Content uploaded by Eleonora Pustovalova
Author content
All content in this area was uploaded by Eleonora Pustovalova on Aug 23, 2022
Content may be subject to copyright.
Submitted 23 June 2022
Accepted 6 August 2022
Published 23 August 2022
Corresponding author
Dmitrij Dedukh,
dmitrijdedukh@gmail.com
Academic editor
Carlos Eurico Fernandes
Additional Information and
Declarations can be found on
page 15
DOI 10.7717/peerj.13957
Copyright
2022 Pustovalova et al.
Distributed under
Creative Commons CC-BY 4.0
OPEN ACCESS
The high diversity of gametogenic
pathways in amphispermic water frog
hybrids from Eastern Ukraine
Eleonora Pustovalova1,2,3, Lukaš Choleva1,2, Dmytro Shabanov3and
Dmitrij Dedukh1
1Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics of the CAS, v.v.i.,
Libechov, Czech Republic
2Department of Biology and Ecology, Faculty of Science, University of Ostrava, Ostrava, Czech Republic
3Laboratory of Amphibian Population Ecology, Department of Zoology and Animal Ecology, School of
Biology, V. N. Karazin Kharkiv National University, Kharkiv, Ukraine
ABSTRACT
Interspecific hybridization can disrupt canonical gametogenic pathways, leading to the
emergence of clonal and hemiclonal organisms. Such gametogenic alterations usually
include genome endoreplication and/or premeiotic elimination of one of the parental
genomes. The hybrid frog Pelophylax esculentus exploits genome endoreplication and
genome elimination to produce haploid gametes with chromosomes of only one
parental species. To reproduce, hybrids coexist with one of the parental species and form
specific population systems. Here, we investigated the mechanism of spermatogenesis
in diploid P. esculentus from sympatric populations of P. ridibundus using fluorescent
in situ hybridization. We found that the genome composition and ploidy of germ cells,
meiotic cells, and spermatids vary among P. esculentus individuals. The spermatogenic
patterns observed in various hybrid males suggest the occurrence of at least six
diverse germ cell populations, each with a specific premeiotic genome elimination and
endoreplication pathway. Besides co-occurring aberrant cells detected during meiosis
and gamete aneuploidy, alterations in genome duplication and endoreplication have
led to either haploid or diploid sperm production. Diploid P. esculentus males from
mixed populations of P. ridibundus rarely follow classical hybridogenesis. Instead,
hybrid males simultaneously produce gametes with different genome compositions
and ploidy levels. The persistence of the studied mixed populations highly relies on
gametes containing a genome of the other parental species, P. lessonae.
Subjects Aquaculture, Fisheries and Fish Science, Genetics, Zoology, Freshwater Biology
Keywords Gametogenesis, Spermatid, Meiosis, Pelophylax, Amphispermy, FISH, Bivalents,
Hybridogenesis
INTRODUCTION
Meiosis is a conserved process for all eukaryotic organisms and represents a hallmark
of sexual reproduction (Lenormand et al., 2016). Chromosome conjugation during
meiosis relies on sufficient homology between chromosomes (McKee, 2004), whereas
insufficient pairing may lead to meiotic abruption and formation of aneuploid gametes.
These mechanisms keep taxa prezygotically reproductively isolated (Zong & Fan, 1989;
How to cite this article Pustovalova E, Choleva L, Shabanov D, Dedukh D. 2022. The high diversity of gametogenic pathways in amphis-
permic water frog hybrids from Eastern Ukraine. PeerJ 10:e13957 http://doi.org/10.7717/peerj.13957
Borodin et al., 1988;Ishishita et al., 2015;Torgasheva & Borodin, 2016;Dedukh et al., 2020).
Interspecific hybridization has both positive (Mallet, 2007;Abbot et al., 2013) and negative
impacts (Arnold & Hodges, 1995;Rieseberg, 2001;Coyne & Orr, 2004) and plays a key role
in evolution. One of the outcomes of hybridization is the creation of individuals with
clonal and hemiclonal reproductive modes (Dawley & Bogart, 1989;Schön, Martens &
Van Dijk, 2009;Neaves & Baumann, 2011;Stöck et al., 2021). Hybrid clonal animals form
gametes with a chromosomal composition identical to that of their somatic cells (Dawley
& Bogart, 1989;Schön, Martens & Van Dijk, 2009;Neaves & Baumann, 2011;Stöck et al.,
2021). Hybrid hemiclonal animals produce unrecombined haploid gametes that require
fertilization to restore diploid chromosomal sets in their offspring (Dawley & Bogart, 1989;
Schön, Martens & Van Dijk, 2009;Stöck et al., 2021;Dedukh & Krasikova, 2021). A switch to
asexual reproduction requires significant modifications to gametogenesis, rescuing hybrids
from sterility, and the creation of alternative pathways for successful reproduction. Thus,
our understanding of reproductive ability and evolutionary potential of hybridization lies
in our understanding of hybrid gametogenesis.
Hemiclonal reproduction, also known as hybridogenesis, has been found in European
water frogs of the genus Pelophylax (Tunner, 1974). This animal system includes two
parental species: P. lessonae (Camerano, 1882) (LL genotype) and P. ridibundus (Pallas,
1771) (RR genotype), and their hybrid P. esculentus (Linnaeus, 1758). Hybrids can be
represented in diploid (RL) and triploid (LLR, LRR) forms (Günther, Uzzell & Berger, 1979;
Berger, 1983). The classical model of hybridogenetic reproduction states that one parental
genome is eliminated during gametogenesis while the other is duplicated and transmitted
to gametes, which appear to be clonal (Tunner, 1973;Tunner & Heppich, 1981;Tunner
& Heppich-Tunner, 1991;Chmielewska et al., 2018;Doležálková-Kaštánková et al., 2021).
Triploid hybrids usually eliminate a genome present in one copy, whereas the genome
present in two copies enters meiosis and forms recombinant gametes (Günther, Uzzell
& Berger, 1979;Graf & Polls-Pelaz, 1989;Ogielska, 1994;Plötner, 2005;Christiansen &
Reyer, 2009;Dedukh et al., 2015;Dufresnes & Mazepa, 2020;Dedukh et al., 2020;Dedukh &
Krasikova, 2021). However, the detailed principles of genome elimination and duplication
during hybrid gametogenesis remain unknown.
Hybridogenetic gametogenesis makes hybrids dependent on parental species and leads
to the formation of population systems where hybrids coexist with one or both parental
species, or for all-hybrid populations with various ploidy and genomic compositions (Graf
& Polls-Pelaz, 1989;Plötner, 2005;Christiansen & Reyer, 2009). In most of the distribution
range, P. esculentus coexists with P. lessonae, creating the L-E system (Graf & Polls-Pelaz,
1989;Plötner, 2005;Pruvost, Hoffmann & Reyer, 2013;Svinin et al., 2013;Svinin et al., 2021;
Hoffman et al., 2015;Dufresnes & Mazepa, 2020). Here, hybrids have a typical hemiclonal
gametogenesis with preferential elimination of the P. lessonae genome, followed by the
transmission of P. ridibundus genome to gametes (Günther, 1983;Bucci et al., 1990;Pruvost,
Hoffmann & Reyer, 2013;Dedukh et al., 2019;Svinin et al., 2021). The R-E system forms
hybrids mixed in populations with P. ridibundus.P. esculentus from this system is specific
to significant alterations in gametogenic pathways, resulting in decreased fertility and
increased numbers of aneuploid gametes (Uzzell, Günther & Berger, 1977;Günther, 1983;
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 2/21
Vinogradov et al., 1991;Borkin et al., 2004;Ragghianti et al., 2007;Doležálková et al., 2016;
Dedukh et al., 2015;Dedukh et al., 2017;Biriuk et al., 2016). Studies of geographic variation
showed that in Central Europe (Doležálková et al., 2016;Doležálková-Kaštánková et al.,
2018;Doležálková-Kaštánková et al., 2021), P. esculentus is present only in a male sex,
and both sexes of P. ridibundus coexist in Eastern Europe. P. esculentus syntopic with P.
ridibundus is present in both sexes and at two ploidy levels (RL, RRL, and LLR) (Borkin et
al., 2004;Shabanov et al., 2020).
