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Taxonomic composition and ploidy level among European water frogs (Anura: Ranidae: Pelophylax) in eastern Hungary
Journal of Zoological Systematics and Evolutionary Research
Supplementary resources (4)
Data
December 2016
... Modern molecular methods are now employed to study the genetic population structure of water frog hybridization sites in Europe. These studies have revealed the clonal transmission of different types of genomes within various hemiclonal population systems (Biriuk et al., 2015;Dedukh et al., 2013Dedukh et al., , 2015Dedukh et al., , 2017Doležálková et al., 2016;Dufresnes et al., 2018;Herczeg et al., 2017;Quilodran et al., 2015). Some studies have also focused on investigating the population structure of the edible frog (Christiansen, 2009) and populations with the presence of parental species alongside exclusively male individuals of hybrid forms . ...
... To accurately evaluate the diversity of clonal individuals, it is necessary to use microsatellite markers with high polymorphism and even distribution (Doležálková-Kaštánkova et al., 2018). For the group of water frogs, several speciesspecific loci have been described (Doležálková et al., 2016;Dufresnes et al., 2018;Garner et al., 2000;Herczeg et al., 2017;Holm, 1979;Hotz & Uzzel, 1982;Pruvost et al., 2013;Zeisset et al., 2000). In this study, we present the results of population structure analysis for 174 individuals using two loci, Rrid059A and RlCA1b5. ...
... Population structure analysis was conducted using Bayesian methods (emplo ying the MCMC algorithm) in the NewHybrids 1.1 (Anderson & Thompson, 2002). The NewHybrids 1.1 program incorporates species-specific alleles as initial conditions (Dufresnes et al., 2017;Falush et al., 2007;Herczeg et al., 2017;Pritchard et al., 2000). The NewHybrids1.1 accurately identifies parental species (P) and hybrids resulting from their crossbreeding (F1), as well as hybrids crossing with each other (F2) or with parental species (Bx). ...
Background. Two species of water frogs, Pelophylax ridibundus and Pelophylax lessonae, and their hybrid, Pelophylax kl. esculentus, are widespread in Ukraine. The objective of this study was to investigate the population structure of various types of hemiclonal population systems (HPS) of water frogs, formed due to the coexistence of frogs in the same territory. In Ukraine, a hybridization centre with the spread of triploid individuals of hybrid nature has been identified. Triploid hybrids are an intriguing research subject due to the diverse hypotheses about their origin and role in HPS. Outside the hybridization centre in Kharkiv Region, triploids are not commonly found. In our study, we describe the initial findings of triploid specimens in Lviv Region and analyze the genetic structure of the HPS where such individuals were detected.
Methods. In total, 193 specimens of green frogs were collected between 2011 and 2015. Here we present population structure analysis which was conducted using two microsatellite loci, Rrid059A and RlCA1b5. A wide range of software programs were utilized for processing the genetic analysis data, including GenePop 4.7.5, Micro-Checker and NewHybrids 1.1.
Results. Three types of hemiclonal population systems were identified: R-E type in Perekalky and Lake Pisochne, L-E type in Lake Luky, and R-E-L type in Nyzhankovychi, Velykyi Lyubin`, Zhovtantsi and Cholgyni. Additionally, population systems with hybrids of mixed ploidy (diploids and triploids) were found in Perekalky, Velykyi Lyubin` and Zhovtantsi.
Conclusions. Genetic diversity analysis revealed variations in the number of alleles per population. P. ridibundus individuals exhibited higher genetic diversity compared to P. lessonae individuals, whereas hybrids showed intermediate genetic diversity. Further investigations of the localities where potential triploids were detected are necessary to assess the survival and reproductive potential of hybrid individuals and determine all types of hybrids and individuals of both marsh and pool frogs.
... RL and EE systems are rare in the Eastern European part but are found in Ukraine, Belarus and Russia (Lada et al., 1995;Borkin et al., 2002;Svinin et al., 2013). REL are rare throughout the frogs range and, with the exception of the East part, are confirmed with a low percentage in the Czech Republic, Poland, Hungary (Herczeg et al., 2016), Romania, Germany (Plötner, 2005), Slovakia , the Netherlands, and Serbia (Hoffmann et al., 2015). To a lesser extent RE systems were also found in Germany, Denmark, Poland, Hungary and Czech Republic (Plötner, 2005;Dolezalkovâ-Kastankovâ et al., 2018). ...
... In different parts of the range, they have a certain percentage, genetic structure and ploidy, sexual structure and other specific population characteristics. The study of the genetic structure allows determining the complex mechanisms of genome elimination of one of the parent species or the possible recombination of genetic material and a number of environmental and evolutionary consequences formed due to these complex processes Herczeg et al., 2016;Dubey et al., 2019). ...
... In the western European part, within the nature protected areas of Hungary, RE and REL systems were determined. Females predominantly represent the hybrids there in contrast to our data (Herczeg et al., 2016). ...
Ecological and evolutionary consequences of population-genetic processes that occur because of natural cross-species hybridization can show mechanisms of overcoming the reproductive barrier and obtaining the species status by a hybrid taxon. This is clearly seen in the population systems of Eurasian water frogs – Pelophylax esculentus complex. The P. esculentus (E) hybrid usually discards one of the parental genomes of P. lessonae (L) or P. ridibundus (R) and reproduces semi-clonally. The genetic structure and direction of gene flows precisely depend on the type and distribution of mixed or pure population systems of water frogs. Three population systems in the south of Ukraine were identified and confirmed as RR, RE and REL. The populations of P. ridibundus are most common (76.2%). A mixed population systems of P. ridibundus and P. esculentus (20.0%) are concentrated in the floodplains of large rivers where triploids were found and the unisexual hybrids (1.0♂ : 0.1♀) were proved. Parent species populations having different ploidy of P. esculentus such as 3n and for the first time 4n were found. A mixed system of three taxa (REL) is rare (3.8%) and locally concentrated in the lower Danube and Dnieper with the smallest proportion of P. lessonae. We did not find populations of P. lessonae (LL), P. esculentus (EE, very rare system of hybrids only), and two mixed populations of parental species RL and semi-clonal LE in the south of Ukraine, but they are known for northern areas. The high number of P. ridibundus tends to decrease; the scarce P. esculentus and the extremely rare P. lessonae require special conservation measures. P. ridibundus (RR) occupies a wide range of diverse natural, permanent, temporary, coastal, continental, and artificial freshwater bodies, including synanthropic ecosystems. Mixed population systems inhabit willow and poplar forests in the floodplains of large rivers. In the south of Ukraine rare and isolated populations of the water frogs occurring outside the main range can be relict. Biotopic preferences, ratio and number of constituent taxa are crucial for an adequate assessment of biological (taxonomic) diversity and development of an appropriate strategy for the population systems’ conservation. Such characteristics as unisexuality of hybrids, their spreading patterns, specific sex structure and ploidy in different population systems of the P. esculentus complex contribute to the understanding of the hybridogenetic dynamics; produce new tendencies of becoming independent hybridogenous taxa and emergence of new evolutionary relationships.
... The scenario of the expansion of the kurtmuelleri lineage is supported by the presence of kurtmuelleri haplotypes in the northern part of the peninsula and rarely also in central, northern, and eastern Europe. However, its frequency is decreasing northwards as is evident from own and published data (Herczeg et al. 2017;Kolenda et al. 2017;Litvinchuk et al. 2020). Kolenda et al. (2017) predict the occurrence of kurtmuelleri haplotypes and alleles in the geographic region between the northern Balkans and Poland. ...
... Apart from the natural range, haplotypes and alleles of kurtmuelleri lineage were recorded in several European countries in the last decades (e.g., Herczeg et al. 2017;Kolenda et al 2017;Bruni et al. 2020;Litvinchuk et al. 2020). Kolenda et al. (2017) proposed three hypotheses for the origin of P. kurtmuelleri haplotypes in central and northern Europe: (1) recent introduction followed by hybridization of autochthonous and allochthonous populations, (2) incomplete lineage sorting or, (3) hybridization before or during the post-glacial expansion. ...
The genus Pelophylax (water frogs) includes relatively common, widely distributed, and even invasive species, but also endemic taxa with small ranges and limited knowledge concerning their ecology and evolution. Among poorly studied species belong endemics of the southwestern Balkans, namely Pelophylax shqipericus, P. epeiroticus and P. kurtmuelleri. In this study, we focused on the genetic variability of these species aiming to reveal their phylogeographic patterns and Quaternary history. We used 1,088 published and newly obtained sequences of the mitochondrial ND2 gene and a variety of analyses, including molecular phylogenetics and dating, historical demography, and species distribution modeling (SDM). We revelated the existence of two mitochondrial lineages within P. epeiroticus and P. shqipericus that diverged at ~ 0.9 Mya and ~ 0.8 Mya, respectively. Contrarily, no deeply diverged lineages were found in P. kurtmuelleri. Pelophylax kurtmuelleri also shows a close phylogenetic relationship with widely distributed P. ridibundus, suggesting that both represent one evolutionary clade called here P. ridibundus/kurtmuelleri. The estimated split between both lineag-es in the clade P. ridibundus/kurtmuelleri date back to ~ 0.6 Mya. The divergence between the ridibundus and kurtmuelleri lineages on the ND2 gene is thus lower than the divergence between the two lineages found in P. epeiroticus and P. shqipericus. According to haplotype networks, demographic analyses, and SDM, endemic water frogs survived the last glacial maximum (LGM) in Balkan in the Balkans microrefugia, and their distribution has not changed significantly or even retracted since the LGM. Haplotypes of the kurtmuelleri lineage were also found in northern parts of Europe, where haplotype diversity is however much lower than in the Balkans, suggesting the possible hypothesis of their postglacial expansion to the north.
