UM researchers John McCutcheon (top) and Toby Spribille, shown here examining lichen in Pattee Canyon near Missoula, made a discovery that added to our understanding of the forest-loving life form.

Overturning 150 Years of Science

UM lichen discovery rocks the research world

By Marina Richie

Who’s smarter? A lichen or us? We might have big brains, yet for 150 years these complex and mysterious life forms outsmarted scientists.

That changed in 2016. Two University of Montana researchers, working with collaborators scattered across the globe, toppled the long-standing idea that lichens are a product of one alga for photosynthesis and one fungus for structure. They found a second fungus that had escaped detection. The most successful of lichens, it turns out, are a threesome. Or, as The New York Times headline put it, “Two’s Company, Three’s a Lichen?”

Toby Spribille and John McCutcheon’s groundbreaking discovery made the July 29 cover of Science and was featured in The Washington Post and The Atlantic magazine, among many other media outlets.

The study of lichens helped form the foundation for a branch of biology called symbiosis — how different organisms cooperate to do things the individuals alone could not. Today, we know human health depends on symbiosis, from mitochondria powering our cells to gut bacteria aiding digestion. The new finding transformed how scientists perceive the lichen symbiosis.

“The people who have the hardest time understanding our work are lichen specialists, because they’ve spent so much time thinking that a lichen is defined by its single fungus,” says Spribille, a research associate in McCutcheon’s microbial genomics and symbiosis lab. “There is a huge historical inertia that says a lichen is made up of two things — one fungus and one alga — and it is often hard to overcome this. It was for us.”

He feels empathy for the Swiss botanist Simon Schwendener, whose 1867 concept that lichens consist of two partners was met with disbelief by esteemed scientists. True breakthroughs are rare and serve as reminders that taking a fresh view can swing the floodgates wide to an outpouring of more discoveries.

“We are starting to ask what organisms actually make a lichen, rather than being told what organisms are there,” McCutcheon says. “We are giving ourselves permission to go out in nature and look again.”

The duo experienced every scientist’s dream. Their finding was nowhere on the Internet. It was in no textbook. Maybe it’s no accident that one source of funding for the new lichen research comes from NASA, a grant funded through the space program to understand complex organisms in order to detect alien life on other planets. The Austrian Science Foundation also played a key funding role for the four-year study.

Ten lichen species are found in this photo of a ponderosa pine branch. Can you find them? (Lichen photos by Tim Wheeler, timwheelerphotography.com)

A ‘Eureka!’ Moment

When the two researchers discuss the lead-up to the breakthrough, they tell the whodunit tale with relish. Their complementary talents are another kind of symbiosis. Spribille studies the evolution of lichen symbiosis, a journey that’s taken him across North America and Eurasia, including completing a Ph.D. in 2011 from the University of Graz, Austria. He’s collected more than 40,000-plus specimens in the field over the past 20 years, starting as a U.S. Forest Service botanist in northwest Montana.

McCutcheon is a symbiosis whiz, known for revealing such microscopic bizarreness as bacteria living within bacteria inside a sap-sucking insect. Both are fascinated by what McCutcheon jokingly refers to as “weird biology.”

Lichens so far have not been assembled in the lab. Scientists have tried for years but could never get a single fungus and a single alga to make the complex structures seen in lichens. In addition to this problem, Spribille had noted other findings related to lichens that didn’t seem to add up. So Spribille and McCutcheon teamed up to ask a simple question about lichens that live close to the UM campus in the ponderosa pine, Douglas fir and larch forests of Pattee Canyon. Their initial research started with a small incubation grant from the University.

One lichen is brown and looks like a beard hanging off a tree limb. It’s known as the edible horsehair lichen (Bryoria fremontii), or wila, a traditional Native American food for making pemmican cakes. The second lichen is yellowish brown and called inedible horsehair lichen (Bryoria tortuosa). Its color marks it as poisonous. The two species make excellent lichens for study because they don’t have to be attached to a surface to live and can be snipped off easily for lab analysis.

When scientists from Finland and Canada previously examined the two lichens, they found no difference in the genetic makeup. So how could one be poisonous and the other not? Spribille brought the question to McCutcheon.

“I told Toby, ‘I guarantee you, this is an example of a classic microbiological problem,’” says McCutcheon. The two suspected the methods used by their Finnish and Canadian colleagues were simply too coarse to detect fine differences. They were confident that looking at whole genomes would reveal the answer. McCutcheon adds, “I was pretty sure what the answer would be. But as it turned out, I was very wrong.”

The two proceeded to sequence 4,000 to 9,000 genes for the alga and fungus of both lichens rather than relying on the four sampled genes of each that are typically used as molecular “bar codes” for species. The prior analysis was correct: McCutcheon was mistaken. No genetic differences existed.

That first finding led them to the next question. Is it a difference in gene expression? Perhaps, they thought. Environmental conditions can cause a single species to produce toxins in some situations but not others. That analysis likewise came up with a resounding answer: No.

There was a third possibility. In Spribille’s initial analysis, he had found a second fungus that he’d eliminated as a contaminant. He assumed a rogue fungus must have fallen upon lichens outdoors, which could be expected.

