f u n g a l b i o l o g y 1 1 7 ( 2 0 1 3 ) 5 8 4 e5 9 8 journal homepage: www.elsevier.com/locate/funbio Starting from scratch: Evolution of the lichen thallus in the basidiolichen Dictyonema (Agaricales: Hygrophoraceae) Manuela DAL-FORNOa, James D. LAWREYa, Masoumeh SIKAROODIa, Smriti BHATTARAIa, Patrick M. GILLEVETa, Marcelo SULZBACHERb, c, € * Robert LUCKING a Department of Environmental Science and Policy, George Mason University, 4400 University Drive, Fairfax, VA 22030-4444, USA b Departamento de Micologia/CCB, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves, s/n, Recife, Pernambuco CEP 50670-901, Brazil c Botany, The Field Museum, 1400 South Lake Shore Drive, Chicago, IL 60605-2496, USA article info abstract Article history: Phylogenetic studies indicate that the basidiolichen genus Dictyonema s.lat., often thought Received 28 March 2013 to represent only a single genus with few species, includes several well-supported genus- Accepted 31 May 2013 level clades, all of which form associations with a unique lineage of obligately lichenized Available online 28 June 2013 cyanobacteria (Rhizonema). In an attempt to elucidate the evolution and genus- and Corresponding Editor: species-level diversification in Dictyonema s.lat., we generated 68 new sequences of the nu- Martin Grube clear large subunit rDNA (nuLSU), the internal transcribed spacer (ITS), and the RNA polymerase II subunit (RPB2), for 29 species-level lineages representing all major clades of Keywords: Dictyonema s.lat. and most of the species currently known. The multilocus phylogeny ob- Basidiocarp tained via maximum likelihood and Bayesian approaches indicates the presence of five Basidiolichens genus-level groups: a basal clade, Cyphellostereum, that is sister to the rest of the species, Cyanolichens a paraphyletic grade representing Dictyonema s.str., and three clades representing the gen- Mushrooms era Acantholichen, Cora, and Corella. To determine the evolutionary transformations of the lichenized thallus in the group, ancestral character state reconstruction was done using six characters (lichenisation, thallus type, cortex type, hyphal sheath and haustorial type, photobiont morphology, and basidiocarp type). Our analysis indicates a progressive development of the lichenized thallus from loosely organized filamentous crusts with separate, cyphelloid basidiocarps in Cyphellostereum, to filamentous crusts with derived hyphal sheath and cyphelloidestereoid basidiocarps partially incorporated into the lichen thallus in Dictyonema, to squamuloseefoliose thalli with corticioid basidiocarps entirely supported by the lichen thallus in Cora. These results indicate a remarkable evolutionary integration of lichenized and reproductive tissues in Dictyonema s.lat., supporting the hypothesis that, at least in this case, lichenized thalli may have evolved from reproductive structures in their nonlichenized ancestors. ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: þ1 312 665 7154; fax: þ1 312 665 7158. E-mail addresses: mdalforn@gmu.edu (M. Dal-Forno), marcelo_sulzbacher@yahoo.com.br (M. Sulzbacher), rlucking@fieldmuseu€ cking). m.org (R. Lu 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.05.006 Evolution in Dictyonema Introduction Lichens represent the most remarkable and successful of living symbiotic organisms in terms of numbers of species, diversity and complexity of symbiotic morpho-anatomical features, and the number of lineages that have achieved this type of symbiosis (Ahmadjian 1993; Kirk et al. 2008; Nelsen et al. 2009; Schoch et al. 2009). Yet, the origin of lichenisation is a matter of debate, as are the evolutionary steps that have led to the development of the integrated lichen thallus (Gargas et al. 1995; Lutzoni et al. 2001; Eriksson 2005; € cking et al. 2009; Schoch et al. 2009). Hawksworth 2005; Lu One theory suggests that, because the lichen thallus is structurally complex, whereas the mycelium of nonlichenized fungi is usually undifferentiated, the lichen thallus evolved from fungal stromata or reproductive fungal tissue (Poelt & Wunder 1967; Moser-Rohrhofer 1969; Jahns 1988). Indeed, in lichens such as Cladonia, the vertical thallus is derived from reproductive tissue (Jahns 1970, 1988). Several studies have shown that the photobiont can affect lichen thallus morphogenesis, to the point that the same lichen fungus is able to form morphologically distinct thalli with different photosynthetic partners (James & Henssen 1976; Brodo & Richardson 1978; Jahns 1988; Armaleo & Clerc 1991; Tschermak-Woess 1995; Sanders 2001a; Tønsberg & Goward 2001; Stocker€ rgo € tter 2002; Takahashi et al. 2006). These theories focus Wo on lichenized Ascomycota, but except for supported sistergroup relationships between rock-inhabiting fungi and lichenized forms in the Arthoniales and Verrucariales (Gueidan et al. 2008; Ruibal et al. 2009), no lineages are known in this phylum that show a direct transition from nonlichenized to lichenized forms. While nearly all of the approximately 18 000 species of lichenized fungi belong in Ascomycota, few members of Basidiomycota are lichenized, being chiefly found in the orders Agaricales, Cantharellales, Hymenochaetales, and Lepidostromatales (Oberwinkler 1970, 2012; Hibbett et al. 2007; Lawrey € cking 2013). Most lichenized Basiet al. 2009; Hodkinson & Lu diomycota concentrate in the family Hygrophoraceae in the Agaricales, forming two diverse clades: Lichenomphalia s.lat. and Dictyonema s.lat. (Lawrey et al. 2009). Despite their phylogenetic affinities, the two differ in nearly every possible way. Lichenomphalia forms agaricoid-omphalinoid mushrooms that arise from lichenized granules or squamules containing a green algal (Coccomyxa) photobiont, whereas Dictyonema forms cyphelloid to stereoidecorticioid basidiocarps and a lichenized, filamentous to squamuloseefoliose thallus containing a cyanobacterial photobiont (Oberwinkler 1970, 2012; Redhead et al. 2002; Chaves et al. 2004; Lawrey et al. 2009). Historically, some authors considered Dictyonema s.lat. to represent several different genera in different families, based on features related to growth form, presence of clamp connections, and nature of the photobiont (Hariot 1891, 1892; Metzner 1934). Foliose forms were usually treated as Cora, whereas filamentous forms were assigned to either Laudatea, Dictyonema, or Rhipidonema, depending on appressed or shelf-like growth and the absence or presence of clamp connections. The other extreme was the inclusion of different growth forms into a sin€ ller gle species as ontogenetic and ecological variation (Mo 585 1893; Oberwinkler 1970; Larcher & Vareschi 1988). Parmasto (1978) chose a middle ground in accepting five ‘lichen’ species in a single genus, but he stated that only two fungal species were involved. This is inconsistent from a nomenclatural point of view, since names of lichens always apply to the fungal component [ICN Art. 13.1(d)] and hence there cannot be two ‘fungal’ and five ‘lichen’ species at the same time. Zmitrovich et al. (2006) even went as far as including Dictyonema in the nonlichenized genus Byssomerulius based on shared mycological characters, disregarding the fact that Byssomerulius, based on its type species, belongs in the unrelated Polyporales (Larsson et al. 2004; Larsson 2007; Oberwinkler 2012). Oberwinkler (1970, 1980, 1984, 2001, 2012) provided several morphological and anatomical treatments of Dictyonema and related groups, discussing the value of certain characters for taxonomy and pointing out that filamentous (Dictyonema s.str.), squamulose (Acantholichen), and foliose (Cora) forms share the same type of unique haustoria (Roskin 1970; Slocum & Floyd 1977; Slocum 1980; Oberwinkler 1980, 1984, 2001, 2012). Until recently, Dictyonema s.lat. was best known by the two supposedly common and widespread tropical montane species, the foliose Dictyonema glabratum (¼Cora pavonia) and the filamentous Dictyonema sericeum (Oberwinkler 1970, 1984, 2001; Parmasto 1978), with the first being a popular object for biological studies (Mitidieri et al. 1964; Feige 1969; Coxson 1987a, 1987b, 1987c; Fritz-Sheridan & Portecop 1987; Iacomini et al. 1987; Fritz-Sheridan 1988; Wolf 1993; Lange et al. 1994; Thomas et al. 1997; Azenha et al. 1998; Trembley et al. 2002a, 2002b). In addition to these two, Parmasto (1978) accepted three more ‘lichen’ species, focussing on mycological characters and regarding variation in thallus morphology as infraspecific and habitat-induced. However, taxonomic and phylogenetic studies suggest that Dictyonema s.lat. is comprised of a fairly large number of species, many undescribed  nez € cking 2008; Lawrey et al. 2009; Ya (Chaves et al. 2004; Lu et al. 2012). This has implications on the commonly used epithets glabratum and pavonia for foliose forms, for which Hawksworth (1988) pointed out that glabratum is the correct usage. However, based on our studies, including revision of € cking et al. most types of names described in this clade (Lu 2013a), glabratum and pavonia are different species, only pavonia being widespread and common. The first detailed molecular study suggested that this group can be divided