Salamandrid Species Research Articles

Genomic insights into the Montseny brook newt (Calotriton arnoldi), a Critically Endangered glacial relict

The Montseny brook newt (Calotriton arnoldi), considered the most endangered amphibian in Europe, is a relict salamandrid species endemic to a small massif located in northeastern Spain. Although conservation efforts should always be guided by genomic studies, those are yet scarce among urodeles, hampered by the extreme sizes of their genomes. Here, we present the third available genome assembly for the order Caudata, and the first genomic study of the species and its sister taxon, the Pyrenean brook newt (Calotriton asper), combining whole-genome and ddRADseq data. Our results reveal significant demographic oscillations which accurately mirrored Europe's climatic history. Although severe bottlenecks have led to depauperate genomic diversity and long runs of homozygosity along a gigantic genome, inbreeding might have been avoided by assortative mating strategies. Other life history traits, however, seem to have been less advantageous, and the lack of land dispersal has driven to exceptional levels of population fragmentation.

Histological changes of the skin during postembryonic development of the crested newt Triturus ivanbureschi (Urodela, Salamandridae)

BackgroundAmphibian skin has been studied for many decades, especially the metamorphic changes in the skin of frogs. Less attention has been paid to salamander skin. Here, we describe changes in the skin structure during postembryonic development in a salamandrid species, the Balkan crested newt Triturus ivanbureschi. MethodUsing traditional histological techniques we examined the skin in the trunk region of three premetamorphic larval stages (hatchling, mid larval and late larval) and two postmetamorphic stages (juvenile, just after metamorphosis, and adult). ResultsIn larval stages, skin consists only of the epidermis, which gradually develops from the single epithelial cell layer in hatchlings, to a stratified epidermis with gland nests and characteristic Leydig cells at the late larval stage. During metamorphosis, Leydig cells disappear, and the dermal layer develops. In postmetamorphic stages, skin is differentiated on stratified epidermis and the dermis with well-developed glands. Three types of glands were observed in the skin of the postmetamorphic stages: mucous, granular and mixed. Gland composition appears to be stage- and sex-specific, with juveniles and adult female being more similar to each other. In juveniles and adult female, there are a similar proportion of glands in both dorsal and ventral skin, whereas in adult male granular glands dominated the dorsal skin, while mixed glands dominated the ventral skin. ConclusionOur results provide a baseline for future comparative research of skin anatomy in salamanders.

Courtship behaviour and male sexual competition of the Taliang crocodile newt, Liangshantriton taliangensis

Abstract The Taliang crocodile newt, Liangshantriton taliangensis, which is endemic to south-western China, is a rare salamandrid species with a distinct evolutionary history. Using combined field observations and captive experiments, we studied the courtship behaviour and male sexual interference of this species and composed a complete ethogram of its courtship behaviour. Unlike previous reports on sperm transfer, male L. taliangensis did not deposit spermatophores during ventral amplexus and females did not pick up sperm masses in this courtship phase. Sperm transfer was only performed during arm-hooking pin-wheel circling, which distinguishes L. taliangensis from Tylototriton species and supports the validity of genus Liangshantriton. Whether L. taliangensis shows bimodality in sperm transfer needs to be explored in additional populations. In the presence of sexual competitors, male L. taliangensis adjusted the duration of their behaviour. We found that males with longer snout-vent length, heavier mass, and higher tailfins may have an advantage in intrasexual competition.

Batrachochytrium salamandrivorans is the predominant chytrid fungus in Vietnamese salamanders

The amphibian chytrid fungi, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), pose a major threat to amphibian biodiversity. Recent evidence suggests Southeast Asia as a potential cradle for both fungi, which likely resulted in widespread host-pathogen co-existence. We sampled 583 salamanders from 8 species across Vietnam in 55 locations for Bsal and Bd, determined scaled mass index as a proxy for fitness and collected environmental data. Bsal was found within 14 of the 55 habitats (2 of which it was detected in 2013), in 5 salamandrid species, with a prevalence of 2.92%. The globalized pandemic lineage of Bd was found within one pond on one species with a prevalence of 0.69%. Combined with a complete lack of correlation between infection and individual body condition and absence of indication of associated disease, this suggests low level pathogen endemism and Bsal and Bd co-existence with Vietnamese salamandrid populations. Bsal was more widespread than Bd, and occurs at temperatures higher than tolerated by the type strain, suggesting a wider thermal niche than currently known. Therefore, this study provides support for the hypothesis that these chytrid fungi may be endemic to Asia and that species within this region may act as a disease reservoir.

