Newt Species Research Articles

Genetic pollution of a threatened native crested newt species through hybridization with an invasive congener in the Netherlands

Genetic pollution of a native species through hybridization with an invasive species poses an insidious conservation threat. To expose genetic pollution, molecular methods employing multilocus data are required. We present a case study of genetic pollution via hybridization of a native crested newt species, Triturus cristatus, by the invasive Triturus carnifex on the Veluwe in the Netherlands. We sequenced 50 nuclear markers by next generation sequencing and one mitochondrial marker by Sanger sequencing for four populations from the native range of both parent species and eleven ponds on the Veluwe. We use three population genetic approaches (HIest, BAPS and Structure) to determine the genetic composition of the Veluwe newts based on all nuclear markers, a subset of 18 diagnostic markers and the complementary 32 non-diagnostic markers, with and without parental populations. BAPS underestimates genetic pollution, whereas Structure is comparatively accurate compared to HIest, although Structure’s relative advantage decreases with the diagnosticity of the markers. Data simulation confirms these findings. Genetic composition of the Veluwe ponds ranges from completely native, via different degrees of genetic admixture, to completely invasive. The observed hybrid zone appears to be bimodal, suggesting negative selection against hybrids. A genetic footprint of the native species is present in invasive populations, evidencing that the invasive locally replaced the native species. Genetic pollution is currently confined to a small area, but the possibility of further expansion cannot be excluded. Removal of genetic pollution will not be easy. We emphasize the need for legal guidance to manage genetic pollution.

Composition and variation of the skin microbiota in sympatric species of European newts (Salamandridae)

The mucous skin of amphibians provides a habitat for microorganisms which may interact with their hosts and thereby affect their condition and health. Cultivation-independent analyses of the bacterial communities based on the detection of PCR-amplified bacterial 16S rRNA genes provides a direct approach to characterize their diversity. In the present pilot study we utilized this approach in combination with a high-throughput DNA sequencing technology (454 pyrosequencing), to characterize the bacterial community structure of the skin of three newt species (Lissotriton vulgaris, Ichthyosaura alpestris, Triturus cristatus), collected near Braunschweig, Germany. 16S rDNA sequences were obtained from 19 unique samples. On average, 6113 amplicon sequences were obtained per sample and these could phylogenetically be assigned to a total of 1615 different operational taxonomic units (OTUs). Altogether, most samples were rather similar in their dominant bacterial taxa. Most represented were Betaproteobacteria (46%; mostly Janthinobacterium), Gammaproteobacteria (28%; mostly Pseudomonas), Flavobacteria (phylum Bacteroidetes: 19%, mostly Flavobacterium), and Sphingobacteria (Bacteroidetes: 5%, mostly Pedobacter). We found no significant differences between the three newt species, or between hemi-nested vs. non-nested PCR, but a strong difference among sampling dates (15 and 17 April 2013) which might be explained by the ongoing transition of the newts from their terrestrial to aquatic phase which coincided with this period, or by differences between sexes as these were unevenly sampled on the two dates. 16S rRNA gene sequences retrieved in this study in several cases were identical or very similar to those previously found on the skin of North American salamanders.

Beam Brook revisited: a molecular study of a historically introduced non-native amphibian (Triturus carnifex) and its limited introgression into native UK Triturus cristatus populations

Human mediated introductions of non-native species can pose a major threat to global biodiversity on several accounts i.e. through competition, the introduction of novel pathogens, and genetic pollution. Where hybridization occurs between two closely related species the F1 offspring are usually phenotypically discernible whereas F2 hybrid, backcrossed or admixed individuals become more difficult to separate. At this point the utilization of molecular methods is required in conservation efforts to differentiate and manage populations. This study demonstrated how a possible threat of hybridization from an introduced non-native (T. carnifex) with a protected native newt species (T. cristatus) could be investigated with molecular tools, and examined the current extent of its genetic introgression over an 80 years period. The results confirmed that hybridization had taken place at the site of introduction (and continues to do so), and that historically limited local dispersal of both non-natives and/or hybrids had occurred sometime in the past. However, the data suggests that although dispersal of hybrids into a local satellite site may still be occuring, hybridization with native species appears limited.

