Performance of different automatic photographic identification software for larvae and adults of the European fire salamander.

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.

Divergent population responses following salamander mass mortalities and declines driven by the emerging pathogen Batrachochytrium salamandrivorans.

Understanding wildlife responses to novel threats is vital in counteracting biodiversity loss. The emerging pathogen Batrachochytrium salamandrivorans (Bsal) causes dramatic declines in European salamander populations, and is considered an imminent threat to global amphibian biodiversity. However, real-life disease outcomes remain largely uncharacterized. We performed a multidisciplinary assessment of the longer-term impacts of Bsal on highly susceptible fire salamander (Salamandra salamandra) populations, by comparing four of the earliest known outbreak sites to uninfected sites. Based on large-scale monitoring efforts, we found population persistence in strongly reduced abundances to over a decade after Bsal invasion, but also the extinction of an initially small-sized population. In turn, we found that host responses varied, and Bsal detection remained low, within surviving populations. Demographic analyses indicated an ongoing scarcity of large reproductive adults with potential for recruitment failure, while spatial comparisons indicated a population remnant persisting within aberrant habitat. Additionally, we detected no early signs of severe genetic deterioration, yet nor of increased host resistance. Beyond offering additional context to Bsal-driven salamander declines, results highlight how the impacts of emerging hypervirulent pathogens can be unpredictable and vary across different levels of biological complexity, and how limited pathogen detectability after population declines may complicate surveillance efforts.

A pilot study to estimate the population size of endangered Galápagos marine iguanas using drones.

BACKGROUND: Large-scale species monitoring remains a significant conservation challenge. Given the ongoing biodiversity crisis, the need for reliable and efficient methods has never been greater. Drone-based techniques have much to offer in this regard: they allow access to otherwise unreachable areas and enable the rapid collection of non-invasive field data. Herein, we describe the development of a drone-based method for the estimation of population size in Galápagos marine iguanas, Amblyrhynchus cristatus. As a large-bodied lizard that occurs in open coastal terrain, this endemic species is an ideal candidate for drone surveys. Almost all Amblyrhynchus subspecies are Endangered or Critically Endangered according to the IUCN yet since several colonies are inaccessible by foot, ground- based methods are unable to address the critical need for better census data. In order to establish a drone-based approach to estimate population size of marine iguanas, we surveyed in January 2021 four colonies on three focal islands (San Cristobal, Santa Fe and Espanola) using three techniques: simple counts (the standard method currently used by conservation managers), capture mark-resight (CMR), and drone-based counts. The surveys were performed within a 4-day window under similar ambient conditions. We then compared the approaches in terms of feasibility, outcome and effort. RESULTS: The highest population-size estimates were obtained using CMR, and drone-based counts were on average 14% closer to CMR estimates-and 17-35% higher-than those obtained by simple counts. In terms of field-time, drone-surveys can be faster than simple counts, but image analyses were highly time consuming. CONCLUSION: Though CMR likely produces superior estimates, it cannot be performed in most cases due to lack of access and knowledge regarding colonies. Drone-based surveys outperformed ground-based simple counts in terms of outcome and this approach is therefore suitable for use across the range of the species. Moreover, the aerial approach is currently the only credible solution for accessing and surveying marine iguanas at highly remote colonies. The application of citizen science and other aids such as machine learning will alleviate the issue regarding time needed to analyze the images.

Context-dependent dispersal determines relatedness and genetic structure in a patchy amphibian population.

Dispersal is a central process in ecology and evolution with far reaching consequences for the dynamics and genetics of spatially structured populations (SSPs). Individuals can adjust their decisions to disperse according to local fitness prospects, resulting in context-dependent dispersal. By determining dispersal rate, distance and direction, these individual-level decisions further modulate the demography, relatedness and genetic structure of SSPs. Here, we examined how context-dependent dispersal influences the dynamics and genetics of a great crested newt (Triturus cristatus) SSP. We collected capture-recapture data of 5564 individuals and genetic data of 950 individuals across an SSP in northern Germany. We added genetic data from six sites outside this SSP to assess genetic structure and gene flow at a regional level. Dispersal rates within the SSP were high but dispersal distances were short. Dispersal was context-dependent: individuals preferentially immigrated into high-quality ponds where breeding probabilities were higher. The studied SSP behaved like a patchy population, where subpopulations at each pond were demographically interdependent. High context-dependent dispersal led to weak but significant spatial genetic structure and relatedness within the SSP. At the regional level, a strong hierarchical genetic structure with very few first-generation migrants as well as low effective dispersal rates suggest the presence of independent demographic units. Overall, our study highlights the importance of habitat quality for driving context-dependent dispersal and therefore demography and genetic structure in SSPs. Limited capacity for long-distance dispersal seems to increase genetic structure within a population and leads to demographic isolation in anthropogenic landscapes.

