Molecular phylogeny and biogeography of West Indian toads (Anura: Bufonidae)

Toads of the Bufo peltocephalus Group (Anura: Bufonidae) occur throughout the Greater Antilles (Cuba to the Virgin Islands), a geographic region of relatively high endemicity. Previous morphological and immunological studies suggested that the West Indian toads are a monophyletic lineage derived from Neotropical Bufo but were unable to clarify relationships within the group. We examined the evolutionary relationships and biogeography of this group of frogs by collecting approximately 2 kb of mitochondrial DNA sequence data from eight West Indian species and selected non-West Indian species from the New World and the Old World. Our analyses support the monophyly of native West Indian toads and a New World origin for the group. Relationships among the West Indian species are less certain, but a Cuban lineage is defined in most analyses.     

Positive Darwinian selection results in resistance to cardioactive toxins in true toads (Anura: Bufonidae)

Members of the Family Bufonidae, true toads, are famous for their endogenously synthesized cardioactive steroids that serve as defensive toxins. Evolution of resistance to these toxins is not understood. We sequenced a key region of the toxin''s binding site in the Na⁺/K⁺ ATPase for relevant taxa representing Hyloidea (including bufonids), Ranoidea and Archaeobatrachia and tested for positive selection in a phylogenetic context. Bufonidae were distinct from other Hyloidea at 4–6 of 12 sites and, with one exception, had a homologous amino acid sequence. Melanophryniscus stelzneri had a distinct sequence, consistent with other independent evidence for a differentiated toxin. Tests within Bufonidae detected positive selection within the binding region, providing, to our knowledge, the first evidence of this type for positive selection within Amphibia. There was no evidence for positive selection on Bufonidae or M. stelzneri lineages. Sequence change in Leptodactylus ocellatus, a leptodactylid predator of Bufonidae, provides a molecular basis for predator resistance possibly associated with gene duplication.     

Interspecific patterns for egg and clutch sizes of African Bufonidae (Amphibia: Anura)

Little is known about reproductive trade-offs in African amphibians, but such data, particularly in the form of quantitative measurements, are a key for investigating life history evolution. Here we compile and analyze known data on African bufonids from published material and new data from preserved museum specimens, to investigate interspecific patterns of egg and clutch sizes variation. Our data is a composite of mixed sources, including ova data from dissected females and laid clutches from observations in the field. Our study shows that, as body size increases, clutch size increases but egg size decreases, and when correcting for body size, egg size is inversely correlated with clutch size. These parameter interactions however, are different for different reproductive modes. In free-swimming larval developing species, the same trends are recovered, but for lecithotrophic viviparous species no significant correlations could be recovered for clutch size and body size nor for the trade-off between clutch size and egg size, and egg size is positively related to body size. The egg size of Nimbaphrynoides occidentalis (Angel, 1943) is a clear outlier, which may be due to its matrotrophic viviparous reproduction. In addition, we observed no statistical difference between ova data collected from dissections and laid clutch data from field observations, which suggests that such a mixed dataset has utility in comparative analyses.     

Molecular systematics of the Middle American genus Hypopachus (Anura: Microhylidae)

We present the first phylogenetic study on the widespread Middle American microhylid frog genus Hypopachus. Partial sequences of mitochondrial (12S and 16S ribosomal RNA) and nuclear (rhodopsin) genes (1275 bp total) were analyzed from 43 samples of Hypopachus, three currently recognized species of Gastrophryne, and seven arthroleptid, brevicipitid and microhylid outgroup taxa. Maximum parsimony (PAUP), maximum likelihood (RAxML) and Bayesian inference (MrBayes) optimality criteria were used for phylogenetic analyses, and BEAST was used to estimate divergence dates of major clades. Population-level analyses were conducted with the programs NETWORK and Arlequin. Results confirm the placement of Hypopachus and Gastrophryne as sister taxa, but the latter genus was strongly supported as paraphyletic. The African phrynomerine genus Phrynomantis was recovered as the sister taxon to a monophyletic Chiasmocleis, rendering our well-supported clade of gastrophrynines paraphyletic. Hypopachus barberi was supported as a disjunctly distributed highland species, and we recovered a basal split in lowland populations of Hypopachus variolosus from the Pacific versant of Mexico and elsewhere in the Mesoamerican lowlands. Dating analyses from BEAST estimate speciation within the genus Hypopachus occurred in the late Miocene/early Pliocene for most clades. Previous studies have not found bioacoustic or morphological differences among these lowland clades, and our molecular data support the continued recognition of two species in the genus Hypopachus.     

The release call of Rhaebo guttatus (Anura: Bufonidae)

The bufonid genus Rhaebo contains 13 species, Rhaebo guttatus being one of the two species that occur in Brazil. Here we describe for the first time the release call of R. guttatus from the Brazilian Amazonia. Release calls are highly variable both in the temporal and in the frequency domains. The advertisement and release calls of Rhaebo guttatus are distinguished from each other by their temporal envelope, distribution of sound energy, and calling emission patterns. The acoustic characterization of release calls in Rhaebo could be potentially informative in the taxonomy of this genus, as previously reported for members of the bufonid genus Rhinella.     

