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

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

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Here we compare the release calls of the hybrid P. escu- Ploidy identification
lentus with its parental species P. ridibundus of both sexes
in a Ukrainian population to find specific traits that can help To identify ploidy, we measured erythrocyte lengths for
distinguish between hybrids and their parental species. It was all studied frogs. We sampled blood either from the facial
shown that advertisement call characteristics of Pelophylax vein (Forzán et al. 2012) or cut fingertips while marking
frogs could depend on geography and types of population individuals for annual monitoring and studying population
system (Wycherley et al. 2002; Hoffmann and Reyer 2013). size. We prepared an air-dried blood smear for each frog
Since our research was exclusively carried out in the Siver- and measured lengths of at least 30 erythrocytes under × 40
skyi Donets River basin (Eastern Ukraine, Kharkiv region), magnification, either with a Carl Zeiss Amplival or Leica
where the other parental species P. lessonae is absent (Biriuk DM 2000 microscope. Bondarieva et al. (2012) identified
et al. 2016), we did not include P. lessonae in our analysis. 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
Materials and methods population systems of this river basin, and attention should
be paid to the “gap” in cell lengths measured for all the
Sample collection individuals in a sample (Drohvalenko et al. 2019).
Some frogs had erythrocyte lengths close to the mar-
Frogs were collected at four localities in the National Park gin or “gap”. For those individuals, we performed kar-
“Homilshanski lisy” in Ukraine (Table 1). Frogs were col- yoanalysis. Frogs were injected with 0.05% colchicine
lected from 2014 to 2021 during the breeding season (spring solution for 12–18 h and then sacrificed with vapors
and summer). Taxonomical and sex identification was car- of ETAC or injection of 2% lidocaine. Further, we dis-
ried out by examining external morphology (Atemasova sected intestine tissue and bone marrow, put it into a
et al. 2019). We examined the shape of the metatarsal tuber- hypotonic solution of 0.07 M KCl for 30 min, centri-
cule, coloration of inguinal region, underside of thighs and fuged at 3000 rpm, and replaced the hypotonic solution
vocal sacs, and spotting pattern. While taxonomical identifi- with Carnoy’s fixative (3 parts of ethanol or methanol
cation is relatively easy for diploid frogs, it could be errone- and 1 part of glacial acetic acid). Small pieces (roughly
ous for triploid hybrids. Therefore, we also identified ploidy 5 ­m m 2) of intestine tissue or bone marrow were homog-
for all the frogs, even those identified as parental species. enized in 100 µl of 70% acetic acid. The resulting cell
For sex identification, we examined the presence of vocal suspension was put on slides heated to 60° C and left to
sacs and nuptial pads. dry. Slides were stained with A ­ gNO 3 following Birstein
Frogs were fed and kept in 20–50 L plastic tanks for (1984) with modifications and analyzed under × 100
1–2 days. Recordings were performed under laboratory con- magnification, either with a Carl Zeiss Amplival or
ditions at ~ 25 °C. Calls were recorded with Nikon Coolpix Leica DM 2000 microscope.
P500 and Nikon D3100 cameras placed at a distance of Pelophylax species used in this study are not listed in
20 cm from frogs. Release calls were stimulated by gently the IUCN Red List of Threatened Species or Red Data
pressing the frog flanks to simulate amplexus. Since it was Book of Ukraine. Tissue collection was performed to mini-
shown that call traits could depend on the body size (e.g., mize animal harm according to the Guidelines for Use of
Davies and Halliday 1978; Gingras et al. 2013), ninety-one Live Amphibians and Reptiles in Field and Laboratory
frogs were measured for snout-vent length (SVL) with a Research of HACC and The Ukrainian Law on the Protec-
vernier caliper. tion of Animals from Cruelty.

Table 1  Frogs collected and coordinates of the studied localities

Geographical coordinates P. esculentus P. ridibundus All
male, 2n fem, 2n male, 3n fem, 3n male fem

Siversky Donets River 49°37′23ʺN; 36°19′53ʺE 26 5 - - 44 16 91

Iskiv pond 49°37′40ʺN; 36°16′58ʺE 2 3 - - - - 5
Lower Dobrytskiy pond 49°33′24ʺN; 36°18′35ʺE 28 15 2 1 - 14 60
Koriakiv pond 49°36′57ʺN; 36°18′45ʺE 17 5 3 2 - - 27
All 79 31 5 3 44 29 183

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Acoustic analysis For measuring spectral parameters, we performed FFT with

50% of window overlap and measured the following param-
Here we use a call-centered approach describing a call as a eters: frequency range, min frequency, max frequency, and
“main coherent sound unit (longer than a typical pulse), sepa- peak (dominant) frequency. We analyzed four repeated calls
rated from other such units by a distinct period of silence” for each frog and calculated means for further statistical analy-
(Köhler et al. 2017). Sound files were amplitude-normalized sis. Coefficients of variation (CV) were calculated according
and cleared from background noise with Audacity® v. 2.1.2 to Gerhardt (1991). Parameters with less than 5% CV val-
software (Audacity Team 2015). Acoustic analysis was car- ues are described as static, and those with CV above 10%
ried out with Raven Pro 1.6.1 (Bioacoustics Research Pro- as dynamic. We used the Kruskal–Wallis test to compare
gram 2019; Cornell Lab of Ornithology). Call duration and groups of frogs by acoustic parameters. Principal component
numbers of peaks in each call were measured on oscillograms. (PCA) and discriminant analysis were used for multivariate

