Serous gland dimorphism in the skin of Melanophryniscus stelzneri (Anura: Bufonidae)

Two serous gland types (I and II) in the skin of the Argentine toad Melanophryniscus stelzneri were discovered using light and electron microscopy. Glands of the two types differ in several traits: features of the products (both mature and immature), organelles involved in biosynthesis, and paths of serous maturation. No consistent differences, however, were detected between the myoepithelial sheaths encircling the secretory units. Type I glands manufacture vesicles containing a single dense body with a repeating substructure and conform to the fundamental secretory line of bufonid skin, a secretory line involved in biosynthesis of steroids. Type II glands synthesize granules of varying densities and seem to belong to a line of glands that secrete proteinaceous products. The occurrence of the two serous gland types in Melanophryniscus stelzneri is discussed in a comparison with current literature on the morphofunctional characteristics of anuran poison glands, which perform both regulative and defensive roles. It is suggested that di- or polymorphism in serous glands is an adaptive trait that allows differential release of active molecules on the body surface. J. Morphol. 237:19–32, 1998. © 1998 Wiley-Liss, Inc.     

Reproductive biology of Melanophryniscus montevidensis (Anura: Bufonidae) from Uruguay: reproductive effort,fecundity, sex ratio and sexual size dimorphism

We analyzed the reproductive traits of a population of Melanophryniscus montevidensis. The reproductive events were associated to rainfalls. Males called from dawn to late night, were more abundant than females, and adopted alternative tactics to find partners (active search). Females were larger than males, and had mature oocytes and lipid reserves after spawning; they showed no association between size and fecundity. As an explosive breeder, females are prepared all year long for reproduction. Several individuals revealed high site-fidelity for breeding. Knowledge about breeding pattern could help to propose suitable and efficient in situ conservation measures for this endangered species.     

Three New Species of Phytotelm-Breeding Melanophryniscus from the Atlantic Rainforest of Southern Brazil (Anura: Bufonidae)

Three new species of Melanophryniscus are described from the Serra do Mar mountain range of the state of Santa Catarina, southern Brazil. All species are found at intermediate to high altitudes and share phytotelm-breeding as their reproductive strategy. The new species are distinguished from other phytotelm-breeding Melanophryniscus based on different combinations of the following traits: snout-vent length, presence of white and/or yellow spots on forearms, mouth, belly and cloaca, pattern and arrangement of warts, and presence and number of corneous spines. The discovery of these species in a rather restricted geographical area suggests that the diversity of phytotelm-breeding species of Melanophryniscus might be severely underestimated. The conservation status of these species is of particular concern, given that one of them is at risk of extinction not only due to its restricted habitat, but also because of anthropogenic disturbances.     

Polyploidy and deviations in the gonadal morphology of tetraploid toads, Bufo danatensis (Amphibia: Anura: Bufonidae)]

Morphological features of gonads of diploid Bufo viridis and tetraploid B. danatensis from 15 localities of USSR (453 spec.) were investigated. Females with the rudimentary ovaries were found among polyploid toads from south Turkmenia. The toads from Tajikistan having the external features of males usually demonstrated a specific kind of lateral gynandromorphism: a single side testes and rudimentary ovaries (with the normal ovarioles). The observed facts are treated either as a result of hybridization of recent species or as a result of originality of genomes organization.     

New species of Aplectana (Ascaridida: Cosmocercidae) in the toads, Rhinella granulosa and Rhinella schneideri (Anura: Bufonidae) from northern Argentina

Aplectana adaechevarriae n. sp., from the large intestine of Rhinella granulosa and Rhinella schneideri, is described and illustrated. Of the 43 nominal species of Aplectana, A. adaechevarriae n. sp. represents the 23rd Neotropical species and the third species reported from Argentina. Of the Neotropical species, only 4, Aplectana elenae, Aplectana hylambatis, Aplectana raillieti, and A. adaechevarriae n. sp., have 8 pairs of preanal papillae. Aplectana adaechevarriae lacks a median preanal papilla; the other 3 species possess a median preanal papilla.     

