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Home , Acarosporaceae, Acarosporales, Ainoa, Alectoria (fungus), Amandinea, Arthrorhaphis

Mycologia, 98(6), 2006, pp. 1088–1103. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897

New insights into classification and evolution of the (, ) from phylogenetic analyses of three ribosomal RNA- and two protein-coding genes

Jolanta Miadlikowska1 Soili Stenroos Frank Kauff Botanical Museum, Finnish Museum of Natural Vale´rie Hofstetter History, University of Helsinki, P.O. Box 7, FI-00014 Emily Fraker Finland Department of Biology, Duke University, Durham, Irwin Brodo North Carolina 27708-0338 Canadian Museum of Nature, P.O. Box 3443, Station Martin Grube D, Ottawa, Ontario, K1P 6P4 Canada Josef Hafellner Gary B. Perlmutter Institut fu¨ r Botanik, Karl-Franzens-Universita¨t, North Carolina Botanical Garden, University of North Holteigasse 6, A-8010, Graz, Carolina at Chapel Hill, CB 3375, Totten Center, Chapel Hill, North Carolina 27599-3375 Vale´rie Reeb Brendan P. Hodkinson Damien Ertz Department of Biology, Duke University, Durham, National Botanic Garden of Belgium, Department of North Carolina 27708-0338 Bryophytes-Thallophytes, Domaine de Bouchout, B-1860 Meise, Belgium Martin Kukwa Department of Plant and Nature Paul Diederich Conservation, Gdansk University, A. Legionow 9, Muse´e national d’histoire naturelle, 25 rue Munster, 80-441 Gdansk, Poland L-2160 Luxembourg, Luxembourg Robert Lu¨cking James C. Lendemer Field Museum of Natural History, 1400 South Lake Department of Botany, Academy of Natural Sciences of Shore Drive, Chicago, Illinois 60605-2496 Philadelphia, 1900 Benjamin Franklin Parkway, Philadelphia, Pennsylvania 19103 Geir Hestmark Department of Biology, University of Oslo, P.O. Box Philip May 1066 Blindern, NO-0316 Oslo, Norway Farlow Herbarium, Harvard University, 22 Divinity Avenue, Cambridge, Massachusetts 02138 Monica Garcia Otalora A´ rea de Biodiversidad y Conservacio´n, ESCET, Conrad L. Schoch Universidad Rey Juan Carlos, c/ Tulipa´n s/n, Department of Botany and , Oregon 28933-Mo´stoles, Madrid, Spain State University, Corvallis, Oregon Alexandra Rauhut A. Elizabeth Arnold Burkhard Bu¨del Department of Plant Sciences, University of Arizona, Fachbereich Biologie, Abt. Pflanzeno¨kologie und 1140 E. South Campus Drive, Forbes 204, Tucson, Systematik, University of Kaiserslautern, Postfach Arizona 85721 3049, 67653 Kaiserslautern, Ce´cile Gueidan Christoph Scheidegger Erin Tripp Swiss Federal Institute for Forest, Snow and Landscape Rebecca Yahr Research (WSL/FNP), Zu¨ rcherstrasse 111, 8903 Connie Robertson Birmensdorf, Switzerland Franc¸ois Lutzoni Einar Timdal Department of Biology, Duke University, Durham, North Carolina 27708-0338 Botanical Museum, University of Oslo, Sars’ gate 1, N-1162 Oslo, Norway

Abstract: The Lecanoromycetes includes most of the -forming fungal (.13 500) and is therefore one of the most diverse class of all Fungi Accepted for publication 15 October 2006. in terms of phenotypic complexity. We report phylo- 1 Corresponding author. E-mail: [email protected] genetic relationships within the Lecanoromycetes

1088 MIADLIKOWSKA ET AL: OF THE LECANOROMYCETES 1089 resulting from Bayesian and maximum likelihood ascal wall of which two layers are thick enough to be analyses with complementary posterior probabilities visible with light microscopy and display different types and bootstrap support values based on three com- of dehiscence (predominantly rostrate but also semi- bined multilocus datasets using a supermatrix ap- fissitunicate or bilabiate), however some members proach. Nine of 10 orders and 43 of 64 families (calicioid lichen-forming fungi, such as and currently recognized in Eriksson’s classification of the ) produce asci with a single evanescent Lecanoromycetes (Outline of Ascomycota—2006 My- layer (Luttrell 1955; Eriksson 1981; Reynolds 1981, conet 12:1–82) were represented in this sampling. Our 1989; Tibell 1984; Hafellner 1988). analyses strongly support the Acarosporomycetidae Members of the Lecanoromycetes form bipartite and as monophyletic, whereas the symbiotic associations with a broad range of photo- delimitation of the largest subclass, the Lecanoromy- bionts, representing chlorococcalean (Astero- cetidae, remains uncertain. Independent of future chloris, s.l., Dictyochloropsis s.l. and Tre- delimitation of the , the Rhizocar- bouxia, are the most frequent genera), filamentous paceae and should be elevated to the algae (Trentepohlia, Phycopeltis) and ordinal level. This study shows that recent classifica- (Calothrix, , Scytonema and Stigonema) (e.g. tions include several nonmonophyletic taxa at differ- Tschermak-Woess 1988; Rikkinen 1995; Beck et al ent ranks that need to be recircumscribed. Our 1998, 2002; Rambold et al 1998; Persˇoh et al 2004; phylogenies confirm that morphology cannot Cordeiro et al 2005). Tripartite symbioses with be applied consistently to shape the classification of cyanobacteria as the secondary photobiont (in terms lichen-forming fungi. The increasing amount of of relative abundance in mature thalli) occur in missing data associated with the progressive addition several unrelated genera within the Lecanoromycetes, of taxa resulted in some cases in the expected loss of however they are particularly common in peltiger- support, but we also observed an improvement in alean (). Rambold et al (1998) statistical support for many internodes. We conclude suggested that photobionts associated with lichen- that a phylogenetic synthesis for a chosen taxonomic forming fungi could be used in lichen systematics. group should include a comprehensive assessment of These authors detected a strong selectivity of myco- phylogenetic confidence based on multiple estimates bionts with respect to their photobionts at the rank of using different methods and on a progressive families and genera in the . sampling with an increasing number of taxa, even if it Most members of the Lecanoromycetes are known involves an increasing amount of missing data. to produce a wide variety of unique secondary Key words: