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 cranial morphology and relationships of the aberrant Carboniferous amphibian Spathicephalus mirus Watson

The skull of Spathicephalus mirus Watson, an amphibian from the Namurian, basal Upper Carboniferous, of Scotland is described. It shares with the Loxommatoidea a closed palate, palatal ornament and antorbital vacuities, and the family Spathicephalidae is recognized as the sister group of the Loxommatidae. A new diagnosis of the Loxommatoidea is presented together with one of the Spathicephalidae. An analysis of the functional morphology of the Spathicephalus skull suggests that it was incapable of rapid jaw closure required for catching fish. Instead it is proposed that Spathicephalus was a sluggish bottom-dwelling filter-feeder of small, soft invertebrates.     

The morphology and relationships of the Carboniferous amphibian Eoherpeton watsoni Panchen

The oldest recorded anthracosaur amphibian Eoherpeton watsoni Panchen, from localities in the Visean and Namurian of the Scottish Carboniferous, is described. It lacks a distinct otic notch, and reconstruction of the position of cranial ligaments and muscles suggest that a tympanum was never developed. On the basis of these observations it is concluded that arguments used to refute a relationship between anthracosaurs and amniotes, based on apparent differences in the morphology of their otic regions, are invalid. The order Anthracosauria is considered to be a monophyletic group characterized by the development of a sutural connection between the tabular and parietal bones on a skull table which includes both the intertemporal and supratemporal as well as the tabular. It is divided into two suborders: Seymouriamorpha characterized by a sutural connection between the squamosal and intertemporal and the presence of a lateral otic tube, and Anthracosauroideae characterized by the absence of post-temporal fossae. The Discosauriscidae, Kotlassiidae and Seymouriidae are included in the Seymouriamorpha, and the Eoherpetontidae, Gephyrostegidae and Embolomeri comprise the Anthracosauroideae. The Proterogyrinidae are considered the most primitive family of embolomeres. A new phylogeny of Amphibia is presented. The old groupings of Labyrinthodontia, Lepospondyli and Lissamphibia are rejected and Amphibia is divided, on the basis of differences in morphology of the neurocranium and its relationship with the surrounding bones of the skull, into two major groups, the first including the Ichthyostcgalia, Temnospondyli, Microsauria and the extant Anura, Apoda and Urodela, while the Aistopoda, Nectridea, Palaeostegalia, Loxommatoidea, Anthracosauria and Diadectomorpha, together with the Amniota, form the second. Similarities in the dentition of the palaeostegalian Crassigyrinus and loxommatoids suggest the two taxa are sister-groups, but the immediate relationships of anthracosaurs remain indeterminate and they here form an unresolved trichotomy with diadectomorphs and amniotes.     

Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)

Aim  The present-day geographical distribution of parasites with a direct biological life cycle is guided mostly by the past dispersal and vicariance events that have affected their hosts. The Amphibia– Polystoma association (which satisfies these criteria) also exhibits original traits, such as host specificity and world-wide distribution. This biological model was thus chosen to investigate the common historical biogeography of its widespread representatives.
Location  North and South America, Eurasia and Africa.
Methods  We investigated the phylogeny of 12 species of neobatrachian parasites sampled from North and South America, Eurasia and Africa. Hosts belonged mostly to hyloids and ranoids of families Bufonidae, Hylidae, Leptodactylidae, Ranidae and Hyperoliidae. Phylogenetic reconstructions were inferred from maximum likelihood and maximum parsimony analyses from complete ITS1 sequences.
Results  The group of American species appeared paraphyletic with one species at the base of a Eurafrican clade, within which two lineages were seen: one composed of only Eurasian species, and the other of European and African species, with the two European species basal to an African clade.
Main conclusions  The route of Polystoma evolution is deduced from the phylogenetic tree and discussed in the light of host evolution. We conclude that Polystoma originated in South America on hyloids, after the separation of South America from Africa. The genus must have colonized North America in Palaeocene times and Eurasia by the mid-Cainozoic, taking advantage of the dispersal of either ancestral bufonids or hylids. Africa, however, appears to have been colonized more recently, during the Messinian period.     