She gets many and she chooses the best: polygynandry inSalamandrina perspicillata(Amphibia: Salamandridae)

Polyandry is a widespread mating strategy, found in a broad number of taxa. Among amphibians, polyandry, and multiple paternity as its direct consequence, is quite common in salamanders, especially within Ambystomatidae and Plethodontidae. In the suborder Salamadroidea the existence of two different types of spermatheca allows several kinds of polyandry strategies to appear. We used multilocus microsatellite genotyping to investigate the presence of polyandry and its effects on the paternity in a previously unstudied species with a terrestrial habit, Salamandrina perspicillata. We collected gravid females in their natural habitat and analysed the paternity of the offspring by using the software COLONY and GERUD. We found that all the analysed clutches had been fertilized by 2–4 males and that in every clutch one male had sired most of the offspring. Our results confirmed that polyandry is an important component of the mating system of this species, suggesting that females are able to recognize the sperm of the male that will provide a genetic benefit for their offspring. We found evidence of female cryptic choice based on males' genetic dissimilarity: (1) males who sire most of the offspring of a given female tend to be genetically different from their sexual partner; (2) a same male, when mated with two females, sired a proportion of the offspring inversely correlated with his genetic similarity to the female; (3) genetic dissimilarity between mating partners is positively correlated with offspring heterozygosity. According to the genetic compatibility model, we hypothesized that in the observed non resource-based mating system the indirect benefit for the offspring should reflect interactions between paternal and maternal genomes rather than the inheritance of the so-called ‘good genes’. This study suggests a polygynandrous mating system for the study species and provides the first report in a salamandrid species in natural condition that reproductive success of males is correlated with genetic dissimilarity between mates. Moreover, we found evidence of an offspring benefit (higher heterozygosity) derived from the most genetically dissimilar father.

Origin and diversification of a salamander sex pheromone system.

Sex pheromones form an important facet of reproductive strategies in many organisms throughout the Animal Kingdom. One of the oldest known sex pheromones in vertebrates are proteins of the Sodefrin Precursor-like Factor (SPF) system, which already had a courtship function in early salamanders. The subsequent evolution of salamanders is characterized by a diversification in courtship and reproduction, but little is known on how the SPF pheromone system diversified in relation to changing courtship strategies. Here, we combined transcriptomic, genomic, and phylogenetic analyses to investigate the evolution of the SPF pheromone system in nine salamandrid species with distinct courtship displays. First, we show that SPF originated from vertebrate three-finger proteins and diversified through multiple gene duplications in salamanders, while remaining a single copy in frogs. Next, we demonstrate that tail-fanning newts have retained a high phylogenetic diversity of SPFs, whereas loss of tail-fanning has been associated with a reduced importance or loss of SPF expression in the cloacal region. Finally, we show that the attractant decapeptide sodefrin is cleaved from larger SPF precursors that originated by a 62 bp insertion and consequent frameshift in an ancestral Cynops lineage. This led to the birth of a new decapeptide that rapidly evolved a pheromone function independently from uncleaved proteins.

Facultative paedomorphosis and the pattern of intra- and interspecific variation in cranial skeleton: lessons from European newts (Ichthyosaura alpestris and Lissotriton vulgaris)

Paedomorphosis, the presence of ancestral larval and juvenile traits that occur at the descendent adult stage, is an evolutionary phenomenon that shaped morphological evolution in many vertebrate lineages, including tailed amphibians. Among salamandrid species, paedomorphic and metamorphic phenotypes can be observed within single populations (facultative paedomorphosis). Despite wide interest in facultative paedomorphosis and polymorphism produced by heterochronic changes (heterochronic polymorphism), the studies that investigate intraspecific morphological variation in facultative paedomorphic species are largely missing. By quantifying the cranium size and development (bone development and remodeling), we investigated the variation at multiple levels (i.e., between sexes, populations and species) of two facultatively paedomorphic European newt species: the alpine and the smooth newt. The pattern of variation between paedomorphs (individuals keeping larval traits at the adult stage) and metamorphs (metamorphosed adult individuals) varied between species and among populations within a single species. The patterns of variation in size and skull formation appear to be more uniform in the alpine than in the smooth newt, indicating that developmental constraints differed between species (more pronounced in alpine than in smooth newt). Our study shows that the cranial skeleton provides detailed insight in the pattern of variation and divergence in heterochronic polymorphism within and between species and open new questions related to heterochronic polymorphism and evolution of cranial skeleton.