The effects of forest management on terrestrial habitats of a rare and a common newt species

Many amphibian species spend part of their life cycle in terrestrial habitats. However, their exact requirements regarding terrestrial habitats have often remained enigmatic. We aimed to compare the terrestrial habitat use of a common (smooth newt) and a rare newt (great crested newt) species around their breeding ponds, to see whether habitat preferences are linked to the rarity of a species. We tested the effects of forest habitat characteristics on newt populations of seven ponds. The ponds were monitored with terrestrial drift fences at different distances and in different habitats surrounding seven ponds in southeastern Finland during 2 years. We fitted all models using a Poisson generalized linear mixed model. Both newt species distinctly favored forests with high understory vegetation cover. Captures were most numerous in the old forests directly adjacent to the ponds. Both the common species and the rare species showed similar habitat use, but there was an indication that the rare species avoided clear-cut areas and had more need for the shelter provided by canopy and field-layer vegetation, especially when the distance to pond increased. Based on our findings, the maintenance of sheltering vegetation around the ponds is very important, especially for the rare species. However, this requires the retention of both field-layer and canopy coverage, and specific management principles—such as avoiding clear-cuts—should be adopted to maintain favorable conditions.

C5-C10 directly bonded tetrodotoxin analogues: possible biosynthetic precursors of tetrodotoxin from newts.

The identification of novel tetrodotoxin (TTX, 1) analogues would significantly contribute to the elucidation of its biosynthetic pathway. In this study, the first C5-C10 directly bonded TTX analogues, 4,9-anhydro-10-hemiketal-5-deoxyTTX (2) and 4,9-anhydro-8-epi-10-hemiketal-5,6,11-trideoxyTTX (3), were found in the newt Cynops ensicauda popei by using a screening method involving HILIC-LC-MS/MS focused on the fragment ions of TTX analogues, and their structures were elucidated by spectroscopic methods. Compound 2 was detected in a wide range of newt species, and the 2 and TTX contents of 22 newt specimens were correlated (rs =0.88). Based on these results and its structural features, 2 was predicted to serve as a precursor of TTX that would be directly converted into 4,9-anhydroTTX (4) by Baeyer-Villiger-like oxidation or via 4,9-anhydro-5-deoxyTTX formed by cleavage of the C5-C10 bond. The bicyclic carbon skeletons of 2 and 3 suggested a possible monoterpene origin for TTX.

C5–C10 Directly Bonded Tetrodotoxin Analogues: Possible Biosynthetic Precursors of Tetrodotoxin From Newts

AbstractThe identification of novel tetrodotoxin (TTX, 1) analogues would significantly contribute to the elucidation of its biosynthetic pathway. In this study, the first C5–C10 directly bonded TTX analogues, 4,9‐anhydro‐10‐hemiketal‐5‐deoxyTTX (2) and 4,9‐anhydro‐8‐epi‐10‐hemiketal‐5,6,11‐trideoxyTTX (3), were found in the newt Cynops ensicauda popei by using a screening method involving HILIC‐LC–MS/MS focused on the fragment ions of TTX analogues, and their structures were elucidated by spectroscopic methods. Compound 2 was detected in a wide range of newt species, and the 2 and TTX contents of 22 newt specimens were correlated (rs=0.88). Based on these results and its structural features, 2 was predicted to serve as a precursor of TTX that would be directly converted into 4,9‐anhydroTTX (4) by Baeyer–Villiger‐like oxidation or via 4,9‐anhydro‐5‐deoxyTTX formed by cleavage of the C5–C10 bond. The bicyclic carbon skeletons of 2 and 3 suggested a possible monoterpene origin for TTX.

