Male-male amplexus between two amphibian families

The amplexus between two different anuran males is observed very rarely. Therefore, here we provide the first documented observation of a long-lasting male-male amplexus between Bombina bombina (Bombinatoridae) and Hyla arborea (Hylidae) together with an overview of the data published in literature. The observed mating pattern is reported from Southern Moravia, the Czech Republic. The possible reasons for its occurrence during the mass breeding season are discussed.

Hematological parameters of a Neotropical wild frog population, with a phylogenetic perspective on blood cell composition in Anura

Hematological parameters can provide key information to an animal health status. However, this information is usually hard to obtain. Here, we described hematological parameters of Leptodactylus podicipinus in the Brazilian Pantanal. We measured red blood cell morphometrics, erythrogram, and leukogram. We also tested for phylogenetic signal in erythrogram and leukogram of 48 frog species from 15 families, testing if body size explains their variation. Lymphocytes were the most abundant leukocytes (> 60%) in Leptodactylus podicipinus, followed by neutrophils (∼10%). Given that L. podicipinus is an abundant and widely distributed species in central Brazil, knowing its hematological pattern can help stablish a baseline and improve its use as a bioindicator of environmental degradation. Mean corpuscular hemoglobin and value contributed more to the phylomorphospace of erythrogram, in which Leptodactylus spp. and Hypsiboas raniceps had lower values of these variables, while Bufotes viridis and Hyla arborea had high values. The phylogenetic signal was spread throughout the dimensions of leukogram phylomorphospace. The variables that most contributed to it were total leukocytes counts, lymphocytes and neutrophils. We also found a moderate phylogenetic signal for both the erythrogram and leukogram. Accordingly, body size accounted for a low proportion of variation in both the leukogram (4.7%) and erythrogram (0.57%). By combining phylogenetic comparative methods with hematological parameters, our results add a new perspective on the evolution of blood cell physiology in frogs.

Construction of individual ddRAD libraries v2

This protocol describes a double digested restriction-site associated DNA (ddRADseq) procedure, that is a variation on the original RAD sequencing method (Davey & Blaxter 2011), which is used for de novo SNP discovery and genotyping. This protocol differs from the original ddRADseq protocol (Peterson et al 2012), in which the samples are pooled just after the ligation to adaptors (i.e. before size selection and PCR). The present ddRAD protocol as been slightly adapted from Alan Brelsford's protocol published in the supplementary material of this paper: Brelsford, A., Dufresnes, C. & Perrin, N. 2016. High-density sex-specific linkage maps of a European tree frog (Hyla arborea) identify the sex chromosome without information on offspring sex. Heredity 116, 177–181 (2016). https://doi.org/10.1038/hdy.2015.83 In the present protocol, all samples are treated separately, in a microplate, until final PCR amplification performed before pooling. Despite being slightly more costly and time-consuming in the lab, it allows for fine adjustement of each sample representation in the final library pool, ensuring similar number of sequencing reads per sample in the final dataset. Briefly, genomic DNA from the samples are individually digested with 2 restriction enzymes (one rare-cutter and one more frequent cutter) then ligated to a barcoded adaptor (among 24 available) at one side, and a single adaptor at the other side, purified with magnetic beads, and PCR-amplified allowing the addition of a Illumina index (among 12 available) for multiplexing a maximum of 288 sample per library. Samples are then pooled in equimolar conditions after visualisation on an agarose gel. Purification and size selection is then performed before final quality control of the library and sequencing.

Expanded host and geographic range of tadpole associations with the Severe Perkinsea Infection group

Severe Perkinsea infection is an emerging disease of amphibians, specifically tadpoles. Disease presentation correlates with liver infections of a subclade of Perkinsea (Alveolata) protists, named Pathogenic Perkinsea Clade (PPC). Tadpole mortality events associated with PPC infections have been reported across North America, from Alaska to Florida. Here, we investigate the geographic and host range of PPC associations in seemingly healthy tadpoles sampled from Panama, a biogeographic provenance critically affected by amphibian decline. To complement this work, we also investigate a mortality event among Hyla arborea tadpoles in captive-bred UK specimens. PPC SSU rDNA was detected in 10 of 81 Panama tadpoles tested, and H. arborea tadpoles from the UK. Phylogenies of the Perkinsea SSU rDNA sequences demonstrate they are highly similar to PPC sequences sampled from mortality events in the USA, and phylogenetic analysis of tadpole mitochondrial SSU rDNA demonstrates, for the first time, PPC associations in diverse hylids. These data provide further understanding of the biogeography and host range of this putative pathogenic group, factors likely to be important for conservation planning.