Bayesian inference, Lecanoromy- compounds, especially polyketide derivatives (such cetes, lichen-forming ascomycetes, maximum likeli- as depsides and depsidones, anthraquinones and hood, missing data, mitochondrial ribosomal small xanthones) terpenes and pulvinic acid derivatives subunit (mitSSU), molecular phylogenetic classifica- (e.g. Elix 1996). These chemical compounds are of tion, nuclear ribosomal large subunit (nucLSU), biological and ecological importance (especially if nuclear ribosomal small subunit (nucSSU), phenoty- present in the upper cortex of lichen thalli; e.g. pic characters, photobionts, phylogenetic confiden- Rundel 1978, Lawrey 1986, Solhaug and Gauslaa ce, phylogeny, RNA polymerase II largest subunit 1996, Po¨ykko¨ et al 2005) and of systematic (e.g. Elix (RPB1), RNA polymerase II second largest subunit 1993, Culberson and Culberson 1994, Schmitt and (RPB2) Lumbsch 2004) and evolutionary significance (e.g. Culberson 1986). Circumscription and ranking of subgroups within INTRODUCTION the Lecanoromycetes varied in previous classifications, The Lecanoromycetes, as recognized in Eriksson’s and the last major improvement before molecular (2006) classification, is the largest class of Fungi. It phylogenetic studies was derived from the study of includes the majority (about 90%) of all described reproductive structures, in particular the apical struc- lichen-forming Ascomycota (estimated to be . 13 500 tures of asci. These characters were used to delineate species, Kirk et al 2001). A common character uniting groups of lichen-forming fungi and resulted in a high members of this class is their ascohymenial ascomatal number of families in the Lecanorales (Hafellner ontogeny, with a predominance of apothecial fruiting 1984). Because of controversy associated with the bodies, although of diverse construction and shape. uniform implementation of these characters to cir- Perithecioid ascomata are known in only four of 64 cumscribe families across the Lecanorales (e.g. Timdal families (Grube et al 2004, Schmitt et al 2005) of 1991) many families were redefined (e.g. Rambold and Eriksson’s (2006) classification and in a few unclassi- Triebel 1992, Hafellner 1993). fied genera. In most lineages asci have a multilayered Molecular studies have substantially challenged 1090 MYCOLOGIA phenotypically based groupings applied to previous small subunit [mitSSU]) (e.g. Lumbsch et al 2001, classifications, as well as resolved placement of many 2004a; Lutzoni et al 2001; Ekman and Tønsberg 2002; sterile taxa, and taxa with uncertain taxonomic Kauff and Lutzoni 2002; Lumbsch 2002; Lu¨cking et al affiliation. The Lecanoromycetes include a minimum 2004; Wedin et al 2005) with only four phylogenetic of three subclasses, the Acarosporomycetidae, Ostro- studies using at least one protein coding gene (RPB2: pomycetidae and Lecanoromycetidae, according to Liu and Hall 2004, Lutzoni et al 2004 and Reeb et al Reeb et al (2004) and Lutzoni et al (2004). The 2004; RPB1andRPB2: Hofstetter et al 2007). Acarosporomycetidae encompasses a single , Hofstetter et al (2007) evaluated the phylogenetic the , defined in most cases by the contribution (resolving power and statistical confi- presence of a crustose or squamulose thallus, a chlor- dence) provided by protein-coding (RPB1 and RPB2) ococcoid photobiont (), apothecia of vari- and ribosomal RNA-coding (nucSSU, nucLSU and ous structures, generally more than a hundred simple mitSSU) loci in a phylogenetic study of 82 members per ascus, and functionally unitunicate ascus of the Lecanoromycetes. This study provided a robust with non- or slightly amyloid tholus and ocular phylogenetic framework and useful guidance for chamber. The Ostropomycetidae includes lichenized selecting loci in future multilocus studies on Leca- and nonlichenized fungi (including lichenicolous noromycetes and Pezizomycotina in general. taxa, Lu¨cking et al 2005) with crustose, squamulose Two studies, Lumbsch et al (2004a) and Wedin et al and filamentous thalli, trentepohlioid and chlorococ- (2005), were designed specifically to reconstruct coid photobionts, ascomata of apothecial or perithe- phylogenetic relationships within the Lecanoromy- cial type, eight or fewer spores per ascus and cetes at the family and higher levels as a framework functionally unitunicate asci. Eriksson (2006) recog- for the evaluation of existing classifications. Although nizes five orders in this subclass, Agyriales (two these studies, as well as Lutzoni et al (2004, 83 taxa families), (two families), (sev- using nucSSU+nucLSU), substantially increased tax- en families), (three families) and Tri- on sampling compared to previously published two- chotheliales (two families). The subclass Lecanoro- gene phylogenies, many internodes including deep mycetidae currently (Eriksson 2006) accommodates relationships among major groups in the Lecanor- three recognized orders: Lecanorales (29 families), omycetes remained poorly supported when using the most speciose group of the Lecanoromycetes; ribosomal genes exclusively. Nevertheless they con- Peltigerales (seven families); and vincingly argued that ascus and ascoma characters (three families). Six families (Brigantiaceae, Elixia- should not be applied consistently to the same ceae, , , Umbilicariaceae hierarchical levels across the Lecanoromycetes. We- and Vezdaeaceae) are of uncertain position within din et al (2005) also provided an overview of the the Lecanoromycetidae and 30 genera could not be recent major phylogenetic analyses of the Lecanor- placed with certainty in any of the three existing omycetes. A recent overview of coexisting classifica- subclasses of the Lecanoromycetes, according to tions of the Lecanoromycetes at the order level also Eriksson (2006). All members of this largest subclass can be found in Lumbsch et al (2004a). within the Lecanoromycetes are with The main objectives of this study were to (i) apotheciate fruiting bodies and most species have increase significantly both taxon and character chlorococcoid or cyanobacterial (in Peltigerales) sampling to diminish phylogenetic