Pheromones are an important component of sexual communication in courting salamanders, but the number of species in which their use has been demonstrated with behavioral evidence remains limited. Here we developed a behavioral assay for demonstrating courtship pheromone use in the aquatically courting Iberian ribbed newt Pleurodeles waltl. By performing an in-depth study of the courtship behavior, we show that females invariably open their cloaca (cloacal gaping) before engaging in pinwheel behavior, the circling movement that is the prelude to spermatophore uptake. In contrast, cloacal gaping was not observed in failed courtships, where females escaped or displayed thanatosis. Since gaping mainly occurred during male amplexus and cloacal imposition, which is the obvious period of pheromone transfer, we next investigated whether male courtship water (i.e., water holding courtship pheromones) alone was able to induce this reaction in females. These tests showed that courtship water induced cloacal gaping significantly more than water, even in the absence of a male. Cloacal gaping thus provides a simple and robust test for demonstrating courtship pheromone use in the Iberian ribbed newt. Since opening the cloaca is an essential prerequisite for spermatophore pick-up in all internally fertilizing salamanders, we hypothesize that variations on this assay will also be useful in several other species.

Two and perhaps three taxa of Lissotriton newt occur in Turkey. Their species status is controversial. The distribution of these taxa and the taxonomic status of each are reviewed and discussed. A database of 128 Turkish Lissotriton localities was compiled and species distribution models were constructed. We reiterate that the presence of Lissotriton (vulgaris) lantzi in Turkey is disputed and needs confirmation. The range of Lissotriton (vulgaris) kosswigi is restricted to north-western Anatolia - given the small global range of this Turkey endemic, a closer look at its conservation status is warranted. The distribution of Lissotritonvulgarisschmidtleri covers western Asiatic and European Turkey. The findings support an allopatric distribution of the Turkish Lissotriton species. We reflect on the biological significance of previously reported morphological intermediates between Lissotriton (vulgaris) kosswigi and Lissotritonvulgarisschmidtleri in the light of the recent proposal to recognize kosswigi at the species level. The available data are in line with species status for Lissotriton (vulgaris) lantzi and Lissotriton (vulgaris) kosswigi. Although Lissotritonvulgarisschmidtleri is a genetically diverged taxon as well, the extent of gene flow with parapatric European Lissotriton taxa is as yet unknown.

The fluorescence analysis carried out in Triturus alpestris alpestris and Triturus alpestris cyreni subspecies has revealed differences related to the content and distribution of AT-rich, GC-rich and non-fluorescent heterochromatic bands. These results provide new evidence on the chromosome differentiation undergone in their phylogenetic splitting.

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.

Species often exhibit different levels of genetic structuring correlated to their environment. However, understanding how environmental heterogeneity influences genetic variation is difficult because the effects of gene flow, drift and selection are confounded. We investigated the genetic variation and its ecological correlates in an endemic and critically endangered stream breeding mountain newt, Neurergus kaiseri, within its entire range in southwestern Iran. We identified two geographic regions based on phylogenetic relationships using Bayesian inference and maximum likelihood of 779 bp mtDNA (D-loop) in 111 individuals from ten of twelve known breeding populations. This analysis revealed a clear divergence between northern populations, located in more humid habitats at higher elevation, and southern populations, from drier habitats at lower elevations regions. From seven haplotypes found in these populations none was shared between the two regions. Analysis of molecular variance (AMOVA) of N. kaiseri indicates that 94.03% of sequence variation is distributed among newt populations and 5.97% within them. Moreover, a high degree of genetic subdivision, mainly attributable to the existence of significant variance among the two regions is shown (θCT = 0.94, P = 0.002). The positive and significant correlation between geographic and genetic distances (r = 0.61, P = 0.002) following controlling for environmental distance suggests an important influence of geographic divergence of the sites in shaping the genetic variation and may provide tools for a possible conservation based prioritization policy for the endangered species.

Male salamanders (Urodela) often make use of pheromones that are produced in sexually dimorphic glands to persuade the female into courtship and mating. The mental gland of lungless salamanders (Plethodontidae) and dorsal cloacal glands (or abdominal glands) of newts (Salamandridae) have been particularly well studied in that respect. In both families, sodefrin precursor-like factor (SPF) proteins have been identified as major components of the courtship pheromone system. However, similar to plethodontids, some newts also make use of subtle head glands during courtship, but few pheromones have been characterized from such structures. Males of red-spotted newts (Notophthalmus viridescens, Salamandridae) have both cloacal and cheek (genial) glands, and are known to apply secretions to the female's nose by both tail-fanning and cheek-rubbing. Here we combined transcriptomic and phylogenetic analyses to investigate the presence, diversity and evolution of SPF proteins in the cloacal and cheek glands of this species.

