Tremella Macrobasidiata and Tremella Variae Have Abundant and Widespread Yeast Stages in Lecanora Lichens

Environmental Microbiology (2021) 00(00), 00–00 doi:10.1111/1462-2920.15455

Tremella macrobasidiata and Tremella variae have abundant and widespread yeast stages in Lecanora lichens

Veera Tuovinen ,1,2* Ana Maria Millanes,3 3 1 2 Sandra Freire-Rallo, Anna Rosling and Mats Wedin Introduction 1Department of Ecology and Genetics, Uppsala University, Uppsala, Norbyvägen 18D, 752 36, Sweden. The era of molecular studies has shown that most of the 2Department of Botany, Swedish Museum of Natural diversity of life is microbial (Pace, 1997; Castelle and History, Stockholm, P.O. Box 50007, SE-104 05, Banfield, 2018). Lichens, symbiotic consortia formed by Sweden. at least two different microorganisms, are a great mani- 3Departamento de Biología y Geología, Física y Química festation of microbial diversity in a unique package; sev- Inorgánica, Universidad Rey Juan Carlos, Móstoles, eral studies in the last decade have continued to E-28933, Spain. demonstrate the since long recognized presence of diverse fungal communities within lichen thalli (U’Ren et al., 2010, 2012; Fleischhacker et al., 2015; Grube and Summary Wedin, 2016; Noh et al., 2020). In recent years, attention Dimorphism is a widespread feature of tremellalean has been drawn especially to the presence of previously fungi in general, but a little-studied aspect of the biol- undetected basidiomycete yeasts in different ogy of lichen-associated Tremella. We show that macrolichens (Spribille et al., 2016; Cernajováˇ and Tremella macrobasidiata and Tremella variae have an Škaloud, 2019, 2020; Tuovinen et al., 2019; Mark abundant and widespread yeast stage in their life et al., 2020). However, little is still known about the ubiq- cycles that occurs in Lecanora lichens. Their sexual uity of most of the individual organisms and their potential filamentous stage is restricted to a specific lichen: contributions or consequences for the lichen symbiosis T. macrobasidiata only forms basidiomata on (Spribille et al., 2016, 2020; Spribille, 2018). Lecanora chlarotera hymenia and T. variae only on Yeasts as a life cycle stage has originated in multiple Lecanora varia thalli. However, the yeast stage of distantly related clades of Fungi, and potential for yeast T. macrobasidiata fi is less speci c and can occur in growth evolved early in their evolution (Kurtzman L. varia T. variae lichens, whilst all life stages of may et al., 2011; Nagy et al., 2014). Dimorphic fungi have the fi L. varia be speci cto . Contrary to the hyphal stages, ability to switch between an unicellular yeast form and a the yeasts are distributed across the thalli and filamentous stage, sometimes in response to environ- Lecanora hymenia of lichens, and not limited to spec- mental cues (Sánchez-Martıneź and Pérez-Martın,́ 2001; Tremella macrobasidiata imens with basidiomata. Lin, 2009). To date, one of the most studied and species L. chlarotera was present in all studied , and in 59% of rich group of lichen-associated basidiomycetes is L. varia specimens. Only in 8% of the L. varia thalli Tremellomycetes (Agaricomycotina), with over could none of the two Tremella species be detected. 60 described species belonging to Tremella s. lat. Our results indicate that lichen-associated Tremella (Diederich et al., 2018) (http://www.lichenicolous.net). may be much more abundant and widespread than Many Tremellomycetes have a dimorphic life cycle previously assumed leading to skewed estimations (Bandoni, 1995), but lichen-associated Tremella species about their distribution ranges and lichen specificity, have been mainly studied when they form sexual fruiting and raise new questions about their biology, ecology bodies (basidiomata) on the lichen thalli or on/in the and function in the symbiosis. fruiting bodies (apothecia) of the ascomycete lichen symbiont. Thus, for most species only the filamentous life stage is well known and has been studied in detail Received 29 January, 2021; revised 3 March, 2021; accepted 3 March, 2021. *For correspondence. E-mail [email protected]. (Diederich, 1996; Millanes et al., 2021). However, in a se; Tel. (+46) 184714263 recent study Tuovinen and colleagues (2019) showed

© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 2 V. Tuovinen et al. that Tremella lethariae has a common and widespread aimed to investigate if (i) also T. variae has a yeast stage, yeast stage in thalli of Letharia and seems to enter the (ii) the yeast stage is restricted to occur in the hymenium sexual cycle only rarely. It is probable, that other lichen- or thallus parts of L. chlarotera and L. varia lichens, associated Tremella species likewise have a yeast stage respectively, (iii) the yeast stage of both T. macrobasidiata in their life cycle, but it has not been detected due to and T. variae is restricted to lichen specimens that have technical difficulties. Observations of basidiospores ger- Tremella basidiomata and (iv) the yeast stage is restricted minating by repetition or budding, presence of conidia in to the respective lichen. We also aimed to study whether Tremella basidiomata (Diederich, 1996; Zamora the different Tremella species differ in their physical inter- et al., 2011; Millanes et al., 2015), Fellomyces yeasts iso- actions with other symbionts in the thalli. lated from lichens (Prillinger et al., 1997), as well as Tremellomycete DNA records from lichen thalli without visible basidiomata (Ekman, 1999; Lindgren et al., 2015; Results Zhang et al., 2015; Wang et al., 2016; Fernández- Life cycle stages of Tremella observed by FISH Mendoza et al., 2017; Banchi et al., 2018), support this and CLSM hypothesis. Nevertheless, complete life cycle and distribution of the different life stages and their prevalence in We used fluorescent in situ hybridization (FISH) to specif- lichens remain unknown for most Tremella species. ically stain Tremella cells in the L. chlarotera and L. varia Lichens as symbiotic entities do not have names thalli with and without basidiomata and visualized the (Goward, 2008), and in the following text we use the cells in different parts of the specimens with confocal name of the ascomycete fungal symbiont followed by the laser scanning microscope (CLSM; Fig. S1). FISH and term ‘lichen’ when referring to a whole lichen thallus CLSM verified that Tremella species occur and are abun- including all its symbionts. Letharia lichens are dant in a yeast form in both studied Lecanora lichens, macrolichens with a stratified thallus, in which Tremella also in specimens without basidiomata (Figs 1–3, cells are embedded in a thick polysaccharide matrix in Figs. S2 and S3), we observed them in every studied the cortex (Tuovinen et al., 2019). However, several specimen. Tremella yeasts were intermixed with Tremella species have been described from crustose Lecanora hyphae in the areolas and often occurred in lichens that lack a clear thallus stratification and a thick, patches. The yeasts were often concentrated, but not well-defined cortex, such as some representatives of restricted, to the upper parts of the thallus (Figs 1A, B Lecanora s. lat. and Lecidea s. lat (Diederich, 1996; and 2A, C, G, Fig. S2 G). We observed Tremella yeasts Zamora et al., 2011, 2016, 2018). Tremella species asso- also in the apothecium thalline margin (Fig. 1B and C, ciated with these two genera are nested within a group of Fig. S3G) as well as in the upper parts of the hymenium Tremella species associated with stratified macrolichens of the apothecia (Fig. 1C and D, 2B, H, Fig S2F, Fig. S3F in the Parmeliaceae, including also T. lethariae (Zamora and G) in both Lecanora species. In L. chlarotera thalli et al., 2011, 2016, 2018; Tuovinen et al., 2019). For this without basidiomata, we observed germinating Tremella study, we chose two Tremella species that produce yeasts, often with multiple projections (Fig. 1E–G, basidiomata on different and well-defined parts of two dif- Videos S1–S2). In one of the L. varia specimens with ferent Lecanora lichens: Tremella macrobasidiata forms T. variae basidiomata, in a thallus part where basidiomata basidiomata only on the apothecial hymenium of were not yet formed, we observed Tremella hyphae Lecanora chlarotera, and Tremella variae only on the (Fig. 2C–E, Video S3). We observed germinating thallus of Lecanora varia, including the thalline margin of Tremella yeasts in L. varia specimens with basidiomata, the apothecium (Zamora et al., 2011, 2016). This diver- both in the thalli (Fig. 2F) and hymenia (Fig. 2H). gent location on the two lichens is remarkable, particu- In basidiomata of both Tremella species, we observed larly as the two Tremella species are phylogenetically basidia, basidiospores, yeasts and hyphae of Tremella very closely related. On the other hand, the studied (Fig. 3, Fig. S2A–E, Fig. S3A–E). Tremella hyphae of Lecanora species are part of different species complexes both studied species grow around and between algal and phylogenetically not very closely related with each cells in the basidiomata, but we did not observe them other (Zhao et al., 2016). penetrating the algal cell walls (Fig. 3F, H, Fig. S3E). We We wanted to study how, and at which life cycle stage, observed tremelloid haustoria in both species, but never Tremella occurs in the thalli of these crustose lichens. clearly attached to any other cells (Figs 3E and 4E, Tremella macrobasidiata basidiospores have been Videos S4, S5). In basidiomata of T. macrobasidiata,we observed to germinate by ballistoconidia and blastoconidia observed conidiogenous cells (Fig. 3C) similar to those in basidiomata formed on L. chlarotera, but for T. variae described by Zamora et al., 2011, which stained with the asexual stages has not yet been observed (Zamora Tremella specific probe, verifying that these cells belong et al., 2011, 2016). In this study, more specifically, we to Tremella and not to any other fungus in the thalli.

