Slippery when wet: Phylogeny and character evolution in the gelatinous cyanobacterial lichens (Peltigerales, Ascomycetes)

Molecular Phylogenetics and Evolution 53 (2009) 862–871

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Slippery when wet: Phylogeny and character evolution in the gelatinous cyanobacterial lichens (Peltigerales, Ascomycetes) Mats Wedin a,*, Elisabeth Wiklund b, Per Magnus Jørgensen c, Stefan Ekman d a

The Swedish Museum of Natural History, P.O. Box 50007, SE-104 05 Stockholm, Sweden Department of Ecology and Environmental Science, Umeå University, SE-901 87 Umeå, Sweden c Museum of Natural History, University of Bergen, Allégaten 41, N-5007 Bergen, Norway d Museum of Evolution, Uppsala University, Norbyvägen 16, SE-752 36 Uppsala, Sweden b

a r t i c l e

i n f o

Article history: Received 9 March 2009 Revised 27 July 2009 Accepted 12 August 2009 Available online 18 August 2009 Keywords: Ascus apex Character state transformation Coccocarpiaceae Collemataceae Heteromerous Homoiomerous Lecanoromycetes Lobaria Pannariaceae Placynthiaceae

a b s t r a c t Many lichen fungi form symbioses with filamentous Nostoc cyanobacteria, which cause the lichen to swell and become extremely gelatinous when moist. Within the Lecanoromycetes, such gelatinous lichens are today mainly classified in the Collemataceae (Peltigerales, Ascomycota). We performed Bayesian MCMC, maximum likelihood, and maximum parsimony analyses of three independent markers (mtSSU rDNA, nuLSU rDNA, and RPB1), to improve our understanding of the phylogeny and classification in the Peltigerales, as well as the evolution of morphological characters that have been used for classification purposes in this group. The Collemataceae and the non-gelatinous Pannariaceae are paraphyletic but can be re-circumscribed as monophyletic if Leciophysma, Physma, Ramalodium and Staurolemma are transferred to the Pannariaceae. The gelatinous taxa transferred to the Pannariaceae deviate from other Collemataceae in having simple ascospores, and several also have a ring-shaped exciple as in other Pannariaceae, rather than the disc-shaped exciple found in the typical Collemataceae. Both Collema and Leptogium are non-monophyletic. The re-circumscribed Collemataceae shares a distinct ascus type with the sister group Placynthiaceae and the Coccocarpiaceae, whereas Pannariaceae includes a variety of structures. All Pannariaceae have one-celled ascospores, whereas all Collemataceae have two- or multi-celled spores. Reconstructions of the number of character state transformations in exciple structure, thallus gelatinosity, and ascus apex structure indicate that the number of transformations is distinctly higher than the minimum possible. Most state transformations in the exciple took place from a ring-shaped to a disc-shaped exciple. Depending on the reconstruction method, most or all transformations in thallus structure took place from a non-gelatinous to a gelatinous thallus. Gains and losses of internal structures in the ascus apex account for all or a vast majority of the number of transformations in the ascus, whereas direct transformations between asci with internal structures appear to have been rare. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Lichenization, the symbiosis where a fungus utilises algae or cyanobacteria as source of carbohydrates, is one of the most important ascomycete life strategies, comprising approximately 50% of all euascomycetes (Pezizomycotina) (Tehler and Wedin, 2008). A comparatively small number of ascomycete lichen fungi (8%, Ahmadjian, 1993; c. 1700 species, Rikkinen, 2002) utilise cyanobacteria as photobionts. These species may have a substantial impact on ecosystems they inhabit by fixing atmospheric nitrogen (Belnap, 2002; Cornelissen et al., 2007; Nash, 2008). Many * Corresponding author. E-mail address: [email protected] (M. Wedin). 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.08.013

cyanobacterial lichens appear to be very sensitive to habitat disturbance such as changes in forest age, structure and composition (e.g. McCune, 1993; Price and Hochachka, 2001; Hedenås and Ericson, 2008). Lichens featuring cyanobacteria as the main or sole photobiont are currently classified in three distantly related taxonomic groups (Lumbsch and Huhndorf, 2007; Tehler and Wedin, 2008): Arctomiaceae (Ostropomycetideae; Lumbsch et al., 2005), Lichinales (Lichinomycetes) and Peltigerales (often treated at subordinal level, Peltigerineae, within Lecanorales; e.g. Lumbsch et al., 2004; Wedin et al., 2005; Tehler and Wedin, 2008) in the Lecanoromycetes. As shown in several studies (e.g. Wiklund and Wedin, 2003; Miadlikowska and Lutzoni, 2004; Wedin and Wiklund, 2004), the lecanoromycetean lichen fungi with cyanobacterial symbioses form a monophyletic group, the Peltigerales. Most

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871

of these cyanolichens produce foliose, lobate thalli with the cyanobacterial photobiont organised in a more or less distinct layer beneath the cortex. Others produce conspicuously gelatinous thalli without a specialised photobiont layer (‘homoiomerous thalli’), and usually without a differentiated cortex. The gelatinous habit is due to the photobiont, Nostoc, having a sheet around the filamentous thread-like colonies that swells and becomes extremely gelatinous when wet. The gelatinous cyanolichens in Peltigerales have traditionally been classified in Collemataceae, the ‘jelly lichens’. The classification of fungi in the Lecanoromycetes has for a long time been strongly influenced by assumptions on the systematic value of certain morphological character complexes. In particular, considerable a priori weight has been given to structures in the fungal sporangium (the sac-like ascus) and to developmental patterns and structures in the fruiting body in which the asci are produced (the disc-like ascoma). These structures have been assumed to transform slowly over evolutionary time and thus reflect phylogenetic patterns. Moreover, similarities in ascus and ascoma structures have been utilised as a priori criteria to delimit higher taxa and for assigning newly described taxa to higher groups. Reviews and criticisms of the usage of ascus structures in the systematics of the Lecanoromycetes have been provided by Wedin et al. (2005) and Ekman et al. (2008). Ascoma and ascus characteristics have been heavily utilised in the classification of the Collemataceae (e.g. as summarised in Henssen et al., 1981) although the group is rather difficult to characterise in these respects, as both ascus and ascoma characteristics are comparatively diverse. Collemataceae currently (Lumbsch and Huhndorf, 2007) comprises eight genera, all with Nostoc as photobiont. This family is—in addition to the more or less gelatinous habit—traditionally characterised by having a similar development of the ascomata, a disc-shaped proper wall (exciple) structure, and asci with an internal amyloid tube structure (Henssen, 1965; Henssen et al., 1981). Few discussions about the relationships within the family are available. Degelius (1954) considered Collema most closely related to Leptogium. These two genera include foliose species with multi-celled ascospores, Leptogium differing from Collema by having a distinct cortex on both thallus surfaces. Degelius (1954) expressed doubt about the monophyly of Collema and suggested that various species of Leptogium had evolved from species of Collema. If this is correct then Leptogium and Collema are non-monophyletic, as suggested by Wiklund and Wedin (2003), Wedin and Wiklund (2004), Miadlikowska and Lutzoni (2004), and Miadlikowska et al. (2006). Although the Collema representatives were nested within Leptogium, the number of taxa included in these studies was limited, and did not include Collema nigrescens, the type of Collema. In addition to the two large genera Collema and Leptogium, which have multi-celled ascospores, Collemataceae currently includes six smaller genera with single-celled ascospores: Homothecium, Leciophysma, Leightoniella, Physma, Ramalodium and Staurolemma. Homothecium and Leciophysma differ from the rest of the family by the ring-shaped proper exciple rather than a disc-shaped exciple in the mature apothecia. They are distinguished from each other by the shape and size of the thalli (Henssen, 1965, 1979; Henssen et al., 1981). The type of amyloid tube in Homothecium and Leciophysma is also different from the ordinary Collemataceae-type (Rambold, in Hafellner et al., 1993). The monotypic Leightoniella differs from other Collemataceae with simple ascospores in the anatomy of the ascoma, where the proper exciple is composed of periclinally arranged hyphae, and has a distinct supporting tissue. Leightoniella is considered most closely related to Collema and Leptogium (Henssen, 1965), which share a similar type of proper exciple. Physma forms comparatively large thalli and produces a conspicuous ascoma wall of lichen thalline

