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Zoological Journal of the Linnean SocietyZoologyPeer-reviewed journal article

Hybridogenetic reproduction of Pelophylax water frogs from different R-E hemiclonal population systems from Eastern Ukraine: selective mortality, clonal and ploidy diversity

Anna FedorovaiD, Eleonora PustovalovaiD, Mykola DrohvalenkoiD, Olha BiriukiD, Marie Doležálková‐KaštánkováiD, Maryna Kravchenko, Olexii Korshunov, Peter MikulíčekiD, Lukáš CholevaiD, Dmytro Holovnia, Dmitrij DedukhiD, Dmytro ShabanoviD

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Abstract

European water frogs from the Pelophylax esculentus complex include two sexual species, P. ridibundus and P. lessonae, and their hybrids, which usually clonally transmit one of the parental species genomes. This unique reproductive strategy allows hybrids to reproduce with one or both parental species, creating diverse population systems. Unlike most well-studied population systems in Europe, the Siverskyi Donets River basin in Eastern Ukraine features diploid and polyploid hybrids coexisting with P. ridibundus, while P. lessonae is absent (R-E systems). To reveal diverse system compositions, genetic divergence, and tadpole selective mortality, we combined novel data from over a decade of observations with previous research on population systems in the Siverskyi Donets River. Two main types of R-E systems were identified: those with diploid hybrids in northern localities and those with both diploid and triploid hybrids, extending from the mainstream of the Siverskyi Donets River to its tributaries. Additionally, we found higher genetic diversity in R-genomes compared to L-genomes, likely due to the absence of P. lessonae and the ongoing input of recombined R-genomes from P. ridibundus and triploid hybrids. This study highlights the importance of continuous monitoring and research to unravel the dynamics and complexity of water frog population systems.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (6 Dec 2024) · CC BY 4.0

Siverskyi Donets water frog systems lacking P. lessonae split into diploid-only and diploid-plus-triploid hybrid types, with clonal, low-diversity L-genomes and steep tadpole-to-adult loss of P. ridibundus.

Central European R-E systems hold only diploid hybrid males with P. ridibundus, whereas the Siverskyi Donets basin hosts diploid and triploid hybrids of both sexes, yet these systems had not been examined with combined molecular and cytogenetic methods. The study maps system types, measures clonal diversity of each parental genome, and tests which offspring genotypes survive to adulthood.

Key results

  1. ShownNo P. lessonae was found across 14 Siverskyi Donets systems. Hybrids dominated, and the systems split into a diploid-hybrid-only type (R-E) and a diploid-plus-triploid type (R-E-Ep). Triploids were restricted to the main river and the Mozh tributary basin.

    1848 new plus 519 published adults: 227 P. ridibundus, 2140 P. esculentus (1926 diploid, 214 triploid, both LRR and LLR); eight R-E and six R-E-Ep systems; diploids prevailed in 13 of 14; triploids ranged 2% to 14%; the Udy and Oskil tributaries held only diploid hybrids. · Compositions and dynamics of HPSs; HPSs compositions and dynamics in the Siverskyi…

  2. ShownIn the Siverskyi Donets basin, where P. lessonae is absent, hybrid L-genomes were much less diverse than R-genomes. In the Dnipro outgroup basin, where P. lessonae occurs, the two genomes had similar diversity.

    193 frogs from 15 localities, 12 of 14 microsatellite loci retained; 37 L-genome vs 77 R-genome alleles overall. Siverskyi Donets: L-genome Na ~4 times and He ~2.8 times lower than R-genome (p=0.0086). Dnipro: both indexes only ~1.1 times lower for L-genomes (p=0.7914). · Genetic diversity; Genetic diversity and clonal lineages in the…

  3. ShownHybrid L-genomes formed many shared multilocus genotypes (clonal lineages), all in Siverskyi Donets frogs, while R-genomes were almost all unique. This fits multiple L-genome clonal origins and a steady input of recombined R-genomes.

    158 L-genomes (153 frogs): 52 MLGs, 17 shared by two to 27 individuals; all Dnipro MLGs unique. 187 R-genomes (185 frogs): 180 MLGs, one shared by five frogs and three by two each. Probability of identity 1.3×10^-5 (P. lessonae) and 7.8×10^-7 (P. ridibundus). · Population genetic analysis based on…; Clonal lineages; Genetic diversity and clonal lineages in the…

  4. ShownIn all three sampled systems, P. ridibundus was much more common among tadpoles and froglets than among adults, consistent with selective loss of hybridolytic parental-species offspring. No P. lessonae juveniles were detected.

    DOB (96) and KOR (39) tadpoles: 75 P. ridibundus, 44 diploid and 16 triploid hybrids. ISK froglets (31): 21 P. ridibundus (all female), 10 diploid hybrids. The P. ridibundus share was lower in adults in every system (p<0.0001); the triploid share differed only in KOR (p=0.0172; ISK p=0.6216, DOB p=0.0850). · Genetic composition and selective mortality of…; Selective mortality of progeny

  5. SuggestedAcross years of monitoring, diploid hybrids were the leading form every year in each R-E-Ep system while triploid and P. ridibundus shares fluctuated. Separately, the spatial survey plus earlier data define three regions: northern R-E, R-E-Ep along the river, and eastern R-Epf.