Sodefrin, a decapeptide isolated from the male dorsal gland of the Japanese fire belly newt Cynops pyrrhogaster, was the first peptide pheromone identified from a vertebrate. The fire belly salamander and sodefrin have become a model for sex pheromone investigation in aquatically courting salamanders ever since. Subsequent studies in other salamanders identified SPF protein courtship pheromones of around 20 kDa belonging to the same gene-family. Although transcripts of these proteins could be PCR-amplified in Cynops, it is currently unknown whether they effectively use full-length SPF pheromones next to sodefrin. Here we combined transcriptomics, proteomics and phylogenetics to investigate SPF pheromone use in Cynops pyrrhogaster. Our data show that not sodefrin transcripts, but multiple SPF transcripts make up the majority of the expression profile in the dorsal gland of this newt. Proteome analyses of water in which a male has been courting confirm that this protein blend is effectively secreted and tail-fanned to the female. By combining phylogenetics and expression data, we show that independent evolutionary lineages of these SPF's were already expressed in ancestral Cynops species before the origin of sodefrin. Extant Cynops species continue to use this multi-component pheromone system, consisting of various proteins in addition to a lineage-specific peptide.

A new subspecies within the Salamandra algira species complex from north-western Africa is described. Previous molecular analysis showed that the populations of S. algira splendens from north-western Morocco consisted of two well supported clades: clade 1 distributed in the Rif Mountains, from Chefchaouen (type locality) to Al Hoceima, and clade 2, located southern from clade 1 being isolated in the northern and central Middle Atlas Mountains. Clade 2 is herein described as a distinct subspecies: Salamandra algira atlantica ssp. nov. based on morphological data, allopatric range and molecular divergence. This new subspecies shows an uncorrected pairwise distance of 0.0265 from clade 1 based on cytochrome b DNA sequences. Salamandra algira atlantica ssp. nov. is a slender and large sized salamander with a highly variable colouration pattern. It can be distinguished from S. algira splendens by the greater proportion of coral red in the background colouration, being the only known subspecies of S. algira in which coral red can exceed the proportion of black. Variable number (0-5) of yellow to golden yellow dorsal blotches, but usually in lower numbers than the nominotypical subspecies. Salamandra algira atlantica ssp. nov. inhabits subhumid to humid forests and karstic systems at mid to high elevations. We briefly discuss the phylogenetic and taxonomic issues among the genus Salamandra which encompasses more valid species than currently recognised.

We describe a new species of the genus Tylototriton from Ingyin Taung Mt., Mohnyin Township, Kachin State, Myanmar, based on morphological and molecular evidence. The new species is assigned to the subgenus Tylototriton s. str. and is clearly distinct from all known congeners by the following characters: medium body size; thin, long tail, lacking lateral grooves; rough skin; truncate snout; wide, protruding supratemporal bony ridges on head, beginning at anterior corner of orbit; weak, almost indistinct sagittal ridge; long, thin limbs, broadly overlapping when adpressed along body; distinct, wide, non-segmented vertebral ridge; 13 or 14 rib nodules; brown to dark-brown background coloration with dull orange-brown to yellowish-brown markings on labial regions, parotoids, rib nodules, whole limbs, vent, and ventral tail ridge. We also briefly discuss biogeography and species diversity of the genus Tylototriton in Myanmar.

The genus Calotriton includes two species of newts highly adapted to live in cold and fast-flowing mountain springs. The Pyrenean brook newt (Calotriton asper), restricted to the Pyrenean region, and the Montseny brook newt (Calotriton arnoldi), endemic to the Montseny massif and one of the most endangered amphibian species in Europe. In the present manuscript, we use an integrative approach including species distribution modeling (SDM), molecular analyses of mitochondrial and nuclear DNA sequence data and morphology to unravel the historical processes that have contributed to shaping the biogeography and genetic structure of the genus Calotriton, with special emphasis on the conservation of C. arnoldi. The results of the molecular analyses confirm that, despite having originated recently, being ecologically similar and geographically very close, there is no signal of hybridization between C. asper and C. arnoldi. SDM results suggest that tough environmental conditions on mountains tops during glacial periods, together with subsequent warmer periods could have prevented the contact between the two species. Within the critically endangered C. arnoldi, a high genetic structure is revealed despite its extremely small distribution range compared to C. asper. Haplotype networks, AMOVA and SAMOVA analyses suggest that two distinct groups of populations can be clearly differentiated with absence of gene flow. This is in concordance with morphological differentiation and correlates with its geographical distribution, as the two groups are situated on the eastern and western sides of a river valley that acts as a barrier. The genetic and morphological results are highly important for the ongoing conservation program of C. arnoldi and strongly justify the management of this species into at least two independent evolutionary significant units (eastern and western sectors) to guarantee the long-term population viability.

