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Insects Conquered a Watery Realm With Just TwoNew GenesMinor genetic changes can have big evolutionary consequences. When a gene duplication gave somewater striders a novel leg part, it opened up a new world for them.

By Viviane Callier

Courtesy of Dr E. Santos and Dr A. Khila

This delicate fanlike organ is a unique addition to the legs of one group of water striders.

Ever since Darwin articulated his theory of natural selection, the question of evolutionary novelties

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has intrigued biologists. It’s relatively easy to understand how natural selection can reshape anexisting trait — to make antlers bigger, legs longer or wings more colorful. But sometimes a fullyformed trait appears seemingly out of the blue, without any apparent antecedent. Where did it comefrom?

Part of the answer can be found in a new study appearing today in Science that shows how thesudden emergence of just one or two new genes can profoundly transform organisms’ appearance,behavior and ecological niche. Using developmental genetics, evolutionary analysis, biomechanicsand ecology, the researchers paint a picture of how a vital novelty evolved within one group ofaquatic insects. But the significance of the discovery as a model for evolutionary innovation couldextend throughout the animal kingdom.

“People have shown with comparative genomics that novel genes can be involved in novelstructures. But this is the first time, to my knowledge, that the direct link is established from a novelgene to a novel structure to the invasion of a completely new ecological opportunity,” saidAbderrahman Khila, an evolutionary and developmental genomicist at the Institute of FunctionalGenomics of Lyon, who led the study on the delicate insects called water striders.

Carnegie Museum of Natural History (left); Dr E. Santos and Dr A. Khila (right)

A water strider of the genus Rhagovelia (left) has a specialized structure on its middle pair of legs that waterstriders from other groups lack. This novel feature enhances the ability of Rhagovelia to navigate across fast-moving water (right).

Water striders are adept at navigating the surface of still water; they glide across ponds and lakesaround the world. Those in the tropical genus Rhagovelia, however, have also figured out how to

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walk across fast-flowing streams and turbulent whitewater. Their secret asset is a special extendablestructure on their middle leg resembling a Japanese fan, which no other water striders have. Bydeploying the fan, the insects can increase their leg’s contact with the water surface and can pushagainst the water more forcefully. It’s a clever adaptation, but how did Rhagovelia acquire it when ithas no precursors in other water striders?

To understand where the leg fan came from, Khila and his postdoctoral fellow Emília Santos, alongwith a couple of students, first had to figure out how to rear the water striders in the lab — a non-trivial task with what turned out to be a finicky species. It took about three years to figure out howto maintain the insects throughout the entire life cycle, from egg to adult. Once the colony wasestablished, the team was ready for experiments.

Khila and Santos ground up the developing legs of the Rhagovelia water striders and sequenced thetranscriptome — the complete suite of genes active in those tissues. The fan is only present on thesecond pair of legs, so the researchers compared the gene activity in the second pair to that of thefirst and third pairs. They discovered about 80-90 genes that were overexpressed only in the secondlegs.

Isabelle Cordeiro

Abderrahman Khila, an evolutionary and developmental genomicist at the Institute of Functional Genomics of Lyon,studies how genomic changes in water striders correspond to their evolution of new capabilities. Here he is showndoing field work in Belem, Brazil.

Next, they used a method called in situ hybridization to pin down where in the leg those 80-90 geneswere active. Khila and Santos identified five genes from that group that were expressed specifically

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in the tip of the second leg, where the fan develops. Three of the five were related to the structure ofthe cuticle, the protective outer layer of the insect’s exoskeleton. The other two genes appeared tobe paralogs — genes that are the result of a duplication event in the DNA. The function of theparalogs was unknown.

In search of clues about the function of the two paralogs, the researchers looked for the genesacross many water strider species. By looking at the evolutionary history of genes in the lineage ofwater striders, the researchers uncovered the ancestral copy of the gene (distinguished from themore recent duplicate) and nailed down the moment in evolutionary time when the duplicateappeared: at the origin of the Rhagovelia genus. Only water striders in the Rhagovelia genus havethe duplicate gene, and they are also the only ones to have a leg fan.

“The evolution of the fan coincides with the duplication of that gene,” Khila said, also noting that theexpression of the gene in the tip of the leg is significant. “It’s a big smoking gun.”

Because the leg fan reminded the researchers of a Japanese fan, they decided to call the newer genegeisha and its ancestral version mother of geisha. To learn what the genes were doing, they used amethod called RNA interference to “knock down” (or turn off) the expression of those genes.Knockdown of geisha and mother of geisha caused the resulting insects to make only small,rudimentary fans. (Because of the strong sequence similarities between the two genes, the scientistshave not yet been able to turn off one without the other, so differences between their functions arestill unclear.)

Dr E. Santos and Dr A. Khila

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The second leg of a Stridulivelia water strider (left) ends in a simple claw, while that of a Rhagovelia water strideralso carries an extensible fanlike structure. The fan improves the ability of Rhagovelia to push against the surfacetension of water.

