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The declining Spadefoot toad, Pelobates fuscus (Pelobatidae): Paleo and recent environmental changes as a major influence on current population structure and status


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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 b gene 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.
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The declining Spadefoot toad, Pelobates fuscus (Pelobatidae): paleo and recent
environmental changes as a major influence on current population structure
and status
Christophe Eggert
*, 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
Cedex, France;
Faculty of Biology, Department of Ecology, Bucharest University, Splaiul Independen t
ßei 91-
95, 050095, Bucharest, Romania;
Institut fu
¨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
5553, Universite
´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
DOI 10.1007/s10592-006-9124-y
(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 _
e 1982;
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
0.038 0.00142
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
0.00313 0.00073
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-
quencies: pA¼0:2377;pC¼0:3150;pG¼0:1309;
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
and (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
larger (
) 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-
page (
_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
past fragmentation.
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.
Historical biogeography
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
W2 ..........T......
W3 .....C...........
W4 ..C..............
W5 .......C.........
W6 .........T....A..
W7 ..... . . . . T. . G. A. .
W8 .........T.....C.
W9 .C.......T.....C.
W10 .........T.....CT
W11 C........T.....C.
W12 C........T...T.C.
W13 ...TG...CT.C.....
W14 ...TG....T.C.....
W15 ...TG.T..T.C.....
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
W 2
W 4
W 5
W 1
W 3
W 7
W 6
W 13
W 15
W 14
W 10
W 8
W 9
W 11
W 12
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
1-2 2-1
2-2 W12
1-1 1-5 1-6
1-21-2 2-12-1
total clade
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
= distance
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
Clade v2Pv2D
(I-T)/ D
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
W4 T0.00
W5 T466.16
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
1-1 T137.87
1-2 T138.61
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
2-2 T74.32
Table 4. Result of the inference key
Clade Inferred phylogeographic scenario
Clade 1-1 1-2
-5-6-not applicable
Clade 2-1 1-2
-3-4-NO restricted gene flow with isolation by distance
Total clade 1-2
-13-YES long 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
Conservation implications
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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... sandy) soils with freshwater ponds for breeding and have a semi-fossorial lifestyle, thanks to well-known adaptations such as metatarsal spades (to dig themselves in) and a strongly ossified skull (to dig themselves out) (Székely et al. 2017;Dufresnes 2019). They are threatened in many parts of their fragmented ranges due to land-use changes, wetland destruction, pollution, species introduction, and ongoing changes in climate, which already led to population extinctions and contractions of geographic ranges (Nyström et al. 2002(Nyström et al. , 2007Džukić et al. 2005;Eggert et al. 2006). Mediterranean regions, where most of the diversity is located (Litvinchuk et al. 2013;Dufresnes et al. 2019b), could be particularly threatened (Iosif et al. 2014). ...
... The species is also present in a large area of northern Italy, especially in the Po Valley (Andreone 2006 (Agasyan et al. 2009a). Declines have been reported for more than a century in various parts of Europe, which have caused a regression of the distribution limits (Džukić et al. 2005;Eggert et al. 2006). ...
... This seems supported by weak genomic differentiation among Central-European samples (Dufresnes et al. 2019b). The refugial areas bear nearly all the genetic diversity of the species, which was lost in the derived northern populations, following post-glacial colonizations (Eggert et al. 2006). ...
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The genomic era contributes to update the taxonomy of many debated terrestrial vertebrates. In an accompanying work, we provided a comprehensive molecular assessment of spadefoot toads (Pelobates) using genomic data. Our results call for taxonomic updates in this group. First, nuclear phylogenomics confirmed the species-level divergence between the Iberian P. cultripes and its Moroccan relative P. varaldii. Second, we inferred that P. fuscus and P. vespertinus, considered subspecies until recently, feature partial reproductive isolation and thus deserve a specific level. Third, we evidenced cryptic speciation and diversification among deeply diverged lineages collectively known as Pelobates syriacus. Populations from the Near East correspond to the Eastern spadefoot toad P. syriacus sensu stricto, which is represented by two subspecies, one in the Levant (P. s. syriacus) and the other in the rest of the range (P. s. boettgeri). Populations from southeastern Europe correspond to the Balkan spadefoot toad, P. balcanicus. Based on genetic evidence, this species is also polytypic: the nominal P. b. balcanicus inhabits the Balkan Peninsula; a new subspecies P. b. chloeae ssp. nov. appears endemic to the Peloponnese. In this paper, we provide an updated overview of the taxonomy and distribution of all extant Pelobates taxa and describe P. b. chloeae ssp. nov.
