Morphing of the phylogeographic lineages of the Balkan alpine newts (Ichthyosaura alpestris, Caudata, Salamandridae): in situ morphological diversification

Numerous alpine newt (Ichthyosaura alpestris) populations from the Balkans, representing all the previously established phylogeographic lineages, were studied for variations in various morphological characteristics (body size and shape, skull qualitative traits and number of trunk vertebrae). Here, we present a decoupling of morphological and mtDNA phylogeographic substructuring in the alpine newt on the Balkan Peninsula. In sharp contrast to other European newts (Triturus spp., Lissotriton spp.), the vast majority of morphological variation in the alpine newt is concentrated at the population level indicating an in situ morphological diversification. We found that the rate of morphological change is similar to the rate of mtDNA change. We hypothesize that the alpine newts are characterized by non-adaptive morphological evolution.     

Testing the evolutionary constraints of metamorphosis: The ontogeny of head shape in Triturus newts

In vertebrates with complex, biphasic, life cycles, larvae have a distinct morphology and ecological preferences compared to metamorphosed juveniles and adults. In amphibians, abrupt and rapid metamorphic changes transform aquatic larvae to terrestrial juveniles. The main aim of this study is to test whether, relative to larval stages, metamorphosis (1) resets the pattern of variation between ontogenetic stages and species, (2) constrains intraspecific morphological variability, and (3) similar to the “hour‐glass” model reduces morphological disparity. We explore postembryonic ontogenetic trajectories of head shape (from hatching to completed metamorphosis) of two well‐defined, morphologically distinct Triturus newts species and their F1 hybrids. Variation in head shape is quantified and compared on two levels: dynamic (across ontogenetic stages) and static (at a particular stage). Our results show that the ontogenetic trajectories diverge early during development and continue to diverge throughout larval stages and metamorphosis. The high within‐group variance and the largest disparity level (between‐group variance) characterize the metamorphosed stage. Hence, our results indicate that metamorphosis does not canalize head shape variation generated during larval development and that metamorphosed phenotype is not more constrained relative to larval ones. Therefore, metamorphosis cannot be regarded as a developmental constraint, at least not for salamander head shape.     

Metamorphosis of cranial design in tiger salamanders (Ambystoma tigrinum): A morphometric analysis of ontogenetic change

While ontogenetic analyses of skull development have contributed to our understanding of phylogenetic patterns in vertebrates, there are few studies of taxa that undergo a relatively discrete and rapid change in morphology during development (metamorphosis). Morphological changes occurring in the head at metamorphosis in tiger salamanders (Ambystoma tigrinum) were quantified by a morphometric analysis of cranial osteology and myology to document patterns of change during metamorphosis. We employed a cross-sectional analysis using a sample of larvae just prior to metamorphosis and a sample of transformed individuals just after metamorphosis, as well as larvae undergoing metamorphosis. There were no differences in external size of the head among the larval and transformed samples. The hyobranchial apparatus showed many dramatic changes at metamorphosis, including shortening of ceratobranchial 1 and the basibranchial. The subarcualis rectus muscle increased greatly in length at metamorphosis, as did hypobranchial length and internasal distance. A truss analysis of dorsal skull shape showed that at metamorphosis the snout becomes wider, the maxillary and squamosal triangles rotate posteromedially, and the neurocranium shortens (while maintaining its width), resulting in an overall decrease in skull length at metamorphosis. These morphometric differences are interpreted in light of recent data on the functional morphology of feeding in salamanders. Morphological reorganization of the hyobranchial apparatus and shape changes in the skull are related to the acquisition of a novel terrestrial feeding mode (tongue projection) at metamorphosis. Metamorphic changes (both internal and external) that can be used to judge metamorphic condition are discussed.     

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.     

Further observations on the fate of morphological variation in a population of Smooth newt larvae (Triturus vulgaris)

Sixteen morphometric characters were studied in a population of Smooth newt larvae. Variation was found to decrease in all characters during the course of larval life. This finding is consistent both with earlier work on newt larvae in the same population and with the results of other authors. There is no direct evidence to show that this decline was caused by selection rather than by developmental canalization, although a theoretical argument suggests that canalization may not be very important in animals as fecund as newts. In the case of vertebral number, it seems likely that developmental processes act to reduce variation in very young larvae, but that selection is important in later life. Variation was also studied in this population at and just after metamorphosis, when about 40% of the emerging juveniles were killed by shrews, by scoring 25 morphometric characters. Despite difficulties of interpretation caused by growth and by the morphogenetic changes associated with metamorphosis, a clear case of stabilizing selection was discovered.     

