Some features of the behaviour of the marbled newt Triturus marmoratus (Latreille, 1800) in captivity

The behaviour in groups of both sexes of marbled newts (Triturus marmoratus) from Sintra, Portugal, kept for a few weeks in a large aquarium, is described. Behaviour patterns described for this species and for other species in this genus were recognised. The following behaviours are described for the first time: Walk-over, Land-over, Stand-on, Show, Erect-body, Yawn, Circle, and Dig. Females seem to have a more active role in courtship than is usually considered. The behaviour frequencies for each sex were analysed using simulation statistics. Some elements usually classified as male sexual behaviours occur in both sexes and are likely to play other social roles. Received in revised form: 31 May 2001 Electronic Publication     

Use of a magnetic compass for Y-axis orientation in premetamorphic newts (Triturus boscai)

Experiments were carried out to investigate whether premetamorphic larvae of Boscas newt (Triturus boscai) are capable of using the geomagnetic field for Y-axis orientation (i.e., orientation toward and away from shore). Larvae were trained outdoor in two different training configurations, using one training tank aligned along the magnetic north–south axis, with shore facing north, and another training tank positioned with its length along the east–west axis, with shore located west. After training, premetamorphic newts were tested in an outdoor circular arena surrounded by a pair of orthogonally aligned cube-surface coils used to alter the alignment of the Earths magnetic field. Each newt was tested only once, in one of four magnetic field alignments: ambient magnetic field (i.e., magnetic north at North), and three altered fields (magnetic north rotated to East, West, South). Distributions of magnetic bearings from tested larvae indicated that they oriented bimodally along the magnetic direction of the trained Y-axis. These findings demonstrate that T. boscai larvae are sensitive to the geomagnetic field and can use it for orienting along a learned Y-axis. This study is the first to provide evidence of Y-axis orientation, accomplished by a magnetic compass, in larval urodeles.     

The annual number of breeding adults and the effective population size of syntopic newts (Triturus cristatus, T. marmoratus)

Pond-breeding amphibians are deme-structured organisms with a population genetic structure particularly susceptible to demographic threats. We estimated the effective number of breeding adults (Nb) and the effective population size (Ne) of the European urodele amphibians Triturus cristatus (the crested newt) and T. marmoratus (the marbled newt), using temporal shifts in microsatellite allele frequencies. Eight microsatellite loci isolated from a T. cristatus library were used, five of which proved polymorphic in T. marmoratus, albeit with high frequencies of null alleles at two loci. Three ponds in western France were sampled, situated 4-10 kilometers apart and inhabited by both species. Parent-offspring cohort comparisons were used to measure Nb; samples collected at time intervals of nine or 12 years, respectively, were used to measure Ne. The adult population census size (N) was determined by mark-recapture techniques. With one exception, genetic distances (FST) between temporal samples were lower than among populations. Nb ranged between 10.6 and 101.8 individuals, Ne ranged between 9.6 and 13.4 individuals. For the pond where both parameters were available, Nb/N (overall range: 0.10-0.19) was marginally larger than Ne/N (overall range: 0.09-0.16), which is reflected in the temporal stability of N. In line with the observed differences in reproductive life-histories between the species, Nb/N ratios for newts were about one order of magnitude higher than for the anuran amphibian Bufo bufo. Despite of the colonization of the study area by T. cristatus only some decades ago, no significant genetic bottleneck could be detected. Our findings give rise to concerns about the long-term demographic viability of amphibian populations in situations typical for European landscapes.     

Characterization of the lampbrush chromosomes of the marbled newt Triturus marmoratus (Latreille, 1800)

The maps of the lampbrush chromosomes of Triturus marmoratus oocytes were constructed on the basis of their lengths and major morphological characters such as giant fusing loops, dense matrix loops, lumpy objects, axial granules, lateral globules and reflected fusions; a nucleolus organizing region occurs subterminally on the right side of chromosome X. — Bivalent I appears morphologically asymmetrical, its two partners being of different lengths and bearing heteromorphic loops and other heterozygous structures: this heteromorphism may indicate that the two partners of bivalent I represent the ZW heterochromosomes of the species. Finally, an autoradiographic study has been performed in order to ascertain the pattern of ³H-uridine incorporation shown by the most typical landmarks and nucleoli.This work was financially supported by C.N.R., Roma.     

