Systematics of treefrogs of the Hypsiboas calcaratus and Hypsiboas fasciatus species complex (Anura,Hylidae) with the description of four new species

We review the systematics of the Hypsiboas calcaratus species complex, a group of widely distributed Amazonian hylid frogs. A comprehensive analysis of genetic, morphological, and bioacoustic datasets uncovered the existence of eleven candidate species, six of which are confirmed. Two of them correspond to Hypsiboas fasciatus and Hypsiboas calcaratus and the remaining four are new species that we describe here. Hypsiboas fasciatus sensu stricto has a geographic range restricted to the eastern Andean foothills of southern Ecuador while Hypsiboas calcaratus sensu stricto has a wide distribution in the Amazon basin. Hypsiboas almendarizae sp. n. occurs at elevations between 500 and 1950 m in central and northern Ecuador; the other new species (H. maculateralis sp. n., H. alfaroi sp. n., and H. tetete sp. n.) occur at elevations below 500 m in Amazonian Ecuador and Peru. The new species differ from H. calcaratus and H. fasciatus in morphology, advertisement calls, and mitochondrial and nuclear DNA sequences. Five candidate species from the Guianan region, Peru, and Bolivia are left as unconfirmed. Examination of the type material of Hyla steinbachi, from Bolivia, shows that it is not conspecific with H. fasciatus and thus is removed from its synonymy.     

Conserved karyotypes in the Hyla pulchella species group (Anura, Hylidae)

Cytogenetic analyses were done on specimens of Hyla marginata and on three populations of H. semiguttata differing in morphology and in the physical parameters of their advertisement call, as well as in individuals of Hyla sp. (aff. semiguttata). All specimens had 2n=24 chromosomes with a morphology very similar to that of other 24-chromosome Hyla species. Hyla semiguttata and H. marginata showed the same C-banding pattern but were distinguished by the location of the NOR on pair 1 in H. semiguttata (in the three populations) and Hyla sp. (aff. semiguttata), and on pair 10 in H. marginata. The H. semiguttata populations did not differ cytogenetically, despite variations in their morphology and advertisement calls. Similarly, H. semiguttata and H. p. joaquini studied previously had identical C-banding patterns and NOR locations, suggesting that they are very closely related.     

Cytogenetic analysis of two species in the Phyllomedusa hypochondrialis group (Anura, Hylidae)

The genus Phyllomedusa has been the target of regular taxonomic investigations. The species Phyllomedusa nordestina was recently separated from P. hypochondrialis. Morphological variations in the P. rohdei interpopulation have already been reported, suggesting the existence of more than one taxon under that name. In the present study, we have cytogenetically characterized two populations of P. nordestina and one of P. rohdei. Both species displayed 2n = 26 chromosomes with 12 metacentric, 12 submetacentric and 2 subtelocentric chromosomes. The C-banding analyses revealed discrete differences in the quantity of centromeric heterochromatin between the two species. The nucleolus organizer region (NOR) was detected in pair 9 of both species, but is located in the pericentromeric region of the short arm in P. nordestina and in the long arm subtelomeric region of P. rohdei. Chromosomal data from this study indicate karyotypic homeology between the two groups of P. hypochondrialis species and suggest the existence of more than one taxon under the P. rohdei name.     

The tadpole of Hypsiboas leptolineatus (Braun and Braun, 1977), a species in the Hypsiboas polytaenius clade (Anura; Hylidae).

The larval morphology of Hypsiboas leptolineatus was studied. The tadpole has an ovoid body in lateral view, wider than deep; snout rounded with dorsal reniform nostrils; spiracle sinistral with lateral wall attached to body; anal tube dextral; tail fins convex with acuminate tip; oral disc ventral; labial tooth row formula is 2(2)/3(1); moderately developed beaks with serrated jaw sheaths. These external oral features are compared with those of the known tadpoles in the Hypsiboas polytaenius clade. The oral cavity was studied using an electron microscope. Life history aspects are commented.     

Vocalizations of Hypsiboas goianus (Lutz, 1968) (Anura: Hylidae) in Central Brazil

Environmental and morphological factors and social context influence the vocalizations of anuran species. Herein, we observed the acoustic behavior of Hypsiboas goianus males from Central Brazil and analyzed the following calls: advertisement, short aggressive and long aggressive. The acoustic parameters of advertisement calls were not correlated with air temperature, mass or snout–vent length; however, the acoustic parameters of short aggressive calls were influenced by these variables. The temporal parameters of advertisement and short aggressive calls were classified as dynamic properties, while the dominant frequency was considered a static property. Dominant frequency is the most important variable to discriminate calls among individuals.     