Previous studies from Eastern Ukraine have shown that hybrid females frequently
produce haploid gametes with the R genome and diploid gametes with the RL genome,
whereas gametes with L genomes have never been detected (Dedukh et al., 2015;Dedukh
et al., 2017). Additionally, diploid hybrid males usually simultaneously produce a mixture
of gametes with the L and R genomes. This phenomenon, called hybrid amphispermy
(Vinogradov et al., 1991), includes the simultaneous formation of L and R sperms, and
was first observed in Central Europe (Vinogradov et al., 1991;Doležálková et al., 2016).
Vinogradov et al. (1991) suggested the existence of at least two germ cell populations that can
eliminate either P. ridibundus or P. lessonae genome during amphispermic reproduction.
An alternative hypothesis proposed the absence of premeiotic genome elimination and a
different separation of the L and R genomes in the first meiotic division (Doležálková et al.,
2016).
In the current study, we analyzed hybridogenetic gametogenesis in Eastern Europe. Using
fluorescent in situ hybridization (FISH) with probe RrS1 specific to centromeric regions of
P. ridibundus chromosomes, we identified the genomes of P. ridibundus during metaphase
of meiosis I, spermatids, and mitotic spreads on chromosomal spreads from hybrid
male gonads. Combining these data, we tested (i) whether amphispermy is widespread
gametogenesis in hybrid males over R-E systems from Eastern Ukraine. Further, we tested
(ii) whether premeiotic genome elimination of both L and R genomes occurs in different
gonial cells of amphispermic males, or not.
MATERIALS AND METHODS
Samples
Sampling was conducted in Kharkiv Oblast, Eastern Ukraine, during 2016–2019. We
collected six adult P. esculentus males from the Mozh River (49.749167; 36.162778), five
males from the Iskiv water body (49.627778; 36.282778), and one male from the Udy River
(49.968333; 36.136944) (Fig. S1). These geographically isolated population systems are
characterized by the coexistence of di- and triploid hybrids of both sexes, represented by
LR, LLR, and LRR genotypes, and P. ridibundus of both sexes. Animals were caught at night
using a torch. All specimens were collected outside of the protected areas within Eastern
Ukraine and therefore, no specific permissions were required. All animal manipulations
were performed according to national and international guidelines. Standard techniques
for capture, tissue sampling, and euthanasia were used to minimize animal suffering.
Before euthanasia, each individual was anesthetized by submersion in ethyl ethanoate
(ETAC). All procedures were approved by the Committee on Bioethics of the V. N.
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 3/21
Karazin Kharkiv National University (minutes No 4, 21.04.2016). The previous species
and ploidy identification were determined by a complex of morphological features and
Ag-staining (Birstein, 1984) with some modification and further confirmed within the
preparation of somatic tissue chromosomes followed by fluorescent in situ hybridization
(FISH) with species-specificity (Ragghianti et al., 1995;Dedukh et al., 2015;Dedukh et al.,
2017).
Preparation of mitotic and meiotic chromosomes
Before euthanasia in ETAC, each frog was injected with 0.05% colchicine for 12 h. The
intestines and testes were dissected, cleaned, and treated hypothonically (0.07M KCl) for
20 min. The tissues were transferred to Carnoy’s fixative (3:1 methanol: glacial acetic
acid), and the solution was changed thrice. To prepare chromosomal spreads, the tissue
fragments were transferred to 70% acetic acid solution for maceration in a suspension of
cells and dropped onto slides pre-heated to 60 ◦C (Biriuk et al., 2016). The chromosomal
and cell nuclei spreads were dried on a heating table at 60 ◦C for 1 h.
Fluorescent in situ hybridization
Male gametogenesis was further analyzed using the FISH method on mitotic and meiotic
chromosomes, following Dedukh et al. (2015) and Dedukh et al. (2017). The slides were
treated with RNAse (100–200 µg/ml) for 1 h and pepsin D (0.005%, diluted in 0.01
N HCl) for 3 min. The probe was labelled with biotin l from the genomic DNA of
P. ridibundus by PCR using the following primers to RrS1 centromeric repeat: 50-
AAGCCGATTTTAGACAAGATTGC- 30; 50-GGCCTTTGGTTACCAAATGC- 30. The
probe was added to the hybridization mixture (50% formamide, 1 µl 2xSSC and tRNA,
10% dextran sulphate, 1.5 µl labelled probe). Slides containing mitotic and meiotic
chromosomes were denatured at 77 ◦C for 3 min and incubated at room temperature for
12–18 h. The slides were then washed thrice in 0.2xSSC at 60 ◦C. Biotin was detected using
avidin conjugated with the fluorochrome Alexa 488 or Cy3. After washing in 4xSSC slides,
they were dehydrated in an ethanol series, air-dried, and mounted in DABCO antifade
solution containing 1 µg/ml DAPI.
Image processing
Mitotic and meiotic chromosomes were inspected after FISH using Provis AX70 Olympus
microscopes and Leica DM 2000 equipped with standard fluorescence filter sets.
Microphotographs of chromosomes were captured with a CCD camera (DP30W Olympus)
using Olympus Acquisition Software and a Leica DFC3000 G camera using Leica LASX
Software. Microphotographs were adjusted and arranged in the Adobe Photoshop CS6
software. FISH-based mapping of RrS1 pericentromeric repeats visualizes the centromeric
regions of P. ridibundus chromosomes (Ragghianti et al., 1995), but cannot identify P.
lessonae genome during interphase. The analysis allowed us to discriminate different
gametogenic stages, as we identified the presence of P. ridibundus genome in mitotic (from
both somatic and germ cells) and meiotic chromosome plates as well as in the nuclei of
somatic and germ cells and spermatids (Table S1). Interphase cells and spermatids with
5–13 signals were discriminated as cells with P. ridibundus genome. Among these signals
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 4/21
five were usually bright and clearly distinguishable while remaining eight signals were
either weak or absent. Cells with 1–4 signals were not taken into account. Five signals
observed in interphase cells and spermatids corresponded to the haploid P. ridibundus
chromosomal set, where we observed five bright signals on all large chromosomes and one
small chromosome while signals on the other chromosomes were either weak or absent.
Ragghianti et al.;Ragghianti et al. (1995 and 2007) observed six signals in interphase cells
of diploid hybrids. Interestingly, Dedukh et al.;Dedukh et al. (2019 and 2020) detected 13
signals in a haploid set of P. ridibundus chromosomes, while they also found a difference in
the signal intensity. A signal variation and polymorphism of the studied pericentromeric
repeat may explain technical differences in laboratory protocol used, the source of genomic
DNA used for probe preparation or the interpopulation polymorphism.
RESULTS
The two geographically isolated populations of P. esculentus were characterized by the
coexistence of diploid and polyploid hybrids. Here, we used FISH with the RrS1 probe
to identify the genome composition of interphase nuclei, spermatids, and meiotic and
mitotic chromosomal plates obtained from the testes of 11 diploid P. esculentus males. The
hybrid testes were round in shape without any visible anomalies. In nine males, the left
testis was larger than the right (left mean 5.8 mm; right mean 4.1 mm) and two males
had testes of equal sizes (frogs’ ID: 19I-60, 19I-62) (Table S2). Testes size difference is
common in P. esculentus and might be accompanied by decreased fertility (Berger, 1970;
Ogielska & Bartmańska, 1999). Data from a single male from the Udy River (17U−4.2)
were insufficient to evaluate hybrid gametogenesis in this locality. Raw data on the number
of each type of gametes produced by this male are presented in Table S1.
