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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.

Satellite DNAs (satDNAs) play a crucial role in understanding chromosomal evolution and the differentiation of sex chromosomes across diverse taxa, particularly when high karyotypic diversity occurs. The Physalaemus cuvieri–Physalaemus ephippifer species complex comprises at least seven divergent lineages, each exhibiting specific karyotypic signatures. The group composed of Ph. ephippifer, Lineage 1B of ‘Ph. cuvieri’ (L1B), and a lineage resulting from their secondary contact is especially intriguing due to varying degrees of sex chromosome heteromorphism. In this study, we characterized the satellitome of Ph. ephippifer in order to identify novel satDNAs that may provide insights into chromosomal evolution, particularly concerning sex chromosomes. We identified 62 satDNAs in Ph. ephippifer, collectively accounting for approximately 10% of the genome. Notably, nine satDNA families were shared with species from distantly related clades, raising questions about their potential roles in anurans genomes. Among the seven satDNAs mapped via fluorescent in situ hybridization, PepSat3 emerged as a strong candidate for the centromeric sequence in this group. Additionally, PepSat11 and PepSat24 provided evidence supporting a translocation involving both arms of the W chromosome in Ph. ephippifer. Furthermore, a syntenic block composed of PepSat3, PcP190, and PepSat11 suggested an inversion event during the divergence of Ph. ephippifer and L1B. The variation in signal patterns of satDNAs associated with nucleolar organizer regions (NORs) highlights the complexity of NOR evolution in this species complex, which exhibits substantial diversity in this genomic region. Additionally, our findings for PepSat30-350 emphasize the importance of validating the sex-biased abundance of satDNAs.

The differentiation of sex chromosomes is thought to be interrupted by relatively frequent sex chromosome turnover and/or occasional recombination between sex chromosomes (fountain-of-youth model) in some vertebrate groups as fishes, amphibians, and lizards. As a result, we observe the prevalence of homomorphic sex chromosomes in these groups. Here, we provide evidence for the loss of sex chromosome heteromorphism in the Amazonian frogs of the genus Engystomops, which harbors an intriguing history of sex chromosome evolution. In this species complex composed of two named species, two confirmed unnamed species, and up to three unconfirmed species, highly divergent karyotypes are present, and heteromorphic X and Y chromosomes were previously found in two species. We describe the karyotype of a lineage estimated to be the sister of all remaining Amazonian Engystomops (named Engystomops sp.) and perform chromosome painting techniques using one probe for the Y chromosome and one probe for the non-centromeric heterochromatic bands of the X chromosome of E. freibergi to compare three Engystomops karyotypes. The Y probe detected the Y chromosomes of E. freibergi and E. petersi and one homolog of chromosome pair 11 of Engystomops sp., suggesting their common evolutionary origin. The X probe showed no interspecific hybridization, revealing that X chromosome heterochromatin is strongly divergent among the studied species. In the light of the phylogenetic relationships, our data suggest that sex chromosome heteromorphism may have occurred early in the evolution of the Amazonian Engystomops and have been lost in two unnamed but confirmed candidate species.

Crossodactylodes is a poorly known genus of small-sized bromeligenous frogs, endemic to Brazil. They have a patchy distribution across the mountains of the Atlantic Forest and the “campo rupestre” ecosystem. To better resolve their evolutionary relationships, we performed phylogenetic analyses using a multigene DNA matrix and representative sampling within the genus. We then evaluated the evolution of phenotypical and natural history traits with the inferred phylogeny. We recovered Crossodactylodes as monophyletic, diagnosed by seven putative synapomorphies in morphological and natural history characters. Evidence supports some morphological synapomorphies as adaptations to the bromeligenous habit. We found high genetic distances among closely distributed lineages within C. bokermanni and C. izecksohni. Some of these lineages might represent undescribed cryptic species. We provide detailed accounts for each species including data on their geographic range, conservation, and natural history. All species of Crossodactylodes occur in highly threatened environments, are restricted to very small geographic ranges, and probably have limited dispersal capacity due to their small body size and dependence on bromeliads. These factors emphasize the need for habitat protection to safeguard species viability.