Landscape epidemiology of Batrachochytrium salamandrivorans: reconciling data limitations and conservation urgency.

Starting in 2010, rapid fire salamander (Salamandra salamandra) population declines in northwestern Europe heralded the emergence of Batrachochytrium salamandrivorans (Bsal), a salamander-pathogenic chytrid fungus. Bsal poses an imminent threat to global salamander diversity owing to its wide host range, high pathogenicity, and long-term persistence in ecosystems. While there is a pressing need to develop further research and conservation actions, data limitations inherent to recent pathogen emergence obscure necessary insights into Bsal disease ecology. Here, we use a hierarchical modeling framework to describe Bsal landscape epidemiology of outbreak sites in light of these methodological challenges. Using model selection and machine learning, we find that Bsal presence is associated with humid and relatively cool, stable climates. Outbreaks are generally located in areas characterized by low landscape heterogeneity and low steepness of slope. We further find an association between Bsal presence and high trail density, suggesting that human-mediated spread may increase risk for spillover between populations. We then use distribution modeling to show that favorable conditions occur in lowlands influenced by the North Sea, where increased survey effort is needed to determine how Bsal impacts local newt populations, but also in hill- and mountain ranges in northeastern France and the lower half of Germany. Finally, connectivity analyses suggest that these hill- and mountain ranges may act as stepping stones for further spread southward. Our results provide initial insight into regional environmental conditions underlying Bsal epizootics, present updated invasibility predictions for northwestern Europe, and lead us to discuss a wide variety of potential survey and research actions needed to advance future conservation and mitigation efforts.

No impact of a short-term climatic "El Niño" fluctuation on gut microbial diversity in populations of the Galápagos marine iguana (Amblyrhynchus cristatus).

Gut microorganisms are crucial for many biological functions playing a pivotal role in the host's well-being. We studied gut bacterial community structure of marine iguana populations across the Galápagos archipelago. Marine iguanas depend heavily on their specialized gut microbiome for the digestion of dietary algae, a resource whose growth was strongly reduced by severe "El Niño"-related climatic fluctuations in 2015/2016. As a consequence, marine iguana populations showed signs of starvation as expressed by a poor body condition. Body condition indices (BCI) varied between island populations indicating that food resources (i.e., algae) are affected differently across the archipelago during 'El Niño' events. Though this event impacted food availability for marine iguanas, we found that reductions in body condition due to "El Niño"-related starvation did not result in differences in bacterial gut community structure. Species richness of gut microorganisms was instead correlated with levels of neutral genetic diversity in the distinct host populations. Our data suggest that marine iguana populations with a higher level of gene diversity and allelic richness may harbor a more diverse gut microbiome than those populations with lower genetic diversity. Since low values of these diversity parameters usually correlate with small census and effective population sizes, we use our results to propose a novel hypothesis according to which small and genetically less diverse host populations might be characterized by less diverse microbiomes. Whether such genetically depauperate populations may experience additional threats from reduced dietary flexibility due to a limited intestinal microbiome is currently unclear and calls for further investigation.

Phylogenomic inference of species and subspecies diversity in the Palearctic salamander genus Salamandra.