... Genetic markers that proved most informative for phylogenetic inferences were several mitochondrial DNA (mtDNA) sequences such as cytochrome b, 16S rRNA (Lymberakis et al., 2007), NADH dehydrogenase subunit genes (ND2 and ND3) (Plötner et al. 2008;Akin et al. 2010a, b;Domeneghetti et al. 2013;Hotz et al. 2013), and nuclear DNA (nuDNA) sequences like serum albumin intron-1 (SAI-1) (Plötner et al. 2009;Hauswaldt et al. 2012;Vucić et al. 2018) as well as microsatellites (Dufresnes et al. 2017a(Dufresnes et al. , 2017b(Dufresnes et al. , 2018 and Restriction-site Associated DNA (RAD) loci (Dufresnes and Dubey 2020). The analysis of length differences of the SAI-1 intron region became widely used for distinguishing species and hybrids within Pelophylax genus (Hauswaldt et al. 2012;Mayer et al. 2013;Herczeg et al. 2016). ...
... The length variation of SAI-1 region was recognized as a useful genetic tool for the detection of water frog species and hybrids (Plötner et al. 2012(Plötner et al. , 2015Herczeg et al. 2016;Vucić et al. 2018). In this study, we detected four species-unique SAI-1 amplicons lengths ( Fig. S1; Table S5): approximately 0.3 kb allele for P. lessonae (referred in the further text as allele L), a 0.7 kb allele for P. kurtmuelleri (K), a 0.85 kb allele for P. ridibundus (R), and a 1.3 kb allele for P. shqipericus (S). ...
Spreading of alien water frog species (genus Pelophylax) has been reported across Europe, posing a severe risk for the replacement or genetic swamping of indigenous species. Crna Mlaka fishponds in Central Croatia were an experimental station for freshwater fish aquaculture during the first half of 20th century and hosted fish from different parts of Balkans. Crna Mlaka fishponds are thus estimated to be at higher risk of non-native frogs, likely introduced with the aquaculture industry, causing introgressive hybridization and genetic admixture of native Pelophylax species. We used nuclear and mitochondrial DNA sequences, in combination with microsatellite markers, to characterize water frogs of Crna Mlaka. We detected alleles of two native species, the Eurasian marsh frog Pelophylax ridibundus (Pallas, 1771) and the pool frog Pelophylax lessonae (Camerano, 1882), as well as non-native Albanian pool frog Pelophylax shqipericus (Hotz, Uzzell, Gunther, Tunner et Heppich, 1987) and Balkan pool frog Pelophylax kurtmuelleri (Gayda, 1940). Moreover, we found five hybrid forms: P. kurtmuelleri x P. ridibundus, P. kurtmuelleri x P. shqipericus, P. lessonae x P. kurtmuelleri, P. ridibundus x P. shqipericus, and P. lessonae x P. shqipericus indicating the existence of a hybrid swarm. The last three hybrids have never been reported in natural populations. Interestingly, although expected, no P. kl. esculentus (P. ridibundus x P. lessonae) was detected. Additionally, water frogs with introgressed mtDNA were detected, which results in cyto-nuclear discordance. The results of this study suggest that this is a unique case of species melting pot, a hybrid swarm emerging from two alien and two native frog species resulting in more than 50% of water frogs at this location being hybrids and/or having introgressed mtDNA and the loss of the hybridogenetic reproductive system of Pelophylax kl. esculentus.
... An intriguing population is the L-E-R type, very rare in western Europe, where all members of the P. esculentus complex exist together. Although the L-E-R type is widely distributed in eastern Europe (Lada, Borkin and Vinogradov, 1995;Borkin et al., 2002) and was occasionally found in central Europe (Mikulíček et al., 2015;Herczeg et al., 2017), little is known about how hybrids are perpetuated in these populations. ...
... 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). ...
... Because this reproductive mode is not restricted to males, it was proposed to be called amphigameticity (Pustovalova et al., 2022). There is also the L-E-R HPS, where both parental species coexist with hybrids (Günther, 1982;Mikulíček et al., 2015;Herczeg et al., 2017;Hermaniuk et al., 2020). Both diploid and triploid hybrids may be involved in any mentioned system type, thus forming L-E-Ep HPS or R-E-Ep-HPS, where Ep indicates the presence of triploids (Shabanov et al., 2020). ...
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.
... Accurate identification of waterfrogs (Pelophylax esculentus complex) using morphological traits is problematic in areas where hybrid frogs occur, requiring the use of molecular methods to distinguish between taxa (e.g. Herczeg et al. 2017, Jośko & Pabijan 2020, Mezhzherin et al. 2023. We used a molecular technique based on amplification of the nuclear serum albumin intron 1 (SAI-1) described by Hauswaldt et al. (2012) and modified by Ermakov et al. (2019). ...
The chytrid Batrachochytrium dendrobatidis
(Bd) is a widespread fungus causing amphibian
declines across the globe. Although data on Bd occurrence
in Eastern Europe are scarce, a recent species
distribution model (SDM) for Bd reported that western
and north-western parts of Ukraine are highly suitable
to the pathogen. We verified the SDM-predicted
range of Bd in Ukraine by sampling amphibians across
the country and screening for Bd using qPCR. A total
of 446 amphibian samples (tissue and skin swabs) from
11 species were collected from 36 localities. We obtained
qPCR-positive results for 33 samples including
waterfrogs (Pelophylax esculentus complex) and fireand
yellow-bellied toads (Bombina spp.) from 8
localities. We found that Bd-positive localities had
significantly higher predicted Bd habitat suitability
than sites that were pathogen-free. Amplification and
sequencing of the internal transcribed spacer (ITS)
region of samples with the highest Bd load revealed
matches with ITS haplotypes of the globally distributed
BdGPL strain, and a single case of the BdASIA-2/
BdBRAZIL haplotype. We found that Bd was nonrandomly
distributed across Ukraine, with infections
present in the western and north-central forested
peripheries of the country with a relatively cool, moist
climate. On the other hand, our results suggest that Bd
is absent or present in low abundance in the more continental
central, southern and eastern regions of Ukraine,
corroborating the model-predicted distribution
of chytrid fungus. These areas could potentially serve
as climatic refugia for Bd-susceptible amphibian hosts.
... Despite its widespread use in phylogeographic surveys (e.g., Plötner et al. 2012), for characterizing the genomes involved in hybridogenetic systems (e.g., Miura et al. 2021), to screen for native (e.g., Herczeg et al. 2017;Vucić et al. 2018) and introduced taxa (e.g., Plötner et al. 2015;Kolenda et al. 2017;Litvinchuk et al. 2020;Jelić et al. 2022), and even for a taxonomic description (Plötner et al. 2012), the diagnosticity and reliability of SAI-1 have repeatedly been questioned (Mayer et al. 2013;Dubey et al. 2019;Cuevas et al. 2022). For instance, amplicon length profiles failed to distinguish the water frogs found in hybridogenetic populations from southern Germany (Mayer et al. 2013). ...
Water frogs (genus Pelophylax) are one of the most widespread and diverse, but also most invasive amphibians of the Western Palearctic region. As such, Pelophylax studies face the challenge of identifying similar taxa that hybridize in sympatry. For this purpose, the nuclear marker serum albumin intron 1 (SAI-1) has been used for over a decade in Pelophylax. Initially praised for its diagnosticity, notably to discriminate common species such as the pool frog (P. lessonae), the marsh frog (P. ridibundus) and their hybridogenetic hybrid the edible frog (P. esculentus) without sequencing (by amplicon length polymorphism), SAI-1 was later questioned due to misidentifications and doubtful patterns of genetic divergence. In this study, we incorporate an up-to-date multilocus phylogeographic framework spanning the entire Pelophylax diversification, to reassess the performance of SAI-1 for lineage identification and discovery. We show that SAI-1 sequences discriminate all Palearctic water frog species and most of their phylogeographic lineages, enabling us to map their distributions and identify the genomes of hybridogenetic hybrids. However, the phylogeny of SAI-1 is aberrant and unrepresentative of the evolution of the genus. In particular, differentiated P. l. lessonae alleles segregating in the Alpine region mimic a species-level divergence that is not recovered by any other marker. Moreover, the indel polymorphism that supposedly distinguishes P. lessonae from P. ridibundus, as well as the main P. ridibundus lineages from the Balkans (P. r. ridibundus vs kurtmuelleri), are not diagnostic across the entire range of these taxa. Hence, SAI-1 is neither the panacea for nor the nemesis of Pelophylax genotyping. Sequencing SAI-1 shall continue to offer a reliable and informative preliminary approach of single-gene barcoding identification of lineages, but analyses without sequencing, and other applications such as phylogenetic and taxonomic inferences, should be avoided.
... Most of the P. esculentus esculentus sequences phased and analyzed contained a marsh frog copy of the ridibundus lineage and pool frog copy of the lessonae lineage, as expected. However, the marsh frog copy of a few individuals corresponded to other lineages, namely kurtmuelleri in the Balkans (Herczeg et al., 2016) or cf. ridibundus G in Eastern Europe (Miura et al., 2021). ...
Palearctic water frogs (genus Pelophylax) are an outstanding model in ecology and evolution, being widespread, speciose, either threatened or threatening to other species through biological invasions, and capable of siring hybrid offspring that escape the rules of sexual reproduction. Despite half a century of genetic research and hundreds of publications, the diversity, systematics and biogeography of Pelophylax still remain highly confusing, in no small part due to a lack of correspondence between studies. To provide a comprehensive overview, we gathered >13,000 sequences of barcoding genes from >1700 native and introduced localities and built multigene mitochondrial (~17 kb) and nuclear (~10 kb) phylogenies. We mapped all currently recognized taxa and their phylogeographic lineages (>40) to get a grasp on taxonomic issues, cyto-nuclear discordances, the genetic makeup of hybridogenetic hybrids, and the origins of introduced populations. Competing hypotheses for the molecular calibration were evaluated through plausibility tests, implementing a new approach relying on predictions from the anuran speciation continuum. Based on our timetree, we propose a new biogeographic paradigm for the Palearctic since the Paleogene, notably by attributing a prominent role to the dynamics of the Paratethys, a vast paleo-sea that extended over most of Europe. Furthermore, our results show that distinct marsh frog lineages from Eastern Europe, the Balkans, the Near East, and Central Asia (P. ridibundus ssp.) are naturally capable of inducing hybridogenesis with pool frogs (P. lessonae). We identified 14 alien lineages (mostly of P. ridibundus) over ~20 areas of invasions, especially in Western Europe, with genetic signatures disproportionally pointing to the Balkans and Anatolia as the regions of origins, in line with exporting records of the frog leg industry and the stocks of pet sellers. Pelophylax thus emerges as one of the most invasive amphibians worldwide, and deserves much higher conservation concern than currently given by the authorities fighting biological invasions.
