European Tree Frog Hyla Arborea Research Articles

The European Tree Frog Hyla arborea as a Probable Reason for Nest Failure in the Eurasian Reed Warbler Acrocephalus scirpaceus

We describe a case in which occupation by the European Tree Frog Hyla arborea caused the nest failure of a pair of Eurasian Reed Warblers Acrocephalus scirpaceus. The frog was observed several times sitting on the reed warblers clutch, on the day when the third egg was laid, thereby preventing the parental birds from incubating. Although small frog species have been reported to rest during the day in bird nests (mainly empty ones), to our knowledge this is the first published report providing evidence for avian nest failure due to nest occupation by an amphibian species.

Arboreal foraging and ambush by grass snakes Natrix natrix on European treefrogs Hyla arborea

Arboreal foraging and ambush by grass snakes Natrix natrix on European treefrogs Hyla arborea

Herpetofauna of the Pieprzowe Mountains Nature Reserve and adjacent areas

Abstract The study was focused on determining the sites of amphibian and reptile occurrence in the Pieprzowe Mountains Nature Reserve (area 18.01 ha) and adjacent areas (total area 58.81 ha). The investigations also involved the biology of breeding in the selected species, as well as determining threats and protection measures. Situated in the eastern part of the Świętokrzyskie Voivodeship, the steppe-like Pieprzowe (Pepper) Mountains Nature Reserve was established in 1979. Its aim is to protect xerothermic assemblages inhabited by many species of rare plants including the largest European aggregation of roses growing wild. The area is located on a steep, sometimes rugged escarpment, which is part of the Vistula River erosional margin. The escarpment exposes Cambrian black pepper-like shales. The slope base is overgrown by a narrow belt of willow thickets. Above occur assemblages of xerothermic thickets with a few trees. The highest parts of the nature reserve are overgrown with xerothermic grasslands with the prevalence of feathergrass steppe (Festuco-Stipion class). The reserve is surrounded by riparian forests, waterlogged meadows and reservoirs of the Vistula former riverbed, the largest of which is an oxbow lake (5.16 ha). The following species were documented in 2016-2017: smooth newt Lissotriton vulgaris L., great crested newt Triturus cristatus Laur., European fire-bellied toad Bombina bombina L., 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., marsh frog Pelophylax ridibundus Laur., moor frog Rana arvalis Nilss., common frog Rana temporaria L., sand lizard Lacerta agilis L., grass snake Natrix natrix L. and common European adder Vipera berus L. The studies were focused on biology of breeding and phenology in common toad and common frog. The main threats posed on herpetofauna include: human presence, littering, fire raising and changes of water balance. © IOŚ-PIB

Herpetofauna of the Podkielecki Landscape Protection Area

Abstract The study was conducted in 2016-2017 in the Podkielecki Landscape Protection Area (area 26,485 ha). It was focused on the occurrence and distribution of amphibians and reptiles, the biology of the selected species and the existing threats. Established in 1995, the Podkielecki Landscape Protection Area surrounds the city of Kielce from the north, east and south-east, and adjoins several other protected areas. It covers the western part of the Świętokrzyskie Mountains (part of the Klonowskie and Masłowskie ranges) and the southern part of the Suchedniów Plateau. The studied area is mostly covered by forest and thicket communities (48.1%) and farmlands (39.9%), followed by built-up areas (7.8%), industrial areas (0.5%), roads and railways (2.7%), and surface water bodies (1%). The protected area is developed mainly on Palaeozoic rocks, including Cambrian and Ordovician sandstones, Silurian and Carboniferous shales, and Devonian marls. Podzolic soils predominate among soils. The largest rivers include Lubrzanka, Czarna Nida, Bobrza and Belnianka. There are no natural lakes within the PLPA limits, and the largest artificial reservoirs include the Cedzyna Reservoir, Morawica Reservoir, Suków Sandpit and two sedimentation reservoirs of the Kielce Power Plant. The area includes 2 nature reserves: Barcza and Sufraganiec. The following amphibian species were recognised during the investigations within the borders of the studied area: alpine newt Ichthyosaura alpestris Laur., great crested newt Triturus cristatus Laur., smooth newt Lissotriton vulgaris L., European fire-bellied toad Bombina bombina L., common spadefoot toad Pelobates fuscus Laur., common toad Bufo bufo L., natterjack toad Epidalea calamita Laur., European green toad Bufotes viridis Laur., European tree frog Hyla arborea L., pool frog Pelophylax lessonae Cam., edible frog Pelophylax esculentus L., marsh frog Pelophylax ridibundus Pall., moor frog Rana arvalis Nilss., and common frog Rana temporaria L. The reptiles were represented by sand lizard Lacerta agilis L., viviparous lizard Zootoca vivipara Jacquin, slow worm Anguis fragilis L., grass snake Natrix natrix L. and common European adder Vipera berus L. The study also included the phenology and breeding biology of the common toad and common frog. The most crucial herpetofauna conservation problems identified here include amphibians killed on roads by vehicles. The study area is intersected by very busy roads, in particular: European route no. E77, national roads nos. 73, 74 and S74, and regional roads nos. 745, 750 and 764. For this reason, future road reconstruction projects should consider the assembly of various crossing roads for wildlife, particularly on the 600 m long section of national road no. 74 near Cedzyna Reservoir. Other threats include illegal waste dumping, pollution of surface waters, fire setting, overgrowing and desiccation of small water bodies. © IOŚ-PIB

