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Received: 06.03.2022 Corresponding editor: W. Böhme
Accepted: 01.09.2022 Published: 15.09.2022
Bonn zoological Bulletin 71 (2): 105–108 ISSN 2190–7307
2022 Drohvalenko M. & Fedorova A. http://www.zoologicalbulletin.de
https://doi.org/10.20363/BZB-2022.71.2.105
Hybridogenetic species complexes are great models for
studying the evolution of reproduction due to hybrids’
capacity to reproduce themselves. The Pelophylax es-
culentus complex is one of the most studied (Dedukh &
Krasikova 2021). It consists of pool frog Pelophylax
lessonae (Camerano, 1882), marsh frog Pelophylax ridi-
bundus (Pallas, 1771), and their hemiclonal hybrid, edi-
ble frog Pelophylax esculentus (Linnaeus, 1758), whose
range approximately coincides with the range of pool
frogs (Hoffmann et al. 2015). Hybrids that transmit paren-
tal genomes clonally are presented as both sexes and two
ploidies (Tunner 1974; Berger 1977; Plötner 2005; Jakob
2007). Hybrids typically coexist with parental and oth-
er hybrid forms in mixed population systems, leading to
formation of a new hybrid generation. Each such system
has evolved the mechanisms to maintain its composition,
like different ontogenetic strategies and selective survival
(e.g., Berger 1973; Hoffmann et al. 2015; Shabanov et al.
2015). It is caused by the variety of ways different forms
contribute to reproduction, mainly the types of gametes:
not only haploid L and R (from hybrids and parental
species), but also occasionally diploid LL, RR, and LR
gametes from hybrids (Dedukh et al. 2013; Pruvost et al.
2015). The most complicated mechanisms are in the sys-
tems with triploids since they include the highest number
of forms (parental species, 2n and 3n hybrids) balancing
with each other (Pruvost 2013; Mikulíček et al. 2015). In
Ukraine, such complex systems were known to be wide-
spread in the eastern part only, in Siverskyi Donets river
basin (Borkin et al. 2004; Shabanov et al. 2020).
Chornobyl Exclusion Zone per se possesses many oppor-
tunities in herpetology, at least due to long-term decreased
anthropogenic pressure. Its radioactive contamination
is already known for affecting amphibians (Gashchak
et al. 2009), though some species remain thriving under
its impact (Burraco et al. 2021a; Burraco 2021b). Radi-
ation also affects amphibian populations. For instance,
the changes in local evolution were described for Hyla
orientalis Bedriaga, 1890 in the Zone, though without
obvious harmful consequences (Car et al. 2022). How-
ever, the impact of some registered effects is not so clear.
Water frogs of the P. esculentus complex from the area
of the Chornobyl fallout impact in Belarus (Briansk re-
gion) were shown to have reduced genome size (Vinogra-
dov & Chubinishvili 1999). Despite the found inuence
on their genomes (Rozanov et al. 1990; Vinogradov et al.
1990), all the previously examined water frogs from the
Zone were diploids. Therefore, it was assumed that only
diploid population systems exist here (S. Litvinchuk, St.
Petersburg, pers. comm.). Herein, we report the evidence
on the previously unrecorded presence of triploid P. escu-
lentus in the Chornobyl Exclusion Zone.
The amphibian survey in the Chornobyl Exclu-
sion Zone was carried out on 9–12 of August 2021 in
collaboration with and legal permission from Chor-
nobyl Radiation and Ecological Biosphere Reserve
(http://zapovidnyk.org.ua) and aimed to explore the
Pelophylax populations within the Zone. The region
of the study included: the Prypiat River (in Chornobyl;
51.272483, 30.244840) and its oodplain (51.341432,
30.199178), the Uzh River (51.273852, 29.741894) and
Abstract. Pelophylax esculentus complex hemiclonal systems are a unique evolutionary object due to intricate mecha-
nisms of their sustainability at genomic, gamete, and developmental levels. Chornobyl Exclusion Zone (Ukraine) presents
a no less unique object of nature evolution under the unprecedented radiation impact and human pressure decrease. By
measuring erythrocyte lengths, we report the rst ndings of triploid hybrid frogs P. esculentus from two localities in
Chornobyl Exclusion Zone, where diploid hybrids only were reported before. The presence of triploids implies high com-
plexity of the local water frog population systems, enabling new research opportunities.
Key words. Water frog, hybrid, population system, erythrocyte.
Scientific note
urn:lsid:zoobank.org:pub:AAABACA0-585E-42B8-8568-42A218E782BB
The rst evidence of triploidy among Pelophylax esculentus (Linnaeus, 1758)
(Anura: Ranidae) in the Chornobyl Exclusion Zone
Mykola Drohvalenko1 & Anna Fedorova2,*
1,2
Department of Zoology and Animal Ecology, V. N. Karazin Kharkiv National University, maidan Svobody 4, Kharkiv, Ukraine
*Corresponding author: Email: anna.fedorova@karazin.ua
1
urn:lsid:zoobank.org:author:D5C5AACE-2A0E-4137-B99B-46083135CA73
2 urn:lsid:zoobank.org:author:5DE735D6-891B-47AB-BB07-60BC76145368
Bonn zoological Bulletin 71 (2): 105–108 ©LIB
Mykola Drohvalenko & Anna Fedorova106
its oodplain (51.256711, 30.222522), isolated old me-
lioration channels (51.245596, 30.169778) and those
in Ilia River valley (51.278037, 29.808185), and the
bypass channel of Chornobyl nuclear plant’s cooling
pond (51.396829, 30.141884). The frogs were caught by
hands, dip-net, and using ashlights at night. Species and
sex were identied morphologically. The presence of vo-
cal sacs and nuptial pads indicated males, while species
were identied by body coloration, metatarsal tubercle
shape, and hindlimbs proportions. Juveniles had weakly
expressed species- and sex-specic features so they were
described as a homogenous group. Snout-vent length
(SVL) was measured via scale photographing and later
measured on photographs. The blood samples were taken
from each frog by cutting the ngertips; wounds were
treated with the antibiotic Vetbicilin-3 (Вasalt, Ukraine).
