High diversity of 5S ribosomal DNA and evidence of recombination with the satellite DNA PcP190 in frogs.

Distinct types of 5S rDNA repeats, differing by size and composition of its non-transcribed spacer (NTS), have been found in diverse taxa. Both concerted evolution and birth-and-death evolution have proven to play important roles in the evolution of the 5S rDNA family. In anurans, however, this subject has been underexplored as only a few anuran species had their 5S rDNA characterized and evolutionary analyzed to date. In the present study, we characterized the 5S rDNA sequences from species of two anuran families using classical molecular biology techniques and bioinformatic approaches. Based on the NTS, more than one type of 5S rDNA was identified in each analyzed species, which suggests that birth-and-death processes take part in the evolution of these sequences along the Anura tree of life. In addition, closely related species shared the same types of sequences, in accordance with the model of concerted evolution. We also found evidence of recombination between 5S rDNA and PcP190 satellite DNA, a repetitive sequence that is derived from the 5S rDNA. The interplay between 5S rDNA and PcP190 satellite DNA might favor the maintenance of the latter in the genome and respond to its presence in several species of frogs. The analysis of 5S rRNA transcripts confirmed the type I 5S rDNA of leiuperine as a functional 5S rRNA gene. Finally, the chromosome mapping of 5S rDNA sequences allowed some inferences of chromosome homology in Leiuperinae. In conclusion, our study provides additional information about the organization, differentiation and functionality of 5S rDNA in anuran species, revealing the potential participation of satellite DNA in the evolution of this family of rDNA.

Non-random distribution of microsatellite motifs and (TTAGGG)n repeats in the monkey frog Pithecopus rusticus (Anura, Phyllomedusidae) karyotype.

The monkey frog, Pithecopus rusticus (Anura, Phyllomedusidae) is endemic to the grasslands of the Araucarias Plateau, southern Brazil. This species is known only from a small population found at the type locality. Here, we analyzed for the first time the chromosomal organization of the repetitive sequences, including seven microsatellite repeats and telomeric sequences (TTAGGG)n in the karyotype of the species by Fluorescence in situ Hybridization. The dinucleotide motifs had a pattern of distribution clearly distinct from those of the tri- and tetranucleotides. The dinucleotide motifs are abundant and widely distributed in the chromosomes, located primarily in the subterminal regions. The tri- and tetranucleotides, by contrast, tend to be clustered, with signals being observed together in the secondary constriction of the homologs of pair 9, which are associated with the nucleolus organizer region. As expected, the (TTAGGG)n probe was hybridized in all the telomeres, with hybridization signals being detected in the interstitial regions of some chromosome pairs. We demonstrated the variation in the abundance and distribution of the different microsatellite motifs and revealed their non-random distribution in the karyotype of P. rusticus. These data contribute to understand the role of repetitive sequences in the karyotype diversification and evolution of this taxon.

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.

Genetic diversity of Morato's Digger Toad, Proceratophrys moratoi: spatial structure, gene flow, effective size and the need for differential management strategies of populations.

The Morato's Digger Toad, Proceratophrys moratoi, is a critically endangered toad species with a marked population decline in southern Brazilian Cerrado. Despite this, new populations are being discovered, primarily in the northern part of the distribution range, which raises a number of questions with regard to the conservation status of the species. The present study analyzed the genetic diversity of the species based on microsatellite markers. Our findings permitted the identification of two distinct management units. We found profound genetic structuring between the southern populations, on the left margin of the Tietê River, and all other populations. A marked reduction was observed in the contemporary gene flow among the central populations that are most affected by anthropogenic impacts, such as extensive sugar cane plantations, which presumably decreases habitat connectivity. The results indicated reduced diversity in the southern populations which, combined with a smaller effective population size, may make these populations more susceptible to extinction. We recommend the reclassification of P. moratoi as vulnerable and the establishment of a special protection program for the southern populations. Our results provide important insights about the local extinction of southern populations of this toad.

Genetic diversity of Morato's Digger Toad, Proceratophrys moratoi: spatial structure, gene flow, effective size and the need for differential management strategies of populations

Abstract The Morato's Digger Toad, Proceratophrys moratoi, is a critically endangered toad species with a marked population decline in southern Brazilian Cerrado. Despite this, new populations are being discovered, primarily in the northern part of the distribution range, which raises a number of questions with regard to the conservation status of the species. The present study analyzed the genetic diversity of the species based on microsatellite markers. Our findings permitted the identification of two distinct management units. We found profound genetic structuring between the southern populations, on the left margin of the Tietê River, and all other populations. A marked reduction was observed in the contemporary gene flow among the central populations that are most affected by anthropogenic impacts, such as extensive sugar cane plantations, which presumably decreases habitat connectivity. The results indicated reduced diversity in the southern populations which, combined with a smaller effective population size, may make these populations more susceptible to extinction. We recommend the reclassification of P. moratoi as vulnerable and the establishment of a special protection program for the southern populations. Our results provide important insights about the local extinction of southern populations of this toad.

