Increasingly cautious sampling, not the black colouration of unpalatable prey, is used by fish in avoidance learning.

The efficiency of aposematic colouration of prey is based on the innate bias or facilitation of avoidance learning of predators. In many toxic bufonids, larvae are uniformly black, which is considered a warning signal. We compared fish predation on normal (black) and 'transient albino' (greyish) common toad Bufo bufo tadpoles that did not differ in toxicity or activity. In a two-stage experiment, each fish was presented with tadpoles of one colour in the first trial and the other colour in a subsequent trial. While tadpoles sampled by fish were typically not ingested, some died from injuries. The attack rate did not differ between tadpole phenotypes nor trials, irrespective of which phenotype was the first exposed to the fish. However, during the second trial, the sampled tadpoles, independent of colouration, were mouthed by fish for shorter periods and tadpole mortality decreased. The duration of mouthing also declined with an increasing number of attacks during subsequent trials. We conclude that in single-species prey populations, black tadpole colouration is not a warning signal as it does not accelerate predator learning about prey unprofitability. Our results indicate that with growing experience, predators sample potentially toxic prey more cautiously. This may explain why natural selection does not eliminate aposematic morphs even if predators continuously sample conspicuous prey.

Potential hotspots of amphibian roadkill risk in Spain.

We test a forecasting strategy to identify potential hotspots of amphibian roadkill, combining the spatial distribution of amphibians, their relative risk of collision with vehicles and data on road density in Spain. We extracted a large dataset from studies reporting road casualties of 39 European amphibian species and then estimated the 'relative roadkill risk' of species as the frequency of occurrence of casualties for each amphibian and standardized by the range of distribution of the species in Europe. Using a map with the spatial distribution of Spanish amphibians at a spatial resolution of 10 × 10 Km squares, we estimated the 'cumulative relative risk of roadkill' for each amphibian assemblage as the sum of risk estimates previously calculated for each species. We also calculated the total length of roads in each square (road density). Finally, combining all layers of information, we elaborated a forecasting map highlighting the potential amphibian roadkill risk across Spain. Our findings are relevant to suggest areas that should be focused on at more detailed spatial scales. Additionally, we found that the frequency of roadkill was unrelated to the evolutionary distinctiveness score and conservation status of amphibian species, while was positively correlated with their distribution range.

Emerging infectious diseases of amphibians in Poland: distribution and environmental drivers.

Emerging infectious diseases are a threat to biodiversity and have taken a large toll on amphibian populations worldwide. The chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and the iridovirus Ranavirus (Rv), are of concern as all have contributed to amphibian declines. In central and eastern Europe, their geographical and host distributions and main environmental drivers determining prevalence are poorly known. We screened over 1000 amphibians from natural and captive populations in Poland for the presence of Bd, Bsal and Rv. In wild amphibian populations, we found that Bd is widespread, present in 46 out of 115 sampled localities as well as 2 captive colonies, and relatively common with overall prevalence at 14.4% in 9 species. We found lower prevalence of Rv at 2.4%, present in 11 out of 92 sampling sites, with a taxonomic breadth of 8 different amphibian species. Bsal infection was not detected in any individuals. In natural populations, Pelophylax esculentus and Bombina variegata accounted for 75% of all Bd infections, suggesting a major role for these 2 species as pathogen reservoirs in Central European freshwater habitats. General linear models showed that climatic as well as landscape features are associated with Bd infection in Poland. We found that higher average annual temperature constrains Bd infection, while landscapes with numerous water bodies or artificial elements (a surrogate for urbanization) increase the chances of infection. Our results show that a combination of climatic and landscape variables may drive regional and local pathogen emergence.

Digit ratio in the common toad Bufo bufo: the effects of reduced fingers and of age dependency.