into at least four genera, Cyphellostereum, Dictyonema s.str., Acantholichen, and Cora (Lawrey et al. 2009), and this concept was employed for a treatment of Galapagos  nez et al. 2012). The putative genera are basidiolichens (Ya well-distinguished morphologically and anatomically, as also noted by Oberwinkler (1984, 2012): Cyphellostereum and Dictyonema have filamentous thalli with the mycobiont forming a hyphal sheath around the cyanobacterial filaments, the sheath consisting of irregular hyphae (lacking haustoria) in Cyphellostereum and of jigsaw-puzzle-shaped cells (forming haustoria) in Dictyonema s.str.. In addition, Cyphellostereum has cyphelloid basidiocarps emerging from the undifferentiated lichenized thallus, whereas Dictyonema s.str. has stereoidecorticioid basidiocarps that develop from the underside of the lichenized thallus. Acantholichen forms 586 M. Dal-Forno et al. a microsquamulose thallus, while species of Cora have folioseemacrosquamulose thalli forming distinct layers (cortex, photobiont layer, medulla) and producing corticioid basidiocarps on the lobe underside. Corella brasiliensis, a basidiolichen morphologically similar to Cora (Vainio 1890; Metzner 1934; Xavier Filho & Vicente 1979), had not yet been sequenced, but was assumed to be in or near the Cora clade based on morphological evidence (Oberwinkler 1970, 1984). Although morphologically and anatomically quite distinct from D. glabratum, Parmasto (1978) even included C. brasiliensis in the synonymy of the latter, regarding it a juvenile or underdeveloped form of no taxonomic value. The transition from unstructured to complex vegetative thalli and the simultaneous gradual incorporation of the basidiocarps into thallus formation lend support to the hypothesis that the lichen thallus evolved from fruiting body structures of nonlichenized ancestors in these basidiolichens. This view was already expressed by Oberwinkler (1970), who regarded the Dictyonema thallus as lichenized fruiting body in his depictions of thallus sections. Oberwinkler (1984: 748) also regarded the foliose thallus (Cora) as ‘. ecologically very well adapted symbiotic structure’. Since Dictyonema s.lat. is hitherto the only lichenized lineage for which direct, extant, nonlichenized ancestors have been established in the paraphyletic genus Arrhenia (Lawrey et al. 2009), this clade is an excellent model to investigate the evolution of the lichen thallus. We therefore expanded our sampling of this group both taxonomically and geographically and used a combination of the large subunit (nuLSU) and the internal transcribed spacer (ITS) partition of the nuclear ribosomal DNA and sequences between the conserved domains 6 and 7 of the protein-coding second largest subunit of the RNA polymerase II gene (RPB2) to expand our previous phylogenetic analyses (Lawrey et al. 2009). Our objectives were to: (1) produce a comprehensive multigene phylogeny of Dictyonema s.lat. using representatives of all the major clades, including for the first time also the genus Corella; (2) test hypotheses about the evolution of the lichen thallus in the clade using ancestral character state reconstructions; and (3) examine current genus concepts. Material and methods Taxon sampling The dataset consisted of 29 ingroup species-level terminals plus the outgroup Eonema pyriforme (Lawrey et al. 2009), including 68 new sequences obtained from specimens of Acantholichen, Cyphellostereum, Cora, Corella, and Dictyonema species collected from 13 countries in North, Central, and South America, Europe, and southeast Asia (Table 1). Table 1 e Specimens, collection information, and GenBank accession numbers of fungi used in this study. Species Eonema pyriforme Cyphellostereum imperfectum Cyphellostereum nitidum Cyphellostereum phyllogenum Cyphellostereum pusiolum Cyphellostereum sp. Dictyonema aeruginosulum Dictyonema hernandezii Dictyonema interruptum Dictyonema irpicinum Dictyonema metallicum Dictyonema obscuratum Dictyonema phyllophilum Dictyonema schenkianum 1 Dictyonema schenkianum 2 Dictyonema sericeum 1 Dictyonema sericeum 2 Dictyonema sericeum 3 Acantholichen pannarioides Corella brasiliensis Cora arachnoidea Cora aspera Cora byssoidea Cora hirsuta Cora inversa Cora minor Cora pavonia Cora reticulifera Cora squamiformis Cora strigosa Strain or Herbarium accession number Collection location Genbank number ITS Genbank number nuLSU Genbank number RBP2 Hjm 18581 € cking 25588 Lu Rivas Plata 1130 Lumbsch s.n. € cking s.n. Lu Rivas Plata 2183b Nelsen 3754 € cking 26258 Lu Ertz 10475 Lumbsch 19837e € cking 26255 Lu € cking 23025 Lu Lumbsch 19821 € cking 30062 Lu € cking 17200 Lu Wilk 8868 Fuentes 4788 € cking 25551b Lu Bungartz 5593 Dal-Forno 1271 ndez 1779 Herna € cking 29128 Lu € cking s.n. Lu € cking s.n. Lu € cking s.n. Lu Navarro s.n. € cking s.n. Lu € cking 26201 Lu Wilk 7577 Paz 3 Sweden Guatemala Philippines Fiji Costa Rica Philippines Costa Rica Ecuador Madeira Fiji Ecuador Brazil Fiji Brazil Costa Rica Bolivia Bolivia Guatemala  pagos Gala Brazil Venezuela Bolivia Colombia Colombia Colombia Costa Rica Ecuador Ecuador Bolivia Peru EU118605 KF443218 e KF443219 EU825976 KF443220 EU825955 KF443221 e e KF443222 KF443223 KF443224 KF443225 EU825972 KF443226 KF443227 KF443228 EU825953 KF443229 KF443232 KF443230 KF443234 KF443235 KF443236 EU825968 KF443238 KF443239 KF443240 KF443241 EU118605 KF443243 EU825970 KF443244 EU825976 KF443245 EU825955 KF443246 EU825967 KF443247 KF443248 KF443249 KF443250 KF443251 EU825972 KF443252 KF443253 KF443254 EU825953 KF443255 KF443256 KF443257 KF443258 KF443259 KF443260 EU825968 KF443261 KF443262 KF443263 KF443264 e KF443277 KF443278 e KF443279 e KF443280 KF443281 KF443282 KF443283 KF443284 e e KF443285 KF443286 e KF443287 e KF443265 KF443276 KF443266 KF443267 KF443268 KF443270 KF443271 KF443272 KF443275 KF443269 KF443273 KF443274 Evolution in Dictyonema Light microscopy All sequenced specimens were examined with an LEICA MS5 (Wetzlar, Germany) and an OLYMPUS SZX12 (Shinjuku, Japan) dissecting microscope and a ZEISS Axioskop 2 (Jena, Germany) and an OLYMPUS BH-2 (Shinjuku, Japan) compound microscope. Sections of the thallus including the photobiont were studied in water without any staining or dyes, as well as in lactophenol cotton blue, Lugol solution, and 10 % potassium hydroxide. Microphotographs were taken with DAGE MTI DC-330 3CCD (Michigan City, IN, USA) and JENOPTIK ProgRes C3 and C5 (Jena, Germany) digital microscope cameras attached to the aforementioned microscopes. Macrophotos were taken in situ with CANON Powershot SX20IS (Ota, Japan) and NIKON F301 (Tokyo, Japan) digital cameras. In addition to sequenced specimens, to test for variation and consistently in morphological, anatomical, and chemical features, we studied several hundred specimens of Dictyonema s.lat. both directly in the field (Mexico, Costa Rica, Colombia, Ecuador, Galapagos, Bolivia, Brazil, Thailand, Philippines) and deposited as herbarium material in BM, F, NY, PC, UDBC, US, W, and herb. Kalb, plus type material of most of the names described in this group (Parmasto 1978). 587 type (crustose filamentous ¼ Laudatea type, shelf-like filamentous ¼ Dictyonema type, microsquamulose ¼ Acantholichen type, macrosquamuloseefoliose ¼ Cora type), (3) thallus cortex (absent, loose corticiform with palisadic ‘medullary’ layer ¼ Cora type, compact paraplectenchymatous ¼ Corella type), (4) photobiont morphology (distinctly filamentous ¼ ‘Scytonema’ type, in irregular, coiled threads or groups ¼ ‘Chroococcus’ type), (5) hyphal sheath and haustorial type (irregular hyphae lacking haustoria ¼ Cyphellostereum type, closed sheath of jigsaw-puzzle-shaped cells forming tubular haustoria ¼ Dictyonema s.lat. type), and (6) integration of the basidiocarps into the thallus (basidiocarp with hymenophore separated from basal thallus by a stipe ¼ Cyphellostereum type, basidiocarp partially integrated into thallus with upper part overgrown by cyanobacterial filaments ¼ Laudatea type, basidiocarp fully supported underneath the vegetative thallus structure ¼ Cora type). For species in which basidiocarps are yet unknown (including Acantholichen and Corella), character 6 was scored as missing data (‘?’). We did not use the character clamp connections, since this is a strictly mycological feature not related to thallus morphology per se and species with clamp connections occurred very scattered in our dataset. Chemical data Morphological data Each sequenced species, including the outgroup, was scored for six morpho-anatomical characters that determine the development of the lichen thallus (Table 1; see also Fig 3): (1) lichenisation (absent, present, and with Rhizonema), (2) thallus In addition to morphology and anatomy, we analysed 64 samples of Dictyonema (8), Acantholichen (1), Cora (50), and Corella (5) by means of thin-layer chromatography (Orange et al. 2001) to detect the possible presence of secondary substances, as suggested by an earlier study (Piovano et al. 1995). Fig 1 e Best-scoring ML tree obtained from a three-gene dataset via RAxML. Supported branches are indicated by thick lines and bootstrap support values as well as posterior probabilities from a separate Bayesian analysis are given. The five genuslevel clades and grades (Cyphellostereum, Dictyonema, Acantholichen, Corella, Cora) are highlighted. The columns to the right indicate the CMI reflecting thallus development and the number of nodes from the root to the terminal leaves. 