Salamandridae Research Articles

Foraging success is differently affected by local climate in two syntopic forest-dwelling salamanders

Amphibians are small ectothermic vertebrates with high permeable skins that are highly constrained by environmental conditions, such as humidity and temperature. Therefore, terrestrial salamanders regulate their surface activity, including foraging, to minimize evaporative water loss. The influence of local micro-climatic factors on the foraging success of European forest dwelling salamanders has rarely been analysed in detail. We examined the role of local climatic conditions on the foraging success, measured by the total number of prey items, of two syntopic forest-dwelling salamanders: Strinati’s cave salamander, Speleomantes strinatii (family Plethodontidae), and Northern spectacled salamander, Salamandrina perspicillata (family Salamandridae). We evaluated the effects of rainfall, temperature and body size on the foraging success of these two species, in eight sampling sites in the Northern Apennines of Italy, for a total of 11 salamander populations. Our findings show that different climatic variables affected the two species differently. Salamandrina perspicillata foraged more intensively during periods of prolonged rainfall with higher temperatures. On the contrary, S. strinatii foraging success was negatively influenced by rising temperature, while rainfall seemed to have no significant effect. Interestingly, body size increased success in S. perspicillata but not in S. strinatii. Overall, our findings show that, when foraging in the same environment, these two species are conditioned by different climatic factors possibly due to their differences in overall body size and their ability to resist dehydration.

Toxic Relationships and Arms-Race Coevolution Revisited.

Toxin evolution in animals is one of the most fascinating and complex subjects of scientific inquiry today. Gaining an understanding of toxins poses a multifaceted challenge given the diverse modes of acquisition, evolutionary adaptations, and abiotic components that affect toxin phenotypes. Here, we highlight some of the main genetic and ecological factors that influence toxin evolution and discuss the role of antagonistic interactions and coevolutionary dynamics in shaping the direction and extent of toxicity and resistance in animals. We focus on toxic Pacific newts (family Salamandridae, genus Taricha) as a system to investigate and better evaluate the widely distributed toxin they possess, tetrodotoxin (TTX), and the hypothesized model of arms-race coevolution with snake predators that is used to explain phenotypic patterns of newt toxicity. Finally, we propose an alternative coevolutionary model that incorporates TTX-producing bacteria and draws from an elicitor-receptor concept to explain TTX evolution and ecology.

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.

Detecting inter- and intra-island genetic diversity: population structure of the endangered crocodile newt, Echinotriton andersoni, in the Ryukyus

The endangered crocodile newt, Echinotriton andersoni, is a relatively large species of the family Salamandridae and is distributed on six islands in the central part of the Ryukyu Archipelago, Japan. Because of an originally small distribution range and recent habitat loss, this species has been steadily declining in number. To elucidate fine-scale population structure, which is essential for effective conservation management, we analyzed genetic diversity and gene flow based on nine microsatellite loci. Our results identified three different island groups (Amamioshima, Tokunoshima, and Okinawajima) and multiple genetic assemblages within the Amami and Okinawa island groups. The gross genetic variation within each island was positively correlated with island size. Population structure followed a latitudinal cline and isolation by distance, even among geographically isolated islands. In northern Okinawajima, relatively complex genetic structure was observed. This unexpected population structure seems to reflect historical migration and distribution expansion through the formation of land bridges and shifted coastlines in the Pleistocene. We also found that small islands showed little genetic variation (Ukeshima, Sesokojima, and Tokashikijima). In particular, our findings revealed that the Tokashikijima population is at greater risk for extinction than the other populations because it has the smallest effective population size.

Slow life-history strategies are associated with negligible actuarial senescence in western Palaearctic salamanders.

