Distal dendrites of frog motor neurons: a computer-aided electron microscopic study of cobalt-filled cells.

With the aid of the cobalt labelling technique, frog spinal cord motor neuron dendrites of the subpial dendritic plexus have been identified in serial electron micrographs. Computer reconstructions of various lengths (2.5-9.8 micron) of dendritic segments showed the contours of these dendrites to be highly irregular, and to present many thorn-like projections 0.4-1.8 micron long. Number, size and distribution of synaptic contacts were also determined. Almost half of the synapses occurred at the origins of the thorns and these synapses had the largest contact areas. Only 8 out of 54 synapses analysed were found on thorns and these were the smallest. For the total length of reconstructed dendrites there was, on average, one synapse per 1.2 micron, while 4.4% of the total dendritic surface was covered with synaptic contacts. The functional significance of these distal dendrites and their capacity to influence the soma membrane potential is discussed.

A concentric analysis of the impact of urbanization on the threatened European tree frog in an agricultural landscape

Pond-breeding amphibians are affected by site-specific factors and regional and landscape-scale patterns of land use. Recent anthropogenic landscape modifications (drainage, agriculture intensification, larger road networks, and increased traffic) affect species by reducing the suitable habitat area and fragmenting remaining populations. Using a robust concentric approach based on permutation tests, we evaluated the impact of recent landscape changes on the presence of the endangered European tree frog (Hyla arborea.) in wetlands. We analyzed the frequency of 1 traffic and 14 land-use indices at 20 circular ranges (from 100-m up to 2-km radii) around 76 ponds identified in western Switzerland. Urban areas and road surfaces had a strong adverse effect on tree frog presence even at relatively great distances (from 100 m up to 1 km). When traffic measurements were considered instead of road surfaces, the effect increased, suggesting a negative impact due to a vehicle-induced effect. Altogether, our results indicate that urbanization and traffic must be taken into account when pond creation is an option in conservation management plans, as is the case for the European tree frog in western Switzerland. We conclude that our easy-to-use and robust concentric method of analysis can successfully assist managers in identifying potential sites for pond creation, where probability of the presence of tree frogs is maximized.

Genetic differentiation in two European tree frog (Hyla arborea) metapopulations in contrasted landscapes of Western Switzerland

The survival of threatened species as the European tree frog (Hyla arborea) is strongly dependent on the genetic variability within populations, as well as gene flow between them. In Switzerland, only two sectors in its western part still harbour metapopulations. The first is characterised by a very heterogeneous and urbanized landscape, while the second is characterised by a uninterrupted array of suitable habitats. In this study, six microsatellite loci were used to establish levels of genetic differentiation among the populations from the two different locations. The results show that the metapopulations have: (i) weak levels of genetic differentiation (FST within metapopulation ≈ 0.04), (ii) no difference in levels of genetic structuring between them, (iii) significant (p = 0.019) differences in terms of genetic diversity (Hs) and observed heterozygozity (Ho), the metapopulation located in a disturbed landscape showing lower values. Our results suggest that even if the dispersal of H. arborea among contiguous ponds seems to be efficient in areas of heterogeneous landscape, a loss of genetic diversity can occur.

