Hyla Arborea Research Articles

Successful development of European tree frog (Hyla arborea Linnaeus, 1758) populations in the south-west and west of Luxembourg following reintroduction

In this article, we provide a comprehensive overview of our European tree frog reintroduction, from the sourcing and captive-rearing of donor animals, through reintroduction and population development, to the current status of the project. Before the start of the project, only one isolated population remained in the east of the country. Starting with two initial reintroductions (2012 to 2014), two tree frog populations have developed over the past decade and we have managed to save the species from extinction on the national level. While both populations have been expanding rapidly in recent years, they show noticeable differences regarding initial development and spread. Further reintroductions to reinforce these populations were carried out from 2019 to 2024. In total, more than 7,000 juveniles and tadpoles were released over the entire duration of the project, resulting in ca. 830 calling males at 61 sites in 2024. Our long-term annual monitoring provides detailed data on population development and spread, which are presented and discussed. In addition, comparisons with similar tree frog reintroduction projects in other countries are made and logistical and epidemiological aspects of the reintroduction project are highlighted. Finally, we give an outlook on practical implications for conservation management, upcoming challenges and future focal points of the project.

Not so cryptic-differences between mating calls of Hyla arborea and Hyla orientalis from Bulgaria.

Anurans are among the most vocally active vertebrate animals and emit calls with different functions. In order to attract a mate, during the breeding season male frogs produce mating calls which have species-specific structure and parameters, and have been successfully used to resolve issues in taxonomy and phylogenetic relations. This is particularly useful when closely related taxa are concerned, as many species are morphologically almost identical, but still their status is well-supported by molecular and genetic data, suggesting the existence of mechanisms for reproductive isolation. Such is the case for treefrogs from the Hyla arborea group, which are now recognized as several distinct species. The present study aims to establish differences in call parameters between the European tree frog, Hyla arborea, and the Eastern tree frog, Hyla orientalis, which both occur on the territory of Bulgaria. Using autonomous audio loggers, calls from six sites (three in the range of H. arborea and three in the range of H. orientalis) were recorded between 7 p.m. and 12 a.m. during the breeding season in 2020-2023. The following parameters in a total of 390 mating calls were analyzed: call count, pulse count, call series duration, call period, peak (dominant) frequency, entropy. Results indicated that sites formed two distinct groups, which corresponded to the known distribution ranges of H. arborea and H. orientalis. The first two components of the PCA explained 71% of the total variance, with variables call count, call series duration, peak frequency and entropy being most important for differentiation between the sites. This study presents the first attempt to differentiate between the calls of these two sister taxa, which both fall within the "short-call treefrogs" group, and results are discussed in terms of known data for mating calls in Hyla sp., as well as limitations and future perspectives.

The lateral stripe – a reliable way for software assisted individual identification for Hyla arborea

The lateral stripe – a reliable way for software assisted individual identification for Hyla arborea

Genetic diversity of European tree frogs (Hyla arborea group): A systematic review

Amphibian populations are increasingly threatened by global change and the study of their genetic diversity is a major conservation priority. Western palearctic tree frog species of the Hyla arborea group are commonly distributed across Europe and the Middle East and many have declining populations. We performed a PRISMA systematic review to gain insight into the genetic diversity of H. arborea group. Sixteen published studies were included in the final qualitative analysis. While the genetic diversity of H. arborea group species was widely variable, it could often be explained by phylogeographic history. Populations in Western and Northern Europe had lower genetic diversity, with some populations also affected by habitat fragmentation. However, important regions of high genetic diversity were found in the Balkan peninsula for H. arborea sensu stricto and around the Black Sea for H. orientalis. Genetic diversity of H. molleri, H. savignyi, H. meridionalis, H. felixarabica, H. intermedia, H. sarda has been investigated only across extensive phylogeographical studies, while data regarding their genetic diversity at the local level are missing. Through our review, we identify knowledge gaps about the genetic diversity of the H. arborea group that require further investigation, of and illustrate how filling these gaps might translate into future conservation efforts.

Does the anesthetic tricaine methanesulfonate (MS-222) distort oxidative status parameters in tadpoles?

