Hyla Arborea Research Articles

Mitochondrial uniformity in populations of the treefrog Hyla molleri across the Iberian Peninsula

AbstractBased on DNA sequences of fragments of the mitochondrial COI and 16S rRNA genes we provide a first assessment of genetic variability of Iberian treefrog populations that have been historically allocated to Hyla arborea, partly as subspecies H. a. molleri. Our data from 147 specimens and 33 populations confirm strong divergence between these frogs and Central European H. arborea but relatively low differentiation across their range, supporting their status as a separate species, H. molleri. Preliminary phylogeographic data indicate a possible weak genetic differentiation of populations from the northern coast of the region of Galicia. We suggest inclusion of nuclear markers and an extension of the sampling into the coastal regions of Asturias and Cantabria, as well as the identification of the contact zone between H. molleri and H. arborea in either the Spanish Basque country or in France, as priorities for future research on this species.

Amphibian phenotypic variation along a gradient in canopy cover: species differences and plasticity

The distributions of many freshwater organisms correlate with a gradient in canopy cover, ranging from sunny wetlands to closed woodland ponds. Little is known about mechanisms that exclude species from some sections of the gradient while allowing persistence in others. I addressed this question by manipulating shading in 740‐l outdoor mesocosms and measuring several ecologically‐relevant traits in three species of amphibian larva (Rana temporaria and Triturus alpestris, generalists occupying the entire gradient; and Hyla arborea, a specialist in open habitats). Shading caused delayed development, but had no effect on survival and increased the growth rate of R. temporaria. Body and tail color were darker in the shade. Plasticity in morphological shape, consisting of reduced gut width and increased tail size under shaded conditions, may reflect poor food availability and low dissolved oxygen. The canopy generalist R. temporaria increased activity in the shade, spent more time basking in shallow water, and maintained high larval performance. Unexpectedly, the specialist H. arborea was also highly plastic. These results describe extensive phenotypic plasticity induced by shade, and highlight traits that may influence performance along the canopy gradient.

Genetic Bottlenecks Driven by Population Disconnection

Connectivity among populations plays a crucial role in maintaining genetic variation at a local scale, especially in small populations affected strongly by genetic drift. The negative consequences of population disconnection on allelic richness and gene diversity (heterozygosity) are well recognized and empirically established. It is not well recognized, however, that a sudden drop in local effective population size induced by such disconnection produces a temporary disequilibrium in allelic frequency distributions that is akin to the genetic signature of a demographic bottleneck. To document this effect, we used individual-based simulations and empirical data on allelic richness and gene diversity in six pairs of isolated versus well-connected (core) populations of European tree frogs. In our simulations, population disconnection depressed allelic richness more than heterozygosity and thus resulted in a temporary excess in gene diversity relative to mutation drift equilibrium (i.e., signature of a genetic bottleneck). We observed a similar excess in gene diversity in isolated populations of tree frogs. Our results show that population disconnection can create a genetic bottleneck in the absence of demographic collapse.

Consequences of genetic erosion on fitness and phenotypic plasticity in European tree frog populations (Hyla arborea)

The detrimental effects of genetic erosion on small isolated populations are widely recognized contrary to their interactions with environmental changes. The ability of genotypes to plastically respond to variability is probably essential for the persistence of these populations. Genetic erosion impact may be exacerbated if inbreeding affects plastic responses or if their maintenance were at higher phenotypic costs. To understand the interplay 'genetic erosion-fitness-phenotypic plasticity', we experimentally compared, in different environments, the larval performances and plastic responses to predation of European tree frogs (Hyla arborea) from isolated and connected populations. Tadpoles from isolated populations were less performant, but the traits affected were environmental dependant. Heterosis observed in crosses between isolated populations allowed attributing their low fitness to inbreeding. Phenotypic plasticity can be maintained in the face of genetic erosion as inducible defences in response to predator were identical in all populations. However, the higher survival and developmental costs for isolated populations in harsh conditions may lead to an additional fitness loss for isolated populations.

