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(American Journal of Botany. 1998;85:1548-1559.)
© 1998 Botanical Society of America, Inc.

SSU rDNA phylogeny of cladoniiform lichens1

Soili K. Stenroos2,,3,a and Paula T. DePriesta

a Department of Botany, NHB-166, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To examine phylogenetic relationships among the "cladoniiform" lichenized fungi, i.e., the families Cladoniaceae, Baeomycetaceae, Icmadophilaceae, Stereocaulaceae, and Siphulaceae, and to provide evidence for the anticipated independent origins of podetia and pseudopodetia, we conducted phylogenetic analyses of SSU (small subunit) rDNA sequences from 39 lichen-forming fungi. These fungi represent all of the major growth forms of lichen associations, fruticose (including "cladoniiform"), foliose, and crustose. Our analysis suggests that lichen-forming fungi with a "cladoniiform" morphology arose multiple times within the ascomycetes. Additionally, each of the other thallus growth forms, crustose, foliose, and fruticose, have originated multiple times. It also seems to be clear that neither all podetiate nor all pseudopodetiate taxa form a monophyletic group. Therefore the term "podetium" should be restricted to homologous structures that are most probably limited to the genera Cladonia, Cladina, Pycnothelia, and allies. The "pseudopodetia" of Stereocaulon (Stereocaulaceae) and Cladia (Cladiaceae) may represent different states of the same homologous character. Our phylogenetic hypothesis supports the monophyletic origin of the order Lecanorales sensu stricto, including representatives of five suborders Cladoniineae, Lecanorineae, Teloschistineae, Agyriineae and Peltigerineae, but excluding representatives of the suborders Acarosporineae (Acarospora schleicheri and Megaspora verrucosa), Pertusariineae (Pertusaria trachythallina), and Umbilicarineae. The suborder Cladoniineae and the family Cladoniaceae both appear to be polyphyletic assemblages.

Key Words: ascomycetes • evolution • lichen-forming fungi • phylogeny • small subunit (SSU) ribosomal DNA • 18S rDNA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The numerous fungi that form lichens by association with photosynthetic algae or cyanobacteria have been artificially categorized by growth forms, i.e., crustose, foliose, or fruticose. These forms, representing lichen thalli that are either crust-like, leaf-like, or shrub-like, respectively, provide an easy teaching tool and memory device for beginning students and professional systematists alike. However, despite the rampant use of these terms, the three categories are far from natural in a phylogenetic sense and some lichen associations cannot be assigned readily to a single category. For example, some lichens produce a composite of these growth forms. An erect fruticose thallus, the thallus verticalis, may develop upwards from a crustose, granulose, or foliose structure, the thallus horizontalis (Ahti, 1982b). In some obviously related taxa the horizontal thallus may be lacking, or at least never described. Conversely, other taxa are known primarily as horizontal thalli, and rarely if ever produce vertical thalli. Furthermore, many vertical thalli bear sexual structures, although some have never been found fertile. These "cladoniiform" lichens, the composites of horizontal and vertical thalli and their presumed relatives, form elaborate and attractive forms (Figs. 1–2) that are compelling subjects for evolutionary analyses.



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  		Figs. 1–2. "Cladoniiform" growth forms. 1. Reindeer lichen Cladina densissima forming rounded heads (photo Soili Stenroos). 2. Dorsiventral "pseudopodetia" of Stereocaulon saxatile forming dense clusters (photo Mauri Korhonen).

 
The "cladoniiform" lichens are currently distributed among a diverse group of ascomycete families. The best known and the most speciose of these is the family Cladoniaceae. The diverse and ecologically important Cladoniaceae sensu Ahti (1993) comprise 11 genera and over 500 species of lichen-forming fungi. Almost 400 of these species are in the largest genus, Cladonia, which includes taxa such as the British soldier lichen (C. cristatella) and the pyxie cup lichen (C. pyxidata), followed by 40 species in its segregate genus Cladina, which includes the reindeer lichens (e.g., C. rangiferina). Other lichens with similar fruticose thalli are currently (Hawksworth et al., 1995) referred to the families Baeomycetaceae (~12 species), Icmadophilaceae (seven species), Stereocaulaceae (~130 species) and – provisionally because it is known only in an asexual state – Siphulaceae (~25 species). For the traditional classification of these groups, see Table 1. All taxa included in these five families, at one time or another, have been classified in the Cladoniaceae or proposed to be closely related to that family mainly on the basis of their "cladoniiform" thalli (e.g., Zahlbruckner, 1926). Under modern classification systems Cladoniaceae and Stereocaulaceae are placed in the suborder Cladoniineae of the large order Lecanorales, Icmadophilaceae is placed in the order Leotiales and Baeomycetaceae is placed either in Lecanorales or Leotiales (Hafellner et al., 1993; Hawksworth et al., 1995; Tehler, 1996; Eriksson and Hawksworth, 1996). The asexual genera previously grouped in the Siphulaceae, Siphula and Thamnolia, cannot be classified conclusively, but are provisionally placed in the Lecanorales (Hawksworth et al., 1995).


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  		Table 1. Traditional classification of "cladoniiform" lichens.

