So it turns out to be a threesome

Lichens are amazing organisms, drawing out our curiosity both because of their aesthetic appeal and their novel ecological and evolutionary characteristics. For over a hundred years, biologists have supposedly known about their basic composition: they are one of nature’s best examples of a symbiosis, a tight coupling of two or more evolutionarily distinct organisms that can function like a single unit. In the case of lichens, the two coupled organisms are a fungus and a photosynthetic alga.

Encrusting lichens at Newberry National Monument, Deschutes Co., OR, June 2011.

That basic understanding of lichens as an evolutionary marriage between alga and fungus remains fact. However, a paper published this summer significantly expanded the overall picture. Lead author Toby Spribille and his colleagues provide evidence in the journal Science that many lichens are symbioses of not just two groups of organisms, but three! How could such a basic aspect of lichen biology go undetected for so long?

Letharia vulpina on tree bark at Diamond Peak
Wilderness, OR, May 2013.

Their surprising discovery came about by careful observation and testing of what might otherwise pass as a rather mundane question in lichen biology. The authors were investigating differences in two closely related species of lichen, Bryoria tortuosa and B. fremontii. The former species produces a compound called vulpinic acid that causes the lichen to have a yellowish color. B. fremontii, however, lacks (or has reduced production of) the acid and is brown in color. What had baffled researchers, however, was the failure to find genetic differences between the two supposed species. Both the ascomycete fungus and the photosynthetic partner (a green alga called Trebouxia simplex) in the two species had identical sequences when several genes were studied. Species are expected to have fixed genetic differences even if their phenotype (their appearance) is very similar.

Spribille and colleagues decided to study the genetic structure again of the two species by sequencing the mRNA transcriptome. They confirmed the genetic similarity that had been observed before and also found little difference in gene expression between B. tortuosa and B. fremontii. However, when the researchers broadened their search to consider whether the transcripts they sequenced might match any other types of fungi, they found that the acid-producing B. tortuosa produced sequences associated with basidiomycetes, a very different group of fungi than are typically seen in lichens. The evidence pointed to a third symbiont in B. tortousa!

Apothecia (spore-producing structures) on encrusting lichens. Left: Alpine bloodspot, Ophioparma ventosa, Deschutes National Forest, OR, May 2012. Right: Ochrolechia sp., Bear Valley, Tahoe National Forest, CA, Dec 2009.

Usnea longissima, hanging from a vine maple in the
Menagerie Wilderness, Linn Co., OR, Oct 2012.

With this new finding, the researchers asked whether other lichen species might potentially harbor basidiomycetes as well. Surveying a variety of other lichen lineages, they found basidiomycete sequences in 52 different genera distributed across six continents! The basidiomycete lineages in the lichens seemed to be diverse group of fungi, but they were associated with specific species. The new group of basidiomycete symbionts is called the Cyphobasidiales.

Up until now, it had been known that some lichens deviated from the classic model of two partners in the lichen symbiosis. For instance, some species have two algal hosts in addition to the fungal partner – both a green alga and a cyanobacterium (Henskens et al. 2012). However, Spribille et al.’s research suggests that the typical lichen association is made up of two distinct fungal lineages and one or two algae. Thus, lichens may typically be threesomes, and sometimes, even foursomes. As if this wasn’t complicated enough, DNA sequencing work by Bates et al. (2011) showed that lichens can also have bacterial communities associated with them. They found unique groups of alpha proteobacteria associated with the lichen body in 4 species they studied. Apparently the more that lichens are studied, the more we could describe these remarkable organisms as comprising their own little ecological worlds!

The lichen Cladonia in a residential yard, Humboldt
Co., CA, Dec 2009.

With the relative ubiquity of the third symbiont evident in sequence data, why haven’t previous researchers seen these basidiomycete symbionts when looking through the microscope at lichen specimens? It turns out that the cells are difficult to detect by microscopy. They are small, and on the periphery of the lichen body, being embedded in a matrix of polysaccharides. Using florescent molecular tags on rRNA sequences specific to the basidiomycete partner, however, the new cells and their location in the lichen thallus (body) became readily apparent.

