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We examined the swimming abilities of tadpoles exposed to the organophosphate chlorpyrifos for 24 h (at concentrations of 0, 1, 5 and 10 µg L-1). Scinax x - signatus, Physalaemus cf cuvieri, and Leptodactylus latrans tadpoles were used as biological models. Our findings evidenced decreased swimming speeds in all tadpole species exposed to chlorpyrifos as compared to the control group, although with significant statistical differences only with P. cuvieri and L. latrans. Changes in swimming ability may indirectly lead to death or alter important growth and development parameters, as the reduced ability to swim can result in increased vulnerability to predators and impacts on feeding, thus altering their fitness.

Physalaemus cuvieri (Fitzinger) Physalaemus cuvieri is the only member of Leptodactylidae family, recorded in five different ponds (Table 2; Figure 17; SM I). The advertisement calls were described based on 170 calls from 17 individuals and are formed by one note with a sparce harmonic sound (Köhler et al. 2017)., Published as part of Manzano, Maria Carolina Rodella, Takeno, Michele Fernandes & Sawaya, Ricardo J., 2022, Advertisement calls of 18 anuran species in the megadiverse Atlantic Forest in southeastern Brazil: review and update, pp. 453-472 in Zootaxa 5178 (5) on page 469, DOI: 10.11646/zootaxa.5178.5.3, http://zenodo.org/record/7033920, {"references":["Kohler, J., Jansen, M., Rodriguez, A., Kok, P. J. R., Toledo, L. F., Emmrich, M., Glaw, F., Haddad, C. F. B., Rodel, M. O. & Vences, M. (2017) The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice. Zootaxa 4251, 001 - 124. https: // doi. org / 10.11646 / zootaxa. 4251.1.1"]}

Although mate searching behavior in female túngara frogs (Physalaemus pustulosus) is nocturnal and largely mediated by acoustic cues, male signaling includes visual cues produced by the vocal sac. To compensate for these low light conditions, visual sensitivity in females is modulated when they are in a reproductive state, as retinal thresholds are decreased. This study tested whether estradiol (E2) plays a role in this modulation. Female túngara frogs were injected with either human chorionic gonadotropin (hCG) or a combination of hCG and fadrozole. hCG induces a reproductive state and increases retinal sensitivity, while fadrozole is an aromatase inhibitor that blocks hCG-induced E2 synthesis. In an analysis of scotopic electroretinograms (ERGs), hCG treatment lowered the threshold for eliciting a b-wave response, whereas the addition of fadrozole abolished this effect, matching thresholds in non-reproductive saline-injected controls. This suggests that blocking E2 synthesis blocked the hCG-mediated reproductive modulation of retinal sensitivity. By implicating E2 in control of retinal sensitivity, our data add to growing evidence that the targets of gonadal steroid feedback loops include sensory receptor organs, where stimulus sensitivity may be modulated, rather than more central brain nuclei, where modulation may affect mechanisms involved in motivation.

Studies on the use of food resources and trophic niche of different species can help understand ecological relationships. In this study, we evaluated the diet of three sympatric species of Physalaemus from the Brazilian semiarid. We analyzed a total of 264 individuals, being 100 specimens of Physalaemus albifrons, 93 of P. cicada, and 71 of P. cuvieri. Seven prey categories were recorded: three for P. cicada and P. albifrons, and six distinct categories for P. cuvieri. The orders Coleoptera, Isoptera and the family Formicidae were the most representative categories in the diet of P. cicada, P. cuvieri, and P. albifrons, respectively. Although there is a high trophic niche overlap, differences in preference for food categories may reduce potential competition in these frog species from the Caatinga.

Advertisement signals can convey information about a sender’s characteristics, such as body size. The reliability of signals, however, can be reduced when signal production is partially dependent on the environment. Here, we assess the effect of display-site properties on the production, attractiveness and honesty of sexual signals. We recorded male túngara frogs (Physalaemus pustulosus) while manipulating water levels in order to constrain calling. We found that water level affected male call properties in a size-dependent manner, with call amplitude being less affected in smaller males when forced to call in shallow water. Next, we tested how size-dependent and display-site-dependent signaling affected female choice and rival competition. Both males and females showed the strongest response to the call of a large male when he was calling at the deepest water levels. However, females showed no preference for large over small males when both were recorded in shallow water levels, or, depending on the call rate and timing of calls, even preferred small males. Likewise, males responded equally to large and small rivals recorded calling during shallow water level trials. Our experiments show that display-site properties can influence signal production and attractiveness in a size-dependent manner. These results can have important consequences for the evolution of signaling, as small males may be able to use their size to their advantage when selecting appropriate display sites and thereby outcompete large males.