INTRODUCTION: Despite the growing number of studies describing digit ratio patterns in tetrapods, knowledge concerning certain basic issues is still scarce. In lower vertebrates such as tailless amphibians (Anura), the numbering of individual fingers on the forelimbs and their homology with the fingers of other vertebrates pose an unsolved problem. Based on reviewed data on anuran limb development, we argue that the correct finger numbering scheme should be based on the assumption that the first finger, not the fifth finger, was reduced on the forelimbs. We analyzed the digit ratio in the common toad (Bufo bufo, Bufonidae), a species characterized by well-developed sexual dimorphism whereby females are larger than males, using both numbering schemes present in the literature. RESULTS: We found that the digit ratio on hindlimbs differed significantly between the sexes only in the cases of left 2D:3D, with lower digit ratios in females, and of left 3D:4D, with lower digit ratios in males. We found that sex was the only significant variable for forelimbs, differentiating 2D:3D on the left forelimb, with lower digit ratios in females; 2D:4D on the right forelimb, with lower digit ratios in males; and 3D:4D on both forelimbs, with lower digit ratios in males. These results relate to variant II reflecting the hypothesis that the first digit was reduced during phylogenesis. There was no relationship between the body size (SVL) of individuals and any digit ratio, excluding 2D:4D on the right forelimbs in models with age variables. Additionally, for a subset of data where individual age was known, the models indicated that age was linked to significant differences in 2D:4D and 3D:4D on the left hindlimbs, while age, SVL, and sex influenced 2D:4D on the right forelimbs. CONCLUSION: We emphasize the importance of the problem of the correct numbering of forelimb digits in Anura and, under the assumption that it was the fifth digit that was reduced, argue that earlier results on digit ratio in this group should be interpreted with caution. The detected relationship between digit ratio and age in amphibians expands our knowledge, indicating that the age of individuals should be included in future digit ratio studies. This relationship may also apply to studies using digit ratio as a noninvasive indicator of endocrine disruption in amphibians.

Sand lizards Lacerta agilis with higher digit ratios are more likely to autotomy.

Digit ratio is a morphological feature regarded as a biomarker of the balance of sex hormones during early development. The exposure of embryos to a set of sex hormones and the mutual relations between those hormones cause the emergence of individual morphological and/or behavioural characteristics as well as differences between sexes. We have thus hypothesised that differences in one of these morphological traits-digit ratio-may be a proxy representing a tendency towards tail autotomy. The aim of this study is to investigate the digit ratio (2D:3D, 2D:4D, 3D:4D) of the sand lizard, Lacerta agilis, Lacertidae, a species characterised by well-developed sexual dimorphism, whereby females are larger than males. We also tested associations between patterns in digit ratio and caudal autotomy, a common defensive mechanism among lizards. To our knowledge, the relationship between a tendency towards autotomy and digit ratio pattern has never been researched. To date, studies on autotomy have mainly focused on the consequences, costs or evolutionary background of tail loss. Hence, researchers examined mostly the frequency of autotomy in the context of predatory pressure or habitat conditions, omitting an individual's behavioural tendency to shed its tail. However, behavioural traits can affect an individual's exposure to predator attack and consequently the need to use an anti-predator strategy. Thus, following this logic, dropping the tail may be the result of the lizard's intraspecific personality characteristics, resulting from the effect of hormones on behaviour or innate traits. Therefore, we suggest that the inclusion of autotomy as a factor explaining observed digit ratio patterns and their variability between taxa has great potential. We used computerised measurements of photographed limbs to determine the length of digits. We found that the digit ratios for all four limbs were significantly lower in females than in males, excluding the 3D:4D ratio for the right hindlimbs. Therefore, the results confirmed the pattern already observed for most lizards. The novel element in our study is the detection of the relationship between a tendency towards caudal autotomy and digit ratio. Individuals with a tendency towards autotomy have a higher 2D:4D ratio in the right forelimbs and a lower 2D:3D ratio in the right hindlimbs. Obtained results suggest that these morphological characteristics are most likely related to intraspecific differences (between bold and shy individuals) which consequently may determine an individual's reaction or susceptibility to be a prey and escape behaviour. Thus, our results are probably the first attempt to link digit ratio to the susceptibility of lizards to tail autotomy.

Numbers, neighbors, and hungry predators: What makes chemically defended aposematic prey susceptible to predation?

