Unraveling the variability and genetic structure of barker frog Physalaemus cuvieri (Leiuperinae) populations from different regions of Brazil.

The barker frog Physalaemus cuvieri is widely distributed in South America and is found in all regions of Brazil. Significant intraspecific morphological variation in this species has been reported. To determine the genetic structure of the natural Brazilian populations of P. cuvieri, 10 different populations geographically separated by 99.41 to 2936.75 km were evaluated using 10 polymorphic microsatellite loci. In addition, mitochondrial DNA data were analyzed to determine genetic distance between the populations. The genetic variation was found to be significant in most of the populations (HE ranged from 0.40 to 0.59, and allelic richness ranged from 2.07 to 3.54). An FST value of 0.27 indicated that high genetic structure was present among the P. cuvieri populations. STRUCTURE analyses grouped the 10 populations into nine clusters and indicated that only two of the populations were not genetically differentiated. The genetic distance calculated from the mitochondrial DNA data showed values <0.03 for seven of the populations.

Study of closely related species within the Physalaemus cuvieri group (Anura): contribution of microsatellite markers.

Various species of the Physalaemus cuvieri group of frogs are difficult to distinguish morphologically, making molecular analysis an attractive alternative for indentifying members of this group, which is considered to be at risk because of loss of habitat. The genetic structure of natural populations of P. ephippifer and P. albonotatus species was investigated and analyzed, together with that of five previously studied populations of P. cuvieri. Nine microsatellite loci were used in the analyses. The overall G(ST) value (0.46) revealed high genetic variation among the populations, as expected for different species. Bayesian analysis implemented by the STRUCTURE software clustered the seven populations into seven groups (K = 7). All the P. albonotatus and P. ephippifer specimens were grouped into a single cluster, both species showing clear differentiation from P. cuvieri. The different grouping based on these microsatellites of some P. cuvieri individuals from Porto Nacional and from Passo Fundo suggests that they could be a new species, indicating a necessity for taxonomic reevaluation. Despite the intrinsic difficulties in analyzing closely related species, the nine microsatellite loci were found to be adequate for distinguishing these three species of the P. cuvieri group and their populations.

Satellite DNA derived from 5S rDNA in Physalaemus cuvieri (Anura, Leiuperidae).

In the present study, we describe for the first time a family of 190-bp satellite DNA related to 5S rDNA in anurans and the existence of 2 forms of 5S rDNA, type I (201 bp) and type II (690 bp). The sequences were obtained from genomic DNA of Physalaemus cuvieri from Palmeiras, State of Bahia, Brazil. Analysis of the nucleotide sequence revealed that the satellite DNA obtained by digestion with EcoRI, called PcP190EcoRI, is 70% similar to the coding region of type I 5S rDNA and 66% similar to the coding region of type II 5S rDNA. Membrane hybridization and PCR amplification of the sequence showed that PcP190EcoRI is tandemly repeated. The satellite DNA as well as type I and type II 5S rDNA were localized in P. cuvieri chromosomes by fluorescent in situ hybridization. The PcP190EcoRI sequence was found in the centromeres of chromosomes 1-5 and in the pericentromeric region of chromosome 3. Type I 5S rDNA was detected in chromosome 3, coincident with the site of PcP190EcoRI. Type II 5S rDNA was located interstitially in the long arm of chromosome 5. None of these sequences co-localized with nucleolar organizer regions. Our data suggests that this satellite DNA originates from the 5S ribosomal multigene family, probably by gene duplication, nucleotide divergence and sequence dispersion in the genome.

Cytogenetic contributions for the study of the Amazonian Engystomops (Anura; Leiuperidae) assessed in the light of phylogenetic relationships.

