The macroevolutionary dynamics of symbiotic and phenotypic diversification in lichens Matthew P. Nelsena,1, Robert Lückingb, C. Kevin Boycec, H. Thorsten Lumbscha, and Richard H. Reea aDepartment of Science and Education, Negaunee Integrative Research Center, The Field Museum, Chicago, IL 60605; bBotanischer Garten und Botanisches Museum, Freie Universität Berlin, 14195 Berlin, Germany; and cDepartment of Geological Sciences, Stanford University, Stanford, CA 94305 Edited by Joan E. Strassmann, Washington University in St. Louis, St. Louis, MO, and approved July 14, 2020 (received for review February 6, 2020) Symbioses are evolutionarily pervasive and play fundamental roles macroevolutionary consequences of ant–plant interactions (15–19). in structuring ecosystems, yet our understanding of their macroevo- However, insufficient attention has been paid to one of the most lutionary origins, persistence, and consequences is incomplete. We iconic examples of symbiosis (20, 21): Lichens. traced the macroevolutionary history of symbiotic and phenotypic Lichens are stable associations between a mycobiont (fungus) diversification in an iconic symbiosis, lichens. By inferring the most and photobiont (eukaryotic alga or cyanobacterium). The pho- comprehensive time-scaled phylogeny of lichen-forming fungi (LFF) tobiont supplies the heterotrophic fungus with photosynthetically to date (over 3,300 species), we identified shifts among symbiont derived carbohydrates, while the mycobiont provides the pho- classes that broadly coincided with the convergent evolution of phy- tobiont minerals, water, and a conducive growing environment in logenetically or functionally similar associations in diverse lineages the form of a thallus. Collectively, lichens dominate ∼7% of the (plants, fungi, bacteria). While a relatively recent loss of lichenization earth’s terrestrial surface (22), and play important roles in hy- in Lecanoromycetes was previously identified, our work instead sug- drological (23) and biogeochemical cycles through weathering gests lichenization was abandoned far earlier, interrupting what had previously been considered a direct switch between trebouxiophy- (24, 25), carbon uptake, and nitrogen-fixation (24, 26), as well as cean and trentepohlialean algal symbionts. Consequently, some of albedo modification (27, 28) and food or nesting sources for the most diverse clades of LFF are instead derived from nonliche- diverse animals (29). Lichen thalli may be large or small and nized ancestors and re-evolved lichenization with Trentepohliales harbor a eukaryotic green alga (from the order Trentepohliales or algae, a clade that also facilitated lichenization in unrelated lineages class Trebouxiophyceae) or cyanobacteria as the primary photo- of LFF. Furthermore, while symbiont identity and symbiotic pheno- biont. Compared to microlichens (typically forming small, crust- EVOLUTION type influence the ecology and physiology of lichens, they are not like [crustose] or microlobed [squamulose] thalli), macrolichens correlated with rates of lineage birth and death, suggesting more (typically forming larger, leaf-like [foliose] or tufted [fruticose] complex dynamics underly lichen diversification. Finally, diversifica- thalli) may have a competitive advantage due to their ability to tion patterns of LFF differed from those of wood-rotting and ecto- overgrow microlichens. However, physiological and ecological mycorrhizal taxa, likely reflecting contrasts in their fundamental requirements associated with carbon uptake, light capture, hy- biological properties. Together, our work provides a timeline for dration, and desiccation may restrict their distribution and ulti- the ecological contributions of lichens, and reshapes our understand- mately influence diversification rates. Similarly, cyanobacteria may ing of symbiotic persistence in a classic model of symbiosis. supply a constant source of fixed N to the mycobiont, and ulti- mately to the ecosystem, but are ecologically restricted due to symbiosis | macroevolution | diversification their dependence on liquid water for photosynthesis (30–33). ymbiotic associations permeate the Tree of Life and form the Significance Sbasis upon which diverse ecosystems are founded (1–5). Over geological timescales, interacting lineages may evolve mutualistic associations with one another from free-living or symbiotic an- Symbioses are evolutionarily and ecologically widespread, yet cestors (6); however, since these require services from all part- we lack a robust understanding of their origins, losses, and ners to increase the overall fitness, they are vulnerable over macroevolutionary consequences. We traced the evolution of partner choice and phenotype in lichens—a classic model of evolutionary time to the