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J Zool Syst Evol Res. 2021;00:1–17.
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1wileyonlinelibrary.com/journal/jzs
Received: 23 February 2020
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Revised: 15 November 2020
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Accepted: 21 November 2020
DOI : 10.1111/jzs.124 47
ORIGINAL ARTICLE
Genetic structure, morphological variation, and gametogenic
peculiarities in water frogs (Pelophylax) from northeastern
European Russia
Anton O. Svinin1,2 | Dmitrij V. Dedukh3 | Leo J. Borkin4 | Oleg A. Ermakov5 |
Alexander Y. Ivanov5 | Julia S. Litvinchuk3 | Renat I. Zamaletdinov6 |
Regina I. Mikhaylova7 | Aleksey B. Trubyanov1 | Dmitriy V. Skorinov8 |
Yurij M. Rosanov8 | Spartak N. Litvinchuk8,9
This is an op en access arti cle under the ter ms of the Creative Commons Attribution L icense, which pe rmits use, dis tribution and reproduction in any medium,
provide d the original wor k is properly cited.
© 2021 The Authors. Journa l of Zoological Systematics and Evolutionary Research published by Wiley-VCH GmbH
Contri buting autho rs: Dmitrij V. Dedu kh (dmitrijde dukh@gmail. com), Leo J. Borki n (leo.borkin @zin.ru), Ole g Aleksandr ovich Er makov (oae rmakov@list. ru), Aleksan der Yurievich Iva nov
(akella5 8@mail .ru), Julia Spa rtako vna Litvinch uk (varisha22@m ail.ru), Renat I rekovich Zamal etdinov (i.ric inus@rambl er.ru), Re gina Ipp olitovna Mikh aylova (s tudy@kazanv eterinary.ru ),
Aleks ey B. Trubyanov (tru e47@mail.ru), Dm itriy V ladimirovich S korinov (skorin ovd@yandex. ru), Yurij Mikhayl ovich Rosanov (ro za-yur @yandex.ru ), and Spa rtak Nikolae vich Litvinc huk
(litvinchukspartak@yandex.ru)
1Mari State Univer sity, Yoshkar-Ola,
Russia
2Nationa l Research Tomsk State
University, Tomsk, Russia
3Saint-Petersburg State University, Saint-
Petersburg, Russia
4Zoological Institute, Russian Academy of
Sciences, Saint-Petersburg, Russia
5Penza State University, Penza, Russia
6Kazan ( Volga Region) Feder al Unive rsity,
Kazan, Russia
7Bauman K azan State Academy of
Veterinary Medicine, Kazan, Russia
8Institute of Cytology, Russian Academy of
Sciences, Saint-Petersburg, Russia
9Dagest an State University, Makhachkala,
Russia
Correspondence
Anton Olegovich Svinin, Mari State
University, 1, Lenin sq., Yoshkar-Ola, Mari
El 42400 0, Russia.
Email: ranaesc@gmail.com
Funding information
Labor atory Projec t of Zoological
Institute of Russian Academy of
Science s, Grant/Award Number:
ААА А-А19-119020590095-9; Russian
Science Fo undation, Grant/Award Number:
18-74-00115; Russian Fou ndatio n for Basic
Research, Grant/Award Number: 18- 04-
00640 and 20 -04 -00918
Abstract
The edible frog, Pelophylax esculentus, is a hybr id fo rm tha t rep roduces via cl ona l pro p-
agation of only one of the parental genomes through generations of hybrids while the
genome of other parental species is eliminated during gametogenesis. Such reproduc-
tive ability requires hybrids to coexist with one of the parental species or rarely both
parental species causing the formation of so-called population systems. Population
systems and reproductive biology of water frogs from the east of the range remained
partially unexplored. In this study, we investigated the distributions, population sys-
tems, genetic structure, types of gametes, and morphological variability of water
frogs of the genus Pelophylax from the northeastern parts of their ranges (Mari El
Republic and adjacent territories, Russia). We examined 1,337 individuals from 68 lo-
calities using morphological traits combined with DNA flow cytometry and a multilo-
cus approach (fragments of a nuclear and two mitochondrial genes). We revealed five
types of population systems: “pure” populations of the parental P. ridibundus (R) and
P. lessonae (L), mixed populations of parental species (R-L) along and with their hybrids
(R-E-L), as well as mixed populations of P. lessonae and P. esculentus (L-E). However, the
“pure” hybrid (E) and the mixed P. ridibundus and P. esculentus (R-E) population systems
were not found. All hybrids studied by DNA flow cytometry were diploid. Analysis of
gametogenesis showed that the majority of hybrid males, as well as hybrid females
from the L-E system, produced gametes with the P. ridibundus genome. However, in
the R-E-L system, hybrid females were usually sterile. The reproduction of hybrids
in such systems is primarily based on crosses of P. esculentus males with P. lessonae
females. Molecular analysis showed the presence of mitochondrial and nuclear DNA
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1 | INTRODUCTION
Interspecific hybridization is a widespread phenomenon among an-
imals. The characteristic of interactions among species (within gen-
era) at the boundaries of their dist ri bu ti on al range s usua ll y depends
on their divergence time. As a rule, closely related species more
easily hybridize than distant species. In cases when species are
strongly diverged and differences between their genomes are al-
ready too large, hybridization becomes impossible (e.g., Dufresnes,
Mazepa, et al., 2019; Dufresnes, Strachinis, et al., 2019). However,
hybrids between distant species may successfully overcome this
barrier when reproduced clonally. In this case, parental genomes
of hybrids remain non-recombining. Such hybrids carry new char-
acteristics that can be supported by adaptive selection. Of ten,
polyploid individuals arise in hybrid lines (Hermaniuk et al., 2013;
Litvinchuk et al., 2016, 2019). The reproduction of the clonal hy-
brids is associated with numerous difficulties, which can strongly
reduce their fertility. Therefore, tetraploid lines, which are charac-
terized by the transition from clonal to sexual reproduction, usually
complete a cycle of reticulate evolution (Borkin & Darevsky, 1980).
Hybridization, asexual reproduction, and polyploidization
are well-known attributes of Palaearctic water frogs of the genus
Pelophylax Fitzinger, 1843 (Berger, 2008; Graf & Pelaz, 1989; Plӧtner,
2005; Polls, 1994) consisted of about 22 species (Frost, 2020).
Therefore, this group has attracted the attention of many research-
ers as a fascinating model to study reticulate evolution (Borkin &
Darevsky, 1980; Litvinchuk, Borkin, et al., 2016). Some species in
this genus can easily hybridize with each other, leading not only to
introgression of genetic material into the gene pool of various spe-
cies but also to hybrids reproducing hemiclonally. Moreover, hemi-
clonal reproduction of such hybrids is frequently accompanied by
polyploidization (Plötner, 2005). The Western Palearctic species
(about 15 species) are usually divided into three groups (P. sahari-
cus, P. lessonae, and P. ridibundus). The representatives of different
groups can hybridize and form several hybridogenetic complexes:
P. e sc u le n tu s, P. g raf i, P. hispanicus, and unnamed hybrid species
with parental P. kurtmuelleri and P. perezi (Dufresnes et al., 2017).