Immune challenges and visual signalling in tree frogs.

In animals, mate-choice is often based on sexual signals that carry information and help the receiver make the best choice to improve the receiver's fitness. Orange visual sexual signals have been hypothesised to carry immune information because they are often due to carotenoid pigments which are also involved in immunity response. Although many studies have focused on the direct relationships between coloration and immunocompetence, few studies have simultaneously studied immunocompetent response and coloration variation after an immune challenge. We tested this hypothesis on starved and ad libitum-fed males of the European tree frog Hyla arborea. Our results show that male coloration is not a reliable indicator of its immune response capacity in this species. However, after an immune challenge induced by a PHA (Phaseolus vulgaris phytohaemagglutinin) injection, starved males presented a significant coloration loss and this alteration was related to the immune response intensity. Taken together, these results suggest that the brighter (lighter) coloration may be used as a cue by female to exclude males with a recent immune challenge, due to diseases or parasites for example.

High-density sex-specific linkage maps of a European tree frog (Hyla arborea) identify the sex chromosome without information on offspring sex.

Identifying homology between sex chromosomes of different species is essential to understanding the evolution of sex determination. Here, we show that the identity of a homomorphic sex chromosome pair can be established using a linkage map, without information on offspring sex. By comparing sex-specific maps of the European tree frog Hyla arborea, we find that the sex chromosome (linkage group 1) shows a threefold difference in marker number between the male and female maps. In contrast, the number of markers on each autosome is similar between the two maps. We also find strongly conserved synteny between H. arborea and Xenopus tropicalis across 200 million years of evolution, suggesting that the rate of chromosomal rearrangement in anurans is low. Finally, we show that recombination in males is greatly reduced at the centers of large chromosomes, consistent with previous cytogenetic findings. Our research shows the importance of high-density linkage maps for studies of recombination, chromosomal rearrangement and the genetic architecture of ecologically or economically important traits.

A phytohaemagglutinin challenge test to assess immune responsiveness of European tree frog Hyla arborea

Immune responsiveness, one measure of individual quality, can be used as a sensitive, non-lethal variable that may be negatively affected in animals exposed to degraded, contaminated or otherwise disturbed areas. One frequently used technique to measure immune responsiveness is the phytohaemagglutinin (PHA) challenge test. Swelling occurring at the injection sites are measured before and 24 h after PHA injection. The immune response is considered to be the difference between the two measures. Although this method is easily performed with wild animals, it has been rarely used on small amphibians. Here, we test the possibility of using a PHA test with the European tree frog, Hyla arborea, and we identify the optimal procedure for measuring immune responsiveness in this species. The results allowed us to simplify the procedure in eliminating phosphate-buffered saline (PBS) injection and reducing the duration of the experiment. Injection of PHA into the leg of H. arborea triggered an immune response with a peak of swelling 14 h after injection. A second injection of PHA into the same animal induced more intense leg swelling. In addition, haematological responses showed that the total number of leucocytes increased after PHA injection. A link between the leg swelling and the total leucocytes count recorded in blood has been found. Consequently, this method may provide a useful tool for predicting the pro-inflammatory capacity of field populations of small amphibians.

Immunocompetence handicap hypothesis in tree frog: trade-off between sexual signals and immunity?