Fig 1. Tremella in Lecanora chlarotera specimens without basidiomata, volume rendering except for C, which is a maximum intensity projection with transmitted light. Green: Tremella, magenta: algal autofluorescence and Lecanora. A. Thallus with three different areoles with Tremella yeasts and algae (*). B. Tremella yeasts in thallus (arrowhead) and thalline margin (TM) of an apothecium. Woody substrate (W), cross section. C. Apothecium thalline margin (TM) with Tremella yeasts, Tremella yeasts also in hymenium (H). Arrow points to dead algal cells that autofluoresce on green wavelengths, live algae (*). D. Hymenium of L. chlarotera apothecium seen from above with Tremella yeasts, paraphyses in magenta, and algae (*). E–G. Tremella yeasts germinating in thallus (arrows), live algae (*). E Related to Video S1. F related to Video S2. Scale 30 μminA,B,D,10μm in C, E–G. [Color figure can be viewed at wileyonlinelibrary.com]

Fig 2. Tremella in Lecanora varia specimens, volume rendering. A–B: specimens without basidiomata, C–H: specimens with T. variae basidiomata, parts of thallus without basidiomata. Green: Tremella, magenta: algal autofluorescence and Lecanora. A. Thallus with Tremella yeasts and algae (*), woody substrate (W). B. Tremella yeasts in the hymenium of L. varia, some of them budding. Asci and paraphyses clearly visible. C–D. Thallus with Tremella yeasts, hyphae and algae (*), C overlay of both channels, D showing only the green channel. The white rectangle shows the Tremella hyphae. E. Close-up of a subset of focal plains of the white rectangle in C and D, showing clamped hyphae with a tremelloid haustorium; the filament (arrow) not attaching to L. varia hyphae. Related to Video S3. F. Germinating Tremella yeast (arrow) in a thallus, surrounded by L. varia hyphae, alga (*). G. Thallus with Tremella yeasts and algae (*). H. Hymenium of L. varia with paraphyses (magenta and green autofluorescence), asci, and Tremella yeasts, one of which is germinating (arrow). Scale 30 μm in A-D; 3 μminE;4μminF;20μminG–H. [Color figure can be viewed at wileyonlinelibrary.com]

Fig 3. Tremella basidiomata in Lecanora chlarotera (A–E) and L. varia specimens (F–G), volume rendering. Green: Tremella, magenta: algal autofluorescence and Lecanora. A. Cross section of T. macrobasidiata basidioma with basidia and Tremella hyphae intermixed with Lecanora hyphae and algae (*). Close-up of the white rectangle in C. B. Tremella yeasts and hyphae deeper down in the basidioma, in a layer below basidia. C. Conidiogenous cell (arrow) of T. macrobasidiata, which gives rise to asteroconidia. This conidiogenous cell is located within the white rectangle in A. Only the green channel and a subset of focal plains shown for clarity. D. Tremella yeasts on thallus, algae (*). E. Tremelloid haustoria of T. macrobasidiata, filament (arrow) not attaching to L. chlarotera hyphae. Related to Video S4. F. T. variae basidioma in cross section. Basidia (arrow), and Tremella hyphae intermixed with L. varia hyphae and algae (*). The Close-up of the white rectangle in H. G. T. variae basidioma on L. varia apothecium margin. Basidia (arrow), basidiospores/yeasts, asci (arrowhead) and paraphyses (PF) intermixed, some basidia clearly in hymenium. Related to Video S5. H. Close-up of F. Tremella variae hyphae (arrow) growing between and around algal cells (*). Only the green channel shown for clarity. Scale 20 μminA–B, F–H; 5 μmin C: 30 μminD,G;4μm in E. [Color figure can be viewed at wileyonlinelibrary.com]

However, we did not observe asteroconidia in the each other, and some of the basidia were clearly formed basidiomata. In the only studied T. variae basidiomata inside the hymenium surrounded by paraphyses that was formed on the thalline margin of a L. varia apo- (Fig. 3G, Video S5). No Tremella hyphae was observed thecium, both basidia and asci were observed next to in samples from specimens without basidiomata.

© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology 6 V. Tuovinen et al. In addition to the specific staining with probes, we used Table S1). At least in some of these specimens, the lack the universal cell wall stain calcofluor white, which binds of detection of T. variae, or any of the two Tremella spp., to β-(1,3) and β-(1,4) polysaccharides, e.g., chitin, for bet- respectively, could be because of divergent Lecanora lin- ter separation of fungal interactions. Our tests revealed eages, as Tremella are assumed to be more or less spe- that calcofluor white binds only weakly or not at all to cies specific (Fig. S4B). Lack of detection could also be Tremella cell walls and was hence not usable for the because of limited sample size for DNA extraction and study of the interspecies cell wall–cell wall contacts in low template concentration for PCR. Despite the in silico this study system. species-specific Tremella primers, several PCR reactions resulted in multiple bands and mixed signal in sequencing, especially in extractions from L. varia specimens, Molecular evidence for the presence of Tremella in the suggesting that even other Tremella species may be pre- Lecanora lichens sent in these thalli. In addition to the observations with FISH, we could verify the presence of both Tremella macrobasidiata and Discussion T. variae in Lecanora specimens without basidiomata by sequencing PCR amplicons produced with in silico Our study gives new and important insights into earlier species-specific primers (Fig. 4, Fig. S1, Table 1). We overlooked aspects of the life cycle of two Tremella spe- conclude that these DNA records correspond to occur- cies associated with crustose Lecanora lichens. Our rence of Tremella yeasts, since we observed only the results show that both T. macrobasidiata and T. variae yeast stage and no hyphae in samples without indeed have a widespread and abundant yeast stage in basidiomata using CLSM. DNA of T. macrobasidiata was their life cycle, and that this life stage is not limited to present in each studied L. chlarotera specimen, but also Lecanora specimens, nor lichen parts, with Tremella in 73% of the studied L. varia specimens without basidiomata. These and previous results by Tuovinen basidiomata and even in 27% of the L. varia specimens et al., 2019 support the hypothesis that dimorphism is that had T. variae basidiomata. DNA of T. variae was pre- common also in lichen-associated Tremella species, con- sent in 58% of the L. varia specimens without sistent with interpretation of yeasts as a typical life cycle basidiomata. We could not verify the presence of either stage of tremellalean fungi in general (Chen, 1998; of the two studied Tremella species in three L. varia Millanes et al., 2011). Following the hypothesis that also specimens (i.e., 92% of L. varia specimens had at least other lichen-associated Tremella species may have a one of the studied Tremella species), nor could we detect prevalent yeast stage in their life cycles, they may be T. variae in any of the L. chlarotera specimens. In two of much more common and abundant symbionts of lichens the L. varia specimens without basidiomata, that we stud- than assumed based on observations of basidiomata ied by FISH and CLSM, T. macrobasidiata and T. variae only, and their distribution ranges may be severely under- were both present according to PCR. The identity of the estimated. Furthermore, assumptions about their lichen visualized Tremella structures in those specimens could specificity and ecology will need to be revisited. unfortunately not be verified, since the probe stains both The association with different, evolutionary distant Tremella species. However, in one of the L. varia speci- groups of lichens has evolved several times in the mens without T. variae basidiomata, and in two speci- Tremellales (Millanes et al., 2011; Liu et al., 2015). The mens with T. variae basidiomata, only T. variae was studied Lecanora lichens are non-stratified crusts and detected with PCR and Tremella yeasts were observed in lack the thick cortex layer in which basidiomycete yeasts all these specimens with CLSM further indicating that have previously been observed to be concentrated in also T. variae occurs in a yeast form in L. varia thalli. some stratified macrolichens (Spribille et al., 2016; The 18 ITS haplotypes of L. chlarotera were mostly not Tuovinen et al., 2019). In the studied Lecanora lichens, structured by geographic location, although some were the Tremella yeast cells are not restricted to the top or only found in Spain and some only in Sweden (Fig. S4A). upper parts of thalli but occur vertically distributed within Our L. varia samples were more variable in their ITS the areoles and can be found even close to the woody (33 haplotypes of which most were singletons) than our substrate. Thus, the space for interaction between L. chlarotera samples (Fig. S4B). The presence or Tremella yeasts and other organisms within the thallus is absence of T. macrobasidiata in L. varia specimens did less limited in these crusts than what has been not strongly depend on the L. varia ITS haplotype. How- suggested for macrolichens (Spribille et al., 2020). ever, the six L. varia specimens where we could detect Bergmann and Werth (2017) studied the intrathalline dis- only T. macrobasidiata, and the three L. varia specimens tribution of Tremella lobariacerum in three Lobaria where neither of the targeted Tremella species could be lichens by quantitative real time PCR, but could detect detected, had a unique L. varia ITS haplotype (Fig. S4B, Tremella DNA only inside or close to basidiomata and

Fig 4. ML tree from ITS sequences of Lecanora, showing the occurrence of associated Tremella species in each sample verified by sequencing. Grey labels represent sequences derived from GenBank for reference of the molecular identity of Lecanora. Samples with visible Tremella basidiomata are marked with a star. Black labels refer to samples where none of the target Tremella could be amplified. Branch lengths are scaled in terms of expected numbers of nucleotide substitutions per site. Maximum likelihood bootstrap percentages are shown above the bra- nches when > 55%. [Color figure can be viewed at wileyonlinelibrary.com]

Table 1. The identity of Tremella in the studied specimens veriﬁed by PCR and sequencing.

Specimens Tremella macrobasidiata Tremella variae Both

Without basidiomata Lecanora chlarotera (n = 23) 100% 0% 0% Lecanora varia (n = 26) 73% 58% 42% With basidiomata Lecanora chlarotera (n = 10) 100% 0% 0% Lecanora varia (n = 11) 27% 100% 27% In total Lecanora chlarotera (n = 33) 100% 0% 0% Lecanora varia (n = 37) 59% 70% 38%

not in thalli without visible basidiomata. Their results indi- We could observe T. variae basidia intermixed with cate that either T. lobariacerum lacks a yeast stage, the asci and paraphyses in the hymenium of one L. varia, yeast stage is restricted to the vicinity of basidiomata, or indicating that the basidium formation is not strictly the non-species specific primers used for qPCR could restricted to the thalline parts of L. varia, although the not detect Tremella in these specimens due to, e.g., low major portion of the basidiomata is formed on the thalline template concentration. In our study, we needed species- parts based on macroscopic observations (Zamora specific primers to amplify T. variae and et al., 2016). In addition, Tremella yeasts were present in T. macrobasidiata by PCR. Microscopic studies, together L. varia hymenia, suggesting that these may germinate with molecular methods, are essential for interpreting the after conjugation and eventually the development of location and life cycle stage of fungal symbionts within basidiomata may start even in the hymenium. The prop- the lichen thallus. erties of a lichen that guide the place for development of As reported here and by Tuovinen et al. (2019), more Tremella basidiomata remain unknown, but different sec- than one Tremella species can co-occur in the same thal- ondary metabolite and polysaccharide composition in dif- lus. Sequences belonging to Tremellales have been ferent parts of the thallus, and different lichens, may play detected from lichen thalli in many metabarcoding studies a role in this. Particularly, the concentration of secondary (Fleischhacker et al., 2015; Zhang et al., 2015; Wang metabolites has been previously reported to correlate et al., 2016; Zhao et al., 2016; Fernández-Mendoza with the presence of a lichen-associated Ascomycete et al., 2017; Banchi et al., 2018), but these studies have Plectocarpon scrobiculatae (Merinero et al., 2015). focused on describing the lichen-inhabiting fungal com- Many Tremellomycetes species are either known or munities only at higher taxonomic levels. Deep, targeted suspected mycoparasites (Spatafora et al., 2017), but the high throughput amplicon sequencing and detailed taxo- nature of the interactions between Tremella and associ- nomic assignments could give a more comprehensive ated lichens remains elusive. The presence of tremelloid picture of the amount of lichen-associated Tremella spe- haustoria in the basidiomata has been considered indica- cies in each lichen specimen, and further shed light on tive of a mycoparasitic lifestyle in lichen-associated the frequency and abundancy of Tremella in lichen sym- Tremella, but not all lichen-associated Tremella species biosis in general. form haustoria (Diederich, 1996). Haustoria-hyphae inter- Regarding the location and distribution of the filamen- actions have been studied in detail in Tremellomycetes tous stage in the lichen thallus, Grube and de los associated with non-lichenized fungi (reviewed in Bauer Ríos (2001) studied microscopically the anatomy and and Oberwinkler, 2008), but rarely in lichens. However, in development of the basidiomata of Biatoropsis usnearum B. usnearum basidiomata, Grube and de los Ríos (2001) (Tremellales) on the stratified macrolichen Usnea rigida, observed B. usnearum haustorial filaments reaching to and could not detect any hyphae connecting the different U. rigida cell walls. While we also did observe typical basidiomata in Usnea thalli. This observation is in line tremelloid haustoria inside the basidiomata of both stud- with ours. In the light of our results, and those of ied Tremella species, we could not trace them clearly Tuovinen and colleagues (2019), it seems probable that attaching to Lecanora hyphae when inspecting the 3D at least the studied Tremella species disperse within and reconstructions. Notably, the cell walls of the fungi are between thalli as basidiospores, conidia or yeasts, and not stained in our study design, which complicates inter- that hyphae are restricted to the basidiomata and its pretation of wall-wall contacts in cases where the dis- vicinity and formed only when the sexual cycle is tance between the stained cytosols is small. In induced. mycoparasitic Tremella, haustorial filaments attach to the

© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Tremella yeasts are abundant in Lecanora lichens 9 cell wall of their host, and a micropore connection staining and CLSM that allows non-disruptive sampling between the cytosols of the two fungi is formed and observation of the structures in 3D has shown to be (Zugmaier et al., 1994; Bauer and Oberwinkler, 2008). a powerful tool in the study of lichen-associated microor- Transmission electron microscopy studies are needed to ganisms in their natural environment (de los Ríos determine whether such connections between Tremella et al., 2002; Cardinale et al., 2008; Aschenbrenner and Lecanora cells occurs, which would shed light on the et al., 2016; Spribille et al., 2016; Tuovinen et al., 2019). functionality of the haustoria in the studied Tremella The method is especially valuable for the study of lichen- species. associated Tremellomycetes, as they are generally miss- Interestingly, in basidiomata of both studied Tremella ing from the cultivable microbiome extracted from lichen species, Tremella hyphae were regularly observed grow- thalli (Muggia et al., 2017; Oh et al., 2020), except for ing around algae, similar to observations of T. lethariae in Fellomyces species (Prillinger et al., 1997; Lopandic wolf lichens (Tuovinen et al., 2019). However, we could et al., 2005). Unfortunately, our attempt to use calcofluor not observe Tremella penetration into the algal cell similar white for identification of cell–cell interactions of different to Lecanora hyphae. Yet again, ultrastructural studies fungi and alga failed, since Tremella cell walls did not would be needed for the interpretation of the wall-wall stain with this widely used fungal cell-wall stain. Notably, interactions between these organisms. Since Tremella Koch and Pimsler (1987) reported the lack of, or un-uni- and Lecanora are indeed distantly related fungi, the type form, binding of calcofluor white to Cryptococcus of contact between each of them and the alga can be (Tremellales). Cryptococcus is known to secrete an expected to be different. We cannot verify or discard exopolysaccharide capsule of glucuronoxylomannans whether there is acquisition and utilization of sugars from (GXMs) around its cell walls (Martinez and the algae by the hyphal stage of Tremella, and to test Casadevall, 2015), and Harrington and Hageage (2003) such a hypothesis more specific studies of the nutrient reported that this polysaccharide capsule did not stain transfer are needed. Moreover, the nutrition source of the with calcofluor white. Spribille et al. (2020) hypothesized Tremella yeasts remains unstudied. In many dimorphic that also lichen-associated Tremella species could species, the nutritional strategy of the yeast and hyphal secrete GMXs to the extracellular interaction matrix in stage is different (Begerow et al., 2017), and may be that macrolichens. Interestingly, Grube and de los Ríos (2001) also for lichen-associated Tremella. observed acidic polysaccharides on the outer parts of The modern view of symbiotic relationships in nature in hyphae of lichen-associated B. usnearum. The lack of general is that they are often observed to be dynamic on calcofluor white binding in the cell walls of the studied a continuum from mutualistic to parasitic, affected both Tremella species may be indicative of presence of such by external factors and by the life cycle stage of the polysaccharides, which may hinder the access of the organism (Johnson et al., 1997; Klironomos, 2003; Jones stain to the cell wall. The presence of relatively thick and Smith, 2004; Sapp, 2004; Johnson, 2010; extracellular material around the Tremella hyphae was Zook, 2015). Furthermore, lichen-associated Tremella lin- clearly visible in CLSM in basidiomata. Alternatively, the eages form several unrelated groups that may potentially cell walls of the studied Tremella species may be poor in have sister taxa with life strategies other than myco- β-(1,3) and β-(1,4) polysaccharides, and rather consist of parasitism (Millanes et al., 2011; Liu et al., 2015). It is α-(1,3) and β-(1,3), (1,6) polysaccharides as in possible that also the nature of symbiotic relations of dif- T. mesenterica (Reid and Bartnicki-Garcia, 1976), leading ferent Tremella species associated with divergent to bad staining with calcofluor white. lichens, which neither form a natural group, varies more Another intriguing aspect of the association between than previously thought. The individual evolutionary paths Tremella and lichens is specificity. Lichen-associated of all symbiotic partners of any given lichen may have Tremella species have been considered to be rather spe- contributed to and resulted in different outcomes of the cific regarding their ascomycete lichen symbionts, a trait symbiotic relationships between Tremella and the other that has been used as an initial character for the species organisms present in a lichen thallus. identification of Tremella basidiomata occurring on a cer- The study of microorganisms in their natural environ- tain lichen (Diederich, 1996; Millanes et al., 2014, 2015, ments is complicated in part due to their tiny size, and 2016; Diederich et al., 2018). To our knowledge, lichen- lichen-associated Tremella species are no exception in associated Tremella for which reference sequences are this regard. They have most often been studied by con- available are largely lacking from environmental ventional bright field microscopy and disruptive sample sequence data (Li et al., 2020), which supports the preparation with KOH, which has provided morphological hypothesis that at least some of them indeed are information useful for taxonomical purposes, but hinders restricted in their occurrence to the lichen symbiosis and the study of spatial structure and physical interactions do not likely occur freely in the surrounding environment. between different organisms in the lichen. Specific In the B. usnearum species complex, the sexual stage of

© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology 10 V. Tuovinen et al. some species is specific to a certain Usnea/Protousnea undertaken using cultures produced in the lab (Begerow species whilst some are generalists within the genera et al., 2017). Understanding an organism requires, how- (Millanes et al., 2014). Genus-level specificity was also ever, knowledge of its whole life cycle under natural con- observed for T. lethariae, which associates in the yeast ditions. Our results add to the growing evidence of the stage with all described Letharia species and for which presence of basidiomycete yeasts in lichens in general basidiomata has been observed on thalli of several of the (Spribille et al., 2016; Cernajováˇ and Škaloud, 2019, taxa (Tuovinen et al., 2019). On the other hand, previous 2020; Tuovinen et al., 2019; Mark et al., 2020), showing molecular studies have detected the yet formally that their occurrence is more common and widespread undescribed species Tremella sp. B in both Bryoria and than previously thought, and the dimorphic life cycle of Letharia lichens without visible basidiomata (Lindgren lichen-associated Tremella species in particular. We have et al., 2015; Tuovinen et al., 2019), indicative of the pres- further shown that, despite growing inside a lichen in ence of a yeast stage of this species in those lichens. seemingly similar microenvironments, the ecological The yeast stage of Tremella sp. B thus seems to be a requirements of the yeast and filamentous stages are not generalist not restricted to a certain lichen genus but may necessarily the same, as both the lichen specificity and still be restricted to lichens belonging to Parmeliaceae. location within the thallus can vary depending on the life No basidiomata has yet been observed for this species, cycle stage in different Tremella species. While it is not and conclusions about the specificity of its sexual cycle possible to make conclusions about the ecological func- can hence not be made. tion of the studied Tremella species or their different life Basidiomata formation in the two Tremella species stages in lichens with the data at hand, it is clear that the studied here seems limited to the respective Lecanora frequency and abundancy at which tremellalean yeasts species, suggesting that the environment provided by a are being recovered in lichen symbiosis deserves deep- certain Lecanora lichen is needed for the completion of ened attention in future studies. the sexual life cycle. However, our results suggest that as the yeast stage of T. macrobasidiata is common even in L. varia lichens, it is less specific than T. variae, which Experimental procedures appears truly specifictoL. varia. Our observation that Lichen material some of the L. varia specimens where we could not detect T. variae were divergent in the Lecanora ITS We collected seven L. chlarotera specimens with sequence may further indicate the higher species speci- Tremella macrobasidiata basidiomata and 12 specimens ficity of T. variae. Millanes and colleagues (2014) showed without basidiomata from Spain. In all of these localities that switching association from one Usnea species to specimens with and without basidiomata were present. In another has promoted speciation in B. usnearum species addition, we collected three L. chlarotera specimens with complex, but the factors driving speciation in other T. macrobasidata basidiomata and 11 without lichen-associated Tremella are still largely unknown. The basidiomata from Sweden. No basidiomata were lack of species-specificity observed for the yeast stage of observed on other L. chlarotera lichens in the Swedish T. macrobasidiata could be related to a relatively recent localities where specimens without basidiomata were col- speciation history within the group of Lecanora- and lected, but no thorough inventory was made. All L. varia Lecidea-associated Tremella species (Zamora specimens were collected from Spain, as T. variae is not et al., 2018). Increased screening for these Tremella spe- known from Sweden, 11 of them with and 26 without cies in other Lecanora lichens and populations will help T. variae basidiomata. In one of the localities only speci- to better understand their specificity and general distribu- mens without basidiomata were observed but in all others tion patterns. Taken together, it seems that some lichen- both specimens with and without basidiomata were pre- associated Tremella species are species specific, whilst sent. Detailed information on the sampling design and others are generalists associated with several related the specimens can be found in (Fig. S1, Table S1). species. In order to verify the identity of the studied Lecanora and Tremella species, we extracted DNA from all of the Lecanora specimens and used it for PCR and Sanger Conclusions sequencing, detailed below. From specimens without In general, the ecology of the yeast stage of dimorphic Tremella basidiomata, we used part of the thallus basidiomycetes with conspicuous fruiting bodies – spe- together with apothecia for DNA extraction. From speci- cies traditionally studied by mycologists and lichenolo- mens with Tremella basidiomata, we made two separate gists instead of microbiologists – is much less DNA extractions: one with a basidioma and one with a understood compared with their filamentous stages, thallus piece together with apothecia without since investigations on the yeast stage have mainly been basidiomata. For the study of the occurrence and location

© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Tremella yeasts are abundant in Lecanora lichens 11 of different life stages of Tremella in the Lecanora speci- designed four different probes for the two Lecanora spe- mens, we used FISH and CLSM. For these, we chose six cies, each mono labelled with Cy5 at 50 end, and evalu- specimens of each lichen, three with basidiomata and ated their performance in apothecia of both Lecanora three without. More specifically, for each L. chlarotera species. Unfortunately, the intensity of the specimen with basidiomata we studied apothecia with autofluorescence of hyphae of both Lecanora species on and without basidiomata as well as thallus parts. For red wavelengths (based on comparisons with negative L. chlarotera specimens without basidiomata we studied controls with no probes) varied considerably between dif- both apothecia and thallus parts. For each L. varia speci- ferent parts of thalli, as well as between different specimen with basidiomata we studied basidiomata, thallus mens, and in most cases separation between the weak parts without basidiomata and apothecia, and for speci- signal intensity from the probe and the autofluorescence mens without basidiomata thallus parts and apothecia with certainty was not possible. Notably, hyphae close to (Fig. S1). algae are most intensely autofluorescent on red wavelengths. Also secondary metabolites, basidia, and Tremella yeasts to some degree, autofluoresce red. Fixation Lecanora varia hyphae and apothecia have stronger Directly after collection, we placed the specimens for autofluorescence than L. chlarotera. The woody sub- FISH on moisturized filter paper on Petri dishes, sprayed strate is highly autofluorescent on both the green and red them with ddH2O, and covered loosely with a lid. We let wavelengths. In addition, most of the fungal cells, and the specimens dry, and moisturized them again, repeat- especially the paraphyses, are autofluorescent on green. ing this cycle for up to 5 days in order to make the lichens We used each probe in combination with helper probes physically active. Then, we cut pieces of moist thalli with (Table S2). attached woody substrate by hand and placed them in 1× PBS (pH 7.4) in 0.2 ml test tubes in a vacuum desic- cator. We applied the vacuum for c.a. 10 s at a time, FISH and cell wall staining removed the tubes and gently tapped them against a We performed the permeabilization, hybridization and bench, and repeated the procedure until the air from the washing steps for the fixed and acetone treated samples tissues was removed and the pieces sunk to the bottom in 0.2 ml test tubes as described in Tuovinen et al., 2019. of the tubes. We replaced the PBS with 4%, methanol We included a negative control without probes for each free, formaldehyde for fixation at +4C for 2 h. After fixa- hybridization reaction and each species. In addition to tion, we washed the samples in 1× PBS three times, the specific staining with probes, we tried the universal 10 min each. We then incubated the samples in acetone stain calcofluor white (Sigma-Aldrich) on seven thalli for 2–8 h in order to dissolve secondary metabolites with (three L. chlarotera with T. macrobasidiata and four strong autofluorescence, followed with PBS wash as L. varia with T. variae). Calcofluor white binds to β-1, above. The PBS was removed, and the sample frozen at 3 and β-1, 4 polysaccharides like chitin and cellulose, −20C until FISH. staining in general both fungal and algal cells, and we aimed for better separation of wall-wall interactions FISH probe design between the different fungi. We added calcofluor white to samples after the washing step after FISH, incubated for We designed a FISH probe matching the studied 10 min in darkness at room temperature, and washed the Tremella species based on the available LSU sequences samples with 1X PBS. We mounted all samples on (T. macrobasidiata: GenBank: KT334594.1, KT334595.1 microscopy slides with SlowFade Diamond (Thermo and T. variae: KT334598.1, KT334599.1), with no match Fisher). for Lecanora (Table S2). Note that this probe matches several other, but not all, Tremellales species, and cannot differentiate between T. macrobasidiata and CLSM T. variae, for which purpose we used PCR and sequencing. The probe was first labelled with 6-FAM at 50 end, We studied the hybridized slides by Zeiss LSM710 confo- but once the function of the probe was verified it was cal microscope, with Plan-Apochromat 63x NA 1.4 oil labelled also with FITC at the 30 end in order to enhance DIC M27 objective. We used 488 and 633 laser lines for the fluorescent signal. We evaluated the performance of excitation of 6-FAM + FITC labelled and Cy5-labelled the Tremella probe in both T. macrobasidiata and probes, respectively. The detection wavelengths were T. variae basidiomata and could achieve a high enough 493–598 nm for green and 638–797 nm for red wave- signal intensity on green wavelengths with the double- lengths. We used one Airy unit pinhole and simultaneous labelled Tremella probe to detect hybridized cells. We scanning. We acquired Z stacks of lichen thalli with

© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology 12 V. Tuovinen et al. optimal settings calculated by the Zen blue software of was tried. We sent the samples for Sanger sequencing to the microscope (Zeiss). Macrogen in the Netherlands.