Crossodactylodes is a poorly known genus of small-sized bromeligenous frogs, endemic to Brazil. They have a patchy distribution across the mountains of the Atlantic Forest and the “campo rupestre” ecosystem. To better resolve their evolutionary relationships, we performed phylogenetic analyses using a multigene DNA matrix and representative sampling within the genus. We then evaluated the evolution of phenotypical and natural history traits with the inferred phylogeny. We recovered Crossodactylodes as monophyletic, diagnosed by seven putative synapomorphies in morphological and natural history characters. Evidence supports some morphological synapomorphies as adaptations to the bromeligenous habit. We found high genetic distances among closely distributed lineages within C. bokermanni and C. izecksohni. Some of these lineages might represent undescribed cryptic species. We provide detailed accounts for each species including data on their geographic range, conservation, and natural history. All species of Crossodactylodes occur in highly threatened environments, are restricted to very small geographic ranges, and probably have limited dispersal capacity due to their small body size and dependence on bromeliads. These factors emphasize the need for habitat protection to safeguard species viability.

Physalaemus barrioi is a poorly known frog endemic to the highlands of Serra da Bocaina in the Atlantic Forest of southeastern Brazil. Herein, we redescribe the species based on the type series and new specimens collected at the type locality. We also present additional data on its vocalization, karyotype, and tadpole, including the external morphology, internal oral features, and chondrocranium. Physalaemus barrioi can be diagnosed by the following combination of characters: SVL in males larger than 24 mm; inguinal glands associated with dark ocelli; throat and vocal sac dark brown; and chest dark with small light blotches. Bioacoustical characteristics also distinguish P. barrioi from all other species of Physalaemus. The tadpole of P. barrioi is very similar to those of the P. gracilis group, from which it can be distinguished by several characteristics, including marginal papillae in a single row, absence of submarginal papillae, elliptical nares, and upper jaw sheath in a wide arch.

The nucleolus organizer regions (NORs) of both karyotypes I and II of Physalaemus petersi (Jiménez de la Espada, 1872) from the Brazilian Amazon were studied by Giemsa staining, and by the Ag-NOR method. Karyological group I specimens were also studied by the fluorescence in situ hybridization (FISH) technique. Multiple NOR-bearing chromosomes were detected in both karyotypes. The coincident results of the Ag-NOR and FISH methods rule out the occurrence of silent NORs in this anuran. There was no intraindividual NOR variability in either group, but interindividual variability of NORs was high in group I. Seven different patterns of active NOR distribution were definitely recognized among fifteen specimens. This was considered to be a NOR site polymorphism. These results, combined with the C-band polymorphism previously reported for P. petersi, demonstrate a high rate of chromosome evolution in this group.

Crossodactylodes is a poorly known genus of small-sized bromeligenous frogs, endemic to Brazil. They have a patchy distribution across the mountains of the Atlantic Forest and the “campo rupestre” ecosystem. To better resolve their evolutionary relationships, we performed phylogenetic analyses using a multigene DNA matrix and representative sampling within the genus. We then evaluated the evolution of phenotypical and natural history traits with the inferred phylogeny. We recovered Crossodactylodes as monophyletic, diagnosed by seven putative synapomorphies in morphological and natural history characters. Evidence supports some morphological synapomorphies as adaptations to the bromeligenous habit. We found high genetic distances among closely distributed lineages within C. bokermanni and C. izecksohni. Some of these lineages might represent undescribed cryptic species. We provide detailed accounts for each species including data on their geographic range, conservation, and natural history. All species of Crossodactylodes occur in highly threatened environments, are restricted to very small geographic ranges, and probably have limited dispersal capacity due to their small body size and dependence on bromeliads. These factors emphasize the need for habitat protection to safeguard species viability.

Doc number: 17 Abstract Background: For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi , and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Results: Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Conclusions: Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the Chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others.

Cytogenetic analyses were performed on specimens from two populations of Physalaemus petersi from three locations in Brazilian West Amazon. Chromosomes from the testis and intestinal epithelium were stained conventionally with Giemsa or C-banded. All animals studied showed a full chromosome complement of 2n = 22, but two distinct karyotypes (I and II) were detected among specimens from one of the populations. Karyotype I specimens showed a XX/XY sex chromosome system and C-band polymorphism. Bivalent chromosomes with heterozygous C-banding frequently lacked chiasmata in the region of this heterochromatin during the first meiotic division. The less common karyotype (II) had a heteromorphic pair of chromosomes, but the relationship of this pair to sex determination could not be elucidated because of the absence of female specimens. Karyotype II was observed in males whose call differed from those of other males in the same population, suggesting that a reevaluation of the taxon P. petersi may be necessary. These results suggest that, in these populations, karyological evolution occurs faster than anatomical evolution.