... These changes have caused a decrease in and range constriction of several amphibian species, including the common spadefoot toad (Pelobates fuscus) and the northern crested newt (Triturus cristatus). Both of these species currently have declining population trends in Europe (Eggert et al. 2006;IUCN 2009;Denoël 2012), including in Estonia (Red Data Book of Estonia 2008), and they are strictly protected within the European Union (EU Habitats Directive 92/43/ EEC). ...
... Thus the availability of sandy soils which can easily be dug by the toad is essential for the species (Eggert and Guyétant 1999;Eggert 2002). In addition, the existence of P. fuscus tadpoles in the pond could also be predicted by land cover types in the vicinity of the breeding site-open areas with grazing were preferred, whereas forested and mire areas avoided, which is corroborated by previous studies (Kauri 1947;Eggert 2002;Eggert et al. 2006;Rannap et al. 2015). Similarly to T. cristatus, the terrestrial habitat preferences of P. fuscus also vary across its range-the species inhabits mostly open agricultural areas in Western-Europe, although at the edges of its range it also occurs in forested areas (Nyström et al. 2002;Rannap et al. 2013). ...
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Ponds—small, isolated freshwater bodies—have vanished in large numbers during the last decades. Despite such great loss, the number of natural small water bodies has still remained quite high in Estonia. Nevertheless, many pond-related species, including amphibians such as the northern crested newt Triturus cristatus and the common spadefoot toad Pelobates fuscus—are in decline in Estonia, suggesting that the conditions of extant natural ponds might not be optimal. However, these conditions have never been examined. To halt the decline of these two pond-breeding species, more than 400 ponds have been constructed or restored from 2004 to 2014 in Estonia. In this study we compared 85 natural and 85 constructed ponds (which were created or restored especially for T. cristatus and/or P. fuscus) to find out: (i) what the main differences are between natural ponds and ponds specially created for threatened species; (ii) whether natural ponds provide breeding conditions for local amphibians; (iii) given the decline of T. cristatus and P. fuscus, what are the characteristics lacking in natural ponds, due to which they are not providing quality breeding habitats for these species. Whereas the constructed ponds were located in open habitats with mineral soils, the natural ponds were mainly in mires and forests, being thus more shaded. Amphibian diversity was higher in the constructed ponds and was positively related to the depth of the pond, the clarity of the water, the presence of slanting slopes, the absence of fish and the presence of nearby fields. T. cristatus preferred constructed ponds for reproduction, while the breeding site selection of P. fuscus was determined mainly by terrestrial habitat characteristics. Importantly, when the threatened species were removed from the sample, the diversity of common amphibians did not differ between natural and constructed ponds, suggesting that in our study sites natural water bodies act still as breeding sites for common species, but not for threatened ones. We conclude that pond construction is an important tool to halt the decline of threatened species, even in landscapes where natural ponds are still preserved.
... In der Schweiz gilt sie als ausgestorben. Europaweit sind Bestandsrückgänge der Art zu beobachten, wobei der Rückgang am gravierendsten in Nord-und Westeuropa ist (EGGERT et al. 2006 ...
... Nutzungsänderung in landwirtschaftlich genutzten Gebieten (grabbare Böden) von einem weiteren Verlust an Lebensräumen bzw. Populationen sowie deren zunehmenden Isolation auszugehen (EGGERT et al. 2006). ...
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... As a result of land-use changes, pollution, destruction of wetlands, changes in climate, and the introduction of invasive species, the ranges of Pelobates species are fragmented and threatened in many countries. There are already observations of contractions of their geographic distribution and population extinctions (Nyström et al., 2002(Nyström et al., , 2007Džukić et al., 2005;Eggert et al., 2006). In Bulgaria, the Balkan Spadefoot Toad is included on the local Red List as an endangered (EN) species (Beshkov, 1985). ...
