Macroenvironmental factors as ultimate determinants of distribution of common toad and natterjack toad in the south of Spain

We have analysed the relations between the distribution areas of Bufo bufo and Bufo calamita in the south of the Iberian Peninsula. In order to characterise the localities where the species were found, we used 24 environmental variables, and we tested their influence on the distribution of both species by nonparametric methods. By means of logistic regression we calculated the odds of finding one or other species in a locality. The environmental parameters that increase the probability of presence of each species and of both species together are related with the climatic stability and with the climatic subregions. Bufo bufo is more likely to be found in areas where the climate is more predictable, probably because in these areas it may exert its competitive superiority over B. calamita . In zones with very low climatic stability B. calamita is more likely to be found than B. bufo , probably because B. bufo lacks the ability to adapt to unpredictable conditions. In areas with intermediate climatic predictability B. bufo is present, but it would be prevented from removing B. calamita , and so the odds of finding each species are equilibrated. In these areas there would be a balance between the superior competitiveness of B. bufo and the higher adaptability of B. calamita . With the same logistic equation we characterise the typical habitats of each species, and also the environments shared by both species.     

A comparison of the fecundities of two species of toad (Bufo bufo and B. calamita) from different habitat types in Britain

The fecundities of Natterjack toads, Bufo calamita , on a Cumbrian dune system and a relict heathland site in Hampshire were compared and contrasted with the fecundity of Common toads, B. bufo , on the Cumbrian dunes. For both species, fecundity was positively correlated with length, while in B. calamita length was also correlated with size of eggs produced, and age of the female. Bufo calamita was found to invest more energy exponentially in egg production with increasing length, and therefore age. This species also laid more than twice as many eggs as comparably-sized female B. bufo , and some probably produced two clutches of spawn per year in southern England. The enhanced fecundity of the species was believed to be an adaptation to living in areas with unpredictable spawning sites. Small female B. bufo on the dunes appeared to put more energy into egg production when small than individuals of the same species on other habitat types in Britain, possibly as a result of reduced life expectancy on the dune habitat. There was a link between old age and the production of non-viable spawn in B. calamita , and males of this species which spawned at the end of the prolonged breeding season were found to have reduced fertilization efficiency when spawning on consecutive evenings.     

Morphological and life history responses to predators in larvae of seven anurans

Amphibian larvae often face two major sources of mortality: pond desiccation and predation. Tadpoles of seven anuran species with different preferences for type of breeding habitat, on a hydroperiod scale, were tested for responses to the presence of predators by raising them experimentally in the presence and absence of a separately caged invertebrate predator that was fed on conspecific tadpoles. The species typically breeding in temporary or semi-permanent ponds ( Rana temporaria , Rana arvalis , Rana dalmatina and Hyla arborea ) – where invertebrate predator populations are predicted to vary considerably spatiotemporally – all showed marked induced increases in tail fin depth in response to predator presence. These species also tended to respond by reduced growth rates. The representative of the most ephemeral habitats, Bufo calamita , did not respond in any of these traits. Its congeneric, Bufo bufo , a toxic inhabitant of permanent ponds and lakes, tended to respond to predator presence by reducing its growth rate, though not by a tail depth increase. I argue that the rather poor swimming performance in Bufo tadpoles may opt for defences other than locomotor ability. The palatable, permanent pond species Pelobates fuscus did not alter either its growth rate or tail morphology. Possible explanations for this result are discussed.     

Factors influencing breeding site choice by the pioneering amphibian Bufo calamita

We have investigated environmental factors influencing the choice of breeding pools by Bufo calamita in two different habitat types in Britain, using a number of methods including the construction of artificial pools and the translocation of spawn laid by competitively superior anuran species. On heathland, major determinants were aspects of pond chemistry especially pH, Na and SO₄. On coastal dunes other criteria were important, notably maximum water temperatures achieved the day prior to spawning. These in turn related to the precise shape and size of the pond. In addition, natterjacks were able to discriminate against ponds which already contained large numbers of competitor species Rana temporaria and Bufo bufo larvae. Possible mechanisms underlying site choice by B. calamita are discussed.     