uncertainty within primary photobionts. The lichenicolous living strate- the Lecanoromycetes, (ii) evaluate Eriksson’s classifi- gy (lichenized and nonlichenized fungi growing on cation (2006) at the family and higher ranks, (iii) lichens) is found in many groups of the Lecanor- resolve the phylogenetic placement of taxa with omycetidae, whose members also serve frequently as unknown affinities and (iv) revisit the distribution hosts for other such fungi (e.g. Clauzade et al 1989, and evolution of selected phenotypic characters Rambold and Triebel 1992, Kirk et al 2001, Lawrey (including photobionts and ascus structure) across and Diederich 2003). the major groups within the Lecanoromycetes and Many recent phylogenetic studies have explored their utility in lichen systematics. relationships within the Lecanoromycetes to evaluate Using a supermatrix approach we assembled three delimitations of particular taxa and less frequently the datasets with a progressively higher number of validity of diagnostic features (especially ascomata taxa and missing data. Internodal support estimated and ascus characters) used to circumscribe taxa (e.g. with maximum likelihood bootstrap (with RAxML Grube et al 2004, Schmitt et al 2005, Wedin et al and PHYML) and Bayesian posterior probabilities 2005). Most of these studies were based on different (with MrBayes) are compared and discussed in the combinations of two or three nuclear ribosomal genes context of missing data and phylogenetic reconstruc- (i.e. nucSSU, nucLSU and mitochondrial ribosomal tions. MIADLIKOWSKA ET AL:SYSTEMATICS OF THE LECANOROMYCETES 1091

MATERIALS AND METHODS closely related have very different types of asci and differ considerably also in other characters such as Because of space limitation associated with this issue of hamathecium and secondary chemistry (including Mycologia, this section is presented in SUPPLEMENT 1 (http//www.mycologia.org). Timdalia, a member of the Acarosporaceae in Wedin et al 2005).

RESULTS AND DISCUSSION Candelariomycetidae/.—One of the most surprising outcomes of all three dataset studies Phylogenetic reconstructions and confidence.—Missing (although the strongest support came from the 5- data in the 5+4-gene supermatrix (26%) and the gene analyses) is the placement of 5+4+3-gene supermatrix (37%) datasets overall did (FIG. 3) and (former , not have a negative affect on phylogenetic resolution Hakulinen 1954) outside the Lecanorales and Leca- and support when using maximum likelihood noromycetidae (FIG. 1). Owing to the ascus type these (RAxML) and Bayesian methods (MrBayes) (see also genera often were considered close relatives of the Wiens 2006). However noticeably lower bootstrap and currently are classified in this values for several nodes were obtained from PHYML family (Eriksson 2006). This unexpected placement analyses on the 5+4-gene and 5+4+3-gene datasets of Candelariaceae also was found and discussed by (FIG. 1, second column vs. first and third columns of Wedin et al (2005) and Hofstetter et al (2007), grid showing support for each internode). All although in the latter study the Candelariaceae is phylogenies were concordant with the tree based on strongly supported as the first phylogenetic split the most complete 5-gene dataset (the 5+4+3-gene before the divergence of the Acarosporomycetidae. tree is shown in FIG. 1). Only a few branches that were We confirm that this group should be recognized as highly supported in the 5-gene phylogeny received no a major independent lineage within the Lecanoromy- significant support (based on two or all three cetes by classifying it in its own subclass (Candelar- methods) in the 5+4-gene or 5+4+3-gene reconstruc- iomycetidae) the same way it was done to accommo- tions (e.g. the of the group delimited by date the unique phylogenetic placement of the and Mycoblastaceae in the Lecanorales, Acarosporaceae. No morphological features are FIG. 1). Adding taxa with missing data to the 5-gene known to confirm the separation of these two genera and 5+4-gene datasets often improved phylogenetic from the Lecanoraceae and the Lecanoromycetidae. confidence (e.g. the monophyly of the Collematineae A revision of the genera Candelaria and Candelariella and the ). Comparing the three meth- is needed, given that Candelaria concolor was found ods used to estimate phylogenetic confidence, we nested within Candelariella (FIG. 1). found that support provided by MrBayes generally was congruent with RAxML bootstrap values, whereas Ostropomycetidae.—As revealed from analyses on 5+4- PHYML seems to require more data (less efficient) gene and 5+4+3-gene datasets, the subclass Ostropo- than the other two methods to provide significant mycetidae is well supported as monophyletic (except support values and seems the least stable as the by PHYML-BS). The phylogenetic tree presented here number of taxa and missing data increased. includes members of these four of five orders part of the current classification of the Ascomycota (Eriksson Acarosporomycetidae/.—The phylogenet- 2006): Agyriales, Gyalectales, Ostropales and Pertu- ic distinctiveness of the Acarosporaceae was shown by sariales (FIG. 1). The and Loxospor- Reeb et al (2004), who suggested recognizing this aceae need to be recognized as members of the family at the subclass level (Acarosporomycetidae). Ostropomycetidae, based on our results. This result was confirmed by Lutzoni et al (2004), The Ostropales and Gyalectales are treated usually Miadlikowska and Lutzoni (2004), Hofstetter et al as Ostropales s.l. (Kauff and Lutzoni 2002, Lu¨cking et (2007) and this study. In agreement with Reeb et al al 2004) due in part to the poor taxon sampling and (2004) neither nor are mono- support these relationships received in past studies. In phyletic (FIG. 1). In our analyses (P. this study we show that the order Ostropales as simplex) diverged earlier than and the circumscribed by Eriksson (2006) is nonmonophy- remaining genera of the Acarosporales, a significant letic due to the inclusion of the Gyalectales. For this result based on all nine support values (but see Wedin reason the Gyalectales should be subsumed within the et al 2005). Pleopsidium (FIG. 2), with -type Ostropales s.l. as proposed by Kauff and Lutzoni asci and ascomata that resemble those of Lecanora, (2002) and Lu¨cking et al (2004). Because is was expected to be closely related to Lecanora classified within the , the Ostropales s.str. (Hafellner 1993). The Acarosporales represent could be restricted to this family (well supported in a strong case where taxa that appear phylogenetically FIG. 1, Ostropales 1), which would allow the recogni- 1092 MYCOLOGIA

