Enantioselective formal total synthesis of the Dendrobatidae frog toxin, (+)-pumiliotoxin B, via O-directed alkyne free radical hydrostannation

Herein we describe our application of the O-directed free radical hydrostannation of disubstituted alkyl-acetylenes (with Ph3SnH and Et3B) to the (+)-pumiliotoxin B total synthesis problem. Specifically, we report on the use of this method in the synthesis of the Overman alkyne 8, and thereby demonstrate the great utility of this process in a complex natural product total synthesis setting for the very first time. We also report here on a new, stereocontrolled, and highly practical enantioselective pathway to Overman's pyrrolidine epoxide partner 9 for 8, which overcomes the previous requirement for use of preparative HPLC to separate the 1:1 mixture of diastereomeric epoxides that was obtained in the original synthesis of 9.

Sélection et polymorphisme chez des grenouilles mimétiques du Pérou (Dendrobatidae)

La diversification des signaux aposématiques dans un cadre de mimétisme müllérien est un phénomène intrigant. Alors que la théorie relative à l'aposématisme et au mimétisme suggère l'évolution vers un signal aposématique unique, d'impressionnantes variations peuvent être observées entre les populations, et cela à petite échelle spatiale. Il a été supposé que la variation spatiale des pressions de sélection engendrées par différents prédateurs puisse être à l'origine de ce phénomène. Afin de tester cette hypothèse, nous avons étudié la transition entre deux systèmes géographiques caractérisés par des patrons aposématiques distincts chez des grenouilles mimétiques et toxiques du nord du Pérou (Dendrobatidae) en combinant les outils de génétique des populations aux outils écologiques. Dans chacun de ces systèmes, Ranitomeya imitator vit en sympatrie avec R. ventrimaculata ou R. variabilis. Il s'agit du principal exemple empirique suggérant que dans un cadre de mimétisme müllérien, il n'y a pas convergence des signaux aposématiques des deux espèces, mais plutôt convergence unidirectionnelle où R. imitator, étant polymorphe, imite des espèces monomorphes avec lesquelles elle est sympatrique. Premièrement, les résultats réfutent les prémisses qui suggèrent que R. imitator converge vers le signal aposématique d’une autre espèce. La haute similarité génétique entre les espèces modèles suggère qu'elles ont divergé plus récemment que les populations de R. imitator ou qu'elles sont encore connectées par du flux génique. Ces résultats indiquent que ces espèces ont été identifiées à tort comme des espèces différentes. De fait, l'identification de l'espèce imitatrice basée sur la variabilité phénotypique est invalidée dans ce système puisque R. imitator et R. variabilis/ventrimaculata démontrent la même variabilité. Deuxièmement, nos résultats démontrent que la prédation varie spatialement, autant en intensité qu'en direction, créant ainsi un paysage hétérogène de pressions de sélection. Ainsi, de fortes pressions de prédation stabilisatrice permettent le maintien de l'organisation géographique de différents signaux aposématiques et expliquent l'uniformité de ces signaux ainsi que les relations mimétiques. Par contre, le relâchement temporaire des pressions de prédation permet l'apparition de nouveaux phénotypes aposématiques via les processus évolutifs neutres, conduisant à un haut polymorphisme au niveau de ces populations. L'interaction de ces modes sélectifs nous a permis de démontrer pour la première fois comment la théorie évolutive de Wright (shifting balance theory) permet la diversification adaptative dans un système naturel. Pour conclure, cette étude a permis de mettre en évidence à quel point les systèmes de mimétisme müllérien peuvent être dynamiques. L'alternance spatiale entre les processus évolutifs neutres et la sélection naturelle permet l'émergence de nouveaux phénotypes aposématiques à une échelle locale, ainsi que l'apparition d'une organisation géographique des signaux d'avertissement et des relations de mimétisme müllérien.

4 SPECIES OF BRAZILIAN POISON FROGS RELATED TO EPIPEDOBATES-PICTUS (DENDROBATIDAE) - TAXONOMY AND NATURAL-HISTORY OBSERVATIONS

We present morphological and natural history data on Epipedobates pictus and three additional taxa currently regarded as synonyms of the former: Epipedobates braccatus, E. flavopictus, and E. hahneli. Topotypes of the four species were examined. Morphological data on tadpoles and adults, in addition to distribution patterns and vocalizations, indicate that the four taxa represent distinct species. Two groups are evident within them: (1) the slender-bodied, Amazonian E. hahneli with small flash marks on the thighs and a low-finned tadpole, and (2) the robust bodied, almost extra-Amazonian E. braccatus, E. flavopictus, and E. pictus with large flash marks on the thighs and tadpoles with fins of moderate height.

