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Systematics |
2Department of Biology, Duke University, Durham, North Carolina 27708-0338 USA; 3Plant Taxonomy and Nature Conservation, Gdansk University, Al. Legionow 9, 80-441 Gdansk, Poland
Received for publication July 1, 2003. Accepted for publication October 24, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: ancestral state reconstruction • Collematineae • Lecanoromycetidae • lichen symbiosis • molecular phylogenetic classification of ascomycetes • nuclear ribosomal large (LSU nrDNA) and small (SSU nrDNA) subunits • Peltigerales • Peltigerineae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Rai, 1990
; Schenk, 1992
). Bipartite fungal symbioses with green algae (usually Coccomyxa Léger & Hesse, Dictyochloropsis Geitler, or Myrmecia Printz) are relatively rare in these lichens (e.g., Tschermak-Woess, 1988
; Ahmadjian, 1993
). The mycobiont of several tripartite peltigeralean lichens [Nephroma Ach., Sticta (Schreber) Ach., Lobaria (Schreber) Hoffm., Psoroma Michaux, and Peltigera Willd.] can also form a cyanomorph thallus, in which the same fungus is associated with only the cyanobacterial partner, forming a bipartite thallus that is attached or growing separately from the mother tripartite thallus (Tønsberg and Holtan-Hartwig, 1983
; White and James, 1988
; Armaleo and Clerc, 1991
; Goffinet and Bayer, 1997
; Paulsrud et al., 1998
, 2000
; Holien and Jørgensen, 2000
). Peltigeralean lichens are worldwide in distribution; however, the Lobariaceae Chevall. are most abundant and diverse in the Southern Hemisphere, whereas other families (e.g., Peltigeraceae Dumort.) are more common and diverse in the Northern Hemisphere. These lichens grow on various substrates: mosses, rocks, soil, or bark, and mostly in moist to humid wooded habitats.
All lecanoralean taxa having cyanobacteria as their main photobiont are part of the suborder Peltigerineae (recognized also at the ordinal level Peltigerales; Kirk et al., 2001
) when broadly delimited. This broad circumscription of the Peltigerineae/Peltigerales includes 7–8 families (Rambold and Triebel, 1992
; Tehler, 1996
; J. Hafellner, H. Hertel, G. Rambold, and E. Timdal, unpublished manuscript), whereas the narrow delimitation of this taxonomic group (Table 1) includes only 3–5 families (Henssen and Jahns, 1973
; Poelt, 1974
; Hafellner, 1988
; Kirk et al., 2001
; Eriksson et al., 2003
). Differences in family numbers within the two major types of delimitations are due in part to the recognition of separate families for Solorina Ach. and Nephroma (Solorinaceae Bayrh. and Nephromataceae Wetm. ex J. C. David & D. Hawksw., respectively) instead of being classified within the Peltigeraceae sensu lato (Table 1). No matter how the Peltigeraceae and Lobariaceae (= Stictaceae sensu Henssen and Jahns, 1973
) have been delimited, they have been always included within the Peltigerineae/Peltigerales. All authors listed in Table 1 also consider the family Placynthiaceae Å. E. Dahl as part of the Peltigerineae/Peltigerales; however, Hafellner (1988
; = Lecotheciaceae Körber) suggested that this taxon may not belong in this group. Three additional families, Coccocarpiaceae (Mont. ex Müll. Stuttg.) Henssen, Collemataceae Zenker, and Pannariaceae Tuck., are sometimes considered as members of the Peltigerineae/Peltigerales (Rambold and Triebel, 1992
; Tehler, 1996
; J. Hafellner, H. Hertel, G. Rambold, and E. Timdal, unpublished manuscript). Otherwise, these three families have been placed within the heterogenous Lecanorales Nannf. (Poelt, 1974
; Hafellner, 1988
; Kirk et al., 2001
), or more specifically in the suborder Lecanorineae (Eriksson et al., 2003
).
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; Jørgensen, 1998
; Kirk et al., 2001
; Eriksson et al., 2003
).
Since the early 1980s (Hafellner, 1984
), ascus structure has been regarded as a highly relevant character for revealing relationships among lichen-forming ascomycetes at the genus and family levels. Asci with an amyloid (I+) tubelike structure in the tholus are present in members of the Peltigeraceae (Peltigera-type), Coccocarpiaceae, Collemataceae (Collema-type), and Placynthiaceae (Placynthium-type). In general, the tholus of the Lobariaceae and Pannariaceae has a more or less diffuse amyloid (I+) caplike structure. The Nephromataceae, with its nassasceous ascus, is the only family lacking an amyloid apical structure within the Peltigerineae/Peltigerales (Poelt, 1974
; Bellemère and Letrouit-Galinou, 1981
; Rambold and Triebel, 1992
). The fissitunicate type of dehiscence detected in Peltigera using transmission electron microscopy (Honegger, 1978
) is believed to occur also in the Nephromataceae (Nephroma), Solorina saccata (L.) Ach., and Lobariaceae (Lobaria and Sticta) based on evidence obtained with light microscopy (Richardson, 1970
; Eriksson, 1981
).
The distribution of secondary compounds across families within the broad selection of peltigeralean lichens sensu lato (s.l.) is heterogeneous. A great diversity of secondary compounds is found within the Peltigeraceae, Nephromataceae, and Lobariaceae (e.g., Culberson, 1969
, 1970
; Yoshimura, 1971
; James and White, 1987
; Galloway, 1988
, 1991
; White and James, 1988
; Holtan-Hartwig, 1993
; Kondratyuk and Galloway, 1995
; Miadlikowska and Lutzoni, 2000
). Galloway (1991)
suggested that triterpenoids, which are widespread within Peltigera, Pseudocyphellaria, and Lobaria, could be especially useful in detecting evolutionary relationships within and among these genera. A distinctly smaller number of lichen substances (e.g., beta-orcinol depsidones, xanthones, anthraquinones, and triterpenoids) are found in selected genera from the Pannariaceae (e.g., Jørgensen and Galloway, 1992
) and Coccocarpiaceae (e.g., Arvidsson, 1982
). Taxonomically diagnostic compounds were not reported from members of the Collemataceae and Placynthiaceae (C. Culberson, personal communication).
