Water Frogs Research Articles

New records of the anomaly P syndrome in two water frog species (Pelophylax ridibundus and P. lessonae) in Russia

The “anomaly P” was described in Palearctic water frogs of the genus Pelophylax by Jean Rostand as complex morphological anomalies of water frogs, including polydactyly, brachymely, hind limb oedema, bone outgrowths, spikes, flexions and additional limbs in the inguinal region. In 2016, the anomaly P syndrome was rediscovered in central Russia, confirming the hypothesis concerning its wider distribution. Here, three new records of this syndrome in two species of western Palearctic water frog from Russia are described.

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.

Identification of Antibacterial Molecule(s) from Animals Living in Polluted Environments.

Snakes feed on germ-infested rodents, while water monitor lizards thrive on rotten matter in unhygienic conditions. We hypothesize that such creatures survive the assault of superbugs and are able to fend off disease by producing antimicrobial substances. In this study, we investigated the potential antibacterial activity of sera/lysates of animals living in polluted environments. Snake (Reticulatus malayanus), rats (Rattus rattus), water monitor lizard (Varanus salvator), frog (Lithobates catesbeianus), fish (Oreochromis mossambicus), chicken (Gallus gallus domesticus), and pigeon (Columba livia) were dissected and their organ lysates/sera were collected. Crude extracts were tested for bactericidal effects against neuropathogenic E. coli K1, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus cereus and Klebsiella pneumoniae. To determine whether lysates/sera protect human cells against bacterialmediated damage, cytotoxicity assays were performed by measuring lactate dehydrogenase release as an indicator of cell death. Lysates/sera were partially characterized using heat-treatment and pronasetreatment and peptide sequences were determined using the Liquid Chromatography Mass Spectrometry (LC-MS). Snake and water monitor lizard sera exhibited potent broad-spectrum bactericidal effects against all bacteria tested. Heat inactivation and pronase-treatment inhibited bactericidal effects indicating that activity is heat-labile and pronase-sensitive suggesting that active molecules are proteinaceous in nature. LCMS analyses revealed the molecular identities of peptides. The results revealed that python that feeds on germ-infested rodents and water monitor lizards that feed on rotten organic waste possess antibacterial activity in a heat-sensitive manner and several peptides were identified. We hope that the discovery of antibacterial activity in the sera of animals living in polluted environments will stimulate research in finding antibacterial agents from unusual sources as this has the potential for the development of novel strategies in the control of infectious diseases.

Population genomics of an exceptional hybridogenetic system of Pelophylax water frogs

BackgroundHybridogenesis can represent the first stage towards hybrid speciation where the hybrid taxon eventually weans off its parental species. In hybridogenetic water frogs, the hybrid Pelophylax kl. esculentus (genomes RL) usually eliminates one genome from its germline and relies on its parental species P. lessonae (genomes LL) or P. ridibundus (genomes RR) to perpetuate in so-called L-E and R-E systems. But not exclusively: some all-hybrid populations (E-E system) bypass the need for their parental species and fulfill their sexual cycle via triploid hybrid frogs. Genetic surveys are essential to understand the great diversity of these hybridogenetic dynamics and their evolution. Here we conducted such study using RAD-sequencing on Pelophylax from southern Switzerland (Ticino), a geographically-isolated region featuring different assemblages of parental P. lessonae and hybrid P. kl. esculentus.ResultsWe found two types of hybridogenetic systems in Ticino: an L-E system in northern populations and a presumably all-hybrid E-E system in the closely-related southern populations, where P. lessonae was not detected. In the latter, we did not find evidence for triploid individuals from the population genomic data, but identified a few P. ridibundus (RR) as offspring from interhybrid crosses (LR × LR).ConclusionsAssuming P. lessonae is truly absent from southern Ticino, the putative maintenance of all-hybrid populations without triploid individuals would require an unusual lability of genome elimination, namely that P. kl. esculentus from both sexes are capable of producing gametes with either L or R genomes. This could be achieved by the co-existence of L- and R- eliminating lineages or by “hybrid amphigamy”, i. e. males and females producing sperm and eggs among which both genomes are represented. These hypotheses imply that polyploidy is not the exclusive evolutionary pathway for hybrids to become reproductively independent, and challenge the classical view that hybridogenetic taxa are necessarily sexual parasites.

