These are the questions base on the article that I attached below. 1) What is the implication that there are no 'fixed sequence differences' between Bryoria fremontii and B. tortuosa? 2) Why is the ri

fremontii , which is dark brown (11 ). Recent phylogenetic analyses have failed to detect any fixed sequence differences between the two species in either the mycobiont (Ascomyc o- ta, Lecanoromycetes, Bryoria ) or the photobiont (Vi- ridiplantae, Trebouxia simplex ) when considering four and two loci, respectively ( 12, 13 ). We hypothesized that differe n- tial gene expression might account for the increased pr o- duction of vulpinic acid in B. tortuosa.

We first selected 15 thalli (six from B. fremontii and nine from B. tortuosa , all free from visible parasitic infection) from sites across western Montana, USA, for mRNA tra n- scriptome sequencing. An initial transcriptome -wide anal y- sis of single nucleotide polymorphisms (SNPs) for Ascomycota and Viridiplantae transcript subsets showed no correlation between genotype and phenotype in B. fremontii and B. tortuosa , confirming previous results ( 12, 13 ) (Fig. 1, A and B). Next, we estimated transcript abundances by mappin g raw reads back to a single, pooled metatranscri p- Basidiomycete yeasts in the cortex of ascomycete macrolichens Toby Spribille, 1,2* Veera Tuovinen, 3,4 Philipp Resl, 1 Dan Vanderpool, 2 Heimo Wolinski, 5 M. Catherine Aime, 6 Kevin Schneider, 1† Edith Stabentheiner, 1 Merje Toome- Heller, 6‡ Göran Thor, 4 Helmut Mayrhofer, 1 Hanna Johannesson, 3 John P. McCutcheon 2,7 1Institute of Plant Sciences, NAWI Graz, University of Graz, 8010 Graz, Austria. 2Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA. 3Department of Organismal Biology, Uppsala University, Norbyvägen 18D, 752 36 Uppsala, Sweden. 4Department of Ecology, Swedish University of Agricultural Sciences, Post Office Box 7044, SE -75007 Uppsala, Sweden. 5Institute of Molecular Biosciences, BioTechMed -Graz, University of Graz, 8010 Graz, Austria. 6Purdue University, Department of Botany and Plant Pathology, West Lafayette, IN 47907, USA. 7Program in Integrated Microbial Biodiversity, Canadian Institute for Advanced Research, Toronto, Ontario, Canada. *Corresponding author. Email: [email protected] †Present address: Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Gla sgow, Glasgow G12 8QQ, UK. ‡Present address: Plant Health and Environmental Laboratory, Ministry for Primary Industries, Auckland, New Zealand. For over 140 years, lichens have been regarded as a symbiosis between a single fungus, usually an ascomycete, and a photosynthesizing partner. Other fungi have long been known to occur as occasional parasites or endophytes, but the one lichen –one fungus paradigm has seldom been questioned. Here we show that many common lichens are composed of the known ascomycete, the photosynthesizing partner, and, unexpectedly, specific basidiomycete yeasts. These yeasts are embedded in the cortex, and their abundance correlates with previously unexplained variations in phenotype. Basidiomycete lineages maintain close associations with specific lichen species over large geographical distances and have been found on six continents. The structurally important lichen cortex, long treated as a zone of differentiated ascomycete cells, appears to consistently contain two unrelated fungi. First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 1 on September 26, 2017 http://science.sciencemag.org/ Downloaded from tome assembly and binning by taxon. Restricting our anal- yses to Ascomycota and Viridiplantae revealed little diffe r- ential transcript abundance associated with phenotype (Fig.

1, C and E). Taken together, th ese analyses confirm previous conclusions that the two lichen species are nomenclatural synonyms ( 12) but still provide no explanation for the u n- derlying phenotypes (which we shall continue to refer to by their species names for convenience). However, by e xpand- ing the taxonomic range to consider all Fungi, we found 506 contigs with significantly higher abundances in vulpinic acid –rich B. tortuosa thalli. A majority of these contigs were annotated as Basidiomycota (Fig. 1D). These data suggested that a previ ously unrecognized basidiomycete was present in thalli of both species but was more abundant whenever vulpinic acid was present in large amounts. We next sought to determine whether this uncharacte r- ized basidiomycete was specific to the studied Bryoria sp e- cies or could be found in other lichens. From metatranscriptome contigs containing ribosomal RNA (rRNA) basidiomycete sequences, we designed specific pri- mers for ribosomal DNA [rDNA; 18S , internal transcribed spacer (ITS), and D1D2 domains of 28S ) to screen lichens growing physically adjacent to Bryoria in Montana forests.