Four species of Brazilian poison frogs related to Epipedobates pictus (Dendrobatidae): taxonomy and natural history observations

Presents morphological and natural history data on Epipedobates pictus and three additional taxa currently regarded as synonyms of the former: Epipedobates braccatus, E. flavopictus and E. hahneli. Morphological data on tadpoles and adults, in addition to distribution patterns and vocalizations, indicate that the four taxa represent distinct species. -from Authors

Phylogenetic systematics of dart-poison frogs and their relatives (Amphibia: Athesphatanura: Dendrobatidae)

The known diversity of dart-poison frog species has grown from 70 in the 1960s to 247 at present, with no sign that the discovery of new species will wane in the foreseeable future. Although this growth in knowledge of the diversity of this group has been accompanied by detailed investigations of many aspects of the biology of dendrobatids, their phylogenetic relationships remain poorly understood. This study was designed to test hypotheses of dendrobatid diversification by combining new and prior genotypic and phenotypic evidence in a total evidence analysis. DNA sequences were sampled for five mitochondrial and six nuclear loci (approximately 6,100 base pairs [bp]; x=3,740 bp per terminal; total dataset composed of approximately 1.55 million bp), and 174 phenotypic characters were scored from adult and larval morphology, alkaloid profiles, and behavior. These data were combined with relevant published DNA sequences. Ingroup sampling targeted several previously unsampled species, including Aromobates nocturnus, which was hypothesized previously to be the sister of all other dendrobatids. Undescribed and problematic species were sampled from multiple localities when possible. The final dataset consisted of 414 terminals: 367 ingroup terminals of 156 species and 47 outgroup terminals of 46 species. Direct optimization parsimony analysis of the equally weighted evidence resulted in 25,872 optimal trees. Forty nodes collapse in the strict consensus, with all conflict restricted to conspecific terminals. Dendrobatids were recovered as monophyletic, and their sister group consisted of Crossodactylus, Hylodes, and Megaelosia, recognized herein as Hylodidae. Among outgroup taxa, Centrolenidae was found to be the sister group of all athesphatanurans except Hylidae, Leptodactyidae was polyphyletic, Thoropa was nested within Cycloramphidae, and Ceratophryinae was paraphyletic with respect to Telmatobiinae. Among dendrobatids, the monophyly and content of Mannophryne and Phyllobates were corroborated. Aromobates nocturnus and Colostethus saltuensis were found to be nested within Nephelobates, and Minyobates was paraphyletic and nested within Dendrobates. Colostethus was shown to be rampantly nonmonophyletic, with most species falling into two unrelated cis- and trans-Andean clades. A morphologically and behaviorally diverse clade of median lingual process-possessing species was discovered. In light of these findings and the growth in knowledge of the diversity of this large clade over the past 40 years, we propose a new, monophyletic taxonomy for dendrobatids, recognizing the inclusive clade as a superfamily (Dendrobatoidea) composed of two families (one of which is new), six subfamilies (three new), and 16 genera (four new). Although poisonous frogs did not form a monophyletic group, the three poisonous lineages are all confined to the revised family Dendrobatidae, in keeping with the traditional application of this name. We also propose changes to achieve a monophyletic higher-level taxonomy for the athesphatanuran outgroup taxa. Analysis of character evolution revealed multiple origins of phytotelm-breeding, parental provisioning of nutritive oocytes for larval consumption (larval oophagy), and endotrophy. Available evidence indicates that transport of tadpoles on the dorsum of parent nurse frogs-a dendrobatid synapomorphy-is carried out primitively by male nurse frogs, with three independent origins of female transport and five independent origins of biparental transport. Reproductive amplexus is optimally explained as having been lost in the most recent common ancestor of Dendrobatoidea, with cephalic amplexus arising independently three times. © American Museum of Natural History 2006.

