A genomic survey of LINE elements in Pipidae aquatic frogs shed light on Rex-elements evolution in these genomes.

The transposable elements (TE) represent a large portion of anuran genomes that act as components of genetic diversification. The LINE order of retrotransposons is among the most representative and diverse TEs and is poorly investigated in anurans. Here we explored the LINE diversity with an emphasis on the elements generically called Rex in Pipidae species, more specifically, in the genomes ofXenopus tropicalis, used as a model genome in the study of anurans,the allotetraploid sister species Xenopus laevis and theAmerican species Pipa carvalhoi. We were able to identify a great diversity of LINEs from five clades, Rex1, L2, CR1, L1 and Tx1, in these three species, and the RTE clade was lost in X. tropicalis. It is clear that elements classified as Rex are distributed in distinct clades. The evolutionary pattern of Rex1 elements denote a complex evolution with independent losses of families and some horizontal transfer events between fishes and amphibians which were supported not only by the phylogenetic inconsistencies but also by the very low Ks values found for the TE sequences. The data obtained here update the knowledge of the LINEs diversity in X. laevis and represent the first study of TEs in P. carvalhoi.

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.

Comparative cytogenetics of nine populations of the Rhinella genus (Anura, Bufonidae) with a highlight on their conservative karyotype.

The genus Rhinella is one of the most diverse groups of bufonid toads, currently composed by 93 valid species and naturally distributed throughout different Neotropical ecoregions. Here, we analyze nine Brazilian populations of toads representing species of the Rhinella margaritifera and Rhinella marina groups. These new data include the first description of the R. hoogmoedi and R. proboscidae karyotypes, as well as other taxonomically unresolved forms. Chromosomal analysis of the populations revealed pronounced chromosomal uniformity (2n=22), including the diploid number and chromosomal morphology. Three different NOR-bearing chromosomes were identified: in the subterminal region of pair 10q in R. hoogmoedi, Rhinella sp. 1 and Rhinella sp. 2, in subterminal region of 7p in R. proboscidae and Rhinella cf. margaritifera while in R. henseli and R. icterica was detected in interstitial region of 7p. Karyotypic uniformity of the genus permits the inference of interspecific chromosome homologies and evolutionary changes in the NOR-bearing chromosome may represent an informative character in species group level. The review of the cytogenetic data of the Rhinella species together with the new karyotypes reported here contributes to the understanding of the chromosomal evolution of these toads, which karyotypes are highly conserved despite the ample distribution of many forms.

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.

Evaluation of the taxonomic status of populations assigned to Phyllomedusa hypochondrialis (Anura, Hylidae, Phyllomedusinae) based on molecular, chromosomal, and morphological approach.

BACKGROUND: The taxonomic and phylogenetic relationships of the genus Phyllomedusa have been amply discussed. The marked morphological similarities among some species hamper the reliable identification of specimens and may often lead to their incorrect taxonomic classification on the sole basis of morphological traits. Phenotypic variation was observed among populations assigned to either P. azurea or P. hypochondrialis. In order to evaluate whether the variation observed in populations assigned to P. hypochondrialis is related to that in genotypes, a cytogenetic analysis was combined with phylogenetic inferences based on mitochondrial and nuclear sequences. RESULTS: The inter- and intra-population variation in the external morphology observed among the specimens analyzed in the present study do not reflect the phylogenetic relationships among populations. A monophyletic clade was recovered, grouping all the specimens identified as P. hypochondrialis and specimens assigned P. azurea from Minas Gerais state. This clade is characterized by conserved chromosomal morphology and a common C-banding pattern. Extensive variation in the nucleolar organizing region (NOR) was observed among populations, with four distinct NOR positions being recognized in the karyotypes. Intra-population polymorphism of the additional rDNA clusters observed in specimens from Barreiras, Bahia state, also highlights the marked genomic instability of the rDNA in the genome of this group. Based on the topology obtained in the phylogenetic analyses, the re-evaluation of the taxonomic status of the specimens from the southernmost population known in Brazil is recommended. CONCLUSIONS: The results of this study support the need for a thorough revision of the phenotypic features used to discriminate P. azurea and P. hypochondrialis. The phylogenetic data presented here also contribute to an extension of the geographic range of P. hypochondrialis, which is known to occur in the Amazon basin and neighboring areas of the Cerrado savanna, where it may be sympatric with P. azurea, within contact zones. The misidentification of specimens may have led to inconsistencies in the original definition of the geographic range of P. azurea. The variability observed in the NOR of P. hypochondrialis reinforces the conclusion that these sites represent hotspots of rearrangement. Intraspecific variation in the location of these sites is the result of constant rearrangements that are not detected by classical cytogenetic methods or are traits of an ancestral, polymorphic karyotype, which would not be phylogenetically informative for this group.

