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Home , Lichen, Lichina, Peltigerales, Pertusariales

The macroevolutionary dynamics of symbiotic and phenotypic diversification in

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– 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 (20, 21): Lichens. traced the macroevolutionary history of symbiotic and phenotypic Lichens are stable associations between a mycobiont () diversification in an iconic symbiosis, lichens. By inferring the most and photobiont (eukaryotic alga or cyanobacterium). The pho- comprehensive time-scaled phylogeny of -forming fungi (LFF) tobiont supplies the heterotrophic fungus with photosynthetically to date (over 3,300 ), 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, , and a conducive growing environment in logenetically or functionally similar associations in diverse lineages the form of a . Collectively, lichens dominate ∼7% of the (, fungi, bacteria). While a relatively recent loss of lichenization earth’s terrestrial surface (22), and play important roles in hy- in was previously identified, our work instead sug- drological (23) and biogeochemical cycles through 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 Trentepohliales or , a clade that also facilitated lichenization in unrelated lineages class ) or 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 and physiology of lichens, they are not like [] or microlobed [squamulose] thalli), macrolichens correlated with rates of lineage birth and death, suggesting more (typically forming larger, -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 (30–33).

ymbiotic associations permeate the Tree of 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 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

www.pnas.org/cgi/doi/10.1073/pnas.2001913117 PNAS Latest Articles | 1of9 Downloaded by guest on September 27, 2021 Most lichens occur as bipartite (two partner) associations be- or near the (), 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 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 of cephalodia (outer) for individual species. Branches are shaded according to BAMM-derived rate estimates, and pie charts on nodes indicate the probability of each state as estimated for a single multistate character. Pie charts are only shown for nodes in which the most-likely state differs from that of the parent node. Geological periods (concentric rings) underlie the phylogeny, and dashed lines are plotted at 50-My intervals. Presence of locus sequence data and bootstrap support values, tip labels, and node states for all nodes are shown in SI Appendix, Figs. S1–S3. The classification used reflects a pre-2018 clade delimitation included in SI Appendix, Tables S24 and S25 and reflects the classification used in our analyses. Recent work has elevated or deprecated specific clades labeled in this figure (for example, now includes , and and have been segregated into numerous families and orders, respectively) (113), but the used in our analyses (which were initiated prior to these changes) is instead reflected in Fig. 1 to maintain consistency.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.2001913117 Nelsen et al. Downloaded by guest on September 27, 2021 occurrence in geological time, yields crucial insight into the the conferred at the organismal-level are linked to the macroevolu- evolutionary persistence of symbioses, the evolution of their tionary dynamics of this clade. complexity, and ecological function. Moreover, it provides criti- The fossil record of lichens is both taxonomically and tem- cal insight into whether phenotypic and symbiotic benefits porally fragmentary (45, 46), thereby restricting our ability to

Photobiont Losses Trebouxiophyceae Photobiont 40 40 TTJJ K P ggNNg TJT J K PPggNNg

30 Bootstrap Replicate 30 Bootstrap Replicate

20 ML Tree 20 ML Tree

10 10 Cumulative Gains Cumulative Losses 0 0 250 200 150 100 50 0 250 200 150 100 50 0 Node Change Age (Ma) Node Change Age (Ma) Trentepohliales Photobiont Cyanobacteria Photobiont 40 40 TTJJ K P ggNNg TJT J K PPgg

30 Bootstrap Replicate 30 Bootstrap Replicate

20 ML Tree 20 ML Tree EVOLUTION 10 10 Cumulative Gains Cumulative Gains 0 0 250 200 150 100 50 0 250 200 150 100 50 0 Node Change Age (Ma) Node Change Age (Ma) Cyanobacteria (Photobiont or Cephalodia) Cephalodia 40 40 TTJJ K P ggNNg TJT J K PPggNNg

30 Bootstrap Replicate 30 Bootstrap Replicate

20 ML Tree 20 ML Tree

10 10 Cumulative Gains Cumulative Gains 0 0 250 200 150 100 50 0 250 200 150 100 50 0 Node Change Age (Ma) Node Change Age (Ma) Macrolichen Growth Form 40 TTJJ K P g

