Analysis in Proceratophrys boiei genome illuminates the satellite DNA content in a frog from the Brazilian Atlantic forest.

Satellite DNAs (satDNAs) are one of the most abundant elements in genomes. Characterized as tandemly organized sequences that can be amplified into multiple copies, mainly in heterochromatic regions. The frog P. boiei (2n = 22, ZZ♂/ZW♀) is found in the Brazilian Atlantic forest and has an atypical pattern of heterochromatin distribution when compared to other anuran amphibians, with large pericentromeric blocks on all chromosomes. In addition, females of Proceratophrys boiei have a metacentric sex chromosome W showing heterochromatin in all chromosomal extension. In this work, we performed high-throughput genomic, bioinformatic, and cytogenetic analyses to characterize the satellite DNA content (satellitome) in P. boiei, mainly due to high amount of C-positive heterochromatin and the highly heterochromatic W sex chromosome. After all the analyses, it is remarkable that the satellitome of P. boiei is composed of a high number of satDNA families (226), making P. boiei the frog species with the highest number of satellites described so far. Consistent with the observation of large centromeric C-positive heterochromatin blocks, the genome of P. boiei is enriched with high copy number of repetitive DNAs, with total satDNA abundance comprising 16.87% of the genome. We successfully mapped via Fluorescence in situ hybridization the two most abundant repeats in the genome, PboSat01-176 and PboSat02-192, highlighting the presence of certain satDNAs sequences in strategic chromosomal regions (e.g., centromere and pericentromeric region), which leads to their participation in crucial processes for genomic organization and maintenance. Our study reveals a great diversity of satellite repeats that are driving genomic organization in this frog species. The characterization and approaches regarding satDNAs in this species of frog allowed the confirmation of some insights from satellite biology and a possible relationship with the evolution of sex chromosomes, especially in anuran amphibians, including P. boiei, for which data were not available.

A new reproductive mode in anurans: Natural history of Bokermannohyla astartea (Anura: Hylidae) with the description of its tadpole and vocal repertoire.

Anurans have the greatest diversity of reproductive modes among tetrapod vertebrates, with at least 41 being currently recognized. We describe a new reproductive mode for anurans, as exhibited by the Paranapiacaba Treefrog, Bokermannohyla astartea, an endemic and poorly known species of the Brazilian Atlantic Forest belonging to the B. circumdata group. We also describe other aspects of its reproductive biology, that are relevant to understanding the new reproductive mode, such as courtship behavior, spawning, and tadpoles. Additionally, we redescribe its advertisement call and extend its vocal repertoire by describing three additional call types: courtship, amplectant, and presumed territorial. The new reproductive mode exhibited by B. astartea consists of: (1) deposition of aquatic eggs in leaf-tanks of terrestrial or epiphytic bromeliads located on or over the banks of temporary or permanent streams; (2) exotrophic tadpoles remain in the leaf-tanks during initial stages of development (until Gosner stage 26), after which they presumably jump or are transported to streams after heavy rains that flood their bromeliad tanks; and (3) tadpole development completes in streams. The tadpoles of B. astartea are similar to those of other species of the B. circumdata group, although with differences in the spiracle, eyes, and oral disc. The vocal repertoire of B. astartea exhibits previously unreported acoustic complexity for the genus. Bokermannohyla astartea is the only bromeligenous species known to date among the 187 known species within the tribe Cophomantini. We further discuss evolutionary hypotheses for the origin of this novel reproductive mode.

Quaternary climatic fluctuations influence the demographic history of two species of sky-island endemic amphibians in the Neotropics.