The P. es cul ent us complex is the most widespread and well-stud-
ied (Plötner, 20 05). This complex consists of two parental species,
the pool frog, P. lessonae (Camerano, 1882), and the marsh frog,
P. ridibundus (Pallas, 1771), as well as their hybrid, the edible frog,
P. e sc ulent us (Linnaeus, 1758).
Pelophylax esculentus is characterized by a special mechanism of
hemiclonal reproduction, known as hybridogenesis, during which
one genome is eliminated from gonial cells, while the second dupli-
cates and transmits to gametes (Tunner, 1974). During gametogen-
esis, the hybrids usually exclude the genome of one of the parental
species and produce gametes with the genome of another; thus,
hybrids require crosses with parental species to emerge and repro-
duce. Such a reproductive strategy resulted in a variety of popula-
tion systems, where hybrids are able to reproduce with one of the
parental species (Berger, 20 08; Borkin et al., 1987, 200 4; Dedukh
et al., 2015; Hoffmann et al., 2015; Plӧtner, 2005; Raghianti et al.,
2007; Vinogradov et al., 1988). Depending on the coexistence of
these parental species hybrids, three types of mixed population
systems can be revealed. In particular, the L-E system includes
P. lessonae and P. es cul ent us, the R-E system consists of P. ridibun-
dus and P. e scu len tus , and the R-E-L system unites both parental
and hybrid species (Plötner, 20 05; Rybacki & Berger, 2001; Uzzell
& Berger, 1975). Moreover, throughout the distribution range, pop-
ulations can be represented by hybrids with only one sex or include
not only diploid but also triploid and, rarely, tetraploid individuals
(Borkin et al., 2004, 2006; Doležálková-Kaštánková et al., 2018;
Hoffmann et al., 2015; Plötner, 2005; Rybacki & Berger, 2001). The
hybrid P. e sc u len tu s can also form “pure” population systems known
mainly from the northwestern edge of the species range where hy-
brids live without parental species (Berger, 2008; Plötner, 2005).
Extensive data concerning the distribution of population sys-
tems and gametogenesis of hybrids in Western and Central Europe
were previously published (Daf et al., 2006; Krizmanić & Ivanović,
2010; Mikulíček et al., 2014; Pagano et al., 2001; Rybacki, 1994a,b;
Sas, 2010; Mayer et al., 2013; Tunner & Heppich-Tunner, 1992;
Zavadil, 1994). However, the frog populations in the East European
(Russian) Plain have not been thoroughly investigated.
Parental species, as well as their hybrids, are frequently ob-
served across the Volga River drainage in the European part of
Russia. However, the local population systems are characterized
by some peculiarities that were collectively named “the Volga
River paradox” (Borkin et al., 2003). First, polyploid individuals
of P. es cul e nt us were not observed in these populations. Second,
introgression of the Anatolian marsh frog (P. cf. bedriagae) into both P. ridibundus and
P. esculentus. The observations of alleles and haplotypes of P. cf. bedriagae in P. ri-
dibundus and P. esculentus individuals from the same localities suggest de novo forma-
tion of local hybrids. However, the presence of the Balkan marsh frog (P. kurtmuelleri)
haplotypes in local hybrids supports the hypothesis regarding the migration of old
hemiclonal lineages from glacial refugia. Finally, the diagnostic value of various mor-
phological characteristics was discussed.
KEYWORDS
amphibia, morphology, Pelophylax esculentus complex, population systems, Ranidae
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mixed R-E-L and R-L systems are considerably more common
than in Western and Central Europe (Borisovsky et al., 2001;
Borkin et al., 2002; Borkin et al., 2003; Ruchin et al., 20 05; Lada
et al., 2011; Svinin et al., 2013; Svinin et al., 2016). Third, P. es cu-
lentus is less frequently observed than in Western and Central
Europe (Borkin et al., 2003). Fourth, introgression of alleles of
the Anatolian marsh frog (P. cf. bedriagae) was widely detected
in local populations of P. ridibundus and P. esc ule ntu s (Ermakov
et al., 2013; Svinin et al., 2016; Ivanov et al., 2019), though the
native range of P. cf. bedriagae covers western Iran, Turkey,
southern Bulgaria, eastern Greece, the Caucasus, and the Crimea
(Akin et al., 2010; Ermakov, Fayzulin, Zaks, et al., 2014; Ermakov,
Fayzulin, Askenderov, et al., 2016; Ermakov, Simonov, et al.,
2016; Fayzulin et al., 2017; Ivanov, 2019; Ivanov et al., 2015;
Kukushkin et al., 2018; Plötner et al., 2012). It should be noted
that P. cf. bedriagae was widely introduced to Italy, Belgium,
France, Switzerland, Germany, and Russia (Bellati et al., 2019;
Dubey & Dufresnes, 2017; Dubey et al., 2014; Dufresnes
et al., 2018; Hoffmann et al., 2015; Holsbeek et al., 2009, 2008,
2010; Litvinchuk et al., 2020; Lyapkov et al., 2018; Vershinin
et al., 2019). Therefore, the detailed distribution of the species in
the Russian Plain has not been elucidated.
In this paper, we described the distribution, population systems,
genetic structure, types of gametes, and morphological variability of
water frogs of the P. e scu len tu s complex in the northeaster n par ts of
their ranges.
2 | MATERIALS AND METHODS
2.1 | Studied sites
Du rin g the pe rio d of 20 0 8–2 019, we exa m in e d 68 lo c alitie s thr oug h-
out the territory of the Mari El and Tatarstan republics, as well as
adjacent territories in the Kirovskaya Oblast (= Kirov Province) in the
northeastern part of European Russia (Figure 1; Table S1). The region
is located at the northeastern border of the distributional ranges of
all three species of the P. e scu len tus complex. Data concerning an
additional 11 localities were taken from the literature (Efremov et al.,
1984; Garanin, 2000; Kuzmin, 2012). The observed localities were
predominantly situated on the watershed between the Volga and
Kama rivers (the Volga River drainage). Several types of water bod-
ies were identified: trenches, ponds, rivers, oxbows, sand quarries,
water reser voirs, and lakes (Table S2). The studied territory is mostly
FIGURE 1 (a) Distributional ranges of three species of the Pelophylax esculentus complex in Europe; the studied area marked by red
square. (b) Sample localities with identified frog species: red—Pelophylax esculentus, yellow—Pelophylax lessonae, and violet—Pelophylax
ridibundus. Forests and cultivated lands are marked by green and rose, respectively
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covered by deciduous broadleaf, coniferous, and mixed forests. The
presence of surrounding forest vegetation was registered for each
water body. Additionally, we estimated the percentage of forest
vegetation and agricultural environments on a square land area of
1 km2 in localities of water frogs using the QG IS Point Sampling Tool
Plugin (https://plugi ns.qgis.org/plugi ns/point sampl ingto ol/), which
extracted data from the global 1 km consensus land-cover maps
(Tuanmu & Jetz, 2014).
2.2 | Samples and methods of species
identification
In total, 1,337 adult water frogs from 68 localities were identified
bas ed on external charac te ri stics (Table 1; Table S1). Amo ng all spe c-
imens, 556 were preserved in 70% ethanol and deposited to collec-
tions (97 in the herpetological collection of Mari State University,
459 in the herpetological collection of Institute of Cytology of the
Russian Academy of Sciences) and 781 individuals were released
to the field sites (Table S8). Among preser ved individuals (n = 556),
blood samples of 460 individuals (before their preservation) were
studied by DNA flow cytometry for reliable determination of species.
Individuals were euthanized by 1% solution of 3-aminobenzoic acid
ethyl ester (MS 222). All procedures were carried out to minimize
suffering. Among these specimens, 222 individuals were measured
and used in the analysis of morphometric characters, 162 individuals
were additionally identified with the use of multiplex PCR, and the
type of gametes was studied for 195 individuals (Table 1).
