A record of alien Pelophylax species and widespread mitochondrial DNA transfer in Kaliningradskaya Oblast' (the Baltic coast, Russia)

Abstract

Alien species can strongly impact local environments and compete against native species, which can lead to their extinction. Marsh frogs of the Pelophylax ridibundus complex are one of the most invasive amphibians in Northern Eurasia. It was previously thought that three water frog species of the genus Pelophylax (the marsh frog, P. ridibundus, the pool frog, P. lessonae and their hemiclonal hybrid, the edible frog, P. esculentus) inhabited Kaliningradskaya Oblast' along the Russian Baltic coast. However, based on our study of the intron-1 of the nuclear serum albumin gene, two other marsh frog species were detected (the Balkan marsh frog, P. kurtmuelleri, and the Anatolian marsh frog, P. cf. bedriagae) as well as putative hybrids between P. ridibundus and P. cf. bedriagae. The majority of individuals of P. ridibundus and hybrids between P. ridibundus and P. cf. bedriagae had mitochondrial (mt) DNA of P. lessonae, while all others featured the P. kurtmuelleri mtDNA. The prevalence of P. lessonae mtDNA haplotypes in populations of P. ridibundus from the Baltic Coast of Russia suggests that local individuals of the latter species originated from crosses between P. esculentus individuals. Two hypotheses could explain the records of P. kurtmuelleri and P. cf. bedriagae in the region. The establishment of local populations of the first species could have occurred via postglacial dispersal from the Balkan refugium. The origin of local P. cf. bedriagae could be an occasional introduction of individuals from the Ponto-Caspian region. Since our study is preliminary (19 individuals), in the future it would be important to continue the study of water frogs in Kaliningradskaya Oblast' and neighboring countries by applying multiple genetic markers. Additional genetic markers will enable researchers to study routes of dispersal and introductions of marsh frogs, to clarify peculiarities of their hybridization and distribution, and to evaluate the impact of P. kurtmuelleri and P. cf. bedriagae on the reproduction success of hybridogenous populations and abundance of local amphibians.

Full text

BioInvasions Records (2020) Volume 9, Issue 3: 599–617
Litvinchuk et al. (2020), BioInvasions Records 9(3): 599–617, https://doi.org/10.3391/bir.2020.9.3.16 599
CORRECTED PROOF
Research Article
A record of alien Pelophylax species and widespread mitochondrial DNA
transfer in Kaliningradskaya Oblast’ (the Baltic coast, Russia)
Spartak N. Litvinchuk1,2,*, Alexander Yu. Ivanov3, Svetlana A. Lukonina3 and Oleg A. Ermakov3
1Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg, 194064, Russia
2Dagestan State University, Gadzhiyev str. 43-a, Makhachkala, 3367000, Russia
3Penza State University, Krasnaya str. 40, Penza, 440026, Russia
Author e-mails: litvinchukspartak@yandex.ru (SNL), akella58@mail.ru (AYuI), lanochkal@yandex.ru (SAL), oaermakov@list.ru (OAE)
*Corresponding author
Abstract
Alien species can strongly impact local environments and compete against native
species, which can lead to their extinction. Marsh frogs of the Pelophylax ridibundus
complex are one of the most invasive amphibians in Northern Eurasia. It was previously
thought that three water frog species of the genus Pelophylax (the marsh frog,
P. ridibundus, the pool frog, P. lessonae and their hemiclonal hybrid, the edible frog,
P. esculentus) inhabited Kaliningradskaya Oblast’ along the Russian Baltic coast.
However, based on our study of the intron-1 of the nuclear serum albumin gene, two
other marsh frog species were detected (the Balkan marsh frog, P. kurtmuelleri, and
the Anatolian marsh frog, P. cf. bedriagae) as well as putative hybrids between
P. ridibundus and P. cf. bedriagae. The majority of individuals of P. ridibundus
and hybrids between P. ridibundus and P. cf. bedriagae had mitochondrial (mt)
DNA of P. lessonae, while all others featured the P. kurtmuelleri mtDNA. The
prevalence of P. lessonae mtDNA haplotypes in populations of P. ridibundus from
the Baltic Coast of Russia suggests that local individuals of the latter species
originated from crosses between P. esculentus individuals. Two hypotheses could
explain the records of P. kurtmuelleri and P. cf. bedriagae in the region. The
establishment of local populations of the first species could have occurred via
postglacial dispersal from the Balkan refugium. The origin of local P. cf. bedriagae
could be an occasional introduction of individuals from the Ponto-Caspian region.
Since our study is preliminary (19 individuals), in the future it would be important
to continue the study of water frogs in Kaliningradskaya Oblast’ and neighboring
countries by applying multiple genetic markers. Additional genetic markers will
enable researchers to study routes of dispersal and introductions of marsh frogs, to
clarify peculiarities of their hybridization and distribution, and to evaluate the
impact of P. kurtmuelleri and P. cf. bedriagae on the reproduction success of
hybridogenous populations and abundance of local amphibians.
Key words: Pelophylax cf. bedriagae, Pelophylax kurtmuelleri, invasive species,
introduction, hybridolysis, hybridization, postglacial dispersal
Introduction
Alien species, which were introduced by man outside their natural ranges,
can strongly impact local environments and compete against native
species, which can lead to their extinction (Kraus 2009, 2015; Bucciarelli et
al. 2014). Several nonnative amphibian species are known in Europe, among
Citation: Litvinchuk SN, Ivanov AYu,
Lukonina SA, Ermakov OA (2020) A
record of alien Pelophylax species and
widespread mitochondrial DNA transfer in
Kaliningradskaya Oblast’ (the Baltic coast,
Russia). BioInvasions Records 9(3): 599–
617, https://doi.org/10.3391/bir.2020.9.3.16
Received: 11 November 2019
Accepted: 31 March 2020
Published: 21 May 2020
Handling editor: John Measey
Thematic editor: Amy Fowler
Copyright: © Litvinchuk et al.
This is an open access article distributed under terms
of the Creative Commons Attribution License
(Attribution 4.0 International - CC BY 4.0).
OPEN ACCESS.

