Out of Asia: Mitochondrial DNA Evidence for an Oriental Origin of Tiger Frogs, Genus Hoplobatrachus

Most examples of intercontinental dispersal events after the Miocene contact between Africa and Asia involve mammal lineages. Among amphibians, a number of probably related groups are known from both continents, but their phylogenies are so far largely unresolved. To test the hypothesis of Miocene dispersal against a Mesozoic vicariance scenario in the context of Gondwana fragmentation, we analyzed fragments of the mitochondrial 16S rRNA gene (572 bp) in 40 specimens of 34 species of the anuran family Ranidae. Results corroborated the monophyly of tiger frogs (genus Hoplobatrachus), a genus with representatives in Africa and Asia. The African H. occipitalis was the sister group of the Asian H. crassus, H. chinensis, and H. tigerinus. Hoplobatrachus was placed in a clade also containing the Asian genera Euphlyctis and Nannophrys. Combined analysis of sequences of 16S and 12S rRNA genes (total 903 bp) in a reduced set of taxa corroborated the monophyly of the lineage containing these three genera and identified the Asian genus Fejervarya as its possible sister group. The fact that the African H. occipitalis is nested within an otherwise exclusively Asian clade indicates its probable Oriental origin. Rough molecular clock estimates did not contradict the assumption that the dispersal event took place in the Miocene. Our data further identified a similar molecular divergence between closely related Asian and African species of Rana (belonging to the section Hylarana), indicating that Neogene intercontinental dispersal also may have taken place in this group and possibly in rhacophorid treefrogs.     

Adhesion and allometry from metamorphosis to maturation in hylid tree frogs: a sticky problem

Many species of climbing frog possess large disc-like digital pads, which facilitate adhesion. The consensus of earlier studies is that frogs depend on a wet adhesive mechanism, chiefly influenced by pad area and surface tension properties of the mediating adhesive fluid. The relationship between morphology, size and adhesion is of particular interest for tree frogs, because evolution of adhesive ability has facilitated niche expansion into arboreal habitats. If frogs are geometrically similar, and growth occurs isometrically, then mass will increase at a higher rate than toe-pad area, and an adhesive system directly dependent on area may be adversely affected. We investigated scaling of adhesive ability with ontogeny in seven species of hylid tree frog to test whether the responses to the challenges of maintaining adhesion with growth were sufficient to allow adult frogs to stick as effectively on smooth substrates as juvenile frogs. In all species, mass increased at a lower rate than expected with isometric growth. This was less pronounced in larger species, perhaps owing to the demands of locomotory modes such as jumping. In smaller hylids, this non-isometric increase in mass was sufficient to enable adult frogs to adhere as effectively as juveniles. In larger species, however, the ability of adult frogs to adhere was significantly lower than that of juveniles, despite evidence of increased toe-pad efficiency. Within all species adhesive forces increased at a greater rate than toe-pad area, suggestive of changes in the relative influence of the contributory components of the wet adhesive mechanism with growth.     

Genetic constraints at the metamorphic boundary: Morphological development in the wood frog,Rana sylvatica

Organisms with complex life-cycles often experience very different environments in different phases of their life. Genes expressed in more than one phase could potentially create a conflict or constraint on evolutionary change if the pattern of selection on those genes were different in the different phases. The potential importance of this type of constraint across metamorphosis in frogs was assessed by measuring the genetic correlation between several morphological traits in both larval and juvenile Rana sylvatica. Genetic correlations within a stage tended to be moderately high and significant whereas correlations across stages were low and not significant. Errors on the genetic parameters make it impossible to prove that there are no genetic constraints across metamorphosis in this population of frogs, but the results are consistent with the hypothesis that gene expression and developmental regulation are partitioned separately before and after metamorphosis.     

PATTERNS OF POSTZYGOTIC ISOLATION IN FROGS

From literature data on 116 taxa crosses involving 46 species of frogs, we found a positive correlation between degree of divergence (measured as Nei's genetic distance, D) and degree of postzygotic isolation. In anurans, hybrid sterility appears to evolve more quickly than inviability, which is consistent with the conclusions of other studies that involved Drosophila species. The lower threshold of D = 0.30 for evolution of hybrid inviability that we found is similar to that observed for Drosophila. This consistency suggests that there may be a general pattern in the acquisition of reproductive isolation in animals.     

