Neocampanella, a new corticioid fungal genus, and a note on Dendrothe/e bispora

The new genus Neocampanella (Agaricales, Agaricomycetes, Basidiomycota) is established for Dentocorticium blastanos Boidin & Gilles, a crustose species, and the new combination, Neocampanella blastanos, is proposed. Morphological and molecular studies support the recognition of the new genus and its close ties to Campanella, a pleurotoid agaric. The recently described Brunneocorticium is a monotypic, corticioid genus closely related to Campanella also. Brunneocorticium pyrifonne S.H. Wu is conspecific with Dendrothele bispora Burds. & Nakasone, and the new combination, Brunneocorticium bisporum, is proposed.


Introduction
Corticioid basidiomycetes have simple, reduced fruiting bodies that often appear as thin, crustose areas on bark and woody substrates. This simple morphology belies their boundless variability at the microscopic level. Originally assumed to be closely related, molecular studies show that corticioid species are distributed throughout the Agaricomycetes, formerly the Hymenomycetes or Homobasidiomycetes Binder et al. 2005;Larsson 2007b). Many genera of corticioid fungi are defined by just a few morphological traits, but molecular techniques and analyses have repeatedly demonstrated that this simplistic taxonomic approach is woefully inadequate and often misleading. For example, familiar and easily recognized corticioid genera such as Hyphodenna (Larsson 2007a), Phanerochaete (De Koker et al. 2003), Gloeocystidiellum (Larsson and Larsson 2003), Leucogyrophana (Jarosch and Besl 2001), and Aleur-odiscus (Wu et al. 2001) were shown to be polyphyletic by molecular methods and analyses.
Introduced in 1907, Dendrothele Hohn. & Litsch. is a corticioid genus with discoid or crustose basidiomes with a smooth hymenial surface, occasionally with small, sterile spines, and a monomitic hyphal system. Dendrohyphidia and gloeocystidia are usually present, and basidiospores are globose to allantoid with nonamyloid, smooth, hyaline, thin or thickened walls. Lemke (1964Lemke ( , 1965 included 18 species in his comprehensive study of Dendrothele (syn. Aleurocorticium P.A. Lemke). In subsequent years, additional species of Dendrothele have been described by various authors (Viegas 1945;Burdsall and Nakasone 1983;Gilbertson and Blackwell 1985;Boidin et al. 1986Boidin et al. , 1996Hjortstam 1987Hjortstam , 1997Greslebin and Rajchenberg 1998;Pouzar 2001;Nakasone 2006;Duhem and Michel 2007). Presently, there are 41 accepted species of Dendrothele listed in CortBase (Parmasto et al. 2004). Goranova (2003) tested the monophyly of Dendrothele by sequencing and analyzing nuclear and mitochondrial ribosomal DNA gene regions. She discovered that Dendrothele is highly polyphyletic with taxa occurring in 11 lineages distributed among the Hymenochaetales, Russulales, Corticiales, and Agaricales. She concluded that convergence in morphology and habitat occurred repeatedly in this group. In this paper, we investigate one of the lineages in the Agaricales that shows a close relationship to Campanella Henn. and describe a new, monotypic genus, Neocampanella. Coincidently, Wu et al. (2007)

Taxon sampling
The nuclear large subunit ribosomal RNA (nLSU rRNA) gene sequence of Neocampanella blastanos was used as a BLAST query of the GenBank database (blast.ncbi.nlm.nih. govlBlast.cgi). Thirty-two of the top-scoring sequences were combined with 9 additional sequences from GenBank, including Campanella eberhardtii (Pat.) Singer, Campanella sp. (two sequences), Caripia montagnei (Berk.) Kuntze, Crinipellis campanella (Peck) Singer, Gymnopus dryophilus (Bull.) Murrill, Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora and Tetrapyrgos sp., as well as one unpublished sequence of an undescribed lachnoid species from the Dongling Mountains, generously provided by M. Binder (Table 1). Based on preliminary analyses (not shown) and the higher-level phylogeny of Agaricales of Matheny et al. (2006), five sequences corresponding to the Omphalotaceae sensu Matheny (including Campanella eberhardtiii were selected as the outgroup. DNA extraction, amplification, and sequencing Goranova (2003) described in detail DNA extraction, amplification, purification, and sequencing procedures followed. These protocols are available as supplementary material.'.

Alignment and phylogenetic analysis
The sequences were aligned with the MAFFT version 6.624b server (align.bmr.kyushu-u.ac.jp/matlt/online/server) using the Q-INS-I algorithm (Katoh and Toh 2008), followed by manual adjustment in MacClade version 4.08 (Maddison and Maddison 2005) (see supplementary data.' Fig. S1). The alignment was converted to PHYLIP format and uploaded to the CIPRES Portal version 1.14 at the San Diego Supercomputing Center (8ball.sdsc.edu:8889/ cipres-web/Home.do) for maximum likelihood (ML) analysis using RAxML version 7.0.4 (Stamatakis 2006;Stamatakis et al. 2008). The ML analysis used 100 rapid bootstrap (RBS) replicates followed by ML optimization (Stamatakis et al. 2008). The general time reversible (GTR) nucleotide substitution model was employed, with among-site rate heterogeneity modeled with CAT approximation during RBS and the initial ML optimization, switching to the discretegamma model with four rate categories during the final ML optimization. A phylogram of the optimal ML tree was produced using FigTree version 1.0 (Rambaut 2006). Maximum parsimony (MP) analyses were conducted using the "portable" version of PAUP* 4.0b10 running in OSX (Swofford 2002). One hundred heuristic MP searches were conducted with the cost of transversions weighted twice that of transitions, with starting trees generated by random taxon Lampteromyces japonicus addition sequences, MAXTREES set to 1000, and tree bisection-reconnection (TBR) branch swapping. One thousand bootstrapped MP analyses were conducted, with MAX-TREES set to 10 and other settings unchanged.

