Article

A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax)

Molecular Ecology Resources

June 2012
12(5):797-805

DOI:10.1111/j.1755-0998.2012.03160.x

Source
PubMed

Authors:

Susanne Hauswaldt

Technische Universität Braunschweig

Maria Ogielska

University of Wrocław

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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.

Hybrid swarm as a result of hybridization between two alien and two native water frog species (genus Pelophylax) in Central Croatia: Crna Mlaka fishpond acting as a species melting pot?

Article

Full-text available

Jun 2022
BIOL INVASIONS

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.

Will there be a second extinction? Molecular identification of multiple alien water frogs (Pelophylax ridibundus sensu lato) in Tuscany, Central Italy, reveals genetic pollution within a unique hybridogenetic system

Article

Full-text available

Jul 2020
HERPETOL J

The introduction of alien water frogs is perhaps one of the most underestimated herpetological conservation issues in Europe. The identification of distinct species is highly challenging at the phenotypic level, and artificial syntopy between various taxa and lineages may lead to diverse outcomes, including hybridisation and local extinction. In central Italy the native synklepton of Pelophylax bergeri (the parental taxon) and P. kl. (klepton) hispanicus (the hybridogenetic hybrid, which clonally transmits the genome of an extinct ridibundus-like taxon) is present. Until recently, data regarding the presence of alien water frogs in central Italy was scarce, and no alien taxa have been reported for Tuscany. In this study, four distinct non-native Pelophylax lineages have been identified via molecular analysis in the Cecina and Arno river basins and ascribed to the Marsh frog group (P. ridibundus sensu lato). Alien Pelophylax ridibundus, P. kurtmuelleri, and P. cf. bedriagae sensu stricto currently appear to be widespread in the Cecina basin. Furthermore, evidence of hybridisation with autochthonous taxa has been suggested by genetic analyses in four out of eight sampling localities. With a view to evaluate urgent conservation strategies, a greater sampling effort is required to assess the actual distribution and ecology of the alien lineages, and further research is necessary to measure their impact on the native hybridogenetic system of the central-southern Italian pool frogs.

A record of alien Pelophylax species and widespread mitochondrial DNA transfer in Kaliningradskaya Oblast' (the Baltic coast, Russia)

Article

Full-text available

Jun 2020

Alien species can strongly impact local environments and compete against native species, which can lead to their extinction. Marsh frogs of the Pelophylax ridibundus complex are one of the most invasive amphibians in Northern Eurasia. It was previously thought that three water frog species of the genus Pelophylax (the marsh frog, P. ridibundus, the pool frog, P. lessonae and their hemiclonal hybrid, the edible frog, P. esculentus) inhabited Kaliningradskaya Oblast' along the Russian Baltic coast. However, based on our study of the intron-1 of the nuclear serum albumin gene, two other marsh frog species were detected (the Balkan marsh frog, P. kurtmuelleri, and the Anatolian marsh frog, P. cf. bedriagae) as well as putative hybrids between P. ridibundus and P. cf. bedriagae. The majority of individuals of P. ridibundus and hybrids between P. ridibundus and P. cf. bedriagae had mitochondrial (mt) DNA of P. lessonae, while all others featured the P. kurtmuelleri mtDNA. The prevalence of P. lessonae mtDNA haplotypes in populations of P. ridibundus from the Baltic Coast of Russia suggests that local individuals of the latter species originated from crosses between P. esculentus individuals. Two hypotheses could explain the records of P. kurtmuelleri and P. cf. bedriagae in the region. The establishment of local populations of the first species could have occurred via postglacial dispersal from the Balkan refugium. The origin of local P. cf. bedriagae could be an occasional introduction of individuals from the Ponto-Caspian region. Since our study is preliminary (19 individuals), in the future it would be important to continue the study of water frogs in Kaliningradskaya Oblast' and neighboring countries by applying multiple genetic markers. Additional genetic markers will enable researchers to study routes of dispersal and introductions of marsh frogs, to clarify peculiarities of their hybridization and distribution, and to evaluate the impact of P. kurtmuelleri and P. cf. bedriagae on the reproduction success of hybridogenous populations and abundance of local amphibians.

Extended distribution of Phtisica marina Slabber, 1769 (Crustacea: Amphipoda): first observation of alien Caprellid in the coastal waters of Indian subcontinent

Article

Feb 2019

Phtisica marina, originally described by Slabber, 1769 from Netherlands, is one of the most abundant caprellid amphipod species reported from numerous regions around the globe and is primarily associated with fouling communities in harbours. This study chronicles the further spreading out of P. marina into the Indian coastal waters. During a regular coastal survey in Gulf of Kachchh (Northwest coast of India), an established population of P. marina was observed in the subtidal regions of Vadinar and Sikka during April 2017. The caprellid could be a possible alien species in Indian waters as the species met the criteria for exotic taxa. The most possible introductory vectors and pathways are discussed in this study. As this species can survive in the stressed environments of low hydrodynamics and it has opportunistic behaviour, further studies are necessary to reveal its potential impact on local communities.

Detection of alien Pelophylax species in Sardinia (western Mediterranean, Italy)

Article

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Jan 2019

Adriana Bellati

Westward range expansion of the Indo-Pacific nakedband gaper Champsodon nudivittis (Ogilby, 1895) in Saronikos Gulf, Greece

Article

Feb 2019

Methodological approaches for studying the european water frog Pelophylax esculentus complex

Article

Full-text available

Aug 2017

The European water frog (Pelophylax esculentus) complex represents a unique and adequate model system for the study of interspecific hybridization and the mechanisms enabling interspecific hybrids to overcome the reproductive barriers. The difficulties in the study of individuals from the P. esculentus complex are associated with high polymorphism of morphological characters in parental species and interspecific hybrids, as well as with the presence of polyploid hybrid forms. From the discovery of the phenomenon of interspecific hybridization and the demonstration of successful reproduction of interspecific hybrids, researchers constantly searched for the methods necessary for the most accurate identification of parental species and various hybrid forms. This review describes biochemical, cytogenetic, and molecular methods and approaches used to identify individuals from the European water frog complex, as well as to analyze the genomes transferred with the gametes of hybrids. The advantages and disadvantages of these approaches are discussed. The presented methods can be used for studying other hybrid complexes of fish, amphibians, and reptiles.

Preliminary genetic data suggest the occurrence of the Balkan water frog, Pelophylax kurtmuelleri, in southwestern Poland

Article

Full-text available

May 2017

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.

New Multiplex PCR Method for Identification of East European Green Frog Species and Their Hybrids

Article

Full-text available

Dec 2019

A molecular multiplex PCR method for identification of East European green frog species (Pelophylax ridibundus, P. cf. bedriagae and P. lessonae) and their hybrids was developed. This simple and rapid method can be used for identification of species-specific mitochondrial and nuclear DNA. The method is based on species-specific differences in primary structure of the subunit 1 of the mitochondrial cytochrome C oxidase gene (COI) and the intron-1 of the nuclear serum albumin gene (SAI-1). Based on the method, we analyzed numerous individuals of these species and their hybrids from East European Plain, the Crimea, the Caucasus, the Ural, as well as introduced populations from Western Siberia and the Kamchatka. In all cases, identification of species performed by use of the multiplex PCR method coincided with results of study of primary nucleotide sequences.

Discovery of alien water frogs (gen. Pelophylax) in Umbria, with first report of P. shqipericus for Italy

Article

Full-text available

Dec 2013

Allochthonous water frogs (gen. Pelophylax) have been repeatedly introduced in several European coun-tries, causing dramatic consequences for the conservation of indigenous taxa. In Italy, invasive populations are known for northern regions, where they were introduced mainly for edible and scientific purposes. Here, we report the first detection of an alien population of water frogs in Central Italy, along the Resina valley (Umbria). Genetic analysis of the mitochondrial ND3 gene polymorphism assigned some specimens to two different Pelophylax ridibundus clad-es widespread in Central and Eastern Europe. By contrast, two samples matched the mitochondrial DNA (mtDNA) sequence of Pelophylax lessonae bergeri, an autochthonous taxon widespread in Central Italy, suggesting possible hybridization between alien and indigenous frogs. Finally, the specific haplotype of Pelophylax shqipericus, the Alba-nian Pool Frog, was also identified according to mtDNA polymorphism. This record, firstly reported for Italy, poses concerns for the conservation of this cryptic taxon, suggesting that international water frog trade may involve also particularly endangered species.

Inheritance patterns of male asexuality in hybrid males of a water frog Pelophylax esculentus

Article

Full-text available

Sep 2024

Gametogenesis produces gametes as a piece of genetic information transmitted to the offspring. While during sexual reproduction, progeny inherits a mix of genetic material from both parents, asexually reproducing organisms transfer a copy of maternal or paternal DNA to the progeny clonally. Parthenogenetic, gynogenetic and hybridogenetic animals have developed various mechanisms of gametogenesis, however, their inheritance is not fully understood. Here, we focused on the inheritance of asexual gametogenesis in hybrid Pelophylax esculentus (RL), emerging after crosses of P. lessonae (LL) and P. ridibundus (RR). To understand the mechanisms of gametogenesis in hybrids, we performed three-generation experiments of sexual P. ridibundus females and hybrids from all-male hybrid populations. Using fluorescent in situ hybridization, micronuclei analysis, flow cytometry and genotyping, we found that most adult hybrid males simultaneously produced two types of clonal sperm. Also, most male tadpole progeny in two successive backcrossed generations simultaneously eliminated L and R parental genomes, while some progeny produced only one type of sperm. We hypothesize that the reproductive variability of males producing two kinds of sperm is an adaptive mechanism to reproduce in mixed populations with P. ridibundus and may explain the extensive distribution of the all-male lineage across the European River Basin.

Countrywide screening supports model-based predictions of the distribution of Batrachochytrium dendrobatidis in Ukraine

Article

Full-text available

Aug 2024
DIS AQUAT ORGAN

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.

Detection of Glacial Refugia and Post-Glacial Colonization Routes of Morphologically Cryptic Marsh Frog Species (Anura: Ranidae: Pelophylax) Using Environmental Niche Modeling

Article

Full-text available

Feb 2024
Diversity

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.