A molecular perspective on the phylogeny of the Hyla pulchella species group (Anura, Hylidae)

A molecular phylogenetic analysis of the Hyla pulchella species group was performed to test its monophyly, explore the interrelationships of its species, and evaluate the validity of the taxa that were considered subspecies of H. pulchella. Approximately 2.8 kb from the mitochondrial genes 12s, tRNA valine, 16s, and Cytochrome b were sequenced. The analysis included 50 terminals representing 10 of the 14-15 species currently recognized in the H. pulchella group, including samples from several localities for some taxa, several outgroups, as well as two species previously suspected to be related with the group (Hyla guentheri and Hyla bischoffi). The results show that the H. pulchella and Hyla circumdata groups are distantly related, and, therefore, should be recognized as separate groups. As currently defined, the H. pulchella group is paraphyletic with respect to the Hyla polytaenia group; therefore, we recognize the Hyla polytaenia clade in the H. pulchella group. Two subspecies of H. pulchella recognized by some authors are considered full species including Hyla pulchella riojana because it is only distantly related to H. pulchella, and Hyla pulchella cordobae because molecular and non-molecular evidence suggests that it is specifically distinct. With the inclusion of the H. polytaenia clade, H. guentheri, and H. bischoffi, and the recognition of the two former subspecies of H. pulchella as distinct species, the H. pulchella group now comprises 25 described species. All representatives of the H. pulchella group with an Andean distribution are monophyletic and nested within a clade from the Atlantic forest from south-southeastern Brazil/northeastern Argentina, and Cerrado gallery forest from central Brazil.     

Systematics of the Osteocephalus buckleyi species complex (Anura,Hylidae) from Ecuador and Peru

We present a new phylogeny, based on DNA sequences of mitochondrial and nuclear genes, for frogs of the genus Osteocephalus with emphasis in the Osteocephalus buckleyi species complex. Genetic, morphologic, and advertisement call data are combined to define species boundaries and describe new species. The phylogeny shows strong support for: (1) a basal position of Osteocephalus taurinus + Osteocephalus oophagus, (2) a clade containing phytotelmata breeding species, and (3) a clade that corresponds to the Osteocephalus buckleyi species complex. Our results document a large proportion of hidden diversity within a set of populations that were previously treated as a single, widely distributed species, Osteocephalus buckleyi. Individuals assignable to Osteocephalus buckleyi formed a paraphyletic group relative to Osteocephalus verruciger and Osteocephalus cabrerai and contained four species, one of which is Osteocephalus buckleyi sensu stricto and three are new. Two of the new species are shared between Ecuador and Peru (Osteocephalus vilmae sp. n. and Osteocephalus cannatellai sp. n.) and one is distributed in the Amazon region of southern Peru (Osteocephalus germani sp. n.) We discuss the difficulties of using morphological characters to define species boundaries and propose a hypothesis to explain them.     

A new species of Osteocephalus (Anura: Hylidae) from Guyana.

A new member of the genus Osteocephalus is described from the Pakaraima mountains of western Guyana. This species is the smallest known member of the genus and is probably closely related to O. subtilis. Both share a small size (less than 40 mm snout-vent length), large and bulgy eyes directed somewhat rostrally, green bones, smooth and brownish dorsal skin, relatively short and truncate snout, small tympanum, subgular and laterally expanded vocal sac, poorly developed subarticular and supernumerary tubercles, a supra-anal glandular ridge, and cream-white venter and subocular region. The new species can be distinguished from O. subtilis by the Buff iris (vs black), smaller overall size (32.7 vs 35.8-38.8 mm snout-vent length), relatively larger toe disks, and less developed foot webbing. The cranium of the new species is well ossified, relatively reduced in width between the orbits, without an exposed frontoparietal fontanelle and with the anterior arm of the squamosal extending to about half the distance to the maxillary. The vocal sac is subgularly poorly developed and possess lateral extensions to the area behind the jaw angles. Well developed supraocular and suprasquamosal cartilages give support to the enlarged eyes of this species.     