Each assayed lichen species carried a genetically distinct strain of the basidiomycete, indicating a high degree of specificity. Furthermore, we found that Letharia vulpina, a common lichen species growing intermixed with Bryoria , maintained basidiomycete genotypes that were distinct from those in Bryoria , not only in Montana but also in northern Europe (fig. S1). When assaying for the basidiom y- cete across the seven main radiations of macr olichens in the class Lecanoromycetes, we found related basidiomycete li n- eages associated with 52 lichen genera from six continents, including in 42 of 56 sampled genera of the family Pa r- meliaceae (fig. S2). As a whole, these data indicate that b a- sidiomyce te fungi are ubiquitous and global associates of the world’s most speciose radiation ( 14) of macrolichens. To place the basidiomycete lineages in a phylogenetic context, we generated a 349 -locus phylogenomic tree by u s- ing gene sequences inferred from our transcriptome data set and other available genomes (table S1). This analysis placed the basidiomycete as sister to Cystobasidium minutum (class Cystobasidiomycetes, subphylum Pucciniomycotina) with high support (Fig. 2A). The only previously known l i- chen -associated members of Cystobasidiomycetes are two species of Cyphobasidium , which is hypothesized to cause galls on species of Parmeliaceae ( 15). When incorporated into a broader sample of published cystobasidiomycete rD NA sequence data ( 16–18 ), the majority of our lichen- derived sequences form a strongly supported monophyletic clade with Cyphobasidium (Fig. 2B). Using current classif i- cation criteria ( 18), the lichen -associated lineages would include numerous new family -level lineages, and we reco g- nize this set of subclades as the new order Cyphobasidiales ( 19 ). Applying a relaxed molecular clock to our phyl o- genomic tree (Fig. 2A) shows the Cystobasidium - Cyphobasidium split occurring around the same time as the origin of three of the main groups of lecanoromycete ma c- rolichens in which Cyphobasidiales species occur, suggest- ing a long, shared evolutionary history. Two fossil calibrations place this split at around 200 million years be- fore the present (figs. S4 and S5). Our in itial microscopic imaging failed to reveal any cells that we could assign to Basidiomycetes with certainty. Fu r- thermore, attempts to culture the basidiomycete from fresh thalli were unsuccessful. We therefore developed protocols for fluorescent in situ hybridization (FISH) targeting specif- ic ascomycete and cystobasidiomycete rRNA sequences. Cy s- tobasidiomycete -specific FISH probes unambiguously hybridized round, ~3 - to 4 -μ m- diameter cells embedded in the peripheral cortex of both B. fremontii and B. tortuosa (Fig. 3 and movie S1). Consistent with the transcript abu n- dance data, these cells were more abundant in thalli of B.

tortuosa (Fig. 3), where they were embedded in secondary metabolite residues (movie S1). Imaging of other lichen sp e- cies likewise revealed cells of similar morphology in the p e- ripheral cortex (fig. S6). Composite three -dimensional FISH images from B. capillaris show the cells occurring in a zone exterior to the lecanoromycete (Fig. 4 and movie S2) and embedded in polysaccharides (Fig. 4C), explaining why these cells are not observed in scanning electron microscopy (Fig. 4A). In some species, such as L. vulpina , the abun- dance of hybridized living cells was low, but selective r e- moval of the polysaccharide layer through washing revealed high den sities of collapsed, apparently dead cells within the cortex (fig. S7). These dead cells may explain the paucity of the FISH signal in some experiments. The mononucleate single cells (fig. S8C), evidence of budding, and absence of hyphae or clamp connectio ns are consistent with an an a- morphic or yeast state in Cystobasidiomycetes. FISH imag- ing of Cyphobasidium galls on the lichen Hypogymnia physodes , obtained from Norway, confirmed the link to the sexual or teleomorphic state (fig. S8), which appears to d e- ve lop rarely ( 15). These data suggest that the gall -inducing form of Cyphobasidium completes its life cycle entirely within lichens. It is remarkable that Cyphobasidium yeasts have evaded detection in lichens until now, despite decades of molecular and micro scopic studies of the Parmeliaceae ( 20–22 ). It seems likely that the failure to detect Cyphobasidium in both Sanger and amplicon sequencing studies (8 ) is due to multi -template polymerase chain reaction bias. The most widespread clade of Cyphobasidium poss esses a 595 –base pair group I intron inserted downstream of the primer bin d- First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 2 on September 26, 2017 http://science.sciencemag.org/ Downloaded from ing site ITS1F, doubling the template length of ITS, a popu- lar fungal barcode ( 23). This, combined with low bac k- ground abundance, can push a template below detection thresholds ( 24). Also, we cannot rule out that Cyphobasidi- um yeasts have actually been sequenced and discarded as presumed contaminants. The lichen cortex layer has long been considered to be key for structural stabilization of macrolichens, as well as for water and nutrient transfer into the thallus interior ( 6, 25 ). Most macrolichens possess a basic two- layer cortex scheme consisting of conglutinated internal hyphae and a thin, polysaccharide -rich peripheral layer ( 25). However, the internal cellular structure is not un iform across lichens (26), and the composition of extracellular polysaccharides is poorly known ( 27). In Bryoria , the layer in which Cy- phobasidium yeasts occur has not been recognized as dis- tinct from the cortex ( 11), although in other parmelioid lichens, a seemingly homologous layer has sometimes been referred to as the “epicortex” ( 20). The discovery of ubiqu i- tous yeasts embedded in the cortex raises the prospect that more than one fungus may be involved in its construction, and it could explain why liche ns synthesized in vitro from axenically grown ascomycete and algal cultures develop only rudimentary cortex layers ( 5). In many lichens, the peripheral cortex layer in which Cyphobasidium yeasts are embedded is enriched with sp e- cific secondary metabolites ( 25), the production of which often does not correlate with the lecanoromycete phylogeny ( 28). The assumption that these substances are exclusively synthesized by the lecanoromycete must now be considered untested. In B. fremontii, differential transcript and cell abundance data, along with physical adjacency to crystalline residues, implicate Cyphobasidium in the production of vulpinic acid, either directly or by inducing its synthesis by the lecanoromycete. Confirming a link by using transcrip- tome or geno me data is impossible until the enzymatic sy n- thesis pathway of vulpinic acid is described. However, related pulvinic acid derivatives are synthesized by other members of Basidiomycota (29 ).