Characterization of Albanian water frog, Pelophylax shqipericus, sperm traits and morphology, by using phase contrast microscopy

Mature spermatozoa traits and morphology of endangered Albanian water frog, Pelophylax shqipericus, have been characterized for the first time through phase contrast microscopy, as part of successful implementation of in vitro fertilization technique for this species. The basic morphology of P. shqipericus spermatozoa consists of an elongated, thick, smooth-edged, and solid-staining head, continuing with a thin and long tail which usually extends 2.48 times the head length. The acrosome was not clearly discernible so the measurements were done on the head as a whole, while the middle section was better visible. Average length of head, including the acrosome and midsection was estimated to be 11.78 μm ± 0.32, while the tail length resulted 29.24 ± 1.75 μm. The average thickness of the head was shown to be 3.45 μm. The total sperm length resulted to be 41.02 ± 1.83 μm. The average sperm concentration was estimated of 25.5 × 106 /ml. Sperm amount, survival rate and motility were also measured. The sperm survival rate was maximal immediately after preparation of the suspension and tended to decrease over time of storage, reaching 50% after 72 hr. Decreased sperm motility seemed to follow the same trend as sperm viability. Sperm traits resulted to be very similar both in size and in shape with those of "Lessonae" frog group, one of the lineages of Western Palearctic species complex, suggesting a strong phylogenetic relationship among these species.

Activity Patterns and Feeding Ecology of the Semi-Aquatic <i>Varanus flavescens</i> (Reptilia: Varanidae)

The study was conducted on activity patterns and feeding ecology of semi-aquatic yellow monitor ( Varanus flavescens ) in the Chalan Beel area of Bangladesh by following focal sampling method. Overall, V. flavescens spent highest proportion (37.73 ± 0.65) of their active time in moving in search of food and lowest proportion (3.05 ± 0.05) of time in conflicting. Maximum (22.80 ± 3.19) and minimum (5.82 ± 1.06) moving activity were recorded in the month of August and December respectively. Two peak feeding times; from 6.30 AM to 10.30 AM and from 3.00 p.m. to 4.30 p.m. were recorded throughout the day. Total 15 prey groups were recorded as yellow monitor’s diet. The diet consisted mainly of beetles, which comprised 15% of the number of prey items in stomach samples. Others diet comprises of spider, dragonfly, water scorpions, mole crickets, crickets, ants, fresh water crabs, grasshopper, frog, small fish, house mouse and bird eggs. Most of the activities of V. flavescens varied significantly (χ 2 test, P < 0.05) in different month in response to changing weather conditions. During heavy cold V. flavescens shows basking activity to regulate their body temperature and the highest proportion (50.48 ± 3.03) of basking activity was recorded in December. The population of V. flavescens is decreasing at an alarming rate all over Bangladesh. This warrants for further study regarding the ecology and conservation of the yellow monitor.

Widespread occurrence of the antifungal cutaneous bacterium Janthinobacterium lividum on Andean water frogs threatened by fungal disease.

Amphibian diversity has declined due to the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Coexistence between amphibian hosts and this pathogen in some locations is attributed to the presence of the cutaneous bacterium Janthinobacterium lividum (Jliv). This microbe inhibits the growth of Bd on the host, reduces morbidity, and improves survival. Andean water frogs in the genus Telmatobius seem to be particularly vulnerable to the disease yet populations of T. intermedius and T. marmoratus persist in southern and central Peru. We investigated the presence of Jliv on these 2 frog species and assessed the relationship of Jliv presence with prevalence and intensity of Bd infection. By sampling 125 frogs from 7 streams (3323-3950 m a.s.l.) and 27 from a city market, we found spatial variation in the mutualism among populations (range 0-40% proportion of Jliv-positives). Overall, 57% of frogs were infected with Bd, 12.5% of frogs hosted both Jliv and Bd, while 7.2% hosted just Jliv. We found that the probability of an individual being infected with Bd was independent of the presence of Jliv; however, we did detect a protective effect of Jliv with respect to intensity of infection. The extent of Jliv distribution in the high Andes stands in stark contrast to the rarity of Jliv on frogs in lower elevation cloud forest biomes.

Allurin and egg jelly coat impact on in-vitro fertilization success of endangered Albanian water frog, Pelophylax shqipericus

Amphibian egg-jelly coat plays an important role in successful fertilization and development. Here, we ask whether proteins like allurin in the jelly coats of frog eggs might influence fertilization rate success. Using in vitro fertilization of Albanian water frog, Pelophylax shqipericus, we found that body cavity eggs or eggs deprived of jelly coat were not fertilized, compromising the success of in vitro fertilization procedure. When de-jellied eggs were inseminated with sperm suspension, the fertilization efficiency is dramatically decreased even inhibited, suggesting that the gel structure is one of the major factors in the achievement of fertilization in the frogs. Fertilization of de-jellied eggs with sperm pre-treated with egg jelly coat, restored the fertilization competency. Such a result suggests that egg jelly coat probably guides the sperm to the egg surface while maintaining the fertilization ability, contributing to a successful in vitro fertilization of Pelophylax shqipericus.

