The Pezizales are the basal order of Euascomycetes (Lutzoni et al., 2004) and comprise 1125 species (Kirk et al., 2001). Most Pezizales form cup-shaped fruit bodies (apothecia) on soil, dung or plant debris. However, several soil-inhabiting taxa have developed hypogeous fruiting in parallel with the loss of forcibly discharged ascospores. In these taxa, small mammals act as vectors for spore dispersal. Hypogeous fruiting has evolved multiple times independently from apothecial ancestors (O'Donnell et al., 1997; Percudani et al., 1999; Hansen et al., 2001, 2005). The trophic status of pezizalean species ranges from saprobic to mycorrhizal, with a few known to be parasitic (Egger & Paden, 1986). Overall, however, very little is known about the trophic status of most taxa. The majority of species are considered to be saprobic, although most hypogeous (previously in the order Tuberales, now spread across the Pezizales) and a few epigeous taxa are claimed, or have been shown, to be mycorrhizal.
The ectomycorrhizal (EcM) lifestyle has developed in several distantly related lineages of ascomycetes, including Elaphomyces within Eurotiales; Cenococcum within Loculoascomycetes; the Rhizoscyphus (syn. Hymenoscyphus) ericae complex within Helotiales; and Tuber within Pezizales (LoBuglio et al., 1996; Percudani et al., 1999; Vrålstad et al., 2000). Maia et al. (1996) reviewed numerous reports of the EcM status of pezizalean taxa. However, these reports rely predominantly on observations of fruitbody habitats rather than on resynthesis or identification of the EcM-forming fungi on root tips. In addition to EcM, ascomycetes also form ectendomycorrhiza. The pezizalean genera Wilcoxina, Sphaerosporella, Balsamia and Geopora have been reported to form ectendomycorrhiza with a thin or fragmented mantle, poorly developed Hartig net, and intracellular colonization on Pinus and Larix spp. (Mikola, 1965; Danielson, 1984; Palfner & Agerer, 1998; Yu et al., 2001; Fujimura et al., 2005). Similarly, Terfezia spp. have been shown to form ectendomycorrhiza on Cistaceae (Dexheimer et al., 1985) and possibly on some nonEcM plant genera (Bratek et al., 1996). Danielson (1982) hypothesized that ectendomycorrhizal associations on conifer roots involve several putative genera of Pezizales. Some pyrophilous members of the Pyronemataceae display a continuum of biotrophic interactions with conifer seedlings, including both pathogenic and EcM associations (Egger & Paden, 1986).
Identification of pezizalean EcM from root tips has remained problematic at both species and genus levels. Septal pore ultrastructure, in particular the presence of simple septa and Woronin bodies, has traditionally been studied to confirm ascomycete EcM (Berndt et al., 1990) and orchid mycorrhiza (Selosse et al., 2004). However, septal pore ultrastructure provides low resolution at the genus level (Kimbrough, 1994). Unlike many basidiomycetes, EcM ascomycetes lack hyphal strands that can be followed from the base of a fruit body to an EcM (Agerer, 1991, 2001). Many pezizalean taxa produce inconspicuous or hypogeous sporocarps that are easily overlooked unless specifically searched for. Identification of EcM fungi has benefited from molecular tools, including restriction fragment-length polymorphism (RFLP) and sequencing of the rDNA internal transcribed spacer (ITS) region (reviewed by Horton & Bruns, 2001). But ‘universal’ primer mismatches and length polymorphism of the ITS region still hamper amplification and sequencing of some pezizalean taxa (Aviram et al., 2004; L.T., personal observation). Primarily because of identification problems, EcM anatomy of pezizalean taxa is little studied. Previously published morphological and anatomical descriptions of pezizalean EcM cover only a few genera, including Tuber, Wilcoxina and Genea (Berndt et al., 1990; Ingleby et al., 1990; Ursic & Peterson, 1997; Jakucs et al., 1998). Generally, pezizalean EcM possess a thin pseudoparenchymatous mantle, infrequent emanating hyphae (EmH), no rhizomorphs and no clamp connections (Agerer, 1991, 2001).
Recent sequencing studies have revealed a high diversity of pezizalean EcM (Tedersoo et al., 2003; Izzo et al., 2005) and orchid mycorrhizal symbionts (Selosse et al., 2004; Julou et al., 2005), which have remained unidentified at the genus level. The purpose of this study is to uncover the EcM-forming taxa within the Pezizales. We identify EcM Pezizales on root tips using direct sequencing of the rDNA ITS region and/or large-subunit (LSU) fragment. We demonstrate that the EcM lifestyle is present in several lineages of Pezizales, including both epigeous and hypogeous fruiting species. Most lineages or genera of Pezizales possess distinct EcM anatomy. To improve the identification of pezizalean EcM, we provide morphological and anatomical EcM descriptions.