The assumption that stratified lichens are constructed by a single fungus with differentiated cell types is so central to the definition of the lichen symbiosis that it has been cod i- fied into lichen nomenclature (30 ). This definition has brought order to the field, but may also have constrained it by forcing untested ass umptions about the true nature of the symbiosis. We suggest that the discovery of Cyphobasid- ium yeasts should change expectations about the potential diversity and ubiquity of organisms involved in one of the oldest known and most recognizable symbioses in science. REFERENCES AND NOTES 1. A. De Bary, Die Erscheinung der Symbiose (Verlag Karl Trübner, 1879). 2. A. Gargas, P. T. DePriest, M. Grube, A. Tehler, Multiple origins of lichen symbioses in fungi suggested by SSU rDNA phylogeny. Science 268 , 1492–1495 (1995). Medline doi:10.1126/science.7770775 3. F. Lutzoni, M. Pagel, V. Reeb, Ma jor fungal lineages are derived from lichen symbiotic ancestors. Nature 411 , 937–940 (2001). Medline doi:10.1038/35082053 4. D. L. Hawksworth, The variety of fungal- algal symbioses, their evolutionary significance, and the nature of lichens. Bot. J. Linn. Soc. 96, 3 –20 (1988). doi:10.1111/j.1095 -8339.1988.tb00623.x 5. V. Ahmadjian, The Lichen Symbiosis (John Wiley & Sons, 1993). 6. R. Honegger, Developmental biology of lichens. New Phytol. 125 , 659–677 (1993). doi:10.1111/j.1469 -8137.1993.tb03916.x 7. I. A. Aschenbrenner, T. Cernava, G. Berg, M. Grube, Understanding microbial multi- species symbioses. Front. Microbiol. 7, 180 (2016). Medline doi:10.3389/fmicb.2016.00180 8. S. T. Bates, D. Berg -Lyons, C. L. Lauber, W. A. Walters, R. Knight, N. Fierer, A preliminary survey of lichen associated eukaryotes using pyrosequencing.

Lichenologist 44, 137–146 (2012). doi:10.1017/S0024282911000648 9. O. Petrini, U. H ake, M. M. Dreyfuss, An analysis of fungal communities isolated from fruticose lichens. Mycologia 82, 444–451 (1990). doi:10.2307/3760015 10. J. M. U’Ren, F. Lutzoni, J. Miadlikowska, A. E. Arnold, Communit y analysis reveals close affinities between endophytic and endolichenic fungi in mosses and lichens. Microb. Ecol. 60, 340–353 (2010). Medline doi:10.1007/s00248 -010 - 9698 -2 11. I. M. Brodo, D. L. Hawksworth, Alectoria and allied genera in North America. Opera Bot. 42, 1–164 (1977). 12. S. Velmala, L. Myllys, P. Halonen, T. Goward, T. Ahti, Molecular data show that Bryoria fremontii and B. tortuosa (Parmeliaceae) are conspecific. Lichenologist 41, 231–242 (2009). doi:10.1017/S0024282909008573 13. H. Lindgren, S. Velmala, F. Högnabba, T. Goward, H. Holien, L. Myllys, High fungal selectivity for algal symbionts in the genus Bryoria . Lichenologist 46, 681–695 (2014). doi:10.1017/S0024282914000279 14. G. Amo de Paz, P. Cub as, P. K. Divakar, H. T. Lumbsch, A. Crespo, Origin and diversification of major clades in parmelioid lichens (Parmeliaceae, Ascomycota) during the Paleogene inferred by Bayesian analysis. PLOS ONE 6, e28161 (2011). Medline doi:10.1371/journal.pone.0028161 15. A. M. Millanes, P. Diederich, M. Wedin, Cyphobasidium gen. nov., a new lichen- inhabiting lineage in the Cystobasidiomycetes (Pucciniomycotina, Basidiomycota, Fungi). Fungal Biol. 10.1016/j.funbio.2015.12.003 (2015). 16. M. C. Aime, P. B. Matheny, D. A. Henk, E. M. Frieders, R. H. Nilsson, M.

Piepenbring, D. J. McLaug hlin, L. J. Szabo, D. Begerow, J. P. Sampaio, R. Bauer, M. Weiss, F. Oberwinkler, D. Hibbett, An overview of the higher level classification of Pucciniomycotina based on combined analyses of nuclear large and small subunit rDNA sequences. Mycologia 98, 896–905 (2006). Medline doi:10.3852/mycologia.98.6.896 17. Q.-M. Wang, M. Groenewald, M. Takashima, B. Theelen, P. -J. Han, X. -Z. Liu, T.

