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Biological Journal of the Linnean Society, 2014, 112, 442–449. With 3 figures
Multiple origins of invasive and ‘native’ water frogs
(Pelophylax spp.) in Switzerland
SYLVAIN DUBEY*†, JULIEN LEUENBERGER† and NICOLAS PERRIN
Department of Ecology and Evolution, Biophore Bld, University of Lausanne, CH 1015 Lausanne,
Switzerland
Received 16 January 2014; revised 10 February 2014; accepted for publication 10 February 2014
The marsh frog (Pelophylax ridibundus) has been introduced in many areas in Central and Western Europe as a
result of commercial trade with Eastern Europe, and is rapidly replacing the native pool frog (P. lessonae). A large
number of Pelophylax species are distributed in Eastern Europe and the strong phenotypic similarity between
these species is rendering their identification hazardous. Consequently, alien populations of Pelophylax might not
strictly be composed of P. ridibundus as previously suspected. In the present study, we analysed the cytochrome-b
and NADH dehydrogenase subunit 3 genes of introduced and native Pelophylax species from Switzerland (299
individuals) in order to properly identify the source populations of the invaders and the genetic status of the native
species. Our study highlighted the occurrence of several genetic lineages of invasive frogs in western Switzerland.
Unexpectedly, we also showed that several populations of the native pool frog (P. lessonae) cluster with the Italian
pool frog P. bergeri from central Italy (considered by some authors as a subspecies of P. lessonae). Hence, these
populations are probably also the result of introductions, meaning that the number of native P. lessonae
populations is fewer than expected in Switzerland. These findings have important implications concerning the
conservation of the endemic pool frog populations, as the presence of multiple alien species could strongly affect
their long-term subsistence. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society,
2014, 112, 442–449.
ADDITIONAL KEYWORDS: amphibians – conservation – cytochrome-b – invasive species – NADH
dehydrogenase subunit 3 – Ranidae.
INTRODUCTION
The determination of the identity of non-indigenous
invasive species is fundamental to assess their origin,
and hence to understand the characteristics that
have facilitated their colonization and establishment
success, as well as to allow their potential control or
eradication (Stepien & Tumeo, 2006; Dubey & Shine,
2008; Krug et al., 2012). However, the correct identification of alien species is not always evident,
particularly when it is based on morphological characteristics. Indeed, such taxa sometimes belong to
widely distributed species complexes exhibiting
very few diagnostic interspecific differences and/or
important intraspecific phenotypic variations (e.g.
*Corresponding author. E-mail: [email protected]
†These authors contributed equally to this work.
442
Dubey, Ursenbacher & Fumagalli, 2006; Stepien &
Tumeo, 2006). In these circumstances, the use of
genetic analyses is the only way to unravel this
problem.
Water frogs of the genus Pelophylax (since Frost
et al., 2006; formerly genus Rana) are widespread in
Eurasia, with numerous species distributed around
the Mediterranean basin, e.g. P. bedriagae, P. bergeri,
P. cretensis, P. epeiroticus, P. shqipericus, P. perezi,
P. ridibundus, P. cerigensis and P. kurtmuelleri
[Lymberakis et al., 2007; see Figure 1A, B for information about the distribution of species, according to
the IUCN Red List of Threatened Species, 2013
(IUCN, 2013)]. However, the species-specific status
of several species is unclear. The marsh frogs
(P. ridibundus) have been introduced in many areas
in Central and Western Europe as a result of international commercial trade (frog leg consumption)
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 442–449
MULTIPLE ORIGINS OF SWISS PELOPHYLAX
Figure 1. Geographical distribution of Pelophylax species
in Europe and the Near East (A, P. bergeri, P. bedriagae,
P. kurtmuelleri, P. lessonae and P. perezi; B, P. cerigensis,
P. cretensis,
P. epeiroticus,
P. ridibundus
and
P.
shqipericus) and location of the Swiss populations analysed in the present study [with information about the
cytochrome-b gene (cyt-b) genetic lineages; C].
