The declining Spadefoot toad, Pelobates fuscus (Pelobatidae): paleo and recent
environmental changes as a major inﬂuence on current population structure
*, Dan Coga
, Michael Veith
, Georg Dzukic
& Pierre Taberlet
Laboratory of Alpine Ecology, CNRS UMR 5553, Universite
´de Savoie, CISM, F-73376, Le Bourget du Lac
Faculty of Biology, Department of Ecology, Bucharest University, Splaiul Independen t
95, 050095, Bucharest, Romania;
¨r Zoologie, Abteilung O
¨kologie, Johannes Gutenberg-Universita
Mainz, Saarstraße 21, D-55099, Mainz, Germany;
Institute for Biological research ‘‘Sinisa Stankovic’’
Bulevar despota Stefana 142, 11060, Belgrade FR, Serbia;
Laboratory of Alpine Ecology, CNRS UMR
´Joseph Fourier, BP 53, F-38041, Grenoble Cedex 9, France (*Corresponding author : E-mail:
Received 5 July 2005; accepted 27 November 2005
Key words: amphibian decline, Dryas, global changes, low polymorphism, mtDNA, nested clade
phylogeographic analysis, Pelobates fuscus, Spadefoot toad
Populations of the European Spadefoot toad (Pelobates fuscus) have experienced recent declines all over
Europe, but these appear to be more intense in north and western Europe. Due to the toad’s fossorial
nature and speciﬁc habitat requirements, environmental conditions have played a major role in structuring
current populations. We examined the phylogeographic structure in P. fuscus from 16 localities throughout
Europe using mitochondrial cytochrome bgene sequence analysis. Sequence divergence among haplotypes
was low (0.54±0.15%). Three very closely related haplotypes occupy northern and western parts of Europe
whereas 12 others were observed among samples from south-eastern Europe, including the Balkans. Our
results suggest that toads only recently colonized the northern and western parts of Europe following
glacial retreat. This expansion probably took place in steppic-like areas during the younger Dryas cold
interval, about 12,900–11,500 years ago. Restricted gene ﬂow with an isolation-by-distance population
structure characterises a major part of its distribution range. Based on our results we suggest that the
northern and western lineages should be considered as distinct conservation units, while the south-eastern
populations from the refugial areas, where nearly all genetic polymorphism occurs and populations appear
less vulnerable, should receive special attention.
Climatic changes during the Pleistocene had a
profound impact on the distributional and genetic
patterns of many species living in temperate zones
(Hewitt 2000). During the coldest periods many
Palaearctic species were conﬁned to southern
refugia, subsequently expanding their ranges again
in warmer periods with retreat of the ice. Evidence
for range expansions and contractions can be de-
tected in current population genetic structures and
used have been used to reconstruct population
history (e.g. Avise 1992; Taberlet et al. 1998).
Lower genetic diversity typiﬁes recently colonized
areas more so than refugial areas, mainly due to
repeated founding events over colonizing routes
Conservation Genetics (2006) 7:185–195 Springer 2006
(Hewitt 1996). Because amphibians are ectother-
mic and adapted to speciﬁc climates, they are
known to be very sensitive to environmental con-
ditions and have even provided reliable informa-
tion concerning palaeoenvironmental conditions
(Bailon and Rage 1992). Therefore, palaeoenvi-
ronmental ﬂuctuations could lead to population
expansion or isolation in amphibian species that
could be inferred from current population genetic
structure (e.g. Barber 1999; Riberon et al. 2001).
Three species of the Spadefoot toad genus
Pelobates occur in Europe (P. cultripes, P. fuscus
and P. syriacus) and a fourth in north-western
Africa (P. varaldii). The Spadefoot toads have
narrow habitat requirements, due to their fossorial
behaviour (Shpun et al. 1993; Martinez-Rica 1997;
¨llert 1997). Toads inhabit preferentially sandy
areas, in which they burrow during daytime by
digging with metatarsal tubercles of their hind
limbs. They breed in permanent or temporary
ponds and the larval period lasts for 2–4 months.