Desiccation may increase egg and tadpole mortality of anurans that reproduce in temporary ponds. Tadpoles of some species, however, can survive temporarily out of water. Among leptodactylids, desiccation-resistant tadpoles were reported only for species that use terrestrial foam nests for reproduction. To test whether Leptodactylus tadpoles' ability to survive out of water represents an exclusive trait of species with terrestrial nests, we compared survival and resistance to hydric stress of tadpoles of six different species of the Leptodactylus groups and one Physalaemus. We also present an exploratory discussion about the evolutionary association between dehydration-tolerant tadpoles, egg-laying microhabitat, and nitrogen excretion type. Tadpoles were divided into two groups, one staying in 100 mL of water and another subjected to hydric stress on moist paper substrate. Our results indicate that tadpoles resistant to desiccation are not common for all species tested, as only the species with terrestrial nests survived longer and lost less body mass than the other related species. As an estimate, tadpoles of terrestrial-nesting frogs were seven times more resistant to desiccation than those with eggs/nests placed directly into the water. A possible dehydration-resistance mechanism is the premetamorphosis ureotelism that has been shown in terrestrial breeding species with tadpoles that survive some time out of water. Therefore, the capacity of tadpoles to survive a few days after pool drying probably arose from the evolutionary association between terrestrial reproduction and the tadpole ability to prevent body dehydration by a mechanism such as ureotelism.

Orientadores: Shirley Maria Recco Pimentel, Anete Pereira de Souza Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Muitas espécies de anfíbios estão entre as mais ameaçados de extinção, e no entanto, informações sobre a variabilidade genética e a estruturação genética das suas populações são ainda limitadas. No presente trabalho, a variabilidade genética de populações da espécie Physalaemus cuvieri foi analisada utilizando microssatélites como marcadores moleculares. Considerando-se sua ampla distribuição geográfica, variação morfológica interpopulacional e variação intra- e interpopulacional em algumas características citogenéticas, espécimes de dez populações de P. cuvieri foram amostradas, em São Pedro da Água Branca (MA), Urbana Santo (MA), Porto Nacional (TO), Uberlândia (MG), Vitória Brasil (SP), Palestina (SP), Nova Itapirema (SP), Chapada dos Guimarães (MT), Vitória da Conquista (BA) e Passo Fundo (RS). Dez locos de microssatélites de P. cuvieri foram isolados a partir de uma biblioteca enriquecida em repetições (CA)8 e (GT)8. A amplificação desses locos em 160 indivíduos de P. cuvieri revelou uma média de 5,7 alelos por loco e uma heterozigosidade esperada variando de 0,30 a 0,85. Embora a maior parte da variação genética esteja dentro das populações, o valor global do FST encontrado indica uma alta diferenciação entre essas populações. O baixo fluxo gênico encontrado sugere baixa capacidade dispersiva, no padrão de escala geográfica usado neste trabalho, e condiz com o comportamento filopátrico atribuído aos anuros. Nesse estudo foi ainda verificado que não existe correlação entre distância geográfica e distância genética das populações amostradas, sugerindo uma alta fidelidade dessa espécie aos seus locais de desova. Os resultados indicam que a distância geográfica sozinha não explica a variabilidade genética dessas populações. Duas populações, São Pedro da Água Branca (MA) e Nova Itapirema (SP), possivelmente passaram por efeito de gargalo populacional (bottleneck), que pode ser resultado, entre outros fatores, da destruição de habitat levando à fragmentação da população. Dos dez locos de microssatélites desenvolvidos para P. cuvieri, nove foram utilizados para verificar a possibilidade de amplificação e sua aplicabilidade no estudo da estrutura genética de duas outras espécies do grupo cuvieri muito proximamente relacionadas à P. cuvieri. Essas espécies são: P. ephippifer (15 indivíduos) e P. albonotatus (11). As duas populações estudadas ficaram isoladas das cinco de P. cuvieri, mostrando um alto grau de diferenciação genética, e indicando claramente sua condição de espécie distinta de P. cuvieri. A população de Crateús - CE agrupou com Urbano Santos - MA (ambas P. cuvieri). Este