Proteomic Analysis of Heloderma horridum horridum Venom: Assessment to Its Transcriptome and Newfound Proteins.

Heloderma horridum horridum, a venomous reptile native to America, has a venom with potential applications in treating type II diabetes. In this work, H. h. horridum venom was extracted, lyophilized, and characterized using enzymatic assays for hyaluronidase, phospholipase, and protease. Proteomic analysis of the venom was conducted employing bottom-up/shotgun approaches, SDS-PAGE, high-pH reversed-phase chromatography, and fractionation of tryptic peptides using nano-LC-MS/MS. The proteins found in H. h. horridum venom were reviewed according to the classification of the transcriptome previously reported. The proteomic approach identified 101 enzymes, 36 other proteins, 15 protein inhibitors, 11 host defense proteins, and 1 toxin, including novel venom components such as calcium-binding proteins, phospholipase A2 inhibitors, serpins, cathepsin, subtilases, carboxypeptidase-like, aminopeptidases, glycoside hydrolases, thioredoxin transferases, acid ceramidase-like, enolase, multicopper oxidases, phosphoglucose isomerase (PGI), fructose-1,6-bisphosphatase class 1, pentraxin-related, peptidylglycine α-hydroxylating monooxygenase/peptidyl-hydroxyglycine α-amidating lyase, carbonic anhydrase, acetylcholinesterase, dipeptidylpeptidase, and lysozymes. These findings contribute to understanding the venomous nature of H. h. horridum and highlight its potential as a source of bioactive compounds. Data are available via PRoteomeXchange with the identifier PXD052417.

Weak warning signals can persist in the absence of gene flow.

Aposematic organisms couple conspicuous warning signals with a secondary defense to deter predators from attacking. Novel signals of aposematic prey are expected to be selected against due to positive frequency-dependent selection. How, then, can novel phenotypes persist after they arise, and why do so many aposematic species exhibit intrapopulation signal variability? Using a polytypic poison frog (Dendrobates tinctorius), we explored the forces of selection on variable aposematic signals using 2 phenotypically distinct (white, yellow) populations. Contrary to expectations, local phenotype was not always better protected compared to novel phenotypes in either population; in the white population, the novel phenotype evoked greater avoidance in natural predators. Despite having a lower quantity of alkaloids, the skin extracts from yellow frogs provoked higher aversive reactions by birds than white frogs in the laboratory, although both populations differed from controls. Similarly, predators learned to avoid the yellow signal faster than the white signal, and generalized their learned avoidance of yellow but not white. We propose that signals that are easily learned and broadly generalized can protect rare, novel signals, and weak warning signals (i.e., signals with poor efficacy and/or poor defense) can persist when gene flow among populations, as in this case, is limited. This provides a mechanism for the persistence of intrapopulation aposematic variation, a likely precursor to polytypism and driver of speciation.

Gene Flow in the Müllerian Mimicry Ring of a Poisonous Papuan Songbird Clade (Pitohui; Aves).

Müllerian mimicry rings are remarkable symbiotic species assemblages in which multiple members share a similar phenotype. However, their evolutionary origin remains poorly understood. Although gene flow among species has been shown to generate mimetic patterns in some Heliconius butterflies, mimicry is believed to be due to true convergence without gene flow in many other cases. We investigated the evolutionary history of multiple members of a passerine mimicry ring in the poisonous Papuan pitohuis. Previous phylogenetic evidence indicates that the aposematic coloration shared by many, but not all, members of this genus is ancestral and has only been retained by members of the mimicry ring. Using a newly assembled genome and thousands of genomic DNA markers, we demonstrate gene flow from the hooded pitohui (Pitohui dichrous) into the southern variable pitohui (Pitohui uropygialis), consistent with shared patterns of aposematic coloration. The vicinity of putatively introgressed loci is significantly enriched for genes that are important in melanin pigment expression and toxin resistance, suggesting that gene flow may have been instrumental in the sharing of plumage patterns and toxicity. These results indicate that interspecies gene flow may be a more general mechanism in generating mimicry rings than hitherto appreciated.

Tempo and Mode of the Evolution of Venom and Poison in Tetrapods.

Toxic weaponry in the form of venom and poison has evolved in most groups of animals, including all four major lineages of tetrapods. Moreover, the evolution of such traits has been linked to several key aspects of the biology of toxic animals including life-history and diversification. Despite this, attempts to investigate the macroevolutionary patterns underlying such weaponry are lacking. In this study we analyse patterns of venom and poison evolution across reptiles, amphibians, mammals, and birds using a suite of phylogenetic comparative methods. We find that each major lineage has a characteristic pattern of trait evolution, but mammals and reptiles evolve under a surprisingly similar regime, whilst that of amphibians appears to be particularly distinct and highly contrasting compared to other groups. Our results also suggest that the mechanism of toxin acquisition may be an important distinction in such evolutionary patterns; the evolution of biosynthesis is far less dynamic than that of sequestration of toxins from the diet. Finally, contrary to the situation in amphibians, other tetrapod groups show an association between the evolution of toxic weaponry and higher diversification rates. Taken together, our study provides the first broad-scale analysis of macroevolutionary patterns of venom and poison throughout tetrapods.

Mimetic Divergence and the Speciation Continuum in the Mimic Poison Frog Ranitomeya imitator.

While divergent ecological adaptation can drive speciation, understanding the factors that facilitate or constrain this process remains a major goal in speciation research. Here, we study two mimetic transition zones in the poison frog Ranitomeya imitator, a species that has undergone a Müllerian mimetic radiation to establish four morphs in Peru. We find that mimetic morphs are strongly phenotypically differentiated, producing geographic clines with varying widths. However, distinct morphs show little neutral genetic divergence, and landscape genetic analyses implicate isolation by distance as the primary determinant of among-population genetic differentiation. Mate choice experiments suggest random mating at the transition zones, although certain allopatric populations show a preference for their own morph. We present evidence that this preference may be mediated by color pattern specifically. These results contrast with an earlier study of a third transition zone, in which a mimetic shift was associated with reproductive isolation. Overall, our results suggest that the three known mimetic transition zones in R. imitator reflect a speciation continuum, which we have characterized at the geographic, phenotypic, behavioral, and genetic levels. We discuss possible explanations for variable progress toward speciation, suggesting that multifarious selection on both mimetic color pattern and body size may be responsible for generating reproductive isolation.