Ontogeny of skull size and shape changes within a framework of biphasic lifestyle: a case study in six <Emphasis Type="Italic">Triturus</Emphasis> species (Amphibia,Salamandridae)

As with many other amphibians, Triturus species are characterized by a biphasic life cycle with abrupt changes in the cranial skeleton during metamorphosis. The post-metamorphic shape changes of the cranial skeleton were investigated using geometric morphometric techniques in six species: Triturus alpestris, T. vulgaris, T. dobrogicus, T. cristatus, T. carnifex, and T. karelinii. The comparative analysis of ontogenetic trajectories revealed that these species have a conserved developmental rate with divergent ontogenetic trajectories of the ventral skull shape that mainly reflect phylogenetic relatedness. A striking exception in the ontogenetic pattern was possibly found in T. dobrogicus, characterized by a marked increase in the developmental rate compared to the other newt species. The size-related shape changes explained a large proportion of shape change during post-metamorphic growth within each species, with marked positive allometric growth of skull elements related to foraging.     

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.     

The ontogenetic shift in thermoregulatory behaviour of newt larvae: testing the 'enemy-free temperatures' hypothesis

Growth and development affect life-history traits, and consequently organismal fitness. The inevitable increase in body size during ontogeny is associated with changes in both resource use and predation risk, which results in the ontogenetic shift in habitat preferences. In this study, we examined whether the shift in preferred body temperatures ( T _ps) of newt larvae Triturus alpestris increases the T _ps deviation of the most vulnerable stages (after hatching and during metamorphosis) from the T _ps range of their main predator, dragonfly larvae Aeshna cyanea – the 'predator-free temperatures' hypothesis. Analyses of thermoregulatory behaviour in the laboratory thermal gradient showed that freshly hatched newt larvae maintained lower water temperatures than larvae in later stages, whose T _ps largely fell into the T _ps range of dragonfly larvae. With respect to the thermal quality of natural habitat, the anti-predator effectiveness of the T _ps shift decreased during development. Water temperatures in natural habitats were located largely below the preferred body temperature range of both newt and dragonfly larvae, which limits their potential thermal niche partitioning. We conclude that factors other than predator avoidance drive the ontogenetic T _ps shift in our model system.     

Functional morphogenesis from embryos to adults: Late development shapes trophic niche in coral reef damselfishes

The damselfishes are one of the dominant coral reef fish lineages. Their ecological diversification has involved repeated transitions between pelagic feeding using fast bites and benthic feeding using forceful bites. A highly‐integrative approach that combined gene expression assays, shape analyses, and high‐speed video analyses was used to examine the development of trophic morphology in embryonic, larval, juvenile, and adult damselfishes. The anatomical characters that distinguish pelagic‐feeding and benthic‐feeding species do not appear until after larval development. Neither patterns of embryonic jaw morphogenesis, larval skull shapes nor larval bite mechanics significantly distinguished damselfishes from different adult trophic guilds. Analyses of skull shape and feeding performance identified two important transitions in the trophic development of a single species (the orange clownfish; Amphiprion percula): (a) a pronounced transformation in feeding mechanics during metamorphosis; and (b) more protracted cranial remodeling over the course of juvenile development. The results of this study indicate that changes in postlarval morphogenesis have played an important role in damselfish evolution. This is likely to be true for other fish lineages, particularly if they consist of marine species, the majority of which have planktonic larvae with different functional requirements for feeding in comparison to their adult forms.     