Effects of Predation and Trophic Conditions on the Reproduction of Tylototriton verrucosus (Caudata: Salamandridae) in Darjeeling

The facts that small hatchlings emerged from small eggs laid under high predation levels prevailing at the lower altitudes of distribution of this species in Darjeeling while larger hatchlings emerged from larger eggs laid under lower levels of predation at higher altitudes, show that predation is not selected for large egg and initial hatchling size in this salamandrid species. Metamorphic size was small under high predation rates because this species relied on crypsis for evading predators. Egg and hatchling size are related inversely to levels of primary productivity and zooplankton abundance in lentic habitats. Hatchling sizes are related positively to egg size and size frequency distribution of zooplankton. Small egg and small hatchling size have been selected for at the lower altitudes of distribution of this salamandrid in Darjeeling because predation rates increased in step with improvement in trophic conditions at the lower altitudes.

Intraspecific variation in the egg-wrapping behaviour of female smooth newts, Lissotriton vulgaris

AbstractIn many species of the Salamandridae family, females provide parental care by carefully wrapping plant material around their eggs. As this behaviour has been shown to have a large effect on offspring survival, variation in this trait is expected to be low. Detailed investigations are, however, lacking. In the present study, we analyzed the consistency of egg-wrapping behaviour in two time periods within a breeding season in female smooth newts (Lissotriton vulgaris). We found a surprisingly low proportion of wrapped eggs of around 47% during the first period (when males were present) and an almost doubled ratio of around 92% in the second period (when males were absent). Also, the variation between individuals was significantly lower in the second period than in the first one. Furthermore, the bigger the females were, the more the proportion of wrapped eggs increased day by day within the first period; however, this relationship did not fully explain the observed difference between the two periods. Our results suggest that parental care in smooth newts can be influenced by the presence of mating partners and body size, and provide the first empirical evidence for within-individual variation in egg-wrapping in a salamandrid species.

Correlations of geographic distribution and temperature of embryonic development with the nuclear DNA content in the Salamandridae (Urodela, Amphibia)

We used flow cytometry to measure the nuclear DNA content in erythrocytes of 27 salamandrid species. Across these species, diploid genome size varied more than 2 fold (51.3-104.4 pg). According to genome size and geographic distribution, 3 groups of newt species were recognized: West Palearctics with smaller amounts of nuclear DNA; Nearctic, with intermediate values; and East Asiatic, with higher genome sizes. Viviparous West Palearctic salamanders differed from most of the oviparous West Palearctic newts in possessing larger genome sizes. The nuclear DNA content strongly correlates with species range limits. At the same temperature, embryos of salamandrid species with larger genome sizes have a markedly longer developmental time than those with smaller genomes. We present an analysis of the relationships between the amount of nuclear DNA and water temperature at the breeding sites.

A Bayesian approach on molecules and behavior: reconsidering phylogenetic and evolutionary patterns of the Salamandridae with emphasis on Triturus newts

The monophyly of European newts of the genus Triturus within the family Salamandridae has for decades rested on presumably homologous behavioral and morphological characters. Molecular data challenge this hypothesis, but the phylogenetic position of Triturus within the Salamandridae has not yet been convincingly resolved. We addressed this issue and the temporal divergence of Triturus within the Salamandridae with novel Bayesian approaches applied to DNA sequence data from three mitochondrial genes (12S, 16S and cytb). We included 38 salamandrid species comprising all 13 recognized species of Triturus and 16 out of 17 salamandrid genera. A clade comprising all the "Newts" can be separated from the "True Salamanders" and Salamandrina clades. Within the "Newts" well-supported clades are: Tylototriton-Pleurodeles, the "New World Newts" (Notophthalmus-Taricha), and the "Modern Eurasian Newts" (Cynops, Pachytriton, Paramesotriton=together the "Modern Asian Newts", Calotriton, Euproctus, Neurergus and Triturus species). We found that Triturus is a non-monophyletic species assemblage, which includes four groups that are themselves monophyletic: (i) the "Large-Bodied Triturus" (six species), (ii) the "Small-Bodied Triturus" (five species), (iii) T. alpestris and (iv) T. vittatus. We estimated that the last common ancestor of Triturus existed around 64 million years ago (mya) while the root of the Salamandridae dates back to 95 mya. This was estimated using a fossil-based molecular dating approach and an explicit framework to select calibration points that least underestimated their corresponding nodes. Using the molecular phylogeny we mapped the evolution of life history and courtship traits in Triturus and found that several Triturus-specific courtship traits evolved independently.

