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Geographical Distribution of Triploidy in Natural Populations of European Hybrid Green Frogs Rana esculenta and R. grafi, and Methods of Its Identification (occasional occurrences of triploids are not included)
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In eastern Ukraine, the Rana esculenta complex consists of three species: R. lessonae, R. ridibunda, and hybrid R. esculenta. The first one was rare, whereas two latter frog taxa were very common. Based on DNA flow cyto-metry, mass occurrence of the triploidy in Rana esculenta has been revealed in 14 localities of Kharkov, Do-netsk, and Lugansk Pro...
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... an enigmatic situation is without serious ex- planation. Indeed, triploids were often revealed in many localities of northern France, the Netherlands, northern Germany, Denmark, southern Sweden, Poland, western Slovakia, and western Hungary (Table 5). However, triploids are virtually absent in surveyed areas of Aus- tria, Italy, ex-Yugoslavia, and the former Soviet Union ( Graf and Polls Pelaz, 1989a). ...
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... authors applied different techniques to check ploidy level in green frog samples (Table 5). To evaluate the reliability and importance of the published data, we give the brief comparison of methods. ...
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... tantly, such an approach allows working with animals in the field and laboratory, without killing them. Therefore, it was no wonder that this very easy and cheap tool has been widely accepted as a good indicator of ploidy level, and the majority of triploidy records was evidenced by erythrocye size only (Table 5). ...
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... fact, in sev- eral studies, morphological identification did not corre- spond with electrophoretic data, and geographic varia- tion in external traits has been suggested (e.g., Hemmer, 1977;Regnier and Neveu, 1986;Günther et al., 1991). Rybacki (1995aRybacki ( , 1995b provided the ranges of DP/CI and T/CI indices, with obvious overlap between diploid and triploid R. esculenta, as well as triploids with R. ri- dibunda (for LR and LLR also see Table 5 in Berger and Truszkowski, 1980). According to Schröer (1997), the results of this method varied strongly in different populations, and in- creased overlap between R. lessonae, diploid and tri- ploid R. esculenta was observed (Schröer, 1996). ...
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Citations
... The other type, R-E HPS (P. esculentus coexists with P. ridibundus) was described from Germany, Poland, and the Czech Republic (Uzzell et al., 1977;Günther and Lubcke, 1979;Günther and Plötner, 1988;Rybacki and Berger, 2001;Doležálková-Kaštánková et al., 2018) and further found to be widespread in Eastern Europe (Borkin et al., 2004;Dufresnes and Mazepa, 2020). In the R-E HPS, most hybrids transmit the (L)genome but diploid hybrids here often produce a mix of gametes with either (L)-or (R)genome Doležálková et al., 2016;Pustovalova et al., 2022). ...
... Our study concerns the region of the Siverskyi Donets River basin (Eastern Ukraine), which received the name "Siverskyi Donets center of water frog diversity" for its great diversity of HPSs types (Shabanov et al., 2009). No adult individuals of P. lessonae have ever been recorded in this region (Borkin et al., 2004;Biriuk et al., 2016;Shabanov et al., 2020). All L-genomes here are provided by diploid and triploid P. esculentus individuals. ...
... The estimated mean number of L-specific alleles (Na) in the L-genome was 3.38 times lower than the mean number of R-specific alleles in the R-genome. Such a low diversity of the L-genome in the studied hybridogenetic lineages is likely associated with the absence of P. lessonae in the Siverskyi Donets River basin (Borkin et al., 2004;Biriuk et al., 2016). The diversity observed in the L-genome of hybrids may reflect the diversity of P. lessonae individuals involved in primary hybridization events whose genes were "frozen" in hybridogenetic lineages. ...