Actuarial senescence has been viewed for a long time as an inevitable and uniform process. However, the work on senescence has mainly focused on endotherms with deterministic growth and low regeneration capacity during the adult stage, leading to a strong taxonomic bias in the study of ageing. Recent studies have highlighted that senescence could indeed display highly variable trajectories that correlate with species life-history traits. Slow life histories and indeterminate growth seem to be associated with weak and late senescence. Furthermore, high regenerative abilities could lead to negligible senescence in ectotherms. However, demographic data for species that would allow testing of these hypotheses are scarce. Here, we investigated senescence patterns in 'true salamanders' from the western Palaearctic. Our results showed that salamanders have slow life histories and that they experience negligible senescence. This pattern was consistent at both intra- and interspecific levels, suggesting that the absence of senescence may be a phylogenetically conserved trait. The regenerative capacities of salamanders, in combination with other physiological and developmental features such as an indeterminate growth and a low metabolic rate, probably explain why these small ectotherms have lifespans similar to that of large endotherms and, in contrast with most amniotes, undergo negligible senescence. Our study seriously challenges the idea that senescence is a ubiquitous phenomenon in the tree of life.

Phylogeny and species delimitation of near Eastern Neurergus newts (Salamandridae) based on genome-wide RADseq data analysis

We reconstruct the molecular phylogeny of Near Eastern mountain brook newts of the genus Neurergus (family Salamandridae) based on newly determined RADseq data, and compare the outcomes of concatenation-based phylogenetic reconstruction with species-tree inference. Furthermore, we test the current taxonomy of Neurergus (with four species: Neurergus strauchii, N. crocatus, N. kaiseri, and N. derjugini) against coalescent-based species-delimitation approaches of our genome-wide genetic data set. While the position of N. strauchii as sister species to all other Neurergus species was consistent in all of our analyses, the phylogenetic relationships between the three remaining species changed depending on the applied method. The concatenation approach, as well as quartet-based species-tree inference, supported a topology with N. kaiseri as the closest relative to N. derjugini, while full-coalescent species-tree inference approaches supported N. crocatus as sister species of N. derjugini. Investigating the individual signal of gene trees highlighted an extensive variation among gene histories, most likely resulting from incomplete lineage sorting. Coalescent-based species-delimitation models suggest that the current taxonomy might underestimate the species richness within Neurergus and supports seven species. Based on the current sampling, our analysis suggests that N. strauchii, N. derjugini and N. kaiseri might each be subdivided into further species. However, as amphibian species are known to be composed of deep conspecific lineages that do not always warrant species status, these results need to be cautiously interpreted in an integrative taxonomic framework. We hypothesize that the rather shallow divergences detected within N. kaiseri and N. derjugini likely reflect an ongoing speciation process and thus require further investigation. On the contrary, the much deeper genetic divergence found between the two morphologically and geographically differentiated subspecies of N. strauchii leads us to propose that N. s. barani should be considered a distinct species, Neurergus barani Öz, 1994.

Non-native Amphibian Pet Trade via Internet in Poland

Abstract Overharvesting and trade in amphibian populations is one of the causes of their global decline. Online trade not only encourages the exploitation of an increasing number of rare and endangered amphibian species from all over the world but also influences the spread of invasive species. The aim of our research was to investigate the amphibian pet trade conducted in online stores and portals in Poland and determine its potential impact on native species. Between November 2013 and October 2014, we regularly (on a monthly basis) checked sale offers on the websites of the 18 biggest pet shops in the country specialised in exotic animals, on a nationwide auction portal and on three exotic pet fan portals. During the study, we reported 486 offers of 112 amphibian species in online stores and on portals. Most of the offers involved one of the four families of amphibians: poison dart frogs (Dendrobatidae), tree frogs (Hylidae), true toads (Bufonidae) and true salamanders (Salamandridae). Our data show increased interest in amphibians as pets in Poland. At least half of the offered species are possible hosts for the chytrid fungus Batrachochytrium dendrobatidis. However, only one species, the American bullfrog Lithobates catesbeianus (Shaw, 1802), appears to be a potential invasive species. To summarise, the species offered in Poland that are characterised as threatened are predominantly those that are relatively easy to breed and that are popular as pets. Further studies are required to investigate the real threat to wild amphibian populations caused by the pet trade

The changing views on the evolutionary relationships of extant Salamandridae (Amphibia: Urodela).