Gametogenesis in diploid hybrid males in Mozh River
Analysis of 436 interphase nuclei from four diploid hybrid males (17T-5, 17T-10, 18T-8,
18T-7) showed the presence of interphase nuclei with 3–18 signals (Figs. 1D,1E,1G,
1H and 1J) along with interphase nuclei without signals (Fig. 1H). Interphase nuclei
without signals were those with exclusive content of P. lessonae chromosomes. Nuclei
with 5–13 signals contained at least a haploid set of P. ridibundus chromosomes, whereas
nuclei with more than 13 signals contained an aneuploid or diploid chromosomal set of
P. ridibundus. The analysis of 79 metaphase plates during mitosis showed 0–24 signals,
among which most metaphase plates had 12–13 signals (Fig. 1E). These results fit well with
the interphase nuclei analysis, suggesting at least three cell populations: cells with 26 P.
lessonae chromosomes, cells with 13 P. ridibundus and 13 P. lessonae chromosomes, and
cells with 26 P. ridibundus chromosomes. Distinguishing germ cells from somatic cells is
difficult. However, as genome elimination and endoreplication occur only in germ cells, we
considered cells with P. lessonae chromosomes as germ cells. During meiosis I, we observed
spermatocytes with 13 bivalents of P. ridibundus and spermatocytes with 13 bivalents of
P. lessonae in all four males analyzed (Figs. 1F and 1G). In two of these males (18T-7,
17T-10), bivalents with P. ridibundus chromosomes dominated (87% and 77%). During
meiosis II, we detected spermatocytes with 13 univalents of P. ridibundus chromosomes
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 5/21
(Fig. 1H) and 13 univalents of P. lessonae chromosomes (Fig. 1I). Additionally, we observed
many cells with aberrant pairing in all analyzed males. The observed hybrids potentially
eliminated different genomes in different cells premeiotically, or had some problems with
selective elimination. We detected spermatids in which the signal of P. ridibundus probe
varied from 0 to 12, suggesting the presence of spermatids in P. lessonae and P. ridibundus
genomes (Figs. 1D and 1J). These males transmitted two parental genomes in their cells
simultaneously, i.e., they were amphigametic.
Fifty-four examined interphase cells of one male (18-T6) had at least five signals,
indicating the presence of the haploid P. ridibundus genome (Fig. 1C). The analysis
of 14 mitotic chromosomal plates showed 8 plates with 26 chromosomes, of which
13 belonged to P. ridibundus and 13 to P. lessonae, the other six mitotic chromosomal
plates were aneuploid. During the analysis of 32 metaphases of meiosis I, we detected 13
bivalents of P. ridibundus (Fig. 1A). We also detected five metaphases of meiosis II with 13
univalents of P. ridibundus (Fig. 1B). In addition, 24 aneuploid chromosomal plates (Fig.
1C) were observed. The analyzed spermatids (n=48) exclusively exhibited the presence
of P. ridibundus chromosomes. We suggest that during gametogenesis in this male, the
genome of P. lessonae was premeiotically eliminated, followed by endoreplication of the P.
ridibundus genome.
In one individual (17T-8), we observed interphase nuclei with 3–26 signals (Figs. 2A,
2B and 2D). Haploid P. ridibundus genome was suggested in cells with 5-13 signals;
diploid P. ridibundus genome was suggested in cells with 15–26 signals. The analysis of 14
mitotic chromosomal plates from this individual showed 3 mitotic chromosomal plates
with approximately 52 chromosomes, including chromosomes exclusive to P. ridibundus
(Fig. 2B) and chromosomes exclusive to P. lessonae (Fig. 2C). In 8 metaphase plates,
we observed 26 chromosomes exclusive to P. ridibundus (Fig. 2D) as well as both P.
ridibundus and P. lessonae chromosomes (Fig. 2I). In meiosis I, we detected chromosomal
plates with 13 tetravalents of P. ridibundus and metaphase plates with 13 tetravalents
of P. lessonae (Fig. 2G) (23% of the total amount). One of the genomes was eliminated
to form spermatocytes with genome-specific tetravalents, whereas the other underwent
two rounds of genome endoreplication. We also found metaphase plates of meiosis I
with approximately 13 tetravalents, including 26 chromosomes of P. ridibundus and 26
chromosomes of P. lessonae (Figs. 2C and 2F). Spermatids of this male had 3–19 signals,
suggesting the presence of two P. ridibundus genomes at least in some spermatids (Figs.
2F–2H). This pattern also supports the amphigametic production.
Gametogenesis in diploid hybrid males in Iskiv pond
Analysis of interphase nuclei of one male (19I-60) revealed both interphase cells without
signals and those with RrS1 signals (Fig. S2J). Some cells had, therefore, chromosomes
exclusive to P. lessonae, and some cells had at least one haploid genome of P. ridibundus.
Mitotic metaphase plates of this individual were represented by 26 chromosomes, with 13
P. ridibundus chromosomes, 13 P. lessonae chromosomes, and 26 chromosomes exclusive
to P. ridibundus (Fig. S2J). Our metaphase inspection of meiosis I clearly distinguished
13 P. ridibundus bivalents (Figs. S2K–S2L). To form such spermatocytes, P. lessonae
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 6/21
Figure 1 Identification of ploidy level and genome composition of gonocytes, spermatocytes, and
spermatids from P. esculentus males collected from the Mozh river basin. FISH with RrS1 probe helps
distinguish pericentromeric regions only of P. ridibundus chromosomes (indicated by thin arrows). (A–C)
Somatic cells (C), spermatids (B, C), and spermatocytes in (continued on next page... )
Full-size DOI: 10.7717/peerj.13957/fig-1
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 7/21
Figure 1 (...continued)
meiosis I (A) and II (B) had only P. ridibundus chromosomes suggesting the presence of premeiotic
genome elimination of P. lessonae genome and endoreplication of P. ridibundus genome. (D–J) Germ
line cells (gonocytes, spermatocytes, and spermatids) with different ploidies suggesting the presence of
premeiotic elimination and endoreplication of different genomes in various cell lines. Interphase cells
(indicated by thick arrows) with a haploid set of P. ridibundus chromosomes (D, E, G, H, J) and with P.
lessonae chromosomes (I). Mitotic metaphase cell with 13 P. ridibundus chromosomes and 13 P. lessonae
chromosomes (E). Meiotic metaphase I with 13 bivalents of P. ridibundus (D, F, J) and 13 bivalents of P.
lessonae (G). Meiotic metaphase II with 13 univalents of P. ridibundus (H) and 13 univalents of P. lessonae
(I). Spermatids (indicated by arrowheads) with haploid set of P. ridibundus chromosomes (D, J) and P.
lessonae (D, J). Scale bar =10 µm.
genome must have been premeiotically eliminated, whereas P. ridibundus genome was
endoreplicated. Additional aneuploid cells (n=30) suggest aberrant genome elimination
and endoreplication. The analysis of spermatids (n=29) revealed that most spermatids
had P. lessonae genome, and only a few spermatids had P. ridibundus genome (Fig. S2L).
Though we observed both interphase nuclei and spermatids exclusively in the P. lessonae
genome, we did not detect meiotic plates with P. lessonae bivalents. Therefore, we suggest
that spermatocytes with P. lessonae must be present in this individual, i.e., the individual
was amphispermic with the prevalence of L-gametes.
The analysis of interphase nuclei (n=307) from two males (19I-62 and 18I-90) showed
some interphase nuclei only in P. lessonae chromosomes and others in P. ridibundus
chromosomes (Fig. S2A–S2C). During the analysis of mitotic metaphases (n=44), we
detected metaphase plates with 26 chromosomes, including 13 P. ridibundus and 13 P.
lessonae chromosomes (Fig. S2B). Most spermatocytes had 13 bivalents of P. ridibundus
(Fig. S2C) while only a few spermatocytes had 13 P. lessonae bivalents. We detected
58 aneuploid chromosome plates in both males (Fig. S2D). In meiosis II, we observed
spermatocytes with 13 univalent P. ridibundus and 13 univalent P. lessonae (Fig. S2A). In
spermatids (n=114), we found those with P. ridibundus chromosomes and exclusive P.
lessonae chromosomes (Fig. S2B), supporting the pattern of amphigametic production.