443
with Eastern Europe (Nöllert & Nöllert, 2003;
Schmeller et al., 2007). Hence, most of the Central
European populations of alien Pelophylax are currently considered to belong to this species (Nöllert &
Nöllert, 2003; Holsbeek et al., 2008). However, the
occurrence of different close relative alien species,
such as P. bedriagae (from Turkey) and P. kurtmuelleri (from Greece, considered as P. ridibundus by
some authors; see, for example, Akin et al., 2010), has
been documented in several western European countries (e.g. Lanza & Corti, 1993; Holsbeek et al., 2008;
Amphibiaweb, 2013). Based on genetic analyses,
Holsbeek et al. (2008) revealed the occurrence of both
P. ridibundus and P. bedriagae lineages in Belgium.
These results suggest that several alien species are
probably co-occurring in many European countries.
This finding is not surprising considering the important number of species distributed in south-eastern
Europe and the Near East, and the strong phenotypic similarity between these species, rendering
their identification hazardous when their origin is
unknown. Consequently, additional genetic analyses
are needed to clarify the situation throughout the
distribution of these invasive water frogs.
Most importantly, the marsh frog is replacing the
native pool frog (P. lessonae) where it is introduced,
through competition for resources (food and space),
predation and a genetic mechanism involving hybridogenesis (e.g. Vorburger & Reyer, 2003). In the case of
hybridogenesis, the hybrid between P. lessonae and
P. ridibundus (the edible frog, P. esculentus) is eliminating the P. lessonae genome from its germ line and
hence is strictly transmitting the P. ridibundus
genome (Tunner, 1974). According to Vorburger &
Reyer (2003), the mechanism of eradication involves
mating between: (1) invasive P. ridibundus and
native P. esculentus, which is expected to produce
viable P. ridibundus offspring; (2) P. lessonae and
P. ridibundus, which generates P. esculentus primary
hybrids; and (3) primary hybrids, which should restore
pure and viable ridibundus offspring. Consequently,
the P. ridibundus genome is expected to progressively
replace the P. lessonae genome, and to potentially
eliminate the native species.
In Switzerland, the first record of the invasive
marsh frog was documented in 1950 (Grossenbacher,
1988) and the species is currently widely distributed
at low elevation, leading to the progressive rarefaction and disappearance of most pool frog populations.
Hence, the pool frog is considered to be ‘near threatened’ in this country (Schmidt & Zumbach, 2005).
In this article, we analyse the mitochondrial
cytochrome-b and NADH dehydrogenase subunit 3
genes of pool, marsh and edible frogs from Switzerland in order to properly identify the source populations of the invaders and the genetic status of the
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 442–449
444
S. DUBEY ET AL.
native species. Comparisons were performed based
on previously published phylogeographical studies of
the genus Pelophylax (see Sumida, Ogata & Nishioka,
2000; Lymberakis et al., 2007; Plötner et al., 2008;
Akin et al., 2010).
MATERIAL AND METHODS
SAMPLING, EXTRACTION AND GENETIC ANALYSES
A total of 299 individuals of water frogs considered as
P. lessonae, P. ridibundus and P. esculentus (based on
the shape of the metatarsal tubercle, as in Nöllert &
Nöllert, 2003) was sampled (with buccal swabs) in
different Swiss populations (six main areas; Supporting Information, Table S1). Our dataset included two
of the largest wetlands in Switzerland, which are
characterized by large populations of P. lessonae (La
Grande Cariçaie, Lake Neuchâtel; Les Grangettes,
Lake Geneva; Fig. 1C). Total cellular DNA was
extracted using the QIA Amp DNA Mini Kit (Qiagen,
Valencia, PA, USA) and double-stranded DNA amplification of the cytochrome-b gene (cyt-b) and NADH
dehydrogenase subunit 3 (ND3) was performed with
the primer pairs cytbPelophylax_F1 (5′-CTCCTGGG
AGTCTGCCTAAT-3′) and cytbPelophylax_R1 (5′-CG
AAGCCTAGAAGATCTTTG-3′), specifically designed
for this study, and ND3L/ND3H (see Plötner et al.,
2008). Polymerase chain reaction amplifications were
performed in a 9800 Fast Thermal Cycler (Applied
Biosystems, Carlsbad, CA, USA) with 0.05 U of Taq
DNA polymerase (Qiagen), and consisted of 35 cycles of
30 s denaturation at 94 °C, 40 s annealing at 50 °C
(cyt-b) or 52 °C (ND3) and 90 s extension at 72 °C.
