Although some species groups have been recognized in the leiuperine genus Physalaemus, no phylogenetic analysis has previously been performed. Here, we provide a phylogenetic study based on mitochondrial and nuclear DNA sequences from 41 of the 46 species of Physalaemus. We employed the parsimony criterion using the software TNT and POY and the Bayesian criterion using the software MrBayes. Two major clades were recovered inside the monophyletic Physalaemus: (i) the highly supported Physalaemus signifer Clade, which included P. nattereri and the species previously placed in the P. deimaticus and P. signifer Groups; and (ii) the Physalaemus cuvieri Clade, which included the remaining species of Physalaemus. Five species groups were recognized in the P. cuvieri Clade: the P. biligonigerus Group, the P. cuvieri Group, the P. henselii Group, the P. gracilis Group and the P. olfersii Group. The P. gracilis Species Group was the same as that previously proposed by Nascimento et al. (2005). The P. henselii Group includes P. fernandezae and P. henselii, and was the sister group of a clade that comprised the remaining species of the P. cuvieri Clade. The P. olfersii Group included P. olfersii, P. soaresi, P. maximus, P. feioi and P. lateristriga. The P. biligonigerus Species Group was composed of P. biligonigerus, P. marmoratus, P. santafecinus and P. riograndensis. The P. cuvieri Group inferred here differed from that recognized by Nascimento et al. (2005) only by the inclusion of P. albifrons and the exclusion of P. cicada. The paraphyly of P. cuvieri with respect to P. ephippifer was inferred in all the analyses. Distinct genetic lineages were recognized among individuals currently identified as P. cuvieri and they were congruent with cytogenetic differences reported previously, supporting the hypothesis of occurrence of formally unnamed species.

Here, we present a molecular phylogenetic analysis of the Neotropical genus Pseudopaludicola focusing on species relationships including 11 of the 17 known species of Pseudopaludicola; several samples of Pseudopaludicola are not assigned to any species; and 34 terminal species as an outgroup. The study was based on the analysis of approximately 2.3 kb of the sequence of the mitochondrial 12S rRNA, tRNAval and 16S rRNA genes through maximum parsimony and Bayesian phylogenetic reconstruction approaches. Our results showed that Pseudopaludicola is a well‐supported monophyletic group organized into four major clades and confirmed that the assemblage of species that lack T‐shaped terminal phalanges is paraphyletic with respect to the P. pusilla Group. Chromosomal data mapped on the cladogram showed a direct correlation among the four clades and observed chromosome numbers (2n = 22, 20, 18 and 16) with a progressive reduction in the chromosome number. Overall, our findings suggest that some taxonomic changes are necessary and reinforce the need for a revision of the genus Pseudopaludicola.

Pseudis paradoxa paradoxa, P. p. platensis, P. bolbodactyla, P. fusca and P. tocantins were analyzed cytogenetically by conventional chromosomal staining, C-banding, silver staining and fluorescent in situ hybridization with an rDNA probe. Pseudis tocantins chromosomes were also stained with distamycin A/DAPI. All of the species had a diploid number of 2n = 24 chromosomes and the nucleolar organizer region (NOR) was located on pair 7. However, the karyotypes could be differentiated based on the morphology of chromosomal pairs 2 and 8, the region that the NORs occupied on the long arms of the homologous of pair 7, and the pattern of heterochromatin distribution. The subspecies P. p. paradoxa and P. p. platensis had identical karyotypes. Heteromorphism in NOR size was seen in P. p. paradoxa, P. p. platensis, P. bolbodactyla and P. fusca. Heteromorphic sex chromosomes (ZZ/ZW) were identified in P. tocantins. The W chromosome was subtelocentric and larger than the metacentric Z chromosomes. The differences observed in the C-banding pattern and in the position of the NOR on the sex chromosomes suggested that inversions and heterochromatinization were responsible for the morphological differentiation of these chromosomes.

In the present study, we describe for the first time a family of 190-bp satellite DNA related to 5S rDNA in anurans and the existence of 2 forms of 5S rDNA, type I (201 bp) and type II (690 bp). The sequences were obtained from genomic DNA of Physalaemus cuvieri from Palmeiras, State of Bahia, Brazil. Analysis of the nucleotide sequence revealed that the satellite DNA obtained by digestion with EcoRI, called PcP190EcoRI, is 70% similar to the coding region of type I 5S rDNA and 66% similar to the coding region of type II 5S rDNA. Membrane hybridization and PCR amplification of the sequence showed that PcP190EcoRI is tandemly repeated. The satellite DNA as well as type I and type II 5S rDNA were localized in P. cuvieri chromosomes by fluorescent in situ hybridization. The PcP190EcoRI sequence was found in the centromeres of chromosomes 1–5 and in the pericentromeric region of chromosome 3. Type I 5S rDNA was detected in chromosome 3, coincident with the site of PcP190EcoRI. Type II 5S rDNA was located interstitially in the long arm of chromosome 5. None of these sequences co-localized with nucleolar organizer regions. Our data suggests that this satellite DNA originates from the 5S ribosomal multigene family, probably by gene duplication, nucleotide divergence and sequence dispersion in the genome.

BackgroundThe taxonomic and phylogenetic relationships of the genus Phyllomedusa have been amply discussed. The marked morphological similarities among some species hamper the reliable identification of specimens and may often lead to their incorrect taxonomic classification on the sole basis of morphological traits. Phenotypic variation was observed among populations assigned to either P. azurea or P. hypochondrialis. In order to evaluate whether the variation observed in populations assigned to P. hypochondrialis is related to that in genotypes, a cytogenetic analysis was combined with phylogenetic inferences based on mitochondrial and nuclear sequences.ResultsThe inter- and intra-population variation in the external morphology observed among the specimens analyzed in the present study do not reflect the phylogenetic relationships among populations. A monophyletic clade was recovered, grouping all the specimens identified as P. hypochondrialis and specimens assigned P. azurea from Minas Gerais state. This clade is characterized by conserved chromosomal morphology and a common C-banding pattern. Extensive variation in the nucleolar organizing region (NOR) was observed among populations, with four distinct NOR positions being recognized in the karyotypes. Intra-population polymorphism of the additional rDNA clusters observed in specimens from Barreiras, Bahia state, also highlights the marked genomic instability of the rDNA in the genome of this group. Based on the topology obtained in the phylogenetic analyses, the re-evaluation of the taxonomic status of the specimens from the southernmost population known in Brazil is recommended.ConclusionsThe results of this study support the need for a thorough revision of the phenotypic features used to discriminate P. azurea and P. hypochondrialis. The phylogenetic data presented here also contribute to an extension of the geographic range of P. hypochondrialis, which is known to occur in the Amazon basin and neighboring areas of the Cerrado savanna, where it may be sympatric with P. azurea, within contact zones. The misidentification of specimens may have led to inconsistencies in the original definition of the geographic range of P. azurea. The variability observed in the NOR of P. hypochondrialis reinforces the conclusion that these sites represent hotspots of rearrangement. Intraspecific variation in the location of these sites is the result of constant rearrangements that are not detected by classical cytogenetic methods or are traits of an ancestral, polymorphic karyotype, which would not be phylogenetically informative for this group.