The phylogenetic relationships among members of the family Salamandridae have been repeatedly investigated over the last 90 years, with changing character and taxon sampling. We review the changing composition and the phylogenetic position of salamandrid genera and species groups and add a new phylogeny based exclusively on sequences of nuclear genes. Salamandrina often changed its position depending on the characters used. It was included several times in a clade together with the primitive newts (Echinotriton, Pleurodeles, Tylototriton) due to their seemingly ancestral morphology. The latter were often inferred as a monophyletic clade. Respective monophyly was almost consistently established in all molecular studies for true salamanders (Chioglossa, Lyciasalamandra, Mertensiella, Salamandra), modern Asian newts (Cynops, Laotriton, Pachytriton, Paramesotriton) and modern New World newts (Notophthalmus, Taricha). Reciprocal non-monophyly has been established through molecular studies for the European mountain newts (Calotriton, Euproctus) and the modern European newts (Ichthyosaura, Lissotriton, Neurergus, Ommatotriton, Triturus) since Calotriton was identified as the sister lineage of Triturus. In pre-molecular studies, their respective monophyly had almost always been assumed, mainly because a complex courtship behaviour shared by their respective members. Our nuclear tree is nearly identical to a mito-genomic tree, with all but one node being highly supported. The major difference concerns the position of Calotriton, which is no longer nested within the modern European newts. This has implications for the evolution of courtship behaviour of European newts. Within modern European newts, Ichthyosaura and Lissotriton changed their position compared to the mito-genomic tree. Previous molecular trees based on seemingly large nuclear data sets, but analysed together with mitochondrial data, did not reveal monophyly of modern European newts since taxon sampling and nuclear gene coverage was too poor to obtain conclusive results. We therefore conclude that mitochondrial and nuclear data should be analysed on their own.

Habitat suitability prediction for Salamandra infraimmaculata (Caudata: Amphibia) in western Iran based on species distribution modeling

Salamandra infraimmaculata is a member of family Salamandridae that is distributed in Iran, Turkey, and Lebanon. We surveyed habitat suitability predictions for the species in Kurdistan province, Western Iran. Distribution prediction showed excellent performance (area under the curve = 0.962), with temperature seasonality (47.8%) and precipitation of coldest quarter (38.4%) being two main bioclimatic variables predicting species presence. Temperature and precipitation are two dependent variables on which vegetation and habitat type depend. Slopes of the western Zagros Mountains have moist and temperate oak forests that contain many springs. The presence of both oak forests and water resources may allow the establishment of populations of fire salamanders in the region. As the forests are decreasing because of human activities, the amount of suitable habitats for this species is also decreasing. It is necessary to develop and implement conservation programs to protect the oak forests on the western slopes of the Zagros.

A biogeographic and ecological perspective to the evolution of reproductive behaviour in the family Salamandridae

Amphibians have a complex reproductive behaviour, which shows the highest diversity among tetrapodes. The family Salamandridae, distributed across the entire Holarctic, is one of the most diverse groups of extant salamanders comprising 114 species in 21 genera. The family has a remarkable diversity of courtship modes, amplexus and sperm transfer. It is often hypothesised that this diversity has evolved in adaptation to a specific mating and/or breeding habitat. We test this hypothesis based upon a phylogenetic reconstruction using the complete mitochondrial genome sequences of 45 Salamandridae species, representing all existing genera. We used ancestral character state reconstruction methods and geographic range models and applied relaxed Bayesian molecular clock models to discuss the results in a temporal framework of Salamandridae evolution. Our results show that the family Salamandridae started to diversify in the Late Cretaceous (ca. 87 mya) and is of Western Palearctic origin. Ancestral character state reconstruction predicts that its common ancestor was oviparous, mated on land without amplexus and probably showed a pin wheel spermatophore transfer, which is still found in the Italian endemic Salamandrina terdigidata. Our results suggest that several colonization of continents with subsequent radiations took place, once to the Nearctic and twice into Eastern Asian realms. However, these events were only in one case associated with a change in mating behaviour (dorsal amplexus in Nearctic newts). Around the Cretaceous-Paleogene boundary (K-Pg boundary) several Salamandridae lineages further diverged, again with no obvious changes in mating behaviour. Overall, there is no significant signal for mating character evolution being caused by changes in habitat type, with only a slight tendency that changes in mating habitat might have led to changes in the type of sperm transfer which in turn was associated with changes in the presence or absence of amplexus.