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in pelvic nerves supplying the posterior large intestine of the toad,Bufo marinus

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in the posterior portion of the large intestine of the toad was studied using single- and dual-label immunohistochemistry. Neurons containing colocalized galanin/somatostatin or vasoactive intestinal peptide alone were observed along intramural pelvic nerves. Some of the galanin/somatostatin neurons also contained 5-hydroxytryptamine. Synaptic boutons containing colocalized calcitonin gene-related peptide/vasoactive intestinal peptide were associated with the galanin/somatostatin neurons. The muscle of the large intestine was also innervated by axons containing galamin/somatostatin, vasoactive intestinal peptide/calcitonin gene-related peptide or vasoactive intestinal peptide alone. Nerve fibres containing calcitonin gene-related peptide/substance P, probably representing primary afferent nerves, were also associated with muscle bundles. Submucosal blood vessels carried dense plexuses of fibres containing vasoactive intestinal peptide alone or and calcitonin gene-related peptide/substance P. Adrenergic perivascular nerves also contained galanin and neuropeptide Y.     

Ultrastructural radioautographic analysis of neurogenesis in the hypothalamus of the adult frog,Rana temporaria,with special reference to physiological regeneration of the preoptic nucleus

The localization and fine structure of proliferating cells in the hypothalamic preoptic area were studied by light-and electron-microscopic radioautography 1–2 h following single application of ³H-thymidine to adult Rana temporaria taken from their natural habitat in the spring and autumn. ³H-thymidine uptake by proliferating cells was much more pronounced in frogs caught in May/June, i.e., a month after the breeding period (labeled cells represent about 10% of the total ventricular zone cell population), compared to animals caught in mid-September, when it was very low. In both ³H-thymidine treatment groups the vast majority of labeled cells are found exclusively within the preoptic recess ventricular zone. With regard to ultrastructure, it contained proliferating cells of at least 4 types, ranging from immature forms (bipolar stem cells) to more differentiated elements (tanycyte-like ependymoblasts, classical ependymoblasts). All of them showed label over their nuclei indicating that these cells are capable of DNA synthesis and mitosis. The possible role of the preoptic recess ventricular zone as a source of precursor cells for new peptidergic neurosecretory cells, conventional neurons and glial cells in the hypothalamic preoptic area of the adult frog is discussed.     

Distribution of NCAM-180 and polysialic acid in the developing tectum mesencephali of the frog Discoglossus pictus and the salamander Pleurodeles waltl

The 180 kDa component of the neural cell adhesion molecule (NCAM-180), total NCAM (NCAM-total) and the polysialic acid modification of NCAM (PSA) show similar temporal and spatial regulation in the developing tecta of Pleurodeles waltl (salamander) and Discoglossus pictus (frog). Whereas NCAM-total is found throughout the tectal tissue on neurons and glia, NCAM-180 is only found on nonproliferating neurons and in fiber layers. PSA is expressed by a subset of NCAM-180-positive cells. Western blots show that there is little polysialylated NCAM-140 in the developing amphibian tectum. Regions unstained for PSA and NCAM-180 correspond precisely to the growth zones of the tectum. NCAM-180 and PSA are not present in tecta of early larvae. Staining intensity is strongest at midlarval stages for both antigens. At metamorphosis, PSA is strongly downregulated, whereas NCAM-180 is downregulated in juvenile animals. Both antigens are still present in fiber layers of adult animals. In dissociated tissue culture of the frog tectum, NCAM-180 is not present on astrocytes, but on neuronal cells. Expression is enhanced at cell contact sites, suggesting that NCAM-180 is involved in cell contact stabilization. This study shows that general features of temporal and spatial regulation of NCAM isoforms and PSA are highly conserved in frog and salamander tecta, despite large differences in the rate of cell migration and the degree of lamination in these homologous brain regions.     

