Bull. Natl. Mus. Nat. Sci., Ser. B, 34(4), pp. 161–173, December 22, 2008 Molecular Phylogenetics of Geastrales with Special Emphasis on the Position of Sclerogaster Kentaro Hosaka1 and Michael A. Castellano2 1 Department of Botany, National Museum of Nature and Science, 4–1–1 Amakubo, Tsukuba 305–0005, Japan 2 USDA, Forest Service, Northern Research Station, Forestry Sciences Laboratory, 3200 Jefferson Way, Corvallis, OR 97331, USA Abstract Molecular phylogenetic analyses for the order Geastrales (Agaricomycetes, Basidiomy- cota, Fungi) were conducted based on a two gene dataset with increased taxon sampling of the genus Sclerogaster. The placement of Sclerogaster in Geastrales, and the monophyly of the genus were strongly supported in all analyses. Sclerogaster forms one of the distinct lineages within Geastrales, but its exact position within Geastrales remains unresolved. Bayesian analyses support the sister relationship between Geastraceae and Sclerogaster without significant posterior probabil- ities. Most analyses under parsimony criterion supported the sister relationship between Sclero- gaster and Schenella, but lack significant bootstrap values. The topology of Geastrales indicated that the truffle-like form may be an ancestral morphology of the earthstar fruit body-type. Biogeo- graphical patterns of Sclerogaster indicate that long distance dispersal may be the most important factor, and this may be associated with its saprotrophic habit. Key words : atp6, Basidiomycota, fungi, LSU, Phallomycetidae, Phylogeny, Sclerogaster, Sys- tematics. fungi. Their results clearly showed that there are Introduction four major clades within the larger gomphoid- The subclass Phallomycetidae (Agarico- phalloid clade, and each of the smaller clades mycetes, Basidiomycota) is commonly known as were well-supported and in two cases, i.e., Phal- the gomphoid-phalloid fungi (Hibbett and Thorn, lales and Gomphales, with a long history of ordi- 2001). Because of its morphological diversity, nal status. Hosaka et al. (2006) elevated Phallales traditional taxonomy failed to recognize the gom- sensu Kirk et al. to subclass status, and proposed phoid-phalloid fungi as a single entity. Hibbett et the new subclass Phallomycetidae. Four major al. (1997) first demonstrated the monophyly of clades within the Phallomycetidae each received the gomphoid-phalloid fungi, which were subse- ordinal status; Phallales, Gomphales, Hysteran- quently supported by additional studies (Binder giales, and Geastrales. and Hibbett, 2002; Hibbett and Binder, 2002; Hi- These phylogenetic analyses revealed many bbett and Thorn, 2001; Hosaka et al., 2006; previously unexpected lineages, and taxonomic Humpert et al., 2001; James et al., 2006; Lutzoni revisions for several taxa were made accordingly. et al., 2004; Matheny et al., 2007; Moncalvo et However, the taxonomic revisions for many taxa al., 2002; Pine et al., 1999). The first attempt to have not been conducted yet. The order Geas- incorporate these results into a formal classifica- trales is especially problematic because several tion scheme by treating the gomphoid-phalloid genera, such as Phialastrum and Trichaster, were fungi as a single order Phallales was made by not included in the phylogenetic analyses al- Kirk et al. (2001). Later Hosaka et al. (2006) an- though they traditionally have been placed in alyzed a 5-gene dataset of the gomphoid-phalloid Geastraceae (Sunhede, 1989). In addition, Zeller 162 Kentaro Hosaka and Michael A. Castellano (1948) described Broomeiaceae in Lycoperdales, included. Additional taxon sampling is necessary but it is unclear whether this family is more for a more definitive placement of the genus. closely related to Geastrales or to other homoba- This study attempts to answer the questions of: sidiomycetes. (1) monophyly of the genus Sclerogaster, and (2) One of the unexpected lineages within Geas- phylogenetic placement of the genus. trales includes the genus Sclerogaster Hesse, which possesses hypogeous (truffle-like) basid- Materials and Methods iomata with a yellow or green tinted gleba (Fig. 3) and yellow to brown, warty basidiospores (Fig. Taxon sampling, PCR, and DNA sequencing 4) (Castellano et al., 1989). When the genus was Taxa sampled, along with GenBank accession described by Hesse (1891), it was placed in the numbers, are listed in Table 1. A total of 51 taxa “Hymenogastreen”, which was considered an ar- (four outgroup and 47 ingroup taxa) were sam- tificial assemblage of truffle-like basidiomycetes pled for this study. The selection of ingroup and (Clémençon et al., 2007). Fischer (1900) later outgroup taxa was based on the phylogeny of placed the genus in the family Hymenogas- previous studies (Hosaka et al., 2006) to cover traceae, but its phylogenetic affinity remained un- the diversity of Geastrales. resolved. Since then, several attempts were made DNA was extracted from glebal tissue of fresh to phylogenetically place the genus. Some au- or dried fruiting bodies. The protocol generally thors (Jülich, 