Around the world in 10 million years: biogeography of the nearly cosmopolitan true toads (Anura: Bufonidae)

Aim The species‐rich family of true toads (Anura: Bufonidae) has been the focus of several earlier studies investigating the biogeography of geographically widespread taxa. Herein, we employ newly developed Bayesian divergence estimate methods to investigate the biogeographical history of this group. Resulting age estimates are used to test several key temporal hypotheses including that the origin of the bufonid clade pre‐dates Gondwanan vicariance (~105 million years ago, Ma). Area cladograms are also invoked to investigate the geographical origin of the family. Location Worldwide, except the Australia–New Guinea plate, Madagascar and the Antarctic. Methods A phylogenetic hypothesis of the relationships among true toads was derived from analysis of 2521 bp of DNA data including fragments from three mitochondrial (12S, tRNA^val, 16S) and two nuclear (RAG‐1, CXCR‐4) genes. Analysis of multiple, unlinked loci with a Bayesian method for estimating divergence times allowed us to address the timing and biogeographical history of Bufonidae. Resulting divergence estimates permitted the investigation of alternative vicariance/dispersal scenarios that have been proposed for true toads. Results Our area cladogram resulting from phylogenetic analysis of DNA data supports a South American origin for Bufonidae. Divergence estimates indicate that the family originated earlier than had been suggested previously (78–99 Ma). The age of the enigmatic Caribbean clade was dated to the late Palaeocene–early Eocene. A return of bufonids to the New World in the Eocene was followed by rapid diversification and secondary expansion into South America by the early Oligocene (Rupelian). Main conclusions The South American origin of Bufonidae in the Upper Cretaceous was followed by relatively rapid expansion and radiation around the globe, ending with a return to the Americas via a Eurasian/North American land bridge in the Eocene. Though the exact route of this dispersal (Beringia or North Atlantic) remains unclear, an argument is made for the less frequently invoked North Atlantic connection. The origin of the enigmatic Caribbean lineage was found to be consistent with colonization following the bolide impact at the K/T boundary. These findings provide the first, firm foundation for understanding true toad divergence times and their truly remarkable and global radiation.     

Comparative morphology of pond,stream and phytotelm‐dwelling tadpoles of the South American Redbelly Toads (Anura: Bufonidae: Melanophryniscus)

We present a comprehensive review of larval morphology in the Neotropical toad genus Melanophryniscus. The taxa studied included 23 species with representatives of recognized phenetic groups and different larval ecomorphological guilds: pond, stream, and phytotelm‐dwelling tadpoles. Their external morphology variation is congruent with current phenetic arrangement based on adult features, but also reflects the habitat where larvae develop. Lotic tadpoles (i.e. M. tumifrons group and M. krauczuki) in general exhibit a more depressed body, a longer tail with lower fins, and larger oral discs than lentic forms (i.e. M. stelzneri group, M. moreirae, M. sanmartini, and M. langonei). Despite their peculiar, confined microhabitat, phytotelm larvae do not diverge markedly from non‐arboreal species. The distinctive features of all species are the presence of a pineal end organ and the placement of the intestinal reversal point at the left of the abdomen in typical larval stages. The buccal cavity and musculoskeletal anatomy are quite conserved between species, yet some characteristics differ from those of other bufonids. The presence of one pair of subhyoid muscles is apparently an exclusive trait of Melanophryniscus among Bufonidae. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 417–441.     

Batrachochytrium dendrobatidis Infection in the Recently Rediscovered Atelopus mucubajiensis (Anura, Bufonidae), a Critically Endangered Frog from the Venezuelan Andes

The harlequin frog, Atelopus mucubajiensis, is a critically endangered species that was believed to be extinct until 2004, when a single adult female was found in the Venezuelan Andes. Its disappearance after 1994 has been attributed to an increase in the prevalence of the fungal pathogen, Batrachochytrium dendrobatidis, in populations of these frogs. Using histology and Real-Time PCR assays, we tested this adult female positive for B. dendrobatidis. We report for the first time, to our knowledge, infection in a live specimen of A. mucubajiensis. The presence of this pathogen in remaining individuals poses a problem for the long-term persistence of this critically endangered species, as new epidemic episodes of chytridiomycosis could jeopardize the survival of remnant populations.     