863

origin (Henssen, 1965; Henssen et al., 1981). Ramalodium and Staurolemma are considered closely related to each other. They are similar in general gross morphology and both lack an amyloid tube-structure in the asci, but differ in having slightly different ascoma ontogeny and morphology (Jørgensen and Henssen, 1999; Jørgensen and Henssen, 1993, 1999; Henssen et al., 1981). In Ramalodium the proper exciple is ring-shaped early in the development and becomes disc-shaped later on, whereas in Staurolemma a disc-shaped exciple surrounds the hymenium already in the young apothecium (Henssen, 1999). No representatives of these smaller genera with single-celled ascospores have been included in any of the major phylogenies focussing on relationships in the Peltigerales. Species of two further more or less gelatinous genera, Polychidium and Leptochidium, have been placed in Collema or Leptogium in early classifications. These, however, form a wellcircumscribed group together with Massalongia (Wedin et al., 2007) and are only distantly related to Collemataceae. The closest relative to the Collemataceae are instead the non-gelatinous Placynthiaceae (Wiklund and Wedin, 2003; Miadlikowska and Lutzoni, 2004; Wedin and Wiklund, 2004; Wedin et al., 2007). Rambold (in Hafellner et al., 1993) noted that many Collemataceae and Placynthiaceae share a similar ascus type with a conical axial body surrounded by a strongly amyloid layer (Rambold and Triebel, 1992, Fig. 19g). This could be seen as a putative morphological synapomorphy for this group. Furthermore, both Collemataceae and Placynthiaceae lack the otherwise commonly occurring lichen secondary compounds, unique organic substances suggested to provide advantages such as protection against ultraviolet radiation, antibiotic activity, and detoxification (Lawrey, 1986; Purvis, 2000). Some representatives of Peltigeraceae and Lobariaceae are also more or less gelatinous, e.g. the aquatic Peltigera hydrothyrea (also first described in the Collemataceae; Russell, 1856) and some free-living cyanobacterial morphs of Peltigera, Sticta and Lobaria like ‘‘Dendriscocaulon umhausense”. Many of these free-living cyanobacterial morphs, not uncommon in taxa forming symbioses with green algae and cyanobacteria simultaneously, seem to be initial stages where symbiosis is first established (Peltigera venosa; Ott, 1988) or at least partly function as asexual reproductive propagules (Lobaria/‘‘Dendriscocaulon”). The other major family in the Peltigerales, which currently includes some gelatinous representatives, is the Pannariaceae. Seventeen genera are classified in this family (Jørgensen, 2003; Lumbsch and Huhndorf, 2007), three of which contain only gelatinous taxa. In the Pannariaceae in the sense of Jørgensen (1978, 1994), the ascus structures vary, also if the recently described Vahliella (Jørgensen, 2008) is removed from the family. Many Pannariaceae have amyloid internal tube-structures in the ascus apices. Santessoniella (Henssen, 1997) is the only larger gelatinous genus currently classified in Pannariaceae. Henssen (1997) suggested, based on studies of ascoma ontogeny and ascus structure, that Santessoniella is more similar to Parmeliella than to Pannaria, and that these two genera possibly should be excluded from the family. Ekman and Jørgensen (2002), however, found the type species S. polychidioides to be nested within Psoroma and thus to clearly belong to the Pannariaceae in a strict sense. Psoroma hypnorum, the type species of Psoroma, typically has green algae as main photobiont and cyanobacteria as an additional photobiont in distinct structures (‘‘cephalodia”). Recently, however, a cyanobacterial gelatinous morph has been reported to occur also in this species (Holien and Jørgensen, 2000). Finally, two small and very rarely collected tropical gelatinous genera are included in Pannariaceae; Kroswia and Lepidocollema. Kroswia was for some time only known sterile and then considered closely related to Pannaria (Jørgensen, 2002), which also contains some gelatinous representatives. Kroswia was recently found fertile, and the ascoma characteristics suggested that it is closely related to Fuscopannaria (Jørgensen,

864

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871

2007). Lepidocollema, finally, has apothecial characters similar to Parmeliella (Henssen and Jahns, 1973; Jørgensen, 2003). The aims of this study were to improve understanding of phylogenetic relationships among the gelatinous lichens in the Peltigerales and to provide basic knowledge of the evolution of those morphological characters that have been extensively employed for classification purposes in this group. We address a series of specific evolutionary questions that have remained unanswered: Are the gelatinous Collemataceae, as currently circumscribed, composing a monophyletic group, or are some of the genera more closely related to Pannariaceae? Are Collema and Leptogium as currently circumscribed reciprocally monophyletic groups? Did the gelatinous habit, typical of lichen fungal symbioses with Nostoc in combination with a homoiomerous thallus, originate more than once in Peltigerineae? Are ascus apex structures and ascoma characteristics reliable predictors of phylogenetic relationships, i.e. do the various states of these characters have single origins, or did they transform several times? Ascus apex characters in particular have played a major role in the systematics of the Lecanoromycetes, although recent molecular phylogenies indicate that some ascus-based classifications are doubtful (Wedin et al., 2005; Ekman et al., 2008). We achieved our aims by estimating a phylogeny on the basis of DNA sequence data from multiple genes and by applying comparative phylogenetic methods. The sampling of taxa and DNA sequence data was considerably extended compared to the investigations of Ekman and Jørgensen (2002), Wiklund and Wedin (2003), and Wedin and Wiklund (2004). In fact, taxon sampling in Peltigerales is the most extensive one so far published, covering most currently accepted genera and all currently accepted families.

2. Materials and methods 2.1. DNA extractions, amplification, and sequencing Total DNA was extracted using the Qiagen DNeasy Plant Mini Kit according to the manufacturer’s instructions, with the exception that the DNA was eluted in sterile water. Three genomic regions potentially useful for the study were selected: the nuclear LSU rDNA, the mitochondrial SSU rDNA, and the gene coding for the RNA polymerase II largest subunit, (RPB1). Polymerase chain reaction (PCR) amplifications were performed using Amersham Pharmacia Biotech Ready-To-Go PCR Beads; the fungal nuLSU and mtSSU rDNA were amplified with the following settings: initial denaturation 94 °C for 5 min, five cycles 94 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s, 30 cycles 94 °C for 30 s, 52 °C for 30 s and 72 °C for 60 s, followed by a final extension of 72 °C for 5 min. The primers used were ITS1F (Gardes and Bruns, 1993), nu-LSU-155-50 (Döring et al., 2000), LR0R, LR3, LR5 and LR6 (Vilgalys’ website: http://www.biology.duke.edu/fungi/mycolab/primers.htm) for the nu rDNA, and mrSSU1, mrSSU3R (Zoller et al., 1999) and MSU 7 (Zhou and Stanosz, 2001), for the mt rDNA, in different combinations. Fungal RNA polymerase II largest subunit (RPB1) was amplified with the following settings: initial denaturation 95 °C for 5 min followed by 7 cycles of 95 °C for 30 s, 58 °C for 40 sec (1°/cycle), 72 °C for 30 s, 33 cycles of 95 °C for 30 s, 51 °C for 40 s, 72 °C for 30 s, followed by a final extension of 72 °C for 8 min. The primers used were gRPB1-A (Stiller and Hall, 1997) and fRPB1-C (Matheny et al., 2002) or the following new primers designed here to fit members of the Pannariaceae: RPB1-BCF GACCAAGATCAAGAARTTRCTGGAG and RPB1-BCR CGNACRTTGCCATTTGCSCG. The PCR products were sequenced with the PCR primers using the DYEnamicET terminator cycle sequencing kit (Amersham Pharmacia Biotech) with the following settings: 28 cycles of 95 °C for 20 s, 50 °C for 15 s and 60 °C for

