Rana Ridibunda Research Articles

Determinations of Erythrocyte Sizes in Adult Pelophylax ridibundus (Amphibia: Anura: Ranidae) Inhabiting Industrial Area in Southern Bulgaria

In this study, basic erythrocyte sizes in P. ridibundus individuals inhabiting a polluted area (heavy metals, nitrates, nitrites, and ammonium) in southern Bulgaria were evaluated. The erythrocyte cells’ parameters, erythrocyte length (EL), erythrocyte width (EW), length/width ratio (EL/EW), and erythrocyte size (ES), and nuclear parameters, nucleus length (NL), nucleus width (NW), NL/NW ratio, nucleus size (NS) and NS/ES ratio were either measured or calculated. The data obtained were compared with the data from identical measurements made in frogs inhabiting a reference site. In this study, an authentic reduction was established, of the basic erythrocyte-metric cells (EL, EW, EL/EW ratio, and ES) and nuclear (NL, NW, NL/NW, and NS) parameters in P. ridibundus individuals that inhabit the polluted industrial area east of the city of Plovdiv. In those frogs’ blood, oval and spherical erythrocytes circulate. For the erythrocytes from frogs in the polluted site, a significant reduction was found in the nucleus/cytoplasm ratio. Those changes can be seen as adaptations enlarging the contact surface and the oxygen capacity of the erythrocytes in conditions of hypoxia. The results of our research once again emphasize the importance of hematological investigations and specifically of the blood cell morphology as reliable biomarkers in ecotoxicological research in the field.

Biological aspectss of the Marsh frog Pelophylax ridibundus population from the Shadegan Wetland, Khuzestan Province, Iran

The Marsh frog, Pelophylax ridibundus (Pallas) is distributed in Central Europe from northeastern France, north to the southern shorelines of the Baltic Sea (and extreme southern Finland), south to northeastern Spain, northern Italy and the Balkans including eastern Greece, east to approximately 81° E in Asiatic Russia, and south to western Iran and Afghanistan. The present study has been conducted on populations of P. ridibundus in the northern, eastern and southern regions of the Shadegan Wetland of Khuzestan Province, Iran with the aim of examining some aspects of its morphology and karyotype. Frog specimens were collected from different portions of the Shadegan Wetland during spring and autumn 2016 and transferred to the laboratory. According to the morphological results, three color patterns are identified among the samples in terms of the morphology, morphometry and sex - according to the sex independency test. T-test results between males and females indicate a significant difference in all traits and the results of a T2 Hotelling test show that there is sexual dimorphism between males and females. However, the results of multivariate analysis of variance (MANOVA) illustrate a separation between males of the northern region and the two other regions, and no differentiation between females in the eastern and southern regions of the wetland. The karyotype of the species in the wetland included 2n= 26, one pair of which had a sex chromosome.

Exposure to common pesticides utilized in northern rice fields of Iran affects survival of non-target species, Pelophylax ridibundus (Amphibia: Ranidae).

Amphibians are the most important vulnerable non-target vertebrate group that are affected by pesticides. Most previous studies have confirmed the destructive effects of pesticides. But, so far, no comprehensive studies have been carried out in Iran. Therefore, to estimate the mortality rate of frogs during the growing season in different cultivating systems, we examined the presence of pesticides in water and substrate as indicators of habitat quality and in the liver tissue of Marsh frog Pelophylax ridibundus (Pallas, 1771), enclosed in the prepared cages at five rice paddy fields in Mazandaran province, Iran. The measurement of pollution was done using mass gas chromatography method and statistical analyses by Minitab software. Furthermore, the probable movement pattern of free frogs was analyzed using capture-mark-recapture method. Thirteen pesticides were detected both in the habitat and in frogs' liver tissue. Among them ß-Mevinphos, Fenitrothion, Bromofos, and Trifluralin had the most frequent occurrence in liver tissue, and Diazinon with concentrations up to 517.8μg/Kg had the highest concentration. Furthermore, there is a significant correlation (R2 > 0.96) between water quality and frogs' contamination, whereas, no correlation was observed between substrate pollution and frogs' contamination. Pesticide concentrations were higher in two stations but lower than lethal doses to frogs, so that no mortality was observed at any of the stations. However, some specimens had a considerable muscle atrophy. Despite no significant movement pattern was detected, we can expect that if this trend continues, in a long term, they will face a reduction in the survival rate.