Testing the hybrid superiority hypothesis in crested and marbled newts

We tested the hybrid superiority hypothesis in the zone of overlap and hybridization of the newts Triturus cristatus and T. marmoratus. To do so, we compared size, age, and growth-related parameters in F₁ hybrids and both parental species in Mayenne, France. We found significant differences in snout-vent length (SVL), body mass and average lifespan between the parental species and hybrids, increasing from T. cristatus – T. marmoratus – hybrids. The relation between age and SVL fitted von Bertalanffy's growth model and showed that SVL_max was significantly larger in hybrids than in the parental species, while the growth coefficient was lower in hybrids and T. marmoratus than in T. cristatus. Triturus cristatus appears to be the better competitor, since it attains sexual maturity faster and thus achieves more annual breeding opportunities. At the evolutionary level, the observed heterosis appears not to have further consequences as the hybrids are largely infertile. Our results support the hypothesis raised for the genus Triturus, that infertile hybrids allocate resources to growth.     

Contemporary gene flow and the spatio-temporal genetic structure of subdivided newt populations (Triturus cristatus, T. marmoratus)

Gene flow and drift shape the distribution of neutral genetic diversity in metapopulations, but their local rates are difficult to quantify. To identify gene flow between demes as distinct from individual migration, we present a modified Bayesian method to genetically test for descendants between an immigrant and a resident in a nonmigratory life stage. Applied to a metapopulation of pond-breeding European newts (Triturus cristatus, T. marmoratus) in western France, the evidence for gene flow was usually asymmetric and, for demes of known census size (N), translated into maximally seven reproducing immigrants. Temporal sampling also enabled the joint estimation of the effective demic population size (Ne) and the immigration rate m (including nonreproductive individuals). Ne ranged between 4.1 and 19.3 individuals, Ne/N ranged between 0.05 and 0.65 and always decreased with N; m was estimated as 0.19-0.63, and was possibly biased upwards. We discuss how genotypic data can reveal fine-scale demographic processes with important microevolutionary implications.     

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.     

The cycle of follicular and interstitial cells (Leydig cells) in the testis of the marbled newt, Triturus marmoratus (Caudata, Salamandridae)

Ultrastructural examination of the marbled newt (Triturus marmoratus) testis throughout the annual cycle revealed that during the period of testicular quiescence (November-February), primordial germ cells proliferate within cords of filament-rich epithelial cells that will become follicular cells (FCs). Fibroblast-like cells surround the FCs and form the lobule-boundary interstitial cells (ICs). During the period of germ cell development from primordial germ cells to round spermatids (March-June), the FCs surrounding the developing germ cells contain scanty cytoplasm with abundant rough endoplasmic reticulum and scarce filaments. With spermatid elongation (July-August), the FC size grows, its nucleus becomes irregularly outlined, and its cytoplasm displays abundant smooth endoplasmic reticulum, residual bodies, lipid droplets, and large vacuoles. After spermatozoon release by the FCs (August-September), the adjacent ICs increase their size and transform into Leydig cells with abundant smooth endoplasmic reticulum, mitochondria with tubular cristae, and lipid droplets. During the period of testicular quiescence (November-February), the Leydig cells undergo involution, eventually developing the morphological attributes of mesenchymal cells. Intermingled among these cells, cords of filament-rich cells are observed. During this period of the cycle, spermatozoon cysts supported by FCs are present. At the beginning of the germ cell proliferation period (March), these spermatozoa are released, and the adjacent ICs undergo a transformation into Leydig cells similar to those observed in August-September. Maturation and involution of ICs occur when testosterone levels are known to be rising and falling, respectively.     

High aquatic niche overlap in the newts Triturus cristatus and T. marmoratus (Amphibia,Urodela)

We studied spatial niche metrics of large-bodied newts (Triturus cristatus and T. marmoratus) in three breeding ponds in western France. Adults and larvae were sampled with underwater funnel traps. Larvae were identified to the species with diagnostic microsatellite DNA markers. The distribution of adult T. cristatus and T. marmoratus across pond regions differed in one out of six cases, no differences were observed between larvae (two ponds studied). Niche overlap and niche breadth indices across resource states defined as pond regions or individual traps were high (Schoener's C: pond regions 0.60–0.98, traps 0.35–0.71; Levins' B: pond regions 0.71–0.98, traps 0.35–0.76). Adults of large-bodied newts significantly differed in resource use from small-bodied newts (T. helveticus). The results are discussed in view of the occurrence of interspecific breeding attempts, and the unpredictable ecological characteristics of newt breeding ponds.     