Saprolegnia diclina: another species responsible for the emergent disease 'Saprolegnia infections' in amphibians.

Many amphibians are known to suffer embryonic die-offs as a consequence of an emergent disease known as 'Saprolegnia infections'. Thus far, the only species of Saprolegnia shown to be involved in natural infections is Saprolegnia ferax. In this study, we have isolated and characterized another Saprolegnia species responsible for 'Saprolegnia infections' on embryos of Bufo calamita in mountainous areas of central Spain. The strain was identified as belonging to Saprolegnia diclina based on morphological, physiological and molecular characters (sequencing of the internal transcribed region of ribosomal DNA). Zoospores of the new strain were able to infect embryos of Bufo calamita, and the symptoms observed were the same as those observed in natural infections. The results presented emphasize the need to carry out isolations and characterizations of the species and/or strains involved in this emergent disease. This will be important in order to design strategies to prevent the impact and spread of species (or strains) pathogenic to amphibians.     

Developmental alterations and osmoregulatory physiology of a larval anuran under osmotic stress

Water salinity represents an environmental stress for many species. Amphibians are particularly sensitive because they are generally poor osmoregulators, and most species are completely absent from brackish and saline environments. We experimentally examined the effect of different salinity levels on larvae of the toad Bufo calamita L., a species that occupies freshwater ponds but can also breed in brackish ponds. Two independent experiments are reported here. In both experiments, tadpoles under saline conditions (ranging between 85 and 200 mOsm) showed a slower developmental rate, metamorphosing between 4 and 9 d later than the controls. Bufo calamita tadpoles reared in brackish water increased their osmolality and solute concentration (mainly sodium and chloride), decreased their levels of glucose, and decreased the total protein content, all measured from whole-animal extracts. Although most larval anurans are strictly ammoniotelic until the completion of metamorphosis, a few species exposed to dehydrating environments have evolved the ability to use urea as an osmolyte during the larval phase. The data presented here reveal that although B. calamita seems to be yet another exception to the rule of larval strict ammoniotelism, the tadpoles are not able to use urea as an osmolyte and rely on sodium-chloride balance instead. Preliminary immunoassays of thyroid hormone content suggest a possible decrease in hormone levels induced in water salinity conditions that correlate with a decreased developmental rate.     

Cytoplasmic Influence on Lethality in Nucleocytoplasmic and Natural Hybrids between Bufo Calamita Laur and Bufo Bufo L. (Amphibia, Anura)

Nucleocytoplasmic interactions have been studied through nuclear and cytoplasmic grafts. Bufo bufo nuclei transplanted in Bufo calamita eggs provoked 95% lethality during gastrulation. Influence of the cytoplasm in such a nucleocytoplasmic incompatibility has been studied through cytoplasmic fractionation and grafting of the fractions obtained. Fractionation of egg cytoplasm gave three fundamental fractions: lipid, hyaline cytoplasm and vitellus. After injection of B. calamita fractions into B. bufo fertilized eggs, the hyaline cytoplasmic fraction provoked a gastrulation block in the same ratio (95%) as in the interspecific nuclear graft experiment. Influence of the hyaline cytoplasmic fractions of both species has been tested on nucleocytoplasmic and natural hybrid developments. The B. calamita hyaline cytoplasmic fraction provoked in each case a developmental block at the beginning of gastrulation. The B. bufo hyaline cytoplasmic fraction acts later at the yolk plug stage and in some cases partially corrects the developmental bolck or exogastrulation of young hybrid gastrula. It can be generalized that in both kinds of hybrids, the hyaline cytoplasmic fraction of B. calamita exerted a lethal effect on B. bufo nucleus.     

