We studied ten specimens of Physuluemus cutiieri collected at different localities in Brazil using conventional staining and banding techniques. All specimens had 2n = 22. There were karyotypic variants: distinct patterns in the number and chromosome localization of Ag-NORs as well as in the corresponding secondary constrictions. Preliminary C-banding patterns obtained for specimens from two localities are also suggestive of karyotypic differentiation in P. cucieri.Sunue Kasahara,

BackgroundNatural polyploidy has played an important role during the speciation and evolution of vertebrates, including anurans, with more than 55 described cases. The species of the Phyllomedusa burmeisteri group are mostly characterized by having 26 chromosomes, but a karyotype with 52 chromosomes was described in P. tetraploidea. This species was found in sintopy with P. distincta in two localities of São Paulo State (Brazil), where triploid animals also occur, as consequence of natural hybridisation. We analyse the chromosomes of P. distincta, P. tetraploidea, and their triploid hybrids, to enlighten the origin of polyploidy and to obtain some evidence on diploidisation of tetraploid karyotype.ResultsPhyllomedusa distincta was 2n = 2x = 26, whereas P. tetraploidea was 2n = 4x = 52, and the hybrid individuals was 2n = 3x = 39. In meiotic phases, bivalents were observed in the diploid males, whereas both bivalents and tetravalents were observed in the tetraploid males. Univalents, bivalents or trivalents; metaphase II cells carrying variable number of chromosomes; and spermatids were detected in the testis preparations of the triploid males, indicating that the triploids were not completely sterile. In natural and experimental conditions, the triploids cross with the parental species, producing abnormal egg clutches and tadpoles with malformations. The embryos and tadpoles exhibited intraindividual karyotype variability and all of the metaphases contained abnormal constitutions. Multiple NORs, detected by Ag-impregnation and FISH with an rDNA probe, were observed on chromosome 1 in the three karyotypic forms; and, additionally, on chromosome 9 in the diploids, mostly on chromosome 8 in the tetraploids, and on both chromosome 8 and 9 in the triploids. Nevertheless, NOR-bearing chromosome 9 was detected in the tetraploids, and chromosome 9 carried active or inactive NORs in the triploids. C-banding, base-specific fluorochrome stainings with CMA3 and DAPI, FISH with a telomeric probe, and BrdU incorporation in DNA showed nearly equivalent patterns in the karyotypes of P. distincta, P. tetraploidea, and the triploid hybrids.ConclusionsAll the used cytogenetic techniques have provided strong evidence that the process of diploidisation, an essential step for stabilising the selective advantages produced by polyploidisation, is under way in distinct quartets of the tetraploid karyotype.

Cryptic species, i.e. morphologically undistinguishable ones, are relatively common among anurans of the Brazilian fauna. The characters generally used to separate these species have been, e.g., bioacoustics, colour patterns, ecology, and ethology of live animals (Cardoso,

Specimens of Leptodactylus mystacinus from Brazil were karyotyped with conventional and differential staining. The 2n = 22 karyotype is similar to that found for the majority of the Leptodactylus, the karyotypic conservatism also confirmed by the similarity of the replication banding patterns with those previously described. L. mystacinus has a small amount of C-banded heterochromatin, located mainly at the centromeres, although telomeric or interstitial bands have also been noticed. With DA/CMA(3) some chromosome regions showed slightly bright fluorescence, and with DA/DAPI, no particular AT-rich repetitive region was observed. Silver staining showed an extensive inter- and intraindividual variation in the number and position of Ag-positive regions, in 1p, 4p, 8p, 8q, and 11p. Nevertheless, FISH using rDNA probes confirmed only the signals on the short arms of chromosomes 4 and 8 as true NORs. The remaining silver stained regions are probably due to the heterochromatin with some affinity to the Ag-staining. Phylogenetic analysis based on partial cytochrome b sequence revealed that L. mystacinus forms a basal branch, so that the presence of multiple NORs in pairs 4 and 8 in this species indicates an autapomorphy.

We describe the standardization of lymphocyte culture procedures in order to improve cytological preparations of anuran species. This methodology permits the use of 5-bromodeoxyuridine (BrdU) treatment to obtain replication banding patterns in the chromosomes of these species.
Descreve-se a padronização da cultura de linfócitos com a finalidade de melhorar as preparações citológicas de espécies de anfíbios anuros. Esta metodologia permite o uso do tratamento pelo BrdU para se obter padrões de bandas de replicação nos cromossomos dessas espécies