Then, PCR fragments were purified and sequenced at
GATC Sequencing Service (Cologne, Germany; http://
www.gatc-biotech.com). Concerning ND3, only a total
of 39 individuals was analysed, representing all the
different haplotypes found in cyt-b.
PHYLOGENETIC
ANALYSES
We aligned sequences of cyt-b and ND3 by eye
(GenBank accession numbers: KC495115–KC495119
and KF959564–KF959571 for cyt-b and KJ152758–
KJ152766 for ND3) and tests were conducted on both
total fragments separately (cyt-b, 466 bp; ND3,
305 bp; all codon positions were used). The trees were
rooted using a sequence of P. saharicus. In addition,
we included previously published sequences of
European and Near East Pelophylax species, i.e.
P. bedriagae, P. bergeri, P. cerigensis, P. cretensis,
P. epeiroticus, P. kurtmuelleri and P. ridibundus
(Sumida et al., 2000; Lymberakis et al., 2007; Akin
et al., 2010; see Fig. 1A, B for the distribution of the
different species).
We used jModelTest 2.1 (Guindon & Gascuel, 2003;
Darriba et al., 2012) to select the model of DNA
substitution for maximum likelihood (ML) analyses.
The HKY + G and TrN + I + G models (Hasegawa,
Kishino & Yano, 1985; Tamura & Nei, 1993; Yang,
1993) best fitted the cyt-b and ND3 datasets, respectively, with Akaike’s Information Criterion (AIC). Then
ML heuristic searches and bootstrap analyses (1000
replicates) were performed using Phyml (Guindon &
Gascuel, 2003).
RESULTS AND DISCUSSION
Our study based on cyt-b and ND3 sequences highlighted the occurrence of several distinct genetic lineages of invasive frogs in Switzerland (Figs 2, 3).
According to the phylogeographical study of Akin
et al. (2010) (partly based on ND3), our samples
belong to three geographically distinct groups of
the P. ridibundus species complex, named ‘Cilician
west’ [MHG4; see Akin et al. (2010) for more details;
Fig. 3) from south-eastern Turkey, ‘cf. bedriagae s.s.’
(MHG6c) from Turkey, Georgia, Russia and Ukraine
(mainly around the Black Sea) and ‘ridibundus,
Europe’ (MHG1) from eastern and south-eastern
Europe. In addition, our cyt-b analyses revealed that
samples from the ND3 ‘ridibundus, Europe’ lineage
are made up of samples identical to previously
published sequences of P. ridibundus, but also to
P. kurtmuelleri from Greece (see Lymberakis et al.,
2007; Fig. 2), the latter species being considered
as P. ridibundus in Akin et al. (2010). Consequently,
Swiss populations previously considered as P.
ridibundus are, in fact, a mosaic of several lineages,
a pattern that has probably resulted from multiple
introductions since the first observation of a specimen
in 1950 (Grossenbacher, 1988). Hence, the situation
is very similar to that observed in Belgium, where
P. ridibundus and P. bedriagae have been introduced
in recent decades (Holsbeek et al., 2008).