A hotchpotch of water frogs in northern Italy

Article

Full-text available

May 2023
BIOL INVASIONS

Water frogs of the genus Pelophylax have been widely traded by humans across all European countries for decades, and so far they have been shown to be invasive in most of them. The spread of alien Pelophylax threatens the persistence of native populations mainly via competition and hybridization. Particularly, the latter is worrying for the persistence of the native hybridogenetic systems, as it may induce hybrid swarms whose final outcome is hard to foresee. Alien water frogs have been already detected across several Italian regions, but the extension of the invasion is still unknown, in terms of both species composition and distribution. Here, we carried out the most extensive molecular survey of alien water frog populations across northern Italy, the main invaded area, and obtained a worrying scenario of multiple introduced alien taxa, often co-occurring at the same site. Frogs carrying the native mitochondrial haplotypes were found mainly at the edge of the invaded range. The most widespread taxa turned out to be P. ridibundus and P. kurtmuelleri, but members of the highly diversified P. bedriagae species complex also occurred in the western (P. bedriagae) and the eastern sector (P. cf. bedriagae sensu stricto and P. cf. bedriagae “Cilician West”) of the range. We inferred the geographic origin of alien taxa according to mitochondrial haplotype variation and provide the fact-finding background to investigate the impact of alien introductions on native populations, in order to evaluate effective and reliable conservation strategies for native hybridogenetic systems.

Numerous uncertainties in the multifaceted global trade in frogs’ legs with the EU as the major consumer

Article

Full-text available

Feb 2023

The commercial trade in frogs and their body parts is global, dynamic and occurs in extremely large volumes (in the thousands of tonnes/yr or billions of frogs/yr). The European Union (EU) remains the single largest importer of frogs’ legs, with most frogs still caught from the wild. Amongst the many drivers of species extinction or population decline (e.g. due to habitat loss, climate change, disease etc.), overexploitation is becoming increasingly more prominent. Due to global declines and extinctions, new attention is being focused on these markets, in part to try to ensure sustainability. While the trade is plagued by daunting realities of data deficiency and uncertainty and the conflicts of commercial interests associated with these data, it is clear is that EU countries are most responsible for the largest portion of the international trade in frogs’ legs of wild species. Over decades of exploitation, the EU imports have contributed to a decline in wild frog populations in an increasing number of supplying countries, such as India and Bangladesh, as well as Indonesia, Turkey and Albania more recently. However, there have been no concerted attempts by the EU and present export countries to ensure sustainability of this trade. Further work is needed to validate species identities, secure data on wild frog populations, establish reasonable monitored harvest/export quotas and disease surveillance and ensure data integrity, quality and security standards for frog farms. Herein, we call upon those countries and their representative governments to assume responsibility for the sustainability of the trade. The EU should take immediate action to channel all imports through a single centralised database and list sensitive species in the Annexes of the EU Wildlife Trade Regulation. Further, listing in CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) can enforce international trade restrictions. More joint efforts are needed to improve regional monitoring schemes before the commercial trade causes irreversible extinctions of populations and species of frogs.

Emerging infectious diseases of amphibians in Poland: distribution and environmental drivers

Article

Full-text available

Nov 2021

Emerging infectious diseases are a threat to biodiversity and have taken a large toll on amphibian populations worldwide. The chytrid fungi Batrachochytrium dendrobatidis ( Bd ) and B. salamandrivorans ( Bsal ), and the iridovirus Ranavirus ( Rv ), are of concern as all have contributed to amphibian declines. In central and eastern Europe, their geographical and host distributions and main environmental drivers determining prevalence are poorly known. We screened over 1000 amphibians from natural and captive populations in Poland for the presence of Bd , Bsal and Rv . In wild amphibian populations, we found that Bd is widespread, present in 46 out of 115 sampled localities as well as 2 captive colonies, and relatively common with overall prevalence at 14.4% in 9 species. We found lower prevalence of Rv at 2.4%, present in 11 out of 92 sampling sites, with a taxonomic breadth of 8 different amphibian species. Bsal infection was not detected in any individuals. In natural populations, Pelophylax esculentus and Bombina variegata accounted for 75% of all Bd infections, suggesting a major role for these 2 species as pathogen reservoirs in Central European freshwater habitats. General linear models showed that climatic as well as landscape features are associated with Bd infection in Poland. We found that higher average annual temperature constrains Bd infection, while landscapes with numerous water bodies or artificial elements (a surrogate for urbanization) increase the chances of infection. Our results show that a combination of climatic and landscape variables may drive regional and local pathogen emergence.

A reference DNA barcode library for Austrian amphibians and reptiles

Article

Full-text available

Mar 2020
PLOS ONE

In the last few years, DNA barcoding became an established method for species identification in biodiversity inventories and monitoring studies. Such studies depend on the access to a comprehensive reference data base, covering all relevant taxa. Here we present a comprehensive DNA barcode inventory of all amphibian and reptile species native to Austria, except for the putatively extinct Vipera ursinii rakosiensis and Lissotriton helveticus, which has been only recently reported for the very western edge of Austria. A total of 194 DNA barcodes were generated in the framework of the Austrian Barcode of Life (ABOL) initiative. Species identification via DNA barcodes was successful for most species, except for the hybridogenetic species complex of water frogs (Pelophylax spp.) and the crested newts (Triturus spp.), in areas of sympatry. However, DNA barcoding also proved powerful in detecting deep conspecific lineages, e.g. within Natrix natrix or the wall lizard (Podarcis muralis), resulting in more than one Barcode Index Number (BIN) per species. Moreover, DNA barcodes revealed the presence of Natrix helvetica, which has been elevated to species level only recently, and genetic signatures of the Italian water frog Pelophylax bergeri in Western Austria for the first time. Comparison to previously published DNA barcoding data of European amphibians and reptiles corroborated the results of the Austrian data but also revealed certain peculiarities, underlining the particular strengths and in the case of the genus Pelophylax also the limitations of DNA barcoding. Consequently, DNA barcoding is not only powerful for species identification of all life stages of most Austrian amphibian and reptile species, but also for the detection of new species, the monitoring of gene flow or the presence of alien populations and/or species. Thus, DNA barcoding and the data generated in this study may serve both scientific and national or even transnational conservation purposes.

Mitochondrial Heteroplasmy in Marsh Frog (Pelophylax ridibundus Pallas, 1771)

Article

Full-text available

Aug 2019

The population features of nuclear and mitochondrial genomes of P. ridibundus Pallas, 1771 and related species of green frogs from Nizhny Novgorod and Sverdlovsk oblasts were investigated for the first time. The existence of the R–E–L population system on the territory of Nizhny Novgorod oblast was confirmed. The presence of heteroplasmy was found in all examined samples of marsh frogs, as well as in hybrid, edible, frogs. These findings indicate the presence of hybridization and introgressions that took place in the history of the studied forms of each of the species. Considerable heterogeneity of animals from the territory of Sverdlovsk oblast in the nuclear and to a higher degree in the mitochondrial genome was demonstrated, which with high probability indicates that the populations existing in this territory originated from multiple introductions. The obtained data characterize the state of population systems, as well as the history of the formation of the modern phylogenogeographic pattern of green frogs on the studied territories.

Small heat shock proteins in the amphibian Pelophylax bergeri: Cloning and characterization of Hsp27 and Hsp30 cDNAs and their expression analysis in ex vivo skin exposed to abiotic stresses

Article

May 2019

Small Heat Shock Proteins (sHSP) are molecular chaperones that play an essential role in maintaining protein homeostasis and promoting cell survival. In this work, for the first time, multiple cDNAs encoding for small Hsp27 and Hsp30, designed, respectively, as PbHsp27-(1-2) and PbHsp30-(1-5), were cloned and characterized in the amphibian Pelophylax bergeri, which is a suitable model for studying biological responses to environmental perturbations. Domain architecture analysis showed that PbHsp27 and PbHsp30 cDNAs displayed the typical signature motifs of the sHSP family such as the conserved α-crystallin domain flanked by variable N-terminal and C-terminal regions. Phylogenetic analysis revealed that PbHsp27 and PbHsp30 clustered, respectively, with Hsp27 and Hsp30 members of other vertebrates, but more closely with amphibians. Overall PbHsp27 and PbHsp30 transcriptional activity, analyzed by qRT-PCR, evidenced that, in ex vivo skin exposed to thermal shock and cadmium treatment, PbHsp27 and PbHsp30 mRNAs were inducible and regulated differently. This study provides the basis for future research on the potential use of PbHsp27 and PbHsp30 as biomarkers of proteotoxic stress in amphibians.

Distribution and Origin of Two Forms of the Marsh Frog Pelophylax ridibundus Complex (Anura, Ranidae) from Kamchatka Based on Mitochondrial and Nuclear DNA Data

Article

Full-text available

Dec 2018

The formation of the first populations of the marsh frog (the Pelophylax ridibundus complex) near Petropavlovsk-Kamchatsky and in the Paratunka River valley must have been the result of human introduction in the late 1980s. At present, more than 20 localities of this species are recorded in Kamchatka. For a more precise definition of the taxonomic status of P. ridibundus sensu lato, samples from five populations (altogether, 30 individuals) from southeastern and central Kamchatka are analyzed using molecular methods. In all frogs, a mitochondrial DNA type specific for the “eastern” form (= the Anatolian P. cf. bedriagae), but not for the “western” form (=the Central European P. ridibundus), is revealed. However, the results of nuclear DNA analysis of marsh frogs from Kamchatka reveal alleles specific for both of the forms, “eastern” and “western,” with a frequency ratio of about 2 : 1. The results of sequencing the mitochondrial ND2 gene and nuclear SAI-1 gene suggest that the “ancestor” individuals might have been introduced into Kamchatka from the Volga–Don interfluve or Ciscaucasia. The absence of both haplotype and nucleotide diversity in the samples studied suggests a single successful introduction that involved a low number of frogs stemming from a single locality.

Batrachochytrium dendrobatidis in Hungary: an overview of recent and historical occurrence

Article

Full-text available

Sep 2018

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.

On distribution of the edible frog Pelophylax esculentus (Linnaeus, 1758) on the territory of the Volga River drainage

Article

Full-text available

Nov 2017

New data about distribution of the edible frog Pelophylax esculentus, diagnosed by the methods of morphometry, flow DNA-cytometry and molecular genetic analysis in 10 regions of the Volga River drainage are presented. Possible ways of the establishment of P. esculentus populations and factors limiting the distribution and abundance of these species in the eastern part of the studied region are analyzed.

First results of green frog (Pelophylax esculentus complex) species diagnostics in Latvia. Первые результаты видовой диагностики зеленых лягушек (Pelophylax esculentus complex) в Латвии [In Russian with English Abstract]

Conference Paper

Full-text available

Mar 2017

Three species of water frogs (Pelophylax esculentus complex) inhabit the territory of Latvia. Using PCR method the species affiliation was performed for 50 frogs from 5 water bodies. P. ridibundus was found in two sites: the Kish and the Jugla Lakes in Riga city. P. esculentus was identified in four habitats: the Jugla Lake in Riga city, small pond and Borne River in Daugavas Loki Nature Park and big fish ponds complex in Nagli. P. lessonae was found only in one biotop: big fish ponds complex in Nagli.

