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Research Review: Molecular Ecology article on Cardenolide and Butterflies

by Professor Francis X. Villablanca
Cal Poly Biological Sciences
Monarch Alert Project Director

Original Article Information: The evolution of cardenolide-resistant forms of Na+,K+-ATPase in Danainae butterflies. Matthew L. Aardema, Ying Zhen, and Peter Andolfatto.
Molecular Ecology (2012) 21, 340–349.

In an article published in the journal Molecular Ecology, researchers report on a monarch butterfly gene that has experienced two mutations. Previous research proposed that both of these mutations may provide monarch butterflies with resistance to cardenolide.

First, something about cardenolides and then back to mutations. Cardenolides are a type of steroid. Many plants contain its derivatives, collectively known as cardenolides, including many that contain structural groups derived from sugars. Cardenolide glycosides are often toxic; specifically, they can stop hearts.

Some plant and animal species use cardenolides as a defense mechanism, most notably the monarch butterflies. Adult monarch butterflies store the cardenolides they have built up as larvae while feeding on milkweeds (plants in the genus Asclepias). The cardenolide content in monarch butterflies deters birds and other vertebrate predators. Notable exceptions are the Black-backed Orioles (Icterus abeillei) and Black-headed Grosbeaks (Pheucticus melanocephalus), and some rodents in the genera Peromyscus and Microtus, all of which are cardenolide-tolerant butterfly predators.

Ok, back to genetics. The gene under scrutiny is named alpha subunit of Na+, K+-ATPase, or ATPa for short. Two of its mutations are associated with the monarch butterflies’ own tolerance to the cardenolides. The shorthand names for the two mutations are N122H and Q111L. The number (122) refers to the mutation’s position in the gene. The letters that bookend the number (N122H) are the specific amino acids within the gene that have been mutated (for example a mutation from N to H). 

The authors argue that earlier research supporting the interpretation that both mutations provide an advantage to monarch butterflies is wrong. 

Their logic is as follows: the Q111L mutation occurred in the distant past of the Lepidoptera (the order of insects that include moths and butterflies). In fact, long before milkweed butterflies (and therefore resistance to milkweed) could have evolved. So, they propose, this mutation could not provide an advantage because at the time the mutation occurred, there was no need for it.

In contrast, the more recent N122H mutation is present in three species of Danaus, (the genus of butterflies including Tigers, Milkweeds, Monarchs, and Queens) and not in other Lepidopterans. Interestingly, this same mutation is present in a beetle that is also resistant to cardenolides. The authors regard this as evidence of convergent evolution (independent evolutionary lineages ending up with the same traits in response to the same natural selection pressure) between the beetle and the three Danaus milkweed butterfly species. The recentness of the mutation and the fact that it also occurs in other cardenolide-resistant invertebrates seems like solid evidence.

The authors are likely correct in their interpretation of the importance of the N122H mutation (the recent one that shows convergence). Yet their interpretation of the importance of the Q111L mutation is not as clear. It is safe to say the mutation is not an “adaptation” because it occurred before milkweed butterflies ever arose and ever encountered milkweed. In other words, Lepidoptera would not have made use of this mutation.

Yet, the mutation could still provide an advantage if it is an “exaptation.”  An exaptation is a trait that currently provides an advantage, but the advantage is for a function that is different from its original function. You might think of this as sort of a “pre-adaptation” because it provides an advantage, but long after the trait arises.

The best way to test the authors’ ideas is to use genetic engineering technology to make “gene knock out” monarch butterflies that have the N122H mutation, or the Q111L, or both. In this manner the effects of each of these mutations could be tested directly by seeing how they affect the ATPa.  This would be like redoing nature’s experiment to see if the mutation actually has the effect we think it has.

This article is important because it shows us how something as simple as a single mutation could allow an organism to evolve an entirely new strategy for dealing with its predators. (Go nature!)  It also clearly shows us that scientists need to know what has already been discovered, but also be willing to test those discoveries in order to “prove it to themselves” and make sure the original conclusions were correct.  In the end, the data don’t interpret themselves; we have to interpret them.