However, we also revealed that, in addition to the
presence of these different lineages of invasive frogs
in Switzerland, several populations generally considered to be native P. lessonae can be assigned, on the
basis of their mtDNA, to an alien species, the Italian
pool frog P. bergeri (considered by some authors to be
a subspecies of P. lessonae; Canestrelli & Nascetti,
2008). Indeed, only haplotypes of the Italian pool frog
P. bergeri, which is morphologically very similar to
P. lessonae (Wycherley, Doran & Beebee, 2002; Nöllert
& Nöllert, 2003), have been found in the natural
reserve of La Grande Cariçaie (the largest wetland in
Switzerland), Lausanne area, and La Combe. In addition, the populations of Aarau and Les Grangettes
present a mixture of P. lessonae and P. bergeri mtDNA
haplotypes (Fig. 1C). The only analysed population
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 442–449
MULTIPLE ORIGINS OF SWISS PELOPHYLAX
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Figure 2. Phylogeny of the cytochrome-b gene (cyt-b) fragment of European and Near East Pelophylax spp. analysed
using a maximum likelihood procedure (bold, water frogs from Switzerland). Values on the branches are indices of support
for the major branches (percentage of 1000 replications; /, support < 50). GenBank accession numbers are given for
previously published sequences. For samples from this study, only haplotypes are provided (H1–H12).
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 442–449
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S. DUBEY ET AL.
Figure 3. Phylogeny of the NADH dehydrogenase subunit 3 (ND3) fragment of European and Near East Pelophylax spp.
analysed using a maximum likelihood procedure (bold, water frogs from Switzerland). Values on the branches are indices
of support for the major branches (percentage of 1000 replications; /, support < 50). Names of lineages are as in Akin et al.
(2010). GenBank accession numbers are given for previously published sequences. For samples from this study, only
haplotypes are provided (H1–H9).
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 442–449
MULTIPLE ORIGINS OF SWISS PELOPHYLAX
showing strict haplotypes of P. lessonae is Finges,
where, unfortunately, only the invasive and hybrid
Pelophylax species still coexist (P. lessonae now being
extinct). In summary, 76%, 73% and 17% of frogs
morphologically identified as P. lessonae, P. esculentus
and P. ridibundus in our present study possess a
P. bergeri haplotype (see Table S1 for more details).
To date, the Italian pool frog is known from the
central and southern part of the Italian peninsula
(south of the Po Plain; see Figs 1, 2), Sicily, Corsica
and Sardinia, and hence its presence in Switzerland
seems to be the result of an introduction. In addition,
its natural colonization from central and southern
Italy is unlikely as P. lessonae is replacing P. bergeri
south of the Alps in the Po Plain (Fig. 1). This result
is also supported by the presence of one single cyt-b
haplotype of P. bergeri (179 samples from five populations), whereas five different haplotypes are present in the endemic P. lessonae (36 samples from
three areas; Fig. 1C). This pattern is very similar to
that observed in different amphibian and reptile
species [Triturus carnifex (Meyer et al., 2009), Hyla
intermedia (Dubey et al., 2006), Lissotriton vulgaris
meridionalis (Meyer et al., 2009) and Natrix tesselata
(Metzger, Ursenbacher & Christe, 2009)] which are
native to Italy and the southern part of Switzerland
(Ticino), and which have been introduced to the
northern part of Switzerland (north of the Alps).
Interestingly, this unexpected result is also supported by previous studies. Indeed, Spolsky & Uzzell
(1986) described the presence of a genetic lineage
(based on electrophoretic analysis of restriction
enzyme mitochondrial fragments) called ‘type D’
within P. lessonae from Switzerland. This type D is
considered to be characteristic of Italian water frogs
(Plötner et al., 2008), and differs from type B and C,
which are characteristic of P. lessonae from central
and northern Europe. In addition, it is also found in
southern Germany close to the Swiss border (T. Ohst
& J. Plötner, unpubl. data; see Plötner et al., 2008).
According to Plötner et al. (2008), the sequence divergence, calculated from shared restriction fragments
between the mtDNAs of P. lessonae (type B and C)
and type D (3.4%), is congruent with genetic distances
obtained from ND2 + ND3 sequences (3.8–5.0%).
