Development and characterization of highly polymorphic microsatellite loci for the Western Spadefoot toad, Pelobates cultripes

Nine highly polymorphic microsatellite markers were isolated and characterized for the Western Spadefoot, Pelobates cultripes. Remarkably, for this amphibian species high numbers of microsatellites were found as part of larger repeat containing regions, making primer design difficult. For nine loci, primers were designed successfully and genotyping of individuals was reliable and consistent. Number of alleles and heterozygosity for these loci ranged from 9 to 34 and from 0.72 to 0.94, respectively. The high levels of polymorphism revealed by our developed loci should provide insight into population genetic structure and levels of dispersal for this typical Mediterranean temporary pond-breeding amphibian.     

High aquatic niche overlap in the newts Triturus cristatus and T. marmoratus (Amphibia,Urodela)

We studied spatial niche metrics of large-bodied newts (Triturus cristatus and T. marmoratus) in three breeding ponds in western France. Adults and larvae were sampled with underwater funnel traps. Larvae were identified to the species with diagnostic microsatellite DNA markers. The distribution of adult T. cristatus and T. marmoratus across pond regions differed in one out of six cases, no differences were observed between larvae (two ponds studied). Niche overlap and niche breadth indices across resource states defined as pond regions or individual traps were high (Schoener's C: pond regions 0.60–0.98, traps 0.35–0.71; Levins' B: pond regions 0.71–0.98, traps 0.35–0.76). Adults of large-bodied newts significantly differed in resource use from small-bodied newts (T. helveticus). The results are discussed in view of the occurrence of interspecific breeding attempts, and the unpredictable ecological characteristics of newt breeding ponds.     

Estimating hydroperiod suitability for breeding amphibians in southern Rhode Island seasonal forest ponds

In New England, seasonal forest ponds provide primary breeding habitat for several amphibian species, including Rana sylvatica (LeConte) and Ambystoma maculatum (Shaw). Because each species requires a minimum duration of inundation to complete its breeding cycle, one of the most important factors determining habitat suitability is a pond’s hydroperiod. The objective of this research was to develop a method for estimating pond hydroperiod from site characteristics such as pond morphology, geology, chemistry, and vegetation structure, and to use the estimates to assess the suitability of individual ponds for breeding amphibians. We monitored the duration of surface inundation in 65 ponds in the Pawcatuck River watershed of southern Rhode Island during 2001 and 2002. Pond hydroperiods, measured from 1 March, ranged from 19 to 44 weeks in 2001 and from 2 to 44 weeks in 2002; mean values were 30 and 21 weeks, respectively. Akaike’s Information Criterion was used to select a multivariate hydroperiod estimation model (R² = 0.59, p < 0.0001) that permitted identification of ponds with hydroperiods suitable for breeding by R. sylvatica (95.4% correct classification rate [CCR]) and A. maculatum (75.4% CCR). Canopy cover, open basin depth, and specific conductance of surface water were among the most useful site characteristics for estimating hydroperiod, while surficial geology and the texture of soil parent material made smaller contributions. The CCR using open basin depth alone was 95.4 and 73.8%, respectively. These findings indicate that it is possible to estimate the hydroperiod of seasonal ponds – and to assess their suitability for individual species of breeding amphibians – without prolonged periods of hydrologic monitoring. Such techniques could have considerable value to wetland regulatory agencies and for planning amphibian habitat management and acquisition at the landscape scale.     

The juxtaglomerular apparatus in the mesonephros of Newt (Triturus cristatus)

Summary 1)As in mammals, the juxtaglomerular apparatus of the Newt (Triturus cristatus) is composed by cells of the media of the afferent glomerular arteriole and by cells of the intermediary tubule. 2) The cells of the media of the glomerular arteriole are of two different types: granular and agranular cells. 3) The intermediary tubule is formed by dark and light cells. 4) Part of interrenal body is located close to glomerular arteriole and intermediary tubule.This work was supported by grant of Consiglio Nazionale delle Ricerche of Italy (C.N.R.) N. 115/815/04677.     

