Two lichens were stubbornly stumping scientists. Genetic tests of Bryoria tortuosa and Bryoria fremontii could not show any reason they should be separate species; their DNA revealed no consistent differences. But in the real world, the stringy, lanky lichens that festoon conifers in the Pacific Northwest like Spanish moss are almost, but not quite, identical. One you can eat. And one can kill you.
There are a few other less momentous differences between the two lichens, called horsehair or treehair lichens. B. tortuosa is usually sallow green, while the B. fremontii is typically dark-chocolate brown, although there are color variations that overlap within both species. And B. tortuosa contains structures called pseudocyphellae – in this case, long yellow slits where the stuffing of the lichen pokes out of its skin. B. fremontii never has these, according to Lichens of North America, perhaps the definitive (and heaviest) text on the subject.
These differences were reflected in how the lichen was used by native peoples. They wove B. fremontii into clothing or baked it in earthen pits into a black gelatinous mass, cooking or serving it with wild onions, blue camas bulbs, fish eggs, or Saskatoon berries. B. tortuosa, on the other hand, was left untouched. That’s because it contains large amounts of a toxin called vulpinic acid, a chemical that gets its name from its use in other lichens (notably Letharia vulpina) as a fox or wolf poison.
Clearly, something must separate these two species, in spite of DNA evidence to the contrary, but what that might be remained ineffable.
Meanwhile, scientists were puzzling over another, larger mystery. Lichens have long been defined as a relationship between two (and sometimes three) partners: a fungus that builds the “house” and an alga living inside it that makes the food through photosynthesis, the same way that plants win their daily bread. The photosynthetic partner can be a green alga – an aquatic, early evolved green plant – or sometimes cyanobacteria, also called blue-green algae. In some lichens, both green algae and cyanobacteria are present (Interestingly, when this happens, they do not seem to get along. In lichens with both, the fungus builds a special compartment called a cephalodium for the blue-green alga to inhabit. They are not sister wives.)
The great mystery was this: when scientists took pure cultures of the two participants in a lichen – the fungus and the food-making partner -- and mixed them together in the lab, they could almost never get them to produce the delicate, frilly, exquisitely crafted lichens that encrust rocks, trees, and soil on nearly the world over. In nature, making these structures seems to be a snap. In the lab, scientists couldn’t seem to make the magic happen. No one knew why.
Now, it turns out the solution to both these mysteries may be one in the same.
In a new study published in July in the journal Science, scientists who set out to understand why B. tortuosa and B. fremontii behave so differently despite their apparent genetic uniformity describe a radical new idea: that the fungus that builds the lichen superstructure is not the only fungus that has a hand in creating it. It was not the idea they set out to test, however. Not by a long shot.
They had first hypothesized that the difference between the two lichens was simply that they were using the same genes differently, expressing some more and other less, and producing different amounts of proteins other biochemicals as a result.
They studied six specimens of B. fremontii and nine B. tortuosa all collected in western Montana. But when they examined what genes were being used by each lichen, they found little difference. That could have meant yet another dead end had they not decided to try something new.
They ran the analysis a second time, but this time they expanded the range of organisms whose genes they surveyed. Suddenly, out popped an anomaly: genes from a very distant group of fungi – the basidiomycetes – were being expressed in both lichens, but vastly more abundantly in B. tortuosa than B. fremontii. Oh my. There weren't supposed to be any of those in there.
Where Have You Been All My Life?
Basidiomycetes, as a group, are famous for making most of the structures we call mushrooms, and rarely form lichens of their own (only 50 of the 18,000 known lichens are basidiolichens). And no one had ever seen a structure remotely resembling a basidiomycete inside Bryoria. Was there really another distantly-related fungus inside there somewhere? If so, where? And how had it hidden from the diligent probings of lichenologists for hundreds of years?
First, the scientists cut cross sections of Bryoria and tried to see this new fungus with their own eyes. But no cells that were unquestionably basidiomycetes presented themselves. Usually, filamentous basidiomycetes are easy to distinguish because their cells form structures called “clamp connections” between each other. I assume they could not see anything like that. They then tried to culture the basidiomycetes -- that is, to get them to grow in a food-filled dish on their own. That effort failed too.
Finally, they tried making fluorescently-labeled proteins that would bind specifically to basidiomycete cells. At last they struck pay dirt. They observed clearly fluorescent, spherical, 3- to 4- micrometer wide cells embedded in the most exterior layer of the lichen. And they observed many more of these cells in B. tortuosa than in B. fremontii.