Evolution of skull shape in the family Salamandridae (Amphibia: Caudata).

We carried out a comparative morphometric analysis of 56 species of salamandrid salamanders, representing 19 out of 21 extant genera, with the aim of uncovering the major patterns of skull shape diversification, and revealing possible trends and directions of evolutionary change. To do this we used micro-computed tomography scanning and three-dimensional geometric morphometrics, along with a well-resolved molecular phylogeny. We found that allometry explains a relatively small amount of shape variation across taxa. Congeneric species of salamandrid salamanders are more similar to each other and cluster together producing distinct groups in morphospace. We detected a strong phylogenetic signal and little homoplasy. The most pronounced changes in the skull shape are related to the changes of the frontosquamosal arch, a unique feature of the cranial skeleton for the family Salamandridae, which is formed by processes arising from the frontal and squamosal bones that arch over the orbits. By mapping character states over the phylogeny, we found that a reduction of the frontosquamosal arch occurs independently in three lineages of the subfamily Pleurodelinae. This reduction can probably be attributed to changes in the development and ossification rates of the frontosquamosal arch. In general, our results are similar to those obtained for caecilian amphibians, with an early expansion into the available morphospace and a complex history characterizing evolution of skull shape in both groups. To evaluate the specificity of the inferred evolutionary trajectories and Caudata-wide trends in the diversity of skull morphology, information from additional groups of tailed amphibians is needed.

Inferring the shallow phylogeny of true salamanders (Salamandra) by multiple phylogenomic approaches

The rise of high-throughput sequencing techniques provides the unprecedented opportunity to analyse controversial phylogenetic relationships in great depth, but also introduces a risk of being misinterpreted by high node support values influenced by unevenly distributed missing data or unrealistic model assumptions. Here, we use three largely independent phylogenomic data sets to reconstruct the controversial phylogeny of true salamanders of the genus Salamandra, a group of amphibians providing an intriguing model to study the evolution of aposematism and viviparity. For all six species of the genus Salamandra, and two outgroup species from its sister genus Lyciasalamandra, we used RNA sequencing (RNAseq) and restriction site associated DNA sequencing (RADseq) to obtain data for: (1) 3070 nuclear protein-coding genes from RNAseq; (2) 7440 loci obtained by RADseq; and (3) full mitochondrial genomes. The RNAseq and RADseq data sets retrieved fully congruent topologies when each of them was analyzed in a concatenation approach, with high support for: (1) S. infraimmaculata being sister group to all other Salamandra species; (2) S. algira being sister to S. salamandra; (3) these two species being the sister group to a clade containing S. atra, S. corsica and S. lanzai; and (4) the alpine species S. atra and S. lanzai being sister taxa. The phylogeny inferred from the mitochondrial genome sequences differed from these results, most notably by strongly supporting a clade containing S. atra and S. corsica as sister taxa. A different placement of S. corsica was also retrieved when analysing the RNAseq and RADseq data under species tree approaches. Closer examination of gene trees derived from RNAseq revealed that only a low number of them supported each of the alternative placements of S. atra. Furthermore, gene jackknife support for the S. atra - S. lanzai node stabilized only with very large concatenated data sets. The phylogeny of true salamanders thus provides a compelling example of how classical node support metrics such as bootstrap and Bayesian posterior probability can provide high confidence values in a phylogenomic topology even if the phylogenetic signal for some nodes is spurious, highlighting the importance of complementary approaches such as gene jackknifing. Yet, the general congruence among the topologies recovered from the RNAseq and RADseq data sets increases our confidence in the results, and validates the use of phylotranscriptomic approaches for reconstructing shallow relationships among closely related taxa. We hypothesize that the evolution of Salamandra has been characterized by episodes of introgressive hybridization, which would explain the difficulties of fully reconstructing their evolutionary relationships.