Alien Pelophylax species on the Baltic Coast
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Figure 1. Native ranges of P. ridibundus (green), P. kurtmuelleri (blue), P. cf. bedriagae (red), and P. esculentus (hashed black).
The studied area is in black quadrangle.
which the most well known are the African clawed frog, Xenopus laevis
(Daudin, 1802), and the American bullfrog, Lithobates catesbeianus (Shaw,
1802) (Ficetola et al. 2007; Measey et al. 2012).
The genus Pelophylax Fitzinger, 1843 consists of about 22 water frog
species distributed predominantly throughout the Palearctic (Frost 2020).
The taxonomic status of some of them is under discussion (i.e., Lymberakis
et al. 2007; Largen and Spawls 2010). The most complicated situation is
with marsh frogs of the P. ridibundus complex, which includes numerous
closely-related cryptic lineages (Plötner and Ohst 2001; Akin et al. 2010;
Plötner et al. 2012). Several of these lineages (e.g., Syrdaryan, Anatolian,
Euphrates, Cilician, Iranian) are yet to receive a formal taxonomic description
(Mezhzherin and Peskov 1992; Plötner and Ohst 2001; Pesarakloo et al.
2016). Marsh frogs are considered one of the most invasive amphibians of
Northern Eurasia (Zeisset and Beebee 2003; Duysebaeva et al. 2005;
Bashinskiy et al. 2018; Bellati et al. 2019). Several cryptic marsh frog species
were introduced to European countries (Supplementary material Table S1).
For example, the Balkan marsh frog, P. kurtmuelleri (Gayda, 1940), was
recorded in the Czech Republic, Switzerland, France, Italy, Ukraine, and
some regions of Russia (Lanza 1962; Bellati et al. 2013; Laghi et al. 2013;
Akin Peksen 2015; Dufresnes et al. 2017, 2018; Bellati et al. 2019; Bisconti
et al. 2019; Ivanov 2019; Vershinin et al. 2019), despite the fact that the
native range of the frog is restricted to the Balkan Peninsula (Figure 1).
Some authors have indicated the presence of alleles and/or haplotypes of
the species in the Baltic Region in Latvia, Lithuania and Poland (Plötner et
al. 2008; Hauswaldt et al. 2012; Kolenda et al. 2017).
Another species, the Anatolian marsh frog (P. cf. bedriagae), was
introduced to Italy, Belgium, France, Switzerland, Germany, and some

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regions of Russia (Holsbeek et al. 2008, 2009, 2010; Ohst 2008; Dubey et al.
2014; Dufresnes et al. 2018; Lyapkov et al. 2018; Bellati et al. 2019; Ivanov
2019; Vershinin et al. 2019), including the vicinities of St. Petersburg City
in the Baltic region of Russia (Ohst 2008; Akin et al. 2010). The species
naturally occurs in western Iran, Turkey, the Caucasus, Bulgaria, eastern
Greece, western Kazakhstan, southern and eastern Ukraine, the Crimea,
and the Volga River region of Russia (Figure 1).
Hybridization between various water frog species is quite common
(Plötner et al. 2010). Some events can lead to mitochondrial (mt) DNA
transfer into other species. Such transfer is mediated by fertile hybrids that
transmit their maternal mtDNA to the paternal gene pool via backcrosses
with males of the paternal parental species (Plötner et al. 2008). In western
Poland, Spolsky and Uzzell (1984) were the first to reveal individuals of the
marsh frog, P. ridibundus (Pallas, 1771), with mtDNA of the pool frog,
P. lessonae (Camerano, 1882). Plötner et al. (2008) noted that 34% of
individuals of P. ridibundus in Europe were characterized by the P. lessonae
mtDNA. In Belgium, Ukraine and European Russia numerous populations
of P. ridibundus possessed mtDNA of P. cf. bedriagae and vice versa, i.e.
P. cf. bedriagae can have the mtDNA of P. ridibundus (Holsbeek et al.
2008, 2009; Ermakov et al. 2013, 2014; Ivanov et al. 2015; Svinin et al. 2015;
Hoffmann et al. 2015; Zamaletdinov et al. 2015). The mtDNA of the
Karpathos marsh frog, P. cerigensis (Beerli, Hotz, Tunner, Heppich & Uzzell,
1994), was found in a population of P. cf. bedriagae from Kaş in southwestern
Turkey (Ohst 2008; Akin et al. 2010; Plötner et al. 2012). Sánchez-Montes
et al. (2016) reported that two populations of P. ridibundus in northwestern
Spain (Prades and Oix) have the mtDNA of the Iberian frog, P. perezi
(López-Seoane, 1885). In southern France (Lac de Condamine), Dufresnes
et al. (2017) found an introduced population of P. kurtmuelleri with the
mtDNA of P. perezi. Finally, in a population from European Russia, Ivanov
et al. (2019) recently described a case of mtDNA transfer from P. cf.
bedriagae into P. lessonae.
Three species of water frogs (P. ridibundus, P. lessonae, and their
hemiclonal hybrid, the edible frog, P. esculentus (Linnaeus, 1758)) inhabit
Kaliningradskaya Oblast’ of Russia (the northern part of the historical East
Prussia) (Litvinchuk et al. 2015). The pool and edible frogs are widespread
and form hybridogenous systems throughout Kaliningradskaya Oblast’.
The marsh frog is rarer. Its populations in the region are located on the
northern border of the species range. The species distribution is restricted
to the westernmost part of the Oblast’ and consists of two isolated parts.
The northern part extends along the shores of the Curonian Lagoon and
Neman River, where the species forms mixed hybridogenous systems with
P. esculentus and sometimes P. lessonae (Litvinchuk et al. 2015). The
southern part is located near the Vistula Lagoon where local marsh frogs
did not usually co-occur with P. esculentus and P. lessonae (Litvinchuk et al.