Interspecific sensitivity of European amphibians towards two pesticides and comparison to standard test species

Background Although debates about the assessment of potential effects of pesticides on amphibians are ongoing, amphibians are not yet considered in the current EU environmental risk assessment of pesticides. Instead, the risk assessment of potential effects on aquatic amphibian life stages relies on use of data of surrogate species like the standard temperate fish species rainbow trout (Oncorhynchus mykiss). This assumption is mainly based on the comparison to amphibian species not native to Europe such as the aquatic African clawed frog (Xenopus laevis). It remains unclear whether these surrogate species cover semi-aquatic Central European amphibian sensitivities. Therefore, we assessed the acute sensitivity of aquatic stages of eight European amphibian species native in Germany (Bufo bufo, Bufotes viridis, Epidalea calamita, Hyla arborea, Pelobates fuscus, Pelophylax sp., Rana dalmatina, R. temporaria) towards commercial formulations of the fungicide folpet (Folpan® 500 SC, Adama) and the insecticide indoxacarb (Avaunt® EC, Cheminova). The determined acute sensitivities (median lethal concentration, LC50) were included in species sensitivity distributions and compared to experimentally determined LC50 values of X. laevis and literature values of O. mykiss. Results The results showed that native amphibian sensitivities differed between the tested pesticides with a factor of 5 and 11. Depending on the pesticide, X. laevis was five and nine times more tolerant than the most sensitive native amphibian species. Comparing literature values of O. mykiss to the experimentally determined sensitivities of the native amphibian species showed that the O. mykiss sensitivity was in the same range as for the tested amphibians for the formulation Folpan® 500 SC. The comparison of sensitivities towards the formulation Avaunt® EC showed an eight times lower sensitivity of O. mykiss than the most sensitive amphibian species. Conclusions A risk assessment using the 96-h LC50 values for fish covers the risk for the assessed aquatic stages of European amphibians after the application of the recommended uncertainty factor of 100 and thus may be adequate for lower tier risk assessment of the studied pesticides. If aquatic amphibian testing will be required for pesticide risk assessment nevertheless, acute tests with the model organism X. laevis and the application of an appropriate uncertainty factor might be a promising approach.

Amphibians in an urban environment: a case study from a central European city (Wrocław, 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.

Herpetofauna of the Kozubów Landscape Park

The research was conducted in 2016–2017 in the 6,613-ha Kozubów Landscape Park (KLP). It focused on the occurrence and distribution of amphibians and reptiles, the biology and phenology of selected species and the existing threats. The KLP, established in 1986, is located in the Nida Basin, covering the eastern part of the Wodzisławski Ridge. This area is characterised by diverse landscape features formed on a chalk substrate. A significant part of the KLP (48.8%) is covered by forests growing on the tops and slopes of loess hills intersected by valleys and gorges. Hornbeam forests and fragments of riparian forests have retained a semi-natural character. The steep, non-forested slopes with a southern and south-western exposure are overgrown by thermophilic xerothermic grasslands. Xerothermic plant communities show a relatively low species diversity, because the main type of substrate in this area is loess. Two nature reserves, Polana Polichno and Wroni Dół, are located within the KLP borders. The following amphibian species were found in the KLP: smooth newt (Lissotriton vulgaris L.), great crested newt (Triturus cristatus Laur.), European fire-bellied toad (Bombina bombina L.), common spadefoot toad (Pelobates fuscus Laur.), common toad (Bufo bufo L.), European green toad (Bufotes viridis Laur.), European tree frog (Hyla arborea L.), edible frog (Pelophylax esculentus L.), pool frog (Pelophylax lessonae Cam.), moor frog (Rana arvalis Nilss.) and common frog (Rana temporaria L.). Reptiles are represented by the sand lizard (Lacerta agilis L.), viviparous lizard (Zootoca vivipara Jacquin), slow worm (Anguis fragilis L.), grass snake (Natrix natrix L.), smooth snake (Coronella austriaca Laur.) and common European adder (Vipera berus L.). The study also included the phenology and reproductive biology of common toad and common frog. Major threats to the herpetofauna are posed by grass burning. Unfavourable changes include decreasing water levels and drying out of water bodies.