30 Bootstrap Replicate

20 ML Tree

10

Cumulative Gains 0 250 200 150 100 50 0 Node Change Age (Ma)

Fig. 2. The evolution of symbiotic associations, growth forms, and tripartite (cephalodiate) interactions through time. Each step indicates a node that possesses the trait of interest and differs in state from its parent node. Terminal taxa that possessed a change between the tip and their parent node were not included to facilitate the visualization of earlier transitions. Results from ASR on the ML tree are shown in black, while results from 100 BSRT are shown in color. Vertical shading corresponds to geological period.

Nelsen et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 27, 2021 determine when and how these evolutionary transitions oc- sensu lato; recent work has divided this clade into several orders) curred. However, linking fossils with molecular sequence data (SI Appendix, Table S8). Ancestral state estimates derived from facilitates the synthesis of time-scaled phylogenies that may be binary hidden state speciation and extinction (HiSSE) analyses of used to address evolutionary questions in the absence of a robust, the Lecanoromycetes-wide ML tree also suggested this node was continuous, and well-annotated fossil record. Previous work has not associated with cyanobacteria, trebouxiophycean, or trente- begun to address questions related to symbiont-switching and pohlialean algae (SI Appendix,Figs.S9–S11), consistent with a loss symbiotic complexity (47–50), but these have typically been re- of the lichen habit. Lichen associations were also secondarily stricted to specific clades and conducted in an atemporal frame- reacquired by some of these delichenized lineages, primarily with work, thereby limiting our ability to assess the uniformity of these Trentepohliales and trebouxiophycean photobionts during the processes across lichens or relate these changes to broader trends. Jurassic–Cretaceous (Figs. 1–3andSI Appendix,Fig.S3). Here we circumvent these by inferring a densely sampled, time- The macrolichen growth form was acquired numerous times scaled phylogeny of 3,373 species of Lecanoromycetes fungi, the across Lecanoromycetes, with the earliest gains occurring in the most diverse and one of the oldest clades of LFF (35–40), and use Late Jurassic–Early Cretaceous with most transitions confined to this to reconstruct transitions in symbiotic association and thallus the Late Cretaceous and Cenozoic (Figs. 1–3andSI Appendix, Fig. phenotype. This ultimately facilitates a clearer understanding of S3 and Tables S9–S12). Lineages associated with cyanobacteria, the evolutionary retention of these symbioses, the roles these traits either as a primary photobiont or in tripartite associations with play in regulating the macroevolutionary dynamics of LFF, and cephalodia, gave rise to macrolichens at the highest rate; however, how lichens have contributed to ecosystem function over time. most macrolichen lineages evolved from bipartite microlichens More broadly, our study sheds light on the macroevolutionary associated with a trebouxiophycean photobiont (Figs. 1 and 3 and dynamics of this classic model of symbiosis (20, 21). SI Appendix, Fig. S3 and Tables S9–S12). Our Bayesian analysis of macroevolutionary mixtures (BAMM) Results analysis revealed that median rates of speciation, extinction, net Multistate, maximum-likelihood (ML) ancestral state reconstruc- diversification, and turnover across Lecanoromycetes in aggregate tions (ASR) under several models (SI Appendix,TablesS1–S5) varied through time, and generally exhibited an increasing trend were conducted on the timescaled ML tree (SI Appendix,Figs.S1 (SI Appendix,Fig.S4). Closer inspection of individual clades and S2) and bootstrap replicate topologies (BSRT). The most demonstrated substantial rate variation (Fig. 1 and SI Appendix, parameter-rich model was considered the best fit (SI Appendix, Figs. S5–S7). However, this variation could not be strictly linked to Tables S6 and S7), and ASR (ML and BayesTraits) of the ML tree any single trait at this broad phylogenetic scale. Photobiont type and BSRT suggested the MRCA of Lecanoromycetes was a bi- and presence of cyanobacterial associations of any kind were not partite microlichen with a trebouxiophycean photobiont (Fig. 1 and significantly linked to differential levels of speciation, extinction, SI Appendix,Fig.S3andTableS8). Associations with Trente- or net diversification, as indicated by structured rate permutations pohliales algae originated in the Jurassic–Cretaceous and were rarely on phylogenies (STRAPP) and HiSSE analyses conducted across lost once acquired (Figs. 2 and 3 and SI Appendix,TablesS9–S12). Lecanoromycetes (SI Appendix,Figs.S8–S12 and Tables S13 and Cyanobacterial symbionts were first acquired during the Jurassic– S14). Taxa forming large thalli possessed marginally higher net Cretaceous when