Boekhout, F.- Y. Bai, Phylogeny of yeasts and related filamentous fungi within Pucciniomycotina determined from multigene sequence analyses. Stud. Mycol. 81, 27–53 (2015). Medline doi:10.1016/j.simyco.2015.08.002 18. Q.- M. Wang, A. M. Yurkov, M. Göker, H. T. Lumbsch, S. D. Le avitt, M. Groenewald, B. Theelen, X. -Z. Liu, T. Boekhout, F. -Y. Bai, Phylogenetic classification of yeasts and related taxa within Pucciniomycotina. Stud. Mycol. 81, 149–189 (2015). Medline doi:10.1016/j.simyco.2015.12.002 19. Cyphobasidiales T. Sprib. & H. Mayrhofer, ord. nov. (MB 816120); diagnosis is the same as type family Cyphobasidiaceae T. Sprib. & H. Mayrhofer, fam. nov. (MB 816119); embedded in lichen thalli; teleomorph filamentous, rarely observed; when fertile, basidia develop thick- walled probasidium and thin-walled, cylindrical meiosporangium; anamorph is the prevalent known form, consisting of budding yeast with round, thin -walled cells, 2.5 to 4.5 μm in diameter, embedded in the upper cortex of lichens, especially Parmeliaceae; exogenous compound utilization not characterized; cell wall constituents unknown. Type genu s, Cyphobasidium Millanes, Diederich, and Wedin, Fungal Biology doi:10.1016/j.funbio.2015.12.003, p. 4 (2015). 20. M. E. Hale Jr., Pseudocyphellae and pored epicortex in the Parmeliaceae: Their delimitation and evolutionary significance. Lichenologist 13, 1–10 (1981). doi:10.1017/S0024282981000030 First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 3 on September 26, 2017 http://science.sciencemag.org/ Downloaded from 21. A. Thell, A. Crespo, P. K. Divakar, I. Kärnefelt, S. D. Leavitt, H. T. Lumbsch, M. R. D. Seaward, A review of the lichen family Parmeliaceae – history, phylogeny and current taxonomy. Nord. J. Bot. 30, 641–664 (2012). doi:10.1111/j.1756 - 1051.2012.00008.x 22. A. Crespo, F. Kauff, P. K. Divakar, R. del Prado, S. Pérez -Ortega, G. Amo de Pa z, Z. Ferencova, O. Blanco, B. Roca -Valiente, J. Núñez -Zapata, P. Cubas, A.

Argüello, J. A. Elix, T. L. Esslinger, D. L. Hawksworth, A. Millanes, C. Molina, M.

Wedin, T. Ahti, A. Aptroot, E. Barreno, F. Bungartz, S. Calvelo, M. Candan, M.

Cole, D. Ertz, B. Goffinet, L. Lindblom, R. Lücking, F. Lutzoni, J. -E. Mattsson, M. I.

Messuti, J. Miadlikowska, M. Piercey -Normore, V. J. Rico, H. J. M. Sipman, I.

Schmitt, T. Spribille, A. Thell, G. Thor, D. K. Upreti, H. T. Lumbsch, Phylogenetic generic classification of parmelioid lichens (Parmeliaceae, Ascomycota) based on molecular, morphological and chemical evidence. Taxon 59, 1735 –1753 (2010). 23. C. L. Schoch, K. A. Seifert, S. Huhndorf, V. Robert, J. L. Spouge, C. A. Levesque, W. Chen, E. Bolchacova, K. Voigt, P. W. Crous, A. N. Miller, M. J. Wingfield, M. C.

Aime, K. -D. An, F. -Y. Bai, R. W. Barreto, D. Begerow, M. -J. Bergeron, M. Blackwell, T. Boekhout, M. Bogale, N. Boonyuen, A. R. Burgaz, B. Buyck, L. Cai, Q. Cai, G.

Cardinali, P. Chaverri, B. J. Coppins, A. Cr espo, P. Cubas, C. Cummings, U.

Damm, Z. W. de Beer, G. S. de Hoog, R. Del -Prado, B. Dentinger, J. Dieguez - Uribeondo, P. K. Divakar, B. Douglas, M. Duenas, T. A. Duong, U. Eberhardt, J. E.

Edwards, M. S. Elshahed, K. Fliegerova, M. Furtado, M. A. Garcia, Z .-W. Ge, G. W.

Griffith, K. Griffiths, J. Z. Groenewald, M. Groenewald, M. Grube, M. Gryzenhout, L. -D. Guo, F. Hagen, S. Hambleton, R. C. Hamelin, K. Hansen, P. Harrold, G.

Heller, C. Herrera, K. Hirayama, Y. Hirooka, H. -M. Ho, K. Hoffmann, V. Hofstetter, F. Hognabba, P. M. Hollingsworth, S. -B. Hong, K. Hosaka, J. Houbraken, K.

Hughes, S. Huhtinen, K. D. Hyde, T. James, E. M. Johnson, J. E. Johnson, P. R.

Johnston, E. B. G. Jones, L. J. Kelly, P. M. Kirk, D. G. Knapp, U. Koljalg, G. M.

Kovacs, C. P. Kurtzma n, S. Landvik, S. D. Leavitt, A. S. Liggenstoffer, K.

Liimatainen, L. Lombard, J. J. Luangsa- ard, H. T. Lumbsch, H. Maganti, S. S. N.

Maharachchikumbura, M. P. Martin, T. W. May, A. R. McTaggart, A. S. Methven, W. Meyer, J.- M. Moncalvo, S. Mongkolsamrit, L . G. Nagy, R. H. Nilsson, T.

Niskanen, I. Nyilasi, G. Okada, I. Okane, I. Olariaga, J. Otte, T. Papp, D. Park, T.

Petkovits, R. Pino -Bodas, W. Quaedvlieg, H. A. Raja, D. Redecker, T. L. Rintoul, C.

Ruibal, J. M. Sarmiento -Ramirez, I. Schmitt, A. Schussler, C. Shearer, K. Sotome, F. O. P. Stefani, S. Stenroos, B. Stielow, H. Stockinger, S. Suetrong, S. -O. Suh, G.- H. Sung, M. Suzuki, K. Tanaka, L. Tedersoo, M. T. Telleria, E. Tretter, W. A.

Untereiner, H. Urbina, C. Vagvolgyi, A. Vialle, T. D. Vu, G. Walther, Q. -M. Wang, Y.

Wang, B. S. Weir, M. Weiss, M. M. White, J. Xu, R. Yahr, Z. L. Yang, A. Yurkov, J. -C.