Sequence analyses Image processing We used Geneious 10.2.3 or R11.1.5 (Biomatters) to We processed the 2D-images using Fiji (Schindelin check the chromatograms and trim the sequence ends, et al., 2012, 2015) or Zen blue (Zeiss), and prepared the and Aliview for initial sequence alignment volume rendering with Zen blue. We used median filtering (Larsson, 2014). For the Lecanora ITS, we aligned our for noise reduction and adjusted colour balance for the newly generated sequences with closely related clarity of presentation. sequences available in GenBank (Table S4) with MAFT v.7 online service (Kuraku et al., 2013; Katoh et al., 2019), using the G-INS-I algorithm and otherwise Molecular methods default parameters. We performed a ML analysis of the We pulverized apothecia together with a small piece of alignment with raxmlGUI 2.0.0-beta.6 (Stamatakis, 2014; thallus, and a basidioma in separate test tubes with a Edler et al., 2021) with the GTRGAMMAI substitution pestle after freezing in liquid nitrogen. Subsequently, we model, 100 runs, and 1000 bootstrap replicates. We used extracted the total DNA using the DNeasy Plant Mini kit Lecidella patavina as an outgroup based on the phylog- (QIAGEN) following the manufacturer’s instructions with eny by Zhao and colleagues (2016). We constructed ITS- a prolonged incubation at 65 C for 1 h and elution of the haplotype networks for the Lecanora species with DNA in 50 μl of the elution buffer. haploNet in Pegas (Paradis, 2010) in R. The newly gen- We used primers ITS1F (Gardes and Bruns, 1993), erated sequences are available in GenBank with acces- ITS4 (White et al., 1990), LR3 (Vilgalys and sion numbers MW374997-MW375029 (Table S1). Hester, 1990) and TelLSU3-3 (Table S3) for PCR and sequencing of the Lecanora ITS. We used HotStart RTG Acknowledgements beads (Illustra™) for the PCR with the following touch down program: four cycles of initial annealing at 60C, This study was funded by the Swedish Research Council, four cycles at 58C and 32 cycles at 56C. We used the grant VR 2016-03589, the Spanish Ministry of Science (for- PCR primers as sequencing primers. mer Ministry of Economy and Competitiveness) We designed specific PCR and sequencing primers for CGL2016-80371-P, and the Swedish Taxonomy Initiative (administered by the Swedish Species Information Centre) each Tremella species (Table S3). The most successful grant STI 2016-27 4.3. We thank the Uppsala University Bio- primer pairs were TmM_ITS_970F for T. macrobasidiata Vis facility for access to CLSM. We thank Juan Carlos and TmV_ITS_1008F for T. variae in combination with Zamora for discussions on Tremella macrobasidiata and are Basid-LSU3-3 (Millanes et al., 2011) (Table S3). From grateful to Bodil Cronholm (The Laboratory of Molecular Sys- samples that resulted in multiple bands, we extracted the tematics (MSL) at the Swedish Museum of Natural History) band of correct size with MinElute Gel Extraction kit for her skilful lab assistance. We thank two anonymous (Qiagen). Based on comparison with band size from reviewers for their valuable comments to improve this manuscript. other samples verified to be T. macrobasidiata and T. variae by sequencing, only bands with completely different lengths were excluded from sequencing reactions. Author contributions We re-amplified samples with negative PCR results with AMM, MW, and VT designed the project. VT, AMM and different PCR primer combinations (Table S3). The PCR SF-R collected the specimens. VT and SF-R conducted reaction for Tremella was done with Phusion HF DNA sample preparation, and VT conducted FISH, CLSM, the Polymerase kit (ThermoFisher). For the primer pair data analyses and wrote the original draft. MW, AMM TmM_ITS_970F/BasidLSU3-3, we used touch down and AR financed and supervised the project, and all co- PCR with four cycles of annealing at 64C, four cycles at authors contributed with writing to the final version. 62C and 32 cycles at 60C. For the primer pair TmV_ITS_1008F/BasidLSU3-3, we used the same touch down cycle as for the Lecanora ITS. Tremella was most References often sequenced only with BasidLSU3-3 due difficulties in Aschenbrenner, I.A., Cernava, T., Berg, G., and Grube, M. acquiring usable sequences with the forward PCR (2016) Understanding microbial multi-species symbioses. primer. When clean sequence (i.e. no double peaks) was Front Microbiol 7: 180. not received with BasidLSU3-3, any of the newly Banchi, E., Stankovic, D., Fernández-Mendoza, F., designed primers or BasidLSU1-3 (Millanes et al., 2011) Gionechetti, F., Pallavicini, A., and Muggia, L. (2018) ITS2

metabarcoding analysis complements lichen mycobiome the identification of mycorrhizae and rusts. Mol Ecol 2: diversity data. Mycol Prog 17: 1049–1066. 113–118. Bandoni, R.J. (1995) Dimorphic heterobasidiomycetes, tax- Goward, T. (2008) Nameless little things. Evansia 25:54–56. onomy and parasitism. Stud Mycol 38:13–27. Grube, M., and de los Ríos, A. (2001) Observations on Bauer, R., and Oberwinkler, F. (2008) Cellular Biatoropsis usnearum, a lichenicolous heterobasidiomycete, basidiomycete–fungus interactions. In Plant Surface and other gall-forming lichenicolous fungi, using different Microbiology, Varma, A., Abbott, L., Werner, D., and microscopical techniques. Mycol Res 105: 1116–1122. Hampp, R. (eds). Berlin: Springer, pp. 267–279. Grube, M., and Wedin, M. (2016) Lichenized fungi and the Begerow, D., Kemler, M., Feige, A., and Yurkov, A. (2017) evolution of symbiotic organization. Microbiol Spectr Parasitism in yeasts. In Yeasts in Natural Ecosystems: 4:6. Ecology, Buzzini, P., Lachance, M.-A., and Yurkov, A. Harrington, B.J., and Hageage, G.J. (2003) Calcofluor white: (eds). Cham: Springer International Publishing, a review of its uses and applications in clinical mycology pp. 179–210. and parasitology. Lab Med 34: 361–367. Bergmann, T.C., and Werth, S. (2017) Intrathalline distribu- Johnson, N.C. (2010) Resource stoichiometry elucidates the tion of two lichenicolous fungi on Lobaria hosts — an anal- structure and function of arbuscular mycorrhizas across ysis based on quantitative real- time PCR. Herzogia, 30: scales. New Phytol 185: 631–647. 253–271. Johnson, N.C., Graham, J.-H., and Smith, F.A. (1997) Func- Cardinale, M., Vieira de Castro, J., Müller, H., Berg, G., and tioning of mycorrhizal associations along the mutualism– Grube, M. (2008) In situ analysis of the bacterial commu- parasitism continuum. New Phytol 135: 575–585. nity associated with the reindeer lichen Cladonia Jones, M.D., and Smith, S.E. (2004) Exploring functional def- arbuscula reveals predominance of Alphaproteobacteria: initions of mycorrhizas: are mycorrhizas always mutual- lichen-associated bacterial community. FEMS Microbiol isms? Can J Bot 82: 1089–1109. Ecol 66:63–71. Katoh, K., Rozewicki, J., and Yamada, K.D. (2019) MAFFT Castelle, C.J., and Banfield, J.F. (2018) Major new microbial online service: multiple sequence alignment, interactive groups expand diversity and alter our understanding of the sequence choice and visualization. Brief Bioinform 20: tree of life. Cell 172: 1181–1197. 1160–1166. Cernajová,ˇ I., and Škaloud, P. (2019) The first survey of Klironomos, J.N. (2003) Variation in plant response to native Cystobasidiomycete yeasts in the lichen genus Cladonia; and exotic arbuscular mycorrhizal fungi. Ecology 84: with the description of Lichenozyma pisutiana gen. nov., 2292–2301. sp. nov. Fungal Biol 123: 625–637. Koch, H.H., and Pimsler, M. (1987) Evaluation of Uvitex 2B: fi fl Cernajová,ˇ I., and Škaloud, P. (2020) Lessons from culturing a nonspeci c uorescent stain for detecting and identifying – lichen soredia. Symbiosis 82: 109–122. fungi and algae in tissue. Labmedicine, 18: 603 606. Chen, C.J. (1998) Morphological and molecular studies in Kuraku, S., Zmasek, C.M., Nishimura, O., and Katoh, K. the genus Tremella. Bibl Mycol 174:1–225. (2013) aLeaves facilitates on-demand exploration of meta- de los Ríos, A., Ascaso, C., and Grube, M. (2002) Infection zoan gene family trees on MAFFT sequence alignment mechanisms of lichenicolous fungi studied by various server with enhanced interactivity. Nucleic Acids Res 41: – microscopic techniques. Bibl Lichenol 82: 153–161. W22 W28. Diederich, P. (1996) The lichenicolous heterobasidiomycetes. Kurtzman, C.P., Fell, J.W., and Boekhout, T. (2011) The Bibl Lichenol 61:1. Yeasts: A Taxonomic Study, 5th ed. San Diego: Elsevier Diederich, P., Lawrey, J.D., and Ertz, D. (2018) The 2018 Science. classification and checklist of lichenicolous fungi, with Larsson, A. (2014) AliView: a fast and lightweight alignment 2000 non-lichenized, obligately lichenicolous taxa. The viewer and editor for large datasets. Bioinformatics 30: – Bryologist 121: 340. 3276 3278. Edler, D., Klein, J., Antonelli, A., and Silvestro, D. (2021) Li, A.-H., Yuan, F.-X., Groenewald, M., Bensch, K., raxmlGUI 2.0 beta: a graphical interface and toolkit for Yurkov, A.M., Li, K., et al. (2020) Diversity and phylogeny phylogenetic analyses using RAxML. Methods Ecol Evol of basidiomycetous yeasts from plant leaves and soil: pro- 12: 373–377. posal of two new orders, three new families, eight new Ekman, S. (1999) Pcr optimization and troubleshooting, with genera and one hundred and seven new species. Stud – special reference to the amplification of ribosomal dna in Mycol 96:17 140. lichenized fungi. The Lichenologist 31: 517–531. Lin, X. (2009) Cryptococcus neoformans: morphogenesis, – Fernández-Mendoza, F., Fleischhacker, A., Kopun, T., infection, and evolution. Infect Genet Evol 9: 401 416. Grube, M., and Muggia, L. (2017) ITS1 metabarcoding Lindgren, H., Diederich, P., Goward, T., and Myllys, L. highlights low specificity of lichen mycobiomes at a local (2015) The phylogenetic analysis of fungi associated with scale. Mol Ecol 26: 4811–4830. lichenized ascomycete genus Bryoria reveals new line- Fleischhacker, A., Grube, M., Kopun, T., Hafellner, J., and ages in the Tremellales including a new species Tremella – Muggia, L. (2015) Community analyses uncover high huuskonenii hyperparasitic. Fungal Biol 119: 844 856. diversity of lichenicolous fungi in alpine habitats. Microb Liu, X.-Z., Wang, Q.-M., Göker, M., Groenewald, M., Ecol 70: 348–360. Kachalkin, A.V., Lumbsch, H.T., et al. (2015) Towards an fi Gardes, M., and Bruns, T.D. (1993) ITS primers with integrated phylogenetic classi cation of the – enhanced specificity for basidiomycetes - application to Tremellomycetes. Stud Mycol 81:85 147.