... The ranges of these hybridizing species overlap widely (Beebee et al. 2005). As a result a hybrid form is generated, traditionally named as Pelophylax esculentus (Herczeg et al. 2017;Jośko and Pabijan 2020;. These hybrids are mostly allodiploids (Vorburger 2001a, b); allotriploids are less common (Christiansen 2009;Christiansen et al. 2005Christiansen et al. , 2010Gṻnther 1970). ...
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.
... A total of 161 Pelophylax water frogs from 13 localities in the Balkans and Slovakia were examined for the presence of parasites ( Fig. 1, Table 1) Trematodes were collected from 4 host species: P. esculentus (n = 50) and P. ridibundus (n = 72) in Slovakia, and P. epeiroticus (Schneider, Sofianidou & Kyriakopolou-Sklavounou, 1984) (n = 30) and P. kurtmuelleri (Gayda, 1940) (n = 9) in Greece and Albania, respectively. Although the latter 2 species have been considered as endemics of the Balkan Peninsula since description, haplotypes and alleles of P. kurtmuelleri have been recorded in several European countries in the last few decades (e.g., Dufresnes et al., 2017Dufresnes et al., , 2018Herczeg et al., 2017;Kolenda et al., 2017;Bellati et al., 2019 and literature therein;Litvinchuk et al., 2020). Water frog species were identified according to morphological traits (Günther, 1990;Plötner, 2005) and molecular markersspecifically, sequences of the mitochondrial ND2 fragment and microsatellites. ...
Parasites might represent a helpful tool in understanding the historical dispersion and phylogeography of their hosts. In order to reveal whether the migration routes and diversification of hosts can be traceable in the genetic structures of their parasites, we investigated the diversity of paramphistomoid trematodes of Pelophylax frogs in 2 geographically distant European regions. Water frogs belonging to the genus Pelophylax represent a striking example of a species with a high variety of ecological adaptations and a rich evolutionary history. The parasites were collected from 2 Balkan endemic species, P. epeiroticus and P. kurtmuelleri, and 2 species in Slovakia, P. esculentus and P. ridibundus. While in Slovakia, Pelophylax frogs harboured 2 species, the diplodiscid Diplodiscus subclavatus and the cladorchiid Opisthodiscus diplodiscoides, only the former was recorded in the south-western Balkans. Remarkably high genetic diversity (16 unique mitochondrial cox1 haplotypes, recognized among 60 novel sequences) was observed in D. subclavatus, and subsequent phylogenetic analyses revealed a strong population-genetic structure associated with geographical distribution. We also evidenced the existence of 2 divergent D. subclavatus cox1 haplogroups in the south-western Balkans, which might be associated with the historical diversification of endemic water frogs in the regional glacial microrefugia.
... The species is also present in Albania; however, the main part of its range is within Greece [3]. The western Palaearctic water frog of the genus Pelophylax includes several distinct species and has been investigated in several phylogenetic, morphological, and ecological studies [4][5][6][7][8][9][10]. The populations of P. epeiroticus (Epirus Water Frog) are suspected to be decreasing due to ongoing decline in the extent and quality of its habitat, thus improved management measures are required for the protected areas and Natura 2000 sites where the species appears [3]. ...
The edible water frog Pelophylax epeiroticus, distributed mainly in Northwest Greece and utilized commercially as food, was investigated in lake Pamvotida (Ioannina). The objective was to assess aspects of population structure (sex ratio, morphometric characteristics, allometric relationships) and proximate composition of the Epirus water frog (Pelophylax epeiroticus). Commercial samples (31 females and 54 males) were obtained and sex ratio, morphometric characteristics, allometric relationships and proximate composition were assessed. A significantly lower abundance of females was indicated (31 females and 54 males). Body length range was higher in females (females 3.4 mm, males 2.6 mm), whereas total weight range was higher in males (females 45.08 gr, males 48.35 gr). Differences in allometric relationships were indicated between sexes. The high protein (15.93 ± 3.32) and low lipid (0.25 ± 0.13) contents indicated that P. epeiroticus is an excellent food source of high nutritional value. A tree classification algorithm indicated that the principal contributing component for sex classification was dry matter, followed by a proportion of edible flesh and protein content. A predicted future increase in demand for wild-caught individuals requires the use of a suitable management plan, coupled with the development of farming practices aiming to assure the sustainable exploitation of this important resource and alleviate the pressure on its populations.
... Adult P. ridibundus usually reproduce in large water bodies (Berger & Rybacki 1994), including fish farming ponds, where the density of large, omnivorous fish is high (Socha & Ogielska 2010;Herczeg et al. 2017;Kolenda et al. 2017). Moreover, tadpoles of P. ridibundus have been shown to survive better in short-term predation trials with fish than other syntopic frog species (Teplitsky et al. 2003). ...
Associational effects such as “associational resistance” or “aposematic commensalism” may facilitate the subsequent evolution of mimicry. However, such effects are usually expected to be contingent on a spatiotemporal co-occurrence of defended and undefended species. Associational resistance may emerge in communities of larval anurans where tadpoles of different species share a generalized morphology but vary in chemical defences. In Europe, the marsh frog Pelophylax ridibundus reproduces in fish-containing habitats that earlier in the season can be used by the common toad Bufo bufo, a species with chemically defended larvae. In a mesocosm experiment, we investigated if the associational protection against predation provided by the defended species could be strong enough to last even after the defended prey metamorphose and leave the system. We expected that the higher abundances (both absolute and relative) of chemically defended B. bufo in the tadpole assemblage exposed to predation by the common carp Cyprinus carpio prior to the occurrence of P. ridibundus tadpoles would be related to the increased later survival of P. ridibundus in the presence of the same fish. The tadpoles of P. ridibundus were highly vulnerable to predation during a 2-week cohabitation with fish. We found no relationship between the survival of P. ridibundus and the absolute density or relative proportion of B. bufo. Although associational effects may be ecologically relevant in aquatic animal communities, they apparently require the co-presence of the defended prey in fish-tadpole interactions.
... Thus, P. kurtmuelleri (or its allelic markers, naturally encompassing at least part of the same polymorphism as P. ridibundus -i. e. not very specific -or derived from backcross hybridization with P. ridibundus) may be native, naturally spread in Eastern Europe, north of the "classic" areal (Herczeg et al. 2016;Kolenda et al. 2017;Litvinchuk et al. 2020). Moreover, P. kurtmuelleri and P. ridibundus (and European Pelophylax bedriagae) are sometimes seen as biologically conspecific, with no reproductive barrier (Hotz et al. 2013, on a molecular basis only) and sometimes as distinct, with P. kurtmuelleri -P. ...
The paper presents a review of literature data, supplemented with original observations, on the presence, establishment, distribution and invasive status of alien fish, amphibian and reptile species in Romania. Consistent criteria were followed in defining alien species records, establishment and invasive status. From the 48 alien fish species, 1 fish hybrid, 1 amphibian and 18 reptile species recorded, only 16 fishes and 3 reptiles can be regarded as established. Of these we consider the criteria for invasive status as being probably fulfilled by one fish species ( Perccottus glenii ), and less likely by six more fish species. The presence and the alien status of the one amphibian are debatable. No reptile species can be considered invasive at present.
... klepton, ami a hibridekt l eltér en a szül fajoktól elszakadva, akár önálló, szaporodóképes állomány formájában is képes fennmaradni . Hazánkban a kecskebéka jellemz en valamelyik, vagy mindkét fajjal együtt fordul el (Herczeg et al. 2016). A zöldbékák er sen vízhez kötött életmódot folytatnak, többnyire a vizekben, az iszapban telelnek át. ...
... To screen for ranaviruses, we used DNA samples extracted from tail or toe clips belonging to 137 individuals from 8 different amphibian species, hosted by the Collection of Genetic Resources of the Hungarian Natural History Museum ( Fig. 1., Table 1.). These samples were collected for phylogeographic and population genetic studies (Vörös et al., 2006;Recuero et al., 2012;Vörös et al., 2017;Herczeg et al., 2017), and were stored in -80 °C. In all cases DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) was used for DNA extraction, following the manufacturer's protocol. ...
... A három zöldbékafaj lokális populációs rendszereinek pontos viszonyait célzott, nagy mintaszámú és lefedettségű genetikai vizsgálatokkal lehet csak megállapítani a fajkomplexumra jellemző összetett hibridizációs mechanizmusok miatt (Vitt & Caldwell 2014). Herczeg et al. (2016) vizsgálatai alapján az Egyek-pusztakócsi mocsárrendszerre a kis tavibéka és kecskebéka által alkotott "LE" típusú populációs rendszer lehet jellemző, amely egybeesik saját megfigyeléseinkkel. A vizuális módszerekkel (nappali és esti vizuális keresés és kézi hálózás) minden faj esetében találtunk egyedeket. ...
A kétéltűek (Amphibia) globálisan az egyik legveszélyeztetettebb élőlénycsoport, ezért elterjedésük ismerete alapvetően fontos, különösen olyan védett területeken, ahonnan kevés a korábbi információ és aktív beavatkozások zajlanak. Jelen munkánkban összesítjük az Egyek-pusztakócsi mocsarak területén több korábbi vizsgálatban végzett felméréseink faunisztikai eredményeit, melyeket ötféle mintavételi módszerrel gyűjtöttünk 15 darab 2,5x2,5 kilométeres UTM cellában. Összesen 11 kétéltűfaj (pettyes gőte, dunai tarajosgőte, vöröshasú unka, barna ásóbéka, barna varangy, zöld varangy, zöld levelibéka, mocsári béka, kis tavibéka, nagy tavibéka, kecskebéka) 14 362 egyedét figyeltük meg. A hat legjobban felmért UTM négyzet között nem volt jelentős eltérés a diverzitás tekintetében. Két 10x10 kilométeres UTM négyzet (DT86 és 96) esetében öt és hét faj jelenlétével bővültek ismereteink. Nagy állományokkal volt jelen két Natura 2000-es jelölőfaj, a vöröshasú unka és a dunai tarajosgőte. Eredményeink szerint a mocsárrendszer a tájrehabilitációs beavatkozásoknak köszönhetően értékes és diverz kétéltűfaunának szolgál otthonául, ezért megfelelő kezelése és védelme kiemelt fontossággal bír.