resultado corrobora os resultados de estudos citogenéticos. As populações de Porto Nacional (TO) e Passo Fundo (RS) formaram dois grupos de indivíduos cada uma. Parte dos indivíduos de Porto Nacional agrupou com a população de Uberlândia (MG) de P. cuvieri, e o restante dos indivíduos não mostrou nenhum agrupamento com qualquer outra população e espécies analisadas neste trabalho. Resultados semelhantes foram obtidos com a população de Passo Fundo, que não mostrou agrupamento com nenhuma outra população analisada. Estudos adicionais dessas populações serão necessários para melhor compreender esses dados. O conhecimento do padrão de dispersão da variabilidade genética de P. cuvieri, avaliada por microssatélites, somado ao conhecimento acumulado, obtido por meio de outras metodologias, poderá contribuir para a definição de estratégias de conservação e contribuir para o delineamento entre as fronteiras taxonômicas dessa espécie. Abstract: Even though many amphibian species are among the most endangered animals, the knowledge about their genetic variability and population genetic structure is still limited. In the present work, natural populations of the barker frog Physalaemus cuvieri were analyzed for genetic variability and differentiation using microsatellites, considering its wide geographic distribution, interpopulational morphological variation and cytogenetic intra- and interpopulational variation. Analyzes comprised specimens from ten P. cuvieri populations sampled in the following regions of Brazil: São Pedro da Água Branca (MA), Urbano Santo (MA), Porto Nacional (TO), Uberlândia (MG), Vitória Brasil (SP), Palestina (SP), Nova Itapirema (SP), Chapada dos Guimarães (MT), Vitória da Conquista (BA) and Passo Fundo (RS). Ten microsatellite loci were isolated for P. cuvieri from a library enriched in (CA)8 and (GT)8 repeats. In 160 P. cuvieri individuals, the average number of alleles per locus was 5.7 and the expected heterozygosity ranged from 0.30 to 0.85. Although most of the genetic variation was found within populations, the overall FST value indicated high genetic differentiation among the populations. The low gene flow among populations suggested low dispersion capability within the geographic scale pattern used in this work, which is compatible with the anuran philopatric behavior. In addition, there was no correlation between geographic and genetic distances of these populations, suggesting fidelity of P. cuvieri populations to their spawning place. These results indicated that the geographic distance alone does not explain the genetic variability observed among these populations. The data also indicated that the populations from São Pedro da Água Branca (MA) and Nova Itapirema (SP), respectively in the Northeast and Southeast regions of Brazil, have likely gone through a recent population bottleneck effect. Destruction of natural habitats leading to population fragmentation could be a major cause of this suggested bottleneck effect. Of the ten microsatellite loci developed for P. cuvieri, nine were used to investigate cross-amplification and suitability for population genetic structure studies in two other species of the cuvieri group closely related to P. cuvieri. These species are: P. ephippifer (15 specimens) and P. albonotatus (11 specimens). These two populations remained isolated from all populations of P. cuvieri, demonstrating its high genetic differentiation, pointing that these two species are distinct from P. cuvieri. The Crateús (CE) population was grouped with Urbano Santos (MA) population (both P. cuvieri). This result corroborated previous cytogenetic results. The Porto Nacional (TO) and Passo Fundo (RS) populations were divided in two sets of individuals each one. Part of Porto Nacional individuals were clustering with P. cuvieri from Uberlândia (MG), and the remaining did not show any grouping with none of other analyzed species. Similar results were found to Passo Fundo population, which also did not show any group with the analyzed species. The knowledge of the dispersion pattern of the P. cuvieri genetic variability evaluated by microsatellites, in addition to the genetic variability data obtained using other methodologies, will contribute for the development of future ecological and conservation strategies and to improve the taxonomy of this species. Doutorado Biologia Celular Doutor em Biologia Celular e Estrutural