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.

Cytogenetics and sperm ultrastructure of Atelopus spumarius (Anura, Bufonidae) from the Brazilian Amazon.

The current taxonomy of most Atelopus species is based on morphological and color data only. Recent studies suggest that A. spumarius may represent a species complex assigned under the same name. Karyotypic data and description of sperm ultrastructure for 13 specimens of A. spumarius are presented here for the first time. A chromosomal analysis revealed 2n = 22 chromosomes, with centromeric heterochromatin in all pairs and a nucleolar organizer region (NOR) on the telomere of pair 7. The sperm was of the bufonoid type, presenting a filiform nucleus covered by an acrosomal complex and a mitochondrial collar in the neck region. The tail was composed of an axoneme, an undulating membrane and an axial rod. A karyotype analysis of A. spumarius showed the same chromosome number and similar chromosomal morphology as described for congeneric species, with slight differences probably resulting from pericentric inversions. The NOR location (on pair 7) was the same as that observed for species belonging to the genus Rhinella. The spermatological findings indicate a close relationship between Atelopus and the bufonoid lineage. The present data are useful for reference in future studies to determine whether more than one species are assigned to A. spumarius.

Cytogenetic analysis of two species in the Phyllomedusa hypochondrialis group (Anura, Hylidae).

The genus Phyllomedusa has been the target of regular taxonomic investigations. The species Phyllomedusa nordestina was recently separated from P. hypochondrialis. Morphological variations in the P. rohdei interpopulation have already been reported, suggesting the existence of more than one taxon under that name. In the present study, we have cytogenetically characterized two populations of P. nordestina and one of P. rohdei. Both species displayed 2n = 26 chromosomes with 12 metacentric, 12 submetacentric and 2 subtelocentric chromosomes. The C-banding analyses revealed discrete differences in the quantity of centromeric heterochromatin between the two species. The nucleolus organizer region (NOR) was detected in pair 9 of both species, but is located in the pericentromeric region of the short arm in P. nordestina and in the long arm subtelomeric region of P. rohdei. Chromosomal data from this study indicate karyotypic homeology between the two groups of P. hypochondrialis species and suggest the existence of more than one taxon under the P. rohdei name.

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.

Sperm ultrastructure of Mytella (Bivalvia) populations from distinct habitats along the northern coast of São Paulo State, Brazil.

Ultrastructural analyses of bivalve spermatozoa are relevant in studies that aim to identify taxonomic traits for the purposes of discriminating species and conducting phylogenetic studies. In the present work, spermatozoa of mussel specimens of the genus Mytella, collected from two populations living in distinct habitats, were examined by electron microscopy. The objective was to identify sperm ultrastructural taxonomic traits that could be used to differentiate Mytella species. The specimens were from populations that live in intertidal zones on the southeast coast of Brazil, either buried in muddy-sand sediment or anchored to rocky substrates. The acrosomal vesicle was conical and long, the axial rod extended from the nucleus to the acrosome, the nucleus was an oblate spheroid with a condensed chromatin, the intermediate portion contained mitochondria encircling a pair of centrioles, and there was a single flagellum. The sperm was of a primitive type. The spermatozoon ultrastructure did not distinguish the specimens buried in muddy-sand sediment from those anchored to rocky substrates. The data suggest that the specimens analyzed, despite living in distinct habitats, belong to the same species, which conchological analyses identified as M. charruana. The presence of an axial rod in their sperm cells supports the inclusion of M. charruana in the subfamily Mytilinae.

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.

Comparative sperm morphology of Proceratophrys and Odontophrynus (Anura, Odontophrynidae).