... Taxonomic status of P. esculentus is still being discussed; however, most studies demonstrated the genetic heterogeneity of this form [22,30,31,34,36]. Pelophylax ridibundus × P. lessonae hybrids, therefore, cannot be considered as a biological species. On the other hand, populations of hybrids do exist in particular biotopes, separately [8] or, more often, along with one or both parental species [21,34]. These populations interact in a particular way with other components of biocenoses, including the parasites. ...
PurposeThe study aims to characterise and compare the helminth assemblages and helminth infracommunities in the marsh frog, Pelophylax ridibundus and the edible frog, P. esculentus collected in the northern part of Ukraine.Methods
Occurrence and abundance of the helminths were analysed by calculating the prevalence, intensity, and mean abundance of infection; similarities between the infracommunities were estimated by the Bray–Curtis index and visualised using nMDS plots. Dissimilarities were estimated using the ANOSIM and SIMPER routines.ResultsIn total, 27 helminth species were found in 143 frogs. Pelophylax ridibundus (n = 86) harboured 20 species of helminths, 24 species were found in P. esculentus (n = 57), and 17 species were shared by the two hosts. Oswaldocruzia bialata and larval Strigea sp. were absent in P. ridibundus, while they reached the prevalence of 30% and 10%, respectively, in P. esculentus. Cosmocerca ornata, Diplodiscus subclavatus, Opisthioglyphe ranae, and Codonocephalus urniger had significantly larger prevalence in P. ridibundus, whereas Haematoloechus asper was found to be more prevalent in P. esculentus. Acanthocephalus ranae, Icosiella neglecta, Haematoloechus variegatus, Pleurogenes claviger, Pleurogenoides medians, and Prosotocus confusus were equally common in both hosts. Helminth infracommunities in the two hosts had identical species richness (1–10 species, 4 on average); abundance was significantly higher in P. ridibundus.Conclusions
Helminth assemblages of the two hosts in northern Ukraine are rather similar; however, small but significant differences were found in their species composition, parameters of infection in some species, and structure of helminth infracommunities.
... For instance, barcoding by cytochrome b [14] or electrophoresis of allozyme loci [13] allows precise taxon identification. Also, the method of microsatellite DNA analysis is widespread and available [13,14,21,24,28,43,49]. It is frequently used to analyze population structure of green frogs. ...
The results of the analysis of the genetic structure in population systems of green frogs on the territory of Lviv and Volyn regions are presented. The material was collected in 2011–2012 in water bodies representing three nature regions of Ukraine – Forecarpathians, Roztochia, and Western Polissia. Three taxonomic groups of green frogs were in a focus of the study: Marsh frog – Pelophylax ridibundus (Pallas, 1771), Pool frog – Pelophylax lessonae (Camerano, 1882) and their hybrid – Edible frog – Pelophylax esculentus (Linnaeus, 1758). DNA was extracted from 91 individuals and analyzed using of 10 pairs of primers: Rrid059, Rrid082, Rrid171, Res5, Res14, Res16, Res22, RlCA1b5, RlCA18, RlCA19. A majority of those are highly polymorphic and diagnostic for species identification. During the analysis, we used programs based on principles of Bayesian statistics and Monte-Carlo Markov Chain algorithms: Structure, BAPS, and NewHybrids. Linkage groups were searched using the GenePop software, and hidden null-alleles were detected using Micro-Checker program. For the first time, in the studied area the genetic structure of populations and population systems were described. After the analysis of genetic diversity of frogs sampled from the Pelophylax ridibundus population and from hemiclonal population systems of mixed R-E-L type, we found that the smallest genetic diversity is observed in the population of Marsh frog from the Nyzhankovychi area (Forecarpathians). More diverse are hemiclonal population systems of green frogs sampled in water bodies of “Cholgynskyi” ornithological reserve (Ukrainian Roztochia) and Shatsk National Nature Park (Western Polissia). Also, for the first time, the hybrid composition of studied localities is described. Hybrids of the first generation (F1) and backcrosses were detected in water bodies of Shatsk National Nature Park and ornithological reserve “Cholgynskyi”.
... Water frog taxon identification was determined using the technique described by Hauswaldt et al. (2012), and is based on allele-size polymorphism in intron-1 of the serum albumin gene (SAI-1; Plötner et al., 2009) named RanaCR1, was identified in the serum albumin intron-1 (SAI-1, with a slight modification in PCR protocol (Herczeg et al., 2017). To verify SAI-1 fragments we sequenced representative alleles on a Hitachi 3130 Genetic Analyzer (Applied Biosystems, UK). ...
Batrachochytrium dendrobatidis (Bd) is a fungal pathogen which causes the emerging infectious disease chytridiomycosis. Bd presents low host specificity and threatens amphibians worldwide thus systematic inventory is the key in order to detect and mitigate the effects of the disease. Extensive data collection was conducted in Hungary in 2009-2015 from fourteen different areas. Combined data – recent field sampling on sixteen taxa and the examination of archived Bombina spp. specimens – from 1360 individuals were analysed with qPCR. Two sentinel taxa, Bombina variegata and the members of the Pelophylax esculentus complex were marked to monitor the occurrence of Bd in two core areas (Bakony Mts and Hortobágy National Park, respectively) of sampling. Climatic variables were also examined in core areas to test their effect on prevalence and infection intensity. Among the sixteen sampled amphibian taxa seven tested positive for Bd and the overall prevalence in Hungary was 7.46%. Among the ethanol-fixed Bombina spp. individuals Bd was not detected. In the first core area (Bakony Mts) the overall prevalence in B. variegata was 10.32% and juvenile individuals showed significantly higher prevalence than adults. On the other hand there was a significant negative relationship between infection prevalence and monthly mean air temperature. Finally, in the other core area (Hortobágy National Park) the overall prevalence in P. esculentus complex was 13.00%, and no differences were found in prevalence or infection intensity between sexes, sampling years or age classes.
... serum albumin intron-1 (SAI-1) (Plötner et al. 2009, Hauswaldt et al. 2012. Also, the difference in lengths of the SAI-1 region has become a widely used method for distinguishing among species and hybrids within Pelophylax species (Hauswaldt et al. 2012, Mayer et al. 2013, Herczeg et al. 2016. Therefore, one of the main focuses of this research was to obtain sequences of the SAI-1 nuclear region of P. shqipericus from its type locality. ...
Species composition of Eurasian water frogs, genus Pelophylax, in Lake Skadar (Montenegro) was analysed using mitochondrial (mtDNA) and nuclear (nuDNA) markers. Specimens were characterised at first using mtDNA sequences of NADH dehydrogenase subunit 3 (ND3) gene. Based on their mitochondrial genomes, 49 specimens were determined as Pelophylax kurtmuelleri while 39 specimens were identified as Pelophylax shqipericus. The systematic affiliation was evaluated further using serum albumin intron-1 (SAI-1) nuDNA. The results of SAI-1 analyses confirmed identification of both species but also their hybrids. The SAI-1 variant of P. shqipericus was ~600 nucleotide base pairs longer compared to P. kurtmuelleri. Five specimens contained both variants, indicating their hybrid origin. However, population allotment of hybrids was low, suggesting normal Mendelian inheritance in the interspecific mating of P. kurtmuelleri and P. shqipericus rather than a hybridogenetic mode of reproduction. Mito-nuclear discordance as a result of backcross hybridization was not observed in this study. However, the absence of mito-nuclear discordance is not surprising since backcross specimens were rarely detected in previous research of water frogs from Lake Skadar. This study showed that species composition in Lake Skadar is the same compared to 30 years ago, when a previous study analysed the water frogs using protein electro-phoresis.
... The Taxonomic and genotypic composition of water frogs ( Herczeg et al. 2017) Objectives ...
... Despite potential differences in microhabitats, all three taxa may thus coexist and hybridize in some populations, as they sometimes do in natural ranges (e.g. Herczeg et al., 2017). ...
The marsh frog (Pelophylax ridibundus sensu lato) is the number one amphibian invader in Western Europe. In Switzerland, marsh frogs were introduced in the 1950-1960s and progressively colonized most of the northern parts of the country. We investigated this invasion using molecular tools. We mapped the cryptic presence of three monophyletic mitochondrial lineages (P. ridibundus, P. kurtmuelleri and P. c. f. bedriagae from southeastern Europe) consistent with registered importations by a local frog-leg industry. High nuclear diversity supports that invasive frogs probably originated from genetically-rich import batches and patterns of population differentiation confirm that multiple independent introduction sites were involved. Moreover, several lines of evidence suggest occasional hybridization with local hybridogenetic water frogs. This invasion emphasizes the issues of frequent amphibian releases and translocations at the international and regional scale for commercial and recreational purposes, and stresses for more adequate legislation, control and information to the general public. Given the parallel invasion by alien pool frogs (i. e., the Italian P. bergeri has replaced the local P. lessonae), the situation of water frogs in Switzerland is critical. The water frog complex makes an alarming symbol of the anthropogenic mark left on wildlife diversity and distributions.
... For hypotheses (ii) and (iii) we predict that genetic variants typical for P. kurtmuelleri will be found in areas between southwestern Poland and northwestern Greece, e.g. in Austria, the Czech Republic or Slovakia. The low frequency of the P. kurtmuelleri SAI-1 variant in central Europe (Hauswaldt et al., 2012;Herczeg et al., 2016) lends support to these hypotheses. Whether P. kurtmuelleri is an independent genetic lineage (irrespective of taxonomic status) or a phylogeographic variant of P. ridibundus can be tested using a multilocus species delimitation approach (implemented in e.g. ...
