Biologia

https://doi.org/10.1007/s11756-022-01256-8

ORIGINAL ARTICLE

Differences in release calls of the hybrid water frog Pelophylax

esculentus and its parental species Pelophylax ridibundus (Anura:
Ranidae) in Ukraine
Anna Fedorova1   · Dmytro Shabanov1

Received: 20 January 2022 / Accepted: 24 October 2022

© The Author(s), under exclusive licence to Plant Science and Biodiversity Centre, Slovak Academy of Sciences (SAS), Institute of Zoology, Slovak Acad-
emy of Sciences (SAS), Institute of Molecular Biology, Slovak Academy of Sciences (SAS) 2022

Abstract
Vocalization in anurans is mainly studied in the context of male-specific advertisement calls, while release calls – charac-
teristic of both sexes – usually attract less attention. Here we compare release calls of both sexes of hybrid Pelophylax escu-
lentus (considering triploid hybrids) and its parental species P. ridibundus in Ukrainian population systems. The principal
component analysis resulted in 83.3% of total variance explained by the first three components, while discriminant analysis
correctly differentiated 69.6% of frogs only. Triploid male hybrids had intermediate positions between diploid hybrids
and parental species. Triploid females had intermediate positions only in terms of temporal parameters because frequency
parameters mainly depended on their body size. A significant correlation between peak frequency and body size was shown
for females of both taxonomical groups and P. ridibundus males. Although we identified differences between the studied
groups, these differences were not unambiguous, with overlapping frequency parameters in males. Including more triploids
and the other parental species is important for further analysis.

Keywords  Release calls · Bioacoustics · Pelophylax · Vocalization

Introduction forms are also found, possessing either one P. ridibundus

Anurans generally have species-specific vocalizations, which
is especially interesting when studying hybrids. Pelophy-
lax esculentus (Linnaeus, 1758), the edible frog, is a hybrid
water frog usually living together with one or both parental
species – P. ridibundus (Pallas, 1771), the marsh frog, and
P. lessonae (Camerano, 1882), the pool frog. Hybrids can
crossbreed with one of the parental species (depending on
which parental species they live in syntopy with) or within
themselves. Reproduction of hybrids occurs by hybridogen-
esis: during gametogenesis, they eliminate one of the paren-
tal genomes (chromosomal sets), reduplicate the other, and
transmit it clonally, without recombination. In many popu-
lation systems, except typical diploid hybrids (possessing
one genome from each parental species), triploids of two
* Anna Fedorova
anna.fedorova@karazin.ua
1 the first to include females in release call analyses; however,
Department of Zoology and Animal Ecology, V. N. Karazin
Kharkiv National University, Kharkiv, Ukraine
and two P. lessonae genomes, or two P. ridibundus and one
P. lessonae genomes (Graf and Polls Pelaz 1989). Mating
calls of both di- and triploid hybrids as well as their parental
species were well studied and showed a genomic dosage
effect (Hoffmann and Reyer 2013), which may be useful for
taxonomical identification. However, the main limitation of
mating calls is their sex-specificity, which neglects all the
data concerning female individuals.
Release calls, on the other hand, are characteristic of both
sexes and were also shown to be a useful taxonomic trait
(Grenat and Martino 2013). Release calls of the Pelophylax
hybrid frogs and their parental species are studied less than
advertisement calls. Wahl (1969) followed by Weber (1976)
described the release calls of P. esculentus based on the calls
of several male frogs only. Weber (1976) also described
release calls of the parental species P. ridibundus, but also
only for males. Furthermore, several studies were performed
on male P. esculentus compared to both their parental spe-
cies (Zamfirescu 2000, 2001, 2002). Chernyshov (2005) was
he worked only with parental species.