A phylogenetic analysis of species in the Bufo crucifer group (Anura: Bufonidae), based on indolealkylamines and proteins from skin secretions

This research tested the utility of two classes of skin secretion compounds to the phylogeny of the Bufo crucifer group. Skin secretions from specimens of nine populations of B. crucifer group were obtained and submitted to qualitative analysis. We observed a clear difference in the composition of the skin secretion molecules obtained from the species of Bufo studied. Fifty-nine molecules, 16 indolealkylamines and 43 proteins, were used as characters, and 39 of these were parsimonious informative. The tree topology of the skin secretion combined data showed areas of congruence and conflict when compared to an mtDNA phylogeny of the B. crucifer group. We used the Templeton test to evaluate the heterogeneity between the skin secretion and mtDNA data. Although not recommended, we performed a combined analysis with the two partitions. The skin secretion characters from the species of Bufo studied have phylogenetic signal. These data are indicative, at least as a preliminary study, of the phylogenetic relationships among the B. crucifer group taxa.     

A new Ansonia from the Isthmus of Kra, Thailand (Amphibia, Anura, Bufonidae)

A new species of torrent-dwelling bufonid frog of the genus Ansonia is described from the Isthmus of Kra, Thailand. Ansonia kraensis is morphologically similar to Malaysian A. malayana, but differs from it in ventral coloration and larval morphology. Occurrence of A. kraensis in this region suggests a heterogeneous nature of the anuran fauna between northern and southern regions of the Malay Peninsula.     

Morphology of the kidney in larvae of Bufo viridis (Amphibia, Anura, Bufonidae)

This study deals primarily with the morphology and ultrastructure of the pronephros in the green toad Bufo viridis during prometamorphosis when the pronephros and the developing mesonephros function simultaneously. Furthermore, the mesonephros was studied during pro- and postmetamorphosis with emphasis on the distal segments of the nephron. The paired kidneys consist of two cranial pronephroi immediately behind the gill region and two more caudal elongated mesonephroi. Each pronephros consists of a single convoluted tubule which opens into the coelom via three nephrostomes. This tubule is divided into three ciliated tubules, three proximal tubule branches, a common proximal tubule and a distal tubule, which in turn continues into the nephric duct. No intermediate segment is present. The length of the pronephric tubule is 12 mm, including the three branches of the ciliated tubules and proximal tubules. Primary urine is formed upon filtration from an external glomerulus, which is a convoluted capillary lined by podocytes, a specialization of the coelomic epithelium. From the coelom the filtrate is swept into the ciliated tubules. In the collecting duct system of the developing mesonephric nephron epithelial cells with conspicuous, apical osmiophilic granules appear in larvae of 9-10 mm. Heterocellularity of mixed intercalated (mitochondria rich) cells and principal cells is observed in the collecting duct system and nephric duct from a larval body length of 14 mm. As the proliferation of mitochondria-rich cells proceeds, the osmiophilic granules disappear and are completely absent from the adult amphibian mesonephros.     

Morphology of the Nephron in the Mesonephros of Bufo bufo (Amphibia,Anura, Bufonidae)

The present study deals with the morphology and ultrastruclure of the nephron in the mesonephros of the toad, Bufo bufo (Linnaeus, 1758). Based on serial sections in paraffin, Araldite and Epon, the position of the different segments of the nephron within the kidney tissue was determined, and a nephron subsequently reconstructed. The nephron consists of the following parts: Malpighian corpuscle, neck segment, proximal tubule, intermediate segment, early distal tubule, late distal tubule and collecting tubule. The late distal tubule was subdivided into three morphologically different sections. The total number of nephrons in the toad mesonephros was estimated at 6000 units. The length of the segments in the reconstructed nephron was calculated. The cytology of the epithelial cells constituting the segments was described using transmission and scanning electron microscopy. Heterocellularity was found in the late distal tubule section I and III and in the collecting tubule. The proportional distribution and number of intercalated (mitochondria-rich) cells in the late distal tubule and collecting tubule was calculated. Only one morphological type of intercalated cell could be distinguished. Late distal tubules were removed from fresh Bufo kidneys for preliminary studies of the intercalated cells with Nomarski optics.     