Zamora, N. Zhang, W. -Y. Zhuang, D. Schindel, Fungal Barcoding Consortium, Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA ba rcode marker for Fungi. Proc. Natl. Acad. Sci. U.S.A. 109 , 6241 –6246 (2012). Medline doi:10.1073/pnas.1117018109 24. E. Kalle, M. Kubista, C. Rensing, Multi- template polymerase chain reaction.

Biomol. Detect. Quantif. 2, 11–29 (2014). doi:10.1016/j.bdq.2014.11.002 25. R. Honegger, “The symbiotic phenotype of lichen -forming ascomycetes and their endo - and epibionts,” in Fungal Associations , B. Hock, Ed., vol. IX of The Mycota , K. Esser, Ed. (Springer, ed. 2, 2012), pp. 287 –339.

26. D. Anglesea, C. Veltkamp, G. H. Gre enhalgh, The upper cortex of Parmelia saxatilis and other lichen thalli. Lichenologist 14, 29–38 (1982). doi:10.1017/S0024282982000048 27. E. S. Olafsdottir, K. Ingólfsdottir, Polysaccharides from lichens: Structural characteristics and biological activity. Planta Med. 67, 199–208 (2001). Medline doi:10.1055/s -2001 -12012 28. C. G. Boluda, V. J. Rico, A. Crespo, P. K. Divakar, D. L. Hawksworth, Molecular sequence data from populations of Bryoria fuscescens s. lat. in the mountains of central Spain indicates a mismatch between haplotypes and chemotypes.

Lichenologist 47, 279–286 (2015). doi:10.1017/S0024282915000274 29. N. Arnold, W. Steglich, H. Besl, Zum vorkommen von pulvinsäure- derivaten in der gattung Scleroderma . Z. My kol. 62, 69 –73 (1996). 30. W. L. Culberson, Proposed changes in the international code governing the nomenclature of lichens. Taxon 10, 161–165 (1961). doi:10.2307/1216004 31. M. G. Grabherr, B. J. Haas, M. Yassour, J. Z. Levin, D. A. Thompson, I. Amit, X.

Adiconis, L. Fan, R. Raychowdhury, Q. Zeng, Z. Chen, E. Mauceli, N. Hacohen, A.

Gnirke, N. Rhind, F. di Palma, B. W. Birren, C. Nusbaum, K. Lindblad -Toh, N. Friedman, A. Regev, Full -length transcriptome assembly from RNA -Seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011). Medline doi:10.1038/nbt.1883 32. B. J. Haas, A. Papanicolaou, M. Yassour, M. Grabherr, P. D. Blood, J. Bowden, M.

B. Couger, D. Eccles, B. Li, M. Lieber, M. D. Macmanes, M. Ott, J. Orvis, N.

Pochet, F. Strozzi, N. Weeks, R. Westerman, T. William, C. N. D ewey, R. Henschel, R. D. Leduc, N. Friedman, A. Regev, De novo transcript sequence reconstruction from RNA -seq using the Trinity platform for reference generation and analysis.

Nat. Protoc. 8, 1494–1512 (2013). Medline doi:10.1038/nprot.2013.084 33. C. Stubben, “genomes: Genome sequencing project metadata,” R package version 2.16.1 (2015); www.bioconductor.org/packages/release /bioc/html/genomes.html . 34. A. Dobin, C. A. Davis, F. Schlesinger, J. Drenkow, C. Zaleski, S. Jha, P. Batut, M. Chaisson, T. R. Gingeras, STAR: Ultrafast universal RNA -seq aligner.

Bioinformatics 29, 15–21 (2013). Medline doi:10.1093/bioinformatics/bts635 35. M. D. Robinson, A. Oshlack, A scaling normalization method for differential expression analysis of RNA -seq data. Genome Biol. 11, R25 (2010). Medline doi:10.1186/gb -2010 -11-3- r25 36. M. D. Robinson, D. J. McCarthy, G. K. Smyth, edg eR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). Medline doi:10.1093/bioinformatics/btp616 37. A. McKenna, M. Hanna, E. Banks, A. Sivachenko, K. Cibulskis, A. Kernytsky, K.

Garimella, D. Altshuler, S. Gabriel, M. Daly, M. A. DePristo, The Genome An alysis Toolkit: A MapReduce framework for analyzing next -generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010). Medline doi:10.1101/gr.107524.110 38. P. De Wit, M. H. Pespeni, J. T. Ladner, D. J. Barshis, F. Seneca, H. Jaris, N. O.

Therkildsen, M. Morikawa, S. R. Palumbi, The simple fool’s guide to population genomics via RNA -Seq: An introduction to high -throughput sequencing data analysis. Mol. Ecol. Resour. 12, 1058–1067 (2012). Medline doi:10.1111/1755 - 0998.12003 39. L. Li, C. J. Stoeckert Jr., D. S. Roos, OrthoMCL: Identification of ortholog groups for eukaryotic genomes. Genome Res. 13, 2178–2189 (2003). Medline doi:10.1101/gr.1224503 40. S. van Dongen, “Graph clustering by flow simulation,” thesis, University of Utrecht (2000); www.library.uu.nl/digiarchief/dip/diss/1895620/full.pdf . 41. K. Katoh, D. M. Standley, MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). Medline doi:10.1093/molbev/mst010 42. J. Castresana, Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552 (2000). Medline doi:10.1093/oxfordjournals.molbev.a026334 43. G. Talavera, J. Castresana, Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments.

