise re-discovered. J. Chelon. Herpetol. 1(1): 13-22.

Highfield, A.C., Martin, J. (1989c): Description of a minia-ture tortoise Testudo flavominimaralis n. species fromNorth Africa. London, The Tortoise Trust/Tortoise Sur-vival Project.

Hundsdörfer, A.K., Wink, M. (2005): New source of geneticpolymorphisms in Lepidoptera? Z. Naturforsch. 60c:618-624.

Jaccard, P. (1901): Étude comparative de la distributionflorale dans une portion des Alpes et des Jura. Bull. Soc.Vaud. Sci. Nat. 37: 547-579.

Jaccard, P. (1908): Nouvelles recherches sur la distributionflorale. Bull. Soc. Vaud. Sci. Nat. 44: 223-270.

Korsunenko, A., Vasilyev, V., Pereshkolnik, S., Mazanaeva,L., Lapid, R., Bannikova, A., Semyenova, S. (2005):DNA polymorphism and genetic differentiation of Tes-tudo graeca L. In: Herpetologica Petropolitana. Pro-ceedings of the 12th Ordinary General Meeting of theSocietas Europaea Herpetologica, August 12-16, 2003.Ananjeva, N., Tsinenko, O., Eds, Russ. J. Herpetol. 12(Suppl.): 40-42.

Mayr, E. (1942): Systematics and the Origin of Speciesfrom the Viewpoint of a Zoologist. New York, ColumbiaUniversity Press.

Mayr, E. (1963): Animal Species and Evolution. Cam-bridge, Massachusetts, Belknap Press.

Mayr, E. (2000): The Biological Species Concept. In:Species Concepts and Phylogenetic Theory. A Debate,p. 17-29. Wheeler, Q.D., Meier, R., Eds, New York,Columbia University Press.

Mertens, R. (1946): Über einige mediterrane Schildkröten-Rassen. Senckenbergiana 27: 111-118.

Mertens, R., Müller, L. (1928): Liste der Amphibien undReptilien Europas. Abh. Senckenberg. Naturforsch. Ges.41: 1-62.

Mishler, B.D., Theriot, E.C. (2000): The PhylogeneticSpecies Concept (sensu Mishler and Theriot): mono-phyly, apomorphy, and phylogenetic species concepts.

In: Species Concepts and Phylogenetic Theory. A De-bate, p. 44-54. Wheeler, Q.D., Meier, R., Eds, New York,Columbia University Press.

Myers, N., Mittermeier, R.A., Mittermeier, C.G., da Fon-seca, G.A.B., Kent, J. (2000): Biodiversity hotspots forconservation priorities. Nature 403: 853-858.

Nagy, Z.T., Joger, U., Guicking, D., Wink, M. (2003):Phylogeography of the European whip snake Coluber(Hierophis) viridiflavus as inferred from nucleotide se-quences of the mitochondrial cytochrome b gene andISSR genomic fingerprinting. Biota 3: 109-118.

Nei, M., Li, W.-H. (1979): Mathematical model for studyinggenetic variation in terms of restriction endonucleases.PNAS 76: 5269-5273.

Palkovacs, E.P., Gerlach, J., Caccone, A. (2002): The evo-lutionary origin of Indian Ocean tortoises (Dipsochelys).Mol. Phylogenet. Evol. 24: 216-227.

Palkovacs, E.P., Marschner, M., Ciofi, C., Gerlach, J., Cac-cone, A. (2003): Are the native giant tortoises from theSeychelles really extinct? A genetic perspective basedon mtDNA and microsatellite data. Mol. Ecol. 12: 1403-1413.

Parham, J.F., Macey, J.R., Papenfuss, T.J., Feldman, C.R.,Türkozan, O., Polymeni, R., Boore, J. (2006): The phy-logeny of Mediterranean tortoises and their close rel-atives based on complete mitochondrial genome se-quences from museum specimens. Mol. Phylogenet.Evol. 38: 50-64.

Perälä, J. (1996): Etelä-Turkin maakilpikonnista. Morfolo-gisia ja ekologisia eroja (Testudo ibera Pallas 1814 &Testudo ibera anamurensis Weissinger 1987) sekä uudenmaakilpikonnalajin kuvaus + 15 kuvaa. In: Virallinenkongressijulkaisu, p. 14-26. Herpetokongressi I. Perälä,J., Vikberg, J., Kanza, M., Eds, Helsinki, Suomen her-petologinen yhdistys ry.

Perälä, J. (2002a): Morphological variation among MiddleEastern Testudo graeca L., 1758 (sensu lato), with afocus on taxonomy. Chelonii 3: 78-108.

