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Made available in DSpace on 2018-11-26T17:41:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-10 Fundacao Grupo Boticario de Protecao a Natureza Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Melanophryniscus is a bufonid frog genus with a broad geographic distribution over southeastern South America. In recent years, several new species of Melanophryniscus have been discovered in southern Brazil showing a distinctive life-history strategy for the genus - breeding in phytotelmata - as well as a strong association with high-altitude regions. In this study, we use mitogenomic data to infer the phylogenetic relationships among diurnal, phytotelm-breeding Melanopluyniscus and to determine the timing of their divergence. We obtained the mitochondrial genomes (not including the control region) for eight individuals of Melanophryniscus representing all three described species (M. alipioi, M. milanoi, and M. xanthostomus), as well as some recently-discovered and potentially new species. Gene order was conserved in all species and corresponded to the general order found in bufonids. Although the phylogenetic relationships among the studied species was poorly supported, dating confirmed that they diverged during the Pleistocene, suggesting that phytotelm breeding could have arisen during drier periods in the glacial/interglacial cycles due to a decrease in the availability of permanent streams or ephemeral/temporary streams or ponds in which Melanophryniscus species commonly breed. Univ Fed Parana, Dept Zool, CP 19020, BR-81531980 Curitiba, Parana, Brazil Mater Natura Inst Estudos Ambientais, BR-80250020 Curitiba, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Praca Infante Dom Henrique S-No, BR-11330900 Sao Vicente, SP, Brazil Pontificia Univ Catolica Parana, Curitiba, Parana, Brazil Louisiana State Univ, Dept Biol Sci, Baton Rouge, LA 70803 USA Occidental Coll, Moore Lab Zool, 1600 Campus Rd, Los Angeles, CA 90041 USA Univ Estadual Paulista, Inst Biociencias, Praca Infante Dom Henrique S-No, BR-11330900 Sao Vicente, SP, Brazil Fundacao Grupo Boticario de Protecao a Natureza: 0895_20111 Fundacao Grupo Boticario de Protecao a Natureza: A0010_2014 CNPq: 571334/2008-3 CAPES: 108/2014

Ethnopharmacological relevance Among amphibians, 15 of the 47 species reported to be used in traditional medicines belong to the family Bufonidae, which demonstrates their potential in pharmacological and natural products research. For example, Asian and American tribes use the skin and the parotoid gland secretions of some common toads in the treatment of hemorrhages, bites and stings from venomous animals, skin and stomach disorders, as well as several types of cancers. Overarching objective In addition to reviewing the occurrence of chemical constituents present in the family Bufonidae, the cytotoxic and biomedical potential of the active compounds produced by different taxa are presented. Methodology Available information on bioactive compounds isolated from species of the family Bufonidae was obtained from ACS Publications, Google, Google Scholar, Pubmed, Sciendirect and Springer. Papers written in Chinese, English, German and Spanish were considered. Results Recent reports show more than 30% of amphibians are in decline and some of bufonid species are considered to be extinct. For centuries, bufonids have been used as traditional folk remedies to treat allergies, inflammation, cancer, infections and other ailments, highlighting their importance as a prolific source for novel drugs and therapies. Toxins and bioactive chemical constituents from skin and parotid gland secretions of bufonid species can be grouped in five families, the guanidine alkaloids isolated and characterized from Atelopus , the lipophilic alkaloids isolated from Melanophryniscus , the indole alkaloids and bufadienolides known to be synthesized by species of bufonids, and peptides and proteins isolated from the skin and gastrointestinal extracts of some common toads. Overall, the bioactive secretions of this family of anurans may have antimicrobial, protease inhibitor and anticancer properties, as well as being active at the neuromuscular level. Conclusion In this article, the traditional uses, toxicity and pharmacological potential of chemical compounds from bufonids have been summarized. In spite of being reported to be used to treat several diseases, neither extracts nor metabolites from bufonids have been tested in such illness like acne, osteoporosis, arthritis and other illnesses. However, the cytotoxicity of these metabolites needs to be evaluated on adequate animal models due to the limited conditions of in vitro assays. Novel qualitative and quantitative tools based on MS spectrometry and Nuclear Magnetic Resonance spectroscopy is now available to study the complex secretions of bufonids.

Toads of the genus Melanophryniscus contain toxic alkaloids sequestered from a diet of mainly ants and mites. Wild-caught specimens of M. klappenbachi were fed an alkaloid-free diet and their alkaloid profile was analyzed during 38 months in captivity. Individual alkaloid patterns varied considerably. Over time, the concentration of two alkaloids, pumiliotoxin 251D and 3,5-disubstituted indolizidine 195B, significantly declined, suggesting that the toads depend on continuous access to alkaloid-containing prey to maintain natural levels of their chemical defense.

Melanophryniscus admirabilis (admirable red-belly toad) is a microendemic and critically endangered species found exclusively along 700 m of the Forqueta River, in a fragment of the Atlantic Forest of southern Brazil. One of the greatest concerns regarding the conservation of this species is the extensive use of pesticides in areas surrounding their natural habitat. In recent years, the adaptation and persistence of animal species in human-impacted environments have been associated with microbiota. Therefore, the present study aimed to characterize the oral bacterial community of wild M. admirabilis and to address the question of how this community might contribute to this toad&rsquo, s adaptation in the anthropogenic environment as well as its general metabolic capabilities. A total of 11 oral samples collected from wild M. admirabilis were characterized and analyzed via high-throughput sequencing. Fragments of the 16S rRNA variable region 4 (V4) were amplified, and sequencing was conducted using an Ion Personal Genome Machine (PGM) System with 316 chips. A total of 181,350 sequences were obtained, resulting in 16 phyla, 34 classes, 39 orders, and 77 families. Proteobacteria dominated (53%) the oral microbiota of toads, followed by Firmicutes (18%), Bacteroidetes (17%), and Actinobacteria (5%). No significant differences in microbial community profile from among the samples were reported, which suggests that the low dietary diversity observed in this population may directly influence the bacterial composition. Inferences of microbiome function were performed using PICRUSt2 software. Important pathways (e.g., xenobiotic degradation pathways for pesticides and aromatic phenolic compounds) were detected, which suggests that the bacterial communities may serve important roles in M. admirabilis health and survival in the anthropogenic environment. Overall, our results have important implications for the conservation and management of this microendemic and critically endangered species.