Parasitism with Acanthocephalus Ranae in Frogs (Pelophylax Ridibundus Pallas 1771), from North-east Romania.

SummaryThe present study was carried out on 10 frogs, Pelophylax ridibundus (Pallas, 1771), captured near the river Bahlui, Iasi, between April and May, 2019, for the study of digestive helminths and their effect on the digestive tract. Macroscopic examination of the general cavity revealed distension of the small intestine. The sectioning of the intestinal wall revealed a variable number of parasites (1 – 12) with a whitish cylindrical body, anchored in the duodenal mucosa. The morphological study of the parasites confirmed the species as Acantocephalus ranae. The prevalence of the infection was 60 % and the average intensity was 4.83 ± 4.15. Lesions of the small intestine were characterized by catarrhal enteritis, hemorrhagic spots and ulcers on the mucosa, occlusion and obstruction of the intestinal lumen. Histologically, eosinophilic inflammatory infiltrate in the glandular crypts, mucosal and submucosal edema, fibrosis and conjunctival hyperplasia, total mucosal atrophy, were found. The infection with Acanthocephalus ranae in Pelophylax ridibundus caused severe pathological changes, conditioned by the intensity of the infection, aspects revealed for the first time in Romania.

Hybridogenesis in the Water Frogs from Western Russian Territory: Intrapopulation Variation in Genome Elimination.

Hybridogenesis in an interspecific hybrid frog is a coupling mechanism in the gametogenic cell line that eliminates the genome of one parental species with endoduplication of the remaining genome of the other parental species. It has been intensively investigated in the edible frog Pelophylax kl. esculentus (RL), a natural hybrid between the marsh frog P. ridibundus (RR) and the pool frog P. lessonae (LL). However, the genetic mechanisms involved remain unclear. Here, we investigated the water frogs in the western Russian territory. In three of the four populations, we genetically identified 16 RL frogs living sympatrically with the parental LL species, or with both parental species. In addition, two populations contained genome introgression with another species, P. bedriagae (BB) (a close relative of RR). In the gonads of 13 RL frogs, the L genome was eliminated, producing gametes of R (or R combined with the B genome). In sharp contrast, one RL male eliminated the L or R genome, producing both R and L sperm. We detected a variation in genome elimination within a population. Based on the genetic backgrounds of RL frogs, we hypothesize that the introgression of the B genome resulted in the change in choosing a genome to be eliminated.

The development of method of hormonal stimulation of N.L.Gerbilsky and appearance of contemporary preparations of long-term storage

The The main discovery of the 20th century in the field of fish breeding is hormonal stimulation of final stages of maturation of fish at hatcheries. The «father» of method of pituitary injections was Leningrad ichthyologist N.L. Gerbilsky. The main achievements of N.L. Gerbilsky and his school are methods of mass extraction of fish pituitaries, their dehydration by acetone for later storage and use, testing of pituitary gonadotropic activity on different objects. The induction of ovulation in female loaches Misgurnus fossilis or spermiation reaction of Galli-Mainini in male frogs Rana ridibunda or R. temporaria were used for pituitary testing. The effective doses of dried pituitaries for sturgeons were determined in milligrams and loach units or frog units. The method of pituitary injections was used for functioning of sturgeon breeding facilities at Volga, Don, Kuban and Kura and other rivers. For guaranteed answer fish breeders used hyperdoses of dry pituitary powder because its gonadotropic activity varied in dependence on stage of maturity of fish-“donors”. The followers of N.L. Gerbilsky led by Prof. I.A. Barannikova developed the technique of production of special glycerol pituitary extract (GPE) with determined gonadotropic activity. The main role in formulation of the recipe of GPE belongs to A.A. Boyev. The use of GPE has removed the problem of hyperdosing leading to worsening quality of mature germinal cells of sturgeons. Later, A.A. Boyev on the basis of this recipe with replacement of glycerol to propylene glycol created stabilizing solution of analogue of mammal LH-RH (“surphagon”) of long-term storage with concentration of active substance from 20 to 500 micrograms/ml.

