The crested newt Triturus cristatus recolonized temperate Eurasia from an extra‐Mediterranean glacial refugium

We assess the role of the Carpathians as an extra‐Mediterranean glacial refugium for the crested newt Triturus cristatus. We combine a multilocus phylogeography (one mitochondrial protein‐coding gene, three nuclear introns, and one major histocompatibility complex gene) with species distribution modelling (projected on current and Last Glacial Maximum climate layers). All genetic markers consistently show extensive genetic variation within and genetic depletion outside the Carpathians. The species distribution model suggests that most of the current range was unsuitable at the Last Glacial Maximum, but a small suitable area remained in the Carpathians. Triturus cristatus dramatically expanded its postglacial range, colonizing much of temperate Eurasia from a glacial refugium in the Carpathians. Within the Carpathians, T. cristatus persisted in multiple geographically discrete regions, providing further support for a Carpathian ‘refugia within refugia’ scenario. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 574–587.     

Morphological integration and ontogenetic niche shift: a study of crested newt limbs

This study deals with the ontogenetic and evolutionary aspects of integration patterns in the limbs of crested newt species, which, like most amphibians, have a biphasic life history with two morphologically distinct stages (larval vs. juvenile and adult) that occupy different environments (aquatic vs. terrestrial). We analyzed the structure and pattern of correlation between limb skeletal elements at three ontogenetic stages (larval, juvenile, and adult) of four closely related species that differ in their preferences of aquatic habitats (more terrestrial and more aquatic). We found dynamic changes in the pattern of morphological integration between successive ontogenetic stages, as well as changes over the course of crested newt phylogeny. Generally, equivalent ontogenetic stages of different species of crested newts show higher concordance in the correlation pattern than successive ontogenetic stages within species. Among species, two opposing correlation patterns were observed: in more terrestrial species, homologous limb elements are less correlated and within-limb elements are more correlated; in aquatic species, the reverse pattern occurs. These results indicate that the function seems to be the covariance-generating factor, which has shaped the patterns of morphological integration of crested newt limbs.     

The evolution of the adult body form of the crested newt (Triturus cristatus superspecies,Caudata, Salamandridae)

We characterized the adult body form of the crested newt (Triturus cristatus superspecies) and explored its evolution. From seven morphometric traits, we determined that body size, interlimb distance and head width define the body form. None of the morphometric traits showed a phylogenetic signal. Three body‐shape morphotypes (Triturus dobrogicus + T. cristatus, Triturus carnifex + Triturus macedonicus and Triturus karelinii + Triturus arntzeni) and three body‐size morphotypes (T. dobrogicus, T. cristatus and all other crested newts) could be recognized. The ancestral phenotype (a large body with a short trunk and a wide head) characterized T. karelinii and T. arntzeni. Triturus carnifex and T. macedonicus had a somewhat different phenotype (large body and wide head, accompanied by mild body elongation). The most derived phenotype included body size reduction and more pronounced body elongation in T. cristatus and, especially, in T. dobrogicus. Body elongation occurred by trunk lengthening but not head and tail lengthening. Additionally, contrary to other tetrapods, evolutionary axis elongation in crested newts was followed by a decrease in body size. We advocate the hypothesis that ecology drives the evolution of body form in crested newts.     

Brain natriuretic peptide (BNP)-like immunoreactivity in the central nervous system of the crested newt.

The distribution of brain natriuretic peptide (BNP)-like immunoreactivity (ir) was studied in the brain of a urodele amphibian, the crested newt Triturus carnifex Laur. BNP-like immunoreactive neurons were found mainly in the caudal hypothalamus (retro- and supra-chiasmatic areas) and in the preoptic area. A widespread innervation throughout the brainstem as far as the spinal cord was also observed. By double immunostaining (after section incubation with a-BNP and a-tyrosine hydroxylase-TH-antibodies), close topographical relationships between BNP-like and TH-like immunoreactive neurons within the hypothalamus were found.     

Relationships among mammalian gonadotropin-releasing hormone, prostaglandins, and sex steroids in the brain of the crested newt, Triturus carnifex.