Sexual dimorphism of skull shape in a lacertid lizard species (Podarcis spp., Dalmatolacerta sp., Dinarolacerta sp.) revealed by geometric morphometrics

Geometric morphometric techniques were used to examine allometric and non-allometric influences on sexual shape dimorphism (SShD) in the ventral cranium (skull base, palate and upper jaw) of four species of lacertid lizards (Podarcis muralis, Podarcis melisellensis, Dalmatolacerta oxycephala, Dinarolacerta mosorensis). These species differ in body shape, ecology and degree of phylogenetic relatedness. The structures of the ventral cranium that were studied are directly involved in the mechanics of feeding and are connected to the jaw musculature; these structures are potentially subject to both sexual and natural selection. Allometry accounted for a considerable degree of cranial shape variation between the sexes. Allometric shape changes between individuals with smaller cranium size and individuals with larger cranium size are mostly related to changes in the skull base showing pronounced negative allometry. The rostral part, however, either scaled isometrically or showed less pronounced negative allometry than the skull base. Non-allometric intersexual shape variation predominantly involved changes related to the jaw adductor muscle chamber, i.e., changes that are associated with biomechanically relevant traits of the jaw system in females and males. Both allometric and non-allometric shape changes appeared to be species-specific. Our results indicate that natural and sexual selection may be involved in the evolution of SShD.     

Metamorphosis inhibition: an alternative rearing protocol for the newt, Cynops pyrrhogaster

The newt is an indispensable model animal, of particular utility for regeneration studies. Recently, a high-throughput transgenic protocol was established for the Japanese common newt, Cynops pyrrhogaster. For studies of regeneration, metamorphosed animals may be favorable; however, for this species, there is no efficient protocol for maintaining juveniles after metamorphosis in the laboratory. In these animals, survival drops drastically after metamorphosis as their foraging behaviour changes to adapt to a terrestrial habitat, making feeding in the laboratory with live or moving foods more difficult. To elevate the efficiency of laboratory rearing of this species, we examined metamorphosis inhibition (Ml) protocols to bypass the period (four months to two years after hatching) in which the animal feeds exclusively on moving foods. We found that approximately 30% of animals survived after 2-year Ml, and that the survivors continuously grew, only with static food while maintaining their larval form and foraging behaviour in 0.02% thiourea (TU) aqueous solution, then metamorphosed when returned to a standard rearing solution even after 2-year-MI. The morphology and foraging behavior (feeding on static foods in water) of these metamorphosed newts resembled that of normally developed adult newts. Furthermore, they were able to fully regenerate amputated limbs, suggesting regenerative capacity is preserved in these animals. Thus, controlling metamorphosis with TU allows newts to be reared with the same static food under aqueous conditions, providing an alternative rearing protocol that offers the advantage of bypassing the critical period and obtaining animals that have grown sufficiently for use in regeneration studies.     

Ontogeny of cranial ossification in the eastern newt, Notophthalmus viridescens (Caudata: Salamandridae), and its relationship to metamorphosis and neoteny

The ontogenetic sequence of cranial osteogenesis through adulthood is described in samples of newts from completely metamorphosing and partially neotenic populations. Cranial ossification proceeds in the same sequence in both samples. Seven stages of cranial development are described on the basis of conspicuous events that occur during ontogeny. These include four larval stages, metamorphs, efts, and adults. Neotenic adults have skulls that are metamorphosed completely and indistinguishable from the skulls of non-neotenic adults. Neoteny in these newts does not involve the skull and is limited to the postmetamorphic retention of some gill structures and, thus, is termed "limited neoteny." The evolution of limited neoteny in newts as a correlated response to the inhibition of land-drive behavior is discussed.     

Morphological and biochemical changes in carotenoid granules in the ventral skin during growth of the Japanese newt Cynops pyrrhogaster

