Esculenta Complex Research Articles

Micronuclei in germ cells of hybrid frogs from Pelophylax esculentus complex contain gradually eliminated chromosomes

In most organisms, cells typically maintain genome integrity, as radical genome reorganization leads to dramatic consequences. However, certain organisms, ranging from unicellular ciliates to vertebrates, are able to selectively eliminate specific parts of their genome during certain stages of development. Moreover, partial or complete elimination of one of the parental genomes occurs in interspecies hybrids reproducing asexually. Although several examples of this phenomenon are known, the molecular and cellular processes involved in selective elimination of genetic material remain largely undescribed for the majority of such organisms. Here, we elucidate the process of selective genome elimination in water frog hybrids from the Pelophylax esculentus complex reproducing through hybridogenesis. Specifically, in the gonads of diploid and triploid hybrids, but not those of the parental species, we revealed micronuclei in the cytoplasm of germ cells. In each micronucleus, only one centromere was detected with antibodies against kinetochore proteins, suggesting that each micronucleus comprises a single chromosome. Using 3D-FISH with species-specific centromeric probe, we determined the role of micronuclei in selective genome elimination. We found that in triploid LLR hybrids, micronuclei preferentially contain P. ridibundus chromosomes, while in diploid hybrids, micronuclei preferentially contain P. lessonae chromosomes. The number of centromere signals in the nuclei suggested that germ cells were aneuploid until they eliminate the whole chromosomal set of one of the parental species. Furthermore, in diploid hybrids, misaligned P. lessonae chromosomes were observed during the metaphase stage of germ cells division, suggesting their possible elimination due to the inability to attach to the spindle and segregate properly. Additionally, we described gonocytes with an increased number of P. ridibundus centromeres, indicating duplication of the genetic material. We conclude that selective genome elimination from germ cells of diploid and triploid hybrids occurs via the gradual elimination of individual chromosomes of one of the parental genomes, which are enclosed within micronuclei.

A procedure for taxon assessment based on morphological variation in European water frogs (Pelophylax esculentus complex)

Methods for morphological identification of water frogs from the Pelophylax esculentus complex have changed over the decades. Still, without resort to genetic analyses, water frogs remain among the most difficult European amphibians to distinguish due to their hybrid nature. The aim of this study was to identify a reliable set of quantitative and qualitative morphological characters that objectively resolve species? identification in the Pelophylax esculentus complex and provide a rapid and robust procedure for taxon identification. Our results showed a substantial difference in morphological characteristics among taxa. The shape and size of the metatarsal tubercle along with the coloration of the hind limbs and flanks appear to be the most important diagnostic traits. Linear discriminant analysis with both quantitative and qualitative traits as a rapid method for the assessment of water frog taxa showed satisfactory accuracy and prediction classification for all 3 members of the complex.

Body size variation in hybrids among populations of European water frogs (Pelophylax esculentus complex) with different breeding systems

Abstract In some populations, hybrids reproduce with a parental species by eliminating the genome of this species from their own germline and produce gametes that only contain the genome of the other parental species (sexual host). This mode of reproduction, known as hybridogenesis, leads to a conflict of interest between the two parties because the sexual host should avoid mating with the hybrid to prevent a reduction in reproductive success, whereas the hybrid depends on such matings for survival. We investigated European water frogs (Pelophylax esculentus complex), including hybrids (P. esculentus, genotype LR) and two sexual host species (P. lessonae, LL and P. ridibundus, RR). We hypothesized that to maximize fitness, hybrid males should be morphologically more similar to the sexual host that is preferred by females for successful reproduction. To test this hypothesis, we compared hybrid males in two different population types, L-E (hybrids coexist with LL) and L-E-R (hybrids coexist with both LL and RR). The latter was described in terms of genome composition, sex ratio, and mate choice preferences; the sex ratio of hybrids was significantly male-biased. We found that LR males from the L-E-R populations were significantly larger than those from the L-E, which makes them more similar to P. ridibundus, the largest species within the P. esculentus complex. We suggest that a larger body size of hybrid males may provide a reproductive advantage in the L-E-R population type, where the most common type of pair caught in the breeding season was LR males × RR females.

