Molecular phylogenetics uncovers two new species in the genus Phyllobates (Anura, Dendrobatidae): the terrible frog gets two new sisters.

True poison-dart frogs (Phyllobates, Dendrobatidae) evolved the ability to secrete batrachotoxins, the most powerful alkaloids known to date. The genus comprises five species whose systematics, at first glance, appeared clear. The most derived clade would include two Colombian species (P.terribilis and P.bicolor) with the highest toxicity, the largest body size, and predominantly yellow body colouration. The other three species (P.aurotaenia, P.vittatus, and P.lugubris) are less toxic on average, have smaller size, and are predominantly black with bright dorsolateral stripes. Recent research has revealed the existence of two major lineages among the three Colombian species. The northern lineage appears to result from a complex evolutionary history, including perhaps introgression among yellow and black taxa. The southern lineage instead revealed the existence of new clades closely related to P.terribilis, black and yellow, that arguably deserve their recognition as new species. Here, available evidence is combined to support the erection of southern populations of P.aurotaenia as a new highly toxic species, sister to P.terribilis, and much closer to it than to any other yellow or black-bodied species, Phyllobatessamperi sp. nov. Their common ancestor is sister to an additional yellow species, which we also describe here as Phyllobatesbezosi sp. nov. Both new species can be externally diagnosed using colouration. Our previous and current analyses also suggest the existence of additional taxa and corroborate multiple transitions in colouration across these hypertoxic taxa.

ResumenLas verdaderas ranas de dardo venenoso (género Phyllobates) evolucionaron la capacidad de secretar batracotoxinas, los alcaloides más poderosos que se conocen hasta hoy. El género comprende cinco especies, cuya taxonomía parecía clara a primera vista. El clado más derivado incluye dos especies colombianas (P.terribilis and P.bicolor) con la mayor toxicidad, mayor tamaño y coloración predominantemente amarilla. Las otras tres especies (P.aurotaenia, P.vittatus and P.lugubris) son en general menos tóxicas, menores en tamaño y predominantemente negras con conspicuas manchas dorsolaterales.Una reciente investigación confirmó la existencia dos linajes mayores entre las tres especies colombianas. El del norte parece ser el producto de una historia evolutiva compleja, incluyendo quizás introgresión entre especies amarillas y negras. El del sur reveló en cambio la existencia de nuevos clados filogenéticamente cercanos a P.terribilis, uno negro y dos amarillos, que requieren reconocimiento formal como nuevas especies. Esta investigación combina la evidencia disponible para apoyar el reconocimiento de las poblaciones del sur de P.aurotaenia como una nueva especie negra, hermana de P.terribilis, y mucho más cercana a ella que a las otras especies negras o amarillas, Phyllobatessamperi sp. nov. Su ancestro común es a su vez el grupo hermano de una especie adicional, amarilla, que también describimos como Phyllobatesbezosi sp. nov. Las dos nuevas especies pueden ser externamente reconocidas por la coloración. Nuestros análisis previos y actuales también sugieren la existencia de otras especies no descritas y corroboran la existencia de múltiples transiciones de coloración en la evolución de las especies hipertóxicas.

First characterization of toxic alkaloids and volatile organic compounds (VOCs) in the cryptic dendrobatid Silverstoneia punctiventris.

BACKGROUND: Poison frogs are known for the outstanding diversity of alkaloid-based chemical defences with promising therapeutic applications. However, current knowledge about chemical defences in Dendrobatoidea superfamily has two sources of bias. First, cryptic, brown-colored species have been neglected in comparison to those conspicuously colored, and second, there has been little interest in characterizing metabolites other than alkaloids mediating defensive functions. In an effort to contribute to fill the gap of knowledge about cryptic species and broadening the spectrum of compounds analyzed we have applied head-space solid phase microextraction coupled to gas chromatography and mass spectrometry (HS-SPME/GC-MS) for extracting amphibian alkaloids and volatile organic compounds (VOCs) from Silverstoneia punctiventris. RESULTS: Using the skin from 8 specimens in 4 biological replicates we have found 33 different compounds. Twenty of them were classified as VOCs into 15 chemical classes including alkanes, alcohols, carbonyl compounds, methylpyridines, benzothiazoles, N-alkylpyrrolidines, pyrazines, and sesquiterpenoids, some of which were previously reported as repellents, defence compounds or defence pheromones in other organisms, and as sex pheromones in a treefrog. Interestingly, six of the remaining compounds were identified as alkaloids previously reported in other toxic/unpalatable dendrobatid frogs. CONCLUSIONS: This is the first report of alkaloids and VOCs found in the Silverstoneia genus, which has been assumed for decades as non-chemically defended. This study establishes HS-SPME/GC-MS as a new application for a simultaneous approach to amphibian alkaloids and VOCs in poison frogs while opens up new research questions to assess the co-occurrence of both type of compounds and to investigate the evolutionary significance of a defence gradient that includes olfactory avoidance, unpalatability, and toxicity in dendrobatids. In addition, our results show that amphibian alkaloids could have a dual function (olfactory at distance, taste by contact) never explored before neither in Silverstonaeia nor in any other dendrobatid species.

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Lambert et al question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

A new species of Pristimantis (Anura: Craugastoridae) from the cloud forests of Colombian western Andes.

The frogs of the genus Pristimantis are recognized for their characteristic reproductive mode and for their incredible diversity of species, becoming the genus with the highest number of species within tetrapod vertebrates. We describe here a new species of Pristimantis from the northwestern Andes of Colombia. The species was found between 2000-2500 m.a.s.l., mostly within moss hanging of tree branches in a tropical cloud forest. It can be easily distinguished from other Pristimantis species of the western Andes by the unique black and white patterning in the ventral surface and the flanks, the hourglass-shaped dorsal folds, and the prominent conical tubercles on eyelids, heels, and outer edge of tarsus. A phylogenetic analysis further supports its status of a lineage reciprocally monophyletic to P. satagius and separated by a genetic distance of 0.03; the latter species bear whitish rather than predominantly black ventral coloration. To the best of our knowledge, this species is only known from the 2,500 ha nature reserve Mesenia-Paramillo, despite other research on this area of the country. Therefore, the species is declared vulnerable while new evidence on its distribution range is accumulated.

Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.

Symbiotic skin bacteria as a source for sex-specific scents in frogs.

Amphibians are known to possess a wide variety of compounds stored in their skin glands. While significant progress has been made in understanding the chemical diversity and biological relevance of alkaloids, amines, steroids, and peptides, most aspects of the odorous secretions are completely unknown. In this study, we examined sexual variations in the volatile profile from the skin of the tree frog Boana prasina and combined culture and culture-independent methods to investigate if microorganisms might be a source of these compounds. We found that sesquiterpenes, thioethers, and methoxypyrazines are major contributors to the observed sex differences. We also observed that each sex has a distinct profile of methoxypyrazines, and that the chemical origin of these compounds can be traced to a Pseudomonas sp. strain isolated from the frog's skin. This symbiotic bacterium was present in almost all individuals examined from different sites and was maintained in captive conditions, supporting its significance as the source of methoxypyrazines in these frogs. Our results highlight the potential relevance of bacteria as a source of chemical signals in amphibians and contribute to increasing our understanding of the role that symbiotic associations have in animals.