European Spadefoot toads Pelobates fuscus, al-
though widely distributed across Europe (No
1997), shows a patchy distribution restricted to
speciﬁc friable soils (e.g. No
¨llert 1997; Kuzmin
1999). The species is strictly protected by both the
Bern Convention and Habitat Directive 92/43/
EEC, is included in most local Red Data Book lists
¨llert 1997; Kuzmin 1999), and is the object of
many local conservation eﬀorts. It is nevertheless
considered a least concern species by the last
IUCN assessment due to its wide range and pre-
sumably large populations (Baillie et al. 2004).
Recently a cryptic species, representing popula-
tions in the former Soviet Union, was detected
with DNA ﬂow cytometry (Borkin et al. 2001).
Declining amphibian populations have a
worldwide occurrence, with various causes
hypothesised to explain the phenomenon (e.g.
Gardner 2001; Collins and Storfer 2003). Spade-
foot toad population declines have been observed
or suspected over major portions of their geo-
graphic range (No
¨llert 1997). Declines were par-
ticularly striking in the western part of their range,
where population extinctions were clearly docu-
mented during the last century (see Lescure 1984;
Parent 1985; Dubois 1998 for France, Rapp _
Perczy 1994 for Belgium, Pelt and Van Bree 1965
for Netherlands, Fog et al. 1997 for Denmark,
Gislen and Kauri 1959; Berglund 1998 for Swe-
den), but also in the north-eastern and eastern
parts (Kuzmin 1999) or in the south-eastern parts
in Serbia (Dzˇ ukic
´et al. 2005). As an example of
local extirpation, toads disappeared from 98% of
the breeding ponds over a 45 year period in Den-
mark (Fog et al. 1997). The fact that strong
regression has occurred mainly in this part of the
species distribution has been singled out as an
important observation in need of scientiﬁc expla-
nation (Dubois 1998).
Because of their speciﬁc habitat requirements,
we argue that environmental conditions strongly
inﬂuence Spadefoot toad population dynamics.
We assume that palaeoenvironmental conditions,
especially repartition of palaeobiomes, have
played a major role in structuring current popu-
lations, making the European Spadefoot toad a
good candidate for illustrating the contraction–
expansion model (Taberlet and Cheddadi 2002).
Furthermore, we hypothesise that toads from
declining areas represent a distinct lineage. For the
present study, we compared samples from the
declining area with samples from other areas
where large populations still persist. We focused
on a sequence of cytochrome b(mtDNA) to
investigate current phylogeographic structure and
estimate the impacts of palaeoenvironmental
Material and methods
We obtained samples from 16 localities through-
out Europe. Since this species is strictly protected,
rare and secretive, collection of large samples has
generally been precluded. Tissue samples in etha-
nol came from a variety of sources: adults killed by
road traﬃc, clipped toes, larvae or larval tailtips
and museum specimens (Figure 1; Table 1).
DNA was isolated from skin or muscle using
a standard proteinase K digestion protocol
) for animal tissues, except that centri-
fugation was performed after digestion (12,000 g,
3-min) to eliminate undigested tissue. Sample
extracts 2 (ll) were ampliﬁed using PCR with
cytochrome bprimers H15915 (Irwin et al. 1991)
and L15162 (Taberlet et al. 1992) in a ﬁnal volume
of 25 ll (200 lM of primers, 2 mM of MgCl
unit of Taq Gold
, Perkin Elmer polymerase). A
10-min initial denaturation at 95C was followed
with 45 thermal cycles of 1-min denaturation at
95 C, 1-min annealing at 50 C and 3-min
extension at 72 C. PCR products were puriﬁed on
QiaQuick PCR columns. Sequencing was per-
formed with a Perkin Elmer ABI PRISM
automated DNA sequencer in 5% LongRanger
gels (FMC) after preparation with ABI PRISM
Dye Terminators Sequencing Ready Reaction Kit
Figure 1. Map of the sampled locations for Pelobates f. fuscus. Population codes correspond to the localities listed in Table 1.