 
As persuasively stated by Ahti (1982a), the "cladoniiform" lichens are a growth-form or life-form group, a loose and superficial concept relative to the rigorous criteria of monophyly in modern systematics. This term "cladoniiform" does not circumscribe a natural group, although some natural groups may be contained within its amorphorus boundaries. In comparing the morphology of "cladoniiform" lichens, Ahti (1982a, b) differentiated those whose vertical thalli are considered podetia (lichenized stipes of generative tissue) vs. pseudopodetia (lichenized stipes of vegetative tissue), distinctions based on the development of generative tissues (Galloway, 1966; Jahns, 1970a; Henssen and Jahns, 1973). Cladonia and Cladina in the Cladoniaceae define true podetia, while the structures of Cladia (family Cladiaceae sensu Filson) and family Sterocaulaceae by comparison are called pseudopodetia (Galloway, 1966, p. 162).

The podetia of the Cladoniaceae sensu Ahti (excluding Cladia) are thought to arise from horizontal thalli, whether or not the latter structures have been observed. This "primary thallus" may be granular, squamulose, or foliose, and some cases may be ephemeral. An inclusive definition of the Cladoniaceae podetium was formulated by Ahti (1982a): "A podetium is a lichenized stem-like portion (stipe, or discopodium) bearing the hymenial discs and sometimes conidiomata in a fruticose apothecium." The normally hollow, stem-like portion is interpreted as a part of the ascoma, developing vertically from generative tissue (sensu Henssen and Jahns, 1973) of the horizontal thallus. This stem may raise the spore-bearing hymenial disc as much as 20 cm above the level of the horizontal thallus. This hymenial disc, traditionally referred to as an "apothecium" (Jahns, Sensen, and Ott, 1995), is usually a bright red or dark brown, but occasionally pale brown or pale pink. Fruticose structures in the families Baeomycetaceae and Icmadophilaceae (Dibaeis) are also called true podetia on the basis of their development, but they are very distinctive in appearance since they are not hollow and not always lichenized. These families differ fundamentally from Cladonia and Cladina in their ascal structure and function (Letrouit-Galinou, 1973) and have more recently been excluded from the family Cladoniaceae and the order Lecanorales and placed in the Leotiales (Gierl and Kalb, 1993; Rambold, Triebel, and Hertel, 1993; however, see Hawksworth et al., 1995, p. 555).

In contrast to the true podetia of Cladonia and Cladina, some "cladoniiform" lichens produce stem-like portions that differ from podetia in the ontogeny of their generative tissues. Traditionally (e.g., Galloway, 1966) the term pseudopodetium has been applied to the stem-like structures of Cladia that bear generative tissues that form ascoma at their tips. These generative tissues are not associated with the horizontal thalli, but with vegetative vertical thalli. This interpretation of pseudopodetia in Cladia was supported by Jahns (1970a, b) and Filson (1981), the latter using the distinctive ontogeny and morphology of the genus Cladia to place it in its own family Cladiaceae. Similarily, the stem-like structures in Stereocaulon of the family Stereocaulaceae are defined as vegetative and called pseudopodetia (Lamb, 1951; Jahns, Sensen, and Ott, 1995). Leprocaulon, Siphula, and Thamnolia lack reproductive structures and, therefore, their vertical thalli are considered vegetative (see Ahti, 1982b). As these selected examples attempt to show, the distinction between podetia and pseudopodetia is not entirely clear and in some cases the distinction may be arbitrary. In fact, fruticose stems occur in groups known to have originated independently within the order Lecanorales, Allocetraria in the Parmeliaceae, Santessonia in the Physciaceae, and Sphaerophorus in the Sphaeophoraceae, as well as within other orders. Furthermore, these terms cannot be adequately applied among groups since these superficial similarities may not be homologous (Lamb, 1951; Ahti, 1982a).

Earlier studies by the senior author (e.g., Hyvönen et al., 1995; Stenroos, Ahti, and Hyvönen, 1997) show that phylogenetic analyses based solely on available morphological, developmental, or chemical characters do not resolve the relationships among "cladoniiform" lichens. For these characters assigning character state homology is difficult or misleading – the organisms are morphologically simple and their structures environmentally modified. In this study we used phylogenetic analyses of nucleotide sequences from the nuclear small subunit ribosomal DNA (SSU rDNA; 18S rDNA) of 73 ascomycetous fungi to examine relationships among representatives of "cladoniiform" growth forms and compared the DNA-based phylogeny with the traditional "cladoniiform" grouping, or the separation of "cladoniiform" lichens into podetiate and pseudopodetiate. Our analysis of SSU rDNA sequences produces phylogenies that predict independent origins of the "cladoniiform" habit and podetiate ontogeny.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collections
We used thalli from either freshly collected or herbarium material (not older than 10–12 yr) for extracting total DNA. Collections of the total 31 taxa examined are listed in Table 2. Voucher specimens are deposited in the U.S. National Museum of Natural History (US) and the Finnish Museum of Natural History, Helsinki (H).


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  		Table 2. Representative collections of taxa used for sequencing.