A Geometric Morphometric Analysis for Ranid Taxonomy: Chondrocranial Shape Variation of Five Ranid Species (Anura: Ranidae)

A landmark-based geometric morphometrics of the dorsal view of the chondrocranium was analysed for 188 specimens to investigate the hypothesis of species shape and size differences among Rana tavasensis, R. macrocnemis, Pelophylax ridibundus, P. bedriagae and P. caralitanus. Canonical variate analysis (CVA) is applied to determine whether the shape and size differences in the chondrocranium of five ranids can be used to classify those at a taxonomic level. The result of CVA analysis shows that five ranid species examined here discriminate as mountain (Rana sp.) and water (Pelophylax sp.) frogs. In discriminant function analysis (DFA), each pair of species was analyzed and the results show that there is no interspecific shape difference among Pelophylax species. However, the interspecific shape variations in the cornua trabeculae, articular process of the palatoquadrate and processus muscularis quadrati, were present between Rana species (R. macrocnemis and R. tavasensis).

Temperature exposure and possible thermoregulation strategies in the Titicaca water frog Telmatobius culeus, a fully aquatic frog of the High Andes

Temperature has an important effect on amphibians, influencing virtually all physio logical systems. Thermoregulation is used to manage unfavourable thermal conditions, but has been poorly studied in aquatic amphibians, and no information is available for adult aquatic anurans. We studied the temperatures to which the Critically Endangered and high Andean fully aquatic Titicaca water frog Telmatobius culeus is exposed and investigated possible thermoregulation strategies of the species in the wild. We measured water temperature in different seasons, microhabitats and depths at which T. culeus is most abundant. We established transects to obtain population densities and activity patterns of the species during the day. Seasonal and daily temperature variations as well as temperature variations among depths and microhabitats were noted. These variations were associated with densities of visible T. culeus, where adult densities were higher at depths with more stable temperatures. T. culeus behaviour associated with thermo regulation strategies in the aquatic habitat included depth selection and movement of individuals between different microhabitats. Selection of microhabitat depended on the temperature at a site and the hour of the day. These results indicate possible behavioural thermoregulation strategies such as movement to different sites or basking used by a highly specialized amphibian, and provide insights into how aquatic benthic organisms use strategies for thermoregulation in aquatic heterogeneous environments. These strategies are probably also used by other aquatic organisms from the tropical Andes. Knowledge of how these organisms respond to temperature could also help to understand the impact of global warming on their status.

Taxonomic survey of water frog populations of Pelophylax bedriagae (Anura: Ranidae) in western Iran: a morphometric and bioacoustic approach

We explored the population systems of Pelophylax bedriagae (Camerano, 1882) in northwestern, western and southwestern Iran. Statistical significance for a hypothesis of sexual dimorphism of the frogs was tested using t-test and multivariate analysis of variances (MANOVA). Due to lack of sexual differences between the male and female frogs, the data of both sexes were pooled for further analysis. Taxonomic relationships among local populations were investigated using discriminate analysis (by stepwise method) with iterative sample assignment. The multivariate morphometric analyses revealed that the range of P. bedriagae extends to western Iran. The next step was to analyze the advertisement call (five standard parameters: call duration, number of pulse groups per call, duration of pulse groups, interval between pulse groups, and intercall interval) of the water frogs inhabiting three sites in western Iran. Rigorous application of the bioacoustic method removes all doubt that these three sites are inhabited by the same species, which on the basis of priority of nomenclature should be called P. bedriagae.