Variability of NOR patterns in European water frogs of different genome composition and ploidy level

Article

Full-text available

Apr 2017
COMP CYTOGENET

We studied water frogs from a complex composed of two species: Pelophylax lessonae (Camerano, 1882) (genome LL, 2n = 26) and P. Ridibundus (Pallas, 1771) (RR, 2 = 26), and their natural hybrid P. Esculentus (Fitzinger, 1843) of various ploidy and genome composition (RL, 2n = 26, and RRL or RLL, 3n = 39). Tetraploids RRLL were found (4n = 52) in juveniles. We applied cytogenetic techniques: AgNO3, chromomycin A3, PI and fluorescent in situ hybridization with a 28S rDNA probe. Results obtained by silver staining corresponded well with those stained with CMA3, PI and FISH. As a rule, NORs are situated on chromosomes 10. The number of Ag-NORs visible on metaphase plates was the same as the number of Ag-nucleoli present in interphase nuclei of the same individual. In all analyzed metaphases, NORs exhibited variations in size after AgNO3 and CMA3 stainings. Sixty-six individuals (out of 407 analyzed) were polymorphic for the localization and number of NORs. Fifty-one diploids had NORs only on one chromosome of pair 10. Three triploids (LLR and RRL) displayed two NORs, and two other triploid RRL individuals displayed one, instead of expected three NORs. In ten individuals extra NORs were detected on chromosomes other than 10 (chromosomes 2 and 9).

ГЕНЕТИЧЕСКИЕ ФОРМЫ ОЗЁРНОЙ ЛЯГУШКИ (PELOPHYLAX RIDIBUNDUS COMPLEX) ЗАПАДНОГО КАВКАЗА ПО ДАННЫМ АНАЛИЗА МИТОХОНДРИАЛЬНОЙ И ЯДЕРНОЙ ДНК / GENETIC CHARACTERISTICS OF MARSH FROG (PELOPHYLAX RIDIBUNDUS COMPLEX) FROM THE WESTERN CAUCASUS BASED ON MITOCHONDRIAL AND NUCLEAR DNA DATA

Article

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Jan 2016

Molecular genetic analysis of 73 specimen of Pelophylax ridibundus complex from 14 locations in Russia (Krasnodarskiy Krai, Republic of Adygea), Georgia and Armenia were conducted using COI and SAI-1 DNA markers. The mtDNA haplotype typical for “eastern” form of marsh frog (= Anatolian P. cf. bedriagae) is prevalent in west Caucasus, while haplotype peculiar to the “western” form (= Central European P. ridibundus) is fully absent. Two specimens carried a haplotype typical for “Euphrates” line were found in northern Armenia. Analysis of nuDNA nevertheless revealed remarkable presence of alleles belonging to the “western” form of marsh frog in the studied area. The mean ratio of “western” and “eastern” allele frequencies is close to 1:3.

Helminth Parasites of the Pelophylax esculentus Complex (Anura: Ranidae) in Hortobágy National Park (Hungary)

Article

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Jan 2016

The Document, Assess, Monitoring, Act (DAMA) protocol details an approach to integrating information about parasites into large-scale studies of biodiversity, climate change, and emerging diseases. This study represents an effort to put the DAMA protocol into practice. We collected 101 individuals of protected ranid frogs belonging to the Pelophylax esculentus complex during 2012 and 2013 in the Hortoba ´gy National Park (HNP) in eastern Hungary in an area where an inventory of amphibian helminths had been conducted 40 yr previously. Collecting sites included flowing water, a fish pond system, and a wetland marsh system. We found the following helminth species: Digeneans: Diplodiscus subclavatus, Haematoloechus variegatus, Opisthioglyphe ranae, Pleurogenes claviger, Pleurogenoides medians; Nematodes: Oswaldo- cruzia filiformis, Rhabdias esculentarum; and Acanthocephala: Acanthocephalus ranae. Rhabdais esculentarum is a new species for the Hungarian fauna and P. ridibundus represents a new host record for R. esculentarum while D. subclavatus, P. claviger, and P. medians are new species for the helminthofauna of the HNP. Our findings showed a significant discrepancy from the results of baseline inventories carried out 40 yr ago, although the reasons for this discrepancy are not clear.Wesuspect that the previously reported helminth species that we did not encounter are restricted to Pelophylax lessonae, a host we have not yet collected at this location, but factors associated with climate change or anthropogenic impacts cannot be ruled out.

2014-Tognarelli et al-morphology genetics green frogs

Data

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May 2014

Surveys on populations of green frogs (Pelophylax) of Western Tuscany sites with molecular and morphometric methods

Article

Jan 2014

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.

Decline of Pelophylax lessonae in mixed populations of water frogs over the last 50 years

Article

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Jan 2024

Two water frog species: the pool frog Pelophylax lessonae (L) and the marsh frog P. ridibundus (R) occur sympatrically in Central Europe and form mixed populations (genetic systems) with their hybrid, the edible frog P. esculentus (E). The aim of the study was to assess the species composition of water frogs in urban and rural populations and compare our current findings with the results of previous studies. We surveyed the same sites that were investigated by Berger et al. in 1962-1970 (Poznań, urban landscape) and 1977-1997 (Dezydery Chłapowski Landscape Park, rural landscape). Because some ponds surveyed in the past were destroyed or dried-up, we also explored others located in the adjacent areas. We captured frogs during breeding seasons 2020 and 2021 and identified them by the nuclear marker gene SAI-1. We found three types of populations in the urban area: RE , E-E and RE -L and four in the rural area: RE , L-E, E-E and RE -L. Compared to the historical data, we found a drastic decrease in the frequency of P. lessonae in urban and rural landscapes: from 89.1% and 68% to 2.7% and 1.8%, respectively. At the same time, the frequency of P. ridibundus increased from 2.2% and 0.1% to 40% and 29.6%, respectively. A similar pattern was found for P. esculentus whose frequency increased from 8.7% and 31.9% to 57.3% and 68.6%, respectively. Additionally, we confirmed the presence of a cryptogenic Balkan water frog, P. kurtmuelleri, which was recently discovered in southwestern Poland. The frequency of SAI-1 allele specific for this taxon reached 7.3%. The patterns found in both types of landscapes are in line with the current situation of both parental species in Europe. Such dynamic changes show the need for long-term monitoring of the population compositions of water frogs, what is crucial for their conservation management.

Detection of environmental DNA from amphibians in Northern Europe applied in citizen science

Article

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Aug 2023

Many species of amphibians in Northern Europe are threatened and the local distributions are rarely described in detail. Application of modern molecular methods provides an important supplementary tool for monitoring the distribution and diversity of amphibians. For this purpose, we designed, tested, validated, and optimized 14 species‐specific assays on genomic DNA extracted from tissue samples to use for quantitative polymerase chain reaction (qPCR) setups targeting mitochondrial DNA from amphibians in freshwater samples. The tests confirmed species specificity for all assays. Considering a systematic definition of the limit of detection for each of the assays, the presented qPCR assays are unlikely to return false positive detection from any co‐occurring species in northern Europe. For field validation, the qPCR assays were applied in a large‐scale nationwide citizen science project in which sampling and qPCR analysis was carried out by high school students. Data from the citizen science project returned the expected results when compared to the known regional distribution of the target species and confirmed the presence of nine out of 14 Danish species of amphibians in the collected freshwater samples. Four out of 2550 qPCR test sets carried out by the high school students required a professional reanalysis in multiple replicates due to initial unexpected results. This emphasizes that efforts from citizen science may generate large amounts of valuable data, as long as the results are carefully scrutinized by experts.

Population structure, mate choice, and genome transmission in naturally formed pairs in a Pelophylax lessonae–Pelophylax esculentus hybridogenetic system

Article

Full-text available

Jul 2023
BEHAV ECOL SOCIOBIOL

Central European water frog Pelophylax esculentus is a hybrid that lives sympatrically and forms genetic systems with one of its parental species, in this case L-E (P. lessonae - P. esculentus). Hybrids are restored in each generation due to hybridogenesis, a unique mode of hemiclonal reproduction. We investigated changes in the species composition, mate choice, and genome transmission in naturally formed pairs in the L-E hybridogenetic system from the Raków fish pond complex (SW Poland). Our observations showed that the genetic system remained the same for over a decade but the share of the taxa and the sex ratios within the species changed. The frequency of P. lessonae decreased by about 20% in favor of P. esculentus. The proportion of males to females of P. lessonae increased twofold and of P. esculentus fourfold. We also found that the stability of the L-E population was ensured by a specific female mate choice pattern with the dominance of homotypic P. lessonae and heterotypic ♀P. esculentus × ♂P. lessonae pairs (almost 90% of the pairs collected from nature). The youngest females and males that formed a successful amplexus were 3 years old. We noted a male-male competition manifested by the exclusion of young males (before the third hibernation) from the participation in the mating. An analysis of genome transmission to gametes revealed that females P. lessonae transmitted Lx, while P. esculentus transmitted the Rx and/or Lx genomes. Males of P. lessonae transmitted the Lx or Ly genomes, while P. esculentus transmitted the Lx, Ly, and/or Rx genomes. The high proportion of the L genomes transferred to gametes enables both the restoration of the parental species P. lessonae and the regular renewal of a new generation of hybrids. Significance statement Central European water frog complex includes two species: Pelophylax ridibundus (genotype RR) and P. lessonae (LL), and their natural hybrid, P. esculentus (RL). Hybrid individuals of both sexes reproduce due to a specific mode of reproduction, i.e., hybridogenesis, in which hybrids eliminate one of the parental genomes (R or L) from the germ line prior to meiosis and transmit the other one (not recombined, i.e., clonal) to the gametes. The hybrids live in mixed populations with one of the parental species and are restored in each generation. This, in turn, allows a kind of balance to be maintained in a population due to specific mate choice and genome transmission. Our research showed that the stability of the studied population was ensured by female preference to P. lessonae and male-male competition manifested by the exclusion of males under 3 years of age from the participation in the mating.