We evaluated the role of Quaternary climatic fluctuations on the demographic history and population structure of amphibian species endemic to the 'campo rupestre' in the Neotropics, evaluating their distributional shifts, demographic changes, and lineage formation from the end of Pleistocene to present. We chose two anurans endemic to the high-elevation 'campo rupestre' in the Espinhaço Range (ER) in northeastern and southeastern Brazil (Bokermannohyla alvarengai and Bokermannohyla oxente), as models to test the role of Quaternary climatic fluctuations over their distribution range in this region. We collected tissue samples throughout their distribution range and used statistical phylogeography to examine processes of divergence and population demography. We generated spatial-temporal reconstructions using Bayesian inference in a coalescent framework in combination with hind-cast projections of species distribution models (SDMs). We also used the results and literature information to test alternative diversification scenarios via approximate Bayesian computation (ABC). Our results show that Quaternary climatic fluctuations influenced the geographic ranges of both species showing population expansion during the last glacial maximum (LGM) and range contraction during interglacial periods, as inferred from selected ABC models and from past projections of SDMs. We recovered Pleistocene diversification for both species occuring in distinctly unique periods for each taxon. An older and range-restricted lineage was recovered in a geographically isolated geological massif, deserving conservation and further taxonomic study. The diversification and distribution of these amphibian species endemic to the Neotropical 'campo rupestre' were influenced by Quaternary climatic fluctuations. The expansion of cold adapted species restricted to higher elevations during glacial periods and their concomitant retraction during interglacial periods may have been crucial for producing patterns of species richness and endemism along elevation gradients in tropical and subtropical domains. Such processes may influence the evolution of the biota distributed in heterogeneous landscapes with varied topography.

Lost and found: Frogs in a biodiversity hotspot rediscovered with environmental DNA.

Declines and extinctions are increasing globally and challenge conservationists to keep pace with biodiversity monitoring. Organisms leave DNA traces in the environment, e.g., in soil, water, and air. These DNA traces are referred to as environmental DNA (eDNA). The analysis of eDNA is a highly sensitive method with the potential to rapidly assess local diversity and the status of threatened species. We searched for DNA traces of 30 target amphibian species of conservation concern, at different levels of threat, using an environmental DNA metabarcoding approach, together with an extensive sequence reference database to analyse water samples from six montane sites in the Atlantic Coastal Forest and adjacent Cerrado grasslands of Brazil. We successfully detected DNA traces of four declined species (Hylodes ornatus, Hylodes regius, Crossodactylus timbuhy, and Vitreorana eurygnatha); two locally disappeared (Phasmahyla exilis and Phasmahyla guttata); and one species that has not been seen since 1968 (putatively assigned to Megaelosia bocainensis). We confirm the presence of species undetected by traditional methods, underscoring the efficacy of eDNA metabarcoding for biodiversity monitoring at low population densities, especially in megadiverse tropical sites. Our results support the potential application of eDNA in conservation biology, to evaluate persistence and distribution of threatened species in surveyed habitats or sites, and improve accuracy of red lists, especially for species undetected over long periods.

Redescription of the tadpole of Leptodactylus flavopictus Lutz, 1926 (Anura: Leptodactylidae) from Pilar do Sul, São Paulo, Brazil.

Frogs of the subfamily Leptodactylinae Werner can be found from southern Texas (USA) to southern Brazil, including the northern Antilles (Frost 2019). This subfamily is composed of 98 recognized species and the genus Leptodactylus Fitzinger contains the largest number of species (74 sp.) (Frost 2019). A study conducted by de Sá et al. (2014) provided a phylogeny of Leptodactylus based on molecular analysis, adult and larval morphology, and behavioral data. Currently, four species groups are recognized within this genus: L. fuscus group, L. pentadactylus group, L. latrans group, and L. melanonotus group (de Sá et al. 2014).

Notes on vocalizations of Brazilian amphibians IV: advertisement calls of 20 Atlantic Forest frog species.

Bioacoustics is a powerful tool used for anuran species diagnoses, given that advertisement calls are signals related to specific recognition and mate attraction. Thus, call descriptions can support species taxonomy. In spite of that, call descriptions are lacking for many species, delaying advances in biodiversity research. Here, we describe the advertisement calls of 20 anuran species from the Brazilian Atlantic Forest. We accessed 50 digital recordings deposited in the Fonoteca Neotropical Jacques Vielliard. Acoustic analyses were carried out in the software Raven pro 1.5. We provide a general comparison of call structure among species inside taxonomic groups and genera. The vocalizations described here belong to poorly known species, which are representatives of six families: Brachycephalidae, Bufonidae, Ceratophryidae, Cycloramphidae, Hylidae, and Phyllomedusidae. Despite this, still there are 163 species of anurans from Atlantic Forest with calls not formally described. Our work represents an important step in providing data for a taxonomic perspective and improving the knowledge of the Atlantic Forest anuran diversity.