A comparative ultrastructural analysis of spermatozoa in Pleurodema (Anura, Leptodactylidae, Leiuperinae).

This study describes the spermatozoa of 10 of the 15 species of the Neotropical frog genus Pleurodema through transmission electron microscopy. The diversity of oviposition modes coupled with a recent phylogenetic hypothesis of Pleurodema makes it an interesting group for the study of ultrastructural sperm evolution in relation to fertilization environment and egg-clutch structure. We found that Pleurodema has an unusual variability in sperm morphology. The more variable structures were the acrosomal complex, the midpiece, and the tail. The acrosomal complex has all the structures commonly reported in hyloid frogs but with different degree of development of the subacrosomal cone. Regarding the midpiece, the variability is given by the presence or absence of the mitochondrial collar. Finally, the tail is the most variable structure, ranging from single (only axoneme) to more complex (presence of paraxonemal rod, cytoplasmic sheath, and undulating membrane), with the absence of the typical axial fiber present in hyloid frogs, also shared with some other genera of Leiuperinae. J. Morphol. 277:957-977, 2016. © 2016 Wiley Periodicals, Inc.

Phylogeny of frogs from the genus Physalaemus (Anura, Leptodactylidae) inferred from mitochondrial and nuclear gene sequences.

Although some species groups have been recognized in the leiuperine genus Physalaemus, no phylogenetic analysis has previously been performed. Here, we provide a phylogenetic study based on mitochondrial and nuclear DNA sequences from 41 of the 46 species of Physalaemus. We employed the parsimony criterion using the software TNT and POY and the Bayesian criterion using the software MrBayes. Two major clades were recovered inside the monophyletic Physalaemus: (i) the highly supported Physalaemus signifer Clade, which included P. nattereri and the species previously placed in the P. deimaticus and P. signifer Groups; and (ii) the Physalaemus cuvieri Clade, which included the remaining species of Physalaemus. Five species groups were recognized in the P. cuvieri Clade: the P. biligonigerus Group, the P. cuvieri Group, the P. henselii Group, the P. gracilis Group and the P. olfersii Group. The P. gracilis Species Group was the same as that previously proposed by Nascimento et al. (2005). The P. henselii Group includes P. fernandezae and P. henselii, and was the sister group of a clade that comprised the remaining species of the P. cuvieri Clade. The P. olfersii Group included P. olfersii, P. soaresi, P. maximus, P. feioi and P. lateristriga. The P. biligonigerus Species Group was composed of P. biligonigerus, P. marmoratus, P. santafecinus and P. riograndensis. The P. cuvieri Group inferred here differed from that recognized by Nascimento et al. (2005) only by the inclusion of P. albifrons and the exclusion of P. cicada. The paraphyly of P. cuvieri with respect to P. ephippifer was inferred in all the analyses. Distinct genetic lineages were recognized among individuals currently identified as P. cuvieri and they were congruent with cytogenetic differences reported previously, supporting the hypothesis of occurrence of formally unnamed species.

Molecular and morphological evidence reveals a new species in the Phyllomedusa hypochondrialis group (Hylidae, Phyllomedusinae) from the Atlantic Forest of the highlands of southern Brazil.

The taxonomic status of a disjunctive population of Phyllomedusa from southern Brazil was diagnosed using molecular, chromosomal, and morphological approaches, which resulted in the recognition of a new species of the P. hypochondrialis group. Here, we describe P. rustica sp. n. from the Atlantic Forest biome, found in natural highland grassland formations on a plateau in the south of Brazil. Phylogenetic inferences placed P. rustica sp. n. in a subclade that includes P. rhodei + all the highland species of the clade. Chromosomal morphology is conservative, supporting the inference of homologies among the karyotypes of the species of this genus. Phyllomedusa rustica is apparently restricted to its type-locality, and we discuss the potential impact on the strategies applied to the conservation of the natural grassland formations found within the Brazilian Atlantic Forest biome in southern Brazil. We suggest that conservation strategies should be modified to guarantee the preservation of this species.

Possible introgression of B chromosomes between bee species (Genus Partamona ).

