BackgroundThe 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.ResultsAplastodiscus 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.ConclusionsBased 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.

BackgroundThe karyotypes of Leptodactylus species usually consist of 22 bi-armed chromosomes, but morphological variations in some chromosomes and even differences in the 2n have been reported. To better understand the mechanisms responsible for these differences, eight species were analysed using classical and molecular cytogenetic techniques, including replication banding with BrdU incorporation.ResultsDistinct chromosome numbers were found: 2n = 22 in Leptodactylus chaquensis, L. labyrinthicus, L. pentadactylus, L. petersii, L. podicipinus, and L. rhodomystax; 2n = 20 in Leptodactylus sp. (aff. podicipinus); and 2n = 24 in L. marmoratus. Among the species with 2n = 22, only three had the same basic karyotype. Leptodactylus pentadactylus presented multiple translocations, L. petersii displayed chromosome morphological discrepancy, and L. podicipinus had four pairs of telocentric chromosomes. Replication banding was crucial for characterising this variability and for explaining the reduced 2n in Leptodactylus sp. (aff. podicipinus). Leptodactylus marmoratus had few chromosomes with a similar banding patterns to the 2n = 22 karyotypes. The majority of the species presented a single NOR-bearing pair, which was confirmed using Ag-impregnation and FISH with an rDNA probe. In general, the NOR-bearing chromosomes corresponded to chromosome 8, but NORs were found on chromosome 3 or 4 in some species. Leptodactylus marmoratus had NORs on chromosome pairs 6 and 8. The data from C-banding, fluorochrome staining, and FISH using the telomeric probe helped in characterising the repetitive sequences. Even though hybridisation did occur on the chromosome ends, telomere-like repetitive sequences outside of the telomere region were identified. Metaphase I cells from L. pentadactylus confirmed its complex karyotype constitution because 12 chromosomes appeared as ring-shaped chain in addition to five bivalents.ConclusionsSpecies of Leptodactylus exhibited both major and minor karyotypic differences which were identified by classical and molecular cytogenetic techniques. Replication banding, which is a unique procedure that has been used to obtain longitudinal multiple band patterns in amphibian chromosomes, allowed us to outline the general mechanisms responsible for these karyotype differences. The findings also suggested that L. marmoratus, which was formerly included in the genus Adenomera, may have undergone great chromosomal repatterning.

Heteromorphic sex chromosomes are common in eukaryotes and largely ubiquitous in birds and mammals. The largest number of multiple sex chromosomes in vertebrates known today is found in the monotreme platypus (Ornithorhynchus anatinus, 2n = 52) which exhibits precisely 10 sex chromosomes. Interestingly, fish, amphibians, and reptiles have sex determination mechanisms that do or do not involve morphologically differentiated sex chromosomes. Relatively few amphibian species carry heteromorphic sex chromosomes, and when present, they are frequently represented by only one pair, either XX:XY or ZZ:ZW types. Here, in contrast, with several evidences, from classical and molecular cytogenetic analyses, we found 12 sex chromosomes in a Brazilian population of the smoky jungle frog, designated as Leptodactylus pentadactylus Laurenti, 1768 (Leptodactylinae), which has a karyotype with 2n = 22 chromosomes. Males exhibited an astonishing stable ring-shaped meiotic chain composed of six X and six Y chromosomes. The number of sex chromosomes is larger than the number of autosomes found, and these data represent the largest number of multiple sex chromosomes ever found among vertebrate species. Additionally, sequence and karyotype variation data suggest that this species may represent a complex of species, in which the chromosomal rearrangements may possibly have played an important role in the evolution process.

Most eukaryotic genomes contain substantial portions of repetitive DNA sequences. These are located primarily in highly compacted heterochromatin and, in many cases, are one of the most abundant components of the sex chromosomes. In this sense, the anuran Proceratophrys boiei represents an interesting model for analyses on repetitive sequences by means of cytogenetic techniques, since it has a karyotype with large blocks of heterochromatin and a ZZ/ZW sex chromosome system. The present study describes, for the first time, families of satellite DNA (satDNA) in the frog P. boiei. Its genome size was estimated at 1.6 Gb, of which 41% correspond to repetitive sequences, including satDNAs, rDNAs, transposable elements, and other elements characterized as non-repetitive. The satDNAs were mapped by FISH in the centromeric and pericentromeric regions of all chromosomes, suggesting a possible involvement of these sequences in centromere function. SatDNAs are also present in the W sex chromosome, occupying the entire heterochromatic area, indicating a probable contribution of this class of repetitive DNA to the differentiation of the sex chromosomes in this species. This study is a valuable contribution to the existing knowledge on repetitive sequences in amphibians. We show the presence of repetitive DNAs, especially satDNAs, in the genome of P. boiei that might be of relevance in genome organization and regulation, setting the stage for a deeper functional genome analysis of Proceratophrys.

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