The color of the ventral skin of the Japanese adult newt Cynops pyrrhogaster is red, whereas that of the small juvenile newts at metamorphosis is creamy. Xanthophores in the red skin have many ring carotenoid vesicles (rcv) and a few homogenous carotenoid granules (hcg), as reported earlier. To understand the reason for this change in coloration of the ventral skin of the newt, we carried out histological and biochemical studies to see whether the size and the number of carotenoid granules (hcg and rcv) in the xanthophores and also carotenoid content in the ventral skin change during the growth of this animal. By electron microscopic observation, only hcg were observed in the creamy skin of larvae at stage 59. The diameter of the hcg in the skin of the larvae was approximately 0.85 microm, but significantly decreased to 0.35 microm in the skin of the small juvenile newt. However, the number of the hcg/100 microm (2) of a xanthophore in the ventral skin was very low in the larva at stage 59, but increased in the small juvenile. The carotenoid content was very low in the creamy skin of small juveniles, but dramatically high in the red skin of the adult newts. In the red skin of the adult newt, many rcv (85%) and a few hcg (15%) were observed. However, the number of carotenoid granules (rcv and hcg)/100 microm(2) of a xanthophore in the red skin of adult newts was not different from that of hcg/100 microm (2) of a xanthophore in the creamy skin of small juveniles. The results, taken together, suggest that the increase in the size and the number of carotenoid granules and also carotenoid content in the ventral skin is very important for red body coloration during the growth of the Japanese newt Cynops pyrrhogaster.     

Expression of sexual ornaments in a polymorphic species: phenotypic variation in response to environmental risk

Secondary sexual traits may evolve under the antagonistic context of sexual and natural selection. In some polymorphic species, these traits are only expressed during the breeding period and are differently expressed in alternative phenotypes. However, it is unknown whether such phenotypes exhibit phenotypic plasticity of seasonal ornamentations in response to environmental pressures such as in the presence of fish (predation risk). This is an important question to understand the evolution of polyphenisms. We used facultative paedomorphosis in newts as a model system because it involves the coexistence of paedomorphs that retain gills in the adult stage with metamorphs that have undergone metamorphosis, but also because newts exhibit seasonal sexual traits. Our aim was therefore to determine the influence of fish on the development of seasonal ornamentation in the two phenotypes of the palmate newt (Lissotriton helveticus). During the entire newt breeding period, we assessed the importance of phenotype and fish presence with an information‐theoretic approach. Our results showed that paedomorphs presented much less developed ornamentation than metamorphs and those ornamentations varied over time. Fish inhibited the development of sexual traits but differently between phenotypes: in contrast to metamorphs, paedomorphs lack the phenotypic plasticity of sexual traits to environmental risk. This study points out that internal and external parameters act in complex ways in the expression of seasonal sexual ornamentations and that similar environmental pressure can induce a contrasted evolution in alternative phenotypes.     

Pervasive genetic integration directs the evolution of human skull shape

It has long been unclear whether the different derived cranial traits of modern humans evolved independently in response to separate selection pressures or whether they resulted from the inherent morphological integration throughout the skull. In a novel approach to this issue, we combine evolutionary quantitative genetics and geometric morphometrics to analyze genetic and phenotypic integration in human skull shape. We measured human skulls in the ossuary of Hallstatt (Austria), which offer a unique opportunity because they are associated with genealogical data. Our results indicate pronounced covariation of traits throughout the skull. Separate simulations of selection for localized shape changes corresponding to some of the principal derived characters of modern human skulls produced outcomes that were similar to each other and involved a joint response in all of these traits. The data for both genetic and phenotypic shape variation were not consistent with the hypothesis that the face, cranial base, and cranial vault are completely independent modules but relatively strongly integrated structures. These results indicate pervasive integration in the human skull and suggest a reinterpretation of the selective scenario for human evolution where the origin of any one of the derived characters may have facilitated the evolution of the others.     

Competitive and predatory interactions between invasive mosquitofish and native larval newts

Invasive fish have a high disruptive potential in aquatic ecosystems, in which amphibians may be highly impacted due to intense competition and/or predation on their eggs and larvae. Most studies have focused on the effect of large invasive fishes such as salmonids, whereas the effect of smaller fish on amphibians has been seldom investigated. We experimentally studied effects of the invasive Eastern mosquitofish (Gambusia holbrooki) on pygmy newts (Triturus pygmaeus), a species endemic to the Iberian Peninsula. We set up outdoor mesocosms in Doñana National Park with native aquatic flora and invertebrate fauna, and containing larval newts at two experimental densities. Density of larval newts was also crossed with presence or absence of mosquitofish, either free-swimming or caged, in order to distinguish consumptive and non-consumptive effects. Increased density of coexisting larval newts did not reduce their survival, but reduced their growth and development. Newt survival and size at metamorphosis were dramatically reduced in the presence of free-swimming mosquitofish, whether at low or high fish densities. Caged mosquitofish, however, had no effect on larval newts. In laboratory trials, mosquitofish preyed more efficiently on insect larvae than did larval pygmy newts, highlighting the high competitive potential of mosquitofish. This was confirmed by the depletion of zooplankton that free fish caused in the experimental outdoor mesocosms. Our study suggests that invasive mosquitofish exert a high negative impact on coexisting newt populations. Such effects can be explained by a combination of direct predation, injuries caused by predation attempts, and intense competitive exploitation of common food resources.     