Ticks, Amblyomma rotundatum (Acari: ixodidae), on toads, Chaunus schneideri and Chaunus granulosus (Anura: bufonidae), in northern Argentina

This communication provides notes on 2 species of toads, Chaunus schneideri and Chaunus granulosus, infested with ixodid ticks, Amblyomma rotundatum, from the provinces of Corrientes and Formosa in northern Argentina. Chaunus schneideri is a new amphibian host record for A. rotundatum, a species previously reported to parasitize other anurans and also reptiles. We examined 74 ticks on 5 toads. All ticks were A. rotundatum; all adults were females, and all developmental stages were randomly attached to host body parts. Ticks remained attached to one of the toads for from 7 to 17 days after the host was captured. One toad, encumbered with 33 ticks, was moribund when found and died shortly thereafter.     

Biosynthesis of bufadienolides in toads. V. The origin of the cholesterol used by toad parotid glands for biosynthesis of bufadienolides

Sodium (1-14C)acetate and (5-3H) mevalonate were incubated with Bufo arenarum toad parotid gland and liver tissues. Both labelled compounds were incorporated into cholesterol produced by liver while the incubations with parotid gland produced no labelled cholesterol. Low and high density lipoproteins isolated from toad plasma were iodinated and used for binding studies. Membrane preparations of parotid gland showed high affinity binding sites for 125I-LDL and 125I-HDL. In addition, while colchicine inhibits the in vitro uptake of (3H)cholesteryl linoleate-LDL into parotid gland tissue an opposite effect was seen with (3H)cholesteryl linoleate-HDL. The above mentioned results would support the hypothesis that the cholesterol used by the parotid gland for the biosynthesis of bufadienolides would be produced in the liver, transported by the circulating lipoproteins and incorporated by the glands by a receptor-mediated mechanism.     

Histological examination of the effects of corticosterone in larvae of the western toad,Bufo boreas (Anura: Bufonidae), and the oriental fire-bellied toad,Bombina orientalis (Anura: Discoglossidae)

The effects of corticosterone (CORT)-treatment on various tissues were examined in two species of anuran larvae, the discoglossid Bombina orientalis, and the bufonid Bufo boreas. Corticosterone was administered directly into aquarium water for 15 days. After treatment, histological analyses were conducted on skin, gut, spleen, thymus, and neural and muscle tissue. Corticosterone treatment prevented sloughing of the skin, which resulted in a build-up of stratum corneum, and inhibited the development of gland nests and the subsequent formation of dermal granular and mucous glands in both species. Corticosterone treatment also decreased epithelial folding in the gut and caused vesiculation of the gut epithelial cells. The thymus of CORT-treated animals was significantly reduced in size (P < .05) and cell density (P < .05), and the spleen of CORT-treated animals was completely involuted. The brain and pituitary of CORT-treated animals had a decreased cell density (P < .05) and many pyknotic cells. An examination of muscle revealed that muscle fibers of CORT-treated animals had a decreased cross-sectional area (P < .05). The dose of CORT used (1.1 μM) was within the range used in other studies in the literature and resulted in tissue levels within the range experienced by larvae at metamorphic climax. Thus, this study is appropriate to address the histological effects of CORT in experimental manipulations and the role of increasing CORT at metamorphic climax. The data suggest that increasing endogenous CORT at metamorphosis may be involved in degeneration of larval tissue, prior to regeneration, which is stimulated by thyroid hormones. © 1995 Wiley-Liss, Inc.     

Development of the nasal chemosensory organs in two terrestrial anurans: the directly developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), and the metamorphosing toad, Bufo americanus (Anura: Bufonidae)