Arctomiales forged bipartite cyanobacterial asso- diversification and speciation rates in STRAPP analyses than ciations through the replacement of a trebouxiophycean photobiont, those producing small thalli (P = 0.084 and P = 0.092, respec- and when acquired them through the same pathway or tively) (SI Appendix,TableS13). via the addition of cyanobacteria to a bipartite trebouxiophycean Tests for associations between traits and diversification rates association (Figs. 1 and 2 and SI Appendix,Fig.S3). Other associa- were also performed on individual Lecanoromycetes subclades. tions with cyanobacterial symbionts largely evolved during the STRAPP analyses conducted in smaller clades that were poly- Paleogene through the addition of cyanobacteria to bipartite morphic for individual traits again suggested no significant dif- trebouxiophycean associations (Figs. 1 and 2 and SI Appendix, ferences were observed, with the exception of , where Fig. S3). However, complex (tripartite) associations evolved at taxa forming larger thalli possessed marginally higher net diver- the greatest rate from bipartite cyanobacterial associations sification and speciation rates (SI Appendix, Table S15). Similarly, (Fig. 3 and SI Appendix,TablesS9–S12). These associations HiSSE analyses on subclades (SI Appendix, Figs. S13–S20 and were initiated during the Jurassic to mid-Cretaceous, but largely Table S16) recovered character-independent diversification (CID) evolved during the Late Cretaceous–Cenozoic and were lost at a models as the best fit (Macrolichen: , Telo- relatively high rate, exceeding the rate at which bipartite associa- schistales), or revealed a more complex pattern, suggesting rate tions were lost to nonlichenized taxa or switched photobiont differences between character states were due in part to a hidden classes (Figs. 1 and 2 and SI Appendix, Fig. S3 and Tables S9–S12). state, rather than a strict presence of the trait itself (Macrolichen: While cyanobacterial associations were lost at a low rate from Peltigerales, , Cladoniineae; Cephalodia: Cladoniineae). bipartite lichens, losses of cyanobacterial symbionts from tripartite The only exception was the presence of complex associations in associations varied by growth form: Microlichens lost cyano- Peltigerales, where a binary state speciation and extinction model bacterial partners, while macrolichens typically retained a cyano- suggested lineages forming tripartite associations had higher net bacterial partner (Fig. 3 and SI Appendix,TablesS9–S12). turnover rates (0.056 vs. 0.035) and extinction fractions (0.190 vs. Across Lecanoromycetes, symbiotic abandonment (loss of li- 2.06e-9), which translated to higher extinction rates (0.009 vs. 7.22e-7), chen habit) occurred at a low rate from bipartite lichen associ- and comparable, but slightly higher speciation (0.047 vs. 0.035) ations, which included trebouxiophycean algae (for ML analysis, and net diversification rates (0.038 vs. 0.035). However, lineages see Fig. 3 and SI Appendix, Tables S9 and S10), or from lichens with tripartite associations were lost in an approximately equal with either trebouxiophycean or cyanobacterial (for BayesTraits, amount through extinction (0.009) and transitions to the bipar- see Fig. 3 and SI Appendix, Tables S11 and S12) primary pho- tite state (0.010), and these lineages were gained at a greater rate tobionts. Most losses of the lichen habit were relatively recent through speciation (0.047) than transitions to the tripartite state (between the tip and parent node of individual taxa, such as (0.0006). In contrast, the favored HiSSE model for tripartite as- in ); however, the lichen symbiosis was also sociations in Cladoniineae suggested these were lost at a higher abandoned relatively early in the evolution of Lecanoromycetes rate through transitions (0.003) to the bipartite state than through (Fig. 2 and SI Appendix, Fig. S3). Both ML and BayesTraits ASR extinction (<1.78e-10). Taken together, these analyses reveal that on the ML and BSRT suggested that lichenization was lost en- in most cases, these traits alone are unlikely to strictly regulate tirely, deep within the Ostropomycetidae (MRCA of Ostropales diversification dynamics within these clades.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.2001913117 Nelsen et al. Downloaded by guest on September 27, 2021 Fig. 3. Transition rate frequencies estimated for the (A) ML tree or (B) BSRT using ML or (C and D) reversible jump Markov chain Monte Carlo (RJMCMC) approaches. Multistate character state coloring follows that outlined in Fig. 1. Shapes represent growth form, and primary photobiont type is listed in each state. States with cephalodia are indicated. The thickness of lines connecting character states is proportional to the transition rate frequency. SI Appendix, Tables S9–S12 further summarize transition rate frequencies. EVOLUTION