Morphological studies
Freehand sections and squash mounts of basidiomes were examined microscopically in 2% (wlv) KOH and 1% (wlv) aqueous phloxine or Melzer's reagent (Kirk et al. 2001). Sections were mounted in 0.1% (wlv) cotton blue in 60% lactic acid to determine cyanophily of the cell walls (Kotlaba and Pouzar 1964;Singer 1986). Drawings of microscopic features were aided with a camera lucid a attachment on an Olympus BH2 compound microscope. Q is the ratio of the average length to width measurements of basidiospores (Kirk et al. 2001). Color descriptions were taken from Kornerup and Wan scher (1978), and herbarium designations follow Holmgren and Holmgren (1998).

Description of taxa
Neocampanella is characterized by thin, effuse, nongelatinized basidiomes with dendrohyphidia, cylindrical to capitate cystidia, and ellipsoid to pyriform basidiospores with a distinct, blunt apiculus. Mature basidia are difficult to observe for they probably collapse soon after discharging spores. The characteristic spore shape, slightly tapering toward the prominent apiculus, is reminiscent of basidiospores found in species of Campanella and related genera. Neocampanella, with its corticioid habit and smooth hymenial surface (see supplementary data;' Figs. S2 and S3), however, would never be confused with Campanella's pleurotoid form and reticulate lamellae. Dendrohyphidia in the hymenium of Neocampanella may be homologous to the Rameales-like structures developed in the pileal epicutis of Campanella and allied genera.
Among corticioid taxa, the distinctive pyriform basidiospores of Neocampanella are present also in Brunneocortidum and Cvlindrobasidium Julich. Cylindrobasidium, in the Physalacriaceae Corner (Matheny et al. 2006), is characterized by a monomitic hyphal system with clamp connections, abundant, oil-like globules in the hyphae, and thin-walled, fusoid cystidia. Dendrohyphidia are absent in Cylindrobasidium but are present in Neocampanella, Brunneocorticium, and many other genera and species of corticioid fungi. The combination of dendrohyphidia and pyriform basidiospores, however, is unique to Neocampanella and Brunneocortidum. Neocampanella has a monomitic hyphal system, whereas Brunneocorticium has a dimitic hyphal system with the brown-pigmented skeletal hyphae dominating the context (see supplementary data;' Fig. S5) and margin. The distinct, sterile brown margin in Brunneocorticium is composed

Marasmiaceae 2
Fig. 1. Phylogenetic placement of Neocampanella blastanos based on maximum likelihood (ML) and maximum parsimony (MP) analyses of nuclear large subunit ribosomal RNA gene sequences. This is the optimal tree obtained with ML. Filled dots on branches indicate groups that are also present in the strict consensus of the 706 equally most parsimonious trees. Numbers along branches before slash marks (I) are frequencies from 100 ML bootstrap analyses. Numbers after slash marks are frequencies from 1000 bootstrapped MP analyses. Bootstrap frequencies below 70% are not shown. Group names are discussed in the text. Scale bar units represent numbers of substitutions per site.  entirely of skeletal hyphae that develop in advance of the fertile, white hymenium (see supplementary data," Fig. S4). Phylogenetic analyses of sequence data from the LSD rRNA gene also support the erection of the new genus Neo-campanella. The precise placement of Neocampanella within the Marasmiaceae 1 clade is not well resolved, but it is strongly supported as a member of that group and it does not appear to be nested within any established genus, includ-ing Brunneocorticium (Fig. 1). Uncorrected sequence divergence between Brunneocorticium and Neocampanella is 3.18%. This value exceeds the levels of sequence divergence observed among members of the core Campanella and Tetrapyrgos clades, which ranged from 0.31% to 2.96% and from 0.27% to 2.7%, respectively, among the taxa.
HABITAT: on wood and bark of small hardwood twigs. Neocampanella blastanos is characterized by a thin, effuse, white basidiome with capitate cystidia, dendrohyphidia, and pyriform basidiospores. This is a rare species but probably occurs worldwide in subtropical to tropical regions. Microscopically, N. blastanos compares favorably with the holotype of Campanella elongatispora Singer (Argentina, Jujuy, Lagunas de Yala, 11 February 1965, R. Singer T4025 at F) with respect to the capitate cystidia and pyriform basidiospores.  Voucher specimens of the B. pyriforme DNA sequences used in this study were examined morphologically and confirmed to be conspecific with D. bispora. For descriptions and illustrations, see Burdsall and Nakasone (1983), Boidin et al. (1996), Maekawa (1998), andWu et al. (2007). This species is characterized by a yellowish white to grayish brown, effuse basidiome with distinct, sterile, brown margins and a smooth hymenial surface. Microscopically, it has a dimitic hyphal system with clamped generative hyphae and aseptate, brown skeletal hyphae, dendrohyphidia, 2-sterigmate basidia, and pyriform basidiospores with a distinct apiculus. Average basidiospore size for three specimens, 9-15 spores, range 11.9-12.5 urn x 6.4-6.7 urn, Q = 1.80-1.90. Cylindrical cystidia with adventitious septa were found in the Chinese specimen only. The colorless dendrohyphidia are often obscured by encrusting hyaline crystals and were overlooked by Wu et al. (2007). Mature basidia were rare and probably developed in a catahymenium as reported by Boidin et al. (1996). It occurs primarily on corticate branches of angiosperms and rarely on gymnosperms. Originally described from the United States in Florida, B. bisporum has a pantropical distribution. Wu et al. (2007) showed, using partial LSU rRNA gene sequences, that B. bisporum (as B. pyriforme) is embedded in the Agaricales and related to Marasmiellus candidus and Tetrapyrgos subdendrophora. Our results confirm this and place Brunneocorticium in the Marasmiaceae 1 clade with Neocampanella, Campanella, and Tetrapyrgos (Fig. 1).

