Changes in Structure and Function of Ventral Epidermis in Hyla arborea savignyi Aud. (Anura; Hylidae) Throughout Metamorphosis

The changes in epidermal ultrastructure during the metamorphic cycle of Hyla arborea are described. The number of cell layers increased from two to four in the late tadpole stages. The cell layers flatten and the process of stratification reaches its peak after the completion of metamorphosis. The mitochondria-rich cell appears early in the tadpole stages. Numerous flask cells are noticeable in the post-metamorphic stages. K+–p-NPPase activity was localized cytochemically in the epidermis of H. arborea during its metamorphic cycle. In the epidermis of the legless tadpole, evidence for K+–p-NPPase activity was confined intracellularly. During the later tadpole stages, preceding metamorphic climax, the main ATPase activity shifted to the baso-lateral cell membranes bordering with the intercellular spaces under the surface and later the stratum corneum. This continued after metamorphic climax in the juvenile toadlets, diminishing later in the adult stage.     

Morphologie des Larynx von Hyla a. arborea (L.) und Hyla meridionalis Boettger (Amphibia,Anura)

The laryngi of the Central European and the Mediterranean treefrog exhibit a great morphological similarity. It suggests that mechanisms of the CNS and not the laryngi determine the patterns of the mating calls which are very different in these two species of treefrogs.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft     

Genetic differentiation of the Iberian tree frogs Hyla arborea molleri and Hyla meridionalis (Amphibia: Anura)

Genetic differentiation of Hyla arborea molleri and H. meridionalis was studied through allozyme electrophoresis and through essays of artificial hybridization. Samples were taken at several localities in Portugal, where these species occur either in sympatry or allopatry, and at one locality in France. Of the 21 presumptive loci analyzed, 6 were monoallelic, while 9 loci were discriminative between both species. A relatively high level of genetic differentiation was found between the two species (NEI's D = 0.725), contrasting with their morphological similarity. Remarkably low levels of genetic variability were found within species, with greater differentiation found among samples of H. meridionalis than of H. a. molleri. Although the capacity for interspecific hybridization was low, hybrid embryos resulted from both reciprocal crosses, but with much greater success in the cross ♂H. a. molleri x δH. meridionalis. Hybrids showed greater similarity with the male parent, and showed heterozygote allozymic phenotypes for discriminative loci. Results are compared with those of similar studies on the same and different species of Hyla, and differentiation of H. a. molleri and H. meridionalis is discussed in relation to their taxonomic status and origin.     

A comparative ultrastructural study on ultimobranchial glands of some Israeli anurans (Bufo viridis,Rana ridibunda and Hyla arborea)

Summary The comparative fine structure of ultimobranchial (UB) glands of adult Israeli anurans (Bufo viridis, Rana ridibunda, Hyla arborea) taken in the wild during the breeding season is presented. Common aspects of the UB secretory cells are considered with especial reference to secretory granules, lipid droplets and tonofilaments. In B. viridis a second cell type with large electron-dense cytoplasmic granules is found in UB follicles. R. ridibunda and H. arborea UB follicles have a second cell type similar to goblet cells in appearance and these appear to be discharging their mucoid contents into the lumina of the follicles. The possible significance of these various cell types is considered.I am indebted to the Central Research Fund of London University for an award for apparatus and travel enabling a short research visit to the Dept. of Zoology, The Hebrew University, Jerusalem and am particularly indebted to Dr. Dvorah Boschwitz and her colleagues for their enormous help during my visit. I would also like to thank Alan D. Phillips for his assistance in the analysis of the material made possible by an award from the Science Research Council. My thanks are also due to Raynor L. Jones for his excellent technical assistance.     

Ultrastructure of Spermiogenesis in Hyla japonica (Anura,Amphibia)

Spermiogenesis in Hyla japonica was examined using transmission electron microscopy. Features which have not been reported previously during spermiogenesis of anurans include: (1) the neck of pericentriolar material is well developed, (2) the perforatorium contains many microtubules, (3) the tail is composed of axoneme and axial rod, but has no undulating membrane. Such a structure has not been described in amphibians, although it occurs in fish and mammals, and (4) the modified microtubules have one to two long projections. So far they have not been observed in any species except Gerris and Aphyosemion. With the exception of the above characteristics, the spermatozoa of Hyla japonica are remarkably similar to those of Bufo sp.     