Finally, we assessed whether the lack of strong correlations facilitated the early diversification of Lecanoromycetes as it between traits and diversification rates could be due to a biased diversified prior to or loosely coincident with Lecanoromycetes understanding of the number of species possessing specific traits. (40), and forms frequent and widespread associations with all Our STRAPP analyses suggested marginally significant differ- four Lecanoromycetes subclasses (54, 56, 57), far more than ences in diversification rates of taxa in specific states when sam- other trebouxiophycean LFA clades (Symbiochloris,andthe pling fractions were assumed to be underestimated (SI Appendix, +Elliptochloris clade) with ages comparable to that of Tables S17–S22). Macrolichen lineages had significantly higher Lecanoromycetes. speciation or net diversification rates only when it was assumed that macrolichen richness estimates represented 80% or less of the Differential Retention of Symbiotic Associations. Tripartite associ- anticipated diversity. Subclade analyses revealed significantly ations were disrupted at a higher rate than bipartite associations. higher speciation and net diversification rates in Lecanorales only This conflicts with previous work suggesting tripartite lichen when macrolichen richness estimates represented 60% or less of associations are relatively stable (50, 55), and likely reflects the the anticipated diversity. However, HiSSE analyses under these comparatively limited sampling (55) or focus on an individual sampling fractions consistently recovered the highest Akaike In- fungal (50). At this broad phylogenetic scale, our findings formation Criterion weights from the CID models (SI Appendix, are consistent with complex associations exhibiting reduced re- Table S23). Consequently, we suggest that the absence of strong tention. While the underlying source of this differential retention associations between these traits and diversification rates are un- among associations of varying complexity is unclear, several likely to be due to an incomplete understanding of the magnitude nonexclusive explanations exist. Mutualistic symbioses require of diversity. services from all partners to increase overall fitness; however, this also makes them vulnerable to destabilization through pro- Discussion cesses such as extinction, evolution of , and shifts in Early Lichen Evolution. Our analyses suggest that associations be- cost:benefit ratios (7, 8). Shifts in levels (especially N) tween Lecanoromycetes fungi and these broadly defined symbiont among other symbioses may alter the nature of the symbiosis and groups were occasionally interrupted by instances of symbiont contribute to its breakdown (58, 59). In lichens, thallus water replacement or abandonment, similar to that observed in hemi- content and nutrient levels may differentially favor individual pteran–endosymbiont associations and plant root symbioses (10, symbionts or symbiont combinations and potentially contribute 51). However, these broadly defined photobiont classes mask to the breakdown or destabilization of the symbiosis (32, 60–62). substantial diversity, and a more refined coding at a lower taxo- Maintenance of this symbiotic balance and overlapping geo- nomic rank reveals increased symbiont-switching (43, 52–54). graphic distributions (11) of constituent species may become Thus, when taken together, transitions between major photobiont increasingly difficult as the number of symbionts increases. Thus, types are rare in most of Lecanoromycetes, while shifts among processes contributing to symbiotic destabilization may be ex- species or genera within these broad photobiont classes are far acerbated with increased complexity and lead to the reduced more frequent. Our work refines previous findings (55) and sug- retention of complex associations. gests that the MRCA of Lecanoromycetes formed a bipartite as- Our analyses illustrated that fungal associations with these sociation with a trebouxiophycean alga. Trebouxiales emerges as three groups of symbionts were lost at a low rate, especially from perhaps the most likely group of trebouxiophycean LFA to have Trentepohliales associations. Cyanobacterial associations have

