The historv of introductions of Bufo mariuk (Amphibia: Anura); a natural experiment in evolution

The Central American toad, Bufo marinus , has been extensively introduced throughout the Caribbean and Pacific regions and is now one of the most widespread of terrestrial vertebrates. Details, such as the sources and dates of introductions, the number of individuals introduced and the fates of the introduced populations have been documented and are described. The availability of this historical information makes the introductions of great potential value as a series of evolutionary experiments. They can be used to study the way in which allopatric populations diverge genetically and the effects of population bottlenecks of known size on the genetic characteristics of populations, and to examine theories of rapid speciation caused by genetic revolutions associated with founder events.     

Plant dispersal, neighbourhood size and isolation by distance

A theoretical relationship between isolation by distance or spatial genetic structure (SGS) and seed and pollen dispersal is tested using extensive spatial-temporal simulations. Although for animals Wright's neighbourhood size N(e) = 4pisigma(2)(t) has been ascertained also, where sigma(2)(t) is the axial variance of distances between parents and offspring, and it was recently confirmed that N(e) = 4pi(sigma(2)(f) + sigma(2)(m))/2 when dispersal of females and males differ, the situation for plants had not been established. This article shows that for a very wide range of conditions, neighbourhood size defined by Crawford's formula N(e) = 4pi(sigma(2)(s) + sigma(2)(p)/2) fully determines SGS, even when dispersal variances of seed (sigma(2)(s)) and pollen sigma(2)(p)) differ strongly. Further, self-fertilization with rate s acts as zero-distance pollen dispersal, and N(e) = 4pi[sigma(2)(s) + sigma(2)(p)(1 - s)/2] fully determines SGS, for most cases where there are both likely parameter values and substantial SGS. Moreover, for most cases, there is a loglinear relationship, I(1) = 0.587 - 0.117 ln(N(e)), between SGS, as measured by I(1), Moran's coefficient for adjacent individuals, and N(e). However, there are several biologically significant exceptions, namely for very low or large N(e), SGS exceeds the loglinear values. There are also important exceptions to Crawford's formula. First, plants with low seed dispersal, high outcross pollen dispersal and high selfing rate show larger SGS than predicted. Second, in plants with very low (near zero) seed dispersal, selfing decreases SGS, opposite expectations. Finally, in some cases seed dispersal is more critical than pollen dispersal, in a manner inconsistent with Crawford's formula.     

Evaluation of the toxicity of eggs,hatchlings and tadpoles of the introduced toad Bufo marinus (Anura: Bufonidae) to native Australian aquatic predators

Abstract The early life history stages of anurans in the Family Bufonidae often possess chemicals that are noxious or toxic to predators. Predators with no evolutionary history of exposure to bufomds may be particularly susceptible to these toxins. We conducted a series of laboratory experiments to investigate the toxic effects of eggs, hatchlings and tadpoles of the introduced toad, Bufo marinus (Linnaeus), on native Australian aquatic predators. There was considerable interspecific and intraspecific variation in these effects. Bufo marinus were highly toxic to some predator species, but were readily consumed by other species without apparent ill effect. Interspecific variation in toxic effects was not related to predator feeding mode or the number of B. marinus ingested by predators, and there was no clear pattern of distribution of vulnerability among species within higher taxa. Intraspecific variation in responses to toxins may result from individual variation in the resistance of predators to B. marinus toxins, or from individual variation in toxicity among B. marinus. Some native species adversely affected by B. marinus appeared unable to detect and avoid B. marinus toxins. This may result from a general inability to assess the toxicity of food items or from a lack of evolutionary exposure to B. marinus toxins.     

Geographical patterns of genetic variability in introduced Australian populations of the marine toad,Bufo marinus: Sorbitol dehydrogenase,Sdh

The marine toad, Bufo marinus, was introduced to Australia from Hawaii in 1935. From 1935 to 1974, the toad population expanded exponentially to occupy 584,000 km², and now has a continuous distribution from Cape York to Tweed River on the eastern coast of the continent. Genetic analysis of the population indicates a difference in allele frequency at the sorbitol dehydrogenase locus. There are two alleles segregating at the locus (NAD-Sdh^a and NAD-Sdh^b). The NAD-Sdh^aa homozygote is common in the two southern populations, but uncommon in northern populations. The north-south difference has been established in less than 25 generations.     