588 Molecular data Genomic DNA was extracted from lichenized thalli using the Bio 101 Fast DNA Spin Kit for tissue (Qbiogene, Illkirch, France) according to the manufacturer’s protocol with slight modifications. About 10 ng of extracted DNA were subjected to a standard PCR in a 25 mL reaction volume using either Taq Gold polymerase (Applied Biosystems, Foster City, CA, USA) or Bio-X-Act Long Mix (Bioline USA, Taunton, MA, USA) according to manufacturer’s protocols. Sequence data were obtained from ITS (ITS1, 5.8S, and ITS2), and nuLSU nuclear rDNA (approximately 600 and 1400 bp, respectively), as well as RPB2 (between the 6 and 7 conserved domains). After visualising the PCR products on a 1 % agarose gel with ethidium bromide and confirming the size, the products were purified with magnetic beads (Agencourt Bioscience, Beverly, MA, USA). The purified PCR products were used in standard sequencing reactions with BigDye Terminator Ready Reaction Mix v3.1 (Applied Biosystems). The primers used were LR0R, LR3R, LR5, LR7, LR16, ITS4, and ITS5 (http://www.biology.duke.edu/ fungi/mycolab/primers.htm) and basidiomycete specific primers bRPB2-6F or bRBP2-5F and bRBP2-7R, bRBP2-7R2 or bRPB2-7.1R (Denton et al. 1998; Liu et al. 1999; Matheny 2005). The sequencing reactions were then purified using Sephadex G-50 (SigmaeAldrich, St. Louis, MO, USA), dried in a speedvac, denatured in HiDi Formamide (Applied Biosystems) and run on an ABI3130-xl capillary sequencer (Applied Biosystems). The data collected were analysed using ABI software, and 500e700 bases were collected for each primer used. These sequences were then assembled together with the software Sequencher version 5.0 (Gene Codes, Ann Arbor, MI, USA) for manual corrections in base calling and to make contiguous alignments of overlapping fragments. M. Dal-Forno et al. We used the Shimodaira-Hasegawa (SH) test incorporated in RAxML 7.2.6 to test hypotheses about putative monophyly of certain groups, particularly Dictyonema s.str.. The Heads-or-Tails (HoT) scores provided by the GUIDANCE web server (Penn et al. 2010a 2010b) were used to quantify differences in evolution of ITS length variation between clades. For this purpose, the ITS alignment for each clade was separately submitted to the GUIDANCE web server and both the overall alignment confidence scores and the confidence scores for each sequence were retrieved. The scores were compared among clades using a nonparametric KruskaleWallis ANOVA in STATISTICA 6.0. Ancestral character state reconstruction and lichen thallus development Ancestral character state reconstruction was done using the morphological dataset and both the best-scoring ML tree and the Bayesian tree sample (1500 trees). The Trace Character History and Trace Characters over Tree functions in MESQUITE 2.75 (Maddison & Maddison 2012) were used to visualize character evolution, employing likelihood ancestral character states. In addition, we applied a linear correlation test between the combined score of all morphological characters for each species (CMI ¼ combined morphology index) and the number of nodes for each species from the root to the terminal leaf in the best-scoring ML tree, to test whether the lichen thallus is progressively more complex along the phylogeny of the clade. The linear correlation was done in STATISTICA 6.0 employing both the parametric Pearson and the nonparametric Spearman rank correlation coefficients. Sequence alignment and phylogenetic analysis Results Newly generated sequences were assembled with sequences from Genbank using BIOEDIT 7.09 (Hall 1999) and automatically aligned with the program MAFFT using the eauto option (Katoh & Toh 2005). The individual nuLSU and ITS alignments were subjected to analysis of ambiguously aligned regions using the GUIDANCE web server (Penn et al. 2010a, 2010b) and introns and regions aligned with low confidence (below 0.90), particularly in the ITS, were removed. This resulted in an alignment length of 1327 for the nuLSU, 600 for the ITS, and 1021 for the RPB2 partition, for a total of 2948 sites in the combined dataset. The alignments were subjected to maximum likelihood (ML) search using RAxML 7.2.6 (Stamatakis et al. 2005; Stamatakis 2006), with parametric bootstrapping using 500 replicates under the GTRGAMMA model. Each gene was first analysed separately and all data were eventually combined since no conflict was detected after removal of ambiguously aligned regions. The dataset was also analysed under a Bayesian framework using MrBAYES 3.1.2 (Huelsenbeck & Ronquist 2001), with two independent runs, a total chain length of one million generations, and four separate chains each, resampling every 1000 trees and generating a majority rule consensus tree from the tree sample after discarding 25 % burnin to obtain posterior probability estimates. Phylogenetic analysis ML analysis of the combined three-gene dataset of the selected species sequences resulted in a well-supported topology in which Cyphellostereum was monophyletic and sister to the remaining species (Fig 1). Dictyonema s.str. formed a paraphyletic grade with Acantholichen, Cora, and Corella emerging as a supported, monophyletic clade. Acantholichen plus Corella formed a supported, monophyletic clade sister to Cora. No support was found in the backbone of the Dictyonema s.str. grade and, consequently, monophyly of Dictyonema s.str. could not be rejected by the SH test (at both the 0.05 and 0.01 level). Bayesian analysis of the same dataset resulted in a congruent topology with strong support for the aforementioned clades (tree not shown but posterior probabilities plotted on the ML tree in Fig 1). Thus, the phylogenetic analysis supports the recognition of up to five genera: Cyphellostereum, Dictyonema s.str., Acantholichen, Corella, and Cora. Ancestral character state reconstruction and lichen thallus development Ancestral character state analysis showed a well-supported progression of morphological and anatomical features in Evolution in Dictyonema Fig 2 e Ancestral character state reconstruction based on the best-scoring ML tree. Bayesian tree sample analysis (not shown) resulted in identical reconstructions. the clade, both based on ML tree reconstruction (Fig 2) and Bayesian tree sampling (results identical, not shown). The lichenized thallus progresses from appressed, crustose filamentous forms in Cyphellostereum (Fig 3A, B) and Dictyonema (Fig 3EeI) to microsquamulose thalli in Acantholichen (Fig 3M, N) and macrosquamulose to large foliose forms in Corella (Fig 3O) and Cora (Fig 3Q, R, W, X). Cora develops a loose, corticiform layer of basally anticlinal and peripherally perpendicular hyphae (Fig 3U), while Corella has a true, 589 paraplectenchymatous cortex (Fig 3P). The photobiont maintains its filamentous morphology in the filamentous thallus types (Fig 3C, K, L) and breaks into irregular groups of cells in the squamulose and foliose forms (Fig 3P, V). The hyphal sheath in Cyphellostereum is composed of irregular, cylindrical hyphae leaving large interspaces (Fig 3C, D), whereas in all other taxa it forms a closed, paraplectenchymatous layer composed of jigsaw-puzzle-shaped cells (Fig 3L, P, V). This correlates with the absence of tubular intracellular haustoria in Cyphellostereum and their presence in the other genera. Coincidentally, the cyanobacterial hyphae are narrow in Cyphellostereum (5e8 mm) and broad in the other genera [8e12(e20) mm]. Cyphellostereum has cyphelloid basidiocarps emerging from the otherwise undifferentiated lichenized thallus (Fig 3A), with the entire basidiocarp free of photobiont filaments; instead, the hyphae associated with the cyanobacterial filaments appear to represent vegetative hyphae, as they are thinner and different from the generative hyphae forming the basidiocarp. In contrast, most species of Dictyonema have stereoidecorticioid basidiocarps emerging from the underside of the lichenized thallus and usually at least partly overgrown with photobiont filaments (Fig 3G, H); the hyphae associated with the cyanobacterial filaments are thicker (4e6 mm) than in Cyphellostereum (2e3 mm) and indistinguishable from the generative hyphae supporting the hymenophore. Most species of Cora and also Dictyonema sericeum have flat, corticioid hymenophores developing on the lobe underside and completely incorporated into the thallus (Fig 3J, S, T), meaning that the thallus entirely supports the hymenophore but is in itself not transformed in shape when fertile. The progressive development of the lichen thallus, as represented by the CMI, is strongly and significantly correlated with the number of nodes from the root to the terminal leaves; i.e., early-diverging species closer to the root have a lessdeveloped thallus, with clear differentiation of thallus and basidiocarps, compared to late-diverging species far from the root (Fig 4). In addition to the phylogenetic progression