Lopandic, K., Molnár, O., and Prillinger, H. (2005) Fel- Sánchez-Martınez,́ C., and Pérez-Martın,́ J. (2001) Dimor- lomyces mexicanus sp. nov., a new member of the yeast phism in fungal pathogens: Candida albicans and genus Fellomyces isolated from lichen Cryptothecia Ustilago maydis—similar inputs, different outputs. Curr rubrocincta collected in Mexico. Microbiol Res 160:1–11. Opin Microbiol 4: 214–221. Mark, K., Laanisto, L., Bueno, C.G., Niinemets, Ü., Sapp, J. (2004) The dynamics of symbiosis: an historical Keller, C., and Scheidegger, C. (2020) Contrasting co- overview. Can J Bot 82: 1046–1056. occurrence patterns of photobiont and cystobasidiomycete Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., yeast associated with common epiphytic lichen species. Longair, M., Pietzsch, T., et al. (2012) Fiji: an open-source New Phytol 227: 1362–1375. platform for biological-image analysis. Nat Methods 9: Martinez, L.R., and Casadevall, A. (2015) Biofilm formation 676–682. by Cryptococcus neoformans. Microbiol Spectr 3:1–11. Schindelin, J., Rueden, C.T., Hiner, M.C., and Eliceiri, K.W. Merinero, S., Bidussi, M., and Gauslaa, Y. (2015) Do lichen (2015) The ImageJ ecosystem: an open platform for bio- secondary compounds play a role in highly specific fungal medical image analysis. Mol Reprod Dev 82: 518–529. parasitism? Fungal Ecol 14: 125–129. Spatafora, J.W., Aime, M.C., Grigoriev, I.V., Martin, F., Millanes, A.M., Diederich, P., Ekman, S., and Wedin, M. Stajich, J.E., and Blackwell, M. (2017) The fungal tree of (2011) Phylogeny and character evolution in the jelly fungi life: from molecular systematics to genome-scale phyloge- (Tremellomycetes, Basidiomycota, fungi). Mol Phylogenet nies. Microbiol Spectr 5:1–32. Evol 61:12–28. Spribille, T. (2018) Relative symbiont input and the lichen Millanes, A.M., Diederich, P., Westberg, M., Pippola, E., and symbiotic outcome. Curr Opin Plant Biol 44:57–63. Wedin, M. (2015) Tremella cetrariellae (Tremellales, Spribille, T., Tagirdzhanova, G., Goyette, S., Tuovinen, V., Basidiomycota, Fungi), a new lichenicolous fungus on Case, R., and Zandberg, W.F. (2020) 3D biofilms: in sea- Cetrariella delisei. Lichenologist 47: 359–368. rch of the polysaccharides holding together lichen symbio- Millanes, A.M., Diederich, P., Westberg, M., and Wedin, M. ses. FEMS Microbiol Lett 367:1–17. (2016) Three new species in the Biatoropsis usnearum Spribille, T., Tuovinen, V., Resl, P., Vanderpool, D., complex. Herz 29: 337–354. Wolinski, H., Aime, M.C., et al. (2016) Basidiomycete Millanes, A.M., Diederich, P., Westberg, M., and Wedin, M. yeasts in the cortex of ascomycete macrolichens. Science (2021) Crittendenia gen. Nov., a new lichenicolous lineage 353: 488–492. in the Agaricostilbomycetes (Pucciniomycotina), and a Stamatakis, A. (2014) RAxML version 8: a tool for phyloge- review of the biology, phylogeny and classification of netic analysis and post-analysis of large phylogenies. Bio- lichenicolous heterobasidiomycetes. Lichenologist 53 informatics 30: 1312–1313. ,103–116. Tuovinen, V., Ekman, S., Thor, G., Vanderpool, D., Spribille, T., Millanes, A.M., Truong, C., Westberg, M., Diederich, P., and and Johannesson, H. (2019) Two basidiomycete fungi in the Wedin, M. (2014) Host switching promotes diversity in host- cortex of wolf lichens. Curr Biol 29:476–483.e5. specialized mycoparasitic fungi: uncoupled evolution in the U’Ren, J.M., Lutzoni, F., Miadlikowska, J., and Arnold, A.E. Biatoropsis-Usnea system. Evolution 68: 1576–1593. (2010) Community analysis reveals close affinities Muggia, L., Kopun, T., and Grube, M. (2017) Effects of between endophytic and endolichenic fungi in mosses growth media on the diversity of culturable fungi from and lichens. Microb Ecol 60: 340–353. lichens. Molecules 22: 824. U’Ren, J.M., Lutzoni, F., Miadlikowska, J., Laetsch, A.D., Nagy, L.G., Ohm, R.A., Kovács, G.M., Floudas, D., Riley, R., and Arnold, A.E. (2012) Host and geographic structure of Gácser, A., et al. (2014) Latent homology and convergent endophytic and endolichenic fungi at a continental scale. regulatory evolution underlies the repeated emergence of Am J Bot 99: 898–914. yeasts. Nat Commun 5: 4471. Vilgalys, R., and Hester, M. (1990) Rapid genetic identifica- Noh, H.-J., Lee, Y.M., Park, C.H., Lee, H.K., Cho, J.-C., and tion and mapping of enzymatically amplified ribosomal Hong, S.G. (2020) Microbiome in Cladonia squamosa is DNA from several Cryptococcus species. J Bacteriol 172: vertically stratified according to microclimatic conditions. 4238–4246. Front Microbiol 11: 268. Wang, Y., Zheng, Y., Wang, X., Wei, X., and Wei, J. (2016) Oh, S.-Y., Yang, J.H., Woo, J.-J., Oh, S.-O., and Hur, J.-S. Lichen-associated fungal community in Hypogymnia (2020) Diversity and distribution patterns of endolichenic hypotrypa (Parmeliaceae, Ascomycota) affected by geo- fungi in Jeju Island, South Korea. Sustainability 12: 3769. graphic distribution and altitude. Front Microbiol 7: 1231. Pace, N.R. (1997) A molecular view of microbial diversity White, T.J., Bruns, T., Lee, S., and Taylor, J. (1990) Amplifi- and the biosphere. Science 276: 734–740. cation and direct sequencing of fungal ribosomal rna Paradis, E. (2010) pegas: an R package for population genes for phylogenetics. In PCR Protocols, Innis, M.A., genetics with an integrated–modular approach. Bioinfor- Gelfand, D.H., Sninsky, J.J., and White, T.J. (eds). San matics 26: 419–420. Diego: Academic Press, pp. 315–322. Prillinger, H., Kraepelin, G., Lopandic, K., Schweigkofler, W., Zamora, J.C., Millanes, A.M., Etayo, J., and Wedin, M. Molnár, O., Weigang, F., and Dreyfuss, M.M. (1997) New (2018) Tremella mayrhoferi, a new lichenicolous apecies species of Fellomyces isolated from epiphytic lichen spe- on Lecanora allophana. Herzogia 31: 666–676. cies. Syst Appl Microbiol 20: 572–584. Zamora, J.C., Millanes, A.M., Wedin, M., Rico, V.J., and Reid, I.D., and Bartnicki-Garcia, S. (1976) Cell-wall composi- Pérez-Ortega, S. (2016) Understanding lichenicolous tion and structure of yeast cells and conjugation tubes of heterobasidiomycetes: new taxa and reproductive innova- Tremella mesenterica. J Gen Microbiol 96:35–50. tions in Tremella s.l. Mycologia 108: 381–396.