Perälä, J. (2002b): The genus Testudo (Testudines: Testu-dinidae): phylogenetic inferences. Chelonii 3: 32-39.

Perälä, J. (2004a): Tortoise systematics: a critique of arecent paper by van der Kuyl et al. (2002). Herpetol. J.14: 51-53.

Perälä, J. (2004b): Testudo [graeca] buxtoni Boulenger,1920. In: Numéro spécial Testudo. Guyot-Jackson, G.,Ed., Manouria 7 (22): 11-12.

Perälä, J., Bour, R. (2004): Neotype of Testudo terrestrisForsskål, 1775. Asiatic Herpetol. Res. 10: 114-119.

Perälä, J., Shacham, B. (2004): Testudo [graeca] floweriBodenheimer, 1935. In: Numéro spécial Testudo. Guyot-Jackson, G., Ed., Manouria 7 (22): 15-16.

Pieh, A. (2001): Testudo graeca soussensis, eine neue Un-terart der Maurischen Landschildkröte aus dem Sousstal(Nordwest-Marokko). Salamandra 36 (2000): 209-222.

Pieh, A., Fritz, U., Berglas, R. (2002): New data onmorphology, distribution and nomenclature of Testudograeca armeniaca Chkhikvadze & Bakradze, 1991.Faun. Abh. Mus. Tierkd. Dresden 22: 329-345.

Pieh, A., Perälä, J. (2001): Eine ungewöhnliche Landschild-kröte des Testudo graeca-Komplexes aus Krasnowodsk(Turkmenien). Herpetozoa 14: 65-73.


Pieh, A., Perälä, J. (2002): Variabilität von Testudo graecaLinnaeus, 1758 im östlichen Nordafrika mit Beschrei-bung eines neuen Taxons von der Cyrenaika (Nord-ostlibyen). Herpetozoa 15: 3-28.

Pieh, A., Perälä, J. (2004): Variabilität der MaurischenLandschildkröten (Testudo graeca Linnaeus, 1758 –Komplex) im zentralen und nordwestlichen Marokkomit Beschreibung zweier neuer Taxa. Herpetozoa 17:19-47.

Posada, D., Crandall, K.A. (1998): Modeltest: testing themodel of DNA substitution. Bioinformatics 14: 817-818.

Prior, K.A., Gibbs, H.L., Weatherhead, P.J. (1997): Popula-tion genetic structure in the black rat snake: implicationsfor management. Conserv. Biol. 11: 1147-1158.

Rögl, F. (1998): Paleogeographic considerations for Medi-terranean and Paratethys seaways (Oligocene toMiocene). Ann. Naturhist. Mus. Wien 99A: 279-310.

Rögl, F. (1999): Mediterranean and Paratethys. Facts andhypotheses of an Oligocene to Miocene palaeogeogra-phy (short overview). Geologica Carpathica 50: 339-349.

Ronquist, F., Huelsenbeck, J.P. (2003): MrBayes 3:Bayesian phylogenetic inference under mixed models.Bioinformatics 19: 1572-1574.

Satunin, K.A. (1910): Nekotorye soobrazheniya o prois-khozhdenii fauny Kavkazkogo kraya. Izv. Kavkazsk.otd. Russk. Geogr. obshch. 20: 1-12.

Schilde, M., Barth, D., Fritz, U. (2004): An Ocadia sinensis× Cyclemys shanensis hybrid. Asiatic Herpetol. Res. 10:120-125.

Semyenova, S.K., Korsunenko, A.V., Vasilyev, V.A.,Pereschkolnik, S.L., Mazanaeva, L.F., Bannikova, A.A.,Ryskov, A.P. (2004): RAPD variation in Mediterraneanturtle Testudo graeca L. Russ. J. Genetics 40: 1348-1355.

Swofford, D.L. (2002): PAUP*. Phylogenetic Analysis Us-ing Parsimony (*and Other Methods), Version 4.0b10.Sunderland, Massachusetts, Sinauer Associates.

Tassanakajon, A., Pongsomboon, S., Rimphanitchayakit, V.,Jarayabhand, P., Boonsaeng, V. (1997): Random ampli-fied polymorphic DNA (RAPD) markers for determina-tion of genetic variation in wild populations of the blacktiger prawn (Penaeus monodon) in Thailand. Mol. Mar.Biol. Biotechnol. 6: 110-115.

Tautz, D., Renz, M. (1984): Simple sequences are ubiq-uitous repetitive components of eukaryontic genomes.Nucl. Acids Res. 12: 4127-4138.

Tsumura, Y., Ohba, K., Strauss, S.H. (1996): Diversity andinheritance of inter-simple sequence repeat polymor-phisms in Douglas fir (Pseudotsuga menziesii) and sugi(Cryptomeria japonica). Theor. Appl. Genet. 92: 40-45.