Cryptic species complexes,widespread species and conservation: lessons from Amazonian frogs of the Leptodactylus marmoratus group (Anura: Leptodactylidae)

Morphologically cryptic species act as a wild card when it comes to biodiversity assessments and conservation, with the capacity to dramatically alter our understanding of the biological landscape at the taxonomic, ecological, biogeographic, evolutionary, and conservation levels. We discuss the potential effects that cryptic species may have on biodiversity assessments and conservation, as well as some of the current issues involving the treatment of cryptic species both at taxonomic and conservation levels. In addition, using a combination of advertisement call and morphological data, we describe a new species of the Leptodactylus marmoratus group from the upper Amazon basin, and we assess how cryptic species can affect conservation assessments of species in the Leptodactylus marmoratus group by examining how recent findings affect our understanding of the distribution of what is assumed to be a widespread Amazonian species, Leptodactylus andreae.     

Comparative cytogenetic analysis on four tree frog species (Anura, Hylidae, Hylinae) from Brazil

A comparative cytogenetic analysis was carried out on four Hylinae tree frogs from Brazil ((Aparasphenodon brunoi, Corythomantis greeningi, Osteocephalus langsdorffii, and Scinax fuscovarius) using Giemsa staining, BrdU replication banding, Ag-NOR staining, C-banding, DAPI and CMA(3) fluorochrome staining, and fluorescence in situ hybridization (FISH) with an rDNA probe. All the species share closely similar 2n = 24 karyotypes, almost indistinguishable by standard staining. The technique of BrdU incorporation allowed the identification of each pair of homologs and the establishment of extensive homeology for the great majority of the chromosomes, mainly of A. brunoi, C. greeningi, and O. langsdorffii. Despite highly conserved replication banding patterns, the use of the other banding techniques disclosed some minor differences, which reinforces the importance of extensive cytogenetic analyses for the karyotypic characterization of Anuran species. The present cytogenetic data confirm the closer proximity of A. brunoi, C. greeningi, and O. langsdorffii, whereas S. fuscovarius is phylogenetically more distant.     

Phylogeny and biogeography of paradoxical frogs (Anura, Hylidae, Pseudae) inferred from 12S and 16S mitochondrial DNA

We used mitochondrial DNA sequences of 12S and 16S ribosomal RNA genes to reconstruct phylogenetic relationships of the nine species of South American aquatic hylids known as paradoxical frogs. Pseudis minuta and P. cardosoi form the sister-group to a clade comprising Lysapsus and the remaining Pseudis. We suggest the resurrection of Podonectes, including P. minutus and P. cardosoi, to avoid the nonmonophyly of Pseudis. Some doubt is cast on the species status of P. cardosoi. Lysapsus limellum, P. bolbodactyla, and P. paradoxa each may comprise more than one species, but lack of comprehensive geographic and morphological appraisals precludes taxonomic changes. Biogeographic implications of the phylogeny are discussed. The correlation between hydrographic basins and Pseudis species is not fully supported, and a recent contact between Amazon populations in eastern Bolivia and western Brazil (Rond?nia) and Paraná basin populations in the Pantanal is suggested. A dispersal-vicariance analysis showed that major diversification of Pseudis and Lysapsus species occurred in the Brazilian Shield, and that the presence of P. paradoxa and L. limellum in the Amazon and Paraná basins is due to recent dispersals. Evidence suggests a vicariant event, most likely caused by Miocene marine introgressions, as the cause for the restricted distribution of L. laevis in the Guiana Shield.     