60 s. The samples were run on an automated sequencer (ABI Prism 377, PE Biosystems or an Applied Biosystems 3130xl Genetic Analyzer). 2.2. Sequence alignments Protein coding RPB1 sequences were unambiguously aligned by translating the DNA sequences to amino acids and manually minimizing the transformation cost according to the BLOSUM62 amino acid substitution matrix in the first place and BLOSUM 80 in the second place (Henikoff and Henikoff, 1992). This alignment contained very few gaps (six internal amino acid alignment sites) and most of the variation was found to be synonymous. Non-coding nuclear LSU rDNA and mitochondrial SSU rDNA sequences varied a lot more in length and were aligned in two steps. First, short sequences (defined as the ones with 6456 nucleotides in the mitochondrial SSU rDNA and 6827 nucleotides in the nuclear LSU rDNA) were removed and the remaining full-length sequences were aligned using the Q-INS-i algorithm (Katoh and Toh, 2008a) of the multiple alignment software MAFFT version 6.611 (Katoh et al., 2002; Katoh and Toh, 2008b) The gap opening cost was set to 1.53 and the offset to 0 (i.e., default parameters). MAFFT has repeatedly been suggested to provide the most accurate DNA and amino acid sequence alignments among currently available alignment software (Wilm et al., 2006; Ahola et al., 2006; Nuin et al., 2006; Carroll et al., 2007; Golubchik et al., 2007). Using the resulting alignments as profile, short sequences were finally aligned to their respective profile using ClustalX 2.0 (Larkin et al., 2007) with gap opening cost 6.0 and gap extension cost 3.0. The reason for not aligning all sequences in a single step using MAFFT was that short sequences were obviously displaced near their ends, possibly owing to a problem with handling terminal gap costs for sequences that are much shorter than the alignment. No manual adjustments were made. This alignment procedure combined objectivity and repeatability with the quality of a manual alignment. Ambiguous alignment in the nuclear LSU rDNA and mitochondrial SSU rDNA was identified and removed using Gblocks version 0.91b (Castresana, 2000) with the relaxed condition parameters suggested by Talavera and Castresana (2007). Including ambiguous alignment in phylogenetic analyses has been suggested to adversely affect phylogenetic analyses by increasing support for the wrong relationships (Talavera and Castresana, 2007). 2.3. Phylogenetic analyses To estimate dataset incongruence between the single-gene matrices the three markers were analysed separately by maximum parsimony bootstrapping and compared. Incongruence was defined as high bootstrap support (P70%; Hillis and Bull, 1993) for incompatible associations (i.e. groups in conflict with each other between the single-gene analyses). No incongruence was identified and we consequently proceeded with a phylogenetic analysis of the concatenated data. The three-gene matrix was then subject to maximum parsimony, Bayesian MCMC, and maximum likelihood analyses. Maximum parsimony and parsimony bootstrap analyses were performed using PAUP* 4.0b10 (Swofford, 2002), with the following settings; Parsimony analyses: heuristic search settings: gaps are treated as ‘‘missing”, 1000 random addition sequence replicates, TBR branch swap, steepest descent off, collapse branches if minimum length is 0, MulTrees on; bootstrap (Felsenstein 1985): heuristic search settings: 10 random addition replicates; bootstrap settings: 1000 bootstrap replicates, full heuristic search, retain groups with frequency >50%. Uninformative characters were excluded from the analyses.

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871

Bayesian MCMC phylogenetic analyses were performed using the MPI version of MrBayes 3.2 (Ronquist and Huelsenbeck, 2003; Altekar et al., 2004). The procedure, including also the choice of likelihood model and priors, closely followed that of Ekman et al. (2008) with the following exceptions: (1) best-fitting 3-rate models were approximated with 2-rate models to fit the constraints in model choice imposed by MrBayes, (2) the appropriate partitioning was selected by choosing between a model with three (corresponding to the genes) and a model with five partitions (also partitioning RPB1 into its codon positions), (3) six discrete categories were used for modelling the gamma distributed rate heterogeneity between sites, and (4) six chains, one cold and five heated, were employed in each of the three parallel runs. Following this scheme, the analyses employed five partitions with the following models: HKY+I+C for RPB1 first codon positions, K80+I+C for RPB1 second codon positions, GTR+I+C for RPB1 third codon positions, GTR+I+C for the nuclear LSU rDNA, and HKY+I+C for the mitochondrial SSU rDNA, model denotations following the documentation accompanying the software ModelTest 3.7 (Posada and Crandall, 1998). All model parameters except topology and branch lengths were unlinked across partitions. Maximum likelihood bootstrap support was estimated using the MPI version of RAxML 7.0.4 (Stamatakis, 2006) in rapid hillclimbing mode (Stamatakis et al., 2007). We used independent GTR+I+dC4 models for the same partitions as in the Bayesian analysis. The number of bootstrap replicates was 1000, each based on a single search for an optimal tree. Each search was started from a maximum parsimony tree. Likelihood optimization was interrupted when the ln likelihood difference between consecutive iterations fell below 0.001. All parameters of the model were estimated from the data. The number of character state transformations in three discrete morphological characters was reconstructed across the part of the phylogeny including Pannariaceae, Collemataceae, Placynthiaceae, and Coccocarpiaceae: (A) thallus heteromerous and non-gelatinous (0) or homoiomerous and gelatinous (1), (B) proper exciple disc-shaped (0) or ring-shaped (1), and (C) ascus apex internally without amyloid structures (0), with a conical axial body surrounded by a strongly amyloid layer (Rambold and Triebel, 1992, Fig. 19g) (1), with a distinct amyloid tube reaching from the ascospore mass to the upper ascus wall (2), or with an amyloid, convex and sickle-shaped sheet (3). Transformations were counted using maximum parsimony and maximum likelihood as implemented in Mesquite version 2.6 (Maddison and Maddison, 2009) across the entire Bayesian posterior tree sample. In the parsimony calculations, character states were treated as unordered. In the likelihood calculations, single-rate models were used in combination with the lowest likelihood decision threshold allowed by the software (108), the latter to avoid artifacts with fewer than the most parsimonious number of transformations. Transformations were also counted using Bayesian stochastic character mapping as implemented in SIMMAP version 1.0 beta 2.4 (Huelsenbeck et al., 2003; Bollback, 2006). Single-rate models were used throughout (and are the only ones allowed for multistate characters). A gamma distributed prior on tree length was obtained by fitting the distribution of parsimony tree lengths (obtained via Mesquite) to a gamma distribution using the software EasyFit 5.0 (MathWave Technologies). Consequently, the following shape and inverse scale parameters of the gamma distribution were used (5-digit precision; A–C corresponding to the characters above): A (25944; 6485.1), B (12165; 4054.0), and C (252.91; 37.383). The gamma distribution was discretised into 60 categories. Trees were rescaled to a total length of 1 before applying the prior. For each tree in the posterior tree sample, 25 draws from the prior were made.