Amphibian diversity in Polish cities: Taxonomic diversity, functional diversity and evolutionary distinctiveness

As a process affecting animal communities, urbanization has been the subject of numerous studies. However, amphibians are still among the least studied vertebrate groups in urbanized landscapes. Generally, it has been found that the process of loss of amphibian diversity is nonrandom, with species from older evolutionary lines at greater risk. Regional data on amphibian assemblages in urban areas is a very useful tool for assessing how these assemblages react to changes.The aim of the present paper is to assess the diversity of amphibians in Polish cities based on data in the relevant literature, exploring different metrics (e.g., taxonomic, functional, and evolutionary diversity) calculated in amphibian species assemblages. We used data from 18 articles (including grey literature), characterized by comparable research methods and published between 1999 and 2017.Overall, amphibian species richness (ASR) amounted to an average of 9 species, ranging from 5 to 11 species per city. The higher species richness occurred in Białystok in North-west of Poland. Functional evenness (FEve), evolutionary distinctiveness (ED sum), and functional richness (FRic) were strongly positively correlated with ASR. However, ED mean was not significantly correlated with the total number of species in the community. Three taxa, the hybridogenetic water frogs Pelophylax esculentus complex, the common toad Bufo bufo, and the common frog Rana temporaria, occurred in all analyzed amphibian assemblages. Our study is one of the first attempts to compare urban amphibian assemblages, using different and complementary diversity metrics on a large spatial scale. In conclusion, we highlight that urban areas play an important role for conservation of amphibians, because they support amphibian assemblages characterized by a high level of overall diversity.

Amphibians in an urban environment: a case study from a central European city (Wroc\u0142aw, Poland)

In 2016, we studied the occurrence of amphibians in 231 selected ponds in the city of Wrocław (Lower Silesia, Poland) and confirmed the occurrence of 10 species: Bombina bombina, Bufo bufo, Bufotes viridis, Hyla arborea, Pelobates fuscus, Pelophylax esculentus complex, Rana arvalis, Rana temporaria, Lissotriton vulgaris and Triturus cristatus. Among all studied sites, the most common were P. esculentus complex (146 occupied sites, 63.2%) and B. bufo (119, 51.5%), while the rarest was P. fuscus (8, 3.5%). The number of species at a single site varied from 0 (28 sites, 12.1%) to 9 (1 site, 0.4%). Frequency and mean amphibian species richness reached 87.9% and 2.7 ± 1.9, respectively. The composition of amphibian species did not change in comparison to previous studies conducted in the years 1997–2009, but declining trends in five species (B. bombina, B. viridis, H. arborea, P. esculentus complex, P. fuscus) were observed (28 compared sites). The results revealed that the permanency of ponds, their occurrence in the vicinity of river valleys, and a high ratio of watercourse length and green area around ponds are positively correlated and have a significant influence on amphibian species richness within the city. Thus, these identified factors should be considered in the course of sustainable urban planning in order to avoid potential conflicts between nature conservation and urban development.

Variation in hybridogenetic hybrid emergence between populations of water frogs from the Pelophylax esculentus complex.

Many closely related species are capable of mating to produce hybrid offspring, which are usually sterile. Nevertheless, altering the gametogenesis of hybrid offspring can rescue hybrids from sterility by enabling asexual reproduction. Hybridogenesis is one of the most complicated asexual reproductive modes, and it includes drastic genome reorganization only in the germline; this is achieved through elimination of one parental genome and duplication of the remaining one to restore diploid chromosomal set and overcome blocks in meiotic progression. We investigated a model of hybridogenesis, namely, water frogs from the Pelophylax esculentus complex, for the emergence of asexual reproduction. Further, we assessed the impact of its asexual reproduction on the maintenance of interspecies hybrids from two populations on the western edge of the P. esculentus range, in which hybrids coexist with either both parental species or with only one parental species. After analysing tadpole karyotypes, we conclude that in both studied populations, the majority of diploid hybrid males produced haploid gametes with the P. ridibundus genome after elimination of the P. lessonae genome. Hybrid females exhibited problems with genome elimination and duplication; they usually produced oocytes with univalents, but there were observations of individual oocytes with 13 bivalents and even 26 bivalents. In some hybrid tadpoles, especially F1 crosses, we observed failed germ cell development, while in tadpoles from backcrosses, germ cells were normally distributed and contained micronuclei. By identifying chromosomes present in micronuclei, we estimated that the majority of tadpoles from all crosses were able to selectively eliminate the P. lessonae chromosomes. According to our results, hybridogenesis in hybrids can appear both from crosses of parental species and crosses between sexual species with hybrid individuals. The ability to eliminate a genome and perform endoreplication to ensure gamete formation differed between male and female hybrids from the studied populations. Some diploid hybrid females can rarely produce not only haploid gametes but also diploid gametes, which is a crucial step in the formation of triploid hybrids.

