The multi-level regulation of clownfish metamorphosis by thyroid hormones.

Most marine organisms have a biphasic life cycle during which pelagic larvae transform into radically different juveniles. In vertebrates, the role of thyroid hormones (THs) in triggering this transition is well known, but how the morphological and physiological changes are integrated in a coherent way with the ecological transition remains poorly explored. To gain insight into this question, we performed an integrated analysis of metamorphosis of a marine teleost, the false clownfish (Amphiprion ocellaris). We show how THs coordinate a change in color vision as well as a major metabolic shift in energy production, highlighting how it orchestrates this transformation. By manipulating the activity of liver X regulator (LXR), a major regulator of metabolism, we also identify a tight link between metabolic changes and metamorphosis progression. Strikingly, we observed that these regulations are at play in the wild, explaining how hormones coordinate energy needs with available resources during the life cycle.

Co-option of the same ancestral gene family gave rise to mammalian and reptilian toxins.

BACKGROUND: Evolution can occur with surprising predictability when organisms face similar ecological challenges. For most traits, it is difficult to ascertain whether this occurs due to constraints imposed by the number of possible phenotypic solutions or because of parallel responses by shared genetic and regulatory architecture. Exceptionally, oral venoms are a tractable model of trait evolution, being largely composed of proteinaceous toxins that have evolved in many tetrapods, ranging from reptiles to mammals. Given the diversity of venomous lineages, they are believed to have evolved convergently, even though biochemically similar toxins occur in all taxa. RESULTS: Here, we investigate whether ancestral genes harbouring similar biochemical activity may have primed venom evolution, focusing on the origins of kallikrein-like serine proteases that form the core of most vertebrate oral venoms. Using syntenic relationships between genes flanking known toxins, we traced the origin of kallikreins to a single locus containing one or more nearby paralogous kallikrein-like clusters. Additionally, phylogenetic analysis of vertebrate serine proteases revealed that kallikrein-like toxins in mammals and reptiles are genetically distinct from non-toxin ones. CONCLUSIONS: Given the shared regulatory and genetic machinery, these findings suggest that tetrapod venoms evolved by co-option of proteins that were likely already present in saliva. We term such genes 'toxipotent'-in the case of salivary kallikreins they already had potent vasodilatory activity that was weaponized by venomous lineages. Furthermore, the ubiquitous distribution of kallikreins across vertebrates suggests that the evolution of envenomation may be more common than previously recognized, blurring the line between venomous and non-venomous animals.

An ancient, conserved gene regulatory network led to the rise of oral venom systems.

Oral venom systems evolved multiple times in numerous vertebrates enabling the exploitation of unique predatory niches. Yet how and when they evolved remains poorly understood. Up to now, most research on venom evolution has focused strictly on the toxins. However, using toxins present in modern day animals to trace the origin of the venom system is difficult, since they tend to evolve rapidly, show complex patterns of expression, and were incorporated into the venom arsenal relatively recently. Here we focus on gene regulatory networks associated with the production of toxins in snakes, rather than the toxins themselves. We found that overall venom gland gene expression was surprisingly well conserved when compared to salivary glands of other amniotes. We characterized the "metavenom network," a network of ∼3,000 nonsecreted housekeeping genes that are strongly coexpressed with the toxins, and are primarily involved in protein folding and modification. Conserved across amniotes, this network was coopted for venom evolution by exaptation of existing members and the recruitment of new toxin genes. For instance, starting from this common molecular foundation, Heloderma lizards, shrews, and solenodon, evolved venoms in parallel by overexpression of kallikreins, which were common in ancestral saliva and induce vasodilation when injected, causing circulatory shock. Derived venoms, such as those of snakes, incorporated novel toxins, though still rely on hypotension for prey immobilization. These similarities suggest repeated cooption of shared molecular machinery for the evolution of oral venom in mammals and reptiles, blurring the line between truly venomous animals and their ancestors.

Toxin expression in snake venom evolves rapidly with constant shifts in evolutionary rates.