It Takes Two to Tango: Relative Influence of Male and Female Identity and Morphology on Complex Courtship Display in a Newt Species

AbstractConsistency in behaviour is currently receiving a renewed interest. Although courtship display is generally consistent in terms of behavioural sequence and structure, there is also commonly important variation in the intensity of courtship display between and within males of a given species. Indeed, not all males have the same ability to perform courtship display (variation between males), and each male can potentially adjust his courtship effort in response to the environment (variation within a male). Although the study of male courtship display has received considerable attention in recent years, it is still unclear which part of the variation can be explained by male ability or motivation. We investigated this issue on two phases of the complex courtship display of the palmate newt Lissotriton helveticus. Overall, we found that both male and female identities affected courtship behaviour, but the relative influence of each sex depended on the courtship phase. Male identity explained variation in fan and creep‐quiver display, whereas female identity explained variation in creep‐quiver only. Interestingly, we did not find any link between the expression of courtship display and male or female morphological traits. Our study showed consistency of male courtship display in newts and successfully dissects the different sources of variation that can affect behavioural repeatability/consistency of courtship display.

The ontogenetic origins of skull shape disparity in the Triturus cristatus group.

Comparative studies of ontogenies of closely related species provide insights into the mechanisms responsible for morphological diversification. Using geometric morphometrics, we investigated the ontogenetic dynamics of postlarval skull shape and disparity in three closely related crested newt species. The skull shapes of juveniles just after metamorphosis (hereafter metamorphs) and adult individuals were sampled by landmark configurations that describe the shape of the dorsal and ventral side of the newt skull, and analyzed separately. The three species differ in skull size and shape in metamorphs and adults. The ontogenies of dorsal and ventral skull differ in the orientation but not lengths of the ontogenetic trajectories. The disparity of dorsal skull shape increases over ontogeny, but that of ventral skull shape does not. Thus, modifications of ontogenetic trajectories can, but need not, increase the disparity of shape. In species with biphasic life-cycles, when ontogenetic trajectories for one stage can be decoupled from those of another, increases and decreases in disparity are feasible, but our results show that they need not occur.

Kicking Triturus arntzeni when it's down: large-scale nuclear genetic data confirm that newts from the type locality are genetically admixed.

We collected nuclear DNA data (52 markers) with next-generation sequencing for nine Triturus newt specimens, including the holotype and two of the paratypes of T. arntzeni, from the type locality at Vrtovać in eastern Serbia. We compare these data to a reference set composed of the four crested newt species distributed in eastern Serbia namely T. cristatus, T. dobrogicus, T. ivanbureschi and T. macedonicus to determine to which of these species the newts from the type locality of T. arntzeni should be attributed. The majority of alleles in individuals from Vrtovać is derived from T. macedonicus, but a considerable number of T. ivanbureschi alleles is also present; alleles typical for T. cristatus and T. dobrogicus are found at low frequency. Accordingly, we interpret Vrtovać as a T. macedonicus - T. ivanbureschi hybrid population, albeit not composed of F1 hybrids but of genetically admixed individuals derived through multiple generations of backcrossing. The data support the notion that the name T. arntzeni should not be applied to a species newly distinguished in T. karelinii sensu lato (to which the name T. ivanbureschi has been given). We conclude that because of the hybrid nature of the individuals from Vrtovać, the name T. arntzeni should be placed not only in the synonymy of T. macedonicus but also in the synonymy of T. ivanbureschi. In this study we demonstrate that next-generation sequencing can provide high quality data for type material with degraded DNA and therefore can play an important role in taxonomy.

Exploring the effect of asymmetric mitochondrial DNA introgression on estimating niche divergence in morphologically cryptic species.

If potential morphologically cryptic species, identified based on differentiated mitochondrial DNA, express ecological divergence, this increases support for their treatment as distinct species. However, mitochondrial DNA introgression hampers the correct estimation of ecological divergence. We test the hypothesis that estimated niche divergence differs when considering nuclear DNA composition or mitochondrial DNA type as representing the true species range. We use empirical data of two crested newt species (Amphibia: Triturus) which possess introgressed mitochondrial DNA from a third species in part of their ranges. We analyze the data in environmental space by determining Fisher distances in a principal component analysis and in geographical space by determining geographical overlap of species distribution models. We find that under mtDNA guidance in one of the two study cases niche divergence is overestimated, whereas in the other it is underestimated. In the light of our results we discuss the role of estimated niche divergence in species delineation.

