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).

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