The origin of supernumerary (B) chromosomes is still a debated topic, with intra- and interspecific origins being the most plausible options. In the bee Partamona helleri, a sequence-characterized amplified region (SCAR) marker being specific to B chromosomes suggested the possibility of interspecific origin. Here, we search for this marker in 3 close relative species and perform DNA sequence comparison between species. The SCAR sequence does not show homology with other sequences in the databases, but does contain an open reading frame with sequence homology with a reverse transcriptase. Dot-blot hybridization using the SCAR marker as a probe confirmed that it is present in B-carrying, but not B-lacking larvae of P. helleri, and indicated its presence in adult individuals of P. cupira and P. criptica. Additionally, PCR amplification of the SCAR marker was successful on genomic DNA obtained from P. helleri and P. rustica larvae carrying B chromosomes, and on genomic DNA obtained from adult individuals of P. cupira, P. criptica and P. rustica. Finally, a comparison of the DNA sequence of the SCAR markers amplified from these 4 species showed very few nucleotide differences between the species. The complete association between B chromosome and SCAR presence and the scarce divergence observed for this DNA sequence between the 4 species analyzed suggest the possibility that this B chromosome has recently been transferred between species through several episodes of interspecific hybridization.

Chromosome analysis in Pseudopaludicola (Anura, Leiuperidae), with description of sex chromosomes XX/XY in P. saltica.

Taxonomic changes have frequently occurred in the anuran genus Pseudopaludicola as a consequence of high morphological similarity among its species. The present work reports karyotypic analysis of three Pseudopaludicola species sampled in their type locality and four Pseudopaludicola populations from distinct localities, aiming at contributing to the systematics of this genus. Chromosomes were stained with Giemsa or submitted to the silver staining (Ag-NOR) and C-banding techniques. The karyotype was 2n=22 in P. mineira, Pseudopaludicola sp. and two populations of P. saltica. The chromosome pair 8 was heteromorphic in P. saltica, characterizing a XX/XY sex-determination system with telocentric X and submetacentric Y. Highly similar karyotypes with 2n=18 chromosomes were observed in P. canga, P. aff. canga from Barreirinhas, State of Maranhão, Uberlândia, State of Minas Gerais and Icém, State São Paulo. The high similarity among the karyotypes 2n=18 suggested that the populations of P. aff. canga belong to the group 'pusilla', the same group of P. canga. The data demonstrated also that P. aff. canga from Barreirinhas (northeast region) is cytogenetically identical to P. canga with regarding the NOR site position in pair 3 and the presence of a heterochromatic block in the pair 2, whereas P. aff. canga from Uberlândia and Icém (southeast) had the NOR in the pair 9. Moreover, the cytogenetic data discriminated P. mineira and Pseudopaludicola sp. from the previously analyzed species with 22 chromosomes, and suggested that Pseudopaludicola sp. is an undescribed species. Sexual heteromorphic chromosomes are firstly reported in Pseudopaludicola and the data indicated the need of an extensive taxonomic review in this genus.

Unusual intra-individual karyotypical variation and evidence of cryptic species in Amazonian populations of Pristimantis (Anura, Terrarana).

We report a cytogenetic analysis of three Pristimantis fenestratus frog populations of the Amazon region in Brazil. The specimens were sampled in Borba and Manaus, Amazonas State, and in Rio Branco, Acre State. They were analyzed using Giemsa, silver staining, C-banding and FISH with rDNA probes. The karyotypes of the three populations revealed 2n = 34 chromosomes, but they differed in the number and position of Ag-NORs and in the heterochromatin pattern as well. The NOR was located on the pairs 05 and 07 in the Rio Branco specimens, pair 10 in the Manaus specimens, and pair 1 in the Borba specimens. A small C-band was detected on the telomeric region of the pair 05 in the Borba population, while in the Manaus there was a heterochromatic block adjacent to the centromere of pair 9. An unusual intra-individual variation of chromosome number was observed in metaphases of Rio Branco specimens, comprising fundamental numbers of 33, 34 and 35. Additionally, interchromosome thread connections were detected between telomere - telomere, centromere - telomere and centromere - centromere regions, and among chromosomal heterochromatin-rich sites. The NOR sites were also involved in those connections. We hypothesize that this variation is due to chromosome missegregation during mitosis. The inter- and intraindividual variation in chromosome number suggests chromosomal instability in P. fenestratus, which has not been detected in any other anuran group. Since Borba is the type-locality of P. fenestratus, a taxonomic review of the Manaus and Rio Branco populations should be done, as indicated by the cytogenetic evidence that they could be new species of Pristimantis.

