Kevin Arbuckle

Widespread convergence in toxin resistance by predictable molecular evolution

Significance Convergence has strong bearing on the fundamental debate about whether evolution is stochastic and unpredictable or subject to constraints. Here we show that, in certain circumstances, evolution can be highly predictable. We demonstrate that several lineages of insects, amphibians, reptiles, and mammals have utilized the same molecular solution, via the process of convergence, to evolve resistance to toxic cardiac glycosides produced defensively by plants and bufonid toads. The repeatability of this process across the animal kingdom demonstrates that evolution can be constrained to proceed along highly predictable pathways at molecular and functional levels. Our study has important implications for conservation biology by providing a predictive framework for assessing the vulnerability of native fauna to the introduction of invasive toxic toads. The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na+/K+-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of 2 of the 12 amino acids comprising the Na+/K+-ATPase H1–H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na+/K+-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.

The biology of color

In living color Animals live in a colorful world, but we rarely stop to think about how this color is produced and perceived, or how it evolved. Cuthill et al. review how color is used for social signals between individual animals and how it affects interactions with parasites, predators, and the physical environment. New approaches are elucidating aspects of animal coloration, from the requirements for complex cognition and perception mechanisms to the evolutionary dynamics surrounding its development and diversification. Science, this issue p. eaan0221 BACKGROUND The interdisciplinary field of animal coloration is growing rapidly, spanning questions about the diverse ways that animals use pigments and structures to generate color, the underlying genetics and epigenetics, the perception of color, how color information is integrated with information from other senses, and general principles underlying color’s evolution and function. People working in the field appreciate linkages between these parallel lines of enquiry, but outsiders need the easily navigable roadmap that we provide here. ADVANCES In the past 20 years, the field of animal coloration research has been propelled forward by technological advances that include spectrophotometry, digital imaging, computational neuroscience, innovative laboratory and field studies, and large-scale comparative analyses, which are allowing new questions to be asked. For example, we can now pose questions about the evolution of camouflage based on what a prey’s main predator can see, and we can start to appreciate that gene changes underlying color production have occurred in parallel in unrelated species. Knowledge of the production, perception, and evolutionary function of coloration is poised to make contributions to areas as diverse as medicine, security, clothing, and the military, but we need to take stock before moving forward. OUTLOOK Here, a group of evolutionary biologists, behavioral ecologists, psychologists, optical physicists, visual physiologists, geneticists, and anthropologists review this diverse area of science, daunting to the outsider, and set out what we believe are the key questions for the future. These are how nanoscale structures are used to manipulate light; how dynamic changes in coloration occur on different time scales; the genetics of coloration (including key innovations and the extent of parallel changes in different lineages); alternative perceptions of color by different species (including wavelengths that we cannot see, such as ultraviolet); how color, pattern, and motion interact; and how color works together with other modalities, especially odor. From an adaptive standpoint, color can serve several functions, and the resulting patterns frequently represent a trade-off among different evolutionary drivers, some of which are nonvisual (e.g., photoprotection). These trade-offs can vary between individuals within the same population, and color can be altered strategically on different time scales to serve different purposes. Lastly, interspecific differences in coloration, sometimes even observable in the fossil record, give insights into trait evolution. The biology of color is a field that typifies modern research: curiosity-led, technology-driven, multilevel, interdisciplinary, and integrative. Spectacular changes to color and morphology in a cuttlefish. Color can conceal or reveal. The giant Australian cuttlefish (Sepia apama) alters the relative size of its pigment-bearing chromatophores and warps its muscular skin to switch between camouflage mode (top) and communication mode (bottom) in under a second. Photos:

