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.     

MITOCHONDRIAL-DNA VARIATION IN THE CRESTED NEWT SUPERSPECIES: LIMITED CYTOPLASMIC GENE FLOW AMONG SPECIES

The crested newt has a widespread European distribution and encompasses four taxa recently elevated to full species: Triturus cristatus, T. carnifex, T. dobrogicus, and T. karelini. These are distinct on morphological, chromosomal, and isozymic grounds and have fairly sharp transition zones. A widespread survey (12 countries, 49 geographic sites, 210 individuals) of mtDNA variation (20–27 restriction enzyme sites mapped per individual) was made in order to 1) correlate mtDNA variation with morphological features defining the species, 2) determine the degree of differentiation within and among species, and 3) detect any introgression among species. The mtDNAs of these species were clearly differentiated (d = 3.9–7.1%). Additionally, geographic structuring was observed within T. carnifex and T. karelini, each displaying two divergent mitochondrial genome types (d = 3.5% and 4.7%, respectively). The other two (more northerly distributed) species were genetically homogeneous over most (T. cristatus) or all (T. dobrogicus) of their ranges. In the case of T. cristatus, one may infer bottlenecking as a result of Pleistocene glaciation events. This may also apply in part to T. dobrogicus, but high population connectedness and gene flow in this lowland river species may alone be sufficient for homogenization of mtDNA. Patterns of mtDNA variation were largely concordant with morphology; some interspecific mitochondrial gene flow was observed, but only close to or in the transition zones. Analyses of mapped restriction-site data by UPGMA and parsimony methods (using the closely related T. marmoratus as an outgroup) produce very similar dendrograms. The levels of divergence found concur with the systematics of the group, but the differentiation within T. carnifex and T. karelini is notable.     

Stabilization of a salamander moving hybrid zone

When related species meet upon postglacial range expansion, hybrid zones are frequently formed. Theory predicts that such zones may move over the landscape until equilibrium conditions are reached. One hybrid zone observed to be moving in historical times (1950–1979) is that of the pond‐breeding salamanders Triturus cristatus and Triturus marmoratus in western France. We identified the ecological correlates of the species hybrid zone as elevation, forestation, and hedgerows favoring the more terrestrial T. marmoratus and pond density favoring the more aquatic T. cristatus. The past movement of the zone of ca. 30 km over three decades has probably been driven by the drastic postwar reduction of the “bocage” hedgerow landscape, favoring T. cristatus over T. marmoratus. No further hybrid zone movement was observed from 1979 to the present. To explain the changing dynamics of the hybrid zone, we propose that it stalled, either because an equilibrium was found at an altitude of ca. 140 m a.s.l. or due to pond loss and decreased population densities. While we cannot rule out the former explanation, we found support for the latter. Under agricultural intensification, ponds in the study area are lost at an unprecedented rate of 5.5% per year, so that remaining Triturus populations are increasingly isolated, hampering dispersal and further hybrid zone movement.     

Delineating fine-scale genetic units in amphibians:Probing the primacy of ponds

The population structure of pond-breeding amphibians is shaped by their distinct breeding foci, but it is unclear to what extent this is reflected in the fine-scale distribution of genetic diversity. We used microsatellite genotypes to investigate the genetic signatures of 24 populations of European newts, Triturus cristatus and T. marmoratus, inhabiting 21 ponds in a confined study area (7.5×3.5km) in western France. Employing a Bayesian clustering approach based on individual genotypes that minimises departures from Hardy–Weinberg equilibrium and linkage disequilibrium, no evidence was found for within-pond substructuring. Subjecting all sampled ponds simultaneously to this procedure revealed a clear signal of partitioning, with the most likely number of clusters however below the actual number of ponds (seven in T. cristatus, three in T. marmoratus). A more hierarchical Bayesian approach, with pond as analysis unit, was achieved to separate ponds from genetically more meaningful units, and reduced the T. cristatus populations to 11 clusters, and the T. marmoratus populations to five clusters. We were unable to specify a minimum nearest-neighbour distance where ponds are separate units, probably due to both historical and current demographic processes. The implications for strategies to manage and conserve endangered amphibians in human-altered landscapes are discussed.     

Distribution of satellite DNA TkS1 in genomes of salamanders (Salamandridae)

Genomes of 22 species and subspecies of salamandrids (Triturus, Cynops, Neurergus, Notophthalmus, Pachytriton, and Pleurodeles ) were studied. Satellite TkS1 (32-33 bp) was found in all examined species, both in direct and reversed orientations. The use of single-primer PCR with oligonucleotides, which are homologous to the most conservative part of satellite TkS1, allowed us to reveal DNA fragments, flanked by single copies (or small tandem repeats) of satellites TkS1 having various (5' and 3') orientation. Such fragments were observed in Pleurodeles waltl, Triturus a. alpestris, T. vulgaris lantzi, T. v. vulgaris, Neurergus crocatus and T. vulgaris graecus. The length of amplified DNA fragments in three subspecies of T. vulgaris differed. This might be connected with unequal amounts and different distribution of 5' and 3' copies of satellite TkS1 in their genomes. Patterns of DNA amplification in Triturus a. alpestris and Neurergus crocatus were quite similar. Only two species (Triturus a. alpestris and T. v. vulgaris) had approximately equal amounts and similar distribution of 5' and 3' copies of satellite TkS1 in their genomes.     