Interestingly, the new results explain one of the mysteries that lichen biologists have confronted for a long time, namely that it has been difficult to recreate the lichen symbiosis in the lab by combining only a single fungal host and alga. The typical lichen shape was hard to reproduce with only two partners.

It is fascinating to ponder the evolutionary history that could lead to such a complex and intimate association of distinct organisms. Green algae, cyanobacteria, and fungi are not closely related. During the early evolution of lichens, how did algae first become associated with the body of fungi? Did early lichens start with two fungal partners plus an alga, or were today’s lichen ancestors more simple in composition?

Lichens covering a rock in coastal scrub near
Muir Beach, Marin Co., CA, Oct 2016.

Whatever the exact evolutionary sequence of events, the lichen symbiosis appears to have proved to be very successful in terms of survival and reproductive success. In fact, fossils suggest that associations of algae and fungi are at least a half billion years old, stretching back to about the time that invertebrate animals diversified in the Cambrian Explosion, and before the arrival of vascular plants on land (Yuan et al. 2005). Today, there are many thousands of species of lichens, inhabiting ecosystems as diverse as deserts, coniferous rainforests, and coastlines.

References

Bates ST, Cropsey GW, Caporaso JG, Knight R, Fierer N. 2011. Bacterial communities associated with the lichen symbiosis. Applied and Environmental Microbiology 77:1309-1314.

Brodo IM, Sharnoff SD, Sharnoff S. 2001. Lichens of North America. Yale University Press.

Henskens FL, Green TG, Wilkins A. 2012. Cyanolichens can have both cyanobacteria and green algae in a common layer as major contributors to photosynthesis. Annals of Botany 110:555-563.

Spribille T, Touvinen V, Resl P, et al. 2016. Basidiomycete yeasts in the cortex of ascomycete lichens. Science 353:488-492.

Yuan X, Xiao S, Taylor TN. 2005. Lichen-like symbiosis 600 million years ago. Science 308:1017-1020.

Lichen (perhaps Amandinea) on wood fence, Pt. Reyes National Seashore, CA, Nov 2008.

Fruticose lichen in redwood forest, Van Duzen County Park, Humboldt Co., CA, 2006.

Lichens: You scratch my back, I'll feed your hyphae

Letharia columbiana from California.

Winter in the Pacific Northwest is the season for lichens. Deciduous trees have lost their foliage, revealing a colorful “understory” of a diverse lichen flora that clings to the branches and trunks of trees. For these organisms, resources for growth seem to be abundant at this time of year: there is no shortage of moisture from rain and snow, and despite shorter days and abundant cloud cover, the absence of leaves probably means there is a fair amount of light that penetrates tree canopies.

Lichens are composite organisms. The bulk of the tissue consists of the body of a fungal host (the “mycobiont”). Fungi are composed of chains of cells known as hyphae. The other half of the lichen partnership is a photosynthetic alga or cyanobacterium (the “phycobiont” or “photobiont”). The cells of the photosynthetic partner are embedded within the body of the fungus. The fungus is responsible for the structure (shape) of the lichen body. About 90% of lichens are host to green algae, including the genera Trebouxia and Trentepohlia (Purvis 2000). The rest have cyanobacterial partners (or on rare occasions, both green algae and cyanobacteria). Cyanobacteria are oxygen-producing photosynthetic bacteria. Worldwide, there are about 14,000 species of lichens (Brodo et al. 2001).



In this fruticose lichen, it appears that small greenish areas where the photobiont may be present can be seen through the fungal tissues on the main axis of this specimen.

Lichens represent a classic example of a symbiosis: two organisms that live in close association, often in inter-dependency. In ecology, symbiosis is a broad term that encompasses a range of relationships between two or more partners – everything from parasitism (one partner benefits at the expense of another) to mutualism (both partners are positively affected by the association). One view of the lichen symbiosis is that it is a mutualism. For instance, a potential benefit to the algal partner by living with its fungal host may be amelioration of desiccation stress. Just as the fungal partner could allow the alga to thrive in very dry places (e.g., the surface of desert rocks) by providing a home for growth, the alga might allow the fungus to live in environments with less organic matter (again, think barren rocks) than would otherwise be tolerable because it provides food (Purvis 2000). Sugars produced by photosynthesis in the photobiont are incorporated into the fungal tissue (Smith et al. 1969). By making dry places hospitable for the alga or carbon-poor places hospitable for the fungus, the lichen symbioses is an interesting example of one organism expanding the realized niche space of another (Purvis 2000). 

