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

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

Maintenance of pure hybridogenetic water frog populations: Genotypic variability in progeny of diploid and triploid parents.

An intriguing outcome of hybridisation is the emergence of clonally and hemiclonally reproducing hybrids, that can sustain, reproduce, and lead to the emergence of polyploid forms. However, the maintenance of diploid and polyploid hybrid complexes in natural populations remains unresolved. We selected water frogs from the Pelophylax esculentus complex to study how diploid and triploid hybrids, which reproduce hemiclonally via hybridogenesis, are maintained in natural populations. During gametogenesis in diploid hybrids, one of the parental genomes is eliminated, and the remaining genome is endoreplicated. In triploid hybrids, the single-copy genome is typically eliminated, while genome endoreplication does not occur. To investigate how diploid and triploid hybrid frogs reproduce in populations without parental species, we crossed these hybrid animals from two separate pure hybrid populations located in Poland. Using cytogenetic analysis of tadpoles that emerged from the crosses, we established which gametes were produced by parental hybrids. The majority of hybrid females and hybrid males produced one type of gamete with the P. ridibundus genome. However, in both studied populations, approximately half of the diploid and triploid hybrids simultaneously produced gametes with different genome compositions and ploidy levels, specifically, the P. ridibundus and P. lessonae genomes, as well as diploid gametes with genomes of both parental species. Triploid hybrid males and females mostly produced haploid gametes with the P. lessonae genome; however, gametes with the P. ridibundus genome have also been observed. These results suggest that not all hybrids follow the classical hybridogenetic reproduction program and reveal a significant level of alterations in the gametogenesis pathways. In addition, we found a variable survival rate of particular progeny genotypes when we crossed hybrid females with different males suggesting the important role of postzygotic barriers on the maintenance of pure hybrid systems. We suggest that the observed variability in produced gametes and the different survival rate of the progeny with certain genotypes is crucial for the existence of pure hybrid systems.

Bottled & canned - Anthropogenic debris as an understudied ecological trap for small animals.

Nowadays, littering is one of the biggest challenges that environmental conservation is facing. Although beverage containers, such as bottles and cans, belong to the most common threats in this context, their effect on animals has been poorly studied. The aim of this study was to assess the diversity and mortality level of the animal taxa entering discarded containers and to investigate which container features influence the number and functional composition of the trapped animals. The study was conducted in 10 urban woodlands in the city of Wroclaw, Poland. In total, 939 open containers were collected. In 56% of them, a total number of 10,162 dead individuals (10,139 invertebrates and 23 vertebrates) was found. The most common amongst them were insects (orders: Coleoptera, Diptera, Hymenoptera), malacostracans (Isopoda), arachnids (Opiliones, Sarcoptiformes) and gastropods (Stylommatophora). The number of dead animals was affected positively by the container capacity and was significantly higher in glass and plastic bottles when compared to aluminium cans. At the same time, the presence of a neck negatively affected the number of dead animals. Container capacity was also positively correlated with the abundance of the most common functional groups: predators, phytophages and saprophages. Moreover, colourless and green, but not brown, containers were a significant predictor for the abundance of the latter two groups. Our study revealed that discarded containers constitute an ecological trap for many groups of animals. There is an urgent need to reduce the amount of rubbish in the environment by, for example, the implementation of regional and international regulations addressing the problem of littering, or organising repeated clean-up and educational activities.

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

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

Digit ratio in the common toad Bufo bufo: the effects of reduced fingers and of age dependency.

INTRODUCTION: Despite the growing number of studies describing digit ratio patterns in tetrapods, knowledge concerning certain basic issues is still scarce. In lower vertebrates such as tailless amphibians (Anura), the numbering of individual fingers on the forelimbs and their homology with the fingers of other vertebrates pose an unsolved problem. Based on reviewed data on anuran limb development, we argue that the correct finger numbering scheme should be based on the assumption that the first finger, not the fifth finger, was reduced on the forelimbs. We analyzed the digit ratio in the common toad (Bufo bufo, Bufonidae), a species characterized by well-developed sexual dimorphism whereby females are larger than males, using both numbering schemes present in the literature. RESULTS: We found that the digit ratio on hindlimbs differed significantly between the sexes only in the cases of left 2D:3D, with lower digit ratios in females, and of left 3D:4D, with lower digit ratios in males. We found that sex was the only significant variable for forelimbs, differentiating 2D:3D on the left forelimb, with lower digit ratios in females; 2D:4D on the right forelimb, with lower digit ratios in males; and 3D:4D on both forelimbs, with lower digit ratios in males. These results relate to variant II reflecting the hypothesis that the first digit was reduced during phylogenesis. There was no relationship between the body size (SVL) of individuals and any digit ratio, excluding 2D:4D on the right forelimbs in models with age variables. Additionally, for a subset of data where individual age was known, the models indicated that age was linked to significant differences in 2D:4D and 3D:4D on the left hindlimbs, while age, SVL, and sex influenced 2D:4D on the right forelimbs. CONCLUSION: We emphasize the importance of the problem of the correct numbering of forelimb digits in Anura and, under the assumption that it was the fifth digit that was reduced, argue that earlier results on digit ratio in this group should be interpreted with caution. The detected relationship between digit ratio and age in amphibians expands our knowledge, indicating that the age of individuals should be included in future digit ratio studies. This relationship may also apply to studies using digit ratio as a noninvasive indicator of endocrine disruption in amphibians.

Online media reveals a global problem of discarded containers as deadly traps for animals.