FIG.1. MIADLIKOWSKA ET AL:SYSTEMATICS OF THE LECANOROMYCETES 1093

FIG. 1. Continued. 1094 MYCOLOGIA

FIG. 1. Phylogenetic relationships among 264 putative members of the Lecanoromycetes based on Bayesian analyses of the combined nucSSU, nucLSU, mitSSU, RPB1 and RPB2 sequences (5+4+3-gene dataset) and 10 species used as outgroup (, and ). This cladogram resulted from a 50% majority rule consensus of 30 000 trees sampled with Bayesian MCMCMC (SUPPLEMENT 1). Numbers in parentheses after taxon names indicate the dataset in which they were included: 5 refers to taxa present in the 5-, 5+4- and 5+4+3-gene datasets, and 4 refers to taxa present in the 5+4- and 5+4+3-gene datasets. When no numbers are found after names, taxa were included only in the 5+4+3-gene supermatrix. Stars indicate genera and families with lichenicolous members. Taxa at the tip of the tree shown in blue indicate phylogenetic placements that are newly revealed or significantly supported compared to previous studies. Taxa in blue at the family and higher levels indicate suggested changes in their circumscription and ranking that needs to be incorporated in future classifications of the Ascomycota. Names followed by a question mark indicate potential changes for future consideration. The nine-box grids on internodes indicate support with different phylogenetic methods (column 1 [boxes 1, 4, 7] 5 bootstrap values calculated with RAxML, column 2 [boxes 2, 5, 8] 5 bootstrap values calculated with PHYML, column 3 [boxes 3, 6, 9] 5 posterior probabilities calculated with MrBayes) based on different datasets (top row [boxes 1–3] being the smallest dataset [111 taxa] but with the least amount of missing data, and the bottom row [boxes 7–9] being the largest dataset [274 taxa] with the largest amount of missing data). Red boxes indicate cases where internodal support is not applicable due to at least one of the (usually two) immediately downstream branches being absent in the 188 or the 111 taxa datasets compared to the 274 taxa dataset. Black boxes indicate RAxML bootstrap values $ 70% (column 1), PHYML bootstrap values $ 70% (column 2) or MrBayes posterior probability values $ 95% (column 3). White boxes indicate RAxML bootstrap values , 70%, PHYML bootstrap values , 70% or MrBayes posterior probability values , 95%. Colors on the right side of the figure indicate major types of primary photobionts associated with mycobionts within an order/family/monophyletic group based on available records for members classified in these taxa, even if not included in the tree. Presence of secondary photobionts (different genera of cyanobacteria) is indicated by a dark blue box (Scytonema/Stigonema), a circle (Nostoc) and an oval (Calothrix). tion of the Graphidales (Ostropales 2; well supported (1974) and Sherwood (1977). However this three- monophyletic group including , Aster- order classification would remove the use of Ostro- othyriaceae [5 Solorinellaceae; Henssen and Lu¨cking pales s.l. for a well supported monophyletic group of 2002] and in FIG.1)andthe lichen-forming fungi preferentially associated with Gyalectales (a poorly supported monophyletic group Trentepohlia,(FIG. 1), which would leave this impor- in FIG. 1, that would include the , tant internode and associated putative synapomorphy and Phlyctidaceae), thus partly reflect- without a name and commonly used rank. An ing the earlier classifications by Henssen and Jahns alternative solution to this problem would be the MIADLIKOWSKA ET AL:SYSTEMATICS OF THE LECANOROMYCETES 1095 use of suborders Graphidineae (Ostropales 2), ) in the Ostropomycetidae, where two of Gyalectineae (Gyalectales) and Stictidineae (Ostro- its hosts belong, also was unexpected given the pales 1) within the Ostropales s.l. as phylogenetically different ascomatal structures (see also Wedin et al circumscribed here. Simultaneous inclusion of Odon- 2005). totremataceae and in phylogenetic Our study also shows that Loxospora is part of the studies is necessary before any changes to the most basal divergence within the Ostropomycetidae classification of the Ostropales s.l. are made. (with significant support values, FIG. 1). Members of The Ostropales s.l. includes morphologically and this have coccalean and somewhat ecologically diverse lichens. Lu¨cking et al (2004) spirally arranged ascospores. This novel phylogenetic demonstrated that the Gomphillaceae, with anasto- affinity revealed by this study is not surprising due to mosing paraphyses, are part of this group, and Grube Loxospora’s (Loxosporaceae) greater similarity (thal- et al (2004) have shown that the perithecial - lus structure) to Pertusariaceae than to Lecanorales, ceae, with unitunicate asci and unbranched true where this taxon is classified currently (Eriksson paraphyses, also belongs to this . Lumbsch et al 2006). Loxospora was re-established as a genus by (2004b) confirmed that the mazaediate genus Nad- Hafellner (1984) and previously was classified in the vornikia is a member of the Thelotremataceae and Haematommataceae. Staiger and Kalb (1995) no- thus the Ostropales s.l. The genus was placed ticed anatomical characters that were not shared by traditionally in the Lecanorales due to its amyloid other members of this family (e.g. the genus hymenium and chlorococcoid photobiont; however ) and created a separate family to its thallus and apothecial structure are more reminis- accommodate Loxospora. Loose and thick paraphy- cent of the Ostropales s.l. (RL unpublished) there- ses, predominance of elatinic acid and the presence fore supporting its placement in the latter group of wide and grouped ascogenous hyphae in ascoma- (Gyalectaceae). tal primordia (cf. Brodo and Henssen 1995) are Our study shows a well supported sister clade further characters that circumscribe this newly relationship of Pertusariaceae (with nonmonophy- reconstructed lineage in the Ostropomycetidae. letic and ) and - Because none of the members of the Haematomma- ceae, to form the order Pertusariales (reconstructed ceae has been included in phylogenetic analyses we as paraphyletic in Wedin et al [2005]) and the cannot justify the exclusion of the Loxosporaceae unexpected placement of (Hymeneliaceae) from the Haematommaceae. nested within the Pertusariaceae. Wedin et al (2005) and Hofstetter et al (2007) also suggested a close Lecanoromycetidae.