Population M was too close to population N to be shown. The dotted line indicates 19th century western edge of expansion (Lescure
1984); the solid line indicates current expansion (No
¨llert 1997). The isolated black point in France represents an extant remnant
population. The shaded area reveals region where current population declines have been documented (see text for references).
Table 1. List of samples used in this study with population codes, locality, sample sizes, mitochondrial haplotypes and nucleotide
diversity Pof Nei and it standart deviation
Country Locality Latitude Longitude n Haplotypes PSD ( PÞ
A France Saint-Avold 4908 N 643 E 5 W14 0 0
B Germany Hannover Circa 5220 N 940 E 2 W14 0 0
C Germany Mainz 4950 N 820 E 3 W14 0 0
D Germany Emsland-Werlte 5250 N 740 E 1 W14 – –
E Sweden South Scania Circa 5610 N 1340 E 1 W14 – –
F Netherlands Nijmegen-Ewijk Circa 5150 N 535 E 2 W13 W14 0.00142 0.00071
G Austria Vienna 4815 N 1624 E 12 W2 (n=4);
W6 (n=7); W7
H Hungary Nagybajom 4626 N 1728 E 1 W1 – –
I Hungary Dabas-Gyon 4709 N 1918 E 1 W4 – –
J Hungary Kunadacs Circa 4604 N 19E1W6 – –
K Romania Bucharest 4429 N 2558 E 11 W1; W8 (n=2);
W9 W11 (n=6); W12
L Romania Bra
˘ila 4447 N 2749 E 4 W2; W10; W11 (n=2) 0.00475 0.00148
M Serbia Kladovo Circa 4540 N 1940 E 2 W1 0 0
N Serbia Deliblato 4550 N 1950 E 3 W3 (n=2); W6 0.00427 0.00214
N Serbia Hrastovaca 4609 N 1941 4 W5 (n=3); W6 0.00427 0.00214
O Polonia Torun 5301 N 1837 E 2 W14; W15 0.00142 0.00071
P Estonia Piirissaar 5822 N 2729 E 5 W14 0 0
(Perkin Elmer). Fragment sequences were aligned
using the SEQUENCE NAVIGATOR program
(version 1.0.1, Perkin Elmer). The sequences are
deposited in EMBL, GenBank and DDBJ
(Accession numbers DQ333357 to DQ333373).
With MODELTEST version 3.06 (Posada
and Crandall 1998) we determined the HKY
substitution model as the best supported by our
data (hLRT test with lnL=)1069.2742; base fre-
pT¼0:3165 with equal rates for all sites and the
number of invariable sites I=0). Phylogenetic
relationships were determined by the neighbour-
joining method using PAUP* (version 4) (Swof-
ford 1998), with 2000 bootstrap replicates to
assess topology robustness. P. syriacus from
Deliblato (Serbia) and P. cultripes from Argeles-
sur-Mer (France) were used for outgroup rooting.
Average molecular distances and substitution
rates between P. fuscus haplotypes and outgroups
as well as among P. fuscus haplotypes were cal-
culated with MEGA 3 (Kumar et al. 2003), with
standard errors calculated for 2000 bootstrap
Nucleotide diversity Pof Nei (1987), the
average number of nucleotide diﬀerences per site
between sequences, was calculated for all samples
with n>1 using DnaSP version 4.10.3 (Rozas
et al. 2003). We compared nucleotide diversity
among geographic groups of populations using the
non-parametric Mann and Whitney U-test.