Individual fluctuations in toxin levels affect breeding site fidelity in a chemically defended amphibian.

Behaviours that influence habitat selection strongly determine species movement patterns. One component of animal behaviour that largely influences movement patterns and habitat choice is site fidelity. California newts (family Salamandridae) demonstrate remarkable site fidelity, typically homing to the same pool of a stream each breeding season. Individuals often occupy a specific pool throughout the breeding season, but some males shift among breeding pools, altering their set of potential mates, competitors, and predators. In this study, we measured dermal concentrations of the chemical defence compound tetrodotoxin (TTX) in recaptured male California newts (Taricha torosa) over five breeding seasons to evaluate whether relative TTX concentrations are associated with breeding site fidelity in the field. Our five years of field sampling indicates that TTX concentrations of individuals and group means fluctuate tremendously, implying that TTX is not a stable phenotypic trait. Despite such fluctuations, we found that an individual's relative TTX concentration explains fidelity to a breeding pool and suggests that newts may be able to assess both their own concentrations of TTX and that of conspecifics to make decisions about remaining in or abandoning a breeding pool. These results provide us a novel dimension to chemical defence phenotypes in nature and their ecological consequences, potentially requiring a re-evaluation of the coevolutionary dynamics of predation pressure on toxin-laden organisms.

Vertebral number is highly evolvable in salamanders and newts (family Salamandridae) and variably associated with climatic parameters

In vertebrates, the relative proportion of the number of trunk and caudal vertebrae is an important determinant of body shape. While among amphibians frogs and toads show low variation in vertebrae numbers, in salamanders the numbers of trunk and caudal vertebrae vary widely, giving rise to phenotypes in the range from short-bodied and long-tailed to long-bodied and short-tailed. We analysed vertebral numbers in the family Salamandridae in a phylogenetic context and calculated the relationship between vertebral changes and changes in climate and other environmental parameters. A significant association was found between morphological change with precipitation and temperature. However, annual precipitation affected the two main groups of salamandrid salamanders differently, with trunk elongation in the terrestrial ‘true salamanders’ and tail elongation in the more aquatic ‘newts’. A - male biased - sexual dimorphism was only observed in Lissotriton vulgaris vulgaris in the number of trunk vertebrae and in Ommatotriton ophryticus and Lissotriton species for the number of caudal vertebrae. Our data indicated that the number of trunk and caudal vertebrae are highly evolvable traits with frequent evolutionary reversals. In some groups (e.g. Cynops, Lyciasalamandra, Neurergus and the Laotriton- Pachytriton-Paramesotriton clade) the number of trunk vertebrae is stable, while in many groups it is subject to change (e.g. Tylototriton). This latter, species-rich genus appears to be an excellent group to further test effects of the environment on body shape.

A New Species of the Genus Tylototriton (Urodela: Salamandridae) from Northeastern Hunan Province, China

We describe a new species from the family Salamandridae from northeastern Hunan Province of China based on morphological and molecular genetic evidences. The new species, Tylototriton liuyangensis, is a member of the T asperrimus group, and shares a number of similarities with T wenxianensis and T broadoridgus. The dorsal surface of the new Tylototriton species is completely black, with nodule-like warts distributed evenly along the lateral margin of dorsal body, and there is no transverse striae between the warts. There are several diagnostic characters of the new species that differ from those of T wenxianensis and T broadoridgus as follows: (1) the interorbital space (IOS) is comparatively wide; (2) there is a bony ridge present on the dorsal head surface, running along the upper eyelids; (3) the space between axilla and groin (AGS) is comparatively large, with the mean male AGS approximately equal to 50% of the snout-vent length (SVL) and the mean female AGS being 53% of SVL; and (4) there is a small genital armature on the anterior angle of labial folds present inside the anal fissure of adult males during the breeding season. The result from the molecular phylogenetic analysis of the genus Tylototriton (including the type specimen of the new species) based on the complete DNA sequence of the mitochondrial ND2 gene (1035 bp) indicated that the new species was similar to T wenxianensis, T dabienicus, and T broadoridgus, but formed an independent clade. This result was consistent with the morphological analysis above, supporting that the Liuyang Population represented a distinct species.