—The delimitation of the Leca- affinity among Aspicilia and members of the Pertusar- noromycetidae is ambiguous due to a lack of support iaceae and but without obtaining for the phylogenetic placement of the strong support for this relationship. Aspicilia was (Sporastatia; Rambold and Triebel 1992, Eriksson shown to be outside the family Hymeneliaceae more 2006), Fuscideaceae, Ophioparmaceae, Rhizocarpa- than 10 y ago based on morphological and isozyme ceae and Umbilicariaceae (FIG. 1). An early diver- data (Lutzoni and Brodo 1995). The current circum- gence of the as revealed here and in scription of the Hymeneliaceae and Pertusariaceae previous studies (Reeb et al 2004, Lutzoni et al 2004) needs to be updated accordingly. was postulated by Honegger (1980) based on char- The Baeomycetaceae (with an uncertain placement acters of the ascus tip. Our phylogeny confirms that in the Ascomycota according to Eriksson 2006) is the narrowest delimitation of the Lecanoromycetidae delimited as monophyletic and a highly supported contains at least three main lineages (Miadlikowska lineage (Phyllobaeis [FIG. 4] and Baeomyces) in our and Lutzoni 2004, Hofstetter et al 2007): the phylogeny (FIG. 1). is also part of this Lecanorales, Peltigerales (and most closely related subclass, although an accurate placement in the group, including the and Porpidiaceae) Ostropomycetidae remains unresolved. The latter is and Teloschistales. If the current topology receives true for the Hymeneliaceae and Agyriales. Based on high support values in future studies, the - ribosomal genes Kauff and Lutzoni (2002) proposed ceae-Ophioparmaceae-Umbilicariaceae and the Rhi- an elevation of the Baeomycetaceae (represented in zocarpaceae-Catillariaceae monophyletic groups their tree by Baeomyces placophyllus) to the order level should be classified as separate orders ( (Baeomycetales) in the Ostropomycetidae. This sug- and Rhizocarpales) within the Lecanoromycetidae. If gestion is confirmed by our study; however some future studies show that it is not possible to putative close relatives of the Baeomycetaceae ( encompass these two new orders within a monophy- and Anamylopsora) were not included. The placement letic Lecanoromycetidae it is likely that each group of Arthrorhaphis citrinella (a juvenile parasite of would have to be recognized at the subclass level 1096 MYCOLOGIA

FIGS. 2–11. Lichen-forming members of the Lecanoromycetes. 2. , Acarosporomycetidae, Acarosporales, Acarosporaceae (photo by E. Timdal). 3. Candelariella lutella, Candelariaceae (photo by E. Timdal). 4. Phyllobaeis imbricata. Close-up of fruiting bodies, Ostropomycetidae, Baeomycetaceae (photo by R. Lu¨cking). 5. ventosa, Ophioparmaceae (photo by E. Timdal). 6. burgessii. Close-up of fruiting bodies, Lecanoromycetidae, MIADLIKOWSKA ET AL:SYSTEMATICS OF THE LECANOROMYCETES 1097

(Umbilicariomycetidae and Rhizocarpomycetidae). and corroborated by Buschbom and Mueller (2004) Therefore no matter how these two monophyletic and Lutzoni et al (2004). Both taxa are strictly entities will be resolved in future studies, they both crustose and distinctly areolate. While members of need to be recognized at least at the ordinal level, as the Rhizocarpaceae often form large, hyaline to proposed here. brown, transversely septate to distinctly muriform ascospores, Sporastatia develops multiple, hyaline Umbilicariomycetidae?/Umbilicariales.—Some novel ascospores. The placement of within and interesting relationships are found in the group (FIG. 1) is interesting because this genus, containing the Umbilicariaceae. Placement of the with thick squamulose thalli, reveals a strong pigmen- Fuscideaceae outside was suggested tation of the wall around the septum of mature by Lutzoni et al (2004), Reeb et al (2004) and Wedin ascospores (torus) but lacks the typical gelatinous et al (2005) and is confirmed in this study. We found perispore (‘‘halo’’). A closer relationship of Catole- it interesting that Fuscidea and , despite chia and Rhizocarpon rather than with was striking differences in anatomy and spore assumed by Hafellner (1978). Noteworthy in this number per ascus, share a unique ascus type and group is also the tendency toward lichenicolous a distinctive type of epihymenial pigmentation, which growth (e.g. in Rhizocarpon and ; Hafellner led Hafellner (1984) to the description of the family and Poelt 1976, Lawrey and Diederich 2003, Santes- Fuscideaceae. Therefore similarities in the ascus son et al 2004) in several parallel lineages however in structure between Teloschistales and Fuscideaceae none of them is the lichenization lost completely. were misleading and turned out to be homoplastic. Three other groups of lichen-forming fungi fall Peltigerales.