Polytypic and polymorphic cytogenetic variations in the widespread anuran Physalaemus cuvieri (Anura, Leiuperidae) with emphasis on nucleolar organizing regions.

We investigated the NOR distribution in ten populations of Physalaemus cuvieri from different regions of Brazil and Argentina. A high variability in NOR pattern was observed and provided a useful tool in grouping several populations. The specimens from the state of Tocantins, northern Brazil, could easily be distinguished from all the other analyzed populations, since its karyotype presented NORs in the chromosome pairs 1,3,4 and 10 (and sometimes also in chromosome 5), and several pericentromeric C-bands. A NOR-site in chromosome 9 characterized three populations from the northeastern region of Brazil. Interestingly, the P. cuvieri populations located in opposite extremes of the geographic distribution had, as a fixed condition, the presence of NORs in 8q int and llp. Besides interpopulational divergences, intrapopulational variability was observed in the number of NORs, except for populations from the states of Bahia and Minas Gerais, which are boundary states respectively in the northeastern and southeastern regions of Brazil. In relation to NOR size, interindividual variations occurred in all Brazilian and Argentinean populations. Additionally, intraindividual variability in NOR size was detected in specimens from Minas Gerais. The data presented herein revealed substantial geographic polytypic variation in P. cuvieri and indicated that a taxonomic reexamination of this species is necessary.

The karyotype of three Brazilian Terrarana frogs (Amphibia, Anura) with evidence of a new Barycholos species.

A recent substantial rearrangement of the 882 described eleutherodactyline frog species has considerably improved the understanding of their systematics. Nevertheless, many taxonomic aspects of the South American eleutherodactyline species remain unknown and require further investigation using morphological, cytogenetic and molecular approaches. In this work, the karyotypes of the Brazilian species Ischnocnema juipoca (Atibaia and Campos do Jordão, SP), Barycholos cf. ternetzi (Uberlândia, MG, and Porto Nacional, TO), and Pristimantis crepitans (Chapada dos Guimarães and São Vicente, MT) were analyzed using Giemsa staining, Ag-NOR labeling, and C-banding techniques. All individuals had a diploid number of 22 chromosomes, but the Fundamental Numbers were different among species. The herein described low chromosome number of Pristimantis crepitans is unique within this genus, suggesting that cytogenetically this species is not closely related either to its congeneric species or to Ischnocnema. In addition, karyotype differences, mainly in the NOR position, clearly distinguished the two Barycholos populations, besides indicating the existence of a so far undescribed species in this genus. A taxonomic review could clarify the systematic position of P. crepitans and verify the hypothetic new Barycholos species.

Polytypic and polymorphic cytogenetic variations in the widespread anuran Physalaemus cuvieri (Anura, Leiuperidae) with emphasis on nucleolar organizing regions

We investigated the NOR distribution in ten populations of Physalaemus cuvieri from different regions of Brazil and Argentina. A high variability in NOR pattern was observed and provided a useful tool in grouping several populations. The specimens from the state of Tocantins, northern Brazil, could easily be distinguished from all the other analyzed populations, since its karyotype presented NORs in the chromosome pairs 1,3,4 and 10 (and sometimes also in chromosome 5), and several pericentromeric C-bands. A NOR-site in chromosome 9 characterized three populations from the northeastern region of Brazil. Interestingly, the P. cuvieri populations located in opposite extremes of the geographic distribution had, as a fixed condition, the presence of NORs in 8q int and llp. Besides interpopulational divergences, intrapopulational variability was observed in the number of NORs, except for populations from the states of Bahia and Minas Gerais, which are boundary states respectively in the northeastern and southeastern regions of Brazil. In relation to NOR size, interindividual variations occurred in all Brazilian and Argentinean populations. Additionally, intraindividual variability in NOR size was detected in specimens from Minas Gerais. The data presented herein revealed substantial geographic polytypic variation in P. cuvieri and indicated that a taxonomic reexamination of this species is necessary.

