The larvae of the European fire salamander (Salamandra salamandra) can inhabit two different habitats: streams and ponds. Streams are characterized by lower predation risks and higher food availability. Thus, ponds are considered a less suitable habitat. To investigate the differential impacts of these two habitats on larval physiology, we measured the stress response of larvae. After successfully validating the measure of water-borne corticosterone release rates in fire salamander larvae, we measured the baseline and stress-induced corticosterone of 64 larvae from ponds and streams in the field. We found that larvae in ponds have a higher baseline and stress-induced corticosterone levels. Additionally, we performed a reciprocal transplant experiment (RTE) and tested whether larvae can adapt their stress responses to changing habitats. After two weeks, we did not find an increase in corticosterone levels when comparing stress-induced corticosterone values with baseline corticosterone values in larvae transferred into ponds, irrespective of their habitat of origin. However, larvae transferred into streams still exhibited an increase in the stress-induced corticosterone response in comparison with the baseline values. These results show that non-invasive hormone measurements can provide information on the habitat quality and potential adaptation and thus emphasize the potential for its use in conservation efforts.

For many species, population sizes are unknown despite their importance for conservation. For population size estimation, capture-mark-recapture (CMR) studies are often used, which include the necessity to identify each individual, mostly through individual markings or genetic characters. Invasive marking techniques, however, can negatively affect the individual fitness. Alternatives are low-impact techniques such as the use of photos for individual identification, for species with stable distinctive phenotypic traits. For the individual identification of photos, a variety of different software, with different requirements, is available. The European fire salamander (Salamandra salamandra) is a species in which individuals, both at the larval stage and as adults, have individual specific patterns that allow for individual identification. In this study, we compared the performance of five different software for the use of photographic identification for the European fire salamander: Amphibian & Reptile Wildbook (ARW), AmphIdent, I3S pattern+, ManderMatcher and Wild-ID. While adults can be identified by all five software, European fire salamander larvae can currently only be identified by two of the five (ARW and Wild-ID). We used one dataset of European fire salamander larval pictures taken in the laboratory and tested this dataset in two of the five software (ARW and Wild-ID). We used another dataset of European fire salamander adult pictures taken in the field and tested this using all five software. We compared the requirements of all software on the pictures used and calculated the False Rejection Rate (FRR) and the Recognition Rate (RR). For the larval dataset (421 pictures) we found that the ARW and Wild-ID performed equally well for individual identification (99.6% and 100% Recognition Rate, respectively). For the adult dataset (377 pictures), we found the best False Rejection Rate in ManderMatcher and the highest Recognition Rate in the ARW. Additionally, the ARW is the only program that requires no image pre-processing. In times of amphibian declines, non-invasive photo identification software allowing capture-mark-recapture studies help to gain knowledge on population sizes, distribution, movement and demography of a population and can thus help to support species conservation.

Population monitoring is a crucial method for conservation projects, especially for the highly endangered clade of amphibians which is threatened by habitat loss and emerging infectious diseases such as the chytrid fungus Batracho-chytrium salamandrivorans (Bsal). Bsal has led to massive population declines of European fire salamanders in Belgium, the Netherlands and parts of Germany and population trends are decreasing. Thus, regular population monitoring is es-sential to keep track of population dynamics and detect potential Bsal outbreaks, especially since valid population esti-mates for many species and/or populations are scarce. In recent years, photographic mark-recapture studies have gained importance enabling researchers to keep track of individuals without the use of harmful marking techniques that might influence behaviour or survival. We monitored a European fire salamander population over four years in the Kottenforst forest, Germany, by combining a mark-recapture approach with a new photo-recognition software, the Amphibian and Reptile Wildbook We investigated potential differences of two ecotypes, i.e., larval populations bred in ponds and streams. Furthermore, we compared the observed number of larvae and the estimated population sizes between two consecutive years. There was a year effect on the observed number of larvae, the percentage of injured larva and estimated population size. The habitat type affected apparent survival rates. There was also a habitat effect on the percentage of injured larvae, but this was only existent in some years. The mean larval size, water temperature and the superpopulation size were not affected by any of the aforementioned factors. Although the population was free from Bsal, the high variation in the popu-lation estimates emphasize the need for a regular and standardised monitoring to assess the current population status and detect early population declines that might otherwise remain overlooked.

Prey organisms need to detect predators and respond with adequate antipredator behaviours. To recognize predators, chemosensory cues play a pivotal role, particularly in aquatic ecosystems, where sight may be restricted. However, it is less known whether the ability to use these cues, enabling appropriate antipredator behaviour, varies between subpopulations occupying different habitats with different predation risks. We examined antipredator behaviour of wild-caught larval fire salamanders (Salamandra salamandra) from two different habitats, ponds and streams. Ponds and streams are inhabited by habitat-specific predators, such as alpine newts (Ichthyosaura alpestris), which only occur in ponds. We confronted larvae from both habitats with either tap water (control) or tap water that had contained alpine newts (newt treatment) and investigated potential behavioural differences in activity and shelter emergence. In the activity test, pond larvae, but not stream larvae, were significantly less active when faced with chemical cues (potentially kairomones) from newts than those faced with a control stimulus, but stream larvae generally exhibited significantly lower activity than pond larvae. In the shelter-emergence test, larvae from both habitats spent significantly less time outside a shelter in the newt treatment than in control water. This suggests that larval fire salamanders, independent of their habitat, recognize chemical cues from potential predators and alter their behaviour accordingly. Larvae of both habitats were able to detect and use chemical cues released from potential predators, but they differed slightly in their response to these chemical cues. This finding reflects varying antipredator strategies in response to the habitat-specific differences.

