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 ribosomal DNA/RNA used or studied so often for fungi?
3) What does it mean for ‘majority of our lichen-derived sequences form a strongly supported monophyletic clade with Cyphobasidium”? What does this signify about the evolution of these the lichen-derived Basidiomycota fungi?
4) Explain the y-axis of Fig. 1C-F. What does ‘logFC’ stand for? What does each dot represent? And what does it mean to be above or below the zero line?
5) If Bryoria tortuosa and fremontii were genetically distinct, how should the colors on the tips of the trees be distributed in Fig. 1 a & b?
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
REPORTS Cite as: Spribille et al ., Science 10.1126/science.aaf8287 (2016). Most definitions of the lichen symbiosis emphasize its dual nature: the mutualism of a single fungus and single photo- synthesizing symbiont, occasionally supplemented by a se c- ond photosynthesizing symbiont in modified structures ( 1– 4 ). Together, these organisms form stratified, often leafy or shrubby body plans (thalli) that resemble none of the sym- bionts in isolation, a feature thought to be unique among symbioses ( 1). Attempts to synthesize lichen thalli from the accepted two components in axenic conditions, however, have seldom produced structures that resemble natural tha l- li ( 5, 6). Notably, a critical structural feature of stratified lichens, the cortex, typically remains rudimentary in labora- tory- generated symbioses ( 5). Recently, it has been sugges t- ed that microbial players, especially bacteria, may play a role in forming complete, functioning lichen thalli (7 ). How- ever, although culturing and amplicon sequencing have r e- vealed rich communities of microbes (7 , 8), including other fungi (8 –10 ), no new st ably associated symbiotic partners have been found. The recalcitrance of lichens to form thalli in vitro means that characterizing symbiont gene activity (e.g., through transcriptomics) requires an approach that works with na t- ural thalli. We used metatrans criptomics to better under-stand the factors involved in forming two macrolichen sy m- bioses, Bryoria fremontii and B. tortuosa . These two species have been distinguished for 90 years by the thallus -wide production of the toxic substance vulpinic acid in B. t ortu- osa , causing it to appear yellowish, in contrast to B. 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. 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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. 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