Relevance of multiple spatial scales in habitat models: A case study with amphibians and grasshoppers

Habitat models for animal species are important tools in conservation planning. We assessed the need to consider several scales in a case study for three amphibian and two grasshopper species in the post-mining landscapes near Leipzig (Germany). The two species groups were selected because habitat analyses for grasshoppers are usually conducted on one scale only whereas amphibians are thought to depend on more than one spatial scale. First, we analysed how the preference to single habitat variables changed across nested scales. Most environmental variables were only significant for a habitat model on one or two scales, with the smallest scale being particularly important. On larger scales, other variables became significant, which cannot be recognized on lower scales. Similar preferences across scales occurred in only 13 out of 79 cases and in 3 out of 79 cases the preference and avoidance for the same variable were even reversed among scales. Second, we developed habitat models by using a logistic regression on every scale and for all combinations of scales and analysed how the quality of habitat models changed with the scales considered. To achieve a sufficient accuracy of the habitat models with a minimum number of variables, at least two scales were required for all species except for Bufo viridis, for which a single scale, the microscale, was sufficient. Only for the European tree frog ( Hyla arborea), at least three scales were required. The results indicate that the quality of habitat models increases with the number of surveyed variables and with the number of scales, but costs increase too. Searching for simplifications in multi-scaled habitat models, we suggest that 2 or 3 scales should be a suitable trade-off, when attempting to define a suitable microscale.

Climate and environment of the earliest West European hominins inferred from amphibian and squamate reptile assemblages: Sima del Elefante Lower Red Unit, Atapuerca, Spain

The Sima del Elefante cave, in the Sierra de Atapuerca (Burgos, Spain), is famous for the fact that level TE9 of its Lower Red Unit recently delivered the oldest hominin remains of Western Europe, identified as Homo antecessor and dated by biostratigraphy and radiometric methods to ca 1.2 Ma. Given the importance of this discovery, every effort is being made to reconstruct the landscapes where these hominins once thrived. The amphibian and squamate reptile assemblage of the Sima del Elefante Lower Red Unit is here studied for the first time. The faunal list comprises at least 17 species (roughly 12,000 bone fossil remains): Salamandra salamandra, Triturus cf. marmoratus, Alytes obstetricans, Pelobates cultripes, Pelodytes punctatus, Bufo bufo, Bufo calamita, Hyla arborea, Rana sp., cf. Pelophylax sp., Lacerta s.l., small-sized indeterminate lacertids, Anguis fragilis, Natrix cf. natrix, Natrix cf. maura, Coronella cf. girondica and Vipera sp. As the amphibians and squamate reptiles do not differ at species level from the extant herpetofauna of the Iberian Peninsula, they can contribute to the reconstruction of the landscape and climate. In this paper, the mutual climatic range and habitat weighting methods are applied to the amphibian and squamate reptile assemblages in order to estimate quantitative data. The results from the squamate and amphibian study indicate that during the hominin presence the mean annual temperature (MAT = 10–13 °C) was always slightly warmer than at present and the mean annual precipitation (MAP = 800–1000 mm) was greater than today in the Burgos area. The landscape had open habitats in the vicinity of the Atapuerca caves throughout the sequence, with wet points in the surrounding area, and a predominance of humid meadows and open woodlands. These results mainly agree with those for large mammals, small mammals and the pollen analysis. The climate and landscape of TE-LRU are very similar to those reconstructed for the TD6 “Aurora Stratum” level of the Gran Dolina cave (Atapuerca) that has yielded H. antecessor remains at ca 960 ky. When compared with Orce localities of similar age (i.e. Barranco León D and Fuente Nueva 3) that have yielded strong evidence of hominin presence (mainly lithic industries), it appears that they have a fairly similar environmental pattern, having in common a good representation of woodland and water-edge areas, even though dry environments are more developed in Barranco León D and Fuente Nueva 3 than in Sima del Elefante and Gran Dolina. H. antecessor may have thus lived under a warm-humid climate in a patchy landscape mainly composed of humid meadows and riparian woodlands. Such a landscape may have provided favourable conditions for a high diversity of large mammals, as well as for hiding and escaping from large carnivores.