Genetic variability in geographic populations of the natterjack toad (Bufo calamita)

Across altitudinal and latitudinal gradients, the proportion of suitable habitats varies, influencing the individual dispersal that ultimately can produce differentiation among populations. The natterjack toad (Bufo calamita) is distributed across a wide geographic range that qualifies the species as interesting for a geographic analysis of its genetic variability. Five populations of B. calamita in the Sierra de Gredos (Spain) were studied in an altitudinal gradient ranging from 750 to 2270 m using microsatellite markers. In addition, we analyzed the latitudinal genetic variation in B. calamita within a global European distribution using genetic diversity parameters (mean number of alleles per locus [M(a)] and expected heterozygosity [H(E)]) obtained from our results and those published in the literature. The low level of genetic differentiation found between populations of B. calamita (F(st) ranging from 0.0115 to 0.1018) and the decreases in genetic diversity with altitude (M(a) from 13.6 to 8.3, H(E) from 0.82 to 0.74) can be interpreted by the combined effects of discontinuous habitat, produced mainly by the high slopes barriers and geographic distance. In the latitudinal gradient, genetic diversity decreases from south to north as a consequence of the colonization of the species from the Pleistocene refugium. We conclude that the genetic variability in B. calamita along its wide altitudinal and latitudinal geographic distribution mainly reflects the colonization history of the species after the last glacial period.     

Suspension feeding of anuran larvae at low concentrations of Chlorella algae (Amphibia,Anura)

Summary Ingestion and filtering rates in larval Xenopus laevis, Bufo calamita, Rana temporia and Bufo bufo fed suspensions of Chlorella fusca were investigated. Concentrations were measured with a Coulter Counter. (1) For all species, filtration occurred at concentrations far below those reported by other authors for Rana sylvatica feeding on Chlorella pyrenoidosa. For Bufo bufo, only larvae near metamorphosis showed ingestion at low particle concentrations. Since buccopharyngeal ventilation continues even in the absence of food particles, this threshold feeding behaviour in the younger larvae must be due to different mechanisms to those found in Daphnia and Calanus studied by other authors: probably reduction of the buccal pumping rate and the mucus production of the filter apparatus. (2) For B. calamita, R. temporaria and X. laevis the highest suspension feeding efficiency was at early tadpole stages, corresponding with the high growth rate of these stages. (3) The life histories of the species provide the basis for understanding their different retention efficiencies and functional responses.Supported by the Deutsche Forschungsgemeinschaft-DFG     

Variability in timing of larval season in an amphibian community in SW Spain

Timing of larval periods of 9 species of amphibians (Hyla meridionalis, Pelobates cultripes, Discoglossus galganot, Bufo calamita, Bufo bufo, Rana perezi, Triturus marmoratus, Triturus boscat, Pleurodeles waltl) was followed in 7 different years Total larval season may last from autumn to summer, whereas in low rainy years it may last from late winter to summer In rainy years, two species are early breeders, starting to breed m autumn, six species commonly start in mid-winter and one species always breeds later, in spring The widest interannual variability m the onset and duration of larval season occurs in the earliest breeders In general, the more rainy the autumn and the higher temperatures were reached, the earlier the season started In contrast, duration of the season was not correlated with rainfall and temperature Interannual variability is interpreted as the possibility of adjusting the reproduction of the species to the availability of breeding habitats, constrained by physiological features of the species and interspecific relationships     

Age Determination of Frogs and Toads (Amphibia, Anura) from North-western Europe

Age was determined in three anuran species inhabiting a temperate oceanic climate ( Rana temporaria, Bufo calamita and Bufo bufo ) by means of the histological structure of the phalanges. Narrow, haematoxylinophilic lines were visible in these, and evidence from the examination of subadults and frog ( R. temporaria ) recaptures showed the lines to be annual. The validity of using the lines as age indicators was tested most extensively on natural frog populations and the effects of endosteal resorption were not found to be significant. The evidence for this conclusion was provided directly by recapture data and indirectly by an analysis of histological measurements made on the phalanges.     

Enzyme activities in red and white muscles of guinea-pigs and rabbits indigenous to high altitude.