Acute and chronic effects of Cr(VI) on Hypsiboas pulchellus embryos and tadpoles

In the last few years there has been great concern about declines in the abundance of several species of amphibians around the world. Among amphibians, anurans have a biphasic life cycle, with aquatic tadpoles and generally terrestrial adults, and they have an extremely permeable skin, making them excellent indicators of the health of the environment. A number of different causes have been suggested for the global decline of anurans, the pollution of their habitat by chemical stressors being considered one of the major factors. Among chemical stressors, heavy metals are known for their high toxicity at very low concentrations. This study assessed short- (96 h, 'acute') and long-term (1272 h, 'chronic') exposure to Cr(VI) at lethal (3 to 90 mg 1(-1)) and sublethal concentrations (0.001 to 12 mg 1(-1)) on Hypsiboaspulchellus (previously called Hyla pulchella; see Faivovich et al. 2005) tadpoles (Fam. Hylidae) from central eastern Argentina. Fertilized eggs collected from a clean pond near La Plata (Buenos Aires Province) were used for acute and chronic toxicity testing. Assays were done under controlled laboratory conditions. Results of chronic exposure were used to assess the effect of factors such as toxicant concentration and age of organisms at the beginning of exposure on the response variables (growth, development and survival until metamorphosis). Results indicated a higher sensitivity to Cr(VI) of individuals first exposed as tadpoles than those first exposed as embryos during acute and chronic exposure. Exposure to the highest sublethal concentrations (6 to 12 mg 1(-1)) of the toxicant showed early inhibitory effects on growth of all treated organisms compensated at longer exposure periods with an increase in the growth rate compared to the control groups.     

Chromosome analysis of five Brazilian species of poison frogs (Anura: Dendrobatidae)

Dendrobatid frogs have undergone an extensive systematic reorganization based on recent molecular findings. The present work describes karyotypes of the Brazilian species Adelphobates castaneoticus, A. quinquevittatus, Ameerega picta, A. galactonotus and Dendrobates tinctorius which were compared to each other and with previously described related species. All karyotypes consisted of 2n = 18 chromosomes, except for A. picta which had 2n = 24. The karyotypes of the Adelphobates and D. tinctorius species were highly similar to each other and to the other 2n = 18 previously studied species, revealing conserved karyotypic characteristics in both genera. In recent phylogenetic studies, all Adelphobates species were grouped in a clade separated from the Dendrobates species. Thus, we hypothesized that their common karyotypic traits may have a distinct origin by chromosome rearrangements and mutations. In A. picta, with 2n = 24, chromosome features of pairs from 1 to 8 are shared with other previously karyotyped species within this genus. Hence, the A. picta data reinforced that the C-banding pattern and the NOR location are species-specific traits in the genus Ameerega. Moreover, the Ameerega monophyletism proposed by previous phylogenetic studies indicates that the karyotypic differences among species in this genus result from a long divergence time.     

Meiotic chromosomes of the tree frog Smilisca baudinii (Anura: Hylidae)

The Mexican tree frog Smilisca baudinii, is a very common frog in Central America. In spite their importance to keep the ecological equilibrium of the rainforest, its biology and genetics are poorly known. In order to contribute with its biological knowledge, we described the typical meiotic karyotype based in standard cytogenetic protocols to specimens collected in Tabasco, Mexico. The study was centered in the analysis of 131 chromosome spreads at meiotic stage from two adults of the species (one female and one male). The metaphase analysis allowed the establishment of the modal haploid number of 1n = 12 bivalent chromosomes. The chromosomic formulae from the haploid bivalent karyotype was integrated by 12 biarmed chromosomes characterized by twelve pairs of metacentric-submetacentric (msm) chromosomes. The meiotic counting gives the idea that diploid chromosome number is integrated by a complement of 2n = 24 biarmed chromosomes. The presence of sex chromosomes from female and male meiotic spreads was not observed. Current results suggest that S. baudinii chromosome structure is well shared among Hylidae family and "B" chromosomes are particular structures that have very important evolutionary consequences in species diversification.     

Observations on the polyphyodont dentition of Hemiphractus proboscideus (Anura: Hylidae)

In Hemiphractus fang–like teeth are ankylosed to the premaxilla, maxilla and prevomer, and bony odontoids are found on the dentary, angular and palatine bones. The odontoids are small, but a larger pair at the front of the lower jaw project upwards and backwards into the mouth and fit into a diastema between the anterior premaxillary teeth when the mouth is closed.
The teeth are unipartite and monocuspid, and each consists of a strongly recurved and elongated cone of orthodentine, capped at the tip by a thin layer of enamel. The inner circumpulpal layer of the dentine is tubular, but no tubules are present in the outer pallial layer. During tooth development, dentine is formed before the enamel matrix is produced, and the tooth germs lie horizontally beneath the ventral surface of each dentigerous bone. On eruption, the tooth germs migrate horizontally and become ankylosed to the outer edge of the jaw bone by a layer of cellular cementum.
During tooth replacement, the vast majority of the dentine of each tooth, and the cementum at the tooth base, are resorbed by osteoclasts. It is not clear whether the tips of the teeth are shed or not.