The karyotype of three Brazilian Terrarana frogs (Amphibia, Anura) with evidence of a new Barycholos species

A recent substantial rearrangement of the 882 described eleutherodactyline frog species has considerably improved the understanding of their systematics. Nevertheless, many taxonomic aspects of the South American eleutherodactyline species remain unknown and require further investigation using morphological, cytogenetic and molecular approaches. In this work, the karyotypes of the Brazilian species Ischnocnema juipoca (Atibaia and Campos do Jordão, SP), Barycholos cf. ternetzi (Uberlândia, MG, and Porto Nacional, TO), and Pristimantis crepitans (Chapada dos Guimarães and São Vicente, MT) were analyzed using Giemsa staining, Ag-NOR labeling, and C-banding techniques. All individuals had a diploid number of 22 chromosomes, but the Fundamental Numbers were different among species. The herein described low chromosome number of Pristimantis crepitans is unique within this genus, suggesting that cytogenetically this species is not closely related either to its congeneric species or to Ischnocnema. In addition, karyotype differences, mainly in the NOR position, clearly distinguished the two Barycholos populations, besides indicating the existence of a so far undescribed species in this genus. A taxonomic review could clarify the systematic position of P. crepitans and verify the hypothetic new Barycholos species.

B-chromosomes in two Brazilian populations of Dendropsophus nanus (Anura, Hylidae)

We report on the presence of B-chromosomes in two populations of Dendropsophus nanus (= Hyla nana Boulenger, 1889) from São Paulo State, Brazil. Such chromosomes were observed in 4 out of 43 specimens (9.3 percent) and in 9 out of 15 specimens (60 percent) from the municipalities of Nova Aliança and Botucatu, respectively. The karyotype 2n = 30 + 1B found in D. nanus was similar to that of other species with 2n = 30 chromosomes, except for the presence of an additional small telocentric chromosome. In one specimen from Botucatu, cells with one to three extra chromosomes were observed. These B-chromosomes appeared as univalent in meiosis I and did not bear a nucleolar organizer region or exhibit constitutive heterochromatin.

Conserved karyotypes in the Hyla pulchella species group (Anura, Hylidae).

Cytogenetic analyses were done on specimens of Hyla marginata and on three populations of H. semiguttata differing in morphology and in the physical parameters of their advertisement call, as well as in individuals of Hyla sp. (aff. semiguttata). All specimens had 2n=24 chromosomes with a morphology very similar to that of other 24-chromosome Hyla species. Hyla semiguttata and H. marginata showed the same C-banding pattern but were distinguished by the location of the NOR on pair 1 in H. semiguttata (in the three populations) and Hyla sp. (aff. semiguttata), and on pair 10 in H. marginata. The H. semiguttata populations did not differ cytogenetically, despite variations in their morphology and advertisement calls. Similarly, H. semiguttata and H. p. joaquini studied previously had identical C-banding patterns and NOR locations, suggesting that they are very closely related.

Cytogenetics of three Brazilian species of Eleutherodactylus (Anura, Leptodactylidae) with 22 chromosomes and re-analysis of multiple translocations in E. binotatus

In this paper, we provide a cytogenetic analysis of Eleutherodactylus guentheri, E. parvus and E. binotatus. All of the species had a diploid chromosomal number of 2n = 22. The karyotypes of E. guentheri and E. parvus were very similar and differed only slightly in the morphology of pair 2. These two species also had an NOR-bearing secondary constriction on the long arms of pair 6. The karyotype of E. binotatus differed from those of E. guentheri and E. parvus in the morphology and size of the chromosomes, in the number of chromosomal arms, in the NOR location (detected on the short arms of pair 1), and in the pattern of heterochromatin. These results reinforce the differences between E. guentheri and E. binotatus and support the existence of two species group. Five individuals of E. binotatus showed morphs for pairs 2 and 3. These morphs probably arose from the translocation of a segment from one chromosome of pair 3 to a homologue of pair 2. In addition, some mitotic metaphases of E. binotatus showed spontaneous chromosomal breaks which suggested that there were sites of fragility. Meiotic diakinesis showed multiple chromosomal rings, indicating the occurrence of multiple translocations, as previously reported by other investigators. These data suggest that, in addition to fission and fusion, other chromosomal rearrangements were probably involved in the differentiation of the karyotypes of these species of Eleutherodactylus, especially E. binotatus.

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae).

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20 C) and old (20 to 42 h, 28 C) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.

A technique for preparing polytene chromosomes from Aedes aegypti (Diptera, Culicinae).

Polytene chromosome preparations were obtained from larval, pupal and adult female Malpighian tubules of Aedes aegypti. The Malpighian tubules of the pupae (0-4 h old) from larvae reared at 20 C provided the best cytogenetic analysis. The interaction of nucleic acids and proteins that influence the spreading of the chromosomes could be reduced with the preparation technique of the sheets submitted to a stronger treatment starting with the hypotony of tissue and successive bathings with acetic acid. A simple technique should facilitate molecular cytogenetics used in the location of resistance and vector competence genes.