Hawksworth (1982)
proposed that the peltigeralean lichens were part of an ancient lineage within the Pezizomycotina O. E. Erikss. & Winka (Euascomycetes). This hypothesis was based mainly on the following observations: (1) the semifissitunicate ascus, which was thought to be ancestral to unitunicate and bitunicate asci; (2) the exceptionally high number of obligatory lichenicolous fungi growing on Peltigeraceae and Lobariaceae; (3) the wide distribution of many peltigeralean species; (4) the great diversity of secondary metabolites; and (5) the cyanobacterium being one of the earliest potential photobionts available for the origin of the lichens (Hawksworth, 1982
, 1988a
, b
; Galloway, 1991
). Broad molecular phylogenetic studies of the Ascomycota (Eriksson and Strand, 1995
; Lutzoni et al., 2001
; Kauff and Lutzoni, 2002
) refuted this hypothesis.
The first molecular phylogenetic study to include peltigeralean species [Peltigera neopolydactyla (Gyelnik) Gyelnik, Nephroma arcticum (L.) Torss., and Solorina crocea (L.) Ach.] was carried out by Eriksson and Strand (1995)
using 28 nucleotide sequences from the nuclear ribosomal subunit (SSU and nrDNA) and representing a broad spectrum of the Ascomycota. The reconstructed relationships allowed the authors to conclude that: (1) the Peltigerales are more closely related to Lecanorales than to, for instance, Helotiales Nannf. (
Leotiales Carpenter) or Pezizales C. Bessey; (2) the genus Solorina should be included in the family Peltigeraceae; and (3) the genus Nephroma deserves to be placed in its own family (Nephromataceae). Based on a combined analysis of the SSU and large subunit (LSU) nrDNA for a broad selection of species from the Ascomycota (52–60 species), Lutzoni et al. (2001)
and Kauff and Lutzoni (2002)
have demonstrated that the order Peltigerales is nested within the bitunicate ascohymenials ( Lecanoromycetidae; Taylor et al., in press
). Moreover, the phylogenetic trees published by Kauff and Lutzoni (2002)
show the Peltigeraceae (Peltigera), Lobariaceae (Lobaria), Collemataceae [Leptogium (Ach.) Gray], and Placynthiaceae [Placynthium (Ach.) Gray] forming a monophyletic and highly supported group.
The only molecular phylogenetic study to date specifically addressing relationships across the Peltigerineae was recently published by Wiklund and Wedin (2003)
. Based on a combined mitochondrial SSU and nuclear LSU rDNA data set for a broad sampling from the Lecanorales s.l., including all putative peltigeralean families, they concluded that the Peltigerineae (including the Coccocarpiaceae, Collemataceae, Lobariaceae, Nephromataceae, Pannariaceae, Peltigeraceae, and Placynthiaceae) is a monophyletic group. This well-supported suborder was sister to the rest of Lecanorales, suggesting that this was the first evolutionary split, taking place after the origin of the Lecanorales. The parsimony and jackknifing analyses resolved the Lobariaceae as a monophyletic group. Within this family, Lobaria and Sticta were monophyletic, whereas relationships among Pseudocyphellaria species were shown to be unresolved in their two majority-rule consensus trees resulting from parsimony jackknife analyses. They also concluded that the Placynthiaceae is a sister group to the Collemataceae and that Leptogium may be paraphyletic. Furthermore, they confirmed the nonmonophyly of the Pannariaceae that was first revealed by the ITS-based phylogenetic study of Ekman and Jørgensen (2002)
, who also showed that Degelia sect. Amphiloma (Fr.) P. M. Jørg. & P. James, Fuscopannaria P. M. Jørg. subg. Micropannaria P. M. Jørg., and Moelleropsis humida Gyelnik are not part of the Pannariaceae.
Phylogenetic relationships within the Lobariaceae inferred from ITS sequences were the focus of a recent study by Thomas et al. (2002)
and by Stenroos et al. (2003)
. Based on selected taxa from New Zealand, Thomas et al. (2002)
demonstrated that the Lobariaceae and genera Nephroma, Peltigera, and Sticta were monophyletic. These results confirm an earlier report that Peltigera is monophyletic (Miadlikowska and Lutzoni, 2000
). They have also shown that the genus Pseudocyphellaria is a nonmonophyletic entity consisting of three independent lineages, including a monophyletic group of species with a white medulla and two groups of taxa with a yellow medulla. Contrary to Wiklund and Wedin (2003)
, Thomas et al. (2002)
reported that the genus Lobaria is not monophyletic. The nonmonophyletic status of Lobaria and Pseudocyphellaria, as well as the monophyletic delimitation of Sticta, was also confirmed by Stenroos et al. (2003)
. Although molecular phylogenetic studies to date support the broad recognition of the Peltigerineae/Peltigerales including the Coccocarpiaceae, Collemataceae, Placynthiaceae, and Pannariaceae, none of these studies provided well-supported sister-group relationships of these taxa with other members of the Lecanorales s.l.
Because the delimitation of the Peltigerineae/Peltigerales varies remarkably among recent classifications, the main goal of our study was to provide a comprehensive molecular phylogeny for the peltigeralean fungi and establish a new classification if needed. By using a different selection of taxa, molecular markers, and phylogenetic methods that were used by Thomas et al. (2002)
, Stenroos et al. (2003)
, and Wiklund and Wedin (2003)
, we conducted an independent reassessment of the relationships within the Peltigerineae. Our specific objectives were to: (1) circumscribe the Peltigerineae/Peltigerales using monophyly as the grouping criterion; (2) provide a robust estimation of the phylogenetic placement of these lichen-forming fungi within the Ascomycota; (3) test the monophyly of selected genera and families of peltigeralean fungi; (4) evaluate the taxonomic rank (order to genus level) that should be applied to peltigeralean fung; and (5) reconstruct the evolution of symbiotic associations for peltigeralean fungi.