Parallel tagged amplicon sequencing of transcriptome‐based genetic markers for Triturus newts with the Ion Torrent next‐generation sequencing platform

Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. baps allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. newhybrids, a hybrid index and hiest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus.

Disentangling prey-handling efficiency of larval newts through multivariate prey trait analysis

Multivariate prey trait analysis is a functional approach to understand predator–prey relationships. In the present study, six prey macroinvertebrate ecological traits were used to identify several key factors in the handling efficiency of seven predatory larval newt species. The results revealed a remarkable similarity in the feeding behaviour among species, suggesting a foraging behaviour convergence among species in the prey-handling efficiency of larval newts. Nevertheless, larvae of some newt species showed clear preferences for particular categories of ecological trait; for example, Lissotriton vulgaris tended to feed on macroinvertebrate taxa with random trajectory, and Calotriton asper fed on macroinvertebrate taxa with high agility. In contrast, Mesotriton alpestris showed a clear ability to feed on prey with movable accessories (cases/tubes) and patterned concealment. This study shows how multivariate approaches can complement traditional diet analyses, and the method has wide applicability across life-stages and species.

Subtractive screen of potential limb regeneration related genes from Pachytriton brevipes

Regeneration capacity varies greatly among different animal species. In vertebrate, amphibian especially the Urodela, has been used as a powerful model system to study the mechanism of tissue regeneration because of the strong ability to regenerate their damaged or lost appendages. Pachytriton brevipes, a species of newt, which is widely distributed in south of China, can completely restore their damaged limbs within several months. In this study, we use modified suppression subtractive hybridization assay and dot-blot screening to identify candidate genes involved in tissue regeneration in P. brevipes. We successfully isolated 81 ESTs from a forward regeneration subtraction library. And we further verified the differential expression of four candidate genes, Rpl11, Cirbp, Ag2 and Trimx, between regenerating blastema and non-regeneration tissues by in situ hybridization. These genes were also be further characterized by phylogenetic and bioinformatic analysis. In general, we provided a comparative experimental approach to study the mechanisms of vertebrate regeneration.

Does intraspecific and intersexual attraction or avoidance influence newt abundance estimates based on fish funnel trap records?

We experimentally investigated whether intraspecific and intersexual attraction (or avoidance) by chemical signals may bias newt trapping success. We installed commercially available fish funnel traps which are often used in newt monitoring studies. We tested if capture rates differed between traps with or without newts. One experimental trap set comprised five traps, one trap being empty and four traps containing one male or female Lissotriton vulgaris or Ichthyosaura alpestris, respectively. Capture rates of newts of neither species nor sex was significantly affected by the presence of a particular newt species or sex in the traps, compared to control traps without newts. Trapping success thus seems not to be biased beyond random effects.

Phylogenetic Relationships of the Genus Paramesotriton(Caudata: Salamandridae) with the Description of a New Species from Qixiling Nature Reserve, Jiangxi, Southeastern China and a Key to the species

The matrilineal genealogy of the genus Paramesotriton is hypothesized based on DNA sequences from mitochondrial NADH subunit two(ND2) and its flanking tRNAs(tRNATrp and a partial tRNAAla). The genealogy identifies a highly divergent, unnamed lineage from Qixiling Nature Reserve, Jiangxi, China and places it as the sister taxon of P. chinensis. The newly discovered population differs from other congeners by several features of external morphology including having large clusters of dark brown conical warts on the dorsum of the head, lateral surface of the body and dorsolateral ridges. Its intermittent dorsal vertebral ridge is the same color as other parts of the dorsum and tail narrows gradually from the base to the tip. Further, the new population differs from all congeners by an uncorrected P-distance of more than 9.38% in ND2. Consequently, we describe the new species of Asian warty newt(Salamandridae) as Paramesotriton qixilingensis sp. nov.