Thyroid Hormone Mediation in Skull Development of Siberian Newt, Salamandrella keyserlingi (Urodela: Hynobiidae), with Comparison to Other Species

Sequence and timing of cranial bones appearance and metamorphic transformation were compared in larvae of Siberian newt, Salamandrella keyserlingi (Urodela, Hynobiidae), reared under different levels of thyroid hormone (TH): i) normal, ii) high (in 1 and 10 ng/ml T 3 ), and iii) low (in 0.02% thiourea, TU, which inhibits thyroid gland activity resulting in TH deficiency). Cranial bones exhibit differential reaction to changes in TH level. Bones appearing in early larval ontogeny display no or only slight response to TH changes. At different TH regimes, they arise at the same stages and at the same age. Bones appearing later show reaction to TH changes: their appearance is accelerated and retarded in TH- and TU-treated larvae, correspondingly. The major response to TH alterations is displayed by ontogenetic events involved into metamorphic remodeling: they are greatly accelerated under TH-treatment and retarded in TH-deficiency. Most metamorphic events fail to occur under TH-deficiency. Comparison of TH-related changes in skull development of Ambystoma mexicanum ( Ambystoma tidae), Triturus vulgaris (Salamandridae), and S. keyserlingi showed that in these species the same bones differ in reaction to TH changes. A corollary of these differences is that in urodeles TH-dependence and TH-sensitivity of cranial bones change both in ontogeny and phylogeny.

Development of the bony skeleton in two salamander species (Mertensiella caucasica and Chioglossa lusitanica: Salamandridae) with partial embryonization

168 The Caucasian salamander (Mertensiella cauca sica) and golden striped salamander (Chioglossa lusi tanica) are the most closely related taxa in the group of true salamanders belonging to the family Salaman dridae [1, 2]. In addition, they stand out among the other salamanders by a considerable similarity in their morphological constitution [3] and life style [3–5]. A study on the reproduction biology of these species has also demonstrated that they are very similar in a num ber of reproductive parameters, namely, both species reproduce in brooks and lay large (5–7 mm in diame ter) eggs in small clutches (on average, 17–18 eggs) [4–6]. These traits distinguish M. caucasica and C. lusitanica from the majority of the other true sala manders, characteristic of which is ovoviviparity or viviparity, and the remaining members of the family Salamandridae, newts, which lay considerably smaller eggs. On the other hand, the large size of the eggs and hatching of larvae at advanced development stages (as was demonstrated for M. caucasica [7]) suggest a pro nounced embryonization of development, i.e., transi tion of the initial stages in larval development to the embryonic period. Among amphibians, this evolutionary process leads to establishment of a direct development (lacking the larval stage), which is present only in members of the family Plethodontidae within the order Urodela. The effect of the degree of embryonization on the specific morphoge netic traits and, in particular, craniogenesis, has been also demonstrated for these amphibians [8, 9]. As we see it, a study of the skeletal development in the amphibian spe cies with partial embryonization of development will help to shed light onto the initial stages in the evolution path way that had led to direct development. For this purpose, we have studied the skeletal development of M. caucasica and C. lusitanica in the ontogenesis. Clutches of both salamander species were incubated under laboratory conditions; the embryos at late developmental stages (staring from limb differen tiation), larvae, and metamorphosing animals were regularly fixed with 10% formaldehyde and stained with alizarin red and alcian blue to detect bones and cartilages, respectively. The developmental stages were determined according to the table of M. caucasica normal development [7], which, except for some col oration traits, equally well described the C. lusitanica developmental stages. GENERAL BIOLOGY

