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.

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.

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.

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, 20ºC) and old (20 to 42 h, 28ºC) 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 20ºC 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

Sexual dimorphism in Ramphastos toco and Ramphastos dicolorus (Piciformes, Aves)

Phenotypic sexual dimorphism seems to be rare in the Ramphastidae family, except in Pteroglossus viridis and in the genus Selenidera. Many breeders of wild birds believe that specimens of Ramphastos toco can be sexed using bill characteristics. In this study, various discriminant phenotypic variables were analyzed in birds which were sexed cytogenetically. Fifty-one specimens of R. toco and 20 R. dicolorus were studied. The statistically significant parameters which served to distinguish the sex in these species were the length of the culmen and tomium, length of the lower corneous beak and the cloaca. Using these parameters, captive bird breeders can determine sex of R. toco specimens by phenotypic analysis and form breeding couples more quickly.

Restriction fragment analysis of the ribosomal DNA of Paratelmatobius and Scythrophrys species (Anura, Leptodactylidae)

Physical maps of the ribosomal RNA gene 28S of species belonging to the genera Paratelmatobius and Scythrophrys were constructed, using five restriction endonucleases. The restriction sites for Bam HI, Bgl II, Bst EII, and Eco RI had similar positions in all species, although there were interspecific differences in the size of the restriction fragments obtained. An additional Pvu II site was found in Scythrophrys specimens from Piraquara (State of Paraná, Brazil) and from Säo Bento do Sul (State of Santa Catarina, Brazil), but not in the Scythrophrys specimens from Rancho Queimado (State of Santa Catarina, Brazil). This finding is in agreement with the hypothesis regarding the existence of two species in the genus Scythrophrys. On the other hand, the extra Bst EII site considered in the literature to be a synapomorphy for the subfamilies Leptodactylinae and Telmatobiinae was not observed in the genera Paratelmatobius and Scythrophrys, which brings new questions about some taxonomic classifications that include Paratelmatobius in Leptodactylinae and Scythrophrys in Telmatobiinae. Interspecific variation was observed in the size of the restriction fragments analyzed and, in the case of group I Scythrophrys, there was also a variation between the individuals of the two populations. These data suggest that sequencing of the rDNA segments studied here may be useful in phylogenetic studies of the genera Paratelmatobius and Scythrophrys

Karyotypic characterization of Ramphastidae (Piciformes, Aves)

The karyotypes of nine species of the family Ramphastidae were determined and compared with that of the toco toucan (Ramphastos toco), the only ramphastid karyotype so far reported in the literature. Differences in the morphology of the largest chromosomes allowed to identify three karyotype groups. The first group included the species R. toco, Baillonius bailloni, Pteroglossus castanotis, P. aracari and Selenidera maculirostris, in which only the first pair of chromosomes was metacentric. The second group included four Ramphastos species (R. dicolorus, R. ariel, R. vitellinus, R. tucanus cuvieri) with two pairs of metacentric macrochromosomes (the first and the seventh). The third group was represented by a single species, A. laminirostris, in which all the autosomal chromosomes were telocentric. All of the species had subtelocentric Z chromosomes, similar in size to homologues of the first pair. Sex chromosome W was a small chromosome. The chromosome number ranged from 2n = 62 in P. aracari to 114 in R. toco. The cytotaxonomic relationships among toucan species are discussed, based on chromosome analysis

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