The distribution of the crested and marbled newt species (Amphibia: Salamandridae: Triturus) – an addition to the New Atlas of Amphibians and Reptiles of Europe

In the recently published New Atlas of Amphibians and Reptiles of Europe (Sillero et al., 2014a), the distribution of the newt genusTrituruswas not resolved at the level of the species. The main reason for this was the lack of high quality distribution data from in and around the parapatric contact zones between species, where interspecific hybridization occurs. We are working extensively onTriturusand the (particularly genetic) data we have accumulated allow us to map the individualTriturusspecies at the appropriate scale. We here provide a database composed of distribution data for the individual species, at generally high resolution, particularly from in and around contact zones. Based on this database we produce maps at the 50 × 50 km UTM grid resolution as used in the new atlas and highlight those grid cells in which more than oneTriturusspecies occurs.

Variation of ventral color pattern in two cryptic newt species, Lissotriton vulgaris and L. lantzi (Amphibia: Salamandridae)

Peculiarities of the ventral color pattern variation in two cryptic species of newts of the genus Lissotriton (L. vulgaris and L. lantzi) have been examined. The belly and throat of these species have black spots on the pale-grey, yellow or orange background. 626 specimens, including 279 males and 244 females of L. vulgaris (11 samples from Russia, Ukraine, Romania, and Turkey), and 52 males and 51 females of L. lantzi (7 samples from Russia and Abkhazia) were used in this study. It was shown that, as a rule, the pattern of ventral coloration in these two species is different.

Development, validation and high‐throughput analysis of sequence markers in nonmodel species

DNA sequences derived from multiple regions of the nuclear genome are essential for historical inferences in the fields of phylogeography and phylogenetics. The appropriate markers should be single-copy, variable, easy to amplify from multiple samples and easy to sequence using high-throughput technologies. This may be difficult to achieve for species lacking sequenced genomes and particularly challenging for species possessing large genomes, which consist mostly of repetitive sequences. Here, we present a cost-effective, broadly applicable framework for designing, validating and high-throughput sequencing of multiple markers in nonmodel species without sequenced genomes. We demonstrate its utility in two closely related species of newts, representatives of urodeles, a vertebrate group characterized by large genomes. We show that over 80 markers, c. 600 bp each, developed mainly from 3' untranslated transcript regions (3'UTR) may be effectively multiplexed and sequenced. Data are further processed using standard, freely available bioinformatic tools, producing phase-resolved sequences. The approach does not require barcoded PCR primers, and the cost of library preparation is independent of the number of markers investigated. We hope that this approach will be of broad interest for researchers working at the interface of population genetics and phylogenetics, exploring deep intraspecific genetic structure, species boundaries and phylogeographies of closely related species.

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.

What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper, Lissotriton helveticus, and Triturus cristatus) and comparison with Illumina‐based approaches

The development of microsatellite loci has become more efficient using next-generation sequencing (NGS) approaches, and many studies imply that the amount of applicable loci is large. However, few studies have sought to quantify the number of loci that are retained for use out of the thousands of sequence reads initially obtained. We analyzed the success rate of microsatellite loci development for three amphibian species using a 454 NGS approach on tetra-nucleotide motif-enriched species-specific libraries. The number of sequence reads obtained differed strongly between species and ranged from 19,562 for Triturus cristatus to 55,626 for Lissotriton helveticus, with 52,075 reads obtained for Calotriton asper. PHOBOS was used to identify sequences with tetra-nucleotide repeat motifs with a minimum repeat number of ten and high quality primer binding sites. Of 107 sequences for T. cristatus, 316 for C. asper and 319 for L. helveticus, we tested the amplification success, polymorphism, and degree of heterozygosity for 41 primer combinations each for C. asper and T. cristatus, and 22 for L. helveticus. We found 11 polymorphic loci for T. cristatus, 20 loci for C. asper, and 15 loci for L. helveticus. Extrapolated, the number of potentially amplifiable loci (PALs) resulted in estimated species-specific success rates of 0.15% (T. cristatus), 0.30% (C. asper), and 0.39% (L. helveticus). Compared with representative Illumina NGS approaches, our applied 454-sequencing approach on specifically enriched sublibraries proved to be quite competitive in terms of success rates and number of finally applicable loci.