Sequencing of laser captured Z and W chromosomes of the tocantins paradoxical frog (Pseudis tocantins) provides insights on repeatome and chromosomal homology.

Pseudis tocantins is the only frog species of the hylid genus Pseudis that possesses highly heteromorphic sex chromosomes. Z and W chromosomes of Ps. tocantins differ in size, morphology, position of the nucleolar organizer region (NOR) and the amount and distribution of heterochromatin. A chromosomal inversion and heterochromatin amplification on the W chromosome were previously inferred to be involved in the evolution of this sex chromosome pair. Despite these findings, knowledge related to the molecular composition of the large heterochromatic band of this W chromosome is restricted to the PcP190 satellite DNA, and no data are available regarding the gene content of either the W or the Z chromosome of Ps. tocantins. Here, we sequenced microdissected Z and W chromosomes of this species to further resolve their molecular composition. Comparative genomic analysis suggests that Ps. tocantins sex chromosomes are likely homologous to chromosomes 4 and 10 of Xenopus tropicalis. Analyses of the repetitive DNA landscape in the Z and W assemblies allowed for the identification of several transposable elements and putative satellite DNA sequences. Finally, some transposable elements from the W assembly were found to be highly diverse and divergent from elements found elsewhere in the genome, suggesting a rapid amplification of these elements on the W chromosome.

Rapid karyotypic evolution with high diploid number variation in a rare genus of bromeligenous frogs.

Bromeligenous Crossodactylodes is a leptodactylid genus closely related to Paratelmatobius and Scythrophrys. The diploid number in all karyotyped species of these two latter genera is 24, which diverges from the modal diploid number (2n = 22) in the family. Here, we analyzed three species of Crossodactylodes and found karyotypes with 2n = 30, 2n = 32, and 2n = 36, diploid numbers that have not been reported in any other diploid leptodactylid species to date. Reconstruction of the ancestral chromosome number indicated that the diploid number changed from 22 to 24 in the common ancestor of Crossodactylodes, Paratelmatobius, and Scythrophrys, and that progressive increases in diploid number have occurred in Crossodactylodes. The large number of telocentric/subtelocentric chromosomes in karyotypes with higher diploid numbers raises the possibility that centric fissions may have occurred during the evolution of Paratelmatobiinae. Three metacentric chromosomes, probably involved in fission events, were inferred to be present in the common ancestor of all species of Crossodactylodes, but in C. bokermanni. Chromosome mapping of the satellite DNA PcP190 suggests homology between one arm of metacentric chromosome 1 of Crossodactylodes sp. 3 and telocentric chromosome 2 of C. itambe, supporting one of the presumed centric fission events.

Comparative mapping of a new repetitive DNA sequence and chromosome region-specific probes unveiling rearrangements in an Amazonian frog complex.

The frog species Physalaemus ephippifer exists in the Amazonian region and harbors heteromorphic Z and W chromosomes. A genetic lineage closely related to this species was recognized based on its mitochondrial DNA and RADseq-style markers, but its taxonomic status is still unclear and has been referred to as Lineage 1 of "P. cuvieri". The heteromorphic sex chromosomes found in P. ephippifer are not present in this lineage and which of its chromosome pairs is homologous to the sex chromosomes of P. ephippifer remain to be elucidated as well as the role of such a karyotypic divergence in the evolution of these frogs. Here, we described a new family of repetitive DNA and used its chromosomal sites along with the markers detected by a probe constructed from the microdissected segment of the Z chromosome of P. ephippifer to infer chromosomal homology. We also analyzed an unnamed species that is considered to be the sister group of the clade composed of Lineage 1 of "P. cuvieri" and P. ephippifer. Our results suggest that complex rearrangements involving the chromosomes that were inferred to be homeologous to the sex chromosomes of P. ephippifer have occurred during the divergence of this group of frogs.

Cytogenetic and genetic data support Crossodactylus aeneus Müller, 1924 as a new junior synonym of C. gaudichaudii Duméril and Bibron, 1841 (Amphibia, Anura).