RESTRICTED GENE FLOW IN A MOVING HYBRID ZONE OF THE NEWTS TRITURUS CRISTATUS AND T. MARMORATUS IN WESTERN FRANCE

Two hybridizing species of newts, Triturus cristatus and T. marmoratus, with overlapping distributions show a parapatric distribution when surveyed in detail. The factors that govern the distribution of cristatus vs. marmoratus in the département (province) of Mayenne in western France are identified as forestation and relief. The parapatric hybrid zone running through Mayenne is narrow but widens to approximately 20 km in an area with mixed habitat. In this area most breeding sites are shared and F₁ hybrids form about 4% of the total population. Analysis of survey data collected about 30 years previously also shows an essentially parapatric distribution. Comparison of past and present distribution maps reveals that cristatus has superseded marmoratus over large areas in the south of Mayenne. An area where marmoratus replaced cristatus also exists, but it is more limited in size. Gene flow between cristatus and marmoratus is analyzed using 10 diagnostic genetic markers [9 protein loci and mitochondrial (mt) DNA]. In syntopic populations nuclear gene flow is bidirectional with a mean frequency of introgressed alleles (f) of 0.3%. In allotopic populations of cristatus and marmoratus gene flow is present in areas of species replacement (f = 0.3%), while gene flow appears to be absent in those areas that have been continuously occupied by a single species. At the biogeographic level, the presence or absence of introgression is paralleled by the persistence or absence, respectively, of pockets of cristatus–marmoratus syntopy. All F₁ hybrids possess the cristatus type mtDNA. This may be due to asymmetric interspecific mate choice and would explain the observed absence of introgression of the maternally inherited mtDNA genome in areas where cristatus replaced marmoratus. The cristatus–marmoratus hybrid zone bears characteristics of both the clinal (parapatric) hybrid zone model and the mosaic hybrid zone model. Such a mixed model—for which we propose the term “reticulate hybrid zone”—can be appreciated only if studied over a two-dimensional geographic area and also through time.     

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.     

Histological,immuno- and enzyme-histochemical investigations on the adenohypophysis of the urodeles,Mertensiella caucasica and Triturus cristatus and the caecilian,Chthonerpeton indistinctum

Summary The pituitary glands of two urodelan species (Mertensiella caucasica, Triturus cristatus) and one caecilian species (Chthonerpeton indistinetum) were examined with histological (Alcian blue, Brookes' trichrome stain), enzyme histochemical (acid phosphatase, -naphthylacetate-esterase, acetylcholinesterase) and immunofluorescence techniques (anti-carp GTH, anti-ovine prolactin, anti-synthetic -MSH). In the pituitary gland of Mertensiella and Triturus six chromophilic cell types could be distinguished. A strong fluorescence was observed in the MSH-, GTH- and TSH-cells.In the pituitary gland of Chthonerpeton only five chromophilic cell types could be distinguished: in the rostral part of the pituitary gland the B₃-cell; in the basal region of the central area the B₂-cell; dorsocaudally the B₁-cell. The acidophilic cells were found in the central and caudal part of the pars distalis. The basophils of the pars intermedia could be observed in the dorsocaudal part of the pituitary gland surrounding the neurohypophysis. All acidophilic cells showed a strong immunofluorescence with anti-ovine prolactin (LTH).     

Heterochromatic DNA in Triturus (Amphibia,Urodela)

The MspI family of highly repeated sequences is a centromeric satellite DNA representing about 1% of the genome of the Italian smooth newt, Triturus vulgaris meridionalis. We have studied the structure, genomic organization, chromosomal localization and conservation across species of this family. MspI sequences are around 197 bp long, as shown by sequencing of three cloned units. The family is organized in large clusters of tandemly arrayed units, present at almost all the centromeres of T.v. meridionalis, and is well conserved in the T.v. vulgaris subspecies. Conserved MspI sequences are also present in the related species T. helveticus, where they appear to be clustered at the centromeres of only a few chromosomes. MspI sequences are not found in other Triturus species analysed. The correlation of these sequences with the overall distribution pattern of heterochromatin and the extent of their conservation within the genus Triturus, are discussed.     

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.     

