The next time you slather mustard on your hotdog or horseradish on your bun, thank caterpillars and brassica for that extra flavor.

While these condiments might be tasty to you, the mustard oils that create their flavors are the result of millions of years of plants playing defense against pests. But at the same time, clever insects like cabbage butterflies worked to counter these defenses, which then started an arms race between the plants and insects.

An international research team led by University of Missouri Bond Life Sciences Center researchers recently gained insight into a genetic basis for this co-evolution between butterflies and plants in Brassicales, an order of plants in the mustard family that includes cabbage, broccoli and kale.

Chris Pires | Image by Roger Meissen, Bond LSC

Chris Pires | Image by Roger Meissen, Bond LSC

The team published these new insights online in Proceedings of the National Academy of Sciences (PNAS) in June.

“We found the genetic evidence for an arms race between plants like mustards, cabbage and broccoli and insects like cabbage butterflies,” said Chris Pires, an MU Bond Life Sciences Center researcher and associate professor of biological sciences in the College of Arts and Sciences. “These plants duplicated their genome and those multiple copies of genes evolved new traits like these chemical defenses and then cabbage butterflies responded by evolving new ways to fight against them.”

A biting taste

While you might like the zing in mustard, insects don’t.

Compounds, called glucosinolates, create these sharp flavors in plants to defend against caterpillars, butterflies and other pests. Brassicales species first evolved glucosinolate defenses around the KT Boundary — when dinosaurs went extinct — and eventually diversified to synthesize more than 120 different types of this compound.

For most insects, these glucosinolates prove toxic, but certain ones like the cabbage butterfly evolved ways to detoxify the compounds.

“Seeing the variation in the detoxification mechanisms among species and their gene copies gave us important evolutionary insights,” said Hanna Heidel-Fischer, a lead author on the study based at the Max Plank Institute for Chemical Ecology in Germany.

To look at these genetic differences, the team used 9 existing Brassicales genomes and also generated transcriptomes — the set of all RNA in a cell — across 14 Brassicales families. This allowed the team to map an evolutionary family tree of sorts over the millennia, seeing where major defense changes occurred. This family tree was compared with the family tree of 9 key species of Pieridae butterflies, which includes the cabbage butterfly.

Pires and his colleagues identified three significant evolutionary waves over 80 million years, where plants developed defenses and insects evolved counter tactics.

Pat Edger | Image by Roger Meissen, Bond LSC

Pat Edger | Image by Roger Meissen, Bond LSC

“We found that the origin of brand-new chemicals in the plant arose through gene duplications that encode novel functions rather than single mutations,” said Pat Edger, a former MU post doc and lead author on the study. “Given sufficient amounts of time the insects repeatedly developed counter defenses and adaptations to these new plant defenses.”

This back-and-forth pressure resulted in the evolution of many more species of plants and butterflies than in other groups without glucosinolate pressures.

Proving an old concept

Co-evolution is not a new idea.

About 50 years ago two now-renowned biologists, Peter Raven and Paul Erhlich, introduced the idea of co-evolution to science. Using cabbage butterflies and Brassica plants as a prime example, the two published a landmark study in 1964 advancing the idea that two species can mutually influence the development and evolution of each other.

To explore the genetics of how this works, Pires’ lab partnered with Chris Wheat, professor of population genetics in the Department of Zoology at Stockholm University.

“Using Ehrlich and Raven’s principles and models, we looked at the evolutionary histories of these plants and butterflies side-by-side and discovered that major advances in the chemical defenses of the plants were followed by butterflies evolving counter-tactics that allowed them to keep eating these plants,” Wheat said.

Chris Pires and colleagues mapped the evolution of Brassicales and butterflies to find how each evolved to combat the defenses of the other. | Courtesy Chris Pires

Chris Pires and colleagues mapped the evolution of Brassicales and butterflies to find how each evolved to combat the defenses of the other. | Courtesy Chris Pires

This research provides striking support for the ideas of Ehrlich and Raven published 50 years ago.

“We looked at the patterns 50 years ago, and found conclusions that proved correct,” said Peter Raven, professor emeritus of the Missouri Botanical Garden and a former University of Missouri Curator. “The wonderful array of molecular and other analytical tools applied now under leadership of people like Chris Pires, provide verification and new insights that couldn’t even have been imagined then.”

Understanding more about how plants and insects co-evolve could one day lead to advances in crops.

“If we can harness the power of genetics and determine what causes these copies of genes, we could produce plants that are more pest-resistant to insects that are co-evolving with them—it could open different avenues for creating plants and food that are more efficiently grown,” said Pires.

Proceedings of the National Academy of Sciences (PNAS) published the study, “The butterfly plant arms-race escalated by gene and genome duplications,” in June. The National Science Foundation (PGRP 1202793), the Knut and Alice Wallenberg Foundation and the Academy of Finland provided the funding for this research.