The widespread occurrence of litter is a severe threat to global ecosystems. We have analyzed online media, to assess the diversity of animals that are prone to getting trapped in discarded containers and check which kind of containers is the most common trap for animals. A total of 503 records from around the world (51 countries, 6 continents) have been found. These include invertebrates (17 taxa, ca.1050 dead individuals), and vertebrates (98 taxa, 496 individuals including 44 carcasses). The latter group was most frequently represented by mammals (78.5% of all cases), then reptiles (15.3%), birds (1.2%), fish (1.0%) and amphibians (0.4%). Nearly 12.5% of the determined vertebrates are classified as vulnerable, endangered or critically endangered, according to the IUCN. Although most trapped individuals were smaller animals, bigger ones such as monitor lizards (Varanus spp.) or large carnivores were also recorded. In most cases, animals were trapped in glass or plastic jars (32.4%), drink cans (16.5%), and steel cans (16.3%). Our results demonstrate that discarded containers can be a threat to all major groups of animals. In order to address this phenomenon, it is necessary to decrease a global production of debris, implement container deposit legislation and organize repeatable cleanup actions.

Microplastic ingestion by tadpoles of pond-breeding amphibians-first results from Central Europe (SW Poland).

Microplastics (MPs) are one of the major threats to aquatic ecosystems. Surprisingly, our knowledge of its occurrence and its impact on the organisms that dwell in small water bodies is still scarce. The aim of this study was to investigate the occurrence and chemical composition of MPs in tadpoles of pond-breeding amphibians. In total, 201 tadpoles belonging to 5 species were collected from 8 ponds located in southwestern Poland. MPs were found in all examined sites and in all studied species. Among those tested, 53 (26%) tadpoles ingested a total of 71 MPs. IR-ATR analysis revealed that particles were of anthropogenic origin and included nylon, polyurethane, polyisoprene and 1,2 polybutadiene.

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates).

Cryptic phylogeographic diversifications provide unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecological and behavioural differentiation. Yet, to date very few comparative studies have been attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (restriction site associated DNA sequencing and mitochondrial DNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species-level cryptic divergences (>5 million years ago [My]) between populations distributed in the Near-East (hereafter P. syriacus sensu stricto [s.s.]) and southeastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10 My), parapatry with highly restricted hybridization (P. balcanicus and P. syriacus s.s., >5 My), narrow hybrid zones (~15 km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3 My), to extensive admixture between Pleistocene and refugial lineages (≤2 My). This full spectrum empirically supports a gradual build up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3 My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.

Recent Findings of Potentially Lethal Salamander Fungus Batrachochytrium salamandrivorans.

The distribution of the chytrid fungus Batrachochytrium salamandrivorans continues to expand in Europe. During 2014-2018, we collected 1,135 samples from salamanders and newts in 6 countries in Europe. We identified 5 cases of B. salamandrivorans in a wild population in Spain but none in central Europe or the Balkan Peninsula.

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.

Common occurrence of Sharpey's fibres in amphibian phalanges.

Sharpey's fibres are known mainly as providing anchorage between tooth and the periodontal ligament but they occur also in other types of bones. In the postcranial skeleton these fibres are usually present at the muscle or tendon attachment sites. They were reported in all major groups of extant vertebrates, as well as in putative lissamphibian ancestors-temnospondyls and lepospondyls. However, it was recently stated that their presence was very rarely described in extant amphibians. In limbs, they were reported predominantly from proximal bones. They have not yet been reported from phalanges, which are the most commonly sectioned amphibian bones. Here, we describe phalangeal histology of nine species representing most major clades of lissamphibians. These results show that Sharpey's fibres occur commonly in lissamphibian phalanges. In shaft, they are radially oriented and occur in the periosteal bone, at sites of tendon attachment. They can also occur in the metaphysis and contact the cartilage. This may provide a basis for foot muscle reconstructions in fossil amphibians.

Batrachochytrium dendrobatidis is present in Poland and associated with reduced fitness in wild populations of Pelophylax lessonae.

The fungus Batrachochytrium dendrobatidis (Bd) is a pathogen associated with global declines of amphibians. We used qPCR to detect Bd in 255 samples from 10 Polish populations of 8 species. We found Bd infection in 3 species (Bombina variegata, Pelophylax lessonae, P. esculentus). The infection intensity in P. lessonae reached a maximum of 58400 genomic equivalents of zoospores (GE), and the 2 most heavily infected individuals died. Previous observations of the populations that included infected individuals showed reduced body size, failure to reproduce, and mortalities of adults. These data highlight the importance of emerging diseases, and the need to recognize them as an important factor in conservation of the genus Pelophylax in Poland and Central and Eastern Europe.

Phalangeal bone anomalies in the European common toad Bufo bufo from polluted environments.

Every spring, many of amphibians are killed by motor vehicles on roads. These road-killed animals can be used as valuable material for non-invasive studies showing the effect of environmental pollution on amphibian populations. The aims of our research were to check whether the phalanges of road-killed toads may be useful as material for histological analysis, and whether various degrees of human impact influence the level in bone abnormalities in the common toad. We also examined whether the sex and age structure of toads can differ significantly depending in the different sites. We chose three toad breeding sites where road-killed individuals had been observed: near the centre of a city, the outskirts of a city, and a rural site. We collected dead individuals during spring migration in 2013. The sex of each individual was determined and the toes were used to determine age using the skeletochronology method. While performing age estimates, we looked for abnormalities in relation to normal bone tissue structure. In urban site, females dominate males (sex ratio 2.6:1), but in populations from rural and semi-urban sites, sex ratio was reverse (1:2.2 and 1:1.4, respectively). However, we did not find any significant differences between age structure of all populations (average age of each population: approximately 4 years). We observed abnormalities in more than 80 % of all toads from the city, compared to approximately 20 % from the rural and semi-urban sites. In particular, we found hypertrophic bone cells, misaligned intercellular substance, and irregular outer edges of bones. We suggest that these malformations are caused by different pollution, e.g. with heavy metals.