Evolutionary history of a complex adaptation: Tetrodotoxin resistance in salamanders

Understanding the processes that generate novel adaptive phenotypes is central to evolutionary biology. We used comparative analyses to reveal the history of tetrodotoxin (TTX) resistance in TTX-bearing salamanders. Resistance to TTX is a critical component of the ability to use TTX defensively but the origin of the TTX-bearing phenotype is unclear. Skeletal muscle of TTX-bearing salamanders (modern newts, family: Salamandridae) is unaffected by TTX at doses far in excess of those that block action potentials in muscle and nerve of other vertebrates. Skeletal muscle of non-TTX-bearing salamandrids is also resistant to TTX but at lower levels. Skeletal muscle TTX resistance in the Salamandridae results from the expression of TTX-resistant variants of the voltage-gated sodium channel Na_V 1.4 (SCN4a). We identified four substitutions in the coding region of salSCN4a that are likely responsible for the TTX resistance measured in TTX-bearing salamanders and variation at one of these sites likely explains variation in TTX resistance among other lineages. Our results suggest that exaptation has played a role in the evolution of the TTX-bearing phenotype and provide empirical evidence that complex physiological adaptations can arise through the accumulation of beneficial mutations in the coding region of conserved proteins.     

Olfactory metamorphosis in the Coastal Giant Salamander (Dicamptodon tenebrosus)

This study examined the gross morphology and ultrastructure of the olfactory organ of larvae, neotenic adults, and terrestrial adults of the Coastal Giant Salamander (Dicamptodon tenebrosus). The olfactory organ of all aquatic animals (larvae and neotenes) is similar in structure, forming a tube extending from the external naris to the choana. A nonsensory vestibule leads into the main olfactory cavity. The epithelium of the main olfactory cavity is thrown into a series of transverse valleys and ridges, with at least six dorsal and nine ventral valleys lined with olfactory epithelium, and separated by ridges of respiratory epithelium. The ridges enlarge with growth, forming large flaps extending into the lumen in neotenes. The vomeronasal organ is a diverticulum off the ventrolateral side of the main olfactory cavity. In terrestrial animals, by contrast, the vestibule has been lost. The main olfactory cavity has become much broader and dorsoventrally compressed. The prominent transverse ridges are lost, although small diagonal ridges of respiratory epithelium are found in the lateral region of the ventral olfactory epithelium. The posterior and posteromedial wall of the main olfactory cavity is composed of respiratory epithelium, in contrast to the olfactory epithelium found here in aquatic forms. The vomeronasal organ remains similar to that in large larvae, but is now connected to the mouth by a groove that extends back through the choana onto the palate. Bowman's glands are present in the main olfactory cavity at all stages, but are most abundant and best developed in terrestrial adults. They are lacking in the lateral olfactory epithelium of the main olfactory cavity. At the ultrastructural level, in aquatic animals receptor cells of the main olfactory cavity can have cilia, short microvilli, a mix of the two, or long microvilli. Supporting cells are of two types: secretory supporting cells with small, electron-dense secretory granules, and ciliated supporting cells. Receptor cells of the vomeronasal organ are exclusively microvillar, but supporting cells are secretory or ciliated, as in the main olfactory cavity. After metamorphosis two distinct types of sensory epithelium occur in the main olfactory cavity. The predominant epithelium, covering most of the roof and the medial part of the floor, is characterized by supporting cells with large, electron-lucent vesicles. The epithelium on the lateral floor of the main olfactory cavity, by contrast, resembles that of aquatic animals. Both types have both microvillar and ciliated receptor cells. No important changes are noted in cell types of the vomeronasal organ after metamorphosis. A literature survey suggests that some features of the metamorphic changes described here are characteristic of all salamanders, while others appear unique to D. tenebrosus.     

Immunohistochemical localization of a gastrin-like peptide in the brain of an amphibian,Xenopus laevis Daud.