In order to free our phylogeographic interpre-
tation of P. fuscus haplotype distribution from a
priori hypotheses of lineage evolution, we applied
a nested clade phylogeographic analysis, NCPA
(Templeton et al. 1995; Templeton 2004). It links
the evolutionary position of haplotypes within a
minimum spanning tree with their geographic
location via rigorous testing of diﬀerent causes of
geographical association of haplotypes. A 95%
plausible set of all haplotype linkages in an un-
rooted haplotype tree (program TCS of Clement
et al. 2000) provided the basis for nested clade
deﬁnition following the basic nesting rules as de-
scribed in Templeton et al. (1987). Geographical
distances (in kilometers) were calculated from
geographical co-ordinates. D
(X) quantiﬁes the
average distances of single clade X haplotypes
from their respective geographical centre; whereas
(X) quantiﬁes the nested clade distances of all
nested clade X haplotypes from their geographical
centre. Diﬀerences of (I)T)D
quantify equivalent measures for tip (T) and inte-
rior (I) nested clades. All measures were calculated
using GeoDis (Posada et al. 2000). This program
also calculates tests for geographic association as
described by Templeton and Sing (1992). The
distribution of distance measures under the null
hypothesis of no geographic association was
determined after 10,000 random permutations of
clades against sampling locations and recalculating
the distances after each permutation (Templeton
et al. 1995). This allows testing for signiﬁcantly
) and smaller (
) distances for each clade
within a group of nested clades with respect to the
null hypothesis. We interpreted NCPA results using
the inference key provided at the GeoDis home-
_lab/geodis.htm; inquiry date 14th of July 2004).
This inference key addresses some recent criticism
to NPCA (Knowles and Maddison 2002).
The alignment of a 702 bp fragment of cytochrome
bsequenced for 60 individuals was straightfor-
ward; there were no indels, and all sequences were
translated without nonsense codons. A total of 15
mitochondrial haplotypes was recovered, with 17
(2.4%) variable sites (Table 2). Levels of diver-
gence among P. fuscus haplotypes were quite low
(HKY molecular distance: 0.0054±0.0015),
whereas molecular divergence between P. fuscus
and outgroup species was 0.1728±0.0232 for P.
syriacus and 0.1932±0.0253 for P. cultripes. All
samples from the north-western populations
(codes A to F) shared the same haplotype (W14),
except for one individual from locality F showing
one nucleotide sequence mutation haplotype
(W13). W14 was also found in Poland, together
with W15 (only one substitution diﬀerence). A
total of 14 distinct haplotypes was found for
individuals from populations G to P. Most phy-
logenetic relationships among haplotypes were
poorly resolved (Figure 2).
Nucleotide diversity of populations ranged
from 0 to 0.00427. Populations from formerly
glaciated or permafrost areas in the north-west
and north (A–F, O and P) showed a signiﬁcant
lower Pthan those from south-eastern Europe (U-
test; U=4, Z=2.24179, P<0.05).
While haplotype W12 appears to be basal in
the NJ tree, it becomes a terminal haplotype in the
minimum spanning tree (Figure 3). When rooted
with additional P. fuscus haplotypes from Italy
and eastern Europe (Crottini et al. unpublished),
the wide-spread W14 becomes the root of the
minimum spanning tree, making clades 1-6 and 2-1
interior clades, which has consequences for NPCA
Signiﬁcant deviation from random association
of haplotypes and localities is indicated only for
clades 1-1, 2-1 (which covers the whole study area),
and the total clade. For all other clades the out-
come is inconclusive. However, interpreting clade
1-1 also gives no result. The pattern observed for
clade 2-1 in the rooted tree can be explained by
restricted gene ﬂow with isolation-by-distance.
Level 2 clades probably have spread over western
Europe in a process of long distance colonisation
(total clade; Table 4). Alternatively, each subclade
of the total clade may have expanded its area after
Nucleotide divergence among haplotypes
This study revealed a surprisingly low level of se-
quence divergence between haplotypes (0.54±
0.15%). In amphibians, intraspeciﬁc sequence
divergence ranges from 0 to 16.2% (review in
´ron et al. 2001). Avise et al. (1998) reported
for amphibians and reptiles a median value of
3.1% in mtDNA (except for the control region)
sequence divergence between phylogroup pairs
(i.e. at intraspeciﬁc level). Slow mtDNA clocks
have also been suggested for some poikilotherms
(Johns and Avise 1998; Avise et al. 1998), but it is
possible that the rate of mtDNA evolution in
Spadefoot toads is unusually slow. Nevo and Be-
iles (1991) reported that among amphibians Pelo-
bates species have among the lowest levels of
allozyme heterozygosity and polymorphism. Based
on an estimated rate of mtDNA divergence in
some amphibians of 0.7–0.8%/Myear (see Ribe
et al. 2001), the low level of diversity in the studied
samples of P. fuscus could result from Pleistocene
divergence, about 1.4–0.18 Myear ago. If however
substitution rates are lower in Pelobates, then
divergence among P. fuscus lineages may well have
started prior to the Pleistocene.