A molecular assessment of phylogenetic relationships and lineage accumulation rates within the family Salamandridae (Amphibia, Caudata)

We examine phylogenetic relationships among salamanders of the family Salamandridae using approximately 2700 bases of new mtDNA sequence data (the tRNA Leu, ND1, tRNA Ile, tRNA Gln, tRNA Met, ND2, tRNA Trp, tRNA Ala, tRNA Asn, tRNA Cys, tRNA Tyr, and COI genes and the origin for light-strand replication) collected from 96 individuals representing 61 of the 66 recognized salamandrid species and outgroups. Phylogenetic analyses using maximum parsimony and Bayesian analysis are performed on the new data alone and combined with previously reported sequences from other parts of the mitochondrial genome. The basal phylogenetic split is a polytomy of lineages ancestral to (1) the Italian newt Salamandrina terdigitata, (2) a strongly supported clade comprising the “true” salamanders (genera Chioglossa, Mertensiella, Lyciasalamandra, and Salamandra), and (3) a strongly supported clade comprising all newts except S. terdigitata. Strongly supported clades within the true salamanders include monophyly of each genus and grouping Chioglossa and Mertensiella as the sister taxon to a clade comprising Lyciasalamandra and Salamandra. Among newts, genera Echinotriton, Pleurodeles, and Tylototriton form a strongly supported clade whose sister taxon comprises the genera Calotriton, Cynops, Euproctus, Neurergus, Notophthalmus, Pachytriton, Paramesotriton, Taricha, and Triturus. Our results strongly support monophyly of all polytypic newt genera except Paramesotriton and Triturus, which appear paraphyletic, and Calotriton, for which only one of the two species is sampled. Other well-supported clades within newts include (1) Asian genera Cynops, Pachytriton, and Paramesotriton, (2) North American genera Notophthalmus and Taricha, (3) the Triturus vulgaris species group, and (4) the Triturus cristatus species group; some additional groupings appear strong in Bayesian but not parsimony analyses. Rates of lineage accumulation through time are evaluated using this nearly comprehensive sampling of salamandrid species-level lineages. Rate of lineage accumulation appears constant throughout salamandrid evolutionary history with no obvious fluctuations associated with origins of morphological or ecological novelties.

Mitochondrial DNA sequence analysis of the spectacled salamander, Salamandrina terdigitata (Urodela: Salamandridae), supports the existence of two distinct species

Salamandrina is a monotypic genus of the family Salamandridae endemic to Italy. Forty five individuals of the spectacled salamander, Salamandrina terdigitata, representing 11 populations throughout the whole distribution range were examined for sequence variation of three mitochondrial DNA genes encoding the 12S and 16S ribosomal RNA’s and cytochrome b (1324 bp). The results indicate the existence of two genetically distinct and geographically non-overlapping mtDNA lineages. The first lineage includes the southern populations and the second one comprises the central-northern populations. The degree of genetic divergence between the two groups is high and comparable to distances calculated from homologous sequences available in GenBank between other Salamandrid species. As a result, the genus Salamandrina probably requires splitting into two species. We also compare substitution rates associated with the mitochondrial genes employed across all Salamandrids studied so far, and discuss two possible palaeogeographic scenarios which could have shaped the splitting of the two Salamandrina lineages. These findings have also important implications for management and conservations of the spectacled salamander, which is protected under several international and regional conventions and directives.

Age, growth, and survivorship in the viviparous salamander Mertensiella luschani from southwestern Turkey

We describe the life-history traits (body size, age at maturity, longevity, and survivorship) of a terrestrial salamander, Mertensiella luschani, from a population living in the Mediterranean environment of southwestern Turkey. We used phalangeal skeletochronology to estimate the age of juveniles and adults. Lines of arrested growth, deposited during the winter cessation of bone growth, allowed us to estimate the age at maturity to be 3 years old in both sexes. Males attained a maximum age of 8, whereas females attained a maximum age of 10. Juveniles ranged from 1 to 3 years old. Males were smaller than females. Growth curves fitted to von Bertalanffy's equation revealed similar growth coefficients in adults and larger average maximum body lengths in males. Adult annual survivorship and adult life expectancy were 0.79 and 5.4 years, respectively. Knowledge of age, growth, and survivorship in this species under the constraints of a seasonal Mediterranean climate, and a comparison of similar traits in other salamandrid species, allow us to test predictions regarding optimal timing of growth and reproduction in these species.