Genome elimination from the germline cells in diploid and triploid male water frogs Pelophylax esculentus

Article

Full-text available

Oct 2022

Hybridogenesis is a hemiclonal reproductive strategy in diploid and triploid hybrids. Our study model is a frog P. esculentus (diploid RL and triploids RLL and RRL), a natural hybrid between P. lessonae (LL) and P. ridibundus (RR). Hybridogenesis relies on elimination of one genome (L or R) from gonocytes (G) in tadpole gonads during prespermatogenesis, but not from spermatogonial stem cells (SSCs) in adults. Here we provide the first comprehensive study of testis morphology combined with chromosome composition in the full spectrum of spermatogenic cells. Using genomic in situ hybridization (GISH) and FISH we determined genomes in metaphase plates and interphase nuclei in Gs and SSCs. We traced genomic composition of SSCs, spermatocytes and spermatozoa in individual adult males that were crossed with females of the parental species and gave progeny. Degenerating gonocytes (24%–39%) and SSCs (18%–20%) led to partial sterility of juvenile and adult gonads. We conclude that elimination and endoreplication not properly completed during prespermatogenesis may be halted when gonocytes become dormant in juveniles. After resumption of mitotic divisions by SSCs in adults, these 20% of cells with successful genome elimination and endoreplication continue spermatogenesis, while in about 80% spermatogenesis is deficient. Majority of abnormal cells are eliminated by cell death, however some of them give rise to aneuploid spermatocytes and spermatozoa which shows that hybridogenesis is a wasteful process.

A development of a PCR-RFLP test system for the identification of mitochondrial lines of the Pelophylax ridibundus lake frog in Kazakhstan

Method

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May 2022

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.

Genomic basis for an informed conservation management of Pelophylax water frogs in

Article

Full-text available

Apr 2022

Genetic identification methods have become increasingly important for species that are difficult to identify in the field. A case in point is Pelophylax water frogs. While their morphological determination is highly complex, they include species protected under EU law and some that are classified as invasive. Additionally, genetic data can provide insights into their complex breeding systems, which may or may not involve the reproductive dependency of one species on another. Here, we generate baseline data for water frog monitoring in Luxembourg. We applied a countrywide sampling approach and used SNPs generated by ddRAD sequencing to identify individuals and infer the breeding systems present in the country. We found Pelophylax lessonae and P. kl. esculentus throughout Luxembourg, mostly living in syntopy. In general, a reproductive dependency of P. kl. esculentus on P. lessonae (L-E system) was revealed. Besides this general system, we detected triploid P. kl. esculentus in six ponds. This indicates a modified L-E system with reproductive dependency of the triploids on the diploid P. kl. esculentus. The invasive P. cf. bedriagae was detected in three ponds in southern Luxembourg, with evidence for hybridization with native water frogs. In addition to the ddRAD data, we tested a simple genetic method for future monitoring based on the MND1 marker. It showed in almost all cases, an identical species identification as the ddRAD data and was successfully applied to DNA extracts from mouth swabs. Combining this method with our baseline data will enable informed choices for the protection of native water frog species in Luxembourg.

Przegląd "nowych" gatunków płazów i gadów występujących w Polsce / A review of "new" species of amphibians and reptiles occurring in Poland

Article

Full-text available

Apr 2019

Several species of amphibians and reptiles new to Poland were described recently. However, data on their origin, distribution and taxonomic status are often limited and ambiguous. Here, we summarize the current state of knowledge on these species and propose some changes to the national list of protected species. The eastern tree frog and eastern slow worm are native to Poland and should be under strict and partial protection, respectively. We recommend inclusion of these species in the regional lists of protected species until the national list is updated. The status of four other taxa (Balkan frog, toad Bufotes variabilis, common wall lizard and dice snake) is uncertain and needs further study. Until the status of the dice snake is established, it should be removed from the list of protected species.

Species composition and distributional peculiarities of green frogs (Pelophylax esculentus complex) in Protected Areas of the Middle Volga Region (Russia)

Article

Full-text available

Sep 2018

A high level of biological diversity, including amphibians, is characteristic for Protected Areas. The group of the European green frogs has a special interest because they are characterised by unique genetic and ecological interactions in the process of hybridogenic reproduction. In 2000–2018 in the Middle Volga Region we studied green frogs in five nature reserves (Mordovia State Nature Reserve, State Nature Reserve «Prisurskiy», Volzhsko-Kamskiy State Nature Biosphere Reserve, Zhiguli State Nature Biosphere Reserve, and State Nature Reserve «Privolzhskaya lesostep'») and four national parks (National Park «Smolny», National Park «Chavash Varmane», National Park «Samarskaya Luka», and National Park «Buzulukskiy Bor»). These Protected Areas are located in a zone of overlap of ranges of Pelophylax lessonae and P. ridibundus, i.e. in area of potential spread of their meroclonal hybrid P. esculentus. Pelophylax ridibundus inhabits eight studied Protected Areas, while P. lessonae has been registered in seven and P. esculentus only in five of them. Two molecular-genetic markers were used in the study: mitochondrial COI and nuclear SAI-1. Mitotypes and alleles of two cryptic forms of P. ridibundus were revealed here, a «western» (P. ridibundus) and «eastern» (P. cf. bedriagae). In the Protected Areas we found four (of six which are possible) combinations of mitotypes of mitochondrial DNA and alleles of nuclear DNA of these two forms, and only one combination in P. esculentus. Six types of population systems of green frogs are presented in the studied Protected Areas. Single-species systems, including P. ridibundus (n = 16; 41.0%) or P. lessonae (n = 10; 25.6%), predominated. Among mixed systems a type including all three species was most common (n = 6; 15.4%). Two-species systems were the rarest: P. ridibundus and P. lessonae (n = 3; 7.7%), P. esculentus and P. lessonae (n = 3; 7.7%), P. ridibundus and P. esculentus (n = 1; 2.6%).

The programmed DNA elimination and formation of micronuclei in germ line cells of the natural hybridogenetic water frog Pelophylax esculentus

Article

Full-text available

May 2018

DNA elimination is a radical form of gene silencing and occurs both in somatic and germ cells. The programmed DNA elimination occurs during gametogenesis in interspecies hybrids that reproduce by hybridogenesis (stick insects, fishes, and amphibians) and concerns removal of whole genomes of one of the parental species and production of clonal gametes propagating the genome of the other species. The cellular mechanisms differ considerably in hybridogenetic insects and fishes but remains unknown in edible frogs Pelophylax esculentus, natural hybrids between Pelophylax lessonae and Pelophylax ridibundus. Here we report DNA elimination mechanism in early developing gonads of diploid and triploid hybrid frogs, studied by TEM, immunofluorescence, and cytochemistry. In gonocytes of both sexes (primary oogonia and prespermatogonia), micronuclei emerge as detached nuclear buds formed during interphase. We found depletion of nuclear pore complexes in micronuclear membrane and chromatin inactivation via heterochromatinization followed by degradation of micronuclei by autophagy. Micronuclei formation does not lead to apoptotic cell death showing that genome elimination is a physiological process. Chromatin elimination via micronuclei in P. esculentus is unique among hybridogenetic animals and contributes to broadening the knowledge about reproductive modes in animals.

МЕТОДИЧЕСКИЕ ПОДХОДЫ К ИЗУЧЕНИЮ КОМПЛЕКСА СРЕДНЕЕВРОПЕЙСКИХ ЗЕЛЕНЫХ ЛЯГУШЕК , "Генетика"

Article

Jan 2017
Genetika

Parasites, taxonomic composition and ploidy level of the Pelophylax esculentus complex

Thesis

Full-text available

Nov 2017

Dávid Herczeg

Taxonomic composition and ploidy level among European water frogs (Anura: Ranidae: Pelophylax) in eastern Hungary

Article

May 2017

The frequency of degenerating germ cells in the ovaries of water frogs (Pelophylax esculentus complex)

Article

Full-text available

Mar 2017
ZOOMORPHOLOGY

Pelophylax esculentus is the fertile hybrid of P. ridibundus and P. lessonae. During gametogenesis, one of the parental genomes is removed from the germ line cells, whereas the other one is clonally transmitted to the gametes. In hybrids, development of gonads is delayed in comparison with parental species. This may result from complex processes of genome elimination in female tadpoles at Gosner stages 28–46, potentially responsible for increased degeneration of germ cells in developing gonads from the very beginning of sexual differentiation to ovaries with diplotene oocytes, respectively. In this work, we revealed that germ cells died by apoptosis, as detected by expression of active caspase-3 using immunohistochemical method. The main group of degenerating germ cells was primary oogonia, however, in P. lessonae and P. ridibundus also secondary oogonia and diplotene oocytes were found. The number of degenerating germ cells was significantly higher in ovaries of P. esculentus. In hybrids, positive correlations were demonstrated between Gosner stage and gonadal volume, Gosner stage and the number of degenerating germ cells, gonadal volume and number of degenerating germ cells. These observations suggest that increased rate of apoptosis in germ cells, probably as the result of improper genome elimination, may be responsible for delayed maturation of ovaries in P. esculentus.

Composition of twenty Green Frog populations (Pelophylax) across Bavaria, Germany

Article

Full-text available

May 2013

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

Applications and limits of morphological methods for species determination of European water frogs (Pelophylax esculentus complex)

Article

Full-text available

Oct 2010

Jörg Plötner

Despite the enormous progress of molecular biology, morphological methods still play an important role in systematic zoology and population biology; many species are still discovered, described, and determined on the basis of their external morphology. Even closely related species and hybrids, such as the various forms of the Central European water frog complex, can almost always be determined using morphological characters alone. In Central European water frogs, the size and shape of the inner metatarsal tubercle in concert with coloration characters and different morphometric parameters are sufficient to determine the majority of indigenous water frogs correctly. Determination errors may particularly arise in respect to triploid hybrids, which exhibit character states similar to the parental species P. lessonae or P. ridibundus. Morphological characters are not useful, however, either for distinguishing between diploid and triploid hybrids or for drawing conclusions on inheritance patterns of P. esculentus.