2.3 | Species identification by DNA flow cytometry
We ana l y ze d 460 speci m ens of thr e e spec ies us ing DN A flow cy t om-
etry (238 were studied for the first time; Table S4). This method ena-
bles the precise identification of species and ploidy of hybrids and
parental species from the P. e scu len tus complex (Borkin et al., 1987;
Vinogradov et al., 1990). The details of the method were previously
published (Borkin et al., 1987; Vinogradov et al., 1990, 1991).
2.4 | Morphological analysis
In total, 222 adults from 18 localities, which were previously stud-
ied by DNA flow cytometry, were used for detailed morphometric
treatment (Tables S1 and S3). We analyzed seven morphological
characteristics in specimens preserved in 70% ethanol: L. is body
length (from tip of snout to center of cloacal opening); Lt.c.—head
width (distance between posterior edge of jaw articulations); F.—
femur length (from center of cloacal opening to distal end of the
femur bone); T.—tibia length (from knee to heel); C.s.—length of tar-
sus; D.p.—length of the first toe; and C.int.l.—length of internal meta-
tarsal tubercle. Measurements were made with a digital caliper to
the nearest 0.1 mm for each specimen by the first author. Based on
these parameters, nine ratios (indices) were calculated (L./Lt.c., L./F.,
L./T., L./C.s., L./D.p., L./C.int.l., F./T. T./C.int.l., and D.p./C.int.l.). In
addition, we used two multiplicative indices. Tarashchuk’s (1989)
index was calculated as Ta r = T.2 × D.p./C.int.l.2 × C.s. Hemmer’s
(1979) index (following Korshunov, 2010) was estimated according
to the formula Hem = D.p./C.int.l. + T./C.int.l.
2.5 | Statistical analysis of morphological
characteristics
Statistic al analysis was performed using st andard procedure s (So kal
& Rohlf, 1981). The Levene and Brown–Forsythe tests were applied
fo r comparison of variance s. We used the Fisher test wit h Bo nferroni
correction for the post hoc comparison of disparate variances. We
applied two-way ANOVA for comparison of means. The Sheffe test
was used for post hoc comparisons. The Mann–Whitney test was
used for comparisons of morphological indices between sexes and
species. For the test, the natural logarithm conversion was made for
all not normally distributed morphometric indices. Determination
of most diagnostic characteristics was evaluated using principal
component analysis (PCA). The level of morphological differences
between species was estimated by squared Mahalanobis distances
in canonical discriminant analysis following Peskov et al. (2009).
Analysis was perfor me d with the us e of St atistic a 8.0 (Stat Soft Inc.).
TABLE 1 Sample size, methods of diagnostics of water frog species
Methods N (localities) n (individuals) RR RL LL Suppl. Inform.
Diagnostics by morphological trait s 68 1,337 587 210 540 Table S1
Morphometry 18 222 57 67 98 Table S3
DNA flow cytometry 28 460 129 169 162 Table S4
Multiplex PCR, markers COI and S A I -1 18 162 66 86 10 Table S5
Sequences of a fragment of the ND2 gene 13 57 28 29 — Table S6
Allozyme analysis of sperm 20* 195* 233 6Table S7
DNA flow cytometry of sperm 20 69 9
Allozyme analysis of oocytes 911 54
Note: Genotypes of Pelophylax ridibundus are designated as RR, Pelophylax esculentus as RL, and P. lessonae as LL. * The number is given for all samples
for which gametes were studied.
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2.6 | Species identification by multiplex PCR
method and sequencing a fragment of the ND2 gene
Pieces of femur muscle or toe tips were fixed by 96% ethanol and
stored in 70% ethanol were used as tissue samples. In total, 162
specimens (10—P. lessonae, 66—P. ridibundus, 86—P. e s cu l entus )
were analyzed (Table S5). The identification of alleles of the in-
tron-1 of the nuclear serum albumin (SA I -1) and the subunit 1 of
the mitochondrial cytochrome-c-oxidase (COI) gene fragments of
water frog species was performed using the methods described
by Ermakov et al. (2019). The annealing temperature, PCR prod-
uct length, and primer structure are given in Table 2. The DNA
was extracted by the standard salt-extraction method (Aljanabi
& Mar tinez, 1997). The PCR 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 ( Th ermo Scientific). In e ach mtDNA and
nDNA PCR mixtures, primer concentrations were equal. PCR was
performed at 94°C for 30 s, 60 and 62°C (for SAI-1 and COI, re-
spectively) for 30 s, and 72°C for 30 s (30 cycles). Notable differ-
ences between amplified species-specific fragments (80–306 bp)
enabled us to visually identify species and their hybrids after
electrophoresis of PCR products in polyacrylamide gels. Species
identif ication of 161 individuals was per for med by both DNA fl ow
cytometry and multiplex PCR methods. It should be noted that
the method does not allow distinguishing alleles and haplotypes
of closely related P. ridibundus and P. kurtmuelleri.
Additionally, selective sequencing was used to verify the pri-
mary identification results. The subunit 2 of mitochondrial NADH
dehydrogenase (ND2) gene (1,038 bp) in 57 specimens (28—P. ri-
dibundus, 29—P. e scu len tus ) was sequenced (Table S6). Before
the primers were removed, the individual amplicon ND2 had the
length 1,168 bp. After primer trimming, the obtained ND2 com-
plete sequences had a length of 1,038 bp. Sequencing of the
ND2 gene was performed on an ABI 3500 automatic sequencer
(Applied Biosystems) using the BigDye® Terminator 3.1 (Applied
Biosystems) kit and the same primers that were used for ampli-
fication. The ND2 gene sequence was amplified with use of the
universal primer ND2L1 5′-AAG CT T TTG GGC CCA TAC CCC-3′
(Meyer, 1993) and developed the specific primer ND2H1 5′-GCA
AGT CCT ACA GAA ACT GAA G-3′. The following amplification
conditions were used: initial denaturation for 1 min at 95°C fol-
lowed by 32 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 60 s,
and final extension for 5 min at 72°C. The PCR mixture propor-
tions were the same as for amplification of the COI gene fragment.
The sequences obtained have been deposited in GenBank (nos.
MN808383-MN808439; Table S6).
The nucleotide sequences were aligned both with BioEdit
(Hall, 1999) software and manually. We used MEGA v. 7.0. sof t-
ware (Kumar et al., 2016) for data processing. For constructing
the phylogenetic tree, the maximum-likelihood (ML) method was
used. The most appropriate D NA substitution mo del for the data-
sets was established using jModelTest 2.1.10 (Posada, 2008). The
ML trees were created with the Tamura–Nei model with the fre-
quency of invariable sites set at 0.5570 (TN93 + I) (–lnL = 4,471.99,
BIC = 13,692.06, AICc = 9,723.51). Node support values in phy-
logenetic trees were estimated according to bootstrap sup-
port (200 replicates). Haplotype networks were constructed
using the median-joining method in PopART software (Leigh &
Bryant, 2015). Haplot ype diversit y (h) and nucleotide diversity (π)
within each species were calculated in DnaSP v. 5.10.01 (Librado
& Rozas, 2009).
2.7 | Identification of gamete types in hybrids
Gamete types were determined in 111 individuals of hybrid P. e s-
culentus (84 males and 11 females; see Table S7). As a control, we
additionally analyzed gametes of 32 P. ridibundus (22 males and
nine females) and 69 P. lessonae (15 males and 54 females) indi-
viduals. To identify the genome composition of gametes, we ap-
plied two methods: DNA flow cytometr y and allozyme analysis.