The Hengduan Mountains Region (HMR) is the largest "evolutionary frontier" of the northern temperate zone, and the origin and maintenance of species in this area is a research hotspot. Exploring species-specific responses to historical and contemporary environmental changes will improve our understanding of the role of this region in maintaining biodiversity. In this study, mitochondrial and microsatellite diversities were used to assess the contributions of paleogeological events, Pleistocene climatic oscillations, and contemporary landscape characteristics to the rapid intraspecific diversification of Liangshantriton taliangensis, a vulnerable amphibian species endemic to several sky-island mountains in the southeastern HMR. Divergence date estimations suggested that the East Asian monsoon, local uplifting events (Xigeda Formation strata), and Early-Middle Pleistocene transition (EMPT) promoted rapid divergence of L. taliangensis during the Pleistocene, yielding eight mitochondrial lineages and six nuclear genetic lineages. Moreover, population genetic structures were mainly fixed through isolation by resistance. Multiple in situ refugia were identified by ecological niche models and high genetic diversity, which played crucial roles in the persistence and divergence of L. taliangensis during glacial-interglacial cycles. Dramatic climatic fluctuations further promoted recurrent isolation and admixing of populations in scattered glacial refugia. The apparent mitonuclear discordance was likely the result of introgression by secondary contact and/or female-biased dispersal. Postglacial expansion generated two major secondary contact zones (Ganluo (GL) and Chuhongjue (CHJ)). Identification of conservation management units and dispersal corridors offers important recommendations for the conservation of this species.

A new species of the genus Tylototriton is described from Ngoc Linh Mountain, Kon Tum Province, in the Central Highlands of Vietnam based on integrative taxonomy, namely by combining molecular and morphological evidence. Tylototritonngoclinhensissp. nov. differs from all other congeners based on morphological data, allopatric distribution, and molecular divergence. In terms of genetic divergence, Tylototritonngoclinhensissp. nov. distinctly differs from the sister species T.panhai (6.77%) and from T.ngarsuensis (12.36%) based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. Tylototritonngoclinhensissp. nov. is a moderate sized and robust salamander species with large cephalic edges, parotoids, and vertebral ridge orange in coloration. The new taxon differs from its congeners by a combination of the following morphological characteristics: size medium (SVL 60.8-66.5 mm, TL 57.6-61.8 mm in males, and SVL 72.5-75.6 mm, TL 62.9-67.9 mm in females); head longer than wide; parotoids very prominent and enlarged, projecting backwards; tail length shorter than snout-vent length; vertebral ridge large, high and glandular in appearance; 14 large and distinct dorsolateral glandular warts; gular fold present; tips of fore and hind limbs overlapping when adpressed along the body; tips of fingers reaching between eye and nostril when foreleg is laid forward; dorsal surface and lateral sides of the head, upper and lower lips, dorsolateral glandular warts, vertebral ridge, the peripheral area of the cloaca and the ventral edge of the tail orange in coloration; the presence of a distinct black line extending from the posterior end of the eye towards the shoulder. Tylototritonngoclinhensissp. nov. is restricted to evergreen montane forests near water bodies on Ngoc Linh Mountain. We suggest that the new species should be classified as Endangered (EN) in the IUCN Red List. This new important discovery represents the eighth Tylototriton taxon described from Vietnam, and at the same time constitutes the southernmost distributional record for the whole genus in Asia.

The black-spotted stout newt (Pachytriton brevipes) is widely distributed in the mountains of southeastern China. In this study, we sequenced and determined the complete mitochondrial genome (mitogenomes) of two P. brevipes samples collected in Mount Wuyi. The assembled mitogenomes were 16,298 bp and 16,301 bp in length, and contained 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, one non-coding region, and one control region. The phylogenetic analysis indicated the two samples of black-spotted stout newt grouped together and are the sister group of P. feii.