Recent molecular studies have detected the occurrence of exotic water frog species (Pelophylax sp.) in central and western European populations. Here, we report genetic evidence for the occurrence of the Balkan water frog, Pelophylax kurtmuelleri, in southwestern Poland. We found a high frequency of an allele of serum albumin intron-1 and a mitochondrial cytochrome b haplotype specific for this southern taxon in frogs from the Barycz river drainage system. We interpret this finding as evidence of admixture between P. kurtmuelleri and the local ridibundus-esculentus water frog population. The origin of the exotic P. kurtmuelleri mitochondrial and nuclear alleles in southwestern Poland could be due to (i) hybridization after a human-mediated introduction of P. kurtmuelleri, (ii) the persistence of ancestral polymorphism in central European P. ridibundus, or (iii) hybridization between P. kurtmuelleri and P. ridibundus in the Balkans followed by the northward expansion of admixed P. ridibundus. Identical mtDNA haplotypes found in southwestern Poland and localities on the borders between Greece, Albania and Macedonia suggest that this region harboured the source population of P. kurtmuelleri at the studied site.
Studying the distribution of morphologically cryptic animal species is always a very difficult task. Because most marsh frog species (the Pelophylax ridibundus complex) are cryptic, we used molecular markers to identify them. Three marsh frog species (P. ridibundus, P. kurtmuelleri and P. cf. bedriagae) inhabit the northern part of Western Palearctic. We created a database of localities and built models of their modern distribution. These models showed that the most suitable habitats are on the north of the Mediterranean region for P. cf. bedriagae, temperate Europe for P. ridibundus, and the Balkan coastal areas for P. kurtmuelleri. The projection of the modern ecological niches under the late-Quaternary climatic conditions showed that the range of P. kurtmuelleri remained largely unchanged during the period, whereas the ranges of P. cf. bedriagae and especially P. ridibundus changed greatly over time. During the Last Glacial Maximum, the presumed range of P. cf. bedriagae covered a relatively large area in the north of the Mediterranean region and the south of European Russia. Glacial refugia of P. ridibundus were apparently located in the northern Balkans, the northern coast of the Black and Azov seas, and possibly in Western Europe. The northward long-distance post-glacial dispersal of P. ridibundus occurred from refugia in the northeastern Balkans and the Black-Azov seas region. Since the Late Pleistocene, suitable habitats for P. cf. bedriagae in southern Russia began to decline, but local habitats for P. ridibundus become more suitable. Therefore, a mosaic of populations consisting of these both species and their hybrids has now been found here.
Background. Molecular typing by PCR-RFLP method allows to identify the specific attribution of an organism that has a weak phenotypic difference. The main advantage of this research method is the capacity to analyze a large number of samples without applying the sequencing method. The purpose of the work is to develop a test-system for the identification the matrilines of marsh frogs of the Pelophylax ridibundus complex from Kazakhstan. Materials and methods. The analysis was based on the variability of the primary structure of the mitochondrial gene the second subunit of dehydrogenase (ND2) which is a species-specific marker. Subsequently a search for marker nucleotide substitutions specific for each lineage and recognition sites for the HaeIII and TasI restriction endonucleases was conducted. Results. As a result of the research, it was confirmed that on the territory of Kazakhstan inhabiting three main forms of lake frogs where two of them are native (Balkhash, Syrdarya), and invasive Anatolian form-P. cf. bedriagae. Conclusions. The authorsʼ PCR-RLFP test technique is a straightforward and reliable tool for detecting mitochondrial lineages in the Pelophylax ridibundus complex marsh frogs and may be used successfully in mass screening investigations.
Hybridogenesis is a special mode of hybrid reproduction where one parental genome is eliminated and the other is transmitted clonally. We propose that this mechanism can perpetuate the genome of extinct species, based on new genetic data from Pelophylax water frogs. We characterized the genetic makeup of Italian hybridogenetic hybrids (P. kl. hispanicus and esculentus) and identified a new endemic lineage of Eastern-Mediterranean origin as one parental ancestor of P. kl. hispanicus. This taxon is nowadays extinct in the wild but its germline subsists through its hybridogenetic descendant, which can thus be considered as a “semi living fossil”. Such rare situation calls for realistic efforts of de-extinction through selective breeding without genetic engineering, and fuels the topical controversy of reviving long extinct species. “Ghost” species hidden by taxa of hybrid origin may be more frequent than suspected in vertebrate groups that experienced a strong history of hybridization and semi-sexual reproduction.
Wykazanie przez Profesora Bergera, że żaba wodna Pelophylax esculentus nie jest gatunkiem lecz hybrydogenetycznym mieszańcem oraz rozwój technik molekularnych przyczyniły się do rozwoju badań nad systematyką żab zielonych zachodniej Palearktyki. Według obecnej wiedzy, dzielą się ona na gatunki hybrydogenetyczne (P. esculentus, P. grafi, P. hispanicus) oraz gatunki biologiczne wpadające do trzech głównych grup genetycznych (kladów), wyznaczonych na podstawie zmienności w mitochondrialnym DNA: 1) saharicus/perezi: P. saharicus, P. perezi, 2) lessonae: P. lessonae, P. bergeri, P. shqipericus, 3) ridibundus: P. ridibundus, P. bedriagae, P. kurtmuelleri, P. epeiroticus, P. cerigensis, P. cretensis, P. caralitanus, P. cypriensis. W Polsce potwierdzono występowanie gatunków P. lessonae i P. ridibun-dus oraz ich naturalnego mieszańca P. esculentus, natomiast w południowozachodniej części kraju stwierdzono wysoką częstość występowania mieszańców pomiędzy P. ridibundus, a pochodzącym z Bałkanów P. kurtmuelleri. Badania nad rozmieszczeniem taksonów w Europie wykazały, że część z nich została introdukowana przez człowieka poza obszarem ich naturalnego występowania, co może zaburzać naturalne systemy genetyczne żab zielonych. Słowa kluczowe: rozmieszczenie gatunków, nowe taksony, introdukcje, mitochondrialny DNA, Pelo-phylax kurtmuelleri WSTĘP Odkrycie zjawiska hybrydogenezy oraz wykazanie, że najpospolitsza żaba Europy żaba wodna, Rana esculenta Linnaeus, 1758, obecnie Pelophylax esculentus, nie jest gatunkiem tylko mieszańcem, zapoczątkowało badania nad biologią i systematyką całej grupy żab zielonych, dzięki czemu dowiedziono, że opisany przez Linneusza gatunek R. esculenta, składa się w rzeczywistości z ponad 20 taksonów, a kolejne wciąż są opisywane.
The increasing prevalence of morphological anomalies has recently been suspected as a potential mechanism in the global decline of amphibian populations and species. We studied the frequency of morphological anomalies in postmetamorphic amphibians in the Egyek-Pusztakócs marshes (Hortobágy National Park, east Hungary). The site was a wetland and grassland complex used heavily by intensive agriculture until 1973 when it was protected, restored, and managed for biodiversity. We examined 4,953 individuals of 11 species encountered in four monitoring schemes (pitfall traps, bottle traps, dip netting, visual surveys) during 2010–2013. We found no evidence of malformation and a low frequency (0.3%) of abnormalities, which did not differ from the background frequency of 0 to 5% estimated in previous studies for wild populations in natural habitats. All observed abnormalities were found in 15 individuals of four species and were consistent with injuries caused by predators, although the effects of parasites could not be excluded. It remains uncertain whether the absence of malformations and the observed low frequency of abnormalities are related to the 40 years of protection and the long-term decrease in agrochemical use or to the more recent grassland restoration and marsh management actions. Nevertheless, our study provides an example that large, healthy populations of amphibians can exist in large protected wetland complexes restored and managed for biodiversity.
Two species of green frogs exist in the state of Bavaria (Germany), the Marsh Frog (Pelophylax ridibundus) and the Pool Frog (Pelophylax lessonae). The Edible Frog (Pelophylax esculentus) is a hybridogenetic hybrid originally resulting from Marsh Frogs mating with Pool Frogs and therefore sometimes not considered a true species. Its distribution covers large parts of Bavaria, occurring either together with P. lessonae in P. lessonae-P. esculentus-systems (LE-system) or with P. ridibundus in P. ridibundus-P. esculentus-systems (RE-system). In order to obtain more precise data on their distribution and population structure, we examined 20 green frog populations in Bavaria, focusing on the possible existence of pure hybrid populations, the occurrence of allochthonous species of green frogs, and the distribution of P. lessonae, the most threatened species of green frogs. A total of 200 frogs were sampled, from which 173 could be reliably identified on the basis of their morphology. Species were identified via morphometric measurements and serum albumin PCR. Potentially triploid specimens were identified on the basis of their increased erythrocyte size. Sequencing of mitochondrial ND2 haplo types allowed identification of allochthonous frogs in three study sites. P. esculentus was the most common green frog species at the study sites, representing 47% of the sample size. Four potentially triploid individuals were detected; however , pure triploid hybrid populations could not be confirmed. Allochthonous frogs were identified in three RE-systems. Pelophylax ridibundus made up 38% of the reliably identified frogs. The percentage of P. lessonae was only 15% of the sampled frogs, possibly due to the decline of suitable habitats and the introduction of allochthonous P. ridibundus. In all three taxa, male frogs always dominated in the sex ratio. Out of the 200 observed frogs, 58 were female (29%), 109 were male (54%) and 33 were subadult (17%). This study showed that in 86.5% it was possible to distinguish between the three species by means of morphometric measurements. However, serum albumin PCR did not always correctly identify specimens. Introduction Green frogs are amongst the most common amphibians in Bavaria. Two species are present here, the Marsh Frog Pelophylax ridibundus (Pallas, 1771) and the Pool Frog P. lessonae (Camerano, 1882). The Edible Frog P. esculen-tus (Linnaeus, 1758) is not considered a true species but a hybridogenetic hybrid resulting from Marsh Frogs mating with Pool Frogs. However, for convenience we consider this form as a species in the following, although it is usually claimed to represent a klepton, P. kl. esculentus. Reproduction in P. lessonae and P. ridibundus follows Men-delian rules of inheritance whereas P. esculentus originates from hybridogenesis: During gametogenesis, one parental genome is deleted completely, so that only one parental set of uncombined chromosomes is transmitted to the next generation (Tunner 1973). Due to the hybridogenetic system , P. esculentus can live and reproduce in the presence of only one parental species. Green frog populations can be pooled as population systems according to their gen-otypic structure and specific reproduction modes. Designation as a single system occurs when the involved species is not limited by primary hybridisation, but can also hybridise hybridogenetically (Plötner 2005). Distinction is thus made between the P. lessonae-P. esculentus-system (LE-system) and the P. ridibundus-P. esculentus-system (RE-system). Their occurrences depend mainly on ecological predilections of the single parental species. P. lesso-nae is more demanding in this respect, needing sunlit waters with rich vegetation (Zahn 1997). P. ridibundus prefers larger water bodies and hibernates in the water, in contrast to P. lessonae. P. esculentus is more flexible than its parental species and poses fewer demands on its habitat (Plötner 2005). Detailed information on the morphology and ecology of the three green frog forms can be found in Günther (1990) and Plötner (2005). Massive differences
The European water frog Pelophylax esculentus is a natural hybrid between P. lessonae (genotype LL) and P. ridibundus (RR). It reproduces through hybridogenesis, eliminating one parental genome from its germline and producing gametes containing the genome of the other parental species. According to previous studies, this elimination and transmission pattern is very diverse. In mixed populations, where only diploid hybrids (LR) live in sympatry and mate with one or both parental species, the excluded genome varies among regions, and the remaining genome is transmitted clonally to haploid gametes. In all-hybrid populations consisting of diploid (LR) and triploid (LLR and/or LRR) frogs, diploid individuals also produce gametes clonally (1n in males, 2n in females), whereas triploids eliminate the genome they have in single copy and produce haploid gametes containing the recombined other genome. However, here, too, regional differences seem to exist, and some triploids have been reported to produce diploid gametes. In order to systematically study such regional and genotype differences in gamete production, their potential origin, and their consequences for the breeding system, we sampled frogs from five populations in three European countries, performed crossing experiments, and investigated the genetic variation through microsatellite analysis. For four populations, one in Poland, two in Germany, and one in Slovakia, our results confirmed the elimination and transmission pattern described above. In one Slovakian population, however, we found a totally different pattern. Here, triploid males (LLR) produce sperm with a clonally transmitted diploid LL genome, rather than a haploid recombined L genome, and LR females clonally produce haploid R eggs, rather than diploid LR eggs. These differences among the populations in gamete production go along with differences in genomotype composition, breeding system (i.e., the way triploids are produced), and genetic variation. These differences are strong evidence for a polyphyletic origin of triploids. Moreover, our findings shed light on the evolutionary potential inherent to the P. esculentus complex, where rare events due to untypical gametogenetic processes can lead to the raise, the perpetuation, and the dispersion of new evolutionary significant lineages which may also deserve special conservation measures.