Many chemically defended aposematic species are characterized by relatively low toxin levels, which enables predators to include them in their diets under certain circumstances. Knowledge of the conditions governing the survival of such prey animals-especially in the context of the co-occurrence of similar but undefended prey, which may result in mimicry-like interactions-is crucial for understanding the initial evolution of aposematism. In a one-month outdoor experiment using fish (the common carp Cyprinus carpio) as predators, we examined the survival of moderately defended aposematic tadpole prey (the European common toad Bufo bufo) with varying absolute densities in single-species prey systems or varying relative densities in two-species prey systems containing morphologically similar but undefended prey (the European common frog Rana temporaria). The density effects were investigated in conjunction with the hunger levels of the predator, which were manipulated by means of the addition of alternative (nontadpole) food. The survival of the B. bufo tadpoles was promoted by increasing their absolute density in the single-species prey systems, increasing their relative density in the two-species prey systems, and providing ample alternative food for the predator. Hungry predators eliminated all R. temporaria individuals regardless of their proportion in the prey community; in treatments with ample alternative food, high relative B. bufo density supported R. temporaria survival. The results demonstrated that moderately defended prey did benefit from high population densities (both absolute and relative), even under long-term predation pressure. However, the physiological state of the predator was a crucial factor in the survival of moderately defended prey. While the availability of alternative prey in general should promote the spread and maintenance of aposematism, the results indicated that the resemblance between the co-occurring defended and undefended prey may impose mortality costs on the defended model species, even in the absence of actual mimicry.

Urban plums and toads: do fleshy fruits affect the post-metamorphic growth of amphibians?

BACKGROUND: The main aim of the study was to analyse the influence of fleshy fruits (plums) on the post-metamorphic growth and feeding behaviour of the green toad Bufotes viridis. We tested the following two hypotheses: (1) juveniles of the green toad are characterised by faster growth in conditions involving fallen plums Prunus cerasifera due to the associated presence of more varied food such as invertebrates; (2) green toads exhibit more active feeding behaviour in the presence of fleshy fruits. METHODS: A total of 120 fresh metamorphs of the green toad were randomly assigned to one of four groups: two experimental groups with fleshy plums and two other groups as controls (without fruits). Each group was kept in an enclosure to which wild invertebrates had free access. Each individual toad was measured for snout-vent length (mm) and body mass (g) every other day for 30 days. In order to determine whether fallen plums influence the feeding behaviour of toads, the number of active and hidden (under an artificial shelter) individuals was also noted. RESULTS: The results showed that green toads from both enclosures with plums were characterised by more rapid growth than individuals from the control treatments. Simultaneously, in the enclosure with fleshy fruits, greater species richness of wild invertebrates was observed. No differences in active feeding behaviour were noted between control groups and groups with plums. DISCUSSION: Fleshy fruits, upon falling, attract many types of invertebrates; thus they may represent good dietary supplements for fresh amphibian metamorphs. Therefore, the presence of fruit trees close to a breeding site might influences the post-metamorphic growth of amphibians, but not their feeding behaviour. The presence of insects associated with fallen fruit seems to favour the occurrence of amphibian populations, which is particularly important, since, due to political and social pressure, numbers of fruit trees are currently being reduced. To our knowledge, no previous study has investigated the potential influence of the presence of fruit trees on the growth and behaviour of anurans.

A matter of proportion? Associational effects in larval anuran communities under fish predation.

In Batesian mimicry, a species lacking defences against predators benefits from mimicking the aposematic signal of a defended species, while the model may incur the costs of reduced defensive efficacy. Similar reciprocal indirect effects may emerge even when the signal is not mimicked; termed associational effects, such interactions are well known in plants sharing herbivores but have received little attention in animal studies. We investigated associational interactions in a system where unequally defended prey (chemically defended Bufo bufo and undefended Rana temporaria tadpoles), sharing general morphology but not an aposematic signal, were exposed to predation by the carp Cyprinus carpio along a gradient of relative prey abundance. In the absence of fish, the assemblage composition had no effect on the survival of Rana, while that of Bufo decreased with increasing abundance of Rana. Fish reduced the survival of tadpoles from both species. However, increased relative abundance of Bufo in the community led to enhanced survival in both Bufo and Rana. Increasing relative proportions of heterospecifics reduced metamorph mass only in Bufo, indicating greater sensitivity to interspecific competition compared to Rana; the effect was reduced in the presence of fish. Our results show that undefended non-mimetic prey enjoy reduced predation with increasing relative abundance of chemically defended prey, which in turn suffer greater mortality with an increasing proportion of the undefended species. Associational resistance/susceptibility, driven by current assemblage composition, not by selection for resemblance, can shape the dynamics of mixed communities of defended and undefended prey in the absence of mimicry.