Chromosomal differentiation of Hyla nana and Hyla sanborni (Anura,Hylidae) with a description of NOR polymorphism in H. nana

Specimens of Hyla nana and Hyla sanborni from a syntopic population were studied cytogenetically. These species are morphologically very similar and are frequently misidentified, confused with each other. Both species had a diploid chromosome number, 2n = 30. However, the karyotypes of H. nana and H. sanborni differed considerably from each other in the number of submetacentric and telocentric chromosomes. The two species also differed in their primary NOR-bearing chromosomes (metacentric pair 13 in H. nana and telocentric pair 12 in H. sanborni). Additional nucleolus organizer regions (NORs) were detected by Ag-NOR staining and FISH in chromosome pairs 1, 5, 6, 12, and 14 in seven specimens of H. nana. Thus, a total of six patterns of NOR were identified. These differences in karyotype and in NOR location allowed the unambiguous identification of syntopic individuals of the two species. However, the chromosomal morphology of both species differed from that reported for populations from other geographic regions, suggesting that a systematic reevaluation of this group of Hyla may be necessary.     

Allozyme variation among Rana balcanica,R. levantina,and R. ridibunda (Amphibia: Anura)1

The allozyme variation among water frogs of the species R. balcanica, R. levantina and R. ridibunda, all formerly considered as one species (R. ridibunda Pallas, 1771), was studied using horizontal starch gel electrophoresis. Blood samples (N # 63) of frogs were collected from five populations in Greece and Israel. Samples (N = 9) of the hybrid frog R. esculenta collected from a locality in Germany were used as an outgroup for phylogenetic analyses. Fifteen enzymes controlled by twenty presumptive loci were identified. Thirteen loci were polymorphic within or among the studied populations. Genetic differentiation among the species was considerably greater than among populations of the same species. Even at Nestos River where R. ridibunda and R. balcanica occur in the same habitats, individuals could be assigned to either species due to characteristic differences of the genotypes (GPI1). This indicates reproductive isolation among these species. The reconstruction of phylogenetic relationships among the three species based on the allozyme data corroborated the model presented on the basis of bioacoustic data: R. ridibunda and R. balcanica (Nei's genetic distance D = 0.0820) are sibling species pertaining to an Eurasian lineage, whereas R. levantina (distance to the European species D = 0.1780 - 0.1955) together with R. perezi represent an independent afroasian lineage.     

Conserved karyotypes in the Hyla pulchella species group (Anura, Hylidae)

Cytogenetic analyses were done on specimens of Hyla marginata and on three populations of H. semiguttata differing in morphology and in the physical parameters of their advertisement call, as well as in individuals of Hyla sp. (aff. semiguttata). All specimens had 2n=24 chromosomes with a morphology very similar to that of other 24-chromosome Hyla species. Hyla semiguttata and H. marginata showed the same C-banding pattern but were distinguished by the location of the NOR on pair 1 in H. semiguttata (in the three populations) and Hyla sp. (aff. semiguttata), and on pair 10 in H. marginata. The H. semiguttata populations did not differ cytogenetically, despite variations in their morphology and advertisement calls. Similarly, H. semiguttata and H. p. joaquini studied previously had identical C-banding patterns and NOR locations, suggesting that they are very closely related.     

Proximate developmental causes of limb length variation between Hyla cinerea and Hyla gratiosa (Anura: Hylidae)

Hyla cinerea and H. gratiosa are closely related treefrogs that differ at metamorphosis in the length of the hind limb relative to snout vent length. A developmental series of larvae of the two species raised under uniform laboratory conditions reveals that H. cinerea have proportionately longer legs at metamorphosis than H. gratiosa for the following reasons: 1) Hyla cinerea initiate limb growth at a smaller body size than H. gratiosa, 2) H. cinerea have a faster rate of limb growth, and 3) body growth rates in H. cinerea and H. gratiosa are nearly identical until just before metamorphosis.     