However, these ND2 and ND3 sequences are unpublished, and hence their similarities with published
sequences of P. bergeri (Akin et al., 2010) cannot be
established. In addition, even if P. bergeri is also
present in southern Germany, its distribution is probably restricted. Indeed, a genetic study performed
on 176 individuals across Bavaria (south-eastern
Germany) strictly revealed mitochondrial haplotypes
(ND2) of P. lessonae (Mayer et al., 2013).
This finding has several implications. First, the
process of hybridogenesis involving P. bergeri and
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P. ridibundus is poorly known (Holsbeek & Jooris,
2010) and may be different from that involving
P. ridibundus and P. lessonae. Second, it is likely that
hybrids resulting from crossing between different
eastern European lineages and pool frogs (P. bergeri
and P. lessonae) are not equally fit, which could affect
the efficiency of hybridogenesis and, in turn, the
eradication of pool frogs, as suggested by Holsbeek
et al. (2008), considering Belgian populations of
P. lessonae, P. ridibundus and P. bedriagae. However,
the situation in Switzerland is more complex as at
least three different lineages of the P. ridibundus
species complex are present, leading to numerous
crossing combinations with the pool frogs (P. lessonae
and P. bergeri) and their hybrids. Moreover, several
studies have highlighted that hybrids might have
advantages compared with parental species or lineages (through a higher genetic diversity), such as an
increased potential to local adaptation (Ellstrand &
Schierenbeck, 2000; Kolbe et al., 2004; Holsbeek
et al., 2008; Liebhold & Tobin, 2008). If the same is
true for water frogs, the presence of multiple invasive
Pelophylax lineages in Switzerland might have a dramatic impact on native populations, which could be
replaced even more rapidly by the invaders.
Third, this finding has important implications concerning the conservation of the endemic pool frog
(P. lessonae) in Switzerland, which now seems to be
extinct in most of its native range, where alien frogs
have been introduced. Indeed, the population of La
Grande Cariçaie was considered as one of the largest
in Switzerland, meaning that the status of P. lessonae
is worse than previously thought. Hence, its status
of ‘near threatened’ in this country (Schmidt &
Zumbach, 2005) should be reconsidered.
As a conclusion, future studies should explore this
problem in more detail and clarify the distribution
and frequency of the different alien species, as well as
the level of hybridization and introgression between
P. bergeri and P. lessonae, via extensive sampling in
Switzerland (as well as in surrounding countries).
Indeed, only nuclear markers will allow a full understanding of this phenomenon. For conservation purposes, it is urgent to understand to what extent
P. bergeri has replaced P. lessonae north of the Alps,
and if the presence of P. bergeri in Switzerland is
recent or ancient. Samples of museum collections
should help to clarify this latter point.
ACKNOWLEDGEMENTS
We thank Séverine Antille, Baudoin Des Monstiers
Merinville, Jean-Marc Fivat, Marie Gallot Lavallée,
Antoine Gander, Florent Goetschi, Honorine Lovis,
Paul Marchesi, Thomas Martignier, Nicolas
Rodrigues, Théodora Steiner, Johan Schuerch and
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 442–449
448
S. DUBEY ET AL.
Yvan Vuille for their help during fieldwork, Andrea
Vaupel, Sabine Brodbeck and Felix Gugerli for providing samples from the Aarau area, Philippe Christe
and Jörg Plötner for helpful comments, and the Swiss
National Science Foundation (grants PZ00P3_136649
to SD and 31003A_129894 to NP) for funding. The
samples were collected with the authorization of the
‘Canton de Vaud’ (N°2012-24).
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Table S1. Location of samples collected in Switzerland, with information on their morphological identification,
cytochrome-b gene (cyt-b) and NADH dehydrogenase subunit 3 (ND3) haplotypes, and corresponding lineages.
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 442–449