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 Biologia
Here we compare the release calls of the hybrid P. escu-
lentus with its parental species P. ridibundus of both sexes
in a Ukrainian population to find specific traits that can help
distinguish between hybrids and their parental species. It was
shown that advertisement call characteristics of Pelophylax
frogs could depend on geography and types of population
system (Wycherley et al. 2002; Hoffmann and Reyer 2013).
Since our research was exclusively carried out in the Siver-
skyi Donets River basin (Eastern Ukraine, Kharkiv region),
where the other parental species P. lessonae is absent (Biriuk
et al. 2016), we did not include P. lessonae in our analysis.
Materials and methods
Sample collection
Frogs were collected at four localities in the National Park
“Homilshanski lisy” in Ukraine (Table 1). Frogs were col-
lected from 2014 to 2021 during the breeding season (spring
and summer). Taxonomical and sex identification was car-
ried out by examining external morphology (Atemasova
et al. 2019). We examined the shape of the metatarsal tuber-
cule, coloration of inguinal region, underside of thighs and
vocal sacs, and spotting pattern. While taxonomical identifi-
cation is relatively easy for diploid frogs, it could be errone-
ous for triploid hybrids. Therefore, we also identified ploidy
for all the frogs, even those identified as parental species.
For sex identification, we examined the presence of vocal
sacs and nuptial pads.
Frogs were fed and kept in 20–50 L plastic tanks for
1–2 days. Recordings were performed under laboratory con-
ditions at ~ 25 °C. Calls were recorded with Nikon Coolpix
P500 and Nikon D3100 cameras placed at a distance of
20 cm from frogs. Release calls were stimulated by gently
pressing the frog flanks to simulate amplexus. Since it was
shown that call traits could depend on the body size (e.g.,
Davies and Halliday 1978; Gingras et al. 2013), ninety-one
frogs were measured for snout-vent length (SVL) with a
vernier caliper.
Table 1  Frogs collected and coordinates of the studied localities
Geographical coordinates P. esculentus
male, 2n
Siversky Donets River 49°37′23ʺN; 36°19′53ʺE 26
Iskiv pond 49°37′40ʺN; 36°16′58ʺE 2 3
Lower Dobrytskiy pond 49°33′24ʺN; 36°18′35ʺE 28
Koriakiv pond 49°36′57ʺN; 36°18′45ʺE 17
All 79
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Ploidy identification
To identify ploidy, we measured erythrocyte lengths for
all studied frogs. We sampled blood either from the facial
vein (Forzán et al. 2012) or cut fingertips while marking
individuals for annual monitoring and studying population
size. We prepared an air-dried blood smear for each frog
and measured lengths of at least 30 erythrocytes under × 40
magnification, either with a Carl Zeiss Amplival or Leica
DM 2000 microscope. Bondarieva et al. (2012) identified
the margin between diploid and triploid erythrocytes of
hybrids in the Siversky Donets River as 26 µm. However,
our further studies showed that this margin differs between
population systems of this river basin, and attention should
be paid to the “gap” in cell lengths measured for all the
individuals in a sample (Drohvalenko et al. 2019).
Some frogs had erythrocyte lengths close to the mar-
gin or “gap”. For those individuals, we performed kar-
yoanalysis. Frogs were injected with 0.05% colchicine
solution for 12–18 h and then sacrificed with vapors
of ETAC or injection of 2% lidocaine. Further, we dis-
sected intestine tissue and bone marrow, put it into a
hypotonic solution of 0.07 M KCl for 30 min, centri-
fuged at 3000 rpm, and replaced the hypotonic solution
with Carnoy’s fixative (3 parts of ethanol or methanol
and 1 part of glacial acetic acid). Small pieces (roughly
5 ­m m 2) of intestine tissue or bone marrow were homog-
enized in 100 µl of 70% acetic acid. The resulting cell
suspension was put on slides heated to 60° C and left to
dry. Slides were stained with A ­ gNO 3 following Birstein
(1984) with modifications and analyzed under × 100
magnification, either with a Carl Zeiss Amplival or
Leica DM 2000 microscope.
Pelophylax species used in this study are not listed in
the IUCN Red List of Threatened Species or Red Data
Book of Ukraine. Tissue collection was performed to mini-
mize animal harm according to the Guidelines for Use of
Live Amphibians and Reptiles in Field and Laboratory
Research of HACC and The Ukrainian Law on the Protec-
tion of Animals from Cruelty.
P. ridibundus All
fem, 2n male, 3n fem, 3n male fem
5 - - 44 16 91
- - - - 5
15 2 1 - 14 60
5 3 2 - - 27
31 5 3 44 29 183
Biologia
Acoustic analysis
Here we use a call-centered approach describing a call as a
“main coherent sound unit (longer than a typical pulse), sepa-
rated from other such units by a distinct period of silence”
(Köhler et al. 2017). Sound files were amplitude-normalized
and cleared from background noise with Audacity® v. 2.1.2
software (Audacity Team 2015). Acoustic analysis was car-
ried out with Raven Pro 1.6.1 (Bioacoustics Research Pro-
gram 2019; Cornell Lab of Ornithology). Call duration and
numbers of peaks in each call were measured on oscillograms.
Fig. 1  Oscillograms and spectrograms of release calls of Pelophy-
lax males (a) and females (b): Ef 2n – P. esculentus diploid
females, Em 2n – P. esculentus diploid males, Ef 3n – P. esculen-
For measuring spectral parameters, we performed FFT with
50% of window overlap and measured the following param-
eters: frequency range, min frequency, max frequency, and
peak (dominant) frequency. We analyzed four repeated calls
for each frog and calculated means for further statistical analy-
sis. Coefficients of variation (CV) were calculated according
to Gerhardt (1991). Parameters with less than 5% CV val-
ues are described as static, and those with CV above 10%
as dynamic. We used the Kruskal–Wallis test to compare
groups of frogs by acoustic parameters. Principal component
(PCA) and discriminant analysis were used for multivariate
tus triploid females, Em 3n – P. esculentus triploid males, Rf – P.
ridibundus females, Rm – P. ridibundus males
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 Biologia