The ultrastructure of the spermatozoa of seven species of Proceratophrys and one of Odontophrynus was analyzed in the present study. The sperm of Odontophrynus juquinha is similar to that of Odontophrynus cultripes, previously described, and to those of Proceratophrys branti, Proceratophrys bigibbosa, Proceratophrys affinis concavitympanum, Proceratophrys huntingtoni, Proceratophrys conformis moratoi, P. moratoi, and Proceratophrys palustris, in relation to the acrosomal vesicle and the subacrosomal cone that covers the anterior region of the nucleus in the sperm head. We observed transverse striations in the midpiece of the Odontophrynus juquinha sperm, a feature not observed in any Proceratophrys species. The sperm tail of all the studied species had a simple flagella and single axoneme associated with auxiliary structures (juxta-axonemal + undulating membrane + axial fiber). However, the juxta-axonemal fiber is thicker and the undulating membrane is shorter in Proceratophrys in comparison with Odontophrynus. In addition to differentiating these two genera, these features represent a possible autapomorphy in Proceratophrys.

Recurrent variation in the active NOR sites in the monkey frogs of the genus Pithecopus Cope, 1866 (Phyllomedusidae, Anura).

Treefrogs of the genus Pithecopus Cope, 1866 exhibit expressive chromosomal homogeneity which contrasts with a high variation frequency of the nucleolus organizer region (NOR) related to the group. Currently, the genus contains eleven species and no chromosomal data are available on P. palliatus Peters, 1873, P. ayeaye Lutz, 1966 and P. megacephalus Miranda-Ribeiro, 1926. Here, we describe the karyotypes of these three species based on Giemsa staining, C-banding, silver impregnation and in situ hybridization (FISH). We were also analyze the evolutionary dynamic of the NOR-bearing chromosome in species of genus under a phylogenetic view. The results indicate that P. palliatus, P. ayeaye, and P. megacephalus have similar karyotypes, which are typical of the genus Pithecopus. In P. palliatus the NOR was detected in the pericentromeric region of pair 9p whereas in P. ayeaye and P. megacephalus we report cases of the multiple NOR sites in karyotypes. In P. ayeaye the NOR was detected in the pericentromeric region of pair 9p in both homologues and additional sites was detected in pairs 3q, 4p, and 8q, all confirmed by FISH experiments. Already in P. megacephalus the NOR sites were detected in pericentromeric region homologues of pair 8q and additionally in one chromosome of pair 13q. A comparative overview of all the Pithecopus karyotypes analyzed up to now indicates the recurrence of the NOR-bearing chromosome pairs and the position of the NORs sites on these chromosomes. We hypothesized that this feature is a result of a polymorphic condition present in the common ancestor of Pithecopus. In such case, the lineages derived from polymorphic ancestor have reached fixation independently after divergence of lineages, resulting in a high level of homoplasy observed in this marker. Our findings help to fill the gaps in the understanding of the karyotype of the genus Pithecopus and reinforce the role of the evolutionary dynamics of the rDNA genes in karyotype diversification in this group.

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.

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.

Unrevealing the leaf frogs Cerrado diversity: A new species of Pithecopus (Anura, Arboranae, Phyllomedusidae) from the Mato Grosso state, Brazil.

The Neotropical frog genus Pithecopus comprises currently 10 species. A recent molecular phylogeny suggested the existence of two subclades within it, one of them including P. palliatus, P. azureus, P. hypochondrialis, and P. nordestinus (lowland species). Herein we describe a new species of this subclade from Pontal do Araguaia, in the Brazilian Cerrado in the Mato Grosso state. Recognition of the new species is supported by adult morphology, advertisement call and molecular data. The new species differs from Pithecopus highland species by its smaller head width and lack of the reticulate pattern on flanks. From lowland species, the new form differs by being significantly smaller in snout vent-length, advertisement call with the greatest number of pulses, and high genetic distance. Interestingly, we also report on occurrence of P. hypochondrialis (its sister species) at an adjacent site (about 3km). Also, we report on the occurrence of the new species in the Chapada dos Guimarães and Santa Terezinha, both also in the Mato Grosso state.

Chromosomal differentiation of populations of Lysapsus limellus limellus, L. l. bolivianus, and of Lysapsus caraya (Hylinae, Hylidae).