Pelophylax esculentus (genotype LR), an interspecific hybrid of P. ridibundus (genotype RR) and P. lessonae (genotype LL), is one of the most studied examples of hemiclonal hybridogenic vertebrates. In most populations, hybrids produce clonal gametes of one parental species and backcross with the other parental species, forming a new generation of hybrids. In this study, we focused on the population system of water frogs in the Siverskyi Donets river basin (eastern Ukraine) with the aim to determine the genetic diversity of parental genomes and clarify how such a system is maintained in nature. Of the 55 frogs analyzed, only one individual was P. ridibundus, 48 belonged to diploid LR and five to triploid LRR hybrids. Using 16 microsatellite loci, we found significantly lower genetic diversity and significantly higher clonality in the L-genome of hybrids compared to the R-genome. This contrasting diversity might be attributed to the absence of P. lessonae in the studied region and the clonal inheritance of the L-genome. Our population-genetic results are partially inconsistent with previous studies focusing on hybrid gametogenesis in the Siverskyi Donets river basin, according to which hybrids form both clonal R and L gametes. We hypothesize that discrepancies between the type of clonally formed gametes and the genotypic composition of the studied population may be due to the selective mortality of some genotypes during larval development and metamorphosis. Selective mortality thus may play an important role in the dynamics of the hybridogenetic systems in eastern Ukraine.
... Наши исследования подтвердили, что использование этого метода позволяет надежно определить плоидность у каждой из изученных особей скальных ящериц. Такие же результаты ранее нами были получены при изучении диплоиднополиплоидных комплексов, например, у амфибий (Borkin et al., 2004;Litvinchuk et al., 2010;Biriuk et al., 2016;Литвинчук и др., 2018, 2019. ...
The study of genome size variation in cells of vertebrates using the DNA flow cytometry makes it possible to precisely determine polyploid individuals, which is extremely important when studying the processes of reticulate speciation. In addition, in many groups of vertebrates, closely related species often differ in the nuclear DNA content. Therefore, the purpose of our study was to explore the variability of genome size and ploidy in populations of 29 species and subspecies of mountain lizards of the genus Darevskia, as well as their hybrids. As a result of the study, the range of variability in individuals of different ploidy (91% of diploid and 9% of triploid individuals) was established not to overlap. Among diploid species, no correlation was found between the nuclear DNA content and phylogenetic relationships, geographic coordinates, altitude, average annual temperatures and precipitation. Representatives of all studied species complexes (with the exception of D. adjarica) had approximately the same limits of variability. Two species (D. derjugini and D. saxicola) show significant intraspecific variability. Comparisons of the genome size of parthenogenetic and bisexual species generally revealed no noticeable differences between them. Studies of triploid hybrids have shown that their genome size as a whole roughly corresponds to the sum of the average size of the diploid genome of the maternal parthenogenetic species and the haploid genome of the paternal species. The variability of genome sizes within samples of triploid hybrids was on average slightly higher than in most parthenogenetic species, but some lower than in bisexual species. The paper discusses peculiarities of reticulate speciation in this group of animals.
... system have been documented in Eastern Ukraine [33][34][35][36][37] and Central Europe [38][39][40][41][42][43] . Eastern Ukrainian populations of P. esculentus include diploid and triploid individuals of both sexes 33,36,37,44 , while in Central Europe, populations only comprise diploid hybrid males coexisting with P. ridibundus. ...
... system have been documented in Eastern Ukraine [33][34][35][36][37] and Central Europe [38][39][40][41][42][43] . Eastern Ukrainian populations of P. esculentus include diploid and triploid individuals of both sexes 33,36,37,44 , while in Central Europe, populations only comprise diploid hybrid males coexisting with P. ridibundus. A common feature of the R-E system is the presence of hybrid males exhibiting two types of gametogenesis. ...