Now they expanded their analysis to see if they could find these same basidiomycete gene signatures in other lichens. Was this a phenomenon exclusive to Bryoria, or something much bigger and more important?
Using DNA-binding molecules that matched the basidiomycete signatures inside Bryoria, they screened other lichens that were growing right next to it in Montana forests. Each had their own characteristic but closely-related basidiomycete signal, including Letharia vulpina that grew intermixed with Bryoria.
What about outside Montana? They expanded their search to all lichens in the major group that Bryoria belongs too – the Lecanoromycetes, itself the largest group of lichen-making fungi on Earth. All together, they found sequences from the new basidiomycetes in 52 lichen genera from six continents. “As a whole, these data indicate that basidiomycete fungi are ubiquitous and global associates of the world’s most speciose radiation of macrolichens,” they wrote.
That means there's an organism inside lichens that's been hiding right in front of our eyes for centuries. What the heck are they? And just what are they doing in there?
Yeast, as I’ve discussed here before, is a way of life, not a taxonomic group. Yeast are fungi growing as single cells rather than as chains of cells in fuzzy filaments. Standard baker’s and brewer’s yeast are Saccharomyces cerevisiae, an ascomycete fungus. The fungi that form most lichens are also ascomycetes – but filamentous forms. But basidiomycetes can live as yeast as well, and this seems to be the case here.
The fact that the cells that glowed were solitary, had only one nucleus (a DNA storage compartment; filamentous fungi can often have multiple nuclei in the same cell), showed evidence of budding (how yeast cells reproduce), and lacked clamp connections all support the idea that the basidiomycetes hiding inside lichens are yeast. But because the cells are embedded in a layer of organic chemicals on the lichen’s surface, they remain invisible when scanning electron micrographs are taken of the lichen’s body, which explains how they hid so long.
In this video produced by Science magazine, you can see a 3D scan toward the end that shows the position of the yeast in green in relation to the fungus and its algal partner.
These lichen-dwelling basidiomycete yeast belong to a genus of obscure fungi called Cyphobasidium. The only two previously known species in this genus were just described in the last year, after they were discovered living in galls on lichens in Norway.
Galls are bit like tumors, so it is possible that these gall-forming fungi evolved from lichen-participating species that turned into pathogens. Or it’s possibly the other way around: the numerous lichen-forming species we’ve just discovered are descended from pathogens that evolved into a more harmonious relationship with their hosts.
In any case, the scientists used a molecular clock (based on assumptions about how frequently mutations occur in fungal genomes) to estimate how long ago the new group of lichen-dwelling yeasts they have dubbed the Cyphobasidiales evolved. According to their calculations, it happened around the same time that the lichens in which they live evolved, about 200 million years ago. If that is true, it implies that the partnership is as old as the lichens themselves, and makes it even more remarkable that no one has detected them until now.
The authors themselves speculate on the reasons for that. We have already discussed they they might have eluded detection in the microscope-only age. But what about the molecular age? The region of DNA that scientists usually use to detect basidiomycete fungi has an unusual piece of DNA inserted into it in most of these new Cyphobasidium species that may have made them hard to detect. It is also possible, they say, that they have been previously detected, but were discarded as contaminants.
Brewing Up a Lichen
The outermost layer of the lichen in which the yeast dwell was previously known to contain complex chemicals whose presence did not seem to always correlate with the fungus that had built the lichen. If the yeasts that live there make these chemicals, either directly or by inducing the lichen's structural fungus to make them, that would go a long way toward explaining this strange phenomenon.
Certainly, in B. tortuosa, the yeast seem to be the ones responsible for vulpinic acid production. Are the two lichens really one species, then, with individuals that fall along a spectrum of how much yeast they host? Or are they really still two species, one friendly to Cyphobasidium, and one more hostile? That question remains unanswered.
The discovery of this second fungus could also explain why building lichens in the lab has proven almost impossible so far. It will be interesting to see if adding a sprinkle of yeast to the lichen forming-brew leads to different results.
That one of the oldest and most well-studied symbioses on the planet contained a partner who escaped detection this long should be a humbling and exciting realization. Once again, life surprises. Once again, Earth shows us there remains so much left to discover right here in our own back yards.
Spribille, Toby, Veera Tuovinen, Philipp Resl, Dan Vanderpool, Heimo Wolinski, M. Catherine Aime, Kevin Schneider et al. "Basidiomycete yeasts in the cortex of ascomycete macrolichens." Science 353, no. 6298 (2016): 488-492.