Robustness of newt heads in condition of co-existence: a case of the Carpathian newt and the alpine newt.

Co-existence between potentially competing newt species can lead to niche differentiation (e.g., in terms of diet shifts). This may cause adaptive responses involving changes in head shape. Here, we tested the hypothesis: the head shape of Lissotriton montandoni is different in conditions of co-occurrence with Ichthyosaura alpestris than in conditions in which other newt species are absent. We analysed images depicting head shape of specimens of I. alpestris and L. montandoni from a museum collection. All specimens of I. alpestris originated in a habitat where L. montandoni also occurred, whereas specimens of L. montandoni derived from populations that cohabited with I. alpestris and populations in which the presence of another newt species was not recorded. In each image, landmarks and semilandmarks were digitised. Females of L. montandoni from the population where I. alpestris also occurred were characterised by more massive heads and longer mouths in lateral views than females from sites where no other newt species occurred. Significant differences in head shape were also found when analysing ventral views between these species when they occupied the same habitat. We confirmed that the head shape of female L. montandoni differs between conditions of co-occurrence and absence of I. alpestris; no differences were found for males. A differently shaped head may be an adaptation to diet; L. montandoni females with longer mouths and more robust basal parts of the head can feed on larger invertebrates and compete more effectively with I. alpestris. The co-existence of newt species should be taken into account in future ecomorphological studies.

Phalangeal bone anomalies in the European common toad Bufo bufo from polluted environments.

Every spring, many of amphibians are killed by motor vehicles on roads. These road-killed animals can be used as valuable material for non-invasive studies showing the effect of environmental pollution on amphibian populations. The aims of our research were to check whether the phalanges of road-killed toads may be useful as material for histological analysis, and whether various degrees of human impact influence the level in bone abnormalities in the common toad. We also examined whether the sex and age structure of toads can differ significantly depending in the different sites. We chose three toad breeding sites where road-killed individuals had been observed: near the centre of a city, the outskirts of a city, and a rural site. We collected dead individuals during spring migration in 2013. The sex of each individual was determined and the toes were used to determine age using the skeletochronology method. While performing age estimates, we looked for abnormalities in relation to normal bone tissue structure. In urban site, females dominate males (sex ratio 2.6:1), but in populations from rural and semi-urban sites, sex ratio was reverse (1:2.2 and 1:1.4, respectively). However, we did not find any significant differences between age structure of all populations (average age of each population: approximately 4 years). We observed abnormalities in more than 80 % of all toads from the city, compared to approximately 20 % from the rural and semi-urban sites. In particular, we found hypertrophic bone cells, misaligned intercellular substance, and irregular outer edges of bones. We suggest that these malformations are caused by different pollution, e.g. with heavy metals.

Females Have Larger Ratio of Second-to-Fourth Digits Than Males in Four Species of Salamandridae, Caudata.

Digit ratio (2D:4D) denotes the relative length of the second and fourth digits. It is considered to be a suitable biomarker of the in utero balance of fetal sex hormones, which affect early development of individuals behavioral and morphological characteristics. In recent decades, digit ratio attracted a great attention in biology and psychology. However, for unmasking the biological basis of the phenomenon, extensive studies on non-human animals are necessary. Despite it was hypothesized that digit ratio is well conserved in all Tetrapoda, and there exist studies on mammals, birds, and reptiles, there are only two such study on anuran amphibians. Therefore, the aim of this study is to investigate the 2D:4D in the most basal salamanders, Caudata. We have studied digit ratio in four species of newts: Triturus cristatus, Mesotriton alpestris, Lissotriton montandoni, and Lissotriton vulgaris, using museum collection. We used computerized measuring of each limbs photos. We have found out that, in M. alpestris, females 2D:4D of all four limbs were significantly larger than in males. In L. montandoni and L. vulgaris, only 2D:4D of rear limbs significantly differed, in females being larger. In T. cristatus, digit ratios of males and females did not statistically differ. Thus, the results confirmed our hypothesis that at least in M. alpestris, L. montandoni, and L. vulgaris, females seem to have larger 2D:4D comparing to males, the pattern known from most mammals and opposite to birds, reptiles and anuran amphibians.