Syst. Biol. 56, 564–577 (2007). Medline doi:10.1080/1063515 0701472164 44. A. Stamatakis, RAxML version 8: A tool for phylogenetic analysis and post - analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014). Medline doi:10.1093/bioinformatics/btu033 45. D. H. Huson, C. Scornavacca, Dendroscope 3: An interactive tool for rooted phylogenetic trees and networks. Syst. Biol. 61, 1061–1067 (2012). Medline doi:10.1093/sysbio/sys062 46. N. Lartillot, N. Rodrigue, D. S tubbs, J. Richer, PhyloBayes MPI: Phylogenetic reconstruction with infinite mixtures of profiles in a parallel environment. Syst.

Biol. 62, 611– 615 (2013). Medline doi:10.1093/sysbio/syt022 47. N. Lartillot, H. Philippe, A Bayesian mixture model for across -site heterogeneities in the amino -acid replacement process. Mol. Biol. Evol. 21, 1095 –1109 (2004). Medline doi:10.1093/molbev/msh112 48. S. Tavaré, “Some probabilistic and statistical problems on the analysis of DNA sequences,” in Lectures in Mathematics in the Life Sciences (American Mathematical Society, vol. 17, 1986), pp. 57 –86.

49. J. L. Thorne, H. Kishino, I. S. Painter, Estimating the rate of evolution of the rate of molecular evolution. Mol. Biol. Evol. 15, 1647–1657 (1998). Medline doi:10.1093/oxfordjournals.molbev.a025892 50. L. Salichos, A. Stamatakis, A. Rokas, Novel information theory -based measures for quantifying incongruence among phylogenetic trees. Mol. Biol. Evol. 31, 1261– 1271 (2014). Medline doi:10.1093/molbev/msu061 First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 4 on September 26, 2017 http://science.sciencemag.org/ Downloaded from 51. D. S. Hibbett, P. B. Matheny, The relative ages of ectomycorrhizal mushrooms and their plant hosts estimated using Bayesian relaxed molecular clock analyses.

BMC Biol. 7, 13 (2009). Medline doi:10.1186/1741- 7007-7-13 52. T. N. Taylor, H. Hass, H. Kerp, M. Krings, R. T. Hanlin, Perithecial ascomycetes from the 400 million year old Rhynie chert: An example of ancestral polymorphism. Mycologia 97, 269–285 (2005) . Medline doi:10.3852/mycologia.97.1.269 53. J. W. Taylor, M. L. Berbee, Datin g divergences in the Fungal Tree of Life: Review and new analyses. Mycologia 98, 838–849 (2006). Medline doi:10.3852/mycologia.98.6.838 54. H. Dörfelt, A. R. Schmidt, A fossil Aspergillus from Baltic amber. Mycol. Res. 109 , 956 –960 (2005). Medline doi:10.1017/S0953756205003497 55. D. Hibbett, D. Grimaldi, M. Donoghue, Fossil mushrooms from Miocene and Cretaceous ambers and the evolution of Homoba sidiomycetes. Am. J. Bot. 84, 981 (1997). Medline doi:10.2307/2446289 56. S. P. Stubblefield, T. N. Taylor, C. B. Beck, Studies of Paleozoic fungi. IV. Wood - decaying fungi in Callixylon newberryi from the Upper Devonian. Am. J. Bot. 72, 1765–1774 (1985). doi:10.2307/2443734 57. T. Feuerer, D. L . Hawksworth, Biodiversity of lichens, including a world -wide analysis of checklist data based on Takhtajan's floristic regions. Biodiversity Conserv. 16, 85–98 (2007). doi:10.1007/s10531- 006-9142 -6 58. J. Miadlikowska, F. Kauff, F. Högnabba, J. C. Oliver, K. Molnár, E. Fraker, E. Gaya, J. Hafellner, V. Hofstetter, C. Gueidan, M. A. Otálora, B. Hodkinson, M. Kukwa, R.

Lücking, C. Björk, H. J. Sipman, A. R. Burgaz, A. Thell, A. Passo, L. Myllys, T.

Goward, S. Fernández -Brime, G. Hestmark, J. Lendemer, H. T. Lumbsch, M.

Schmull, C. L. Schoch, E. Sérusiaux, D. R. Maddison, A. E. Arnold, F. Lutzoni, S.

Stenroos, A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric taxa, 317 genera and 66 families. Mol. Phylogenet. Evol. 79, 132–168 (2014). Medline doi:10.1016/j.ympev.2014.04.003 59. Y. Ohmura, K. Uno, K. Hosaka, T. Hosoya, “DNA fragmentation of herbarium specimens of lichens, and significance of epitypification for threatened species of Japan,” The 10th Internat ional Mycological Congress, Bangkok, Thailand, 4 to 8 August 2014 (2014), p. 151. 60. R. Lanfear, B. Calcott, S. Y. W. Ho, S. Guindon, Partitionfinder: Combined selection of partitioning schemes and substitution models for phylogenetic analyses. Mol. Biol. Evol. 29, 1695 –1701 (2012). Medline doi:10.1093/molbev/mss020 61. S. Altermann, S . D. Leavitt, T. Goward, M. P. Nelsen, H. T. Lumbsch, How do you solve a problem like Letharia? A new look at cryptic species in lichen -forming fungi using Bayesian clustering and SNPs from multilocus sequence data. PLOS ONE 9, e97556 (2014). Medline doi:10.1371/journal.pone.0097556 62. E. Paradis, pegas: An R package for population genetics with an integrated - modular approach. Bioinformatics 26, 419–420 (2010). Medline doi:10.1093/bioinformatics/btp696 63. Y. Yamamoto, R. Mizuguchi, Y. Yamada, Tissue cultures of Usnea rubescens and Ramalina yasudae and production of usnic acid in their cultures. Agric. Biol.