The effect of anesthesia/euthanasia with ethyl 3-aminobenzoate methanesulfonate (MS-222) on the oxidative status of Hyla arborea tadpoles was examined to determine whether the use of the anesthetic can confound the experimental results of the oxidative stress-based investigation. The experiment was conducted on two groups of tadpoles reared at different temperatures to produce differences in antioxidant capacity between the groups. After development at different temperatures (20 °C and 25 °C), the animals were exposed to different concentrations of MS-222 (0, 0.1, 1, and 5 g/L) for 15 min. The higher temperature decreased catalase activity, glutathione and protein carbonyl levels and increased glutathione reductase activity. The glutathione level and glutathione/thiol-related parameters were significantly changed after MS-222 exposure. However, individuals from the different temperature groups responded differently to the tested anesthetic, pointing to the possible influence of the initial levels of antioxidant capacity. The analysis of the interaction between the factors (temperature and MS-222) confirmed that the anesthetic can confound the results regarding the effects of temperature on the oxidative status parameters. The concentration of 0.1 g/L MS-222 had the lowest influence on the alterations in oxidative status and the results of the effect of temperature. A brief review of the current literature on the use of MS-222 in tadpoles made clear the absence of precise information on anesthetic concentration and exposure time. Similar studies should be repeated and extended to other amphibian species and other factors of interest to provide better guidance on tadpole anesthesia/euthanasia for future experiments that consider oxidative status parameters.

Effects of background color on pigmentation, morphological traits, and behavior in the European tree frog (Hyla arborea, Hylidae, Anura) tadpoles

Abstract Amphibian tadpoles are capable of avoiding threats (predators, uv radiation, etc.) through changes in coloration, behavior, and shape. In this paper, we tested how quickly European tree frog (Hyla arborea) tadpoles can change body pigmentation to achieve crypsis and whether color change is reversible. Additionally, we tested how different environmental background colorations affect the body length, shape, and ontogenetic trajectories of tadpoles. We also analyzed if tadpoles can relate to their coloration and choose the appropriate background to enhance crypsis. For this purpose, we reared tadpoles on white and black backgrounds for 36 days. Halfway through the experiment, half of the tadpoles from each treatment were placed on the alternative background. Our results suggest that H. arborea tadpoles are capable of rapidly responding to color changes in their environment, however, color-matching with the white background is poor. These quick color changes are reversible. Rearing in different background coloration and rapid color changes do not affect tadpoles’ length variation but affect tadpoles’ shape. Tadpoles introduced to the white background at the start of the experiment developed deeper tail fins and more pronounced snouts. We also found that H. arborea tadpoles actively choose an appropriate background to achieve maximum crypsis. This study represents the basis for the future analysis of adaptive coloration in tadpoles as it has a very complex function in anurans.

What coloration brings: Implications of background adaptation to oxidative stress in anurans

BackgroundUrban development results in habitat destruction, affecting populations of amphibians, the most fragile group of vertebrates. With changes in the environment, these animals become more exposed to light and predators. To enhance their chances of survival, they display plasticity of body coloration. Aside from adaptive benefits, animals exhibiting background matching meet the energetic costs and restrictions of changing body tones. To study the physiological consequences of Hyla arborea tadpole adaptation to background color, we followed oxidative stress parameters after rearing larvae on a constant background (black/white) and after changing the background color.ResultsLarvae cultivated for 20 days on constant substrate color exhibited differences in body coloration but without differences in lipid peroxidation (LPO) concentration between dark and pale individuals, suggesting that coloration investment during this period did not induce higher oxidative damage in darker tadpoles. Prolonged exposure of larvae (37 days) to a dark habitat increased antioxidative system defense and LPO concentrations, compared to animals reared permanently in the white surroundings. The positive correlation of oxidative damage with color intensity of individuals points to the physiological consequences of higher investment in the number of pigment cells necessary for dark pigmentation. In individuals faced with non-matching background and change in body coloration, defense system declined and LPO occurred relative to individuals cultivated in white habitat.ConclusionHere, we have pointed to consequences related to background matching and stress that amphibians experienced during chromatic adaptations. Background color change causes a complex physiological response affecting the antioxidative defense parameters. This investigation elucidates the accompanying cost of amphibiansʼ adjustment to an altered environment.

