Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae,Anura)

Using histological techniques and computer‐aided three‐dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the “ventromedial diverticulum”) is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Phylogeography of Iberian Discoglossus (Anura: Discoglossidae)

The genus Discoglossus (Anura, Discoglossidae) comprises five morphologically similar species. Molecular analyses have found high values of genetic differentiation among these cryptic taxa and confirmed the existence of two Iberian endemic lineages ( Discoglossus galganoi and D. jeanneae ) and a distinct lineage of uncertain origin representing a third species in NE Spain. In order to analyse the pattern of geographical distribution of haplotype diversity within Iberian Discoglossus and test the hypothesis of an Algerian origin for populations in NE Spain sequence data has been resolved from 35 populations of D. galganoi and D. jeanneae on the Iberian Peninsula and samples from NE Spain and north Africa. I analysed 959 bp corresponding to partial sequences of cytochrome b and nad 4 and found extremely low values of sequence divergence among populations of D. jeanneae . Three clades can be recognized within D. galganoi , however, and a statistically significant association with geography can be explained as a result of past fragmentation. Sequence data strongly support an Algerian origin for populations of Discoglossus from NE Spain. Finally, the taxonomic status of Discoglossus in Morocco is discussed and its elevation to species status as Discoglossus scovazzi Camerano, 1878 is suggested.     

Developmental origin of the frontoparietal bone in Bombina variegata (Anura: Discoglossidae)

Development of the frontoparietal bone in the European yellow-bellied toad, Bombina variegata, was followed on the basis of histological analysis of transverse serial sections through the larval skulls to recognize early stages of ossification represented by osteoid (uncalcified bone matrix) and on cleared and stained specimens to investigate more advanced stages. Ossification of the frontal begins as three tiny areas of osteoid (F(1), F(2), F(3)) adjoining the dorsal surface of the orbital cartilage, which are separated by areas without osteoid. F(3) is the largest (most advanced). Prior to calcification, F(3) extends to fuse with F(2) and then with F(1), but it does not expand over the prootic fissure posteriorly. As calcification begins the strip of bone is joined posteromedially by F(4). Only then does a single ossification center corresponding to the parietal arise on the anterodorsal surface of the otic capsule. This ossification sequence corresponds to those observed in the Actinopterygii and in caudate amphibians.     

Advertisement calls of the midwife toads Alytes (Amphibia, Anura, Discoglossidae) in continental Spain

The advertisement calls of six Spanish populations of midwife toads (genus Alytes ) are described presenting characteristic audiospectrograms and oscillograms of the calls. Numerical data concerning the spectral and temporal features of the calls in relation to caller size (SVL) and temperature are also compared. The regression temperature vs. call duration groups the different populations according to the extant recognized phylogeny of the genus. Based on this character, the montane population of A. obstetricans from Penalara (Madrid) appears to be more closely related to the Pyrenean populations of A. o. almogavarii than to the more western populations of A. o. boscai in Central Spain, which are closer geographically.

Zusammenfassung

Paarungsrufe der Geburtshelferköten Alytes (Amphibia, Anura, Discoglossidae) aus zentralspanischen Populationen
Die Paarungsrufe von sechs Geburtshelferkröten aus sechs spanischen Populationen werden hinsichtlich ihrer charakteristischen Tonfrequenz-Spektrogramme und Oszillogramme beschrieben.
Die numerischen Daten der spektralen und zeitlichen Eigenheiten der Rufmerkmale wurden mit der Größe der Männchen (SVL) und der Temperatur verglichen. Die Regression zwischen Temperatur und Dauer des Rufes ordnet die verschiedenen Populationen in der gleichen Weise ein, wie sie schon aus der allgemein anerkannten Phylogenetik der Gattung bekannt ist. Aufgrund dieser Untersuchungen, kann davon ausgegangen werden, daß die Gebirgspopulation A. obstetricans von Pealara (Madrid) enger mit der Pyrenäen-Population A. o. almogavarii verwandt ist als mit der westlichen Population A. o. boscai in Zentralspanien, obwohl sie zu jener geographisch näher liegt.     

Advertisement calls of the midwife toads Alytes (Amphibia, Anura, Discoglossidae) in continental Spain

The advertisement calls of six Spanish populations of midwife toads (genus Alytes) are described presenting characteristic audiospectrograms and oscillograms of the calls. Numerical data concerning the spectral and temporal features of the calls in relation to caller size (SVL) and temperature are also compared. The regression temperature vs. call duration groups the different populations according to the extant recognized phylogeny of the genus. Based on this character, the montane population of A. obstetricans from Penalara (Madrid) appears to be more closely related to the Pyrenean populations of A. o. almogavarü than to the more western populations of A. oboscai in Central Spain, which are closer geographically.     

