Expression patterns of melanin-related genes are linked to crypsis and conspicuousness in a pumpkin toadlet.

Colour signals play pivotal roles in different communication systems, and the evolution of these characters has been associated with behavioural ecology, integumentary production processes and perceptual mechanisms of the species involved. Here, we present the first insight into the molecular and histological basis of skin colour polymorphism within a miniaturized species of pumpkin toadlet, potentially representing the lowest size threshold for colour polytypism in tetrapods. Brachycephalus actaeus exhibits a coloration ranging from cryptic green to conspicuous orange skin, and our findings suggest that colour morphs differ in their capability to be detected by potential predators. We also found that the distribution and abundance of chromatophores are variable in the different colour morphs. The expression pattern of coloration related genes was predominantly associated with melanin synthesis (including dct, edn1, mlana, oca2, pmel, slc24a5, tyrp1 and wnt9a). Up-regulation of melanin genes in grey, green and brown skin was associated with higher melanophore abundance than in orange skin, where xanthophores predominate. Our findings provide a significant foundation for comparing and understanding the diverse pathways that contribute to the evolution of pigment production in the skin of amphibians.

Convergence of gut microbiota in myrmecophagous amphibians.

The gut microbiome composition of terrestrial vertebrates is known to converge in response to common specialized dietary strategies, like leaf-eating (folivory) or ant- and termite-eating (myrmecophagy). To date, such convergence has been studied in mammals and birds, but has been neglected in amphibians. Here, we analysed 15 anuran species (frogs and toads) representing five Neotropical families and demonstrated the compositional convergence of the gut microbiomes of distantly related myrmecophagous species. Specifically, we found that the gut microbial communities of bufonids and microhylids, which have independently evolved myrmecophagy, were significantly more similar than expected based on their hosts' evolutionary divergence. Conversely, we found that gut microbiome composition was significantly associated with host evolutionary history in some cases. For instance, the microbiome composition of Xenohyla truncata, one of the few known amphibians that eat fruits, was not different from those of closely related tree frogs with an arthropod generalist diet. Bacterial taxa overrepresented in myrmecophagous species relative to other host families include Paludibacter, Treponema, and Rikenellaceae, suggesting diet-mediated selection and prey-to-predator transmission likely driving the observed compositional convergence. This study provides a basis for examining the roles of the gut microbiome in host tolerance and sequestration of toxic alkaloids from ants and termites.

Initial Phylotranscriptomic Confirmation of Homoplastic Evolution of the Conspicuous Coloration and Bufoniform Morphology of Pumpkin-Toadlets in the Genus Brachycephalus.

The genus Brachycephalus is a fascinating group of miniaturized anurans from the Brazilian Atlantic Forest, comprising the conspicuous, brightly colored pumpkin-toadlets and the cryptic flea-toads. Pumpkin-toadlets are known to contain tetrodotoxins and therefore, their bright colors may perform an aposematic function. Previous studies based on a limited number of mitochondrial and nuclear-encoded markers supported the existence of two clades containing species of pumpkin-toadlet phenotype, but deep nodes remained largely unresolved or conflicting between data sets. We use new RNAseq data of 17 individuals from nine Brachycephalus species to infer their evolutionary relationships from a phylogenomic perspective. Analyses of almost 5300 nuclear-encoded ortholog protein-coding genes and full mitochondrial genomes confirmed the existence of two separate pumpkin-toadlet clades, suggesting the convergent evolution (or multiple reversals) of the bufoniform morphology, conspicuous coloration, and probably toxicity. In addition, the study of the mitochondrial gene order revealed that three species (B. hermogenesi, B. pitanga, and B. rotenbergae) display translocations of different tRNAs (NCY and CYA) from the WANCY tRNA cluster to a position between the genes ATP6 and COIII, showing a new mitochondrial gene order arrangement for vertebrates. The newly clarified phylogeny suggests that Brachycephalus has the potential to become a promising model taxon to understand the evolution of coloration, body plan and toxicity. Given that toxicity information is available for only few species of Brachycephalus, without data for any flea-toad species, we also emphasize the need for a wider screening of toxicity across species, together with more in-depth functional and ecological study of their phenotypes.