Novel natural peptides from Hyla arborea schelkownikowi skin secretion

Hyla arborea schelkownikowi is one of the leaf frog species inhabiting the southern territories of Russia and the former USSR. This frog species is a member of the Hylidae Rafinesque, 1815 batrachians family. The present study deals with the previously uninvestigated peptidome of the Hyla arborea schelkownikowi skin secretion. Nano-electrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS) of the skin secretion, in the intact form and after acetylation, was selected as the general method of analysis. Electron-capture dissociation (ECD) and collision-induced dissociation (CID) fragmentation were both employed, while de novo sequencing was performed by manual interpretation of the MS data. The suppression of the cyclization of b-ions in the mass spectrometer by the acetylation reaction proved to be very efficient for the de novo sequencing of short peptides. Ten skin peptides were found and all of them, except for bradykinin, had not previously been reported. Six of the peptides belong to the tryptophyllins and related peptides, while three peptides are similar to the aureins.

Pond‐based survey of amphibians in a Saxon cultural landscape from Transylvania (Romania)

Habitat‐based inventories provide critical reference data that are essential to track changes in amphibian communities and their habitats. We present the results of a pond inventory in a cultural landscape from central Romania. The presence/absence of amphibians was assessed through multiple‐year surveys during the breeding season and larval development. Ten amphibian species and a species complex were identified: Triturus cristatus, T. vulgaris, Bombina variegata, Bufo bufo, B. viridis, Rana dalmatina, R. temporaria, R. arvalis, Hyla arborea, Pelobates fuscus and the R. esculenta complex. The species richness is larger in the permanent ponds than in the temporary ones. Rana dalmatina, B. bufo and the R. esculenta complex are the most frequent in the permanent ponds, while Bombina variegata and R. temporaria were the most common in temporary ponds. The scarcity of B. viridis and R. arvalis is explained by the lack of available habitats. Our data allow a more complex analysis of the spatial and temporal determinants of amphibian habitat use in this cultural landscape, and provide a consistent baseline for future surveys and monitoring programmes.

Support for a role of colour vision in mate choice in the nocturnal European treefrog (Hyla arborea)

Summary Although nocturnal anurans use vision for reproductive communication, it remains unknown whether they see colours at night. Here, we explored this question in the European treefrog (Hyla arborea), by conducting two mate choice experiments under controlled light conditions. Experiments involved static male models with identical calls but different vocal sac colouration combining chromatic (red/orange) and brightness (dark/light) information. We found that females preferred dark red over light orange, evidencing for the first time a visually-guided mate choice in nocturnal diffuse light conditions. Conversely, females did not discriminate between dark orange and light red. The preference for dark over light in the first but not in the second experiment suggested that females had not only access to brightness cues but also to chromatic cues. The absence of preference may originate from females choosing at random in a situation where colour and brightness cues may convey contradictory information about male quality or from individual heterogeneity in the type of cues used for mate choice. Overall, these experiments provide the first support for the use of colour vision in a nocturnal amphibian.

Amphibians of Oława

Observations were conducted of 28 water bodies from 2006 to 2009 in Olawa, a mid-sized town (of about 31,000 residents) in southwestern Poland. 13 amphibian taxa were confirmed. These same taxa were also confirmed in the surrounding areas of the town. Twice as many amphibian species were found in areas affected to a lesser degree by urbanization than in heavily urbanized areas. Two groups of amphibians were distinguished, depending on their tolerance of urbanization at breeding ponds and surroundings: 1) species preferring only natural terrain or areas close to being natural - Bombina bombina, Pelobates fuscus and Rana arvalis; 2) taxa breeding in both natural areas as well as those under the pressure of urbanization - Tritunis cristatus, Tritunis vulgaris, Bufo bufo, Bufo viridis, Hyla arborea, Rana esculenta complex and Rana temporaria.