Cell suspension obtained from testes of adult animals was ana-
lyzed. To obtain the sperm suspension, testes were dissected in
a drop of the Versene solution (Biolot, St. Petersburg), and then,
the suspension was selectively examined under a light micro-
scope (phase contrast). Sperm of 69 P. es cul ent us males (includ-
ing a hermaphrodite), 20 P. ridibundus males, and nine P. lessonae
TABLE 2 Primers of multiplex PCR test systems for identification of water frog species
Primer Position Sequence (5′-3′)
Annealing
temperature, °C
PCR product
length, bp Specificity
COIR-Pu 624-601 CCTGCRGGATCAAAAAATGTTGT 63.6 –All three species
COIF-Pl 329-349 GA ACTGTGTACCCCCCACTAG 63.7 294 P. lessonae
COIF-Pr 4 09-429 GCTGGGG TT TCATCA ATTC TG 61. 8 214 P. ridibundus
COIF-Pb 183-204 CT TTGGA AATTGACTCGTGCC A 63.8 440 P. cf. bedriagae
SA1F-Pu 25-59 C CATAC A A ATGT GCTAAG TAG G T T 61. 3 –All three species
SA1R-Pl 140 -1 19 TACCGTA CC GATATTTG TATG C 60.2 10 9 P. lessonae
SA1R-Pr 245-221 GATAC A A ATGATAC AT TC CC A CC T 61 .0 210 P. ridibundus
SA1R-Pb 450-429 T TGT T CC C TATAC TA A GG TCAC 59. 3 415 P. cf. bedriagae
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males were analyzed by DNA flow cytometr y (see details in Biriuk
et al., 2016). Sperm suspension of 33 males of P. e scu len tu s, 2 P. ri-
dibundus, and 6 P. lessonae was analyzed by allozyme analysis (18
P. e sc u le n tu s were studied by both DNA flow cytometry and al-
lozyme analyses). The suspension (stored at –80°C) was used as a
tissue sample and vertical polyacrylamide (6%) gel electrophoresis
was performed using Tris-citric pH 8.0 buffer. The Ldh-A (lactate
dehydrogenase-1) locus with species-diagnostic polymorphism
was visualized by the standard technique (Shaw & Prasad, 1970).
The same method was used for the analysis of genome composi-
tion in oocytes of 11 P. esc ule ntu s, 9 P. ridibundus, and 54 P. le sso -
nae (for details, see Uzzell et al., 1980).
3 | RESULTS
3.1 | Distribution of species and population
systems
Among 1,337 individuals of water frogs determined by morphologi-
cal traits, 587 (44%) were P. ridibundus, 540 (40%) were P. lessonae,
and 210 (16%) were P. esc ule ntu s. We registered the pool frog in 37
localities (54%), the marsh frog in 47 localities (69%), and hybrids in
18 (27%) localities. Pelophylax lessonae was predominantly found in
ponds located in a fore st. The species inhabits forest biotopes more
often than others (41%; Table 3). Pelophylax ridibundus is usually ob-
served in open water bodies and rivers that flow down through for-
ests on agricultural land (68%). Hybrid frogs were registered in the
transition zone of these biotopes (Figure 1). We detected hybrids
co-occurring with P. lessonae individuals (L-E systems) and wit h both
pare nta l spe cie s (R-E-L syst ems). The L-E sys te ms we re cons ide rab ly
more com mo n in fore st (70%; Table 3) than the R-E-L sys te ms (25%).
Marsh frogs inhabited dams on rivers (27%), rivers (27%), ponds
(23%), lakes (7%), carriers (5%), and oxbows (4%). Among these bod-
ies of water, artificial and natural bodies of water accounted for 67%
and 33% , respectively. Both pool and edible frogs preferred artificial
water bodies (76 an d 68%, respect ively) an d ma inly occurred in ponds
(49% and 40%). Parental species were found to co-occur syntopically
(mixed R-L and R-E-L populations) in various types of water bodies.
Population systems with only one parental species (68%) were
most frequent. Pure P. ridibundus populations occurred in 44% (n = 30)
localities, and pure P. lessonae populations occurred in 22% (n = 15).
We found only 4 (6%) localities, including both parental species with-
out hybrids (mixed R-L systems). Population systems including hybrids
were obser ved in 19 localities (28%): L-E (n = 6; 9%) and R-E-L (n = 13;
19%). We did not find the E and R-E population systems.
3.2 | Genome size variation
After measuring the ploidy level by DNA flow cytometr y, we
found that all studied water frogs were diploids (Table S4). The
mean genome size (the nuclear DNA content, 2C) in P. ridibundus
(n = 125) was 16.22 ± 0.01 pg (15.89–16.52), P. esc ule ntu s (n = 176)
was 15.05 ± 0.01 pg (14.81–15.61), and P. lessonae (n = 167) was
13.82 ± 0.01 pg (13.62–14.10). The genome size values among spe-
cies did not overlap.
3.3 | Genetic structure
According to the multiplex PCR test system, all pool frogs (n = 10)
and most marsh frogs (53 out of 66 individuals) had species-specific
markers for both mtDNA and nDNA (L/LL and R/RR consequently).
Other marsh frogs had introgressive genot ypes, including mtDNA of
P. cf. bedriagae in the P. ridibundus nDNA background (B/RR—7 indi-
viduals) or had their own mtDNA but were heterozygous by nDNA
(R/RB—6 individuals). In a total sample of marsh frogs, mtDNA and
nDNA of P. cf. bedriagae had relatively small contributions (0.11 and
0.05%, respectively).
In edible frogs, we found five out of six possible combinations
of mtDNA and nDNA of parental species, except genotype L/BL
(Table 4). Most P. e scu len tus (74 ou t of 86) had ge not ype s L/R L or R/RL.
Others (n = 12) carried genes of P. cf. bedriagae (B/RL, R/BL, B/BL). In
R-E-L systems, mtDNA haplotypes of Anatolian and Central European
marsh frogs (R or B—79%) were prevalent, while in L-E systems, edible
frogs had predominantly mtDNA of the pool frog (L—79%).
The study of the nuclear SAI-1 marker showed the presence of
alleles of P. cf. bedriagae in six marsh frogs from Medvedevo only
(13% for the locality and 5% for the species in total). In P. esculen-
tus, alleles of P. cf. bedriagae were revealed in two individuals (2%
for the species) from Medvedevo (13%) and Chermyshevo (3%).
The mitochondrial DNA of P. cf. bedriagae was more frequent. We
found it in 11% individuals of the marsh frogs (four localities; Table 4;
Suppl. Table 5) and 11% edible frogs (three localities). Haplotypes of
P. lessonae wer e det ect ed in 47% ind ivi dua ls of P. es cul ent us (Table 4).
The result s of species id en ti fic at io n us in g both DNA fl ow cytom-
etry and multiplex PCR methods for the same individuals (n = 161)
coincided completely.
