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Traditional methods of phylogenetic reconstruction and species delimitation may be impeded by frequent hybridization among lineages. In this study, we conducted phylogenetic and clustering analyses of ddRAD genomic data on the entire genus Salamandra, which includes six species and over 25 subspecies of terrestrial salamanders. We expanded previous datasets to include missing subspecies and incorporated new samples, with an emphasis on secondary contact zones. Results obtained from a full dataset of 392 individuals (356,874 bp; 24,192 SNPs) were compared with those obtained after excluding substantially admixed individuals (n = 95; 835,467 bp; 51,557 SNPs) to explore the consequences of introgression on phylogenetic inference and taxonomic arrangement of subspecies. We found conflicting phylogenetic placements for taxa represented by many admixed individuals (identified by clustering ancestries). In contrast, a time-calibrated tree constructed without hybrids largely agrees with previous phylogenetic hypotheses. Within S. atra, we found paraphyly of S. atra atra, suggesting an additional candidate subspecies. Within S. infraimmaculata, two lineages are assignable to known subspecies and we additionally identified a third, deeply diverged lineage sampled near the Turkish/Syrian border. In S. algira, we found limited admixture between the subspecies S. a. tingitana and S. a. splendens despite their geographic proximity. Finally, within S. salamandra, we detected significant levels of hybridization between subspecies, which blurred their phylogenetic relationships, although the removal of admixed samples in subset analyses clarified the situation in most cases. Monophyly was recovered for subspecies that were previously found paraphyletic, including S. s. salamandra, S. s. gallaica, and S. s. fastuosa. Salamandra s. "alfredschmidti" was confirmed to be a junior synonym of S. s. bernardezi. Previously disputed subspecies, like S. s. "molleri" and S. s. "hispanica", correspond to separated lineages but are affected by admixture with other lineages. Further newly identified candidate subspecies in S. salamandra included a southern lineage within S. s. wer-neri and a western lineage within S. s. bernardezi. Finally, we re-evaluate the status of recognized subspecies in Salamandra, based on evidence from multiple delimitation criteria. Given that the evolutionary history could not be resolved for all subspecies, we highlight taxa within Salamandra that warrant further molecular examination and taxonomic revision, notably within the S. s. gallaica/"molleri"/bejarae complex. This study illustrates the impact of hybridization in phylogenetic analyses and its downstream effects in the identification of conservation units and their naming in the Linnean classification. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The composition of cutaneous (skin-associated) bacterial communities of amphibians has been intensively studied in light of the potential of some of these commensal bacterial taxa to mitigate infection with the chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). However, surprisingly, the absolute densities in which these bacteria occur on the skin are only poorly known. We here combine quantification of bacterial 16SrDNA copies from skin swabs by quantitative PCR with counts from scanning electron microscopy (SEM) images to assess and compare bacterial abundances on the skin of various central European amphibians. We focus on the fire salamander (Salamandra salamandra) which is severely threatened by the spread of Bsal. Based on counts from SEM pictures of selected skin parts, local densities of ~43,000 bacteria per mm² were ascertained in frogs, although the data are insufficient to understand whether these values apply to the whole body surface. Bacterial densities are doubtless much lower in fire salamanders in which we observed almost no bacteria in SEM examination. From qPCR data, we find: (i) statistically relevant differences in bacterial abundances among species, with the lowest abundances in terrestrial salamanders and the highest abundances in toads; (ii) higher bacterial loads in captive compared to wild fire salamanders, and much higher loads in Bsal-infected captive specimens, in agreement with the hypothesis that Bsalinfection leads to blooming of opportunistic bacteria that may cause secondary infection; (iii) an only weak decrease of bacterial load after repeated swabbing, in agreement with the hypothesis that skin swabs capture only a part of the bacteria of the swabbed skin surface. We discuss the multiple sources of uncertainty in absolute estimates of abundances of cutaneous bacteria and suggest further research to clarify and reduce these uncertainties. [ABSTRACT FROM AUTHOR]

Amphibians provide an iconic example of diseasedriven global loss in biodiversity. The recently described chytrid fungus Batrachochytrium salamandrivorans (Bsal) is an emerging pathogen that is driving amphibian populations to local [...]

Besides immediate effects, early nutritional conditions also have an impact on the entire life history of individuals. We tracked a cohort of captive fire salamanders that experienced only during their early larval phase, either rich or poor nutritional conditions, and thereafter intermediate standard conditions for almost 10 years. We measured on a regular basis, their morphometric variables, noted behavioural and colouration developments, and monitored their survival. While deficits in body mass and colouration from initially poorly raised individuals vanished in adults, differences in size in comparison to initially richly raised larvae remained throughout life. Behaviour at adulthood did not differ any further from that during early conditions, nevertheless it tended to be consistent in certain exploratory traits over several years. After almost ten years, the overall survival rate was about 50%, but so far, no effect of early living conditions on subsequent survival became apparent. Taken together, our long-term study on the effects of early nutritional conditions on fire salamanders revealed interesting insights into the life trajectory of these animals. Immediate effects from early nutritional conditions were found in almost all aspects of behaviour and physiology. The majority of effects are compensated within the lifetimes of individuals, apparently without detectable costs. [ABSTRACT FROM AUTHOR]

The Near Eastern fire salamander (Salamandra infraimmaculata) reaches its southern distribution range in Israel. Although the population structure has been analysed in central Israel and at the southern distribution limit, we lack knowledge on populations in the northern area, such as along Mount Hermon. S. infraimmaculata occurs at Mt. Hermon along an altitudinal gradient and appears to be fragmented by urban and agricultural landscape. We studied the genetic structure of four populations based on microsatellite loci and the mitochondrial D-loop to determine the genetic diversity and connectivity between populations. We observed moderate gene flow at lower parts, i.e. from Tel Dan and Nimrod Castle to Banias indicating extant but limited connectivity. Genetic diversity and gene flow declined along the altitudinal gradient at Mt. Hermon, reaching rock-bottom levels in the highest located population of Nimrod Pool. The observed isolation-by-elevation gradient might induce a higher extinction risk for the highland populations of S. infraimmaculata. [ABSTRACT FROM AUTHOR]