Orientador: Luciana Bolsoni Lourenço Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Recentes análises filogenéticas e citogenéticas têm revelado grande diversidade dentre leiuperídeos do grupo Physalaemus cuvieri. O parafiletismo de P. cuvieri em relação a Physalaemus ephippifer foi inferido com base na análise de sequências de DNA, e linhagens reconhecidas dentre indivíduos de P. cuvieri parecem representar espécies ainda não descritas. A espécie Physalaemus ephippifer ocorre na foz do Rio Amazonas no Brasil e possivelmente na Guiana, mas sua correta distribuição geográfica permanece desconhecida. Análises citogenéticas de indivíduos de P. ephippifer da localidade-tipo (Belém-PA) revelaram um interessante heteromorfismo entre os homólogos do par cromossômico 8 de fêmeas, caracterizando a presença de um sistema de determinação sexual cromossômico ZZ/ZW. Os cromossomos Z e W apresentam uma NOR distal no braço longo, adjacente a uma banda heterocromática terminal, e o cromossomo W difere do Z por apresentar um segmento terminal no braço curto que inclui uma NOR e uma banda heterocromática. As NORs de P. ephippifer se mostraram co-localizadas, em experimentos de hibridação in situ, com as sequências de DNA repetitivo Pep194. Com o objetivo de contribuir para a investigação da diversidade genética e citogenética de populações amazônicas de Physalaemus do grupo P. cuvieri, analisamos as relações filogenéticas e os cariótipos de indivíduos de P. ephippifer de Santa Isabel-PA e exemplares coletados em localidades do Pará (Monte Alegre, Óbidos, Alenquer, Curuá, Parauapebas e Prainha) e de Roraima (Viruá), cuja identificação taxonômica é incerta. Para tanto, as preparações cromossômicas obtidas de machos a partir de suspensões de células do intestino e testículo foram submetidas à coloração convencional por Giemsa, bandamento C, coloração com DAPI, impregnação por prata e experimentos de hibridação in situ com sondas de DNA repetitivo Pep194 e DNA ribossomal 28S. Além disso, utilizamos uma sonda, chamada de Zqter, produzida a partir de um segmento microdissecado da região terminal do braço longo do cromossomo Z de P. ephippifer. As relações filogenéticas foram inferidas com base em sequências de DNA mitocondrial dos genes 12S, RNAt-val e 16S, utilizando o critério de máxima parcimônia. Os cariótipos dos indivíduos de Physalaemus sp. em análise foram semelhantes entre si, porém três citótipos foram reconhecidos devido ao padrão diferencial na distribuição das NORs no par 8. Os cariótipos dos espécimes de Monte Alegre, Óbidos, Alenquer, Prainha e Curuá (Citótipo I) e Parque Nacional do Viruá (Citótipo II) mostraram uma NOR pericentromérica no braço curto do par 8, porém apenas o Citótipo I foi portador de uma NOR terminal no braço longo desse mesmo par. O exemplar de Parauapebas (Citótipo III) mostrou duas NORs no braço longo dos cromossomos do par 8. Nas inferências filogenéticas, os exemplares de Physalaemus sp. portadores dos Citótipos I e II foram recuperados no mesmo clado (Clado A). Neste clado, foi possível observar uma subestruturação que acompanha a variação cariotípica detectada, no entanto a distância genética entre os dois grupos em questão, inferida com base em sequências parciais do gene 16S, foi baixa (0,1%). É provável, portanto, que os dois citótipos representem uma variação intraespecífica. O exemplar de Parauapebas incluído nas análises filogenéticas (Clado B) apresentou-se como grupo-irmão de um clado que incluiu P. ephippifer (Clado C) e o clado correspondente a uma das linhagens de P. cuvieri (Clado D, equivalente à Linhagem 1 de P. cuvieri, que agrupa indivíduos do norte e do nordeste do Brasil). A alta distância genética do Clado A em relação aos Clados B-D, inferida com base em segmento do gene 16S, sugere que o Clado A possa representar uma espécie ainda não descrita. Nas metáfases de indivíduos de Viruá, Alenquer e Curuá a sonda Zqter foi detectada na região pericentromérica do braço curto do par 8, adjacente às NORs detectadas pela sonda Pep194, sugerindo que esta região é homeóloga a região terminal ao braço longo do Z de P. ephippifer. As homelogias encontradas sugerem um possível evento de inversão cromossômica durante diferenciação desses cariótipos. A sonda Pep194 detectou, além das NORs dos cromossomos Z e W de P. ephippifer e da NOR pericentromérica de Physalaemus sp., as NORs dos cromossomos 8 e 9 de indivíduos de P. cuvieri de Urbano Santos-MA e os cromossomos 8 e 11 de indivíduos de P. cuvieri de Palestina-SP. Em contraste, essa sonda não detectou a NOR pericentromérica do cromossomo 9 de P. centralis, uma espécie mais distantemente