A low level of intraspeciﬁc polymorphism at
higher latitudes, particularly in areas glaciated
during the Pleistocene cold periods, and a higher
level of genetic variation in areas that corre-
sponded to refugia is a trend described in many
species (Taberlet 1998). Limited mitochondrial
Table 2. Variable sites from the aligned 702 base pair sequences
Base position Haplotypes
16 79 88 97 136 148 184 202 250 292 307 331 433 481 505 574 694
W1 TTTCA T C T T C C T A C G T C
W7 ..... . . . . T. . G. A. .
variation over a wide area usually suggests a recent
range expansion from a single source area (e.g.
Phillips 1994; Barber 1999; Phillips et al. 2000). In
this very specialized species, landscape features are
known to strongly inﬂuence the dispersal and
abundance of populations. Fossil data from the
Quaternary suggest ﬂuctuations in Pelobates pop-
ulation abundance. During the Neolithic period
remains of Pelobates fuscus were scarce in a
southern France locality, but became the most
abundant species during the medieval period, in a
more agricultural landscape (Bailon and Rage
1992). Range extensions were noticed by Kauri
(1946) in north Estonia following deforestation
and expansion of agricultural landscape associated
with pond cleaning practices. Andreone et al.
(1993) reported that Italian Spadefoot toads
(Pelobates fuscus insubricus) prefer the alluvial and
morainal plains, and that colonization was faster
in regions with morainal debris from glaciers. Also
Meissner (1970) argued that river banks with
alluvial sand deposits are the most suitable
Spadefoot toad habitat. Kauri (1946) observed
that glacial drift areas promoted population
spreading in northern countries. Steppic-like areas
with short vegetation are more likely used by
toads, while adults seem to avoid shrub-covered
areas (Eggert 2002).
The present north-western populations proba-
bly originated during a range expansion in the
younger Dryas cold and dry interval (12,900–
11,500 year bp). There are several facts that sup-
port this hypothesis. (I) During this period dry
steppe and steppe-tundra replaced, mainly in
loessic soil, the previously extended woodland
covered much of Europe (Figure 4; Frenzel et al.
1992). (II) From the debate about the natural or
the introduced origin of Spadefoot toads in Swe-
den, the natural origin hypothesis was retained
(Gislen 1938). Swedish toads could not have des-
cended from Pleistocene populations (120,000–
80,000 years), because Scandinavia was mostly
covered by ice for the last time 22,500 to about
15,000 years ago (Adams and Faure 1997). During
Dryas southern Sweden was free of ice and con-
nected for the last time to Denmark. As Gislen and
Figure 2. Neighbour joining tree of P. fuscus haplotypes; the
root was located using P. syriacus and P. cultripes as outgroups;
only bootstrap support values ‡50 are given.
1-1 1-5 1-6
1-1 1-5 1-6
Figure 3. Minimum spanning tree and nested clade structure.