To achieve these goals, phylogenetic analyses of the nuclear LSU and SSU rDNA were conducted using maximum parsimony (MP) and maximum likelihood (ML) as optimality criteria for 113 specimens (59 within the Peltigerineae/Peltigerales), representing 108 species (54 within the Peltigerineae/Peltigerales), and 59 genera (12 within the Peltigerineae/Peltigerales). Representatives of virtually all major lineages of the Ascomycota were included in this study. Bootstrap proportions (BP; Felsenstein, 1985
) and posterior probabilities (PP) obtained with Bayesian-Metropolis coupled Markov chain Monte Carlo (B-MCMCMC) tree sampling (Larget and Simon, 1999
; Huelsenbeck, 2000
) were used to estimate levels of confidence for topological bipartitions revealed by phylogenetic analyses. Symbiotic ancestral states for selected lineages within the Peltigerineae/Peltigerales were reconstructed using ML (Pagel, 1999
) as implemented in Mesquite 0.995 (Maddison and Maddison, 2003a
, b
) on 20 000 trees sampled with B-MCMCMC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Kauff and Lutzoni, 2002
) to provide a core phylogenetic framework of the Ascomycota for assessing phylogenetic placement(s) of the peltigeralean fungi. These 53 sequences represent 51 species from 13 of 15 ascomycete orders that include lichen-forming species, 11 of 31 orders incorporating only nonlichenized species (according to the classification of Hawksworth et al., 1995
), and two basidiomycetes as outgroup. To these 53 core sequences, we added five LSU and SSU sequences from Schmitt et al. (2001)
representing members of the Pertusariineae/Pertusariales M. Choisy ex D. Hawksw. & O. E. Erikss.: two Ochrolechia A. Massal. species (Pertusariaceae Körber ex Körber), and three Coccotrema Müll. Arg. species (Coccotremataceae Henssen ex J. C. David & D. Hawksw.). This core sampling of 58 specimens (56 species) included four genera from the Peltigerineae/Peltigerales: Leptogium (Collemataceae), Placynthium (Placynthiaceae), Lobaria (Lobariaceae), and Peltigera (Peltigeraceae).
We sequenced the same portion of the LSU and SSU nrDNA for an additional 55 specimens (51 species and 10 genera) representing six putative peltigeralean families (Collemataceae, Lobariaceae, Nephromataceae, Pannariaceae, Peltigeraceae, and Solorinaceae). All families included in the Peltigerineae/Peltigerales (except Coccocarpiaceae) under various classifications (Table 1) were represented in our overall sampling of 107 species. Of the 113 specimens included in our study, the 59 peltigeralean specimens (54 species and 12 genera) were considered part of the ingroup. Nine of the 12 genera were represented by at least two species. All putative genera (except Siphulastrum Müll. Arg. and Dendriscocaulon Nyl.) from Nephromataceae, Lobariaceae, Solorinaceae, and Peltigeraceae (sensu Eriksson et al., 2003
) were part of our sampling. For the remaining three families, two genera were selected from the Pannariaceae and Collemataceae (2/14, Psoroma and Pannaria Del. ex Bory; 2/8, Collema F. H. Wigg. and Leptogium), and one of seven genera was sampled from the Placynthiaceae (Placynthium; following the classification of Eriksson et al., 2003
). To test if phenotypic circumscriptions of widely recognized genera are monophyletic, several putative North American and European species from Lobaria, Pseudocyphellaria, Sticta (Lobariaceae), Nephroma (Nephromataceae), and Peltigera (Peltigeraceae) were selected. For the purpose of this study, we kept the generic concept of Lobaria s.l. including Lobarina (Vainio) Nyl. ex Cromb. [= Lobaria scrobiculata (Scop.) Nyl. ex Crombie group] from before the taxonomic changes introduced by Yoshimura (1998a)
. Because we wanted to evaluate the intrageneric classification of the genus Peltigera that we proposed in a previous paper (eight monophyletic sections based on morphological, chemical, and LSU data; Miadlikowska and Lutzoni, 2000
), this genus was represented by the highest number of species (28). Voucher information and GenBank accession numbers for the 59 LSU and SSU nrDNA sequences from the Peltigerineae/Peltigerales included in this study have been archived as supplemental data accompanying the online version of this article.
To diminish the amount of ambiguity in the 113-specimen alignments and to improve phylogenetic resolution and support, we removed 36 sequences belonging to taxa that are clearly outside the Lecanoromycetidae for a second set of analyses. The two Acarospora A. Massal. species were used to root the ingroup of the trees derived from these 77-specimen data sets.
Molecular data
Genomic DNA was obtained from fresh samples and herbarium specimens. DNA isolation, symmetric PCR amplification, asymmetric PCR sequencing, PCR product purification, and automated sequencing were performed as presented in Miadlikowska and Lutzoni (2000)
and Miadlikowska et al. (2003)
. The SSU symmetric PCR were done using primers nssu97a, nssu97b, or nssu131 (Kauff and Lutzoni, 2002
) at the 5' end in combination with NS22 (Gargas and Taylor, 1992
) at the 3' end. In addition to these primers, the following set of primers was used for cycle sequencing reactions: SR7, SR7R, SR11R, nssu634, nssu897R, nssu1088, and nssu1088R (Kauff and Lutzoni, 2002
). The LSU and SSU sequences were subjected to BLAST searches for a first verification of their identities. They were assembled using Sequencher 4.1 (Gene Codes Corporation, Ann Arbor) and aligned manually with McClade 4.01 (Maddison and Maddison, 2001
). The delimitation of ambiguous regions within alignments, their unequivocal coding, and the elaboration of symmetric step matrices for each of these coded characters were done using a method developed by Lutzoni et al. (INAASE 2.3b; 2000).
Phylogenetic analyses
Weighted maximum parsimony (MP) and maximum likelihood (ML) analyses were performed with PAUP* 4.0b.8a (Swofford, 2001
). Analyses were done on separate and combined LSU and SSU data sets of 113 and 77 specimens. Parsimony ratchet search strategy (PAUPRat; Nixon, 1999
; Sikes and Lewis, 2001
) and its modified version for the likelihood search were implemented in PAUP* on selected MP and ML data sets.