TAB LE 3 Percentage (mean ± standard deviation and range) of
forest vegetation and agricultural land in areas (1 km2) associated
with water frog population systems
Population
system nForest Agricultural
R30 11.7 ± 23.2 (0–82) 73.5 ± 35.3 (0–100)
R-L 427.0 ± 48.2 (0–99) 69.8 ± 46.8 (0–100)
R-E-L 13 25.4 ± 35.6 (0–100) 54.2 ± 41.4 (0–100)
L15 46.4 ± 41.5 (0–100) 46.9 ± 43.0 (0–100)
L-E 669.8 ± 32.9 (14–100) 17.8 ± 27.2 (0–66)
P. lessonae 38 40.9 ± 40.6 (0–100) 47.2 ± 41.8 (0–100)
P. esculentus 19 39.4 ± 39.9 (0–100) 46.7 ± 40.6 (0–100)
P. ridibundus 43 15.8 ± 27.8 (0–100) 67.7 ± 37.8 (0–100)
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The phylogenetic analysis based on the mitochondrial ND2 gene
fragment showed that the local P. ridibundus mtDNA was closely re-
lated to P. kurtmuelleri. The second cluster formed P. cf. bedriagae,
and P. lessonae was most distant (Figure 2). Sequencing of the ND2
gene of mtDNA in 28 individuals of the marsh frog and 29 individuals
of the edible frog revealed a low level of genetic diversity (Table 5).
Genetic variability was lacking in P. ridibundus (n = 21) and P. es cu-
lentus with mtDNA of the pool frog (n = 8). These frogs had one
of the most frequent Europe-specific haplotypes (Figure 2), which
were found in genetic pools of marsh and edible frogs across all their
ranges. Intermediate values of haplotype diversity, low frequency of
nucleotide diversity, and positive results of the Fs test (which showed
a deficit of haplotypes in investigated population systems) were ob-
served in marsh frogs with mtDNA of P. cf. bedriagae (n = 7) and
edible frogs with haplotypes of P. ridibundus (n = 16) and P. cf. bedria-
gae (n = 5). One individual of the edible frog from Chermyshevo had
genetic markers of three taxa. This frog had mtDNA of the Balkan
water frog (P. kurtmuelleri) and nuDNA alleles of P. cf. bedriagae and
P. lessonae.
3.4 | Gamete type variation in hybrids
Based on DNA flow cytometry data (Table 6), the proportion of
haploid sperm in a mixture of cells obtained from testes of P. es -
culentus was 37% on average (SD = 26%, range 0%–85%, n = 69),
P. ridibundus was 64% (SD = 28%, range 0%–94%, n = 20), and
P. lessonae was 87% (SD = 4%, range 77%–91%, n = 9). The major-
ity of studied hybrid males (from both the L-E and R-E-L popula-
tion types) and one hermaphrodite from Myamikeevo (from the
R-E-L population) produced sperm with the genome of P. ridibun-
dus (n = 52; 75%). Three hybrid males (4%) yielded sperm with the
P. lessonae genome, two hybrid males (3%) produced sperm with
intermediate (presumably non-clonal) genomes with the nuclear
DNA content average between parental species, and 11 males
(16%) did not have sperm (and other haploid cells). Among these
presumably sterile individuals, only four (36%) had normal-sized
testes. A hybrid male (1%) from Chermyshevo (the R-E-L popula-
tion) produced a mixture of sperm cells containing intermediate
and P. ridibundus geno me s (25% and 75% of ana lyz ed ha plo id ce lls ,
respectively).
Similar data were obtained after allozyme analysis. The majority
(n = 9; 60%) of hybrids produced sperm with the genome of P. ridib-
undus (Table 6). Four males of P. esc ule ntu s (27%) had gametes with
the P. lessonae genome. Spe rm of two hybrids (13%) from Nol'ka (the
L-E population) gave allozyme spectra characteristic of both parental
species.
In contrast to males, hybrid females produced different types
of gametes in the studied populations (Table 6). In the R-E-L sys-
tem (Chermyshevo), the majority of females (n = 7; 88%) produced
oocytes with a mixture of two parental genomes. Only one P. es cu-
lentus female (13%) gave gametes with the P. lessonae genome. In
the L-E system (Kuguvan), all three studied hybrid females produced
gametes with the P. ridibundus genome.
TAB LE 4 Genetic features of the edible frog Pelophylax esculentus according to the multiplex PCR data
Population
system
Number of
populations n
COI mtDNA
L R B
SA I −1 nDNA
RL RL BL RL BL
R-E-L 748 10 26 110 1
L-E 438 30 8 — — —
Tot al 11 86 40 (47%) 34 (39%) 1 (1%) 10 (12%) 1 (1%)
Note: B, mtDNA haplotypes and nDNA alleles of P. cf. bedriagae; L, P. lessonae; R, P. ridibundus.
TAB LE 5 Genetic diversity in marsh and edible frogs from northeastern European Russia
Types of mtDNA n H h ± SD π ± SD S/ηK
Marsh frogs
ridibundus 21 1— — — —
cf. bedriagae 7 3 0.524 ± 0.209 0.0019 ± 0.0 009 7/7 2.0 0
Edible frogs
ridibundus 16 40.350 ± 0.148 0.0016 ± 0.0011 13/13 1.62
cf. bedriagae 530.700 ± 0.218 0.0021 ± 0.0 00 8 5/5 2.20
lessonae 81— — — —
Note: h, haplotype diver sity; H, number of haplotypes; K, mean number of nucleotide substitutions; n, sample size; S, total number of polymorphic
positions; η, total number of mutations; π, nucleotide diver sity (per site).
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3.5 | Variation of morphological characters
The means of all 11 morphological indices for P. esc ule ntu s were inter-
mediate between parental species, but the limits overlapped (Table 7).
Two-way ANOVA showed the absence of sexual dimorphism for
all studied indices. However, significant species-specific differences
were revealed for all indices (two-way ANOVA; p < .001). The post hoc
comparison of all indices showed that differences between parental
species were significant (p < .001). After comparison of hybrids with
parental species, we detected four insignificant differences (L./Lt.c.,
L./F., L./C.s., and L./D.p.) between P. ridibundus and P. e scu le ntu s, as well
as two indices (L./F. and L./C.s.) between P. lessonae and P. esculentus.
A comparison of variance for indices L./C.int.l., D.p./C.int.l.,
T./C.int.l., Hem, and Tar showed significant differences (p < .001).
A comparison of variance for all three species using Levene and
Brown–Forsythe tests showed significant dif ferences (p < .001) for
five indices: L./C.int.l., D.p./C.int.l., T./C.int.l., Hem, and Tar. The post
hoc pairwise comparison of variances by the Fisher test (with the
Bonferroni correction for p-value) revealed the following differences
between variances. Males and females of P. esculentus differed from
females of P. ridibundus. The hybrids also had equal variances with
both males and females of P. lessonae. Sexual dimorphism in body
size (pairwise comparison by Fisher's test with the Bonferroni cor-
rection) for all three species was not revealed.
FIGURE 2 Phylogenetic tree inferred by ML analysis (a) and haplotype network (b) using a fragment of the ND2 gene sequences for 57
individuals from Mari El Republic (shown in black borders in phylogenetic tree and designated as symbol RU* in haplotype networks) and
130 individuals from various European populations represented in GenBank. Bootstrap values over 70% are shown next to branches. The
out group is not shown. Countries are shown as two-letter codes (ISOs)
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The result s of the PCA demonstrated good separation of the three
species by the first principal component (PC1), which explains 53% of
the total variance (Figure 3, Table 8). The first axis (PC1) had high load-
ings for five indices: L ./C.int.l., T./C.int.l., D.p./C.int.l., Hem, and Tar. All
these characteristics are connected with the length of the inner meta-
tarsal tubercle. The second component (PC2) explained 26% of the
total variance.