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Table 1. List of individuals studied, numbers and names of localities, years when individuals were collected, numbers of
specimens stored in herpetological collections of the Zoological Institute of Russian Academy of Sciences (ZISP), presence of
other water frog species (esc is Pelophylax esculentus and les is P. lessonae) in the locality, geographic coordinates (“Lat” is
latitude and “Long” is longitude), results of marsh frog species identification (rid is P. ridibundus, kurt is P. kurtmuelleri, bedr is
P. cf. bedriagae, and rid/bedr are individuals which have alleles of both P. ridibundus and P. cf. bedriagae), and accession
numbers of sequences. nDNA is nuclear and mtDNA is mitochondrial DNA markers. SAI-1 is the intron-1 of the nuclear serum
albumin gene and ND2 is the subunit 2 of mitochondrial NADH dehydrogenase gene.
N Locality Date ZISP
number
Other
species
Coordinates Results Accession numbers
Lat, N Long, E nDNA mtDNA SAI-1 ND2
1 Sovetsk 2014 14233 esc+les 55.094 21.844 rid kurt – –
2 Morskoe 2018 14244 – 55.228 20.920 rid les – –
3 Rybachiy 2014 14237 esc 55.157 20.844 rid kurt MN497958 MN271952
3 Rybachiy 2018 14241 esc 55.157 20.844 rid kurt MN497958 MN271952
3 Rybachiy 2018 14242 esc 55.157 20.844 rid kurt MN497958 MN271952
3 Rybachiy 2008 14239 esc 55.157 20.844 rid les – –
4 Zelenogradsk 2018 14243 esc 54.952 20.484 rid les – –
5 Kaliningrad 2014 7025.702 – 54.691 20.512 rid/bedr les – MN271954
5 Kaliningrad 2014 14236 – 54.691 20.512 bedr kurt MN497959 MN271953
6 Ushakovo 2014 14238 – 54.612 20.243 kurt kurt MN497957 MN271951
7 Baltiysk 2002 7025.538 – 54.635 19.874 kurt kurt – –
7 Baltiysk 2002 7025.539 – 54.635 19.874 rid kurt – –
7 Baltiysk 2014 7025.605 – 54.635 19.874 rid/bedr les – –
7 Baltiysk 2018 14240 – 54.635 19.874 rid les MN497961 MN271956
8 Mamonovo 2014 14230 – 54.449 19.952 rid les MN497960 MN271955
8 Mamonovo 2014 14231 – 54.449 19.952 rid/bedr les – MN271955
8 Mamonovo 2014 14232 – 54.449 19.952 rid les MN497960 MN271955
8 Mamonovo 2014 14234 – 54.449 19.952 rid les – –
8 Mamonovo 2014 14235 – 54.449 19.952 rid les – –
2015). In addition, a presumably introduced population of the marsh frog
(syntopic with P. esculentus) is known from the southern part of the
Oblast’ in fish ponds near the town of Pravdinsk (Borkin et al. 1986;
Litvinchuk et al. 2015).
No molecular studies have been specifically conducted on marsh frogs
from the Baltic Coast of Russia. Therefore, the aim of our paper was to
study the genetic variation, using a multilocus approach, of local marsh
frogs and describe records of alien marsh frog species and interspecies
mtDNA transfer.
Materials and methods
Pieces of femur muscle from herpetological collections of the Zoological
Institute of Russian Academy of Sciences (fixed by 96% ethanol and stored
in 70% ethanol) were used as tissue samples. We studied 19 marsh frog
specimens collected from 2002 to 2018 in eight localities from Kaliningradskya
Oblast’ (Table 1, Figure 2). The DNA was extracted by the standard salt-
extraction method (Aljanabi and Martinez 1997).
The primary identification of alleles of the intron-1 of the nuclear serum
albumin gene (SAI-1) of three marsh frog species (P. ridibundus,
P. kurtmuelleri and P. cf. bedriagae) was performed using the methods
described by Hauswaldt et al. (2012) and Ermakov et al. (2019). The
method described by Ermakov et al. (2019) was used to identify the mtDNA
(the COI gene fragment) of P. ridibundus and P. cf. bedriagae. To identify

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Figure 2. Proportion of the nuclear SAI-1 gene alleles (A) and haplotypes of mitochondrial
markers (B) in marsh frogs from eight localities in Kaliningradskaya Oblast’, Russia. Lowlands
are in green and hilly areas in brown. Pie charts reflect sample size. rid is Pelophylax
ridibundus, kurt is P. kurtmuelleri, bedr is P. cf. bedriagae, and les is P. lessonae. Numbers for
localities are given in Table 1.
haplotypes of P. kurtmuelleri, we used an endonuclease restriction analysis.
The COI gene fragment (744 bp) was amplified using UTF 5′-TGT AAA
ACG ACG GCC AGT TCT CAA CCA AYC AYA ARG AYA TYG G-3′
and UTR 5′-CAG GAA ACA GCT ATG ACT ARA CTT CTG GRT GKC
CRA ARA AYC A-3′ (Lissovsky et al. 2010) primers at 95 °C for 30 s, 55 °C
for 30 s, and 72 °C for 50 s (30 cycles). The PCR reaction mixture (25 μL)
contained 50–100 ng of DNA, 0.5 μM of each primer, 0.2 mM dNTPs, 1.5 mM
MgCl2, 2.5 μL 10×PCR buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl), and
2 units of Taq polymerase (Thermo Scientific). The PCR fragments obtained
were digested with the restriction endonuclease Bme1390I (site CCTGG at
5917 position in the P. kurtmuelleri mitochondrion (NC_026895); Hofman
et al. 2016) for 2–4 h at 37 °C (2–4 enzyme units to 2–4 μl of amplification
mixture). After restriction obtained fragments had different lengths: 388 bp
for P. kurtmuelleri and 354 bp for P. ridibundus and P. cf. bedriagae.
Selective sequencing was used to verify the primary identification results.
The nuclear SAI-1 gene fragment was sequenced in eight specimens and
the subunit 2 of mitochondrial NADH dehydrogenase (ND2) gene in 10
specimens (Table 1). Sequencing of fragments was performed on an ABI

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3500 automatic sequencer (Applied Biosystems) using the BigDye®
Terminator 3.1 (Applied Biosystems) kit, and the same primers that were
used for amplification. The ND2 gene sequence (1038 bp) was amplified
with use of the universal primer ND2L1 5′-AAG CTT TTG GGC CCA
TAC CCC-3′ (Meyer 1993) and a developed specific primer ND2H1 5′-
GCA AGT CCT ACA GAA ACT GAA G-3′. The following amplification
methods were used: initial denaturation for 1 min at 95 °C, followed by 32
cycles of 94 °C for 30 s, 60 °C for the ND2 and 53 °C for the SAI-1 pair of
primers for 30 s, 72 °C for 60 s, and final extension for 5 min at 72 °C. The
PCR reaction mixture proportions were the same as for amplification of
the COI gene fragment. The sequences obtained have been deposited in
GenBank (ND2 gene no. MN271951–MN271956 and SAI-1 gene no.
MN497957–MN497961).
The nucleotide sequences were aligned both with BioEdit (Hall 1999)
software and manually. We used MEGA v. 7.0. software (Kumar et al. 2016)
for data processing. For constructing the phylogenetic tree, the maximum
likelihood (ML) method was used. The most appropriate DNA substitution
model for the datasets was established using jModelTest 2.1.10 (Posada 2008).
The ML trees were created with the Hasegawa-Kishino-Yano model for
ND2 gene, gamma distributed (HKY+G) (–lnL = 3239.07, BIC = 8313.13)
and Tamura-Nei model for SA gene (T92) (–lnL = 1300.32, BIC = 3611.79).
Node support values in phylogenetic trees were estimated according to
bootstrap support (500 replicates).
Maps of native ranges of P. ridibundus, P. kurtmuelleri, P. cf. bedriagae,
and P. esculentus (Figure 1) are based on previously published nuclear (n)
DNA data (Ohst 2008; Akin et al. 2010; Plötner et al. 2012; Ermakov et al.
2013, 2014, 2016a, b; Akin Peksen 2015; Ivanov et al. 2015, 2019; Svinin et
al. 2015; Zamaletdinov et al. 2015; Fayzulin et al. 2017, 2018; Kolenda et al.
2017; Kukushkin et al. 2018; Ivanov 2019).
Results
Despite the fact that our data are preliminary (only 19 individuals were
studied), based on the analysis of the nuclear SAI-1 fragment, we were able
to detect alleles of the following three marsh frog species in Kaliningradskaya
Oblast’ (Table 1): P. ridibundus (13 individuals; 68%; six localities),
P. kurtmuelleri (n = 2; 11%; two localities ) and P. cf. bedriagae (n = 1; 5%;
three localities). The remaining three individuals (16%; three localities)
contained alleles of both P. ridibundus and P. cf. bedriagae. Putative
hybrids of P. kurtmuelleri with other marsh frog species were not found.
The following pecularities in geographical distribution of the species
were revealed. Populations distributed in the Curonian Lagoon and
Neman River (localities 1–4 in Figure 2) only featured P. ridibundus nDNA
alleles. Populations located around the Vistula Lagoon (localities 5–8 in
Figure 2) were composed of individuals with nDNA alleles of all three marsh