Cloning and characterization of 29 tetranucleotide and two dinucleotide polymorphic microsatellite loci from the endangered marbled murrelet (Brachyramphus marmoratus)

We developed 31 novel, polymorphic microsatellite loci in the marbled murrelet (Brachyramphus marmoratus), a critically endangered seabird. Variability was tested on 15 individuals from the Santa Cruz, California population, with each locus characterized by two to 12 alleles. Observed levels of heterozygosity ranged from 0.13 to 0.93. These loci provide a valuable means of assessing the population structure and demographic parameters of this species, which may be critical to its conservation across a fragmented habitat.     

Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.     

Di‐ and tetranucleotide microsatellite markers for the Alpine newt (Triturus alpestris): characterization and cross‐priming in five congeners

We developed a set of di‐ and tetranucleotide microsatellite markers for the Alpine newt, Triturus alpestris. Polymorphism as detected in 39 individuals ranged from 3 to 32 alleles at a locus. Cross‐priming with samples of five other Triturus species showed extremely poor levels of cross‐species utility. Still, these markers are suitable for studies of inter‐ and intrapopulation genetic diversity in the focal species.     

New polymorphic microsatellite loci for the Macedonian crested newt, Triturus macedonicus, and cross-priming testing in four other crested newt species

Eleven microsatellites have been characterized for Triturus macedonicus. Nine loci showed different variation patterns in a sample of 40 individuals from a single breeding pond in Zagori province (Greece), with an average number of 4.2 alleles per locus and an expected heterozygosity ranging from 0.1199 to 0.8079. Distinct cross-priming amplification rates were recovered on four additional crested newt species. Two monomorphic T. macedonicus loci were polymorphic in other Triturus species. The microsatellites developed herein could be a useful intraspecific genetic tool to undertake fine-scale population genetic analyses as well as in the study of contact zones between crested newt species.     

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt,Triturus alpestris (Urodela)

Summary Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA-and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, orginating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.Abbreviations AM amygdala - CAnt commissura anterior - CH commissura hippocampi - CP commissura posterior - Ctm commissura tecti mesencephali - DH dorsal hypothalamus - DTh dorsal thalamus - FLM fasciculus longitudinalis medialis - Fsol fasciculus solitarius - H habenula - LFB lateral forebrain bundle - ME median eminence - MFB medial forebrain bundle - NID nucleus infundibularis dorsalis - nIP neuropil of nucleus interpeduncularis - NPOP nucleus preopticus posterior - NS nucleus septi - OVLT organum vasculosum laminae terminalis - PD pars distalis - Pdo dorsal pallium - PHi primordium hippocampi - PI pars intermedia - Pl lateral pallium - PN pars nervosa - PRO preoptic recess organ - Ptec pretectal area - PVO paraventricular organ - Ra nucleus raphe - Rm nucleus reticularis medius - SCO subcommisural organ - ST striatum; strm stria medullaris thalami - strt stria terminalis thalami - TM tegmentum mesencephali - TO tectum opticum - TP tuberculum posterius - trch tractus cortico-habenularis - trmp tractus mamillopeduncularis - VH ventral hypothalamus - Vm nucleus motorius nervi trigemini - VTh ventral thalamus - II optic nerve     

A combination of techniques proves useful in the development of nuclear markers in the newt genus Triturus

To increase the number of markers available for study of phylogeny and phylogeography in the newt genus Triturus, we developed and tested 59 primer pairs using three different techniques. Primers were obtained from published sources, by designing exon-primed intron-crossing primers and from randomly cloned anonymous nuclear DNA fragments. Successful polymerase chain reaction products were cloned and sequenced. Five fragments were successfully amplified and sequenced for six species of Triturus: intron 7 of the β-fibrinogen gene (βfibint7), third intron of the calreticulin gene (CalintC), the 11th intron of the α-subunit of the platelet derived growth factor receptor (PDGFRα) and two anonymous markers (Cri1 and Cri4). The average percentage species divergence across all the markers is low (c. 3%), compared to what has been found in mitochondrial DNA (25-30%).     