relacionada filogeneticamente a P. cuvieri e P. ephippifer Abstract: Recent phylogenetic and cytogenetic analyses have revealed high diversity among leiuperids of the Physalaemus cuvieri group. The paraphyly of P. cuvieri with respect to Physalaemus ephippifer was inferred based on the analysis of DNA sequences, and genetic lineages that included individuals at first identified as P. cuvieri may represent undescribed species. P. ephippifer occurs at the mouth of the Amazon River in Brazil and possibly in Guyana, but its geographical distribution is unclear. Cytogenetic analyses of individuals of P. ephippifer from the type locality (Belém-PA) revealed an interesting heteromorphism between the homologous chromosomes of pair 8 in females, which characterized the presence of a chromosomal sex determination system ZZ/ZW. The Z and W chromosomes have a distal NOR in the long arm, adjacent to a terminal heterochromatic band, and the W chromosome differs from Z chromosome by a terminal segment in the short arm that comprises a NOR and a heterochromatic band. The NORs of P. ephippifer are shown co-located with DNA repetitive Pep194 in in situ hybridization experiments. Aiming to contribute to the investigation of the genetic and cytogenetic diversity of Amazonian populations belonging to P. cuvieri group, we analyzed the phylogenetic relationships and the karyotypes of individuals of P. ephippifer from Santa Isabel-PA and specimens collected from seven localities in the states of Pará (Monte Alegre, Óbidos, Alenquer, Curuá, Parauapebas and Prainha) and Roraima (Viruá), for which the taxonomic identification is uncertain. Chromosome preparations obtained from male individuals were subjected to conventional staining with Giemsa, C-banding, DAPI staining, silver impregnation and in situ hybridization with probes for the repetitive DNA Pep194 and ribosomal DNA 28S. In addition, we used a probe, named Zqter probe, produced from a segment microdissected from the terminal region of the long arm of the Z chromosome of P. ephippifer. Phylogenetic relationships were inferred based on mitochondrial DNA sequences of 12S, tRNA-val and 16S genes, using the criterion of maximum parsimony. The karyotypes of the individuals of Physalaemus sp. analyzed were similar, but three cytotypes were recognized with differential distribution of NORs in pair 8. Individuals from Monte Alegre, Óbidos, Alenquer, Prainha and Curuá (Cytotype I) showed a pericentromeric NOR in the short arm of pair 8 and a terminal NORin the long arm. In the male karyotypes of Viruá (Cytotype II), the NOR was located in the pericentromeric region of the short arm of pair 8. The individuals from Parauapebas (Cytotype III) showed two NORs in the long arm of chromosome pair 8. In the phylogenetic inferences, the specimens of Physalaemus sp. with Cytotypes I and II were nested in the same clade (Clade A). This clade showed a substructure that reflected the two karyotypic groups detected. The genetic distance between these two groups, however, inferred from partial sequences of the 16S gene, was low 0,1 %. It is likely, therefore, that the two cytotypes represent an intraspecific variation. The exemplar of Parauapebas included in the phylogenetic analysis (Clade B) was the sister group of a clade that included P. ephippifer (Clade C) and the clade corresponding to one of the lineages of P. cuvieri (Clade D, equivalent to Lineage 1 of P. cuvieri, which groups individuals of northern and northeastern Brazil). The high genetic distance between Clade A and clades B-D, inferred from 16S gene segment, suggests that Clade A may represent an undescribed species. In metaphases of individuals from Viruá, Alenquer and Curuá, the Zqter probe was detected in the pericentromeric region of the short arm of pair 8, adjacent to the NOR detected by the probe Pep194, suggesting that this region is homeologous with the terminal region of the long arm of the Z chromosome of P. ephippifer. The inferred homeologies suggest a possible event of chromosomal inversion along the differentiation of these karyotypes. The Pep194 probe detected, beyond the NORs of the Z and W chromosomes of P. ephippifer and the pericentromeric NOR of chromosomes 8 of the cytotypes of Physalaemus sp., the NORs of chromosomes 8 and 9 of individuals of P. cuvieri from Urbano Santos-MA and chromosomes 8 and 11 of individuals P. cuvieri from Palestina-SP. In contrast, this probe did not detect the pericentromeric NOR of chromosome 9 of P. centralis, which is distantly related phylogenetically to P. cuvieri and P. ephippifer Doutorado Biologia Celular Doutora em Biologia Celular e Estrutural