Oxidative stress in Pelophylax esculentus complex frogs in the wild during transition from aquatic to terrestrial life

During life, anuran individuals undergo drastic changes in the course of transition from aquatic to terrestrial habitat, when they are faced with metabolically demanding processes (growth, responses to developmental pressures), which result in increased production of reactive oxygen species (ROS), signaling molecules involved in development that can induce oxidative damage and stress. This situation can be further complicated by environmental influences. The aim of this study was to investigate oxidative stress parameters in naturally developing Pelophylax esculentus complex frogs during four developmental periods: premetamorphosis, prometamorphosis, metamorphic climax and juvenile stage, in order to examine changes in the response of the antioxidative system (AOS) and oxidative damage during the transition from aquatic to terrestrial life. Results show that ontogenetic shifts in anurans are accompanied by different levels of damage and AOS responses, which vary from the increased first-line enzymatic activities during the early period of development (premetamorphosis), through increased changes in the non-enzymatic complement during the metamorphic climax, to changes in both the enzymatic and non-enzymatic components observed in juvenile individuals. Premetamorphic individuals and individuals in metamorphosis displayed higher levels of lipid peroxidation, indicating that direct exposure to the environment for the first time and the modulation of organs are the most susceptible stages for oxidative damage. On the other hand, lower oxidative damage in juveniles points to the ability of their AOS to efficiently respond to challenges of the terrestrial environment. This study highlights the importance of ROS and the AOS of anurans in response to different developmental and/or environmental pressures that individuals face.

Ants biting amphibians: a review and new observations

Abstract Antagonistic interactions between insects and amphibians are the subject of many scientific articles, mostly concerning amphibian predation on insect, but many fewer examples exist of the opposite situation. In this article we review available information from the literature and add our own observations collected during amphibian pitfall trap monitoring in 2012–2016 in Western Poland, as well as discuss potential conservation implications of observed behavior. We identified a total of 29 cases involving 94 individual ants attacking four species of Anura, Rana temporaria, Pelophylax esculentus complex, Bufo bufo, and Pelobates fuscus, and biting their back, cloaca, armpits, or hind legs. Bites were inflicted by three ant species: Myrmica rubra, Lasius fuliginosus, and Formica polyctena. The number of ants found on an amphibian was positively and significantly correlated with its body length. To date, direct damage by ants on amphibians was reported mainly from the tropics in general predation accident. However, as we document here, it is probably a more common phenomenon, especially in some ecological traps or during pitfall trapping, which is a common method to mitigate road mortality of frogs and toads.

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

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 Hortobagy 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 (Hortobagy 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.

Researching Malformations in Frogs of the Pelophylax esculentus Complex (Amphibia: Anura) in the Natural Populations of the Trakhtemyriv Peninsula (Ukraine)

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Genetic diversity in population systems of green frogs (Pelophylax esculentus complex) in waterbodies of Western Ukraine

The results of the analysis of the genetic structure in population systems of green frogs on the territory of Lviv and Volyn regions are presented. The material was collected in 2011–2012 in water bodies representing three nature regions of Ukraine – Forecarpathians, Roztochia, and Western Polissia. Three taxonomic groups of green frogs were in a focus of the study: Marsh frog – Pelophylax ridibundus (Pallas, 1771), Pool frog – Pelophylax lessonae (Camerano, 1882) and their hybrid – Edible frog – Pelophylax esculentus (Linnaeus, 1758). DNA was extracted from 91 individuals and analyzed using of 10 pairs of primers: Rrid059, Rrid082, Rrid171, Res5, Res14, Res16, Res22, RlCA1b5, RlCA18, RlCA19. A majority of those are highly polymorphic and diagnostic for species identification. During the analysis, we used programs based on principles of Bayesian statistics and Monte-Carlo Markov Chain algorithms: Structure, BAPS, and NewHybrids. Linkage groups were searched using the GenePop software, and hidden null-alleles were detected using Micro-Checker program. For the first time, in the studied area the genetic structure of populations and population systems were described. After the analysis of genetic diversity of frogs sampled from the Pelophylax ridibundus population and from hemiclonal population systems of mixed R-E-L type, we found that the smallest genetic diversity is observed in the population of Marsh frog from the Nyzhankovychi area (Forecarpathians). More diverse are hemiclonal population systems of green frogs sampled in water bodies of “Cholgynskyi” ornithological reserve (Ukrainian Roztochia) and Shatsk National Nature Park (Western Polissia). Also, for the first time, the hybrid composition of studied localities is described. Hybrids of the first generation (F1) and backcrosses were detected in water bodies of Shatsk National Nature Park and ornithological reserve “Cholgynskyi”.