Genetic structure, morphological variation, and gametogenic peculiarities in water frogs ( Pelophylax ) from northeastern European Russia

The edible frog, Pelophylax esculentus, is a hybrid form that reproduces via clonal propagation of only one of the parental genomes through generations of hybrids while the genome of other parental species is eliminated during gametogenesis. Such reproductive ability requires hybrids to coexist with one of the parental species or rarely both parental species causing the formation of so-called population systems. Population systems and reproductive biology of water frogs from the east of the range remained partially unexplored. In this study, we investigated the distributions, population systems, genetic structure, types of gametes, and morphological variability of water frogs of the genus Pelophylax from the northeastern parts of their ranges (Mari El Republic and adjacent territories, Russia). We examined 1,337 individuals from 68 localities using morphological traits combined with DNA flow cytometry and a multilocus approach (fragments of a nuclear and two mitochondrial genes). We revealed five types of population systems: “pure” populations of the parental P. ridibundus (R) and P. lessonae (L), mixed populations of parental species (R-L) along and with their hybrids (R-E-L), as well as mixed populations of P. lessonae and P. esculentus (L-E). However, the “pure” hybrid (E) and the mixed P. ridibundus and P. esculentus (R-E) population systems were not found. All hybrids studied by DNA flow cytometry were diploid. Analysis of gametogenesis showed that the majority of hybrid males, as well as hybrid females from the L-E system, produced gametes with the P. ridibundus genome. However, in the R-E-L system, hybrid females were usually sterile. The reproduction of hybrids in such systems is primarily based on crosses of P. esculentus males with P. lessonae females. Molecular analysis showed the presence of mitochondrial and nuclear DNA introgression of the Anatolian marsh frog (P. cf. bedriagae) into both P. ridibundus and P. esculentus. The observations of alleles and haplotypes of P. cf. bedriagae in P. ridibundus and P. esculentus individuals from the same localities suggest de novo formation of local hybrids. However, the presence of the Balkan marsh frog (P. kurtmuelleri) haplotypes in local hybrids supports the hypothesis regarding the migration of old hemiclonal lineages from glacial refugia. Finally, the diagnostic value of various morphological characteristics was discussed.

Систематический список амфибий Казахстана: краткая история изучения, современная таксономия, вопросы актуализации

The aim of this article is to briefly describe the history of creation of a recent checklist of amphibians of Kazakhstan. This history as an academic phenomenon begun in the second half of the 18th Century – the time of the scientific travels of Peter Simon Pallas in western Kazakhstan. The very first checklist of amphibians developed for the territory of Kazakhstan was by A.M. Nikolsky in a monograph published in 1918. This forms the basis of all future work and contains scientific names which, for the most part, were interpreted in their correct forms: Ranodon sibiricus, Bombina bombina, Pelobates fuscus, Bufo bufo, Bufo viridis, Rana arvalis, R. asiatica, R. esculenta, and R. temporaria (nine species). Over the next 100 years, in quantitative terms, the checklist did not change much and varied between 10 species and 12 species. Qualitative changes were determined by views on the taxonomic position and phylogenetic relationships of specific and genera: R. amurensis – R. asiatica, Hynobius – Salamandrella, Rana – Pelophylax, for example. Taxonomic revisions carried out over the last two decades, based mainly on molecular genetic analysis, have made serious adjustments to the checklist of amphibian fauna of Kazakhstan. The species composition of the genera Pelobates (P. verspetinus) and Bufotes (B. sitibundus, B. perrini, B. pewzowi) has been determined and intraspecific heterogeneity of Pelophylax ridibundus has been confirmed in the modern concept of cryptic species complexes. This updating has further prospects and the most important tasks are to survey the northern territories of Kazakhstan adjacent to Russia to confirm or reject the presence of Lissotriton vulgaris, R. temporaria and R. amurensis as well as to continue taxonomic studies regarding the composition of the Bufotes viridis and Pelophylax ridibundus complexes.