Conservation of a threatened European tree frog (Hyla arborea) metapopulation

Summary: Amphibians are among the most vulnerable animals of the world. One third of all species are currently threatened with extinction. Habitat loss is the major menace to pond- and stream-breeding species in the old world. In highly urbanized landscape like the Swiss Plateau, most species suffer from habitat reduction and fragmentation. Among all indigenous species, the European tree frog (Hyla arborea L., 1758) is one of the most endangered. It experienced an alarming decline during the last century and its regional long-term persistence is not guaranteed. We developed a monitoring framework based on calling male counts which included multiple visits to each wetland during the reproduction period in order to precisely determine its distribution on the Lemanic coast. Our results indicate that visiting populations 3 limes under suitable climatic conditions (temperature >20°C) provides reliable presence/absence data. Based on our monitoring data, we analyzed the species requirements regarding its breeding habitat. It appeared that anthropogenic activities had paradoxical effects on the species. On one hand, urbanization, traffic and intensive agriculture had a strong detrimental effect on tree frog distribution. On the other hand, large tree frog populations were frequently associated with gravel pits and military training grounds. Our results allowed us to create a habitat suitability map taking into account detrimental landscape elements around ponds (>1100m away from urban areas and >500m away from first class roads). In parallel, we developed a metapopulation model of the European tree frog in order to identify the critical threats to the long term persistence of the species. Our results indicated that suitable pond density is at the low end of the species requirements. Pond creation must therefore be considered an essential complementary approach to pond conservation and restoration. Our model also provided a mapping solution permitting the location of the must suitable area for pond creation from a metapopulation perspective. As many other amphibians, the European tree frog is not only exposed to an aquatic habitat (breeding and larval period), but also to a terrestrial stage (summer and overwintering habitats). Unfortunately, animals in their terrestrial phase are less conspicuous and, as a consequence, their terrestrial needs are relatively unknown. Using a recent tracking method (the Harmonic Direction Finder), we followed post-breeding frogs and identified favored terrestrial habitats, thus providing another practical conservation tool. We conclude that only the combination of multiple spatially explicit approaches (landscape-scale habitat suitability, metapopulation dynamics and terrestrial needs) is likely to provide wildlife managers with effective tools for the conservation of highly endangered amphibians. Résumé: Les amphibiens font partie des animaux les plus vulnérables du monde. Un tiers des espèces est actuellement menacé d'extinction. Dans l'ancien monde, la disparition des habitats constitue la principale menace pour les grenouilles, crapauds, tritons et salamandres. Dans les paysages fortement urbanisés comme le Plateau Suisse, la plupart des espèces souffrent d'une réduction et d'une fragmentation de leurs habitats. Parmi toutes les espèces indigènes, la rainette verte (Hyla arborea L., 1758) est l'une des plus menacée. Sa distribution a régressé de manière alarmante durant le siècle passé et sa survie régionale à long terme n'est pas assurée. Nous avons développé une méthode de suivi des populations se basant sur le comptage des mâles chanteurs durant la période de reproduction. Cette méthode requiert plusieurs visites à chaque plan d'eau de manière à déterminer précisément la distribution de l'espèce. Nos résultats démontrent que 3 visites par population dans des conditions climatiques favorable (température >20°C) permettent d'obtenir des données de présence/ absence valables. Sur la base de nos comptages sur la Côte lémanique, nous avons analysé les exigences de l'espèce concernant ses sites de reproduction. Il est apparu que les activités humaines avaient un effet paradoxal sur l'espèce. D'une part, l'urbanisation, le trafic routier et l'intensification de l'agriculture ont un effet fortement préjudiciable, tandis que d'autre part les plus grandes populations sont souvent associées à des gravières et autres places d'armes. Nos résultats ont permis de créer une carte de qualité d'habitat prenant en compte les éléments paysagers préjudiciables à la rainette (situé à plus de 1100m de zones urbaines et à plus de 500m de routes de première classe). En parallèle, nous avons développé un modèle métapopulationnel (incluant l'ensemble des populations) de manière à identifier les menaces prépondérantes sur la survie à long terme de l'espèce. Nos résultats ont permis de déterminer que la densité actuelle de plans d'eau adéquats est à la limite inférieure des exigences de l'espèce. La création d'étangs doit donc être considérée comme une approche indispensable et complémentaire à la protection et à la restauration des sites existants. Notre modèle a également fourni des résultats cartographiables permettant l'identification des sites les plus appropriés dans une perspective métapopulationnelle. Comme de nombreux autres amphibiens, la rainette verte est exposée à un habitat aquatique (reproduction et développement larvaire) ainsi qu'à un habitat terrestre (été et hiver). Les animaux étant particulièrement cryptiques dans cette seconde phase, leurs besoins terrestres sont relativement mal connus. Nous avons donc développé une nouvelle méthode de télémétrie basée sur le goniomètre harmonique. Cette méthode nous a permis de suivre des rainettes dans leurs migrations jusqu'à leurs habitats d'été et d'établir ainsi des recommandations pratiques pour la conservation de la rainette. Nous concluons que la combinaison de multiples approches spatialement explicites (qualité d'habitat, dynamique de métapopulation et habitats terrestres) est seule à même de produire des outils efficaces pour la conservation des espèces menacées d'amphibiens.

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.

Within-population polymorphism of sex-determination systems in the common frog (Rana temporaria).