Variation patterns of allozymes and of ND3 haplotypes of mitochondrial DNA reveal a zone of genetic transition among western Palearctic water frogs extending across northeastern Greece and European Turkey. At the western end of the zone, allozymes characteristic of Central European frogs known as Pelophylax ridibundus predominate, whereas at the eastern end, alleles characteristic of western Anatolian water frogs (P. cf. bedriagae) prevail. The ND3 haplotypes reveal 2 major clades, 1 characteristic of Anatolian frogs, the other of European; the European clade itself has distinct eastern and western subclades. Both the 2 major clades and the 2 subclades overlap within the transition zone. Using Bayesian model selection methods, allozyme data suggest considerable immigration into the Nestos River area from eastern and western populations. In contrast, the ND3 data suggest that migration rates are so high among all locations that they form a single panmictic unit; the best model for allozymes is second best for mitochondrial DNA (mtDNA). Nuclear markers (allozymes), which have roughly 4 times as deep a coalescent history as mtDNA data and thus may reflect patterns over a longer time, indicate that eastern and western refugial populations have expanded since deglaciation (in the last 10 000 years) and have met near the Nestos River, whereas the mtDNA with its smaller effective population size has already lost the signal of partitioning into refugia.
Water frogs inhabiting Cyprus represent a distinct evolutionary species of Messinian origin that is formally described in this paper. The systematic status of Cypriot frogs is evidenced by specific characters in their mitochondrial (mt) and nuclear (nu) DNA sequences, and the fact that they form a well supported monophyletic clade in both mtDNA and nuDNA phylogenies. While genetic data revealed clear and reproducible differences between this new taxon and all other western Palearctic water frog species including Pelophylax bedriagae in the Levant and two Anatolian water frogs lineages (P. cf. bedriagae -1 and P. cf. bedriagae -2), there is no diagnostic morphological or morphometric character that allows a clear discrimination between Cyprus frogs and frogs from the adjacent mainland. If several morphometric indices are combined as predictor variables in a discriminant analysis, however, both females and males of Cypriot water frogs are correctly distinguished from the other eastern Mediterranean lineages. While phylogenies based on concatenated sequences of two mitochondrial genes (ND2 + ND3) suggest a sister group relationship of Cypriot and Anatolian water frog lineages, our nuclear data hypothesize a sister group relationship between Cypriot frogs (sp. n.) and Crete frogs (P. cretensis), thus speaking for the same isolation time of both island populations.
Water frogs inhabiting Cyprus represent a distinct evolutionary species of Messinian origin
that is formally described in this paper. The systematic status of Cypriot frogs is evidenced
by specific characters in their mitochondrial (mt) and nuclear (nu) DNA sequences,
and the fact that they form a well supported monophyletic clade in both mtDNA
and nuDNA phylogenies. While genetic data revealed clear and reproducible differences
between this new taxon and all other western Palearctic water frog species including Pelophylax
bedriagae in the Levant and two Anatolian water frogs lineages (P. cf. bedriagae-
1 and P. cf. bedriagae-2), there is no diagnostic morphological or morphometric
character that allows a clear discrimination between Cyprus frogs and frogs from the adjacent
mainland. If several morphometric indices are combined as predictor variables in a
discriminant analysis, however, both females and males of Cypriot water frogs are correctly
distinguished from the other eastern Mediterranean lineages. While phylogenies
based on concatenated sequences of two mitochondrial genes (ND2 þ ND3) suggest a
sister group relationship of Cypriot and Anatolian water frog lineages, our nuclear data
hypothesize a sister group relationship between Cypriot frogs (sp. n.) and Crete frogs
(P. cretensis), thus speaking for the same isolation time of both island populations.
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 Provinces. One hybrid specimen from Kharkov Province was tetraploid. All polyploids were recorded along the middle part of Seversky Donets River (above 450 km). Triploids comprised two groups with different genome composition (LLR and LRR), and were found in three types of population systems (E, R–E, and L–E–R). Geographic distribution of polyploidy in European green frogs is briefly outlined. Different meth-ods of ploidy level identification are discussed. The chromosome count and nuclear DNA cytometry provide the most reliable data.
Poland is inhabited by the two water frog species Rana ridibunda (genotype RR) and Rana lessonae (LL) and their hybrido-genetic hybrid Rana esculenta. Besides diploid (RL) hybrids two triploid (RRL, RLL) forms were found. In most populations the hybrid occurs syntopically with only one of the parental species. Concerning their genotypic structure water frog populations can be subdivided into three systems: the L-E (lessonae-esculenta) system, the R-E (ridibunda-esculenta) system and the E-E (esculenta-esculenta) system. The composition of water frog populations in Poland is highly differentiated. Among 27 population types described from central Europe 18 types were found in Poland. The highest diversity of population types was seen in the R-E system (8 types). In the L-E system three and in the E-E system four different types were found. Among 11170 investigated adult water frogs 690 (6%) presumptive triploid R. esculenta individuals occurred. In western Poland the proportion of triploid individuals and the diversity of population types was higher (7.7% triploid individuals, 13 population types) than in the eastern parts of the country (0.2% triploid individuals, six population types). The distribution picture of triploid individuals in western Poland is similar to that seen in eastern Germany (as a rule a high proportion of triploids occurs) while in eastern Poland the distribution pattern of triploid genotypes resembles the situation observed in the territory of the former Soviet Union (nearly no triploids appear).
Genotypic variation at six of 67 microsatellite loci we developed from Rana ridibunda, containing di- or trinucleotide simplesequence repeats, confirms the value of microsatellites as an evolutionary genetic tool for studying western Palearctic water frogs, a model system characterized by clonal reproduction in natural hybrid lineages. R. ridibunda, Rana lessonae. and their hemiclonal hybrid Rana esculenta, which transmits only its ridibunda genome to gametes, are highly variable in central Poland and northern Switzerland: with one exception (a locus that is part of a coding region), each locus was polymorphic in each taxon when scorable, and the number of alleles was high (in Swiss lessonae-esculenta populations, 1-6 per locus, total 13, for ridibunda genomes; 1-9 per locus, total 23, for lessonae genomes; 3 shared by both genomes). Estimated repeat numbers ranged from 3 to 14 for trinucleotide- and from 6 to 27 for dinucleotide-repeats. Nonamplifying (null) alleles, problematic because they underestimate genotypic variability if undetected, occur in ridibunda genomes of Swiss R. esculenta at one locus: apparent lessonae null alleles at another locus were subsequently resolved for several segregating alleles evidenced by bands of much lower intensity. Two loci had multiple ridibunda alleles in Swiss R. esculenta and thus discriminate between hemiclones. with a resolution superior to that of allozymes. Adding these two loci to our protein electrophoretic data (7 loci) more than doubled the number of hemiclones detected, showed that several allozyme-defined hemiclones are composites of multiple microsatellite hemiclones, and revealed clonal diversity in populations that are uniclonal by allozyme data. The extent to which microsatellite-defined hemiclones result from primary hybridizations or from subsequent mutations in ridibunda genomes of R. esculenta lineages remains to be determined. A sample of newly metamorphosed R. ridibunda from hybrid × hybrid matings collected at a Swiss population in the fall had a significant heterozygote excess at the two loci that varied in ridibunda genomes; this shows that most, probably all, of them originated from inter- rather than intrahemiclonal matings, in accord with the idea that the usual hybrid × hybrid inviability in lessonae-esculenta systems is caused by deleterious recessive alleles on the clonally inherited ridibunda genomes. Three of four microsatellite loci tested amplified in most species of western Palearctic water frogs.
In order to study the relationship between community organization and spatial-temporal variability, species richness and abundance of amphibian species were estimated in 17 sites of the French Upper Rhone floodplain. These sites were chosen because they were representative of the diversity of successional processes, age and connection with the active channel. Ecological variation among these sites was estimated by the analysis of 14 mesological, hydrological, and biotic variables, by means of correspondence analysis. Level of disturbance, eutrophication, water origin, presence of fish and water persistence were the main determinants of site typology.