A simple measure of the strength of convergent evolution

Summary Convergent evolution, the independent occurrence of phenotypic similarity, is a widespread and common phenomenon. Methods have been developed to identify instances of convergence, but there is a lack of techniques for quantifying the strength of convergence. We therefore investigated whether convergent evolution can be quantified in a meaningful way. We have developed a simple metric (the Wheatsheaf index) that provides an index of the strength of convergent evolution incorporating both phenotypic similarity and phylogenetic relatedness. The index is comparable across any quantitative or semiquantitative traits and thus will enable the testing of various hypotheses relating to convergence. The index performs well over a range of conditions. We apply it to an empirical example using Anolis lizard ecomorphs to demonstrate how it can be used. The Wheatsheaf index provides an additional tool that complements methods aimed at identifying cases of convergent evolution. It will enable cases of convergence to be analysed in more detail, test hypotheses about its mechanics as an evolutionary process and, more generally, the predictability of evolution (how often do we see strong convergence and does this mean evolutionary solutions are limited?).

Antipredator defenses predict diversification rates

Significance Prey use a variety of mechanisms to avoid the risk of predation, including chemical defense, camouflage, and conspicuous coloration. Here we show that variation in these forms of protection can have profound effects on macroevolutionary patterns in amphibians. Chemical defense and conspicuous coloration both increase speciation rates, in line with the “escape-and-radiate” hypothesis. However, chemical defense also increases extinction rates, lowering net diversification. We therefore show that alternative defensive strategies have important macroevolutionary consequences but that these are only partially consistent with standard escape-and-radiate interpretations. Hence, we suggest a conceptual expansion of this highly influential evolutionary hypothesis. In addition, our findings may have implications for endangered species conservation by allowing some predictability of extinction risk from knowledge of antipredator defenses. The “escape-and-radiate” hypothesis predicts that antipredator defenses facilitate adaptive radiations by enabling escape from constraints of predation, diversified habitat use, and subsequently speciation. Animals have evolved diverse strategies to reduce the direct costs of predation, including cryptic coloration and behavior, chemical defenses, mimicry, and advertisement of unprofitability (conspicuous warning coloration). Whereas the survival consequences of these alternative defenses for individuals are well-studied, little attention has been given to the macroevolutionary consequences of alternative forms of defense. Here we show, using amphibians as the first, to our knowledge, large-scale empirical test in animals, that there are important macroevolutionary consequences of alternative defenses. However, the escape-and-radiate hypothesis does not adequately describe them, due to its exclusive focus on speciation. We examined how rates of speciation and extinction vary across defensive traits throughout amphibians. Lineages that use chemical defenses show higher rates of speciation as predicted by escape-and-radiate but also show higher rates of extinction compared with those without chemical defense. The effect of chemical defense is a net reduction in diversification compared with lineages without chemical defense. In contrast, acquisition of conspicuous coloration (often used as warning signals or in mimicry) is associated with heightened speciation rates but unchanged extinction rates. We conclude that predictions based on the escape-and-radiate hypothesis must incorporate the effect of traits on both speciation and extinction, which is rarely considered in such studies. Our results also suggest that knowledge of defensive traits could have a bearing on the predictability of extinction, perhaps especially important in globally threatened taxa such as amphibians.

Quantification provides a conceptual basis for convergent evolution

While much of evolutionary biology attempts to explain the processes of diversification, there is an important place for the study of phenotypic similarity across life forms. When similar phenotypes evolve independently in different lineages this is referred to as convergent evolution. Although long recognised, evolutionary convergence is receiving a resurgence of interest. This is in part because new genomic data sets allow detailed and tractable analysis of the genetic underpinnings of convergent phenotypes, and in part because of renewed recognition that convergence may reflect limitations in the diversification of life. In this review we propose that although convergent evolution itself does not require a new evolutionary framework, none the less there is room to generate a more systematic approach which will enable evaluation of the importance of convergent phenotypes in limiting the diversity of lifes forms. We therefore propose that quantification of the frequency and strength of convergence, rather than simply identifying cases of convergence, should be considered central to its systematic comprehension. We provide a non‐technical review of existing methods that could be used to measure evolutionary convergence, bringing together a wide range of methods. We then argue that quantification also requires clear specification of the level at which the phenotype is being considered, and argue that the most constrained examples of convergence show similarity both in function and in several layers of underlying form. Finally, we argue that the most important and impressive examples of convergence are those that pertain, in form and function, across a wide diversity of selective contexts as these persist in the likely presence of different selection pressures within the environment.