In recent decades, a renewed interest in comparative studies of embryonic ontogeny in anurans is taking place. Toad embryos are often employed as model organisms, and scarce attention has been put on interspecific variations. In this work we analyze the development of transient embryonic and larval structures in 21 species in five genera of Bufonidae. These species vary in their ovipositional mode and the type of environments where the embryos and tadpoles develop, including ponds, streams, and axils of leaves of terrestrial or epiphytic plants. Comparative anatomical studies and sequence heterochrony analyses show that primary morphological variations occur in the morphology at the tail-bud stage, the arrangement and development of the external gills, adhesive gland type and division timing, growth of the dorsal hatching gland on the head, configuration of the oral disc, emergence and development of the hind limbs, and presence of the abdominal sucker. Some of these transformations are best ex...

Made available in DSpace on 2018-11-26T16:04:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-09-01 Fundacao Grupo Boticario de Protecao a Natureza Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Despite encompassing a relatively small geographical area, montane regions harbour disproportionately high levels of species diversity and endemism. Nonetheless, relatively little is known about the evolutionary mechanisms that ultimately lead to montane diversity. In this study, we used target capture of ultraconserved elements to investigate the phylogenetic relationships and diversification patterns of Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae), two frog genera that occur in sky islands of the southern Atlantic Forest of Brazil. Specifically, we tested whether diversification of montane species in these genera could be explained by a single climatic shift leading to isolation in sky islands, followed by climatic stability that maintained populations in allopatry. In both genera, the topologies inferred using concatenation and coalescent-based methods were concordant and had strong nodal support, except for a few recent splits, which nevertheless tended to be supported by more informative loci. Estimation of divergence time of a combined dataset using both genera is consistent with a concordant timing of their diversification. These results support the scenario of diversification by isolation in sky islands and suggest that allopatry attributable to climatic gradients in montane regions is an important mechanism for generating species diversity and endemism in these regions. Univ Fed Parana, Dept Zool, Curitiba, Parana, Brazil Mater Nat Inst Estudos Ambientais, Curitiba, Parana, Brazil Louisiana State Univ, Dept Biol Sci, Baton Rouge, LA 70803 USA Louisiana State Univ, Museum Nat Sci, Baton Rouge, LA 70803 USA Pontificia Univ Catolica Parana, Escola Ciencias Vida, Curitiba, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Sao Paulo, Brazil Occidental Coll, Moore Lab Zool, Los Angeles, CA 90041 USA Univ Estadual Paulista, Inst Biociencias, Sao Paulo, Brazil Fundacao Grupo Boticario de Protecao a Natureza: A0010_2014 CNPq: 301636/2016-8

Las estrategias reproductivas en anfibios varían en función de la duración de la temporada reproductiva, las características de los individuos y de factores vinculados al entorno social (densidad y la proporción de sexos, OSR). El entorno social es predictor de la intensidad de selección sexual ya que modifica los niveles de competencia y la elección de pareja, exacerbando los costos del conflicto sexual y permitiendo la aparición de tácticas reproductivas alternativas. Melanophryniscus rubriventris es un modelo de estudio atractivo ya que presenta un comportamiento reproductivo explosivo, con agregación de gran cantidad de individuos y OSR sesgadas hacia los machos que generan una competencia intensa por la pareja. En esta tesis se describen las estrategias reproductivas de machos y hembras en relación con la dinámica de arribo al sitio reproductivo, las interacciones entre sexos, y las características de los ejemplares reproductores (edad y tamaño corporal) tanto en condiciones naturales como bajo manipulación del contexto social. Los resultados muestran la aparición explosiva de altas densidades de individuos con una fuerte sincronía en la llegada de ambos sexos a los sitios reproductivos. Los machos utilizan mayormente una estrategia de búsqueda activa y lucha directa por el acceso a las hembras, ejerciendo acoso sobre ellas e intentando desmontar a otros machos en amplexo. La edad y el tamaño no mostraron efectos sobre el éxito reproductivo de los machos y no se observaron indicios directos de mecanismos de elección de pareja por parte de las hembras sobre estos rasgos. La estrategia utilizada por las hembras fue evitar el contacto con los machos, ya sea permaneciendo inmóvil o mediante escapes rápidos a densidades altas que se interpreta como un posible mecanismo para reducir la coerción sexual. Ninguno de los sexos mostró cambios en sus estrategias de obtención de pareja ante variaciones en el contexto social. Este sistema de apareamiento, enmarcado en un ambiente con precipitaciones impredecibles y con importantes condicionantes del contexto social (altas densidades y competencia física intensa), parece dificultar el establecimiento de estrategias que podrían maximizar el éxito reproductivo y la eficacia biológica de los individuos como la competencia por cantos, la presencia de machos satélites, el arribo temprano de individuos con ciertos fenotipos, entre otras. Los mecanismos para obtener pareja en la especie estarían esencialmente basados en la chance, con ausencia de elección pre-copulatoria debido al riesgo al que se exponen las hembras por la fuerte coerción. Amphibian reproductive strategies vary according to season length, individual reproductive traits, and factors associated with the social environment such as density and odds sex ratio (OSR). The social environment predicts the intensity of sexual selection due to its effect on mate choice and intra-sexual competition. Moreover, it exacerbates the costs derived from sexual conflict and allows for the existence of alternative reproductive tactics in order to counteract such costs. Melanophryniscus rubriventris is an attractive model for the study of sexual conflict due to its explosive reproductive behavior characterized by the occurrence of a large number of individuals. This situation, together with male-biased sex ratios promotes intense competition between males for the access to females. This thesis describes male and female reproductive strategies of the species related to the dynamics of the arrival, the interactions within and between the sexes, and individual traits (body size and age) under both, natural and experimental conditions with manipulation of the social context. The species showed an explosive pattern, with the gathering of a large number of individuals and the synchronous arrival of both sexes to the reproductive sites. The reproductive strategies of males were mainly based on the active search of the females, scramble competition, and female harassment regardless of the differences in the social context. Male reproductive success was not related to age or body size and no direct evidence of mate choice was observed for the females. Females´ strategy appeared to be the avoidance of male contact by either standing still or by a quick escape at high densities which can be seen as a potential mechanism to reduce sexual coercion. Neither males nor females showed alternative mating strategies related to changes in the social context. This mating system takes place in an environment with unpredictable rain cycles together with strong social determinants (high densities and intense male competence) seems to hamper the occurrence of certain strategies which may help to maximize the individuals’ reproductive success such as vocalization, satellites males, or the early arrival of individuals with certain phenotypic traits, among others. In this context, the mating probability of males would be mainly explained by chance, with the absence of precopulatory mate choice in females which face risks due to strong sexual coercion. Fil: García, Cecilia Graciela.