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Figure 3. Biotopes of Pelophylax cf. bedriagae and its hybrids with P. ridibundus in Yuznyi park in the center of Kaliningrad city (A);
P. kurtmuelleri in the Ushakovo settlement (B); P. ridibundus and its hybrids with P. cf. bedriagae in the vicinities of Mamonovo
Town (C); P. kurtmuelleri, P. ridibundus and its hybrids with P. cf. bedriagae in the Vistula Spit in Baltiysk Town (D).
frog species. Ponds in Yuzhnyi park in the center of Kaliningrad City
(Figure 3A) were inhabited by P. cf. bedriagae and their hybrids with
P. ridibundus (Figure 4). An individual of P. kurtmuelleri was collected in a
drainage channel in Ushakovo settlement (Figure 3B). The system of shallow
quarry ponds in the vicinities of Mamonovo Town (Figure 3C) were
populated by P. ridibundus and their putative hybrids with P. cf. bedriagae.
Individuals of P. kurtmuelleri, P. ridibundus, and putative hybrids
P. ridibundus and P. cf. bedriagae inhabited a brakish fort moat in the
Vistula Spit in Baltiysk Town (Figure 3D).
According to our data, marsh frogs in Kaliningradskaya Oblast’ possessed
the mtDNA of the following two species only: P. kurtmuelleri (n = 8; 42%;
5 localities) and P. lessonae (n = 11; 58%; 6 localities). Only two individuals
identified by the nuclear SAI-1 fragment as P. kurtmuelleri had the
conspecific mtDNA. The other 17 individuals were characterised by non-
conspecific mitochondrial genomes. The majority of individuals of
P. ridibundus (n = 8; 62%; five localities) had the mtDNA of P. lessonae.
Five individuals of P. ridibundus (38%; three localities) and an individual
of P. cf. bedriagae carried the P. kurtmuelleri mtDNA. All three putative

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Figure 4. Individual of Pelophylax cf. bedriagae or its hybrids with P. ridibundus in Yuznyi
park in the center of Kaliningrad city.
hybrids (P. ridibundus × P. cf. bedriagae) had the mtDNA of P. lessonae.
Marsh frogs with mtDNA haplotypes of P. lessonae only were detected in
three localities (Figure 2b: 2, 4 and 8), P. kurtmuelleri in two localities
(1 and 6), and both species in three localities (3, 5 and 7). The P. lessonae
mtDNA was more frequent in populations located around the Vistula
Lagoon (67%) than near the Curonian Lagoon and in Neman River (43%).
The phylogenetic analysis based on the nuclear SAI-1 gene fragment
(Figure 5) showed that local P. kurtmuelleri alleles were most similar to the
diversity found in Poland. The individual of P. cf. bedriagae from
Kaliningradskaya Oblast’ was similar to Anatolian and West-Kazakhstan
individuals of the species. The genetic differences between P. ridibundus
and P. kurtmuelleri (p-distance 0.8 ± 0.3%) were less than between P. cf.
bedriagae with P. ridibundus and P. kurtmuelleri (4.3 ± 0.7% and 4.8 ±
0.8%, respectively).
The phylogenetic analysis of the mitochondrial ND2 gene fragment
(Figure 6) showed that the P. kurtmuelleri mtDNA from Kaliningradskaya
Oblast’ was closely related to haplotypes sequenced in conspecific
individuals from Macedonia, as well as in P. ridibundus from Latvia,
Ukraine and Romania. The P. lessonae mtDNA found in marsh frogs from
the Baltic Coast of Russia was quite similar to the mtDNA in European
populations of the pool frog. The genetic differences between the mtDNA
clades of P. ridibundus and P. kurtmuelleri were 1.2 ± 0.3%. P. lessonae
differed from P. ridibundus and P. kurtmuelleri on 14.1 ± 0.1% и 14.3 ±
0.1%, respectively.

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Figure 5. Dendrogram of phylogenetic relationships within P. kurtmuelleri, P. ridibundus and
P. cf. bedriagae inferred from sequence analysis of the nuclear DNA SAI-1 gene by the
maximum likelihood (ML) method. Bootstrap support values higher than 80% are shown. Full
circles represent our data and empty circles were data obtained from GenBank.
Discussion
The history of marsh frog populations in Kaliningradskaya Oblast’ is
unknown. These populations might have existed in the region for a long
time. The first reliable record could be attributed to Muhling (1898), who
found “Rana esculenta var. ridibunda” in Baltiysk (“Pillau”) Town. Le Roi
(1903) suggested that water frogs from the Rybachiy (“Rossitten”) settlement
might belong to “Rana esculenta var. ridibunda”. Pagast (1941) mentioned
records of marsh frogs (“Rana ridibunda”) in the vicinities of Kaliningrad

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Figure 6. Dendrogram of phylogenetic relationships among haplotypes within P. kurtmuelleri,
P. ridibundus, and P. lessonae inferred from sequence analysis of the mitochondrial ND2 gene
by the maximum likelihood (ML) method. Bootstrap support values higher than 80% are
shown. Full circles represent our data and empty circles were data obtained from GenBank.
Triangles indicate specimens of P. lessonae, while circles indicate specimens of the
P. ridibundus complex.