865

3. Results 3.1. Taxon sampling Sequences were newly produced or retrieved from GenBank (Table 1). We sampled taxa representing most genera currently classified in Collemataceae (Collema, Leciophysma, Leptogium, Physma, Ramalodium, and Staurolemma) and in Pannariaceae (Degelia, Erioderma, Fuscoderma, Fuscopannaria, Leioderma, Pannaria, Parmeliella, Protopannaria, Psoroma, and Vahliella; Jørgensen, 2003, 2008), including the type species of all these genera with the exception of Degelia and Erioderma. We failed to include material of Homothecium and Leightoniella (Collemataceae) and Austrella, Degeliella, Kroswia, Lepidocollema, Santessoniella, and Siphulastrum (Pannariaceae) due to lack of fresh material. Most of these groups are small, often monotypic, and rare. In addition, we included representatives of all other families currently accepted in the Peltigerales covering most of the genera in each group: Coccocarpiaceae (Coccocarpia, Steinera), Lobariaceae (Lobaria, Pseudocyphellaria, and Sticta), Massalongiaceae (Leptochidium, Massalongia, and Polychidium), Nephromataceae (Nephroma), Peltigeraceae (Peltigera, Solorina) and Placynthiaceae (Placynthium). Two representatives of Lecideaceae represent the sister group to Peltigerales according to Miadlikowska et al. (2006), and the outgroup used to root the tree, Cladonia, Hypogymnia, and Stereocaulon, represents the closely related Lecanorales s. str. 3.2. DNA sequencing We obtained 29 new nLSU rDNA, 30 new mtSSU rDNA and 45 RPB1 sequences (Table 1). The sequences were aligned together with sequences from GenBank. The data matrices consisted of bp-sites from the beginning of nLSU up to the LR5-priming site, and from the mrSSU1 to the mrSSU3R priming sites, respectively. 3.3. Phylogenetic analyses We compiled matrices including 70 taxa for which we had exon data of at least two of the three DNA markers. MAFFT and ClustalX alignments of nuLSU and mtSSU were 1017 and 1242 sites in length, 153 and 535 of which were suggested for removal by Gblocks. The concatenated matrix (after alignment and removal of ambiguous) included 2189 characters (RPB1 618, nuLSU rDNA 864 and mtSSU rDNA 707) of which 1112 were variable and 861 parsimony-informative. Of the variable characters, 333 belonged to the RPB1 partition of which 298 were parsimony-informative, 390 belonged to the nuLSU rDNA of which 254 were informative, and 389 to the mtSSU rDNA, of which 309 were parsimonyinformative. The Bayesian analysis halted automatically after 2.7  106 generations. The majority-rule consensus tree (Fig. 1) was based on the 16,200 trees included in the pooled posterior tree sample from all three runs. The harmonic mean ln likelihood across all three runs was 29,546.35. The MP analyses resulted in 30 most parsimonious trees of 5660 steps, CI = 0.28 and RI = 0.62, and differed only the details of the terminal topology within Pannariaceae and Collemataceae. Groupings significantly supported by the ML and MP bootstrap analyses are also indicated in Fig. 1. 3.4. Number of character state transformations The number of character state transformations for three discrete morphological characters using maximum parsimony, maximum likelihood, and stochastic mapping are shown in Table 2. In all characters, the predicted number of transformations is roughly

866

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871

Table 1 Specimens and sequences included (newly produced sequences in bold), and character states of the three characters studied in the Collematineae (scored for this group only; for state explanations, see Table 2). Species

Cladonia rangiferina Hypogymnia physodes Lecidea fuscoatra Lecidea silacea Stereocaulon paschale Coccocarpia domingensis C. erythroxyli C. palmicola Collema cristatum C. curtisporum C. furfuraceum C. multipartitum C. nigrescens C. parvum Degelia durietzii D. plumbea Erioderma leylandii E. verruculosum Fuscoderma amphibolum F. applanatum Fuscopannaria ahlneri F. leucosticta F. praetermissa Leciophysma finmarkicum L. furfurascens Leioderma erythrocarpum Leptochidium albociliatum Leptogium cyanescens L. diffractum L. gelatinosum L. imbricatum L. lichenoides L. plicatile L. saturninum Lobaria amplissima L. hallii L. linita L. pseudoglaberrima L. pulmonaria L. retigera L. scrobiculata L. virens Massalongia carnosa Nephroma bellum N. parile Pannaria rubiginosa P. rubiginella Parmeliella triptophylla Peltigera aphtosa P. horizontalis Physma byrsaeum P. radians P. pseudoisidiatum Placynthium nigrum Polychidium muscicola Protopannaria pezizoides Pseudocyphellaria aurata P. crocata P. divulsa P. dubia Psoroma hypnorum Ramalodium. succulentum Solorina crocea S. saccata Staurolemma omphalarioides

Staurolemma sp. nov. Steinera glaucella Sticta canariensis

Specimen data for newly produced sequences

Sweden, Wedin 6157 (UPS)

Dominica, Ceni & Veˇzda (BM) Veˇzda Lich Rar Exs. 234 Norway, Løfall bpl-L10515 (O) Norway, Hofton et al. 01202 (O) Norway, Wedin 6187 (BM) Norway, Haugan 7015 (O) Sweden, Wedin 7046 (UPS) Sweden, Nordin 5500 (UPS) New Zealand, Wedin 8045 (S) Portugal, Purvis et al. 27/4/1995 (BM)

New Zealand, Wedin 4099 (UPS) New Zealand, Wedin 8016 (S) South Korea, Thor 16992 (UPS) USA, Harris 33159 (S) Sweden, Wedin 7671 (UPS) Sweden, Nordin 5515 (UPS) Sweden, Nordin 5695 (UPS) New Zealand, Wedin 8013 (S) USA, Tønsberg 29087 (BG) Norway, Wedin 6182 (BM) Sweden, Nygren 13 (UPS) Norway, Wedin 6184 (BM) Sweden, Nordin 5505 (UPS) Norway, Wedin 6206 (BM) Sweden, Nordin 5566 (UPS) Sweden, Wedin 7672 (UPS), LSU only; Sweden, Wedin 13/5/1996 (UPS), RPB1 only UK, Wedin 6172 (BM) Canada, Wedin 4801 (UPS) Sweden, Wedin 6911 (UPS) Uruguay, Geymonat 2002 (UPS) Obermayer, Dupl. Lich. Graec. 266 Sweden, Wedin 5092 (UPS) Sweden, Wedin 6168 (UPS) Norway, Wedin 6192 (BM) Sweden, Hermansson 8916 (UPS) Sweden, Wedin 6169 (UPS) Portugal, Purvis et al. 27/4/95 (BM) Canada, Thor 10050 (S) Sweden, Wedin 7037 (UPS) UK, Wedin 6182 (BM) Tahiti, Jones s.n.(BG) Japan, Thor 12547 (UPS) USA, Smith 11.III.05 (S) Sweden, Wedin 6778 (UPS) Austria, Obermayer 8547 (UPS) Obermayer, Lich Exs. Graec 178 Sweden, Wiklund 51 (UPS) Portugal, Purvis, James & Smith 7/5/1995 (BM) Chile, Wedin 6012 (BM) Chile, Wedin 6118 (BM) Sweden, Wiklund 35 (UPS) Australia, Tibell 12239 (UPS)

Spain, Hafellner & Hafellner 41399 (UPS) Obermayer, Lich. Exs. Graec. 255, LSU only. Norway, Tønsberg 17654 (BG), mtSSU only China, Aptroot 55941 (ABL) UK, Wolseley & Orange 1998 (BM)

nuLSU rDNA

mtSSU rDNA

RBP1

Ascus apex structures

Structure of the proper exciple

Thallus gelatinosity

AY300832 AY756338 AY756339 AY756340 AY340568 DQ912346 DQ883800 GQ258987 DQ917408 GQ258988 AY340541 GQ258989 GQ258990 GQ258991 GQ258992 AY340543 DQ900639 DQ973041 GQ258993 GQ258994 GQ258995 DQ900640 GQ258996 GQ258997 GQ258998 — DQ900644 AF356672 GQ258999 GQ259000 GQ259001 DQ900645 GQ259002 GQ259003

AY300881 AY756400 AY756401 AY756402 AY340525 — DQ912294 GQ259016 GQ259017 GQ259018 AY340488 GQ259019 GQ259020 GQ259021 GQ259022 AY340491 AY340492 DQ972990 GQ259023 GQ259024 GQ259025 DQ900630 GQ259026 GQ259027 GQ259028 GQ259031 DQ900633 AY340406 GQ259029 AY340497 GQ259030 GQ259032 GQ259033 AY340499

DQ915595 AY756407 AY756408 AY756409 GQ259046 DQ912372 DQ883743 — — GQ259047 GQ259048 — GQ259049 GQ259050 GQ259051 GQ259052 — DQ973062 — GQ259053 GQ259054 GQ259055 GQ259056 GQ259057 GQ259058 GQ259059 GQ259060 GQ259061 GQ259062 — — — GQ259063 GQ259064

1 1 1 1 1 1 1 1 ? 3 3 3 3 0 0 2 2 2 2 2 0

0 0 0 0 0 0 0 0 ? 1 1 1 1 1 1 1 1 1 1 1 1

0 0 0 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 0

1 1 1 1 1 1 1

0 0 0 0 0 0 0

1 1 1 1 1 1 1

AY340546 GQ259004 GQ259005 GQ259006

AY340500 GQ259034 GQ259035 GQ259036

GQ259065 GQ259066 — GQ259067

AY340549 AY340550 AY340551 AY340553 AY340554 AY300844 AY340557 AY340558 GQ259007 GQ259008 AF286759 GQ259009 GQ259010 GQ259011 GQ259012 AF356674 DQ900647