The immunocompetence handicap hypothesis (ICHH) assumes that testosterone plays a dual role in mediating both sexual signal expression and immunosuppression. Although there is good evidence of the ICHH in birds, it was primarily tested using only one sexual signal approach. By focusing on the European tree frog Hyla arborea, a species for which mate choice is based on both acoustic and visual signals, we test for the first time the ICHH in a multimodal communication system. The ICHH may be viewed as a universal way to explain signal honesty no matter the sensory modality used by animals. After studying the effects of testosterone on the acous- tic and visual sexual signals through a daily testosterone supplementation, the effects of androgen on the immune system response were measured through a phytohaemagglutinin injection. In H. arborea, we demonstrate that both the acoustic and visual sexual sig- nals were testosterone dependent. However, the phytohaemagglutinin injection did not provoke immunosuppression for testosterone- supplemented males. Surprisingly, the testosterone-supplemented males with a high body mass presented an immunoenhancement. Therefore, it seems that ICHH is not an integrating mechanism that is able to explain signal honesty in the multimodal signaling system used by H. arborea in mate choice. On the contrary, our results support the idea that multiple mechanisms (e.g., cost-added and index signals) operating simultaneously may explain signal honesty of both acoustic and visual signals.

Population genetics in a fragmented population of the European tree frog (Hyla arborea)

Habitat degradation and fragmentation are known to be major threats for population persistence in European amphibians. The European tree frog Hyla arborea has suffered from dramatic population declines in the last decades and has therefore been categorised as threatened in many Red Data lists. In the region of Hannover (Germany), the European tree frog has a fragmented distribution. The aim of our study was to infer the genetic consequences of habitat fragmentation in this area by examining genetic variation and population structure. DNA samples from 193 individuals from 11 sampling sites (10 sampling sites located 2 to 32 km apart from each other near Hannover and for comparison one sampling site 140 km northeast) were analysed with eight highly polymorphic microsatellite loci. Bayesian analyses indicated that the tree frog occurrences near Hannover were fragmented into four genetically distinct clusters according to their geographical distribution. Pairwise genetic distances between sampling sites varied between 0 and 0.23 (FST) and 0 and 0.48 (Dest) and indicated high to moderate gene flow within genetic clusters and nearly absent gene flow among genetic clusters. Moreover, we identified a potential source population within the region for an introduced population in the southwest of Hannover. Our data suggest that the genetic structure is influenced in part by isolation by distance and in part by lack of habitat or migration barriers. Habitat fragmentation should by counteracted by targeted conservation measures in areas where gaps in distribution and genetic fragmentation have been revealed.

Within- and among-population impact of genetic erosion on adult fitness-related traits in the European tree frog Hyla arborea

Assessing in wild populations how fitness is impacted by inbreeding and genetic drift is a major goal for conservation biology. An approach to measure the detrimental effects of inbreeding on fitness is to estimate correlations between molecular variation and phenotypic performances within and among populations. Our study investigated the effect of individual multilocus heterozygosity on body size, body condition and reproductive investment of males (that is, chorus attendance) and females (that is, clutch mass and egg size) in both small fragmented and large non-fragmented populations of European tree frog (Hyla arborea). Because adult size and/or condition and reproductive investment are usually related, genetic erosion may have detrimental effects directly on reproductive investment, and also on individual body size and condition that in turn may affect reproductive investment. We confirmed that the reproductive investment was highly size-dependent for both sexes. Larger females invested more in offspring production, and larger males attended the chorus in the pond more often. Our results did not provide evidence for a decline in body size, condition and reproductive effort with decreased multilocus heterozygosity both within and among populations. We showed that the lack of heterozygosity-fitness correlations within populations probably resulted from low inbreeding levels (inferior to ca. 20% full-sib mating rate), even in the small fragmented populations. The detrimental effects of fixation load were either low in adults or hidden by environmental variation among populations. These findings will be useful to design specific management actions to improve population persistence.

Where movement happens: scale-dependent landscape effects on genetic differentiation in the European tree frog

Functional connectivity among fragmented populations depends on the landscape matrix between occupied habitat patches and its effect on the frequency of animal movement and gene flow. The quantification of landscape effects on gene flow should therefore be scale-dependent. Here, we explored the impact of different spatial scales in a landscape genetic analysis of the European tree frog Hyla arborea in a fragmented landscape in Switzerland. We examined the effects of landscape elements and geographic distance on genetic differentiation at three distance classes reflecting varying frequencies of tree frog movement. We calculated pairwise FST-values and assembled 16 landscape elements within 1 km wide corridors between all pairs of tree frog breeding sites. Per distance class, we computed a multiple regression model with stepwise backward elimination and permutation testing. At distances of 2 km, geographic distance had a negative effect on gene flow as had landscape elements such as forests and roads. In general, hedgerows and various structure-rich landscape elements positively affected gene flow. As we found distinct scale-dependent landscape effects on gene flow, future landscape genetic studies should analyse the effects of landscape variables at different spatial dimensions relevant for the movement and dispersal of the study organisms. Corresponding studies should also carefully consider relevant correlations among the landscape elements tested and should preferentially replicate their analysis at the landscape-level in order to avoid idiosyncratic results owing to the particular scale and landscape studied.