Die westpaläarktischen Wasserfrösche. Von Märtyrern der Wissenschaft zur biologischen Sensation

Book

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Jan 2005

Jörg Plötner

Möglichkeiten und Grenzen morphologischer Methoden zur Artbestimmung bei europäischen Wasserfröschen (Pelophylax esculentus-Komplex)

Article

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Jan 2010

Jörg Plötner

On the presence of Rana ridibunda ridibunda Pallas in the Netherlands

Article

Full-text available

Jan 1975

Marinus Steven Hoogmoed

Since long there has been a dispute whether in the Netherlands there occur one or two species of green frogs of the genus Rana. There has never been any doubt concerning the presence of Rana esculenta Linnaeus, a species widely distributed throughout the country (Van de Bund, 1964, 1968). The first to mention Rana ridibunda ridibunda Pallas from the Netherlands were Van Kampen & Heimans ( 1927), who observed that they saw a specimen of Rana esculenta var. ridibunda from Den Horn in the province of Groningen. Unfortunately they did not state where the specimen was deposited and no such specimen could be found in the collections of the Rijksmuseum van Natuurlijke Historie (RMNH) in Leiden or in the Instituut voor Taxonomische Zoölogie in Amsterdam. Since that time there have been several additional records of the species. Van Wijk (1946, 1947, 1951) recorded the species from the provinces of Groningen, Zuid-Holland and Limburg. However, these data are not very reliable as they were based on field-observations by untrained people, mainly persons who keep reptiles and amphibians as pets. The material on which their observations were based was not available for examination, so the identifications could not be checked. Van Wijk (1947, 1951) also stated that he had received specimens from Groningen which he identified as R. ridibunda. As the characters he used to distinguish between the green frogs were correct, his identification probably was correct too. Unfortunately, also this material is not available for examination. The next author dealing with this problem is Van de Bund (1964), who gives more details concerning the localities of the specimens previously recorded by Van Wijk (1946, 1947, 1951): Groningen (Groningen, Haren, Eelde), Zuid-Holland (Sassenheim, Schiedam), Limburg (Meerssen). However, Van de Bund

Large eggs and ploidy of green frog populations in Central Europe

Article

Full-text available

Apr 2011

Green frogs of Central Europe consist of three taxa: Pelophylax ridibundus, P. lessonae and their natural hybridogenetic hybrid, P. esculentus, which forms as a rule mixed populations with its parental species. We examined 659 095 eggs from P. ridibundus (48 females), P. lessonae (133 females) and P. esculentus (170 females) originating from 39 populations in Austria, Denmark, Germany and Poland. Some females of each taxon laid eggs that fell into discrete size classes (small, medium and large). Large eggs were mostly diploid gametes from which triploids developed. They were found in P. esculentus (25 233 large eggs in 152 spawns), P. lessonae (81 in 10 spawns) and P. ridibundus (7 in 3 spawns). The main purpose of the paper was to demonstrate that the numbers of large eggs were clearly associated with triploid P. esculentus frogs. In pure hybrid (esculentus) populations large eggs comprised between 2.44-40.96% of all ova, while triploid adult frogs constituted between 13.9-73.2% of all individuals, in mixed ridibundus-esculentus populations the large eggs and triploid frogs ranged between 0.85-36.6% and 9.2-56.2%, respectively. However, in mixed lessonae-esculentus populations large eggs comprised only 1.74% of the spawns, whereas triploid frogs represented 2.1% of the adults in the population.

Genome Dosage Effect and Hybrid Morphology — The Case of the Hybridogenetic Water Frogs of the Pelophylax esculentus Complex

Article

Full-text available

Jan 2012

Western Palearctic water frogs offer a unique possibility to study the genome dosage effect (GDE). There are two morphologically distinct species, Pelophylax ridibundus RR and Pelophylax lessonae LL, and their hybridogenetic hybrid Pelophylax esculentus (RL, LLR or RRL). It is supposed that RL have intermediate morphological features, LLR are more similar to P. lessonae, and RRL more similar to P. ridibundus. We tested if the morphology of the water frogs reflects the GDE, and whether it can be used in the field for determination of the genome composition. Mean values of the indices DP/CI, T/CI and F/T followed the order LL-LLR-RL-RRL-RR. After applying discriminant and canonical analyses 89% RR, 95% LL, 91% RL, 84% LLR and 52% RRL were correctly classified. Surprisingly, the L haplotype had bigger influence on morphology than the R haplotype — all hybrid genotypes were morphologically closer to P. lessonae than to P. ridibundus.

Morphometric and DNA investigations into European water frogs (Rana kl. esculenta Synklepton (Anura, Ranidae)) from different population systems

Article

Full-text available

Apr 2009
J ZOOL SYST EVOL RES

The population specific variability of diploid and triploid R. kl. esculenta individuals was investigated by means of morphometric methods (canonical discriminant analysis, UPGMA cluster analysis) and DNA fingerprinting. As a result of the morphometric investigations, as well as of the DNA investigations, a clear separation of single populations was possible. However, no correlations between the morphometry and different population systems could be recognized. Clear morphometric differences could be seen between diploid ♀♀ and ♂♂ and triploid ♀♀ on the one hand, and triploid ♂♂ on the other. While the diploid ♀♀ and ♂♂ and the triploid ♀♀ were located in the intermediate area between the parental species R. lessonae and R. ridibunda according to their morphometric parameters, the triploid ♂♂ showed a great overlap with R. lessonae. Up to now, this phenomenon has not been explained.The first results of the DNA investigations provided further hints at the high inter-individual and population-specific variability of R. kl. esculenta. R. kl. esculenta individuals of the R. lessonae/esculenta population Toter See could be distinguished from conspecific individuals of the R. ridibunda/esculenta-♀♀ population Alte Oder according to their fingerprint patterns. Moreover, in the R. lessonae / esculenta population, the fingerprints or the diploid R. kl. esculenta-♀♀ and the investigated R. lessonae-♀ were very similar. Furthermore, in this population, many R. kl. esculenta genotypes resemble R. lessonae in their morphometric features. This finding suggests the occurrence of recombination in R. kl. esculenta.In general, every population seems to have its own genetic background. A classification of water-frog populations according to population systems is only possible under certain conditions.

DNA content and genome composition of diploid and triploid water frogs belonging to the Rana esculenta complex (Amphibia, Anura)

Article

Full-text available

Dec 2004
CAN J ZOOL

The Central European water frog Rana esculenta L., 1758 is a natural hybrid between Rana lessonae Camerano, 1882 (LL) and Rana ridibunda Pallas, 1771 (RR). Hybrids are usually diploid (RL) or triploid (LLR or RRL). Distinguishing LL from RL, RR from RL, and LLR from RRL according to external morphology is ambiguous. In this study we checked whether the DNA content in erythrocyte nuclei measured by image cytometry is useful in de-termination of the taxonomic status of diploids (LL, RR, and RL) and the genome composition of triploids (LLR and RRL). For exact and direct identification of parental species, as well as for determination of genome composition in hybrids, we applied actinomycin D – 4′,6-diamidino-2-phenylindole chromosome staining to metaphase plates. We ana-lyzed 43 LL, 12 RR, and 32 RL diploids, and 37 LLR and 19 RRL triploids. All diploid hybrids had 2n = 26 chromo-somes, and all triploid hybrids had 3n = 39 chromosomes. Neither aneuploid nor mosaic hybrids were detected. The expected numbers of 13 R. lessonae (L) and 13 R. ridibunda (R) chromosomes in RL hybrids were recorded in about 31% of individuals. In the rest of the sample the composition was variable, ranging from 9 to 14 R chromosomes and the corresponding number of L chromosomes. The expected composition of 26 L and 13 R chromosomes was detected in about 32% of LLR triploids, whereas in the rest of the sample the composition of chromosomes ranged from 8 to 15 R chromosomes and the corresponding number of L chromosomes. The expected numbers of 26 R and 13 L chro-mosomes were detected in about 26% of RRL triploids, whereas in the rest of the sample the composition of chromo-somes ranged from 19 to 28 R chromosomes and the corresponding number of L chromosomes. The DNA content densitometry showed that RR and RL diploids had 9.5% and 3.8% more DNA, respectively, than LL diploids. These differences, although statistically significant, were not sufficient to unequivocally discriminate LL from RL and RR from RL. Triploids had about 50% more DNA than LL diploids (49% in LLR and 51% in RRL), but these differences were too small for unequivocal determination of their genome composition. 1901 Résumé : La grenouille verte Rana esculenta L., 1758 d'Europe centrale est un hybride naturel de Rana lessonae Camerano, 1882 (LL) et de Rana ridibunda Pallas, 1771 (RR). Les hybrides sont généralement diploïdes (RL) ou tri-ploïdes (LLR ou RRL). La distinction des LL des RL, des RR des RL et des LLR des RRL d'après les caractères morphologiques externes donne des résultats ambigus. Nous avons vérifié si le contenu de l'ADN des noyaux des éry-throcytes, mesuré par cytométrie par analyse d'images, permettait de déterminer le statut taxinomique des diploïdes (LL, RR et RL) et la composition du génome des triploïdes (LLR et RRL). Afin d'identifier directement et avec précision les espèces parentales et de déterminer la composition du génome des hybrides, nous avons coloré les chromosomes des plaques en métaphase à l'actinomycine D et au 4′,6-diamidino-2-phénylindole. Nous avons analysé 43 diploïdes LL, 12 RR et 32 RL, ainsi que 37 triploïdes LLR et 19 RRL. Tous les hybrides diploïdes possédaient 2n = 26 chromo-somes et tous les hybrides triploïdes 3n = 39 chromosomes. Nous n'avons observé ni hybride aneuploïde, ni hybride en mosaïque. Les nombres attendus de 13 chromosomes de R. lessonae (L) et de 13 de R. ridibunda (R) ont été notés chez environ 31 % des hybrides RL; dans le reste de l'échantillon, la composition était variable, allant de 9 à 14 chro-mosomes R avec un nombre complémentaire de chromosomes L. La composition attendue de 26 chromosomes L et de 13 R a été retrouvée chez environ 32 % des triploïdes LLR; dans le reste de l'échantillon, la composition chromoso-mique variait de 8 à 15 chromosomes R avec un nombre complémentaire de chromosomes L. La composition attendue de 26 chromosomes R et de 13 L a été retrouvée chez environ 26 % des triploïdes RRL; dans le reste de l'échantillon, la composition chromosomique variait de 19 à 28 chromosomes R avec un nombre complémentaire de chromosomes L. La mesure du contenu en ADN par densitométrie indique que les diploïdes RR et RL avaient respectivement 9,5 % et 3,8 % plus d'ADN que les diploïdes LL. Bien que statistiquement significatives, ces différences ne suffisent pas pour sans équivoque les LL des RL et les RR des RL. Les triploïdes avaient environ 50 % de plus d'ADN que les diploïdes LL (49 % chez les LLR et 51 % chez les RRL), mais les différences sont trop faibles pour déterminer de fa-çon non équivoque les compositions génomiques. [Traduit par la Rédaction] Ogielska et al.

Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (Pelophylax esculentus complex)

Article

Full-text available

Jan 2009

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.

A genetic mechanism of species replacement in European waterfrogs?