Reproductive biology of the fire-bellied toads, Bombina bombina and B. variegata (Anura: Discoglossidae): egg size, clutch size and larval period length differences

Clutch size, egg size, larval size and duration of larval period were estimated for samples of Bombina bombina and B. variegata from several populations. Bombina variegata lays fewer eggs per batch (mean 17.4) than B. bombina (mean 32.5). The volume of B. variegata eggs is 2.4 times greater and the dry weight 2.3 times greater than that of B. bombina. As a consequence, the volume of B. variegata hatchlings and their dry weight are respectively 1.9 times and 2.5 times greater than that of B. bombina. Analysis of variance showed that variation of egg size in B. variegata is significantly greater than in B. bombina. When reared under laboratory conditions freshly metamorphosed individuals of both species showed no difference in body size or weight. Although growth rates are equal, tadpoles of B. variegata reached metamorphosis 14 days earlier than B. bombina tadpoles because of differences in size of hatchlings. Differences in reproductive parameters found for the two European Bombina species can be interpreted as adaptations to contrasting environments in which they breed.     

Regional Morphological and Cytochemical Differentiation in the Fertilized Egg of Discoglossus pictus (Anura)

A vertical column of cytoplasm poor in yolk (CPY) is located in the centre of the animal region of the unfertilised and fertilised egg of Discoglossus pictus. At the base of this column is found a central region of CPY designated as "clear cytoplasm". Cytochemical methods show that the CPY in this whole region is rich in glycogen and RNA.
By 60 min post opposition (p.o.) the pigmented cortical layer starts moving towards the future ventral side. It attains its definitive position by 90 min p.o. when the grey crescent, visible from 75 min p.o. onwards, achieves its maximal extension on the future dorsal side. The cytoplasmic column is now tilted towards the future ventral side. It progressively loses its direct contact with the cell membrane and disappears.
From 90 min p.o. onwards, the "clear cytoplasm" is found progressively closer to the dorsal grey crescent cortex. When the first cleavage furrow appears at 135 min p.o., the "clear cytoplasm" is situated very near the dorsal cortex and even extends somewhat below the equator. In places a relatively thin layer of cytoplasm containing medium-sized and a few large yolk granules intervenes between the grey crescent cortex and the "clear cytoplasm".
These displacements suggest that sperm entry evokes a dorsally directed cytoplasmic movement in the animal half of the egg which, among other things, may facilitate an interaction between the vegetative yolk and the grey crescent cortex, or may directly influence the dorso-ventral polarisation of the vegetative yolk.     

Plasma Membrane Domains and the Site of Sperm Entrance in Discoglossus pictus (Anura) Eggs

Freeze-fracture quantitative analysis reveals three different plasma membrane (PM) domains in the unfertilized egg of the anuran Discoglossus pictus . One of these is specific to the sperm entrance site (D1). where the plasma membrane shows a larger number of intramembranous particles (IMP) than the rest of the egg surface. Such an increment is due to a markedly higher number of the IMPs anchored to the P-face. The two other domains (D2 and D3) are characterized by a lower IMP density at the P-face with respect to D1. The IMP density decreased within 10 min after fertilization by about 33% in all domains observed, probably due to the insertion of new membrane through exocytosis. The possibility that the IMPs located in D1 may represent putative plasma membrane proteins playing a role in sperm-egg interaction and/or in egg activation is discussed.     

The larval hyobranchial apparatus of discoglossoid frogs: its structure and bearing on the systematics of the Anura (Amphibia: Anura)

The morphology of the larval hyobranchial apparatus of discoglossoid frog species representing the genera Ascaphus, Alytes, Bombina, and Discoglossus is described and the resulting characters were analysed cladistically. Seven species representing seven major lineages of frogs were included in the cladistic analysis of characters. Several changes in the terminology of the musculature are introduced, and a new interpretation of the subarcualis-muscle system is presented. The phylogenetic analysis suggest that the hyobranchial apparatus was substantially altered in the lineages leading to and within the Pipanura. This notably involved fusion, reduction and loss of skeletal structures and muscles, and splitting of certain muscles into muscle groups. The result confirm previous hypotheses based on the study of adults: discoglossoid species retain the most numlerous plesiomorphic characters among extant ianurans. The larval hyobranchial apparatus is in many features structrually similar to that of urodeles. Many of their character states were most likely present in the most recent common ancestor of all living forgs. The cladistic analysis of 31 characters of ithe larval hyobranchial apparatus supports major clades: Anura, Bombinanura, Pipanura, and Pelobatoidea + Neobatrachia. The cladiostic analysis and interpretation of larval characters is in part compatible with phylogenetic hypotheses based on characters of adults and rRNA sequences, but is in conflict with the Mesobatrachia and Archaeobatrachia concepts of other authors.     

