Interspecific cytogenomic comparison reveals a potential chromosomal centromeric marker in Proceratophrys frog species.

Among the repetitive elements, satellite DNA (SatDNA) emerges as extensive arrays of highly similar tandemly repeated units, spanning megabases in length. Given that the satDNA PboSat01-176, previously characterized in P. boiei, prompted our interest for having a high abundance in P. boiei and potential for centromeric satellite, here, we employed various approaches, including low coverage genome sequencing, followed by computational analysis and chromosomal localization techniques in four Proceratophrys species and, investigating the genomic presence and sharing, as well as its potential for chromosomal centromere marker in Proceratophrys frog species. Our findings demonstrate that PboSat01-176 exhibits high abundance across all four Proceratophrys species, displaying distinct characteristics that establish it as the predominant repetitive DNA element in these species. The satellite DNA is prominently clustered in the peri/centromeric region of the chromosomes, particularly in the heterochromatic regions. The widespread presence of PboSat01-176 in closely related Proceratophrys species reinforces the validity of the library hypothesis for repetitive sequences. Thus, this study highlighted the utility of the satDNA family PboSat01-176 as a reliable centromeric marker in Proceratophrys species, with potential to be applied in other species of anuran amphibians. The observed sharing and maintenance of this sequence within the genus suggest possibilities for future research, particularly through expanded sampling to elucidate parameters that underlie the library hypothesis and the evolutionary dynamics of satDNA sequences.

Analysis in Proceratophrys boiei genome illuminates the satellite DNA content in a frog from the Brazilian Atlantic forest.

Satellite DNAs (satDNAs) are one of the most abundant elements in genomes. Characterized as tandemly organized sequences that can be amplified into multiple copies, mainly in heterochromatic regions. The frog P. boiei (2n = 22, ZZ♂/ZW♀) is found in the Brazilian Atlantic forest and has an atypical pattern of heterochromatin distribution when compared to other anuran amphibians, with large pericentromeric blocks on all chromosomes. In addition, females of Proceratophrys boiei have a metacentric sex chromosome W showing heterochromatin in all chromosomal extension. In this work, we performed high-throughput genomic, bioinformatic, and cytogenetic analyses to characterize the satellite DNA content (satellitome) in P. boiei, mainly due to high amount of C-positive heterochromatin and the highly heterochromatic W sex chromosome. After all the analyses, it is remarkable that the satellitome of P. boiei is composed of a high number of satDNA families (226), making P. boiei the frog species with the highest number of satellites described so far. Consistent with the observation of large centromeric C-positive heterochromatin blocks, the genome of P. boiei is enriched with high copy number of repetitive DNAs, with total satDNA abundance comprising 16.87% of the genome. We successfully mapped via Fluorescence in situ hybridization the two most abundant repeats in the genome, PboSat01-176 and PboSat02-192, highlighting the presence of certain satDNAs sequences in strategic chromosomal regions (e.g., centromere and pericentromeric region), which leads to their participation in crucial processes for genomic organization and maintenance. Our study reveals a great diversity of satellite repeats that are driving genomic organization in this frog species. The characterization and approaches regarding satDNAs in this species of frog allowed the confirmation of some insights from satellite biology and a possible relationship with the evolution of sex chromosomes, especially in anuran amphibians, including P. boiei, for which data were not available.

Cytogenetic approaches provide evidence of a conserved diploid number and cytological differences between Proceratophrys species (Anura: Odontophrynidae).

Taxonomic and cytogenetic aspects of Proceratophrys have not been thoroughly clarified in the literature; thus, the objective of the present study was to provide unprecedent karyotype data regarding P. schirchi, P. laticeps and P. melanopogon. Additionally, the karyotype of P. boiei (2n = 22) and its ZZ / ZW sex chromosome system was analyzed for different populations of southeastern and southern Brazil. All Proceratophrys species have a diploid number of 2n = 22 chromosomes. In P. schirchi, a strong C-band was found in the long arm in one of the homologues of the pair 8 in female metaphasic cells, denoting that this chromosome pair could represent the heteromorphic sex chromosome in a ZZ / ZW sex system. Despite the conserved number of chromosomes, there are considerable chromosomal differences in P. melanopogon and P. boiei (southern Brazil), strongly differentiating them cytogenetically from other species of the genus. Moreover, with the confirmation of chromosomal heteromorphism related to sexual differentiation in P. boiei and the possible description of this system in P. schirchi, the Proceratophrys genus can be regarded as an interesting group for evolutionary studies and sex chromosome differentiation in anurans.

Chromosome Mapping of U2 snDNA in Species of Leptodactylus (Anura, Leptodactylidae).

Small nuclear RNA (snRNA) is a class of molecules involved in the processing of pre-mRNA and in regulatory cell processes. snRNAs are always associated with a set of specific proteins. The complexes are referred to as small nuclear ribonucleoproteins, and spliceosome U RNAs are their most common snRNA components. The repetitive sequences of U snDNAs have been cytogenetically mapped in several species of Arthropoda, fishes, and mammals; however, their distribution remains unknown in amphibians. Here, we show results of FISH mapping of U2 snDNA repetitive sequences in species of the amphibian genus Leptodactylus to reveal the distribution patterns of this sequence in their karyotypes. The probe hybridized in the metacentric chromosome pair 6 in Leptodactylus fuscus, L. gracilis, L. latrans, L. chaquensis, L. petersii, L. podicipinus, and L. brevipes. A different pattern was observed in L. labyrinthicus with hybridization signals in 4 chromosome pairs. The same localization of U2 gene sequences in most of the species analyzed suggests a relatively conserved pattern and a similarity of the chromosome 6 among these species of Leptodactylus.