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

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

Mutual maintenance of di- and triploid Pelophylax esculentus hybrids in R-E systems: results from artificial crossings experiments

BackgroundInterspecies animal hybrids can employ clonal or hemiclonal reproduction modes where one or all parental genomes are transmitted to the progeny without recombination. Nevertheless, some interspecies hybrids retain strong connection with the parental species needed for successful reproduction. Appearance of polyploid hybrid animals may play an important role in the substitution of parental species and in the speciation process.ResultsTo establish the mechanisms that enable parental species, diploid and polyploid hybrids coexist we have performed artificial crossing experiments of water frogs of Pelophylax esculentus complex. We identified tadpole karyotypes and oocyte genome composition in all females involved in the crossings. The majority of diploid and triploid hybrid frogs produced oocytes with 13 bivalents leading to haploid gametes with the same genome as parental species hybrids usually coexist with. After fertilization of such gametes only diploid animals appeared. Oocytes with 26 bivalents produced by some diploid hybrid frogs lead to diploid gametes, which give rise to triploid hybrids after fertilization. In gonads of all diploid and triploid hybrid tadpoles we found DAPI-positive micronuclei (nucleus-like bodies) involved in selective genome elimination. Hybrid male and female individuals produced tadpoles with variable karyotype and ploidy even in one crossing owing to gametes with various genome composition.ConclusionsWe propose a model of diploid and triploid hybrid frog reproduction in R-E population systems. Triploid Pelophylax esculentus hybrids can transmit genome of parental species they coexist with by producing haploid gametes with the same genome composition. Triploid hybrids cannot produce triploid individuals after crossings with each other and depend on diploid hybrid females producing diploid eggs. In contrast to other population systems, the majority of diploid and triploid hybrid females unexpectedly produced gametes with the same genome as parental species hybrids coexist with.

Methodological approaches for studying the european water frog Pelophylax esculentus complex

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.

Oxidative stress parameters in two Pelophylax esculentus complex frogs during pre- and post-hibernation: Arousal vs heavy metals

In spring, frogs from temperate regions are faced with arousal-induced oxidative stress and exposure to various xenobiotics from the environment. The question is whether pollutants can significantly modify the antioxidative defense system (AOS) response of hibernators during recovery from hibernation. If this assumption is true, we would then expect different patterns of seasonal variations in the AOS between individuals exposed to different levels of pollution. To examine this assumption, we determined the relationship between seasonal variations of accumulated metals and AOS parameters in the skin and muscle of two frog species from the Pelophylax esculentus complex (P. ridibundus and P. esculentus) inhabiting two localities (the Danube-Tisza-Danube canal and the Ponjavica River) with different levels of pollution during pre- and post-hibernation periods, respectively autumn and spring. Our results showed that even though there were differences in the concentrations of accumulated metals and AOS parameters between localities and species, the frogs displayed almost the same patterns of AOS variations during seasons, with a higher AOS response observed in spring. The parameters SH groups, GSH, GR and SOD had been contributed most rather than others. Our findings indicate that oxidative stress during the post-hibernation period was mainly caused by the organisms' recovery from hibernation, as the result of natural selection acting on the AOS, and that the accumulated metals did not significantly modify the AOS response. The present study provides new insight into the biological and physiological cellular responses of frogs to arousal stress.

Speciation in Indo-Pacific swiftlets (Aves: Apodidae): integrating molecular and phenotypic data for a new provisional taxonomy of the Collocalia esculenta complex.