Aposematic signals as well as body behaviours may be important anti-predator defences. Species of the genus Melanophryniscus are characterised by having toxic lipophilic alkaloids in the skin and for presenting a red ventral colouration, which can be observed when they perform the behaviour called the unken reflex. Both the reflex behaviour and the colouration pattern are described as defence mechanisms. However, there are currently no studies testing their effectiveness against predators. This study aimed to test experimentally if both ventral conspicuous colouration and the unken reflex in Melanophryniscus cambaraensis function as aposematic signals against visually oriented predators (birds). We simulated the species studied using three different clay toad models as follows: (a) in a normal position with green coloured bodies, (b) in the unken reflex position with green coloured body and extremities and (c) in the unken reflex position with a green body and red extremities. Models were distributed on a known M. cambaraensis breeding site and in the adjacent forest. More than half of the attacks on the models were from birds; however, there was no preference for any model type. Thus, just the presence of the red colour associated with the motionless unken reflex position does not seem to prevent attacks from potential predators. It is possible that the effective aposematic signal in Melanophryniscus is achieved through the unken reflex movement together with the subsequent exhibition of the warning colouration and the secretion of toxins.

Despite encompassing a relatively small geographical area, montane regions harbor disproportionately high levels of species diversity and endemism. Yet, relatively little is known about the evolutionary mechanisms ultimately leading to montane diversity. In this study, we use target capture of ultraconserved elements to investigate the phylogenetic relationships and diversification patterns of Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae), two frog genera that occur in sky islands of the southern Atlantic Forest of Brazil. Specifically, we test whether diversification of montane species in these genera can be explained by a single climatic shift leading to isolation in sky islands, followed by relative climatic stability that maintained populations in allopatry. In both genera, the topologies inferred using concatenation and coalescent-based methods were concordant and had strong nodal support, except for a few recent splits. These recent splits tended to be supported by more informative loci (those with higher average bootstrap support), suggesting that, while individual trees may be well resolved, the relationships they recover are being obscured by non-informative data. Divergence dating of a combined dataset using both genera is consistent with concordant timing of their diversification. These results support the scenario of diversification-by-isolation in sky islands, and suggest that allopatry due to climatic gradients in montane regions are an important mechanism for generating species diversity and endemism in these regions.

Made available in DSpace on 2020-12-10T16:58:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundacao Grupo Boticario de Protecao a Natureza Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The advent of next-generation sequencing allows researchers to use large-scale datasets for species delimitation analyses, yet one can envision an inflection point where the added accuracy of including more loci does not offset the increased computational burden. One alternative to including all loci could be to prioritize the analysis of loci for which there is an expectation of high informativeness. Here, we explore the issue of species delimitation and locus selection with montane species from two anuran genera that have been isolated in sky islands across the southern Brazilian Atlantic Forest: Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae). To delimit species, we obtained genetic data using target enrichment of ultraconserved elements from 32 populations (13 for Melanophryniscus and 19 for Brachycephalus), and we were able to create datasets that included over 800 loci with no missing data. We ranked loci according to their number of parsimony-informative sites, and we performed species delimitation analyses using BPP with the most informative 10, 20, 40, 80, 160, 320, and 640 loci. We identified three types of phylogenetic node: nodes with either consistently high or low support regardless of the number of loci or their informativeness and nodes that were initially poorly supported where support became stronger as we included more data. When viewed across all sensitivity analyses, our results suggest that the current species richness in both genera is likely underestimated. In addition, our results show the effects of different sampling strategies on species delimitation using phylogenomic datasets. Univ Fed Parana, Dept Zool, BR-81531980 Curitiba, Parana, Brazil Mater Nat Inst Estudos Ambientais, BR-80250020 Curitiba, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Praca Infante Dom Henrique S-No,Parque Bitaru, BR-11330900 Sao Paulo, Brazil Pontificia Univ Catolica Parana, Escola Ciencias Vida, BR-80215901 Curitiba, Parana, Brazil Louisiana State Univ, Dept Biol Sci, Baton Rouge, LA 70803 USA Louisiana State Univ, Museum Nat Sci, Baton Rouge, LA 70803 USA Occidental Coll, Moore Lab Zool, 1600 Campus Rd, Los Angeles, CA 90041 USA Univ Estadual Paulista, Inst Biociencias, Praca Infante Dom Henrique S-No,Parque Bitaru, BR-11330900 Sao Paulo, Brazil Fundacao Grupo Boticario de Protecao a Natureza: 0895_20111 Fundacao Grupo Boticario de Protecao a Natureza: A0010_2014 CNPq: 301636/2016-8