Zamora, J.C., Pérez-Ortega, S., and Rico, V.J. (2011) Table S4. Reference sequences acquired from the GenBank Tremella macrobasidiata (Basidiomycota, Tremellales), a for the verification of the identity of the studied Lecanora new lichenicolous fungus from the Iberian Peninsula. The specimens. Lichenologist 43: 407–415. Fig. S1. Schematic of the sampling for DNA extractions used Zhang, T., Wei, X.-L., Zhang, Y.-Q., Liu, H.-Y., and Yu, L.-Y. for PCR, and fluorescent in situ hybridization (FISH). The (2015) Diversity and distribution of lichen-associated fungi arrows in the macrophotographs point to the basidiomata of in the Ny-Ålesund region (Svalbard, high Arctic) as rev- Tremella macrobasidiata on Lecanora chlarotera apothe- ealed by 454 pyrosequencing. Sci Rep 5: 14850. cium and T. variae on L. varia thallus. Scale bars 1 mm. Zhao, X., Leavitt, S.D., Zhao, Z.T., Zhang, L.L., Arup, U., Fig. S2. Lecanora chlarotera specimens with Tremella Grube, M., et al. (2016) Towards a revised generic classi- macrobasidiata basidioma, volume rendering. Green: fi cation of lecanoroid lichens (Lecanoraceae, Ascomycota) Tremella, magenta: algal autofluorescence and Lecanora.A- based on molecular, morphological and chemical evi- B) Cross section of a basidiomata with basidia (arrow), – dence. Fungal Divers 78: 293 304. basidiospores (arrowhead), Tremella hyphae intermixed with — ’ Zook, D. (2015) Symbiosis evolution s co-author. In Reticu- L. chlarotera hyphae. B) As A, only the green channel late Evolution, Gontier, N. (ed). Cham: Springer Interna- shown. C) Basidioma shown from above, slightly tilted, – tional Publishing, pp. 41 80. showing plenty of basidiospores (arrowhead). D) A spore Zugmaier, W., Bauer, R., and Oberwinkler, F. (1994) Myco- germinating to a hypha, which gives rise to a parasitism of some Tremella species. Mycologia 86: ballistoconidium (arrowhead), yeasts possibly conjugating – 49 56. (arrow) inside a basidioma, basidia (B). Only the green channels shown for clarity. E) Germinating yeast inside a Supporting Information basidiomata. F) L. chlarotera hymenium without T. macrobasidiata basidiomata with Tremella yeasts and Additional Supporting Information may be found in the online Lecanora asci (arrow). G) L. chlarotera apothecium and version of this article at the publisher’s web-site: adjacent thallus in cross section. Woody substrate (W), thalline margin (TM), hymenium (H), Tremella yeasts (arrow), Movie S1 Volume rendering of germinating Tremella yeasts algae (*). Scale 30 μm in A-C, F-G; 10 μminD;2μminE. (green) in Lecanora chlarotera (magenta) thallus in a specimen without basidiomata. Related to Fig. 2E. Only the green Fig. S3. Lecanora varia specimens with Tremella variae channel shown for clarity. basidiomata A-F, specimen without basidiomata G, volume rendering, except for E. Green: Tremella, magenta: algal and Movie S2 Volume rendering of germinating Tremella yeasts secondary metabolite autofluorescence and Lecanora. A-B) (green) in Lecanora chlarotera thallus (magenta) in a speci- Cross section of a basidioma with basidia (arrow), basidio- men without basidiomata. Related to Fig. 2F. spores, Tremella hyphae (arrowhead) intermixed with Movie S3 Volume rendering of Lecanora varia thallus part L. chlarotera hyphae and close to algae (*). Note the heavy were basidioma not yet visible from a specimen with visible red autofluorescence from the secondary metabolites on the basidiomata. Tremella hyphae (green) with haustoria, fila- basidioma surface. B) As A, only the green channel shown. ments not attaching to L. varia hyphae (magenta). Related to C) Basidioma shown from above slightly tilted, showing, Fig. 3E. basidia (arrow), plenty of basidiospores (arrowhead) and the Movie S4 Volume rendering of Tremella macrobasidiata heavy autofluorescence from secondary metabolites. D) fi basidioma, with Tremella haustorial laments (green) not As C, only the green channel shown. E) One optical attaching to Lecanora chlarotera hyphae (magenta). Related section of algal layer in T. variae basidiomata, related to to Fig. 4E. Fig. 4F. L. varia haustoria (arrow) penetrates the algae (*). Movie S5 Z-series of optical slices/focal planes of Lecanora Large amount of polysaccharides surround the Tremella varia apothecium with a Tremella variae basidioma visible on hyphae (arrowhead). F) L. varia hymenium with Tremella the thalline margin, from top of the basidiomata to the inner yeasts, Lecanora asci (arrowhead) and paraphyses (PF). G) parts. The first slices show the secondary metabolite L. varia apothecium with Tremella yeasts both in hymenium autofluorescence (magenta) on top of the basidioma. Below (H) and thalline margin (TM), algae. Scale 30 μm in A-B, the surface, first basidiospores (green) emerge. In deeper F,G; 20 μm in C-D; 10 μminE. layers, both asci (magenta) and basidia of T. variae (green) Fig. S4. Lecanora-ITS haplotype networks with the identity next to each other, surrounded by paraphyses (magenta and of the observed Tremella species in the same specimen and fl green auto uorescence). Related to Fig. 4G. the presence of basidiomata. The size of the circle reflects Table S1 Lichen specimens used in this study with informa- the amount of identical ITS haplotypes. A: Lecanora tion on Tremella and Lecanora identity. chlarotera specimens from Spain and Sweden. All speci- Table S2 Probes and fluorophores used to label them, mens had T. macrobasidiata. Individuals with visible unlabeled helper probes and the cell-wall stain used for fluo- basidiomata are marked with a star. B: All L. varia speci- rescent in situ hybridization in this study. mens came from Spain. We could not detect any of the two Table S3. Primers and their targets used in this study. studied Tremella species in three L. varia specimens.