In a remarkable example of convergent evolution, insect species spanning 300 million years of divergence have evolved identical single-amino-acid substitutions that confer resistance to plant cardenolide toxins.
Plants and herbivorous insects are the most diverse groups of multicellular organisms, and understanding this species profusion is a central problem in evolutionary biology. A key explanatory hypothesis1 is that iterative co-evolution between plants that produce toxic compounds2 (such as nicotine and mustard oils) and herbivorous insects that have resistance to these toxins3 drives the diversification of each group. Some toxins have an extremely broad mode of action, leading to the evolution of highly divergent detoxification mechanisms in the specialized herbivores that resist these compounds4. But other toxins have just one molecular target. The medically important cardiac glycosides (cardenolides), for example, block activity of animal (Na++K+)ATPase5, an enzyme that regulates ion gradients across the cell membrane. Writing in Proceedings of the National Academy of Sciences, Dobler et al.6 demonstrate how co-evolution between plants containing toxic cardenolides and the herbivorous insects that feed on them represents an exquisite case of convergent molecular evolution, in which distantly related insect species have evolved a common adaptive response in a single gene.
Cardenolides comprise a diverse group of triterpenoid-derived steroids and are produced by at least 60 genera from 12 families of flowering plants, including Asclepias species, or milkweeds, and Digitalis species, or foxgloves. They disrupt the function of the (Na++K+)ATPase by binding to the first extracellular loop of the enzyme's α-subunit. Dobler and colleagues studied the sequence of the gene encoding the (Na++K+)ATPase in 18 cardenolide-resistant herbivorous insect species from 15 genera in 4 orders (Coleoptera, Lepidoptera, Diptera and Hemiptera). They found sequence changes leading to amino-acid substitutions in the α-subunit's first extracellular loop in 16 of the cardenolide-feeding species. The authors then used in vitro assays to show that cells expressing some of these altered sequences survive in the presence of the cardenolide oubain, which is otherwise toxic to cultured cells.
Remarkably, one of the amino-acid substitutions (at position 122) is present in cardenolide-feeding species of all 4 orders, but not in any of 14 insect species (also from all 4 orders) that do not feed on cardenolide-producing plants. This suggests that resistance-conferring substitutions present in a subset of cardenolide-feeding species are the result of adaptive evolution that occurred repeatedly across 300 million years of insect evolutionary divergence. However, it is worth noting that some cardenolide-resistant species do not show adaptive changes in the (Na++K+)ATPase sequence, suggesting that there are probably other molecular routes to this common trait.
In addition to being a striking case of convergent molecular evolution, this adaptation to cardenolide-producing plants carries ecological implications that extend beyond plant–herbivore interactions (Fig. 1). Some cardenolide-resistant insects sequester these compounds, which provides protection against predators, and some also display warning coloration that advertises this defence7. Indeed, the majority of the cardenolide-feeding insects studied by Dobler et al. exhibit warning coloration and are highly toxic. These herbivore traits in turn have their own evolutionary impacts. Coexisting toxic species often evolve similar warning signals, presumably to spread the costs of 'teaching' predators avoidance — an evolutionary process described as Müllerian mimicry. And, in a process known as Batesian mimicry, palatable species sometimes evolve to mimic toxic ones, in order to usurp predator protection.
Although these phenomena — host plant specialization, sequestration of plant toxins, warning coloration, and mimicry — are quite common across herbivorous insects, cardenolide-feeding species have been central to the study of these dynamics. The work by Dobler et al. therefore adds a crucial molecular-genetic piece to this complex puzzle, establishing cardenolide-feeding herbivores as one of the best examples of how molecular, functional and ecological convergence can be linked.
However, fascinating questions remain. It is unclear whether the evolutionarily important mutations are fixed in each cardenolide-feeding species or whether intraspecies genetic variation exists. Comparing the ratio of non-synonymous changes (mutations that lead to an amino-acid substitution) and synonymous changes (mutations that do not change the amino acid) in the gene encoding the (Na++K+)ATPase within and between species would help to reveal whether natural selection has fixed many other mutations — in addition to the functionally important ones already identified — in the cardenolide-feeding lineages8. Reconstructing the progression of all amino-acid substitutions in each species lineage could reveal whether interactions between mutations have played a part in the adaptive evolution occurring at this sequence9. Furthermore, there is variation in the abundance and structure of the cardenolides produced by plants, but we do not know whether this variation has been driven by population variation in insect resistance or vice versa.
It also remains to be confirmed that the amino-acid substitutions identified by Dobler and colleagues do actually confer resistance to cardenolides in insects, because the authors tested the effects of these changes only in vitro in human cells. One approach to move beyond cell lines would be to engineer Drosophila fruitflies to express the resistance genes, and then conduct oubain-feeding trials. Solving the crystal structure of just one (Na++K+)ATPase from a cardenolide specialist would also allow for a more refined analysis of the molecular mechanisms underlying each substitution that confers resistance.
Finally, the findings of Dobler et al. pose additional ecological questions. For example, have similar convergent evolutionary mechanisms evolved in the predators that feed on cardenolide-laden insects? Black-headed grosbeak birds (Pheucticus melanocephalus) predate overwintering monarch butterflies (Danaus plexippus) in the Oyamel fir forests of Mexico10, but it is not known whether these birds, and other such predators, have also evolved cardenolide-resistant (Na++K+)ATPases.
The patterns identified by Dobler and colleagues in the insect (Na++K+)ATPase gene are likely to become a textbook example of convergent evolution at the molecular level — illuminating how a common selective agent can lead to a common set of evolutionary solutions. Their approach highlights the way in which modern evolutionary biology can pinpoint variations in genotype that lead to variation in molecular and organismal traits in ecologically relevant species, not just in model organisms11. Ultimately, such studies are helping to identify the ecological forces and molecular mechanisms responsible for generating the diversity of life on Earth.
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Ecology Letters (2019)
Advanced Functional Materials (2016)