Maximum parsimony analyses
Two MP searches were carried out, MP1 on the LSU + SSU 113 operational taxonomic unit (OTU) combined data set and MP2 on the LSU + SSU 77 OTU combined data set. Constant sites and ambiguously aligned sites were removed from the MP searches, and phylogenetic signal from ambiguous regions was recovered using INAASE 2.3b. Unambiguously aligned portions of the LSU and SSU alignments were subjected to symmetric step matrices using the computer program STMatrix 2.1 (written by S. Zoller and available upon request from S. Z. or F. L.) as outlined in Gaya et al. (2003)
. Gaps were treated as a fifth character state for the unambiguous portions of the alignments. The two MP analyses were performed using heuristic searches with 550 random-addition-sequences (RAS) for MP1 and 1000 RAS for MP2, TBR (tree bisection reconnection) branch swapping, Multrees option in effect, rearrangement limit = 8, and collapsing branches with maximum branch length equal to zero. PAUPRat was implemented with PAUP* on MP1 and MP2 data sets by performing 200 iterations with 15% of characters perturbed per iteration with the same parameters as for the original MP analyses. Branch support for MP1 and MP2 trees was estimated by bootstrap analyses (BP-MP; Felsenstein, 1985
) by performing 200 bootstrap replicates with four RAS per bootstrap replicate with the same parameters as for the initial MP analyses.
Maximum likelihood analyses
Two ML searches were carried out, ML1 on the LSU + SSU 113 OTU combined data set and ML2 on the LSU + SSU 77 OTU combined data set. Constant sites were part of ML analyses. A hierarchical likelihood ratio test (Modeltest 3.04 PPC; Posada and Crandall, 1998
) was used to select the nucleotide substitution models and parameters for ML searches. The ML1 search was performed using the TIMef+G+I substitution model with equal base frequencies, substitution rate matrix (A
C = 1.0000, A
–
G = 2.8365, A–T = 0.6634, C–G = 0.6634, CT = 5.2300, and G–T = 1.0000), gamma distribution parameter 
= 0.5165, and proportion of invariable sites I = 0.5698. The ML2 search was performed using the TrNef+G+I substitution model with base frequencies (A = 0.2965, C = 0.2039, G = 0.2700, and T = 0.2296), substitution rate matrix (A–C = 1.0000, A–G = 2.7081, A–T = 1.0000, C–G = 1.0000, C–T = 6.0870, and G–T = 1.0000), gamma distribution parameter = 0.5431, and proportion of invariable sites I = 0.4159. The ML1 analysis was performed by TBR swapping on the MP1 tree with the Multrees option in effect, reconnection limit = 8, and all branches of effectively zero length collapsed. The ML2 analyses were done using heuristic searches with 50 RAS and the same settings as for the ML1 analysis. To confirm that the most likely tree was found, an additional TBR swapping on the MP2 tree was completed using the same search parameters as for the initial ML2 analysis. PAUPRat was implemented with PAUP* on the ML2 data set by performing 100 iterations with 15% of characters perturbed per iteration with the same parameters as for the initial ML2 analysis.
We used Bayesian posterior probabilities (PP) as implemented in "MrBayes 2.01" (Huelsenbeck, 2000
) to assess confidence for relationships revealed by ML combined searches. All Bayesian analyses were initiated with random trees and flat priors. One of every 50 trees was sampled for 2 000 000 generations with four chains and substitution parameters updated during the search. To ensure that all chains converged at the same level, majority rule consensus trees were assembled with PAUP* using only the last 20 000 of the 40 000 trees sampled. Internodes with PP 
95% were considered statistically significant. Throughout this paper, we refer to the Bayesian analysis of the ML1 data set and the resulting majority rule consensus tree as the B/ML1 analysis or tree. In addition to Bayesian searches, phylogenetic confidence for the ML2 data set was estimated with bootstrap resampling (BP-ML). We executed 100 bootstrap replicates with three RAS per bootstrap replicate with the same parameters as for the initial ML2 analysis.
Testing for phylogenetic congruence among data partitions
Before combining the LSU and SSU data sets for 113 and 77 specimens, phylogenetic congruence was assessed for each data partition. For MP analyses, this was done by inspecting internodes with bootstrap scores 70% resulting from the separate SSU and LSU bootstrap analyses (Mason-Gamer and Kellogg, 1996
; outlined in Miadlikowska and Lutzoni, 2000
). For ML analyses, congruence was tested by inspecting internodes with posterior probabilities 95% resulting from the separate Bayesian analyses (as outlined in Miadlikowska et al., 2002
). Conflicts between the SSU and LSU data partitions were considered significant only if two different relationships (one monophyletic and the other nonmonophyletic) for the same set of taxa were supported by BP 70% or PP 95%.
Reconstructing symbiotic ancestral states within the Peltigerineae/Peltigerales
Symbiotic ancestral states within the peltigeralean fungi were reconstructed based on the 77-specimen LSU + SSU data set phylogeny (ML2) for nine crucial and significantly supported nodes (PP 95%) within the Peltigerineae/Peltigerales. Three character states representing all possible symbiotic associations among peltigeralean fungi (0 = bimembered with a green alga, 1 = bimembered with a cyanobacterium, and 2 = trimembered with a cyanobacterium and a green alga) were considered putative ancestral symbiotic states. Ancestral states were reconstructed with maximum likelihood as the optimality criterion (Pagel, 1999
) on the same 20 000 trees sampled with B-MCMCMC (as described earlier) using the Trace Character Over Trees option in Mesquite 0.995 (Maddison and Maddison, 2003a
, b
). Using a likelihood ratio test, the asymmetric two-parameter model was selected for this analysis. Only ancestral states reconstructed with raw likelihood scores greater than 2.0 (i.e., the default setting T = 2.0 in Mesquite), corresponding to a conservative approximation of proportional likelihood values >0.95 in our analysis, were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The final size of the combined alignment for the 77-specimen data set was 5218 sites (2130 LSU and 3088 SSU sites). A total of 2968 sites were excluded from the MP2, ML2, and Bayesian analyses (1018 LSU sites representing 10 ambiguous regions and introns at six splicing sites; 1950 SSU sites representing four ambiguous regions and introns at 10 splicing sites). Of the 2250 characters included in the ML2 and Bayesian searches, 1635 were constant (774 LSU and 861 SSU sites) and 615 were variable (338 LSU and 277 SSU sites). For the MP2 analyses, the 1635 constant sites were excluded and seven coded INAASE characters replaced the three and four ambiguously aligned regions from the LSU and SSU data sets, respectively, for a total of 622 variable characters. Of these, 401 were parsimony informative (243 LSU and 158 SSU characters).