Gametogenesis produces gametes as a piece of genetic information transmitted to the offspring. While during sexual reproduction, progeny inherits a mix of genetic material from both parents, asexually reproducing organisms transfer a copy of maternal or paternal DNA to the progeny clonally. Parthenogenetic, gynogenetic and hybridogenetic animals have developed various mechanisms of gametogenesis, however, their inheritance is not fully understood. Here, we focused on the inheritance of asexual gametogenesis in hybrid Pelophylax esculentus (RL), emerging after crosses of P. lessonae (LL) and P. ridibundus (RR). To understand the mechanisms of gametogenesis in hybrids, we performed three-generation experiments of sexual P. ridibundus females and hybrids from all-male hybrid populations. Using fluorescent in situ hybridization, micronuclei analysis, flow cytometry and genotyping, we found that most adult hybrid males simultaneously produced two types of clonal sperm. Also, most male tadpole progeny in two successive backcrossed generations simultaneously eliminated L and R parental genomes, while some progeny produced only one type of sperm. We hypothesize that the reproductive variability of males producing two kinds of sperm is an adaptive mechanism to reproduce in mixed populations with P. ridibundus and may explain the extensive distribution of the all-male lineage across the European River Basin.
... The spatial heterogeneity of the water frog hybridization zone is its universal feature. It is characterized by the alternation of regions with populations containing hybrids having different types of gametogenesis (Christiansen et al. 2005;Morozov-Leonov et al. 2009), ploidy levels (Borkin et al. 2004;Christiansen et al. 2005;Mezhzherin et al. 2010;Vorburger 2001a) and sex composition Mezhzherin et al. 2010). Most of the hybridization zone is occupied by populations with an allodiploid structure of hybrids, bisexual hybrids in approximately equal proportions, producing haploid gametes with a non-recombined P. ridibundus genome. ...
Reproduction of water frog hybrids Pelophylax esculentus (Pelophylax ridibundus x Pelophylax lessonae) is associated with hemiclonal reproduction and backcrossing. The hemiclonal mode of reproduction occurs within P. esculentus allodiploids. In this case, the unrecombined genome of one parental species is transmitted to the offspring after premeiotic elimination of the chromosome set of the second parental species. Usually, the chromosome set of P. lessonae is eliminated, and the altered genome of P. ridibundus is passed on to the progeny. The hemiclonal inheritance within diploid Pelophylax esculentus hybrids may be accompanied by certain aberrations of premeiotic elimination. As a result, the formation of P. ridibundus specimens with introgressions of the P. lessonae genetic material, or the formation of recombinant hybrids occurs, depending on which of the parental species backcrossing takes place. The aim of our study is to describe the aberration of premeiotic elimination within the water frog P. esculentus complex detected by the nuclear gene Ldh-B inheritance, with an attempt to find out the causes of this phenomenon. It has been established that aberrations of premeiotic elimination are widespread, but only within populations of water frog from the river system of Upper Dnieper within Ukraine. The highest level of introgression takes place in the water frog populations within Kiev metropolis under conditions of expressed anthropogenization, while the maximum frequency of recombinants was detected within populations from the basin of Desna River, that has preserved native ecosystems. It was demonstrated that the frequency of premeiotic aberrations does not correlate with the intensity of interspecific water frog hybridization. Populations with introgressions are more common than populations with recombinants, however, within the latter, the frequency of recombination events is higher. The primary factor of gametogenesis aberrations, most likely, is the genetic characteristics of the local populations of parental species, since unambiguous explanations of this phenomenon based on the action of environmental stress (pollution of water systems) are not obvious.
... However, the low level of discrimination obtained by the authors based on the traits used raises the question about the reliability of the identification of single individuals of P. lessonae in this region. In the Donets drainage during the first years of the XXI century, the frequency of pool frog specimens in populations was about 1% (Borkin et al., 2004). The absence of this species during the last two decades against the background of a significant anthropogenic transformation of the floodplain ecosystems of this river allows to believe that P. lessonae in this region has become rarer or even disappeared. ...