The calcified amorphous layer of the skin of Bufo marinus (Amphibia: Anura)

The integument of Bufo marinus is surveyed. It is similar to that of other anurans and contains the three chromatophores common to anurans. The skin of these toads contains more than 28% minerals deposited as small crystals in a mucopolysaccharide-positive amorphous layer between the stratum compactum and the stratum spongiosum. These crystals reveal a high content of calcium and phosphorus aggregated in and near membrane-bound vesicles which have an appearance very similar to matrix vesicles. Electron microscopy gives the appearance that these vesicles are associated with flbroblasts located between alternating bundles of collagen. Histochemical studies indicate that the amorphous layer possesses many of the characteristics commonly associated with mineralized cartilage or bone.     

Vascularization of the pars distalis of the hypophysis in the toad,Bufo bufo (L.) (Amphibia,Anura)

Summary The vascularization of the pars distalis of the hypophysis of the toad, Bufo bufo (L.), was studied by the traditional method of injecting a mixture of India-ink and gelatine into the circulatory system of the head via the arteria carotis communis. Further, methyl-methacrylate corrosion casts of the brains were made; the hypothalamo-adenohypophysial region of these corrosion casts was studied with the scanning electron microscope. The results showed that the portal vessels which arise from the median eminence do not supply distinct areas in the pars distalis as is supposed by the point-to-point-hypothesis. The portal vessels enter the ventro-median region of the pars distalis and branch off into a three-dimensional network of the secondary capillary plexus of the pars distalis. The plexus is made up mostly by four- to six-sided meshes. This angioarchitecture guarantees an optimal supply of the glandular cells of the pars distalis with nutritional factors and releasing hormones, on the one hand, and facilitates the removal of the hormones which are released by these cells, on the other hand. The venous drainage of the pars distalis is exerted mainly by two large veins, which bilaterally leave the dorso-lateral region (venous pole) of the pars distalis and by a few small veins, which drain into the wide, sinus-like vessel, which curves around the dorso-caudal region of the pars distalis and joins bilaterally the vena hypophysea transversa.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (Projekt Nr. 2183/N 39) and by the Stiftungs- und Förderungsgesellschaft der Paris-Lodron-Universität SalzburgThe authors wish to thank Mag. Ursula Albrecht for excellent technical assistance and Mr. Gerhard Sulzer for photographic work     

Structural and mechanical aspects of the skin of Bufo marinus (Anura, Amphibia)

Specific biomechanical characters and some structures possibly related to them were investigated in the skin of the toad Bufo marinus using tensile testing techniques (at constant strain till rupture) as well as morphological methods (histological, immunohistochemical and electronmicroscopical). Mechanical parameters of the native skin varied considerably according to sex, individual variability and/or site of specimen collection. In skin strips of males and females excised from different parts of the body thickness ranged from 0.45 to 0.87 mm, strain (epsilonf) from 96.52 to 211.03, tensile strength (sigmam) from 5.72 to 9.38 MPa, and stiffness (E-modulus) from 5.76 to 6.73. The dermis of B. marinus is provided with a collagenous stratum compactum of considerable thickness, a stratum spongiosum with loosely arranged fibres and a marked calcified layer (substantia amorpha). Collagen appears to be the main determinant of skin mechanics. However, the slope of the J-shaped static stress-strain curves indicates elastin to be responsible for the high values of strain. Contrary to van Gieson and orcein staining, immunostaining with a monoclonal antibody against elastin revealed very few elastic fibers between collagen bundles and in the vertical fiber tracts (perforating bundles), but a considerable amount in the tela subcutanea. This was partly confirmed at the ultrastructural level by tannic acid staining.     