Diversification of the widespread neotropical frog Physalaemus cuvieri in response to Neogene-Quaternary geological events and climate dynamics.

Here we reconstructed the demographical history and the dispersal dynamics of Physalemus cuvieri through the Neogene-Quaternary periods by coupling DNA regions with different mutation rates, ecological niche modelling, reconstruction of spatio-temporal lineage dispersal and coalescent simulations. Still, to test alternative diversification scenarios we used approximate Bayesian computation. Molecular phylogenetic analysis recovered four deep and strongly supported clades, which we interpret as population lineages. The ancestral location reconstruction placed the root in southcentral Amazonia, and the dispersal events indicate that spatial displacement was widespread early in the diversification of this species. The demographical scenario of "Multiple Refugia" with recent lineage admixture was the most likely hypothesis to predict the observed genetic parameters of P. cuvieri. Our results revealed that Neogene orogenic events might have played a prominent role in the early diversification of P. cuvieri. The species shows deep divergences with strong regional population structure, despite its widespread distribution. Final uplift of the central Brazilian Plateau and formation of the river basins in Central South America played an important role in the origin, diversification and the maintenance of P. cuvieri lineages.

A new species of Pseudopaludicola Miranda-Ribeiro (Anura: Leptodactylidae: Leiuperinae) from eastern Brazil, with novel data on the advertisement call of Pseudopaludicola falcipes (Hensel).

The Neotropical genus Pseudopaludicola includes 21 species, which occur throughout South America. Recent studies suggested that the population of Andaraí, in the state of Bahia, is an undescribed species, related to P. pocoto. Herein we formally describe this new species from lowlands of eastern Brazil. Recognition of this new species is supported by adult morphology, advertisement call, karyotype, and molecular data. It is diagnosed mainly by its small size, terminal phalanges knobbed (lack any expansion of the digital tips), proportionally short hindlimbs, 11 pairs of chromosomes, and advertisement call composed of series of three-pulsed notes, emitted at a high rate. In addition, we report for the first time the presence of P. pocoto in the campo rupestre (rupestrian grasslands) of Chapada Diamantina, a population with a much darker dorsal coloration than the population from the type locality. We also redescribed the advertisement call of P. falcipes based on recordings from topotypic males.

The advertisement calls of two species of Brachycephalus (Anura: Brachycephalidae) from southern Atlantic Forest, Brazil.

The advertisement calls are known for only eight species within the genus Brachycephalus. Herein, we describe the advertisement calls of B. olivaceus and B. quiririensis, both species from southern Atlantic Forest, state of Santa Catarina, Brazil. The advertisement calls of these species are similar, composed by one short and high-frequency note. The parameters range overlap between calls, as exemplified by the dominant frequency of 6.4-7.0 kHz in B. olivaceus and 6.2-6.5 kHz in B. quiririensis. Additionally, we provide a compilation of the available bioacoustic information for this genus.

ATLANTIC AMPHIBIANS: a data set of amphibian communities from the Atlantic Forests of South America.

Amphibians are among the most threatened vertebrates in the world and this is also true for those inhabiting the Atlantic Forest hotspot, living in ecosystems that are highly degraded and threatened by anthropogenic activities. We present a data set containing information about amphibian communities sampled throughout the Atlantic Forest Biome in South America. The data were extracted from 389 bibliographic references (articles, books, theses, and dissertations) representing inventories of amphibian communities from 1940 to 2017. The data set includes 17,619 records of 528 species with taxonomic certainty, from 1,163 study sites. Of all the records, 14,450 (82%) were classified using the criterion of endemism; of those, 7,787 (44%) were considered endemic and 6,663 (38%) were not. Historically, multiple sampling methods were used to survey amphibians, the most representative methods being active surveys (82.1%), surveys at breeding sites (20%), pitfall traps (15.3%), and occasional encounters (14.5%). Species richness averaged 15.2 ± 11.3 (mean ± SD), ranging from 1 to 80 species per site. We found a low dominance in the communities, with 10 species occurring in about 26% of communities: Physalaemus cuvieri (4.1%), Dendropsophus minutus (3.8%), Boana faber (3.1%), Scinax fuscovarius (2.8%), Leptodactylus latrans (2.7%), Leptodactylus fuscus (2.6%), Boana albopunctata (2.3%), Dendropsophus nanus (1.6%), Rhinella ornata (1.6%), and Leptodactylus mystacinus (1.6%). This data set represents a major effort to compile inventories of amphibian communities for the Neotropical region, filling a large gap in the data on the Atlantic Forest hotspot. We hope this data set can be used as a credible tool in the proposal of new studies on amphibian sampling and even in the development of conservation planning for these taxa. This information also has great relevance for macroecological studies, being foundational for both conservation and restoration strategies in this biodiversity hotspot. No copyright or proprietary restrictions are associated with the use of this data set. Please cite this data paper when the data are used in publications or teaching events.