... The extinction of mixed populations within the southern limits of the hybridization zone is confirmed by observations of the dynamics of the Donets river water frog populations. According to studies conducted in 1989(Borkin et al., 2004, based on DNA flow cytometry, the structure of water frog samples from the Donets river populations was as follows. The proportion of P. lessonae was about 0.011; proportion of diploid RL-hybrid was equal to 0.336. ...
Geographic peculiarities of population structure and hemicloning reproduction of the water frog Pelophylax esculentus complex of 904 samples within southern East European Plain were analyzed. The proportion of populations of P. ridibundus is 0.667 against those 0.042 of P. lessonae . The RE-type populations are the most common among mixed ones (0.153). The colonies of the two parental species and P. kl. esculentus (REL-type), as well as those of P. lessonae with P. kl. esculentus (LE-type), have the frequencies 0.072 and 0.046, respectively. All-hybrid populations (E-type) are not numerous across the region (0.017). In the populations of Central, Northern and Western Ukraine, the P. lessonae genome is eliminated during gametogenesis within hybrids while in the Lower Dnieper and Lower Danube drainages, genome of P. ridibundus is eliminated. In the Eastern Ukraine populations, hybrids usually produce diploid gametes or haploid gametes with the P. ridibundus genome, less often with the chromosome set of P. lessonae only, and even more rarely a mixture of different types of gametes. The predominance of P. ridibundus in hybrid communities and the elimination of the P. lessonae genome in hybrids inevitably leads to the transformation of hybrid populations into “pure” R-type populations. This circumstance makes hybridization with P. ridibundus a factor in the shrinking of P. lessonae populations. That trend is especially expressed in the Forest steppe zone, where hybridization proceeds most intensively, the P. lessonae genome is eliminated in hybrids, and unstable populations with a numerical superiority of P. ridibundus predominate.
... Günther 1973, 1975b, Günther and Lübcke 1979, Plötner and Klinkhardt 1992, Plötner et al. 1994, Hoffman et al. 2015. Triploid hybrids were also detected in some R-E populations in Germany, Austria, Denmark, Russia, Ukraine, and Poland (Günther 1975b, Rybacki 1994a,b, Rybacki and Berger 2001, Borkin et al. 2004, 2006, Biriuk et al. 2016. As a general rule, LLR hybrids transmit the L and RRL hybrids the R genome to their gametes (Günther and Lübcke 1979, Günther 1983, Vinogradov et al. 1990, Christiansen 2009, Christiansen and Reyer 2009, Pruvost et al. 2013, Deduk et al. 2015, reviewed by Plötner 2005) via a process referred to as meiotic hybridogenesis (Alves et al. 1998, Alves et al. 2001. ...
Der mitteleuropäische Wasserfroschkomplex umfasst Pelophylax lessonae (Genotyp LL), Pelophylax ridibundus (RR) und deren Hybriden Pelophylax esculentus (LR), der sich hemiklonal durch Rückkreuzen mit LL in lessonae-esculentus (L-E)-Populationen oder RR in ridibundus-esculentus (R-E)-Populationen reproduziert. Außerdem können Hybriden in reine Hybridpopulationen (E) bilden, in denen triploide Individuen die Elternarten funktionell ersetzen. Bislang ist wenig über die molekularen Mechanismen bekannt, die der klonalen Gametenbildung in der Keimbahn der Hybridform zugrunde liegen. In dieser Studie wurden erstmalig 160 Gene der Elternarten untersucht, die an der Gametenbildung beteiligt sind. Zusätzlich wurden 131 SNPs von 52 dieser Gene von 652 Wasserfröschen aus 26 Populationen analysiert. Im Ergebnis wurden 14 SNPs von 10 Genen entdeckt, deren Frequenzen mit dem Populationssystem assoziiert waren. In Übereinstimmung mit ihren Funktionen könnten diese Gene im Zusammenheng mit den system-spezifischen hybridogenetischen Reproduktionsmodi stehen. Sowohl transkriptomische als auch SNP-Daten lieferten Hinweise auf genetische Introgression, d. h. einen Transfer lessonae-spezifischer Allelen in den ridibundus-Genpool oder umgekehrt. Außerdem wurde bei P. lessonae eine kryptische genetische Diversität beobachtet. SNP-Analysen ergaben auch, dass LR-Individuen aus E-Populationen eine höhere genetische Ähnlichkeit mit LR-Individuen aus R-E als aus L-E-Populationen aufweisen. Diese Ergebnisse werfen die Frage nach dem Ursprung der E-Populationen auf, von denen bisher angenommen wurde, dass sie aus L-E-Populationen hervorgegangen sind. Die neuen molekularen Daten stehen mit den hemiklonalen Fortpflanzungsmodi von P. esculentus im Einklang und unterstreichen, dass diese wahrscheinlich auf komplexen Wechselwirkungen zwischen verschiedenen Genen und Faktoren basieren.