The introduction of three cryptic tree frog species in the Dutch coastal dunes challenges conservation paradigms

Abstract Invasive species are considered one of the main drivers of the sixth mass extinction. Conservation solutions depend on whether a species is also indigenous to the country it invades (i.e., beyond its native range). In the case of invasive cryptic species, genetic tools are required to establish their identity. We illustrate these issues with the human-mediated colonization of the Dutch coastal dunes by Hyla tree frogs. Although previously assumed to concern the indigenous common tree frog H. arborea, European tree frogs comprise a complex of allopatric cryptic species, meaning the taxonomic identity of introduced Dutch populations warrants investigation. We sequence mtDNA for 173 individuals from native and introduced populations across the Netherlands and compare our dataset with hundreds of Hyla haplotypes previously barcoded in the Western Palearctic. Two of the dune populations carry an mtDNA haplotype of the native species H. arborea that occurs naturally elsewhere in the Netherlands. In contrast, mtDNA assigned to the eastern tree frog H. orientalis was detected in all three other dune populations. In one of these populations mtDNA of the Italian tree frog H. intermedia was also found. Not one, but three species of tree frogs have thus been introduced to the Dutch coastal dunes, only one of them being native to the Netherlands. This situation causes a conservation conundrum as some introduced populations are lawfully protected but could pose a threat to local biodiversity. Regarding the ‘true’ exotic tree frog species, all conservation options should be considered.

Distribucija gatalinki (Hyla SPP.) u Srbiji - implikacije skorašnje taksonomske revizije

We present the updated distribution data of the Hyla arborea species complex in Serbia. Hyla arborea inhabits all biogeographic regions, while Hyla orientalis inhabits only the easternmost parts of northeastern , Eastern and southeastern Serbia. Potential hybrid zone encompasses parts of central and southern Serbia from the Đerdap gorge, over Niš to Bosilegrad. Since the taxonomic separation UROŠEVIĆ, A. ET AL.: DISTRIBUTION OF TREE FROGS (HYLA SPP.) IN Serbia 138 between the tree frog taxa was not yet recognized in national nomenclature, we propose the official Serbian names. The species H. arborea will keep the standard Serbian name "Gatalinka", while for the species H. orientalis we propose the name "Istočna gatalinka".

Morphometry of two cryptic tree frog species at their hybrid zone reveals neither intermediate nor transgressive morphotypes.

Under incomplete reproductive isolation, secondary contact of diverged allopatric lineages may lead to the formation of hybrid zones that allow to study recombinants over several generations as excellent systems of genomic interactions resulting from the evolutionary forces acting on certain genes and phenotypes. Hybrid phenotypes may either exhibit intermediacy or, alternatively, transgressive traits, which exceed the extremes of their parents due to epistasis and segregation of complementary alleles. While transgressive morphotypes have been examined in fish, reptiles, birds, and mammals, studies in amphibians are rare. Here, we associate microsatellite‐based genotypes with morphometrics‐based morphotypes of two tree frog species of the Hyla arborea group, sampled across a hybrid zone in Poland, to understand whether the genetically differentiated parental species also differ in morphology between each other and their hybrids and whether secondary contact leads to the evolution of intermediate or transgressive morphotypes. Using univariate approaches, explorative multivariate methods (principal component analyses) as well as techniques with prior grouping (discriminant function analyses), we find that morphotypes of both parental species and hybrids differ from each other. Importantly, hybrid morphotypes are neither intermediate nor transgressive but found to be more similar to H. orientalis than to H. arborea.

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 the 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 the 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 L. 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 establish 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, whereas Bufotes viridis and Hyla arborea had high values. The phylogenetic signal was spread throughout the dimensions of the 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 applying phylogenetic comparative methods to hematological parameters, our results add a new perspective on the evolution of blood cell physiology in frogs.

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

BackgroundAlthough 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.ResultsThe 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.ConclusionsA 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.

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.