The nominal anuran species Crossodactylus gaudichaudii Duméril and Bibron, 1841 and Crossodactylus aeneus Müller, 1924 are indistinguishable based on adult and larval morphology, being subject of taxonomic doubts. Here, we describe the karyotypes of C. gaudichaudii and C. aeneus, using classical and molecular cytogenetic markers. In addition, we used sequences of the H1 mitochondrial DNA to infer their phylogenetic relationships by Maximum Likelihood (ML) and Maximum Parsimony (MP) approaches and species delimitation test (by bPTP approach). The karyotypic data do not differentiate C. gaudichaudii and C. aeneus in any of the chromosome markers assessed. In both phylogenetic analyses, C. gaudichaudii and C. aeneus were recovered into a strongly supported clade. The species delimitation analysis recovered the specimens assigned to C. gaudichaudii and C. aeneus as a single taxonomic unit. Taken the cytogenetic and genetic results together with previous studies of internal and external morphology of tadpoles and biacoustic pattern, C. gaudichaudii and C. aeneus could not be differentiated, which supports the hypothesis that they correspond to the same taxonomic unit, with C. aeneus being a junior synonym of C. gaudichaudii.

Long-time evolution and highly dynamic satellite DNA in leptodactylid and hylodid frogs.

BACKGROUND: Satellite DNA sequences are the most abundant components of heterochromatin and are repeated in tandem hundreds to thousands of times in the genome. However, the number of repeats of a specific satellite family can vary even between the genomes of related species or populations. The PcP190 satellite DNA family was identified in the genome of the leptodactylid frog Physalaemus cuvieri, which showed to be derived most likely from the 5S rDNA in an ancestral species. In this study, we investigate the presence of the PcP190 satellite DNA in several P. cuvieri populations and in four closely related species at the chromosomal and molecular level. Furthermore, we investigate the occurrence of this satellite DNA in the genomes of P. marmoratus as well as in representative species of the leptodactylid genus Leptodactylus (L. latrans) and the hylodid family (Crossodactylus gaudichaudii), all with the aim of investigating if the PcP190 satellite DNA presents or not a restricted distribution. RESULTS: The PcP190 satellite DNA was detected in all the analyzed species. Some of them exhibited particular sequence differences, allowing the identification of species-specific groups of sequences, but in other species, the sequences were more conserved. However, in a general analysis, conserved and variable domains have been recognized within the PcP190 monomer. The chromosomal analysis performed on P. cuvieri populations and closely related species revealed high variability of the satellite DNA amount and its chromosomal location, which has always been coincident with regions of centromeric/pericentromeric heterochromatin. CONCLUSION: The PcP190 satellite DNA was found in representatives of two families, Leptodactylidae and Hylodidae, indicating that these sequences are widely distributed and conserved in these frogs. There is a pattern of non-random variation within the repeating units, indicating interplay between stochastic events and selective pressure along the PcP190 sequences. Karyotypic differences involving the PcP190 satellite DNA prove to be highly dynamic on the chromosomes of the Physalaemus and its differential accumulation has contributed to the differentiation process of the Z and W sex chromosomes in P. ephippifer.

Comparative cytogenetic analysis of some species of the Dendropsophus microcephalus group (Anura, Hylidae) in the light of phylogenetic inferences.