The LSU and SSU trees were congruent for both the 113- and 77-specimen data sets when using the reciprocal 70% BP and 95% PP criterion. Therefore, the LSU and SSU data sets were combined and analyzed simultaneously using maximum parsimony (MP1 and MP2, respectively) and maximum likelihood (ML1 and ML2, respectively) as optimization criteria.
Comparison of resolution and support among optimality criteria and taxon sampling
The MP1 search on the 113-specimen LSU + SSU combined data set with 24 INAASE characters revealed 60 equally most parsimonious trees (not shown) of 7039.72 steps, which were part of two islands. One island of one tree was hit once out of 550 RAS and the second island of 59 trees was hit 46 times (consistency index [CI, excluding uninformative characters] = 0.3679; retention index [RI] = 0.6751; tree not shown). The 30 most parsimonious trees found with PAUPRat (resulting from 30 of 200 iterations) were the same length as the optimal trees found with the MP1 search and were a subset of the 60 most parsimonious MP1 trees. The level of confidence provided by the bootstrap analysis was relatively low for the deep relationships among ascomycetes and for many internodes within the Peltigerineae/Peltigerales ingroup (Fig. 1).
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95% in ML1 with BP values 70% in MP1. The level of confidence recovered from B/ML1 (Fig. 1) was higher than phylogenetic support obtained by bootstrapping the MP1 data set. Of the 59 nodes with PP 95% in B/ML1, 17 received bootstrap support <70% in the MP1 analysis (Fig. 1). Overall, none of the combined analyses on the 113 specimens provided well-resolved and supported deep relationships within the Pezizomycotina (Euascomycetes).
A single most likely tree (–ln = 12 428.611 70; part of a single island hit 20 times out of 50 RAS) was revealed by the ML2 analysis (Fig. 2A). The same topology was obtained when swapping on the MP2 tree and when implementing PAUPRat, for which the most likely tree was hit 33 times out of 100 iterations. The same monophyletic groups were found by MP2 and ML2 analyses within the Peltigerineae/Peltigerales. However, 14 of 44 nodes with PP 95% in the ML2 received BP-MP support <70% in the MP2 analyses, whereas only eight (mostly short) internodes with significant posterior probabilities received BP-ML support below 70%. Therefore, a maximum likelihood based bootstrap analysis provided confidence values 70% for six additional topological bipartitions compared to a maximum parsimony based bootstrap analysis. Only one node with bootstrap value 70% in the MP2 was not significant (PP <95%) with the Bayesian inference part of ML2 (Fig. 2A). Phylogenetic relationships within the bitunicate ascohymenials (sensu Lutzoni et al., 2001
; Kauff and Lutzoni, 2002
) and the Peltigerineae/Peltigerales were better resolved and supported based on this 77-specimen ML2 analysis than on the 113-specimen B/ML1 analysis (Fig. 2A vs. Fig. 1). However, many of the topological bipartitions revealed exclusively by ML2 were not significantly supported. Five posterior probabilities became significant with ML2. Six of the internodes that were not found with B/ML1 were statistically significant (PP 95%) with the Bayesian inference restricted to 77 taxa. The reverse was also true for five internodes. None of the deep internodes within the Lecanoromycetidae was well supported based on this ML2 analysis.
Phylogenetic relationships within the Ascomycota
In general, the same main Ascomycota lineages as outlined by Lutzoni et al. (2001)
and Kauff and Lutzoni (2002)
are found in our B/ML1 tree (Fig. 1). Our results differ in the phylogenetic placement of the Pertusariales + Icmadophilaceae Triebel clade. We confirm here the monophyletic status of this group (PP = 100% and BP-ML = 96%; Figs. 1 and 2A). This group was sister to and classified within the bitunicate ascohymenials (node BA) by Lutzoni et al. (2001
; but was not significant [PP = 91%]) and Kauff and Lutzoni (2002
; PP = 96%, but BP <50%). In our study this group is sister to the Ostropales s.l. + Baeomycetales (Kauff and Lutzoni, 2002
) group (but not significant [PP = 70%]; Fig. 1). Relationships within the Pertusariales (i.e., among Coccotrema, Ochrolechia, and Pertusaria DC.) are not well supported in our study. The family Coccotremataceae (represented by the genus Coccotrema) was consistently recovered as a highly significant monophyletic group in our study (PP = 100% and BS = 100%), but we could neither confirm nor reject the monophyly of the Pertusariaceae.
When comparing our results to Lutzoni et al. (2001)
and Kauff and Lutzoni (2002)
, statistical significance (PP 95%) was maintained for the following major monophyletic groups within the Ascomycota: Pezizomycotina/Euascomycetes, the next internode after the divergence of the Pezizomycetes O. E. Erikss. & Winka (operculate discomycetes; node 2 in Lutzoni et al., 2001
), Acarosporomycetidae (Acarosporaceae Zahlbr. in Lutzoni et al., 2001
, and Kauff and Lutzoni, 2002
; the recognition of this group at the subclass level will be done formally by V. Reeb, F. Lutzoni, and C. Roux, unpublished manuscript), unitunicate ascohymenials including Ostropales s.l. and Baeomycetales (the latter = Baeomycetaceae Dumort.; node 14 in Lutzoni et al., 2001
), Leotiomycetes O. E. Erikss. & Winka (Helotiales-Rhytismatales M. E. Barr ex Minter in Lutzoni et al., 2001
), Lichinomycetes (= Lichinales Henssen & Büdel in Lutzoni et al., 2001
; the recognition of this group at the subclass level will be done formally by V. Reeb, F. Lutzoni, and C. Roux, unpublished manuscript), Pezizomycetes/operculate discomycetes and Saccharomycotina O. E. Erikss. & Winka (Hemiascomycetes). In our study, posterior probabilities dropped below the 95% level of significance for the Lecanoromycetes O. E. Erikss. & Winka (PP = 51%; node 6 "Lecanoromycotina" in Lutzoni et al., 2001
), Eurotiomycetideae (PP = 51%), and Sordariomycetes O. E. Erikss. & Winka (PP = 51%; node 3 assigned for Arthoniales Henssen ex D. Hawksw. & O. E. Erikss. + Pyrenomycetes-Dothideales Lindau in Lutzoni et al., 2001
, and Pyrenomycetideae + Loculoascomycetes in Kauff and Lutzoni, 2002
). Within the latter group, Sordariomycetidae and Arthoniomycetidae are both well-supported monophyletic entities (PP = 100%, BP = 100%; Fig. 1). None of our analyses revealed the large order Lecanorales (sensu Eriksson et al., 2003
, or Tehler, 1996
) as monophyletic (Figs. 1 and 2A). Relationships among lecanoralean genera are not well established, except for the well-supported basal placement of Porpidia albocoerulescens (Wulfen) Hertel & Knoph (Cladoniineae sensu Tehler, 1996
) in B/ML1 (PP = 99%; Fig. 1) and the close relationship of the "Caliciales" C. Bessey with members of the Teloschistales D. Hawksw. & O. E. Erikss. in ML2 (PP = 98%, Fig. 2A).