The salamander genus Salamandra is widespread across Europe, North Africa, and the Near East and is renowned for its conspicuous and polymorphic colouration and diversity of reproductive modes. The phylogenetic relationships within the genus, and especially in the highly polymorphic species S. salamandra, have been very challenging to elucidate, leaving its real evolutionary history and classification at species and subspecies levels a topic of debate and contention. However, the distribution of diversity and species delimitation within the genus are critically important for identifying evolutionarily significant units for conservation and management, especially in light of threats posed by the pathogenic chytrid fungus Batrachochytrium salamandrivorans that is causing massive declines of S. salamandra populations in central Europe. Here, we conducted a phylogenomic analysis from across the taxonomic and geographic breadth of the genus Salamandra in its entire range. Bayesian, maximum likelihood and network-based phylogenetic analyses of up to 4905 ddRADseq-loci (294,300 nucleotides of sequence) supported the distinctiveness of all currently recognised species (Salamandra algira, S. atra, S. corsica, S. infraimmaculata, S. lanzai, and S. salamandra), and all five species for which we have multiple exemplars were confirmed as monophyletic. Within S. salamandra, two main clades can be distinguished: one clade with the Apenninic subspecies S. s. gigliolii nested within the Iberian S. s. bernardezi/fastuosa; and a second clade comprising all other Iberian, Central and East European subspecies. Our analyses revealed that some of the currently recognized subspecies of S. salamandra are paraphyletic and may require taxonomic revision, with the Central- and Eastern-European subspecies all being poorly differentiated at the analysed genomic markers. Salamandra s. longirostris - sometimes considered a separate species - was nested within S. salamandra, consistent with its subspecies status. The relationships identified within and between Salamandra species provide valuable context for future systematic and biogeographic studies, and help elucidate critical evolutionary units for conservation and taxonomy.

Phylotranscriptomic evidence for pervasive ancient hybridization among Old World salamanders.

Hybridization can leave genealogical signatures in an organism's genome, originating from the parental lineages and persisting over time. This potentially confounds phylogenetic inference methods that aim to represent evolution as a strictly bifurcating tree. We apply a phylotranscriptomic approach to study the evolutionary history of, and test for inter-lineage introgression in the Salamandridae, a Holarctic salamanders group of interest in studies of toxicity and aposematism, courtship behavior, and molecular evolution. Although the relationships between the 21 currently recognized salamandrid genera have been the subject of numerous molecular phylogenetic studies, some branches have remained controversial and sometimes affected by discordances between mitochondrial vs. nuclear trees. To resolve the phylogeny of this family, and understand the source of mito-nuclear discordance, we generated new transcriptomic (RNAseq) data for 20 salamandrids and used these along with published data, including 28 mitochondrial genomes, to obtain a comprehensive nuclear and mitochondrial perspective on salamandrid evolution. Our final phylotranscriptomic data set included 5455 gene alignments for 40 species representing 17 of the 21 salamandrid genera. Using concatenation and species-tree phylogenetic methods, we find (1) Salamandrina sister to the clade of the "True Salamanders" (consisting of Chioglossa, Mertensiella, Lyciasalamandra, and Salamandra), (2) Ichthyosaura sister to the Near Eastern genera Neurergus and Ommatotriton, (3) Triturus sister to Lissotriton, and (4) Cynops paraphyletic with respect to Paramesotriton and Pachytriton. Combining introgression tests and phylogenetic networks, we find evidence for introgression among taxa within the clades of "Modern Asian Newts" and "Modern European Newts". However, we could not unambiguously identify the number, position, and direction of introgressive events. Combining evidence from nuclear gene analysis with the observed mito-nuclear phylogenetic discordances, we hypothesize a scenario with hybridization and mitochondrial capture among ancestral lineages of (1) Lissotriton into Ichthyosaura and (2) Triturus into Calotriton, plus introgression of nuclear genes from Triturus into Lissotriton. Furthermore, both mitochondrial capture and nuclear introgression may have occurred among lineages assigned to Cynops. More comprehensive genomic data will, in the future, allow testing this against alternative scenarios involving hybridization with other, extinct lineages of newts.

Proteomics of Galápagos Marine Iguanas Links Function of Femoral Gland Proteins to the Immune System.

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.

Erratum: The Role of Plasticity and Adaptation in the Incipient Speciation of a Fire Salamander Population (Genes 2019, 10, 875).

In the original article, there was a mistake in Figure 5 as published. When summarizing the results in the scheme, the treatment groups were mixed, and so some of the symbols for morphological and gene expression traits were not in accordance with the results [...].

Functional colour genes and signals of selection in colour-polymorphic salamanders.

Coloration has been associated with multiple biologically relevant traits that drive adaptation and diversification in many taxa. However, despite the great diversity of colour patterns present in amphibians the underlying molecular basis is largely unknown. Here, we use insight from a highly colour-variable lineage of the European fire salamander (Salamandra salamandra bernardezi) to identify functional associations with striking variation in colour morph and pattern. The three focal colour morphs-ancestral black-yellow striped, fully yellow and fully brown-differed in pattern, visible coloration and cellular composition. From population genomic analyses of up to 4,702 loci, we found no correlations of neutral population genetic structure with colour morph. However, we identified 21 loci with genotype-phenotype associations, several of which relate to known colour genes. Furthermore, we inferred response to selection at up to 142 loci between the colour morphs, again including several that relate to coloration genes. By transcriptomic analysis across all different combinations, we found 196 differentially expressed genes between yellow, brown and black skin, 63 of which are candidate genes involved in animal coloration. The concordance across different statistical approaches and 'omic data sets provide several lines of evidence for loci linked to functional differences between colour morphs, including TYR, CAMK1 and PMEL. We found little association between colour morph and the metabolomic profile of its toxic compounds from the skin secretions. Our research suggests that current ecological and evolutionary hypotheses for the origins and maintenance of these striking colour morphs may need to be revisited.