—Strongly supported as monophyletic in within the Fuscideaceae-Umbilicariaceae clade: all analyses, the order Peltigerales comprises two Ophioparmaceae (represented by Ophioparma suborders, the Peltigerineae and Collematineae, as [FIG. 5], Boreoplaca [Lecanoromycetes genera inc. defined by Miadlikowska and Lutzoni (2004) based sed.] and [Lecideaceae] [FIG. 1]). on ribosomal genes and confirmed by Hofstetter et al Presence of an amyloid ascus with a tholus exhibiting (2007) based on a five-locus analysis. Peltigerineae, a strongly amyloid dome in these otherwise morpho- which differs from the Collematineae by the de- logically and anatomically distinct genera was used by velopment of conspicuous heteromerous thalli, the Wedin et al (2005) to support the monophyly (strong common occurrence of tripartite symbioses with PP support) of Boreoplaca, Hypocenomyce and Ophio- Nostoc as a secondary photobiont (cephalodia), the parma to form the extended family Ophioparmaceae. presence of bipartite associations with green algae Lumbsch et al (2004a) found in their study based on (Coccomyxa) and the production of diverse secondary nucLSU and mitSSU data that the family Elixiaceae compounds (mostly triterpenoids), includes Lobar- (with a single species Elixia flexella, not included in iaceae, and . For the this study) formed a well supported monophyletic first time the monophyly of (FIG. 1) is shown group within the family Umbilicariaceae and that the here to receive high bootstrap values. We found that former family circumscription including species with , a member of the (classi- foliose (umbilicate) thalli, possibly comprised a sister fied in the Collematineae), belongs to the Peltiger- group of crustose species. ineae and we confirm that Massalongia is placed also Rhizocarpomycetidae?/Rhizocarpales.—This order pro- in this suborder; however their sister relationship and posed here includes Rhizocarpaceae and Sporastatia. phylogenetic placement within the Peltigerineae While excluding Sporastatia from the family Acaros- remains uncertain. The monophyly of the Collemati- poraceae, Hafellner (1995) already questioned its neae, the second suborder within the Peltigerales, placement in the Catillariaceae, which could not be became significantly supported only when more tested here. A close affiliation between Sporastatia members from each family were incorporated in the and Rhizocarpon first was shown by Reeb et al (2004) phylogenetic analyses (10 taxa in the 5-gene dataset

r Peltigerales, (photo by R. Lu¨cking). 7. dissimilis. Close-up of fruiting bodies, Lecanoromycetidae, Teloschistales, (photo by E. Timdal). 8. russula, Lecanoromycetidae, Lecanorales, Lecanoraceae (photo by R. Lu¨cking). 9. atra, Lecanoromycetidae, Mycoblastaceae (photo by E. Timdal). 10. floerkeana, Lecanoromycetidae, Lecanorales, (photo by E. Timdal). 11. membranacea, Lecanoromycetidae, Lecanorales, (photo by E. Timdal). All photographs by E. Timdal are available at http://www.toyen.uio.no/ botanisk/lav/Photo_Gallery/PG_index.html. 1098 MYCOLOGIA vs. 19 in the 5+4+3-gene dataset). Collematineae as species here, and this phenotypically diverse genus defined here includes the monophyletic - and its relatives, , (both not ceae (, , , , included in this study) and ,areall and ), (only nonmonophyletic genera (Kasalicky et al 2000, Gaya was sampled), Collemataceae ( et al 2003, Søchting and Lutzoni 2003, Gaya 2006). and nonmonophyletic Leptogium [FIG. 6]) and Pla- Our results also confirm that the Caliciaceae, here cynthiaceae (excluding Polychidium). represented by Tholurna (FIG. 7) and Calicium, are nested in the buellioid branch (Buellia and related .—Lecideaceae, intermixed with - genera) of the (Wedin et al 2002) and are ceae (Porpidia), appeared as a sister group of the not supported as a monophyletic group. Loss of active Peltigerales (strongly supported in FIG. 1). This is ascospore dispersal (i.e. evanescent asci) and evolu- surprising because the Lecideaceae seem to share no tion of mazaedial ascomata evidently occurred several common features with members of the Peltigerales. times (e.g. shown to be sister of the Moreover the placement of Porpidia intermixed with by Geiser et al [2006]), including in in the Lecideaceae, detected also by Busch- the buellioid clade. Placement of within bom and Mueller (2004), questions the recognition Buellia (see also Helms et al [2003]) raises doubts of the entire family Porpidiaceae based on ascal about the validity of the former genus, which is structures. A basal position of (former distinguished only by spermatial characters; however Lecidea) to Lecideaceae received only PP support in Amandinea species included in the analysis is not the the 5+4-gene analysis. Both Lecidoma and Porpidia generic type (A. coniops). Although the genus Buellia have asci with amyloid tube structures but of different s.l. is extremely diverse (e.g. Marbach 2000) and features (Hafellner 1984), whereas in Lecidea this pending a proper sampling in phylogenetic study, we tube seems to be reduced to a minute structure in the propose to maintain the current classification. A close tholus tip. These three genera have brown to dark relationship between the genus and ascomata, crustose to adpressed squamulose thalli with coccalean green algae, (Asterochloris and Tre- was mentioned by Scheidegger et al 2001 and bouxia, Rambold et al 1998). In contrast to Lecidea, demonstrated in a phylogenetic study by Helms et al Porpidia species have prominent dark parathecial (2003). Both taxa have delayed ascospore septum margins, halonate ascospores and intensely anasto- formation, shared with the genus , which is mosing paraphyses, while a carbonization of portions nested in the buellioid clade. of the apothecia as in these two genera is not found in We propose here to establish two suborders within Lecidoma.FurthermorePorpidia and Lecidea are the Teloschistales, Physciineae and Teloschistineae. strictly saxicolous whereas Lecidoma grows on . The phylogeny presented here (FIG. 1) shows two Hafellner (1984) had introduced the monotypic main lineages within the Physciineae, a predominantly family Lecidomataceae to accommodate the relatively buellioid clade (Caliciaceae) and a rinodinoid clade unique genus Lecidoma. ( and related genera, Physciaceae). Teloschistales.—The Letroutiaceae, Lecanorales.