... The most complicated mechanisms are in the systems 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 widespread 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. ...
Pelophylax esculentus complex hemiclonal systems are a unique evolutionary object due to intricate mechanisms 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 first findings 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 complexity of the local water frog population systems, enabling new research opportunities.
... Diploid and triploid hybrids can coexist independently from parental species, producing selfmaintaining evolutionary units (known as "E systems") [18,19,25,28,29]. Such populations are widespread in Sweden, Denmark, Germany, Poland, and Ukraine [28][29][30][31][32]. Here, we focused on the reproduction of hybrid frogs in pure hybrid populations to determine contribution of hybrid animals to the progeny. ...
An intriguing outcome of hybridisation is the emergence of clonally and hemiclonally reproducing hybrids, that can sustain, reproduce, and lead to the emergence of polyploid forms. However, the maintenance of diploid and polyploid hybrid complexes in natural populations remains unresolved. We selected water frogs from the Pelophylax esculentus complex to study how diploid and triploid hybrids, which reproduce hemiclonally via hybridogenesis, are maintained in natural populations. During gametogenesis in diploid hybrids, one of the parental genomes is eliminated, and the remaining genome is endoreplicated. In triploid hybrids, the single-copy genome is typically eliminated, while genome endoreplication does not occur. To investigate how diploid and triploid hybrid frogs reproduce in populations without parental species, we crossed these hybrid animals from two separate pure hybrid populations located in Poland. Using cytogenetic analysis of tadpoles that emerged from the crosses, we established which gametes were produced by parental hybrids. The majority of hybrid females and hybrid males produced one type of gamete with the P. ridibundus genome. However, in both studied populations, approximately half of the diploid and triploid hybrids simultaneously produced gametes with different genome compositions and ploidy levels, specifically, the P. ridibundus and P. lessonae genomes, as well as diploid gametes with genomes of both parental species. Triploid hybrid males and females mostly produced haploid gametes with the P. lessonae genome; however, gametes with the P. ridibundus genome have also been observed. These results suggest that not all hybrids follow the classical hybridogenetic reproduction program and reveal a significant level of alterations in the gametogenesis pathways. In addition, we found a variable survival rate of particular progeny genotypes when we crossed hybrid females with different males suggesting the important role of postzygotic barriers on the maintenance of pure hybrid systems. We suggest that the observed variability in produced gametes and the different survival rate of the progeny with certain genotypes is crucial for the existence of pure hybrid systems.
... During gametogenesis, the hybrids usually exclude the genome of one of the parental species and produce gametes with the genome of another; thus, hybrids require crosses with parental species to emerge and reproduce. Such a reproductive strategy resulted in a variety of population systems, where hybrids are able to reproduce with one of the parental species (Berger, 2008;Borkin et al., 1987Borkin et al., , 2004Dedukh et al., 2015;Hoffmann et al., 2015;Plӧtner, 2005;Raghianti et al., 2007;Vinogradov et al., 1988). Depending on the coexistence of these parental species hybrids, three types of mixed population systems can be revealed. ...