1981; Castellano et al., 1989) con- follows that of Doyle and Doyle (1987) but with sidered Sclerogaster to be closely related to Oc- the following modifications. For fresh materials, taviania (Boletales), but others (Dodge and immature glebal tissue was soaked in DMSO Zeller, 1936; Zeller and Dodge, 1935; Kreisel, buffer (Seutin et al., 1991) with an addition of 1969) implied close affinity of Sclerogaster with 100 mM Tris-HCl (pH 8.0) and 0.1 M sodium Hydnangium (Agaricales). Furthermore, sulfite (Na2SO3) under 4°C until extraction was Malençon (1931) and Heim (1971) implied a conducted. For dried materials, immature glebal close affinity of the genus with Russulales. tissue was soaked overnight in modified DMSO Therefore, Sclerogaster have been placed in at buffer under room temperature. least three distinct orders of Basidiomycota. Soaked tissue samples were then ground in liq- Molecular phylogenetic analyses strongly sug- uid nitrogen using mortar and pestle. After grind- gested placement of the genus in Geastrales with ing, samples were immediately transferred to 1.5 close affinity to the Geastraceae and Schenel- mL tubes with 1,000 mL of 2X CTAB buffer laceae (Pyrenogastraceae) (Hosaka et al., 2006). (Doyle and Doyle, 1987) with an addition of This was surprising because a close relationship 0.1M Na2SO3. Some taxa of Geastrales have a between Sclerogaster and Geastraceae had never powdery spore mass at maturity, and the powdery been postulated previously. Morphologically, spore mass cannot easily be ground in liquid ni- Sclerogaster, Geastraceae and Schenellaceae all trogen. If the spore mass was the only tissue possess globose spores with a surface ornament- available for DNA extraction, spores were ed with spines or warts (Domínguez de Toledo ground using the bead beating protocol modified and Castellano, 1996; Sunhede, 1989; Castellano from Munkacsi et al. (2006). Approximately 30 et al., 1989). Sclerogaster differs from Geas- milligrams of dry basidiospores were mixed with traceae and Schenellaceae in having a green to 0.2 mg of 0.1 mm glass beads (BioSpec), 20 g of yellow or orange gleba (Fig. 3) versus brown to 1 mm chrome steel beads (BioSpec) and two 3 black gleba that turns powdery at maturity. How- mm chrome steel beads (BioSpec), and frozen in ever, only one species (Sclerogaster xerophilus) liquid nitrogen for 5 minutes. The mixture was was sampled from approximately 15 described beaten at 30 Hz for 1 minute using the TissueL- species, and more critically, no type species were yser (Qiagen), frozen in liquid nitrogen for 3 Sclerogaster phylogeny 163 minutes, and then beaten again at 30 Hz for 1 ing was performed following the manufacturer’s minute. After grinding, 1,000 mL of 2X CTAB instructions (Big Dye ver. 3.1, Applied Biosys- buffer with an addition of 0.1 M Na2SO3 was tems) using the same primers described above. added directly to the sample tubes. Samples were incubated at 55°C overnight, Phylogenetic analyses and centrifuged at 12,000 rpm for 5 minutes. DNA sequences were initially aligned using Only the aqueous phase was transferred to a new Muscle v.3.6 (Edgar, 2004a, b), followed by man- tube, and precipitated tissue debris were discard- ual alignment in the data editor of BioEdit ver. ed. The equal volume of the mixture of chloro- 7.0.1 (Hall 1999). Ambiguously aligned regions form: isoamylalcohol (24 : 1) was added to the and introns were excluded from the analyses. To buffer, mixed vigorously for two minutes, and test for incongruence between the two individual centrifuged at 12,000 rpm for 15 minutes. The datasets, parsimony analyses of individual loci aqueous phase was pipetted out and transferred were compared. First, 70 % bootstrap (BS) trees to a new tube. This step of using chloroform was were calculated (100 BS replicates with five ran- conducted only once. dom addition sequences, TBR and Multrees op- After transferring ca. 300 mL of the aqueous tions off) including only the taxa with sequences phase, 1,000 mL of 6M sodium idodine buffer (6 from both loci (36 taxa). These trees were used M NaI, 50 mM Tris-HCl (pH 7.4), 10 mM EDTA, as constraints in a different dataset (for example, 0.1 M Na2SO3) was added and mixed gently for 1 parsimony analysis of the atp6 dataset with the minute. Silica mixture was prepared following LSU tree as a constraint), using the ‘Load Con- the protocol of Rogstad (2003), and 25 mL of the straints’ option in PAUP version 4.0b10 (Swof- mixture was added to the samples. Samples were ford 2002). Parsimony analyses were conducted incubated at 55°C for 1 hour, and centrifuged at under these constraints, keeping only the trees full speed for ca. 10 seconds. The supernatant that are compatible with these constraints. Com- was discarded and 750 mL of washing buffer (10 parisons of constraint and unconstraint trees were mL Tris-HCl (pH 7.4), 1mM EDTA, 100 mM made using the ‘Tree Scores’ option in PAUP NaCl, 50% EtOH) was added, mixed briefly, and version 4.0b10 (Swofford 2002). Parsimony centrifuged at full speed for ca.