How repeatable is evolutionary history?

Scientists have identified a ‘weakness’ in the clover genome that biases species to evolve the same trait

Evolutionary biologist Ken Olsen
and Cynthia Vigueira, then a postdoctoral associate in the Olsen lab,
examine white clover in the Washington University greenhouse. One morph
of this species of clover releases cyanide gas to discourage nibbling.
(Credit: Genevieve Hay )

Writing about the weird soft-bodied fossils found in the Burgess
Shale in the Canadian Rockies, paleontologist Stephen Jay Gould noted
that of 25 initial body plans exhibited by the fossils, all but four quickly became extinct. If we rewound the tape, he asked, and cast the
dice once more, would the same four body plans be the ones that survived? He thought
it unlikely.

We can’t repeat the Burgess Shale experiment, but Washington
University in St. Louis biologist Ken Olsen, PhD, says there are other
ways to ask whether evolution is repeatable. One is to look at related
species that have independently evolved the same traits and ask if the
same genes are responsible and, if so, whether the same mutations led to
the trait.

Looking at 27 species in the genus Trifolium (clovers),
Olsen, an associate professor of biology in Arts & Sciences, showed that six of them
displayed what is called a balanced polymorphism.

In some environments,
natural selection favors plants that release hydrogen cyanide to
discourage nibbling, while in others, plants that do not release cyanide
are favored. The polymorphism evolved independently in each of the six

Often, we think of evolution as driven by chance mistakes in DNA
replication, some of which produce novel traits. But in this case,
chance played little part. The clover species are in a sense predisposed
to develop this trait.

“We see exactly the same genetic mechanism — and it’s kind of a weird
mechanism — underlying the repeated evolution of the acyanogenic
(cyanide-less) trait in different clover species,” Olsen said.

The plants that don’t make cyanide have deletions in their genomes in
the spots where the required genes would normally be found. It’s not
that the gene is mutated; it’s missing entirely.

“This is interesting,” he said, “because it gets at the question of
how constrained evolution is. The more it is constrained, the more
predictable it is, but also the less adaptive flexibility there is.”

“If you look at life on the planet, there’s such an incredible
diversity of life forms and traits that we tend to think anything goes,”
Olsen said. “But when we look more carefully, we see there are
constraints. There aren’t any living species of limbed vertebrates with
six toes, for example; it’s five toes or fewer.”

The work appears in a special issue of Philosophical Transactions of
the Royal Society B published online June 23. The issue honors the
scientific contributions of Leslie D. Gottlieb, an early advocate of the
use of biochemical and molecular data to study plant evolution.

The cyanide bomb

Scientists have known that some
forms of white clover release hydrogen cyanide for more than a century.
They also quickly realized that white clover is polymorphic for the
trait, meaning the species includes both cyanogenic and acyanogenic

This polymorphism has been the subject of a large number of studies
to determine both the distribution of the two morphs and the nature of
the selective forces responsible for maintaining the polymorphism.

Plants that release cyanide have a two-chemical “cyanide bomb” that
is activated only when plant cells are crushed and the chemicals come in

Stored in the central vacuole of the plant cells are
cyanogenic glucosides: sugar molecules with an attached cyanide group.
In the plant cell’s wall, an enzyme called linamarase can cleave the
bond attaching the cyanide to the sugar.

When a slug, snail or chewing insect — the major predators on clover
in the seedling lifestage — crushes the tender cells, the enzyme cleaves
the cyanide, which combines with hydrogen to form gaseous hydrogen

Many plants have this ability. It’s what makes peach pits and apple
seeds poisonous, for example. “In the case of clover, cyanide release
probably doesn’t kill herbivores outright,” Olsen said. “It’s more
likely to just taste terrible — so it serves as a feeding deterrent.
The level of cyanide released is much higher in other species, such as
birdsfoot trefoil (the yellow-flowered plant that blooms along highways
in June). In that case, it probably could kill them.”

The clover genus Trifolium is surprisingly varied. Of the clover species shown here, T. repens (bottom right), commonly known as white clover, and T. isthmocarpum
(middle left), a salt-tolerant species known as Moroccan clover,
include both cyanide-producing and cyanide-less plants, although some of
the other species have one of the two genes needed to synthesize
cyanide. (Credit: Ken Olsen)

But white clover plants that make cyanide don’t grow everywhere.
You’re much more likely to find them in warmer climates than in cold
ones. In New Orleans, for example, 85 percent of the white clover
plants growing in lawns might be cyanogenic, while in Wisconsin, only 10
percent might be.

A working hypothesis, Olsen said, is that in cooler climates, there
are fewer herbivores around. “Since making these compounds is
energetically expensive, plants that don’t spend their resources making
them have a competitive advantage in the cooler climates.”

Just press DELETE

Making the bomb requires two
genes that are located in different parts of the clover genome. One of
these genes controls the synthesis of the cyanogenic glucosides, and the
other encodes the linamarase protein.

What happens when a cyanide-less morph pops up, Olsen said, is that
one of these genes is deleted. “We see independent gene deletions
occurring repeatedly in multiple species. So lots and lots of gene

This is not the “normal” way we think of adaptive variation
occurring, Olsen said. Most of the time, random mutational changes
affect one or a few nucleotides within one gene, which might convert one
amino acid to another, which might alter a protein’s function. So the
changes are random and incremental. Instead, in this case, the entire
gene disappears.

In the clover genus, something is making it easier for adaptive
variation to arise through gene deletions than through simple mutations,
Olsen said.

He thinks that “something” might be repetitive nucleotide sequences
(repeats) near the cyanide bomb genes. In that case, chromosomes align
to the “wrong” repeat when they pair during meiosis and swap genetic
material. Unequal swaps caused by the misalignment can delete or add on
extra chunks of DNA within the chromosome.

“Normally, a deletion like this would be detrimental,” Olsen said.
“But when these genes are deleted, the plant is favored in certain
environments, and so this morph is maintained. That’s why we see this
polymorphism so often in natural populations.”

It’s not that evolution, restarted, would repeat itself exactly,
Olsen said. But the closer the evolutionary relationship between
species, the more likely there will be underlying predispositions that
make the same traits pop up repeatedly in the same way.

In some ways, these predispositions are analogous to the crease
patterns in origami paper that make it easier to fold the paper into
some shapes than others. Evolution can fold across a crease — but it is
much easier to fold along one.