Genetic divergence and speciation mechanisms of the Amazonian Engystomops frog have been inferred mainly from mtDNA sequences, microsatellite and male advertisement call. Although many aspects of this divergence remain unclear, cytogenetic analyses of Amazonian Engystomops populations are described and are compared to the relationships inferred from mitochondrial and nuclear DNA sequence data. High cytogenetic variation distinguished karyotypic patterns among the populations, even between populations which had no prezygotic isolation previously inferred from mating call analysis. Interestingly, the Puyo and Acre populations, which are in different clades, showed heteromorphic sex chromosomes (XX/XY), not identified in the other Ecuatorian populations analyzed. The analysis of a specimen collected in a site near Yasuní (Ecuador) which was cytogenetically related to the specimens from Puyo (Ecuador), was also phylogenetically closely related these specimens. In the rhodopsin nucleotide sequences, six polymorphic sites were identified and various specimens were heterozygous for them. All the data presented herein, in conjunction with those previously reported, corroborate the hypothesis that Engystomops petersi is a complex of distinct species. It also indicates that karyotypic evolution patterns may have played a substantial role in the Engystomops speciation and the occurrence of sporadic mating events between divergent evolutive lineages is discussed.

Comparative analysis of the chromosomal positions of rDNA genes in species of the tripunctata radiation of Drosophila.

The location of rDNA on chromosomes has been found to be highly variable in different groups of insect families of various orders. Yet, in other insect families the rDNA position is relatively constant. This contrast so far has received limited attention. We investigated the rDNA position on mitotic chromosomes in 18 species of Drosophila, 16 of which are from the tripunctata radiation, subgenus Drosophila, through fluorescent in situ hybridization (FISH). All species showed fluorescent signals only on the sex chromosomes. On the X chromosome a single fluorescent mark, but with variable locations, was found. On the Y, we observed variation both in location and in number of fluorescent marks (from 1 to 5). This constancy of chromosome location, in contrast to the great variability found in other groups, is consistent with the work carried out in other species of Drosophila. This suggests that the presence of a nucleolus organizer region (NOR) on each sex chromosome is probably an ancestral condition in the genus. Moreover, this difference in the variation of rDNA position among groups points out an interesting evolution question, which deserves further study.

NOR dispersion, telomeric sequence detection in centromeric regions and meiotic multivalent configurations in species of the Aplastodiscus albofrenatus group (Anura, Hylidae).

In the present study, we examined the chromosome number and morphology, heterochromatin pattern, and location of the nucleolar organizer region (NOR) and telomeric sequences in Aplastodiscus albofrenatus, A. arildae, A. ehrhardti and A. eugenioi in order to cytogenetically characterize the Aplastodiscus albofrenatus group. The 4 species analyzed in this study had diploid numbers of 2n = 22 and very similar chromosome morphology. These species could be differentiated based on the distribution and amount of heterochromatin and the location of the nucleolar organizer region (NOR). Six of the 8 A. albofrenatus individuals had an NOR polymorphism previously unknown in anurans since only one of the homologs of pairs 1 and 7 was stained. In the other 2 specimens, the NOR occurred on both homologs of pair 7. In A. ehrhardti, pairs 6 and 10 were stained by the AgNOR technique, but only pair 6 was confirmed by in situ hybridization. The NOR was located on pair 10 in A. arildae and on pair 7 in A. eugenioi. In A. albofrenatus and A. arildae, multivalent rings involving NOR-containing chromosomes were observed during prophase I of meiosis. The telomeric probe identified the telomeres in all species and also centromeric regions in the chromosomes of A. albofrenatus and A. arildae, which were coincident with centromeric heterochromatin. The conserved chromosomal morphology seen mainly in the first 7 pairs among species of the A. albofrenatus group suggests that all of these species probably originated from a common ancestral karyotype and that chromosomal rearrangements resulted in karyotype differentiation, with changes in NOR location, as well as telomeric and heterochromatin dispersal.

Cytogenetic comparison of tree frogs of the genus Aplastodiscus and the Hypsiboas faber group (Anura, Hylidae).

Four species of Aplastodiscus and two species of Hypsiboas were cytogenetically compared. Aplastodiscus perviridis, A. cochranae, H. albomarginaus, and H. faber had 2n = 24 chromosomes, while A. albosignatus and A. leucopygius had 2n = 20 and 2n = 18 chromosomes, respectively. Aplastodiscus perviridis and A. cochranae had identical karyotypes, as indicated by their chromosomal morphology, the location of the nucleolus organizer region (NOR) on chromosome pair 12, and the heterochromatin pattern. The NOR-bearing chromosomes of A. albosignatus and A. leucopygius (pair 9) were very similar in size and morphology (metacentric) when compared to A. perviridis and A. cochranae (pair 12) and to H. faber (pair 11); the NOR of these chromosomes also occurred in the same region, suggesting that these chromosomes are homologous. Although H. albomarginatus differs from the other species with regard to the location of its NOR on pair 2, this species had the same diploid number and a chromosomal morphology similar to that of A. perviridis and A. cochranae. Chromosomal differentiation among the species appears to have occurred by reduction in chromosome number.