Fil: Baldo, Juan Diego. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Nordeste. Instituto de Biologia Subtropical. Instituto de Biologia Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biologia Subtropical. Instituto de Biologia Subtropical - Nodo Posadas; Argentina

Nuptial pads are patches of thickened skin, associated with sexually dimorphic skin glands, present only in the thumbs and fingers of male anurans, and hence considered dimorphic sexual characters. Despite the morphological description for many species, the evolution and function of nuptial pads are still unclear. In this study, we describe the presence of nuptial-pad–like structures in females of two species of Melanophryniscus and compare their morphology and histology to males' nuptial pads. The epidermal modifications found in females are conical papillary epidermal projections, similar to the structures found in males; however, the density of these projections in males is twice the average found in females. Also, the nuptial-pad–like structure found in females cannot be considered an actual nuptial pad, because there are no specialized skin glands associated with the structure. This study brings the first records of epidermal modification in females of Melanophryniscus and provides the descr...

We describe the external morphology, buccal cavity, chondrocranium, hyobranchial skeleton, and musculature of the tadpole of Rhinella achavali (Achaval's Toad), along with its karyotype. Tadpoles were found in a small, permanent stream and were showing schooling behavior. The characterization of the proposed species groups within Rhinella cannot be currently improved using external larval morphology, as it seems to be much conserved within this genus. Buccal cavity morphology confirms the distinctiveness of the Rhinella veraguensis (Veragua Toad) group with respect to other known Rhinella. Musculoskeletal character states are similar among bufonids, although within this family the basal genus Melanophryniscus shows some distinctive states. The karyotype is composed of 22 bi-armed chromosomes, with secondary constrictions in pair 7, as found in the other species in the Rhinella marina (Marine Toad) group.

Presentamos el primer registro de Batrachochytrium dendrobatidis infectando poblaciones amenazadas del sapito de panza roja de Tandil (Melanophryniscus aff. montevidensis). Obtuvimos muestras de hisopados de piel de 32 individuos cuyos resultados indicaron una prevalencia del 35.5% y niveles de infección que variaron entre 0.34-915 equivalentes genómicos de Batrachochytrium dendrobatidis. Este hallazgo representa una nueva amenaza que, conjuntamente con la alta fragmentación del hábitat, podría estar afectando la viabilidad de las poblaciones estudiadas. We present the first record of Batrachochytrium dendrobatidis (Bd) infecting endangered populations of the Tandilean Red-belly toad (Melanophryniscus aff. montevidensis). We obtained skin swab samples of 32 individuals. The prevalence was 35.5% and the infection levels varied between 0.34 and 915 Bd-genomic equivalents. This finding represents a new threat that could be affecting, in conjunction with the high habitat fragmentation, the viability of the studied populations. Fil: Agostini, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina Fil: Cortelezzi, Agustina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Exactas. Instituto Multidisciplinario de Ecosistemas y Desarrollo Sustentable; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Berkunsky, Igor. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Exactas. Instituto Multidisciplinario de Ecosistemas y Desarrollo Sustentable; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soler, Gabriela. Instituto Superior de Formación Docente y Técnica Nº 10 “Dr. Osvaldo Zarini”; Argentina Fil: Burrowes, Patricia. Universidad de Puerto Rico; Puerto Rico

The genus Melanophryniscus Gallardo, 1961 currently comprises 25 recognized species (Frost 2012) arranged in three (Cruz & Caramaschi 2003) to four species groups (Cespedez & Motte 2001, quoted by Maneyro et al. 2008) on the basis of morphological characters. The Melanophryniscus stelzneri species group currently contains nine species, i.e. M. atroluteus (Miranda-Ribeiro, 1920), M. cupreuscapularis Céspedez & Alvarez, 2000, M. dorsalis (Mertens, 1933), M. fulvoguttatus (Mertens, 1937), M. klappenbachi Prigioni & Langone, 2000, M. krauczuki Baldo & Basso, 2004, M. montevidensis (Philippi, 1902), M. rubriventris (Vellard, 1947), and M. stelzneri (Weyenbergh, 1875). So far, advertisement calls of only four of these species have been described, i.e. those of M. atroluteus, M. dorsalis, M. krauczuki, and M. montevidensis (Kwet et al. 2005, Baldo & Basso 2004). Herein, we describe the courtship call and distress call of M. klappenbachi and compare it with the calls of other members of the group (for definitions of the respective call types see below).