Nelsen et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 27, 2021 previously been regarded as relatively stable in comparison with associate with trebouxiophycean algae, Trentepohliales algae have that of (50, 55, 63). Our analyses indicate that cya- also played an important role in facilitating the evolution of lichen- nobacterial associations were lost at a low rate from bipartite ization, and maintaining the subsequent diversification of LFF. lichens; however, losses of cyanobacterial symbionts from tri- Here we suggest cyanobacterial associations in Lecanoromycetes partite associations varied by growth form. Thus, the evolutionary are derived, consistent with recent work (55), but conflicting with retention of fungal–cyanobacterial associations appears to be earlier assertions that cyanobacterial associations were ancestral to regulated in part by the level of symbiotic complexity and growth green algal lichens (79). The timing of these cyanobacterial acqui- form. However, this trend may be disproportionately driven by a sitions by Peltigerales and Arctomiales broadly coincided with the single clade (Peltigerales). Regardless, loss of N-fixing cyanobac- Jurassic–Cretaceous (40) or Triassic–Jurassic (80) age of a separate teria may limit growth of the green algal photobiont and myco- class () of cyanobacteria-associated LFF outside biont. Alternatively, N-requirements may instead potentially be Lecanoromycetes. Furthermore, previous work has suggested that fulfilled through the occupation of N-enriched (64) or symbiotic cyanobacteria—which associates with Peltigerales acquisition of N-fixing bacteria (65). and Arctomiales LFF (54, 56, 57)—diverged from its sister during the Triassic–Jurassic (81); thus, acquisition of Nostoc symbionts by The Breakdown of an Iconic Symbiosis. Mutualistic interactions have Lecanoromycetes LFF clades may have occurred relatively shortly been lost in favor of parasitism or an autonomous existence in a after the origin of this cyanobacterial clade, and is consistent with diverse range of lineages, including bacteria, plants, corals, algae, observations among several other clades of interacting LFF and and fungi (8, 9, 66). Transitions to the nonlichenized state appear LFA (40). relatively rare in Lecanoromycetes—consistent with observations Finally, Cyphobasidium (Cystobasidiomycetes; ) in other ancient symbioses (8, 9)—and were initiated during the is a clade of mycoparasitic fungi most commonly associated with Late Triassic to Early Jurassic at or near the MRCA of Ostropales thalli from a limited number of macrolichens, especially mem- sensu lato (Ostropomycetidae). As modern non-LFF in this clade bers of the LFF family (82–87). We were unable to are a heterogeneous assemblage of saprotrophs, plant parasites, obtain published crown age estimates for Cyphobasidium, but and endophytes, it is not immediately clear what nutritional and stem-based estimates initially suggested a Permian origin (84), ecological mode these nonlichenized ancestors utilized, or what while a more densely sampled dataset instead suggested a Late processes facilitated this transition. This early loss conflicts with Cretaceous divergence from its sister (88). This is broadly con- previous work, which instead suggested lichenization was lost far sistent with the age of Parmeliaceae macrolichens, and suggests more recently—in or near the ostropalean family Stictidaceae— Cyphobasidium evolved after the first Lecanoromycetes macro- and that diverse, ostropalean lineages of LFF (such as Graph- lichens, but may have associated with Parmeliaceae macrolichens idaceae sensu lato) were part of a continuously maintained shortly after their evolution. Further work is required to clarify lichenization event initiated at the MRCA of Lecanoromycetes or the age of the crown node and the taxonomic breadth of lichens earlier (41, 42). Our work instead suggests that the lichen habit with which it associates. was reacquired multiple times following this loss. This discrepancy is likely a consequence of the increased sampling here coupled Complex Factors Underlie Lichen Diversification. While symbiont