Dieta, área de uso e transporte de girinos de Ameerega trivittata (Spix, 1824) (Anura: Dendrobatidae) em uma região de floresta de terra firme na Amazônia Oriental

Este estudo explora aspectos da dieta, área de uso e transporte dos girinos de Ameerega trivittata em uma região de floresta de terra firme na Amazônia Oriental. Nós coletamos 56 espécimes no total (48 machos e oito fêmeas), e destes 44 indivíduos de A. trivittata tiveram aferidos o seu conteúdo estomacal onde, formigas da Subfamília Myrmicinae, foi o táxon mais consumido pelos machos, e Isoptera pelas fêmeas. Isso provavelmente se deve à necessidade da aquisição de alcaloides pelos machos, presentes em formigas, que lhes dá um alto grau de toxicidade, e consequentemente, proteção, já que são os machos que realizam o cuidado parental nessa espécie, transportando os girinos para as poças. Estimamos a área de uso de quatro machos, sendo estas, em média, duas vezes maiores do que a encontrada para a mesma espécie na Amazônia Peruana. Não houve relação entre o tamanho dos machos e abundância e/ou tamanho médio dos girinos carregados e não houve relação entre a área das poças e a abundância de girinos, porém houve uma diminuição na abundância de girinos em relação à abundância de ninfas de Odonata, o que corrobora com outros trabalhos feitos para dendrobatídeos, já que estas são conhecidas por regular as populações de girinos.

The evolution of coloration and toxicity in the poison frog family (Dendrobatidae)

The poison frogs (family Dendrobatidae) are terrestrial anuran amphibians displaying a wide range of coloration and toxicity. These frogs generally have been considered to be aposematic, but relatively little research has been carried out to test the predictions of this hypothesis. Here we use a comparative approach to test one prediction of the hypothesis of aposematism: that coloration will evolve in tandem with toxicity. Recently, we developed a phylogenetic hypothesis of the evolutionary relationships among representative species of poison frogs, using sequences from three regions of mitochondrial DNA. In our analysis, we use that DNA-based phylogeny and comparative analysis of independent contrasts to investigate the correlation between coloration and toxicity in the poison frog family (Dendrobatidae). Information on the toxicity of different species was obtained from the literature. Two different measures of the brightness and extent of coloration were used. (i) Twenty-four human observers were asked to rank different photos of each different species in the analysis in terms of contrast to a leaf-littered background. (ii) Color photos of each species were scanned into a computer and a computer program was used to obtain a measure of the contrast of the colors of each species relative to a leaf-littered background. Comparative analyses of the results were carried out with two different models of character evolution: gradual change, with branch lengths proportional to the amount of genetic change, and punctuational change, with all change being associated with speciation events. Comparative analysis using either method or model indicated a significant correlation between the evolution of toxicity and coloration across this family. These results are consistent with the hypothesis that coloration in this group is aposematic.

Convergent evolution of aposematic coloration in Neotropical poison frogs: a molecular phylogenetic perspective

Poison frogs of the family Dendrobatidae contain cryptic as well as brightly colored, presumably aposematic species. The prevailing phylogenetic hypothesis assumes that the aposematic taxa form a monophyletic group while the cryptic species (Colostethus sensu lato) are basal and paraphyletic. Analysis of 86 dendrobatid sequences of a fragment of the 16S rRNA gene resulted in a much more complex scenario, with several clades that contained aposematic as well as cryptic taxa. Monophyly of the aposematic taxa was significantly rejected by SH-tests in an analysis with additional 12S and 16S rDNA fragments and reduced taxon sampling. The brightly colored Allobates femoralis and A. zaparo (Silverstone) comb. nov. (previously Epipedobates) belong in a clade with cryptic species of Colostethus. Additionally, Colostethus pratti was grouped with Epipedobates, and Colostethus bocagei with Cryptophyllobates. In several cases, the aposematic species have general distributions similar to those of their non-aposematic sister groups, indicating multiple instances of regional radiations in which some taxa independently acquired bright color. From a classificatory point of view, it is relevant that the type species of Minyobates, M. steyermarki, resulted as the sister group of the genus Dendrobates, and that species of Mannophryne and Nephelobates formed monophyletic clades, corroborating the validity of these genera. Leptodactylids of the genera Hylodes and Crossodactylus were not unambiguously identified as the sister group of the Dendrobatidae; these were monophyletic in all analyses and probably originated early in the radiation of Neotropical hyloid frogs.