Cytogenetic analysis were done on specimens from two populations of Lysapsus limellus limellus, three of L. l. bolivianus and of one of Lysapsus caraya. All animals showed a diploid chromosomal number of 2n=24. The karyotypes of the two L. limellus subspecies were very similar, differing only by the larger amount of telomeric heterochromatin and a small pericentromeric C-band on the short arms of pair 2 in L. l. limellus specimens. The karyotype of L. caraya differed from those of the two L. limellus subspecies in terms of chromosomal morphology, C-banding pattern and location of the main NOR on chromosomes 7 and 6, respectively. The karyotype of the L. l. bolivianus population from Guajará-Mirim/RO differed from those of the other populations of the same subspecies in morphology and heterochromatin pattern of chromosomes 7 and 8. Additional NORs were detected by silver staining and confirmed by FISH in one of the homologues of pairs 1 and 8 in L. l. bolivianus and in pair 7 in L. caraya. These results suggest that a reassessment of the taxonomic status of L. limellus subspecies, especially of the L. l. bolivianus populations, may be necessary.

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.

Comparative sperm ultrastructure of twelve leptodactylid frog species with insights into their phylogenetic relationships.

The spermatozoa of representatives of three Neotropical frog subfamilies, Leiuperinae, Leptodactylinae and Paratelmatobiinae, were observed using Transmission Electron Microscopy, with the aim of identifying ultrastructural traits that provide insights into the phylogenetic relationships among these anurans, which are currently unclear. In the leiuperines, spermatozoa of Physalaemus albifrons, P. cicada, P. deimaticus and P. feioi were characterized by an acrosomal vesicle covering the subacrosomal cone that was not observed in the spermatozoa of Physalaemus centralis and P. cuvieri. The tail of the spermatozoa of P. albifrons, P. centralis, P. cicada, P. cuvieri, P. deimaticus, and P. feioi presented a long undulating membrane, whereas Engystomops petersi and E. freibergi, which form a sister clade to Physalaemus, had an axial fiber, which were absent in Physalaemus. Other leiuperine, E. puyango had an abaxonemal bulb-like swelling distally to the paraxonemal rod, which were also absent in Physalaemus. These differences support the revalidation of Engystomops as a true taxon, distinct from Physalaemus. The tail of the spermatozoa of E. petersi and E. freibergi was similar to that of Paratelmatobius poecilogaster (Paratelmatobiinae). The spermatozoa of Leptodactylus natalenis (Leptodactylinae) had undulating membrane and axial fiber, in contrast with Adenomera marmorata, which lacked these structures. Morphological differences between A. marmorata and L. natalensis sperm cells appeared to validate the allocation of A. marmorata into a genus distinct from Leptodactylus. Overall, dissimilarities in the spermatozoa of the leptodactylids provided an important phylogenetic signal for the understanding of their taxonomic relationships.

Ultrastructure variation in the spermatozoa of Pseudopaludicola frogs (Amphibia, Anura, Leptodactylidae), with brief comments on its phylogenetic relevance.

The taxonomic history of the small frogs of the genus Pseudopaludicola from South America has been controversial. Phylogenetic inferences based on molecular data have identified four Pseudopaludicola clades, correlating with the known variation in karyotypes (2n = 22, 20, 18, and 16). In this study, the ultrastructure of the spermatozoa was analyzed in 12 species of the Pseudopaludicola, with the aim of describing their morphology and identifying characters that may contribute to a better understanding of the phylogenetic relationships. The spermatozoa presented marked differences in tail structures. The tails of the spermatozoa of the species with 2n = 22 chromosomes (Pseudopaludicola sp. 1 [P. pusilla group], Pseudopaludicola falcipes, P. mineira, and Pseudopaludicola saltica), as well as Pseudopaludicola ameghini and Pseudopaludicola ternetzi (2n=20), have juxta-axonemal fibers, undulating membranes and axial fibers. In contrast, in the species with 2n = 18 (P. facureae, P. giarettai, Pseudopaludicola canga, P. atragula, and Pseudopaludicola sp. 2) and 2n = 16 (Pseudopaludicola mystacalis), there are no evident axial or juxta-axonemal fibers, but a paraxonemal rod with a thick undulating membrane, which is shorter than that found among Pseudopaludicola species. The ultrastructural morphological differences observed in the spermatozoa of these species may be phylogenetically informative, given that they coincide with the consensus phylogeny of the group and appear to represent a progressive simplification of the spermatozoon.