In nature, a wide variety of cardiotonic steroids is found in plants, the insects that feed on them and in the parotid glands and skin of some toads (family Bufonidae; genera Bufo, Atelopus, Dendrophryniscus and Melanophryniscus). All these natural drugs contain a steroid nucleus with a lactone ring, five-membered in the case of cardenolides, six-membered in bufadienolides. The cardiac glycosides have a carbohydrate or sugar moiety attached through an oxygen bridge to carbon 3 of the `A' ring of the steroid. The myocardial effects of these compounds are attributable to increased intracellular concentrations of Ca2+ and Na+ resulting from inhibition of the transmembrane Na+/K+ ATPase pump. The digitalis glycosides are by far the best known of the cardiac glycosides, but many hundreds of others have been identified in dozens of different species of plants from at least 12 different families. The Apocyanaceae (dog banes) are sources of African arrow poisons (for example Carissa acokanthera , \`bushman's poison' and Strophanthus hispidus ) and also contain many of the most beautiful but deadly tropical flowering shrubs such as Plumeria rubra , \`frangipani', Nerium oleander , \`common, pink or white oleander' and Thevetia peruviana , \`yellow oleander'.1 The yellow oleander contains at least eight different cardiac glycosides, including Thevetin A, Thevetin B (cerberoside), thevetoxin, neriifolin, peruvoside and ruvoside. All parts of the plant are dangerous, especially the seeds.2 Ingestion of oleander seeds or leaves is a common cause of accidental poisoning worldwide, particularly among children.3,,4 Cases have been reported from places as diverse as Hawaii, the Solomon Islands, Southern Africa, Australia, Europe, the Far East and the United States.1,,2 The oleanders have been used for suicide, homicide, abortion and as herbal remedies in India, Thailand, Brazil and elsewhere.2,5,, …

The red-belly toads (Melanophryniscus) of southern South America secrete defensive alkaloids from dermal granular glands. To date, all information on Melanophryniscus alkaloids has been obtained by extraction from either skins or whole organisms; however, in other amphibians, tetrodotoxins, samandarines, and bufadienolides have been detected in both skin and other organs, which raise the possibility that lipophilic alkaloids may occur in non-integumentary tissues in Melanophryniscus as well. To test this hypothesis, we studied the distribution of alkaloids in the skin, skeletal muscle, liver, and mature oocytes of the red-belly toad M. simplex from three localities in southern Brazil. Gas chromatography and mass spectrometry of skin extracts from 11 individuals of M. simplex resulted in the detection of 47 alkaloids (including isomers), 9 unclassified and 38 from 12 known structural classes. Each alkaloid that was present in the skin of an individual was also present in the same relative proportion in that individual’s skeletal muscle, liver, and oocytes. The most abundant and widely distributed alkaloids were the pumiliotoxins 251D, 267C, and 323A, 5,8-disubstituted indolizidines 207A and 223D, 5,6,8-trisubstituted indolizidine 231B, 3,5-disubstituted pyrrolizidines cis-223B and cis- and trans-251K, and izidine 211C. We report the first record of piperidines in Melanophryniscus, bringing the total number of alkaloid classes detected in this genus to 16. Alkaloid composition differed significantly among the three study sites. The functional significance of defensive chemicals in non-integumentary tissues is unknown.

The activity of many species of anurans is affected by variables such as rainfall, air temperature, photoperiod, and food resources. In this study we investigated the activity period of the toad Melanophryniscus moreirae and tested the influence of photoperiod and air temperature on the number of toads seen active. The study was carried out between March 2004 and December 2005 on the elevated area of Parque Nacional do Itatiaia (PNI) southeast Brazil, at elevations varying from 2300 to 2400 m. Twenty 10 × 10 m plots were established as sampling units. To evaluate hourly activity, the plots were visited between 05:00 and 24:00 h, one plot at each hourly interval. Hourly activity was estimated based on the total number of toads seen at hourly intervals, across the study period. Annual and reproductive activities were estimated based on the total number of individuals and number of amplectant pairs, respectively, found within a month. Melanophryniscus moreirae is a strictly diurnal species with two...

Alkaloid profiles in skin of poison frogs/toads (Dendrobatidae, Mantellidae, Bufonidae,and Myobatrachidae) are highly dependent on diet and hence on the nature of habitat.Extracts of the two species of toads (Melanophryniscus klappenbachi and Melanophryniscuscupreuscapularis) from similar habitats in the Corrientes/Chaco Provinces of Argentinahave similar profiles of alkaloids, which differ considerably in profiles from otherMelanophryniscus species from Brazil, Uruguay and Argentina. Structures of two majoralkaloids 239Q (1) and 275I (2) were determined by mass, FTIR, and NMR spectralanalysis as 5Z,9Z-3-(1-hydroxybutyl)-5-propylindolizidine and 6Z,10E-4,6-di(pent-4-enyl)quinolizidine, respectively. A third alkaloid, 249F (3), is postulated to be a homopumiliotoxinwith an unprecedented conjugated exocyclic diene moiety. Fil: Daly, John W.. National Institutes of Health; Estados Unidos Fil: Garraffo, H. Martin. National Institutes of Health; Estados Unidos Fil: Spande, Thomas F.. National Institutes of Health; Estados Unidos Fil: Yeh, Herman J.C.. National Institutes of Health; Estados Unidos Fil: Peltzer, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Saneamiento Ambiental. Cátedra de Ecotoxicología; Argentina Fil: Cacivio, Pedro M.. No especifíca; Fil: Baldo, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Faivovich, Julián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina

A new species of bufonid toad of the genus Melanophryniscus from northern Uruguay is described. It is included in the M. moreirae group and its external morphology is similar to Melanophryniscus sanmartini. Melanophryniscus sp. nov. is distinguished by having a light brown dorsal coloration with six darker longitudinal glandular ridges on the dorsal surface of the body and the gular region uniformly black.

Little is known about the reproductive biology of species of the genus Melanophryniscus. This study provides the first description of amplexus behaviour, egg-laying sites, clutch characteristics and larval development of Melanophryniscus sp., a species of uncertain taxonomic status that is found in the southernmost range of the genus (Sierra de la Ventana, Argentina), and their relationship with habitat features and selection pressures is discussed. Results are compared with the reproductive biology of other species in the genus. Fil: Cairo, Samanta Lis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Departamento de Zoología; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Grupo de Estudios en Conservación y Manejo; Argentina Fil: Zalba, Sergio Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Grupo de Estudios en Conservación y Manejo; Argentina Fil: Úbeda, Carmen A.. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Departamento de Zoología; Argentina

A new species of bufonid toad of the genus Melanophryniscus is described from a mountaintop that is part of the Serra do Mar in the northeastern State of Parana, Brazil. Melanophryniscus alipioi sp. nov. is distinguished from other known species by its uniformly dark brown dorsal color and a unique breeding site. The new species reproduces in bromeliads, a reproductive mode previously unknown for this genus. This species might be susceptible to current habitat lost.