Phylogeography of the natterjack toad Bufo calamita in Britain: genetic differentiation of native and translocated populations

The natterjack toad Bufo calamita is rare in Britain, which is at the northwestern edge of its biogeographical range. We investigated the level of genetic differentiation amongst almost all (34 out of 38) of the surviving British populations of this species, and among six new populations established by translocations during the 1980s. For eight microsatellite loci, allele sizes and frequencies were analysed using samples from each of these populations. The populations clustered into three robustly differentiated groups, each of which corresponded with a geographical region (east/southeast England, Merseyside and Cumbria). The Cumbrian populations showed a further weak geographical substructuring into northern and southern clades. The populations in south Cumbria were genetically more diverse than those in any of the other regions, as judged by the mean numbers of alleles per locus and the mean heterozygosity estimates. The translocated populations clustered close to their founders and, with one exception, did not differ significantly with respect to mean allele numbers, heterozygosity or polymorphism level. However, significant genetic differentiation (as measured by unbiased R _ST) was found between all but one of the founder-translocation pairs. The implications of this phylogeographic study for the future conservation of B. calamita in Britain are discussed.     

The effects of genetic drift during range expansion on geographical patterns of variation: a computer simulation of the colonization of Australia by Bufo marinus

A computer simulation is performed of allele frequency changes resulting from genetic drift at eleven loci during the probable course of colonization of Australia by populations of the Giant Toad, Bufo marinus. The history of twelve populations for which allele frequency data are available is modelled. Account is taken of the likely pattern of relationship among the populations, the effective size of the populations (as indicated by the observed variance of allele frequencies) and the probable isolation of the populations from each other following their separation. In all simulations, allele frequencies at some loci show a significant association with latitude while others do not. In six of ten simulations, there is a significant association between degree of variation at a locus and the presence of a latitudinal cline of allele frequencies. There are also indications of this kind of association in simulations of a uni-directional range expansion. These results demonstrate that such associations, which were also observed in the data from the actual populations, can result from genetic drift during a range expansion, and therefore cannot be taken as evidence of the action of natural selection.     

Effects of the ecological conditions on morphological variations of the green toad, Bufo viridis, in Turkey

Ten populations of the green toad, Bufo viridis, from three areas having different climatic regimes were compared. Multivariate analyses of morphological characteristics revealed three distinct populations of green toads: the first group inhabiting the lowland and humid localities was characterized by a small body size, the second group occurred at higher altitudes and in desert areas had a significantly larger body size, while the third group from the Mediterranean localities was characterized by a large tympanum, a longer tarsus and short first toes. Interestingly, the specimens from Giresun Island were clustered with the second group with a similar pattern of body size. Differences in the morphological characteristics among populations were positively associated with ecological conditions, and not with geographic distance. Some characteristics previously used for taxonomic purposes in B. viridis were shown to be associated with local climatic conditions.     

Fine-scale population structure in a desert amphibian: landscape genetics of the black toad (Bufo exsul)

Environmental variables can strongly influence a variety of intra- and inter-population processes, including demography, population structure and gene flow. When environmental conditions are particularly harsh for a certain species, investigating these effects is important to understanding how populations persist under difficult conditions. Furthermore, species inhabiting challenging environments present excellent opportunities to examine the effects of complex landscapes on population processes because these effects will often be more pronounced. In this study, I use 16 microsatellite loci to examine population structure, gene flow and demographic history in the black toad, Bufo exsul , which has one of the most restricted natural ranges of any amphibian. Bufo exsul inhabits four springs in the Deep Springs Valley high desert basin and has never been observed more than several meters from any source of water. My results reveal limited gene flow and moderately high levels of population structure ( F _ST = 0.051–0.063) between all but the two closest springs. I found that the geographic distance across the arid scrub habitat between springs is significantly correlated with genetic structure when distance accounts for topography and barriers to dispersal. I also found very low effective population sizes ( N _e = 7–30) and substantial evidence for historical population bottlenecks in all four populations. Together, these results suggest that the desert landscape and B.   exsul 's high habitat specificity contribute significantly to population structure and demography in this species and emphasize the importance of considering behavioural and landscape data in conservation genetic studies of natural systems.     