 
DNA isolation and PCR amplification of fungal nuclear SSU rDNA
Total DNA was extracted from young parts of the thalli using the fungal miniprep protocols described by Taylor and Swann (1993; modified by A. Tehler) or Grube et al. (1995). DNA, suspended in dH2O, was amplified in three segments by the polymerase chain reaction (PCR) between the primers corresponding to the following nucleotides in the SSU of the rDNA repeat: NSR 0004–5' (5'-CTGGTTGATTCTGCCAGT-3'; Gargas and DePriest, 1996) and NSR 0871–3' (5'-CGTCCCTATTAATCATTACG-3'; Gargas and Taylor, 1992); NSR 0802–5' (5'-GAATAATAGAATAGGACG-3'; Gargas and Taylor, 1992) and NSR 1312–3' (5'-AATTAAGCAGACAAATCACT-3'; Gargas and Taylor, 1992); NSR 1184–5' (5'-GACTCAACACGGGGAAACTC-3'; Gargas and Taylor, 1992) and NSR 1797–3' (5'-AATGATCCTTCCGCAGGT-3'; DePriest, 1993); or optionally in two segments between the primers NSR 0054–5' (5'-CATGTCTAAGTTTAAGCAA; Gargas and Taylor, 1992) and NSR 0871–3', and NSR 0802–5' and NSR 1750–5' (5'-TAAAAGTCGTAACAAGGTTTCCGTAGG-3'; Gargas and DePriest, 1996). At least one primer in each pair preferentially amplifies fungal SSU rDNA and not that of the green algal partner of lichen-forming fungi. For amplification we used 4.0 units of Dynazyme (Finnzymes, Helsinki, Finland) or 1.25 units Klentaq LA (AB Peptides, St. Louis, Missouri) in 100-µL reactions (10 mmol/L Tris-HCl pH 8.8, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.1% Triton X-100), with 200 µmol/L of dNTP mix and 0.5 µmol/L of primers, with between 0.1 and 10 ng of DNA. The thermal cycler (MJ Research MiniCycler or Perkin-Elmer Cetus) conditions for 30 cycles were: template denaturing at 95°C for 60 s, primer annealing at 50°C for 60 s, and primer extending at 72°C for 2 min (extended by 5 s in each cycle). The resulting PCR products were precipitated with 20% polyethylene glycol (PEG) and 2.5 mol/L NaCl, and quantified by fragments stained by ethidium bromide and separated by agarose gel electrophoresis.

DNA sequencing
The PCR products were sequenced using the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems) with the detection on a 373A Automatic Sequencing Apparatus (Applied Biosystems). Double-strand PCR products were used for sequencing using the following oligonucleotide primers in different combinations (depending on the length of the amplification product due to the presence of sequence insertions): NSR 0004–5', NSR 0054–5', NSR 0802–5', NSR 0871–3', NSR 1184–5', NSR 1312–3', NSR 1750–5' and NSR 1797–3' as described above, and NSR 0323–3' (5'-TCGAAAGTTGATAGGGCAG-3'; Gargas et al., 1995), NSR 0381–5' (5'-CCGGAGAAGGAGCCTGAGAAAC-3'; Gargas and Taylor, 1992), NSR 0553–5' (5'-GCAAGTCTGGTGCCAGCAGCC-3'; White et al., 1990), NSR 0573–3' (5'-GGCTGCTGGCACCAGACTTGC-3'; White et al., 1990), NSR 1203–3' (5'-GAGTTTCCCCGTGTTGAGTC-3'; Gargas et al., 1995), NSR 1410–5' (5'-TTTGAGGCAATAACAGGT-3'; DePriest, 1993), NSR 1482–3' (5'-TTGTCTCTGTCAGTGTAG-3'; DePriest, 1993), and NSR 1597–3' (5'-GATGACTCGCGCTTACTA-3'; DePriest, 1992). The excess DyeDeoxy terminators were removed by filtration through Sephadex G-50 Fine (Pharmacia) in spin columns. Sequencing reactions were run for 48 cm on a 4% polyacrylamide gel.

SSU rDNA alignments
Sequence fragments obtained from each sequencing reaction were assembled into full-length sequences using Sequence Navigator 1.0 (Applied Biosystems); both strands were sequenced from multiple primers. The SSU rDNA sequence of the model organism Saccharomyces cerevisiae (Rubtsov et al., 1980; Mankin, Skryabin, and Rubtsov, 1986) was used to define the exact positions of the insertions. The insertions were removed, and only the conserved sequences were used for the analysis. The 76 sequences were aligned with the program PILEUP; gaps were reduced by manual adjustment.

Taxon selection
To examine phylogenetic relationships among "cladoniiform" fungi and to provide evidence for the anticipated independent origins of podetia and pseudopodetia, we conducted phylogenetic analyses of SSU rDNA sequences from 39 lichen-forming fungi and 37 other fungi. The lichen-forming fungi in the analysis represent examples of all of the major growth forms of lichen associations, fruticose (including "cladoniiform"), foliose, and crustose. The phylogenetic analysis includes new SSU rDNA sequences from 29 lichen-forming fungi, 14 from "cladoniiform" taxa in five families, and 15 from other lichen taxa in 11 families. The "cladoniiform" taxa include representatives from all lichen families with species having this growth form, Cladoniaceae (five taxa, genera Cladonia, Cladia, Pycnothelia, and Gymnoderma), Stereocaulaceae (four taxa, genera Stereocaulon and Pilophorus), Baeomycetaceae (one taxon, genus Baeomyces), Icmadophilaceae (one taxon, genus Dibaeis) and "Siphulaceae" (two taxa, genera Thamnolia and Siphula), and the genus Leprocaulon (unsettled position). The goal of our taxon selection was to represent as many genera as possible within these families.