La estructura de la señal acústica en anuros ha sido considerada especie-específica y utilizada incluso para el reconocimiento de nuevas especies indistinguibles por caracteres morfológicos. En este trabajo, presentamos una descripción de la estructura del canto de Melanophryniscus rubriventris y un análisis comparativo de parámetros espectrales y temporales del canto de anuncio en tres poblaciones argentinas, cada una de ellas asignadas a una de las tres subespecies tradicionalmente reconocidas. El canto de anuncio emitido por los machos de M. rubriventris puede describirse como un trino agudo formado por dos segmentos: el primero está compuesto por una serie de emisiones cortas, aisladas y no pulsadas seguidas por el segundo segmento que conforma un trino rápido con una frecuencia constante de pulsos. La estructura del canto en dos segmentos coincide con las descripciones realizadas para otras cinco especies del género, pero difiere en los componentes temporales y espectrales. Las comparaciones de los cantos de las distintas poblaciones estudiadas de M. rubriventris no revelan ninguna diferencia significativa. Estas evidencias no parecen sustentar la existencia de subespecies ni que se deban efectuar cambios en el status específico de las poblaciones argentinas., The structure of the acoustic signal in anurans has been considered species-specific and used even for the recognition of new indistinguishable species by morphologic characters. In this work, we presented a description of the structure of the call ofMelanophryniscus rubriventris and a comparative analysis of spectral and temporal parameters of the advertisement call in three Argentine populations assigned to three traditionally recognized subspecies. The call emitted by males of M. rubriventris can be described like an acute trill formed by two segments: a first segment with a series of short, unpulsed isolated emissions followed by the second segment: a fast trill of a constant frequency of pulses. The structure of the call in two segments agrees with the descriptions made for other five species of the genus, but it differs in the temporal and spectral components. The call of the different studied populations from M. rubriventris does not reveal any significant difference. These evidences do not seem to sustain the existence of subspecies, or changes in the specific status of the Argentine populations., Asociación Herpetológica Argentina (AHA)

Increasing evidence shows that alkaloids in the skins of poisonous frogs are sequestered from a specialized diet of ants. It is considered likely that in the small aposematic bufonid genus Melano- phryniscus, skin alkaloids are taken up via a similar system. However, quantitative aspects of the diet in this genus remain poorly studied. As a result, studies that correlate diet and skin alkaloids in these toads are precluded by the lack of extensive information on the diet of most species and populations within the genus. We describe diet composition of Melanophryniscus rubriventris with a particular focus on geographic variation over much of its range distribution in northwestern Argentina and compare the diet of this species with that of other members of the genus. The diet of M. rubriventris was composed of 17 arthropod prey categories. The species consumed primarily small, gregarious prey such as ants, mites, aphids, burrowing bugs, and beetles. Diet composition of the populations studied was similar in the number of prey categories, but we found noticeable interpopulation variation in the proportion of each prey item consumed. Earlier works had reported that ants, collembolans, and mites were prominent in the diet of other Melanophryniscus. Our study indicates that, although ants are common prey, they are not the main prey category in all populations.

Bufonid toads of the genus Melanophryniscus represent one of several lineages of anurans with the ability to sequester alkaloids from dietary arthropods for chemical defense. The alkaloid profile for Melanophryniscus stelzneri from a location in the province of Córdoba, Argentina, changed significantly over a 10-year period, probably indicating changes in availability of alkaloid-containing arthropods. A total of 29 alkaloids were identified in two collections of this population. Eight alkaloids were identified in M. stelzneri from another location in the province of Córdoba. The alkaloid profiles of Melanophryniscus rubriventris collected from four locations in the provinces of Salta and Jujuy, Argentina, contained 44 compounds and differed considerably between locations. Furthermore, alkaloid profiles of M. stelzneri and M. rubriventris strongly differed, probably reflecting differences in the ecosystem and hence in availability of alkaloid-containing arthropods.

The South-American red bellied toads (Melanophryniscus spp.) belonging to the Bufonidae family contain toxic alkaloids in their skin, predominantly of the pumiliotoxin group. Whole animal methanolic extracts of individual specimens of three species (Melanophryniscus atroluteus, M. devincenzii, and M. montevidensis) were analyzed for the presence of toad specific bufadienolides and indolalkylamines (serotonin derivatives) by HPLC-electrospray (ESI)-MS-TOF. No bufadienolides, but few bufotenines, mainly dehydrobufotenine, were detected in the extracts in variable amounts. The concentration of the dehydrobufotenine in the extracts seems to be species specific. Whereas M. atroluteus and M. montevidensis contain very low or trace amounts, M. devincenzii specimens exhibit high concentrations of this indolalkylamine. In comparison, analysis of extracts from Bufo arenarum (Uruguay) and from B. bufo (Germany) confirmed the presence of bufadienolides as well as of bufotenine derivatives. Tadpoles of both species exhibited a different pattern: extracts from B. arenarum tadpoles contained only dehydrobufotenine, but those from B. bufo tadpoles bufotoxin and two alkylamines. Melanophryniscus toads appear not to be able to compensate the high variability of toxic skin alkaloids by producing defensive bufadienolides.