Evolutionary Dynamics of the Repetitive DNA in the Karyotypes of Pipa carvalhoi and Xenopus tropicalis (Anura, Pipidae).

The large amphibian genomes contain numerous repetitive DNA components that have played an important role in the karyotypic diversification of this vertebrate group. Hypotheses based on the presumable primitive karyotype (2n = 20) of the anurans of the family Pipidae suggest that they have evolved principally through intrachromosomal rearrangements. Pipa is the only South American pipid, while all the other genera are found in Africa. The divergence of the South American lineages from the African ones occurred at least 136 million years ago and is thought to have had a strong biogeographic component. Here, we tested the potential of the repetitive DNA to enable a better understanding of the differentiation of the karyotype among the family Pipidae and to expand our capacity to interpret the chromosomal evolution in this frog family. Our results indicate a long history of conservation in the chromosome bearing the H3 histone locus, corroborating inferences on the chromosomal homologies between the species in pairs 6, 8, and 9. The chromosomal distribution of the microsatellite motifs also provides useful markers for comparative genomics at the chromosome level between Pipa carvalhoi and Xenopus tropicalis, contributing new insights into the evolution of the karyotypes of these species. We detected similar patterns in the distribution and abundance of the microsatellite arrangements, which reflect the shared organization in the terminal/subterminal region of the chromosomes between these two species. By contrast, the microsatellite probes detected a differential arrangement of the repetitive DNA among the chromosomes of the two species, allowing longitudinal differentiation of pairs that are identical in size and morphology, such as pairs 1, 2, 4, and 5. We also found evidence of the distinctive composition of the repetitive motifs of the centromeric region between the species analyzed in the present study, with a clear enrichment of the (CA) and (GA) microsatellite motifs in P. carvalhoi. Finally, microsatellite enrichment in the pericentromeric region of chromosome pairs 6, 8, and 9 in the P. carvalhoi karyotype, together with interstitial telomeric sequences (ITS), validate the hypothesis that pericentromeric inversions occurred during the chromosomal evolution of P. carvalhoi and reinforce the role of the repetitive DNA in the remodeling of the karyotype architecture of the Pipidae.

Great Abundance of Satellite DNA in Proceratophrys (Anura, Odontophrynidae) Revealed by Genome Sequencing.

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.

Chromosome spreading of the (TTAGGG)n repeats in the Pipa carvalhoi Miranda-Ribeiro, 1937 (Pipidae, Anura) karyotype.

Pipidae is a clade of Anura that diverged relatively early from other frogs in the phylogeny of the group. Pipids have a unique combination of morphological features, some of which appear to represent a mix of adaptations to aquatic life and plesiomorphic characters of Anura. The present study describes the karyotype of Pipa carvalhoi Miranda-Ribeiro, 1937, including morphology, heterochromatin distribution, and location of the NOR site. The diploid number of P. carvalhoi is 2n=20, including three metacentric pairs (1, 4, 8), two submetacentric (2 and 7), three subtelocentric (3, 5, 6), and two telocentric pairs (9 and 10). C-banding detected centromeric blocks of heterochromatin in all chromosome pairs and the NOR detected in chromosome pair 9, as confirmed by FISH using the rDNA 28S probe. The telomeric probes indicated the presence of interstitial telomeric sequences (ITSs), primarily in the centromeric region of the chromosomes, frequently associated with heterochromatin, suggesting that these repeats are a significant component of this region. The findings of the present study provide important insights for the understanding of the mechanisms of chromosomal evolution in the genus Pipa, and the diversification of the Pipidae as a whole.

Chromosome evolution in Cophomantini (Amphibia, Anura, Hylinae).

The hylid tribe Cophomantini is a diverse clade of Neotropical treefrogs composed of the genera Aplastodiscus, Boana, Bokermannohyla, Hyloscirtus, and Myersiohyla. The phylogenetic relationships of Cophomantini have been comprehensively reviewed in the literature, providing a suitable framework for the study of chromosome evolution. Employing different banding techniques, we studied the chromosomes of 25 species of Boana and 3 of Hyloscirtus; thus providing, for the first time, data for Hyloscirtus and for 15 species of Boana. Most species showed karyotypes with 2n = 2x = 24 chromosomes; some species of the B. albopunctata group have 2n = 2x = 22, and H. alytolylax has 2n = 2x = 20. Karyotypes are all bi-armed in most species presented, with the exception of H. larinopygion (FN = 46) and H. alytolylax (FN = 38), with karyotypes that have a single pair of small telocentric chromosomes. In most species of Boana, NORs are observed in a single pair of chromosomes, mostly in the small chromosomes, although in some species of the B. albopunctata, B. pulchella, and B. semilineata groups, this marker occurs on the larger pairs 8, 1, and 7, respectively. In Hyloscirtus, NOR position differs in the three studied species: H. alytolylax (4p), H. palmeri (4q), and H. larinopygion (1p). Heterochromatin is a variable marker that could provide valuable evidence, but it would be necesserary to understand the molecular composition of the C-bands that are observed in different species in order to test its putative homology. In H. alytolylax, a centromeric DAPI+ band was observed on one homologue of chromosome pair 2. The band was present in males but absent in females, providing evidence for an XX/XY sex determining system in this species. We review and discuss the importance of the different chromosome markers (NOR position, C-bands, and DAPI/CMA3 patterns) for their impact on the taxonomy and karyotype evolution in Cophomantini.

Cytogenetic analyses of eight species in the genus Leptodactylus Fitzinger, 1843 (Amphibia, Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations.

BACKGROUND: The 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. RESULTS: Distinct 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. CONCLUSIONS: Species 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.