in morphologicaleanatomical features, the three major clades and grades also exhibit distinctive patterns of sequence evolution, particularly in the ITS1 and ITS2 regions (Fig 5). Species currently assigned to Cyphellostereum have extremely variable, partially unalignable sequence portions in both the ITS1 and ITS2 region (overall alignment confidence score ¼ 0.784). Species contained within Dictyonema s.str. also have highly lengthvariable ITS1 and ITS2 regions but their alignment is less ambiguous (overall alignment confidence score ¼ 0.862). Species classified as Cora have substantially less length variation in the ITS and the level of ambiguity is significantly lower (overall alignment confidence score ¼ 0.954). The differences are highly significant at the p ¼ 0.0001 level (Fig 6). Discussion Lichens, especially ascolichens, exhibit a remarkable diversity of thallus morphologies, and this variation has traditionally provided the basis for delimiting taxonomic groups. However, it is now apparent that thallus morphology is not always a good indicator of phylogenetic relationships among 590 M. Dal-Forno et al. Fig 3 e Thallus development and morphological and anatomical characters in Dictyonema s.lat.. (A) Cyphellostereum pusiolum (Brazil, Sulzbacher & Coelho 1480), filamentousecrustose thallus with emerging, nonlichenized, cyphelloid basidiocarps. (B) € cking 15207a), filamentousecrustose thallus enlarged. (C) C. phyllogenum (Costa Rica, Lu € cking C. phyllogenum (Costa Rica, Lu 15207a), cyanobacterial filaments with loose hyphal sheath formed by irregular hyphae. (D) C. imperfectum (holotype), dense € cking s.n.), filamentousecrustose hyphal sheath formed by irregular hyphae. (E) Dictyonema phyllophilum (Costa Rica, Lu thallus enlarged. (F) D. hernandezii (holotype), filamentousecrustose thallus enlarged. (G, H) D. schenkianum (Costa Rica, € cking 17200), filamentousecrustose thallus with emerging, partially integrated, cyphelloidestereoid basidiocarps. (I) Lu € cking 25551b), filamentous, shelf-like thallus. (J) D. sericeum (Guatemala, Lu € cking 25551b), filaD. sericeum (Guatemala, Lu mentous, shelf-like thallus with fully supported, corticioid basidiocarps on the underside. (K) D. phyllophilum (Costa Rica, € cking 17252i), cyanobacterial filaments with heterocysts. (L) D. schenkianum (Brazil, Lu € cking 30060), cyanobacterial filaments Lu with hyphal sheath of jigsaw-puzzle-shaped cells. (M) Acantholichen pannarioides (Costa Rica, Chaves 3910), microsquamulose thallus competing with other lichens. (N) A. pannarioides (Costa Rica, Sipman 48329), squamules enlarged. (O) C. brasiliensis € cking 35314), section through (Costa Rica, Dal-Forno 1768), macrosquamuloseefoliose thallus. (P) C. brasiliensis (Colombia, Lu thallus showing groups of photobiont cells wrapped in paraplectenchymatous sheath and paraplectenchymatous true cor€ cking s.n.), terrestrial thallus between € cking s.n.), epiphytic thallus. (R) C. pavonia (Ecuador, Lu tex. (Q) Cora aspera (Colombia, Lu Evolution in Dictyonema Fig 4 e Linear correlation between the number of nodes between root and terminal leaf for each species and the CMI. lichenized fungi (Grube & Hawksworth 2007; Rivas Plata & Lumbsch 2011; Lumbsch & Leavitt 2011; Gaya et al. 2012). Even among basidiolichens that generally have simpler and less variable thalli, identical morphologies arose independently in unrelated groups, such as Multiclavula, Lepidostroma, and Lichenomphalia (Oberwinker 1970, 1984, 2012; Ertz et al. € cking 2013). In 2008; Sulzbacher et al. 2012; Hodkinson & Lu the Dictyonema clade, however, thallus morphology is strongly correlated with phylogeny: ancestral character state reconstructions indicate a noticeable progression from a simple, undifferentiated vegetative thallus with separate cyphelloid basidiocarps in the early-diverging Cyphellostereum to forms in which basidiocarps themselves appear to dominate the lichen thallus, with individual hymenophores regularly dispersed over the thallus underside, in the late-diverging Cora. This suggests that the structure of the basidiocarp and its gradual incorporation into the lichen thallus might be responsible for thallus formation in these lichenized species, and it may also explain why the most derived species of Cora and Corella closely resemble shelf-like stereoid macrofungi. Oberwinkler (1970) regarded the Dictyonema s.lat. thallus as lichenized fruiting body, a view shared by other workers (Parmasto 1978; Slocum 1980; Ryan 2002; Trembley et al. mençon et al. (2004) remarked that hyme2002b). Similarly, Cle nium development in Cora glabrata may be interpreted as an aggregation of simple cyphelloid basidiomes (the hymenophore) on the undersurface of a lichenized ‘stroma’. This interpretation is supported by the observation that in Dictyonema s.str., Acantholichen, Corella, and Cora, the hyphae forming the ‘vegetative’ thallus (cortex, medulla, photobiont 591 layer) resemble those producing the hymenophore, and hence can be interpreted as generative hyphae, whereas in Cyphellostereum, the hyphae associated with the photobiont filaments strongly differ from the generative hyphae forming the basidiocarp. As a consequence, it is no longer possible to attribute morphological differentiation in this clade as ontogenetic or € ller ecological variation, as done by previous workers (Mo 1893; Oberwinkler 1970, 2001; Parmasto 1978; Larcher & Vareschi 1988). Instead, this ‘variation’ reflects distinct evolutionary patterns resulting in a large number of species- and genus-level clades, a view also accepted by Oberwinkler (2012). The observed trends in thallus evolution, along with the morphological transformation of the basidiocarps, are restricted to lichenized forms and therefore likely caused by lichenisation. The immediate, nonlichenized ancestors of Dictyonema, Eonema pyriforme, and the genus Arrhenia s.lat. (Redhead et al. 2002; Barrasa & Rico 2003; Lawrey et al. 2009), have mostly dorsiventral, arrhenioid (with gills) or cyphelloid (without gills) basidiocarps, the latter resembling those of Cyphellostereum. Most other members of the family Hygrophoraceae have radially symmetrical, agaricoid-omphalinoid basidiocarps, including the lichenized genus Lichenomphalia, which forms only crustose to microsquamulose thalli in which the basidiocarps are not integrated into the lichen thallus (Oberwinkler 1970, 2001, 2012; Lutzoni & Vilgalys 1995; Lutzoni 1997; Redhead et al. 2002; Lawrey et al. 2009; Lodge et al., in preparation). We conclude that the evolution of dorsiventral basidiocarps in Arrhenia and Eonema facilitated the subsequent formation of an integrated lichen thallus in Dictyonema. Our reasoning is that flattened, dorsiventral basidiocarps can incorporate photobionts readily into dorsal sterile portions exposed to light. In radially symmetrical basidiocarps (as in Lichenomphalia), the hymenophore separates the sterile cap from the sterile stipe, and hence also from the substrate where the interaction between mycobiont and photobiont occurs. The structurally complex thallus in Cora was regarded by Oberwinkler (1984: 748) as ‘. ecologically very well adapted symbiotic structure’, which is reflected in the abundance of these lichens compared to their mushroom-like relatives (i.e., Cyphellostereum and Lichenomphalia). In tropical paramo regions and comparable habitats, Cora lichens occur in dozens to hundreds of individuals per m2 over extensions of many km2 (Fig 7A), values never observed for any other known basidiolichen. While most basidiolichens are pioneer species on open soil, Cora lichens compete successfully with ascolichens with similar morphologies occurring in the same habitats, such as Coccocarpia, Coenogonium, Hypotrachyna, Lobariella, Normandina, Peltigera, and Sticta, among others (Bigelow 1970; bryophytes and small vascular plants. (S) C. strigosa (Peru, Paz 3), thallus underside showing fully supported hymenophore. € cking 21017), hymenophore enlarged. (U) Cora aff. squamiformis (Colombia, Lu € cking 35312), (T) C. arachnoidea (Costa Rica, Lu section through thallus showing loose upper cortex. (V) Same material, section through thallus showing groups of photo€ cking s.n.), undescribed epiphytic species biont cells wrapped in paraplectenchymatous sheath. (W) Cora sp. (Costa Rica, Lu € cking s.n.), undescribed terrestrial species forming with green thallus forming concentric ridges. (X) Cora sp. (Colombia, Lu € cking except A (Marcelo Sulzbacher). Scale in A, I, Q, R, small white squamules with brownish margin. Photographs by R. Lu W [ 20 mm, in J, M, S [ 10 mm, in G, H, O, X [ 5 mm, in B, E, F, N [ 1 mm, in U [ 50 mm, in C, D, K, L, P, V [ 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 592 M. Dal-Forno et al. Fig 5 e Selected ITS alignments obtained via the GUIDANCE web server for Cyphellostereum, Dictyonema, and Cora. Purple colours denote high and blue colours denote low confidence scores, also expressed by the columns (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Barrasa & Rico 2001; Brodo et al. 2001; Chaves et al. 2004; Smith et al. 2009). Within Dictyonema s.lat., all species associate with Scytonema-like Rhizonema