Fig. 1  Oscillograms and spectrograms of release calls of Pelophy- tus triploid females, Em 3n – P. esculentus triploid males, Rf – P.
lax males (a) and females (b): Ef 2n – P. esculentus diploid ridibundus females, Rm – P. ridibundus males
females, Em 2n – P. esculentus diploid males, Ef 3n – P. esculen-

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

Results from the main part of the call by a pause. Such low amplitude
peaks were not observed in P. esculentus female calls.
We analyzed 732 release calls from 183 frogs, among which Principal component (PCA) analysis resulted in three
110 were P. esculentus (79 males and 31 females), and 73 principal components with eigenvalues > 1.0 and 83.3% of
were P. ridibundus (44 males and 29 females). Among the total variance explained (Table 2).
hybrids, eight frogs were identified as triploids. The general The discriminant analysis using all seven temporal and fre-
structure of the release calls differed between the two taxo- quency parameters resulted in two discriminant functions that
nomical groups. Pelophylax esculentus calls of both sexes were significant (peak frequency and peak rates included into
consisted of single or double peaks with approximately the the model). Only 69.6% of frogs were classified correctly, and
same period of silence between them (Fig. 1a, b). Such sepa- all triploid hybrids were wrongly classified (Table 3), which
rate peaks were not observed in P. ridibundus release calls. corresponds to the results of the PCA (Fig. 2).
In most P. ridibundus male calls, groups of peaks could be Higher values of peak numbers and peak rates were typi-
distinguished, as well as in female calls. The number of peaks cal for P. ridibundus of both sexes, while triploid hybrids
was higher for P. esculentus females and lower for males had an intermediate position between diploid P. esculentus
(Fig. 1). Similar observations were made for P. ridibun- and P. ridibundus (Fig. 3). Frequency parameters differed
dus calls: the number of peaks was significantly higher for only between females, with P. ridibundus having higher
females than males. Most P. esculentus male release calls values (Fig. 3). Post hoc analysis with the Kruskal–Wallis
(71%) start with several peaks of low amplitude separated 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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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 A significant correlation between peak frequency and body
peak numbers and peak rates (Fig. 3). size was shown for females (Fig. 4a, b) of both taxonomical
Call durations in males were lower in our study than groups and P. ridibundus males (Fig. 4d), but not for P. escu-
those reported by Zamfirescu (2002): 0.42 ± 0.071 vs. lentus males (Fig. 4c). However, the significance level for P.
0.48 ± 0.119 in P. esculentus (p = 0.0154) and 0.34 ± 0.079 ridibundus was relatively low, which may be the consequence
vs. 0.45 ± 0.051 in P. ridibundus (p < 0.0001). of the low number of individuals included in the analysis. A high
All seven measured parameters were dynamic (evalu- correlation was also shown between body size and minimum fre-
ated according to Gerhardt 1991) with CV values above quency (p < 0.001) in P. esculentus of both sexes and P. ridibun-
15% (Table 4). Only for triploid hybrids, peak frequencies dus females, but not P. ridibundus males (p = 0.3854). No cor-
and minimum frequencies were evaluated as stable param- relation was found between body size and all other parameters.
eters, which might be caused by the low number of sam- Such correlation explains why triploid females differ drastically
ples. In general, females tended to have higher coefficients from other forms by frequency parameters (Fig. 3): all three trip-
of variation, but the difference was not significant (KW- loid females had relatively small body sizes and, consequently,
H(5;42) = 1.7675; p = 0.8803). rather high “peak frequency” and “min frequency” parameters.

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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

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analysis of triploid P. esculentus shows that they have (Fig. 4a). More triploid females with different body sizes are
an intermediate position between P. ridibundus and dip- needed for a comprehensive analysis. Our analysis also did
loid P. esculentus. Still, they were incorrectly differenti- not include the other parental species – P. lessonae – because
ated by the discriminant analysis. The best classification it is absent in the studied territory (Siversky Donets River
worked for P. esculentus males (90.5%) and P. ridibundus basin), but it should be included in further studies.
females (75.9%). Unambiguous differences between the Although this analysis did not reveal any characteristics
release calls of P. ridibundus and P. esculentus should of release calls that can exclusively differentiate between
not be expected since P. esculentus is a hybrid, and its hybrid P. esculentus (both diploid and triploid) and one of
release call may overlap with the parental ones. However, their parental species P. ridibundus, we showed general sex-
for mating calls, a strict difference between hybrids and and species-specific trends in frogs’ calls that may become
parental species was shown (Hoffmann and Reyer 2013). a basis for further analysis.
For the release calls, we see similar tendency, but compar-
Supplementary Information  The online version contains supplemen-
isons with the other parental species P. lessonae and more tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11756-0​ 22-0​ 1256-8.
triploid hybrids are needed. Some temporal parameters
for both parental species were measured by Chernyshov Acknowledgements  The authors thank the students at the School of
(2005). Even though Chernyshov did not compare the two Biology (V. N. Karazin Kharkiv National University) who helped catch
and record frogs during the fieldwork. Anna Fedorova is very grate-
species directly, disparities were observed, e.g., a differ- ful to Eleonora Pustovalova, whose support and interest in this work
ence in call durations of P. ridibundus and P. lessonae, helped to complete this manuscript. The authors are also sincerely
which also depended on frog size and water temperature. grateful to the Armed Forces of Ukraine, whose bravery and profes-
Following Chernyshov, we expect to see a more evident sionalism provided a chance to finish the work on this manuscript.
difference between the release calls of P. lessonae and P.
ridibundus in our further analysis. It is possible that the
Declarations 
temporal and frequency data we extracted are not sufficient Conflict of interest  The authors declare that they have no conflict of
to distinguish between P. esculentus and P. ridibundus of interest.
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. References
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