    DOB over nine years: diploids 62% to 85%, triploids 7% to 26%, P. ridibundus 4% to 21%. ISK over eight years: diploids 82% to 96%, triploids 0% to 10%. ZAM triploids up to 29% in 2013. Across five systems, P. ridibundus ranged 0% to 23%. · Compositions and dynamics of HPSs; HPSs compositions and dynamics in the Siverskyi…

Methods, data and reproducibility

Adults, froglets and tadpoles sampled from 2014 to 2024 were typed by morphology, erythrocyte-length cytometry, Ag-NOR staining, DAPI pericentromeric staining and two-probe FISH (RrS1, PlesSat01-48). Microsatellite genotyping gave per-genome diversity (Na, He) and multilocus-genotype clonal lineages; juvenile and adult genotype ratios were compared.

  • Adult water frogs newly sampled 2014 to 2024 from 14 Siverskyi Donets basin waterbodies, Eastern Ukraine · Pelophylax ridibundus; P. esculentus hybrids · n = 1848 adults
  • Outgroup adults from four Dnipro basin waterbodies (Seym, Udai, Psel and Merla tributary basins) · Pelophylax ridibundus, P. lessonae, P. esculentus · n = 40 adults
  • Previously published frogs from four of the 14 Siverskyi Donets systems, pooled for composition analysis · Pelophylax ridibundus; P. esculentus hybrids · n = 519
  • Tadpoles from DOB, KOR and ISK typed by microsatellites (DOB, ISK) or DAPI staining plus FISH (KOR) · Pelophylax ridibundus; P. esculentus hybrids · n = 140 tadpoles (96 DOB, 39 KOR, five ISK)
  • Froglets (post-metamorphic juveniles before first hibernation) from ISK, karyotyped and FISH-typed · Pelophylax ridibundus; P. esculentus hybrids · n = 31 froglets
  • Adults genotyped at 14 microsatellite loci (12 retained) from 15 localities (11 Siverskyi Donets, four Dnipro) · Pelophylax esculentus complex · n = 193 adults
  • Adult blood smears measured for erythrocyte length to separate diploid from triploid hybrids · Pelophylax esculentus complex · n = 1713 adults
  • Individuals Ag-stained for nucleolus organizer regions to refine ploidy calls · Pelophylax esculentus complex · n = 264 (88 froglets, 140 tadpoles, 36 adults)
Methods & tools
Morphological identification (metatarsal tubercle, thigh and vocal sac coloration, spotting), Erythrocyte-length cytometry for ploidy, Ag+ staining of nucleolus organizer regions (NORs), DAPI staining of pericentromeric loci, FISH with species-specific probes RrS1 and PlesSat01-48, Multiplex PCR microsatellite genotyping (14 loci adults, 18 loci tadpoles), GeneMarker (SoftGenetics) allele scoring, GenAlEx 6.5 for MS Excel, QGIS 3.34.1-Prizren, NucleoSpin Tissue kit (Macherey-Nagel); Geneaid DNA Isolation Kit, Gosner (1960) tadpole staging, Colchicine-based chromosome preparation (Pustovalova et al. 2022a protocol)
Reagents & environment
QGIS 3.34.1-Prizren (maps); GenAlEx 6.5 for MS Excel (diversity indexes, MLGs, probability of identity); GeneMarker (SoftGenetics) for allele scoring.
Reproducibility gaps
The paper cites no code or data repository. Per-locality counts, locus details and MLG tables are in supplementary Tables S1 to S4, which are not included in the text.
Limitations (6)
  • Two systems (LOZ, GRZ) had only n=2 and n=3 frogs, so their R-E status is inferred from neighbouring sites. Nine localities were surveyed only once, so their compositional dynamics could not be described. · stated by the authors
  • Temporal shifts in taxon ratios are hard to pin down because habitat conditions vary, sample sizes are limited and sampling errors occur. Pond drying and repeated introgression may also drive the variation. · stated by the authors
  • The earlier malformation data for DOB metamorphs lacked species identification, so it is unknown whether anomalies drive mortality of specific genotypes. · stated by the authors
  • The authors note it is still not understood why offspring viability drops, irrespective of whether clonal or recombined parental genomes are inherited. · stated by the authors
  • The juvenile-adult comparisons are cross-sectional, and juveniles were typed differently per site (microsatellites for DOB tadpoles, DAPI/FISH for KOR tadpoles, karyotyping/FISH for ISK froglets). The test behind the p-values and the 'significant' year-to-year changes is not described. · noted in this summary
  • Some counts do not reconcile: 17 shared plus 37 unique L-genome MLGs versus 52 reported, and 88 Ag-stained froglets versus 31 froglets collected. Check the supplementary tables before reusing these numbers. · noted in this summary

Read this if you work on hybridogenesis, hemiclonal inheritance or polyploidy in Pelophylax water frogs and want decade-long field, cytogenetic and microsatellite data on R-E systems in Eastern Ukraine.

Citations
5
FWCI
1.75
Field percentile
top 16%

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