Table 2  Results of the PCA: three principal components explaining

most of the variance between acoustic parameters of release calls
PC1 PC2 PC3 Total

Eigenvalue 2.9 1.8 1.05 5.8

% Total vari- 41.8 26.4 15.1 83.3
ance
Correlating Min frequency Peaks number Call duration
values Max fre- Peaks rate
quency
Frequency
range
Peak fre-
quency

comparisons between studied groups of frogs. Pearson cor-
relation was applied to see the connection between frequency
parameters and body size.
Fig. 2  Results of principal component analysis: Ef 2n – P. esculentus
diploid females, Em 2n – P. esculentus diploid males, Ef 3n – P. escu-
lentus triploid females, Em 3n – P. esculentus triploid males, Rf – P.
ridibundus females, Rm – P. ridibundus males

Results
We analyzed 732 release calls from 183 frogs, among which
110 were P. esculentus (79 males and 31 females), and 73
were P. ridibundus (44 males and 29 females). Among the
hybrids, eight frogs were identified as triploids. The general
structure of the release calls differed between the two taxo-
nomical groups. Pelophylax esculentus calls of both sexes
consisted of single or double peaks with approximately the
same period of silence between them (Fig. 1a, b). Such sepa-
rate peaks were not observed in P. ridibundus release calls.
In most P. ridibundus male calls, groups of peaks could be
distinguished, as well as in female calls. The number of peaks
was higher for P. esculentus females and lower for males
(Fig. 1). Similar observations were made for P. ridibun-
dus calls: the number of peaks was significantly higher for
females than males. Most P. esculentus male release calls
(71%) start with several peaks of low amplitude separated
from the main part of the call by a pause. Such low amplitude
peaks were not observed in P. esculentus female calls.
Principal component (PCA) analysis resulted in three
principal components with eigenvalues > 1.0 and 83.3% of
total variance explained (Table 2).
The discriminant analysis using all seven temporal and fre-
quency parameters resulted in two discriminant functions that
were significant (peak frequency and peak rates included into
the model). Only 69.6% of frogs were classified correctly, and
all triploid hybrids were wrongly classified (Table 3), which
corresponds to the results of the PCA (Fig. 2).
Higher values of peak numbers and peak rates were typi-
cal for P. ridibundus of both sexes, while triploid hybrids
had an intermediate position between diploid P. esculentus
and P. ridibundus (Fig. 3). Frequency parameters differed
only between females, with P. ridibundus having higher
values (Fig. 3). Post hoc analysis with the Kruskal–Wallis
test showed that call duration was the least variable between