AY340504 AY340505 AY340506 AY340508 AY340509 AY300895 AY340512 AY340513 GQ259037 AY652623 AY340515 GQ259038 GQ259039 GQ259040 GQ259041 AY340518 DQ900634

GQ259068 — GQ259069 GQ259070 GQ259071 — GQ259072 GQ259073 GQ259074 GQ259075 DQ915598 GQ259076 GQ259077 GQ259078 — GQ259079 GQ259080

0 0 2

1 1 1

0 0 0

2 2 ? 1

0 0 ? 0

1 1 1 0

AY340561 AY340562 AY340563 AY340564 — AY340565 GQ259013 DQ973043 AY300863 GQ259014

AY340519 AY340520 AY340521 AY340522 GQ259042 AY340523 GQ259043 — AY340524 GQ259044

GQ259081 GQ259082 — GQ259083 GQ259084 GQ259085 GQ259086 DQ973066 — —

0

1

0

2 0

1 0

0/1 1

0

0

1

GQ259015 AY300863 AY340570

GQ259045 AY300913 AY340527

GQ259087 — GQ259088

0

0

1

867

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871 Table 1 (continued) Species

Specimen data for newly produced sequences

nuLSU rDNA

mtSSU rDNA

RBP1

Sticta fuliginosa Vahliella leucophaea

UK, Wedin 6078 (BM) Sweden, Wedin 6849 (UPS)

AY340572 DQ900642

AY340529 AY652621

GQ259089 GQ259090

between 2.3 and 6.2 times the minimum possible amount of change (which corresponds to the number of states minus one). The three reconstruction methods agree roughly about the proportions of change contributed by each individual. However, the 95% credible sets of character state transformation types were clearly different between methods. Maximum likelihood reconstructions assigned 95% of the total probability to only four types of transformations, whereas the corresponding credible sets for maximum parsimony and stochastic mapping reconstructions included seven and eight transformation types, respectively.

4. Discussion Two groups corresponding to the two suborders Peltigerineae and Collematineae (sensu Miadlikowska and Lutzoni, 2004) are recovered in all analyses, with the exception that Steinera (ML and Bayesian inference) or Coccocarpiaceae as a whole (MP) falls outside these two groups in the analyses of the combined dataset. In the ML and Bayesian analyses, the Coccocarpiaceae (with the exception of Steinera) falls within the Collematineae-clade with high support, and forms the sister group to the Placynthiaceae– Collemataceae, but this detailed placement is without significant support. In all analyses, Steinera has a basal position in the Peltigerineae. In Wiklund and Wedin (2003) and Wedin and Wiklund (2004) the Coccocarpiaceae grouped within the Collematineae, but without support. In the investigation by Miadlikowska et al. (2006) which was based on a larger number of genes (five) but a smaller taxon sample, Coccocarpia formed the sister group to Pannariaceae. Vahliella leucophaea (Jørgensen, 2008) falls outside of the Pannariaceae in all analyses, similar to the results of Ekman and Jørgensen (2002) and Wedin and Wiklund (2004), but additional samples and species should be included in future analyses to test this further. Should further investigations confirm that Vahliella is the sister group to the rest of Peltigerineae sensu Miadlikowska and Lutzoni (2004), then a new family needs to be described. The most striking, and completely unexpected, result in our study is that all the included representatives of the gelatinous Collemataceae with one-celled ascospores (Leciophysma, Physma, Ramalodium, and Staurolemma) are nested within the Pannariaceae in all analyses. Collemataceae (in our sampling) is left to contain only Collema and Leptogium, both of which also turn out to be highly non-monophyletic in all analyses. Already Watson (1929) suggested excluding the simple-spored gelatinous genera from the Collemataceae, although he proposed that these should be classified in a newly proposed family, the Physmataceae (‘Physmaceae’), a concept that was never widely accepted. Collemataceae in our revised sense thus contains more or less foliose, gelatinous lichens with a thallus either lacking or having a very simple cortex, lacking lichen compounds, having apothecia with a closed cupular exciple, as well as asci with a conical axial body surrounded by a strongly amyloid layer (Magne, 1946; Degelius, 1954, p. 87), a feature shared with the closest relative, the Placynthiaceae, and similar to the homologous structure in Coccocarpiaceae (Rambold and Triebel, 1992). Ascospores in Collemataceae are unpigmented and usually ellipsoid, ovoid, or fusiform, possessing transverse and

Ascus apex structures

Structure of the proper exciple

Thallus gelatinosity

sometimes also longitudinal septa. Collema occultatum and related species have more or less cubical ascospores, but we have not sampled any of these taxa. Leptogium and Collema have traditionally been distinguished on the basis of the presence (Leptogium) or absence (Collema) of a proper cortex. Already Degelius (1954) considered the possibility that the two genera were non-monophyletic relative to each other and suggested, based on similarities in ascospore characteristics, that certain Leptogium species may be more closely related to certain Collema species. This is clearly supported by our results (Fig. 1), but our taxon sampling is obviously not large enough to suggest any new classification. At present we note, however, that the two generic type species (Leptogium lichenoides and Collema nigrescens) appear in two different subgroups within the Collemataceae. It is also noteworthy that the larger species of Leptogium (L. saturninum, L. cyanescens) group with the larger species of Collema s. str. in all analyses, whereas the type of Leptogium forms a group with small squamulose species which are usually regarded as closely related to it (L. gelatinosum, L. imbricatum). Non-monophyly of Collema and/or Leptogium was also found, but with a more limited taxon sampling, by Wiklund and Wedin (2003), Wedin and Wiklund (2004), Miadlikowska and Lutzoni (2004), and Miadlikowska et al. (2006). Pannariaceae includes a well-supported basal group of the foliose Degelia, Erioderma, Leioderma, and Parmeliella triptophylla (the type of Parmeliella) in all analyses. This was also the case in Wedin and Wiklund (2004) and Wedin et al. (2007), but the addition of the RPB1 dataset has increased the support for this relationship considerably. The Degelia–Parmeliella clade shows some diversity in ascus structure. The species of Degelia investigated here have no internal amyloid structures (own observations), but some reports suggest an internal tube or sheet in some species (Arvidsson and Galloway, 1981; Jørgensen and James, 1990). Erioderma has no tube, but often an internal sheet (Keuck, 1977). Parmeliella triptophylla lacks a sheet, but has a distinct tube-structure. The Pannariaceae also includes Leciophysma, Ramalodium, Physma, and Staurolemma, genera formerly referred to the Collemataceae. In addition, Ekman and Jørgensen (2002) showed that the type species of the gelatinous Santessoniella (S. polychidioides), which is lacking in our sampling, is a true member of the Pannariaceae. Ramalodium and Staurolemma are, as expected, closely related. Staurolemma differs from Ramalodium mainly in the presence of an additional ascoma margin of thalline origin in Staurolemma, and in that the exciple is ring-shaped early in the development in Ramalodium but later turns disc-shaped. In Staurolemma, the exciple is disc-shaped already very early on in the development. The detailed placement of Physma, formerly classified in Collemataceae, varies between the model-based ML and Bayesian analyses, and the MP analyses. In all analyses, Physma appears nested within the Pannariaceae. In the ML and Bayesian analyses, Physma is inside the Pannariaceae as the sister group to Fuscoderma, and in the MP analyses, Physma is the sister group to the group including all Pannariaceae except the Degelia–Parmeliella clade, but this detailed placement is without support. Physma is a poorly investigated group, but our own observations suggest that it has ascal characteristics rather similar to many core-Pannariaceae, i.e. a distinct tube. Physma apparently differs from the rest of Pannariaceae