For 47 taxa sequences were obtained from the GenBank (Table 3). These 47 included all of the 41 ascomycete taxa and three zygomycete taxa from Gargas et al. (1995), and three Peltigerineae taxa submitted by O. Eriksson and A. Strand.


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  		Table 3. SSU rDNA sequences obtained from GenBank.

 
Phylogenetic analysis
The aligned set of 2015 nucleotide positions from 76 taxa was used for parsimony analysis with the program PAUP 3.1.1 (Swofford, 1993). None of the characters were excluded, invariant characters were ignored, gaps were treated as missing data, all characters were equally weighted, and branch lengths equal to zero were collapsed to polytomies. The heuristic analysis was repeated 50 times using random addition. The unrooted networks were rooted with three zygomycetous fungi as outgroups. Bootstrap percentages used for assessing support for the branches were determined from 200 resamplings of the data set.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence analysis
For most of the 29 taxa we obtained nearly complete nucleotide sequences of nuclear SSU rDNA (1700 of 1800 nucleotides). Some taxa have SSU rDNA sequences considerably longer than the 1800 nucleotides SSU rDNA of model fungi such as Saccharomyces cerevisiae. For example, one taxon had a more than 4000 nucleotide sequence (Stenroos and DePriest, unpublished data), due to the presence of group I introns, degenerate introns, or other unidentified sequence insertions at 11 distinct positions within the conserved SSU coding region. Introns and sequence interruptions are now known from 21 positions in the SSU rDNA of lichen-forming fungi, 16 of these found in "cladoniiform" lichens (DePriest and Been 1992; DePriest, 1995; Gargas et al., 1995; Grube et al., 1995; Stenroos, DePriest, Ivanova, and Gargas, unpublished data). All introns and sequence interruptions were removed from the SSU rDNA sequences to allow alignment. Sequences from the 76 taxa were aligned to produce a matrix of 2015 nucleotide-position characters (including gaps). Across the alignment, 799 nucleotide positions were variable, and of these 556 were potentially phylogenetically informative.

Phylogenetic analysis
When the aligned sequences of the 76 taxa were subjected to parsimony analysis, two equally parsimonious trees of 2822 steps were found in heuristic searches of 50 random addition repeats. One of the two trees is shown in Fig. 3. The other tree differs only in the polyphyly of the order Leotiales (clade including Leotia lubrica and Sclerotinia sclerotiorum). The consistency index (CI) of the two trees was 0.424, with a retention index (RI) of 0.606. The highly resolved phylogeny is essentially in agreement with that presented in Gargas et al. (1995).



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  		Fig. 3. Preliminary phylogenetic hypothesis for the lichen-forming ascomycetes, as derived from parsimony analysis of SSU rDNA sequences from 74 representative ascomycetes with three zygomycetous fungi as outgroups. The tree is one of the two equally parsimonious cladograms of 2822 steps (CI=424, RI=0.606) found in a heuristic search of 50 random-addition repeats using PAUP 3.1.1. The additional tree differs only in the polyphyly of the nonlichen-forming order Leotiales (clade including Leotia lubrica and Sclerotinia sclerotiorum ). Bootstrap persentages, greater than 10%, from 200 replications are shown under each supported branch. Arrows indicate preliminary placements for two additional lichen-forming taxa.

 
The 39 lichen-forming taxa occur in four clades among the 73 representatives of the ascomycetes. Lichen clade I, the Arthoniales, represented by Arthonia radiata, Schismatomma pericleum, Dendrographa leucophaea, and Lecanactis abietina, was found to be monophyletic, supported by a bootstrap value of 77. As in previous analyses (Gargas et al., 1995), the Arthoniales is a sister clade to the Sordariales and represents an independent origin of lichenization. The fruticose growth form of Dendrographa probably arose from a crustose ancestor. The three additional clades containing lichen-forming taxa are closely related and members of a poorly supported clade (bootstrap value under 50) that includes the nonlichen orders Eurotiales (Eurotium rubrum, Thermoascus crustaceus, and Eremascus albus) and Onygenales (Malbranchea gypsea and Histoplasma capsulatum), and the lichen-containing order Caliciales (Mycocalicium albonigrum). It is equivocal whether lichenization arose multiple times in this clade.

Lichen clade II includes two representatives of the Umbilicariaceae, Umbilicaria subglabra and Lasallia rossica. This family was considered by Hafellner et al. (1994) and Hawksworth et al. (1995) as a member of the Lecanorales, but molecular data have supported its exclusion (Ivanova and Bobrova, 1996). These species form a sister clade to representatives of Caliciales (Mycocalicium albonigrum), the Eurotiales and the Onygenales.

Lichen clade III encompasses the order Lecanorales (sensu stricto) around the type genus of the order, Lecanora, and including Porpidia crustulata and Nephroma arcticum. This monophyletic group includes representatives of five suborders, 14 families, 24 genera, and 27 species. All representatives of the suborder Cladoniineae, and the "cladoniiform" families Cladoniaceae and Stereocaulaceae, are members of this clade. The Lecanorales s.str. includes the fruticose Sphaerophorus globosus, traditionally considered a member of Caliciales (Gargas and Taylor, 1995; Wedin, 1996), and fruticose Leprocaulon sp., an asexual taxon of unsettled position (Hawksworth et al., 1995, p. 603). Three other fruticose taxa, Santessonia namibensis, Allocetraria madreporiformis, and Pseudevernia cladonia, arose from within the Lecanorales s.str. The poor bootstrap support (under 50) of lichen clade III may be a result of the large number of taxa sampled and their low level of variation. As a consequence, the conserved SSU rDNA gene has limited resolution within the order.