Table 3  Results of discriminant analysis based on three temporal and four frequency parameters

Actual Predicted % correctly clas-

sified
Ef 2n Ef 3n Rf Em 2n Em 3n Rm

Ef 2n 15 0 2 6 0 5 53.57
Ef 3n 1 0 0 0 0 2 0.00
Rf 2 0 23 0 0 5 76.66
Em 2n 7 0 0 67 0 0 91.89
Em 3n 0 0 0 4 0 1 0.00
Rm 2 0 7 13 0 22 50.00

Bold face – correctly classified frogs, Ef2n – P. esculentus diploid females, Em2n – P. esculentus diploid males, Ef3n – P. esculentus triploid
females, Em3n – P. esculentus triploid males, Rf – P. ridibundus females, Rm – P. ridibundus males

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Biologia

Fig. 3  Means of the three temporal and four frequency parameters: Ef2n – P. esculentus diploid females, Em2n – P. esculentus diploid males,
Ef3n – P. esculentus triploid females, Em3n – P. esculentus triploid males, Rf – P. ridibundus females, Rm – P. ridibundus males

studied groups (Fig. 3). The most variable parameters were
peak numbers and peak rates (Fig. 3).
Call durations in males were lower in our study than
those reported by Zamfirescu (2002): 0.42 ± 0.071 vs.
0.48 ± 0.119 in P. esculentus (p = 0.0154) and 0.34 ± 0.079
vs. 0.45 ± 0.051 in P. ridibundus (p < 0.0001).
All seven measured parameters were dynamic (evalu-
ated according to Gerhardt 1991) with CV values above
15% (Table 4). Only for triploid hybrids, peak frequencies
and minimum frequencies were evaluated as stable param-
eters, which might be caused by the low number of sam-
ples. In general, females tended to have higher coefficients
of variation, but the difference was not significant (KW-
H(5;42) = 1.7675; p = 0.8803).
A significant correlation between peak frequency and body
size was shown for females (Fig. 4a, b) of both taxonomical
groups and P. ridibundus males (Fig. 4d), but not for P. escu-
lentus males (Fig. 4c). However, the significance level for P.
ridibundus was relatively low, which may be the consequence
of the low number of individuals included in the analysis. A high
correlation was also shown between body size and minimum fre-
quency (p < 0.001) in P. esculentus of both sexes and P. ridibun-
dus females, but not P. ridibundus males (p = 0.3854). No cor-
relation was found between body size and all other parameters.
Such correlation explains why triploid females differ drastically
from other forms by frequency parameters (Fig. 3): all three trip-
loid females had relatively small body sizes and, consequently,
rather high “peak frequency” and “min frequency” parameters.

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 Biologia

Table 4  Coefficients of variation (CV) of seven temporal and frequency parameters calculated for six groups of Pelophylax frogs
Call duration Peak frequency Frequency range Min frequency Max frequency Peaks number Peaks rate

Em 2n 16.92 24.64 27.66 18.25 20.31 32.75 31.40

Ef 2n 29.62 28.68 32.09 25.80 24.19 34.20 37.71
Em 3n 32.29 5.03 28.11 13.57 22.19 32.31 38.81
Ef 3n 38.96 2.35 31.07 4.01 19.53 61.80 47.39
Rm 22.97 21.73 26.83 15.44 17.10 40.16 44.06
Rf 27.91 24.22 45.98 20.80 31.93 37.09 22.14

Bold – CV ≤ 5%. CV = SD*100/mean. Ef 2n – P. esculentus diploid females, Em 2n – P. esculentus diploid males, Ef 3n – P. esculentus triploid
females, Em 3n – P. esculentus triploid males, Rf – P. ridibundus females, Rm – P. ridibundus males

