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.

Heteromorphisms between sex chromosomes are rarely found in anurans and sex chromosome differentiation is considered to be a set of recent recurrent events in the evolutionary history of this group. This paper describes for the first time heteromorphic sex chromosomes Z and W in the leiuperid genus Physalaemus. They were found in P. ephippifer, a species of the P. cuvieri group, and corresponded to the eighth pair of its karyotype. The W chromosome differed from the Z chromosome by the presence of an additional segment in the short arm, composed of a distal NOR and an adjacent terminal DAPI-positive C-band. The identification of this sex chromosome pair may help in future investigations into the sex determining genes in the genus Physalaemus.

BackgroundSatellite DNA sequences are the most abundant components of heterochromatin and are repeated in tandem hundreds to thousands of times in the genome. However, the number of repeats of a specific satellite family can vary even between the genomes of related species or populations. The PcP190 satellite DNA family was identified in the genome of the leptodactylid frog Physalaemus cuvieri, which showed to be derived most likely from the 5S rDNA in an ancestral species. In this study, we investigate the presence of the PcP190 satellite DNA in several P. cuvieri populations and in four closely related species at the chromosomal and molecular level. Furthermore, we investigate the occurrence of this satellite DNA in the genomes of P. marmoratus as well as in representative species of the leptodactylid genus Leptodactylus (L. latrans) and the hylodid family (Crossodactylus gaudichaudii), all with the aim of investigating if the PcP190 satellite DNA presents or not a restricted distribution.ResultsThe PcP190 satellite DNA was detected in all the analyzed species. Some of them exhibited particular sequence differences, allowing the identification of species-specific groups of sequences, but in other species, the sequences were more conserved. However, in a general analysis, conserved and variable domains have been recognized within the PcP190 monomer. The chromosomal analysis performed on P. cuvieri populations and closely related species revealed high variability of the satellite DNA amount and its chromosomal location, which has always been coincident with regions of centromeric/pericentromeric heterochromatin.ConclusionThe PcP190 satellite DNA was found in representatives of two families, Leptodactylidae and Hylodidae, indicating that these sequences are widely distributed and conserved in these frogs. There is a pattern of non-random variation within the repeating units, indicating interplay between stochastic events and selective pressure along the PcP190 sequences. Karyotypic differences involving the PcP190 satellite DNA prove to be highly dynamic on the chromosomes of the Physalaemus and its differential accumulation has contributed to the differentiation process of the Z and W sex chromosomes in P. ephippifer.

BackgroundDendropsophus is a monophyletic anuran genus with a diploid number of 30 chromosomes as an important synapomorphy. However, the internal phylogenetic relationships of this genus are poorly understood. Interestingly, an intriguing interspecific variation in the telocentric chromosome number has been useful in species identification. To address certain uncertainties related to one of the species groups of Dendropsophus, the D. microcephalus group, we carried out a cytogenetic analysis combined with phylogenetic inferences based on mitochondrial sequences, which aimed to aid in the analysis of chromosomal characters. Populations of Dendropsophus nanus, Dendropsophus walfordi, Dendropsophus sanborni, Dendropsophus jimi and Dendropsophus elianeae, ranging from the extreme south to the north of Brazil, were cytogenetically compared. A mitochondrial region of the ribosomal 12S gene from these populations, as well as from 30 other species of Dendropsophus, was used for the phylogenetic inferences. Phylogenetic relationships were inferred using maximum parsimony and Bayesian analyses.ResultsThe species D. nanus and D. walfordi exhibited identical karyotypes (2n = 30; FN = 52), with four pairs of telocentric chromosomes and a NOR located on metacentric chromosome pair 13. In all of the phylogenetic hypotheses, the paraphyly of D. nanus and D. walfordi was inferred. D. sanborni from Botucatu-SP and Torres-RS showed the same karyotype as D. jimi, with 5 pairs of telocentric chromosomes (2n = 30; FN = 50) and a terminal NOR in the long arm of the telocentric chromosome pair 12. Despite their karyotypic similarity, these species were not found to compose a monophyletic group. Finally, the phylogenetic and cytogenetic analyses did not cluster the specimens of D. elianeae according to their geographical occurrence or recognized morphotypes.ConclusionsWe suggest that a taxonomic revision of the taxa D. nanus and D. walfordi is quite necessary. We also observe that the number of telocentric chromosomes is useful to distinguish among valid species in some cases, although it is unchanged in species that are not necessarily closely related phylogenetically. Therefore, inferences based on this chromosomal character must be made with caution; a proper evolutionary analysis of the karyotypic variation in Dendropsophus depends on further characterization of the telocentric chromosomes found in this group.