Orientador: Luciana Bolsoni Lourenço Morandini Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Os primeiros resultados citogenéticos obtidos para a espécie Physalaemus ephippifer, atualmente alocada no grupo P. cuvieri, mostraram um interessante heteromorfismo cromossômico ligado ao sexo. As 14 fêmeas analisadas presentaram um par cromossômico 8 heteromórfico, enquanto nos 7 machos analisados esse par era homomórfico. A diferença entre os cromossomos das fêmeas era devida à presença de um segmento adicional, composto por uma NOR e uma banda heterocromática a ela adjacente, localizado na região terminal do braço curto de apenas um desses homólogos, denominado de morfo 8b. Apesar desse importante achado, o uso de técnicas citogenéticas convencionais nessa e em outras espécies do grupo P. cuvieri não foram suficientes para esclarecer os mecanismos envolvidos na evolução cromossômica, já que poucos caracteres citogenéticos informativos foram evidenciados e também uma grande variação em relação às NORs foi encontrada. Com a intenção de buscar novos marcadores citogenéticos que auxiliem no estudo dos cromossomos sexuais de P. ephippifer e que colaborem na análise citogenética desse grupo de Physalaemus, foram estudadas sequências de DNA repetitivo isoladas desta espécie. Para tanto, o DNA genômico extraído de duas fêmeas de P. ephippifer, provenientes de Belém (Pará), foi digerido com a enzima de restrição BamHI e os fragmentos gerados foram separados por eletroforese em gel de agarose. Fragmentos posteriormente denominados de Pep194, Pep165 e Pep320 isolados a partir desse gel, foram clonados, sequenciados e localizados in situ no cariótipo de P. ephippifer. A seqüência Pep320 mostrou correspondência parcial com o fragmento EU343727.1 isolado de P. cuvieri, cuja sequência está disponível no GenBank. Apesar desses fragmentos não apresentarem similaridade entre si, as sequências Pep165 e Pep320 foram mapeadas no mesmo sítio cromossômico, correspondente à banda pericentromérica do braço curto do cromossomo 3. Esse resultado levanta um questionamento, ainda não respondido, acerca da organização molecular dessas sequências, que podem estar arranjadas em clusters independentes ou representar partes de uma mesma unidade repetitiva. A sequência Pep194 foi mapeada em regiões coincidentes com as NORs, localizadas no braço longo dos cromossomos identificados como Z e W, e no braço curto do cromossomo W. Apesar deste resultado, a análise da sequência Pep194 não apresentou nenhuma similaridade com regiões codificadoras do DNAr nucleolar, nem mesmo com regiões intergênicas associadas a elas já descritas. Tal sequência apresentou interessante arranjo interno, sendo composta de duas repetições diretas terminais, cada uma com 63 pb, sendo ambas flanqueadoras deuma região interna de 68 pb. Para melhor investigar a organização desta sequência no genoma de P. ephippifer, foram analisados produtos resultantes da amplificação por PCR de segmentos do DNA genômico, efetuada com o auxílio de primers construídos especificamente para se anelarem em regiões internas do segmento isolado. Os resultados obtidos por essa análise evidenciaram a presença de uma unidade repetitiva de 131 pb, que representa parte do fragmento Pep194, diferindo desse por não apresentar uma das regiões de 63 pb. No entanto, não é possível descartar a co-existência de uma unidade repetitiva de 194 pb, não detectada nessas análises. Em paralelo a esses experimentos, sequências pertencentes a elementos retrotransponíveis Rex1 foram isoladas do genoma de P. ephippifer por PCR, clonadas, sequenciadas e mapeadas por FISH em uma região heterocromática pericentromérica do braço curto do cromossomo 3. A análise das sequências desses fragmentos comprovou serem correspondentes a parte da sequência codificadora da enzima transcriptase reversa do elemento Rex1. A fim de verificar a presença desse elemento em outras espécies de Physalaemus, os mesmos primers utilizados nos experimentos com P. ephippifer, foram usados para a amplificação de sequências a partir de amostras do DNA genômico de P. albifrons, P. albonotatus, P. henselli e P. spiniger. A sequência isolada de P. albonotatus apresentou uma deleção interna de cerca de 220 pb quando comparada com as sequências correspondentes, aqui descritas ou disponíveis no GenBank. Isso permite sugerir que, embora derivado de um elemento Rex1, esse segmento provavelmente deixou de ser um elemento de transposição ativo. Embora esse seja o primeiro trabalho que descreve a ocorrência de Rex1 em anuros, a presença desse elemento parece comum, pelo menos no gênero Physalaemus. Além de apresentar similaridade com o segmento de Rex1 e com os fragmentos Pep165 e Pep320, a banda heterocromática pericentromérica de 3p é também o sítio de ocorrência de DNAr 5S. Tal região, reconhecida como DAPI-positiva na presente análise, permite clara distinção entre os cromossomos 3 e 4 de P. ephippifer, frequentemente confundidos se analisados apenas em relação à sua morfologia. As quatro sequências repetitivas aqui isoladas apresentaram-se eficientes marcadores citogenéticos e poderão ser utilizadas para futuros estudos comparativos entre espécies do gênero Physalaemus. Abstract: Previous cytogenetic studies of Physalaemus ephippifer, a species currently allocated to the group P. cuvieri, showed an interesting female-specific chromosome heteromorphism. In 14 females of P. ephippifer, chromosome pair 8 was heteromorphic with regard to the occurrence of an NOR anda terminal C-band. Such heteromorphism was not found in the seven analyzed male specimens. These findings suggest that the chromosomes of pair 8 may be sex chromosomes in P. ephippifer, characterizing a ZZ ?/ ZW ? sex-determination system in this species. Despite these important data, conventional cytogenetic techniques performed in this and other species of the Physalaemus group were not sufficient to clarify the processes involved in the karyological divergence in this anuran group. Aiming to look for new cytogenetic markers that may help in the study of P. ephippifer sex chromosomes and in the cytogenetic analysis of this group of Physalaemus, we studied repetitive DNA sequences isolated from P. ephippifer. Genomic DNA extracted from two females of P. ephippifer from Belém (Pará) was digested with the restriction enzyme BamHI. The restriction fragments were separated by electrophoresis in agarose gel. DNA fragments, which were ultimately named Pep194, Pep165, and Pep320, were isolated from the gel, cloned, sequenced, and localized in situ in the karyotype of P. ephippifer. The sequence Pep320 was very similar to the fragment EU343727.1 isolated from P. cuvieri. Although Pep320 and Pep165 were totally different in nucleotide sequence, they were mapped on the same chromosome site, which corresponded to the pericentromeric C-band in the short arm of chromosome 3. This raises doubts about the molecular organization of these sequences, which can be arranged in independent clusters, but can also represent partial regions of the same repeat unit. The sequence Pep194 was mapped in regions that coincided with the NORs, located in the long arm of Z and W chromosomes and in the short arm of the W chromosome. However, the Pep194 sequence had no similarity with the coding regions of the nucleolar rDNA or with intergenic spacers associated to them. The restriction fragment Pep194 had an interesting internal arrangement, being composed of two terminal direct repeats, each with 63 bp, flanking an internal region of 68 bp. To further investigate the organization of this sequence in the genome of P. ephippifer, we amplified some Pep194 segments from genomic DNA by PCR using specific primers designed to anneal in inner Mestrado Biologia Celular Mestre em Biologia Celular e Estrutural