Phylogenetic delimitation of the peltigeralean fungi
No significant discrepancies were found between phylogenetic trees derived from the 113- and 77-specimen data sets in terms of relationships among peltigeralean fungi (Figs. 1 and 2A). All potential families (Table 1) belonging to the Peltigerineae/Peltigerales (Collemataceae, Placynthiaceae, Lobariaceae, Nephromataceae, Pannariaceae, Peltigeraceae, and Solorinaceae) are nested within the Lecanoromycetidae. They form a single monophyletic group with high support values (PP = 100% and BP-ML value = 81%; Fig. 2A). Based on the MP2 and ML2 analyses (Fig. 2A), two major robust monophyletic groups were reconstructed within the Peltigerineae/Peltigerales: the Pannariaceae + Collemataceae + Placynthiaceae group (PP = 100% and BP-ML = 77%) and the Nephromataceae + Massalongia + Lobariaceae + Peltigeraceae + Solorinaceae group (PP = 100% and BP-ML = 84%). The latter group was also revealed in the B/ML1 analysis (PP = 99%; Fig. 1). Based on our taxon sampling, the Collemataceae, Pannariaceae, Lobariaceae, Nephromataceae, and Peltigeraceae were each supported as monophyletic groups (all families PP = 100% and BP-ML 95%; Figs. 1 and 2A). Based on the ML2 data set, we found strong evidence for the Collemataceae and Placynthiaceae sharing a most recent common ancestor (PP = 100% and BP-ML = 91%) and for this group being sister to the family Pannariaceae (PP = 100% and BP-ML = 77%; Fig. 2A). The monogeneric family Solorinaceae is shown here as paraphyletic (but not significant), and together with the family Peltigeraceae, they form a monophyletic group (PP and BP = 100%; Figs. 1 and 2A). Phylogenetic placements of the Nephromataceae and Massalongia carnosa (Dickson) Körber within the Nephromataceae + Massalongia + Lobariaceae + Peltigeraceae + Solorinaceae group could not be resolved here with high confidence.
All analyses revealed Lobaria, Pseudocyphellaria (Lobariaceae), Leptogium (Collemataceae), and Solorina (Peltigeraceae) as being nonmonophyletic genera, whereas Sticta (Lobariaceae), Nephroma (Nephromataceae), and Peltigera (Peltigeraceae) were consistently monophyletic (PP = 100% and BP-ML 99%; Figs. 1 and 2A). All Lobaria species, except L. pulmonaria (L.) Hoffm. and L. hallii (Tuck.) Zahlbr. form a robust monophyletic entity together with Pseudocyphellaria rainierensis Imshaug (PP 98%; Fig. 2A). Lobaria pulmonaria (type species) belongs to a second distinct lineage of Lobaria, whereas the phylogenetic placement of L. hallii is unresolved. Pseudocyphellaria is partitioned into three separate lineages represented by P. anomala Brodo & Ahti (at the base of the Lobariaceae), P. perpetua McCune & Miadlikowska + P. crocata (L.) Vainio (nested within the Lobariaceae; PP = 100% and BP 93%), and P. rainierensis (part of the Lobaria group).
All Peltigera sections represented by more than one specimen were monophyletic and all, except sect. Horizontales Miadlikowska & Lutzoni, were highly supported (PP 95%; Fig. 2A). Phylogenetic relationships among sections are not well supported, except for the sister relationship of sect. Peltigera and Retifoveatae Miadlikowska & Lutzoni (PP >95%) and their sister relationship to sect. Horizontales (PP >95%; Fig. 2A).
Reconstructing symbiotic ancestral states within the Peltigerineae/Peltigerales
We reconstructed ancestral symbiotic states for nine statistically significant nodes within the Peltigerineae/Peltigerales monophyletic group. For four nodes (2, 3, 5, and 6), the bimembered symbiotic state with cyanobacterium was significant (raw likelihood values >2.0) on 100% of the trees sampled from the posterior distribution (Fig. 3). This symbiotic state was also predominantly significant for nodes 1, 7, and 9, with frequencies ranging from 86 to 94%. Nodes 4 and 8 were mostly nonsignificant. The trimembered state was significant only for three nodes: 4, 8, and 9, but at a very low frequency of 1%. The bimembered symbiotic state with a green alga was never found to be highly probable for any of the ancestral nodes within the peltigeralean clade.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Kauff and Lutzoni, 2002
; Lumbsch et al., 2002
) and the phylogenetic summary by Taylor et al. (in press)
or the topological inconsistencies are not significant. For example, some of the highly supported groups recovered by Lutzoni et al. (2001)
and Kauff and Lutzoni (2002)
obtained posterior probabilities that were lower than the 95% PP threshold value in our study (e.g., monophyly of the Eurotiomycetidae and Sordariomycetes). This might be due to our more extensive taxon sampling designed to address phylogenetic relationships among peltigeralean fungi.