Lichen clade IV includes an assortment of taxa that are excluded from their current placements in the Lecanorales and the Leotiales. Pertusaria trachythallina of the Lecanorales forms a monophyletic group with Gyalecta ulmi of the Gyalectales, and Baeomyces rufus and Dibaeis baeomyces of the Leotiales. Siphula ceratites and Thamnolia vermicularis, asexual taxa not classified to order (Tehler, 1996; but see Hawksworth et al., 1995, pp. 421, 453, 558), join this clade. Siphula and Thamnolia form a clade with Dibaeis that is far removed from Baeomyces from which Dibaeis was recently segregated.

SSU rDNA sequences for two additional taxa were not included in the final phylogenetic analysis. The taxa, Acarospora schleicheri and Megaspora verrucosa of Lecanorales suborder Acarosporineae, showed long-branch attraction in a preliminary analysis. Using the tree shown in Fig. 3. as a backbone constraint (PAUP 3.1.1) these taxa could be placed preliminarily outside the Lecanorales and tentatively in lichen clade IV.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Lichen-forming fungi arose multiple times within the ascomycetes (Vainio, 1890: xiv; Gargas et al., 1995). This is also the case with "cladoniiform" lichen-forming fungi. One "cladoniiform" group, including representatives of the Stereocaulaceae (Stereocaulon and Pilophorus) and Cladoniaceae (Cladonia, Pycnothelia, and Cladia), arose within the Lecanorales s.str. This clade also includes two crustose taxa, Squamarina lentigera and Rimularia fuscoatra. The sequences from the former species may be spuriously placed in this clade due to long-branch attraction. However, neither of these two crustose taxa should be excluded a priori from this clade solely based on their growth form; additional taxa from these crustose genera should be analyzed to resolve their relationships. Gymnoderma lineare, currently placed in the family Cladoniaceae, falls outside of this group and its relationship to that family will be examined in future studies. Leprocaulon sp. is a sister taxon to Physcia and Santessonia of the Physciaceae and apparently is unrelated to any other "cladoniiform" lichens. The phylogenetic position of Gymnoderma and Leprocaulon reveals the possibility that a "cladoniiform-like" morphology arose convergently in the Lecanorales. Certainly it arose in a distinct clade containing the "Siphulaceae" (Siphula and Thamnolia), Icmadophilaceae (Dibaeis), and Baeomycetaceae (Baeomyces).

Furthermore, neither all podetiate nor all pseudopodetiate taxa form a monophyletic group. Structures called podetia occur in widely separated clades, in Cladonia and Pycnothelia within the Lecanorales and in Dibaeis and Baeomyces outside the Lecanorales. The pseudopodetia found in Stereocaulon, Cladia, and Pilophorus appear plesiomorphic to the podetia found in Cladonia and Pycnothelia. The genus Cladia, represented by C. aggregata, and the genus Pilophorus, represented by P. acicularis, appear more closely related to the podetiate genera Cladonia and Pycnothelia) than to the pseudopodetiate genus Stereocaulon. Therefore, production of pseudopodetia, in their current definition, cannot be used as a synapomorphy to describe the Stereocaulaceae. Finally, each of the other thallus growth forms, crustose, foliose, and fruticose, are widely represented in our analysis and have originated multiple times. This is indicated by the placement of Arthonia radiata, Porpidia crustulata, Rhizocarpon geographicum, and Pertusaria trachythallina (crustose thalli), Physcia aipolia, Xanthoria elegans, and Peltigera neopolydactyla (foliose thalli), and Teloschistes cf. chrysophthalmus, Sphaerophorus globosus, and Pseudevernia cladoniae (fruticose thalli), for instance.

The phylogenetic hypothesis presented here allows only a provisional classification for the cladoniiform lichens since it is based solely on molecular data from a single gene. The problems caused by single-gene analyses have been discussed. One of the limitations is the potential for differential evolution of a gene or nucleotide sequence and the organisms (Lutzoni and Vilgalys, 1994; Tehler, 1995a, b). A single gene phylogeny may reflect the evolution of the specific gene, and that evolution may differ from the species phylogeny. Furthermore, the rDNA sequences can produce phylogenies that differ from those based on other nuclear genes. In addition, multiple changes at a single nucleotide position among organisms that are phylogenetically distant, may obscure homology (Hillis, 1994). Certainly, multiple genes and diverse character systems should be considered when proposing phylogenetic relationships and natural classifications.