We prepared the air-dried blood smears and photographed
and measured ~50 erythrocytes for each frog using a
Leica DFC3000 G camera with Leica LASX Software.
Triploid water frogs have about 1.5-fold more DNA in
nuclei than diploids, so the ploidy of P. esculentus frogs
can be estimated by mean erythrocytes length (Ogielska
et al. 2004; Bondareva et al. 2012). It is also known that
the exact cut-off value of erythrocyte length for triploids
should be separately estimated for a particular population
system since it varies among them (though often about
26 µm) (Drohvalenko et al. 2019). Techniques used in
the capture and sampling sought to minimize animal suf-
fering according to Directive 2010/63/EU (protection of
animals used for scientic purposes). All the frogs were
released after the sampling.
The total catch was 52 frogs: 1 tadpole (late devel-
opment stage), 31 juveniles, and 19 adults (10 females,
9 males). Adult hybrids were found in Prypiat and Uzh
oodplains, in Ilia River, old melioration channels, and
bypass channel of cooling pond.
The distribution of collected frogs by SVL and eryth-
rocytes lengths are shown in Fig.1. Adults are visually
divided into two groups without any transient state. The
vast majority (with a mean of 21.54 µm, 95% CI [21.06,
22.02]) belong to diploids. Two spike values exceeding
the considered cut-off value putatively mean the triploidy
Fig. 1. A. The distribution of frogs from the Zone by their mean erythrocyte lengths vs body length (SVL). Red dash line marks
putative cut-off value for adult triploids; dash circle marks juveniles of doubtful ploidy. Boxplots denote means (middle line), IQRs
(boxes) and extreme values (whiskers). B. The sample parameters for different groups of studied frogs.
The rst evidence of triploidy among Pelophylax esculentus in the Chornobyl Exclusion Zone
Bonn zoological Bulletin 71 (2): 105–108 ©LIB
107
of these individuals (male from bypass channel and fe-
male from Prypiat oodplain, with 27.03 and 26.67 µm,
respectively). Although the gap between small-cell and
large-cell individuals is obvious, more individuals should
be examined to specify the erythrocyte length ranges for
diploids and triploids, as well as their possible overlap-
ping.
The distribution of erythrocyte sizes for juveniles looks
more intricate. As there is no gap but rather a continuous
set of cell lengths from small to large, we cannot con-
dently dene the cut-off value for juveniles, so we treat-
ed them on the graph as a single group (mean 22.16 µm,
95% CI [21.39, 22.93]). It is known for certain systems
(Drohvalenko et al. 2021) that cell length distribution
could vary much between adults and juveniles, widely
overlapping in the last. Worth noting that the juveniles
with the largest cells (dash circle Fig. 1; mean 25.87 µm,
95% CI [23.57, 28.17]) originated from the bypass chan-
nel, where the triploid male was assumingly found. We
thus can carefully suggest that these juveniles are a new
generation of triploids. At the same site, there were also
caught three individuals with tails remnants (still in the
course of metamorphosis), which SVL weren’t measured
– they, however, had the smallest erythrocytes: 19.79,
19.71, and 18.80 µm. No signicant differences were
found between different ages, neither among diploids
(Student’s t-test p=0.354) nor triploids (Mann-Whitney’s
p = 0.533).
Due to the late summer survey, the catch of frogs ap-
peared relatively small and insufcient to claim neither
the exclusive presence of triploids in two localities only
nor the exact composition of the studied systems. Nev-
ertheless, every presence of hybrids P. esculentus prob-
ably means the presence of the parental species, which
they depend on – and triploids presence implies the pres-
ence of diploid hybrids, producing 2n-gametes (Biriuk
et al. 2016). In certain locations, the presence of some
forms could be suggested by habitat preferences (like
P. lessonae in swampy channels; [Pysanets, 2014]) or
behavioral features (like P. ridibundus possibly migrat-
ing to bypass channel from Prypiat at breeding; [Wells,
2007]). The local absence of any form could indeed be
just an artifact.
The features of triploid P. esculentus from the Chor-
nobyl Exclusion Zone are planned to be examined using
precise methods. Their very presence here would mean
the existence of more complicated hemiclonal popula-
tion systems there than was supposed before. At least
the L-E-R system with triploids (in the bypass channel)
is already more complex than the most complex system
studied in Ukraine (Meleshko et al. 2014). They could
become new models of interspecies evolution due to a
combination of their inherent hemiclonality phenomenon
and external radiation inuence. The absence of previous
data on triploids in the Zone could possibly be explained
by samples too small to record non-numerous triploids.
Or, we can hypothesize the recent emergence of triploids
due to local population systems evolution – in that case,
the role of radiation in this phenomenon is yet to be stud-
ied.
Our next step is to focus on obtained material deep-
ly. As the collection of new bigger samples has become
quite complicated due to ongoing war and following haz-
ardous aftermath, the application of advanced techniques
available for ploidy and genomes identication (karyolo-
gy, microsatellite analysis, FISH) is scheduled.
Acknowledgments. We want to thank the Chornobyl Radiation
and Ecological Biosphere Reserve scientists, without whose
kind assistance the whole research wouldn’t have been per-
formed: Denys Vyshnevskyi and Sergii Domashevskyi. We also
thank Dmytro Shabanov for his help with improving the man-
uscript. And we are enormously grateful to all the defenders
of Ukraine, whose efforts made this paper possible to nalize.
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