with the recent recognition of additional lineages of nonlichenized identity and phenotype may each be expected to differentially ostropalean taxa outside of Stictidaceae (, influence fitness and lineage diversification, and our analyses Claviradulomyces), and further illustrates the lability and evolu- revealed substantial diversification rate heterogeneity across tionary dynamism in some of the best-known fungal symbioses (67, Lecanoromycetes, this variation was not strictly regulated by the 41). This pattern of occasional loss and reacquisition is consistent presence of a single trait. Consequently, our analyses suggest that with observations in a diverse range of biotic interactions including discrepancies in the number of fungal species supported by algal pollination in angiosperms (68–72), arbuscular mycorrhizal groups may not be a strict consequence of symbiont-associated associations in vascular plants (10, 73), mutualistic seed dispersal diversification rate variation and may instead be a function of in primates (74), and the Sulcia primary endosymbiont in he- other variables including but not limited to: Interaction age, mipterans (Onycta) (14). Such regains are often relatively recent, differential compatibility with LFF, algal diversity, and ecological and raise questions about whether these transitions are rare or breadth. This study therefore contributes to a broader set of rarely preserved due to elevated extinction rates. work emphasizing the diverse drivers underlying diversification in mutualisms and large and old clades, and the need to more Convergent and Broad Temporal Concordance among Lichen Associations. fully consider the variable influence of multiple traits and envi- While temporal and topological uncertainty obscure the exact timing ronmental factors both through time and across clades (12, 13, and number of acquisitions of Trentepohliales photobionts within 89, 18, 90). Lecanoromycetes, these occurred at the greatest rate from non- lichenized lineages. This is consistent with a broader pattern seen Contrasting Patterns of Fungal Diversification. Finally, our work across Pezizomycotina, where fungal lineages outside of Lecanor- reveals that the overall pattern of lichen diversification differs omycetes also commonly acquired Trentepohliales algae from the from that of ectomycorrhizal and saprotrophic fungi. Lecanor- nonlichenized state (40). Similarly, these new associations with omycetes and Agaricomycetes represent two of the most diverse Trentepohliales algae likely occurred during the Cretaceous, a time and best-studied clades of fungi, and while both have diversified when several other LFF lineages across Pezizomycotina (Arthonio- through the latter half of the Mesozoic and Cenozoic, the rela- mycetes, , ) were forging associa- tive time course of each radiation is distinct. Recent work has tions with Trentepohliales algae (40), and wet angiosperm-dominated revealed that Agaricomycetes sustained a rapid increase in spe- megathermal —habitats that harbor the greatest diversity ciation and net diversification rates during the Jurassic (91). Our of modern Trentepohliales algae—were originating and diversifying analyses in Lecanoromycetes instead suggest a gradual increase (75, 76). Trentepohliales-associated lichens typically occur on or in speciation and extinction rates that is especially apparent in these habitats, where their ecological contributions are through the mid-Cretaceous to present. Whatever extrinsic fac- primarily limited to serving as food sources or nest for tors may or may not have contributed to a Jurassic initiation of invertebrates (29, 77, 78). Additionally, these associations were lost at the Agaricomycetes radiation, the substantial differences in their a comparatively low rate in Lecanoromycetes once acquired, sug- intrinsic biology and nutritional modes may be relevant to the gesting a higher degree of retention. Taken together with previous different histories: Agaricomycetes are largely wood-rot, litter work (40), our findings highlight that while LFF overwhelmingly decay, and ectomycorrhizal fungi, whereas Lecanoromycetes are