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.     

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.     

Thermal parameters changes in males of Rhinella arenarum (Anura:Bufonidae) related to reproductive periods

The regulation of body temperature in ectotherms has a major impact in their physiological and behavioral processes. Observing changes in thermal parameters related with reproduction allows us to better understand how Rhinella arenarum optimizes a thermal resource. The aim of this study was to compare the thermal parameters of this species between breeding and non-breeding periods. In the field, we recorded the body temperature from captured animals, the air temperature, and the temperature of the substrate. In the laboratory, we measured the temperature R. arenarum selected on a thermal gradient and the critical extreme temperatures. The results of our study show variations in thermal parameters between the two situations studied. This species makes efficient use of thermal resources during the breeding period by basking to significantly increase body temperature. Because calling is energetically costly for males, this behavior results in increased efficiency to callers during the breeding period.     

Molecular systematics of Alethe,Sheppardia and some other African robins (Muscicapoidea)

Phylogenetic relationships between species of akalats (Sheppardia) and alethes (Alethe) were derived from mitochondrial and nuclear sequence data, and phylogeographic pattern was also investigated in three widespread species (two Alethe and one Sheppardia) endemic to the African lowland rainforest biome. Co-distributed species of Alethe and Sheppardia showed similar spatial relationships between the Guineo-Congolian biome and the Western Rift mountains, and preliminary area relationships are shown for species of Sheppardia and Alethe distributed in the Cameroon highlands, the woodland areas north and south of the lowland forest block, and east African montane and lowland forest. Within widespread species, phylogeographic pattern was generally similar, with long branches between Upper and Lower Guinea populations, and between eastern and western Congolian populations. At the interspecific level, the nuclear intron used gave more resolution than the mitochondrial data, which gave poor resolution at basal nodes. While the affinities of some species groups were weakly supported, no support was suggested for the monophyly of Alethe and little for Sheppardia as those genera are currently circumscribed, and taxonomic changes are suggested. As found by previous workers, monophyletic clades corresponding to Muscicapini and Saxicolini of Sibley and Ahlquist (1990) were not recovered, and the membership of any monophyletic 'African forest robin assemblage' needs to be resolved.     

Light at the end of the tunnel: insights into the molecular systematics of East African puddle frogs (Anura: Phrynobatrachidae)

To investigate intra- and interspecific variability of ‘Little Brown Frogs’ (LBFs) from East Africa, we analysed molecular data from 23 species of Phrynobatrachus using 16S rRNA, a mitochondrial gene considered a universal DNA barcoding marker in amphibians. Conspecific populations, excluding P. natalensis, exhibited low (0–0.5%) sequence divergences, while conspecific populations of P. natalensis exhibited a wide range of differentiation (0.2–6.4%) with a distinct bimodal distribution, affirming that this may be a complex of cryptic species. Sister species yielded a range of differentiation from 1.5–8.3% with median of 2.3%. Results revealed that at least three non-sister populations of miniaturized puddle frogs are currently identified as P. mababiensis, and a new species from Ethiopia was identified. This first step into revising East African Phrynobatrachus systematics illustrates the necessity of rigorous taxonomic study, especially for groups with confusing or highly incomplete taxonomy.     

Two new species of cosmocercids (Ascaridida) in the toad Chaunus arenarum (Anura: Bufonidae) from Argentina

Aplectana tarija n. sp. and Cosmocercoides lilloi n. sp., intestinal parasites of the toad Chaunus arenarum, are described. Of the 41 nominal species of Aplectana, A. tarija n. sp. represents the 22nd Neotropical species and the second species reported from Argentina. Of the 14 nominal species of Cosmocercoides, C. lilloi n. sp. represents the 15th species and the first species reported from South America.     

Molecular systematics of Goniodidae (Insecta: Phthiraptera)

The higher level phylogenetic relationships within the avian feather lice (Insecta: Phthiraptera: Ischnocera) are extremely problematic. Here we investigate the relationships of 1 family (Goniodidae), sometimes recognized as distinct within Ischnocera, using parsimony and likelihood analyses of nuclear and mitochondrial DNA sequences. These data support monophyly for a restricted definition of traditional Goniodidae, but recognition of this family would result in paraphyly of the large heterogeneous family Philopteridae. We show that the New World Chelopistes is not related to other members of Goniodidae, despite similarities in morphology, but rather is the sister taxon to Oxylipeurus. Within Goniodidae, genera are divided into those occurring on Galliformes (the Goniodes complex) and those occurring on Columbiformes (the Coloceras complex). Within the well-sampled Coloceras complex, or Physconelloidinae, several groups are identified. However, traditionally recognized genera such as Coloceras and Phvsconelloides appear to be paraphyletic. Whereas the phylogeny of Goniodidae reflects some aspects of host relationships, biogeography also influences coevolutionary history.     