Amphibian species richness was highest in temporary sites experiencing intermediate level of disturbance. Three species (Rana ridibunda, Rana dalmatina, Triturus helveticus) were ubiquitous, three species (Hyla arborea, Bufo calamita, Pelodytes punctatus) were characteristic of habitats with intermediate variability and four (Triturus cristatus, Triturus alpestris, Bufo bufo, Bombina variegata) were rare in the floodplain. The most abundant species in the floodplain appeared to be r-selected and most of them showed a temporal flexibility of reproduction. These reproduction traits were assumed to respond to the balance between habitat unpredictability and interspecific constraint by making the adjustment to both availability and suitability of breeding habitats possible.
Globalization and increasing human impact on natural aquatic systems have facilitated the movement of species and the establishment
of nonindigenous species enhancing hybridisation opportunities between naturally allopatric species. In this review, we focus
on a special case of natural hybrid speciation and the consequences of recent anthropogenic hybridisation in the water frog
complex (Pelophylax esculentus complex), which consists of two parental species, Pelophylax lessonae and Pelophylax ridibundus and a hybrid taxon. The hybrid water frogs reproduce hybridogenetically and eliminate the genome of the syntopic water frog
species. Although the actual cause triggering chromosome exclusion remains elusive, it has been proposed that chromosome elimination
takes place prior to meiosis and may involve enzymatic degradation of the discarded genome. Translocations of water frogs
in Western Europe have become frequent the last decade leading to rapid expansion of the range of the marsh frog P. ridibundus. Subsequent hybridisation of the exotic P. ridibundus may dramatically affect the viability and maintenance of hybrid water frog populations throughout Europe. Interestingly,
the impact of this introduced species may differ depending on their geographic origin, which defines the ability to induce
genome elimination. This may result in fertile or sterile hybrids, making global conservation guidelines challenging. We predict
a severe genetic and ecological impact of nonindigenous P. ridibundus prompting for strict conservation measures to reduce species translocations and for studies on the geographic origin of exotic
frog species.
The complex study, including allozyme variability and cytometry of hybrid populations of green frogs Pelophylax esculentus (L., 1758) complex has confirmed that the only region of Ukraine where allodiploid are encountered frequently is the Severski
Donets basin (9% of all hybrids). In other areas, only two polyploidy hybrids (0.9%) and one probably autopolyploid individual
of each parental species have been registered. According to allozyme specters, all three polyploidy hybrids from the Severski
Donets basin were males and belonged to biotype P. esculentus (=lessonae) — 2 ridibundus, and their population in this region has halved during the past decade.
SYNOPSIS. Recent genetic studies of asexually reproducing fishes in the genus Poeciliopsis (Poeciliidae) revealed abundant variation in the form of multiple sympatric clones. Recur- rent hybridizations between sexual species provides the principal source of clonal variation. The hybrids are spontaneously endowed with a clonal reproductive mechanism that per- petuates a high level of heterozygosity. Migration within and between river systems, and mutations, also contribute to clonal diversity in" these fish. Coexistence among different clones and with the sexual ancestors depends in part upon specializations characteristic of individual clones. Clonal reproduction is an efficient mechanism for freezing a portion of the niche-width variation contained in the gene pool of the more broadly adapted, sexual ancestors. Multiclonal populations achieve significantly higher densities relative to the sexual forms than do monoclonal populations. This relationship is a function of the clonal variability upon which natural selection can act and upon the capacity of a multiclonal population to better exploit a heterogeneous environment through niche diversification. In all-female organisms such as Poeciliopsis, which are dependent upon sexual species for insemination, competitive abilities probably are at a premium in the densely populated pools and arroyos of the Sonoran Desert. Competitive abilities are probably less important for truly parthenogenetic clones which rely on colonization abilities to escape from their sexual ancestors and from other clones.
Hybridization and polyploidy play an important role in animal speciation. European water frogs of the Pelophylax esculentus complex demonstrate unusual genetic phenomena associated with hybridization, clonality and polyploidy which presumably indicate an initial stage of reticulate speciation. The Seversky Donets River drainage in north-eastern Ukraine is inhabited by both sexes of the diploid and triploid hybrid P. esculentus and only one parental species Pelophylax ridibundus. Based on the presence of various types of hybrids, all populations studied can be divided into three geographical groups: I) P. ridibundus-P. esculentus without triploids; II) P. ridibundus-P. esculentus without diploid hybrids; and III) P. ridibundus-P. esculentus with a mixture of diploids and triploids. A study of gametogenesis revealed that diploid P. esculentus in populations of the first type usually produced haploid gametes of P. ridibundus and a mixture of haploid gametes that carried one or another parental genome (hybrid amphispermy). In populations of the second type, hybrids are derived from crosses of P. ridibundus males with triploid hybrid females producing haploid eggs with a genome of P. lessonae. Therefore, we suggest that clonal genome duplication in these eggs might be the result of suppression of second polar body formation or extra precleavage endoreduplication. In populations of the third type, some diploid females can produce diploid gametes. Fertilization of these eggs with haploid sperm can result in triploid hybrids. Other hybrids here produce haploid gametes with one or another parental genome or their mixture giving rise to new diploid hybrids.
Rana esculenta is a hybrid between Rana lessonae (LL) and Rana ridibunda (RR), and hybrids may be diploid (LR) or triploid (LLR or LRR). Genotypes can be roughly determined from erythrocyte size and morphometry in adult frogs, but accurate genotyping requires more labourious methods. Here I demonstrate that both the L and R genomes have specific microsatellite alleles, and that genotype and ploidy can be accurately inferred from the quantitative ratio of PCR-amplified (polymerase chain reaction-amplified) genome-specific alleles. This method greatly facilitates genotyping in DNA studies of the R. esculenta complex and allows analysis of badly preserved samples and embryos.
Water frogs inhabit nearly all of Europe, northern Africa and western Asia to Pakistan. The European forms were named Rana esculenta by Linnaeus in 1758. Since that time many forms have been described, but the systematic status of the central European forms has been much disputed.
Polyploidization is a rare yet sometimes successful way for animals to rapidly create geno- and phenotypes that may colonize new habitats and quickly adapt to environmental changes. In this study, we use water frogs of the Pelophylax esculentus complex, comprising two species (Pelophylax lessonae, genotype LL; Pelophylax ridibundus, RR) and various diploid (LR) and triploid (LLR, LRR) hybrid forms, summarized as P. esculentus, as a model for studying recent hybridization and polyploidization in the context of speciation. Specifically, we compared the geographic distribution and genetic diversity of diploid and triploid hybrids across Europe to understand their origin, maintenance and potential role in hybrid speciation. We found that different hybrid and parental genotypes are not evenly distributed across Europe. Rather, their genetic diversity is structured by latitude and longitude and the presence/absence of parental species but not of triploids. Highest genetic diversity was observed in central and eastern Europe, the lowest in the northwestern parts of Europe. This gradient can be explained by the decrease in genetic diversity during postglacial expansion from southeastern glacial refuge areas. Genealogical relationships calculated on the basis of microsatellite data clearly indicate that hybrids are of multiple origin and include a huge variety of parental genomes. Water frogs in mixed-ploidy populations without any parental species (i.e. all-hybrid populations) can be viewed as evolutionary units that may be on their way towards hybrid speciation. Maintenance of such all-hybrid populations requires a continuous exchange of genomes between diploids and triploids, but scenarios for alternative evolutionary trajectories are discussed.
© 2015 John Wiley & Sons Ltd.
European green frogs include the phenotypically distinct, largely sympatric forms Rana lessonae Camerano 1882, R. esculenta Linnaeus 1758, and R. ridibunda Pallas 1771, the relationships of which is still unsufficiently known. Recent investigations on Polish green frog populations indicate that R. lessonae and R. ridibunda are taxa at species rank, whereas R. esculenta is a hybrid resulting from interspecific crosses between R. lessonae and R. ridibunda. All three forms of green frogs are easily crossable indicating close relationship, and lack of genetic isolation. Ecological isolating mechanisms were probably established during the Pleistocene. In spite of very frequent natural hybridization, very high number of F1 hybrids (= esculanta) in nature, and successful backcrosses especially with lessonae, introgression between lessonae and ridibunda seems not to occur owing to an incompletely understood hereditary mechanism.
The scarcity of parthenogenetic vertebrates is often attributed to their ‘inferior’ mode of clonal reproduction, which restricts them to self-reproduce their own genotype lineage and leaves little evolutionary potential with regard to speciation and evolution of sexual reproduction. Here, we show that for some taxa such uniformity does not hold. By using hybridogenetic water frogs (Pelophylax esculentus) as a model system, we demonstrate that triploid hybrid males from two geographic regions exhibit very different reproductive modes. With an integrative data set combining field studies, crossing experiments, flow cytometry and microsatellite analyses, we found that triploid hybrids from Central Europe are rare, occur in male sex only and form diploid gametes of a single clonal lineage. In contrast, triploid hybrids from North Western Europe are widespread, occur in both sexes and produce recombined haploid gametes. These differences translate into contrasting reproductive roles between regions. In Central Europe, triploid hybrid males sexually parasitize diploid hybrids and just perpetuate their own genotype – which is the usual pattern in parthenogens. In North Western Europe, on the other hand, the triploid males are gamete donors for diploid hybrids, thereby stabilizing the mixed 2n-3n hybrid populations. By demonstrating these contrasting roles in male reproduction, we draw attention to a new significant evolutionary potential for animals with non-sexual reproduction, namely reproductive plasticity.Key words: all-hybrid population; sperm-dependent parthenogenesis, asexual reproduction; gamete production; hemiclone; hybridogenesis; hybridization; polyploidy; Rana; sexual parasite.This article is protected by copyright. All rights reserved.