Article

Full-text available

Mar 2003

Introduced Rana ridibunda currentlyreplace the native waterfrogs R. lessonaeand R. esculenta in several areas ofcentral Europe. The unusual reproductive systemin waterfrogs of the Rana esculentacomplex suggests that this replacement may bedriven by a genetic mechanism: Ranaesculenta, a hybrid between R. ridibundaand R. lessonae, eliminates the lessonae genome from the germline and clonallytransmits the ridibunda genome(hybridogenesis). Hybrids form mixedpopulations with R. lessonae (L-E-system)in which they persist by backcrossing with theparental species. Matings between hybrids areunsuccessful, because their ridibundagenomes contain fixed recessive deleteriousmutations. When introduced into a L-E-system,R. ridibunda can mate with both nativetaxa, producing R. ridibunda offspringwith R. esculenta, and R. esculentaoffspring with R. lessonae (primaryhybridizations). If primary hybrids arehybridogenetic, they produce viable R.ridibunda offspring in matings with otherhybrids, because their clonal genomes areunlikely to share the deleterious allelespresent in the ancient clones. Thus, R.ridibunda will increase in the population atthe expense of both native taxa, eventuallyleaving a pure R. ridibunda population.We provide three lines of evidence for thisprocess from a currently invaded population inSwitzerland: (1) Primary hybridizations takeplace, as roughly 10% of hybrids in thepopulation possess ridibunda genomesderived from the introduced frogs. (2)Hybridogenesis occurs in primary hybrids,although at a low frequency. (3) Many hybrid hybrid matings in the population indeed produceviable offspring. Hence, the proposed geneticmechanism appears to contribute to the speciesreplacement, although its importance may belimited.

Genetic detection of multiple exotic water frog species in Belgium illustrates the need for monitoring and immediate action

Article

Full-text available

Jun 2010

The recent genetic screening of water frogs (genus Pelophylax) in Belgium has shown that the invasion of two water frog species from Eastern Europe and the Mediterranean region, P. ridibundus and P. cf. bedriagae is widespread. Possibly other exotic water frogs are invading and establishing themselves through commercial trade. We used a genetic identification approach to rapidly detect and identify morphologically cryptic exotic water frog species in a large number of populations throughout the northern part of Belgium. Among a total of 944 individuals, we found 506 non-indigenous specimens, seven of which belonged to species not recorded before with certainty in Belgium or neighbouring regions in the wild. One of them was identified genetically as the Iberian green frog (P. perezi), but was most likely a P. perezi×P. esculentus hybrid. Six individuals of the Levantine frog (P. bedriagae) were found in a pond in the vicinity of a pet shop where the species is sold. All other exotic frogs belonged to four different haplotypes of P. ridibundus, established in Belgium since c. 1970, and two haplotypes of P. cf. bedriagae, a poorly-known eastern Mediterranean sister species of the latter. Overall, our study underscores the extent of exotic water frog invasions associated with the pet trade. Although two of the exotic species were recorded in small numbers, their early detection is essential with regards to adequate control and eradication of invasive species. KeywordsEuropean water frog complex-Invasive species- Pelophylax esculentus - Pelophylax ridibundus -Tracing

Interspecific hybridization in oysters: Restriction Enzyme Digestion Chromosome Banding confirms Crassostrea angulata×Crassostrea gigas F1 hybrids

Article

Full-text available

May 2007
J EXP MAR BIOL ECOL

The taxonomic status of the two commercially important cupped oysters, Crassostrea angulata, the Portuguese oyster (Lamarck, 1819) and Crassostrea gigas, the Japanese oyster (Thunberg, 1793) has long been in question. The recent observation of the hybridization between C. gigas and C. angulata and the production of fertile F1s led us to search for cytogenetic evidence of both parental genomes in the interspecific hybrids. The cytogenetic characterization of the hybrids was performed by the use of restriction endonuclease treatments. This technique has recently shown the potential for individual chromosome identification by banding in oysters. Chromosomes of C. gigas, C. angulata and their hybrids were treated with two different restriction enzymes (ApaI and HaeIII), stained with Giemsa, and examined for banding patterns. These chromosome markers allowed the parental haploid sets to be identified in the hybrids. The analysis of the banded karyotypes of the interspecific hybrids showed that for each chromosome pair, one of the homologues presented a banding pattern consistent with that of C. gigas and the other homologue presented a banding pattern consistent with that of C. angulata. These cytogenetic results substantiate the reported interspecific hybridization between C. gigas and C. angulata. In view of these results and taking into account the present expansion of C. gigas aquaculture in southern Europe, the question of the need for preservation of pure C. angulata stocks should be raised as only a few populations remain in the south of Spain and Portugal. Recently, changes in the genetic composition of populations in southern Portugal have indeed been observed, showing that human activities have created contact zones between the two taxa while no natural sympatric zones exist in Europe.

Mitochondrial genome organization and divergence in hybridizing central European waterfrogs of the Pelophylax esculentus complex (Anura, Ranidae)

Article

Full-text available

Sep 2011

Natural transfer of mitochondrial DNA has occurred between three western Palaearctic waterfrog taxa: Pelophylax lessonae, Pelophylax ridibundus and their hybridogenetic hybrid, Pelophylax kl. esculentus. The transfer is asymmetric with most P. kl. esculentus and approximately one third of all central European P. ridibundus having mtDNA derived from P. lessonae (L-mtDNA). We obtained complete nucleotide sequences of multiple mitochondrial genomes (15,376-78 bp without control regions) from all 3 taxa, including a P. ridibundus frog with introgressed L-mtDNA. The gene content and organization of the mitogenomes correspond to those typical of neobatrachians. Divergence between the mtDNAs of P. lessonae and P. ridibundus is high with an uncorrected p-distance of 11.9% across the entire mitogenome. However, the rate of nucleotide substitution depends on the degree of functional constraint with up to 30-fold differences in levels of divergence. In general, mitochondrial genes encoding the translational machinery evolve very slowly, whereas genes encoding polypeptides of the electron transport system, especially the ND genes, evolve rapidly. Only 25 of 211-213 observed amino acid replacements could be classified as radical and are therefore more likely to be exposed to selection. A disproportionately high number of amino acid substitutions has occurred in the ND4, ND4L and cytb genes of the P. lessonae lineage (including 36% of all radical changes). In contrast to the interspecific divergence, nucleotide polymorphism within L- and R-mtDNA is very low: L-mtDNA haplotypes differed on average by only 19 nucleotides, while there was no variation within two mtDNAs derived from P. ridibundus. This is an expected finding considering that we have sampled a post-glacial expansion area. Moreover, the introgressed L-mtDNA on a P. ridibundus background differed from other L-mtDNAs by only a few substitutions, indicative of a very recent introgression event. We discuss our findings in the context of natural selection acting on L-mtDNA and its potential significance in cytonuclear epistasis.

Hemiclone diversity in the hybridogenetic frog Rana esculenta outside the area of clone formation: The view from protein electrophoresis

Article

Full-text available

Sep 2007

European water frog hybrids Rana esculenta reproduce hemiclonally, by hybridogenesis: In the germ line they exclude the genome of the parental species Rana lessonae and produce haploid, unrecombined gametes with a genome of the parental species Rana ridibunda. These hybrids coexist with and depend as sexual parasites on the host parental species R. lessonae (the L-E population system); matings with R. lessonae restore somatic hybridity in each generation of R. esculenta. We investigated 15 L-E system populations in northern Switzerland, which is outside R. ridibunda native range. Frequency of hybrids in samples varied from 8% in marsh ponds to 100% in gravel pits and forest ponds. Clonal diversity (variation among R. ridibunda genomes of hybrids), detected by six protein electrophoretic marker loci, revealed a total of eight hemiclones and locally ranged from uniclonal populations in southern parts of the survey region to six coexisting hemiclones in the north. All alleles distinguishing hemiclones occur commonly in the nearest native R. ridibunda populations of east-central Europe; the most probable source of clonal diversity in our samples is multiple clone formation by primary hybridizations in the sympatry area of R. ridibunda and R. lessonae and subsequent dispersal of hemiclonal lineages. A positive correlation between amount of clonal diversity and hybrid frequency, predicted by the Frozen Niche Variation (FNV) model (each hemiclone is characterized by a relatively narrow niche, coexistence is possible through niche partitioning), was not found; this contrasts with hemiclonally reproducing fish hybrids (Poeciliopsis). Historical factors, such as availability of different colonizing hemiclones may be strong enough to override the signal from operation of the FNV.

A new PCR-RFLP-based method for an easier systematic affiliation of European water frogs

Article

Full-text available

Jan 2011
MOL ECOL RESOUR

We describe a non-invasive, PCR-RFLP-based method that allows reliable determination of the European water frog species Pelophylax lessonae and Pelophylax ridibundus and the hybrid form Pelophylax esculentus. Maximum-likelihood analysis of ITS2 sequences revealed two robust monophyletic clades corresponding to water frogs of the P. lessonae and P. ridibundus groups. Three restriction enzymes (KpnI, HaeII, and SmaI) were used to digest three conserved ITS2 domains. Taxonomic identification was unambiguous; the three restriction enzymes gave the same results. A French reference sample was identified using allozyme electrophoresis. Our PCR-RFLP method confirmed circa 83% of identification of the allozyme method. We conclude that the difference between identifications was caused by introgression.

Evolution of serum albumin intron-1 is shaped by a 5 ‘truncated non-long terminal repeat retrotransposon in western Palearctic water frogs (Neobatrachia)

Article

Full-text available

Sep 2009
MOL PHYLOGENET EVOL

A 5' truncated non-LTR CR1-like retrotransposon, named RanaCR1, was identified in the serum albumin intron-1 (SAI-1) of at least seven species of western Palearctic water frogs (WPWF). Based on sequence similarity of the carboxy-terminal region (CTR) of ORF2 and/or the highly conserved 3' untranslated region (3' UTR), RanaCR1-like elements occur also in the genome of Xenopus tropicalis and Rana temporaria. Unlike other CR1 elements, RanaCR1 contains a CA microsatellite in its 3' UTR. The low nucleotide diversity of the 3' UTR compared to the CTR and to SAI-1 suggests that this region still plays a role in WPWF, either as a structure-stabilizing element, or within a species-specific transcriptional network. Length variation of water frog SAI-1 sequences is caused by deletions that extend in some cases beyond the 5' or 3' ends of RanaCR1, probably a result of selection for structural and functional stability of the primary transcript. The impact of RanaCR1 on SAI-1 evolution is also indicated by the significant negative correlation between the length of both SAI-1 and RanaCR1 and the percentage GC content of RanaCR1. Both SAI-1 and RanaCR1 sequences support the sister group relationship of R. perezi and R. saharica, which are placed in the phylogenetic tree at a basal position, the sister clade to other water frog taxa. It also supports the monophyly of the R. lessonae group; of Anatolian water frogs (R. cf. bedriagae), which are not conspecific with R. bedriagae, and of the European ridibunda group. Within the ridibunda clade, Greek frogs are clearly separated, supporting the hypothesis that Balkan water frogs represent a distinct species. Frogs from Atyrau (Kazakhstan), the type locality of R. ridibunda, were heterozygous for a ridibunda and a cf. bedriagae specific allele.