Summary The indirect immunofluorescence technique was used to demonstrate a substance reacting with gastrin antisera in the brain of Xenopus laevis.Immunoreactive material was found in two sites: (1) In the caudal hypothalamus more precisely in the nucleus infundibularis ventralis, (NIV) of the pars ventralis of the tuber cinereum, (PVTC). The fluorescent axons of the reactive parikarya of the NIV give rise to two symmetrical tracts which run rostro-ventrally and join, in the infundibular floor, the preoptico-hypophysial tract, where they form an uneven median tract coursing caudally and running along the medio-tuberal area before entering the external zone of the median eminence. (2) In the anterior preoptic area (APOA), where numerous nerve fibers and endings form a dense network near the preoptic recess. The exact origin of these terminals has not yet been determined.Control of immunohistochemical specificity shows that the labeling by gastrin antisera is suppressed by gastrin (2–17), but also by cholecystokinin (CCK) and pentagastrin (Peptavlon). These results indicate that the immunoreactive substance revealed belongs to the gastrin group and has an antigenic determinant composed of the amino acid sequence or a portion thereof common to gastrin, CCK and Peptavlon (Trp-Met-Asp-Phe).It should be emphasized that, in the brain of Xenopus laevis, both gastrinimmunoreactive sites correspond to the sites of uptake of steroid hormones (Kelley et al., 1975; Morrell et al., 1975).Supported by the D.G.R.S.T., Contrat n 77.7.0648     

Protein kinases in amphibian ectoderm induced for neural differentiation

Summary Ectoderm explants from early gastrula stages of Xenopus laevis were induced with a neutralizing factor. The factor was isolated from Xenopus gastrulae and partially purified by chromatography on DEAE cellulose. The ectoderm was cultured for different periods of time and then homogenized. Protein kinase activity was determined in the homogenates from induced and control explants with histone H 1 or C-terminal peptide derived from histone H 1 as substrates. The C-terminal peptide is a more specific substrate for protein kinase C, whereas histoneH 1 is a substrate for cAMP/cGMP-dependent protein kinases as well protein kinase C. With both substrates the enzyme activity increases after induction. With the C-terminal peptide as the substrate the protein kinase activity is lower, but its relative increase after induction higher. This suggests that besides cAMP/cGMP dependent protein kinases protein kinase C or related enzymes are involved in the neural induction and differentiation processes. This corresponds to previous experiments which have shown that treatment of ectoderm with phorbol myristate acetate, an activator of protein kinase C and protein kinase C related enzymes, initiates neural differentiation. Endogeneous substrates, which are more intensively phosphorylated after induction are proteins with apparent molecular weights 21 kDa and 31 kDa. Addition of protein kinase C to the induced and control homogenates abolishes the difference in the phosphorylation rate of these proteins.     

Three types of cutaneous glands in the skin of the salamandrid Pleurodeles waltl. A histological and ultrastructural study

Histological and ultrastructural investigations revealed three different multicellular skin gland types in the salamandrid Pleurodeles waltl. The mucous glands are small, with one layer of secretory cells surrounding a central lumen; they produce the viscous and slippery mucus film that has various functions in amphibians. The serous glands can be divided based on their histological and ultrastructural characters into the granular gland Type I (GGI) and the granular gland Type II (GGII). The first type (GGI) is moderately sized and distributed throughout the body surface, with higher concentrations in the parotoid and back regions. In contrast, the second type (GGII) is very large (for Pleurodeles) and was found only in the tail, with highest concentration in the tail dorsum. Both granular gland types contain mainly proteinaceous materials but differ in their morphological features including size, shape, cellular organization and vesicle distribution, vesicle size and vesicle shape. Both GGI and GGII are especially concentrated in body parts that are presented to an attacking predator and are hypothesized to produce repellent to poisonous substances to thwart potential aggressors. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Immunocytochemical localization and spatial relation to the adenohypophysis of a somatostatin-like and a corticotropin-releasing factor-like peptide in the brain of four amphibian species

Summary The distribution of somatostatin (SRIF) — and corticotropin-releasing factor (CRF)-like — immunoreactive material was studied in the brain of four amphibian species (Ambystoma mexicanum, Pleurodeles waltlii, Xenopus laevis, Rana ridibunda) by use of immunocytochemistry. A wide network of SRIF-immunoreactive fibers and numerous perikarya were observed in all amphibians examined, with a dense accumulation of nerve endings in the external layer of the median eminence (ELME). In the representatives of the four amphibian species the CRF-like system was more circumscribed. Immunoreactive perikarya were present in the preoptic area, mainly in a ventrobasal position, and in the interpeduncular nucleus. The tract running along the ventral part of the tuber cinereum ends in the ELME facing the rostroventral lobe of the pars distalis that contains corticotrophs. CRF fibers were scarce or absent in the neural lobe. In all species studied in the present work, CRF fibers end in the area of the ELME close to the pituitary lobe containing corticotrophs. This correlation is similar to that reported for the Japanese quail and several teleosts.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek and the CNRS     