Olfactory and vomeronasal projections and the pathway of the nervus terminalis in ten species of salamanders

Primary olfactory and vomeronasal projections as well as the pathway of the nervus terminalis were studied in 10 representative species of salamandrid and plethodontid salamanders by means of injections of horseradish peroxidase and examination of whole-mount preparations. Olfactory projections are very similar in the different urodeles, but vomeronasal projections differ in shape and number of termination fields. Whereas the direct-developing Plethodontini and Bolitoglossini reveal only one or two fields, the salamandrid species and the members of the plethodontid tribes Desmognathinae and Hemidactyliini, all possessing an aquatic larval stage, exhibit several vomeronasal projection fields. In all species examined centrifugal axons of the nervus terminalis leave the olfactory projection area ventrocaudally and terminate in the preoptic region and the hypothalamus.

The feeding system of terrestrial tiger salamanders (Ambystoma tigrinum melanostictum baird).

High speed cinematography was used to record the feeding activities of terrestrial Ambystoma tigrinum melanostictum. A description of these activities based on films of more than 50 feeding sequences is presented, and the mechanical units involved are defined, described, and functionally analyzed. Evolutionary implications of the feeding system are discussed. In a typical feeding sequence, A. t. melanostictum stations and maintains its lower jaw 3-5 mm from the prey. The mouth is then opened to form a gape of ∼60° by raising the anterior end of the flexed skull and by elevating and advancing the trunk while the mental symphysis of the lower jaw remains stationary. As the mouth opens the bulging tongue is recontoured so that the posterior glandular region becomes the tip of the fully protruded tongue, which may extend 3 to 7 mm beyond the symphysis. Dorsally the protruded tongue has a deep central depression and pronounced anterolateral rims. The anterior rim collapses on contact, thereby engulfing the prey in a sticky trough that retains it during tongue withdrawal. The cervical region is then flexed and the skull snaps downward. If the prey resists the tongue and is captured by marginal teeth, A. t. melanostictum relies on repeated tongue protraction and retraction, in some cases accompanied by inertial feeding. Swallowing involves gular expansion and contraction, and is accompanied by eye depression. When the mouth is opened during ingestive activities, the lower jaw remains in place. Apparently, A. t. melanostictum uses the dorsal trunk, the cucullaris major and the robust heads of the depressor mandibulae muscles to open the mouth. During skull elevation the lower jaw is partially immobilized by the geniohyoideus, and rectus cervicis superficialis muscles. The subarcualis rectus I muscles are prime movers in tongue projection. Hebosteoypsiloideus muscles assist in tongue protrusion by slackening the rectus cervicis profundus muscles that would otherwise restrict anterior displacement of the otoglossal cartilage and copula. Tongue contouring is performed by the complex genioglossus musculature. Sublingual and anterolingual sinuses facilitate protrusion and contouring by providing space and lubrication. Rectus cervicis muscles (profundus and superficialis) are responsible for tongue withdrawal. Closure of the mouth is accomplished by the four levator mandibulae muscles, and again the lower jaw is immobilized, mostly by ventral longitudinal muscles. Skull-trunk elevation during prey capture and ingestion was also observed and filmed in several other species of Ambystoma, in Dicamptodon ensatus, and in two salamandrid species. Apparently raising and straightening the craniovertebral axis, while the mental symphysis retains contact with the substratum, is a common feature of urodele feeding systems, and does not require peculiar morphological adaptations.

The lamphbrush chromosomes of Salamandra salamandra (L.) (Amphibia Urodela).

Lampbrush chromosomes isolated from the germinal vesicle of medium sized oocytes can be individually identified by differences in two characters: (1) chromosome regions rich in well developed loops, and (2) number and position of spheres. Actually the lateral loops are not all equally extended, but those which are inserted in a certain region of the axis of some chromosomes are more developed and sometimes are loaded with dense and copious matrix; chiasmata do not occur inside these regions. One or more spheres are present on eight chromosomes in the complement (chromosomes I–VI, VIII and X): the total number of spheres inserted on S. salamandra lampbrush chromosomes is the highest among the salamandrid species studied so far. These landmarks as well as the maximally developed normal loops are schematically drawn on the maps of the single lampbrush chromosomes. The length of the maps corresponds to the mean value of the lengths of each chromosome relative to that of chromosome XII, taken as 100 units long.