Preference for the other sex: Olfactory sex recognition in terrestrial fire salamanders (Salamandra salamandra)

AbstractFinding a potential mate, that is an individual of the opposite sex, is a fundamental step for sexual reproduction in animal species. Signals involved in the context of mate attraction are mediated by acoustic, visual, and/or chemical signals. For amphibians in general, and especially for many newt and salamander species, chemical cues are known to play important roles in inter- and intraspecific communication. We therefore investigated the use of olfactory cues for sex recognition in terrestrial fire salamanders (Salamandra salamandra) that belong to the group of true salamanders within the Salamandridae. Specifically, we performed odor preference tests with adult salamanders of both sexes and tested whether substrate-borne chemical cues provide sex-specific signals. We found an overall preference for chemical cues of the opposite sex, i.e. males and females differed significantly in their preference for a specific sex. Females spent significantly more time in the compartment with the chemical cues of a male, whereas males did not show a significant preference, but in general more males preferred the compartment with the chemical cues of a female. Our results suggest that fire salamanders are capable to discriminate the sex of conspecific individuals based on pure chemical cues. We discuss our results in the light of the presence of dorsal glands in the males' cloaca of true salamander species, from which pheromones can be directly released onto the substrate.

Strong correlation between cross‐amplification success and genetic distance across all members of ‘True Salamanders’ (Amphibia: Salamandridae) revealed by Salamandra salamandra‐specific microsatellite loci

The unpredictable and low cross-amplification success of microsatellite loci tested for congeneric amphibian species has mainly been explained by the size and complexity of amphibian genomes, but also by taxonomy that is inconsistent with phylogenetic relationships among taxa. Here, we tested whether the cross-amplification success of nine new and 11 published microsatellite loci cloned for an amphibian source species, the fire salamander (Salamandra salamandra), correlated with the genetic distance across all members of True Salamanders (genera Chioglossa, Lyciasalamandra, Mertensiella and Salamandra that form a monophyletic clade within the family of Salamandridae) serving as target species. Cross-amplification success varied strongly among the species and showed a highly significant negative relationship with genetic distance and amplification success. Even though lineages of S.salamandra and Lyciasalamndra have separated more than 30 Ma, a within genus amplification success rate of 65% was achieved for species of Lyciasalamandra thus demonstrating that an efficient cross-species amplification of microsatellite loci in amphibians is feasible even across large evolutionary distances. A decrease in genome size, on the other hand, paralleled also a decrease in amplified loci and therefore contradicted previous results and expectations that amplification success should increase with a decrease in genome size. However, in line with other studies, our comprehensive dataset clearly shows that cross-amplification success of microsatellite loci is well explained by phylogenetic divergence between species. As taxonomic classifications on the species and genus level do not necessarily mirror phylogenetic divergence between species, the pure belonging of species to the same taxonomic units (i.e. species or genus) might be less useful to predict cross-amplification success of microsatellite loci between such species.

Reconstruction of the climate envelopes of salamanders and their evolution through time

Ecological niches evolve through time, but at different rates and to different degrees. An integrated approach using diverse databases, methods, and analytical tools is used to estimate climate envelopes for species of salamanders (family Salamandridae). These species, which range widely across the Holarctic and have a rich and long fossil record, are used to probe the evolutionary dynamics of niches studied in a phylogenetic context through time and across space. Climate data and statistical methods are used to estimate niche dimensions related to precipitation and temperature, in both a phylogenetic and ecogeographic context. Using phylogenetic methods, climate envelopes are estimated for segments of a time-calibrated phylogenetic tree of salamandrids, to explore how far back in time it is reasonable to make such estimates. Our research strategy illuminates some of the challenges and limitations of the available data and methods and identifies potential solutions, such as the need for physiological and behavioral data that may help to better define species' niches or the development of novel evolutionary models that account for paleoclimatic data. We explore and analyze limits to the application of currently available methodologies. Organisms known to have evolved slowly and conservatively, and which are ectotherms and likely to be profoundly affected by climatic variables, may be the most useful for studies of this kind.