“I would do that differently now,” Spribille reflects. “I was looking for two partners, because that is what we had been primed to expect. I didn’t pay much attention to the second fungus and didn’t even bother to look to see if it was the same fungus in various places.”

McCutcheon sees it another way. “That’s the point. Sometimes in research, you have a finding that’s so confusing you just have to let it sit for a few years, and you loop back to it later.”

They re-ran their analysis to see if the genes of the new fungus correlated with the toxic differences of the two lichens.

“There it was,” Spribille says. “There was a clear pattern. It was all in the second fungus. Whenever the lichen produced this toxin, it had 12 times more of this additional fungus.”

The implications of this were enormous, because it suggested that lichen science had been missing something all along. What seemed like a solid result for the horsehair lichens was only the beginning of showing a wider pattern.

“Having gotten this far, I realized that we really had our work cut out for us to find out if this was a wider thing” Spribille says. Only after sampling many different fresh lichens from around the world to sequence for a second fungus did the broader pattern begin to gel.

“This was the eureka moment if there was one,” Spribille says. “It’s not a contaminant. Most lichens have their own second fungus. We’re pulling an organism out of lichens, with almost 100 percent success, that nobody knew existed.”

Spribille has collected more than 40,000 specimens in the field over the past 20 years.

It’s All About Fresh Eyes

At this point they had only ever “seen” this second fungus in DNA sequence. “We had to see the cells in the lichen or no one would believe us,” McCutcheon says.

It turns out that in some cases, the second fungus interlaces with the known fungus in the outer cortex (a waxy outer layer that coats the outside of the lichen) and is hard to tease apart. In others, including the two horsehair lichens, the newly found fungus is the cortex itself.

How could such an obvious fungus be so overlooked? Spribille explained by quickly sketching a classic cross section of a lichen that depicts a stringy fungus layer topped with a middle layer full of colored circles representing alga, and then a third outer (cortex) fungus layer like a line of beads.

“If you were to ask a small child, how many kinds of things do you see, they would tell you three,” Spribille says of the sketch.

It’s all about fresh eyes and a willingness to question what’s presented at face value as correct. If instructed to see one fungus expressing itself in two ways, instead of two unique fungi, most people see what they are taught. Spribille noted that when he leads field trips, the most intriguing questions come from people who haven’t yet been trained to think any one particular way about lichens.

The next step in their work is to delve into the symbiotic role of the second fungus in a host of other lichen species. Spribille believes lichens can help unlock the secrets of communication between eukaryotic cells (those cells with membranes surrounding a nucleus and other organelles).

Another benefit of lichen research is their proximity in nature. Spribille and McCutcheon enjoy stepping out into the forest and savoring the up-close wonder of lichens in all their diversity. A typical Montana watershed, valley bottom to mountain top, may harbor as many as 1,000 lichen species, compared to a dozen or so tree species.

“Lichens are one of the most visible and complex symbioses on Earth,” says Spribille. “Studying this system potentially gives us a window on how eukaryotic cells talk to each other.”

“After this work, the world looks different to me now,” says McCutcheon, “and that’s pretty exciting.” •

The powdered sunshine lichen (Vulpicida pinastri) displays the vibrant yellows of vulpinic acid. (Photo by Tim Wheeler, a graduate student in John McCutcheon’s UM lab)

Likin’ Lichens

Like space exploration looking for alien life, a journey into the world of lichens offers a dizzying array of outlandish shapes. Lichens can be as flat as sandpaper, as long as a wizard’s beard or as crinkled and fantastical as ocean coral. Some hug rocks; others drip from tree branches and trunks. They might be a bright shade of orange or yellow, or a subtle black, gray or tan. An individual lichen may be more than 100 years old. The evolution of lichens traces back some 400 million years, placing them as one of the first land-dwelling organisms.

Lichens self-assemble to form a complex organism able to survive droughts, revive in rain and repel predators. Each one represents a cooperative partnership of an alga and a fungus. Many, it turns out, also harbor a second fungus. The fungi form protective homes for the water-requiring algae, so lichens can thrive in dry climates. The algae, in turn, employ photosynthesis to convert carbon dioxide in the atmosphere into oxygen we breathe. Lichens absorb what’s in the air, including pollutants, which convey to scientists the health of the Earth. More than 20,000 identified lichen species add to our planet’s tremendous diversity of life. Arctic tundra and cold northern forests host some of the highest diversity.

Three strategies ensure lichens keep going on this planet. They may break off pieces that spread and grow into new lichens. Or a few algal cells surrounded by fungal cells disperse. Fungi also can grow fruiting bodies that produce spores and find the right algal partner to form a new lichen. •

Moss or Lichen?

A moss is a plant with parts that work like leaves, stems and roots and contains chloroplasts for photosynthesis. A lichen is a plant-like organism that lacks roots, stems or leaves. The algae provide chloroplasts for photosynthesis. Mosses and lichens are often found growing close together, sharing similar habitats. Lichens benefit from mosses that are like sponges, retaining water. •