—The order Lecanorales includes eight and Teloschistaceae forming a monophyletic group families (FIG. 1), Cladoniaceae, Lecanoraceae, Myco- sister of the monophyletic Physciaceae is confirmed blastaceae, Parmeliaceae, , , here for the first time (FIG. 1). These families differ and Stereocaulaceae. At least one considerably in the cortical pigmentation of their further large family, the emended (not thallus. While the Physciaceae are diverse in their sampled for this study), which also includes members cortical pigmentation (mostly atranorin), predomi- of the Micareaceae and Ectolechiaceae (Eriksson nance of anthraquinones is characteristic for the 2006), was found to be closely related to - Teloschistaceae. Most representatives of the Tel- ceae by Andersen and Ekman (2004, 2005) and oschistaceae and Physciaceae are characterized by therefore is part of the Lecanorales. polar diblastic ascospores, which often display con- The well supported Parmeliaceae is the most spicuous endospore thickenings. Letrouitia (Letroui- speciose family within the Lecanorales and comprises tiaceae) diverged earlier than Megalospora (Megalos- mostly foliose to fruticose lichens associated exclu- poraceae) and Teloschistaceae. Megalospora is sively with coccalean green algae (predominantly phenotypically different from the Teloschistales, Trebouxia, Rambold et al 1998). A diagnostic charac- especially in its peculiar ascus type (i.e. without any ter for this family is the presence of a cupula in the teloschistalean features, ascospores without internal ascomata, a well differentiated cup-shaped hyphal thickenings, thallus chemistry and the lack of quinoid structure beneath the hypothecium (Henssen and substances). is represented only by one Jahns 1974). All members also share a similar type of MIADLIKOWSKA ET AL:SYSTEMATICS OF THE LECANOROMYCETES 1099 ascus with a broadly shaped nonamyloid masse axiale. (Eriksson 2006), is shown here to be a member of A series of papers recently reviewed the previously the Ramalinaceae. Lopezaria is similar in ascospore controversial classification within this family (e.g. type and to Megalospora but apparently not Crespo et al 2001; Blanco et al 2004a, b, 2005, 2006; closely related to the latter, and its large, thick-walled Thell et al 2004). Blanco et al (2006) recently ascospores thus have evolved independently. Apothe- demonstrated that the taxonomic value of key cial features are otherwise similar to those of certain characters (presence of and atranorin in tropical species and support its placement in the cortex of the thallus, occurrence of pseudocy- the Ramalinaceae. Except for Lopezaria, asci in this phellae and pored epicortex) traditionally used to group are of similar type, but diverse growth forms classify members of the Parmeliaceae at generic and include crustose, squamulose and fruticose thalli. suprageneric levels has been overemphasized in Hafellner (1988) regarded Lecaniaceae (as an avail- previous classifications. Most of the cetrarioid genera able family name for crustose bacidioid lichens with are grouped together, sister of (FIG. 1). lecanorine apothecia) and Ramalinaceae as members Another well supported monophyletic group includes of the same evolutionary lineage in term of thallus and , both with fruticose, pendent to evolution. , currently classified within shrubby thalli and distinguished by cortical com- the Lecanoraceae and Strangospora, currently with an pounds (usnic acid vs. amorphous melanin-like uncertain placement in the Lecanoromycetes (Eriks- substances) and hymenial characters including asco- son 2006), are shown here as members of the spore pigmentation (pigmented vs. hyaline). The lack Lecanorales. Hafellner (1984) had introduced the of support for most deep internodes within the family and later (Hafellner 1995) Parmeliaceae is due to the little divergence recorded discussed a possible closer relationship of Strangos- within this strongly supported monophyletic group. pora and Scoliciosporum, both with similar ascomata ITS can be aligned across members of this family for and Lecanora-type asci, but the type species of these example. Therefore the fastest evolving sites of the genera have different ascospores (polyspored one- nucLSU, nucSSU and mitSSU, which would be most celled vs. eight-spored phragmospore). The addition appropriate to increase phylogenetic confidence in of more taxa from these genera is needed to resolve this portion of the tree, had to be excluded from their affiliation within the Lecanorales. these analyses due to the presence of indels rendering Results from this study do not support previous positional homology too uncertain in these regions (a subordinal circumscriptions within the Lecanorales typical problem of broad selection of taxa that also (Hafellner et al 1993). Cladoniineae as shown here include a large group of closely related taxa). includes Cladoniaceae (, Cladonia The Lecanoraceae comprise Lecanora, and [FIG. 10] and ) and Stereocaulaceae (Le- Pyrrhospora (FIG. 8) in our tree. This relationship is praria [FIG. 11], and ). Both supported by tholus amyloidity in the ascus and the families share the same main photobiont type presence of a broad masse axiale, common features in identified as Asterochloris (Rambold et al 1998) and, all three genera. The sister relationship of with the exception of Squamarina, asci with tholi and Tephromela (FIG. 9), as also found by Wedin et al provided with amyloid tube structures. Discovered for (2005), is unexpected. Both genera were classified in the first time by Ekman and Tønsberg (2002) the separate families by Hafellner (1984). They differ close relationship of Lepraria and Stereocaulon was considerably in their ascospores (large and thick- supported only in the 5+4-gene phylogeny. For the walled in Mycoblastus vs. small and thin-walled in first time Squamarina is well supported as being Tephromela) and secondary chemistry (Mycoblastus related to the Stereocaulaceae (shown by Stenroos partly with chinoid substances vs. Tephromela partly and DePriest 1998 but not supported). However the with the rare a-collatolic acid) but share the tar-black inclusion of Squamarina in Stereocaulaceae is in- pigmentation of the epihymenium, which can extend congruent with morpho-anatomical characters, such downward into the hymenium. The inclusion of as ascomatal and ecological attributes (e.g. all further taxa will show whether two separate families, Squamarina grow on calcareous substrates). While Tephromelataceae and Mycoblastaceae, are needed. the Cladoniaceae and Stereocaulaceae previously The monophyletic Psoraceae, Ramalinaceae and were placed in the informal group owing to Sphaerophoraceae as well as their interfamilial ascus characters, Squamarinaceae was recognized as relationships are all well supported. Our analyses a separate group (Hafellner et al 1993). Ekman and support the Ramalinaceae to include the Bacidiaceae, Tønsberg (2002) demonstrated that the Lecanora- as outlined by Ekman (2001, but see Andersen and ceae are more closely related to the Cladoniaceae and Ekman 2005). Lopezaria, considered to be a genus of Stereocaulaceae than suggested by Hafellner et al uncertainpositionwithintheLecanoromycetes (1993), who included the Lecanoraceae together with 1100 MYCOLOGIA the Parmeliaceae in the suborder . Thus ous asci (i.e. . 