Electric circuit theory applied to alien invasions: a connectivity model predicting the Balkan frog expansion in Northern Italy

Invasive species are a major threat to biodiversity and alien herpetofauna already caused conservation problems in Italy and in the world. Pelophylax kurtmuelleri (Gayda, 1940) is a water frog native to Southern Balkans, that was introduced in NW Italy (Liguria) in 1941, from where it has spread to Piedmont, Lombardy and recently to Emilia Romagna. Surprisingly, during its expansion, the Balkan frog seemed not to be able to cross the Po River (with a single exception). So, we investigated the reasons for such directional limitation of range expansion, focusing on the potential role of the Po River. Combining ecological niche and connectivity models, we adopted a two-step process in order to assess: (i) if the habitat suitability differs between the two sides of the Po Plain; (ii) if the Po River could act as a barrier in terms of dispersal. Results showed that the northern side of the plain seems less suitable than the southern one, even if many suitable patches occur along the main left tributaries of the Po River and in the hilly part of the plain. The Po River behaves like a barrier for the Balkan frog, but the only known point on the north bank of the river indicates that it may be able to only slow the dispersal and not to completely stop it. So, the most probable interpretation for the absence of the Balkan frog from the Northern Po Plain, is the combination of habitat suitability and connectivity issues: the former makes less probable that new viable populations can establish in the North, the latter decreases the northwards flux of the frogs. Given these findings, the Balkan frog seems able to spread in the whole Northern Italy and the colonization of the northern Po plain could happen shortly.

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

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.

Cytotoxicity in the frog (Fejervarya limnocharis) after acute cadmium exposure in vivo

Chromosome aberrations (CA) in frogs (Fejervarya limnocharis) exposed to 5, 10 and 20 mg/L of cadmium chloride (CdCl2) for 24, 48 and 72 h were invetegated. Treated frogs were compared to a control group. Cadmium (Cd) was not detected in the water or control frogs. The highest Cd concentrations in water and frog samples were found at 20 mg/L exposed for 72 h. The water samples indicated that exposure to 10 mg/L of Cd for 24 h was significantly different from the control (p < 0.05). Cd concentrations in frogs differed significantly between the control and experimental groups (p < 0.05). The cytotoxicity assessment revealed ten types of CA in the frogs, including single chromatid gap (SCG), isochromatid gap, single chromatid break, isochromatid break, iso-arm fragmentation, single chromatid decomposition, centric fragmentation, centromere gap, deletion and fragmentation. The most common CA in the study was SCG. The statistical analysis indicated significant differences in the percentage of cells with CA for exposures of 20 mg/L (24 h), 10 and 20 mg/L (48 h) and 5, 10 and 20 mg/L (72 h) compared to the control (p < 0.05). The results of this study show that high Cd concentrations and long duration exposure can cause CA in frogs.

Ranavirus genotypes in the Netherlands and their potential association with virulence in water frogs (Pelophylax spp.)

Ranaviruses are pathogenic viruses for poikilothermic vertebrates worldwide. The identification of a common midwife toad virus (CMTV) associated with massive die-offs in water frogs (Pelophylax spp.) in the Netherlands has increased awareness for emerging viruses in amphibians in the country. Complete genome sequencing of 13 ranavirus isolates collected from ten different sites in the period 2011–2016 revealed three CMTV groups present in distinct geographical areas in the Netherlands. Phylogenetic analysis showed that emerging viruses from the northern part of the Netherlands belonged to CMTV-NL group I. Group II and III viruses were derived from the animals located in the center-east and south of the country, and shared a more recent common ancestor to CMTV-amphibian associated ranaviruses reported in China, Italy, Denmark, and Switzerland. Field monitoring revealed differences in water frog host abundance at sites where distinct ranavirus groups occur; with ranavirus-associated deaths, host counts decreasing progressively, and few juveniles found in the north where CMTV-NL group I occurs but not in the south with CMTV-NL group III. Investigation of tandem repeats of coding genes gave no conclusive information about phylo-geographical clustering, while genetic analysis of the genomes revealed truncations in 17 genes across CMTV-NL groups II and III compared to group I. Further studies are needed to elucidate the contribution of these genes as well as environmental variables to explain the observed differences in host abundance.

All-male hybrids of a tetrapod Pelophylax esculentus share its origin and genetics of maintenance