However, we cannot recommend combining this molecular data with available morphological and chemical characters. The classification and systematics of the "cladoniiform" lichens refer to the relationships among lichen-forming fungi, independent of their symbiotic partners (not as in Mindell [1992] who considers symbiotic associations as merged lineages). Most traditional taxonomic characters (gross morphology, vegetative reproduction, etc.) have not been effective tools for classification of these lichen-forming fungi because such characters cannot be attributed to either of the symbionts independently. Characters of the composite organism, the color of the vegetative structures, the branching of the vertical thallus, the placement of squamules, etc., have created confusion over what is being classified; perhaps the most important conceptual issue is that such characters have no equivalent among nonlichen-forming fungi. To compare lichens to other fungi, it is necessary to use characters that can be attributed directly and individually to the fungal partner. C. F. Culberson and Armaleo (1992) showed that secondary product chemistry was genetically controlled by the fungus. However, the taxonomic value of secondary products is not well understood; secondary products are known to vary quantitatively within a population, and qualitatively among single-spore progeny of Cladonia chlorophaea species (Culberson, Culberson, and Johnson, 1988). Convergent evolution of compounds limit the utility of secondary substances as evolutionary markers (Culberson, 1986; Gowan, 1989). Furthermore, our earlier studies demonstrated the unsatisfactory and subjective nature of analyzing morphological and chemical characters (e.g., Stenroos, Ahti, and Hyvönen, 1997). Indeed, divergent morphological and chemical characters provide only an indirect examination of the genomes of lichen-forming fungi, while nucleotide sequences allow a direct look at those genomes. Improved phylogenetic resolution will come from analyzing multiple genes.

Order Lecanorales (supported)
In Tehler's treatment (1996; see also Hafellner et al., 1993) the order Lecanorales has been divided in eight suborders, one of which is the suborder Cladoniineae, incorporating most of the "cladoniiform" fungi. Our phylogenetic hypothesis supports the monophyletic origin of the order Lecanorales s.str., including representatives of the five suborders Cladoniineae, Lecanorineae, Teloschistineae, Agyriineae, and Peltigerineae (DePriest et al., unpublished data; see also Tehler, 1996), but excluding representatives of the suborders Acarosporineae (Acarospora schleicheri and Megaspora verrucosa), Pertusariineae (Pertusaria trachythallina), and Umbilicarineae (Lasallia rossica and Umbilicaria subglabra), sensu Tehler (1996). The exclusion of Pertusariineae from Lecanorales is supported by T. Lumbsch (in Hafellner et al., 1994), who stated that Pertusariales should continue to be accepted as a distinct order.

Suborder Cladoniineae (not supported)
In our study we sampled six of the 15 families of Cladoniineae and found that Cladoniineae, as currently delimited, appears to be a polyphyletic assemblage. A core group of Cladoniineae is formed around the type genus Cladonia by the families Stereocaulaceae, Squamarinaceae, Cladoniaceae, and Rhizocarpaceae. However, the crustose genus Rimularia (currently in the suborder Agyriineae) and the fruticose genera Pseudevernia and Allocetraria (currently in the suborder Lecanorineae) are included in this monophyletic group. Conversely, Megalospora, Porpidia and Lecidea, currently members of Cladoniineae, appear outside of this monophyletic group. Porpidia and Lecidea form sister taxa basal to the order Lecanorales, which supports the classification that has been proposed by Hafellner et al. (1994; unpublished data). As to Megalospora, Sipman also (1983) noted similarities to Teloschistaceae. On the basis of this preliminary analysis, the suborder Cladoniineae should be newly circumscribed relative to suborders Lecanoriineae and Teloschistineae to produce monophyletic taxa.

Cladoniaceae (not supported)
Cladoniaceae sensu Ahti (Ahti, 1993) includes 11 genera (Table 1). Of these we sampled representatives of the genera Cladonia, Cladia, Pycnothelia, and Gymnoderma. Based on these selected taxa, the family Cladoniaceae appears to be a polyphyletic assemblage. The two representatives of the genus Cladonia, viz. Cladonia bellidiflora and C. subcervicornis, are sister taxa. Preliminary data support that representatives of Cladina will join these taxa. The genus Pycnothelia, represented by P. papillaria, forms a sister taxon to the Cladoniae. In fact, Pycnothelia was included in Cladonia, e.g., by Vainio (1887). This close relationship is supported by their similar podetial ontogeny, i.e., their stipe is generative and part of the ascoma (Ahti, 1982a). Furthermore, in both genera their podetia are typically hollow (except for Cladonia solida and C. stereoclada, whose podetia appear to be solid except for some narrow central canals in older specimens).

The status of the genus Gymnoderma has been controversial. Zahlbruckner (1926) distinguished Gymnoderma from Cladonia, although Evans (1947) placed both in Cladonia. Most later authors have treated Gymnoderma as a distinct genus on the basis of a few morphological characters, e.g., solid podetia. Verdon and Elix (1986) state that Gymnoderma has true podetia like Cladonia, although they may be poorly developed (Ahti, 1982a). However, it has been difficult to find many well-defined criteria to clearly distinguish and delimit Gymnoderma from the morphological diversity and plasticity of the genus Cladonia (Yoshimura and Sharp, 1968; Yoshimura, 1973). In the present study Gymnoderma, represented by G. lineare (which is rather different from the type species G. coccocarpum and possibly merits placement in a separate genus) falls well outside the monophyletic group formed by Cladonia and Pycnothelia. Gymnoderma lineare appears basal to the genus Stereocaulon and the selected members of Parmeliaceae. This suggests that G. lineare does not belong to the family Cladoniaceae, or at least it is not a sister taxon to Cladonia and Pycnothelia, a concept supported by the presence of totally solid podetia in G. lineare. The genus Neophyllis has been included in Gymnoderma by some authors (Hawksworth and Yoshimura, 1973; Hawksworth et al., 1995). If G. lineare is a sister taxon to Neophyllis or if these two taxa are congeneric, both may well be quite distant to Cladoniaceae because ontogenetic studies by H. Döring (A. Henssen in Hafellner et al., 1994) are interpreted to suggest that Neophyllis might be closer to Stereocaulaceae. However, we feel that more representatives of Cladoniaceae, especially the genera Neophyllis, Calathaspis, and Myelorrhiza (Verdon and Elix, 1986), need to be included in the analysis to address the classification of Gymnoderma.