Chem. 49, 3347–3348 (1985). 64. M. del Carmen Molina, A. Crespo, Comparison of development of axenic cultures of five species of lichen -forming fungi. Mycol. Res. 104 , 595–602 (2000). doi:10.1017/S0953756299002014 65. M. Gardes , T. D. Bruns, ITS primers with enhanced specificity for basidiomycetes —application to the identification of mycorrhizae and rusts. Mol.

Ecol. 2, 113–118 (1993). Medline doi:10.1111/j.1365 -294X.1993.tb00005.x 66. T. J. White, T. Bruns, S. Lee, J. Taylor, “Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics,” in PCR Protocols: A Guide to Methods and Applications , M. A. Innis, D. H. Gelfand, J. J. Sinsky, T. J. White, Eds.

(Academic Press, 1990), pp. 315 –322. 67. S. Behrens, C. Rühland, J. Inácio, H. Huber, A. Fonseca, I. Spencer -Martins, B. M.

Fu chs, R. Amann, In situ accessibility of small- subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3 -labeled oligonucleotide probes.

Appl. Environ. Microbiol. 69, 1748–1758 (2003). Medline doi:10.1128/AEM.69.3.1748 -1758.2003 68. J. Inácio, S. Behrens, B. M. Fuchs, A. Fonseca, I. Spencer- Martins, R. Amann, In situ accessibility of Saccharomyces cerevisiae 26 S rRNA to Cy3 -labeled oligonucleotide probes comprising the D1 and D2 domains. Appl. Environ.

Microbiol.

69, 2899–2905 (2003) . Medline doi:10.1128/AEM.69.5.2899 - 2905.2003 69. C. Baschien, W. Manz, T. R. Neu, L. Marvanová, U. Szewzyk, In situ detection of freshwater fungi in an alpine stream by new taxon -specific fluorescence in situ hybridization probes. Appl. Environ. Microbiol. 74, 6427–6436 (2008). Medline doi:10.1128/AEM.00815 -08 70. B. M. Fuchs, F. O. Glöckner, J. Wulf, R. Amann, Unlabeled helper oligonucleotides increase the in situ accessibility to 16 S rRNA of fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 66, 3603–3607 (2000). Medline doi:10.1128/AEM.66.8.3603- 3607.2000 71. D. Anglesea, G. N. Greenhalgh, C. Veltkamp, The cortex of branch tips in Usnea subfloridana . Trans. Br. Mycol. Soc. 81, 438–444 (1983). doi:10.1016/S0007 - 1536(83)80109 -0 72. G. N. Greenhalgh, A. Whitfield, Thallus tip structure and matrix development in Bryoria fuscescens . Lichenologist 19, 295–305 (1987). doi:10.1017/S0024282987000264 73. R. Honegger, A . Haisch, Immunocytochemical location of the (1→ 3) (1→4) -β - glucan lichenin in the lichen -forming ascomycete Cetraria islandica (Icelandic moss). New Phytol. 150 , 739–746 (2001). doi:10.1046 /j.1469- 8137.2001.00122.x 74. H. J. Elwood, G. J. Olsen, M. L. Sogin, The small -subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata . Mol. Biol. Evol. 2, 399–410 (1985). Medline ACKNOWLEDGMENTS This project was supported by an incubation grant from the University of Montana to J.P.M. and T.S.; by an Austrian Science Fund grant (P25237) to T.S., H.M., and P.R.; by NSF (IOS- 1256680, IOS-1553529, and EPSCoR award NSF- IIA-1443108) and NASA Astrobiology Institute (NNA15BB04A) grants to J.P.M.; by a NSF Graduate Research Fellowship (DGE -1313190) to D.V.; by a grant from the Swedish University of Agricultural Sciences Council for PhD Education (2014.3.2.5 -5149) to V.T.; and by a grant (DO2011- 0022) from Stiftelsen Oscar och Lili Lamms minne to G.T. Specimens from Glacier Bay National Park, AK, were collected with the support of the U.S. National Park Service as part of CESU (Cooperative Ecosystem Studies Units) project P11AC90513. We thank D.

Armaleo and F. Lutzoni of Duke University for allowing us to use unpublished data from the Cladonia grayi proteome, as well as P. Dyer, P. Crittenden, and D.

Archer (University of Nottingham, UK) for access to unpublished data from the Xanthoria parietina genome project, which is conducted together with the U.S.

Department of Energy Joint Genome Institute (supported by the Office of Science of the U.S. Department of Energy under contract no. DE -AC02 - 05CH11231). We thank T. Goward, M. Grube, P. Lukasik, J. T. Van Leuven, F.

Fernández -Mendoza, A. Millanes, V. Wagner, and M. Wedin for discussions and L.

Bergström, C. Gueidan, J. Hermansson, H. Holien, B. Kanz, E. Lagostina, S.