Indolizidine 239Q and quinolizidine 275I. Major alkaloids in two Argentinian bufonid toads (Melanophryniscus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Composição e diversidade de espécies da anurofauna da Estação Científica Ferreira Penna, Floresta Nacional de Caxiuanã, Pará, Brasil

O presente estudo compara a composição da anurofauna dos 4 principais habitats (mata de terra firme, capoeira, igapó e vegetação aquática) existentes nas áreas de dois igarapés de água preta (igarapé Arauá e Laranjal), pertencentes a Estação Científica Ferreira Penna (ECFPn), localizada na Floresta Nacional de Caxiuanã (FLONA de Caxiuanã), Pará, Brasil. As amostragens nas duas áreas foram feitas através de transectos medindo 850 m de comprimento por 10 m de largura. As duas áreas dos igarapés foram amestradas nos quatro períodos do ano relacionados à precipitação (período de transição chuva/seca, seca, transição seca/chuva e chuva). Para a comparação entre as áreas e entre os habitats foram utilizados os índices de diversidade de Shannon-Weiner e índice de similaridade de Jaccard. No total das amostragens foram registrados 924 indivíduos pertencentes a 29 espécies, distribuídas em 15 gêneros e 5 famílias. Quanto aos habitats a mata de terra firme apresentou maior diversidade. O número de espécies registradas foi maior no período de chuvas. Este estudo associado aos estudos de Ávila-Pires e Hoogmoed (1997), contabilizam 41 espécies de anuros para a região, um número muito alto para Amazônia oriental, reforçando a hipótese de que a baixa diversidade de espécies de anuros normalmente associada a esta região possa ser fruto do baixo número de inventários realizados até o presente.

Batrachochytrium dendrobatidis in amphibians confiscated from illegal wildlife trade and used in an ex situ breeding program in Brazil

This paper describes an outbreak of chytridiomycosis affecting a group of Dendrobates tinctorius, a Neotropical anuran species, confiscated from the illegal wildlife trade and housed in a private zoo in Brazil as part of an ex situ breeding program. We examined histological sections of the skin of 30 D. tinctorius and 20 Adelphobates galactonotus individuals. Twenty D. tinctorius (66.7%) and none of the A. galactonotus were positive for Batrachochytrium dendrobatidis (Bd). Multiple development stages of Bd infection were observed. The reasons for the interspecific difference in the rate of infection could not be determined, and further studies are advised. Because the examined population consisted of confiscated frogs, detailed epidemiological aspects could not be investigated, and the source of the fungus remains uncertain. The existence of ex situ amphibian populations is important for protecting species at higher risk in the wild, and ex situ amphibian conservation and breeding programs in Brazil may be established using confiscated frogs as founders. However, this paper alerts these programs to the urgency of strict quarantine procedures to prevent the introduction of potential pathogens, particularly Bd, into ex situ conservation programs.

The occurrence of defensive alkaloids in non-integumentary tissues of the Brazilian red-belly toad Melanophryniscus simplex (Bufonidae)

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.

Intraspecific and intraspecific views of colour signals in the strawberry poison frog Dendrobates Pumilio

Poison frogs in the anuran family Dendrobatidae use bright colors on their bodies to advertise toxicity. The species Dendrobates pumilio Schmidt 1858, the strawberry poison frog, shows extreme polymorphism in color and pattern in Panama. It is known that females of D. pumilio preferentially choose mates of their own color morph. Nevertheless, potential predators must clearly see and recognize all color morphs if the aposermatic signaling system is to function effectively. We examined the ability of conspecifics and a model predator to discriminate a diverse selection of D. pumilio colors from each other and from background colors. Microspectrophotometry of isolated rod and cone photoreceptors of D. pumilio revealed the presence of a trichromatic photopic visual system. A typical tetrachromatic bird system was used for the model predator. Reflectance spectra of frog and background colors were obtained, and discrimination among spectra in natural illuminants was mathematically modeled. The results revealed that both D. pumilio and the model predator discriminate most colors quite well, both from each other and from typical backgrounds, with the predator generally performing somewhat better than the conspecifics. Each color morph displayed at least one color signal that is highly visible against backgrounds to both visual systems. Our results indicate that the colors displayed by the various color morphs of D. pumilio are effective signals both to conspecifics and to a model predator.