Localization of brush border cytoskeletal proteins in gastric oxynticopeptic cells from the bullfrog Rana catesbeiana

The contribution of brush border cytoskeletal proteins (actin, villin, fimbrin, and brush border myosin-1) to organization of the cytoskeletal network underlying apical plications of oxynticopeptic cells was examined by immunohistochemical techniques in frozen sections of gastric mucosa from the bullfrog, Rana catesbeiana. Apical localization of F-actin with phalloidin in oxynticopeptic cells inhibited with cimetidine revealed small, punctate domains within the apical cytoplasm that were consistent with the presence of short microvilli revealed by electron microscopy. Localization of F-actin in cells stimulated with forskolin was limited to a wide continuous band of cytoplasm corresponding to the location of numerous long surface folds. Inhibition of protein synthesis with cycloheximide did not prevent acid secretion or formation of actin filaments within surface folds in stimulated oxynticopeptic cells, suggesting that the formation of filaments does not require actin synthesis. Staining of gastric mucosae with fluorescent DNase-1 demonstrated that oxynticopeptic cells possess an unusually large pool of non-filamentous actin. Taken together, these results suggest that actin-filament formation in stimulated cells occurs by polymerization of an existing pool of non-filamentous actin. Localization of antibodies specific for villin and fimbrin revealed that these proteins were present within intestinal absorptive cells and gastric surface and neck cells but were not present within inhibited or stimulated oxynticopeptic cells. Brush border myosin-1, present in intestinal absorptive cells, was not present in gastric epithelium. Thus, we propose that actin-containing projections in oxynticopeptic cells are not organized like intestinal microvilli and that filament formation occurs after stimulation by modulating intracellular pools of filamentous and non-filamentous actin.     

Immunohistochemical localisation of substances reactive to antisera against α-and β-endorphin and met-enkephalin in the brain of Rana temporaria L.

Summary In the brain of Rana temporaria, two distinct systems reactive with - and -endorphin antisera, respectively, and with a met-enkephalin antiserum, have been detected immunohistochemically.Neurons reacting with - and -endorphin antisera are located (1) in the preoptic nucleus, and (2) in the pars ventralis of the tuber cinereum. Immunoreactive nerve fibres of both groups of perikarya end in the infundibular floor near the capillaries and the preoptico-hypophysial tract. Control reactions have shown that the immunoreactivity is suppressed by the corresponding antigens, but also by -LPH. In view of these results the immunoreactive systems examined correspond to an /-endorphin system or a lipotropinergic system.Neurons reacting with the met-enkephalin antiserum are located in the paraventricular organ. Intense immunofluorescence was observed in the infundibular floor. Controls show that the labelling by met-enkephalin antiserum is exclusively suppressed by met-enkephalin.In the pituitary gland, on the other hand, - and -endorphin antisera reveal: 1) the MSH/ACTH-like cells of the pars intermedia and 2) the ACTH-like cells of the pars distalis.Supported by the D.G.R.S.T., Contrat no 77.7.0648     

Localization of binding sites for atrial natriuretic factor and angiotensin II in the central nervous system of the clawed toad Xenopus laevis

Summary The distribution of binding sites for atrial natriuretic factor (ANF) and angiotensin II (A II) was investigated in the central nervous system (CNS) of the clawed toad Xenopus laevis by means of in vitro autoradiography using [¹²⁵I]-rat ANF(99–126) or [¹²⁵I] [Val⁵] A II and [¹²⁵I]human A II as labeled ligands. The highest densities of specific ANF-binding were detected in the nucleus habenularis, thalamic regions, hypophyseal pars nervosa and nucleus interpeduncularis. Moderate ANF-binding was found in the bulbus olfactorius, pallium, septum, striatum, lateral forebrain bundle, nucleus infundibularis, hypophyseal pars distalis and tectum. The highest levels of specific A II binding sites were observed in the nucleus praeopticus, nucleus habenularis, hypophyseal pars nervosa and pars distalis, whereas the amygdala contained moderate A II binding. The existence of specific binding sites for ANF and A II in the CNS of Xenopus laevis suggests that both peptides act as neurotransmitters or neuromodulators in the amphibian CNS. The co-localization of dense binding sites for both peptides in the nucleus habenularis, hypophyseal pars nervosa and pars distalis supports the view that ANF and A II have opposite regulatory functions in these regions.