BackgroundSexual parasites offer unique insights into the reproduction of unisexual and sexual populations. Because unisexuality is almost exclusively linked to the female sex, most studies addressed host-parasite dynamics in populations where sperm-dependent females dominate. Pelophylax water frogs from Central Europe include hybrids of both sexes, collectively named P. esculentus. They live syntopically with their parental species P. lessonae and/or P. ridibundus. Some hybrid lineages consist of all males providing a chance to understand the origin and perpetuation of a host-parasite (egg-dependent) system compared to sperm-dependent parthenogenesis.MethodsWe focused on P. ridibundus-P. esculentus populations where P. ridibundus of both sexes lives together with only diploid P. esculentus males. Based on 17 microsatellite markers and six allozyme loci, we analyzed (i) the variability of individual genomes, (ii) the reproductive mode(s) of all-male hybrids, and (iii) the genealogical relationships between the hybrid and parental genomes.ResultsOur microsatellite data revealed that P. esculentus males bear Mendelian-inherited ridibundus genomes while the lessonae genome represents a single clone. Our data indicate that this clone did not recently originate from adjacent P. lessonae populations, suggesting an older in situ or ex situ origin.ConclusionsOur results confirm that also males can perpetuate over many generations as the unisexual lineage and successfully compete with P. ridibundus males for eggs provided by P. ridibundus females. Natural persistence of such sex-specific hybrid populations allows to studying the similarities and differences between male and female reproductive parasitism in many biological settings.

First detection of the amphibian chytrid fungus (Batrachochytridium dendrobatidis) in free- living anuran populations in Greece

The amphibian chytrid fungus (Batrachochytrium dendrobatidis) is a widespread, cosmopolitan pathogen largely affecting free-living amphibian populations. So far there are no published studies for the presence of B. dendrobatidis in Greece. In this preliminary study we sampled 59 metamorphosed anurans from four Greek wetlands. Five samples were positive for the fungus by real-time PCR. B. dendrobatidis was detected in three species (Bufo viridis, Pelophylax epeiroticus, Pelophylax ridibundus) but not in endangered endemic Karpathos water frogs (Pelophylax cerigensis). This is the first report of the amphibian chytrid fungus (Batrachochytrium dendrobatidis) in free-living anuran populations from Greece.

Invasion genetics of marsh frogs (Pelophylax ridibundus sensu lato) in Switzerland

The marsh frog (Pelophylax ridibundus sensu lato) is the number one amphibian invader in Western Europe. In Switzerland, marsh frogs were introduced in the 1950-1960s and progressively colonized most of the northern parts of the country. We investigated this invasion using molecular tools. We mapped the cryptic presence of three monophyletic mitochondrial lineages (P. ridibundus, P. kurtmuelleri and P. c. f. bedriagae from southeastern Europe) consistent with registered importations by a local frog-leg industry. High nuclear diversity supports that invasive frogs probably originated from genetically-rich import batches and patterns of population differentiation confirm that multiple independent introduction sites were involved. Moreover, several lines of evidence suggest occasional hybridization with local hybridogenetic water frogs. This invasion emphasizes the issues of frequent amphibian releases and translocations at the international and regional scale for commercial and recreational purposes, and stresses for more adequate legislation, control and information to the general public. Given the parallel invasion by alien pool frogs (i. e., the Italian P. bergeri has replaced the local P. lessonae), the situation of water frogs in Switzerland is critical. The water frog complex makes an alarming symbol of the anthropogenic mark left on wildlife diversity and distributions.

Neural circuits underlying jaw movements for the prey-catching behavior in frog: distribution of vestibular afferent terminals on motoneurons supplying the jaw.

Coordinated movement of the jaw is essential for catching and swallowing the prey. The majority of the jaw muscles in frogs are supplied by the trigeminal motoneurons. We have previously described that the primary vestibular afferent fibers, conveying information about the movements of the head, established close appositions on the motoneurons of trigeminal nerve providing one of the morphological substrates of monosynaptic sensory modulation of prey-catching behavior in the frog. The aim of our study was to reveal the spatial distribution of vestibular close appositions on the somatodendritic compartments of the functionally different trigeminal motoneurons. In common water frogs, the vestibular and trigeminal nerves were simultaneously labeled with different fluorescent dyes and the possible direct contacts between vestibular afferents and trigeminal motoneurons were identified with the help of DSD2 attached to an Andor Zyla camera. In the rhombencephalon, an overlapping area was detected between the incoming vestibular afferents and trigeminal motoneurons along the whole extent of the trigeminal motor nucleus. The vestibular axon collaterals formed large numbers of close appositions with dorsomedial and ventrolateral dendrites of trigeminal motoneurons. The majority of direct contacts were located on proximal dendritic segments closer than 300µm to the somata. The identified contacts were evenly distributed on rostral motoneurons innervating jaw-closing muscles and motoneurons supplying jaw-opening muscles and located in the caudal part of trigeminal nucleus. We suggest that the identified contacts between vestibular axon terminals and trigeminal motoneurons may constitute one of the morphological substrates of a very quick response detected in trigeminal motoneurons during head movements.

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.