Comparative analysis based on replication banding reveals the mechanism responsible for the difference in the karyotype constitution of treefrogs Ololygon and Scinax (Arboranae, Hylidae, Scinaxinae).

According to the recent taxonomic and phylogenetic revision of the family Hylidae, species of the former Scinax catharinae (Boulenger, 1888) clade were included in the resurrected genus Ololygon Fitzinger, 1843, while species of the Scinax ruber (Laurenti, 1768) clade were mostly included in the genus Scinax Wagler, 1830, and two were allocated to the newly created genus Julianus Duellman et al., 2016. Although all the species of the former Scinax genus shared a diploid number of 2n = 24 and the same fundamental number of chromosome arms of FN = 48, two karyotypic constitutions were unequivocally recognized, related mainly to the distinct size and morphology of the first two chromosome pairs. Some possible mechanisms for these differences had been suggested, but without any experimental evidence. In this paper, a comparison was carried out based on replication chromosome banding, obtained after DNA incorporation of 5-bromodeoxiuridine in chromosomes of Ololygon and Scinax. The obtained results revealed that the loss of repetitive segments in chromosome pairs 1 and 2 was the mechanism responsible for karyotype difference. The distinct localization of the nucleolus organizer regions in the species of both genera also differentiates the two karyotypic constitutions.

Possible interspecific origin of the B chromosome of Hypsiboas albopunctatus (Spix, 1824) (Anura, Hylidae), revealed by microdissection, chromosome painting, and reverse hybridisation.

The B chromosome in the hylid Hypsiboas albopunctatus (2n = 22 + B) is small, almost entirely composed of C-positive heterochromatin, and does not pair with any chromosome of the A complement. B probe, obtained by microdissection and DOP-PCR amplification, was used to search for homology between the B and regular chromosomes of H. albopunctatus and of the related species H. raniceps (Cope, 1862). Reverse hybridisation was also carried out in the investigation. The B probe exclusively painted the supernumerary, not hybridising any other chromosomes in H. albopunctatus, but all H. raniceps chromosomes showed small labelling signals. This result might be an indication that differences exist between the repetitive sequences of A and B chromosomes of H. albopunctatus, and that the chromosomes of H. raniceps and the heterochromatin of the B chromosome of H. albopunctatus are enriched with the same type of repetitive DNA. In meiotic preparations, the B labelled about 30% of scored spermatids, revealing a non-mendelian inheritance, and the painted B in micronucleus suggests that the supernumerary is eliminated from germ line cells. Although our results could suggest an interespecific origin of the B at first sight, further analysis on its repetitive sequences is still necessary. Nevertheless, the accumulation of repetitive sequences, detected in another species, even though closely related, remains an intriguing question.

Cytogenetic analysis of Phyllomedusa distincta Lutz, 1950 (2n = 2x = 26), P. tetraploidea Pombal and Haddad, 1992 (2n = 4x = 52), and their natural triploid hybrids (2n = 3x = 39) (Anura, Hylidae, Phyllomedusinae).

BACKGROUND: Natural 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. RESULTS: Phyllomedusa 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. CONCLUSIONS: All 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.

Comparative karyotype analysis and chromosome evolution in the genus Aplastodiscus (Cophomantini, Hylinae, Hylidae).

