Multiple contact zones and karyotypic evolution in a neotropical frog species complex.

Previous studies of DNA sequence and karyotypic data have revealed high genetic diversity in the Physalaemus cuvieri - Physalaemus ephippifer species complex-a group of small leptodactylid frogs in South America. To date, seven major genetic lineages have been recognized in this group, with species delimitation tests supporting four to seven of them as valid species. Among these, only P. ephippifer shows heteromorphic sex chromosomes, but the implications of cytogenetic divergence for the evolution of this group are unknown. We analyzed karyotypic, mitochondrial DNA, and 3RAD genomic data to characterize a putative contact zone between P. ephippifer and P. cuvieri Lineage 1, finding evidence for admixture and karyotypic evolution. We also describe preliminary evidence for admixture between two other members of this species complex-Lineage 1 and Lineage 3 of P. cuvieri. Our study sheds new light on evolutionary relationships in the P. cuvieri - P. ephippifer species complex, suggesting an important role of karyotypic divergence in its evolutionary history and underscoring the importance of hybridization as a mechanism of sex chromosome evolution in amphibians.

Cytogenetics of four foam-nesting frog species of the Physalaemus gracilis group (Anura, Leptodactylidae).

Intending to increase the knowledge about cytogenetics of Physalaemus and the sparsely studied P. gracilis group, we analyzed the karyotypes of P. carrizorum, P. gracilis, P. lisei, and P. sp. aff. gracilis. We studied chromosome morphology, heterochromatin patterns, Ag-NORs location and mapped the repetitive DNA sequence PcP190. All species showed diploid karyotypes composed of 22 bi-armed chromosomes and similar C- bands and Ag-NOR patterns. C-bands were mainly centromeric and pericentromeric; non-centromeric C-bands were detected on the telomeres of pair 1 in P. lisei, although polymorphic, and interstitially on pair 10 of P. gracilis. This last character is useful to distinguish P. gracilis from its sibling species P. sp. aff. gracilis. The Ag-NOR sites were detected on the long arms of chromosome pair 8 but with a variable position among species. Clusters of PcP190 showed centromeric and pericentromeric positions coincident with conspicuous C-bands, on pairs 2 and 9 in P. gracilis and P. sp. aff. gracilis, pair 3 in P. carrizorum, and pair 7 in P. lisei. These results significantly increase the knowledge about Physalaemus cytogenetics and encourage further studies on the satellite PcP190 in other genera of Leiuperinae to better understand its taxonomic distribution and the evolutionary dynamics.

Sex chromosomes in the Vizcacheras' White-lipped frog, Leptodactylus bufonius (Anura, Leptodactylidae).

Cytogenetic analyses were performed on specimens of Leptodactylus bufonius from different localities in Argentina. Mitotic chromosomes were studied with Giemsa and differential staining techniques (Ag-NOR, C-banding, and CMA3/DAPI) and fluorescence in situ hybridization with the 18S DNAr probe. All specimens showed karyotypes with 2n = 2x = 22 and FN = 44. Secondary constrictions were present in the long arm of chromosome pair 8, coincident with Ag-NOR and hybridization signals of the 18S DNAr probe. The C-banding technique evidenced an important amount of heterochromatin with a sex-linked pericentromeric band in the short arm of chromosome pair 4. This heterochromatic band was heteromorphic in males but present in both homologues of females, and it was CMA3 positive (DAPI negative) at fluorescence staining. The occurrence of heteromorphic XY sex chromosomes in L. bufonius is the second known case in Leptodactylus and the fifth within the speciose family Leptodactylidae.

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

The hyline tribe Lophyohylini includes 87 species of treefrogs, of which cytogenetics aspects have been studied in less than 20% of them. In order to evaluate the evolution of some of its chromosome characters (NOR position, C-bands, and DAPI/CMA3 bands), we studied the karyotypes of 21 lophyohylines, 16 of them for the first time, and analyzed them in a phylogenetic context. Most species showed similar karyotypes regarding chromosome number (2n = 24) and morphology (FN = 48), excepting Phyllodytes edelmoi and Osteocephalus buckleyi with 2n = 22 (FN = 44) and 2n = 28 (FN = 50), respectively. The NOR location was variable among species and provided valuable phylogenetic information. This marker was located in pair 11 in all species of Trachycephalus, Itapotihyla langsdorffii, and Nyctimantis arapapa, representing the plesiomorphic condition of Lophyohylini. Besides, other apomorphic states were recovered for the clades comprising N. rugiceps and N. siemersi (NOR in pair 5), and Dryaderces pearsoni, Osteocephalus, and Osteopilus (NOR in pair 9). Phyllodytes presented variation for NORs position; they were in pair 2 in P. edelmoi, pair 7 in P. melanomystax, and pair 8 in P. gyrinaethes and P. praeceptor. Polymorphisms in size, number, and activity of this marker were observed for N. siemersi, Osteocephalus fuscifacies, and some species of Trachycephalus. Remarkably, in N. siemersi NORs were detected on a single chromosome in the two specimens studied by this technique, raising the question of how this complex polymorphism is maintained. Interstitial telomeric sequences were found in P. edelmoi, P. melanomystax, and Osteocephalus buckleyi, and their presence seems to be not related to the chromosome reorganization events. Finally, some species showed spontaneous rearrangements, possibly as a consequence of an uncommon phenomenon in anuran cytogenetics: the presence of fragile sites or secondary constrictions not associated with NORs. We propose that this rare feature would have played an important role in the evolution of this group of frogs. From the evidence obtained in this and previous studies, we conclude that Lophyohylini presents a complex chromosome evolution.

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.