Release vocalizations in neotropical toads (Bufo): ecological constraints and phylogenetic implications

The release vocalizations of four nominal neotropical toad taxa ( Bufo achalensis , Bufo limensis , Bufo spinulosus , Bufo arenarum ) which compose three sympatric species pairs, were quantitatively analysed and homologous call types statistically compared. The first three taxa are closely related members of the mainly Andean Bufo spinulosus species group in which advertisement calls are absent. The specific vocal repertoire consisted of a uniform, unpulsed release call and one (in B. arenarum, two) pulsed release trill(s) which were given either singly or in series of up to three single calls. The uniform call was similar in structure and dominant (= basic) frequency in all taxa and probably represents an unspecific acoustic signal which is used to avoid heterospecific amplexus between sympatric toads. The release trills significantly differed in most call features among the taxa and, using discriminant analyses, even single calls were correctly assigned to sympatric pairs of species. In contrast, calls of allopatric pairs of species were confounded at rates of up to 36% indicating that selection towards species-specific signals increases when different species live in sympatry. The release trills of two allopatric populations (Perú, Argentina) assigned to B. spinulosus were similar in structure, but nevertheless features of pulse train permitted an unequivocal distinction, suggesting a long-lasting independent evolution. The taxonomic significance of this finding remains to be evaluated in further investigations. The complex vocal repertoire also offered the opportunity to assess phylogenetic relationships among the taxa. The extra-Andean B. achalensis seems to be closer related to the Andean B. spinulosus than this species is to the widely sympatric B. limensis – a species with several primitive character states indicating an early separation from the ancestral stock.     

Checklist of helminth parasites of the cane toad Bufo marinus (Anura: Bufonidae) from Mexico

Thirty-four adult cane toads Bufo marinus L. (12 males and 22 females) collected from 2 localities in Mexico (Cerro de Oro and Temascal Dams, Oaxaca) in September 2003 were examined for helminth parasites. In total, 14,749 helminths belonging to 14 taxa were collected. Included were 2 adult digeneans (Choledocystus hepaticus, Mesocoelium monas); 1 larval cestode (an unidentified pseudophyllidean); and 11 nematodes, including 3 species of larvae (Contracaecum sp., Physaloptera sp., Physocephalus sexalatus) and 8 species of adults (Aplectana itzocanensis, Cosmocerca sp., Cruzia morleyi, Ochoterenella digiticauda, Oswaldocruzia sp., Raillietnema sp., Rhabdias americanus, and Rhabdiasfuelleborni). Higher species richness was recorded in B. marinus from Cerro de Oro (12 taxa versus 9 in those from Temascal); hosts from both localities shared 7 taxa. There were 25 new locality records, and 2 taxa were registered in Mexico for the first time. To date, 112 helminth species have been recorded parasitizing B. marinus along its native and introduced range of distribution, with 40.5% of them reported from Mexico.     

The effects of ecological and genetic neighbourhood size on the evolution of two competing species

Summary Individual-based simulations were conducted to examine the effect of a small ecological neighbourhood (an area in which ecological processes such as density-dependent factors operate) and the genetic neighbourhood size (the size of an area from which the parents may be assumed to be drawn at random) on the coevolution of two competing species. For the simulations, individuals of two consumer species compete for two types of food organisms. Different genotypes (one locus and two alleles) have different efficiencies of food acquisition for different food types. Individual consumer organisms search for food within their home ranges and reproduce depending on the amount of food eaten. The dispersal distance of the offspring follows a normal distribution with a zero mean and _d standard deviation. Simulations were conducted by varying the home range size, mating area (area from where individuals choose their mates), standard deviation of dispersal distance, food generation time, the reproductive rates of food populations and the sizes and number of independent food populations. Food organisms reproduce either within one population or independently within 16 spatially divided populations. For all the simulations, competitive exclusion was the most frequent outcome and character displacement was the least frequent outcome. Through a 200-generation simulation, the two consumer species could co-exist longer and maintain a polymorphic resource use longer when the home range and mating size were small in 16 spatially divided populations than when random mating and homogeneous interaction occurred within a community (perfect mixing population). For perfect mixing populations, the frequency of character displacement increased as the food generation time became short and the reproductive rates of food decreased. It follows from the results that the sizes of the genetic and ecological neighbourhoods and the mode of resource dynamics can affect the evolution of two competing species.     