Tuniyev, B.S. (1990): On the independence of the ColchisCenter of amphibian and reptile speciation. Asiatic Her-petol. Res. 3: 67-84.

Tuniyev, B.S. (1995): On the Mediterranean influence onthe formation of herpetofauna of the Caucasian isthmusand its main xerophylous refugia. Russ. J. Herpetol. 2:95-119.

Türkozan, O., Kumlutas, Y., Arikan, H., Ilgaz, Ç., Avcı,A. (2003): Morphological and serological comparisonof Mediterranean spur-thighed tortoises, Testudo graeca,from the Aegean region and southeastern Turkey. Zool.Middle East 29: 41-50.

van der Kuyl, A.C., Ballasina, D.L.P., Dekker, J.T., Maas,H., Willemsen, R.E., Goudsmit, J. (2002): Phylogeneticrelationships among the species of the genus Testudo(Testudines: Testudinidae) inferred from mitochondrial12S rRNA gene sequences. Mol. Phylogenet. Evol. 22:174-183.

van der Kuyl, A.C., Ballasina, D.L.P., Zorgdrager, F. (2005):Mitochondrial haplotype diversity in the tortoise speciesTestudo graeca from North Africa and the Middle East.BMC Evol. Biol. 5: 29.

Vanlerberghe-Masutti, F., Chavigny, P. (1998): Host-basedgenetic differentiation in the aphid Aphis gossypiiGlover, evidenced from RAPD fingerprints. Mol. Ecol.7: 905-914.

Weissinger, H. (1987): Testudo graeca anamurensis ssp.nov. aus Kleinasien. ÖGH-Nachrichten 10/11: 14-18.

Wermuth, H. (1958): Status und Nomenklatur der Mau-rischen Landschildkröte, Testudo graeca, in SW-Asienund NO-Afrika. Senckenbergiana Biol. 39: 149-153.

Wermuth, H., Mertens, R. (1961): Schildkröten, Krokodile,Brückenechsen. Jena, Fischer.

Wermuth, H., Mertens, R. (1977): Testudines, Crocodylia,Rhynchocephalia. Das Tierreich 100: I-XXVII, 1-174.

Wheeler, Q.D., Meier, R., Eds, (2000): Species Con-cepts and Phylogenetic Theory. A Debate. New York,Columbia University Press.

Wheeler, Q.D., Platnick, N.I. (2000): The PhylogeneticSpecies Concept (sensu Wheeler and Platnick). In:Species Concepts and Phylogenetic Theory. A Debate,p. 55-69. Wheeler, Q.D., Meier, R., Eds, New York,Columbia University Press.

Wiley, E.O., Mayden, R.L. (2000): The EvolutionarySpecies Concept. In: Species Concepts and PhylogeneticTheory. A Debate, p. 70-89. Wheeler, Q.D., Meier, R.,Eds, New York, Columbia University Press.

Wink, M., Sauer-Gürth, H., Martinez, F., Doval, G., Blanco,G., Hatzofe, O. (1998): Use of GACA-PCR for molec-ular sexing of Old World vultures (Aves: Accipitridae).Mol. Ecol. 7: 779-782.

Wink, M., Guicking, D., Fritz, U. (2001): Molecular evi-dence for hybrid origin of Mauremys iversoni Pritchardet McCord, 1991, and Mauremys pritchardi McCord,1997. Zool. Abh. Mus. Tierkd. Dresden 51: 41-49.

Wolfe, A.D., Liston, A. (1998): Contributions of PCR-basedmethods to plant systematics and evolutionary biology,p. 43-86. In: Molecular Systematics of Plants II. DNASequencing. Soltis, D.E., Soltis, P.S., Doyle, J.J., Eds,Boston, Massachusetts, Kluwer Academic Publishers.

Wolfe, A.D., Xiang, Q.-Y., Kephart, S.R. (1998): Assessinghybridization in natural populations of Penstemon (Scro-phulariaceae) using hypervariable intersimple sequencerepeat (ISSR) bands. Mol. Ecol. 7: 1107-1125.

Zietkiewicz, E., Rafalski, A., Labuda, D. (1994): Genomefingerprinting by simple sequence repeat (ISSR)-anchored polymerase chain reaction amplification. Ge-nomics 20: 176-183.

Received: June 19, 2006. Accepted: July 14, 2006.

Amphibia-Reptilia 28 (2007): 97-121 - iucn-tftsg.org · Amphibia-Reptilia 28 (2007): 97-121 Phenotypic plasticity leads to incongruence between morphology-based taxonomy and genetic

Documents