However, the lichen lifestyle may not always be beneficial for the algae. Photobionts like the cyanobacterium Nostoc can make it just fine outside of the lichen association. Brodo et al. (2001) note that the lichen symbiosis actually represents a range of associations from ones in which the algal partner may not be appreciably harmed by the fungus to ones that might be better characterized as a prison for the phycobiont. In fact, as early as 1869, the Swiss botanist Schwendener suggested that lichen fungi may be parasitic on their fungal hosts (Purvis 2000).

I still have much to learn about lichen biology, but here are a few interesting points about the lichen symbiosis that I picked up scanning some research:

- Lichen symbioses have evolved multiple times during the course of fungal evolution (Gargas et al. 1995). So, picking up a photosynthetic partner seems to be an advantageous evolutionary strategy. Most lichen associations are formed with ascomycete fungi, but a few basidiomycetes (mushroom-forming fungi) form lichens as well (Lawrey et al. 2009).

- Lichen symbioses are sometimes more of an extended family gathering than merely a two member partnership. Some fungi have both green algae and cyanobacteria as symbionts, sometimes living in separate places within the fungal tissue, sometimes living in closer proximity (Brodo et al. 2001, Henskens et al. 2012). Moreover, diverse bacteria and even other endophytic fungi can be associated with the lichen microcosm (Arnold et al. 2009, Grube et al. 2009, Bates et al. 2011, Hodkinson et al. 2012). “Hey you, move over! It is getting crowded in here!”

- The presence of one partner may influence the physiology of the other. In a study of the lichen Cladonia, the lichen association caused up-regulation of genes involved in photoprotection and antioxidation pathways in the alga and fungus respectively (Kranner et al. 2005).

- In lichens where the algal partner as been identified down to the species level (only a small percentage thus far), individual fungal species generally only associate with a specific algal species. The alga, on the other hand, shows less fidelity – one species may appear in many fungal hosts (Brodo et al. 2001, Yahr et al. 2004).

- Reproduction of lichens is perhaps a little more complex than in other species because two organisms are involved (Brodo et al. 2001). One way that maintenance of the lichen association from generation to generation is achieved is by vertical “transmission”. In vertical transmission, the propagules of the mycobiont and phycobiont can be produced vegetatively (so parents and daughters are the same genetically) and they disperse together to new living quarters (DalGrande et al. 2012). However, a paper by Wornik and Grube (2010) suggests that young lichen fungi can pick up algae anew from the environment, even if they originally dispersed with a photobiont to begin with. 

In summary, these points emphasize just how complex and diverse the lichen symbiosis can be. There is tremendous variation in the expression of the symbiosis and in the identity and arrangement of the partners involved. I’d bet there is a lot of fascinating biology and ecological insight yet to be gained from studying lichens. If species numbers, geographic distribution and lifestyle diversity are valid measures of evolutionary “success”, then the lichen association has been successful indeed!

References
Arnold et al. 2009. Systematic Biology 58:283.
Bates et al. 2011 Applied and Environmental Microbiology 77:1309.
Brodo et al. 2001. Lichens of North America. Yale University Press.
DalGrande et al. 2012. Molecular Ecology 21:3159.
Gargas et al. 1995. Science 268:1492.
Grube et al. 2009. ISMEJ 3:1105.
Henskens et al. 2012 Annals of Botany 110:555.
Hodkinson et al. 2012. Environmental Microbiology 14:147.
Lawrey et al. 2009. Mycological Research 113:1154.
Kranner et al. 2005 Proc Natl Acad Sci USA
Purvis, W. 2000. Lichens. Smithsonian Institution Press.
Smith et al. 1969. Biological Reviews 44:17.
Wornik and Grube. 2010. Micobial Ecology 59:150.
Yahr et al. 2004. Molecular Ecology 13:3367.

Two crustose lichens. In the bottom photograph, the large disk-like structures are apothecia, sites where fungal spores are produced.