The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus)

Summary The cytological features and membrane specialisations of neuroepithelial cells (apical cells) in direct contact with the lumen of the lung were studied with transmission and scanning electron microscopy. The luminal surface of the apical cell is characterised by microvilli, a cilium with an 8+1 microtubular pattern and numerous coated vesicles. The cytoplasmic region immediately beneath the luminal plasma membrane contains numerous smooth-walled vesicles, tubules and microtubules, a few microfilaments and dense granules (15–20 nm in diameter). The luminal pole of the cell is marked off from the basal or vascular pole by a well-defined terminal web associated with junctional complexes. Protrusion of the luminal pole occurs as a transient phenomenon and is accompanied by a pinching in of the cell at the terminal web. It is proposed that the distinctive features of the luminal pole of the apical cell are comparable to those of recognised chemoreceptor cells. It is also proposed that in view of the common features of apical and basal cells the apical cell functions as a receptor/transducer and the basal cells serve as an accessory source of peptides/5-hydroxytryptamine to be released on stimulation of the apical cell. Furthermore, we have drawn attention to the structural heterogeneity of the neuroepithelial bodies in various vertebrate classes.     

Innervation and cytochemistry of the neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus)

Summary Neuroepithelial bodies (NEB) were identified in the lung of Bufo marinus. The characteristics of the cells and their innervation were studied with electron and fluorescence microscopy before and after close vagosympathetic denervation. The bodies consist of low columnar cells which rest on the epithelial basal lamina. The majority of the cells do not reach the lumen of the lung (basal cells); the few which do (apical cells) are bordered by microvilli and possess a single cilium. The neuroepithelial cell cytoplasm contains a variety of organelles the most characteristic of which are dense cored vesicles. Microspectrofluorometry and electron microscopic cytochemistry indicate significant quantities of 5-hydroxytryptamine in these cells. The neuroepithelial bodies could be divided into three groups on the basis of their innervation: 1) About 60% of the NEBs are innervated solely by nerve fibres containing agranular vesicles which form reciprocal synapses; 2) about 20% are innervated solely by adrenergic nerve fibres which form distinct synaptic contacts; and 3) the remaining 20% are innervated by both types of nerve fibres. It is proposed that the NEBs are receptors monitoring intrapulmonary P_{CO 2} and so leading to modulation of activity in afferent nerve fibres (type containing agranular vesicles). The presence of NEBs solely with an adrenergic (efferent) innervation poses a problem with this interpretation.     

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

The filter apparatus of Rana temporaria and Bufo bufo larvae (Amphibia,Anura)

Summary In larvae of Rana temporaria and Bufo bufo the location of filter apparatus within the larval organization, the arrangement of the morphological parts as branchial food trap, ventral velum, and filter rows, as well as their surface anatomy, are similar to that of other species of Orton's larval type IV. The means by which mucous with its entrapped food particles is transported from the filter rows to the esophagus is finally resolved. The dorsally positioned ciliary cushion extends far ventrally between the filter plates. From their contact with the filter rows, the cilia transport the mucous to Kratochwill's caudally positioned Flimmerrinne and from there to the esophagus. The original chordate principle of mucous entrapment and ciliary transport is thus retained by these anuran larvae. The only modification specific to the latter is the division into a ventral filter apparatus, whose epithelia serve for mucus entrapment, and a dorsal ciliary area.Six different types of cell may be distinguished ultrastructurally: (1) The ubiquitous squamous epithelium with merocrine extrusions; (2) the large supporting cells of the filter rows and of the ventral velum; (3) the ciliary cells of the ciliary cushion; (4) three different types of mucous producing secretory cells: (a) A type of cell similar to the goblet cell is found in the ciliary cushion (merocrine extrusion); (b) The secretory pits of the ventral velum and the secretory ridges have similar bottle-shaped merocrine secretory cells; (c) The merocrine apical cells of the filter rows are the final kind. It is evident that the ciliary cushion epithelium resembles that of both the manicotto glandulare of anuran larvae and the trachea and bronchus of Mammalia.Supported by the Deutsche Forschungsgemeinschaft-DFG