BACKGROUND: Dendropsophus is a monophyletic anuran genus with a diploid number of 30 chromosomes as an important synapomorphy. However, the internal phylogenetic relationships of this genus are poorly understood. Interestingly, an intriguing interspecific variation in the telocentric chromosome number has been useful in species identification. To address certain uncertainties related to one of the species groups of Dendropsophus, the D. microcephalus group, we carried out a cytogenetic analysis combined with phylogenetic inferences based on mitochondrial sequences, which aimed to aid in the analysis of chromosomal characters. Populations of Dendropsophus nanus, Dendropsophus walfordi, Dendropsophus sanborni, Dendropsophus jimi and Dendropsophus elianeae, ranging from the extreme south to the north of Brazil, were cytogenetically compared. A mitochondrial region of the ribosomal 12S gene from these populations, as well as from 30 other species of Dendropsophus, was used for the phylogenetic inferences. Phylogenetic relationships were inferred using maximum parsimony and Bayesian analyses. RESULTS: The species D. nanus and D. walfordi exhibited identical karyotypes (2n = 30; FN = 52), with four pairs of telocentric chromosomes and a NOR located on metacentric chromosome pair 13. In all of the phylogenetic hypotheses, the paraphyly of D. nanus and D. walfordi was inferred. D. sanborni from Botucatu-SP and Torres-RS showed the same karyotype as D. jimi, with 5 pairs of telocentric chromosomes (2n = 30; FN = 50) and a terminal NOR in the long arm of the telocentric chromosome pair 12. Despite their karyotypic similarity, these species were not found to compose a monophyletic group. Finally, the phylogenetic and cytogenetic analyses did not cluster the specimens of D. elianeae according to their geographical occurrence or recognized morphotypes. CONCLUSIONS: We suggest that a taxonomic revision of the taxa D. nanus and D. walfordi is quite necessary. We also observe that the number of telocentric chromosomes is useful to distinguish among valid species in some cases, although it is unchanged in species that are not necessarily closely related phylogenetically. Therefore, inferences based on this chromosomal character must be made with caution; a proper evolutionary analysis of the karyotypic variation in Dendropsophus depends on further characterization of the telocentric chromosomes found in this group.

Molecular organization and chromosomal localization of 5S rDNA in Amazonian Engystomops (Anura, Leiuperidae).

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.

Heteromorphic Z and W sex chromosomes in Physalaemus ephippifer (Steindachner, 1864) (Anura, Leiuperidae).

Heteromorphisms between sex chromosomes are rarely found in anurans and sex chromosome differentiation is considered to be a set of recent recurrent events in the evolutionary history of this group. This paper describes for the first time heteromorphic sex chromosomes Z and W in the leiuperid genus Physalaemus. They were found in P. ephippifer, a species of the P. cuvieri group, and corresponded to the eighth pair of its karyotype. The W chromosome differed from the Z chromosome by the presence of an additional segment in the short arm, composed of a distal NOR and an adjacent terminal DAPI-positive C-band. The identification of this sex chromosome pair may help in future investigations into the sex determining genes in the genus Physalaemus.

Satellite DNA Mapping in Pseudis fusca (Hylidae, Pseudinae) Provides New Insights into Sex Chromosome Evolution in Paradoxical Frogs.

In the frog genus Pseudis, previous works found a sex-linked heteromorphism of the PcP190 satellite DNA in the nucleolus organizer region (NOR)-bearing chromosome pairs of Pseudis bolbodactyla and Pseudis tocantins, which possess a ZZ/ZW sex determination system. A pericentromeric inversion was inferred to have occurred during W chromosome evolution, moving a chromosomal cluster enriched by the PcP190 from the short arm (as observed in P. bolbodactyla) to the NOR-bearing long arm (as observed in P. tocantins). However, whether such an inversion happened in P. tocantins or in the common ancestor of Pseudis fusca and P. tocantins remained unclear. To assess this question, we mapped PcP190 in the karyotype of P. fusca from three distinct localities. Southern blotting was used to compare males and females. The mitochondrial H1 fragment (which contains the 12S ribosomal RNA (rRNA), tRNAval, and 16S rRNA genes) and cytochrome b gene were partially sequenced, and a species tree was inferred to guide our analysis. Pseudis fusca specimens were placed together as the sister group of P. tocantins, but based on genetic distance, one of the analyzed populations is probably an undescribed species. A cluster of PcP190, located in the long arm of chromosome 7, is sex linked in this putative new species but not in the remaining P. fusca. We could infer that the pericentromeric inversion that moved the PcP190 site to the NOR-bearing chromosome arm (long arm) occurred in the common ancestor of P. fusca, the putative undescribed species, and P. tocantins.

Extensive Cryptic Diversity Within the Physalaemus cuvieri-Physalaemus ephippifer Species Complex (Amphibia, Anura) Revealed by Cytogenetic, Mitochondrial, and Genomic Markers.