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(“Königsberg”) City, the Vistula (“Frischen”) and Curonian (“Kurischen”)
lagoons. Thus, we can assume that, at a minimum, P. ridibundus has inhabited
the region for more than a century. Hovewer, it is obvious that the age of
the Baltic Coast populations of P. ridibundus should be much older
because the species inhabits some islands in the Baltic Sea, which have been
isolated from the mainland for more than 9,000 years (Ojaveer 2017).
Two hypotheses could explain the origin of P. kurtmuelleri and P. cf.
bedriagae in the region. They could be recently introduced or a relic of
previous distributions of species in northern Europe. Some indirect
evidence supports the latter proposal. Records of two isolated caudate
amphibian species with a more southern distribution exist in the region
(Litvinchuk 1996; Jakóbik et al. 2019). These are the Alpine newt,
Ichthyosaura alpestris (Laurenti, 1768), and the fire salamander, Salamandra
salamandra (Linnaeus, 1758). Hovewer, these records are exclusively
associated to relic beach forest massifs. Additionally, the Baltic populations
of the green toad, Bufotes viridis (Laurenti, 1768), bear mtDNA of a
southern species, the Anatolian B. sitibundus (Pallas, 1771). Perhaps,
expanding populations of B. viridis captured the B. sitibundus mtDNA in
Balkan refugium before its postglacial dispersal throughout the Baltic Region
(Dufresnes et al. 2019). The same capture of genes of P. kurtmuelleri and
P. cf. bedriagae appears possible for P. ridibundus which have a glacial
refugium in the Balkans.
This hypothesis of a genetic exchange between species before a
postglacial dispersal seems most plausible for P. kurtmuelleri, whose
distributional range in the Balkans overlaps with P. ridibundus (Figure 1).
Recent records of alleles and/or haplotypes of P. kurtmuelleri in populations
of P. ridibundus through European Russia, Ukraine, Belarus’, Lithuania,
Latvia, and Poland (Plötner et al. 2008; Hauswaldt et al. 2012; Kolenda et
al. 2017; Lukonina et al. 2019; Vershinin et al. 2019) could support this
proposal. Hovewer, the relic hypothesis seems unlikely for P. cf. bedriagae
because no records of alleles and haplotypes of the species were found in
Poland or the Baltic Republics located around Kaliningradskaya Oblast’
(Plötner et al. 2008; Hauswaldt et al. 2012; Kolenda et al. 2017). Therefore,
an occasional introduction seems to be more credible for P. cf. bedriagae.
Marsh frogs are often introduced as a food source (i.e., consumption of
frogs legs), to stock garden ponds, a result of dispersal through newly
created waterways, laboratory animals for teaching and study at
universities, and occasionally with fish fry (Duysebaeva et al. 2005; Kuzmin
2013; Bisconti et al. 2019). The first three pathways of introductions are
unrealistic for the species studied here because Kaliningradskaya Oblast’
has not recently created any large water channels and local people do not
use marsh frogs as a food source or to stock garden ponds. However, the
last two pathways could be introduction vectors for these species in this
region. The Baltiyskiy Federal University (Kaliningrad) is the only educational

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institution which uses local and regionally collected water frogs for
teaching purposes, and frogs could be released into natural water bodies in
Kaliningrad. Similar marsh frog introductions were documented in 1961 in
Ust’-Kamennogorsk (Duysebaeva et al. 2005), in 1970 in Gorno-Altaisk
(Yakovlev and Malkov 1985; Yakovlev 1987), and in 1972 in Yakutsk
(Belimov and Sedalischev 1980). Usually, marsh frogs are collected from
the Volga River delta, which has the highest abundances in Russia, for
teaching in universities located in the North-Western Region of European
Russia. According to Ivanov (2019), based on the analysis of the nuclear
SAI-1 fragment, marsh frogs from the Volga River delta region (Republic
of Kalmykia and Astrakhanskaya Oblast’, Russia) are represented by both
P. ridibundus and P. cf. bedriagae (and their hybrids). Therefore, the Volga
River delta region could be a source for the introduction of P. cf. bedriagae
to Kaliningradskaya Oblast’.
The second realistic pathway for the introduction of P. cf. bedriagae to
the Baltic Region of Russia is an occasional release of tadpoles with juvenile
fish. Since the 1940s the European carp (Cyprinus c. carpio) has been
intensively aquacultured throughout Russia. For example, thousands of
fish were released between 1953 and 1955 into the Curonian Lagoon in
Kaliningradskaya Oblast’ (Kudersky 2001; Khainovsky and Ulianov 2015).
The native range of the European carp is the Ponto-Caspian region (Tsepkin
2003), where the majority of fish fry rearing ponds are located; fry produced
in the the Ponto-Caspian region are then transported throughout Russia to
stock fish farms. Marsh frog introductions related to fish reservoir stocking
have been previously observed from the Altayskiy Kray and Alakol and
Issyk-Kul lakes in the 1960s (Yakovlev and Malkov 1985; Duysebaeva et al.
2005; Kuzmin 2013) and the Krasnoyarskiy Kray and Republic of Khakassia
in Siberia in the 1970s and 1980s (Chuprov 2013). Marsh frogs from the
Reftinskoe Reservoir in the Ural Mountains (Russia) were occasionally
introduced with fish fry in the 1970s from Krasnodarskiy Kray in the
Western Caucasus (Ivanova and Berzin 2019). Both P. ridibundus and
P. cf. bedriagae (and their hybrids) inhabit the Ponto-Caspian Region of
Russia (Ermakov et al. 2014; Ivanov et al. 2015; Ermakov et al. 2016a, b;
Ivanov 2019). Therefore, the region could be a source for introduction of
P. cf. bedriagae to Kaliningradskaya Oblast’.
Marsh frogs prefer open lanscapes. Since the start of the Holocene, the
eastern part of the Baltic Region has been covered by closed forest massifs
(Smirnova and Turubanova 2004), which are usually populated by P. lessonae
and P. esculentus. The prevalence of the P. lessonae mtDNA in marsh frogs
from the Baltic Coast of Russia may suggest that the territory was originally
inhabited by mixed populations of P. lessonae and P. esculentus, in which
the latter species usually produced gametes of P. ridibundus (reviewed by
Plötner 2005). Sporadically, viable individuals of P. ridibundus may be
produced in such populations as a result of crosses between P. esculentus

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individuals (hybridolysis; see details in Plötner 2005 and Dubey et al. 2019).
Usually, P. ridibundus, which overwinters in water, do not survive in
waterbodies populated by P. lessonae and P. esculentus (both of which, as a
rule, overwinter on land) due to freezing of the water and low concentrations
of dissolved oxygen (Berger 1984; our data). However, P. ridibundus
tolerates brackish water and can survive in deep lagoons of the Baltic Sea
(Litvinchuk et al. 2015). Such individuals resulting from hybridolysis
should carry the mtDNA of P. lessonae because the parental P. esculentus
have perpetuated through hybridogenesis with P. lessonae only. Similar
populations of P. ridibundus carrying the P. lessonae mtDNA were found
in the Czech Republic, Slovakia, Switzerland, Germany, Poland, and the
Danish Island of Bornholm in the Baltic Sea (Plötner et al. 2008; Hofman
et al. 2012; Mikulíček et al. 2014; Dubey et al. 2014; Hoffmann et al. 2015;
Hawlitschek et al. 2016; Dufresnes et al. 2018). Populations of Kaliningradskaya
Oblast’ are now the northeasternmost records of this phenomenon.
The presumed hybridization of P. kurtmuelleri and P. ridibundus (which
carried the P. lessonae mtDNA) in the Baltic Region of Russia leads to
coexistance of mtDNA genomes of both P. kurtmuelleri and P. lessonae in
local populations of P. ridibundus. The absence of the P. cf. bedriagae
mtDNA in local marsh frog populations could indicate selection due to
local environmental conditions and/or drift. Individuals of P. cf. bedriagae
(and their hybrids) with the mtDNA of P. kurtmuelleri have been previously
detected in introduced marsh frog populations in Belgium (Holsbeek et al.
2008, 2009).
The release of alien water frogs in Kaliningradskaya Oblast’ can have
several negative consequences, but the threat of genetic introgression is the
greatest among them. Hybridization between the alien species (P. cf. bedriagae)
and native species (P. ridibundus and presumably P. kurtmuelleri) can be
common here. The result of this is replacement or local extinction of native
species by introgressive hybridization (see Blackburn et al. 2014). Moreover,
such hybridization can impact the persistence of local water frog
hybridogenetic systems (Holsbeek et al. 2010; Dufresnes et al. 2017; Fayzulin
et al. 2018), since P. esculentus strictly reproduces successfully with only
“true” P. ridibundus and P. lessonae. As shown from laboratory crosses, a low
frequency of nuclear alleles of P. cf. bedriagae among parent P. ridibundus
from Mariy El Republic (Russia) disturbed germ cell development in
hybridogenous P. esculentus (Dedukh et al. 2019). Additional evidence of
the negative impact of P. cf. bedriagae and P. kurtmuelleri on hemiclonal
reproduction of P. esculentus can provide data about their distribution.
P. esculentus is absent in the Balkan mountainous regions (Figure 1), which
is the only region where P. kurtmuelleri is found, and in the eastern part of
European Russia, where P. ridibundus bears some portion of P. cf. bedriagae
alleles (Fayzulin et al. 2018). In Kaliningradskaya Oblast’, the presence of
P. cf. bedriagae and P. kurtmuelleri in water bodies surrounding the Vistula