The present study was carried out to evaluate the in vitro brain release of prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), androgens, and 17 beta-estradiol in male and female crested newt, Triturus carnifex, during three different periods of the annual sexual cycle; in addition, the effects of mammalian gonadotropin-releasing hormone (mGnRH), PGF2 alpha, and PGE2 on prostaglandins and steroids release by the brain were evaluated during the same periods. In brain incubations of both sexes, PGF2 alpha and estradiol were higher during postreproduction, while PGE2 and androgens were higher during reproduction. In both sexes, mGnRH increased PGF2 alpha and estradiol during postreproduction, and PGE2 during reproduction; PGF2 alpha increased estradiol secretion during postreproduction. Only in the male, did both mGnRH and PGE2 increase androgens during reproduction. It could be suggested that in Triturus carnifex, the regulation of the reproductive activity in the central nervous system (CNS) depends on the relationships among mGnRH, prostaglandins and steroids. In particular, PGF2 alpha and PGE2 seem to play different roles in the CNS of the newt: PGF2 alpha is involved in the postreproductive processes, through estradiol secretion, while PGE2 in the reproductive ones (through androgens secretion?).     

Niche position, but not niche breadth, differs in two coexisting amphibians having contrasting trends in Europe

Aim We explored general habitat‐related explanations (niche breadth and niche position) to the contrasting status of two amphibian species that have largely overlapping ranges and habitats – the rare and declining crested newt (Triturus cristatus), and the more common smooth newt (Triturus vulgaris). These closely related and ecologically similar species provide an excellent opportunity to study those methodologically challenging hypotheses, and this is the first such study on amphibians. Location Denmark. Methods We derived multivariate habitat models from 27 characteristics of 210 ponds and their surroundings, and their occupation by newts. In addition to the model performance, niche breadths were compared using the mean beta diversity of amphibian communities in the presence of each newt species. Results For each newt species, the habitat models comprised three variables and correctly classified 74% of observations. Diverse invertebrate fauna (prey base) and shorter distances to other ponds inhabited by conspecifics were positive for both species, while the surrounding habitat (notably dry grasslands with forests) was important for the crested newt and the sediment type of the pond for the smooth newt only. Beta diversity of the amphibian communities of occupied ponds did not differ between the two newt species. Hence, in an area of frequent coexistence, habitat requirements of the species differed in key variables, not in the extent of specialization. Main conclusions Our study supported the niche position rather than the niche‐breadth hypothesis of rarity. We suggest that the rarity and/or continuing decline of the crested newt is related to the degradation of (semi)natural terrestrial habitats around suitable water bodies in Europe. Consequently, special restoration of such habitats has a high potential for the recovery of this rare species, while general pond management appeared more beneficial for the common smooth newt.     

Cranial shape variation and molecular phylogenetic structure of crested newts (Triturus cristatus superspecies: Caudata,Salamandridae) in the Balkans

In the present study, we investigated the degree of congruence between phylogeny, as inferred from mitochondrial (mt)DNA sequences, and cranium shape variation of crested newts (Triturus cristatus superspecies) in the Balkans. These newts belong to four phylogenetic clades defined by mtDNA analysis, and significantly differed in cranial shape. Allometry explained a high percentage of shape variation in crested newts. The clade‐specific allometric slopes significantly diverged for both the ventral cranium and dorsal cranium, indicating that differences in shape between clades could not be a simple consequence of their difference in size. The analysis of hierarchical and spatial variation showed similarity in the patterns of global and spatially localized hierarchical variation of cranial shape. We also found significant congruence between the pattern of cranial shape variation and molecular phylogeny. The differences in morphology of Triturus dobrogicus in comparison to other crested newt clades, including marked differences in cranium shape, is discussed in the context of the evolution and ecology of crested newts. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 348–360.     

PGF2 alpha, PGE2, and sex steroids from the abdominal gland of the male crested newt Triturus carnifex (Laur.).

Prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), progesterone, androgens, and 17 beta-estradiol in vitro release by the abdominal gland of the crested newt, Triturus carnifex (Laur.), was studied during the prereproductive, reproductive and postreproductive periods. In addition, the in vitro effects of the PGF2 alpha and/or PGE2 on progesterone, androgens and estradiol release by the abdominal gland were evaluated. PGF2 alpha, PGE2 and progesterone release was higher during the reproductive period, and in the same period, PGE2 treatment induced a progesterone increase. PGF2 alpha induced an increase of abdominal gland estradiol release at the end of the reproductive period. These results seemed to confirm the pheromonal role assigned to progesterone, and suggested a PGE2 stimulatory role in inducing progesterone release, even if pheromonal activity of PGF2 alpha and PGE2 cannot be excluded. In addition, PGF2 alpha-dependent estradiol increase at the end of reproduction could be interpreted as a mechanism for interruption of the abdominal gland activity.     