We calculated Mahalanobis distances between species and
found that P. e scu len tus was more similar to P. ridibundus than to
P. lessonae for mo s t sim p le in dices : L. /L t .c., L. / T., L ./C .s. , L. /D. p., an d
F./ T. ( Tab le 9). All thes e ind ice s are not co nne c ted with the len gth of
the inner metatarsal tubercle. However, if indices connected with
callus internus length (L./C.int.l., D.p./C.int.l., T./C.int.l.), P. es cul e n-
tus proved to be more similar to P. lessonae than to P. ridibundus.
These results were similar in both males and females ( Table 9).
4 | DISCUSSION
After a 12-year study, we summarized data on the distributions of
water frog species and their population systems in the eastern parts
of their ranges, genetic st ruc ture, types of gametes produced by hy-
brids, and peculiarities of morphology. The concordance between
the molecular structure of P. ridibundus and the hybridogenous P. es-
culentus was tested, as well as the morphological peculiarities of
frogs from different population system types and genotypes in the
studied territory.
4.1 | Morphological peculiarities
For a long time, zoologists were searching for diagnostic mor-
phological traits for reliable identification of water frog species.
Significant differences between parental species by means of indi-
ces selec ted in the current work were revealed in numerous stud-
ies (Berger, 1966; Borisovsky et al., 2000; Gubanyi & Korsos, 1992;
Günther, 1975; Lad a, 2012; L ad a et al. , 1995; Ne kr as ova & Mo rosov-
Leonov, 2001; Okulova et al., 1997; Peskov et al., 2009; Plötner
et al., 1994; Ruchin et al., 20 05). The characteristics and indices for
the hybridogenetic P. e scu len tus were intermediate between paren-
ta l spe ci es. However, th e lim it s of the se va lue s in hybr ids ove rl app ed
with those of parental species (Berger, 1966; Borisovsk y et al., 2001;
TAB LE 6 Gamete types produced by water frogs from studied population systems
Gamete t ype Pelophylax ridibundus Pelophylax lessonae
Pelophylax esculentus
L-E R-E-L
Males
DNA flow cytometry
R20 (1.00)a — 34 (0.87) 17 (0.59)
L — 9 (1.00) 1 (0.03) 2 (0.07)
E (intermediate) — — 1 (0.03) 1 (0.03)
R + E — — — 1 (0.03)
Sterile — — 3 (0.07) 8 (0.28)
Tot al 20 939 29
LDH electrophoresis
R2 (1.00) — 9 (0.64) —
L — 6 (1.00) 3 (0.21) 1 (1.00)
mixture (R + L)b — — 2 (0.14) —
Tot al 2 6 14 1
Females
LDH electrophoresis
R9 (1.00) — 3 (1.00) —
L — 54 (1.00) — 1 (0.13)
Ec — — — 7 (0.87)
Tot al 954 38
aNumber of individuals (share of the total in the group).
bMixture of haploid spermatozoa bearing Pelophylax ridibundus chromosomes and haploid spermatozoa bearing P. lessonae chromosomes.
cThe Ldh-A spectra of both parental species; according to Dedukh et al. (2019), these oocy tes cont ained chromosomes of both parent al species
(conditionally named here as the E type), sometimes, a portion of oocytes from one female contained the mixture of haploid E- and R-oocytes or
haploid E- and diploid RL-oocytes.
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Gubanyi & Korsos, 1992; Lada et al., 1995; Ruchin et al., 2005; and
others). According to the Mahalanobis squared distances calcu-
lated by us, P. es cu l entus was more similar to P. ridibundus than to
P. lessonae. In previous papers (Borisovsky et al., 20 00; Nekrasova
& Morosov-Leonov, 2001; Reminnyi & Matviychuk, 2018;
Tarashchuk, 1985, 1989), the multiplicative index Tar was considered
to be the most reliable for species identification in the P. e sc u le n-
tus complex. We found that the limit values of the index in parental
TAB LE 7 Variability of body length and morphological indices in water frog species studied by DNA flow cytometry and/or molecular COI
and S A I-1 markers analysis
Indices Pelophylax ridibundus (n = 57) Pelophylax esculentus (n = 67 ) Pelophylax lessonae (n = 98)
L. 75.1 ± 1.36
10.26
59.0–100.0 73.9 ± 1.05
8.60
58.0–95. 5 61.6 ± 0.74
7.35
48.0–79.0
L./Lt.c. 2.6 0 ± 0.02
0.13
2.36–2.95 2.64 ± 0.01
0.12
2.42–2.90 2.76 ± 0.01
0.13
2. 52–3.1 5
L./ F. 2.00 ± 0.01
0.10
1. 8 1–2.32 2.06 ± 0.01
0.09
1. 8 7–2 . 27 2.10 ± 0.01
0.10
1. 8 9–2 . 36
L. / T. 1.89 ± 0.01
0.07
1. 74–2. 0 2 2.03 ± 0.01
0.08
1. 8 8–2. 3 3 2.19 ± 0.01
0.10
1. 97–2 . 4 8
L./C.s. 3.59 ± 0.02
0.17
3.16–4.0 0 3.64 ± 0.02
0.16
3.35–4.10 3 .76 ± 0.02
0.21
3.28–4.28
L./D.p. 7.08 ± 0.07
0.54
5.78–8.40 7.22 ± 0.06
0.47
6. 42–8. 33 8.24 ± 0.07
0.70
6.78–10.50
L./C.int.l. 18.19 ± 0 .26
1.97
13.28–23.75 15.00 ± 0.13
1.06
13 . 2 0 –17. 6 9 13.09 ± 0.08
0.78
11.25–15.00
F./ T. 0.95 ± 0.01
0.04
0.84–1.02 0.99 ± 0.01
0.04
0.9 1–1 . 10 1.05 ± 0.00
0.04
0.89–1 . 1 5
D.p./C.int.l. 2.58 ± 0.04
0.33
1.96–3. 31 2.08 ± 0.02
0.16
1. 6 8–2 . 4 0 1.60 ± 0.01
0.12
1.28–1.82
T./C.int.l. 9.61 ± 0.13
1.01
7.6 0 –1 2 .75 7.40 ± 0.06
0.46
6.60–8.68 5.99 ± 0.04
0.38
5.11– 6.91
Hem 12.20 ± 0.17
1.29
9. 6 0–1 6 . 0 0 9.48 ± 0.07
0.57
8.48–11 . 0 3 7.58 ± 0.05
0.45
6.38–8.69
Tar 47.60 ± 1.38
10.43
27. 5 0 –7 7. 5 0 27.80 ± 0.41
3.40
21.40–37.70 16.50 ± 0.20
1.97
11 . 2 0 –19. 6 0
Note: n is number of specimens. Firs t column for species: numerator, mean value ± standard error of mean, denominator, standard deviation; second
column: limits. Italicized values are significant (p < 0.0 01).
FIGURE 3 Principal component analysis results: scatterplot for three water frog species in the space of the first and second principal
components (PC1 and PC2; morphological indices) and contributions of indices to the principal components (gradient colors of vectors)
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species and P. e scu len tus overlapped slightly. Perhaps, high variabil-
ity of marsh frogs in the region is caused by the presence of alleles
of two genetically distant species (P. cf. bedriagae and P. ridibundus).
The combination of indices together with some external morpho-
logical features (coloration of vocal sacks, inner metatarsal tubercle
shape, and relative hind limb length) enables the reliable identifica-
tion of water frog species. Correct diagnostics were made in 99%
of cases: only four individuals of 460 P. esc ule ntu s were determined
incorrectly, while parental species were always correctly identified.