Two dispersed highly repeated DNA families of Triturus vulgaris meridionalis (Amphibia,Urodela) are widely conserved among Salamandridae

Two BamHI families of repeated sequences were characterized from the genome of the Italian smooth newt, Triturus vulgaris meridionalis (Amphibia, Urodela). The first family, which is divided into subfamilies, consists of tandemly arranged arrays whose basic repeat is around 398 bp long; these arrays are dispersed throughout the entire chromosome sets of the various species of Triturus tested. Moreover the family is widely conserved among Salamandridae, being detected by genomic DNA blotting of Notophthalmus viridescens, Taricha granulosa, Salamandrina terdigitata and Euproctus platycephalus. The second BamHI family is represented by a cloned sequence of 419 bp, which is dispersed in the chromosome set of several species of Triturus. The sequence is also conserved in S. terdigitata and in E. platycephalus but is not detectable in N. viridescens or T. granulosa. The cloned sequence is most probably only part of a longer unit interspersed within the Triturus genome.by H.C. Macgregor     

Experimental hybridization within the genus Triturus (Urodela: Salamandridae)

Spermatogenesis in the F₁ hybrid (2n=24=12 +12 ) between the closely related newt species T. cristatus carnifex and T. marmoratus was apparently normal up to pachytene. Many unpaired chromosomes were present at diplotene and a typical diakinesis was lacking. Primary spermatocytes at meta-and meta-anaphase contained up to 12 regular intergenomal bivalents and a corresponding number of univalents when less then 12 II. Most chiasmata were terminal or subterminal, some intercalary. Chiasmata between corresponding heterospecific chromosomes can be reported as true: real crossing over has taken place, proving the presence of primary chromosomal homologies between the 2 sets of the parental species. Evidence for recombination is based on the segregation of particular markers (i.e., subterminal C-bands and NORs) observed in certain chromosomes at metaphase II. One chromatid of single chromosomes can show the T. cristatus phenotype and the other the T. marmoratus phenotype. A few primary spermatocytes contain a certain number of irregular associations (intragenomal or intrahaploid bivalents, irregular intergenomal bivalents, chromosome multivalents) joined by chiasmata which can be defined as anomalous. Other abnormalities concern the occurrence of interlocked bivalents which occasionally show an anomalous exchange between heterologous chromatids. — Cytogenetic criteria useful to evaluate the taxonomic relationships between different species have been discussed as well as some possible trends in chromosome evolution and speciation within the genus Triturus.     

Early Miocene Amphibians (Caudata, Salientia) from the Mokrá-Western Quarry (Czech Republic) with comments on the evolution of Early Miocene amphibian assemblages in Central Europe

The Mokrá-Western Quarry exhibits the rare occurrence of Early Miocene (MN 4) vertebrate fauna within the area of the eastern part of Central Europe. In addition to a rich fauna of reptiles and mammals, two fossiliferous karst joints (Mokrá-Western Quarry, 1/2001 Turtle Joint and Mokrá-Western Quarry, 2/2003 Reptile Joint) yielded a rich fauna of amphibians including 13 amphibian taxa: Salamandridae: Mioproteus sp., Chelotriton sp., type I, Chelotriton sp., type II, Triturus aff. roehrsi, Triturus cf. marmoratus, Triturus sp. (T. cristatus species group), Chioglossa meini, Mertensiella mera, Salamandridae gen. and sp. indet.; Pelobatidae: Pelobates sanchizi; Ranidae: Rana sp. (synklepton Rana esculenta); Bufonidae: Bufo sp. The first records of the West European species Triturus cf. marmoratus and Chioglossa meini are reported from the eastern part of Central Europe indicating the wide distribution of those taxa throughout the whole of Europe as early as MN 4. The oldest known record of Pelobates sanchizi documents the Early Miocene presence of representatives closely related to the extinct Late Oligocene representatives of Pelobates. The slow evolution of amphibian species is documented by the presence of Triturus cf. marmoratus and the oldest known occurrence of the extinct salamander Mertensiella mera.     

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.     

Massive genetic introgression in threatened northern crested newts (<Emphasis Type="Italic">Triturus cristatus</Emphasis>) by an invasive congener (<Emphasis Type="Italic">T. carnifex</Emphasis>) in Western Switzerland

Genetic pollution through introgressive hybridization of local species by exotic relatives is a major, yet neglected aspect of biological invasions, particularly in amphibians where human introductions are frequent. In Western Switzerland, crested newts make an interesting case: the Italian species Triturus carnifex was introduced at least a century ago within the range of the native and threatened T. cristatus. To understand the genetic consequences of this introduction and inform wildlife management authorities, we conducted a genetic survey on the remaining northern crested newt populations known in the area, using newly-developed species-diagnostic nuclear (microsatellites) and mitochondrial (control region) DNA markers. We documented massive nuclear introgression by the T. carnifex genome, which has completely replaced T. cristatus in most populations, especially in the Geneva area where the introduction was originally reported. However, many of these individuals retained the ancestral T. cristatus mtDNA, which could be explained by asymmetric introgression between the two species, stemming from demographic and/or selective processes. Analyses of genetic diversity support multiple events of T. carnifex releases, most-likely of proximate North Italian origin. We pinpointed the last indigenous populations in the region and recommend to prioritize their protection. Our study demonstrates the invasive potential of introduced taxa through introgressive hybridization, alerts about the underestimated rate of illegal amphibian translocations, and emphasizes the need for genetic analyses to monitor such invasions.     

Length and interspersion of repetitive and non repetitive DNA sequences in four Amphibian species with different genome sizes

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.     

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.