Analysis of interphase nuclei (n=110) in two other males (18I-91 and 19I-61)
revealed nuclei exclusively with P. lessonae chromosomes and nuclei with P. ridibundus
chromosomes (Figs. S2E–S2G,S2I). During the analysis of mitotic metaphases (n=13)
obtained from the other male (19I-61), we found metaphase plates with 26 chromosomes,
among which 13 chromosomes were from P. lessonae and 13 were from P. ridibundus (Fig.
S2E), while mitotic chromosomal plates were not detected in one of the males (18I-91).
Both males simultaneously produced spermatocytes with 13 P. ridibundus bivalents (Fig.
S2F) and 13 P. lessonae bivalents. During meiosis II, we detected spermatocytes with 13 P.
lessonae univalents (Figs. S2H and S2I) and with 13 P. ridibundus univalents (Fig. S2G). In
spermatids, the number of signals was varied from 0 to 13. Spermatids with no signal were
considered as bearing P. lessonae genome (Fig. S2I); spermatids with 5-13 were considered
as bearing P. ridibundus genome (Figs. S2F–S2H). These two males (18I-91, 19I-61)
potentially eliminated different genomes in different cells premeiotically and transmitted
the two genomes in their cells, thus being amphigametic.
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 8/21
Figure 2 Identification of ploidy level and genome composition of gonocytes, spermatocytes and sper-
matids from particular P. esculentus male producing diploid spermatids collected from the Mozh river
basin. Interphase cell nuclei (indicated by thick arrows) with diploid P. ridibundus chromosomal set (A,
D). Mitotic metaphases with 26 P. ridibundus chromosomes (D), approximately 47 P. ridibundus chromo-
somes (B), approximately 40 P. lessonae chromosomes (C) and 13 P. ridibundus and 13 P. lessonae chro-
mosomes. Meiotic metaphase I with 13 P. ridibundus bivalents (A, H), approximately 12 tetravalents (or
mixture of bivalents and tetravalents) with chromosomes exclusive to P. ridibundus (E), and with approxi-
mately 11 tetravalents with chromosomes exclusive to P. lessonae (G). Meiotic metaphase I with a mixture
of approximately nine P. lessonae tetravalents and four P. lessonae bivalents as well as four P. ridibundus
tetravalents and four P. ridibundus bivalents. Spermatids (shown by arrowheads) with at least five P. ridi-
bundus chromosomes (designated as haploid P. ridibundus genome) (B, H), with only P. lessonae chromo-
somes (designated as haploid or diploid P. lessonae genome) and at least 14 P. ridibundus chromosomes
and at least 17 P. ridibundus chromosomes (designated as diploid P. ridibundus genome) (F, H). P. ridi-
bundus chromosomes identified using FISH-based detection of pericentromeric RrS1 repeats (indicated by
thin arrows). Scale bar =10 µm.
Full-size DOI: 10.7717/peerj.13957/fig-2
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 9/21
DISCUSSION
Diverse spermatogenesis in diploid hybrids
Our study of hybrid P. esculentus males from Eastern Ukrainian populations revealed
diverse gamete formation (Fig. 3,Fig. S3,Table S1). Nine out of eleven males simultaneously
produced two types of haploid gametes with parental chromosomes (amphispermic male,
Fig. 4, Pathway III), one with P. lessonae genome and one with P. ridibundus genome, free
of recombination and crossover between the genomes of parental species. A single male
represented the second type of spermatogenesis-producing spermatid with P. ridibundus
genome only (Fig. 3B,Table S1). We also found a male suspected to form diploid sperm
based on sperm analysis and tetravalent observations during meiosis, which corresponded
to the third type of spermatogenesis (Figs. 3B and 3D). The simultaneous production of
fertile gametes with P. lessonae and P. ridibundus genomes (amphispermy) was determined
using DNA flow cytometry in the Iskiv pond population (Biriuk et al., 2016) and from
artificial crosses in the Mozh River (Mazepa et al., 2018). By analyzing the process of
gametogenesis in detail, we provide clear pathways on the mechanisms of the origins of
diverse gametes in these tetrapod animals.
Inspecting meiosis, we revealed spermatocytes with 13 univalents or bivalents of P.
ridibundus (39% for Mozh, 47% for Iskiv, 43% for both) as well as 13 univalents or
bivalents of P. lessonae (32% for Mozh, 20% for Iskiv, 26% for both) (Fig. S3A). Interphase
nuclei and mitotic chromosomes from testis cell suspensions often bear either P. ridibundus
or P. lessonae chromosomes (Figs. 3A and 3C). The methodology used cannot distinguish
whether interphase nuclei and metaphase chromosomes belong to germ cells or somatic
cells. However, as genome elimination and endoreplication occur only in the germ cells,
we considered the observed cells as germ cells. As we detected germ cells and spermatocytes
bearing only P. ridibundus or P. lessonae chromosomes, we suggest that genome elimination
and endoreplication occurred in germ cells before meiosis (Fig. 4, Way III). A phenomenon
of premeiotic genome elimination has been described earlier in water frog hybrids during
tadpole development and causes the classical formation of a single gamete type (Tunner
& Heppich-Tunner, 1991;Ogielska, 1994;Dedukh et al., 2017;Dedukh et al., 2019;Dedukh
et al., 2020;Chmielewska et al., 2018). The presence of cells with only P. ridibundus and P.
lessonae genomes indicated the existence of at least two cell population types eliminating
different parental genomes, even in a single individual, as proposed by Vinogradov et al.
(1991). Comparative genomic hybridization on Central-European amphispermic males
has revealed meiotic metaphase I with univalent and bivalent-like configurations, including
bivalent-like configurations between the two parental genomes (Doležálková et al., 2016).
Based on these observations, Doležálková et al. proposed a hypothesis in which premeiotic
elimination would be absent in these cases, followed by segregation of P. ridibundus and
P. lessonae chromosomes during meiosis I. Diploid hybrid males from Eastern Europe
likely do not use this hypothetical strategy, as evidenced by our observation of premeiotic
genome elimination followed by genome duplication in different germ cell populations
(Fig. 4). However, it should be noted that bivalent-like configurations between the two
different parental genomes were not observed in our males. The presence of aneuploid cells
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 10/21
Figure 3 Relative number of normal and aneuploid chromosomal plates during mitosis (A, C) and
meiosis (B, D) from hybrid frogs collected from the R-E system of the Mozh river (A, B) and Iskiv pond
(C, D). R, genome of P. ridibundus; L, genome of P. lessonae; aneuploidy, number of chromosomes more
or less 13 bivalents or univalents.
Full-size DOI: 10.7717/peerj.13957/fig-3
during meiosis (on average 25% for Mozh, 33% for Iskiv, 29% for both) indicates problems
with genome elimination and/or endoreplication (Fig. 4, Way V). Aneuploid meiocytes
and meiocytes with unusual pairings were detected earlier in both hybrid females (Dedukh
et al., 2015;Dedukh et al., 2017) and males (Biriuk et al., 2016) from the same locality and
generally in various population types (Heppich, Tunner & Greilhuber, 1982;Bucci et al.,
1990;Christiansen et al., 2005;Christiansen, 2009;Christiansen & Reyer, 2009;Dedukh et
al., 2019). It should be noted that aberrations were highly numerous in hybrid frogs from
a mixed population of P. ridibundus, suggesting difficulties in genome elimination and
duplication during hybrid gametogenesis (Uzzell, Günther & Berger, 1977;Ragghianti et al.,
2007;Doležálková et al., 2016;Dedukh et al., 2015;Dedukh et al., 2017;Biriuk et al., 2016).