Gamete types, sex determination and stable equilibria of all-hybrid populations of diploid and triploid edible frogs (Pelophylax esculentus)

Article

Full-text available

Jul 2009
BMC EVOL BIOL

Ditte G Christiansen

Triploid individuals often play a key role in speciation by hybridization. An understanding of the gamete types (ploidy and genomic content) and stability of hybrid populations with triploid individuals is therefore of importance for exploring the role of hybridization in evolution. The all-hybrid populations of the edible frog, Pelophylax esculentus, are unique in their composition and genetic dynamics: Diploid (genotype LR) and triploid (LLR and LRR) hybrids depend on each other`s different gamete contributions for successful reproduction and maintenance of the populations, as the parental genotypes P. lessonae (LL) and P. ridibundus (RR) are absent among adults. This study provides data and interpretations on gamete types and sex determination that are essential for understanding the function, evolutionary potential and threats of this intriguing system.

Microsatellite Characterization of Subspecies and Their Hybrids in Culex pipiens Complex (Diptera: Culicidae) Mosquitoes Along a North–South Transect in the Central United States

Article

Full-text available

Mar 2009

Mosquitoes in the Culex pipiens complex, Cx. p. pipiens L. and Cx. p. quinquefasciatus Say are morphologically similar and important vectors of West Nile and St. Louis Encephalitis viruses in the United States. The subspecies differ with respect to overwintering strategies, with Cx. p. pipiens entering diapause in response to winter conditions and Cx. p. quinquefasciatus lacking this ability, yet they hybridize when found in sympatry. Specimens (n = 646) were collected using gravid traps set along a transect from New Orleans, LA, to Chicago, IL. Microsatellite markers were used to: genetically characterize subspecies and hybrids, determine the degree and extent of hybridization to better define the hybrid zone, and examine the efficacy of hybrid detection between microsatellites and a single-gene assay based on the acetylcholinesterase.2 gene (HotAce.2). The results support the presence of two distinct genetic entities, with abroad, stable hybrid zone in between. Admixture analyses classified >40% of individuals as hybrids. Allelic richness was markedly different at the northern and southern ends of the transect, and there was a significant isolation by distance effect. The hybrid zone seems to be wider and extends further to the south than previous work indicated, and as a result, we propose new boundaries compared with those indicated by a previous study. Microsatellites detected more hybrids than the HotAce.2 assay, although the latter assay remains useful as a screening tool. We suggest that the parental subspecies and the hybrid zone are maintained by heterosis combined with selection for diapause at northern latitudes.

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: Consequences of uncontrolled commercial trade and weak international legislation

Article

Full-text available

Dec 2008
MOL ECOL

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non-native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non-native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large-scale hybridization and introgression.

Biochemical variation in the Rana esculenta complex: A new hybrid form related to Rana perezi and Rana ridibunda

Article

Full-text available

Jan 1978
Experientia

Investigations of the green frogs from western Europe for electrophoretic variations at 4 enzyme loci demonstrated a new form which must be considered as a hybrid between Rana ridibunda and R. perezi. Biochemical evidence supports the hypothesis that its reproduction is hybridogenetic, as it is for R. esculenta.

Natural Interspecies Transfer of Mitochondrial DNA in Amphibians

Article

Full-text available

Oct 1984

mtDNAs of two Central European water frog species, Rana ridibunda and Rana lessonae, were examined by electrophoresis of restriction enzyme fragments. Two types of mtDNA occur in R. ridibunda. One shares with mtDNA of R. lessonae 25.8% of 132 fragments generated by 19 enzymes, corresponding to a nucleotide sequence divergence of 8.1%; the other has diverged from R. lessonae mtDNA by only 0.3%. This latter type is a variant R. lessonae mtDNA that has been transferred into R. ridibunda; the introgression may have occurred via the hybridogenetic hybrid lineages collectively known as Rana esculenta. Of 37 R. ridibunda from Poland, 59% had the typical R. ridibunda mtDNA; 41% had the modified R. lessonae mtDNA as did a single individual from Switzerland (introduced). A single R. ridibunda from Turkey, outside the present range of R. lessonae, had the typical R. ridibunda mtDNA phenotype. Discordancies between inheritance of mitochondrial and nuclear genomes point up the danger of relying on a single molecular feature in reconstructing phylogeny. In addition, studies of mtDNA provide otherwise inaccessible information on complex evolutionary histories of closely related species. A knowledge of these complexities is important to an understanding of phylogenetic relationships and of the genetic processes that underlie the evolution of clonal taxa.

Electrophoretic phenotypes of Rana ridibunda, Rana lessonae, and their hybridogenetic associate, Rana esculenta

Article

Jan 1975

Biometrical studies on the population of green frogs from the environs of Poznan

Article

Jan 1966

L. Berger

Inheritance Patterns in TriploidRana “esculenta” (Amphibia, Salientia)

Article

Jul 1979

Premeiotic chromosome doubling after genome elimination during spermatogenesis of the species hybrid Rana esculenta

Article

Jun 1982
THEOR APPL GENET

Gamete production in the hybridogenetic species hybrid Rana esculenta (Rana ridibunda X Rana lessonae) is preceded by a premeiotic elimination of the R. lessonae genome and subsequent duplication of the remaining R. ridibunda genome, so that only ridibunda chromosomes enter a quasi normal meiosis, and only ridibunda gametes are formed. This is demonstrated by differences in genome specific centromere fluorescence and electrophoretic patterns between somatic and gonadal tissue.

Morphometric, enzymological and bioacoustic studies in Italian water frogs (Amphibia, Ranidae)

Article

Jan 1994

Neotropical catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum

Article

Jun 2011

Cat¢sh species Pseudoplatystoma corruscans (pintado) and Pseudoplatystoma reticulatum (cachara) consti-tute large-size migratory ¢sh species of high com-mercial value and ecological importance, and are widely distributed in South American hydrographic basins (Buitrago-Sua Ł rez & Burr 2007). Hybrids be-tween these species have been produced in several Brazilian hatcheries (Godinho 2007; Porto-Foresti, Hashimoto, Alves, Almeida, Senhorini, Bortolozzi & Foresti 2008), mainly with an intention to obtain in-dividuals that present commercial advantages. Be-sides the fact that the production of these ¢sh species has already become a large-scale practice in ¢sh farms, a lack of rigid control and monitoring of production and management is observed. Otherwise, the possibility of genetic contamination of native and cultured stocks can be pointed out as serious problems involving the process when the pro-duction and commercialization of hybrids are carried out in the places of parental native species occurrence (Toledo-Filho, Almeida-Toledo, Foresti, Calcagnotto, Santos & Bernardino 1998). As escapes of individuals produced in ¢sh farms into the natural environment are very frequent (Orsi & Agostinho 1999), the occur-rence of ecological disequilibrium (Einum & Fleming 1997), genetic introgression and even the extinction of native species may occur (Allendorf, Leary, Spruell & Wenburg 2001; Epifanio & Philipp 2001). In this case, beyond the harmful environmental e¡ects of hy-bridization, the loss of the native genetic pool of dis-tinct parental species may preclude the development of future breeding programmes and the commercial production of these species. The identi¢cation of pure parental species and hy-brids is an essential step in order to provide tools for the proper management of these animals and avoid the risks they represent (Toledo-Filho, Almeida-Tole-do, Foresti, Bernardino & Calcagnotto 1994). How-ever, two main facts can be pointed out to reinforce the di⁄culties in the control and management of ¢sh production, as ¢sh are commercialized when they are still juveniles and morphological identi¢cation is dif-¢cult and mostly imprecise. In such a context, genet-ic methodologies can provide diagnostic molecular markers to identify, in a clear and accessible way, par-ents and hybrids (Young, Ostberg, Keim & Thorgaard 2001; Hashimoto, Mendonc° a, Senhorini, Bortolozzi, Oliveira, Foresti & Porto-Foresti 2009). Hybrids between P. corruscans and P. reticulatum have already been captured in nature, probably due to escapes from hatcheries. These ¢sh were shown to be fertile and were able to back-cross with the paren-tal species in captivity (J. A. Senhorini, pers. comm.), thus reinforcing the necessity of adequate

New contact zone and assemblages among frogs of Southern France

Article

May 2002
J ZOOL SYST EVOL RES

Water frogs are involved in several hybridogenetic complexes. However, investigations in Southern and Western France produced evidence of new hybridization events between R. lessonae and R. perezi (some new hybrids tadpoles were discovered), and the existence of new assemblages [R–G, L–P, R–P, P–R–G: (R, ridibunda; G, grafi; L, lessonae; P, perezi)]. In respect to hybridogenesis, these assemblages offer opportunities for both primary and secondary hybridizations. Such peculiarities may be explained either by introductions or by relic populations and may influence water frog evolution.Neue Kontaktzonen und Gruppierungen bei Wasserfröschen in SüdfrankreichWasserfrösche sind vielfach an der Bildung von hybridogenetischen Komplexen beteiligt. Unsere Untersuchungen in Süd- und Westfrankreich zeigten jedich neue Hybridisierungen zwischen Rana lessonae und Rana perezi (einige neue Hybride konnten im Kaulquappenstadium entdeckt werden) und das bestehen von neuen Komplex-Gruppierungen [R–G, L–P, R–P, P–R–G: (R, ridibunda; G, grafi; L, lessonae; P, perezi)]. In Bezug auf den Hybridogenese-Mechanismus lassen diese Komplex-Gruppierungen sowohl primäre als auch sekundäre Hybridisierungen zu. Diese Besonderheiten können entweder durch Introgression oder durch das Bestehen von Reliktpopulationen erklärt werden; sie könnten so die Evolution der Wasserfrösche merklich beeinflussen.

Rana ridibunda varies geographically in inducing clonal gametogenesis in interspecies hybrids

Article

Nov 1985
J Exp Zool

Diploid F1 hybrids between Rana ridibunda from Adriatic SW Yugoslavia and Polish R. lessonae, and between Polish R. ridibunda and an unnamed species from SW Yugoslavia are shown by electrophoresis and examination of lampbrush chromosomes to contain both parental genomes in their diploid oocytes I. In contrast, central European R. ridibunda genomes in diploid hybrids, whether F1s or from natural hemiclonal lineages, induce exclusion of the lessonae genome in the germ line, only ridibunda translation products and endoreduplicated ridibunda chromosomes being detected in their diploid oocytes I. The data demonstrate geographic variation of R. ridibunda in the ability of its genomes to induce clonal gametogenesis in interspecies hybrids. They also suggest that genomes of the unnamed Yugoslavian species may be resistant to such exclusion.