Nearly all vertebrates possess an olfactory organ but the vomeronasal organ is a synapomorphy for tetrapods. Nevertheless, it has been lost in several groups of tetrapods, including aquatic and marine animals. The present study examines the development of the olfactory and vomeronasal organs in two terrestrial anurans that exhibit different developmental modes. This study compares the development of the olfactory and vomeronasal organs in metamorphic anurans that exhibit an aquatic larva (Bufo americanus) and directly developing anurans that have eliminated the tadpole (Eleutherodactylus coqui). The olfactory epithelium in larval B. americanus is divided into dorsal and ventral branches in the rostral and mid-nasal regions. The larval olfactory pattern in E. coqui has been eliminated. Ontogeny of the olfactory system in E. coqui embryos starts to vary substantially from the larval pattern around the time of operculum development, the temporal period when the larval stage is hypothesized to have been eliminated. The nasal anatomy of the two frogs does not appear morphologically similar until the late stages of embryogenesis in E. coqui and the terminal portion of metamorphosis in B. americanus. Both species and their respective developing offspring, aquatic tadpoles and terrestrial egg/embryos, possess a vomeronasal organ. The vomeronasal organ develops at mid-embryogenesis in E. coqui and during the middle of the larval period in B. americanus, which is relatively late for neobatrachians. Development of the vomeronasal organ in both frogs is linked to the developmental pattern of the olfactory system. This study supports the hypothesis that the most recent common ancestor of tetrapods possessed a vomeronasal organ and was aquatic, and that the vomeronasal organ was retained in the Amphibia, but lost in some other groups of tetrapods, including aquatic and marine animals.     

Testicular myxosporidiasis in the flat-backed toad, Bufo maculatus (Amphibia: Bufonidae), from Cameroon, Africa.

Macroscopic cysts measuring less than or equal to 860 x 500 microns were found in the testes of a flat-backed toad, Bufo maculatus, collected in Cameroon, West Africa. On histologic examination, the cysts contained numerous spores of a Myxobolus sp. (Myxozoa: Myxobolidae). Spores in fixed tissues measured 9.2 microns long, 8.9 microns wide, and 4.0 microns thick; the range of values for length, width, and thickness were 8.8 to 9.6 microns, 8.6 to 9.4 microns, 3.6 to 4.4 microns, respectively (n = 20). The shape index (length/width) was 1.03, and ranged from 1.00 to 1.09. Pathology was limited to a slight constriction of adjacent seminiferous tubules by the cysts. No host inflammatory response was noted. This myxozoan is distinct from all other members of the genus infecting anurans and is assigned the name Myxobolus bufonis sp. n.     

Population traits of the burrowing toad Rhinella fernandezae (Gallardo, 1957) (Anura, Bufonidae).

Size distribution, sex ratio and use of burrows of the burrowing toad Rhinella fernandezae were studied in Buenos Aires province, Argentina. Two sites separated by approximately 300 m were studied: one was a road next to a swamp, and the other a garden of a country house located further from the swamp. We identified toad burrows, and individuals were sexed, measured and given an individual mark. Burrows were examined in subsequent months after the first sampling to assess the presence of toads. We found significant differences in the size distribution between areas, being the proportion of juveniles greater at the site next to the swamp where the reproduction of the species was observed. This result may suggest that the site located near to the swamp functions as a source habitat of individuals that migrate to the other site, where recruitment would be very scarce. Sex proportion of adults did not differ from 1:1 in neither the total population nor in each site, suggesting that there was not differential mortality by sex. Some toads changed burrows throughout the study period, but there were not differences in the frequency of change between adults and juveniles.     

Genome size rather than content might affect call properties in toads of three ploidy levels (Anura: Bufonidae: Bufo viridis subgroup)

In vertebrates, genome size has been shown to correlate with nuclear and cell sizes, and influences phenotypic features, such as brain complexity. In three different anuran families, advertisement calls of polyploids exhibit longer notes and intervals than diploids, and difference in cellular dimensions have been hypothesized to cause these modifications. We investigated this phenomenon in green toads (Bufo viridis subgroup) of three ploidy levels, in a different call type (release calls) that may evolve independently from advertisement calls, examining 1205 calls, from ten species, subspecies, and hybrid forms. Significant differences between pulse rates of six diploid and four polyploid (3n, 4n) green toad forms across a range of temperatures from 7 to 27 °C were found. Laboratory data supported differences in pulse rates of triploids vs. tetraploids, but failed to reach significance when including field recordings. This study supports the idea that genome size, irrespective of call type, phylogenetic context, and geographical background, might affect call properties in anurans and suggests a common principle governing this relationship. The nuclear‐cell size ratio, affected by genome size, seems the most plausible explanation. However, we cannot rule out hypotheses under which call‐influencing genes from an unexamined diploid ancestral species might also affect call properties in the hybrid‐origin polyploids. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 584–590.