In sharp contrast with birds and mammals, the sex chromosomes of ectothermic vertebrates are often undifferentiated, for reasons that remain debated. A linkage map was recently published for Rana temporaria (Linnaeus, 1758) from Fennoscandia (Eastern European lineage), with a proposed sex-determining role for linkage group 2 (LG2). We analysed linkage patterns in lowland and highland populations from Switzerland (Western European lineage), with special focus on LG2. Sibship analyses showed large differences from the Fennoscandian map in terms of recombination rates and loci order, pointing to large-scale inversions or translocations. All linkage groups displayed extreme heterochiasmy (total map length was 12.2 cM in males, versus 869.8 cM in females). Sex determination was polymorphic within populations: a majority of families (with equal sex ratios) showed a strong correlation between offspring phenotypic sex and LG2 paternal haplotypes, whereas other families (some of which with female-biased sex ratios) did not show any correlation. The factors determining sex in the latter could not be identified. This coexistence of several sex-determination systems should induce frequent recombination of X and Y haplotypes, even in the absence of male recombination. Accordingly, we found no sex differences in allelic frequencies on LG2 markers among wild-caught male and female adults, except in one high-altitude population, where nonrecombinant Y haplotypes suggest sex to be entirely determined by LG2. Multifactorial sex determination certainly contributes to the lack of sex-chromosome differentiation in amphibians.

Immunocytochemical and pharmacological characterization of metabotropic glutamate receptors of the vestibular end organs in the frog.

Using immunocytochemistry and multiunit recording of afferent activity of the whole vestibular nerve, we investigated the role of metabotropic glutamate receptors (mGluR) in the afferent neurotransmission in the frog semicircular canals (SCC). Group I (mGluR1alpha) and group II (mGluR2/3) mGluR immunoreactivities were distributed to the vestibular ganglion neurons, and this can be attributed to a postsynaptic locus of metabotropic regulation of rapid excitatory transmission. The effects of group I/II mGluR agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) on resting and chemically induced afferent activity were studied. ACPD (10-100 microM) enhanced the resting discharge frequency. MCPG (5-100 microM) led to a concentration-dependent decrease of both resting activity and ACPD-induced responses. If the discharge frequency had previously been restored by L-glutamate (L-Glu) in high-Mg2+ solution, ACPD elicited a transient increase in the firing rate in the afferent nerve suggesting that ACPD acts on postsynaptic receptors. The L-Glu agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA), were tested during application of ACPD. AMPA- and NMDA-induced responses were higher in the presence than absence of ACPD, implicating mGluR in the modulation of ionotropic glutamate receptors. These results indicate that activation of mGluR potentiates AMPA and NMDA responses through a postsynaptic interaction. We conclude that ACPD may exert modulating postsynaptic effects on vestibular afferents and that this process is activity-dependent.

Cryptic diversity among Western Palearctic tree frogs: postglacial range expansion, range limits, and secondary contacts of three European tree frog lineages (Hyla arborea group).

We characterize divergence times, intraspecific diversity and distributions for recently recognized lineages within the Hyla arborea species group, based on mitochondrial and nuclear sequences from 160 localities spanning its whole distribution. Lineages of H. arborea, H. orientalis, H. molleri have at least Pliocene age, supporting species level divergence. The genetically uniform Iberian H. molleri, although largely isolated by the Pyrenees, is parapatric to H. arborea, with evidence for successful hybridization in a small Aquitanian corridor (southwestern France), where the distribution also overlaps with H. meridionalis. The genetically uniform H. arborea, spread from Crete to Brittany, exhibits molecular signatures of a postglacial range expansion. It meets different mtDNA clades of H. orientalis in NE-Greece, along the Carpathians, and in Poland along the Vistula River (there including hybridization). The East-European H. orientalis is strongly structured genetically. Five geographic mitochondrial clades are recognized, with a molecular signature of postglacial range expansions for the clade that reached the most northern latitudes. Hybridization with H. savignyi is suggested in southwestern Turkey. Thus, cryptic diversity in these Pliocene Hyla lineages covers three extremes: a genetically poor, quasi-Iberian endemic (H. molleri), a more uniform species distributed from the Balkans to Western Europe (H. arborea), and a well-structured Asia Minor-Eastern European species (H. orientalis).

The contribution of patch topology and demographic parameters to PVA predictions: the case of the European tree frog

Population viability analyses (PVA) are increasingly used in metapopulation conservation plans. Two major types of models are commonly used to assess vulnerability and to rank management options: population-based stochastic simulation models (PSM such as RAMAS or VORTEX) and stochastic patch occupancy models (SPOM). While the first set of models relies on explicit intrapatch dynamics and interpatch dispersal to predict population levels in space and time, the latter is based on spatially explicit metapopulation theory where the probability of patch occupation is predicted given the patch area and isolation (patch topology). We applied both approaches to a European tree frog (Hyla arborea) metapopulation in western Switzerland in order to evaluate the concordances of both models and their applications to conservation. Although some quantitative discrepancies appeared in terms of network occupancy and equilibrium population size, the two approaches were largely concordant regarding the ranking of patch values and sensitivities to parameters, which is encouraging given the differences in the underlying paradigms and input data.