Long term data on the amphibians of Wrocław

The paper presents information about the amphibian species in Wroclaw – frequency oftheir occurrence as well as number of individuals in water bodies. For the study 31 sites were selected. Only in two sites no amphibians were found. On the rest of the sites in total 11 amphibian taxons were present – Triturus cristatus, Triturus vulgaris, Bombina bombina, Bufo bufo, Bufo viridis, Hyla arborea, Pelobates fuscus, water frogs from the Rana esculenta complex (both a species Rana lessonae and a hybrid Rana kl. esculenta), Rana temporaria and Rana arvalis. The most numerous were water frogs (occurred on 90.3% of all sites), Bufo bufo (48.4% sites), Bombina bombina and Rana temporaria (both species on 45.2% si- tes). The rarest species were Triturus cristatus (16.1% sites) and Pelobates fuscus (9.7% sites). In Wroclaw amphibians are the most abundant in ponds located within river valleys. There are 10 species there (on the average 4.44 species per pond), with Bombina bombina and water frogs being the most numerous. Second amphibian-friendly environment are sites within the city forests, having 8 species (on the average 5.22 species). Water frogs, Rana temporaria and Rana arvalis were most common here, and Triturus cristatus and Triturus vulgaris occurred mostly in this category. The lowest frequency was observed in the city center (on the average 0.66 species), with one site without any amphibians, and with only water frogs in the remaining two sites. After a pond reconstruction populations of Triturus vulgaris and Bufo bufo disappeared, and the number of Bufo viridis increased. For three sites the changes in amphibian species assemblages in consecu- tive years of observations are provided. In two sites the trend of maintaining the same species composition was observed, and on third site the composition changed due to the pond reconstruction (T. vulgaris and B.bufo substituted by B. viridis).

Amphibians of Gniezno

From 2005 to 2007 the water bodies in the area of Gniezno have been controlled to establish amphibian species composition and their number. Thirty four of water bodies were under observation. In 23 among them (67.6%) the following species of amphibians were present: common frog Rana temporaria, moor frog Rana arvalis, “water frogs” Rana esculenta complex, common spadefoot, fire-bellied toad Bom- bina bombina, common toad Bufo bufo; green toad Bufo viridis; and smooth newt Tritunis vulgaris. “Water frogs” and common frog formed the largest populations. Great crested newt Tritunis cristatus, natterjack toad Bufo calamita, and tree frog Hyla arborea were not observed.

Amphibian communities in small water bodies in the city of Olsztyn

The aims of the study were determine the effects of anthropogenic pressure on the species composition and population size of amphibians of small water bodies in Olsztyn city (NE Poland). The presence of 11 amphibian species was noted in 83.5% of water bodies. The dominants were: the common frog Rana temporaria, the edible frog Rana esculenta, the pool frog Rana lessonae, the common toad Bufo bufo and the moor frog Rana arvalis. The fire-bellied toad Bombina bombina was an influent, while the common newt Triturns vulgaris, the common spadefoot Pelobates fnsciis, the tree frog Hyla arborea, the green toad Bufo viridis and the crested newt Tritimis cristatus were the recedents. The most frequent were “green frogs”, which occur in 71.9% of water bodies inhabited by amphibians (60.0% of all water bodies). The species characterized by the lowest occurrence frequency were the tree frog (6.0% and 5.0%, respectively), the green toad (5.4% and 4.5%) and the crested newt (3.6% and 3.0%). In the paper the urban pressure on amphibian communities is widely discussed.

Amphibians of Lublin

The occurrence of amphibians in Lublin city (South-East Poland) was studied in the years1998–2006. At 33 localities, including all water bodies in the Lublin area, 12 taxons of amphibians were found. Common frog Rana temporaria was most frequently observed in studied water bodies (84.7% of all locations). The results indicate that the rarest and extremely threatened with extinction species are: crested newt Triturus cristatus and common tree frog Hyla arborea.