-

Peltigerineae

Physma byrsaeum ? ? Physma pseudoisidiatum Physma radians 1.0 Fuscoderma amphibolum Fuscoderma applanatum Psoroma hypnorum Staurolemma sp. nov. Staurolemma omphalariodes Ramalodium succulentum Pannaria rubiginosa Pannaria rubiginella Fuscopannaria ahlneri 0.96 Fuscopannaria leucosticta Fuscopannaria praetermissa Pannariaceae Protopannaria pezizoides Leciophysma finmarkicum Leciophysma furfurascens Degelia durietzii Leioderma erythrocarpa Erioderma leylandii Erioderma verruculosum Degelia plumbea Parmeliella triptophylla Leptogium imbricatum Leptogium lichenoides Leptogium gelatinosum ? ? Collema parvum Leptogium plicatile Collema cristatum Collema curtisporum Collema nigrescens Collemataceae Collema furfuraceum Leptogium cyanescens Leptogium saturninum Collema multipartitum Leptogium diffractum Placynthium nigrum Coccocarpia domingensis Coccocarpia palmicola Coccocarpia erythroxyli Lobaria amplissima Lobaria virens Lobaria pseudoglaberrima Lobaria scrobiculata Lobaria hallii Lobaria linita Lobaria pulmonaria Lobaria retigera Lobariaceae Sticta canariensis Sticta fuliginosa Pseudocyphellaria divulsa Pseudocyphellaria aurata Pseudocyphellaria dubia Peltigerales Pseudocyphellaria crocata Nephroma bellum Nephromataceae Nephroma parile Peltigera aphtosa Peltigera horizontalis Peltigeraceae Solorina crocea Solorina saccata Massalongia carnosa Massalongiaceae Polychidium muscicola Leptochidium albociliatum Vahliella leucophaea Steinera glaucella Lecidea fuscoatra Lecidea silacea Hypogymnia physodes Cladonia rangiferina Stereocaulon paschale

Collematineae

Thallus gelatinosity

Exciple structure

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871

Ascus apex

868

0.1 Fig. 1. Phylogenetic relationships among the Peltigerales resulting from Bayesian MCMC analysis (majority-rule consensus tree based on 16,200 trees, ln likelihood 29,546.35) based on a combined three-locus data set (nuLSU rDNA, mtSSU rDNA, and RPB1). Thick orange internodes received significant support in all three analyses conducted (Bayesian inference PP P 95%, ML-BP P 70%, MP-BP P 70%), thick black internodes were significant in the two model-based analyses but not under MP. Two nodes, indicated with Bayesian posterior probabilities, were only supported in the Bayesian inference. The quantity and direction of change within Collematineae was investigated in three discrete morphological characters (Table 2), the states of which are indicated with colours and shading: Ascus apex internally without amyloid structures (white), with a conical axial body surrounded by a strongly amyloid layer (orange), with a distinct amyloid tube reaching from the ascospore mass to the upper ascus wall (blue), or with an amyloid, convex and sickle-shaped sheet (grey); proper exciple disc-shaped (white) or ring-shaped (grey); thallus gelatinous (blue) or nongelatinous (white). ‘‘?” denotes taxa where ascoma and asci currently are unknown. The scale is in units of substitutions per nucleotide site.

869

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871

Table 2 (A) Average number of state transformations in three discrete morphological characters across a posterior sample of trees incorporating the Coccocarpiaceae, Pannariaceae, Placynthiaceae, and Collemataceae. (B) Proportion of change (summing to 1) distributed over individual character state changes. Character state transformations were counted using maximum parsimony (MP), maximum likelihood (ML), and Bayesian stochastic mapping (SM). SM results are reported here as marginal posterior probabilities. Character states were coded as follows: ascus apex internally without amyloid structures (0), with a conical axial body surrounded by a strongly amyloid layer (1), with a distinct amyloid tube reaching from the ascospore mass to the upper ascus wall (2), or with an amyloid, convex and sickle-shaped sheet (3); proper exciple disc-shaped (0) or ring-shaped (1); thallus heteromerous and non-gelatinous (0) or homoiomerous and gelatinous (1).

(A) MP ML SM

Ascus apex

Exciple structure

Thallus gelatinosity

6.76 7.00 8.37

3.00 3.03 3.83

4.00 3.98 6.22

(B) From:

To: 0 — — —

1 0.02 0.00 0.06

2 0.25 0.56 0.32

3 0.12 0.14 0.12

From:

MP ML SM

0

MP ML SM

1

0.05 0.14 0.10

— — —

0.06 0.00 0.04

0.00 0.00 0.02

MP ML SM

2

0.43 0.16 0.27

0.02 0.00 0.02

— — —

0.02 0.00 0.01

MP ML SM

3

0.01 0.00 0.01

0.00 0.00 0.01

0.00 0.00 0.01

— — —

in having a rather complex cortex structure, and in having a closed, disc-shaped exciple at least in the mature stage (Henssen and Jahns, 1973, p. 287). Pannariaceae in the new circumscription is rather complex and difficult to characterise morphologically. Pannariaceae mainly contains foliose and squamulose lichens with a thallus with a well-defined, sometimes multilayered, cortex, usually—but not always—containing secondary medullary compounds. The apothecia mostly have a ring-shaped exciple and the asci have rather diverse internal amyloid structures. All Pannariaceae have one-celled ascospores. The reconstructed number of character state transformations within the Coccocarpiaceae, Pannariaceae, Collemataceae, and Placynthiaceae indicate that all three characters investigated are more prone to change than previous classifications have postulated, the number of changes predicted in this study clearly exceeding the minimum allowed by the number of character states (i.e., the number of states minus one, one plesiomorphic state and the rest apomorphic, Table 2). Considerable flexibility in the ascus apex over evolutionary time is a result consistent with the findings reported by Lumbsch et al. (2001, 2007), Wedin et al. (2005), and Ekman et al. (2008). Although most of the change in the proper exciple seems to have taken place from an ordinary ring-shaped exciple (the common state among the Lecanoromycetes) to a disc-shaped exciple, all three reconstruction methods agree that change in the opposite direction has not been uncommon. Reconstructions of thallus gelatinosity are more ambiguous, as maximum likelihood and maximum parsimony agree that all change took place from a non-gelatinous and heteromerous thallus to a gelatinous and homoiomerous thallus, whereas stochastic mapping assigns about one third of the total probability to change in the reverse direction. Change in the ascus apex is more complex owing to the higher number of states (four instead of two in the other characters), leading to twelve possible types of transformations (Table 2. The general pattern emerging is that transformations from any other state to an ascus apex without internal structures (i.e., losses) and transformations in the opposite direction (gaining any ascus apex structure) together make up between 88 and 100% of the total amount of change, depending on the reconstruction method. Direct

0

1

To: 0 — — —

1 0.24 0.30 0.31

From:

0.76 0.70 0.69

— — —

0

1

To: 0 — — —

1 1.00 1.00 0.67

0.00 0.00 0.33

— — —

transformations between asci containing axial bodies, tubes, or sickle-shaped sheets, on the other hand, seem to have been rare. This suggests that the evolution of the ascus apex in this group of lichen fungi has proceeded primarily via a state of absence of amyloid structures in the ascus apex. Furthermore, the loss of an internal ascus structure seems to have limited effect on the subsequent potential to revert to an ascus with internal structures. A few important additional comments regarding the Peltigerineae, the remaining major group in Peltigerales (sensu Miadlikowska and Lutzoni, 2004), are warranted. The pattern in our phylogeny is similar to earlier published analyses. Peltigeraceae, Nephromataceae, and Massalongiaceae together form a monophyletic group. Lobariaceae (Lobaria, Sticta, and Pseudocyphellaria) and Lobaria, as traditionally circumscribed, are monophyletic. The possible non-monophyly of Lobaria has been discussed several times in recent literature. Yoshimura (1998, 2002) accepted Lobarina and Lobariella as segregates from Lobaria, as he presumed that Lobaria s. lat. would be polyphyletic. Yoshimura did not base this statement on any phylogenetic analyses, however. In our present investigation, Lobaria s. lat. is strongly supported as monophyletic in all analyses (Fig. 1). The type of Lobarina (L. scrobiculata) is clearly nested in Lobaria (type L. pulmonaria). The type of Lobariella (L. crenulata) has only been included in the ITS-based phylogeny by Stenroos et al. (2003) where Lobaria was found to be paraphyletic (but this was very poorly supported, see the discussion in Wiklund and Wedin, 2003). Lobariella (under the invalid name Durietzia) was the sister group to the Lobaria amplissima species group in Stenroos et al. (2003). It should thus be noted that no firm evidence suggest anything but that Lobaria s. lat. is a monophyletic group in its traditional circumscription. We have taken here a few important steps to improve the understanding of evolutionary relationships within the Peltigerales, the order to which a majority the lichens with a primary cyanobacterial photobiont belongs. The gelatinous lichens in the Peltigerales turn out to form a non-monophyletic group, and the Collemataceae and Pannariaceae consequently need to be re-circumscribed. The simple-spored gelatinous genera Leciophysma, Physma, Ramalodium, and Staurolemma should be transferred to

