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.

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.

Recently, Physalaemus albifrons (Spix, 1824) was relocated from the Physalaemus cuvieri group to the same group as Physalaemus biligonigerus (Cope, 1861), Physalaemus marmoratus (Reinhardt & Lütken, 1862) and Physalaemus santafecinus Barrio, 1965. To contribute to the analysis of this proposition, we studied the karyotypes of Physalaemus albifrons, Physalaemus santafecinus and three species of the Physalaemus cuvieri group. The karyotype of Physalaemus santafecinus was found to be very similar to those of Physalaemus biligonigerus and Physalaemus marmoratus, which were previously described. A remarkable characteristic that these three species share is a conspicuous C-band that extends from the pericentromeric region almost to the telomere in the short arm of chromosome 3. This characteristic is not present in the Physalaemus albifrons karyotype and could be a synapomorphy of Physalaemus biligonigerus, Physalaemus marmoratus and Physalaemus santafecinus. The karyotype of Physalaemus santafecinus is also similar to those of Physalaemus marmoratus and Physalaemus biligonigerus owing to the presence of several terminal C-bands and the distal localization of the NOR in a small metacentric chromosome. In contrast, the Physalaemus albifrons karyotype has no terminal C-bands and its NOR is located interstitially in the long arm of submetacentric chromosome 8. The NOR-bearing chromosome of Physalaemus albifrons very closely resembles those found in Physalaemus albonotatus (Steindachner, 1864), Physalaemus cuqui Lobo, 1993 and some populations of Physalaemus cuvieri Fitzinger, 1826. Additionally, the Physalaemus albifrons karyotype has an interstitial C-band in chromosome 5 that has been exclusively observed in species of the Physalaemus cuvieri group. Therefore, we were not able to identify any chromosomal feature that supports the reallocation of Physalaemus albifrons.

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All the species of Physalaemus Fitzinger, 1826 karyotyped up until now have been classified as 2n = 22. The species of the Physalaemus cuvieri group analyzed by C-banding present a block of heterochromatin in the interstitial region of the short arm of pair 5. Physalaemus cicada Bokermann, 1966 has been considered to be a member of the Physalaemus cuvieri species group, although its interspecific phylogenetic relationships remain unknown. The PcP190 satellite DNA has been mapped on the chromosomes of most of the species of the Physalaemus cuvieri group. For two species, Physalaemus cicada and Physalaemus kroyeri (Reinhardt & Lütken, 1862), however, only the chromosome number and morphology are known. Given this, the objective of the present study was to analyze the chromosomes of Physalaemus cicada and Physalaemus kroyeri, primarily by C-banding and PcP190 mapping. The results indicate that Physalaemus kroyeri and Physalaemus cicada have similar karyotypes, which were typical of Physalaemus. In both species, the NORs are located on the long arm of pair 8, and the C-banding indicated that, among other features, Physalaemus kroyeri has the interstitial band on chromosome 5, which is however absent in Physalaemus cicada. Even so, a number of telomeric bands were observed in Physalaemus cicada. The mapping of the PcP190 satellite DNA highlighted areas of the centromeric region of the chromosomes of pair 1 in both species, although in Physalaemus kroyeri, heteromorphism was also observed in pair 3. The cytogenetic evidence does not support the inclusion of Physalaemus cicada in the Physalaemus cuvieri group. In the case of Physalaemus kroyeri, the interstitial band on pair 5 is consistent with the existence of a cytogenetic synapomorphy in the Physalaemus cuvieri species group.

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The nominal anuran species Crossodactylus gaudichaudii Duméril and Bibron, 1841 and Crossodactylus aeneus Müller, 1924 are indistinguishable based on adult and larval morphology, being subject of taxonomic doubts. Here, we describe the karyotypes of C. gaudichaudii and C. aeneus, using classical and molecular cytogenetic markers. In addition, we used sequences of the H1 mitochondrial DNA to infer their phylogenetic relationships by Maximum Likelihood (ML) and Maximum Parsimony (MP) approaches and species delimitation test (by bPTP approach). The karyotypic data do not differentiate C. gaudichaudii and C. aeneus in any of the chromosome markers assessed. In both phylogenetic analyses, C. gaudichaudii and C. aeneus were recovered into a strongly supported clade. The species delimitation analysis recovered the specimens assigned to C. gaudichaudii and C. aeneus as a single taxonomic unit. Taken the cytogenetic and genetic results together with previous studies of internal and external morphology of tadpoles and biacoustic pattern, C. gaudichaudii and C. aeneus could not be differentiated, which supports the hypothesis that they correspond to the same taxonomic unit, with C. aeneus being a junior synonym of C. gaudichaudii.