A single hybrid male produced spermatocytes with 13 tetravalents of P. ridibundus and
13 tetravalents of P. lessonae, indicating that it underwent an additional round of genome
duplication (Fig. 3B). To form spermatocytes with 13 tetravalents of P. ridibundus, the
cells must first eliminate P. lessonae chromosomes, followed by two rounds of duplication
of P. ridibundus chromosomes, and vice versa for P. lessonae tetravalents (Fig. 4, Way IV).
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 11/21
Figure 4 Suggested gametogenic pathways in sexual species and hybrid males from studied R-E systems. Pathway I: Genome elimination and
endoreplication (‘classical’ hybridogenesis). During classical genome elimination, one of the parental genomes is eliminated before meiosis, whereas
the other is endoreplicated, allowing the restoration of the diploid chromosome set. These cells undergo meiotic division with 13 bivalents during
meiosis I and 13 bivalents during meiosis II. Subsequent spermatids bear the genomes of only one parental species (P. ridibundus or P. lessonae).
Pathway II: Genome elimination of one of the parental species (P. ridibundus or P. lessonae) during meiosis. This type of gamete formation also in-
volves the elimination of only one parental genome. However, it occurs directly during meiosis. After meiotic divisions I (13 bivalent stages) and II
(13 univalent stages), spermatids bear the endoreplicated genome. Pathway III: The genomes of different parental species were eliminated from dif-
ferent germline populations. Therefore, some gonocytes bear only P. ridibundus chromosomes, whereas some cells have P. lessonae chromosomes
only. Germ cells with both parental genomes duplicated and formed two types of parental species bivalents (2n =26). After meiosis II, the sper-
matids were from both parental species (P. ridibundus and P. lessonae). Pathway IV: Diploid sperm formation. Two rounds of endoreduplication
of one parental species genome resulted in the formation of tetravalents, bearing four sets of P. ridibundus or P. lessonae genomes in meiosis I. Such
cells, which have undergone meiosis II, bear a double chromosome set (RR, LL, or even RL). Pathway V: Abnormal meiosis. Due to disruptions dur-
ing the elimination of P. ridibundus or P. lessonae genome, there are no vital spermatids, so the individual is sterile.
Full-size DOI: 10.7717/peerj.13957/fig-4
Additional detection of spermatocytes with 13 tetravalents during meiosis I with both
genomes of the parental species suggests the absence of genome elimination and two
rounds of genome endoreplication. Interphase cells with 26 P. ridibundus chromosomes
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 12/21
(Fig. 2A) resembled the results obtained for the diploid hybrid males with metaphase plates
and tetravalents (Ragghianti et al., 2007). Similar observations were made by Dedukh et
al. (2015) during lampbrush chromosome analysis, where the authors found one hybrid
female with 26 P. ridibundus bivalents. In addition, such a pattern supports the presence
of two rounds of genome endoreplication preceding meiosis after the elimination of
one of the parental genomes. Chromosomal plates with tetravalents are typically formed
in autopolyploid frogs of the Pleuroderma genus (Salas et al., 2014). Nevertheless, in
these species, bi-, tetra-, and octavalents were also detected among metaphase plates,
suggesting some pairing inaccuracies (Salas et al., 2014). Bi & Bogart (2010) showed
the presence of quadrivalents (the same as tetravalents) in Ambystoma hybrid females
by investigating lampbrush chromosomes, suggesting occasional synapses between
homologous chromosomal regions. Nevertheless, such oocytes are a rare phenomenon in
Ambystoma (Bi & Bogart, 2010), while in water frogs, we provide frequent observations
with numbers of spermatocytes with tetravalents varying in their genome composition. We
hypothesized that these cells could proceed through meiosis and form diploid sperm with
the LL, RL, and RR genomes (Fig. 4, Way IV). Such gametes may lead to the emergence
of triploid frogs (approximately 5%) observed in the Mozh Basin (Drohvalenko et al.,
2022). However, the fertilization success of diploid sperms to compete with haploid sperms
requires further investigation.
As not only hybrid males but hybrid females (Dedukh et al., 2015;Dedukh et al.,
2017;Christiansen & Reyer, 2009;Christiansen & Reyer, 2009;Pruvost, Hoffmann & Reyer,
2013) can also produce gametes of both parental species, Dubey et al. (2019) called
this phenomenon as ‘amphigamy’. However, this term has following interpretations
according to Rieger, Michaelis & Green (1991): (1) the fusion of two sex cells and the
formation of conjugated pairs of nuclei (dikaryophase). If amphigamy immediately follows
karyogamy, the process is referred to as amphimixis (Renner, 1916); and (2) the normal
fertilization process (Battaglia, 1947). Therefore, we considered correcting the term to
‘amphigameticity’ to indicate the ability of interspecific hybrid males and females to
produce gametes of both parental species.
The gain and loss during diverse gamete formation
To establish successful hemiclonal genome propagation, hybrid organisms must adapt
gametogenesis accordingly. The F1 hybrids of P. ridibundus and P. lessonae showed
premeiotic genome elimination and endoreplication, rescuing their fertility (Tunner &
Heppich-Tunner, 1991;Dedukh et al., 2019). However, premeiotic genome elimination and
endoreplication do not occur in all populations of germ cells, causing unusual pairing in
meiosis and abruption of gamete formation, thereby decreasing fertility in otherwise vital
individuals (Vorburger, 2001;Dedukh et al., 2015;Dedukh et al., 2019;Dedukh et al., 2020;
Doležálková et al., 2016). Reported cases of genome elimination and/or endoreplication
failure cause the formation of aneuploid cells during mitosis and meiosis (Fig. 3,Fig.
S3). Not all changes in genome elimination and endoreplication machinery harm the
reproduction of hybrid frogs. At least one hybrid male from Eastern Ukraine potentially
produced diploid spermatozoa with LL, RL, and RR genomes. The formation of diploid
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 13/21
gametes is crucial for the emergence of triploid hybrids in some population systems (Tunner
& Heppich-Tunner, 1992;Brychta & Tunner, 1994;Rybacki & Berger, 2001;Mikulícek &
Kotlík, 2001;Pruvost et al., 2015).
We stress that hybrids have an additional challenge in the selective elimination of P.
ridibundus genome. During the initial crossing of P. ridibundus and P. lessonae, hybrids
usually transmit the P. ridibundus genome and eliminate P. lessonae (Berger, 1971;Dedukh
et al., 2019). Subsequent backcrosses of diploid hybrids with P. lessonae individuals ensure
the maintenance of hybrids and lead to the formation of a mixed population of hybrids
and P. lessonae (Berger, 1971;Günther, 1983;Christiansen & Reyer, 2009). Hybridogenetic
reproduction of hybrid frogs in this population type is characterized by stable propagation
of P. ridibundus genome with relatively rare aberrations in genome elimination and
endoreplication (Berger, 1971;Graf & Müller, 1979;Pruvost, Hoffmann & Reyer, 2013;
Dedukh et al., 2019). Surprisingly, a growing number of evidence shows that also hybrid
frogs in a mixed population with P. ridibundus produced mostly R gametes and/or L
gametes (this study; Uzzell, Günther & Berger, 1977;Graf & Polls-Pelaz, 1989;Vinogradov
et al., 1991;Dedukh et al., 2015;Dedukh et al., 2017;Biriuk et al., 2016, for the exceptions
see Doležálková-Kaštánková et al., 2021), although the L gametes are the crucial cells
for the hybrid’s persistence (Fig. S3C). As haploid gametes with P. ridibundus genome
would not lead to hybrid progeny when coexisting with P. ridibundus, it is clear that
these hybrids have to under absence of P. lessonae produce fertile P. lessonae gametes to
perpetuate themselves. Obvious difficulties in forming gametes with P. lessonae genome
may explain why mixed populations of hybrids and P. ridibundus are rare over continental
Europe compared to mixed hybrid populations with P. lessonae (Uzzell, Günther & Berger,
1977;Graf & Polls-Pelaz, 1989;Plötner, 2005). For example, the evolutionary origin of P.
ridibundus–P. esculentus male populations in Central Europe seems to be rare event in the
past time, as clonally inherited lessonae genomes share their ancestors (Doležálková et al.,
2016;Doležálková-Kaštánková et al., 2018;Doležálková-Kaštánková et al., 2021).