Previous cytogenetic and phylogenetic analyses showed a high variability in the frog taxa Physalaemus cuvieri and Physalaemus ephippifer and suggested the presence of undescribed diversity in this species complex. Here, by 1) adding specimens from the Brazilian Amazon region, 2) employing sequence-based species delimitation approaches, and 3) including RADseq-style markers, we demonstrate that the diversity in the P. cuvieri-P. ephippifer species complex is even greater than previously suspected. Specimens from Viruá and Western Pará, located at the Guiana Amazonian area of endemism, were recovered as distinct from all previously identified lineages by the phylogenetic analyses based on mitochondrial DNA and RAD markers, a PCA from RAD data, and cytogenetic analysis. The sequence-based species delimitation analyses supported the recognition of one or two undescribed species among these Amazonian specimens and also supported the recognition of at least three other species in the P. cuvieri-P. ephippifer species complex. These new results reinforce the need for a comprehensive taxonomic revision.

Signature of the Paleo-Course Changes in the São Francisco River as Source of Genetic Structure in Neotropical Pithecopus nordestinus (Phyllomedusinae, Anura) Treefrog.

Historical processes that have interrupted gene flow between distinct evolutionary lineages have played a fundamental role in the evolution of the enormous diversity of species found in the Neotropical region. Numerous studies have discussed the role of geographic barriers and Pleistocene forest refugia in the diversification of the region's biodiversity. In the present study, we investigated the relative contribution of these different factors to the evolutionary history of Pithecopus nordestinus, a Neotropical tree frog, which is amply distributed in the Brazilian Atlantic Forest and adjacent areas of the Caatinga biome. We used an extensive sample and multilocus DNA sequences to provide an overview of the intraspecific genetic diversity of P. nordestinus, characterize historical diversification patterns, and identify possible phylogenetic splits. We tested different scenarios of diversification based on Pleistocene Refugia and river barrier models using approximate Bayesian computation (ABC) and ecological niche modeling (ENM). The phylogenetic approach indicate the occurrence of processes of phylogeographic divergence in both time and space, related to historical shifts in the course of the São Francisco River during Plio-Pleistocene period, resulting in two principal, highly divergent clades. The ABC model provided strong statistical support for this scenario, confirming the hypothesis that the São Francisco River acted as an effective geographical barrier during vicariant events in the evolutionary history of P. nordestinus. We believe that the climatic changes that occurred during the Pleistocene also played a secondary role in the genetic signatures identified, reinforcing the divergence of populations isolated by physical barriers. These findings reinforce the conclusion that the two models of diversification (geographic barriers and refugia) are not mutually exclusive in the Neotropical domain but may interact extensively during the diversification of species on a regional scale.

Atlantic West Ophiothrix spp. in the scope of integrative taxonomy: Confirming the existence of Ophiothrix trindadensis Tommasi, 1970.

We re-describe and confirm the validity of Ophiothrix trindadensis Tommasi, 1970 (Echinodermata: Ophiuroidea). This is a native species from Brazil, however it lacked a type series deposited in scientific collections. The recognition of O. trindadensis was made possible using integrative taxonomy applied to many specimens from the type locality (Trindade Island) as well as from different locations along the Brazilian coast (Araçá Bay and Estuarine Complex of Paranaguá). Initially, 835 specimens were studied and divided into four candidate species (CS) inferred from external morphological characters. Afterwards, the CSs were compared using integrative taxonomy based on external morphology, arm microstructures morphology (arm ossicle), morphometry, and molecular studies (fragments of the mitochondrial genes 16S and COI). Analyses indicated CS1 and CS2 as O. trindadensis, and CS3 as O. angulata, both valid species. CS4 remains O. cf. angulata as more data, including their ecology and physiology, are needed to be definitively clarified. Our integrative investigation using specimens from the type locality overcame the lack of type specimens and increased the reliable identification of O. trindadensis and O. angulata.

The mitochondrial genome of the endemic Brazilian paradoxical frog Pseudis tocantins (Hylidae).

In this work, we present for the first time the mitochondrial genome of a paradoxical frog (Pseudis tocantins). This genome is 15.56 kb, excluding the control region, and is similar in gene content to other hylid mitogenomes. Maximum likelihood analysis, using the mitogenomes of several anurans, indicated P. tocantins as closely related to other hylid species.

Unraveling the Sex Chromosome Heteromorphism of the Paradoxical Frog Pseudis tocantins.