Micro-habitat use within a guild of newt larvae (Trituridae) in an Alpine lake

Population and ecological parameters such as numbers of larvae, microhabitat use, niche breadth and niche overlap of three species of syntopic larval newts (Alpine newt Triturus alpestris, Italian crested newt T. carnifex, and common newt T. vulgaris) were studied for two years in a small pond at 1160 m a.s.l. in NE Slovenia. Differences in microhabitat partitioning among larval newts were small. The largest niche breadth was estimated for larval T. alpestris, and the narrowest estimate was for larval T. carnifex in both years. Ecological differences seem to be very small and quite variable among sites and years. It appears that the developmental stage and size of newt larvae are more important in explaining resource partitioning than the characteristics of each species. Because of the absence of potential invertebrate predators and adult newts in the second half of the breeding season, the injuries could only be caused by intra-and interspecific predation attempts.     

Postglacial species displacement in Triturus newts deduced from asymmetrically introgressed mitochondrial DNA and ecological niche models

ABSTRACT: BACKGROUND: If the geographical displacement of one species by another is accompanied by hybridization, mitochondrial DNA can introgress asymmetrically, from the outcompeted species into the invading species, over a large area. We explore this phenomenon using the two parapatric crested newt species, Triturus macedonicus and T. karelinii, distributed on the Balkan Peninsula in south-eastern Europe, as a model. RESULTS: We first delimit a ca. 54,000 km2 area in which T. macedonicus contains T. karelinii mitochondrial DNA. This introgression zone bisects the range of T. karelinii, cutting off a T. karelinii enclave. The high similarity of introgressed mitochondrial DNA haplotypes with those found in T. karelinii suggests a recent transfer across the species boundary. We then use ecological niche modeling to explore habitat suitability of the location of the present day introgression zone under current, mid-Holocene and Last Glacial Maximum conditions. This area was inhospitable during the Last Glacial Maximum for both species, but would have been habitable at the mid-Holocene. Since the mid-Holocene, habitat suitability generally increased for T. macedonicus, whereas it decreased for T. karelinii. CONCLUSION: The presence of a T. karelinii enclave suggests that T. karelinii was the first to colonize the area where the present day introgression zone is positioned after the Last Glacial Maximum. Subsequently, we propose T. karelinii was outcompeted by T. macedonicus, which captured T. karelinii mitochondrial DNA via introgressive hybridization in the process. Ecological niche modeling suggests that this replacement was likely facilitated by a shift in climate since the mid-Holocene. We suggest that the northwestern part of the current introgression zone was probably never inhabited by T. karelinii itself, and that T. karelinii mitochondrial DNA spread there through T. macedonicus exclusively. Considering the spatial distribution of the introgressed mitochondrial DNA and the signal derived from ecological niche modeling, we do not favor the hypothesis that foreign mitochondrial DNA was pulled into the T. macedonicus range by natural selection.     

Effects of neurotransmitters upon the discharge of secretory product from the cutaneous glands of the red-spotted newt.

The effects of sympathetic and parasympathetic agonists and antagonists on discharge of secretory product by the granular and mucous glands were examined in the red-spotted newt, Notopthalmus viridescens viridescens. Observations were made also on the South African clawed toad. Xenopus laevis, the grass frog, Rana pipiens, and the crested newt, Triturus cristatus. In contrast to the granular glands of the South African clawed toad and the grass frog, which were stimulated by alpha-adrenergic agents, those of the red-spotted newt discharge in response to acetylcholine, either in vitro when added to the Holtfreter's solution in which explants were incubated, or in vivo when injected subcutaneously. Granular glands of the crested newt were also dischared in response to subcutaneous injection of acetylcholine. Stimulation of the granular glands by acetylcholine was blocked by atopine but not by tubocurarie, indicating that the cholinergic receptors are muscarinic rather than nicotinic. The mucous glands of the red-spotted newt, on the other hand, did not discharge in response to either acetylcholine or to adrenergic agents.     