Evolutionary biologists are always interested in deciphering the geographic context of diversification, therefore they introduced the concept of comparative phylogeography, which helps to identify common mechanisms that contribute to shared genetic structures among organisms from the same region. Here, we used multi-locus genetic data along with environmental data to investigate shared phylogeographic patterns among three Asian-endemic newt genera, Cynops, Paramesotriton and Pachytriton, which occurred in montane/submontane streams or ponds in southern China. Our 222 samples from 78 localities covered the entire range of the three genera and represented the largest dataset of this group to date. We reconstructed matrilineal genealogies from two protein-coding, mitochondrial genes, and gene network from two nuclear genes. We also estimated divergence times of major cladogenetic events and used occurrence data to evaluate niche difference and similarity between lineages. Our results revealed a common basal split in all three genera that corresponds to the separation of two geographic terrains of southern China. Those ancient divergence occurred during middle to late Miocene and likely correlate with paleoclimatic fluctuations caused by the uplift of the Qinghai-Xizang (Tibet) Plateau (QTP). Particularly, the strengthening and weakening of Asian summer monsoons during the Miocene may have profoundly impacted southern China and led to repeatedly vicariance in those newts. However, despite differences in realized niches between lineages, there is no evidence for divergence of fundamental niches. Preservation of old newt matriline lineages in mountains of southern China suggests that the region acts as both museums and cradles of speciation. Based on those results, we advocate a multi-pronged protection strategy for newts in the three genera.

Asian warty newts, genus Paramesotriton, are endemic to southern China and northern Vietnam. Despite the achievements in biodiversity, molecular systematics and biogeography of species in this genus, population genetic diversity studies are lacking due to the lack of economical and available genetic markers. In this study, we developed 17 highly polymorphic microsatellite loci from RAD simplified genomic data for the Asian warty newts, genus Paramesotriton and successfully completed cross-species amplification tests on 20 samples of four species of Paramesotriton. These microsatellite markers can be used as important tools to study population genetic structure, levels of gene flow, population differentiation, mating systems and landscape genetics within the genus Paramesotriton and, thus, to make scientific conservation decisions and actions for the conservation of these rare and endangered amphibians.

Global climatic transitions and Tibetan Plateau uplifts are hypothesized to have profoundly impacted biodiversity in southeastern Asia. To further test the hypotheses related to the impacts of these incidents, we investigated the diversification patterns of the newt genus Tylototriton sensu lato, distributed across the mountain ranges of southeastern Asia. Gene-tree and species-tree analyses of two mitochondrial genes and two nuclear genes revealed five major clades in the genus, and suggested several cryptic species. Dating estimates suggested that the genus originated in the early-to-middle Miocene. Under different species delimitating scenarios, diversification analyses with birth-death likelihood tests indicated that the genus held a higher diversification rate in the late Miocene-to-Pliocene era than that in the Pleistocene. Ancestral area reconstructions indicated that the genus originated from the northern Indochina Peninsula. Accordingly, we hypothesized that the Miocene Climatic Transition triggered the diversification of the genus, and the reinforcement of East Asian monsoons associated with the stepwise uplifts of the Tibetan Plateau promoted the radiation of the genus in southeastern Asia during the Miocene-to-Pliocene period. Quaternary glacial cycles likely had limited effects on speciation events in the genus, but mainly had contributions on their intraspecific differentiations.

The Paramesotriton Chang, 1935 genus of Asian warty newts is the second most diverse genus in the family Salamandridae, currently containing 14 recognized species from northern Vietnam to southwest-central and southern China. Although species of this genus have been included in previous phylogenetic studies, the origin and interspecific relationships of the genus are still not fully resolved, especially at key nodes in the phylogeny. In this study, we sequenced mitochondrial genomes and 32 nuclear genes from 27 samples belonging to 14 species to reconstruct the interspecific phylogenetic relationships within Paramesotriton and explore its historical biogeography in southern China. Both Bayesian inference and maximum-likelihood analyses highly supported the monophyly of Paramesotriton and its two recognized species groups ( P. caudopunctatus and P. chinensis groups) and further identified five hypothetical phylogenetic cryptic species. Biogeographic analyses indicated that Paramesotriton originated in southwestern China (Yunnan-Guizhou Plateau/South China) during the late Oligocene. The time of origin of Paramesotriton corresponded to the second uplift of the Himalayan/Qinghai-Xizang (Tibetan) Plateau (QTP), rapid lateral extrusion of Indochina, and formation of karst landscapes in southwestern China. Principal component analysis (PCA), independent sample t-tests, and niche differentiation using bioclimatic variables based on locations of occurrence suggested that Paramesotriton habitat conditions in the three current regions (West, South, and East) differ significantly, with different levels of climatic niche differentiation. Species distribution model (SDM) predictions indicated that the most suitable distribution areas for the P. caudopunctatus and P. chinensis species groups are western and southern/eastern areas of southern China. This study increases our knowledge of the taxonomy, biodiversity, origin, and suitable distribution areas of the genus Paramesotriton based on phylogenetic, biogeographic, and species distribution models.