Comparison of the spermatozoan morphology of Isognomon bicolor and Isognomon alatus (Mollusca, Bivalvia, Isognomonidae).

In this study we used transmission and scanning electron microscopy to examine the spermatozoan structure of Isognomon bicolor and Isognomon alatus. The spermatozoa of both species were of the primitive or ect-aquasperm type. The acrosomal morphologies were essentially similar but the top of the acrosomal vesicle in I. bicolor sperm had a slightly flattened edge whereas the apex of the acrosomal vesicle of I. alatus sperm had a rounded outline. This difference suggested that acrosomal morphology could be an important character for taxonomic differentiation. In the present work, the results demonstrated that the gamete ultrastructure of the two distinct species I. alatus, from Panama, and I. bicolor, from the southeastern region of Brazil, were similar to the other studied species of the superfamily Pterioidea.

Molecular phylogeny and karyotype differentiation in Paratelmatobius and Scythrophrys (Anura, Leptodactylidae).

Paratelmatobius and Scythrophrys are leptodactylid frogs endemic to the Brazilian Atlantic forest and their close phylogenetic relationship was recently inferred in an analysis that included Paratelmatobius sp. and S. sawayae. To investigate the interspecific relationships among Paratelmatobius and Scythrophrys species, we analyzed a mitochondrial region (approximately 2.4 kb) that included the ribosomal genes 12S and 16S and the tRNAval in representatives of all known localities of these genera and in 54 other species. Maximum parsimony inferences were done using PAUP* and support for the clades was evaluated by bootstrapping. A cytogenetic analysis using Giemsa staining, C-banding and silver staining was also done for those populations of Paratelmatobius not included in previous cytogenetic studies of this genus in order to assess their karyotype differentiation. Our results suggested Paratelmatobius and Scythrophrys formed a clade strongly supported by bootstrapping, which corroborated their very close phylogenetic relationship. Among the Paratelmatobius species, two clades were identified and corroborated the groups P. mantiqueira and P. cardosoi previously proposed based on morphological characters. The karyotypes of Paratelmatobius sp. 2 and Paratelmatobius sp. 3 described here had diploid chromosome number 2n = 24 and showed many similarities with karyotypes of other Paratelmatobius representatives. The cytogenetic data and the phylogenetic analysis allowed the proposal/corroboration of several hypotheses for the karyotype differentiation within Paratelmatobius and Scythrophrys. Namely the telocentric pair No. 4 represented a synapomorphy of P. cardosoi and Paratelmatobius sp. 2, while chromosome pair No. 5 with interstitial C-bands could be interpreted as a synapomorphy of the P. cardosoi group. The NOR-bearing chromosome No. 10 in the karyotype of P. poecilogaster was considered homeologous to chromosome No. 10 in the karyotype of Scythrophrys sp., chromosome No. 9 in the karyotype of Paratelmatobius sp. 1, chromosome No. 8 in the karyotypes of Paratelmatobius sp. 2 and of Paratelmatobius sp. 3, and chromosome No. 7 in the karyotype of P. cardosoi. A hypothesis for the evolutionary divergence of these NOR-bearing chromosomes, which probably involved events like gain in heteochromatin, was proposed.

The sperm of Hylodinae species (Anura, Leptodactylidae): ultrastructural characteristics and their relevance to interspecific taxonomic relationships.