The morphometric and erythrometric analyses of Pelophylax ridibundus living in anthropogenic pollution resources

The morphometric and erythrometric analyses of Pelophylax ridibundus living in anthropogenic pollution resources

Amino Acid Spectrum of Blood of the Lake Frog Pelophylax ridibundus Introduced into the Ponds of the Middle Urals

Amino Acid Spectrum of Blood of the Lake Frog Pelophylax ridibundus Introduced into the Ponds of the Middle Urals

Восприимчивость перепелов обыкновенных (Coturnix coturnix), чижей (Carduelis spinus) и лягушек озёрных (Rana ridibunda) к вирусу Западного Нила

Восприимчивость перепелов обыкновенных (Coturnix coturnix), чижей (Carduelis spinus) и лягушек озёрных (Rana ridibunda) к вирусу Западного Нила

Faunistic review of amphibians in protected areas of Ukrainian Roztochia

Background. Study of amphibian fauna as a necessary component of ecosystems is always relevant and requires a regular renewal of information, especially in protected areas. We conducted a field investigation of amphibians, a literature review and an inventory of the collections of herpetological funds in relation to three protected areas: the Roztochia Biosphere Reserve, the Yavorivskyi National Park, and the Cholgini Ornithological Reserve. Materials and Methods. The field study was performed by classical methods: route method with manual catching and with herpetological nets, vocalization method, sampling of amphibians crushed on the road, and catching individuals with frog fences. We also analyzed the literature and amphibian collections in the Zoological Museum of Ivan Franko National University of Lviv. Results. According to the available literature, the amphibian fauna of these three areas under protection is similar and typical of this region. The results of our field research slightly differ from the literature data, thus, there is a need for further studies. On the study areas, we found 12 species of amphibians, which is more than half of the number of amphibian species in Ukraine, in particular: Smooth Newt Lissotriton vulgaris, Great Cres­ted Newt Triturus cristatus, Fire-bellied Toad Bombina bombina, Eastern Tree Frog Hyla orientalis, Common Spadefoot Toad Pelobates fuscus, Common Toad Bufo bufo, Green Toad Bufotes viridis, Common Frog Rana temporaria, Moor Frog Rana arvalis, Marsh Frog Pelophylax ridibundus, Edible Frog Pelophylax esculentus and Pool Frog Pelophylax lessonae. We detected 2 species of Caudata and 10 species of Anura. 10 species of amphibians were found in the Roztochia Biosphere Reserve, 5 species – in the Yavorivskyi National Park, and 9 species – in the Cholgini Ornithological Reserve. Conclusions. According to the field results, 4 species inhabit all three protected areas – Common Toad, Eastern Tree Frog, Marsh Frog and Edible Frog. A relatively small variety of amphibians found in the Yavorivskyi National Park can be due to the homogeneity of habitats and a proximity of one of the largest military proving grounds in Europe, which limits amphibians in choice of the reproductive and trophic habitats. These habitats deserve special attention because of their fast degradation for the last 10 years. Keywords: amphibians, fauna, protected areas, Ukrainian Roztochia, the Roztochia Biosphere Reserve, the Yavorivskyi National Park, the Cholgini Ornithological Reserve

Heavy Metal Accumulation in the Tissues and Organs of Pelophylax ridibundus (Pallas, 1771) and Pelophylax lessonae (Camerano, 1882) (Amphibia: Ranidae) Living in the Waterbodies of Nizhniy Novgorod

A comprehensive atomic-emission study and comparative assessment were done of the content and distribution of heavy metals (Mn, Cu, Cr, Al, Fe, Zn, and Sr) in the organs and tissues (muscles, skin, bones, gonads, liver, heart, spleen, and blood) of marsh and pool frogs collected in the lakes of a big industrial city (Nizhni Novgorod). High concentrations of heavy metals were found in the spleen (Cr), bones (Zn and Sr), liver (Cu) of lake frogs. High coefficients of the biological absorption of Mn, Sr, Zn (bone tissue), Fe (liver, spleen), and Cu (heart) were found in pool frogs. The cumulative properties of heavy metals were estimated from the accumulation coefficient established. Species regularities of heavy metal accumulation from water are presented in descending order as the following series: Zn > Cr > Al > Cu > Fe > Sr > Mn for pool frogs; and Fe > Zn > Mn > Cu > Cr > Al > Sr for marsh frogs. The priority intake of chromium, manganese, aluminum and strontium from the aqueous medium into the body occurred through the skin. Active zinc accumulation occurred mainly through food, which was confirmed by high values of the biological absorption coefficient of zinc for the liver of Pelophylax ridibundus and Pelophylax lessonae. The dependence of the heavy metal accumulation in the muscles from the organs in contact with the external environment (skin) and the digestive system (liver) was established by regression analysis. The most important adaptation of tailless amphibians is their ability to prevent excessive accumulation of heavy metals in their body, while living in the conditions of increased environmental pollution. Our results obtained speak for the high accumulation of heavy metals in the body of tailless amphibians, determined by the conditions of the aquatic environment and the bioavailability of these metals.