In this light of the evidence, diploid hybrid males persisting within the R-E system in
Eastern Europe in high numbers over decades of observation (Borkin et al., 2004;Shabanov
et al., 2020) remains unclear. As hybrid males produce mainly a mixture of R and L genomes
(Fig. 3,Fig. S3), while female and co-occurring triploid hybrids with the RRL genotype
produce R and RL gametes, the proportion of hybrids that received P. lessonae gametes is
expected to be lower than observed. Moreover, long-term clonal propagation of the genome
may theoretically lead to the accumulation of deleterious mutations, thus decreasing the
survival of hybrids (Tunner & Heppich-Tunner, 1991;Christiansen et al., 2005;Christiansen,
2009;Dubey et al., 2019). The maintenance of these hybrid males may explain different
competition rates between P. esculentus and P. ridibundus tadpoles (Berger, 1977;Hotz et
al., 1999), or a general selection against parental genotypes (Reyer, Arioli-Jakob & Arioli,
2015).
CONCLUSION
We found diverse pathways of hybridogenetic reproduction in diploid hybrid males
from Eastern Ukraine. Investigating gametogenesis, we observed one or another parental
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 14/21
genome elimination followed by endoreplication of the remaining genome in diverse
germ cell populations. These pathways resulted in the simultaneous formation of gametes
with P. ridibundus and P. lessonae genomes in most males. We found these males crucial
for the hybrid’s persistence in these populations because they are the only ones able to
form P. lessonae gametes. However, genome elimination and endoreplication have not
always occurred correctly, resulting in aneuploidy and the abruption of meiosis in some
spermatocytes. We find the gametogenic diversity as the key evolutionary force producing
a variety of gametes with different genome compositions and ploidy levels, maintaining
these populations in particular and increasing global vertebrate diversity in general.
ACKNOWLEDGEMENTS
The authors thank Olexii Korshunov for his help in frog collecting and Olha Biriuk
for providing critical comments during the preliminary manuscript preparation, Anna
Fedorova for her support and help at different stages of work. We are also grateful to the
stuff of the Laboratory of Amphibian Population Ecology and students of VN Karazin
National University who helped with animal care.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
Lukáš Choleva, Eleonora Pustovalova, Dmitrij Dedukh were funded by the Czech Science
Foundation (Grantová Agentura Cbreveeské Republiky; project no. 21-25185S), and IAPG,
AS CR, v.v.i Institutional Research Concept RVO67985904 (Ústav živočišnéfyziologie a
genetiky Akademie věd České republiky, v.v.i). Lukáš Choleva, Dmitrij Dedukh were
funded by the Czech Science Foundation (grant no. GA19-24559S). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Czech Science Foundation: 21-25185S, GA19-24559S.
IAPG, AS CR, v.v.i Institutional Research Concept RVO67985904.
Competing Interests
The authors declare there are no competing interests.
Author Contributions
•Eleonora Pustovalova conceived and designed the experiments, performed the
experiments, analyzed the data, prepared figures and/or tables, and approved the
final draft.
•Lukaš Choleva conceived and designed the experiments, authored or reviewed drafts of
the article, and approved the final draft.
•Dmytro Shabanov conceived and designed the experiments, authored or reviewed drafts
of the article, and approved the final draft.
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 15/21
•Dmitrij Dedukh conceived and designed the experiments, performed the experiments,
prepared figures and/or tables, and approved the final draft.
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
The Committee on Bioethics of the V. N. Karazin Kharkiv National University (minutes
4, 21.04.2016) approved the study.
Data Availability
The following information was supplied regarding data availability:
The raw data are available in the Supplemental Files.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.13957#supplemental-information.
REFERENCES
Abbot R, Albach D, Ansell S, Arntzen JW, Baird SJE, Bierne N, Boughman J, Brelsford
A, Buerkle CA, Buggs R, Butlin RK, Dieckmann U, Eroukhmanoff F, Grill A,
Cahan SH, Hermansen JS, Hewitt G, Hudson AG, Jiggins C, Jones J, Keller B,
Marczewski T, Mallet J, Martinez-Rodriguez P, Möst M, Mullen S, Nichols R, Nolte
AW, Parisod C, Pfennig K, Rice AM, Ritchie MG, Seifert B, Smadja CM, Stelkens R,
Szymura JM, Väinölä R, Wolf JBW, Zinner D. 2013. Hybridization and speciation.
Journal of Evolutionary Biology 26:229–246 DOI 10.1111/j.1420-9101.2012.02599.x.
Arnold ML, Hodges SA. 1995. Are natural hybrids fit or unfit relative to their parents?
Trends in Ecology & Evolution 10(2):67–71 DOI 10.1016/S0169-5347(00)88979-X.
Battaglia E. 1947. Sulla terminologia dei processi apomittici. Nuovo Giornale Botanico
Italiano 54:674–696 DOI 10.1080/11263504709440462.
Berger L. 1970. Sex ratio in the F1 progeny within forms of Rana esculenta complex.
Genetica Polonica 12:87–101.
Berger L. 1971. Viability, sex and morphology of F2 generation within forms of Rana
esculenta complex. Zoologica Poloniae 21(4):345–393.
Berger L. 1977. Systematics and hybridization in the Rana esculenta complex. In: Taylor
DH, Guttman SI, eds. The reproductive biology of amphibians. Boston: Springer,
367–388 DOI 10.1007/978-1-4757-6781-0_12.
Berger L. 1983. Systematyka i systemy genetyczne zab zielonych Europy. Przegrad
Zoologiczny 28:47–61.
Bi K, Bogart JP. 2010. Probing the meiotic mechanism of intergenomic exchanges
by genomic in situ hybridization on lampbrush chromosomes of unisex-
ual Ambystoma (Amphibia: Caudata). Chromosome Research 18:371–382
DOI 10.1007/s10577-010-9121-3.
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 16/21
Biriuk OV, Shabanov DA, Korshunov AV, Borkin LJ, Lada GA, Pasynkova RA,
Rosanov JM, Litvinchuk SN. 2016. Gamete production patterns and mating systems
in water frogs of the hybridogenetic Pelophylax esculentus Complex in northeastern
Ukraine. Journal of Zoological Systematics and Evolutionary Research 54(3):215–225
DOI 10.1111/jzs.12132.
Birstein VJ. 1984. Localization of NORs in karyotypes of four Rana species. Genetica
64:149–154 DOI 10.1007/BF00115338.
Borkin LJ, Korshunov AV, Lada GA, Litvinchuk SN, Rosanov JM, Shabanov DA,
Zinenko AI. 2004. Mass occurrence of polyploid green frogs (Rana esculenta
Complex) in eastern Ukraine. Russian Journal of Herpetology 11:194–213
DOI 10.30906/1026-2296-2004-11-3-203-222.
Borodin PM, Rogatcheva MB, Zhelezova AI, Oda S. 1988. Chromosome pairing
in inter-racial hybrids of the house musk sherew (Suncus murinus, Insectivora,
Soricidae). Genome 41:79–90 DOI 10.1139/g97-103.
Brychta BH, Tunner HG. 1994. Flow cytometric analysis of spermatogenesis in triploid
Rana esculenta.Zoologica Poloniae 39:507.
Bucci S, Ragghianti M, Mancino GL, Hotz H, Uzzell T. 1990. Lampbrush and
mitotic chromosomes of the hemiclonally reproducing hybrid Rana escu-
lenta and its parental species. Journal of Experimental Zoology 255:37–56
DOI 10.1002/jez.1402550107.