Hylodinae leptodactylids (sensu Lynch 1971) form a group of diurnal frogs, which is hypothesized on the basis of morphological traits to be the closest relatives of the dendrobatid frogs. Our study describes ultrastructural characteristics of sperm from three hylodine species (Hylodes phyllodes, Crossodactylus sp. n. and Megaelosia massarti) to reassess the intergeneric relationships within the Hylodinae, as well as the supposed relationship between the Hylodinae and Dendrobatidae. The ultrastructure of the sperm is very similar among the three species and is indicative of its conserved nature within the Hylodinae. The structure of the acrosomal complex was very similar to that of other leptodactylid species, to most of the remaining species included in the Bufonoidea lineage, and also to that observed in the dendrobatid species examined so far. Since such a structure has been considered a plesiomorphic trait, it contributes little to our understanding of the relationships between the Hylodinae and Dendrobatidae. The flagellar apparatus of Crossodactylus sp. n. is very similar to that of most leptodactylids. The sperm of Megaelosia massarti and Hylodes phyllodes display a distinctive condition in their axial and juxtaxonemal fibers. This distinctive flagellar condition expands the already known variability in sperm structure within the Leptodactylidae.

Chromosomal differentiation of Hyla nana and Hyla sanborni (Anura, Hylidae) with a description of NOR polymorphism in H. nana.

Specimens of Hyla nana and Hyla sanborni from a syntopic population were studied cytogenetically. These species are morphologically very similar and are frequently misidentified, confused with each other. Both species had a diploid chromosome number, 2n = 30. However, the karyotypes of H. nana and H. sanborni differed considerably from each other in the number of submetacentric and telocentric chromosomes. The two species also differed in their primary NOR-bearing chromosomes (metacentric pair 13 in H. nana and telocentric pair 12 in H. sanborni). Additional nucleolus organizer regions (NORs) were detected by Ag-NOR staining and FISH in chromosome pairs 1, 5, 6, 12, and 14 in seven specimens of H. nana. Thus, a total of six patterns of NOR were identified. These differences in karyotype and in NOR location allowed the unambiguous identification of syntopic individuals of the two species. However, the chromosomal morphology of both species differed from that reported for populations from other geographic regions, suggesting that a systematic reevaluation of this group of Hyla may be necessary.

Chromosomal comparisons among and within populations of Simulium (Chirostilbia) pertinax (Diptera, Simuliidae).

Chromosomal studies were carried on six larval populations of Simulium (Chirostilbia) pertinax from different locations in Brazil. Larvae were collected in the states of Paraná, Rio Grande do Sul, Rio de Janeiro and São Paulo. Polytene chromosome map comparisons within and among populations showed no differences in banding pattern, except for some limited polymorphism (secondary NOR and four band polymorphisms). There were no chromosomal variations associated with the resistance or susceptibility of the larvae to temephos. The chromosomal homosequentiality found among the six populations suggests that S. pertinax may be a monomorphic species.

Cytogenetics of two species of Paratelmatobius (Anura: Leptodactylidae), with phylogenetic comments.

In this paper we provide a cytogenetic analysis of Paratelmatobius cardosoi and Paratelmatobius poecilogaster. The karyotypes of both species showed a diploid number of 24 chromosomes and shared some similarity in the morphology of some pairs. On the other hand, pairs 4 and 6 widely differed between these complements. These karyotypes also differed in their NOR number and location. Size heteromorphism was seen in all NOR-bearing chromosomes of the two karyotypes. In addition, both karyotypes showed small centromeric C-bands and a conspicuous heterochromatic band in the short arm of chromosome 1, although with a different size in each species. The P. cardosoi complement also showed other strongly stained non-centromeric C-bands, with no counterparts in the P. cardosoi karyotype. Chromosome staining with fluorochromes revealed heterogeneity in the base composition of two of the non-centromeric C-bands of P. cardosoi. Comparison of the chromosomal morphology of these Paratelmatobius karyotypes with that of P. lutzii showed that the P. poecilogaster karyotype is more similar to that of P. lutzii than P. cardosoi. These cytogenetic results agree with the proposed species arrangements in the P. cardosoi and P. lutzii groups based on morphological and ecological data.

Chromosomal rearrangements as the source of variation in the number of chromosomes in Pseudis (Amphibia, Anura).