The Role of Plasticity and Adaptation in the Incipient Speciation of a Fire Salamander Population.

Phenotypic plasticity and local adaptation via genetic change are two major mechanisms of response to dynamic environmental conditions. These mechanisms are not mutually exclusive, since genetic change can establish similar phenotypes to plasticity. This connection between both mechanisms raises the question of how much of the variation observed between species or populations is plastic and how much of it is genetic. In this study, we used a structured population of fire salamanders (Salamandra salamandra), in which two subpopulations differ in terms of physiology, genetics, mate-, and habitat preferences. Our goal was to identify candidate genes for differential habitat adaptation in this system, and to explore the degree of plasticity compared to local adaptation. We therefore performed a reciprocal transfer experiment of stream- and pond-originated salamander larvae and analyzed changes in morphology and transcriptomic profile (using species-specific microarrays). We observed that stream- and pond-originated individuals diverge in morphology and gene expression. For instance, pond-originated larvae have larger gills, likely to cope with oxygen-poor ponds. When transferred to streams, pond-originated larvae showed a high degree of plasticity, resembling the morphology and gene expression of stream-originated larvae (reversion); however the same was not found for stream-originated larvae when transferred to ponds, where the expression of genes related to reduction-oxidation processes was increased, possibly to cope with environmental stress. The lack of symmetrical responses between transplanted animals highlights the fact that the adaptations are not fully plastic and that some level of local adaptation has already occurred in this population. This study illuminates the process by which phenotypic plasticity allows local adaptation to new environments and its potential role in the pathway of incipient speciation.

Patterns, Mechanisms and Genetics of Speciation in Reptiles and Amphibians.

In this contribution, the aspects of reptile and amphibian speciation that emerged from research performed over the past decade are reviewed. First, this study assesses how patterns and processes of speciation depend on knowing the taxonomy of the group in question, and discuss how integrative taxonomy has contributed to speciation research in these groups. This study then reviews the research on different aspects of speciation in reptiles and amphibians, including biogeography and climatic niches, ecological speciation, the relationship between speciation rates and phenotypic traits, and genetics and genomics. Further, several case studies of speciation in reptiles and amphibians that exemplify many of these themes are discussed. These include studies of integrative taxonomy and biogeography in South American lizards, ecological speciation in European salamanders, speciation and phenotypic evolution in frogs and lizards. The final case study combines genomics and biogeography in tortoises. The field of amphibian and reptile speciation research has steadily moved forward from the assessment of geographic and ecological aspects, to incorporating other dimensions of speciation, such as genetic mechanisms and evolutionary forces. A higher degree of integration among all these dimensions emerges as a goal for future research.

Pooling skin swabs does not inhibit qPCR detection of amphibian chytrid infection.

Immediate and reliable pathogen detection in large numbers of samples is essential in wildlife disease monitoring and is often realized by DNA-based techniques. Pooling samples increases processing efficiency and reduces processing costs, and has been suggested as a viable technique for quantitative PCR detection of fungal amphibian pathogens of the genus Batrachochytrium. For these fungi, this diagnostic method has been validated by in vitro set ups that provided controlled test conditions but did not take into account potential effects from amphibian skin compounds (e.g. skin secretions and Microbiota) on the approach. Some of these skin compounds are known to cause PCR inhibition in single sample applications and could lead to false negative reactions and thereby hamper pathogen detection. In this study we examined the effect of skin compounds on the pooled extraction method by swabbing individuals of seven amphibian species (one Anura and six Caudata) prior to the inoculation of the swabs with chytrid zoospores. For each species, swabs were extracted in pools of different sizes (from one to four swabs) with only one swab per pool being inoculated with zoospores. There were no significant differences regarding the ability to detect zoospores when comparing pool sizes for any species, with a tendency for more false negatives when the inoculated swab had been inoculated with a single zoospore. This study provides further in vivo evidence for the viability of the pooled extraction method for DNA-based detection of pathogens.