BACKGROUND: The frogs of the Tribe Cophomantini present, in general, 2n = 24 karyotype, but data on Aplastodiscus showed variation in diploid number from 2n = 24 to 2n = 18. Five species were karyotyped, one of them for the first time, using conventional and molecular cytogenetic techniques, with the aim to perform a comprehensive comparative analysis towards the understanding of chromosome evolution in light of the phylogeny. RESULTS: Aplastodiscus perviridis showed 2n = 24, A. arildae and A. eugenioi, 2n = 22, A. callipygius, 2n = 20, and A. leucopygius, 2n = 18. In the metaphase I cells of two species only bivalents occurred, whereas in A. arildae, A. callipygius, and A. leucopygius one tetravalent was also observed besides the bivalents. BrdU incorporation produced replication bands especially in the largest chromosomes, and a relatively good banding correspondence was noticed among some of them. Silver impregnation and FISH with an rDNA probe identified a single NOR pair: the 11 in A. perviridis and A. arildae; the 6 in A. eugenioi; and the 9 in A. callipygius and A. leucopygius. C-banding showed a predominantly centromeric distribution of the heterochromatin, and in one of the species distinct molecular composition was revealed by CMA3. The telomeric probe hybridised all chromosome ends and additionally disclosed the presence of telomere-like sequences in centromeric regions of three species. CONCLUSIONS: Based on the hypothesis of 2n = 24 ancestral karyotype for Aplastodiscus, and considering the karyotype differences and similarities, two evolutionary pathways through fusion events were suggested. One of them corresponded to the reduction of 2n = 24 to 22, and the other, the reduction of 2n = 24 to 20, and subsequently to 18. Regarding the NOR, two conditions were recognised: plesiomorphy, represented by the homeologous small-sized NOR-bearing pairs, and derivation, represented by the NOR in a medium-sized pair. In spite of the apparent uniformity of C-banding patterns, heterogeneity in the molecular composition of some repetitive regions was revealed by CMA3 staining and by interstitial telomeric labelling. The meiotic tetravalent might be due to minute reciprocal translocations or to non-chiasmatic ectopic pairing between terminal repetitive sequences. The comparative cytogenetic analysis allowed to outline the chromosome evolution and contributed to enlighten the relationships within the genus Aplastodiscus.

Karyotype analysis of seven species of the tribe Lophiohylini (Hylinae, Hylidae, Anura), with conventional and molecular cytogenetic techniques.

Few species of the tribe Lophiohylini have been karyotyped so far, and earlier analyses were performed mainly with standard staining. Based on the analysis of seven species with use of routine banding and molecular cytogenetic techniques, the karyotypes were compared and the cytogenetic data were evaluated in the light of the current phylogenies. A karyotype with 2n = 24 and NOR in the chromosome 10 detected by Ag-impregnation and FISH with an rDNA probe was shared by Aparasphenodon bokermanni Miranda-Ribeiro, 1920, Itapotihyla langsdorffii (Duméril and Bibron, 1841), Trachycephalus sp., Trachycephalus mesophaeus (Hensel, 1867), and Trachycephalus typhonius (Linnaeus, 1758). Phyllodytes edelmoi Peixoto, Caramaschi et Freire, 2003 and Phyllodytes luteolus (Wied-Neuwied, 1824) had reduced the diploid number from 2n = 24 to 2n = 22 with one of the small-sized pairs clearly missing, and NOR in the large chromosome 2, but the karyotypes were distinct regarding the morphology of chromosome pairs 4 and 6. Based on the cytogenetic and phylogenetic data, it was presumed that the chromosome evolution occurred from an ancestral type with 2n = 24, in which a small chromosome had been translocated to one or more unidentified chromosomes. Whichever hypothesis is more probable, other rearrangements should have occurred later, to explain the karyotype differences between the two species of Phyllodytes Wagler, 1830. The majority of the species presented a small amount of centromeric C-banded heterochromatin and these regions were GC-rich. The FISH technique using a telomeric probe identified the chromosome ends and possibly (TTAGGG)n-like sequences in the repetitive DNA out of the telomeres in Itapotihyla langsdorffii and Phyllodytes edelmoi. The data herein obtained represent an important contribution for characterizing the karyotype variability within the tribe Lophiohylini scarcely analysed so far.

Chromosome banding in three species of Hypsiboas (Hylidae, Hylinae), with special reference to a new case of B-chromosome in anuran frogs and to the reduction of the diploid number of 2n = 24 to 2n = 22 in the genus.

The chromosomes of hylids Hypsiboas albopunctatus, H. raniceps, and H. crepitans from Brazil were analyzed with standard and differential staining techniques. The former species presented 2n = 22 and 2n = 23 karyotypes, the odd diploid number is due to the presence of an extra element interpreted as B chromosome. Although morphologically very similar to the small-sized chromosomes of the A complement, the B was promptly recognized, even under standard staining, on the basis of some characteristics that are usually attributed to this particular class of chromosomes. The two other species have 2n = 24, which is the chromosome number usually found in the species of Hypsiboas karyotyped so far. This means that 2n = 22 is a deviant diploid number, resulted from a structural rearrangement, altering the chromosome number of 2n = 24 to 2n = 22. Based on new chromosome data, some possibilities were evaluated for the origin of B chromosome in Hypsiboas albopunctatus, as well as the karyotypic evolution in the genus, leading to the reduction in the diploid number of 2n = 24 to 2n = 22.