Discussion Roberts 2010); moreover, a genome dosage effect was

observed in Pelophylax esculentus complex (Hoffmann
Hybrid anurans usually have intermediate call character- and Reyer 2013). Our analysis shows differences between
istics with respect to those of the parental forms (Ger- the studied groups in both temporal and frequency param-
hardt 1974; Gerhardt et al. 1980; Mable and Bogart 1991; eters. However, these differences are inconclusive. The

Fig. 4  Correlation between peak frequency and snout-vent length (SVL). a P. esculentus females (dot – diploid, circle – triploid), b P. ridibun-
dus females, c P. esculentus males (dot – diploid, circle – triploid), d P. ridibundus males

13
Biologia
analysis of triploid P. esculentus shows that they have
an intermediate position between P. ridibundus and dip-
loid P. esculentus. Still, they were incorrectly differenti-
ated by the discriminant analysis. The best classification
worked for P. esculentus males (90.5%) and P. ridibundus
females (75.9%). Unambiguous differences between the
release calls of P. ridibundus and P. esculentus should
not be expected since P. esculentus is a hybrid, and its
release call may overlap with the parental ones. However,
for mating calls, a strict difference between hybrids and
parental species was shown (Hoffmann and Reyer 2013).
For the release calls, we see similar tendency, but compar-
isons with the other parental species P. lessonae and more
triploid hybrids are needed. Some temporal parameters
for both parental species were measured by Chernyshov
(2005). Even though Chernyshov did not compare the two
species directly, disparities were observed, e.g., a differ-
ence in call durations of P. ridibundus and P. lessonae,
which also depended on frog size and water temperature.
Following Chernyshov, we expect to see a more evident
difference between the release calls of P. lessonae and P.
ridibundus in our further analysis. It is possible that the
temporal and frequency data we extracted are not sufficient
to distinguish between P. esculentus and P. ridibundus of
both sexes. We suggest that more sophisticated compari-
son methods, which will not be based on individual dimen-
sions but the comparison of patterns as a whole, should
be more effective.
There is also a possibility that release calls are less spe-
cies-specific than mating calls (Hoffmann and Reyer 2013).
Also, the function of the release call could be different for
different sexes. Males use the call as a direct release signal
to indicate their sex, so the difference between species might
not be very important. Such differences should arise from
selection against interspecific mating. But such selection was
ineffective in this group of frogs.
According to Gerhardt (1991) static parameters are more
taxonomically relevant than dynamic ones. Call duration and
peak rate were identified as static parameters for many dif-
ferent species (Gerhardt 1991; Castellano and Rosso 2006).
However, in our analysis, these parameters seemed to be
dynamic, i.e., intragroup variation was higher than inter-
group, which was also confirmed by a Kruskal–Wallis test
explaining the absence of differences between the studied
groups.
The significant correlation between body size and fre-
quency parameters corresponds with previous data for anu-
rans (e.g., Davies and Halliday 1978; Gingras et al. 2013).
Such a correlation explains the results of our post hoc analy-
sis (Fig. 3). Moreover, it explains why triploid females dif-
fered so extremely from diploid P. esculentus by frequency
parameters. All three triploid females had rather small body
sizes and, therefore, relatively high frequency parameters
(Fig. 4a). More triploid females with different body sizes are
needed for a comprehensive analysis. Our analysis also did
not include the other parental species – P. lessonae – because
it is absent in the studied territory (Siversky Donets River
basin), but it should be included in further studies.
Although this analysis did not reveal any characteristics
of release calls that can exclusively differentiate between
hybrid P. esculentus (both diploid and triploid) and one of
their parental species P. ridibundus, we showed general sex-
and species-specific trends in frogs’ calls that may become
a basis for further analysis.
Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11756-0​ 22-0​ 1256-8.
Acknowledgements  The authors thank the students at the School of
Biology (V. N. Karazin Kharkiv National University) who helped catch
and record frogs during the fieldwork. Anna Fedorova is very grate-
ful to Eleonora Pustovalova, whose support and interest in this work
helped to complete this manuscript. The authors are also sincerely
grateful to the Armed Forces of Ukraine, whose bravery and profes-
sionalism provided a chance to finish the work on this manuscript.
Declarations 
Conflict of interest  The authors declare that they have no conflict of
interest.
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