Osmium Zinc Iodide staining of Golgi elements in oocytes of Triturus cristatus

Summary Developing oocytes of the newt Triturus cristatus were studied in order to clarify the role played by the Golgi apparatus in the formation of yolk. The cytochemical method used for this purpose was that of Maillet (1968) which employs an Osmium Zinc Iodide (OZI) complex.Previtellogenic oocytes reveal a pattern of OZI staining only after hormonal (HCG) stimulation, following which both the Golgi apparatus and the multivesicular bodies are stained.Vitellogenic oocytes taken from non-hormonally stimulated females reveal OZI deposits in a number of vesicles peripheral to the Golgi apparatus as well as within the superficial layer of the forming yolk platelets. Following hormone stimulation, many of the Golgi apparatus located in the central ooplasm of vitellogenic oocytes have all their cisternae blackened by the OZI deposits; other apparatuses, more peripherally located, remain essentially unchanged in their staining pattern. Further, a large number of OZI stained vesicles becomes visible in the vicinity of the Golgi apparatus and within the superficial layer of the forming yolk platelets.The present findings are interpreted as indicating the occurrence of fusion between Golgi derived vesicles and forming yolk platelets. It is also suggested that the vesicles in question function as carriers of Golgi produced enzymes which are presumably required to accomplish the final elaboration of the yolk material.Supported by a grant from the Consiglio Nazionale delle RicercheWe acknowledge the valuable help received from Prof. G. Mancino throughout this investigation     

Delineating fine-scale genetic units in amphibians:Probing the primacy of ponds

The population structure of pond-breeding amphibians is shaped by their distinct breeding foci, but it is unclear to what extent this is reflected in the fine-scale distribution of genetic diversity. We used microsatellite genotypes to investigate the genetic signatures of 24 populations of European newts, Triturus cristatus and T. marmoratus, inhabiting 21 ponds in a confined study area (7.5×3.5km) in western France. Employing a Bayesian clustering approach based on individual genotypes that minimises departures from Hardy–Weinberg equilibrium and linkage disequilibrium, no evidence was found for within-pond substructuring. Subjecting all sampled ponds simultaneously to this procedure revealed a clear signal of partitioning, with the most likely number of clusters however below the actual number of ponds (seven in T. cristatus, three in T. marmoratus). A more hierarchical Bayesian approach, with pond as analysis unit, was achieved to separate ponds from genetically more meaningful units, and reduced the T. cristatus populations to 11 clusters, and the T. marmoratus populations to five clusters. We were unable to specify a minimum nearest-neighbour distance where ponds are separate units, probably due to both historical and current demographic processes. The implications for strategies to manage and conserve endangered amphibians in human-altered landscapes are discussed.     

Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians

Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation.     

Comparison of melatonin-binding sites in the brain of two amphibians: an autoradiographic study

Neuroanatomic comparison of the binding capability of 2-[¹²⁵I] iodomelatonin in the crested newt Triturus carnifex Laur. and the green frog Rana esculenta, using quantitative autoradiographic techniques, revealed a heterogeneous distribution pattern. The highest and relatively high binding activities were shown to occur in the optic tracts and in the suprachiasmatic area of the hypothalamus and the optic tectum, respectively, of both species. Low or no 2-[¹²⁵I] iodomelatonin binding values were obtained in the preoptic nucleus, the tuberal hypothalamus, the medulla oblongata, the septum and the dorsal pallium. A differential binding pattern was observed in the amygdaloid nucleus pars lateralis, the striatum and the hindbrain of these amphibians. Indeed, notably high binding levels were shown to occur in the former two brain areas of the crested newt, whereas high levels were displayed in the latter brain region of the green frog. On the basis of elevated quantities of melatonin receptors in mesencephalic, hypothalamic and telencephalic sites, it seems plausible to ascribe some important sensory functions to this receptor system in both species. The remarkably different binding activities in the brain of the two amphibians could be correlated with the simpler cytoarchitectonic brain structure of urodeles and with species-specific variations.     