Coexistence of sperm-dependent asexual hybrids with their sexual progenitors depends on genetic and ecological interactions between sexual and asexual forms. In this study, we investigate genotypic composition, modes of hybridogenetic gametogenesis and habitat preferences of European water frogs (Pelophylax esculentus complex) in a region of sympatric occurrence. Pelophylax esculentus complex comprises parental species P. ridibundus and P. lessonae, whose primary hybridization leads to hybridogenetic lineages of P. esculentus. Hybrids clonally transmit one parental genome and mate with the other parental species, forming a new generation of hybrids. In the region of western Slovakia, we found syntopic occurrence of diploid and triploid hybrids with P. lessonae, syntopic occurrence of all three taxa as well as the existence of pure P. ridibundus populations. All triploid hybrids were exclusively male possessing one ridibundus and two different lessonae genomes (RLL). Sex ratio in diploid hybrids was substantially female-biased. Irrespective of the population composition, diploid hybrids excluded the lessonae genome from their germ line and produced ridibundus gametes. Contrarily, RLL males unequivocally eliminated the ridibundus genome and produced diploid lessonae sperms. Perpetuation of RLL males in studied populations is most likely achieved by their mating with diploid hybrid females. The composition of water frog populations is also shaped by taxon-specific habitat preferences. While P. ridibundus preferred larger water bodies (gravelpits, fishery ponds, dead river arms), P. lessonae was most frequently found in marshes and smaller sandpits. Pelophylax esculentus occupied predominately similar habitats as its sexual host P. lessonae.
The water frogs of western Tuscany (Italy) belong to the L-E hybridogenetic system, and comprise one parental species and its hybrid. A stringent morphological approach for discriminating the Italian hybrids from non-hybrids has yet to be established. In this work, using the molecular marker RrS1, we have determined the hybrid versus non-hybrid status of 43 water frogs collected from two sampling sites ("Fiume Morto Vecchio" and "Padule di Bientina"). At "Fiume Morto Vecchio" we determined 25 non-hybrids and nine hybrids and in "Padule di Bientina" we determined eight hybrids and one non-hybrid individual. All individuals of these two frog populations were analyzed morphologically. We used the derived residuals from regression analysis of all normally distributed morphological parameters on callus internus length (snout to vent length, tibia length, head width, distance nostril eye, first toe length and body mass) to build sex independent variables in discriminant analysis providing a valid contribution to morphologically distinguish hybrids from non-hybrid green frogs in Italy.
Urbanization is a pervasive process causing habitat fragmentation, spatial isolation of populations, and reduction of biological diversity. In this study, we applied 11 microsatellite loci and Bayesian analyses to investigate genetic diversity and population structure in marsh frogs (Pelophylax ridibundus) living in two types of environment—highly fragmented urban landscapes, and landscapes characterized by the presence of a river and artificial canals. Our results show reduced genetic diversity, lower effective population sizes, and higher genetic differentiation for spatially isolated urban populations in comparison with populations outside intensely urbanized areas. Reduction of allelic diversity in urban localities isolated for 13–37 generations is more conspicuous than reduction of expected heterozygosity. Populations living close to the River Danube, its branches, and artificial canals are genetically more homogenous. Our results also suggest that the Danube in Bratislava is not a natural barrier to gene flow. In contrast, it acts as a natural corridor for water frog dispersal. Population structure of P. ridibundus also shows higher genetic connectivity within water paths than between them, suggesting limited overland dispersal, and reflects the historical landscape structure associated with the distribution of the lost river branches.
Forty-two water bodies in an agricultural lowland area of northern Italy were investigated with respect to the presence of amphibians and to a number of habitat features. The following species were found: Rana kl. esculenta, R. latastei, Bufo bufo, B. viridis, Hyla arborea, Triturus carnifex and T. vulgaris meridionalis. A discriminant analysis was carried out for each species to test whether there were significant difference between ecological characteristics of used and unused sites; all analyses showed a significant difference, and they also made it possible to identify the most important habitat features for each species found. Vegetational factors seemed to be the most important.
Two water frog populations, Rana lessonae-esculenta systems, have been studied in Hungary from the morphological point of view. Multivariate analyses of selected morphological measurements based on biochemical identification of the species provide two ratios as possible key characters for determination of the two forms.
Rana esculenta is a hybridogenetic hybrid between Rana ridibunda and Rana lessonae. Usually the hybrids, among which diploid and triploid individuals of both sexes were described, live in mixed populations with one of the parental species (L-E or R-E systems). The hybrids also form pure esculenta-esculenta populations (E-E system). 114 individuals from the Baltic Sea region were collected in 1990–1991 and 1998-1999. Blood smears were taken and erythrocyte areas were measured. The erythrocyte area ranged from 318.27 μm2 to 571.82 μ2 and two peaks could be distinguished: 370 μ2 and 460–490 μ2. The smears were then stained according to the Feulgen method and the DNA amount per nucleus was determined by measuring the integrated optical density of the nucleus area. The correlation between erythrocyte size and DNA content was relatively high (0.82). Nevertheless, in some cases the erythrocyte size was not a good indicator of DNA content. Specimens had various DNA content and they formed two distinct groups: one with higher and one with lower DNA content. However, we were not able to discriminate the expected two classes of diploid and triploid individuals. The results indicated that the problem of ploidy is complicated, and we cannot rule out the existence of aneuploids or mosaics among hybrids. The problem needs to be elucidated in combination with other methods, especially because it allows measuring the ploidy level in great number of animals without killing them and thus seems to be very useful for population studies.
Rana esculenta is a hybrid between Rana lessonae (LL) and Rana ridibunda (RR), and hybrids may be diploid (LR) or triploid (LLR or LRR). Genotypes can be roughly determined from erythrocyte size and morphometry in adult frogs, but accurate genotyping requires more labourious methods. Here I demonstrate that both the L and R genomes have specific microsatellite alleles, and that genotype and ploidy can be accurately inferred from the quantitative ratio of PCR-amplified (polymerase chain reaction-amplified) genome-specific alleles. This method greatly facilitates genotyping in DNA studies of the R. esculenta complex and allows analysis of badly preserved samples and embryos.
The diploid hybridogenetic frog Rana esculenta, an interspecific hybrid of R. ridibunda and R. lessonae, usually transmits its ridibunda genome to progeny: the lessonae genome is lost before gametogenesis is completed. Samples of ovary reveal only ridibunda alleles when examined electrophoretically. Cytological and genetic evidence suggests that the lessonae genome is excluded premeiotically. Since recombination between the parental genomes occurs at a low frequency, such recombination should be detectable by examination of the alleles expressed in individual enlarged oocytes.
Examination of electrophoretic markers for four loci from 860 individual oocytes (100 each from eight females, ten each from six females) revealed no evidence of either heterozygosity or recombination, although the eight females for which 100 oocytes were examined were somatically heterozygous 29 times for these loci, so that there were 2,900 possibilities for detecting heterozygosity or recombination in the oocytes.
These data abundantly confirm the inactivity of the lessonae genome during vitellogenesis in Rana esculenta. They are consistent with a premeiotic exclusion of the lessonae genome, but do not require it. The lessonae genome may be present although inactive, which may permit occasional crossing over between the lessonae and ridibunda genomes. The exclusion of the lessonae genome possibly is a consequence of its inactivity. Exclusion of the non-ridibunda genome seems to be under control of loci located in the ridibunda genome. A regulatory endoduplication of the ridibunda genome may be necessary before an orderly meiosis can occur.
We investigated the genomic constitution and the reproduction mode of Rana esculenta from two pure hybrid populations found in western Slovakia, using cytomorphological, karyological and allozyme electrophoretic methods. Both populations were composed of diploid R. esculenta females and diploid and triploid R. esculenta males. A single subadult R. ridibunda female was caught at one of the localities. The females significantly outnumbered the males in both populations. All electro-phoretically investigated triploid R. esculenta males possessed the allopolyploid genomic constitution RLL (one “ridibunda” and two “lessonae” genomes). In the ovaries/testes of diploid females/males, exclusively allelic products characteristic of R. ridibunda were detected, suggesting that they hybridogenetically produced gametes carrying a “ridibunda” genome. On the contrary, the electrophoresis revealed only allelic products characteristic of Rana lessonae in the testes of triploid RLL males from one of the populations. Thus, these males apparently substitute genetically the host species R. lessonae and play the important role in perpetuation of the investigated population.
Western Palearctic water frogs in the genus Pelophylax are a set of morphologically similar anuran species that form hybridogenetic complexes. Fully reliable identification of species and especially of hybrid ploidy depends on karyological and molecular methods. In central Europe, native water frog populations consist of the Pelophylax esculentus complex, that is, P. lessonae (LL), P. ridibundus (RR) and the hybrid form P. esculentus that can have different karyotypes (RL, LLR and RRL). We developed existing molecular methods further and propose a simple PCR method based on size-differences in the length of the serum albumin intron-1 and the RanaCR1, a non-LTR retrotransposon of the chicken repeat (CR) family. This PCR yields taxon-specific banding patterns that can easily be screened by standard agarose gel electrophoresis and correctly identify species in all of the 160 samples that had been identified to karyotype with other methods. To distinguish ploidy levels in LR, LLR and RRL specimens, we used the ratio of the peak heights of the larger (ridibundus specific) to the smaller (lessonae specific) bands of fluorescently labelled PCR products resolved on a capillary DNA sequencer and obtained a correct assignment of the karyotype in 93% of cases. Our new method will cut down time and expenses drastically for a reliable identification of water frogs of the P. esculentus complex and potentially for identification of other hybridogenetic complexes and/or taxa, and it even serves as a good indicator of the ploidy status of hybrid individuals.
Abstract Hybrid zones are either distributed along clines or in a mosaic of patches. This distribution may depend upon variation in taxon habitat use. Habitat use and distribution of diverse taxa of water frogs (Rana ridibunda, R. lessonae, R. perezi, R. kl. grafi and R. kl. esculenta) in France are analysed to determine whether water frog complexes conform to the mosaic or clinal model. Biogeographical scenarios may be invoked in order to explain the distribution of water frogs. However, the distribution of R. perezi and R. kl. grafi, being restricted to regions characterized by Mediterranean or Oceanic climatic conditions, suggests that these frogs do not endure cold winters. R. ridibunda is widespread in Southern France and its distribution suggests multiple introductions. It is concluded that water frogs conform to the mosaic zone model rather than to the tension zone model because: (i) taxa exhibited differences in habitat use, (ii) pure parental species were documented and (iii) hybrids are not unfit relative to parental species.