The paradoxical frog Pseudis tocantins is the only species in the Hylidae family with known heteromorphic Z and W sex chromosomes. The Z chromosome is metacentric and presents an interstitial nucleolar organizer region (NOR) on the long arm that is adjacent to a pericentromeric heterochromatic band. In contrast, the submetacentric W chromosome carries a pericentromeric NOR on the long arm, which is adjacent to a clearly evident heterochromatic band that is larger than the band found on the Z chromosome and justify the size difference observed between these chromosomes. Here, we provide evidence that the non-centromeric heterochromatic bands in Zq and Wq differ not only in size and location but also in composition, based on comparative genomic hybridization (CGH) and an analysis of the anuran PcP190 satellite DNA. The finding of PcP190 sequences in P. tocantins extends the presence of this satellite DNA, which was previously detected among Leptodactylidae and Hylodidae, suggesting that this family of repetitive DNA is even older than it was formerly considered. Seven groups of PcP190 sequences were recognized in the genome of P. tocantins. PcP190 probes mapped to the heterochromatic band in Wq, and a Southern blot analysis indicated the accumulation of PcP190 in the female genome of P. tocantins, which suggests the involvement of this satellite DNA in the evolution of the sex chromosomes of this species.

Comparative cytogenetics of tree frogs of the Dendropsophus marmoratus (Laurenti, 1768) group: conserved karyotypes and interstitial telomeric sequences.

The diploid number 2n = 30 is a presumed synapomorphy of Dendropsophus Fitzinger, 1843, although a noticeable variation in the number of biarmed/telocentric chromosomes is observed in this genus. Such a variation suggests that several chromosomal rearrangements took place after the evolutionary origin of the hypothetical ancestral 30-chromosome karyotype; however, the inferred rearrangements remain unknown. Distinct numbers of telocentric chromosomes are found in the two most cytogenetically studied species groups of Dendropsophus. In contrast, all three species of the Dendropsophus marmoratus (Laurenti, 1768) group that are already karyotyped presented five pairs of telocentric chromosomes. In this study, we analyzed cytogenetically three additional species of this group to investigate if the number of telocentric chromosomes in this group is not as variable as in other Dendropsophus groups. We described the karyotypes of Dendropsophus seniculus (Cope, 1868), Dendropsophus soaresi (Caramaschi & Jim, 1983) and Dendropsophus novaisi (Bokermann, 1968) based on Giemsa staining, C-banding, silver impregnation and in situ hybridization with telomeric probes. Dendropsophus seniculus, Dendropsophus soaresi and Dendropsophus novaisi presented five pairs of telocentric chromosomes, as did the remaining species of the group previously karyotyped. Though the species of this group show a high degree of karyotypic similarity, Dendropsophus soaresi was unique in presenting large blocks of het-ITSs (heterochromatic internal telomeric sequences) in the majority of the centromeres. Although the ITSs have been interpreted as evidence of ancestral chromosomal fusions and inversions, the het-ITSs detected in the karyotype of Dendropsophus soaresi could not be explained as direct remnants of ancestral chromosomal rearrangements because no evidence of chromosomal changes emerged from the comparison of the karyotypes of all of the species of the Dendropsophus marmoratus group.

Chromosomal analysis of Physalaemus kroyeri and Physalaemus cicada (Anura, Leptodactylidae).