Activation of a neuralizing factor in amphibian ectoderm

Summary Isolated gastrula ectoderm has no neural-inducing activity and does not differentiate into neural tissues. It has, however, a high neural-inducing capacity, but the inducing factors are present in a masked, inactive form. The inducing factors are partially activated by homogenization and by freezing of the homogenate and are fully activated by treatment with ethanol. The relative distribution of inducing factors in different subcellular fractions changes after treatment with demecolcine and cytochalasin B or after autolytic incubation of the homogenate. The inducing activity of the high-speed supernatant is enhanced under these conditions. The experiments suggest that the activation of neuralizing factor(s) depends on the release from complex structures. Cytoskeletal elements seem to be involved. When early neural plate homogenate was fractionated, the high-speed supernatant showed neural-inducing activity. This is in contrast to the high-speed supernatant from the ectoderm homogenate, which shows no such activity.     

Embryonic appearance of rod opsin in the urodele amphibian eye

Notophthalmus (Triturus) viridescens, a urodele amphibian (newt) common to the Eastern United States, is a promising subject for developmental and regeneration studies. We have available a monoclonal antibody shown to be specific in many vertebrates for rod opsin, the membrane apoprotein of the visual pigment rhodopsin. This antibody to an N-terminal epitope, by rigorous biochemical and immunological criteria, recognizes only rod photoreceptor cells of the retina in light-and electron-microscopic immunocytochemistry. To determine the ontogeny and localization of rhodopsin in developing rods as an indicator of function in the embryonic urodele retina, we have utilized this antibody in the immunofluorescence technique on sections of developing N. viridescens. It was applied to serial sections of the eye region of Harrison stage 28 (optic vesicle) through stage 43 (most adult retina histology complete) embryos, and subsequently visualized with biotinylated species antibody followed by extravidin fluorescein isothiocyanate. The first positive reaction to rhodopsin could be detected in two to four cells (total) of the stage 37 embryonic eye, in the region of the central retinal primordium where the photoreceptors will be found. Some indications of retinal outer nuclear and inner plexiform layers could be seen at this time. Later embryonic stages demonstrated increasing numbers of positive cells in the future photoreceptor outer nuclear layer and outer and inner segments, spreading even to the peripheral retina. Nevertheless, by stale 43, no positive cells could be found at the dorsal or ventral retinal margins. Thus, biochemical differentiation of a photoreceptor population in the urodele retina occurs at a stage before retinal histogenesis is complete. The total maturation of retinal rods occurs topographically over a long period until the adult distribution is achieved. Correspondence to: D.S. McDevitt     

漆树科植物漆酚的多样性及演化关系研究

漆酚是漆树科植物独有的化学成分,具有良好的抗氧化、抗肿瘤、抗病毒等生物活性,极具药物和工业生产开发价值。该研究通过对国内外有关漆树科各类群有关漆酚数据的搜集和整理,开展漆树科漆酚的多样性与分布情况的综合统计;同时在利用核基因ETS和叶绿体基因rps16和trnL-F重建漆树科系统发育树的基础上,探讨漆酚在漆树科及各属间的起源和演化关系。结果表明:(1)漆树科漆酚具有较高的多样性,在其苯环和-R链上都有很大的变异。(2)发现漆酚仅存在于Anacardioideae亚科中,是该亚科的一个重要界定特征。(3)基于漆酚苯环结构和-R链特征的祖先性状重建分析表明,漆树科的漆酚是由邻苯二酚向间苯二酚演化,而漆酚-R链的十五烃(烯)基则是向十七烃(烯)基平行演化。     

No ecological opportunity signal on a continental scale? Diversification and life‐history evolution of African true toads (Anura: Bufonidae)