Impacts of hydroperiod on growth and survival of larval amphibians in temporary ponds of Central Pennsylvania,USA

The effects of variable hydroperiod (three levels) and initial density of amphibians (two levels) on survival, growth rate, and time to and mass at metamorphosis were studied for wood frogs (Rana sylvatica), Jefferson salamanders (Ambystoma jeffersonianum), and spotted salamanders (A. maculatum). Experiments were carried out in 260-1 mesocosms set up outdoors in a forest. These pond simulations were designed to mimic conditions that occur in palustrine temporary wetlands in central Pennsylvania. No animals reached metamorphosis in the short hydroperiod (56 days). However a greater proportion (66%) of tadpoles of R. sylvatica survived to the end of the 56-day, treatment than the 84- or 158-day treatments (29 and 14%, respectively), from which all survivors metamorphosed. In contrast, neither of the salamanders metamorphosed by 84 days; survival to metamorphosis at 158 days was 15% for A. jeffersonianum and 10% for A. maculatum. Average instantaneous growth rates for A. jeffersonianum decreased with each increase in hydroperiod. Growth of R. sylvatica was greater in the 56-day hydroperiod than in hydroperiods of 84 or 158 days. Initial amphibian density had no effect on growth or survival of any species. It appears that salamander larvae were predatory on tadpoles, since survival of R. sylvatica was negatively correlated with survival of A. jeffersonianum in 84-day treatments and with growth of A. maculatum in 158-day treatments.     

Effets de modifications de la composition électrolytique du plasma sur la structure et la fonction des cellules interrénales (corticosurrénaliennes) d'un Amphibien Urodèle: Triturus cristatus

Résumé Dans le but de modifier la composition électrolytique du plasma, des Tritons à crête (Triturus cristatus) sont placés dans une solution de chlorure de potassium (KCl 5). D'autres Tritons (témoins) sont placés dans de l'eau du robinet. Tous les animaux ont été préalablement hypophysectomisés.8 jours après le début de l'expérience, les cellules interrénales des Tritons hypophysectomisés placés dans la solution de KCl présentent une activité stéroidogénique élevée, se traduisant notamment par une forte déplétion lipidique, une augmentation du nombre des mitochondries et un développement important du réticulum endoplasmique agranulaire et tubulaire; d'autre part, la réaction 5-3-hydroxystéroide deshydrogénasique est intense. Toutes les cellules du tissu interrénal sont fortement stimulées dans ces conditions.Les cellules interrénales des Tritons témoins présentent par contre une activité stéroidogénique très réduite, comme en témoignent leur structure et la faible intensité de la réaction 5-3-hydroxystéroide deshydrogénasique.Les mécanismes précis pouvant être à l'origine de la stimulation des cellules interrénales de Tritons hypophysectomisés placés dans la solution de KCl sont discutés.

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Effects of modifications of plasma electrolyte composition on the structure and function of the interrenal (adrenocortical) cells of the newt, Triturus cristatus

Summary In order to induce a modification of the plasmatic electrolyte composition, newts (Triturus cristatus) are placed into a potassium chloride solution (KCl 5), while controls are kept in tap water. All animals are previously hypophysectomized.8 days after the beginning of the experiment, the interrenal cells of the hypophysectomized newts kept in KCl solution exhibit a very strong steroidogenic activity, as evidenced by a striking lipid depletion, an increase in the number of mitochondria and a considerable development of the agranular tubular endoplasmic reticulum. On the other hand, the 5-3-hydroxysteroid dehydrogenase reaction is notably intensified. All the cells of the interrenal tissue are affected by the stimulatory effect of the KCl solution.Control animals show only reduced steroidogenic activity of their interrenal cells, as demonstrated both by the morphology of these cells and the weak 5-3-hydroxysteroid dehydrogenase reaction.The mechanisms involved in the stimulatory effect on steroidogenesis of the variation of the electrolyte composition of the plasma are discussed.