evolution of cheaters or other forms of symbiosis—and revealed shifts among symbiont groups and destabilization (7, 8). Thus, mutualistic associations may be evo- phenotypic evolution. Symbiont switches broadly coincided with lutionarily transient; they may be replaced with a functionally sim- the convergent acquisition of similar partners by divergent ilar but phylogenetically distinct symbiont, or the symbiosis may be clades. Fungi abandoned the lichen habit far earlier than previ- abandoned entirely (8–10). Ascertaining the timing and pathways by ously understood, and subsequently reacquired it with algae which interactions originate and are severed may yield insight into that have frequently facilitated independent fungal transitions factors facilitating these transitions. Mutualistic interactions may to lichenization. Finally, diversification in lichenized fungi was also directly or indirectly regulate macroevolutionary clade dy- not strictly modulated by partner choice or phenotype, and namics; however, predicting their effects on the macroevolutionary – differed from other fungi, suggesting complex and variable dy- dynamics of these clades is not straightforward (11 13). Which namics within lichen fungi and across fungal nutritional modes. symbiont a mutualistic lineage associates with may also influence its fitness, geographic range, or contribute to density-dependent pro- Author contributions: M.P.N., R.L., C.K.B., H.T.L., and R.H.R. designed research; M.P.N. cesses, and ultimately shape lineage diversification. For example, performed research; M.P.N. analyzed data; and M.P.N., R.L., C.K.B., H.T.L., and R.H.R. symbionts with greater ecological flexibility or resistance to extinc- wrote the paper. tion may confer reduced extinction and higher net diversification to The authors declare no competing interest. their associated lineages (13). To better understand the complex This article is a PNAS Direct Submission. macroevolutionary dynamics of biotic associations, comparative Published under the PNAS license. analysis of especially large and old clades can be particularly re- 1To whom correspondence may be addressed. Email: [email protected]. vealing about the timeline and pathways by which they are gained This article contains supporting information online at https://www.pnas.org/lookup/suppl/ and lost. Such studies have revealed shifts in the type of root doi:10.1073/pnas.2001913117/-/DCSupplemental. symbiosis (10), hemipteran–bacterial endosymbionts (14), and the First published August 13, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2001913117 PNAS | September 1, 2020 | vol. 117 | no. 35 | 21495–21503 Downloaded by guest on October 2, 2021 Most lichens occur as bipartite (two partner) associations be- or near the family Stictidaceae (Ostropomycetidae), resulting in tween a mycobiont and photobiont; however, some lichen- the evolution of saprotrophic or plant parasitic lineages (41, 42). forming fungi (LFF) regularly form complex tripartite (three Among lichens, lichen-forming algae (LFA) vary in their eco- partner) associations that include a green algal photobiont, and logical and geographic preferences, as well as the number of LFF cyanobacteria that are typically restricted to cephalodia, small species supported; consequently, LFA may be expected to indi- structures in or on the thallus in which cyanobacterial function is rectly regulate the diversification dynamics of LFF (43). Simi- restricted to nitrogen fixation and carbon is obtained from the larly, the ecological and geographic preferences of a lichen are green algal photobiont via the mycobiont (34). In contrast to also shaped by thallus growth form (which is determined by the these complex associations, the lichen-forming habit has been LFF), which may also confer competitive advantages that in- abandoned entirely in some fungi. One of the best-known ex- crease fitness. Consistent with this is the enhanced diversification amples of this occurs in the fungal class Lecanoromycetes, the observed in several macrolichen lineages of LFF (44). Together, most diverse (>14,000 species, 80% of known LFF) and one of these evolutionary shifts in photobiont association and growth the oldest clades of LFF (35–40). Lichenization has been con- form may affect the evolutionary trajectories of these lineages tinuously maintained from, or prior to, the most recent common and their ecological contributions. Consequently, identifying the ancestor (MRCA) of Lecanoromycetes, but recently lost within evolutionary pathways underlying these transitions, and their Fig. 1. Time-scaled ML phylogeny of 3,373 Lecanoromycetes fungi. Concentric rings surrounding the phylogeny indicate binary character state for growth form (inner), primary photobiont (middle), and presence