Khila and Santos then challenged the insects in the water to figure out what the fans were doing forthem. They compared four groups: a closely related genus of water strider called Stridulivelia thatdoes not have fans, untreated Rhagovelia, Rhagovelia in which the fan was surgically removed, andRhagovelia in which geisha and mother of geisha were knocked down by RNA interference.

The researchers observed how the insects coped with two different watery environments. In calmwater, the fan did not appear to provide a speed advantage in locomotion, although the waterstriders with fans could go farther with each stroke of the leg, which meant they could paddle moreslowly. “So they can go just as fast without working as hard,” Khila said.

In the challenging environment of flowing water, however, the fans provided a crucial advantage:They enabled the insects to move upstream, while those without fans were swept away by thecurrent. The Rhagovelia with knocked-down genes, which had developed only rudimentary fans,could cope with slowly moving water but failed at higher speeds. In short, the fans — even in aprimitive form — were critical for enabling Rhagovelia species to navigate across running water,where closely related species could not.

Khila said it’s rare for experiments to be able to bring together biomechanics, transcriptomics,evolutionary biology, ecology and developmental genetics, but that combination is what’s needed toprovide a complete answer to how phenotypic evolution occurs.

“The integrative, interdisciplinary nature of this demonstration is what’s striking here,” said EhabAbouheif, an evolutionary developmental biologist at McGill University. (Abouheif was not involvedin the study but was Khila’s postdoctoral adviser between 2006 and 2011.) According to Abouheif,Khila didn’t just show that this new gene makes the water striders’ fan; he also “reconstructs itsevolutionary history, and then does the trademark ecological tests to show that it has an ecologicalfunction.”

“This study is really impressive,” agreed Greg Wray, an evolutionary developmental biologist atDuke University. “Usually something like this gets assembled over a period of time, maybe bydifferent groups, and then there’s one last piece that links them together. It’s impressive to be ableto go basically across the whole arc of the project in one go like this.

“The other thing I think is interesting is that there are not that many cases where people have beenable to use gene expression, absent any genetics, to zoom in so precisely on a genetic change thatcauses an obvious trait,” he added. “So that’s pretty impressive as well.”

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Mélisandre Tefit

Emília Santos, a postdoctoral fellow in Khila’s laboratory, worked with him on identifying the genes responsible forRhagovelia’s leg fan and superior control on moving water.

Of course, the genes geisha and mother of geisha don’t act in isolation. Khila and his team are nowtrying to find out what other genes interact with them, so as to uncover the full developmentalgenetic network that gives rise to the fan structure. Wray suspects that geisha and mother of geishaare co-opting a gene network that has a different purpose. “It’s really hard to imagine that these twogenes by themselves alone are making the structures,” he said. “I’m going to guess that they arepulling in some other genes that have other roles in development.”

That said, biologists familiar with the study also find it interesting that geisha and mother of geishaare novel genes that don’t have a known role in development. “So far, the literature [aboutevolutionary novelties] is dominated by examples of co-option and repurposing of ‘old’ genes andpathways into ‘new’ contexts,” said Armin Moczek, an evolutionary developmental biologist atIndiana University, in an email.

“From butterfly-wing spots to beetles’ horns to numerous other examples, the image that emerges isthat of organisms as Lego creations, as the modified reassemblies of the same and seemingly verylimited pool of genes, developmental pathways and morphogenetic processes,” Moczek continued.“We now expect to see novelty made possible through co-option. What this paper shows is that newgenes continue to matter.” Even between closely related species, he said, the sudden emergence of

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new genes can help to facilitate “ecologically significant innovation.”

Ian Dworkin, a geneticist who studies fly evolution at McMaster University, noted that in theliterature of evo-devo research — which looks at the role of developmental mechanisms inevolutionary changes — examples of co-opted genes are abundant. What is unclear is whether thatabundance reflects the frequency with which co-option occurs in nature or whether it is the result ofascertainment bias (systematic sampling errors introduced by how biologists study evo-devo). “It isnice to see a cool innovation that implicates a novel gene — it’s not simply a co-option story,” hesaid. “That’s why this is really exciting.”

Dworkin wonders whether inserting and activating geisha and mother of geisha in Stridulivelia, thefan-less sister genus of Rhagovelia, would be sufficient to trigger the development of the leg fan.Although the experiment would be very difficult to pull off in a non-model organism, “that would bethe real kicker to demonstrate that that is the key gene for it,” he said.

Evolutionary scientists often get stuck in a rut by thinking of the co-option of old genes or theemergence of novel ones as exclusive alternatives, Abouheif said. But this study is a reminder thatevolution can be a mix of both, through the recycling of the old and the addition of the new intonetworks that create novelty. The question for researchers to ponder, he said, is: “How do thesenetworks get assembled?”

This article was reprinted on ScientificAmerican.com.