870

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871

the Pannariaceae, as also supported by morphological traits such as spore septation and ascus characteristics. Current classifications often assume that gelatinosity, ascus apex structures, as well as exciple construction change only very rarely, a prediction incompatible with our findings of more frequent change. On the other hand, many important issues concerning the evolutionary history of the Peltigerales remain to be clarified with more data than were available to us. The non-monophyly of Coccocarpiaceae s. lat, Collema, Leptogium, and Degelia needs confirmation with additional markers and taxa, and alternative monophyletic constellations need to be identified, and named when appropriate. A number of species groups and small genera are not represented in our phylogeny, and at the moment we can only make educated guesses about their relationships. Furthermore, ascus apex structures as well as ascoma anatomy are multifaceted character complexes, the morphology, function, and evolution of which are incompletely understood. Continued studies of phylogeny and character evolution are essential to enhance the understanding of this ecologically important and fascinating group of lichen-forming fungi. Acknowledgments This study was supported by grants from The Swedish Research Council (VR 629-2001-5756, VR 621-2002-349, VR 621-20033038, VR 621-2006-3760) to M.W. We are grateful to the Directors and Curators of herbaria and museums cited in Table 1 for the loan of specimens. The staff at Umeå University Plant Science Centre and Carin Olofsson provided invaluable laboratory assistance at Umeå University, as did the staff at the Molecular Systematics Laboratory, in particular Bodil Cronholm, at the Swedish Museum of Natural History. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev.2009.08.013. References Ahmadjian, V., 1993. The Lichen Symbiosis. John Wiley & Sons, Inc., New York. Ahola, V., Aittokallio, T., Vihinen, M., Uusipaikka, E., 2006. A statistical score for assessing the quality of multiple sequence alignments. BMC Bioinformatics 7. paper 484. Altekar, G., Dwarkadas, S., Huelsenbeck, J.P., Ronquist, F., 2004. Parallel Metropoliscoupled Markov chain Monte Carlo for Bayesian phylogenetic inference. Bioinformatics 20, 407–415. Arvidsson, L., Galloway, D.J., 1981. Degelia, a new lichen genus in the Pannariaceae. Lichenologist 13, 27–50. Belnap, J., 2002. Nitrogen fixation in biological soil crusts from south-east Utah, USA. Biol. Fert. Soils 35, 128–135. Bollback, J.P., 2006. SIMMAP: stochastic character mapping of discrete traits on phylogenies. BMC Bioinformatics 7, 88–94. Carroll, H., Beckstead, W., O’Connor, T., Ebbert, M., Clement, M., Snell, Q., McClellan, D., 2007. DNA reference alignment benchmarks based on tertiary structure of encoded proteins. Bioinformatics 23, 2648–2649. Castresana, J., 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552. Cornelissen, J.H.C., Lang, S.I., Soudzilovskaia, N.A., During, H.J., 2007. Comparative cryptogam ecology: a review of bryophyte and lichen traits that drive biogeochemistry. Ann. Bot. 99, 987–1001. Degelius, G., 1954. The lichen genus Collema in Europe. Morphology, taxonomy and ecology. Symb. Bot. Ups. 13 (2), 1–499. Döring, H., Clerc, P., Grube, M., Wedin, M., 2000. Mycobiont specific PCR primers for the amplification of nuclear ITS and LSU rDNA from lichenised ascomycetes. Lichenologist 32, 200–204. Ekman, S., Jørgensen, P.M., 2002. Towards a molecular phylogeny for the lichen family Pannariaceae (Lecanorales, Ascomycota). Can. J. Bot. 80, 625–634. Ekman, S., Anderson, H.L., Wedin, M., 2008. The limitations of ancestral state reconstruction and the evolution of the ascus in the Lecanorales (lichenized Ascomycota). Syst. Biol. 57, 141–156. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rust. Mol. Ecol. 2, 113–118.

Golubchik, T., Wise, M.J., Easteal, S., Jermiin, L.S., 2007. Mind the gaps: evidence of bias in estimates of multiple sequence alignments. Mol. Biol. Evol. 24, 2433– 2442. Hafellner, J., Hertel, H., Rambold, G., Timdal, E., 1993. Discussion 4. Lecanorales. In: Hawksworth, D.L. (Ed.), Ascomycete Systematics: Problems and Perspectives in the Nineties. Advanced Science Institutes Series, Plenum Press, New York, pp. 379–387. Hedenås, H., Ericson, L., 2008. Species occurrences at stand level cannot be understood without considering the landscape context: Cyanolichens on aspen in boreal Sweden. Biol. Conserv. 141, 710–718. Henikoff, S., Henikoff, J.G., 1992. Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 89, 10915–10919. Henssen, A., 1965. A review of the genera of the Collemataceae with simple spores (excluding Physma). Lichenologist 3, 29–41. Henssen, A., 1979. New species of Homothecium and Ramalodium from S America. Bot. Not. 132, 257–282. Henssen, A., 1997. Santessoniella, a new cyanophilic genus of lichenized ascomycetes. Symb. Bot. Ups. 32 (1), 75–93. Henssen, A., 1999. New species of Ramalodium and Staurolemma from Australasia (Collemataceae, lichenized ascomycetes). Nova Hedw. 68, 117–130. Henssen, A., Jahns, H.M., 1973 (‘1974’). Lichenes: Eine Einführung in die Flechtenkunde. Georg Thieme Verlag. Stuttgart. Henssen, A., Keuck, G., Renner, B., Vobis, G., 1981. The lecanoralean centrum. In: Reynolds, D.R. (Ed.), Ascomycete Systematics. The Luttrellian Concept. SpringerVerlag, New York, Heidelberg, Berlin, pp. 138–234. Hillis, D.M., Bull, J.J., 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42, 182–192. Holien, H., Jørgensen, P.M., 2000. A blue–green Psoroma hypnorum found in Trøndelag, Central Norway. Graphis Scripta 11, 49–52. Huelsenbeck, J.P., Nielsen, R., Bollback, J.P., 2003. Stochastic mapping of morphological characters. Syst. Biol. 52, 131–158. Jørgensen, P.M., 1978. The lichen family Pannariaceae in Europe. Opera Bot. 45, 1– 123. Jørgensen, P.M., 1994. Studies in the Pannariaceae VI. The taxonomy and phytogeography of Pannaria Del. s. lat. J. Hattori Bot. Lab. 76, 197–206. Jørgensen, P.M., 2002. Kroswia, a new lichen genus in the Pannariaceae (lichenized ascomycetes). Lichenologist 34, 297–303. Jørgensen, P.M., 2003. Conspectus familiae Pannariaceae (Ascomycetes lichenosae). Ilicifolia 4, 1–78. Jørgensen, P.M., 2007. New discoveries in Asian pannariaceous lichens. Lichenologist 39, 235–243. Jørgensen, P.M., 2008. Vahliella, a new lichen genus. Lichenologist 40, 221–225. Jørgensen, P.M., Henssen, A., 1993. Physma omphalarioides—its taxonomic position and phytogeography. Graphis Scripta 5, 12–17. Jørgensen, P.M., Henssen, A., 1999. Further species of the lichen genus Staurolemma (Collemataceae, lichenized Ascomycetes). Bryologist 102, 22–25. Jørgensen, P.M., James, P.W., 1990. Studies in the lichen family Pannariaceae IV: the genus Degelia. Bibl. Lichenol. 38, 253–276. Katoh, K., Misawa, K., Kuma, K., Miyata, T., 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066. Katoh, K., Toh, H., 2008a. Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework. BMC Bioinformatics 9. paper 212. Katoh, K., Toh, H., 2008b. Recent developments in the MAFFT multiple sequence alignment program. Brief. Bioinform. 9, 286–298. Keuck, G., 1977. Ontogenetisch-systematische Studie über Erioderma. Bibl. Lichenologica 6, 1–175. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. Lawrey, J.D., 1986. Biological role of lichen substances. Bryologist 89, 111–122. Lumbsch, H.T., Huhndorf, S.M. (Eds.), 2007. Outline of Ascomycota—2007. Myconet, vol. 13, pp. 1–58. Lumbsch, H.T., del Prado, R., Kantvilas, G., 2005. Gregorella, a new genus to accommodate Moelleropsis humida and a molecular phylogeny of Arctomiaceae. Lichenologist 37, 291–302. Lumbsch, H.T., Schmitt, I., Döring, H., Wedin, M., 2001. ITS sequence data suggest variability of ascus types and support ontogenetic characters as phylogenetic discriminators in the Agyriales (Ascomycota). Mycol. Res. 105, 265–274. Lumbsch, H.T., Schmitt, I., Mangold, A., Wedin, M., 2007. Ascus types are phylogenetically misleading in Trapeliaceae and Agyriaceae (Ostropomycetidae, Ascomycota). Mycol. Res. 111, 1133–1141. Lumbsch, H.T., Schmitt, I., Palice, Z., Wiklund, E., Ekman, S., Wedin, M., 2004. Supraordinal phylogenetic relationships of Lecanoromycetes based on a Bayesian analysis of combined nuclear and mitochondrial sequences. Mol. Phylogenet. Evol. 31, 822–832. Maddison, W.P., Maddison, D.R., 2009. Mesquite: a modular system for evolutionary analysis. Version 2.6. http://mesquiteproject.org. Magne, F., 1946. Anatomie et morphologie comparées des asques de quelques lichenes. Rev. Bryol. Lichénol. 15, 203–209. Matheny, P.B., Liu, Y.J., Ammirati, J.F., Hall, B.D., 2002. Using RPB1 sequences to improve phylogenetic inference among mushrooms (Inocybe, Agaricales). Am. J. Bot. 89, 688–698.