The thermal physiology of two sympatric treefrogs Hyla cinerea and Hyla chrysoscelis (Anura; Hylidae)

Metabolic rates, temperature acclimation, lipid deposition and temperature tolerance were investigated in two species of hylid treefrogs, the green treefrog (Hyla cinerea) and the coastal plain (Cope's) gray treefrog (Hyla chrysoscelis). The rate of oxygen consumption at rest differed between the two species only at 30 degrees C; there was no difference in respiratory metabolism at lower ambient temperatures. Hyla cinerea generally completed metabolic acclimation earlier than H. chrysoscelis, particularly at high temperatures; both species appeared to be fully acclimated in 6 days or less. The gray treefrog is less tolerant of high ambient temperatures than the green treefrog; mean upper lethal temperature was 41.5 degrees C for Hyla chrysoscelis and 43.7 degrees C for H. cinerea. Metabolized energy was higher at high ambient temperatures (i.e. 29 degrees C) for H. chrysoscelis than H. cinerea, while the reverse was true at 19 degrees C. The coefficient of utilization (100 X metabolized energy/gross energy intake) did not vary significantly between species or within species over the ambient temperature range of 19-24 degrees C; H. chrysoscelis had a significantly higher efficiency at 29 degrees C. Lipid reserves were generally similar in the two species throughout the summer. Differences in behavior, seasonal variation in activity and timing of reproduction are all related to thermal physiology and may play a role in determining the distributional limits of the two species.     

Isolation and characterization of microsatellite loci for the European tree frog (Hyla arborea)

We developed 11 new microsatellite markers for the European tree frog (Hyla arborea), and tested patterns of polymorphism in 54 adults (27 males and 27 females) from two ponds close to Lausanne (Western Switzerland). One marker was sex linked and two pairs displayed linkage disequilibrium. Comparisons of allele numbers with heterozygosity values support a stepwise-mutation model at neutral equilibrium, with mutation rates spanning nearly two orders of magnitude. These markers will prove useful for population genetic studies and fine-scale investigations requiring genetic assignment techniques.     

Histochemical and Biometric Study of the Gastrointestinal System of Hyla Orientalis (Bedriaga, 1890) (Anura,Hylidae)

This study was carried out to assess the localization of hyaluronic acid (HA) and the distribution of glycoproteins in the gastrointestinal system of adult Hyla orientalis. Histochemical analysis of the gastrointestinal system in H. orientalis showed that mucous content included glycogene and/or oxidable dioles [periodic acid/Schiff (PAS)+], neutral or acid-rich (PAS/AB pH 2.5+), sialic acid residues (KOH/PAS+) and acid sulphate [Aldehyde fuchsin (AF)+] glycoproteins. However the mucus content was not the same in stomach, small and large intestine. The mucus content of stomach included only glycogene and/or oxidable dioles and sialic acid residues. Besides these histochemical methods, the localization of HA was detected using biotinylated hyaluronic acid binding protein labeled with streptavidin-fluorescein isothiocyanate (FITC). In the extracellular matrix of the submucosa, the reaction for HA was evident. Since HA was located in submucosa beneath the epithelial layer of gastrointestinal system, it has a significant role in hydric balance, and essential to provide the gastrointestinal system integrity and functionality. According to biometric results, there were statistical differences between small and large intestine in terms of the amount of material stained positive with PAS/AB, PAS, KOH/PAS and AF/AB. Additionally, number of goblet cells in the small and large intestine was significantly different.Key words: Gastrointestinal system, goblet cell, glycoproteins, hyaluronic acid, amphibian, Hyla.     

Habitat fragmentation causes bottlenecks and inbreeding in the European tree frog (Hyla arborea)

A genetic study of the European tree frog, Hyla arborea, in Denmark was undertaken to examine the population structure on mainland Jutland and the island of Lolland after a period of reduction in suitable habitat and population sizes. The two regions have experienced the same rate of habitat loss but fragmentation has been more severe on Lolland. Genetic variation based on 12 polymorphic DNA microsatellites was analysed in 494 tree frogs sampled from two ponds in Jutland and 10 ponds on Lolland. A significant overall deviation from Hardy-Weinberg expectations could be attributed to three ponds, all on Lolland. This was most probably caused by an inbreeding effect reducing fitness, which was supported by the observed significant negative correlation between larva survival and mean F(IS) value and mean individual inbreeding coefficient. A significant reduction in genetic variation (bottleneck) was detected in most of the ponds on Lolland. Population-structure analysis suggested the existence of at least 11 genetically different populations, corresponding to most of the sampled population units. The results indicated that the populations were unique genetic units and could be used to illustrate the migration pattern between newly established ponds arisen either by natural colonization of tree frogs or by artificial introduction. A high degree of pond fidelity in the tree frogs was suggested. A severe fragmentation process reducing population size and fitness within some of the populations probably caused the significant reduction in genetic variation of tree frog populations on Lolland.