The activities of several enzymes functioning in different areas of fuel catabolism were measured under standardized conditions, using crude homogenates of sartorius and ventricular muscle from outbred guinea-pigs and rabbits indigenous to high or low altitude. The activities of sartorius and myocardium were found to reflect the metabolic patterns known to be associated with white and red muscle. Both species had right ventricular hypertrophy at high altitude. The enzyme activities in the high altitude guinea-pig were not significantly different from those in the sea level animals. In the high altitude rabbit, compared with the low altitude rabbit, the activities of glyceraldehyde-3-phosphate deydrogenase and phosphofructokinase were greater in both the sartorius and myocardium. In addition, mitochondrial glycerol-3-phosphate dehydrogenase activity was greater in the sartorius at high altitude, while aspartate aminotransferase and beta-hydroxyacylcoenzyme A dehydrogenase activities were greater in the myocardium at high altitude. Succinate dehydrogenase activity was comparable at the two altitudes for both tissues. There was a greater proportion of skeletal muscle type lactate dehydrogenase in the high altitude rabbit myocardium but no difference was found with the guinea-pig.     

Hindlimb muscle fiber types in two frogs (Rana catesbeiana and Litoria caerulea) with different locomotor behaviors: histochemical and enzymatic comparison

To test how differences in locomotor behaviors may be reflected in muscle fiber-type diversity within anurans, a comparison of hindlimb muscles between the powerful terrestrial hopper, Rana catesbeiana, and the tree frog, Litoria caerulea, was done. One postural muscle (tibialis posticus, TP) and one primary hopping muscle (plantaris longus, PL), were characterized to identify muscle fiber types using standard histochemical methods. In addition, spectophotometric analysis of activity levels of the oxidative enzyme citrate synthase (CS) and the glycolytic enzyme lactate dehydrogenase (LDH) were done in each muscle. In spite of presumed differences in behavior between the species, we found no significant differences in the proportions of the identified fiber types when the muscles were compared across species. In addition, there were no significant differences in the proportions of the different fiber types between the postural versus phasic muscles within species. Within Rana, the postural muscle (TP) had greater oxidative capacity (as measured by CS activity) than did the phasic muscle (PL). Both muscles had equivalent LDH activities. Within Litoria, PL and TP did not differ in either LDH or CS activities. Both PL and TP of Litoria had less LDH activity and greater CS activity than their homologs in Rana. Thus, in spite of the uniform populations of fiber types between muscles and species, the metabolic diversity based on enzyme activity is consistent with behavioral differences between the species. These results suggest that the range of functional diversity within fiber types may be very broad in anurans, and histochemical fiber typing alone is not a clear indicator of their metabolic or functional properties.     

Differences in susceptibility to Saprolegnia infections among embryonic stages of two anuran species

Many amphibians are known to suffer embryonic die-offs as a consequence of Saprolegnia infections; however, little is known about the action mechanisms of Saprolegnia and the host-pathogen relationships. In this study, we have isolated and characterized the species of Saprolegnia responsible for infections of embryos of natterjack toad (Bufo calamita) and Western spadefoot toad (Pelobates cultripes) in mountainous areas of Central Spain. We also assessed the influence of the developmental stage within the embryonic period on the susceptibility to the Saprolegnia species identified. Only one strain of Saprolegnia was isolated from B. calamita and identified as S. diclina. For P. cultripes, both S. diclina and S. ferax were identified. Healthy embryos of both amphibian species suffered increased mortality rates when exposed to the Saprolegnia strains isolated from individuals of the same population. Embryonic developmental stage was crucial in determining the sensitivity of embryos to Saprolegnia infection. The mortalities of P. cultripes and B. calamita embryos exposed at Gosner stages 15 (rotation) and 19 (heart beating) were almost total 72 h after challenge with Saprolegnia, while those exposed at stage 12 (late gastrula) showed no significant effects at that time. This is the first study to demonstrate the role of embryonic development on the sensitivity of amphibians to Saprolegnia.     