Melanophryniscus sp. is an endemic toad restricted to Sierra de la Ventana, in the Argentine Pampas. Part of its reproductive habitat is crossed by a paved road. We studied the effects of the road on the mortality and mobility of the toads. During the days following rain, we surveyed transects on both sides of the road, capturing, photographing and immediately releasing each toad. Population size in the area was estimated in 1074 individuals (871 to 1363, 95% CI) by means of mark-recapture analysis. We found fifteen individuals killed by vehicles in the 2003-04 and ten in the 2004-05 reproductive seasons. This mortality represents from 2.5 to 5.9% of the population annually, considering that 73% of the mortality period was sampled and a detection rate of dead frogs of 40%. Other factors associated to roadside habitat, such as rapid drying of roadside ditches, maintenance work, increased frequency of fire, pollution by gas, oil and fuel emissions and losses from vehicles, and poaching as pets by tourists, may also reduce the survival of the toads. We recorded capture sites and calculated the average distance between captures as 33.04 m for males and 22.50 m for females. Only two of the 76 observations of recaptured toads were made on the opposite side of the road. Roads can be considered as having a significant impact on this species by augmenting mortality, hindering the mobility of the species and increasing habitat isolation. Fil: Zalba, Sergio Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Cairo, Samanta Lis. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina

The Brazilian Atlantic Forest (BAF) is recognized as one of the world's biodiversity hotspots, with even more species per unit of area than the Amazon, however the mechanisms that led to such astonishing diversity are yet to be fully understood. In this study, we investigate the diversification of two co-distributed frog genera associated with montane areas of southern BAF: Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae). Species delimitation methods using mitochondrial and nuclear loci supported the existence of a remarkable number of highly endemic species in each genus, most of which occupy only one or a few adjacent mountaintops. Their timing of diversification was highly congruent, supporting recent speciation events within the past 600 thousand years. Extended Bayesian skyline plots indicate that most populations have remained relatively stable in size across the evolutionary past, with recent growth after 0.15My, suggesting that the drastic changes found in previous studies on lowland frog species were not shared by these montane taxa. These results are consistent with the existence of a montane refugium in southern BAF, allowing species persistence through the climatic shifts experienced along the BAF during the Quaternary.

Introduction Amphibians secrete a wide diversity of chemicals from skin glands as defense against predators, parasites, and pathogens. Most defensive chemicals are produced endogenously through biosynthesis, but poison frogs sequester lipophilic alkaloids from dietary arthropods. Alkaloid composition varies greatly, even among conspecific individuals collected at the same time and place, with some individuals having only a few micrograms of one or a few alkaloids and others possessing >1 mg of >30 alkaloids. The paucity of alkaloids in juveniles and their abundance in adults suggests that alkaloids accumulate over time; however, alkaloid diversity is highly variable among adult poison frogs and has never been studied in relation to individual age. Using skeletochronology to infer individual ages and gas chromatography–mass spectrometry and vapor phase Fourier-transform infrared spectral analysis to identify the defensive chemicals of 63 individuals, we tested the relationship between defensive chemicals and age, size, and sex in the Brazilian red-belly toad, Melanophryniscus moreirae, a poison frog that possesses both sequestered alkaloids and the biosynthesized indolealkylamine bufotenine. Results Adult females were, on average, older and larger than adult males. Juveniles were smaller but not necessarily younger than adults and possessed bufotenine and 18 of the 37 alkaloids found in adults. Alkaloid richness was positively related to age, but not size, whereas the quantities of sequestered alkaloids and bufotenine were positively related to size, but not age. Defensive chemicals were unrelated to sex, independent of size. Conclusions The relationship between alkaloid richness and age appears to result from the gradual accumulation of alkaloids over a frog’s lifetime, whereas the relationship between the quantity of defensive chemicals and size appears to be due to the greater storage capacity of larger individuals. The decoupling of age and size effects increases the amount of individual variation that can occur within a population, thereby possibly enhancing anti-predator efficacy. Further, given that both richness and quantity contribute to the overall chemical defense of individual frogs, our results suggest that older, larger individuals are better defended than younger, smaller ones. These considerations underscore the importance of including age in studies of the causes and consequences of variation in poison frog chemical defenses. Electronic supplementary material The online version of this article (doi:10.1186/s12983-015-0120-2) contains supplementary material, which is available to authorized users.

Next-generation sequencing continues to revolutionize biodiversity studies by generating unprecedented amounts of DNA sequence data for comparative genomic analysis. However, these data are produced as millions or billions of short reads of variable quality that cannot be directly applied in comparative analyses, creating a demand for methods to facilitate assembly. We optimized an in silico strategy to efficiently reconstruct high-quality mitochondrial genomes directly from genomic reads. We tested this strategy using sequences from five species of frogs: Hylodes meridionalis (Hylodidae), Hyloxalus yasuni (Dendrobatidae), Pristimantis fenestratus (Craugastoridae), and Melanophryniscus simplex and Rhinella sp. (Bufonidae). These are the first mitogenomes published for these species, the genera Hylodes, Hyloxalus, Pristimantis, Melanophryniscus and Rhinella, and the families Craugastoridae and Hylodidae. Sequences were generated using only half of one lane of a standard Illumina HiqSeq 2000 flow cell, resulting in fewer than eight million reads. We analysed the reads of Hylodes meridionalis using three different assembly strategies: (1) reference-based (using bowtie2); (2) de novo (using abyss, soapdenovo2 and velvet); and (3) baiting and iterative mapping (using mira and mitobim). Mitogenomes were assembled exclusively with strategy 3, which we employed to assemble the remaining mitogenomes. Annotations were performed with mitos and confirmed by comparison with published amphibian mitochondria. In most cases, we recovered all 13 coding genes, 22 tRNAs, and two ribosomal subunit genes, with minor gene rearrangements. Our results show that few raw reads can be sufficient to generate high-quality scaffolds, making any Illumina machine run using genomic multiplex libraries a potential source of data for organelle assemblies as by-catch.