Discussion
Recent molecular studies have established that crustose, corticioid species are distributed throughout the Agaricales (Binder et al. 2005;Larsson et al. 2004;Larsson 2007b). The results of our study confirm and expand on these prior observations. Morphological and molecular studies show that the monotypic, corticioid genera Neocampanella and Brunneocorticium are closely related to the pleurotoid genera Campanella and Tetrapyrgos. Although the species samples were small, previous studies showed that Campanella and Tetrapyrgos were distinct, monophyletic sister clades (Moncalvo et al. 2002;Aime and Phillips-Mora 2005;Matheny et al. 2006). Our results confirm that Tetrapyrgos is monophyletic, but Campanella appears to be polyphyletic. The first indication that Campanella may not be monophy- Botany Vol. 87, 2009 letic came from Wilson and Desjardin's (2005) phylogenetic study of gymnopoid and marsmioid agarics, which showed that Campanella eberhardtii did not cluster with Tetrapyrgos and M. candidus but was in a sister clade, with Marasmiellus ramealis (Bull.) Singer, to Rhodocollybia. In the present study, Campanella eberhardtii was found to be more closely related to the outgroup Omphalotaceae (sensu Matheny) than to the other Campanella species of the ingroup Marasmiaceae.
A comparative discussion of Campanella, Tetrapyrgos, Marasmiellus, and allied genera is beyond the scope of this study. However, general observations on the similarities and differences among the corticoid genera, Neocampanella and Brunneocorticium, and the agaricoid genera, Campanella and Tetrapyrgos, may be useful. These four genera are included in the Marasmiaceae 1 lineage and display a wide range of phenotypes. Besides the obvious basidiome form differences, crustose versus pileate, the dimitic hyphal system with brown pigmented skeletal hyphae of Brunneocorticium is contrasted with monomitic hyphal systems of the other genera. The gelatinous context of Campanella is unlike the membranous texture of the corticioid taxa, and the unusual tetrahedral basidiospores of Tetrapyrgos are unique in Marasmiaceae 1.
Although morphological differences are striking in the genera of the Marasmiaceae 1 clade, some notable morphological similarities include pyriform to fusiform basidiospores found in Campanella, Neocampanella, and Brunneocorticium, and hymenial cystidia with a bulbous apex present in Neocampanella and some Campanella species. Dendrohyphidia developed in the hymenium of the corticioid genera may be homologous to the diverticulate pileocystidia or hyphae of the pileipellis in the agaricoid genera.
In addition to phenotypic differences among species in the Marasmiaceae 1 clade, nutritional requirements differ also. Most species are saprobic on decayed logs and branches or stems of woody grasses. Sterile white basidiomycete 3034, however, was shown to be pathogenic on 12 plant species in Australia (Vinnere et al. 2005).
Several inconsistent results in Fig. 1 deserve comment. First, Marasmiellus candidus is included within the Marasmiaceae 1 clade. Wilson and Desjardin (2005) discussed this anomaly and noted that M. candidus and species in section Candidi are morphologically distinct from other Marasmiellus species. Second, species within the Tetrapyrgos clade have basidiospores that are tetrahedral in shape or have a distinct lateral bulge, except for Campanella junghuhnii. Parmasto (1981) described a faint bulge on the abaxial side of some basidiospores of C. junghuhnii that suggests a transfer to Tetrapyrgos may be appropriate after further study.
Laboratory, and DEB-0732968, which supports the Assembling the Fungal Tree of Life (AFTOL) project. Dr. Harold H. Burdsall, Jr., Dr. Karl-Henrik Larsson, Dr. Scott Redhead, and an anonymous reviewer provided valuable comments and suggestions to improve this manuscript.