A new species of Brachycephalus (Anura, Brachycephalidae) from the coast of Santa Catarina State, southern Atlantic Forest, Brazil.

We describe a new species of Brachycephalus from municipality of São Francisco do Sul and municipality of Itapoá, in the state of Santa Catarina, Brazil, southern Atlantic Forest. The new species is known from six localities from near sea level up to 250 meters and represents the first record of a "pumpkin-toadlet" occurring in the lowlands. Morphological traits and phylogenetic analysis of a fragment the 16S mtDNA gene place the new species in the Brachycephalus pernix group. The new species is supported by external morphology, osteology, advertisement call, and mtDNA divergence. It is characterized, among other traits by a dorsal body color dark green with a dark brown vertebral stripe, and an orange background; snout-vent length of 9.2-10.8 mm in males and 11.1-12.4 mm in females; and advertisement call short (0.02-0.03 seconds), composed of one high-frequency note (dominant frequency 6.6-7.3 kHz). We observed synchronized alternation in the emission of vocalizations among neighbor males, indicating that males of the new species are able to hear and use vocalizations to interact with each other. We provide descriptions of clutch, eggs, and juvenile and observations on parental care. The new species has not been recorded within any protected area and can be threatened by human-induced habitat loss and modification.

Comparative analysis based on replication banding reveals the mechanism responsible for the difference in the karyotype constitution of treefrogs Ololygon and Scinax (Arboranae, Hylidae, Scinaxinae).

According to the recent taxonomic and phylogenetic revision of the family Hylidae, species of the former Scinax catharinae (Boulenger, 1888) clade were included in the resurrected genus Ololygon Fitzinger, 1843, while species of the Scinax ruber (Laurenti, 1768) clade were mostly included in the genus Scinax Wagler, 1830, and two were allocated to the newly created genus Julianus Duellman et al., 2016. Although all the species of the former Scinax genus shared a diploid number of 2n = 24 and the same fundamental number of chromosome arms of FN = 48, two karyotypic constitutions were unequivocally recognized, related mainly to the distinct size and morphology of the first two chromosome pairs. Some possible mechanisms for these differences had been suggested, but without any experimental evidence. In this paper, a comparison was carried out based on replication chromosome banding, obtained after DNA incorporation of 5-bromodeoxiuridine in chromosomes of Ololygon and Scinax. The obtained results revealed that the loss of repetitive segments in chromosome pairs 1 and 2 was the mechanism responsible for karyotype difference. The distinct localization of the nucleolus organizer regions in the species of both genera also differentiates the two karyotypic constitutions.

Model-based analyses reveal insular population diversification and cryptic frog species in the Ischnocnema parva complex in the Atlantic forest of Brazil.