Filson (1981) established a new monotypic family Cladiaceae for the genus Cladia, on the basis of its thallus structure. The thalli of Cladia have been called pseudopodetia. True horizontal thallus is apparently absent in Cladia, and the vertical thallus is hollow and perforate and lacks the supporting stereome typical to Cladonia, Cladina, and Pycnothelia. However, the family Cladiaceae has not been generally accepted (Hawksworth et al., 1995) and Cladia has been placed in Cladoniaceae (Ahti, 1993). Surprisingly, in our analysis, Cladia forms a sister taxon to a crustose genus Rimularia, currently a member of suborder Agyriineae. This relationship is not supported by any other characters and should be re-examined when sequences from more taxa are available. Furthermore, Cladia appears more closely related to Pilophorus (currently in the family Stereocaulaceae) than to Cladonia and Pycnothelia.

Stereocaulaceae (not supported)
The Stereocaulaceae are represented in our analysis by four taxa in two genera, Stereocaulon and Pilophorus. Three representatives of the genus Stereocaulon form a monophyletic assemblage. Together with Squamarina this assemblage is a sister taxon to the clade that includes most Cladoniaceae as well as the genus Pilophorus. The genus Pilophorus has recently been treated as a member of Stereocaulaceae by many authors (Jahns, 1981; Hawksworth et al., 1995; Tehler, 1996), although earlier it had been thought to be related to Cladoniaceae (Zahlbruckner, 1926; Lamb, 1951). The inclusion of Pilophorus in Stereocaulaceae is generally based on the presence of "pseudopodetia" and cephalodia in both Stereocaulon and Pilophorus. Actually, the statement by Lamb (1951, p. 575), who refers to the opinion of Kajanus (1911) and further, to Vainio (1890), is based on the impression that only Stereocaulon has a "pseudopodetium" and therefore differs from Pilophorus. Nevertheless, our analysis based on molecular characters supports the theory that Pilophorus acicularis is more closely related to Cladoniaceae and accordingly should be excluded from Stereocaulaceae.

Although the SSU rDNA provides limited variation at the species level and is probably not reliable in addressing all subgeneric relationships, our analysis showed a predicted phylogeny of the three selected representatives of the genus Stereocaulon. According to our analysis S. vesuvianum and S. taeniarum are sister taxa and more closely related to each other than to S. ramulosum. This conforms with the subgeneric classification of Stereocaulon presented by Lamb (1951, 1977). In Lamb's treatment, S. ramulosum is in the subgenus Holostelidium, while the two others, S. vesuvianum and S. taeniarum, both belong to subgenus Stereocaulon.

Baeomycetaceae and Icmadophilaceae
Traditionally, the family Baeomycetaceae and its recent segregate Icmadophilaceae have been assigned to the order Lecanorales (Henssen and Jahns, 1973; Poelt, 1974; Hale, 1983). In addition, Baeomycetaceae and Cladoniaceae were thought to be closely related and to have their phylogenetic origin in Lecideaceae (see Ahti, 1982a). However, Chadefaud (1960) and Honegger (1983) stated that the genus Baeomyces should be placed near Leotia in the order Leotiales; this opinion was based on the similar ascus structure of these taxa. The idea of including Baeomycetaceae in Leotiales has recently been accepted (Gierl and Kalb, 1993; Rambold, Triebel, and Hertel, 1993; Tehler, 1996). However, Eriksson and Pettersson (in Eriksson and Hawksworth, 1996, p. 106), in an analysis of SSU rDNA sequences of Baeomyces rufus, Icmadophila ericetorum, and 34 other ascomycetes, suggested that Baeomyces and Icmadophila have their closest allies in the Lecanorales, although they could not totally rule out the inclusion of these two genera in Leotiales.

The genus Baeomyces traditionally included some species with rose-colored hymenia. These have been assigned to a distinct genus, Dibaeis. Species with brown hymenia are placed in Baeomyces and Phyllobaeis (Gierl and Kalb, 1993). In the recent classification by Hawksworth et al. (1995) Dibaeis is included in the family Icmadophilaceae, while Baeomyces is in Baeomycetaceae. However, Tehler (1996) does not recognize Icmadophilaceae, but includes the genera Baeomyces and Icmadophila (Dibaeis is not mentioned) in Baeomycetaceae. Our data suggest that Baeomyces rufus and Dibaeis baeomyces (syn. Baeomyces roseus) are phylogenetically distant and more closely related to taxa from other orders and suborders than to each other. Neither belongs to orders Lecanorales or Leotiales. Even if Dibaeis and Baeomyces are placed in a single order, they will belong to different families and different genera. In our analysis, the genus Baeomyces (B. rufus) forms a sister taxon to Gyalecta ulmi, which is currently placed in the order Gyalectales. Dibaeis forms a monophyletic group with Siphula and Thamnolia (see below) and appears to be more closely related to them than to Baeomyces. To conclude, our analysis supports the generic segregation of Baeomyces and Dibaeis, but cannot resolve their ordinal classification.