Molecular systematics of sponges (Porifera)

The first application of molecular systematics to sponges was in the 1980s, using allozyme divergence to dis-criminate between conspecific and congeneric sponge populations. Since this time, a fairly large database has been accumulated and, although the first findings seemed to indicate that sponge species were genetically more divergent than those of other marine invertebrates, a recent review of the available dataset indicates that levels of interspecific gene identities in most sponges fall within the normal range found between species of other invertebrates. Nevertheless, some sponge genera have species that are extremely divergent from each other, suggesting a possible polyphyly of these genera. In the 1990s, molecular studies comparing sequences of ribosomal RNA have been used to reappraise the phylogenetic relationships among sponge genera, families, orders and classes. Both the 18S small subunit and the 28S large subunit rRNA genes have been sequenced (41 complete or partial and 75 partial sequences, respectively). Sequences of 18S rRNA show good support for Porifera being true Metazoa, but they are not informative for resolving relationships among genera, families or orders. 28S rRNA domains D1 and D2 appear to be more informative for the terminal nodes and provide resolution for internal topologies in sufficiently closely related species, but the deep nodes between orders or classes cannot be resolved using this molecule. Recently, a more conserved gene, Hsp70, has been used to try to resolve the relationships in the deep nodes. Metazoan monophyly is very well supported. Nevertheless, the divergence between the three classes of Porifera, as well as the divergence between Porifera, Cnidaria and Ctenophora, is not resolved. Research is in progress using other genes such as those of the homeodomain, the tyrosine kinase domain, and those coding for the aggregation factor. For the moment the dataset for these genes is too restricted to resolve the phylogenetic relationships of these phyla. However, whichever the genes, the phylogenies obtained suggest that Porifera could be paraphyletic and that the phylogenetic relationships of most of the families and orders of the Demospongiae have to be reassessed. The Calcarea and Hexactinellida are still to be studied at the molecular level.     

Molecular systematics of polychaetes (Annelida)

Hydrobiologia - Some progress has been made in the field of molecular systematics of polychaetes over the past couple of years. In particular, phylogenetic analyses of sequence data from the 18S...     

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.     

The ontogeny of kin recognition in tadpoles of the toadBufo melanostictus (Anura; Bufonidae)

The ontogeny of kin recognition and influence of social environment on the development of kin recognition behaviour was experimentally investigated in tadpoles of Bufo melanostictus that lived in aggregations and showed low larval dispersion. Embryos and tadpoles of the toad were reared as (i) kin only, (ii) with kin and non-kin (separated by a mesh screen), and (iii) in isolation. They were tested for the ability to discriminate between (i) familiar siblings and unfamiliar non-siblings, (ii) familiar siblings and familiar non-siblings and, (iii) unfamiliar siblings and unfamiliar non-siblings. All tadpoles were fed on boiled spinach before conducting trials. Preference of test tadpoles to associate near the end compartments whether empty or containing members of specific stimulus groups was assessed using a rectangular choice tank. When tested in tanks with empty end compartments, the test tadpoles showed random distribution and thus no bias for the apparatus or the procedure. In the presence of kin/non-kin in the end compartments a significantly greater number of test tadpoles spent the majority of the time near familiar or unfamiliar kin rather than near familiar or unfamiliar non-kin. Kin discrimination ability persisted throughout larval development. Familiarity with siblings is not required for discriminating kin from non-kin, and kin discrimination ability is not modified following exposure to non-kin. Also, involvement of dietary cues is unlikely to be the prime mechanism of kin recognition inB. melanostictus unlike in some other anurans.     

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.     

Temporal stability in hybridization between Bufo microscaphus and Bufo woodhousii (Anura: Bufonidae): Behavior and morphology

I analyzed temporal variation in hybridization between the southwestern toad (Bufo microscaphus) and Woodhouse's toad (Bufo woodhousii) along the Virgin River in southwestern Utah and northwestern Arizona. Bufo woodhousii is largely restricted to the floodplain of the Virgin River from southern Nevada to the vicinity of St. George, Utah. By contrast, B. microscaphus is confined primarily to the tributaries of the Virgin River, only occupying the Virgin River proper exclusive of B. woodhousii along its upper reaches east of La Verkin Creek. As in all other zones of sympatry for these bufonids, behavior and morphology provide clear evidence of hybridization at a number of sites along the Virgin River. Analysis of morphology and behavior indicates that the distribution of these taxa and their hybrids is largely unchanged relative to that documented forty years ago by Blair (1955). Comparisons of morphological hybrid index scores reveal slight shifts in relative numbers of parental taxa at only one site. Hybridization between these anurans has been relatively stable geographically, and may be related to the nature of the riparian habitat available for breeding.     

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.