The Atlantic Forest (AF) of Brazil has long been recognized as a biodiversity conservation hotspot. Despite decades of studies the species inventory of this biome continues to increase with the discovery of cryptic diversity and the description of new species. Different diversification mechanisms have been proposed to explain the diversity in the region, including models of forest dynamics, barriers to gene flow and dispersal. Also, sea level change is thought to have influenced coastal diversification and isolated populations on continental islands. However, the timing and mode of diversification of insular populations in the AF region were rarely investigated. Here, we analyze the phylogeography and species diversity of the small-sized direct-developing frog Ischnocnema parva. These frogs are independent from water bodies but dependent on forest cover and high humidity, and provide good models to understand forest dynamics and insular diversification. Our analysis was based on DNA sequences for one mitochondrial and four nuclear genes of 71 samples from 18 localities including two islands, São Sebastião, municipality of Ilhabela, and Mar Virado, municipality of Ubatuba, both in the state of São Paulo. We use molecular taxonomic methods to show that I. parva is composed of six independently evolving lineages, with the nominal I. parva likely endemic to the type locality. The time-calibrated species tree shows that these lineages have diverged in the Pliocene and Pleistocene, suggesting the persistence of micro-refuges of forest in the AF. For the two insular populations we used approximate Bayesian computation to test different diversification hypotheses. Our findings support isolation with migration for São Sebastião population, with ∼1Mya divergence time, and isolation without migration for Mar Virado population, with ∼13Kya divergence time, suggesting a combination of different processes for diversification on AF islands.

The 100th: An appealing new species of Dendropsophus (Amphibia: Anura: Hylidae) from northeastern Brazil.

We describe a new species of the Dendropsophus leucophyllatus Group from the Atlantic Forest of the southern region of State of Bahia, Brazil. It can be distinguished from all species of the D. leucophyllatus Group on the basis of morphological characters (especially its unique dorsal pattern and snout in dorsal view), advertisement calls and divergence in mitochondrial DNA gene sequences. The inclusion of D. anceps on the group remains controversial but our phylogenetic analyses do not recover the new species as sister to syntopic species of the D. leucophyllatus Group (with or without D. anceps). These results also highlight the palimpsest that is past relation between the Atlantic and Amazon forests.

Expanding the knowledge about the occurrence of anurans in the highest amphibian diversity area of Atlantic Forest: Parque Estadual da Serra do Mar, São Paulo, Brazil / Expandindo o conhecimento sobre a ocorrência de anuros na área com maior diversidade de anfíbios da Floresta Atlântica: Parque Estadual da Serra do Mar, São Paulo, Brasil

Abstract The vegetation cover in the Atlantic Forest has been converted to human use or degraded by human activities, which declined the vegetation to 16% of its original extent. Although several protected areas have been created in this Neotropical biome over the past decades, our knowledge of the amphibian species within these areas is still far from adequate. Here, we present lists of anuran species from three different areas of Parque Estadual da Serra do Mar: núcleos Curucutu, Santa Virgínia and São Sebastião. To survey anuran species, we visited six sampling units (two ponds, two streams and two trails inside forest fragments) in each locality. Samplings were accomplished from December 2014 to February 2015, December 2015 to February 2016 and December 2016 to February 2017, totalizing 27 days of field samplings in each sampling unit. We recorded 34 anuran species in the Núcleo Curucutu, 44 species in the Núcleo São Sebastião and 42 species in the Núcleo Santa Virgínia, totalizing 65 species belonging to 12 families (number of species in parentheses): Bufonidae (5), Brachycephalidae (6), Centrolenidae (1), Craugastoridae (1), Cycloramphidae (2), Hemiphractidae (1), Hylidae (29), Hylodidae (3), Leptodactylidae (12), Microhylidae (2), Odontophrynidae (2), and Phyllomedusidae (1). We hope that these lists can be useful for future studies as well as helping in the management and conservation planning of these protected areas.

Resumo A cobertura vegetal da Floresta Atlântica foi convertida para uso humano ou degradada pela atividade humana permanecendo atualmente 16% da sua extensão original. Embora muitas áreas protegidas tenham sido criadas neste bioma neotropical nas últimas décadas, nosso conhecimento sobre as espécies de anfíbios nestas áreas ainda está longe do adequado. Aqui, nós apresentamos listas das espécies de anuros para três diferentes áreas do Parque Estadual da Serra do Mar: núcleos Curucutu, Santa Virgínia e São Sebastião. Para amostrar as espécies de anuros, visitamos seis locais de amostragem (duas poças, dois riachos e duas trilhas) em cada localidade. As amostragens foram realizadas de dezembro 2014 a fevereiro de 2015, dezembro de 2015 a fevereiro de 2016 e de dezembro de 2016 a fevereiro de 2017, totalizando 27 dias de amostragens de campo em cada ponto de amostragem. Registramos 34 espécies de anuros no Núcleo Curucutu, 44 espécies no Núcleo São Sebastião, e 42 espécies no Núcleo Santa Virgínia, totalizando 65 espécies pertecentes a 12 famílias (número de espécies entre parênteses): Bufonidae (5), Brachycephalidae (6), Centrolenidae (1), Craugastoridae (1), Cycloramphidae (2), Hemiphractidae (1), Hylidae (29), Hylodidae (3), Leptodactylidae (12), Microhylidae (2), Odontophrynidae (2), e Phyllomedusidae (1). Esperamos que estas listas possam ser úteis para futuros estudos assim como auxiliar os planos de manejo e conservação destas áreas protegidas.