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Litvinchuk et al. (2020), BioInvasions Records 9(3): 599–617, https://doi.org/10.3391/bir.2020.9.3.16 612
Lagoon could led to the disappearance of local hybridogenic population
systems (unlike the Curonian Lagoon, where alleles of both these species
are absent).
In addition, reproductive barriers between marsh frog species could be
altered under the new conditions. The viability of interspecific crossings
between various water frog species have been widely tested by several
authors (see Plötner et al. 2010), and P. ridibundus, P. kurtmuelleri and
P. cf. bedriagae can succesfully hybrize in the Eastern Balkans (Hotz et al.
2013). We assume that releases of alien species can favor production of
new hybrid genotypes, whose invasive potential can be higher than those of
parental species. If they do not suffer from intrinsic incompatibilities,
hybrids might acquire an increased potential for local adaptations and
resistance to diseases, higher survival, growth and development rates
(Grant and Grant 1992; Frankham et al. 2002; Seehausen 2004).
The establishment and spread of P. kurtmuelleri and P. cf. bedriagae
populations in the Baltic Coast of Russia could also threaten the
persistence of native amphibians via competitive interactions in terms of
food resources and breeding sites; they can also prey on larvae, juveniles
and adults. For example, the introduction of marsh frogs in north-eastern
Kazakhstan and Western Siberia (Russia) reduced the abundance of two
native anurans: tetraploid green toad, Bufotes pewzowi (Bedriaga, 1898),
and the moor frog, Rana arvalis Nilsson, 1842 (Berezovikov 2008; our
data). However, it should be noted that in Kaliningradskaya Oblast’
P. kurtmuelleri and P. cf. bedriagae inhabit human-made waterbodies
which are unsuitable for life and reproduction of most local amphibians.
Since our study is preliminary (19 individuals), in the future it would be
very important to continue the study of water frogs in the Baltic Region of
Russia and neighboring countries by applying multiple genetic markers.
This would enable researchers to study routes of dispersal and introductions
of marsh frog species, to clarify peculiarities of their hybridization and
patterns of distribution, and to evaluate the impact of P. kurtmuelleri and
P. cf. bedriagae on reproduction success of hybridogenous populations and
abundance of local amphibians.
Acknowledgements
We are very grateful to N. B. Ananjeva and A. A. Ostroshabov for providing possibility to use
herpetological collections of the Zoological Institute of Russian Academy of Sciences. We are
grateful to J. Measey, A. Fowler and the reviewers who provided useful comments on an earlier
draft of the manuscript.
Funding Declaration
The study was supported by grants of Russian Foundation for Basic Research (20-04-00918 for
SNL and 18-04-00640 for AYuI, SAL and OAE).

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Supplementary material
The following supplementary material is available for this article:
Table S1. Frequencies of occurrence of alleles and haplotypes (in %) of various water frog species in European presumably invasive
populations.
This material is available as part of online article from:
http://www.reabic.net/journals/bir/2020/Supplements/BIR_2020_Litvinchuk_etal_SupplementaryMaterial.xlsx