Geographic variation in sex-chromosome differentiation in the common frog (Rana temporaria).

In sharp contrast with birds and mammals, sex-determination systems in ectothermic vertebrates are often highly dynamic and sometimes multifactorial. Both environmental and genetic effects have been documented in common frogs (Rana temporaria). One genetic linkage group, mapping to the largest pair of chromosomes and harbouring the candidate sex-determining gene Dmrt1, associates with sex in several populations throughout Europe, but association varies both within and among populations. Here, we show that sex association at this linkage group differs among populations along a 1500-km transect across Sweden. Genetic differentiation between sexes is strongest (FST  = 0.152) in a northern-boreal population, where male-specific alleles and heterozygote excesses (FIS  = -0.418 in males, +0.025 in females) testify to a male-heterogametic system and lack of X-Y recombination. In the southernmost population (nemoral climate), in contrast, sexes share the same alleles at the same frequencies (FST  = 0.007 between sexes), suggesting unrestricted recombination. Other populations show intermediate levels of sex differentiation, with males falling in two categories: some cluster with females, while others display male-specific Y haplotypes. This polymorphism may result from differences between populations in the patterns of X-Y recombination, co-option of an alternative sex-chromosome pair, or a mixed sex-determination system where maleness is controlled either by genes or by environment depending on populations or families. We propose approaches to test among these alternative models, to disentangle the effects of climate and phylogeography on the latitudinal trend, and to sort out how this polymorphism relates to the 'sexual races' described in common frogs in the 1930s.

Synapses on motoneuron dendrites in the brachial section of the frog spinal cord: a computer-aided electron microscopic study of cobalt-filled cells.

Cobalt-labelled motoneuron dendrites of the frog spinal cord at the level of the second spinal nerve were photographed in the electron microscope from long series of ultrathin sections. Three-dimensional computer reconstructions of 120 dendrite segments were analysed. The samples were taken from two locations: proximal to cell body and distal, as defined in a transverse plane of the spinal cord. The dendrites showed highly irregular outlines with many 1-2 microns-long 'thorns' (on average 8.5 thorns per 100 microns 2 of dendritic area). Taken together, the reconstructed dendrite segments from the proximal sites had a total length of about 250 microns; those from the distal locations, 180 microns. On all segments together there were 699 synapses. Nine percent of the synapses were on thorns, and many more close to their base on the dendritic shaft. The synapses were classified in four groups. One third of the synapses were asymmetric with spherical vesicles; one half were symmetric with spherical vesicles; and one tenth were symmetric with flattened vesicles. A fourth, small class of asymmetric synapses had dense-core vesicles. The area of the active zones was large for the asymmetric synapses (median value 0.20 microns 2), and small for the symmetric ones (median value 0.10 microns 2), and the difference was significant. On average, the areas of the active zones of the synapses on thin dendrites were larger than those of synapses on large calibre dendrites. About every 4 microns 2 of dendritic area received one contact. There was a significant difference between the areas of the active zones of the synapses at the two locations. Moreover, the number per unit dendritic length was correlated with dendrite calibre. On average, the active zones covered more than 4% of the dendritic area; this value for thin dendrites was about twice as large as that of large calibre dendrites. We suggest that the larger active zones and the larger synaptic coverage of the thin dendrites compensate for the longer electrotonic distance of these synapses from the soma.

Multiscale determinants of tree frog (Hyla arborea L.) calling ponds in western Switzerland

A tree frog (Hyla arborea L., 1758) metapopulation in western Switzerland was studied during spring 2001. All potential calling ponds in an area of 350 km(2) were searched for tree frog calling males. Twenty-nine out of 111 ponds sheltered between 1 and 250 callers. Most ponds were occupied by less than 12 males. Pond parameters were measured at three different levels using field analysis and a Geographical Information System (GIS). The first level was water chemistry and pond-associated measures. The second level was the surrounding land use in a 30 m buffer around the pond. The third level consisted of landscape indices on a broader scale (up to 2 km). Logistic regression was applied to identify parameters that can predict the presence of calling males in a pond. Response variable was the presence or absence of callers. Four significant parameters allowed us to explain about 40% of the total deviance of the observed occupational pattern. Urbanization around the pond had a highly negative impact on the probability of presence of calling males. Hours of direct sunlight on the pond was positively correlated with callers. Higher water conductivity was associated with a lesser probability of species presence. Finally, the further the closest two-lane road, the higher the probability of callers presence. Our results show that presence or absence of callers is influenced by factors acting at various geographical scales.

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.