Our B/ML1 analysis (Fig. 1) indicates that the Acarosporomycetidae (= Acarosporineae sensu Eriksson et al., 2003
) and the Pertusariales might be outside of the Lecanoromycetidae (= Lecanorales sensu Eriksson et al.). The close relationship shown here between the Ostropales (sensu Kauff and Lutzoni, 2002
) and Pertusariales + Icmadophilaceae (= Ostropomycetidae in Figs. 1 and 2), although not well supported (Fig. 1), differs from the results obtained by Kauff and Lutzoni (2002)
in which the latter group was shown to be part of the bitunicate ascohymenials (= Lecanoromycetidae) with significant support. The recognition of the Ostropomycetidae will be done formally by V. Reeb, F. Lutzoni, and C. Roux (unpublished manuscript). This affiliation of the Pertusariales + Icmadophilaceae to the Ostropales + Baeomycetales group was also recovered, but with poor support, by Bhattacharya et al. (2000)
. Lumbsch et al. (2002)
presented some evidence for the close relationship between members of the Pertusariales and Ostropales sensu Kauff and Lutzoni (2002)
, i.e., including Agyriales Clem. & Shear. Ekman and Tønsberg (2002)
have also shown that Coccotrema, Ochrolechia, and Pertusaria are nested within the Ostropales sensu Kauff and Lutzoni (2002)
with high posterior probability (PP = 96%).
The Pertusariaceae is the only family common to all previous classifications of the Pertusariineae/Pertusariales (e.g., Hafellner, 1988
; Tehler, 1996
; Eriksson et al., 2003
). Lumbsch et al. (2001)
, based on a combined LSU and SSU nrDNA data set, found that the Coccotremataceae and Pertusariaceae form two monophyletic sister groups, which they refer to as the Pertusariales. Despite this result, Eriksson et al. (2003)
maintains that the Coccotremataceae is a family with an uncertain position within the Ascomycota. Using a very different taxon sampling than Lumbsch et al. (2001)
, we confirm here the monophyly of the Pertusariaceae + Coccotremataceae group (PP = 100% and BP 85%, Fig. 2A). Therefore, the latter family is clearly part of the Pezizomycotina, outside the Pezizomycetes (PP = 100%; Fig. 1). However, compared to Lumbsch et al. (2001)
, we cannot decipher statistically between Pertusaria being sister to Ochrolechia vs. Coccotrema (Figs. 1 and 2A). Given that the Coccotremataceae is a monogeneric family (Schmitt et al., 2001
) and sometime forms a strongly supported monophyletic group with members of the Pertusariaceae (Fig. 2A), subsuming Coccotremataceae within the Pertusariaceae should be considered in future studies.
We found with high statistical confidence that the Pertusariaceae + Coccotrema form a monophyletic entity with the Icmadophilaceae (PP = 100% and BP-ML = 96%; Figs. 1 and 2A). This robust sister relationship was revealed also by Lutzoni et al. (2001)
and Kauff and Lutzoni (2002)
. Platt and Spatafora (2000)
, in their molecular phylogenetic study of the family Icmadophilaceae, rejected the inclusion of this family within the Lecanorales, but could not provide evidence favoring its affiliation with the Helotiales as was suggested earlier (Rambold et al., 1993
; Tehler, 1996
; Kirk et al., 2001
). Eriksson et al. (2003)
consider this family to be a familiae incertae sedis within the Lecanoromycetes. Based on former phylogenetic treatments that included this family, and based on the results from our study, we conclude that the Icmadophilaceae are part of the Pertusariales, an order that may belong to the Ostropomycetidae (Figs. 1 and 2A).
Phylogenetic relationships among peltigeralean fungi
Analyses of the 113- and 77-specimen LSU + SSU data sets showed that all putative peltigeralean families (Collemataceae, Placynthiaceae, Pannariaceae, Nephromataceae, Lobariaceae, Solorinaceae, and Peltigeraceae) and Massalongia together form a strongly supported monophyletic group within the Lecanoromycetidae (PP = 100% in the ML2 and BP-ML = 81%; Fig. 2A). This placement of the peltigeralean fungi among the derived lineages within the Ascomycota (Fig. 1) provides further evidence for the rejection of the hypothesis of the ancient origin of these lichen-forming fungi introduced by Hawksworth (1982
, 1988a
, b
). Because of the phylogenetic relationships among peltigeralean fungi within the Lecanoromycetidae (Fig. 2A) and the current circumscription of the Lecanoromycetes according to Eriksson et al. (2003)
, we propose here that the monophyletic peltigeralean fungi be recognized at the order level (Peltigerales) as in previous classifications of the Ascomycota (e.g., Hafellner, 1988
; Kirk et al., 2001
; Table 1). With the exception of the Coccocarpiaceae (not part of our study), our revised circumscription of the Peltigerales is identical to the Peltigerineae sensu Wiklund and Wedin (2003)
, a study based on a combined mitochondrial SSU and nuclear LSU rDNA data set. Our phylogenetic delimitation of the peltigeralean fungi supports the broad taxonomic concept of these lichens by Rambold and Triebel (1992)
, Tehler (1996)
, and J. Hafellner, H. Hertel, G. Rambold, and E. Timdal (unpublished manuscript), except that they all recognized this taxonomic entity at the subordinal level (Peltigerineae) instead of the ordinal level (Peltigerales; Table 1).
Our study revealed two strongly supported monophyletic groups at the base of the Peltigerales (Fig. 2A). These two main peltigeralean groups (group 1 = Pannariaceae + Placynthiaceae + Collemataceae and group 2 = Nephromataceae + Massalongia + Lobariaceae + Peltigeraceae + Solorinaceae) differ from the two clades (group 1 = Pannariaceae and group 2 = the rest of the peltigeralean fungi) defined by the basal dichotomy of the study by Wiklund and Wedin (2003)
. However, because basal peltigeralean relationships received low support values or were unresolved by their parsimony jackknife analysis, this descrepancy between their study and our study does not appear to be significant.
We propose that the two well-supported monophyletic groups at the base of the Peltigerales, reported here, be recognized at the suborder level (Fig. 2B). One of these two groups corresponds to the Peltigerineae sensu stricto, which includes the Lobariaceae, Nephromataceae, Peltigeraceae, and Massalongia (Table 1). The second group is a new suborder, Collematineae, which includes the Collemataceae, Pannariaceae, and Placynthiaceae.
Suborder
Collematineae Miadlikowska & Lutzoni subordo nov.
Diagnosis
A subordine Peltigerineae differt in morphologia et anatomia thallorum. In contrario ad ea thalli crustosi et squamulosi aut gelatinosi homoeomericique frequenter occurrentes. Thalli foliosi si presentes numquam heteromerici. Substanciae chimicae utiles in taxonomia plerumque deficientes.