Pathological and mortality findings associated withAeromonas hydrophilafrom frog eggs in Al‐Ahsa region of Saudi Arabia

Background Amphibians decline globally due to infection with virulent bacteria, despite their vital roles in the ecosystem. The current study aimed at addressing the pathological and bacterial findings of the infected Pelophylax ridibundus indigenous to the Al-Ahsa region. Many clinical signs were observed in the eggs, including discoloration, disintegration of the outer jelly coat; defects in morphology, and mortality were reported. Furthermore, neuromasts lacking apical cilia and microvilli were observed in the cleavage stages of the infected developing egg. Additionally, cutaneous hyperaemia, lethargy, peripheral oedema, subcutaneous haemorrhage and cutaneous ulcerations were observed in the infected P. ridibundus adults. Methods Ribbons of frog eggs were collected from Al-Ahsa oasis, the Eastern region, Kingdom of Saudi Arabia (KSA), and examined under light and scanning electron microscope. The developed eggs and adults were further examined using a dissecting microscope to determine signs of infection. A bacterial strain designated as PRE-1 was isolated from the infected eggs, identified genotypically and then characterized phenotypically. Strain PRE-1 was identified as Aeromonas hydrophila based on the comparative gene sequences of the 16S rRNA gene at identity level of (99.92%). Furthermore, phylogenetic relationship based on the 16S rRNA gene sequences of our strain and other relevant strains Aeromonas spp. showed that PRE-1 formed a cluster with A. hydrophila (99% bootstrap support), providing a robustness to the identification of PRE-1 as A. hydrophila. This bacterium was found to be associated with disease outbursts in the marsh frogs from the Al-Ahsa region. Conclusion It is important to continue monitoring the emerging diseases so that their cause can be discerned, and appropriate protocols can be applied to limit the spread of the loss of amphibians, which are vital for our ecosystem.

Effects of Gd3+ and Ca2+ on Frog Heart Muscle Contractility and Respiration, Swelling and Inner Membrane Potential of Rat Heart Mitochondria

We studied the inotropic and chronotropic effects of gadolinium ions (Gd3+) on contractility of frog Rana ridibunda heart muscle preparations. In addition, the influence of Gd3+ on oxygen uptake rates, swelling and the mitochondrial inner membrane potential (ΔΨmito) were studied in glutamate- and malate-energized rat heart mitochondria (RHM). It was found that Gd3+ decreases the amplitude and frequency of spontaneous heart contractions. At the same time, Gd3+ prevents Ca2+-induced swelling of these organelles in saline media and falling of ΔΨmito. In this case, regardless of the presence of Ca2+ in the medium, Gd3+ has a weak effect on mitochondrial state 3 or 3UDNP respiration (in the presence of 2,4-dinitrophenol). In calcium free experiments, Gd3+ stimulates passive swelling of RHM. These effects of Gd3+ may indicate that Gd3+ has no toxic effect on RHM but, on the other hand, inhibits opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane. Our data provide better insight into the mechanisms of action of rare earth elements on Ca2+-dependent processes in the myocardium of vertebrates.