N-Terminal Tagging Strategy for De Novo Sequencing of Short Peptides by ESI-MS/MS and MALDI-MS/MS

The major portion of skin secretory peptidome of the European Tree frog Hyla arborea consists of short peptides from tryptophyllin family. It is known that b-ions of these peptides undergo head-to-tail cyclization, forming a ring that can open, resulting in several linear forms. As a result, the spectrum contains multiple ion series, thus complicating de novo sequencing. This was observed in the Q-TOF spectrum of one of the tryptophyllins isolated from Hyla arborea; the sequence FLPFFP-NH(2) was established by Edman degradation and counter-synthesis. Though no rearrangements were observed in FTICR-MS and MALDI-TOF/TOF spectra, both of them were not suitable for mass-spectrometry sequencing due to the low sequence coverage. To obtain full amino acid sequence by mass spectrometry, three chemical modifications to N-terminal amino moiety were applied. They include acetylation and sulfobenzoylation of N-amino group and its transformation to 2,4,6-trimethylpyridinium by interaction with 2,4,6-trimethylpyrillium tetrafluoroborate. All three reagents block scrambling and provide spectra better than the intact peptide. Unfortunately, all of them also readily react with lysine side chain. Hence, all investigated procedures can be used to improve sequencing of short peptides, while acetylation is the recommended one. It shows excellent results, and it is plain and simple to perform. This is the procedure of choice for MS-sequencing of short peptides by manual or automatic algorithms.

Nuclear and Mitochondrial Patterns of Introgression between the Parapatric European TreefrogsHyla arboreaandH. intermedia

Postglacial northward expansions from distinct southern European refugia have lead to the formation of several secondary contact zones between species. These zones are providing growing insights into the relevance of introgressive hybridization in shaping patterns of genetic diversity, and into the evolutionary processes involved in the completion of reproductive isolation barriers. Using both nuclear (9 allozyme loci) and mitochondrial (PCR-RFLP of a cytochrome b gene fragment) diagnostic markers we investigated the genetic patterns of variation across a zone of parapatry between the two European treefrogs Hyla arborea and H. intermedia. Neither F1 and F2 hybrids nor backcrosses were identified, indicating the lack of current gene exchange between the two species. However, introgressed alleles were observed in both species and in all markers analysed, which testifies to the occurrence of past events of introgressive hybridization. A wide variation in the frequencies of introgressed alleles was observed b...

Conspicuous colouration of the vocal sac of a nocturnal chorusing treefrog: carotenoid-based?

AbstractIn nocturnal chorusing anurans, mate choice has long been considered to be based exclusively on the highly conspicuous male calls. However an additional visual cue, the pulsing male vocal sac, has recently been shown to increase male attractiveness. In several species, including the European treefrog Hyla arborea, this organ is characterized by a bright orange colouration. In the present study, analysis of the pigments extracted from the vocal sacs of European treefrogs revealed the colouration of these organs to be based on several pigments, including three carotenoids, which are highly costly immune function enhancing molecules. This result raises the important question as to whether the carotenoid-based colouration of the vocal sac in H. arborea could constitute an additional visual cue used by females to discriminate male quality.

Supplementary feeding affects the breeding behaviour of male European treefrogs (Hyla arborea)

BackgroundWe investigated the effects of energetic constraints on the breeding behaviour of male European treefrogs Hyla arborea and how calling males allocated additional energy supplied by feeding experiments.ResultsPresence in the chorus was energetically costly indicated by both fed and unfed males losing weight. Males that were supplied with additional energy did not show longer chorus tenure. Instead, fed males returned sooner to the chorus. Additionally, fed males called more often than control males, a novel response for anurans. A significantly higher calling rate was noted from males even 31 nights after supplementary feeding.ConclusionThis strategy of allocating additional energy reserves to increasing calling rate is beneficial given the preference of female hylids for males calling at high rates and a female's ability to detect small incremental increases in calling rate.

Chorus tenure and estimates of population size of male European tree frogs Hyla arborea: implications for conservation

AbstractChorusing male anurans typically spend only a part of the breeding season calling although chorus tenure is often the best predictor of mating success. We determined the number of nights males attended a chorus (chorus tenure) and its influence on mating success in the European tree frog, Hyla arborea. The median chorus tenure was 7.5 nights out of a study season of 38 nights. Males that spent more than two nights in the chorus were present for an average of 47% of the nights between their first and last night in the chorus. Minimum daily temperature, ambient temperature at initiation of calling, and daily rainfall explained 37.8% of the variance in male attendance. Twenty-five males were calling on the night of peak activity, a fraction of the 44 males marked. This suggests that estimates of male population size based on peak activity, widely used by conservation biologists, are inaccurate. We suggest that, when mark-recapture methods cannot be used, male population size be calculated by using a regression model based on the peak number of calling males that can be further developed as more data accumulates.