Population trends of four species of amphibians in western France; results from a 15 year time series derived from road mortality counts

Reports of declining amphibians are a major concern for conservation given their role as indicators of environmental change. This includes potentially impacting on the ecology other species and highlights the importance of monitoring long-term changes in amphibian populations. The core difficulty is gathering long term data sets that as well as being time consuming, may be costly and impractical for logistical reasons. Road mortalities have value in this respect since they are highly visible and have been frequently used as a metric to monitor changes in a variety of animal populations. In this paper they have been employed as proxies to estimate long-term population changes in four European species of amphibians. From January 2005 to December 2019 a total of 747 amphibian mortalities were recorded, the majority of which were urodeles. Most individuals were adults that entered roads during the annual migration period. Long term annual counts fluctuated widely in all species and peaked during the 4-year period from 2009 to 2012 with second period of high numbers in Triturus marmoratus and Hyla arborea during 2018 and 2019. Long-term population trends were evaluated using regression analysis of the logarithmic (loge) transforms of annual counts against year as independent variables, which were then tested against a hypothetical 0 regression coefficient, indicative of population stability. The results indicated long-term stability in T. marmoratus and H. arborea but potential declines in Lissotriton helveticus and Pelophylax lessonae, the latter showing metapopulation extinctions followed by recolonisation. Stepwise regression of potential climate drivers in amphibian numbers suggested a 2-year lag of rainfall during October and December were potential factors involved in population change.

Early Pleistocene amphibians and squamates from Copăceni (Dacian Basin, southern Romania)

This paper describes an Early Pleistocene amphibian and squamate assemblage from the Dacian Basin, southern Romania. The fossil sample allows for the identification of a moderately diverse assemblage, which includes representatives of 13 genera of the families Salamandridae (Lissotriton vulgaris, Triturus sp.), Bombinatoridae (Bombina sp.), Hylidae (Hyla aff. Hyla arborea), Bufonidae (Bufo bufo), Pelobatidae (Pelobates syriacus), Ranidae (Ranidae indet., Pelophylax sp., Rana ?temporaria, Rana sp.), Lacertidae (Lacertidae indet.), Anguidae (Pseudopus sp.), Natricidae (Natrix natrix), and Colubridae (Coronella austriaca, Zamenis longissimus). The taxonomic composition of the Early Pleistocene amphibian and squamate assemblage from Copăceni is similar to the one reported from other Romanian sites of the same age, and also to that of Recent Romanian herpetofaunas. The amphibian and squamate assemblage from Copăceni supports the existence of a permanent freshwater body, of neighbouring forests or shrubberies, and thick soil, in an Early Pleistocene climate that was warmer than the present one.

�������� ������� ������ � ������ � ������ ������ �ᒺ��� ���������� ����� �Samarskyi Lis � UA0000212�

In 2020, waters and territories of the eastern part of Emerald Network object “Samarskyi Lis – UA0000212” were studied by biology specialists. The main purpose of the research was to identify rare species of plants and animals. We found species of flora and fauna protected by European Red List, Red Data Book of Ukraine, Additions to the Bern Convention and Regional Red Data Book. The list of plant species subjected to special protection contains 42 species, including Allium savranicum, Agropyron dasyanthum, and Jurinea cyanoides. It is noted that in the eastern part of the Emerald Network object “Samarskyi Lis ‒ UA0000212” the presence of other rare plant species is assumed (Senecio borysthenicus (D&c.) Andrz., Agropyron dasyanthum Ledeb, Viola lavrenkoana Klokov., Allium savranicum Besser). This matter requires monitoring in various phenological periods of the year. Concerning entomofauna in the eastern part of the Emerald Network object “Samarskyi Lis – UA0000212” it was found 7 species listed in the Red Data Book of Ukraine (2009): Lucanus cervus, Papilio machaon, Iphiclides podalirius, Zerunthia polyxena, Xylocopa valla, Scolіa maculata, Catocala sponsa, as well as 2 species of insects protected by the provisions of the Bern Convention ‒ Lucanus cervus, Lycaena dispar. The ichthyofauna of the eastern part of the Emerald Network object “Samarskyi Lis ‒ UA0000212” includes 4 species that are listed in the Bern Convention: Leucaspius delineatus, Rhodeus amarus, Cobitis taenia, Sykhorn abotter. Analysis of terrestrial vertebrates revealed the presence of 95 species to be protected. There are 5 species of amphibians (three of them are included in the International Red List – Lissotriton vulgaris, Hyla arborea, Bufo bufo), as well as 6 species of reptiles, among which one is protected by IUCN (Emys orbicularis), two species listed in the Red Data Book of Ukraine (Coronella austriaca, Vipera renardi). 74 species of birds were found in the study area. Three of them (Coracias garrulus, Ciconia nigra and Buteo rufinus) are listed in the Red Data Book of Ukraine. In the eastern part of the Emerald Network object “Samarskyi Lis ‒ UA0000212” there are 10 species of mammals, of which nine are protected by the provisions of the Bern Convention, and two species (Eptesicus serotinus, Nyctalus noctula) are listed in the Red Data Book Of Ukraine. It is proposed to conduct further monitoring studies in the eastern part of the Emerald Network “Samarskyi Lis ‒ UA00002122”