A morphometric study of a hybrid newt population (Triturus cristatus) T. carnifex): Beam Brook Nurseries, Surrey, U.K.

The crested or great crested newt ( Triturus cristatus spp) is declining and now considered threatened in many of the countries where it is present. This has resulted in the four members of the superspecies being afforded protection under local, national and international law. This study looks at a possible threat to T. cristatus in southern England through hybridization, by the introduction of a related alien species ( T. carnifex ). The study used multivariate morphometries to discriminate closely related species, and their hybrids. The character set involved both continuous and meristic data, collected through body measurements and colour pattern. The identification of the species and/or hybrids at the introduction site and surrounding areas was mapped. From the results it can be inferred that hybridization has taken place at the introduction site, but there is no morphological evidence for the spread of hybrids/aliens in to the surrounding areas.     

The application of eDNA for monitoring of the Great Crested Newt in the UK

Current ecological surveys for great crested newts are time‐consuming and expensive and can only be carried out within a short survey window. Additional survey methods which would facilitate the detection of rare or protected species such as the great crested newt (Triturus cristatus) would be extremely advantageous. Environmental DNA (eDNA) analysis has been utilized for the detection of great crested newts in Denmark. Here, the same methodology has been applied to water samples taken from UK ponds concurrently with conventional field surveying techniques. Our eDNA analysis exhibited an 84% success rate with a kappa coefficient of agreement between field and eDNA surveys of 0.86. One pond determined to be negative for great crested newt by field survey was positive by eDNA analysis, revealing the potential for improved detection rates using this methodology. Analysis of water samples collected in late summer indicates that eDNA analysis could be used to detect great crested newt after the optimal survey window for current field techniques had passed. Consequently, eDNA analysis could augment currently stipulated techniques for great crested newt surveying as a relatively quick and inexpensive tool for collecting great crested newt presence and distribution data within the UK instead of or prior to full field surveys.     

Genetic structure in northeastern populations of the Alpine newt (Triturus alpestris): evidence for post-Pleistocene differentiation

Genetic variation in 13 populations of the Alpine newt, Triturus alpestris, was assessed at the northeastern margin of its range (southern Poland). Variation at six microsatellite loci was scored in 354 newts, and two mitochondrial DNA fragments (c. 2000 bp) were sequenced in a subset of 27 individuals. Significant differences in allele frequencies and the presence of private alleles determined genetic units corresponding to three separate mountain ranges, i.e. the Carpathian, Sudetes and Holy Cross Mountains. F(ST)'s were three times greater in among than in within mountain range pairwise comparisons. An assignment test and pairwise F(ST)'s suggested relatively high levels of gene flow at the local level, although the Sudetes populations revealed some subtle structuring. Genetic variation was lower in the Carpathians and Holy Cross Mountains. The geographic pattern of mitochondrial DNA variation indicated that these newt populations originated from a single glacial refugium/founder population, and that the colonization of southern Poland took place in an easterly direction. The data show that substantial neutral variation and between group divergence has accumulated relatively quickly in these low-vagility organisms. The Alpine newt case exemplifies species history as a factor determining patterns of genetic diversity in marginal populations.     

Genetic variation in the Italian crested newt, Triturus carnifex, and the origin of a non-native population north of the Alps

Genetic variation over 40 protein loci and 46 populations representing three taxa of crested newts revealed moderate genetic distances between Triturus carnifex carnifex, T. c. macedonicus and T. cristatus. Two populations from the Geneva Basin (presumed to be introduced) were genetically similar to T. c. carnifex and dissimilar to T. c. macedonicus and T. cristatus, showing that they belong to T. c. carnifex and not to native T. cristatus. A significant pattern of spatial genetic variation was found within T. c. carnifex along a north to south axis, from Croatia to Calabria. The Genevan populations showed highest genetic similarity with T. carnifex from Tuscany, suggesting that the propagule originated from that area. Effects of a population genetic bottleneck associated with the introduction could not be documented. The observed high allelic variation in Genevan T. c. carnifex could not be directly explained by introgression from T. cristatus. Comparisons across the range, including zones of hybridization within the T. cristatus superspecies, indicated that some alleles typical for the Genevan population may represent the so-called `hybrizymes'.     