cyanobionts as primary photobionts, and these appear to be obligately lichenized and largely tropical, occurring also in other, unrelated but ecologically similar € cking et al. 2009). In terms of the level of uniquelichens (Lu ness, lichenisation appears to differ in each genus. Species in Cyphellostereum, although always having Rhizonema as primary photobiont, commonly harbour admixtures of cyanobacteria and even unicellular chlorophytes, suggesting a more loose association of the fungus with diverse photobionts. Oberwinkler (2001, 2012) mentioned green algae as primary photobionts of certain Cyphellostereum species. However, in the abundant fresh and herbarium material revised and sequenced by us, all species feature cyanobacterial Rhizonema as primary photobionts (Lawrey et al. 2009; Lawrey € cking et al. 2013b). Most likely, forms et al., in preparation; Lu with a primary green algal photobiont represent species of Semiomphalina, which is more closely related to Lichenomphalia than to Dictyonema (Dal-Forno et al., in preparation). Such a specimen was in fact erroneously depicted as Dictyonema sp. by us (Lawrey et al. 2009: fig 9D). Dictyonema s.str., Acantholichen, Cora, and Corella species always form associations with Evolution in Dictyonema Fig 6 e KruskaleWallis ANOVA of the variation of the individual ITS sequence alignment confidence scores for Cyphellostereum (n [ 4), Dictyonema (n [ 9), and Cora (n [ 10). a single, dominant photobiont, as apparent from 454 sequencing data (Dal-Forno et al., in preparation). Morphologically, photobionts in Cyphellostereum and Dictyonema s.str. resemble free-living Scytonema trichomes, whereas in the derived Cora and Corella clades, the trichomes are coiled and broken, forming coiled aggregates of more rounded cells somewhat similar mençon to Chroococcus (Parmasto 1978; Chaves et al. 2004; Cle € cking et al. 2009). This difference was discussed et al. 2004; Lu € ller 1893; Ma € gdefrau & Winkler 1967; by other authors (Mo Tomaselli & Caretta 1969; Oberwinkler 1970; Ryan 2002) and even led to the establishment of a separate lichenized genus, Wainiocora, supposedly differing from Cora by a Chroococcus photobiont (Tomaselli 1950, 1951). Oberwinkler (1970), Parmasto (1978), and Chaves et al. (2004) suggested that these morphotypes represent the same photobiont, which was confirmed by molecular phylogenetic analysis showing that this variation is not due to a photobiont switch but caused by fungal-specific morphogenetic effects on the photobiont € cking et al. 2009, 2013b). Thus, in addition to progressive (Lu thallus development, the Dictyonema s.lat. clade also exhibits a gradual evolution towards stable symbiotic interactions between its bionts, with an increased morphological dominance of the mycobiont over the photobiont, also expressed by the nature of the haustoria. The particular and unique haustoria of Dictyonema s.lat. have been described and discussed in detail by several authors (Roskin 1970; Slocum & Floyd 1977; Slocum 1980; Oberwinkler 1980, 1984, 2001, 2012). They are present in Dictyonema s.str., Acantholichen, Cora, and Corella, but not in Cyphellostereum. We also found a strict correlation between the presence of haustoria, the shape of the hyphal sheath around the photobiont cells, and photobiont morphology. Taxa with haustoria always feature a hyphal sheath formed by jigsaw-puzzleshaped cells, with the sheath being tubular around the photobiont filaments in Dictyonema s.str. and orbicular around the irregular photobiont cell groups in Acantholichen, Corella, and Cora. Both haustoria and sheath are absent in Cyphellostereum. Remarkably, the cyanobacterial filaments are much narrower in Cyphellostereum than in the other genera, which we initially interpreted as a different photobiont species. However, 454 sequencing data indicate the primary photobiont to represent 593 the same clade in all observed lichens (Lawrey et al., in preparation). This apparently conflicting result is explained by the effect of the intracellular haustoria: apparently, the cyanobacterial filaments in Cyphellostereum, which lack haustoria, represent the natural cell width, whereas the haustorial hyphae in Dictyonema, Acantholichen, Corella, and Cora cause lateral ‘inflation’ of the penetrated photobiont cells. On the other hand, even in taxa with haustoria present, the cell width of the pho€ cking et al. 2013a, tobiont can vary according to species (Lu 2013b). A differentiated cortex is a particular achievement of lichen thalli, as it provides a certain level of protection (Jahns 1988; Honegger 2001; Sanders 2001b). In many lichens, this correlates with the production of particular secondary metabolites in the cortex acting as sun-screens, such as atranorin, usnic acid, parietin, and melanin (Rundel 1978; Lawrey 1986;  ndez et al. 1996; Gauslaa & Solhaug & Gauslaa 1996; Ferna Solhaug 2001; Bjerke & Dahl 2002; Gauslaa 2009). In the Dictyonema clade, only the highly evolved foliose forms represented by the genera Cora and Corella feature a cortex. The cortex is different in both genera, suggesting independent evolution, which is supported by their phylogeny, with Corella being sister to the squamulose, ecorticate Acantholichen, and Cora forming a separate clade. The unique, medullary upper cortex in Cora and the differences compared to Corella were already noted by earlier workers (Zahlbruckner 1926; Metzner 1934; Tomaselli 1950; Ozenda 1963; Tomaselli & Caretta 1969; Xavier Filho & Vicente 1979). Oberwinkler (1970) interpreted the paraplectenchymatous cortex of Corella as ‘. kollabierte Hyphen .’ (collapsed hyphae) and Parmasto (1978) did not recognize the difference as significant or of taxonomic importance. Our results show that this difference is structural and highly consistent, and the phylogeny, with Corella being sister to Acantholichen, even suggests that the foliose thalli of Corella might have evolved independently from those of Cora. The particular cortex of Corella species appears to be directly derived from the paraplectenchymatous sheath enclosing the photobiont, forming an extra, continuous layer on the surface directly above the photobiont layer. Piovano et al. (1995) reported the presence of atranorin and tenuiorin in material of Dictyonema glabratum, now in the genus Cora. These are substances commonly found in large macrolichens in the Ascomycota, such as Lobariaceae, Parmeliaceae, and Peltigeraceae (Huneck & Yoshimura 1996; Miadlikowska & Lutzoni 2000; Moncada et al. 2013), and their presence in Cora would imply a remarkable level of parallel evolution in completely unrelated lineages with comparable ecology. However, our analysis of 64 samples of Cora, Corella, Acantholichen, and Dictyonema, resulted in the complete absence of acetone-soluble compounds deposited extracellularly in the hyphal walls. This is supported by the colour change in wettened Cora lichens, which become much darker than in dry state, very different from Lobariaceae and Parmeliaceae that have atranorin as cortical compound. We therefore consider the result by Piovano et al. (1995) as artifactual, since it is highly unlikely that only a species or population in Chile would produce these substances, whereas samples from Mexico, Costa Rica, Colombia, Ecuador, Bolivia, and Brazil studied by us contained no secondary compounds. Another chemical study (Xavier Filho et al. 1980) reported 594 Fig 7 e (A) A Cora species growing abundantly on the ground in the Colombian paramo, competing with bryophytes, small vascular plants, and other lichens. (B) Cyphellostereum basidiocarps emerging from inconspicuous, terricolous thalli. (C) Cora cyphellifera from Ecuador (holotype), showing cyphelloid hymenophores similar to those of Cyphellostereum. (D) Cladonia squamules growing in the centre of a cyanobacterial Coccocarpia species from the Philippines, resembling a photosymbiodeme and demonstrating how different lichens growing intermingled can misguide even the experienced observer. Photographs by € cking except C (Manuela Dal-Forno). Scale [ 10 mm. R. Lu M. Dal-Forno et al. phytohaemagglutinin from Dictyonema sericeum, D. glabratum, and Corella brasiliensis, but these are intracellular substances not comparable to extracellular secondary compounds in lichens. The same applies to the proteins, lipids, and carbohydrates reported by Elifio et al. (2000), Sassaki et al. (2001), and Carbonero et al. (2002) from D. glabratum. Phytohaemagglutinin, found for example in legumes, triggers blood agglutination and is also assumed to play a role in early stages of lichen symbiosis (Lockhart et al. 1978). The occurrence of this substance has not been much studied, but there are reports from Peltigera (Lockhart et al. 1978), which makes it unlikely that the shared occurrence in Dictyonema, Cora, and Corella has any phylogenetic significance. The Dictyonema clade is a prime example of how interpretation of morphological differentiation as ontogenetic or ecological variation, even if based on detailed field observations, can lead to misinterpretations about the evolution and classification of a group of organisms. In this case, we refer to the stud€ ller (1893) and Larcher & Vareschi (1988), discussed ies by Mo by others (Oberwinkler 1970, 2001, 2012; Parmasto 1978) as potential evidence for ecomorphological and ontogenetic varia€ ller (1893) provided a lengthy tion in these lichens. Mo account on the ontogeny and seasonal variation of Dictyonema s.lat. lichens depending on ecological conditions, based on field observations over several years. While this study is unique in its approach, it merges different taxa, at the time unknown to the author, to document ‘variation’. For example, the nonlichenized, terricolous basidiomata considered by € ller (1893) to represent ‘free-living’ Cora mushrooms are Mo in reality species of Cyphellostereum, which often cooccur with Cora in the same habitat (Fig 7B). He also mentioned that certain Cora lichens, with bluish thalli, produced the same cyphelloid basidiocarps, considering this evidence for the conspecificity of all these elements. These are likely to rep€ cking resent Cora cyphellifera, a new species described by us (Lu € ller (1893) also et al. 2013a) from northern Ecuador (Fig 7C). Mo observed foliose Cora thalli growing out of filamentous Dictyonema and concluded that one and the same fungus was involved and simply changed its morphology due to switching from a Scytonema-like to a Chroococcus-like photobiont. While such statements were revolutionary for his time and actually hold true in several lichen lineages, such as the photosymbiodemes in Lobariaceae and Pannariaceae (James & Henssen 1976; Brodo & Richardson 1978; Jahns 1988; Armaleo & Clerc 1991; Tschermak-Woess 1995; Sanders 2001a; Tønsberg & € rgo € tter 2002; Takahashi et al. 2006), Goward 2001; Stocker-Wo in this particular case they are incorrect and based on accidental observation of two different lichens growing together. Such phenomena are not rare; for example, we observed Cladonia squamules growing in the centre of a Coccocarpia thallus suggesting a photosymbiodeme (Fig 7D). However, in the hundreds of collections and field individuals seen by us, we never found evidence of Cora-like thalli developing from Dictyonema-like forms, and the phylogenetic data clearly do not support this idea: in instances where we collected specimens of Cora, Dictyonema or Cyphellostereum growing closely together, sequence data always showed that they represented different taxa. A similar case is the study by Larcher & Vareschi (1988), who attributed morphological differentiation in four Evolution in Dictyonema populations of what they identified as a single species, D. glabratum, as habitat-induced. Unfortunately, one of their populations, actually labelled by them (Larcher & Vareschi 1988: 272) as f. brasiliense, represents C. brasiliensis and thus not only a different species, but a different genus. They correctly observed the differences in cortex type between true Cora and Corella, but their interpretation of this difference as habitat-induced is misguided. We also suspect that the other populations studied by these authors represent three different Cora species, as € cking et al. this genus is highly speciose in the Andes (Lu 2013a), but without revising their material, this is difficult to ascertain. The observed variation in ITS sequences among the different clades, with reduced variation in more derived clades, suggests that Cyphellostereum represents a group that diverged earlier, whereas Cora is a relatively young clade. A dating € cking et al. study employing a relaxed molecular clock (Lu 2013c) suggests the early divergence of Dictyonema s.lat. to have taken place about 45 mya during the Eocene, with the Cyphellostereum crown node estimated at 35 mya in the late Eocene, whereas Cora diversified during the early Miocene, about 20 mya. These results support our hypothesis that the morphologically primitive Cyphellostereum species are relicts from an earlier radiation, with many species now extinct, whereas Cora represents a more recent radiation, with unrecognized and partially cryptic species diversity. The relatively young age of the Dictyonema clade, together with the evidence of progression in thallus morphology and anatomy, has been viewed as witnessing the ‘birth’ of lichenisation in this clade € cking et al. 2013c). Notably, there are of Basidiomycota (Lu some lineages in Ascomycota, specifically in class Dothideomycetes, which also form filamentous lichens and produce hyphal sheaths comparable to those of Dictyonema s.str.; these are the genera Cystocoleus, Racodium, and Racoleus (Gauckler 1960; Nelsen et al. 2009; Hawksworth et al. 2011). If our viewpoint is correct, these might represent other recently emerging lichenized lineages on their way to evolving competitive lichen thalli. It should, however, be pointed out that basidiolichens, such as the clade studied here, cannot be used as model for thallus evolution in ascolichens, since the underlying conditions are different. In Basidiomycota, the dikaryotic mycelium can live in a vegetative stage and eventually produce basidiocarps, which make the formation of a thallus by integrating the basidiocarp a logical evolutionary step. In Ascomycota, the vegetative mycelium is always formed by a haploid mycelium, whereas dikaryotic hyphae only produce ascocarps. We therefore have to assume that thallus evolution in ascolichens followed different pathways. Acknowledgements Financial support for this study was provided by grant DEB 0841405 from the National Science Foundation to George Mason University: ‘Phylogenetic Diversity of Mycobionts and Photobionts in the Cyanolichen Genus Dictyonema, with Emphasis on the Neotropics and the Galapagos Islands’ (PI: J. € cking, P. Gillevet). Material was also colLawrey; CoPIs: R. Lu lected as part of grant DEB-0715660 to The Field Museum: 595 ‘Neotropical Epiphytic Microlichens e An Innovative Inventory of a Highly Diverse yet Little Known Group of Symbiotic € cking) and DEB-0206125 to The Field MuOrganisms’ (PI: R. Lu € cking), as well as two lichen seum: ‘TICOLICHEN’ (PI: R. Lu courses as part of the Organization for Tropical Studies  ndez, Thorsten  s Herna (OTS) speciality courses syllabus. Jesu Lumbsch, Elias Paz, Luis Salcedo, and Karina Wilk provided some of the material used in this study. We thank William Sanders for fruitful discussions on the evolution of the lichen thallus and we are indebted to Franz Oberwinkler for critical revision of a previous version of this manuscript, which helped to improve it considerably. references Ahmadjian V, 1993. The Lichen Symbiosis. John Wiley & Sons, Inc., NY. Armaleo D, Clerc P, 1991. Lichen chimeras: DNA analysis suggests that one fungus forms two morphotypes. Experimental Mycology 15: 1e10. Azenha G, Iturriaga T, Michelangeli FI, Rodriguez E, 1998. Ethnolichenology, biological activity, and biochemistry of Amazonian lichen species. Emanations from the Rainforest 1: 8e14. Barrasa JM, Rico VJ, 2001. Lichenized species of Omphalina (Tricholomataceae) in the Iberian Peninsula. Lichenologist 33: 371e386. Barrasa J, Rico V, 2003. The non-omphalinoid species of Arrhenia in the Iberian Peninsula. Mycologia 95: 700e713. Bjerke JW, Dahl T, 2002. Distribution patterns of usnic acidproducing lichens along local radiation gradients in West Greenland. Nova Hedwigia 75: 487e506. Bigelow HW, 1970. Omphalina in North America. Mycologia 62: 1e32. Brodo IM, Richardson DHS, 1978. Chimeroid associations in the genus Peltigera. Lichenologist 10: 157e170. Brodo IM, Duran Sharnoff S, Sharnoff S, 2001. Lichens of North America. Yale University Press, New Haven & London. Carbonero ER, Sassaki GL, Gorin PAJ, Iacomini M, 2002. A (1 / 6)linked b-mannopyrananan, pseudonigeran, and a (1 / 4)linked b-xylan, isolated from the lichenised basidiomycete Dictyonema glabratum. FEMS Microbiology Letters 206: 175e178. ~ a L, Navarro E, 2004. A € cking R, Sipman HJM, Uman Chaves JL, Lu first assessment of the Ticolichen biodiversity inventory in Costa Rica: the genus Dictyonema (Polyporales: Atheliaceae). Bryologist 107: 242e247. mençon H, Emmet V, Emmet E, 2004. Cytology and plectology Cle of the Hymenomycetes. In: Bibliotheca Mycologia, vol. 199, pp. 1e488. Coxson DS, 1987a. Effects of desiccation on net photosynthetic activity in the basidiomycete lichen Cora pavonia E. Fries from the cloud/mist zone of the tropical volcano La Soufriere (Guadeloupe). The Bryologist 90: 241e245. Coxson DS, 1987b. Net photosynthetic response patterns of the basidiomycete lichen Cora pavonia (Web.) E. Fries from the tropical volcano La Soufriere (Guadeloupe). Oecologia 73: 454e458. Coxson DS, 1987c. The temperature dependence of photoinhibition in the tropical basidiomycete lichen Cora pavonia E. Fries. Oecologia 73: 447e453. Denton AL, McConaughy BL, Hall BD, 1998. Usefulness of RNA polymerase II coding sequences for estimation of green plant phylogeny. Molecular Biology and Evolution 15: 1082e1085. Elifio SL, Da Silva MLCC, Iacomini M, Gorin PAJ, 2000. A lectin from the lichenized basidiomycete Dictyonema glabratum. New Phytologist 148: 327e334. 