A new species of flea-toad (Anura: Brachycephalidae) from southern Atlantic Forest, Brazil.

We describe a new species of Brachycephalus that is morphologically similar to the flea-toads B. didactylus, B. hermogenesi, and B. pulex. The new species occurs from the sea level up to 1000 m and it is widely distributed throughout southern Atlantic Forest. Brachycephalus sulfuratus sp. nov. is distinguished from all of its congeners by the combination of the following characters: (1) small body size (SVL of adults: 7.4-8.5 mm for males and 9.0-10.8 mm for females); (2) "leptodactyliform" body; (3) pectoral girdle arciferal and less robust compared to the Brachycephalus species with "bufoniform" body; (4) procoracoid and epicoracoid fused with coracoid but separated from the clavicle by a large fenestrae; (5) toe I externally absent; toes II, III, IV, and V distinct; phalanges of toes II and V reduced; (6) skin smooth with no dermal ossifications; (7) in life, general background color brown with small dark-brown spots; skin of throat, chest, arms, and forearms with irregular yellow blotches; in ventral view, cloacal region of alive and preserved specimens surrounded by a dark-brown inverted v-shaped mark outlined with white; (8) advertisement call long, composed of a set of 4-7 high-frequency notes (6.2-7.2 kHz) repeated regularly.

Possible interspecific origin of the B chromosome of Hypsiboas albopunctatus (Spix, 1824) (Anura, Hylidae), revealed by microdissection, chromosome painting, and reverse hybridisation.

The B chromosome in the hylid Hypsiboas albopunctatus (2n = 22 + B) is small, almost entirely composed of C-positive heterochromatin, and does not pair with any chromosome of the A complement. B probe, obtained by microdissection and DOP-PCR amplification, was used to search for homology between the B and regular chromosomes of H. albopunctatus and of the related species H. raniceps (Cope, 1862). Reverse hybridisation was also carried out in the investigation. The B probe exclusively painted the supernumerary, not hybridising any other chromosomes in H. albopunctatus, but all H. raniceps chromosomes showed small labelling signals. This result might be an indication that differences exist between the repetitive sequences of A and B chromosomes of H. albopunctatus, and that the chromosomes of H. raniceps and the heterochromatin of the B chromosome of H. albopunctatus are enriched with the same type of repetitive DNA. In meiotic preparations, the B labelled about 30% of scored spermatids, revealing a non-mendelian inheritance, and the painted B in micronucleus suggests that the supernumerary is eliminated from germ line cells. Although our results could suggest an interespecific origin of the B at first sight, further analysis on its repetitive sequences is still necessary. Nevertheless, the accumulation of repetitive sequences, detected in another species, even though closely related, remains an intriguing question.

Cytogenetic analysis of Phyllomedusa distincta Lutz, 1950 (2n = 2x = 26), P. tetraploidea Pombal and Haddad, 1992 (2n = 4x = 52), and their natural triploid hybrids (2n = 3x = 39) (Anura, Hylidae, Phyllomedusinae).