The genus Physalaemus Fitzinger is composed by 49 species (Frost 2020), which are grouped in two major clades (Lourenço et al. 2015), the Physalaemus cuvieri and P. signifer clades. Within the P. cuvieri clade, five species groups are recognized: P. biligonigerus, P. cuvieri, P. gracilis, P. henselii, and P. olfersii (Lourenço et al. 2015). According to Lourenço et al. (2015), seven species are assigned to the P. olfersii group: P. feioi, P. insperatus, P. lateristriga, P. maximus, P. olfersii, P. orophilus, and P. soaresi. Regarding their larval stage, four species have their tadpoles described for external morphology: P. maximus, P. olfersii, P. orophilus, and P. soaresi (Weber et al. 2005; Baêta et al. 2007; Giaretta et al. 2009; Pezzuti et al. 2019); two of them have their tadpoles described for oral internal morphology: P. maximus and P. soaresi (Ruggeri & Weber 2012). Herein, we describe the external and oral internal morphology of tadpole of P. lateristriga from Paraná State, southern Brazil, and compare them to the known tadpoles of the P. olfersii group.

The anuran larynx is an organ of great evolutionary interest because it impacts male reproductive success in courtships. However, little is known about the diversity of the larynx's anatomy, evolutionary history and systematics importance. Here, we describe and compare the anatomy of the larynx of 10 Physalaemus species of the P. cuvieri clade, focusing on the P. olfersii species group. We also reconstructed the ancestral states and tested the phylogenetic signal for the anatomical features. In all the species, the larynx has a general globular shape with the arytenoid cartilages covering almost its entire dorsal surface, while the anterior process of the cricoid cartilages covers most of the ventral surface. The size of the secondary fibrous mass, the thickness of the vocal membrane, and the attachment position of the vocal membrane's free edge considerably differ among the species. Moreover, only four species of a single clade in the P. olfersii species group have the primary fibrous mass well-developed with a suspended region in the dorsolateral passage. We found a significant phylogenetic signal for all these characters. Ancestral reconstructions pointed to reduction tendencies in the thickness of the vocal membrane and the size of the secondary fibrous mass, and a shift of the ventral attachment of the vocal membrane, increasing the angle of its free edge along the phylogeny. This latter trait can diagnose the entire Physalaemus olfersii group, which has the ventral ends of the arytenoids positioned posteriorly, giving this group the steepest angles for the vocal membrane's free edge in relation to the frontal plane. Based on our results, the larynges can contribute to the Physalaemus olfersii species group's systematics and could be elucidative to understand the evolution of the genus. High levels of anatomical and bioacoustical complexity and diversity observed in the group support the expected correlation between vocal anatomy and bioacoustical signal.