Three new species of Melanophryniscus are described from the Serra do Mar mountain range of the state of Santa Catarina, southern Brazil. All species are found at intermediate to high altitudes and share phytotelm-breeding as their reproductive strategy. The new species are distinguished from other phytotelm-breeding Melanophryniscus based on different combinations of the following traits: snout-vent length, presence of white and/or yellow spots on forearms, mouth, belly and cloaca, pattern and arrangement of warts, and presence and number of corneous spines. The discovery of these species in a rather restricted geographical area suggests that the diversity of phytotelm-breeding species of Melanophryniscus might be severely underestimated. The conservation status of these species is of particular concern, given that one of them is at risk of extinction not only due to its restricted habitat, but also because of anthropogenic disturbances. Fil: Bornschein, Marcos R.. Universidade Federal do Minas Gerais; Brasil. Universidade Federal do Paraná; Brasil. Mater Natura Instituto de Estudos Ambientais; Brasil Fil: Firkowski, Carina R.. Universidade Federal do Paraná; Brasil Fil: Baldo, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Ribeiro, Luiz Fernando. Universidade Federal do Paraná; Brasil. Mater Natura Instituto de Estudos Ambientais; Brasil. Pontificia Universidade Católica do Paraná. Escola de Saúde; Brasil Fil: Belmonte Lopes, Ricardo. Universidade Federal do Paraná; Brasil Fil: Correa, Leandro Javier. Mater Natura Instituto de Estudos Ambientais; Brasil Fil: Morato, Sérgio A. A.. STCP Engenharia de Projetos Ltda.; Brasil Fil: Pie, Marcio R.. Universidade Federal do Paraná; Brasil. Mater Natura Instituto de Estudos Ambientais; Brasil

A new bufonid toad is described. The species belongs to the genus Melanophryniscus, and may be the largest species in this genus. Its size and external features (pale green in dorsal surface) resemble those of Melanophryniscus cambaraensis and Melanophryniscus macrogranulosus, from which it differs by the absence of frontal swelling. At present, the new species is endemic to the southern border of the Brazilian Southern Plateau, in central Rio Grande do Sul state.

Bufonid poison frogs of the genus Melanophryniscus contain alkaloid-based chemical defenses that are derived from a diet of alkaloid-containing arthropods. In addition to dietary alkaloids, bufadienolide-like compounds and indolealkylamines have been identified in certain species of Melanophryniscus. Our study reports, for the first time, the co-occurrence of large quantities of both alkaloids sequestered from the diet and an endogenously biosynthesized indolalkylamine in skin secretions from individual specimens of Melanophryniscus moreirae from Brazil. GC/MS analysis of 55 individuals of M. moreirae revealed 37 dietary alkaloids and the biosynthesized indolealkylamine bufotenine. On average, pumiliotoxin 267C, bufotenine, and allopumilitoxin 323B collectively represent ca. 90 % of the defensive chemicals present in an individual. The quantity of defensive chemicals differed between sexes, with males possessing significantly less dietary alkaloid and bufotenine than females. Most of the dietary alkaloids have structures with branched-chains, indicating they are likely derived from oribatid mites. The ratio of bufotenine:alkaloid quantity decreased with increasing quantities of dietary alkaloids, suggesting that M. moreirae might regulate bufotenine synthesis in relation to sequestration of dietary alkaloids.

A new species of bufonid toad in the genus Melanophryniscus, from the province of Misiones, northeastern Argentina, is described. It is included in the stelzneri group and is most similar to Melanophryniscus atroluteus than to any other species. Melanophryniscus sp. nov. is distinguished, by having a uniformly brown dorsal coloration, numerous white spots along the mandibular arch; the pupil surrounded by a golden ring; the iris gold, finely spotted with dark brown; and the frontoparietal fontanelle widely exposed anteriorly. Moreover, the advertisement call of the new species is noticeable longer than the call of M. atroluteus and has a higher dominant frequency. The Melanophryniscus species present in Misiones are discussed and several areas of syntopy are reported.

Larson, P. M., de Sa, R. O. and Arrieta, D. 2003. Chondrocranial, hyobranchial and internal oral morphology in larvae of the basal bufonid genus Melanophryniscus (Amphibia: Anura). — Acta Zoologica (Stockholm) 84 : 145- 154 Melanophryniscus is a genus of small toads inhabiting the southern portion of South America. This genus is considered basal within the family Bufonidae. Data on larval chondrocranial morphology do not exist for the genus and larval internal oral anatomy has only been described for a single species. Here, we describe chondrocranial and internal oral morphology in Melanophryniscus montevidensis , M. orejasmirandai and M. sanmartini . Chondrocranial mor- phology is similar among the species examined. Comparisons with other bufonids and with outgroup taxa suggest that the following chondrocranial characters may represent synapomorphies for the Bufonidae: free (or absent) ceratobranchial IV, a reduced or absent larval crista parotica, the lack of a larval otic process, and late development of thin, poorly chondrified orbital cartilages. The presence of an elongated processus anterior dorsalis of the suprarostral alae and the absence of a chondrified commissura quad- ratoorbitalis appear to be unique in Melanophryniscus among bufonids. Internal oral anatomy is conserved in Melanophryniscus , and among bufonids in general.

Melanophryniscus moreirae is recorded from a fifth locality. A specimen was collected in Serra do Papagaio, municipality of Alagoa, Minas Gerais, Brazil, 18 km southeast from the nearest previous record in Aiuruoca. This new record fills a gap of about 32 km in the distribution of the species, between Itamonte and Aiuruoca.