     

Greening of the coasts: a review of the Perna viridis success story

The green mussel Perna viridis has been receiving a lot of attention from workers working in the research areas of intertidal ecology, aquaculture, pollution monitoring, biofouling, zoogeography and invasion biology. P. viridis is a remarkable species in terms of its ability to reach very high biomass levels, to withstand environmental fluctuations, to concentrate a variety of organic and inorganic environmental pollutants, to colonise artificial marine habitats and to invade new geographic territories. This review collates data available on salient aspects of the distribution, biology and ecology of P. viridis. It is argued that the remarkable success of P. viridis as an invasive species basically stems from its long larval duration, fast growth rate, high fecundity, early maturity, high productivity and ability to withstand fluctuating environmental conditions (temperature, salinity, water turbidity and pollutants). Relevant aspects of the data are compared with the data available for a similar species Perna perna, which too is an invasive species, but to a more limited extent.     

The declining Spadefoot toad, Pelobates fuscus (Pelobatidae): paleo and recent environmental changes as a major influence on current population structure and status

Populations of the European Spadefoot toad (Pelobates fuscus) have experienced recent declines all over Europe, but these appear to be more intense in north and western Europe. Due to the toad’s fossorial nature and specific habitat requirements, environmental conditions have played a major role in structuring current populations. We examined the phylogeographic structure in P. fuscus from 16 localities throughout Europe using mitochondrial cytochrome b gene sequence analysis. Sequence divergence among haplotypes was low (0.54±0.15%). Three very closely related haplotypes occupy northern and western parts of Europe whereas 12 others were observed among samples from south-eastern Europe, including the Balkans. Our results suggest that toads only recently colonized the northern and western parts of Europe following glacial retreat. This expansion probably took place in steppic-like areas during the younger Dryas cold interval, about 12,900–11,500 years ago. Restricted gene flow with an isolation-by-distance population structure characterises a major part of its distribution range. Based on our results we suggest that the northern and western lineages should be considered as distinct conservation units, while the south-eastern populations from the refugial areas, where nearly all genetic polymorphism occurs and populations appear less vulnerable, should receive special attention.     

Diameter class structure and sprouting characteristics of a northernmost Euptelea pleiosperma population in China: Implications for conservation

Euptelea pleiosperma (Eupteleaceae) is a class III threatened tree species in China. Investigations on structure, regeneration and population dynamics is of great scientific significance to properly conserve, develop and use the resources of this species. This study targeted a small and isolated northernmost E. pleiosperma population in China, which is distributed in Meidigou of Taikuanhe Nature Reserve in Shanxi province. The study aimed at seeking the answers: (1) is the E. pleiosperma population growing or declining? (2) what is the implications of sprouting for the E. pleiosperma population? It is shown through this study that sprouting is a main trait of the population with 49.4% of main stems having sprouted stems. The population is growing rather than declining thanks to the outstanding sprouting trait of E. pleiosperma. The population occurs only in a small shady and moisture valley area most likely because a moist environment is necessary for E. pleiosperma to survive, develop and regenerate. Mainly by rapid producing abundant sprouted stems, E. pleiosperma forms itself as an edifying species and demonstrates strong competition ability with other coexistent canopy species in the limited area. The sprouting characteristics of E. pleiosperma provide a mechanism for the expansion and maintenance of its population.     

The distribution of GABA-like-immunoreactive neurons in the brain of the newt,Triturus cristatus carnifex,and the green frog,Rana esculenta

Summary The distribution of -aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cell bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.Dedicated to Professor Valdo Mazzi (Dipartimento di Biologia Animale, Università di Torino), in honor of his 70th birthday     

Predicting establishment success for alien reptiles and amphibians: a role for climate matching

We examined data comprising 1,028 successful and 967 failed introduction records for 596 species of alien reptiles and amphibians around the world to test for factors influencing establishment success. We found significant variations between families and between genera. The number of jurisdictions where a species was introduced was a significant predictor of the probability the species had established in at least one jurisdiction. All species that had been introduced to more than 10 jurisdictions (34 species) had established at least one alien population. We also conducted more detailed quantitative comparisons for successful (69 species) and failed (116 species) introductions to three jurisdictions (Great Britain, California and Florida) to test for associations with climate match, geographic range size, and history of establishment success elsewhere. Relative to failed species, successful species had better climate matches between the jurisdiction where they were introduced and their geographic range elsewhere in the world. Successful species were also more likely to have high establishment success rates elsewhere in the world. Cross-validations indicated our full model correctly categorized establishment success with 78–80% accuracy. Our findings may guide risk assessments for the import of live alien reptiles and amphibians to reduce the rate new species establish in the wild.     