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The development of genomics in recent years has led to updates in the taxonomy of many terrestrial vertebrates. Following the most recent taxonomic revisions (Dufresnes et al., 2019a, b) of the anuran family Pelobatidae Bonaparte, 1850 (genus Pelobates Wagler, 1830), three species of spadefoot toad are now recognised on the territory of Bulgaria: Pelobates fuscus (Laurenti, 1768), P. syriacus Boettger, 1889, and P. balcanicus Karaman, 1928. According to Dufresnes et al. (2019a), the Balkan Spadefoot Toad has two subspecies: P. b. balcanicus and Pelobates b. chloeae Dufresnes et al., 2019, of which only the former is found in Bulgaria.
... The common spadefoot toad Pelobates fuscus (Laurenti, 1768) is a widespread species ranging throughout most of Europe (Dufresnes et al., 2019a;2019b). It is a highly specialized, nocturnal, burrowing anuran, with strong population declines due to habitat loss (Eggert et al., 2006;Temple & Cox, 2009). Breeding occurs in permanent or temporary ponds. ...
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Urbanization impacts biodiversity both directly through physical expansion over land, and indirectly due to land use conversion and human behaviors associated with urban areas. We assessed the response of a common spadefoot toad population (Pelobates fuscus) to habitat loss and fragmentation resulting from urban development by studying changes in size, body condition and age parameters. We compared samples collected in the early 2000s (sample A) and later on during 2012–2014 (sample B). The terrestrial habitats in the study area were severely reduced and fragmented due to the expansion of the human settlement. We found no significant differences in the age parameters between the two sampling periods; the median lifespan shortened from 3.5 (sample A) to 3.0 years (sample B), while the other age parameters were similar in both samples. In contrast, snout-vent length, body mass and body condition experienced a significant decrease over time. Our results suggest that changes in body size and body condition, rather than age parameters, better reflect the response of the common spadefoot toad population to declining habitat quality. Therefore, body measurements can provide reliable estimates of the impact of habitat degradation in amphibian populations.
... The phylogeography of some European amphibians is characterised by genetic diversity gradients, e.g. declining diversity from south to north and east to west, caused by postglacial recolonization from glacial refuge areas (e.g. the European tree frog Hyla arborea, Dufresnes et al. 2013; the natterjack toad Epidalea calamita, Allentoft et al. 2009;Rowe et al. 1998Rowe et al. , 2006Beebee and Rowe 2000; the spadefoot toad Pelobates fuscus, Eggert et al. 2006; the Italian agile frog Rana latastei, Garner et al. 2004, the common frog Rana temporaria, Palo et al. 2004). As predicted, we observed a decrease of genetic diversity (AR) from south to north and from east to west for B. variegata as well. ...
Full-text available
Amphibian populations worldwide are threatened by declines and extinctions mainly due to habitat loss and fragmentation. Habitat fragmentation threatens the yellow-bellied toad Bombina variegata in the northern and western regions of its distribution where it is strictly protected. We studied the genetic structure and diversity of populations at three geographical scales using microsatellite loci to detect potential threats for population persistence. At the local scale, we sampled four neighbouring localities at 1-2.6 km distance to detect effects of short-term (decades) fragmentation on connectivity. At the regional scale, five additional localities in the mountains of the Westerwald (Rhineland-Palatinate, Germany) were studied at up to 50.1 km distance to analyse genetic diversity and population structure. At the continental scale, we included data from regions in the northern distribution with fragmented populations (Hesse and Lower Saxony, Germany) and more continuous populations in the South (Alsace, France; Geneva, Switzerland; Trentino, Italy) to evaluate variation of genetic diversity. At the local scale, short-term fragmentation caused significant genetic differentiation between breeding assemblages only 1.4 km apart from each other. At the regional scale, we found notable genetic distance among localities. At the continental scale, we identified Alsace, Trentino and Geneva in the South as regions with low genetic structuring and high allelic richness, and the northern remaining regions in Germany as deeply structured with reduced allelic richness. We suggest that reduced genetic diversity and habitat fragmentation in northern regions makes these populations particularly vulnerable to decline. In conclusion, informed conservation management of B. variegata should focus on measures maintaining or improving con-nectivity among neighbouring populations.
