Hundreds of millions of years ago, a tiny green microbe joined forces with a fungus, and together they conquered the world. It's a tale of two cross-kingdom organisms, one providing food and the one other shelter, and it's been our touchstone example of symbiosis for 150 years. The trouble is, that story is nowhere near complete. All Images: Tim Wheeler
A sweeping genetic analysis of lichen has revealed a third symbiotic organism, hiding in plain sight alongside the familiar two, that has eluded scientists for decades. The stowaway is another fungus, a basidiomycete yeast. It's been found in 52 genera of lichen across six continents, indicating that it is an extremely widespread, if not ubiquitous, part of the symbiosis. And according to molecular dating, it's probably been along for the ride since the beginning.
"I think this will require some rewriting of the textbooks," said Catharine Aime, a mycologist at Purdue University and co-author on the study published today in Science.
Toby Spribille, who led the new analysis, has been studying lichens in one way or another for most of his life. He grew up in northwest Montana, where the shrubby, rubbery organisms are a ubiquitous part of the natural landscape. But when Spribille started to get serious about lichen research in grad school, he hit a roadblock.
"Lichens are nearly impossible to re-synthesise in the lab," he told Gizmodo, explaining how the colonies take a long time to grow and the conditions needed to induce symbiosis are not well known. Unable to rear their test subjects in controlled environments, lichen researchers have struggled to perform basic experiments that could shed light the roles of the different symbionts. ***
“I think this will require some rewriting of the textbooks.”
*** But recent advances in metagenomics — tools for extracting and sequencing DNA from environmental samples, no culturing required — offer a new way in. This approach caught Spribille's attention when he learned something very strange about Bryoria, a lichen found throughout conifer forests of the western United States and Canada.
"Bryoria have a long and storied cultural significance," Spribille said, explaining how certain Native American tribes relied on the lichen as winter survival food. "There's also evidence that first peoples would remove the more light coloured ones and wash them, so that certain substances wouldn't make them sick."
Those substances include a toxin called vulpinic acid. The lichen that produces it, Bryoria tortuosa, can be distinguished from its non-toxic cousin, Bryoria fremontii, on the basis of its yellowish hue. But a few years back, when a group of biologists at the University of Helsinki tried uncover the genetic basis for this difference using a targeted approach called barcoding, they were stumped.
"They found that the toxic and non-toxic forms [of the two species] were identical — at least, in the known parts of the lichen," Spribille said. "And they didn't really study it further. We looked at that and said, this is a classic question you could go at with genomics."
Parmeliopsis hyperopta ambigua
When Spribille and his colleagues analysed Bryoria's RNA — the messenger form of DNA — they discovered something amazing. "We found there was this third thing, riding along in every single sample," he said, referring to the previously unknown basidiomycete.
At first, the researchers worried that the extra RNA sequences could be contamination, a common pitfall of genomic research. And so, they decided to see if they could find the basidiomycete in other lichens, too. "We found it in everything," Spribille said. "From Alaska to Ethiopia to Antarctica, it always was there."
The final proof that this was not an elaborate hoax came when the researchers developed green fluorescent markers that attach to specific RNA sequences in the basidiomycete, and blue markers that attach to complementary RNA sequences in the other fungus, an ascomycete. Sure enough, when they added these markers to samples of lichen tissue, the cells of a hidden fungal partner glowed under the microscope.
"This is an exciting discovery that forces us to reconsider what we thought we knew about lichens," said Kathleen Treseder, a fungal ecologist at the University of California Irvine who was not involved with the study. "It would not have been possible without recent technological advances in how we study fungi."
We can't be certain the second fungus is present in all lichens. Spribille's study only looked at lichens in the Parmeliaceae family, the most widespread and successful group on Earth today. But the entire lineage is vast and ancient, and it's possible some groups split off on the evolutionary tree before the basidiomycete arrived on the scene.
We also can't be sure when that third partner joined up. Using an age-estimation method called molecular clock dating, the researchers showed that the basidiomycete lineage is as old as the ascomycete lineage. "By inference, these two lineages arose at the same point in time," Aime said. But we'd need well-preserved fossils to build a case that all three lichen partners have been together since the very beginning.
Aime and her collaborators are now studying the new fungus more closely. The classic view of lichen is that the photosynthetic organism (an algae or a cyanobacteria, occasionally both) provides food, while the ascomycete fungus offers shelter and structure.
The recent studies on Bryoria and vulpinic acid hint at a likely role for the newcomer: Defence. "We can't prove the connection of the yeast to the toxin, but the evidence points overwhelmingly to it being involved in some way," Spribille said. ***
“One thing that sets lichen apart from all other symbioses is that all the components are microbes. But when they come together, they form something self-replicating and beautiful that you can hold in your hand.”
*** "Basidiomycetes have really cool metabolisms," Aime said, describing how these fungi produce all sorts of toxic defence compounds, called secondary metabolites. "It's likely they're producing a lot of the metabolites we've used to diagnose lichens in the past."
Spribille noted that the location of the new fungus, inside a layer of starch that gives lichen its rubbery toughness, suggests it may also play a structural role. Clearly, more research is needed to sort out all the ways this newcomer fits into our picture of lichen — but serendipitously, its discovery could make future studies easier. "One reason we think it may have been so hard to culture lichen is that we were missing a key ingredient," Spribille said.
The nuances of lichen ecology aside, Spribille's research underscores the incredible complexity of the microbial world which is now being revealed through genomics. "One thing that sets lichen apart from all other symbioses is that all the components are microbes," Spribille said. "But when they come together, they form something self-replicating and beautiful that you can hold in your hand. For me, that's something to draw inspiration from."