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.2001913117 Nelsen et al. Downloaded by guest on September 27, 2021 overwhelmingly LFF. The stipitate-pileate sporocarps (fruiting further enhanced and diversified the roles and contributions of bodies) of Agaricomycetes were linked to enhanced diversifica- lichens to ecosystem processes. Maintenance of the lichen sym- tion, but are only involved in reproduction and dispersal, biosis was interrupted earlier in the evolution of Lecanor- while the lichen thallus encompasses all aspects of physiology omycetes than previously appreciated, and lichenization was and function. Lichens also exist on the surfaces of their sub- reacquired with Trentepohliales algae loosely coeval with when strates directly interacting with the increasing complexity of other nonlichenized lineages were forging lichen associations Cretaceous and Cenozoic vegetation and environments, while with Trentepohliales. Finally, our work reveals that the diversi- the mycelium of Agaricomycetes fungi are largely buffered from fication dynamics underlying the evolution of LFF are complex, many aspects of the environment by occurring in or plants. and do not reflect those seen in fungi with contrasting nutritional When epiphytism evolved in Lecanoromycetes is unclear, but modes, illustrating how fundamental biological differences un- many early-diverging clades are primarily rock- or ground- derpin variable evolutionary responses to shared events in inhabiting, consistent with the MRCA of Lecanoromycetes oc- Earth history. cupying these habitats. Thus, Lecanoromycetes may not yet have evolved an epiphytic habit with the later expansion of wet Methods tropical angiosperm-dominated being reflected in later A more detailed description of the methodology employed is included in increases in diversification rates (38, 92, 93). Alternatively, SI Appendix. gymnosperm-dominated forests, or the climates favoring them, may simply have not been conducive to lichen diversification. Matrix Assembly and Phylogeny. Here we utilized an iterative process to infer Further work is required to disentangle the factors that could a megaphylogeny of the fungal class Lecanoromycetes. Seven commonly have contributed to a delayed diversification of lichens relative sequenced loci (18s, 28s, mtSSU, ITS, RPB1, RPB2, MCM7) were selected, and to saprotrophic and mycorrhizal fungi, but the present study matrices assembled in Phlawd 3.4a (105). The final alignment consisted of 3,373 in-group taxa (SI Appendix, Table S24), ∼25% of the extant diversity of reveals that biological differences may in part explain the con- Lecanoromycetes (35). A locus-partitioned ML analysis was conducted in trasting patterns of diversification in these two clades. Together, ExaML v1.0.12 (106). We then generated 100 bootstrap replicates and our work illustrates the complex diversification dynamics un- inferred the ML topology as above. The ML and BSRT were then made derlying one of the largest fungal radiations, while demonstrating ultrametric using penalized likelihood, as implemented in treePL v1.0 (107). conflicting patterns of diversification between two of the largest We identified shared nodes between individual topologies and the maxi- and best-known clades of fungi. mum clade credibility tree from previous work (40) and used age estimates for these nodes as constraints in our megaphylogenies.