596 Eriksson OE, 2005. Origin and evolution of Ascomycotadthe protolichenes hypothesis. Svensk Mykologisk Tidskrift 26: 30e33. Ertz D, Lawrey JD, Sikaroodi M, Gillevet PM, Fischer E, Killmann D, rusiaux E, 2008. A new lineage of lichenized basidiomycetes Se inferred from a two-gene phylogeny: the Lepidostromataceae with three species from the tropics. American Journal of Botany 95: 1548e1556. Feige B, 1969. Stoffwechselphysiologische Untersuchungen an den tropischen Basidiolichene Cora pavonia (Sw.) Fr. Flora 160: 169e180.  ndez E, Quilhot W, Gonza  lez I, Hidalgo ME, Molina X, Ferna Meneses I, 1996. Lichen metabolites as UVB filters. Cosmetics & Toiletries 111: 69e74. Fritz-Sheridan RP, 1988. Nitrogen fixation on a tropical volcano, La Soufriere: nitrogen fixation by the pioneer lichen Dictyonema glabratum. Lichenologist 20: 96e100. Fritz-Sheridan RP, Portecop J, 1987. Nitrogen fixation on the tropical volcano, La Soufriere (Guadeloupe): 1. A survey of nitrogen fixation by blueegreen algal microepiphytes and lichen endophytes. Biotropica 19: 194e199. Gargas A, DePriest PT, Grube M, Tehler A, 1995. Multiple origins of lichen symbiosis in fungi suggested by SSU rDNA phylogeny. Science 268: 1492e1495. € Gauslaa Y, 2009. Okologische Funktionen von Flechtenstoffen. € €che der Kommission fu € r Okologie Rundgespra 36: 95e108. Gauckler K, 1960. Die Flaumflechten Cystocoleus niger und Raco€ nkischen Schichtstufenland Nordbayerns. dium rupestre im fra Berichte der Bayerischen Botanischen Gesellschaft 33: 20e22. Gauslaa Y, Solhaug KA, 2001. Fungal melanins as a sun screen for symbiotic green algae in the lichen Lobaria pulmonaria. Oecologia 126: 462e471. € gnabba F, Holguin A, Molnar K, Ferna  ndez-Brime S, Gaya E, Ho € cking R, Stenroos S, Arup U, Søchting U, Van den Boom P, Lu Sipman HJM, Lutzoni F, 2012. Implementing a cumulative supermatrix approach for a comprehensive phylogenetic study of the Teloschistales (Pezizomycotina, Ascomycota). Molecular Phylogenetics and Evolution 63: 374e387. Grube M, Hawksworth DL, 2007. Trouble with lichen: the reevaluation and re-interpretation of thallus form and fruit body types in the molecular era. Mycological Research 111: 1116e1132. Gueidan C, Ruibal C, De Hoog S, Gorbushina A, Untereiner WA, Lutzoni F, 2008. A rock-inhabiting ancestor for mutualistic and pathogen-rich fungal lineages. Studies in Mycology 61: 111e119. Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95e98. ces du genre Dictyonema. Hariot P, 1891. Observations sur les espe Bulletin de la Societe Mycologique de France 7: 32e41. ces du genre Dictyonema. Hariot P, 1892. Observations sur les espe Beihefte zum Botanischen Centralblatt 2: 19. Hawksworth DL, 1988. A new name for Dictyonema pavonium (Swartz) Parmasto. Lichenologist 20: 101. Hawksworth DL, 2005. Life-style choices in lichen-forming and lichen-dwelling fungi. Mycological Research 109: 135e136. Hawksworth DL, Santesson R, Tibell L, 2011. Racoleus, a new genus of sterile filamentous lichen-forming fungi from the tropics, with observations on the nomenclature and typification of Cystocoleus and Racodium. IMA Fungus 2: 71e79. Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, € cking R, Eriksson OE, Huhndorf S, James T, Kirk PM, Lu Lumbsch HT, Lutzoni F, Matheny PB, McLaughlin DJ, Powell MJ, Redhead S, Schoch CL, Spatafora JW, Stalpers JA, Vilgalys R, Aime MC, Aptroot A, Bauer R, Begerow D, Benny GL, Castlebury LA, Crous PW, Dai Y-C, Gams W, Geiser DM, Griffith GW, Gueidan C, Hawksworth DL, Hestmark G, ~ ljalg U, Hosaka K, Humber RA, Hyde KD, Ironside JE, Ko M. Dal-Forno et al. Kurtzman CP, Larsson K-H, Lichtwardt R, Longcore J, Mia˛dlikowska J, Miller A, Moncalvo J-M, Mozley-Standridge S, Oberwinkler F, Parmasto E, Reeb V, Rogers JD, Roux C, Ryvarden L, Sampaio JP, Schußler A, Sugiyama J, Thorn RG, Tibell L, Untereiner WA, Walker C, Wang Z, Weir A, Weiss M, White MM, Winka K, Yao Y-J, Zhang N, 2007. A higher-level phylogenetic classification of the Fungi. Mycological Research 111: 509e547. € cking R, 2013. Lepidostromatales, a new order of Hodkinson BP, Lu lichenized fungi (Basidiomycota, Agaricomycetes), with two new genera, Ertzia and Sulzbacheromyces, and one new species, Lepidostroma winklerianum. Fungal Diversity (in press). Honegger R, 2001. The symbiotic phenotype of lichen-forming ascomycetes. In: Hock B (ed.), Fungal Associations. The Mycota, vol. IX. Springer, Berlin, Heidelberg, pp. 165e188. Huelsenbeck JP, Ronquist F, 2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17: 754e755. Huneck S, Yoshimura I, 1996. Identification of Lichen Substances. Springer, Berlin, Heidelberg. Iacomini M, Zanin SMW, Fontana JD, 1987. Isolation and characterization of B-D-glucan, heteropolysaccharide, and trehalose components of the basidiomycetous lichen Cora pavonia. Carbohydrate Research 168: 55e65. Jahns HM, 1970. Untersuchungen zur Entwicklungsgeschichte der Cladoniaceen mit besonderer Berucksischtigung des PodetienProblems. Nova Hedwigia 20: 1e177 ievi. Jahns HM, 1988. The lichen thallus. In: Galun M (ed.), CRC Handbook of Lichenology, vol. I. CRC Press, Inc., Boca Raton, pp. 95e143. James PW, Henssen A, 1976. The morphological and taxonomic significance of cephalodia. In: Brown DH, Hawksworth DL, Bailey RH (eds), Lichenology: Progress and Problems. Academic Press, London, pp. 27e77. Katoh K, Toh M, 2005. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33: 511e518. Kirk PM, Cannon PF, Minter DW, Stalpers JA, 2008. Dictionary of the Fungi, 10th edn. CAB International, Wallingford. € del B, Zellner H, Zotz G, Meyer A, 1994. Field Lange OL, Bu measurements of water relations and CO2 exchange of the tropical, cyanobacterial basidiolichen Dictyonema glabratum in a Panamanian rainforest. Botanica Acta 107: 279e290. Larcher W, Vareschi V, 1988. Variation in morphology and functional traits of Dictyonema glabratum from contrasting habitats in the Venezuelan Andes. Lichenologist 20: 269e277. Larsson KH, 2007. Re-thinking the classification of corticioid fungi. Mycological Research 111: 1040e1063. Larsson KH, Larsson E, Koljalg U, 2004. High phylogenetic diversity among corticioid homobasidiomycetes. Mycological Research 108: 983e1002. Lawrey JD, 1986. Biological role of lichen substances. The Bryologist 89: 111e122. € cking R, Sipman HJM, Chaves JL, Redhead SA, Lawrey JD, Lu Bungartz F, Sikaroodi M, Gillevet PM, 2009. High concentration of basidiolichens in a single family of agaricoid mushrooms. Mycological Research 113: 1154e1171. Liu YL, Whelen S, Hall BD, 1999. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799e1808. Lockhart CM, Rowell P, Stewart WDP, 1978. Phytohaemagglutinins from the nitrogen-fixing lichens Peltigera canina and P. polydactyla. FEMS Microbiology Letters 3: 127e130. € cking R, 2008. Foliicolous lichenized fungi. Flora Neotropica Lu Monograph 103: 1e867. € cking R, Lawrey JD, Sikaroodi M, Gillevet PM, Chaves JL, Lu Sipman HJM, Bungartz F, 2009. Do lichens domesticate photobionts like farmers domesticate crops? Evidence from Evolution in Dictyonema a previously unrecognized lineage of filamentous cyanobacteria. American Journal of Botany 96: 1409e1418.  ndez JEM, € cking R, Dal-Forno M, Lawrey JD, Bungartz F, Herna Lu Marcelli MP, Moncada B, Morales E, Nelsen MP, Paz E,  nezSalceco L, Spielmann AA, Wilk K, Will-Wolf S, Ya Ayabaca A, 2013a. Ten new species of lichenized Basidiomycota in the genera Dictyonema and Cora (Agaricales: Hygrophoraceae). Phytotaxa (in press). € cking R, Barrie FR, Genney D, 2013b. Dictyonema coppinsii, a new Lu name for the European species known as Dictyonema interruptum (Basidiomycota: Agaricales: Hygrophoraceae), with a validation of its photobiont Rhizonema (Cyanoprokaryota: Nostocales: Rhizonemataceae). Lichenologist 46 (in press). € cking R, Dal-Forno M, Lawrey JD, 2013c. Last but not least: Lu witnessing the ’birth’ of lichenization in the Basidiomycota. Mycologia (in review). Lumbsch HT, Leavitt SD, 2011. Goodbye morphology? A paradigm shift in the delimitation of species in lichenized fungi. Fungal Diversity 50: 59e72. Lutzoni FM, 1997. Phylogeny of lichen- and non-lichen-forming omphalinoid mushrooms and the utility of testing for combinability among multiple data sets. Systematic Biology 46: 373e406. Lutzoni F, Vilgalys R, 1995. Integration of morphological and molecular data sets in estimating fungal phylogenies. Canadian Journal of Botany 73: 649e659. Lutzoni F, Pagel M, Reeb V, 2001. Major fungal lineages are derived from lichen symbiotic ancestors. Nature 411: 937e940. Maddison WP, Maddison DR, 2012. Mesquite: a modular system for evolutionary analysis. http://mesquiteproject.org. Matheny PB, 2005. Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences (Inocybe; Agaricales). Molecular Phylogenetics and Evolution 35: 1e20. € gdefrau K, Winkler S, 1967. Lepidostroma terricolens n.g.n.sp., Ma eine Basidiolichene der Sierra Nevada de Santa Marta (Kolumbien). Mitteilungen des Instituto Colombo-Aleman de Investigaciones Cientıficas 1: 11e17. Metzner P, 1934. Zur Kenntnis der Hymenolichenen. Berichte der Deutschen Botanischen Gesellschaft 51: 231e240. Miadlikowska J, Lutzoni F, 2000. Phylogenetic revision of the genus Peltigera (lichen-forming Ascomycota) based on morphological, chemical, and large subunit nuclear ribosomal DNA data. International Journal of Plant Science 161: 925e958. Mitidieri J, Joly S, Ferraz EC, 1964. Teste de antibiose exercida pelo extrato do liquens Parmelia tinctorum Desp. e Cora pavonia (Web.) E. Fries. Revista de Agricultura [Piracicaba] 39: 119e121. € ller A, 1893. Ueber die eine Thelephoree, welche die HymeMo nolichenen: Cora, Dictyonema und Laudatea bildet. Flora 77: 254e278. € cking R, Betancourt-Macuase L, 2013. Phylogeny of Moncada B, Lu the Lobariaceae (lichenized Ascomycota: Peltigerales), with a reappraisal of the genus Lobariella. The Lichenologist 45: 203e263. Moser-Rohrhofer M, 1969. Der vegetative Flechenthallus e ein Derivat des Ascophors. Anzeiger der Mathematisch€ Naturwissenschaftlichen Klasse der Osterreichischen Akademie der Wissenschaften 106: 109e120. € cking R, Grube M, Mbatchou JS, Muggia L, Rivas Nelsen MP, Lu Plata E, Lumbsch HT, 2009. Unravelling the phylogenetic relationships of lichenised fungi in Dothideomyceta. Studies in Mycology 64: 135e144. € ge Oberwinkler F, 1970. Die Gattungen der Basidiolichenen. Vortra aus dem Gesamtgebiet der Botanik, N.F. 4: 139e169. Oberwinkler F, 1980. Symbiotic relationships between fungus and alga in basidiolichens. In: Schwemmler W, Schenk HEA (eds), Endocytobiology Endosymbiosis and Cell Biology, vol. 1. De Gruyter, Berlin, New York, pp. 305e315. 597 Oberwinkler F, 1984. Fungusealga interactions in basidiolichens. Beihefte zur Nova Hedwigia 79: 739e774. Oberwinkler F, 2001. Basidiolichens. In: Hock B (ed.), Fungal Associations. The Mycota, vol. IX. Springer, Berlin, Heidelberg, NY, pp. 211e225. Oberwinkler F, 2012. Basidiolichens. In: Hock B (ed.), Fungal Associations. The Mycota, 2nd edn, vol. IX, Springer, Berlin, Heidelberg, NY, pp. 341e362. Orange A, James PW, White FJ, 2001. Microchemical Methods for the Identification of Lichens. British Lichen Society, London. Ozenda P, 1963. Lichens. In: Handbuch der Pflanzenanatomie, Band VI. Borntraeger, Berlin, p. 199 Teil 9, Abteilung: Spezieller Teil. Parmasto E, 1978. The genus Dictyonema (‘Thelephorolichenes’). Nova Hedwigia 29: 99e144. Penn O, Privman E, Landan G, Graur D, Pupko T, 2010a. An alignment confidence score capturing robustness to guidetree uncertainty. Molecular Biology and Evolution 27: 1759e1767. Penn O, Privman E, Ashkenazy H, Landan G, Graur D, Pupko T, 2010b. GUIDANCE: a web server for assessing alignment confidence scores. Nucleic Acids Research 38: W23eW28. Piovano M, Chamy MC, Garbarino JA, Quilhot W, 1995. Studies on Chilean lichens XXIV. Secondary products from Dictyonema glabratum (Basidiomycotina). Boletin de la Sociedad Chilena de Quımica 40: 163e165. Poelt J, Wunder H, 1967. Uber biatorinische und lecanorinische Berandung von Flechtenapothecien untersucht am Beispiel € cher 86: der Caloplaca ferruginea-Gruppe. Botanische Jahrbu 256e265. Redhead SA, Lutzoni F, Moncalvo JM, Vilgalys R, 2002. Phylogeny of agarics: partial systematics solutions for core omphalinoid genera in the Agaricales (Euagarics). Mycotaxon 83: 19e57. Rivas Plata E, Lumbsch HT, 2011. Parallel evolution and phenotypic disparity in lichenized fungi: a case study in the lichenforming fungal family Graphidaceae (Ascomycota: Lecanoromycetes: Ostropales). Molecular Phylogenetics and Evolution 61: 45e63. Roskin PA, 1970. Ultrastructure of the hosteparasite interaction €r in the basidiolichen Cora pavonia (Web.) E. Fries. Archiv fu Mikrobiologie 70: 176e182. Ruibal C, Gueidan C, Selbmann L, Gorbushina AA, Crous PW, Groenewald JZ, Muggia L, Grube M, Isola D, Schoch CL, Staley JT, Lutzoni F, De Hoog GS, 2009. Phylogeny of rockinhabiting fungi related to Dothideomycetes. Studies in Mycology 64: 123e133. Rundel PW, 1978. The ecological role of secondary lichen substances. Biochemical and Systematic Ecology 6: 157e170. Ryan BD, 2002. Dictyonema. In: Nash III TH, Ryan BD, Gries C, Bungartz F (eds), Lichen Flora of the Greater Sonoran Desert Region, vol. 1. Lichens Unlimited, Arizona State University, Tempe, AZ, pp. 169e171. Sanders WB, 2001a. Composite lichen thalli of Sticta sp. from Brazil, with morphologically similar lobes containing either a chlorobiont or a cyanobiont layer. Symbiosis 31: 47e55. Sanders WB, 2001b. Lichens: the interface between mycology and plant morphology. BioScience 51: 1025e1035. Sassaki GL, Gorin PAJ, Iacomini M, 2001. Characterization of lysogalactolipids, C-2 and C-3 O-acyl trigalactosylglycerol isomers, obtained from the lichenized fungus Dictyonema glabratum. FEMS Microbiology Letters 194: 155e158. Schoch CL, Crous PW, Groenewald JZ, Boehm EWA, Burgess TI, de Gruyter J, de Hoog GS, Dixon LJ, Grube M, Gueidan C, Harada Y, Hatakeyama S, Hirayama K, Hosoya T, Huhndorf SM, Hyde KD, Jones EBG, Kohlmeyer J, Kruys  A,  L, Mbatchou JS, € cking R, Lumbsch HT, Marvanova Li YM, Lu McVay AH, Miller AN, Mugambi GK, Muggia L, Nelsen MP, Nelson P, Owensby CA, Phillips AJL, Phongpaichit S, Pointing SB, Pujade-Renaud V, Raja HA, Rivas Plata E, Robbertse B, Ruibal C, Sakayaroj J, Sano T, Selbmann L, 598 Shearer CA, Shirouzu T, Slippers B, Suetrong S, Tanaka K, Volkmann-Kohlmeyer B, Wingfield MJ, Wood AR, Woudenberg JHC, Yonezawa H, Zhang Y, Spatafora JW, 2009. A class-wide phylogenetic assessment of Dothideomycetes. Studies in Mycology 64: 1e15. Slocum RD, 1980. Light and electron microscopic investigations in the Dictyonemataceae (basidiolichens). II. Dictyonema irpicinum. Canadian Journal of Botany 58: 1005e1015. Slocum RD, Floyd GL, 1977. Light and electron microscopic investigations in the Dictyonemataceae (basidiolichens). Canadian Journal of Botany 55: 2565e2573. Smith CW, Aptroot A, Coppins BJ, Fletcher A, Gilbert OL, James PW, Wolseley PA (eds), The Lichens of Great Britain and Ireland. The British Lichen Society, London. Solhaug KA, Gauslaa Y, 1996. Parietin, a photoprotective secondary product of the lichen Xanthoria parietina. Oecologia 108: 412e418. Stamatakis A, 2006. RAxML-VI-HPC: maximum-likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688e2690. Stamatakis A, Ludwig T, Meier H, 2005. RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics 21: 456e463. € rgo € tter E, 2002. Resynthesis of photosymbiodemes. In: Stocker-Wo Kranner I, Beckett RP, Varma AK (eds), Protocols in Lichenology. Culturing, Biochemistry, Ecophysiology and Use in Biomonitoring. Springer, Berlin, Heidelberg, pp. 47e60. € cking R, Parnmen S, Moncada B, Sulzbacher MA, Baseia IG, Lu 2012. Unexpected discovery of a novel basidiolichen in the threatened Caatinga biome of northeastern Brazil. The Bryologist 115: 601e609. Takahashi K, Wang LS, Tsubota H, Deguchi H, 2006. Photosymbiodemes Sticta wrightii and Dendriscocaulon sp. (lichenized Ascomycota) from Yunnan, China. Journal of the Hattori Botanical Laboratory 100: 783e796. Thomas MA, Nash III TH, Gries C, 1997. Ecophysiological comparison of two tropical/subtropical lichen species: Dictyonema glabratum from an alpine habitat and Coenogonium interplexum from a lowland forest. Bibliotheca Lichenologica 67: 183e195. Tomaselli R, 1950. Appunti sulla sistematica e distribuzione geografica dei Basidiolicheni. Archivio Botanico 28: 3e19 [Terza Ser. 10(2)]. M. Dal-Forno et al. Tomaselli R, 1951. Notes sur les Basidiolichens. Revue Bryologique et Lichenologique 20: 212e214. Tomaselli R, Caretta G, 1969. Morphological and anatomical observations on Cora pavonia (Basidiolichenes) and hypotheses of factors determining lichen symbiosis. Atti dell’Istituto  di Pavia, Serie Botanico e Laboratorio Crittogamico dell’Universita 6 5: 11e18. Tønsberg T, Goward T, 2001. Sticta oroborealis sp. nov. and other Pacific North American lichens forming dendriscocauloid cyanotypes. The Bryologist 104: 12e23. Trembley ML, Ringli C, Honegger R, 2002a. Hydrophobins DGH1, DGH2, and DGH3 in the lichen-forming basidiomycete Dictyonema glabratum. Fungal Genetics and Biology 35: 247e259. Trembley ML, Ringli C, Honegger R, 2002b. Differential expression of hydrophobins DGH1, DGH2 and DGH3 and immunolocalization of DGH1 in strata of the lichenized basidiocarp of Dictyonema glabratum. New Phytologist 154: 185e195. Tschermak-Woess E, 1995. The taxonomic position of the green phycobiont of Sticta canariensis (Ach.) Bory ex Delise and its extraordinary modification in the lichenized state. Bibliotheca Lichenologica 58: 433e438. Vainio EA, 1890. Etude sur la classification naturelle et la morsil. Acta Societatis pro Fauna et Flora phologie des lichens du Bre Fennica 7: 1e256. Wolf JHD, 1993. Epiphyte communities of tropical montane rain forests in the northern Andes. I. Lower montane communities. Phytocoenologia 22: 1e52.  gicas sobre Xavier Filho L, Vicente C, 1979. Observaciones morfolo Corella. Boletim da Sociedade Broteriana, Serie 2 53: 7e13. Xavier Filho L, Mendes LCG, Vasconcelos CAF, Costa AC, 1980. Fitohemaglutinina (lectinas) em basidioliquenes. Boletim da Sociedade Broteriana, Serie 2 54: 41e46.  nez A, Dal-Forno M, Bungartz F, Lu € cking R, Lawrey JD, 2012. A Ya first assessment of Galapagos basidiolichens. Fungal Diversity 52: 225e244. Zmitrovich IV, Malysheva VF, Spirin WA, 2006. A new morphological arrangement of the Polyporales. I. Phanerochaetineae. Mycena 6: 4e56. Zahlbruckner A, 1926. Lichenes (Flechten). B. Spezieller Teil. In: € rlichen Pflanzenfamilien, 2nd Engler A, Prantl K (eds), Die natu edn. Engelmann, Leipzig, pp. 61e270.