Battle between molecular evolutionary forces shapes toxin resistance in Neotropical grass frogs

Reporting on:
Mohammadi S, Yang L, Harpak A, Herrera-Álvarez S, Rodríguez-Ordoñez MP, Peng J, Zhang K, Storz JF, Dobler S, Crawford AJ, Andolfatto P. 2021. Concerted evolution reveals co-adapted amino acid substitutions in Na+ K+-ATPase of frogs that prey on toxic toads. Current Biology. [link]

Neotropical grass frog, Leptodactylus insularium. Photo by Brian Gratwicke (2011).

​This month I wanted to write about a study I had the privilege of working on with a talented and diverse group of researchers from around the world. Our multidisciplinary teamwork allowed us to uncover a very cool story of evolution involving Neotropical grass frogs from South America.
 
This story centers around gene duplication – a mutational event wherein an extra copy of a gene is produced. Gene duplication is a powerful tool in evolution because it can liberate one copy of the gene to mutate and produce new roles without causing significant detrimental effects in the organism—the extra copy can act as a sort of safety backup. We explored the evolution of a gene duplication that had been identified in Neotropical grass frogs (genus Leptodactylus) and consequently unraveled the remarkable pathway that led them to gain a specialized adaptation—toxin resistance. The gene in question is ATP1A1, which codes for a protein vital to all animal cells—the sodium-potassium pump (or Na+K+-ATPase).

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We surveyed genetic material from many species of Neotropical grass frogs. We found that the ATP1A1 duplication was present in all species of the genus. We further found that there are 12 shared amino acid differences between the two gene copies. Two of these amino acid substitutions were known in other animals to provide resistance to common toxins known as cardiotonic steroids. These toxins are used as a chemical defense in toads, insects, snakes, and many plants. They target and disable sodium-potassium pumps. Because Neotropical grass frogs are known to feed on toads, having a resistant copy of this gene makes sense. This was a textbook example of neo-functionalization, wherein a gene acquires a new function after a duplication event. In this case, a new function evolved between a resistance-conferring copy, which we dubbed the R copy, and a copy that retained ancestral susceptibility, which we dubbed the S copy

DNA is transcribed into RNA, which is then translated into the amino acids that make a protein. This is known as the Central Dogma of Molecular Biology and it's the process by which genetic information turns into a functional product (protein). Image from Wikimedia Commons.

​To trace the origins of this duplication we constructed a genealogy of the ATP1A1 gene using DNA sequences. However, this genealogy showed that the gene duplication occurred multiple times, once in each species, and was then followed by 12 parallel amino acid substitutions. This didn’t make sense considering how unlikely it is for the identical duplication and subsequent identical mutations to occur multiple times. The true origin of the duplication and subsequent 12 substitutions was revealed when we constructed our genealogy using amino acid sequences (i.e., protein sequences): they occurred once in the shared common ancestor of all Neotropical grass frogs and were subsequently inherited by all the descendent species.
 
Although the DNA-based genealogy didn’t show us the origins of the gene duplication, it exposed another hidden story. The DNA patterns show that the R and S genes remained very similar to each other throughout tens of millions of years because of frequent non-allelic gene conversion (NAGC). NAGC is a molecular mechanism that homogenizes differences between two gene copies. This is known as the nefarious concerted evolution, which can stop neo-functionalization by working to keep duplicated genes identical. Frequent NAGC can result in a genealogy in which duplicated genes (two different genes) in the same species appear to share a more recent common ancestor than with their counterparts (the same genes) in other species. This explains why our DNA-based genealogy showed multiple origins of the gene duplication.

In comes the hero, natural selection, which can oppose the homogenizing force of NAGC by promoting divergence between the duplicated genes. Hidden in the ATP1A1 gene sequences are short segments of divergence between the duplicates, which are strongholds that evaded concerted evolution. There are 12 of them, and they strongly distinguish R and S. We ran a computational model to determine how strong natural selection would have had to be to overcome the forces of concerted evolution at these 12 sites. The results suggested that substantial selection had to act to create such strongholds of “escape” from the hold of frequent NAGC.
 
The fact that strong selection maintained these 12 amino acid substitutions implies that they’re functionally important and collectively contribute to organismal fitness. We knew that 2 of these 12 substitutions were associated with cardiotonic steroid resistance, but we had no clue what the other 10 were doing. To figure this out, we performed protein engineering experiments and functional assays. We produced the S and R copy proteins in the lab, and then added various combinations of the 12 R substitutions onto the S copy gene to measure their effects. Our results confirmed that the 2 resistance-conferring substitutions were indeed responsible for resistance in the R protein, while the additional 10 did nothing to enhance resistance. However, our results revealed that the 2 resistance-conferring substitutions come at a high price. When they’re added both individually and together on the S protein, they drastically reduce the protein’s ability to function. Thus, the resistance-conferring substitutions provide an adaptive advantage at the expense of protein function. This is where the functional importance of the other 10 substitutions is revealed. When these 10 substitutions are added together with the 2 resistance-conferring ones, protein function is rescued.

​Thanks to the genetic signals left behind from a tension between molecular evolutionary forces (concerted evolution vs. natural selection), we were able to trace the functional changes underlying this incredible adaptation in frogs. The multidisciplinary talents of our global research team were essential to unravel this story, and highlight how diverse collaborations can better unravel biological stories.

by Shabnam Mohammadi