Identification and Species Delimitation of the Enigmatic Marsh Frog Pulchrana rawa (Matsui, Mumpuni, and Hamidy, 2012): Second Confirmed Specimen and First Country Record for Malaysia

Pulchrana rawa is a peat swamp specialist that was described from eastern Sumatra in 2012 based on a single specimen. Since then, no confirmed records have been forthcoming; thus, virtually nothing is known about this species. Recent fieldwork at peat swamp forests in southwestern Peninsular Malaysia led to the discovery of a frog that morphologically resembled P. rawa. Despite having relatively high mitochondrial divergence (6.6%) that was consistent with interspecific divergences of other closely related species, results from species delimitation analyses indicated that the Malaysian specimen was conspecific with P. rawa. In addition to extending the geographic distribution of this species to a different country, we also provide the first quantitative characterization of its call. This study provides novel insights into the distribution and natural history of this enigmatic species that will inevitably aid in threat assessments and conservation efforts.

Accumulation of Radionuclides in the Marsh Frog Pelophylax ridibundus in the Zone of a Nuclear Plant

Accumulation of Radionuclides in the Marsh Frog Pelophylax ridibundus in the Zone of a Nuclear Plant

Aematological and Genotoxicological Research on Pelophylax ridibundus and Bufotes variabilis Living Around the Çan (Çanakkale, Turkey)

Haematologic parameters play an important role in the determination of the general health status as well as the physiology of amphibian species and the effects of various stress and poor conditions on some species. With this study, changes in the hematological parameters (red blood cell count, white blood cell count, hemoglobin concentration, hematocrit value, mean cell volume, mean cell hemoglobin and mean cell hemoglobin concentration) on an aquatic (Pelophylax ridibundus) and a terrestrial (Bufotes variabilis) amphibian living around the Can (Canakkale, Turkey) were presented. Also, to reveal the DNA damage on these two amphibian species, various nuclear abnormalities like micronucleus, binucleated nucleus, lobbed nucleus, blebbed, and notched nucleus were analyzed. As a result of this study, the aquatic amphibian species when compared with the terrestrial amphibian species, significant differences in hemoglobin concentration (U= 22.5; W=88.5; Z= -3.446; p= 0.001), and heterophil count (U=34.00; W=205.00; Z= -3.79; p=0.00) were determined. The hemoglobin concentration of the terrestrial species was found higher while the heterophil count of the aquatic species was higher. Total nuclear abnormalities were found higher on the B. variabilis species but there was not a significant statistical difference. The micronucleus and other nuclear abnormalities of these two amphibian species when compared, the frequency of the notched nucleus was found statistically different. The frequency of the notched nucleus was higher in the B. variabilis species.

Histopathologic and genotoxic effects of deltamethrin on marsh frog, Pelophylax ridibundus (Anura: Ranidae)

It is known that amphibians inhabiting agricultural areas are constantly exposed to chemicals such as insecticides. Deltamethrin, a type II pyrethroid insecticide, is widely used in the world. The present study aimed to investigate the toxic effects of different concentrations (0.625, 1.25, and 2.50 mg/kg body wt) of orally applied deltamethrin to marsh frog (Pelophylax ridibundus) after 96 h of treatment. The histopathological abnormalities in liver, gastrointestinal tract, and kidney tissues were evaluated with quantitative analyses. In liver, increase of melanomacrophagic aggregates, non-homogeneous hepatocyte parenchyma, sinusoidal dilatations, infiltration, vascular epithelial degeneration, central vein degeneration, and congestion were determined in exposed frogs. In gastrointestinal tract, vacuolization, hypertrophy, congestion, infiltration, necrosis, and erosion of the epithelial layer, increasing goblet secretion, degeneration in villi, epithelial disorganization, and edema were observed in high-dose groups. In all experimental groups, glomerular shrinkage, hemorrhage, degeneration, infiltration, increase in Bowman distance, and eosinophilic-stained tubular lumens were detected in kidneys. Histopathological changes were more prominent in 1.25 and 2.50 mg/kg groups than the other groups. To determine the genotoxic effects of deltamethrin, the peripheral blood samples of the frogs were used. The erythrocytic nuclear abnormalities such as micro-nucleus, double-nucleus, kidney-shaped nucleus, notched nucleus, and bud nucleus were determined in the frogs, and the genotoxicity did not show a significant increase between control and low-dose groups, statistically. However, the genotoxic effects increased in medium- and especially high-dose groups. The results of this study showed that acute deltamethrin exposure can lead to histopathologic and genotoxic effects in P. ridibundus.

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

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.