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

Comparison of landscape graph modelling methods for analysing pond network connectivity

Landscape fragmentation negatively impacts species populations by isolating them. Assessing landscape connectivity could help to improve biodiversity conservation. Among various methods available to model and analyse connectivity, graph-theoretic approaches are recognized as powerful tools, even if their ecological significance may be questionable in some cases. Indeed, there are many ways to construct a landscape graph and their impacts on the assessment of connectivity are rarely explored. Our aim was to compare three methods of constructing landscape graphs to identify differences and similarities in the resulting network connectivity. The methods can be distinguished according to the type of data used: expert opinions, field data or a combination of the two. The methodological framework was applied to seven pond-dwelling species (Alytes obstetricans, Bufo bufo, Epidalea calamita, Hyla arborea, Natrix natrix, Rana temporaria, Triturus cristatus) in the Ile-de-France region (France). Three common methods were applied to construct landscape graphs: (1) using a land cover map (LM) and expert opinions to define nodes and links; (2) using a habitat suitability model (HSM) and species occurrence data to define nodes and links; and (3) using a HSM to define nodes and a land cover map to define links (HSM_LM). To carry out our study, we produced a land cover map, collected and prepared input data for HSMs, generated HSMs to map the probability of species occurrence and constructed landscape graphs from the three methods. For each of them, several connectivity metrics were calculated and compared. The results revealed large differences in the statistical distribution of connectivity values, even though the spatial location of the main areas of low and high connectivity was roughly the same. In general, the LM method provided lower values of connectivity and smaller areas of high values than the other two, regardless of species. Conversely, the HSM method had the highest connectivity values, while the combined HSM_LM method appeared to be intermediate. Our study was not intended to conclude whether one method is better than another; only to point out that results vary greatly depending on the graph construction method. To evaluate the predictive performance of each model, a validation process should be conducted with another independent biological dataset, which was not available in our study. The high variability of results argues for taking care to ensure that the construction of models is carefully consistent with ecological assumptions and the objective pursued.

Combining spatial modeling tools and biological data for improved multispecies assessment in restoration areas

Habitat restoration is one of the actions to reduce landscape fragmentation by promoting connectivity and thus biodiversity. But knowing where to implement these habitats is a major issue and planners lack guidance for answering this question, in particular when it involves multiple species and over a large area. We proposed to combine biological data, habitat suitability models and spatial graphs to improve multiscale and multispecies connectivity in Ile-de-France, a highly artificialized region of 12,000 km2. The framework consisted of i) modeling habitat suitability for eight pond-dwelling species (Alytes obstetricans, Bufo bufo, Epidalea calamita, Hyla arborea, Rana temporaria, Salamandra salamandra, Triturus cristatus, and Natrix natrix), ii) modeling the ecological network for each species, iii) prioritizing each sampling point depending on the gain in connectivity if a new pond was created there and iv) combining single-species results to identify the areas that could improve multispecies connectivity. The multivariate statistical analysis revealed that transitional forest environments appeared to be the most strategic areas for improving multispecific connectivity (at least for 5 species). Targeted addition of habitat within an ecological network can increase habitat density in deficient areas and reconnect network sub-parts. This approach is therefore promising to guide conservation actions and “no net loss” biodiversity measures, especially the final stage of offset in the mitigation hierarchy.