Key innovations provide ecological opportunity by enabling access to new resources, colonization of new environments, and are associated with adaptive radiation. The most well-known pattern associated with adaptive radiation is an early burst of phenotypic diversification. Venoms facilitate prey capture and are widely believed to be key innovations leading to adaptive radiation. However, few studies have estimated their evolutionary rate dynamics. Here, we test for patterns of adaptive evolution in venom gene expression data from 52 venomous snake species. By identifying shifts in tempo and mode of evolution along with models of phenotypic evolution, we show that snake venom exhibits the macroevolutionary dynamics expected of key innovations. Namely, all toxin families undergo shifts in their rates of evolution, likely in response to changes in adaptive optima. Furthermore, we show that rapid-pulsed evolution modelled as a Lévy process better fits snake venom evolution than conventional early burst or Ornstein-Uhlenbeck models. While our results support the idea of snake venom being a key innovation, the innovation of venom chemistry lacks clear mechanisms that would lead to reproductive isolation and thus adaptive radiation. Therefore, the extent to which venom directly influences the diversification process is still a matter of contention.

Many Options, Few Solutions: Over 60 My Snakes Converged on a Few Optimal Venom Formulations.

Gene expression changes contribute to complex trait variations in both individuals and populations. However, the evolution of gene expression underlying complex traits over macroevolutionary timescales remains poorly understood. Snake venoms are proteinaceous cocktails where the expression of each toxin can be quantified and mapped to a distinct genomic locus and traced for millions of years. Using a phylogenetic generalized linear mixed model, we analyzed expression data of toxin genes from 52 snake species spanning the 3 venomous snake families and estimated phylogenetic covariance, which acts as a measure of evolutionary constraint. We find that evolution of toxin combinations is not constrained. However, although all combinations are in principle possible, the actual dimensionality of phylomorphic space is low, with envenomation strategies focused around only four major toxin families: metalloproteases, three-finger toxins, serine proteases, and phospholipases A2. Although most extant snakes prioritize either a single or a combination of major toxin families, they are repeatedly recruited and lost. We find that over macroevolutionary timescales, the venom phenotypes were not shaped by phylogenetic constraints, which include important microevolutionary constraints such as epistasis and pleiotropy, but more likely by ecological filtering that permits a small number of optimal solutions. As a result, phenotypic optima were repeatedly attained by distantly related species. These results indicate that venoms evolve by selection on biochemistry of prey envenomation, which permit diversity through parallelism, and impose strong limits, since only a few of the theoretically possible strategies seem to work well and are observed in extant snakes.

Population Genomic Analysis of a Pitviper Reveals Microevolutionary Forces Underlying Venom Chemistry.

Venoms are among the most biologically active secretions known, and are commonly believed to evolve under extreme positive selection. Many venom gene families, however, have undergone duplication, and are often deployed in doses vastly exceeding the LD50 for most prey species, which should reduce the strength of positive selection. Here, we contrast these selective regimes using snake venoms, which consist of rapidly evolving protein formulations. Though decades of extensive studies have found that snake venom proteins are subject to strong positive selection, the greater action of drift has been hypothesized, but never tested. Using a combination of de novo genome sequencing, population genomics, transcriptomics, and proteomics, we compare the two modes of evolution in the pitviper, Protobothrops mucrosquamatus. By partitioning selective constraints and adaptive evolution in a McDonald-Kreitman-type framework, we find support for both hypotheses: venom proteins indeed experience both stronger positive selection, and lower selective constraint than other genes in the genome. Furthermore, the strength of selection may be modulated by expression level, with more abundant proteins experiencing weaker selective constraint, leading to the accumulation of more deleterious mutations. These findings show that snake venoms evolve by a combination of adaptive and neutral mechanisms, both of which explain their extraordinarily high rates of molecular evolution. In addition to positive selection, which optimizes efficacy of the venom in the short term, relaxed selective constraints for deleterious mutations can lead to more rapid turnover of individual proteins, and potentially to exploration of a larger venom phenotypic space.