The challenge of finding a high-quality male: a treefrog solution based on female assessment of male calls

In the context of sexual selection through mate choice, it has recently been suggested that the effectiveness of intersexual communication is affected not only by the honesty of male sexual signals but also by the number of signal components simultaneously used to convey information. We investigated female use of acoustic components in the chorusing treefrog Hyla arborea using two-choice phonotaxis tests. Females showed a significant preference for higher call rates and calls of short duration, as well as for higher call amplitudes. They also favoured lower peak frequencies. Since this call characteristic was negatively correlated with body weight, such a preference should lead to their selecting larger males. Females also exhibited a marginal preference for longer call bouts. The reliability of the multiple call components involved in mate choice in H. arborea are discussed, as well as the validity of these results for mate choice in the natural context of a noisy anuran chorus.

Are differences in fitness traits related to genetic clusters? An empirical test on the European tree frog

Microsatellites are one of the preferred marker types to estimate the level of genetic diversity and subdivision in natural populations of endangered species. Many studies thereby attempted to identify genetically defined management units for conservation by applying a variety of genetic clustering methods. It is not clear, however, whether genetic clusters inferred from neutral molecular markers reflect differences in fitness or adaptation. In this study, I conducted a common garden experiment on the endangered European tree frog ( Hyla arborea) to clarify whether fitness-related traits of larval development differed between three genetic groups defined by Bayesian clustering analysis. I reared larvae under semi-natural conditions and measured growth and developmental rates as well as survival at five larval stages from eclosion to completion of metamorphosis. Nested general and generalized linear models showed significant cluster differences for several variables in terms of smaller growth rates and body sizes at early larval stages. These differences were, however, probably not linked to adaptive divergence among clusters but rather to neutral genetic processes in the breeding sites of one cluster, which were spatially isolated and subject of recent bottlenecks. Hence, a genetic load effect (e.g. inbreeding depression) may have acted on the populations of this particular cluster. I advise studies aiming to define management units for conservation to not only use genetic clustering methods but to complement their findings with experimental approaches on fitness-related traits.

Multiple signals and male spacing affect female preference at cocktail parties in treefrogs.

Effective acoustic communication in the face of intense conspecific background noise constitutes a constant sensory challenge in chorusing and colonial species. An evolutionary approach suggests that behavioural and environmental constraints in these species should have shaped signal design and signalling behaviour to enable communication in noisy conditions. This could be attained both through the use of multicomponent signals and through short-term adjustments in the spatial separation of calling males. We investigated these two hypotheses in a chorusing anuran, the hylid Hyla arborea, through a series of phonotaxis experiments conducted within a six-speaker arena in a high background noise situation, by presenting females with male calls containing either single or multiple attractive call components, and by modifying distances between speakers. We found that female ability to discriminate attractive calls increased when several attractive call components were available, providing novel evidence that the use of multicomponent signals enhances communication in complex acoustic conditions. Signal discrimination in females also improved with speaker separation, demonstrating that within natural choruses, spatial unmasking conditioned by male density and spatial separation probably improves female discrimination of competing males. Implications of these results for the accuracy of mate choice within choruses are discussed.

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.

GOOD GENES DRIVE FEMALE CHOICE FOR MATING PARTNERS IN THE LEK-BREEDING EUROPEAN TREEFROG

Investigating the mechanisms underlying female mate choice is important for sexual-selection theory, but also for population-genetic studies, because distinctive breeding strategies affect differently the dynamics of gene diversity within populations. Using field-monitoring, genetic-assignment, and laboratory-rearing methods, we investigated chorus attendance, mating success and offspring fitness in a population of lek-breeding tree-frogs (Hyla arborea) to test whether female choice is driven by good genes or complementary genes. Chorus attendance explained approximately 50% of the variance in male mating success, but did not correlate with offspring fitness. By contrast, offspring body mass and growth rate correlated with male attractiveness, measured as the number of matings obtained per night of calling. Genetic similarity between mating partners did not depart from random, and did not affect offspring fitness. We conclude that females are able to choose good partners under natural settings and obtain benefits from the good genes, rather than compatible genes, their offspring inherit. This heritability of fitness is likely to reduce effective population sizes below values previously estimated.

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...