Chmielewska M, Dedukh D, Haczkiewicz K, Rozenblut-Kościsty B, Kaźmierczak
M, Kolenda K, Serwa E, Pietras-Lebioda A, Krasikova A, Ogielska M. 2018. The
programmed DNA elimination and formation of micronuclei in germ line cells
of the natural hybridogenetic water frog Pelophylax esculentus.Scientific Reports
8(1):1–19 DOI 10.1038/s41598-018-26168-z.
Christiansen DG. 2009. Gamete types, sex determination and stable equilibria of all-
hybrid populations of diploid and triploid edible frogs (Pelophylax esculentus). BMC
Evolutionary Biology 9(1):135 DOI 10.1186/1471-2148-9-135.
Christiansen DG, Fog K, Pedersen BV, Boomsma JJ. 2005. Reproduction and hybrid
load in all-hybrid populations of Rana esculenta water frogs in Denmark. Evolution
59:1348–1361 DOI 10.1111/j.0014-3820.2005.tb01784.x.
Christiansen DG, Reyer HU. 2009. From clonal to sexual hybrids: genetic recombination
via triploids in all-hybrid populations of water frogs. Evolution 63(7):1754–1768
DOI 10.1111/j.1558-5646.2009.00673.x.
Coyne JA, Orr HA. 2004. Speciation. Sunderland: Sinauer Associates, Inc.
Dawley RM, Bogart JP. 1989. Evolution and ecology of unisexual vertebrates. Albany: New
York State Museum Publications.
Dedukh D, Krasikova A. 2021. Delete and survive: strategies of programmed
genetic material elimination in eukaryotes. Biological Reviews 97:195–216
DOI 10.1111/brv.12796.
Dedukh D, Litvinchuk J, Svinin A, Litvinchuk S, Rosanov J, Krasikova A. 2019.
Variation in hybridogenetic hybrid emergence between populations of water
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 17/21
frogs from the Pelophylax esculentus complex. PLOS ONE 14(11):e0224759
DOI 10.1371/journal.pone.0224759.
Dedukh D, Litvinchuk S, Rosanov J, Mazepa G, Saifitdinova A, Shabanov D, Krasikova
A. 2015. Optional endoreplication and selective elimination of parental genomes
during oogenesis in diploid and triploid hybrid European water frogs. PLOS ONE
10(4):e0123304 DOI 10.1371/journal.pone.0123304.
Dedukh D, Litvinchuk S, Rosanov J, Shabanov D, Krasikova A. 2017. Mutual
maintenance of di- and triploid Pelophylax esculentus hybrids in R-E systems:
results from artificial crossings experiments. BMC Evolutionary Biology 17:220
DOI 10.1186/s12862-017-1063-3.
Dedukh D, Riumin S, Chmielewska M, Rozenblut-Kościsty B, Kolenda K, Kaźmierczak
M, Dudzik A, Ogielska M, Krasikova A. 2020. Micronuclei in germ cells of hybrid
frogs from Pelophylax esculentus complex contain gradually eliminated chromo-
somes. Scientific Reports 10(1):1–13 DOI 10.1038/s41598-020-64977-3.
Doležálková M, Sember A, Marec F, Ráb P, Plötner J, Choleva L. 2016. Is pre-
meiotic genome elimination an exclusive mechanism for hemiclonal repro-
duction in hybrid males of the genus Pelophylax?BMC Genetics 17:100
DOI 10.1186/s12863-016-0408-z.
Doležálková-Kaštánková M, Mazepa G, Jeffries DL, Perrin N, Plötner M, Plötner J,
Guex GD, Mikulíček P, Poustka AJ, Grau J, Choleva L. 2021. Capture and return
of sexual genomes by hybridogenetic frogs provides clonal genome enrichment in a
sexual species. Scientific Reports 11(1):1–10 DOI 10.1038/s41598-021-81240-5.
Doležálková-Kaštánková M, Pruvost NBM, Plötner J, Reyer HU, Janko K, Choleva L.
2018. All-male hybrids of a tetrapod Pelophylax esculentus share its origin and genet-
ics of maintenance. Biology of Sex Differences 9:1–13 DOI 10.1186/s13293-018-0172-z.
Drohvalenko M, Pustovalova E, Fedorova A, Shabanov D. 2022. First finding of triploid
hybrid frogs Pelophylax esculentus (Anura: Ranidae) in Mozh river basin (Kharkiv
region, Ukraine). Biodiversity, Ecology and Experimental Biology 23(2):61–67
DOI 10.34142/2708-5848.2021.23.2.04.
Dubey S, Maddalena T, Bonny L, Jeffries DL, Dufresnes C. 2019. Population genomics
of an exceptional hybridogenetic system of Pelophylax water frogs. BMC Evolutionaty
Biology 19:164 DOI 10.1186/s12862-019-1482-4.
Dufresnes C, Mazepa G. 2020. Hybridogenesis in water frogs. eLS 1:718–726
DOI 10.1002/9780470015902.a0029090.
Graf JD, Müller WP. 1979. Experimental gynogenesis provides evidence of hybrido-
genetic reproduction in the Rana esculenta complex. Experientia 35:1574–1576
DOI 10.1007/BF01953200.
Graf JD, Polls-Pelaz M. 1989. Evolutionary genetics of the Rana esculenta Complex.
In: Dawley RM Bogart JP, eds., ed. Evolution and ecology of unisexual vertebrates.
Albany: New York State Museum Publications, 289–302.
Günther R. 1983. Zur populationsgenetik der mitteleuropäischen wasserfrösche des Rana
esculenta—synkleptons (Anura, Ranidae). Zoologischer Anzeiger 211(1/2):43–54.
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 18/21
Günther R, Uzzell T, Berger L. 1979. Inheritance patterns in triploid Rana esculenta
(Amphibia, Salientia). Mitteilungen des Zoologischen Museums Berlin 55:35–57.
Heppich S, Tunner HG, Greilhuber J. 1982. Premeiotic chromosome doubling after
genome elemination during spermatogenesis of the species hybrid Rana esculenta.
Theoretical and Applied Genetics 61:101–104 DOI 10.1007/BF00273874.
Tunner HG, Heppich S. 1981. Premeiotic genome exclusion during oogenesis in the
common edible frog, Rana esculenta. Die Naturwissenschaften 68(4):207–208
DOI 10.1007/BF01047207.
Hoffman A, Plötner J, Pruvost NBM, Christiansen DG, Röthlisberger S, Choleva L,
Mikulíček P, Cogălniceanu D, Sas-Kovács I, Shabanov D, Morozov-Leonov S,
Reyer HU. 2015. Genetic diversity and distribution patterns of diploid and polyploid
hybrid water frog populations (Pelophylax esculentus complex) across Europe.
Molecular Ecology 24:4371–4391 DOI 10.1111/mec.13325.
Hotz H, Semlitsch RD, Gutmann E, Guex GD, Beerli P. 1999. Spontaneous het-
erosis in larval life-history traits of hemiclonal frog hybrids. Proceedings of the
National Academy of Sciences of the United States of America 96(5):2171–2176
DOI 10.1073/pnas.96.5.2171.
Ishishita S, Tsuboi K, Ohishi N, Tsuchiya K, Matsuda Y. 2015. Abnormal pairing of
X and Y sex chromosomes during meiosis I in interspecific hybrids of Phodopus
campbelli and P. sungorus.Scientific Reports 5(1):1–9 DOI 10.1038/srep09435.
Lenormand T, Engelstadter J, Johnston SE, Wijnker E, Haag CR. 2016. Evolutionary
mysteries in meiosis. Philosophical Transactions of the Royal Society B: Biological
Sciences 371(1706):20160001 DOI 10.1098/rstb.2016.0001.
Mallet J. 2007. Hybrid speciation. Nature 446:279–283 DOI 10.1038/nature05706.