... In particular, the L-E system includes P. lessonae and P. esculentus, the R-E system consists of P. ridibundus and P. esculentus, and the R-E-L system unites both parental and hybrid species (Plötner, 2005;Rybacki & Berger, 2001;Uzzell & Berger, 1975). Moreover, throughout the distribution range, populations can be represented by hybrids with only one sex or include not only diploid but also triploid and, rarely, tetraploid individuals (Borkin et al., 2004(Borkin et al., , 2006Doležálková-Kaštánková et al., 2018;Hoffmann et al., 2015;Plötner, 2005;Rybacki & Berger, 2001). The hybrid P. esculentus can also form "pure" population systems known mainly from the northwestern edge of the species range where hybrids live without parental species (Berger, 2008;Plötner, 2005). ...
... Earlier triploids were also not found in adjacent regions (Borkin et al., 2002(Borkin et al., , 2003. The nearest triploids were observed approximately 1,000 km from this location to the south in the Severskiy Donets River drainage in eastern Ukraine and adjacent Rostov Oblast of Russia (Biriuk et al., 2016;Borkin et al., 2004Borkin et al., , 2006Dedukh et al., 2017Dedukh et al., , 2015. However, in studied by us localities, the possibility of triploid emergence is high, as hybrid females are able to produce diploid gametes (Dedukh et al., 2019). ...
The edible frog, Pelophylax esculentus, is a hybrid form that reproduces via clonal propagation of only one of the parental genomes through generations of hybrids while the genome of other parental species is eliminated during gametogenesis. Such reproductive ability requires hybrids to coexist with one of the parental species or rarely both parental species causing the formation of so‐called population systems. Population systems and reproductive biology of water frogs from the east of the range remained partially unexplored. In this study, we investigated the distributions, population systems, genetic structure, types of gametes, and morphological variability of water frogs of the genus Pelophylax from the northeastern parts of their ranges (Mari El Republic and adjacent territories, Russia). We examined 1,337 individuals from 68 localities using morphological traits combined with DNA flow cytometry and a multilocus approach (fragments of a nuclear and two mitochondrial genes). We revealed five types of population systems: “pure” populations of the parental P. ridibundus (R) and P. lessonae (L), mixed populations of parental species (R‐L) along and with their hybrids (R‐E‐L), as well as mixed populations of P. lessonae and P. esculentus (L‐E). However, the “pure” hybrid (E) and the mixed P. ridibundus and P. esculentus (R‐E) population systems were not found. All hybrids studied by DNA flow cytometry were diploid. Analysis of gametogenesis showed that the majority of hybrid males, as well as hybrid females from the L‐E system, produced gametes with the P. ridibundus genome. However, in the R‐E‐L system, hybrid females were usually sterile. The reproduction of hybrids in such systems is primarily based on crosses of P. esculentus males with P. lessonae females. Molecular analysis showed the presence of mitochondrial and nuclear DNA introgression of the Anatolian marsh frog (P. cf. bedriagae) into both P. ridibundus and P. esculentus. The observations of alleles and haplotypes of P. cf. bedriagae in P. ridibundus and P. esculentus individuals from the same localities suggest de novo formation of local hybrids. However, the presence of the Balkan marsh frog (P. kurtmuelleri) haplotypes in local hybrids supports the hypothesis regarding the migration of old hemiclonal lineages from glacial refugia. Finally, the diagnostic value of various morphological characteristics was discussed.
... If the L genome is excluded, the mating produces P. ridibundus females only (right side). Symbols are the same as in fig. 1. 1995; Rybacki and Berger, 2001;Borkin et al., 2002;Borkin et al., 2004;Mikulíček et al., 2015;Herczeg et al., 2017). Regardless of the differences in taxa contribution, at all three sites we found significant male-biased asymmetry in the hybrid sex ratio (table 1). ...