Geographic separation and genetic differentiation of populations are not coupled with niche differentiation in threatened Kaiser's spotted newt (Neurergus kaiseri).

The combination of niche modelling and landscape genetics (genomics) helps to disentangle processes that have shaped population structure in the evolutionary past and presence of species. Herein, we integrate a comprehensive genomic dataset with ecological parameters and niche modelling for the threatened Kaiser's newt, a newt species adapted to mountain spring-ponds in Iran. Genomic analysis suggests the existence of two highly differentiated clades North and South of the Dez River. Genetic variation between the two clades (76.62%) was much greater than within clades (16.25%), suggesting that the Dez River prevented gene flow. River disconnectivity, followed by geographic distance, contributed mostly to genetic differentiation between populations. Environmental niche and landscape resistance had no significant influence. Though a significant difference between climatic niches occupied by each clade at the landscape-scale, habitat niches at the local-scale were equivalent. 'Niche similarity analysis' supported niche conservatism between the two clades despite the southward shift in the climatic niche of the Southern clade. Accordingly, populations of different clades may occupy different climatic niches within their ancestral niche. Our results indicate that the change of climatic conditions of geographically and genetically separated populations does not necessarily result in the shift of an ecological niche.

The conspicuous postmetamorphic coloration of fire salamanders, but not their toxicity, is affected by larval background albedo.

Experimental work in the early 20th century showed that background albedo experienced by larvae of the fire salamander (Salamandra salamandra) induce a durable morphological modification of the postmetamorphic color pattern, which needed confirmation due to the controversies regarding Paul Kammerer's experiments. Such a carry-over effect would be relevant as the black and yellow pattern of the alkaloid-containing adult fire salamanders has been suggested to serve as an aposematic signal. Hence, we hypothesized that (a) adult coloration is conspicuous to potential predators under light conditions at night, given the nocturnal activity of this species, and (b) a condition affecting the salamander's coloration pattern would also affect its toxicity to maintain a quantitatively honest aposematic signal. To test the first hypothesis, we used spectrometry to model the vision of potential avian and snake predators and confirmed that fire salamander's black-and-yellow pattern is contrasting enough against the forest leaf litter to be considered conspicuous at night. To test the second hypothesis, we first confirmed the background carry-over effect on black and yellow proportions in the dorsal skin of experimentally reared fire salamanders, using a rigorous experimental design. Then, we calculated the conspicuousness and determined the alkaloid profiles of these individuals. We did not find a correlation between conspicuousness and toxicity at the intrapopulation level. Moreover, there was no background carry-over effect on the alkaloid profile. We discuss our results in a physiological, ecological, evolutionary, and historical context.

Allopatric diversification and evolutionary melting pot in a North African Palearctic relict: The biogeographic history of Salamandra algira.

North Africa is a climatically and topographically complex region with unique biotic assemblages resulting from the combination of multiple biogeographic realms. Here, we assess the role of climate in promoting intra-specific diversification in a Palearctic relict, the North African fire salamander, Salamandra algira, using a combination of phylogenetic and population genetic analyses, paleoclimatic modelling and niche overlap tests. We used mitochondrial DNA (Cyt-b), 9838 ddRADseq loci, and 14 microsatellite loci to characterize patterns of genetic diversity and population structure. Phylogenetic analyses recover two major clades, each including several lineages with mito-nuclear discordances suggesting introgressive patterns between lineages in the Middle Atlas, associated with a melting pot of genetic diversity. Paleoclimatic modelling identified putative climatic refugia, largely matching areas of high genetic diversity, and supports the role of aridity in promoting allopatric diversification associated with ecological niche conservatism. Overall, our results highlight the role of climatic microrefugia as drivers of populations' persistence and diversification in the face of climatic oscillations in North Africa, and stress the importance of accounting for different genomic regions when reconstructing biogeographic processes from molecular markers.

A salamander's toxic arsenal: review of skin poison diversity and function in true salamanders, genus Salamandra.