Effects of nostril plugging and of habenulectomy on sexual behaviour in the male crested newt

In an attempt to gain some insight into the possible involvement of the habenulae in the control of sexual behaviour in the male crested newt, a comparison was made between the effects of olfactory deprivation by bilateral nostril plugging and of habenulectomy on courtship performance and locomotor activity. Both treatments led to a decrease in spontaneous locomotion and to a drastic abolition of the complex courtship ritual characterized by the sequence of male postures displayed prior to spermatophore deposition. Following nostril plug removal, the animals resumed their normal activities. Unilateral plugging had no effect. These results seem to lend further support to the importance of specific olfactory stimuli in sexual behaviour. The main finding obtained here was the strict comparability between the behavioural changes resulting from habenulectomy and the olfactory-related changes following nostril plugging, linking from a functional point of view the habenulae with the olfactory system. According to recent reports available in the literature, the morphological organization of the habenular nuclei in urodeles still needs further clarification. On the basis of the present results, the possible importance of the habenulae in olfactory integration in the crested newt might be postulated.     

Tyrosine hydroxylase-immunoreactive neurons in the hypothalamus of the crested newt. An electron microscopic study

The fine structure and specialized neuronal, vascular and ventricular relations of tyrosine hydroxylase (TH)-immunopositive neurons and processes were examined in the hypothalamus of the crested newt. TH-immunoreactive neurons form a well developed system. Its possible role in the hypothalamic neuroendocrine mechanisms is discussed.     

Species‐specific habitat preferences do not shape the structure of a crested newt hybrid zone (Triturus cristatus x T. carnifex)

Reproductive isolation barriers maintain the integrity of species by preventing interspecific gene flow. They involve temporal, habitat or behavioral isolation acting before fertilization, and postzygotic isolation manifested as hybrid mortality or sterility. One of the approaches of how to study reproductive isolation barriers is through the analysis of hybrid zones. In this paper, we describe the structure of a hybrid zone between two crested newt species (Triturus cristatus and T. carnifex) in the southern part of the Czech Republic using morphological, microsatellite, and mitochondrial (mtDNA) markers. Specifically, we tested the hypothesis that the structure of the hybrid zone is maintained by species‐specific habitat preferences. Comparing the genetic structure of populations with geographical and ecological parameters, we found that the hybrid zone was structured primarily geographically, with T. cristatus‐like populations occurring in the northeast and T. carnifex‐like populations in the southwest. Despite T. cristatus tending to occur in deeper ponds and T. carnifex on localities with more shading, the effect of both ecological parameters on the structure of the zone was minimal. Next, we corroborated that T. carnifex individuals and some hybrids possess mtDNA of T. dobrogicus, whose nuclear background was not detected in the studied hybrid zone. Hybridization between T. carnifex and T. dobrogicus (resulting in unidirectional mtDNA introgression) had to predate subsequent formation of the hybrid zone between T. cristatus and T. carnifex. Populations of crested newts in the southern part of the Czech Republic thus represent a genetic mosaic of nuclear and mitochondrial genomes of three species.     

The effect of pH on feeding behaviour in newt larvae (Triturus: Amphibia)

The feeding responses of three species of newt larvae were compared under circumneutral and sublethal acid conditions. Under acid conditions (pH 4.5) feeding behaviour was suppressed in palmate newts, Triturus helveticus, and smooth newts. T. vulgaris , but not in crested newts, T. cristatus. At low pH, approach and orientation towards food occurred in T. helveticus and T. vulgaris , but snapping was inhibited; T. cristatus snapped and consumed food immediately it was offered under the same conditions. These differences are not consistent with the apparent greater tolerance of T helveticus for acidified ponds. The observations suggest that the chemosensory system of T. helveticus and T. vulgaris may be impaired at low pH.     

Eight polymorphic microsatellite loci for the critically endangered crested ibis,Nipponia nippon (Ciconiiformes: Threskiornithidae)

The Chinese crested ibis, Nipponia nippon, is a critically endangered bird. The current population of this species has developed from four wild individuals rediscovered in 1981. Given its conservation status, there is considerable interest in assessing the genetic diversity and individual relatedness in this species. For this purpose, a set of eight polymorphic di‐ or trinucleotide microsatellite loci was developed for the crested ibis. The expected heterozygosity at these loci ranges from 0.01 to 0.50, with less than four alleles being observed at individual loci, a reflection of the serious population bottleneck experienced by this species.