Table S1.
Frequencies of occurrence of alleles and haplotypes (in %) of various water frog species in European presumably invasive populations.
Locality
Country
Latitude, N
Longitude, E
Nuclear DNA markers
Mitochondrial DNA markers
Source
Type*
n**
ridibundus
kurtmuelleri
cf.
bedriagae
Cilician undescribed species
n
ridibundus
lessonae
kurtmuelleri
cf.
bedriagae
caralitanus
Cilician undescribed species
perezi
Assebroek Belgium 50,193 3,258 msat 2 50 0 50 0 28 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Hoegaarden Belgium 50,769 4,889 msat 7 29 0 71 0 5 80 0 0 20 0 0 0 Holsbeek et al. (2008, 2009)
Huldenberg Belgium 50,771 4,635 msat 4 25 0 75 0 8 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Anderlecht-2 Belgium 50,806 4,289 msat 4 50 0 50 0 5 100 0 0 0 0 0 0 Holsbeek et al. (2008, 2009)
Tienen Belgium 50,806 4,985 msat 2 50 0 50 0 6 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Haasrode Belgium 50,853 4,723 msat 6 17 0 83 0 8 100 0 0 0 0 0 0 Holsbeek et al. (2008, 2009)
Heverlee-2 Belgium 50,854 4,719 msat 4 75 0 25 0 22 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Attenrode-2 Belgium 50,855 4,920 msat 2 50 0 50 0 14 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Attenrode-1 Belgium 50,855 4,920 msat 3 67 0 33 0 3 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Attenrode-3 Belgium 50,855 4,920 msat 1 0 0 100 0 - - - - - - - - Holsbeek et al. (2009)
Drieslinter Belgium 50,856 5,051 msat 2 50 0 50 0 24 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Kortenaken Belgium 50,856 5,052 msat 10 10 0 90 0 21 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Heverlee-1 Belgium 50,857 4,709 msat 11 64 0 36 0 11 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Kersbeek Belgium 50,884 4,991 msat 5 40 0 60 0 12 100 0 0 0 0 0 0 Holsbeek et al. (2008, 2009)
Molenbeek-Wersbeek-2 Belgium 50,903 4,939 ms at 12 50 0 50 0 6 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Molenbeek-Wersbeek-1 Belgium 50,903 4,938 ms at 10 60 0 40 0 1 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Asse-1 Belgium 50,903 4,192 msat 5 80 0 20 0 5 100 0 0 0 0 0 0 Holsbeek et al. (2008, 2009)
Wilsele Belgium 50,906 4,705 msat 4 50 0 50 0 4 100 0 0 0 0 0 0 Holsbeek et al. (2008, 2009)
Molenbeek-Wersbeek-3 Belgium 50,918 4,942 ms at 2 50 0 50 0 12 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Kiewit Belgium 50,956 5,373 msat 2 50 0 50 0 - - - - - - - - Holsbeek et al. (2009)
Rillaar-1 Belgium 50,973 4,903 msat 2 50 0 50 0 8 100 0 0 0 0 0 0 Holsbeek et al. (2008, 2009)
Mechelen Belgium 51,001 4,510 msat 3 67 0 33 0 18 100 0 0 0 0 0 0 Holsbeek et al. (2008, 2009)
Rijmenam-2 Belgium 51,005 4,619 msat 3 33 0 67 0 18 0 0 0 100 0 0 0 Holsbeek et al. (2008, 2009)
Rijmenam-1 Belgium 51,006 4,576 msat 5 20 0 80 0 37 100 0 0 0 0 0 0 Holsbeek et al. (2008, 2009)
Citov Czechia 50,375 14,442 SAI-1 6 33 50 17 0 - - - - - - - - Akin Peksen (2015)
Efringen-Kirchen Germany 47,650 7,530 SAI-1 6 58 0 42 0 6 67 0 0 33 0 0 0 Ohst (2008)
Steinstadt Germany 47,770 7,530 SAI-1 9 89 0 11 0 9 89 0 0 11 0 0 0 Ohst (2008)
Bremgarten Germany 47,900 7,580 SAI-1 4 67 0 33 0 4 75 0 0 25 0 0 0 Ohst (2008)
Hartheim Germany 47,930 7,600 SAI-1 10 80 0 20 0 10 70 0 0 30 0 0 0 Ohst (2008)
Breisach Germany 48,050 7,580 SAI-1 4 88 0 12 0 4 100 0 0 0 0 0 0 Ohst (2008)
Jechtingen Germany 48,120 7,580 SAI-1 9 88 0 12 0 9 56 0 0 44 0 0 0 Ohst (2008)
Leopoldskanal Germany 48,200 7,680 SAI-1 2 25 0 75 0 2 100 0 0 0 0 0 0 Ohst (2008)
Ettenheim Germany 48,270 7,870 SAI-1 6 75 0 25 0 6 100 0 0 0 0 0 0 Ohst (2008)
Trittenheim Germany 49,830 6,890 SAI-1 5 100 0 0 0 4 25 0 0 75 0 0 0 Ohst (2008)
Bingen Germany 49,980 7,870 SAI-1 3 50 0 50 0 2 100 0 0 0 0 0 0 Ohst (2008)
Aue bei Winz-Hattingen Germany 51,400 7,150 SAI-1 4 50 0 50 0 4 100 0 0 0 0 0 0 Ohst (2008)
Heisinger Aue Germany 51,420 7,070 SAI-1 10 45 0 55 0 9 100 0 0 0 0 0 0 Ohst (2008)
Oder River Germany 52,421 14,533 SAI-1 5 90 0 10 0 - - - - - - - - Akin Peksen (2015)
St Mathieu Tréviers France 43,930 4,050 SAI-1 4 75 0 25 0 4 100 0 0 0 0 0 0 Ohst (2008)
Ramière France 44,770 4,850 SAI-1 10 75 0 25 0 9 100 0 0 0 0 0 0 Ohst (2008)
Gravillère Platière France 45,330 4,770 SAI-1 10 70 0 30 0 7 86 0 0 14 0 0 0 Ohst (2008)
Crolles France 45,600 6,170 SAI-1 2 100 0 0 0 2 50 0 0 50 0 0 0 Ohst (2008)
Chautagnes France 46,050 5,880 SAI-1 26 81 0 19 0 20 80 0 0 20 0 0 0 Ohst (2008)
Etournelles France 46,230 5,930 SAI-1 3 100 0 0 0 2 50 0 0 50 0 0 0 Ohst (2008)
Delta de la Dranse France 46,230 6,350 SAI-1 5 90 0 10 0 5 60 0 0 40 0 0 0 Ohst (2008)
Toulouse France 43,530 1,470 SAI-1 1 100 0 0 0 1 0 0 0 100 0 0 0 Ohst (2008)
Aramon France 43,653 3,328 - - - - - - 1 100 0 0 0 0 0 0 Akin Peksen (2015)
Octon France 43,657 3,407 msat 1 0 100 0 0 1 0 0 0 0 0 0 100 Dufresnes et al. (2017)
Camargue France 43,660 4,670 SAI-1 2 100 0 0 0 2 100 0 0 0 0 0 0 Ohst (2008)
Beauzelle France 43,670 1,390 TYR 8 25 0 75 0 9 67 0 0 11 0 0 22 Sanchez-Montes et al. (2016)
Montpellier France 43,720 4,100 SAI-1 3 33 0 67 0 3 0 0 0 100 0 0 0 Ohst (2008)
Ferrussac France 43,752 3,483 msat 4 0 100 0 0 - - - - - - - - Dufresnes et al. (2017)
Devois la Trivalle France 43,772 3,461 msat 2 0 100 0 0 - - - - - - - - Dufresnes et al. (2017)
St. Etienne du Gres France 43,779 4,653 - - - - - - 1 100 0 0 0 0 0 0 Akin Peksen (2015)
Coulet Tournant France 43,799 3,498 msat 4 0 100 0 0 2 0 0 100 0 0 0 0 Dufresnes et al. (2017)
Natges France 43,818 3,566 msat 2 0 100 0 0 2 0 0 100 0 0 0 0 Dufresnes et al. (2017)
Mare du Goutal France 43,821 3,540 msat 2 0 100 0 0 1 0 0 100 0 0 0 0 Dufresnes et al. (2017)
Coulet France 43,822 3,530 msat 2 0 100 0 0 - - - - - - - - D ufresnes et al. (2017)
Rancas France 43,831 3,560 m sat 1 0 100 0 0 - - - - - - - - Dufresnes et al. (2017)
Besses France 43,843 3,385 msat 4 0 100 0 0 1 0 0 100 0 0 0 0 Dufresnes et al. (2017)
Bairades France 43,848 3,559 msat 1 0 100 0 0 1 0 0 100 0 0 0 0 Dufresnes et al. (2017)
St-Michel Le Laquet France 43,856 3,362 msat 6 100 0 0 0 6 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Source de la Bueges France 43,856 3,666 msat 9 0 100 0 0 3 67 0 33 0 0 0 0 Dufresnes et al. (2017)
Bagnelades France 43,861 3,379 msat 7 100 0 0 0 7 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Bergerie de l'Hopital France 43,861 3,379 msa t 10 100 0 0 0 10 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Source de la Pradasse France 43,863 3,686 msat 2 0 100 0 0 2 0 0 100 0 0 0 0 Dufresnes et al. (2017)
Cros reservoir France 43,866 3,368 msat 3 100 0 0 0 3 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Clapas de Lamathe France 43,867 3,683 msat 8 0 100 0 0 7 0 0 100 0 0 0 0 Dufresnes et al. (2017)
Cros Farm France 43,870 3,370 msat 6 92 8 0 0 6 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Baume Vieille France 43,888 3,399 msat 5 90 10 0 0 5 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Moulin France 43,889 3,397 msat 5 90 10 0 0 3 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Sotch de Caylus France 43,889 3,387 ms at 4 100 0 0 0 4 75 0 25 0 0 0 0 Dufresnes et al. (2017)