Kauri (1959) suggested, toads probably reached
Sweden via Denmark 8–9,000 years ago, before
the land connection broke. (III) After being
unsuitable for herpetofauna during the last glaci-
ation, which ended around 10,000 years ago,
Britain was separated from the continent around
7,000 year ago, but Spadefoot toads have never
been found in Great Britain (Lambeck 1997;
Gleed-Owen 2000). We assume that population
expansion reached this part of western Europe
after the connection was severed, unlike Triturus
vulgaris and T. cristatus, originating from the
Table 3. NCA result for clades with signiﬁcant geographic structure; I=interior clade, T= tip clade; v2=observed chi-square; P
v2=probability of random v2(10,000 permutations) being greater or equal to observed v2;D
=distance within clade; D
within nested clade; (I)T)/D=interior vs. tip clade distances; signiﬁcant values:
=smaller than mean distance;
=larger than mean
distance; the root was set to W14 via multiple outgroup haplotypes
1-1 I/T60 <0.001 )95.92 )105.85
W1 I115.97 132.62
W2 T0.00 58.98
W3 T0.00 94.79
1-2 0.41 1.000 131.05 )95.83
W6 I131.05 135.06
W7 T0.00 230.88
1-3 4.00 0. 500 0.0 )3.17
W8 I0.00 71.83
W9 T0.00 71.83
W10 T0.00 78.17
1-4 0.32 1.000 74.86 8.52
W11 I74.86 74.61
W12 T0.00 66.09
1-6 19.89 0.264 492.63 44.80
W13 T0.00 466.05
W14 I492.63 491.84
W15 T0.00 428.03
2-1 63.76 <0.001 348.06
1-6 I486.24 672.46
2-2 0.012 1.000 0.931 0.543
1-3 I74.86 74.69
1-4 T73.93 74.15
Total clade 50.22 <0.001 501.59
2-1 I575.75 570.31
Table 4. Result of the inference key
Clade Inferred phylogeographic scenario
Clade 1-1 1-2
Clade 2-1 1-2
-3-4-NO ﬁrestricted gene ﬂow with isolation by distance
Total clade 1-2
-13-YES ﬁlong distance colonisation possibly
coupled with subsequent fragmentation
or past fragmentation followed by range expansion
Subsequent fragmentation is not indicated due to only one mutation between clades 2-1 and 2-2.
Balkan refuge (Kalezic
´1984; Crnobrnja et al.
1997), managed to colonize Britain and even Ire-
land (T. vulgaris). The speciﬁc habitat require-
ments of P. fuscus which deﬁnitely limits their rate
of dispersal and choice of migration routes, might
explain their absence from the British Isles. It
comes a bit surprising that our rigorous testing for
historical processes without a priori assumptions
did not select a ‘contiguous range expansion’
model for the wide-spread clade 1-6. This clade
exhibits the typical pattern of an expanding pop-
ulation, with one ancestral wide-spread haplotype
(W14) and few locally restricted haplotypes (W13
and W15) (Avise 2000). Based on outgroup root-
ing of the minimum spanning tree with P. fuscus
haplotypes from Italy and Russia (Crottini et al.
unpublished) the north-western clade 1-6 (haplo-
types W13, W14 and W15) is ancestral to all other
haplotypes. This unusual pattern of north–south
descent with a haplotype that currently is found in
formerly glaciated areas as ancestor to haplotypes
in the presumed glacial refuge argues for a more
comprehensive analysis that covers the whole
range of P. fuscus. Nevertheless, occurrence of the
P. fuscus clade 1-6 in a previously glaciated area
does not necessarily mean that it survived there.
This striking result may therefore indicate that (I)
in this special case NCPA identiﬁed a wrong
hypothesis as being signiﬁcant due to incomplete
geographical sampling (type II error; see Knowles
and Maddison 2002), (II) that the north-western
lineage in fact survived in southern or south-east-
ern European refugium and postglacially reached
the areas now north of their descendants, or that
(III) P. fuscus is another example for a species that
survived glaciations in more northern cryptic
refugia (e.g. Stewart and Lister 2001). For the time
being we are not able to distinguish between theses
variants, although the signiﬁcantly lower nucleo-
tide diversity of northern and north-eastern pop-
ulations is indicative of the second hypothesis.