Sexual dimorphism by morphometric characteristics in water
frogs has been discussed in many studies. However, the dif-
ferences between males and females can vary geographically
(Aleksandrovskaya, 1981; Caune, 1987; Lada, 2012). For example,
significant sexual differences in morphological traits were found for
P. ridibundus (and P. cf. bedriagae) in Kazakhstan, Armenia, Ukraine,
and Russia (Aleksandrovskaya, 1981; Lada, 2012; Nekrasova
& Morozov-Leonov, 2001; Terentjev, 1943), for P. esculentus in
France, Latvia, Ukraine (Caune, 1987; Lada, 2012; Mikitinez &
Suryadna, 2007; Regnier & Neveu, 1986), and, finally, for P. lessonae
was registered more often than in previous two species and ob-
served in France, Poland, Latvia, Hungary, Ukraine, and Russia
(Berger, 1966; Caune, 1987; Gubanyi & Korsos, 1992; Lada, 2012;
Okulova et al., 1997; Regnier & Neveu, 1986). In the northeastern
part of European Russia, we did not reveal sexual dimorphism in
all indices for the three species. However, the dimorphism would
be expressed by a separate comparison of females and males of
P. lessonae. The Mann–Whitney test showed differences in six in-
dices. In contrast, multiple comparisons using two-way ANOVA
showed that these dif ferences become insignificant.
We did not find any significant dif ference between P. es cul en-
tus samples from two various types of population systems: the L-E
(Kuguvan) and R-E-L (Chermyshevo) systems. No differences in body
proportions were registered between P. lessonae samples from these
two population systems, as well. Importantly, these localities are
separated both by a relatively large distance (100 km) and by the
Volga River, which forms a physical barrier. Moreover, both popu-
lations exist in different environmental conditions (small ponds in
the forest in Kuguvan and large open artificially impounded water
bodies in Chermyshevo). Genetically different variants (with the
presence of alleles and haplotypes of P. cf. bedriagae) of hybrids from
these populations, as well as hybrids producing dif ferent types of
gametes, also demonstrated no differences in body proportions.
4.2 | Distribution of population systems, molecular
structure, and gamete types
The distribution of water frog species, types of population sys-
tems, and hybrid gametogenesis in the studied region are highly
similar to adjacent territories in the East European Plain, namely,
in Nizhegorodskaya Oblast (= Nizhny Novgorod Province) and
the republics of Udmurtia, Chuvashia, and Mordovia (Borisovsky
et al., 2001; Borkin et al., 2002; Ruchin et al., 20 05, 2010). This find-
ing indicates the common features of water frogs in the studied re-
gion, making populations from the Volga River drainage distinct from
hybrids from other regions of Europe.
First, we and other researchers (Borkin et al., 2003) noted
that hybrids in the Volga River drainage are not as numerous as
in Western and Central European populations. In the studied lo-
calities, hybrids were found in 26% of the analyzed populations
(Table S1), but compared to parental species, they were notably
rare and observed only in 16% of all water frog individuals. A lower
occurrence of P. es cul e nt us was previously observed in adjacent
territories. For example, in the Udmur tia and Mordovia republics,
as well as Ivanovo and Nizhegorodskaya Oblasts, hybrids were
TAB LE 8 Factor loadings of analyzed morphometric indices
on the first two principal components for water frog species
(Pelophylax ridibundus, Pelophylax esculentus and Pelophylax lessonae)
Index PC1 PC2
L./Lt.c. − 0.295 0.662
L./ F. −0.084 0.739
L. / T. −0. 626 0.701
L./C.s. −0.178 0.823
L./D.p. −0.4 87 0.646
L./C.int.l. 0.967 −0.033
F./ T. −0.729 0. 242
D.p./C.int.l. 0.930 −0. 294
T./C.int.l. 0.953 − 0. 244
Hem 0.957 −0.257
Tar 0.959 −0.226
Expl.Var 5.830 2.894
Prp.Total 0.530 0.263
Note: Value loadings with strong impact on the principal component s
are shown in bold.
TAB LE 9 Squared Mahalanobis distances between three species
of water frogs. R—Pelophylax ridibundus, E—Pelophylax esculentus,
and L—Pelophylax lessonae
Indices
Males (n = 123) Females (n = 99)
E-R E-L R-L E-R E-L R-L
L./Lt.c. 0.13 0.88 1.68 0.06 0.88 1.41
L./ F. 0.48 0.01 0.62 0.29 0.23 1.0 4
L. / T. 3.56 3.12 13.34 2.22 3.06 10.48
L./C.s. 0.14 0.23 0.73 0.03 0 .47 0.73
L./D.p. 0.07 4.00 4.65 0.05 2.58 3.35
L./C.int.l. 6.01 2.05 15.08 6.35 3.30 18.80
F./ T. 0.72 1.94 5.02 1.73 2 .93 9.17
D.p./C.int.l. 6.00 5.57 23.13 5.78 6.65 24. 84
T./C.int.l. 11.98 4.17 30 .27 13 .76 6.95 40.27
Hem 11. 61 5.01 31.89 12.68 7. 67 40.08
Tar 11.50 3.60 27. 9 7 12.68 4. 52 32.33
Note: Lowest values are shown in bold.
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found in 5%–9% of the studied populations (Borkin et al., 2002;
Ruchin et al., 2005). Such a low occurrence of hybrids in local pop-
ulations could be explained by the load of ineffective reproduc-
tion of hybrids that have numerous gametogenesis disorders and
a high proportion of sterile hybrids, while hybridogenetic hybrid
frequency is low (Dedukh et al., 2019; Litvinchuk et al., 2016). This
finding may be attributable to introgression of P. cf. bedriagae al-
leles into the genetic pool of P. ridibundus (Fayzulin et al., 2018;
Svinin et al., 2016). However, it should be noted that the number
of hybrid males lacking or strongly reduced (<3 mm in length) tes-
tes in the studied region (18%; Table S7) was obviously lower in
diploids from most populations inhabiting Eastern Europe (57%;
Litvinchuk, 2018). Therefore, most likely, the reduced reproduc-
tive ability of local hybrids is due to decreased fer tilit y of hybrid
females.
All observed hybrids were diploids, and no triploids were re-
vealed. Earlier triploids were also not found in adjacent regions
(Borkin et al., 2002, 2003). The nearest triploids were observed
approximately 1,000 km from this location to the south in the
Severskiy Donets River drainage in eastern Ukraine and adjacent
Rostov Oblast of Russia (Biriuk et al., 2016; Borkin et al., 2004, 2006;
Dedukh et al., 2017, 2015). However, in studied by us localities, the
possibility of triploid emergence is high, as hybrid females are able
to produce diploid gametes (Dedukh et al., 2019). According to ear-
lier results, the formation of diploid eggs produced by diploid hybrid
females often leads to the emergence of triploids in the majority of
Central European systems (Berger, 2008; Plötner, 2005). However,
the abs en ce of triploids in na tura l po pu lations sug ge sts their mor tal-
ity in initial stages of development or undiscovered mechanisms of
post- or prezygotic elimination.
According to our analysis, we detected five t ypes of population
systems in the studied territory. In particular, we observed popu-
lation systems, including parental species only (R and L systems), a
mixture of parental species without hybrids (R-L systems), two pa-
rental species with hybrids (R-L-E systems), and P. lessonae individu-
als co-occurring with P. es cul ent us (L-E ). We did no t det ect pu re E an d
R-E population systems.