Terrestrial salamanders of the genus Salamandra represent one of the most prominent groups of amphibians. They are mainly distributed across Europe but also reach Northern Africa and the Near East. Members of the six currently accepted species have long been known to be poisonous; however, work on their toxins was mostly published in German language, and therefore, many nuances of these studies have remained hidden from the majority of herpetologists and toxinologists. Several Salamandra species are called fire salamanders due to their highly contrasted, black-yellow colouration which probably serves to deter predators, although thorough evidence for aposematism in Salamandra is still lacking. Salamandra skin toxins do not only represent a potent antipredator defence but may also have antimicrobial effects. A better understanding of this dual function of Salamandra skin secretions is of utmost importance in the face of the emergence of a fungal disease causing catastrophic declines of fire salamanders in Central Europe, caused by the fungus Batrachochytrium salamandrivorans. In this review, we summarize the knowledge on Salamandra toxins, providing a list of the compounds so far isolated from their secretion and focusing on the bioactivity of the major compounds in Salamandra secretions, the steroidal alkaloids. We identify priorities for future research, including a screening of co-occurrence of steroidal alkaloids and tetrodotoxins in salamandrids, chemical characterization of already identified novel steroidal compounds, elucidation of the presence and role of peptides and proteins in the secretion, and experimental in vitro and in vivo study of the interactions between bioactive compounds in Salamandra skin secretions and cutaneous fungal and bacterial pathogens.

Disruption of skin microbiota contributes to salamander disease.

Escalating occurrences of emerging infectious diseases underscore the importance of understanding microbiome-pathogen interactions. The amphibian cutaneous microbiome is widely studied for its potential to mitigate disease-mediated amphibian declines. Other microbial interactions in this system, however, have been largely neglected in the context of disease outbreaks. European fire salamanders have suffered dramatic population crashes as a result of the newly emerged Batrachochytrium salamandrivorans (Bsal). In this paper, we investigate microbial interactions on multiple fronts within this system. We show that wild, healthy fire salamanders maintain complex skin microbiotas containing Bsal-inhibitory members, but these community are present at a remarkably low abundance. Through experimentation, we show that increasing bacterial densities of Bsal-inhibiting bacteria via daily addition slowed disease progression in fire salamanders. Additionally, we find that experimental-Bsal infection elicited subtle changes in the skin microbiome, with selected opportunistic bacteria increasing in relative abundance resulting in septicemic events that coincide with extensive destruction of the epidermis. These results suggest that fire salamander skin, in natural settings, maintains bacterial communities at numbers too low to confer sufficient protection against Bsal, and, in fact, the native skin microbiota can constitute a source of opportunistic bacterial pathogens that contribute to pathogenesis. By shedding light on the complex interaction between the microbiome and a lethal pathogen, these data put the interplay between skin microbiomes and a wildlife disease into a new perspective.

Jailed in the mountains: Genetic diversity and structure of an endemic newt species across the Pyrenees.

The Pyrenees represent a natural laboratory for biogeographic, evolutionary and ecological research of mountain fauna as a result of the high variety of habitats and the profound effect of the glacial and interglacial periods. There is a paucity of studies providing a detailed insight into genetic processes and better knowledge on the patterns of genetic diversity and how they are maintained under high altitude conditions. This is of particular interest when considering the course of past climate conditions and glaciations in a species which is considered site tenacious, with long generation times. Here we analyzed the genetic patterns of diversity and structure of the endemic Pyrenean brook newt (Calotriton asper) along its distribution range, with special emphasis on the distinct habitat types (caves, streams, and lakes), and the altitudinal and geographical ranges, using a total set of 900 individuals from 44 different localities across the Pyrenean mountain range genotyped for 19 microsatellite loci. We found evidence for a negative longitudinal and positive altitudinal gradient of genetic diversity in C. asper populations. The fact that genetic diversity was markedly higher westwards is in accordance with other Pyrenean species. However, the impact of altitudinal gradient on the genetic diversity seems to differ from other species, and mostly from other amphibians. We found that lower altitudes can act as a barrier probably because the lowlands do not provide a suitable habitat for C. asper. Regarding the distinct habitat types, caves had significantly lower values of genetic diversity compared to streams or lakes. The mean FST value was relatively high (0.304) with maximum values as high as 0.771, suggesting a highly structured total population. Indeed, populations were grouped into five subclusters, the eastern populations (cluster 1) remained grouped into two subclusters and the central-western Pyrenees (cluster 2) into three subclusters. The increase of isolation with geographical distance is consistent with the population structure detected. In conclusion, C. asper seems to be adapted to high altitude mountain habitats, and its genetic diversity is higher in the western Pyrenees. In terms of conservation priority, we consider more relevant the populations that represent a reservoir of genetic diversity.