All the species of Physalaemus Fitzinger, 1826 karyotyped up until now have been classified as 2n = 22. The species of the Physalaemus cuvieri group analyzed by C-banding present a block of heterochromatin in the interstitial region of the short arm of pair 5. Physalaemus cicada Bokermann, 1966 has been considered to be a member of the Physalaemus cuvieri species group, although its interspecific phylogenetic relationships remain unknown. The PcP190 satellite DNA has been mapped on the chromosomes of most of the species of the Physalaemus cuvieri group. For two species, Physalaemus cicada and Physalaemus kroyeri (Reinhardt & Lütken, 1862), however, only the chromosome number and morphology are known. Given this, the objective of the present study was to analyze the chromosomes of Physalaemus cicada and Physalaemus kroyeri, primarily by C-banding and PcP190 mapping. The results indicate that Physalaemus kroyeri and Physalaemus cicada have similar karyotypes, which were typical of Physalaemus. In both species, the NORs are located on the long arm of pair 8, and the C-banding indicated that, among other features, Physalaemus kroyeri has the interstitial band on chromosome 5, which is however absent in Physalaemus cicada. Even so, a number of telomeric bands were observed in Physalaemus cicada. The mapping of the PcP190 satellite DNA highlighted areas of the centromeric region of the chromosomes of pair 1 in both species, although in Physalaemus kroyeri, heteromorphism was also observed in pair 3. The cytogenetic evidence does not support the inclusion of Physalaemus cicada in the Physalaemus cuvieri group. In the case of Physalaemus kroyeri, the interstitial band on pair 5 is consistent with the existence of a cytogenetic synapomorphy in the Physalaemus cuvieri species group.

First insights on the retroelement Rex1 in the cytogenetics of frogs.

BACKGROUND: While some transposable elements (TEs) have been found in the sequenced genomes of frog species, detailed studies of these elements have been lacking. In this work, we investigated the occurrence of the Rex1 element, which is widespread in fish, in anurans of the genus Physalaemus. We isolated and characterized the reverse transcriptase (RT)-coding sequences of Rex1 elements of five species of this genus. RESULTS: The amino acid sequences deduced from the nucleotide sequences of the isolated fragments allowed us to unambiguously identify regions corresponding to domains 3-7 of RT. Some of the nucleotide sequences isolated from Physlaemus ephippifer and P. albonotatus had internal deletions, suggesting that these fragments are likely not active TEs, despite being derived from a Rex1 element. When hybridized with metaphase chromosomes, Rex1 probes were revealed at the pericentromeric heterochromatic region of the short arm of chromosome 3 of the P. ephippifer karyotype. Neither other heterochromatin sites of the P. ephippifer karyotype nor any chromosomal regions of the karyotypes of P. albonotatus, P. spiniger and P. albifrons were detected with these probes. CONCLUSIONS: Rex1 elements were found in the genomes of five species of Physalaemus but clustered in only the P. ephippifer karyotype, in contrast to observations in some species of fish, where large chromosomal sites with Rex1 elements are typically present.

Comparative cytogenetics of Physalaemus albifrons and Physalaemus cuvieri species groups (Anura, Leptodactylidae).

Recently, Physalaemus albifrons (Spix, 1824) was relocated from the Physalaemus cuvieri group to the same group as Physalaemus biligonigerus (Cope, 1861), Physalaemus marmoratus (Reinhardt & Lütken, 1862) and Physalaemus santafecinus Barrio, 1965. To contribute to the analysis of this proposition, we studied the karyotypes of Physalaemus albifrons, Physalaemus santafecinus and three species of the Physalaemus cuvieri group. The karyotype of Physalaemus santafecinus was found to be very similar to those of Physalaemus biligonigerus and Physalaemus marmoratus, which were previously described. A remarkable characteristic that these three species share is a conspicuous C-band that extends from the pericentromeric region almost to the telomere in the short arm of chromosome 3. This characteristic is not present in the Physalaemus albifrons karyotype and could be a synapomorphy of Physalaemus biligonigerus, Physalaemus marmoratus and Physalaemus santafecinus. The karyotype of Physalaemus santafecinus is also similar to those of Physalaemus marmoratus and Physalaemus biligonigerus owing to the presence of several terminal C-bands and the distal localization of the NOR in a small metacentric chromosome. In contrast, the Physalaemus albifrons karyotype has no terminal C-bands and its NOR is located interstitially in the long arm of submetacentric chromosome 8. The NOR-bearing chromosome of Physalaemus albifrons very closely resembles those found in Physalaemus albonotatus (Steindachner, 1864), Physalaemus cuqui Lobo, 1993 and some populations of Physalaemus cuvieri Fitzinger, 1826. Additionally, the Physalaemus albifrons karyotype has an interstitial C-band in chromosome 5 that has been exclusively observed in species of the Physalaemus cuvieri group. Therefore, we were not able to identify any chromosomal feature that supports the reallocation of Physalaemus albifrons.