Correlation between ontogenetic dietary shifts and venom variation in Australian brown snakes ( Pseudonaja )

Venom is a key evolutionary trait, as evidenced by its widespread convergent evolution across the animal kingdom. In an escalating prey-predator arms race, venoms evolve rapidly to guarantee predatory or defensive success. Variation in venom composition is ubiquitous among snakes. Here, we tested variation in venom activity on substrates relevant to blood coagulation among Pseudonaja (brown snake) species, Australian elapids responsible for the majority of medically important human envenomations in Australia. A functional approach was employed to elucidate interspecific variation in venom activity in all nine currently recognised species of Pseudonaja. Fluorometric enzymatic activity assays were performed to test variation in whole venom procoagulant activity among species. Analyses confirmed the previously documented ontogenetic shift from non-coagulopathic venom in juveniles to coagulopathic venom as adults, except for the case of P. modesta, which retains non-coagulopathic venom as an adult. These shifts in venom activity correlate with documented ontogenetic shifts in diet among brown snakes from specialisation on reptilian prey as juveniles (and throughout the life cycle of P. modesta), to a more generalised diet in adults that includes mammals. The results of this study bring to light findings relevant to both clinical and evolutionary toxinology.

How the cobra got its flesh-eating venom: cytotoxicity as a defensive innovation and its co-evolution with hooding, aposematic marking, and spitting

The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa.

Differential procoagulant effects of saw-scaled viper (Serpentes: Viperidae: Echis ) snake venoms on human plasma and the narrow taxonomic ranges of antivenom efficacies

Saw-scaled vipers (genus Echis) are one of the leading causes of snakebite morbidity and mortality in parts of Sub-Saharan Africa, the Middle East, and vast regions of Asia, constituting a public health burden exceeding that of almost any other snake genus globally. Venom-induced consumption coagulopathy, owing to the action of potent procoagulant toxins, is one of the most relevant clinical manifestations of envenomings by Echis spp. Clinical experience and prior studies examining a limited range of venoms and restricted antivenoms have demonstrated for some antivenoms an extreme lack of antivenom cross-reactivity between different species of this genus, sometimes resulting in catastrophic treatment failure. This study undertook the most comprehensive testing of Echis venom effects upon the coagulation of human plasma, and also the broadest examination of antivenom potency and cross-reactivity, to-date. 10 Echis species/populations and four antivenoms (two African, two Asian) were studied. The results indicate that the venoms are, in general, potently procoagulant but that the relative dependence on calcium or phospholipid cofactors is highly variable. Additionally, three out of the four antivenoms tested demonstrated only a very narrow taxonomic range of effectiveness in preventing coagulopathy, with only the SAIMR antivenom displaying significant levels of cross-reactivity. These results were in conflict with previous studies using prolonged preincubation of antivenom with venom to suggest effective cross-reactivity levels for the ICP Echi-Tab antivenom. These findings both inform upon potential clinical effects of envenomation in humans and highlight the extreme limitations of available treatment. It is hoped that this will spur efforts into the development of antivenoms with more comprehensive coverage for bites not only from wild snakes but also from specimens widely kept in zoological collections.

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.

windex: Analyzing Convergent Evolution Using the Wheatsheaf Index in R.

Summary windex is a package developed for the R statistical environment to provide novel tools for the analysis of convergent evolution. The recently described Wheatsheaf index provides quantitative measures of the strength of convergence and opens up new possibilities for exploring this evolutionary phenomenon. The windex package allows implementation of this method with additional functions that can be used to create plots and perform statistical tests. R provides compatibility with other packages, and the R environment is familiar to many researchers. Availability The windex package is freely available from CRAN: http://cran.r-project.org/web/packages/windex/. Consequently, windex can be installed directly from R and is distributed under the GNU General Public License 2.0.