BACKGROUND: Natural polyploidy has played an important role during the speciation and evolution of vertebrates, including anurans, with more than 55 described cases. The species of the Phyllomedusa burmeisteri group are mostly characterized by having 26 chromosomes, but a karyotype with 52 chromosomes was described in P. tetraploidea. This species was found in sintopy with P. distincta in two localities of São Paulo State (Brazil), where triploid animals also occur, as consequence of natural hybridisation. We analyse the chromosomes of P. distincta, P. tetraploidea, and their triploid hybrids, to enlighten the origin of polyploidy and to obtain some evidence on diploidisation of tetraploid karyotype. RESULTS: Phyllomedusa distincta was 2n = 2x = 26, whereas P. tetraploidea was 2n = 4x = 52, and the hybrid individuals was 2n = 3x = 39. In meiotic phases, bivalents were observed in the diploid males, whereas both bivalents and tetravalents were observed in the tetraploid males. Univalents, bivalents or trivalents; metaphase II cells carrying variable number of chromosomes; and spermatids were detected in the testis preparations of the triploid males, indicating that the triploids were not completely sterile. In natural and experimental conditions, the triploids cross with the parental species, producing abnormal egg clutches and tadpoles with malformations. The embryos and tadpoles exhibited intraindividual karyotype variability and all of the metaphases contained abnormal constitutions. Multiple NORs, detected by Ag-impregnation and FISH with an rDNA probe, were observed on chromosome 1 in the three karyotypic forms; and, additionally, on chromosome 9 in the diploids, mostly on chromosome 8 in the tetraploids, and on both chromosome 8 and 9 in the triploids. Nevertheless, NOR-bearing chromosome 9 was detected in the tetraploids, and chromosome 9 carried active or inactive NORs in the triploids. C-banding, base-specific fluorochrome stainings with CMA3 and DAPI, FISH with a telomeric probe, and BrdU incorporation in DNA showed nearly equivalent patterns in the karyotypes of P. distincta, P. tetraploidea, and the triploid hybrids. CONCLUSIONS: All the used cytogenetic techniques have provided strong evidence that the process of diploidisation, an essential step for stabilising the selective advantages produced by polyploidisation, is under way in distinct quartets of the tetraploid karyotype.

Comparative karyotype analysis and chromosome evolution in the genus Aplastodiscus (Cophomantini, Hylinae, Hylidae).

BACKGROUND: The frogs of the Tribe Cophomantini present, in general, 2n = 24 karyotype, but data on Aplastodiscus showed variation in diploid number from 2n = 24 to 2n = 18. Five species were karyotyped, one of them for the first time, using conventional and molecular cytogenetic techniques, with the aim to perform a comprehensive comparative analysis towards the understanding of chromosome evolution in light of the phylogeny. RESULTS: Aplastodiscus perviridis showed 2n = 24, A. arildae and A. eugenioi, 2n = 22, A. callipygius, 2n = 20, and A. leucopygius, 2n = 18. In the metaphase I cells of two species only bivalents occurred, whereas in A. arildae, A. callipygius, and A. leucopygius one tetravalent was also observed besides the bivalents. BrdU incorporation produced replication bands especially in the largest chromosomes, and a relatively good banding correspondence was noticed among some of them. Silver impregnation and FISH with an rDNA probe identified a single NOR pair: the 11 in A. perviridis and A. arildae; the 6 in A. eugenioi; and the 9 in A. callipygius and A. leucopygius. C-banding showed a predominantly centromeric distribution of the heterochromatin, and in one of the species distinct molecular composition was revealed by CMA3. The telomeric probe hybridised all chromosome ends and additionally disclosed the presence of telomere-like sequences in centromeric regions of three species. CONCLUSIONS: Based on the hypothesis of 2n = 24 ancestral karyotype for Aplastodiscus, and considering the karyotype differences and similarities, two evolutionary pathways through fusion events were suggested. One of them corresponded to the reduction of 2n = 24 to 22, and the other, the reduction of 2n = 24 to 20, and subsequently to 18. Regarding the NOR, two conditions were recognised: plesiomorphy, represented by the homeologous small-sized NOR-bearing pairs, and derivation, represented by the NOR in a medium-sized pair. In spite of the apparent uniformity of C-banding patterns, heterogeneity in the molecular composition of some repetitive regions was revealed by CMA3 staining and by interstitial telomeric labelling. The meiotic tetravalent might be due to minute reciprocal translocations or to non-chiasmatic ectopic pairing between terminal repetitive sequences. The comparative cytogenetic analysis allowed to outline the chromosome evolution and contributed to enlighten the relationships within the genus Aplastodiscus.