Contributions to Ecological and Biogeochemical Processes. In the ab- EVOLUTION sence of a robust fossil record, our ASRs suggest that lichen con- Ancestral State Reconstruction. Taxa were scored for various traits based on tributions to ecosystem function have increased and become more our personal experience, field work, or examination of material, as diverse through time. The earliest Lecanoromycetes formed bipar- well as monographs, species descriptions, and the broader literature when tite microlichen associations with trebouxiophycean algae that were ambiguous. Primary photobiont identity (cyanobacteria, Trebouxiophyceae, Trentepohliales, none), presence of cephalodia and growth form (macro vs. retained until the Late Jurassic, indicating that early Lecanor- micro) were then condensed into a single multistate that consisted of eight omycetes lichens would not have substantially contributed to ni- state combinations, which was used for ancestral state reconstruction. We trogen cycling, and their roles in carbon-cycling and water retention inferred the ancestral state of the multistate character across all nodes in the may have been limited, depending on the ecosystems and substrates ML tree and BSRT in corHMM v1.13 (108). To further assess the robustness of occupied. Macrolichens first evolved during the Late Jurassic–Early these results, we also performed BayesTraits v3.0.1 (109, 110) analyses on the Cretaceous, with most transitions occurring through the Cenozoic. ML tree and the pool of BSRT to compare transition rate estimates with This agrees with a taxonomically ambiguous lichen fossil from the those obtained in corHMM and evaluate the ancestral state at two crucial Lower Cretaceous, consistent with a foliose growth form (94), Eo- nodes (the root, and MRCA of Ostropales). We also estimated ancestral cene macrolichen (Lecanoromycetidae) fossils (45), and previous states by calculating model-averaged marginal reconstructions based on HiSSE (89) analyses (analyses described below). Finally, we inferred when ASR (44). The evolution of macrolichens likely enhanced con- acquisitions of a character state occurred by slicing the tree into 1-My time tributions to many processes through increased substrate sur- intervals and summing the nonterminal nodes whose most-likely state was face roughness and texture, which would have increased the trait of interest and was descended from a node differing in its character capture of aeolian dust, water interception, and retention, state (18). contributions to C and mineral cycling, and lichen–animal in- teractions, while also reducing albedo (28, 29, 95). The evolu- Diversification Rate Analyses. Species richness for individual families were tion of cyanobacterial associations further enriched lichen initially retrieved from The Dictionary of Fungi (35), which represented the contributions to ecosystem function through the contribution of most complete estimate of species richness at the time this study was initi- fixed nitrogen. Placing the ages of N-focused lichen associa- ated. Richness values were updated, based on families that have since been tions in the context of other N-fixing or -scavenging associa- split or merged up until early 2014. BAMM (111) analyses were conducted on tions reveals that despite the long existence of terrestrial fungi, the ML tree using clade-specific sampling proportions (SI Appendix, Table S25) and assuming time-constant diversification within each rate class. We plants, and diazotrophic bacteria, we find that these N-focused then conducted STRAPP (112) analyses to test for associations between rates associations likely did not evolve or extensively diversify prior and binary trait states (primary photobiont, growth form, presence of any to the Mesozoic (10, 96–104). Further work is required to de- cyanobacterial association). Subclades with substantial variation in trait state termine what underlies this provocative pattern. and relatively low character state imbalance were further analyzed for the effects of growth form and symbiotic complexity. In addition, we utilized Conclusion HiSSE analyses (89) to test for character-dependent diversification. While LFF Symbioses play important roles in modern ecosystem function, are one of the best-studied groups of fungi, the proportion of species yet the age and evolutionary persistence of these associations awaiting discovery varies substantially across groups of LFF. As these biased may vary over geological timescales. By reconstructing the evo- diversity estimates could skew our analyses aimed at detecting correlations between trait states and diversification rates, we conducted supplemental lution of symbiont association and symbiotic phenotype, we BAMM, STRAPP, and HiSSE analyses in which our sampling fractions were provide insight into the macroevolutionary dynamics of the li- multiplied by a growth form-specific (macro vs. micro) scalar to assess the chen symbiosis. The evolutionary preservation of these associa- robustness of our analyses to undocumented diversity. tions varied by complexity, with tripartite associations appearing highly unstable, and facilitating the loss of otherwise stable Data Availability. Code, data, and phylogenetic trees are available on GitHub cyanobacterial associations. The evolution of macrolichens (https://github.com/mpnelsen/Lecanoromycetes_megaphylogeny).

Nelsen et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 27, 2021 ACKNOWLEDGMENTS. We thank Trevor Price and Mark Westneat for University of Chicago Committee on Evolutionary Biology. Analyses comments on an earlier draft of this manuscript. M.P.N. was supported were performed on computer clusters at the University of Chicago, and by a William Harper Rainey Fellowship through the University of the Field Museum, and the Grainger Bioinformatics Center at the Chicago, a Brown Family Fellowship through the Field Museum, the Field Museum.

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