A cytogenetic study of Pseudis specimens from three localities in Rio Grande do Sul State, the southernmost Brazilian, was performed to identify karyotypic characteristics that could account for differences in vocalization pattern and body size. Individuals from around Tainhas were compared to those of São Jerjnimo and Eldorado do Sul. Specimens from these latter two localities were identified as Pseudis minuta, while those from the former were classified as Pseudis sp. (aff minuta). The populations from São Jerjnimo and Eldorado do Sul had 2n = 24 chromosomes, classified as metacentric. submetacentric and subtelocentric. The population from Tainhas had 2n = 28 chromosomes, with four pairs of telocentric chromosomes. Modelling of these 28 chromosomes and testing for fusion in the centromeric/telomeric regions yielded a karyotype of 2n = 24 chromosomes, similar to that of the other populations. The similarity was reinforced by the location of the NORs and heterochromatin. The Tainhas population showed an increase in heterochromatin, as seen by the presence of additional C-bands, especially in the telocentric chromosomes. These data suggest that the two karyotypes described in this work had a common ancestry. There is evidence that the differentiation of these karyotypes may have occurred by chromosome fission and heterochromatin addition. Based on the present karyotype (2n = 28) and on morphological and vocalization studies by other researchers, we conclude that the Tainhas population may represent a new species.

An ultrastructural study of spermiogenesis in three species of Physalaemus (Anura, Leptodactylidae)

Ultrastructural aspects of spermiogenesis and testicular spermatozoa of three anuran amphibians, Physalaemus biligonigerus, P. fuscomaculatus and P. gracilis, were investigated by electron microscopy. The nuclei, middle pieces and tails of the three species were similar. In all cases, the nuclei were elongated and the acrosome consisted of a cone-shaped cap. The connecting pieces located in the flagellum implantation zone had transverse striations. The tails had a 9 + 2 axial filament pattern, a juxtaxonemal fiber and an undulating membrane. In contrast to other Leptodactylidae spermatozoa, no axial rod were observed in these Physalaemus species

An ultrastructural study of spermiogenesis in three species of Physalaemus (Anura, Leptodactylidae)

Ultrastructural aspects of spermiogenesis and testicular spermatozoa of three anuran amphibians, Physalaemus biligonigerus, P. fuscomaculatus and P. gracilis, were investigated by electron microscopy. The nuclei, middle pieces and tails of the three species were similar. In all cases, the nuclei were elongated and the acrosome consisted of a cone-shaped cap. The connecting pieces located in the flagellum implantation zone had transverse striations. The tails had a 9 + 2 axial filament pattern, a juxtaxonemal fiber and an undulating membrane. In contrast to other Leptodactylidae spermatozoa, no axial rod were observed in these Physalaemus species

An ultrastructural study of spermiogenesis in three species of Physalaemus (Anura, Leptodactylidae).

Ultrastructural aspects of spermiogenesis and testicular spermatozoa of three anuran amphibians, Physalaemus biligonigerus, P. fuscomaculatus and P. gracilis, were investigated by electron microscopy. The nuclei, middle pieces and tails of the three species were similar. In all cases, the nuclei were elongated and the acrosome consisted of a cone-shaped cap. The connecting pieces located in the flagellum implantation zone had transverse striations. The tails had a 9 + 2 axial filament pattern, a juxtaxonemal fiber and an undulating membrane. In contrast to other Leptodactylidae spermatozoa, no axial rod were observed in these Physalaemus species.

An ultrastructural study of spermiogenesis in three species of Physalaemus (Anura, Leptodactylidae).

Ultrastructural aspects of spermiogenesis and testicular spermatozoa of three anuran amphibians, Physalaemus biligonigerus, P. fuscomaculatus and P. gracilis, were investigated by electron microscopy. The nuclei, middle pieces and tails of the three species were similar. In all cases, the nuclei were elongated and the acrosome consisted of a cone-shaped cap. The connecting pieces located in the flagellum implantation zone had transverse striations. The tails had a 9 + 2 axial filament pattern, a juxtaxonemal fiber and an undulating membrane. In contrast to other Leptodactylidae spermatozoa, no axial rod were observed in these Physalaemus species.

Polymorphism of the nucleolus organizer regions (NORs) in Physalaemus petersi (Amphibia, Anura, Leptodactylidae) detected by silver staining and fluorescence in situ hybridization.

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.