Cytoskeletal and contractile proteins in coelomic oocytes,unfertilized and fertilized eggs of discoglossus pictus (Anura)

The distribution of actin, myosin, and tubulin has been investigated in coelomic oocytes, unfertilized and fertilized eggs of Discoglossus pictus utilizing: (1) immunofluorescence; (2) electron microscopy; (3) incubation with heavy meromyosin (HMM), and (4) SDS-polyacrylamide gel electrophoresis (PAGE). In coelomic oocytes, the germinative area (GA) has long, irregular microvilli containing microfilaments. In the rest of the oocyte, the microvilli are shallow. During the transit of the oocyte in the oviduct, a dimple forms by the invagination of the GA. A palisade of microfialment bundles is present in the finger-shaped microvilli of the dimple and extends for about 10 μm in the cytoplasm. In the rest of the egg, microvilli are absent and only random filaments appear in the cortex. Following HMM incubation, the dimple microfilaments are decorated with arrowheads pointing toward the bulk of the cytoplasm. SDS-PAGE of egg extracts shows bands co-migrating with actin (43K), pyruvate kinase (57K), and phosphorylase (94K). As result fertilization, the pattern of microfilament bundles in the dimple disappers in parallel with the dimple invergination itself. Generally, the entire oocyte cortex is positive to immunofluorescent staining with anti-actin, antimyosin, and antitubulin antibodies. However, the pattern of distribution and intensity of immunofluorescent staining changes for each antiserum, during different stages. It is concluded that a contractile system is present in Discoglossus eggs, and it is particularly developed in the dimple. The dimple is probably a major compartment for the storage of unpolymerized tubulin.     

Ultrastructural and lectin binding changes during the formation of the animal dimple in oocytes of Discoglossus pictus (Anura)

The numbers of spores, stalk cells, and basal disk cells in fruiting bodies of Dictyostelium discoideum were estimated by direct cell counting. It was found that the ratios of differentiated cells varied with the number of cells in the fruiting body. Hence, this invalidates, in D. discoideum at least, an assumption used in many theories of differentiation that proportions do not vary with size. Simple statistical analysis showed that a semilogarithmic equation could describe the relationship of spore to stalk cell number and spore to basal disk cell number, whereas a double-logarithmic equation described the basal disk and stalk cell number relationship. Studies under different environmental conditions and with different strains suggest that the basic equations describing the relationships are conserved. However, quantitative differences in the proportioning of the cell types have been observed. Previous papers concerning the proportions of D. discoideum are reviewed, and the implications of the results, in regard to theories of differentiation, are analyzed.     

Comparative data on acetylcholinesterase cytochemistry in various chromaffin cell types of Discoglossus pictus (Amphibia, Anura)

The cytochemical localization of acetylcholinesterase (AChE) activity was studied in the adrenal chromaffin cells of Discoglossus pictus. Reaction end-products were associated with all types of chromaffin cells, i.e. adrenaline (A), noradrenaline (N) and small granule chromaffin (SGC-A, SGC-N) cells, and nervous elements present in the gland. The SGC-A and SGC-N showed the same intensity of AChE reaction in A and N cells, respectively. On the whole, the A and SGC-A cells were more reactive than the N and SGC-N cells. The functional role of the SGC cells is discussed on the basis of the cytochemical results.     

Morphometric variation and divergence in the West Mediterranean Discoglossus (Amphibia: Discoglossidae)

Morphometrics of the four species of the genus Discoglossus occurring in the West Mediterranean area D. galganoi, D. montalentii, D. pictus and D. sardus ) was investigated. Univariate and multivariate statistics showed that the species are morphologically diverse and that populations of the same species form distinct and recognizable groups. Our results conflict in part with those derived from allozyme analysis of the same taxa. The major conflict involves the comparisons between D. montalentii and D. sardus , and between D. g. galganoi and D. g. jeanneae. . Whereas D. montalentii is genetically very distinct from D. sardus , their morphometric diversity is relatively low. On the other hand, morphometric divergence between D. g. galganoi and D. g. jeanneae (the latter described as a species by Busack, i.e. D. jeanneae , is high, but they are very similar genetically. Both adaptation to the different climatic regimes characterizing the West Mediterranean area and the phylogenetic history are suggested to account for the observed changes in the morphology.