M. Wedin et al. / Molecular Phylogenetics and Evolution 53 (2009) 862–871 McCune, B., 1993. Gradients in epiphytic biomass in three Pseudotsuga-Tsuga forests of different ages in western Oregon and Washington. Bryologist 96, 405–411. Miadlikowska, J., Lutzoni, F., 2004. Phylogenetic classification of Peltigeralean fungi (Peltigerales, Ascomycota). Am. J. Bot. 91, 449–464. Miadlikowska, J., Kauff, F., Hofstetter, V., Fraker, E., Reeb, V., Grube, M., Hafellner, J., Kukwa, M., Lücking, R., Hestmark, G., Otalora, M.G., Tauhut, A., Büdel, B., Scheidegger, C., Timdal, E., Stenros, S., Brodo, I., Perlmutter, G.B., Ertz, D., Diederich, P., Lendemer, J.C., May, P., Schoch, C.L., Arnold, A.E., Hodkinson, B.P., Gueidan, C., Tripp, E., Yahr, R., Robertson, C., Lutzoni, F., 2006. New insights into classification and evolution of the Lecanoromycetes (Pezizomycotina, Ascomycota) from phylogenetic analyses of three ribosomal RNA- and two protein-coding genes. Mycologia 98, 1088–1103. Nash III, T.H., 2008. Nitrogen, its metabolism and potential contribution to ecosystems. In: Nash, T.H., III (Ed.), Lichen Biology, second ed. Cambridge University Press, pp. 216–233. Nuin, P.A.S., Wang, Z., Tillier, E.R.M., 2006. The accuracy of several multiple sequence alignment programs for proteins. BMC Bioinformatics 7. paper 471. Ott, S., 1988. Photosymbiodemes and their development in Peltigera venosa. Lichenologist 20, 361–368. Posada, D., Crandall, K.A., 1998. ModelTest: testing the model of DNA substitution. Bioinformatics 9, 817–818. Price, K., Hochachka, G., 2001. Epiphytic lichen abundance: effects on stand age and composition in costal British Columbia. Ecol. Appl. 11, 904–913. Purvis, O.W., 2000. Lichens. The Natural History Museum, London. Rikkinen, J., 2002. Cyanolichens: an evolutionary overview. In: Rain, A.N., Bergman, B., Rasmussen, U. (Eds.), Cyanobacteria in Symbiosis. Kluwer Academic Publishers, The Netherlands, pp. 31–72. Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Russell, J.L., 1856. Hydrothyria venosa, a new genus and species of the Collemaceae. Proceed. Essex Inst. 1, 188. Rambold, G., Triebel, D., 1992. The inter-lecanoralean associations. Bibl. Lich. 48, 1– 201. Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–2690. Stamatakis, A., Blagojevic, F., Nikolopoulos, D., Antonopoulos, C., 2007. Exploring new search algorithms and hardware for phylogenetics: RAxML meets the IBM cell. J VLSI Signal Process. 48, 271–286.

871

Stenroos, S., Stocker-Wörgötter, E., Yoshimura, I., Myllys, L., Thell, A., Hyvönen, J., 2003. Culture experiments and DNA sequence data confirm the identity of Lobaria photomorphs. Can. J. Botany 81, 232–247. Stiller, J.W., Hall, B.D., 1997. The origin of red algae: implications for plastid evolution. Proc. Natl. Acad. Sci. USA 94, 4520–4525. Talavera, G., Castresana, J., 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 56, 564–577. Tehler, A., Wedin, M., 2008. Systematics of lichenized fungi. In: Nash, T.H., III (Ed.), Lichen Biology, second ed. Cambridge University Press, pp. 336–352. Watson, W., 1929. The classification of lichens I. New Phytol. 28, 1–36. Wedin, M., Jørgensen, P.M., Wiklund, E., 2007. Massalongiaceae fam. nov., an overlooked monophyletic group among the cyanobacterial lichens (Peltigerales, Lecanoromycetes, Ascomycota). Lichenologist 39, 61–67. Wedin, M., Wiklund, E., 2004. The phylogenetic relationships of Lecanorales suborder Peltigerineae revisited. Symb. Bot. Ups. 34 (1), 469–475. Wedin, M., Wiklund, E., Crewe, A., Döring, H., Ekman, S., Nyberg, Å., Schmitt, I., Lumbsch, H.T., 2005. Phylogenetic relationships of the Lecanoromycetes (Ascomycota) as revealed by analyses of mtSSU and nLSU rDNA sequence data. Mycol. Res. 109, 159–172. Wiklund, E., Wedin, M., 2003. The phylogenetic relationships of the cyanobacterial lichens in the Lecanorales suborder Peltigerineae. Cladistics 19, 419– 431. Wilm, A., Mainz, I., Steger, G., 2006. An enhanced RNA alignment benchmark for sequence alignment programs. Algorithm Mol. Biol. 1. paper 19. Yoshimura, I., 1998. Vainio and Lobaria, old and modern concepts. In: Marcelli, M.P., Ahti, T. (Eds.), Recollecting Edvard August Vainio. CETESB, Sao Paulo, Brazil, pp. 85–94. . Yoshimura, I., 2002. Lobariella. In: Nash, T. H., III, Ryan, B. D., Gries, C., Bungartz, F. (Eds.), Lichen Flora of the Greater Sonoran Desert Region. I. Arizona State University, Tempe, Arizona, pp. 270–272. Zhou, S., Stanosz, G.R., 2001. Primers for amplification of mt SSU rDNA, and a phylogenetic study of Botryosphaeria and associated anamorphic fungi. Mycol. Res. 105, 1033–1044. Zoller, S., Scheidegger, C., Sperisen, C., 1999. PCR primers for the amplification of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetes. Lichenologist 31, 511–516.