Phylogeography is often used to investigate the effects of glacial cycles on current genetic structure of various plant and animal species. This approach can also identify the number and location of glacial refugia as well as the recolonization routes from those refugia to the current locations. To identify the location of glacial refugia of the Yellow-spotted mountain newt, Neurergus derjugini, we employed phylogeography patterns and genetic variability of this species by analyzing partial ND4 sequences (867 bp) of 67 specimens from 15 sampling localities from the whole species range in Iran and Iraq. Phylogenetic trees concordant with haplotype networks showed a clear genetic structure among populations as three groups corresponding to the populations in the north, center, and south. Evolutionary ages of clades north and south ranging from 0.15 to 0.17 Myr, while the oldest clade is the central clade, corresponding to 0.32 Myr. Bayesian skyline plots of population size change through time show a relatively slight increase until about 25 kyr (around the last glacial maximum) and a decline of population size about 2.5 kyr. The presence of geographically structured clades in north, center, and south sections of the species range signifies the disjunct populations that have emerged in three different refugium. This study illustrates the importance of the effect of previous glacial cycles in shaping the genetic structure of mountain species in the Zagros range. These areas are important in terms of long-term species persistence and therefore valuable areas for conservation of biodiversity.

We report on a study that extended mitochondrial transcript information from a recent EST project to obtain complete mitochondrial genome sequence for 5 tiger salamander complex species (Ambystoma mexicanum, A. t. tigrinum, A. andersoni, A. californiense, and A. dumerilii). We describe, for the first time, aspects of mitochondrial transcription in a representative amphibian, and then use complete mitochondrial sequence data to examine salamander phylogeny at both deep and shallow levels of evolutionary divergence. The available mitochondrial ESTs for A. mexicanum (N=2481) and A. t. tigrinum (N=1205) provided 92% and 87% coverage of the mitochondrial genome, respectively. Complete mitochondrial sequences for all species were rapidly obtained by using long distance PCR and DNA sequencing. A number of genome structural characteristics (base pair length, base composition, gene number, gene boundaries, codon usage) were highly similar among all species and to other distantly related salamanders. Overall, mitochondrial transcription in Ambystoma approximated the pattern observed in other vertebrates. We inferred from the mapping of ESTs onto mtDNA that transcription occurs from both heavy and light strand promoters and continues around the entire length of the mtDNA, followed by post-transcriptional processing. However, the observation of many short transcripts corresponding to rRNA genes indicates that transcription may often terminate prematurely to bias transcription of rRNA genes; indeed an rRNA transcription termination signal sequence was observed immediately following the 16S rRNA gene. Phylogenetic analyses of salamander family relationships consistently grouped Ambystomatidae in a clade containing Cryptobranchidae and Hynobiidae, to the exclusion of Salamandridae. This robust result suggests a novel alternative hypothesis because previous studies have consistently identified Ambystomatidae and Salamandridae as closely related taxa. Phylogenetic analyses of tiger salamander complex species also produced robustly supported trees. The D-loop, used in previous molecular phylogenetic studies of the complex, was found to contain a relatively low level of variation and we identified mitochondrial regions with higher rates of molecular evolution that are more useful in resolving relationships among species. Our results show the benefit of using complete genome mitochondrial information in studies of recently and rapidly diverged taxa.

A unique example of phenotypic flexibility of the oral apparatus is present in newts (Salamandridae) that seasonally change between an aquatic and a terrestrial habitat. Newts grow flaps of skin between their upper and lower jaws, the labial lobes, to partly close the corners of the mouth when they adopt an aquatic lifestyle during their breeding season. Using hydrodynamic simulations based on μCT-scans and cranial kinematics during prey-capture in the smooth newt (Lissotriton vulgaris), we showed that this phenotypic flexibility is an adaptive solution to improve aquatic feeding performance: both suction distance and suction force increase by approximately 15% due to the labial lobes. As the subsequent freeing of the corners of the mouth by resorption of the labial lobes is assumed beneficial for the terrestrial capture of prey by the tongue, this flexibility of the mouth fine-tunes the process of capturing prey throughout the seasonal switching between water and land.