Defining population boundaries: use of three Bayesian approaches with microsatellite data from British natterjack toads (Bufo calamita)

Defining boundaries between populations is often difficult in the absence of information about current levels of gene flow. Such definitions can be important, however, both for the understanding of population dynamics and for conservation planning. Recently developed Bayesian methods for analysing genetic data now provide a powerful approach to this problem. Natterjack toads Bufo calamita are endangered in Britain, where their distribution is restricted to four geographically discrete regions. In three of these regions the boundaries between populations are often uncertain. We therefore used Bayesian approaches with microsatellite data to try and define British natterjack population structure, and thus inform conservation management. A large sample of natterjack toads from all 38 locations in Britain where the species is native was genotyped at eight microsatellite loci. The genetic diversity of natterjack populations declined as a function of increasing latitude, echoing postglacial colonization dynamics. Comparisons of three assignment methods (structure, baps and geneland) generated some broad similarities but also some inconsistencies in the definitions of population structure, especially in the most complex region (south Cumbria). Implications of the analyses for the future conservation of Bufo calamita in Britain are discussed.     

Intra- und interspezifische Crowding-Effekte bei Kaulquappen der Kreuzkröte,Bufo calamita Laur.

Summary Tadpoles ofBufo calamita, a species that spawns in little temporary pools, were tested on intra- and interspecific crowding-effects. Water was conditioned by tadpoles of the following species:Bufo calamita (tadpoles of different age),Alytes obstetricans, Bombina variegata, Bufo bufo, Hyla arborea, Rana lessonae, R. ridibunda andR. temporaria. Tadpoles ofBufo calamita are most sensitive to water conditioned by older tadpoles of the own species during the first 2–3 weeks of development. They are also strongly inhibited by tadpoles ofRana temporaria andBufo bufo (that, as a rule, do not occur in the same habitat). Relative little effects are observed in water conditioned by tadpoles of the two species that occur very often in ecological sympatry withBufo calamita: Hyla arborea andBombina variegata. Young tadpoles ofBufo calamita are most tolerant of tadpoles of the same age of the own species. The crowding-effect increases with the number and weight of tadpoles/volume conditioning the water. In the age of 2–3 weeks the sensibility to intra- and interspecific crowding-effects fades. Thus inBufo calamita the age specific sensibility to crowding-effects in tadpoles may act as a density regulation mechanism in temporary pools.

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Mit Unterstützung des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung (Beitrag Nr. 3.256.69).     

Laich-Fressen durch Kaulquappen als mögliche Ursache spezifischer Biotoppräferenzen und kurzer Laichzeiten bei europäischen Froschlurchen (Amphibia,Anura)

Zusammenfassung In einem Kunstweiher aufgezogene Kaulquappen von Rana temporaria fressen in der Versuchsanordnung von Abb. I den Laich folgender sympatrischer Anuren-Arten: Rana temporaria (Kannibalismus), Rana esculenta, Rana ridibunda, Bufo bufo, Bufo calamita, Bombina variegata und Hyla arborea (Laichräubern). — Daß die Kaulquappen der früh im Jahr laichenden Rana temporaria später gelegten arteigenen und artfremden Laich fressen, wirkt als Selektionsdruck in folgende Richtungen: Rana temporaria züchtet sich selbst eine kurze Laichzeit. Spät in seichtem Wasser laichende Arten werden im Biotop von Rana temporaria unterdrückt (Bombina variegata, Bufo calamita, Hyla arborea). Rana esculenta, Rana ridibunda und Bufo bufo sind dem Laichräubern durch Kaulquappen wenig ausgesetzt, weil sie in größeren Wassertiefen laichen. Zudem ist Bufo bufo ebenfalls ein Frühlaicher. Alytes obstetricans steht außer Konkurrenz, weil er die Eier nicht ins Wasser setzt. — Untersuchungen über die Biotoppräferenzen und Laichzeiten der betroffenen Arten bestätigen diese Interpretation.