White-bellied swiftlets of the Collocalia esculenta complex constitute a radiation of colony-breeding swifts distributed throughout the tropical Indo-Pacific region. Resolution of their taxonomy is challenging due to their morphological uniformity. To analyze the evolutionary history of this complex, we combine new biometric measurements and results from plumage assessment of museum specimens with novel as well as previously published molecular data. Together, this body of information constitutes the largest systematic dataset for white-bellied swiftlets yet compiled, drawn from 809 individuals belonging to 32 taxa for which new molecular, biometric, and/or plumage data are presented. We propose changing the classification of white-bellied swiftlets, for which two species are currently recognized, to elevate eight regional forms to species level, and we also describe two new subspecies. The ten taxa we recommend recognizing at the species level are: Collocalia linchi (Java to Lombok, Sumatran hills), C. dodgei (montane Borneo), C. natalis (Christmas Island), C. affinis (Greater Sundas, including the Thai-Malay Peninsula and Andaman-Nicobar Islands), C. marginata (Philippines), C. isonota (Philippines), C. sumbawae (west Lesser Sundas), C. neglecta (east Lesser Sundas), C. esculenta (Sulawesi, Moluccas, New Guinea, Bismarck Archipelago, Solomon Islands), and C. uropygialis (Vanuatu, New Caledonia). Future molecular and morphological work is needed to resolve questions of speciation and population affinities in the Philippines, Christmas Island, Wallacea and central Melanesia, and to shed light on historic diversification and patterns of gene flow in the complex.

Differentiation of frogs from two populations belonging to the Pelophylax esculentus complex by LC-MS/MS comparison of their skin peptidomes.

LC-MS/MS was applied to establish the composition of the skin peptidome of a Slovenian green frog belonging to the Pelophylax esculentus complex. As this was similar to the peptidome of the Moscow population of Pelophylax ridibundus, it allowed us to identify the Slovenian frog from the Pelophylax esculentus complex as Pelophylax ridibundus. The sequences of six new peptides from the brevinin 2 family are reported for the first time on the basis of manual interpretation of their tandem mass spectra. The structural similarity of the brevinin 2 peptides from the Moscow and Slovenian populations of Pelophylax ridibundus enables peptides from this family to be utilized as biomarkers for Pelophylax ridibundus inter- and intraspecies differentiation, and the proposed approach can be used as an analytical tool for differentiating the corresponding species and populations. The potential biological activities of the novel peptides were estimated by 2D mass mapping. The results allowed us to classify all of the available peptides belonging to the brevinin 2 family. Graphical Abstract Intraspecies identification within the green frog complex.

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

The western Palaearctic water frogs in the genus Pelophylax comprise several distinct species and three hybridogenetic hybrid forms. In this study, we focus on the Pelophylax esculentus complex, which consists of two sexual species, Pelophylax ridibundus and Pelophylax lessonae, and their hybridogenetic hybrid, Pelophylax esculentus. Specifically, we investigated taxonomic composition and ploidy level of water frogs sampled in three different types of wetland habitats in the Hortobagy National Park (HNP), eastern Hungary. Using variation in serum albumin intron 1 (SAI-1) and 15 microsatellite loci, we detected the presence of all members of the P. esculentus complex in the studied localities. In one locality, all three taxa occurred syntopically, while in others water frog populations consisted of P. ridibundus and P. esculentus exclusively. The genomic composition of the 63 examined hybrid specimens analysed with microsatellites showed the occurrence of diploid genotypes only. We used a population genetic approach (allelic richness, gene diversity, multilocus genotypes and multilocus disequilibrium) to infer the breeding system of water frogs at HNP. Our data indicate that at least in two populations, hybrids form gametes with clonally transmitted P. ridibundus genome and produce a new hybrid generation by mating with P. lessonae.

Pecular Features of Hematopoiesis in the Liver of Mature and Immature Green Frogs (Pelophylax Esculentus Complex)

Abstract The article describes characteristic features of the hematopoiesis in mature and immature green frogs (Pelophylax esculentus complex). Quantitative differences in liver myelograms were insignificant. However, in a sample of mature animals numerous significant correlations between the number of pigment inclusions in the liver and indicators of erythropoiesis and myelopoiesis were observed. Those correlations were absent in the immature frogs. We concluded that aft er the frogs’ breeding a lack of plastic resources, in particular, hemosiderin remains up to the hibernation.

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

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.