... A barna ásóbékák a rövid, alacsony növényzettel borított élőhelyeket részesítik előnyben. Habár főként az idősebb egyedek kerülik a bokros vegetációkat (Eggert et al. 2006), Farmos határában azonban a cserjékkel borított gyepes élőhelyeken is megtalálható a faj. A barna ásóbéka mellett még jelentős vöröshasú unka és tarajos gőte állomány is él a területen, melyek főként a szikes réteken fordulnak elő. ...
... A barna ásóbékák a rövid, alacsony növényzettel borított élőhelyeket részesítik előnyben. Habár főként az idősebb egyedek kerülik a bokros vegetációkat (Eggert et al. 2006), Farmos határában azonban a cserjékkel borított gyepes élőhelyeken is megtalálható a faj. A barna ásóbéka mellett még jelentős vöröshasú unka és tarajos gőte állomány is él a területen, melyek főként a szikes réteken fordulnak elő. ...
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A Pest megyei Farmos határában 2004 óta zajlik a kétéltűmentési akció, a közúti gázolások csökkentése érdekében. A kétéltűek telelő-és szaporodóhelyét a 311-es műút választja el, óriási veszélynek kitéve a tavasszal nagy tömegben vonuló állatokat. A Duna-Ipoly Nemzeti Park Igazgatóság munkatársai több mint 10 éve szervezik a mentést önkéntesek bevonásával, mely egy-ben számos adatot szolgáltat az itt élő kétéltű populációk monitorozásához. Az egy évtizedes munka során 348 491 kétéltűt gyűjtöttünk be terelőkerítés és vödörcsapdák segítségével és vittünk át az út túloldalára, többségében barna ásóbékákat (97 %). Kulcsszavak: kétéltűek, Farmos, békamentés, barna ásóbéka, környezeti nevelés, közúti gázolások csökkentése
... syriacus) range limits (Džukić et al., 2008;Cogȃlniceanu et al., 2013). P. fuscus suffered multiple dramatic local declines across Europe during the last three decades (Nyström et al., 2002;Eggert et al., 2006), being already extinct in Switzerland (Agasyan et al., 2009), and its southern and western range forecasted to contract under future climate scenarios (Araújo et al., 2006). Both species breed in temporary and/or permanent ponds, apparently sharing the same ecological niches; the morphology of their spawn and tadpoles is almost identical, making them very difficult to differentiate directly in the field within their sympatry zone (Cogȃlniceanu et al., 2000;Sidorovska et al., 2002). ...
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Salinity tolerance is critical during the early ontogeny of amphibians, shaping future population size, health and dispersal in a certain area. We focused our research on two related anurans with similar ecological niches—Pelobates fuscus and P. syriacus—inhabiting the western Black Sea coast, at the limits of their ranges. We hypothesize that their differences in salinity tolerance are shaping the actual range limits in coastal areas, within the sympatry zone. We quantified experimentally the impact of salinity (range 0–9‰) during early ontogeny to ask if salinity can modulate their coexistence, by affecting differently reproductive success and fitness. Exposure to salinity from egg to developmental stage Gosner 25 caused mild to severe malformations and affected survival and size in both species, but the impact was lower in P. syriacus compared to P. fuscus when exposed to salt concentrations of 6‰. Embryos of either species did not survive the 9‰ salinity concentration. We expect that increases in salinization up to 6‰ could severely reduce the range of P. fuscus, but not P. syriacus, in coastal areas. These results are highly relevant for the conservation of P. fuscus, which is already declining across Europe. A full-text view-only version of the paper is available here:
1. In the context of global amphibian decline, monitoring and restoration programmes are important. Acoustic monitoring is a possible approach for underwater vocalising species like the rapidly declining European common spadefoot toad (Pelobates fuscus). In this study, our aim was to design a dedicated software detector to be used in combination with programmable audio recorders to process the large amount of data generated by long-term acoustic monitoring and to use it for investigating the seasonal and circadian patterns of P. fuscus vocal activity. 2. The software detector targets advertisement calls of the species. Based on acoustic analysis of that call, we developed a detector that utilises both frequency and time features of the calls. Data collected during three breeding seasons in four known or potential P. fuscus breeding sites of north-eastern France were used to build a ground truth in order to test the performance of the detector. Then, we used the detector for analysing four acoustic monitoring campaigns conducted in two different sites over two breeding seasons to gain insight into the seasonal and circadian patterns of vocal activity of this species. 