Leavitt, B. McCune, J. Nascimbene, C. Printzen, T. Wheeler, and D. Winston for field support and fresh material. C. Björk, S. Gunnarsson, L. Herritt, M. Hiltunen, W. Obermayer, A. de los Ríos, and E. Timdal provi ded technical help, advice, and photos. We acknowledge the Purdue University Genomic Core Facility for generating transcriptomic data for Cystobasidium minutum and the Institute of Molecular Biosciences –Graz Microscopy Core Facility and S. Kohlwein for providing infrastructural support for imaging. Data are available under accession numbers SRP076577 and SRP073687 in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (transcriptomes), NCBI nucleotide accession numbers KU948728 to KU948928 (single -locus DNA sequences), and the Dryad digital repository at http://dx.doi.org/10.5061/dryad.7qv72 (alignments, scripts, and tree files). SUPPLEMENTARY MATERIALS www.sciencemag.org/cgi/content/full/science.aaf8287/DC1 Materials and Methods Figs. S1 to S16 Tables S1 to S12 References ( 31–74 ) Movies S1 and S2 First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 5 on September 26, 2017 http://science.sciencemag.org/ Downloaded from 6 April 2016; accepted 22 June 2016 Published online 21 July 2016 10.1126/science.aaf8287 First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 6 on September 26, 2017 http://science.sciencemag.org/ Downloaded from Fig. 1. Genome -wide divergence and transcript abundance of fungi and algae, based on symbiont subsets extracted from wild Bryoria metatranscriptomes. (A and B) Unrooted maximum likelihood topologies for (A) the Ascomycota member (lecanoromycete) and (B) the Viridiplantae member (alga) within the lichen pair B.

fremontii and B. tortuosa , based on 30,001 and 25,788 SNPs, respectively. Numbers refer to metatranscriptome sample IDs (table S2). Scale bars indicate the average number of substitutions per site. ( C to E) Logarithm of the fold change (logFC) between vulpinic acid –deficient ( B. fremontii) and vulpinic acid –rich ( B. tortuosa ) phenotypes in 15 Bryoria metatranscriptomes, plotted against transcript abundance (logCPM, logarithm of counts per million reads). Only transcripts found in all 15 samples were included. Ascomycota transcripts only are shown in (C). All fungal transcripts are shown in (D), with taxonomic assignments superimposed; a plot with statistically significant transcript differential abundance is shown as an inset. Viridiplantae transcripts are shown in (E). Red dots indicate a log fold change with P < 0.05 in (C), (E) (highlighted with arrows), and the inset of (D). First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 7 on September 26, 2017 http://science.sciencemag.org/ Downloaded from Fig. 2. Placement of Cyphobasidiales members and their diversity within lic hens. (A) Maximum likelihood phylogenomic tree based on 39 fungal proteomes and 349 single -copy orthologous loci. Dating based on a 58- locus subsample shows relative splits between Cyphobasidiales and Cystobasidium minutum and splits leading to the lecanor omycete genera Xanthoria, Cladonia , and Bryoria (colored bars indicate 95% confidence intervals; fungi occurring in lichens are shown in green). ( B) Maximum likelihood rDNA phylogeny of the class Cystobasidiomycetes, with images of representative lichen species from which sequences were obtained in each clade; thick branches indicate bootstrap support >70%. Shaded triangles are scaled to the earliest branch splits of underlying sequence divergence in each clade. Full versions of the trees are shown in fig. S3. First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 8 on September 26, 2017 http://science.sciencemag.org/ Downloaded from Fig. 3. Differential abundance of Cyphobasidiales yeasts in B. fremontii and B. tortuosa . (A) B.

fremontii, with ( B) few FISH -hybridized live yeast cells at the level of the cortex. ( C) B. tortuosa , with ( D) abundant FISH -hybridized cortical yeast cells (scale bars, 20 μm). First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 9 on September 26, 2017 http://science.sciencemag.org/ Downloaded from Fig. 4. Fluorescent cell imaging of dual fungal elements in lichen thalli. (A) Scanning electron microscopy image of a thallus filament of B.

capillaris (scale bar, 200 μm). ( B) FISH hybridization of B. capillaris thallus, showing Cyphobasidiales yeasts (green) and the lecanoromycete (blue) with algal chlorophyll A autofluorescence (red). The volume within the two vertical lines is visualized on the right; the unclipped frontal view is shown at the top. Movie S2 shows an animation of the three -dimensional ~100 - μm z -stack. ( C) Detail of yeast cells (scale bar, 5 μm). First release: 21 July 2016 www.sciencemag.org (Page numbers not final at time of first release ) 10 on September 26, 2017 http://science.sciencemag.org/ Downloaded from Basidiomycete yeasts in the cortex of ascomycete macrolichens Stabentheiner, Merje Toome-Heller, Göran Thor, Helmut Mayrhofer, Hann\ a Johannesson and John P. McCutcheon Toby Spribille, Veera Tuovinen, Philipp Resl, Dan Vanderpool, Heimo Woli\ nski, M. Catherine Aime, Kevin Schneider, Edith published online July 21, 2016 ARTICLE TOOLS http://science.sciencemag.org/content/early/2016/07/20/science.aaf8287 MATERIALS SUPPLEMENTARY http://science.sciencemag.org/content/suppl/2016/07/20/science.aaf8287.D\ C1 CONTENT RELATED http://science.sciencemag.org/content/sci/353/6297/337.full REFERENCES http://science.sciencemag.org/content/early/2016/07/20/science.aaf8287#B\ IBL This article cites 64 articles, 12 of which you can access for free PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions Terms of Service Use of this article is subject to the registered trademark of AAAS. is a Science American Association for the Advancement of Science. No claim to origina\ l U.S. Government Works. The title Science, 1200 New York Avenue NW, Washington, DC 20005. 2017 © The Au\ thors, some rights reserved; exclusive licensee (print ISSN 0036-8075; online ISSN 1095-9203) is published by the Amer\ ican Association for the Advancement of Science on September 26, 2017 http://science.sciencemag.org/ Downloaded from