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Summary Tadpoles of Rana temporaria which have been raised in an artificial pond prey in the experimental situation shown in Fig. 1 upon the spawn of the following sympatric European Anura species: Rana temporaria (cannibalism), Rana esculenta, Rana ridibunda, Bufo bufo, Bufo calamita, Bombina variegata and Hyla arborea (predation). — Rana temporaria is an early breeder. Thus selection pressure acts in the following ways: Rana temporaria establishes for itself an explosive breeding pattern. Species which breed later in the year in shallow water are suppressed in the biotope of Rana temporaria and develop other biotope preferences (Bombina variegata, Bufo calamita, Hyla arborea). Rana esculenta, Rana ridibunda and Bufo bufo spawn in deeper water. Therefore they are scarcely exposed to the tadpoles of Rana temporaria. Moreover Bufo bufo, too, is an early breeder. Alytes obstetricans laying no spawn in the water is not endangered. — An analysis of biotope preferences and breeding times of the species involved confirms these interpretations.

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Mit Unterstützung des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung.     

Microsatellite analysis of natterjack toad Bufo calamita Laurenti populations: consequences of dispersal from a Pleistocene refugium

Populations of the amphibian Bufo calamita were sampled for genetic analysis in eleven areas distributed across its biogeographical range in Europe. Genetic diversity estimates across eight microsatellite loci showed a decline in polymorphism, numbers of alleles and heterozygosity as a function of distance from the presumed ice-age refugium in Iberia. Trials with a selection of tree-building algorithms indicated mat UPGMA of Cavalli-Sforza chord distances (D_c) generated the tree topology most easily reconciled with other biogeographical information. Genetic distance measures were also calibrated against a postglacial event from which the separation of extant populations could be estimated in real time. D_c again outperformed two other measures (Nei's standard distance, D_s , and δμ²) in producing realistic correlations with minimal variance. The genetic analysis was consistent wim die hypodiesis that B. calamita survived in a single refugium (Iberia) during the Pleistocene glaciation and indicated that it spread north and east from there during the last interstadial which commenced about 14 000 years before present (BP). Microsatellites should provide useful tools for biogeographical investigations of other species, especially with respect to patterns of population dispersal.     

Implication of Cytoplasmic Factors in the Lethality of Bufonidae Nucleocytoplasmic Hybrids

The nucleocytoplasmic hybrid combination performed by nuclear graft between B. bufo nucleus and B. calamita egg is 95% lethal during gastrulation mostly at the young gastrula stage (75%) and to a lesser extent at the yolk plug stage (20%). The heterologous B. calamita cytoplasm was treated or fractioned by different techniques including centrifugation, nucleic acid extraction, ammonium sulfate treatment and heating.
The treated cytoplasmic fractions were injected in B. bufo fertilized eggs in order to check their influence on morphogenetic capacities of the B. bufo nucleus. Injection of hyaline cytoplasmic fraction obtained from B. calamita centrifuged eggs blocks the B. Bufo fertilized egg gastrulation in similar rates as in nucleocytoplasmic association performed by interspecific nuclear grafting. Gastrulation, however, is not affected by injection of total nucleic acids extracted from this hyaline fraction. The soluble cytoplasmic fraction obtained after 60% ammonium sulfate precipitation blocks the development at the beginning of gastrulation, this property being inactivated by heating for 5 min at 60°C. Supernatant from 70% ammonium sulfate precipitation blocks the development at the yolk plug stage and is inactivated by heating for 5 min at 80°C.
These experiments suggest that two distinct factors in B. calamita eggs are involved in developmental arrest of injected B. bufo fertilized eggs, these two factors respectively acting on the beginning and on the end of gastrulation.     

Geographical variations in breeding activity patterns of the Natterjack toad Bufo calamita in Britain

Bufo calamita is on the edge of its range in Britain, and exists only in three widely separated geographical regions of the country. The largest populations with the highest densities of animals are found on the Irish Sea coast. Data on the chronology of breeding activity show that the reproductive season for these thriving populations starts and finishes earlier than that for toads in eastern England, but that first records of metamorphosis occur at similar times throughout the country. The possible significance of these observations to the variable success of the species in the different geographical regions is discussed.