3. Evaluation of the P. fuscus call detector against a ground truth returned falsepositive rates below 1.5% and true-positive rates ranging from 53% to 73%. These figures are compatible with long-term monitoring of the presence of the species. Running the software detector on standard hardware, the computation time for post-processing the 360 hr of a typical 3-month monitoring campaign was less than 1 day. 4. The seasonal pattern of P. fuscus underwater vocal activity is more complex than previously recognised. Over the whole ostensible 3-month breeding season, the actual time window for vocalising and breeding can last from a few days up to several weeks and may be split into clearly distinct episodes. When vocalisations occurred at both night- and daytime, the circadian vocal activity of P. fuscus occasionally proceeded uninterrupted for 24 hr but usually a several hour lull occurred immediately prior to sunset. When vocalisations occurred at both nightand daytime, the vocal activity pattern followed a bimodal distribution with a nocturnal highest peak of activity and a second peak occurring in the morning. 5. Our results demonstrate that it is feasible to monitor presence of P. fuscus in north-eastern France using a dedicated software detector combined with programmable audio recorders. Based on the outcomes of the detector applied to long-term audio data sets, we reveal temporal patterns of the vocal activity of the species and subsequently provide recommendations for attended and unattended acoustic monitoring.
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Amphibians and reptiles frequently oocur as fossils in the Quaternary. These animals, mainly anurans and turtles, made up a part of the diet of Man; turtle shells were used in making ornaments and turtles were put in burials as offerings. Man also took a part in modifying the geographical distributions of species. Amphibians and reptiles provide reliable information concerning palaeoenvironmental conditions. Both groups are ectotherm vertebrates: they use environmental heat, that is heat from outside the body. Therefore, amphibians and reptiles are quite valuable palaeoenvironmental indicators. They have probably the best potential as far as temperature and presence of water are concerned. Fossils belonging to these two groups strongly contribute to the determination of the palaeoenvironments in which Man lived. -from English summary
I analyzed geographic partitioning of mitochondrial DNA (mtDNA) restriction-site variants in the spotted salamander, Ambystoma maculatum. Two highly divergent and geographically separate genetic lineages were identified that differed by a minimum of 19 restriction sites (6% sequence divergence). One of the lineages has a disjunct distribution with very closely related haplotypes occurring in Missouri, Arkansas, North Carolina, and Virginia. The other lineage is found in Michigan, Illinois, and Alabama. The geographic separation of highly divergent mtDNA haplotypes, a pattern that was predicted based on the sedentary nature of these salamanders, is evidence for long-term barriers to gene flow. In contrast, the large-scale disjunction of very similar haplotypes suggests recent, long-distance gene flow and does not match the phylogeographic expectation for a small terrestrial vertebrate. I explain this potential contradiction in the level of importance assigned to gene flow by a scenario in which historical barriers to gene flow account for the two divergent mtDNA assemblages, but stochastic sorting of ancestral polymorphism is responsible for the large-scale geographic disjunction. Ten of 16 populations collected in the Ozark Highlands were fixed for the same haplotype. I attribute this lack of detectable variation to recent colonization of this area, a hypothesis that is supported by paleoecological data and demonstrates the potential benefits of combining data from paleobotany, geology, and other disciplines to reconstruct the historical biogeography of a species.
An electrophoretic survey of proteins from 27 populations examines the level of genetic differentiation among 4 subspecies of T. vulgaris in Yugoslavia. Populations of T. v. vulgaris, T. v. meridionalis and T. v. graecus are genetically the most similar to each other, while T. v. dalmaticus is substantially different. This pattern, which is not in accordance with the pattern of morphological similarities, is discussed with special reference to Pleistocene glaciation.-from AuthorInst. of Zoology, Fac. of Sci., Studentskitrg 16, 11000 Belgrade, & Inst. Biol. Research, Belgrade, Yugoslavia.
Thirty species of labyrinthodonts, 13 species of lepospondyls, 1 species of incertae sedis, 32 anuran species and 13 urodelan species from Tertiary localities in Czechoslovakia are listed. Some of this material still needs modern systematic classification. -from Author