The niche‐filling process predicted by the “ecological opportunity” (EO) model is an often‐invoked mechanism for generating exceptional diversity in island colonizers. Whether the same process governs lineage accumulation and trait disparity during continental colonization events is less clear. Here, we test this prediction by investigating the rate dynamics and trait evolution of one of Africa's most widespread amphibian colonizers, the true toads (Bufonidae). By reconstructing the most complete molecular phylogeny of African Bufonidae to date, we find that the diversification of lineages in Africa best conforms to a constant rate model throughout time and across subclades, with little support for EO. Evolutionary rates of life‐history traits have similarly been constant over time. However, an analysis of generalists and specialists showed a shift toward higher speciation rates associated with habitat specialization. The overall lack of EO signal can be interpreted in a number of ways and we propose several explanations. Firstly, methodological issues might preclude the detection of EO. Secondly, colonizers might not experience true EO conditions and due to the size, ecological heterogeneity and age of landmasses, the diversification processes might be more complex. Thirdly, lower speciation rates of habitat generalists may have affected overall proliferation of lineages.     

Mesoderm differentiation in early amphibian embryos depends on the animal cap

Embryos of Ambystoma mexicanum from the late morula to the late blastula stage were dissected and cultivated in varying combinations. The marginal zone (presumptive mesoderm) when isolated together with the vegetal region differentiated to notochord after dissection from early blastulae, but did not differentiate to other tissues. When isolated from middle to late blastulae, in addition myoblasts and mesenchyme were formed. The marginal zone isolated together with the animal region (presumptive ectoderm) differentiated to notochord, muscle, mesenchyme, renal tubules and mesothelium irrespective of the stage of dissection. Combination of isolated animal and vegetal regions did lead to the induction of mesodermal organs. The experiments suggest that further steps in the differentiation of mesodermal organs after the induction of mesoderm by the vegetalizing factor depend on factors from the animal region, which are involved in pattern formation.     

The origins of graptolites and other pterobranchs: a journey from ‘Polyzoa’

The graptolites, known only from fossils, have been convincingly allied to the pterobranch hemichordates, a group of tiny, mostly colonial marine invertebrates bearing feeding arms. The phylogenetic position of pterobranchs has been subject to debate and revision for over a century. Their colonial lifestyle and feeding arms were originally seen as evidence placing them among the bryozoans, until later and more careful anatomical studies revealed more characters in common with acorn worms. Pterobranchs and acorn worms are now grouped as the phylum Hemichordata. For many decades, it was thought that pterobranchs were closer to the ancestral form of hemichordates, particularly because ‘lophophorate’ invertebrates also possess feeding arms, notably the phoronids and bryozoans, as do crinoid echinoderms. This traditional view has been challenged by recent molecular evidence. First, there is strong molecular evidence to indicate that lophophorates are very distant from hemichordates and echinoderms, in a different major branch of the animal phylogenetic tree. Therefore, similarities between the feeding structures must be due to convergent evolution. Second, there is strong evidence that hemichordates and echinoderms form a clade (Ambulacraria) within the deuterostomes, rather than hemichordates being closer to chordates. Third, there is weaker evidence that pterobranchs may be derived from acorn worms, and hence that the vermiform body plan may be ancestral within hemichordates. This suggestion warrants further testing. Here we review the evidence for these conclusions, highlight strengths and weaknesses in the data and analyses, and consider the implications for the origins of pterobranchs and graptolites.     

Birth and death of orphan genes in Rickettsia

The origin and evolution of the thousands of species-specific genes with unknown functions, the so-called orphan genes, has been a mystery. Here, we have studied the rates and patterns of orphan sequence evolution, using the Rickettsia as our reference system. Of the Rickettsia conorii orphans examined in this study, 80% were found to be short gene fragments or fusions of short segments from neighboring genes. We reconstructed the putative sequences of the full-length genes from which the short orphan fragments are thought to have originated. One of the genes thus reconstructed displays weak similarity to the ankyrin-repeat protein family, an identification that is strongly supported by comparative molecular modeling. Studies of the patterns of gene fragmentation underscore the importance of short repeated sequences as targets for recombination events that result in sequence loss and the formation of short, transient open reading frames. Our analysis demonstrates that gene sequences present in the common ancestor can be inferred even in cases when no full-length open reading frame is present in any of the contemporary species. Such reconstructions support the identification of lost protein functions and hint at important lifestyle changes.