Lac de Condamine France 43,891 3,664 msat 6 0 100 0 0 6 0 0 0 0 0 0 100 Dufresnes et al. (2017)
Chateau de Sorbs France 43,891 3,404 msat 1 100 0 0 0 1 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Four Banal France 43,894 3,402 msat 10 95 5 0 0 6 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Corombelle France 43,894 3,381 msat 1 100 0 0 0 1 100 0 0 0 0 0 0 Dufresnes et al. (2017)
Ville Vieille France 43,899 3,405 msat 8 87 13 0 0 6 83 0 17 0 0 0 0 Dufresnes et al. (2017)
Ikaria Greece 37,608 26,152 - - - - - - 3 0 0 0 0 100 0 0 A kin et al. (2010)
Resina river Italy 43,247 12,488 - - - - - - 4 100 0 0 0 0 0 0 Domeneghetti et al. (2013)
Tula Italy 40,726 8,985 SAI-1 4 0 75 25 0 4 0 0 50 50 0 0 0 Bellati et al. (2019)
Ploaghe Italy 40,658 8,741 SAI-1 2 0 0 100 0 2 0 0 0 100 0 0 0 Bellati et al. (2019)
Pattada Italy 40,576 9,106 SAI-1 4 0 62 38 0 4 0 0 100 0 0 0 0 Bellati et al. (2019)
Pabillonis Italy 39,589 8,719 SAI-1 2 0 0 0 100 2 0 0 0 0 0 100 0 Bellati et al. (2019)
Uta Italy 39,287 8,954 SAI-1 2 0 0 0 100 2 0 0 0 0 0 100 0 Bellati et al. (2019)
Aspromonte NP Italy 38,180 15,848 - - - - - - 4 0 0 100 0 0 0 0 Bisconti et al. (2019)
Aspromonte NP Italy 38,189 15,828 - - - - - - 4 0 0 100 0 0 0 0 Bisconti et al. (2019)
Aspromonte NP Italy 38,193 15,829 - - - - - - 4 0 0 100 0 0 0 0 Bisconti et al. (2019)
Aspromonte NP Italy 38,195 15,832 - - - - - - 4 0 0 100 0 0 0 0 Bisconti et al. (2019)
Aspromonte NP Italy 38,198 15,842 - - - - - - 4 0 0 100 0 0 0 0 Bisconti et al. (2019)
Aspromonte NP Italy 38,200 15,819 - - - - - - 4 0 0 100 0 0 0 0 Bisconti et al. (2019)
Aspromonte NP Italy 38,201 15,799 - - - - - - 4 0 0 100 0 0 0 0 Bisconti et al. (2019)
Aspromonte NP Italy 38,211 15,843 - - - - - - 4 0 0 100 0 0 0 0 Bisconti et al. (2019)
Jurmala Latvia 56,989 25,920 - - - - - - 6 100 0 0 0 0 0 0 Hoffmann et al. (2015)
Bulduri Latvia 57,000 23,900 - - - - - - 4 50 0 50 0 0 0 0 Plötner et al. (2008)
Kruonio Reservoir Lithuania 54,792 24,251 SAI-1 16 97 3 0 0 - - - - - - - - Hauswald et al. (2012)
Niezgoda-1 Poland 51,517 17,038 SAI-1 6 83 17 0 0 - - - - - - - - Hauswald et al. (2012)
Niezgoda-2 Poland 51,518 17,038 SAI-1 1 50 50 0 0 - - - - - - - - Hauswald et al. (2012)
Stawno Poland 51,320 17,210 SAI-1 6 33 67 0 0 6 67 0 33 0 0 0 0 Kolenda et al. (2017)
Potasznia Poland 51,320 17,290 SAI-1 3 50 50 0 0 3 100 0 0 0 0 0 0 Kolenda et al. (2017)
Staryi Petergof Russia 59,885 29,910 SAI-1 1 0 0 100 0 3 0 0 0 100 0 0 0 Akin et al. (2010)
Mamonovo Russia
54,449
19,952
SAI-1
5
70
0
30
0
5
0
100
0
0
0
0
0
Present paper
Ushakovo Russia
54,612
20,243
SAI-1
1
0
100
0
0
1
0
0
100
0
0
0
0
Present paper
Baltiysk Russia
54,635
19,874
SAI-1
4
63
25
13
0
4
0
50
50
0
0
0
0
Present paper
Yuzhnyi park, Kaliningrad Russia
54,691
20,512
SAI-1
2
25
0
75
0
2
0
50
50
0
0
0
0
Present paper
Zelenogradsk Russia
54,952
20,484
SAI-1
1
100
0
0
0
1
0
100
0
0
0
0
0
Present paper
Rybachiy Russia
55,157
20,844
SAI-1
4
100
0
0
0
4
0
25
75
0
0
0
0
Present paper
Morskoe Russia 55,228 20,92 SAI-1 1 100 0 0 0 1 0 100 0 0 0 0 0 Present paper
Sovetsk Russia
55,094
21,844
SAI-1
1
100
0
0
0
1
0
0
100
0
0
0
0
Present paper
Prades Spain 41,310 0,980 TYR 1 100 0 0 0 1 0 0 0 0 0 0 100 Sanchez-Montes et al. (2016)
Oix Spain 42,270 2,530 TYR 2 100 0 0 0 3 0 0 0 0 0 0 100 Sanchez-Montes et al. (2016)
Grand Lancy Switzerland 46,178 6,123 - - - - - - 1 0 0 100 0 0 0 0 Dufresnes et al. (2018)
Triengen Switzerland 47,235 8,076 - - - - - - 4 0 0 0 100 0 0 0 Dufresnes et al. (2018)
Carpière Switzerland 46,234 6,291 - - - - - - 3 67 0 33 0 0 0 0 Dufresnes et al. (2018)
Bavois Switzerland 46,680 6,570 SAI-1 8 75 0 25 0 8 100 0 0 0 0 0 0 Ohst (2008)
Creux de Terre Switzerland 46,720 6,570 SAI-1 7 50 0 50 0 6 50 0 0 50 0 0 0 Ohst (2008)
Grande Cariçaie Switzerland 46,780 6,670 - - - - - - 26 69 27 0 0 0 4 0 Dubey et al. (2014)
Chevroux Switzerland 46,891 6,904 - - - - - - 15 79 7 7 7 0 0 0 Dufresnes et al. (2018)
Aarau area Switzerland 47,390 8,050 - - - - - - 10 70 30 0 0 0 4 0 Dubey et al. (2014)
* TYR is tyrosinase gene; SAI-1 is the intron-1 of the nuclear serum albumin gene; and msat is microsatellites.
** n is number of specimens.
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