Skull size and shape variation versus molecular phylogeny: a case study of alpine newts (<Emphasis Type="Italic">Mesotriton alpestris</Emphasis>, Salamandridae) from the Balkan Peninsula

We explored the phylogenetic signal of skull size and shape in alpine newts from the Balkans, a group of European newts that, in spite of their considerable phylogeographic substructuring (as inferred from previous DNA analyses), maintain a conserved phenotype. In terms of skull shape disparity, geometric morphometrics show that the dorsal cranium carries a significant phylogenetic signal, the most notable evidence in this present study. On the contrary, no phylogenetic signal in the shape of the ventral cranium was found. This result indicates that the variation in the shape of the ventral cranium is more prone to other factors and processes, such as adaptations to local environments rather than phylogenetic constraints. Variation in skull size within alpine newts seems to be independent from phylogenetic constraints.     

Do Experience and Body Size Play a Role in Responses of Larval Ringed Salamanders,Ambystoma annulatum,to Predator Kairomones? Laboratory and Field Assays

Prey may experience ontogenetic changes in vulnerability to some predators, either because of changes in morphology or experience. If prey match their level of antipredator behavior to the level of predatory threat, prey responses to predators should reflect the appropriate level of threat for their stage of development. For larval salamanders, responses to predators may change with body size because larger larvae are less vulnerable to predation by gape‐limited predators or because fleeing responses by large salamanders may be more effective than for smaller salamanders. In a field experiment, small larval ringed salamanders, Ambystoma annulatum, responded to chemical stimuli (‘kairomones’) from predatory newts, Notophthalmus viridescens, with an antipredator response (decreased activity). Laboratory‐reared larvae decreased their activity following exposure to newt kairomones, indicating that larval ringed salamanders do not require experience with newts to recognize them as predators. In both experiments, larvae distinguished between chemical stimuli from newts and stimuli from tadpoles (non‐predators) and a blank control. In a third experiment, field‐caught (experienced) larvae showed a graded response to newt kairomones based on their body size: small larvae tended to decrease their activity while larger larvae showed no change or an increase in activity. This graded response was not observed for neutral stimuli, indicating that it is predator‐specific. Therefore, ringed salamander larvae exhibit threat‐sensitive ontogenetic changes in their response to chemical stimuli from predatory newts.     

Morph switching in a dimorphic population of <Emphasis Type="Italic">Triturus alpestris</Emphasis> (Amphibia,Caudata)

The usual life cycle of Alpine newts comprises an aquatic larval stage and a terrestrial juvenile and adult stage. However, some populations differ from this pattern in exhibiting facultative paedomorphosis where some individuals reach sexual maturity while retaining larval traits such as gills and gill slits. While paedomorphic newts can, in some circumstances, initiate metamorphosis, once a newt has commenced metamorphosis, the state is irreversible. Because the frequency of this switching from one morph to the other has never been quantified in the wild, we attempted to estimate switching rate and survival by carrying out a 3-year monitoring survey of a population inhabiting an alpine lake. While morph switching did occur in this population, it involved a relatively low proportion of the paedomorphs (approx. 12%), suggesting that metamorphosis is not favoured in the study population. The hypothesis of paedomorphic advantage was not supported since neither survival nor body condition differed between morphs. The ontogenetic pathway of wild Alpine newts is thus characterised by two forks in the developmental pathway. The first occurs during the larval stage (metamorphosis vs. paedomorphosis), and the second occurs in paedomorphic adults (switching for metamorphosis vs. continuation of the paedomorphic lifestyle). Such a two-level decision process may allow individuals to cope with environmental uncertainty.