Description
Lichen-forming ascomycetes. Mostly bipartite with cyanobacteria, rarely tripartite. Thalli non- or gelatinous, squamulose, foliose, or subfruticose, rarely crustose; homoiomerous or heteromerous. Cortex present on both sides of the thallus or lower side ecorticated and sometimes with dark prothallus. Vegetative propagules (soredia, isidia, or phyllidia) common. Apothecia immersed or sessile; lecideine, biatorine, or lecanorine. Paraphyses simple or branched, sometimes strongly gelatinous. Ascospores aseptate, one- to multiseptate, or muriform, colorless. Asci with an amyloid external wall layer, I+ blue. Secondary substances uncommon or absent. On various substrates including stones, soil, bark, and mosses, often in moist habitats.
Type
Collemataceae Zenker.
Familiae alterae
Pannariaceae Tuck. and Placynthiaceae Å. E. Dahl.
Remarks
The suborder Collematineae includes peltigeralean fungi of varying morphological forms, but never species with conspicuous heteromerous thalli typical of Peltigerineae species. All species with gelatinous and homoiomerous thalli classified within the Peltigerales (Collemataceae), except the cyanomorph of P. venosa (L.) Hoffm. (Peltigeraceae), are part of the Collematineae. Members of this suborder are predominantly bipartite, forming associations mostly with the cyanobacteria Nostoc and Scytonema, but also with cyanobacteria of the Dichotrix type (e.g., in the Placynthiaceae). Unlike the Peltigerineae, tripartite symbioses are rarely found in this group (only in the Pannariaceae) and bipartite associations with green algae have never been reported. Contrary to the great diversity of secondary compounds found in the Peltigerineae, very few of these substances are detectable by TLC in the Collematineae. Asci with an amyloid tholus and colorless ascospores are two common characteristics of the Collematineae.
In his discussion of morphological and anatomical features of the genus Coccocarpia Pers., Arvidsson (1982)
suggested that members of the Coccocarpiaceae where likely to be closely related to peltigeralean taxa such as Placynthiaceae, Peltigeraceae, and especially Pannariaceae. Based on the phylogenetic placement of Steinera glaucella (Tuck.) Dodge, the Coccocarpiaceae (at least in part) seems to be a member of the Peltigerales (Wiklund and Wedin, 2003
). However, because its relationship within the Peltigerales is unresolved, the classification of this family at the subordinal level is unsettled at this time.
The Arctomiaceae Th. Fr., a small family of two genera (Arctomia Th. Fr. and Wawea Henssen & Kantvilas) associated with Nostoc, could be considered as another member of the Peltigerales. Henssen and Kantvilas (1985)
demonstrated that there were structural similarities between Wawea and members of the Collemataceae and Coccocarpiaceae. Despite this phenotypic evidence, the Arctomiaceae have never been classified within the Peltigerineae or Peltigerales. Currently, this family is classified within the Lecanorales (Kirk et al., 2001
) or in the Lecanorineae (Eriksson et al., 2003
).
With the exception of the Placynthiaceae, our phylogenetically based circumscription of the suborder Peltigerineae is very similar to the narrow delimitation of the Peltigerales sensu Hafellner (1988)
and Kirk et al. (2001)
, as well as of the Peltigerineae sensu Poelt (1974)
and Eriksson et al. (2003
; Table 1). As for the studies by Wiklund and Wedin (2003)
and Thomas et al. (2002)
, the phylogenetic placement of the Nephromataceae sister to the Lobariaceae is not well supported at this time (Fig. 2A). We confirm here the monophyly of four of the six peltigeralean families (Fig. 2A): Collemataceae, Lobariaceae (shown earlier by Thomas et al., 2002
and Wiklund and Wedin, 2003
), Nephromataceae, (shown earlier by Lohtander et al., 2002
and Thomas et al., 2002
), and Peltigeraceae (shown earlier by Miadlikowska and Lutzoni, 2000
). The monogeneric family Solorinaceae is reconstructed here as paraphyletic; however, its monophyly cannot be rejected due to the nonsignificant support obtained for its paraphyly (PP = 94% in B/ML and PP = 68% in ML2; Figs. 1 and 2). The highly supported monophyly of the Solorina + Peltigera group (PP and BP = 100%), which was also reported earlier by Miadlikowska and Lutzoni (2000)
, and the very likely paraphyly of Solorina, strongly support the recognition of the genus Solorina within the family Peltigeraceae. The latter circumscription is in agreement with all classifications of the lichen-forming ascomycetes proposed during the last 10 years (Table 1).
The family Pannariaceae is shown here forming a monophyletic group; however, the inclusion of Degelia in analyses by Wilkund and Wedin (2003; another putative member of the Pannariaceae according to Jørgensen and Galloway, 1992
, and Eriksson et al., 2003
) resulted in a nonmonophyletic delimitation of this family. Based on an extensive sampling, Ekman and Jørgensen (2002)
demonstrated that the Pannariaceae (part of the Collematineae in this study; Fig. 2A and B) represents a polyphyletic assemblage and concluded that Degelia, Fuscopannaria, Moelleropsis humida, and perhaps Parmeliella Müll. Arg. do not belong to this family.
In addition to Solorina (Peltigeraceae), three genera were found to be nonmonophyletic: Leptogium (Collemataceae), Lobaria, and Pseudocyphellaria (Lobariaceae; Fig. 2A and B). In light of this new information, these four genera are in need of a comprehensive phylogenetic revision based on both phenotypic and genotypic evidence. Closer affiliation of Leptogium cyanescens (Rabenh.) Körber with Collema species than with other Leptogium species reported by Wiklund and Wedin is confirmed here with high support values. Based on ITS sequences, Thomas et al. (2002)
and Stenroos et al. (2003)
found that Pseudocyphellaria and Lobaria species are intercalated. In our study, P. rainierensis is nested within the main Lobaria clade, and P. anomala with L. pulmonaria forms a paraphyletic group at the base of the Lobariaceae (Fig. 2A). Contrary to the study by Thomas et al. (2002)
, restricted to Pseudocyphellaria species from New Zealand, our phylogenetic analysis of North American species shows the Pseudocyphellaria species with a white medulla (represented by P. rainierensis, P. crocata, and P. anomala) to be part of at least three separate lineages w
