Comparative landscape genetics reveals the evolution of viviparity reduces genetic connectivity in fire salamanders.

Evolutionary changes in reproductive mode may affect co-evolving traits, such as dispersal, although this subject remains largely underexplored. The shift from aquatic oviparous or larviparous reproduction to terrestrial viviparous reproduction in some amphibians entails skipping the aquatic larval stage and, thus, greater independence from water. Accordingly, amphibians exhibiting terrestrial viviparous reproduction may potentially disperse across a wider variety of suboptimal habitats and increase population connectivity in fragmented landscapes compared to aquatic-breeding species. We investigated this hypothesis in the fire salamander (Salamandra salamandra), which exhibits both aquatic- (larviparity) and terrestrial-breeding (viviparity) strategies. We genotyped 426 larviparous and 360 viviparous adult salamanders for 13 microsatellite loci and sequenced a mitochondrial marker for 133 larviparous and 119 viviparous individuals to compare population connectivity and landscape resistance to gene flow within a landscape genetics framework. Contrary to our predictions, viviparous populations exhibited greater differentiation and reduced genetic connectivity compared to larviparous populations. Landscape genetic analyses indicate viviparity may be partially responsible for this pattern, as water courses comprised a significant barrier only in viviparous salamanders, probably due to their fully terrestrial life cycle. Agricultural areas and, to a lesser extent, topography also decreased genetic connectivity in both larviparous and viviparous populations. This study is one of very few to explicitly demonstrate the evolution of a derived reproductive mode affects patterns of genetic connectivity. Our findings open avenues for future research to better understand the eco-evolutionary implications underlying the emergence of terrestrial reproduction in amphibians.

Distribution of dorsal-forming activity in precleavage embryos of the Japanese newt, Cynops pyrrhogaster: effects of deletion of vegetal cytoplasm, UV irradiation, and lithium treatment.

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.

Evolutionary patterns in ontogenetic transformation: from laws to regularities.

The concept of heterochrony derives from classical approaches to the study of ontogeny and phylogeny. Under the influence of landmark books by deBeer (1930) and Gould (1977), the traditional theories have been revised to fit into the conceptual framework of modern genetics and evolutionary theory. The current scheme, however, suffers from a problem of lack of precise definitions. The term heterochrony is now used to refer to a developmental process as well as to an evolutionary pattern. That is, it refers to a microevolutionary process of adaptation, operating in local populations under selection and to a macroevolutionary pattern based on undefined internal laws of form. Such conceptually contradictory frameworks are a source of confusion and of empirical misuse of concepts. We propose to reduce the dependence of current thinking about heterochrony on the concept of "timing" and instead focus on the organization of sequences of developmental events in ontogeny. Although Haeckelian views have been rejected, most experts would agree that some subtle parallelism between ontogeny and phylogeny does occur. This relationship deserves renewed attention and urodeles are particularly suited to study it due to their variable patterns of ontogeny and complex life cycles. Current reductionist attempts to apply the morphological terminology and postulates of classical heterochrony concepts to cellular and molecular (genetic) aspects of morphogenesis are problematic. Molecular heterochrony requires a linear or strictly hierarchical structure of gene regulation of development. In addition, isomorphism between genetic mutations and morphological changes would be required for the existing terminology to apply. Finally, we caution against a broad interpretation of heterochronic processes at the molecular level, since the approach may end up permitting the meaningless interpretation of any developmental change as heterochrony.

Allometry of red cell oxygen binding and hematology in larvae of the salamander, Ambystoma tigrinum.

Very few studies have attempted to relate blood characteristics to body mass within, rather than between, species. Thus, respiratory and hematological properties of the blood of larval tiger salamanders (Ambystoma tigrinum) have been measured in animals ranging in body mass from approximately 2 to 112 g. This amphibian species was chosen because larvae of very different body mass may be of similar developmental stage, minimizing interference from ontogenetic factors. Mean corpuscular volume, mean corpuscular Hb, hematocrit, blood Hb concentration, blood O2 capacity and Hill's n were all positively correlated with body mass (P less than 0.005). Blood O2 affinity (P50) and Bohr shift were not significantly correlated with body mass (P greater than 0.20). The findings are discussed in the context of the general effect of body mass upon metabolic rate in vertebrates, as well as environmental and biological factors specific to larvae of the tiger salamander.

Epitheliophagy: intrauterine cell nourishment in the viviparous alpine salamander, Salamandra atra (Laur.).

The intrauterine nourishment of the viviparous alpine salamander, Salamandra atra, has been demonstrated to consist of two phases: oophagy - after hatching from the jelly membrane, the developing embryo ingests the remaining disintegrated, unfertilized egg mass. Epitheliophagy - a special cranial portion of the uterus wall, the zona trophica, is stimulated by the presence of the embryo. After the yolk mass has been exhausted, the developing embryo is supplied with epithelial cells as nourishment until the end of pregnancy. The epithelial cells of the zona trophica are released into the uterus lumen by partial necrosis of the underlying connective tissue. Regeneration and detachment of the uterine epithelium occur simultaneously in different regions of the zona trophica. A special dentition enables the embryo, according to its position in the uterus, to feed directly on the zona trophica.

Observations on the migration and differentiation of neural crest cells in somite extirpated salamander larvae.

In order to research the influences of the somites or their derivatives on the migration and differentiation of neural crest cells, unilateral extirpation of somites was performed in the larvae of the salamander (Hynobius lichenatus Boulenger). Two types of neural crest cells appeared in the somite-free environment. They could already be distinguished from each other shortly after beginning of migration. One type of the cells was distributed in the expanded space between the epidermis and the neural tube, with melanocytes differentiated from them. The other type of the cells migrated closely to the lateral wall of the neural tube. They aggregated as a cord on the ventrolateral side of the neural tube and gave rise either to neurons or to satellite cells of spinal ganglia. The sequence of cytological events along the development of the cells and the time required for each event were essentially identical with those of the melanocytes and spinal ganglion cells developing on the control side. The present results suggest not only that migration and differentiation of neural crest cells are independent of the possible regulation of the somites or their derivatives but also that the fate of neural crest cells is determined before the onset of migration.

Hepatic arginase activity in intra- and extrauterine larvae of the ovoviviparous salamander. Salamandra salamandra (L.) (Amphibia, Urodela).

The hepatic arginase activity of Salamandra salamandra was determined at three different stages of intra- and extrauterine larval development and at fully metamorphosed juveniles. The highest enzymatic activity was found in intrauterine larvae in November, the lowest in intrauterine larvae in June of the following year. These data can be correlated with the ureotelism of intrauterine larvae previously described and are discussed with respect to the metabolism of larval and juvenile fire salamanders.

Ultrastructure of epidermis of Salamandra salamandra followed throughout ontogenesis.

Ventral epidermal ultrastructure of the amphibian urodele Salamandra salamandra is described and followed throughout its life cycle. Tadpoles were divided into five categories on the basis of the organization of their epidermis and the ultrastructure of its cells. In newly hatched tadpoles the epidermis is arranged in two layers and four types of cells were recognized. The number of epidermal layers increases in the metamorphosing tadpole. At this stage the layers become organized in four strata. Metamorphosis involves the disappearance of some cell types and the appearance of others, typical of the adult epidermis. The significance of these ontogenetic changes in epidermal ultrastructure is discussed in respect to aquatic and terrestrial life habits.