Long-term persistence of aspen – a key host for many threatened species – is endangered in old-growth conservation areas in Finland

Large, dead and dying European aspens (Populus tremula L.) host many threatened species in Fennoscandian boreal forests. Large aspen trees have mostly disappeared and are being harvested from the managed forests that cover 95% of the forest area in Finland. Due to the small area protected (4.1%), the aspen-associated species may encounter major difficulties in the protected areas if aspen trees disappear due to natural forest succession. The availability of aspens was assessed in the old-growth conservation area network in eastern Finland. We mapped all the living and dead aspens in 15 protected old-growth forests. The total number of counted trees was 32 903 individuals. Current amounts of living (2.7 m³/ha) and especially dead aspens (2.8 m³/ha) in the protected areas were higher than in the surrounding managed forests (1.1 and 0.1 m³/ha for living and dead trees, respectively). However, while saplings (dbh<5 cm) occur in most of the areas (12 individuals/ha on average) they survive poorly and young aspen cohorts (5 cm <dbh<15 cm) are lacking or are very rare. The most likely reason for the poor sapling survival is high browsing pressure by the mammalian herbivores, especially the moose. The moose population has increased many times in Finland during the past decades. The poor regeneration of aspens implies that the value of the old-growth conservation areas for aspen-associated species will face a serious bottleneck within a few decades when the currently middle-aged tree cohorts disappear. If the current high browsing pressure and lack of natural disturbances continue the obligatory aspen-associated species may disappear both locally and regionally from the network of the protected areas.     

Forest fragmentation and plant reproductive success: a case study in four perennial herbs

We assessed the effects of habitat fragmentation on reproductive success in natural populations of four forest herbs with differing life-history traits and whose distribution patterns appeared to be negatively affected by decreased habitat size and/or increased isolation: Carex sylvatica, Galium odoratum, Sanicula europaea and Veronica montana. Our aims were to test (1) whether habitat size and isolation are positively correlated with population size and isolation, respectively, (2) whether plant reproductive success, a major component of plant fitness, is reduced in small and/or isolated populations when also accounting for differences in habitat quality (edaphic conditions, light intensity) and the effects of plant size, and (3) whether species with different life histories are affected differently. There were significant positive relationships between habitat and population size and between habitat and population isolation in some, but not all of the species. We mostly found no negative effects of small population size or isolation on reproduction. However, reproductive success was reduced in small populations of Sanicula, and this effect was independent of differences in plant size and environmental conditions. The reduced fecundity in small populations may be a consequence of the Allee-effect, a possible mechanism being pollen limitation. Furthermore, the proportion of flowering ramets was reduced in small and isolated populations of Galium, which may have been caused by changes in population structure. Lastly, we found some evidence for largely outcrossing, non-clonal species to be more sensitive to reductions in population size, at least in terms of their reproductive success.     

Experimental investigation of the effect of spatial aggregation on reproductive success in a rewardless orchid

Plant reproductive success within a patch may depend on plant aggregation through pollinator attraction. For rewardless plants that lack rewards for pollinators, reproductive success may rely strongly on the learning abilities of pollinators. These abilities depend on relative co-flowering rewarding and rewardless plant species spatial distributions. We investigated the effect of aggregation on the reproductive success of a rewardless orchid by setting up 16 arrays in a factorial design with two levels of intraspecific aggregation for both a rewardless orchid and a rewarding co-flowering species. Our results show that increasing aggregation of both species negatively influenced the reproductive success of the rewardless plants. To our knowledge, this is the first experimental study demonstrating negative effects of aggregation on reproductive success of a rewardless species due both to its own spatial aggregation and that of a co-flowering rewarding species. We argue that pollinator learning behaviour is the key driver behind this result.The authors declare that the experiments presented here comply with the current laws applicable in the country in which they were performed (France).