Genetic diversity and fitness in peripheral and central populations of the European tree frog Hyla arborea.

Genetic diversity is expected to decrease in small and isolated populations as a consequence of founder effects, bottlenecks, inbreeding and genetic drift. In this study we analyse temporal and spatial effects on genetic variation and progeny viability of the European tree frog (Hyla arborea) at two scales. First, the Swedish distribution has been isolated from the continental distribution for more than 8000 thousand years, and secondly, within Sweden, recent habitat alterations that have taken place during this century have increased isolation between local populations. Genetic variation and progeny survival in relation to isolation was studied within the entire Swedish distribution of the tree frog. Allozyme electrophoresis analysis of froglets, sampled across the Swedish distribution, revealed a low overall genetic variation (1.06 alleles/locus) at the protein level in comparison with continental populations (1.54-1.68 alleles/locus). However, egg hatchability (97%) and early larval survival (95%) were not lower than in other parts of the tree frog distribution or in other anuran species. Within the Swedish distribution, early larval survival was lower in isolated breeding ponds than in more central ones. However, no differences in genetic variation were found in relation to isolation. Polymorphism was detected only at a single locus, and was restricted geographically to the eastern part of the Swedish distribution. Bottlenecks due to climatic changes and fragmentation of suitable habitat (primarily natural pastures with ponds) are suggested as possible causes of the low genetic diversity of the Swedish tree frog population.

Inguinal Bodies of Some Bufo Species

Previously undescribed large elongated bodies over the muscles of the groin region (inguinal bodies) have been found in some representatives of the genus Bufo but not in specimens of the genera Rana, Bombina, and Hyla. Anurans were captured in the field in several localities in southern Poland. On the same day they were killed and examined. Three species of the genus Bufo live in Poland: the common toad, Bufo bufo, the green toad, Bufo viridis, and the relatively rare natterjack, Bufo calamita. In May, the following specimens of the genus Bufo were examined: 7 adults (3 females and 4 males) of B. viridis; 1 adult female and 1 adult male of B. calamita; 8 adults (5 females and 3 males) of B. bufo. In July-August the following specimens of this genus were investigated: 13 adults (7 females and 6 males) and 7 juveniles of B. viridis; 2 adult males of B. calamita; 10 adults (6 females and 4 males) and 3 juveniles of B. bufo. In August, the representatives of both sexes of other anurans living in Poland were also examined, namely the European common frogsRana temporaria (7 adults and 10 juveniles); the frogsRana lessonae (4 adults and 6 juveniles); the yellowbellied toads-Bombina variegata (15 adults and 20 juveniles); the fire-bellied toads-Bombina bombina (5 adults and 7 juveniles); the European tree frogs-Hyla arborea (4 adults and 7 juveniles). In each individual investigated the pelvic part of the abdominal skin and the proximal part of the femoral skin were removed and the groin regions were macroscopically observed. A pair of large elongated bodies (inguinal bodies) supplied by pelvic arteries and veins were visible macroscopically in the groins of each investigated individual of B. viridis (Fig. 1A) and B. calamita. In B. bufo such distinctive structures were never observed (Fig. 1B), however, in a few individuals small accumulations of fat were present in either one or both groins along the pelvic vessels. Similar structures were absent in all investigated representatives of the genera Rana, Bombina, and Hyla. Inguinal bodies of B. viridis and B. calamita, as well as fat accumulations of specimens of B. bufo were easily excised, fixed in Bouin's fluid, routinely embedded in paraffin, serially sectioned, and stained with haematoxylin-eosin or May-Griinwald-Giemsa stain. In B. viridis captured in May, the inguinal bodies were macroscopically brownish and histologically classified as lymphomyeloid bodies (Fig. 2A) since they possesed abundant populations of lymphocytes and granulocytes. In B. viridis captured in July-August, the similar structures of both adults and juveniles were macroscopically yellow and fatty, but serial sections revealed within the fat tissue a few lymphoid foci (Fig. 2B). This suggests that the inguinal bodies of B. viridis undergo conspicuous seasonal changes. In FIG. 1. Inguinal regions of Bufo viridis (Fig. 1A) and Bufo bufo (Fig. 1B). Contours of the inguinal body in the left groin of B. viridis have been outlined (Fig. 1A).