Fossil data suggest several expansion–regres-
sion events in western Europe. For example, fossil
remains that resemble specimens of P. fuscus in
both size and morphology were described from
the Pliocene and Early Pleistocene of Poland
(Mlynarski 1962), and from the Pleistocene of
Romania (Venczel 1989) and Czechoslovakia
ˇek 1988). Spadefoot toads populations
reached the south of France during Pleistocene
(Riss III–Wurm II, 120,000–80,000 years, Bailon
1991), which corresponds to the end of the Eemian
interglacial period, with a rapid cooling about
110,000 years ago. A strong regression likely
occurred in central Europe during the next glacia-
tion event, but spadefoots were again present in
southern France since at least the Neolithic period
(Bailon and Rage 1992). This suggests that spade-
foots were relatively constant inhabitants in cen-
tral and eastern Europe, sometimes even during
Spadefoot toads from north-western populations,
including all the declining area, represent a distinct
lineage and therefore should be considered as an
evolutionary signiﬁcant unit (ESU, Ryder 1986;
Moritz 1994a). This ESU’s current repartition may
be the result of a circa younger Dryas biological
process. Our data were consistent with the local
observations of Spadefoot toad populations
spreading in open landscapes (Kauri 1946; Bailon
and Rage 1992) and highlight the inﬂuence of cli-
matic and landscape changes in toad population
expansion process. Since 8,000 years ago temper-
ate forest had returned to most of Europe and
loessic areas became gradually less abundant
(Frenzel et al. 1992). Phylogeographic data sug-
gests that because of their limited dispersal
opportunities due to the narrow habitat require-
ments of adults, Spadefoot toads are especially
Figure 4. Dryas paleobiomes map (12,900–11,500 years ago)
after Adams and Faure (1997). PD: Polar dessert, ST: Steppe-
tundra, WS: Wooded steppe, DS: Dry steppe.
vulnerable to environmental changes. Even so re-
cent P. fuscus population decline may be the result
of many proximate causes that could have various
and complex connections with global environ-
mental changes (Kiesecker et al. 2001). Metapop-
ulation processes have critical inﬂuence on
Spadefoot toad population viability (Eggert 2000;
Hels 2002) and habitat fragmentation, resulting
from human activity or natural environmental
changes, may signiﬁcantly participate in decline of
the toad by further limiting their dispersal. It is
noteworthy that very low mtDNA polymorphism
was observed, especially in areas where popula-
tions have suﬀered recent strong declines. Never-
theless nuclear DNA polymorphisms remain to be
studied, since the survival of anuran populations
could be hindered by low genetic variability and
inbreeding depression (e.g. Rowe et al. 1999). The
observed mtDNA low polymorphism, associated
with the observation of a lower female fecundity in
the border of the declining area (Eggert, unpub-
lished data), underline the need of a study on nu-
clear genetic diversity. Nevertheless we suggest that
future conservation eﬀorts in the declining area
should include management of genetic diversity.
Long-term conservation policies, devoted on
genetic diversity management (see Moritz 1994b),
should clearly consider Balkan region, while al-
most all mitochondrial diversity was found in
southern refugial areas, underscoring the high
conservation value of those areas. Anyway the
biogeographical uniqueness of the Balkan penin-
sula is indisputable, with ca. 28% of amphibians
and 21% of reptiles being endemic (Dzukic and
Kalezic 2004). Identifying areas where populations
are more likely to survive is now more urgent, not
only for short term conservation goals, but for the
long term persistence through climatic oscillations.
This study was mainly supported by the French
Oﬃce National des Foreˆ ts and Electricite
France. One of the author (G.D.) is partly sup-
ported by the Ministry of Science, Technology and
Development of Serbia (project title ‘‘Integrative
study of amphibians and reptiles of the central
Balkans’’, grant No. 1623). We thank C. LeBihan
(ONF), R. Guye
´tant, and A. Ribe
´ron for assis-
tance and R. Jehle, E. Ripﬂ, A. Bitz, B. Ujvari, M.
Puky, A. Stumpel, V. Lacoste and K. Grossenb-
acher for providing us with tissue samples.
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of the world since the Last Glacial Maximum, Oak Ridge
National Laboratory, TN, USA http://www.esd.ornl.gov/
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