100 spores/ascus) evolved indepen- in our analysis the Cladoniineae are nested within dently in several lineages of the Lecanoromycetes the Lecanorineae using the previous subordinal (e.g. in Acarosporaceae, Biatorella, Maronea, Sporasta- concept (Poelt 1974). It is too early to propose tia, Strangospora and Thelocarpon). Less pronounced a revised subordinal classification within the Leca- polyspory is found in many other groups throughout norales. the Lecanoromycetes (e.g. in Candelariella, [species formerly assigned to Solorinella], Gyalideop- Photobiont selectivity as a taxonomic character.—Al- sis, members of Buellia, Caloplaca, , Leca- though photobiont relationships and life strategy nora, Rinodina and other genera). Unusually large characters were mainly disregarded in previous ascospores (e.g. in Asterothyrium, Megalospora, Myco- taxonomic treatments of the lichen-forming fungi, blastus, Pertusaria, Psorotheciopsis and Solorina)or photobiont associations are highly structured across long-filiform ascospores (e.g. in , Bapal- the Lecanoromycetes phylogeny (FIG. 1, similar to muia, and )alsohaveevolved what was anticipated for the Lecanorales by Rambold independently many times within the Lecanoromy- et al 1998) suggesting that these symbiotic interac- cetes. tions are the result of a highly selective process and Our study confirms that different types of asci can where shifts from one main type of photobiont to occur in a single lineage of closely related taxa or that another were rare during the evolution of the lichen the same ascus type can be found in distinct lineages symbiosis. Large monophyletic groups of the Leca- (homoplasy). The widespread occurrence of the noromycetes have preferences for certain types of Lecanora type ascus (in Candelariaceae, Lecanora, photobionts (FIG. 1). For example members of the Parmeliaceae, , Pleopsidium, Scoliciosporum and Ostropales s.l., with mostly crustose thalli and high Strangospora) suggests that this type of ascus could be species diversity in wet tropical , are pre- ancestral (Chadefaud et al 1968) as discussed by dominantly associated with photobionts of the Tren- Wedin et al (2005) and therefore residual in many tepohliaceae, which do not occur as photobionts in lineages of the Lecanoromycetes. the Acarosporomycetidae, Candelariomycetidae, Le- canoromycetidae, Rhizocarpales and Umbilicariales. Characters of ascomatal architecture and pigmen- Only a few lineages in this order, including Gom- tation are also of varying significance for classification phillaceae, Asterothyriaceae (with Gyalidea), Di- in the Lecanoromycetes. While a cupula structure in ploschistes, Phlyctis, and Stictis, were able to switch the ascomata of the Parmeliaceae is a characteristic from filamentous to chlorococcalean green algae or feature of the whole group, such structures also occur in rare cases (Petractis) to cyanobacteria (Scytonema) intermittently in other Lecanoromycetes and can be as their photosynthetic partner. Mycobionts of the found in species of Caloplaca, Collema, Lecanora and Peltigerales (Lecanoromycetidae) have strong prefer- Rinodina. Although perithecioid ascomata character- ences for cyanobacteria (mostly Nostoc). In the ize the large family , such ascomata also Peltigerales bipartite associations with cyanobacteria are found in smaller unrelated genera in the seem to be the ancestral state, which either is Ostropomycetidae (e.g. Belonia, Protothelenella, Thele- maintained or switched repeatedly to coccalean nella or ). green algae (Coccomyxa and Dictyochloropsis in , and ), resulting either in ACKNOWLEDGMENTS phycosymbiodemes, tripartite symbioses or bipartite species that associate only with green algae in the We thank Bill Rankin, Sean Dilda and John Pormann for later stage of development (Miadlikowska and Lut- their assistance with the Duke C.S.E.M. computer cluster, zoni 2004). Lutzoni’s lab members for helpful comments and sugges- tions, Molly McMullen (Duke University Herbarium) for Photobiont-mycobiont patterns of associations curating lichen specimens and for proofreading the can greatly contribute to our understanding of manuscript, William R. Buck and David M. Hillis for relationships and evolution of lichen-forming fungi, providing lichen specimens. This publication resulted as already suggested by Rambold et al (1998). from the Assembling the Fungal Tree of Life (AFTOL) However this will require a re-examination of existing project, which is supported by NSF Assembling the Tree of records of green algae and cyanobacteria reported Life (AToL) award DEB-0228668 to FL. Additional to be associated with lichen-forming taxa based financial support comes from NSF CAREER award DEB- on recent phylogenetic treatments of these photo- 0133891 to FL and from the Academy of Finland bionts. (No. 211172) to SS. We also acknowledge support from NSF 0090301, Research Coordination Network: A Phylog- Ascomatal features as taxonomic characters.—As al- eny for Fungi to M. Blackwell, J.W. Spatafora and ready reported by Reeb et al (2004), highly polyspor- J.W. Taylor. MIADLIKOWSKA ET AL:SYSTEMATICS OF THE LECANOROMYCETES 1101

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