Chromosome 1 in crested and marbled newts (Triturus)

The heteromorphic chromosomes 1 of Triturus cristatus carnifex and T. marmoratus were studied in mitotic metaphase after staining with the Giemsa C-banding technique and with the fluorochromes, DAPI (AT-specific) and mithramycin (GC-specific). They were also examined in the lampbrush form under phase-contrast before fixation and after fixation and staining with Giemsa. Chromosomes 1 of T.c. carnifex are asynaptic and achiasmatic throughout most of their long arms. They are also heteromorphic in most of their long arms for the patterns of Giemsa and fluorochrome staining and the distribution of distinctive lampbrush loops. The heteromorphic regions correspond to the regions that are asynaptic and achiasmatic. They stain more strongly with mithramycin and more weakly with DAPI than the remainder of the chromosomes, signifying that their DNA is relatively rich in GC. The patterns of staining with Giemsa and fluorochromes and the distributions of distinctive lateral loops vary from one animal to another in the same species and even in the same population. The asynaptic and achiasmatic regions of chromosomes 1 in T. marmoratus extend throughout the whole of the long arms and well beyond the heterochromatic region. Chiasmata form only in the short arm and occasionally in the short euchromatic segment at the tip of the long arms. The staining patterns of chromosomes 1 in T. marmoratus differ from those in T.c. carnifex although, like carnifex, their DNA is relatively GC-rich. The chromosomes 1 of T. marmoratus are more submetacentric than those of T.c. carnifex. In T. marmoratus chromosome 1B is about 12% shorter than 1A. There is a short paracentric inversion heterozygosity in the long arm of chromosome 1B in T. marmoratus which probably accounts for the lack of chiasmata in the euchromatin that separates the centromere from the start of the heterochromatin. In both carnifex and marmoratus, embryos that are homomorphic for chromosome 1 arrest and die at the late tailbud stage of development. The same applies to F₁ hybrid embryos T.c. carnifex x T. marmoratus, and this has permitted identification of chromosomes 1A and 1B in both species. There is no correspondence between patterns of Giemsa or fluorochrome staining of the heteromorphic regions of chromosome 1 and any feature of the lampbrush chromosomes. However, the short euchromatic ends of the long arms of chromosomes 1 in both species are distinguished in the lampbrush form by a series of uniformly small loops of fine texture associated with very small chromomeres. The Giemsa C-staining patterns of both chromosomes 1A and 1B are different in each of the four subspecies of T. cristatus. T.c. karelinii stands out by having unusually large masses of Giemsa C-staining centromeric heterochromatin on all but 1 of its 12 chromosomes. A scheme is proposed for the evolution of chromosome 1 in T. cristatus and T. marmoratus, based on all available cytological and molecular data.     

Organization of histamine-containing neurons in the brain of the crested newt,Triturus carnifex

The distribution of immunoreactivity for histamine was studied in the brain of the urodele Triturus carnifex using the indirect immunofluorescence method. Histamine-immunoreactive cell bodies were localized in the caudal hypothalamus within the dorsolateral walls of the infundibular recesses. These immunoreactive cell bodies were pear-shaped, bipolar and frequently of the cerebrospinal-fluid-contacting type. Histaminergic nerve fibers were detected in almost all parts of the brain. Dense innervation was seen in the telencephalic medial pallium and ventral striatum, the neuropil of the preoptic area, the septum, the paraventricular organ, the posterior commissure, the caudal hypothalamus, the ventral and lateral mesencephalic tegmentum. Medium density innervation was observed in the lateral mesencephalic tegmentum and optic tectum. Poor innervation was present in the telencephalic dorsal pallium and in the central gray of the medulla oblongata. Few fibers occurred in the olfactory bulbs and in the telencephalic lateral pallium. Double immunofluorescence staining, using an antibody against tyrosine hydroxylase, showed that histamine-immunostained somata and those containing tyrosine-hydroxylase-like immunoreactivity were co-distributed in the tuberal hypothalamus. No co-occurrence of histamine-like and tyrosine hydroxylase-like immunostaining was seen in the same neuron. The pattern of histamine-immunoreactive neurons in the newt was similar to that described in other vertebrates. Our observations, carried out on the apparently simplified brain of the newt confirm that the basic histaminergic system is well conserved throughout vertebrates.