In the L-E and R-E-L systems, hybrids and parental individuals
were represented by both sexes, which were usually presented in
approximately the same proportion. Nevertheless, in some popula-
tions of P. esculentus (for example, the L-E system in Kuguvan), the
number of males (80%) strongly exceeded the number of females.
The prevalence of the R-E-L type among mixed population sys-
tems in the studied region could be explained by the syntopical oc-
currence of two parental species in the same water bodies formed
after deforestation of surrounding territories. Migration of a paren-
tal species into the preferable biotopes of others can also lead to
their hybridization. Interestingly, we found the dominance of sterile
hybrid females in R-E-L systems in comparison with L-E, which al-
lows us to suggest that the primary hybrid progeny (with disturbed
gametogenesis) predominantly present in R-E-L systems (Dedukh
et al., 2019; present paper). In L-E systems, hybrids are represented
by clonal lineages, mostly producing gametes with the P. ridibundus
genome. The primary hybrids from the nearest R-E-L systems origi-
nally migrated to such L-E populations.
There are two hypotheses about the origin of local hybridoge-
netic P. esc ule ntu s in the studied territory. The first hypothesis sug-
gests that the hybrids can emerge “de novo” after crosses of parental
species. Alternatively, hybridogenetic hybrids could obtain this region
from glacial refugia and colonize the Volga River drainage together
with parental species. The sporadic appearance of alleles and haplo-
types of P. cf. bedriagae in the gene pool of P. ridibundus, and co-oc-
cur ri ng hybrid is a goo d ma rker in dicating the “de novo” origin of local
P. e sc u le n tu s. However, the presence of P. kurtmuelleri haplotypes in
a local hybrid can suppor t the hypothesis about the migration of old
hemiclonal lineages from more southwestern glacial refugia.
The effect of P. cf. bedriagae genome on selective genome elimi-
nation in P. e scu len tus has not been determined; however, our study
showed that hybrids with alleles of P. cf. bedriagae can be found.
Perhaps hybrids between P. lessonae and pure P. cf. bedriagae are
sterile. In our study, we analyzed individuals of P. ridibundus and
P. e sc u le n tu s with an undetermined frequency of P. cf. bedriagae
alleles. Crosses between some other species of water frogs with
P. ridibundus (P. shqipericus × P. ridibundus and P. epeiroticus × P. ridib-
undus) led to progeny unable to reproduce via hybridogenesis (Hotz
et al., 1985).
In the study area, we revealed that the majorit y of individuals
produced gametes with the P. ridibundus genome. Such gametes are
widespread for hybrid frogs from L-E systems in Central Europe, pro-
viding reproduction and maintenance of such systems (Ragghianti
et al., 2007). However, we found that some P. e scu l en tus produced
gametes with the P. lessonae genome. Such hybrids are common for
southern parts of Eastern Europe populated by R-E systems (Biryuk
et al., 2016; Dedukh et al., 2015, 2017; Doležálková-Kaštánková
et al., 2018). However, the prevalence of hybrids producing gametes
with the P. ridibundus genome cannot contribute to continued main te-
nance of R-E systems in Volga River drainage. Hybrids from both the
L-E and R-E-L systems were able to produce not only clonal gametes
with one of the parent al genomes but also gametes with intermediate
betw een parental species the nuclea r DN A co nten t (p re sumab ly, non -
clonal), as well as a mixture of these and clonal gametes.
Molecular studies helped us to disclose the role of species in the
functioning of population systems. Previously, we failed to obtain
progeny from hybrid females (Dedukh et al., 2019), but the pres-
ence of P. ridibundus mtDNA in hybrid females from L-E systems
(Kuguvan; 10-year study) indicates their previous participation in the
reproduction of hybrids. Widespread unidirectional mitochondrial
DNA transfer from P. lessonae to P. ridibundus through hybrids was
found in Western and Central Europe (Plötner et al., 2008). Although
the transfer from P. cf. bedriagae to P. lessonae was recently found in
a water frog population from the Volga River drainage in Mordovia
Republic (Ivanov et al., 2019), this transfer is still a highly rare phe-
nomenon. The lack of introgression from P. lessonae to P. ridibundus
can be explained by the fact that local hybrid males and females
rarely interbreed and/or that progeny of such interhybrid crosses is
unviable.
|
13
SVININ e t al.
4.3 | Conservation status of P. esculentus
populations
In some regions of the Volga River drainage (Samara, Tambov, and
Kursk Oblasts, as well as republics of Udmurtia and Mordovia), the
edible frog is under protection (see review in Lada, 2012). However,
the conser vation status of this frog depends on understanding the
taxonomic status of P. e s cu l entus . If edible frogs are hybrids formed
“de novo,” th eir conservation stat us shou ld be rev iewed becaus e hy-
brids can permanently emerge as a result of human activity (defor-
estation and creation of artificial water bodies). On the other hand,
if hybrids represent long-aged hemiclonal lineages that colonize new
territories from glacial refugia, protection of such populations is
necessary.
Regardless, studies of population systems from the northeastern
part of the P. e scu lentu s comp lex distribution seem to be of consider-
able interest as a model for the study of the first steps of reticulate
speciation and analysis of the effect of P. cf. bedriagae gene intro-
gression on hemiclonal reproduction of P. e scule ntus .
ACKNOWLEDGEMENTS
We are thankful to A.I. Fayzulin and I.V. Chikhlyaev (Tolyatti, Russia)
for the data about Chodrayal settlement. This work was sup-
ported by grants of the Russian Science Foundation Nos. 18-74-
00115 (DDV), 18-04-00640 (OAE, AOS, AYI), and 20-04-00918
(SNL, DVS, JMR). The work was fulfilled under the Laboratory
Project of Zoological Institute of Russian Academy of Sciences No.
AAA A-A19-119020590095-9 (LJB).
DATA AVAIL AB ILI T Y STAT E MEN T
Data available in article supplemenatry material.
ORCID
Anton O. Svinin https://orcid.org/0000-0002-8400-6826
Dmitrij V. Dedukh https://orcid.org/0000-0002-1152-813X
Oleg A. Ermakov https://orcid.org/0000-0003-1486-6799
Renat I. Zamaletdinov https://orcid.org/0000-0001-9153-7820
Aleksey B. Trubyanov https://orcid.org/0000-0002-5432-7231
Dmitriy V. Skorinov https://orcid.org/0000-0002-9916-2098
Spartak N. Litvinchuk https://orcid.org/0000-0001-7447-6691
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
Table S1. Studied localities and sample size of water frogs.
Table S2. Types of biotopes and population systems of water frogs.
Table S3. Morphological traits of studied individuals.
Table S4. Genome size and ploidy level of studied individuals.
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Table S5. Results of genetic (COI and S A I -1) identification of water
frogs from various population systems.
Table S6. GenBank numbers of sequences of the mitochondrial ND2
gene in studied by us water frog specimens (a) and published previ-
ously (b).
Table S7. Types of gametes produced by parental species and hy-
brids: A. Males. B. Females.
Table S8. List of studied individuals of water frogs.
How to cite this article: Svinin AO, Dedukh DV, Borkin LJ, et
al. Genetic structure, morphological variation, and
gametogenic peculiarities in water frogs (Pelophylax) from
northeastern European Russia. J Zool Syst Evol Res.
2021;00:1–17. https://doi.o rg /10.1111/jzs .12447