Mazepa G, Doležálková M, Choleva L, Plötner J, Biriuk O, Drohvalenko M, Korshunov
O, Shabanov D, Wolf J, Perrin N. 2018. Distinct fate of the asexual genomes in
two convergently evolved Pelophylax hybridogenetic systems. In: Sex uncovered: the
evolutionary biology of reproductive systems. Roscoff: Inserm, 57.
McKee BD. 2004. Homologous pairing and chromosome dynamics in meiosis and mito-
sis. Biochimica et Biophysica Acta 1677:165–180 DOI 10.1016/j.bbaexp.2003.11.017.
Mikulícek P, Kotlík P. 2001. Two water frog populations from western Slovakia consist-
ing of diploid females and diploid and triploid males of the hybridogenetic hybrid
Rana esculenta (Anura, Ranidae). Mitteilungen aus dem Museum fuer Naturkunde in
Berlin Zoologische Reihe 77:59–64 DOI 10.1002/mmnz.20010770110.
Neaves WB, Baumann P. 2011. Unisexual reproduction among vertebrates. Trends in
Genetics 27(3):81–88 DOI 10.1016/j.tig.2010.12.002.
Ogielska M. 1994. Nucleus-like bodies in gonial cells of Rana esculenta [Amphibia,
Anura] tadpoles-a putative way of chromosome elimination. Zoologica Poloniae
39:3–4.
Ogielska M, Bartmańska J. 1999. Development of testes and differentiation of germ cells
in water frogs of the Rana esculenta-complex (Amphibia, Anura). Amphibia-Reptilia
20:251–263 DOI 10.1163/156853899X00286.
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 19/21
Plötner J. 2005. Die westpaläarktischen Wasserfrösche: von Märtyrern der Wissenschaft zur
biologischen Sensation. Bielefeld: Laurenti.
Pruvost NBM, Hoffmann A, Reyer HU. 2013. Gamete production patterns, ploidy,
and population genetics reveal evolutionary significant units in hybrid water frogs
(Pelophylax esculentus). Ecology and Evolution 3(9):2933–2946 DOI 10.1002/ece3.687.
Pruvost NBM, Mikulíček P, Choleva L, Reyer HU. 2015. Contrasting reproductive
strategies of triploid hybrid males in vertebrate mating systems. Journal of Evolution-
ary Biology 28(1):189–204 DOI 10.1111/jeb.12556.
Ragghianti M, Bucci S, Marracci S, Casola C, Mancino G, Hotz H, Guex GD, Plötner J,
Uzzell T. 2007. Gametogenesis of intergroup hybrids of hemiclonal frogs. Genetics
Research 89:39–45 DOI 10.1017/S0016672307008610.
Ragghianti M, Guerrini F, Bucci S, Mancino G, Hotz H, Uzzell T, Guex GD. 1995.
Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid
frog Rana esculenta and parental species. Chromosome Research 3(8):497–506
DOI 10.1007/BF00713965.
Renner O. 1916. Zur Terminologie des pflanzlichen Generationswechsels. Biologisches
Zentralblatt 36:337–374.
Reyer HU, Arioli-Jakob C, Arioli M. 2015. Post-zygotic selection against parental
genotypes during larval development maintains all-hybrid populations of the frog
Pelophylax esculentus.BMC Evolutionary Biology 15(1):1–16
DOI 10.1186/s12862-015-0404-3.
Rieger R, Michaelis A, Green MM. 1991. Glossary of genetics classical and molecular. 5th
edn. Berlin Heidelberg New York: Springer.
Rieseberg LH. 2001. Chromosomal rearrangements and speciation. Trends in Ecology &
Evolution 16(7):351–358 DOI 10.1016/S0169-5347(01)02187-5.
Rybacki M, Berger L. 2001. Types of water frog populations (Rana esculenta complex) in
Poland. Mitteilungen aus dem Museum für Naturkunde in Berlin. Zoologische Reihe
77:51–57 DOI 10.1002/mmnz.20010770109.
Salas N, Valetti J, Grenat P, Otero M, Martino A. 2014. Meiotic behavior of two
polyploid species of genus Pleurodema (Anura: Leiuperidae) from central Argentina.
Acta Herpetologica 9(1):109–113 DOI 10.1002/mmnz.20010770109.
Schön I, Martens K, Van Dijk P. 2009. Lost sex. In: The evolutionary biology of partheno-
genesis. Heidelberg: Springer.
Shabanov D, Vladymyrova M, Leonov A, Biriuk O, Kravchenko M, Mair Q, Meleshko
O, Newman J, Usova O, Zholtkevych G. 2020. Simulation as a Method for
Asymptotic System Behavior Identification (e.g., Water Frog Hemiclonal Population
Systems). In: Information and communication technologies in education, research, and
industrial applications. ICTERI 2019. Communications in Computer and Information
Science. 1175. Cham: Springer DOI 10.1007/978-3-030-39459-2_18.
Stöck M, Dedukh D, Reifová R, Lamatsch DK, Starostová Z, Janko K. 2021. Sex
chromosomes in meiotic, hemiclonal, clonal and polyploid hybrid vertebrates: along
the ‘extended speciation continuum’. Philosophical Transactions of the Royal Society B
376(1833):20200103 DOI 10.1098/rstb.2020.0103.
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 20/21
Svinin AO, Dedukh DV, Borkin LJ, Ermakov OA, Ivanov AY, Litvinchuk JS, Zamalet-
dinov RI, Mikhaylova RI, Trubyanov AB, Skorinov DV, Rosanov YM, Litvinchuk
SN. 2021. Genetic structure, morphological variation, and gametogenic peculiarities
in water frogs (Pelophylax) from northeastern European Russia. Journal of Zoological
Systematics and Evolutionary Research 59(3):646–662
DOI 10.1111/jzs.12447.
Svinin AO, Litvinchuck SN, Borkin LJ, Rosanov JM. 2013. Distribution and population
system types of green frogs (Pelophylax Fitzinger, 1843) in Mari El Republic. Current
Study of Herpetology 13(3/4):137–147.
Torgasheva AA, Borodin PM. 2016. Cytological basis of sterility in male and female
hybrids between sibling species of grey voles Microtus arvalis and M. levis.Scientific
Reports 6:36564 DOI 10.1038/srep36564.
Tunner HG. 1973. Demonstration of the hybrid origin of the common green frog Rana
esculenta.Naturwissenschafte 60:481–482 DOI 10.1007/BF00592872.
Tunner H. 1974. Die klonale Struktur einer Wasserfröschpopulation. Journal of Zoologi-
cal Systematics and Evolutionary Research 12:309–314.
Tunner H, Heppich-Tunner S. 1991. Genome exclusion and two strategies of chro-
mosome duplication in oogenesis of a hybrid frog. Naturwissenschaften 78:32–34
DOI 10.1007/BF01134041.
Tunner H, Heppich-Tunner S. 1992. A new population system of water frogs discovered
in Hungary. Proceedings of the Sixth Ordinary General Meeting of the Societas
Europaea Herpetologica 19-23:453–460.
Uzzell T, Günther R, Berger L. 1977. Rana ridibunda and Rana esculenta: a leaky
hybridogenetic system (Amphibia, Salientia). Proceedings of the Academy of Natural
Sciences of Philadelphia 128:147–171.
Vinogradov AE, Borkin LJ, Günther R, Rosanov JM. 1991. Two germ cell lineages with
genomes of different species in one and the same animal. Hereditas 114(3):245–251
DOI 10.1111/j.1601-5223.1991.tb00331.x.
Vorburger C. 2001. Non-hybrid offspring from matings between hemiclonal hybrid
waterfrogs suggest occasional recombination between clonal genomes. Ecology Letters
4:628–636 DOI 10.1046/j.1461-0248.2001.00272.x.
Zong E, Fan G. 1989. The variety of sterility and gradual progression to fertility in
hybrids of the horse and donkey. Heredity 62(3):393–406 DOI 10.1038/hdy.1989.54.
Pustovalova et al. (2022), PeerJ, DOI 10.7717/peerj.13957 21/21