Novel Jefferson salamander, Ambystoma jeffersonianum, microsatellite DNA markers detect population structure and hybrid complexes: PRIMER NOTE

Article

Mar 2003
MOL ECOL NOTES

Twenty polymorphic microsatellite DNA markers were isolated and characterized in Ambystoma jeffersonianum collected from three vernal pools in the mid-Atlantic region of the U.S. These markers revealed a high degree of genetic diversity (7–23 alleles per locus), heterozygosity (46.7% to 100%), and allelic heterogeneity (96% of comparisons were statistically significant). Genetic distances were greatest in comparisons between collections, intermediate within collections, and least among sibling pairs. Six markers were trisomic in A. jeffersonianum-A. laterale hybrids. These microsatellite DNA loci should allow delineation of genetic structure within and among populations of the diploid A. jeffersonianum and provide an effective method for identification of triploid hybrid individuals.

Mable BK, Alexandrou MA, Tayor MI. Genome duplication in amphibians and fish: an extended synthesis. J Zool 284: 151-182

Article

Jul 2011
J ZOOL

Whole genome duplication (leading to polyploidy) is widely accepted as an important evolutionary force in plants, but it is less recognized as a driver of animal diversification. Nevertheless, it occurs across a wide range of animals; this review investigates why it is particularly common in fish and amphibians, while rare among other vertebrates. We review the current geographic, ecological and phylogenetic distributions of sexually reproducing polyploid taxa before focusing more specifically on what factors drive polyploid formation and establishment. In summary, (1) polyploidy is phylogenetically restricted in both amphibians and fishes, although entire fish, but not amphibian, lineages are derived from polyploid ancestors. (2) Although mechanisms such as polyspermy are feasible, polyploid formation appears to occur principally through unreduced gamete formation, which can be experimentally induced by temperature or pressure shock in both groups. (3) External reproduction and fertilization in primarily temperate freshwater environments potentially exposes zygotes to temperature stress, which can promote increased production of unreduced gametes. (4) Large numbers of gametes and group breeding in relatively confined areas could increase the probability of compatible gamete combinations in both groups. (5) Both fish and amphibians have a propensity to form reproductively successful hybrids; although the relative frequency of autopolyploidy versus allopolyploidy is difficult to ascertain, multiple origins involving hybridization have been confirmed for a number of species in both groups. (6) Problems with establishment of polyploid lineages associated with minority cytotype exclusion could be overcome in amphibians via assortative mating by acoustic recognition of the same ploidy level, but less attention has been given to chemical or acoustic mechanisms that might operate in fish. (7) There is no strong evidence that polyploid fish or amphibians currently exist in more extreme environments than their diploid progenitors or have broader ecological ranges. (8) Although pathogens could play a role in the relative fitness of polyploid species, particularly given duplication of genes involved in immunity, this remains an understudied field in both fish and amphibians. (9) As in plants, many duplicate copies of genes are retained for long periods of time, indicative of selective maintenance of the duplicate copies, but we find no physiological or other reasons that could explain an advantage for allelic or genetic complexity. (10) Extant polyploid species do not appear to be more or less prone to extinction than related diploids in either group. We conclude that, while polyploid fish and amphibians share a number of attributes facilitating polyploidy, clear drivers of genome duplication do not emerge from the comparison. The lack of a clear association of sexually reproducing polyploids with range expansion, harsh environments, or risk of extinction could suggest that stronger correlations in plants may be driven by shifts in mating system more than ploidy. However, insufficient data currently exist to provide rigorous tests of these hypotheses and we make a plea for zoologists to also consider polyploidy as a possibility in continuing taxonomic surveys.

New contact zone and assemblages among water frog of Southern France

Article

Mar 2001

Discovery of natural Miscanthus (Poaceae) triploid plants in sympatric populations of Miscanthus sacchariflorus and Miscanthus sinensis in southern Japan

Article

Jan 2011
AM J BOT

Looming petroleum shortages and projected negative impacts of human-induced climate change may be partly alleviated by the development and use of bioenergy feedstock crops. Miscanthus ×giganteus, a highly productive sterile triploid hybrid grass that was discovered in Japan several decades ago, has considerable potential as an alternative source of energy. Given the risks, however, involved in the reliance upon production of one clone of this hybrid, which is a natural cross between Miscanthus sacchariflorus and Miscanthus sinensis, for lignocellulosic bioenergy production, natural occurrences of triploidy were investigated in sympatric populations of tetraploid M. sacchariflorus and diploid M. sinensis in Japan. Seeds were counted and DNA content was estimated by flow cytometry for plants of M. sacchariflorus and M. sinensis in several sympatric populations throughout Japan. Chromosomes were also counted for select plants. Based on seed-set data, M. sacchariflorus has significantly lower seed set than M. sinensis in Japan. Putative triploid seeds were found on M. sacchariflorus plants in southern Japan. This is the first report of the natural occurrence of Miscanthus triploid plants in several decades. If found to be sterile and similar in productivity to the commonly cultivated clone of M. ×giganteus, these triploid plants might serve as additional sources of genetic variation for bioenergy production. Seed set data also indicates that other triploid plants might be found in more northern regions of Japan.

Effects of geographic distance, sea barriers and habitat on the genetic structure and diversity of all-hybrid water frog populations

Article

Apr 2010

The history of population size and migration patterns leaves its mark in the genetics of populations. We investigate the genetic structure of the edible frog, Pelophylax esculentus in the Danish archipelago and adjacent countries. This frog is of particular interest because it is a hybrid that, in this area, forms all-hybrid populations of diploid (LR) and triploid (LLR and LRR) genomotypes with no (or very few) adults of the parental species (LL and RR). This study is the first to cover the entire geographic range of Danish, Swedish and German all-hybrid populations, documenting their extent and providing a broad picture of their diversity of neutral genetic markers and genomotype proportions. With 18 microsatellite markers, we found that genetic diversity declines northwards in agreement with the glacial refuge and central-marginal hypotheses; however, populations on small and mediumsized islands are no less diverse than those on large islands and continental peninsulas. Isolation by distance exists across the archipelago with limited influence of fragmentation by brackish seawater. The extremely low genetic diversity in all-hybrid populations, compared with adjacent populations, may be responsible for the maintenance of their special breeding system. We also show large variation among ponds in proportions of LLR, LR and LRR genomotypes, but little geographic pattern in their distribution. Instead, we found relationships between the genomotype proportions and some of 15 habitat parameters monitored. Body size differences among LLR, LR and LRR further suggest ecological differences.

Premeiotic genome exclusion during oogenesis in the common edible frog, Rana esculenta

Article

May 1981

Identification of interspecific hybrids by amplified fragment length polymorphism: The case of sturgeon

Article

Oct 2001

The identification of interspecific hybrids represents an important issue for conservation biology and trade controls. In Italy, the commercial demand for sturgeon is rapidly increasing and interspecific hybrids represent a relevant part of aquacultural production. In this study we tested the suitability of the amplified fragment length polymorphism (AFLP) technique for sturgeon hybrid detection. Multilocus AFLP profiles were analysed by cluster analysis and assignment tests based on observed and simulated samples. Our results show that this approach can easily identify sturgeon hybrids, encouraging its application not only in sturgeon but also in other systematic groups.

Widespread unidirectional transfer of mitochondrial DNA: A case in western Palaearctic water frogs

Article

Jun 2008
J EVOLUTION BIOL

Interspecies transfer of mitochondrial (mt) DNA is a common phenomenon in plants, invertebrates and vertebrates, normally linked with hybridization of closely related species in zones of sympatry or parapatry. In central Europe, in an area north of 48 degrees N latitude and between 8 degrees and 22 degrees E longitude, western Palaearctic water frogs show massive unidirectional introgression of mtDNA: 33.7% of 407 Rana ridibunda possessed mtDNA specific for Rana lessonae. By contrast, no R. lessonae with R. ridibunda mtDNA was observed. That R. ridibunda with introgressed mitochondrial genomes were found exclusively within the range of the hybrid Rana esculenta and that most hybrids had lessonae mtDNA (90.4% of 335 individuals investigated) is evidence that R. esculenta serves as a vehicle for transfer of lessonae mtDNA into R. ridibunda. Such introgression has occurred several times independently. The abundance and wide distribution of individuals with introgressed mitochondrial genomes show that R. lessonae mt genomes work successfully in a R. ridibunda chromosomal background despite their high sequence divergence from R. ridibunda mtDNAs (14.2-15.2% in the ND2/ND3 genes). Greater effectiveness of enzymes encoded by R. lessonae mtDNA may be advantageous to individuals of R. ridibunda and probably R. esculenta in the northern parts of their ranges.

Unique all‐hybrid populations of the edible frog, Pelophylax esculentus– genotype determination, population structure and reproduction

Jan 2011
29

Christiansen DG

Christiansen DG (2011) Unique all-hybrid populations of the edible frog, Pelophylax esculentus – genotype determination, population structure and reproduction. Flora og Fauna, 117, 29–41

Die westpalä Wasserfrö Von Mä der Wissenschaft zur biologischen Sensation. Laurenti Verlag, Bielefeld, 160 pp. Plö tner J (2010) Mö und Grenzen morphologischer Methoden zur Artbestimmung bei europä Wasserfrö schen (Pelophylax escu-lentus-Komplex) Zeitschrift fü Feldherpetologie

Jan 2005
129-146

Plö

Plö tner J (2005) Die westpalä Wasserfrö. Von Mä der Wissenschaft zur biologischen Sensation. Laurenti Verlag, Bielefeld, 160 pp. Plö tner J (2010) Mö und Grenzen morphologischer Methoden zur Artbestimmung bei europä Wasserfrö schen (Pelophylax escu-lentus-Komplex). Zeitschrift fü Feldherpetologie, 17, 129–146.

Molecular identification of hybrids between Neotropi-cal catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum

Jan 2011
1890-1894

Prado Fd
Hashimoto Dt
Mendonç Ff
Ja Senhorini
F Foresti

Prado FD, Hashimoto DT, Mendonç FF, Senhorini JA, Foresti F, Porto-Foresti F (2011) Molecular identification of hybrids between Neotropi-cal catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum. Aquaculture Research, 42, 1890–1894.

Hemiclonal hybrid water frogs associated with the sexual host species Rana perezi

Jan 1994
243

Hotz H

A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax)

Abstract

No full-text available

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