The reproduction is one of the main events in the life of an organism, and anurans stand out among vertebrates because of the diversity of their reproductive strategies. We studied the reproduction of two syntopic species, Physalaemus aff. albonotatus and P. santafecinus, and comparatively described their reproductive activity pattern, advertisement calls, calling sites, daily calling activity, amplexus behavior, foam nests, and microhabitats in foam nests. In regards to the reproductive activity pattern, both species were defined as prolonged breeders. However, P. santafecinus exhibited a behavior like explosive breeders: it had a faster reproductive response against rains than P. aff. albonotatus. The calling activity was restricted exclusively to night hours in P. santafecinus, whereas P. aff. albonotatus called during both night and day. The advertisement calls of both species showed a rich harmonic structure, and were characterized by a bimodal harmonic dominance. The species differed significantly in microhabitat calling sites, foam nests, and microhabitats in foam nests. Namely, P. santafecinus frequently called and constructed its nests in sites more exposed than those of P. aff. albonotatus. The general differences in reproductive behaviors observed between the species principally agree with their different reproductive activity patterns., Facultad de Ciencias Naturales y Museo

Environmental temperatures are a major constraint on ectotherm abundance, influencing their distribution and natural history. Comparing thermal tolerances with environmental temperatures is a simple way to estimate thermal constraints on species distributions. We investigate the potential effects of behavioral thermal tolerance (i. e. Voluntary Thermal Maximum, VTMax) on anuran local (habitat) and regional distribution patterns and associated behavioral responses. We tested for differences in Voluntary Thermal Maximum (VTMax) of two sympatric frog species of the genus Physalaemus in the Cerrado. We mapped the difference between VTMax and maximum daily temperature (VTMax-ETMax) and compared the abundance in open and non-open habitats for both species. Physalaemus nattereri had a significantly higher VTMax than P. cuvieri. For P. nattereri, the model including only period of day was chosen as the best to explain variation in the VTMax while for P. cuvieri, the null model was the best model. At the regional scale, VTMax-ETMax values were significantly different between species, with P. nattereri mostly found in localities with maximum temperatures below its VTMax and P. cuvieri showing the reverse pattern. Regarding habitat use, P. cuvieri was in general more abundant in open than in non-open habitats, whereas P. nattereri was similarly abundant in these habitats. This difference seems to reflect their distribution patterns: P. cuvieri is more abundant in open and warmer habitats and occurs mostly in warmer areas in relation to its VTMax, whereas P. nattereri tends to be abundant in both open and non-open (and cooler) areas and occurs mostly in cooler areas regarding its VTMax. Our study indicates that differences in behavioral thermal tolerance may be important in shaping local and regional distribution patterns. Furthermore, small-scale habitat use might reveal a link between behavioral thermal tolerance and natural history strategies.

Foam-nesting frogs of the genus Physalaemus Fitzinger, 1826 have been long known for their diverse acoustic patterns (e.g., Barrio 1965; Bokermann 1966). Of the 49 species, the vocalizations of only three remain unknown to date (see Table 1 of Hepp & Pombal 2020): P. caete Pombal & Madureira, 1997, P. deimaticus Sazima & Caramaschi, 1988, and P. insperatus Cruz, Cassini & Caramaschi, 2008. Physalaemus caete is rare and narrowly distributed in Brazil’s northern Atlantic Forest (Santos et al. 2016; Bernardo et al. 2017; Haddad et al. 2018), and an explosive breeder (EMS pers. obs.). For those reasons the species is classified in the endangered category of Brazil’s red list of threatened species (ICMBio, 2018). Physalaemus caete was described in the late 1990s from the coastal region of the Brazilian state of Alagoas (Passo do Camaragibe) and reported for a second locality in Alagoas (Murici; Pombal & Madureira 1997). Recently, the distribution was extended to a third locality (Paulista, state of Pernambuco; Santos et al. 2016). The distribution range of P. caete coincides with the “Pernambuco endemism center”, a biogeographic subregion of coastal Atlantic Forest north of the São Francisco River (sensu Silva & Casteli 2003). Here we describe the advertisement call of the Pernambuco population of P. caete and make comparisons with calls of congeners.

SynopsisThe complexity of an animal’s interaction with its physical and/or social environment is thought to be associated with behavioral flexibility and cognitive phenotype, though we know little about this relationship in amphibians. We examined differences in cognitive phenotype in two species of frog with divergent natural histories. The green-and-black poison frog (Dendrobates auratus) is diurnal, displays enduring social interactions, and uses spatially distributed resources during parental care. Túngara frogs (Physalaemus=Engystomops pustulosus) are nocturnal, express only fleeting social interactions, and use ephemeral puddles to breed in a lek-type mating system. Comparing performance in identical discrimination tasks, we find that D. auratus made fewer errors when learning and displayed greater behavioral flexibility in reversal learning tasks than túngara frogs. Further, túngara frogs preferred to learn beacons that can be used in direct guidance whereas D. auratus preferred position cues that could be used to spatially orient relative to the goal. Behavioral flexibility and spatial cognition are associated with hippocampal function in mammals. Accordingly, we examined differential gene expression in the medial pallium, the amphibian homolog of the hippocampus. Our preliminary data indicate that genes related to learning and memory, synaptic plasticity, and neurogenesis were upregulated in D. auratus, while genes related to apoptosis were upregulated in túngara frogs, suggesting that these cellular processes could contribute to the differences in behavioral flexibility and spatial learning we observed between poison frogs and túngara frogs.

Histology is often underappreciated for the detection of the amphibian pathogenic fungus Batrachochytrium dendrobatidis, the cause of the potentially lethal skin disease chytridiomycosis. We evaluated the sensitivity of histology to detect chytrids in 20 wild specimens of 2 frog species from Uruguay that were clinically normal, but confirmed by PCR to be infected by B. dendrobatidis. We detected maturing and sporulated sporangia in 15 of 20 (75%) frogs, which is more sensitive than previously reported for histology. The effort needed to identify chytrids in histologic skin sections of Physalaemus henselii and Pleurodema bibroni required examination of 3.2 and 8.7 mm of skin sections for each frog species, respectively.