Siphulaceae
The genera Siphula and Thamnolia are invariably sterile and, therefore, cannot be classified using ascal structures as criteria, i.e., in the so-called Hafellner system (Hafellner, 1988). For practical reasons, species sharing morphological and chemical characters have been assigned to entities that are treated as genera. Siphula includes ~25 species; Thamnolia currently includes only one species (if T. vermicularis var. subuliformis and subsp. solida are not accepted at species level; Kärnefelt and Thell, 1995). Some authors have applied the family name Siphulaceae (incl. Thamnoliaceae) to incorporate Endocena, Siphula, and Thamnolia (e.g., Poelt, 1974).

In our analysis Siphula ceratites and Thamnolia vermicularis form a monophyletic assemblage together with Dibaeis baeomyces. Siphula and Thamnolia can therefore be tentatively included in Icmadophilaceae, although we need to add at least the genus Icmadophila and preferably one of the smaller genera of Icmadophilaceae to the analysis to verify the status of all these taxa. Our results, however, indicate that Siphula and Thamnolia are not in Lecanorales. In addition, our preliminary data suggest that the genus Siphula is probably not monophyletic, which can also be suggested on the basis of the secondary chemistry (Santesson, 1967). Our present analysis only includes S. ceratites but when introducing a partial sequence of S. coriacea to the data set it does not join S. ceratites but appears close to the members of Stereocaulon. More species of Siphula need to be added to test the possible polyphyly of the genus.

Other taxa
We have included some additional fruticose or "stipitate" taxa in our analysis. These taxa have not been included in the Cladoniaceae, but have been placed in other families or have remained unclassified. The genus Sphaerophorus traditionally has been placed in the Caliciales on the basis of mazaedium (a spore mass formed by an ascoma) produced by these taxa. In our analysis, S. globosus appears as a sister taxon to Lecanora dispersa, but more taxa need to be included to resolve this relationship. However, its inclusion in the order Lecanorales conforms with the recent results presented by Wedin (1996). The genus Dactylina has been included in Parmeliaceae by Hawksworth et al. (1995) and Tehler (1996). This is supported in our analysis by the sister-taxa relationship of Allocetraria madreporiformis (syn. Dactylina madreporiformis) and Pseudevernia cladoniae; based on conidium form and secondary chemistry. Kärnefelt and Thell (1996) transferred Dactylina madreporiformis to the genus Allocetraria (also Parmeliaceae). Furthermore, the inclusion of the genus Santessonia in Physciaceae (Hawksworth et al., 1995; Tehler, 1996) is supported by the sister-taxa relationship of Santessonia namibensis and Physcia aipolia. The genus Leprocaulon is not known fertile and therefore it has remained unclassified (Tehler, 1996). However, traditionally it was placed near Stereocaulon since it resembles morphologically some of the smaller species of that genus. In our analysis Leprocaulon is a member of Lecanorales, although not related to Stereocaulon. Preliminary results support that it is related to Physciaceae, but we cannot assign it to a family before including more taxa in the analysis.

Concluding remarks
Vainio (1880, p. 2), the monographer of Cladoniaceae, considered Darwinian evolution ("descendensi-teoria") as a major revolution in systematics that would require abandoning traditional ideas:

Descendensi-teorian kannalta ei systematiikin tehtäväksi enään tule järjestää organismia niin että ne, joilla on enimmin yhtäläisyyttä, tulevat rinnatusten, tai niin että vasta-alkavainen vähimmällä vaivalla saapi selkoa niistä; sen tehtäväksi tulee etsiä niiden geneetillistä yhteyttä, - se muuttuu toisilla sanoilla, genealogiaksi. [According to the "descendence" theory systematists should not arrange organisms so that those that are most similar go side by side, or so that a novice can easily get a general idea of them; instead, the goal of systematics should be to find genetic connections between organisms - that is, in other words, genealogy].

The traditional terms fruticose, foliose, and crustose represent such similarities that allow us to easily arrange lichen-forming fungi. However, they are descriptive terms that provide no information on the genetic connections among these organisms. The term "cladoniiform" offers no more phylogenetic precision than fruticose and therefore should be abandoned as a name for a group of lichens. The term "podetium" should be restricted to homologous structures that are most probably limited to the genera Cladonia, Cladina, Pycnothelia, and allies. The "pseudopodetia" of Stereocaulon (Stereocaulaceae) and Cladia (Cladiaceae) may represent different states of the same homologous character. However, without adding further taxa to the analyses we do not know whether podetia arose from some type of pseudopodetia or vice versa. We should not arrange these organisms by their growth forms but rather by their phylogenetic relationships.


   FOOTNOTES
 
1 SS thanks T. Ahti and A. Tehler for discussions and research support during the early phases of this work, T. Ahti for critically reviewing the manuscript, N. Ivanova for invaluable and skillful help in various laboratory duties; and L. Kivistö, S. Lommi, and M. Kuusinen for supplying fresh lichen material for analyses. The project was supported by Smithsonian Institution Postdoctoral Fellowship and Academy of Finland funding to SS and a National Museum of Natural History Research Initiatives Award to PTD and in the later stages of manuscript preparation by NSF-PEET award DEB-9712463. Back

2 Current address: Herbarium, Department of Biology, University of Turku, FIN-20014, Turku, Finland. Back

3 Author for correspondence. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. Hammer
Lateral growth patterns in the Cladoniaceae
Am. J. Botany, May 1, 2001; 88(5): 788 - 796.
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