Individual life histories are strongly influenced by early environmental conditions and experiences. They shape morphology as well as behaviour and can promote adaptive divergence and phenotypic plasticity with regard to different habitat types. The fire salamander (Salamandra salamandra) in the Kottenforst forest in Bonn, Germany, exhibits two genetically distinct ecotypes occurring in two larval habitats, either ponds or streams. In this study, we investigated whether both ecotypes differ in risk-taking behaviour, measured as the behavioural response during a shelter-emergence test and a shelter-seeking test, and whether larval habitat type and size impact these behaviours. Our results revealed an influence of size as well as habitat type. Larger larvae of both habitats appear to be more risk-prone, as they spent more time outside the starting shelter in the shelter-emergence test. Irrespective of size, pond larvae sought shelter more often in the shelter-seeking test and are thus considered to be less risk-prone. These results indicate that larvae conform to a given niche by adjusting their behaviour. Future studies are needed to disentangle the role of genetic adaptation or phenotypic plasticity and to investigate long-term consequences of the larval habitat for the adult phenotype. Thereby, efforts should be made to create a concise set of multiple tests assessing behavioural patterns.

Predation is one of the strongest selection pressures, forcing prey organisms to detect predators and to display various antipredator behaviours, such as refuge-use or decreased activity. To recognise predators, chemosensory cues play a pivotal role, particularly in aquatic ecosystems. However, it is less known whether the ability to use these cues to respond with adequate antipredator behaviour varies between individuals occupying different habitats that are dissimilar in predation risk. Using field experiments, we examined antipredator behaviour of larval fire salamanders ( Salamandra salamandra ) from two different habitats, ponds and streams. Among other differences, ponds and streams are inhabited by habitat-specific predators, such as alpine newts ( Ichthyosaura alpestris ) occurring in ponds. We exposed larvae from both habitats to either chemical cues from alpine newts or a blank control (tap water) and investigated potential differences in their behavioural responses in two experiments. Pond larvae, but not stream larvae, became significantly less active when faced with chemical cues from newts compared to those faced with a control stimulus. Moreover, larvae from both habitats tested in water containing chemical cues spent significantly less time outside a shelter than those in control water. Our results demonstrate that larval fire salamanders recognise predatory newts through kairomones and alter their behaviour accordingly. However, experience with predatory newts may not be necessary to differentiate kairomones from control water, but may be beneficial for larvae to further develop their antipredator behaviour, thus representing conformance to a niche.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) infects numerous amphibian species worldwide and is suggested to drive population declines and extinction events. We report a study of Bd infection at the northernmost distribution of the European yellow-bellied toad Bombina variegata. A total of 577 individuals from ponds in 16 study sites were sampled for DNA and Bd throughout the breeding season. Microsatellite genotyping revealed 3 genetic clusters for the host B. variegata with an overall low genetic diversity. One of the clusters displayed a low microsatellite heterozygosity, a high inbreeding coefficient as well as high Bd infection prevalence and intensities. Multi-model estimates identified site, time of sampling, and heterozygosity to be important predictors of an individual's Bd infection status, and identified a strong effect of site on individual Bd infection intensity. The study site effects are suggestive of localized infection peaks, and the increase of individual Bd infection probabilities towards the end of the sampling period suggests cumulative infection during the breeding season. This study highlights the need for regular monitoring of Bd infection variables at multiple localities and times to gain insights into Bd dynamics. Due to the detected relationship between individual Bd infection status and heterozygosity, conservation measures should focus on the maintenance of high genetic diversity and connectivity within and among amphibian populations.

Population decline and local extinction of amphibian populations have increased in the last decades. The European tree frog (Hyla arborea) is particularly endangered in its northern distribution range. One of the suggested conservation strategies for threatened amphibians is resettlement. The Okologische Schutzstation Steinhuder Meer e.V. (OSSM) started a resettlement project in 2008 for the European tree frog in Lower Saxony and North Rhine-Westphalia, Germany. Hereby, individuals were translocated from a donor population to a location approximately 40 km distant, establishing a mirrored population. The objective of this study was to monitor genetic diversity of these two populations and genetic exchange with a nearby located population at Lake Steinhuder Meer. Therefore, a total of 91 individuals were analysed at 12 species-specific nuclear markers (microsatellite loci). The genetic diversities of the three populations were almost similar but the mirrored population (H-e = 0.66) exhibited a small reduction compared to its donor population (H-e = 0.72). Significant indications for a recent bottleneck were detected for the donor and mirrored population. However, the mirrored population seems to have recovered from founder effects, since the actual number of calling males surveyed at this site since establishment reveals a stable and steadily growing population. F-ST values and D-est values showed significant differentiation between all sites with a global F-ST of 0.08. Likewise, results of a Bayesian clustering analysis indicated the existence of three genetic clusters. The software Geneclass2 assigned nearly half of the individuals of the mirrored population to its source population. Furthermore, the analysis suggested recent migration between the mirrored population and Lake Steinhuder Meer. Compared to other Hyla populations the genetic diversity was high at all localities and population sizes seem to have increased. We conclude from our study that resettlement projects can be efficient measures to counteract amphibian population decline when supported by population genetic analyses.