CORE Metadata, citation and similar papers at core.ac.uk Provided by OpenKnowledge@NAU Mycologia, 106(3), 2014, pp. 553–563. DOI: 10.3852/13-263 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897 An elusive ectomycorrhizal fungus reveals itself: a new species of Geopora (Pyronemataceae) associated with Pinus edulis Lluvia Flores-Renterı´a1 of plant species, particularly trees that dominate Matthew K. Lau woodland and forest communities around the world. Louis J. Lamit Many EcM fungi reproduce sexually through the Catherine A. Gehring production of macroscopic sporocarps. At least 6000 Department of Biological Sciences and Merriam-Powell species of fungi form ectomycorrhizal associations Center for Environmental Research, Northern Arizona (Rinaldi et al. 2008, Brundrett 2009), which is , 0.5% University, Flagstaff, Arizona 86011 of the estimated , 1 500 000 fungal species (Hawks- worth 2001). That number is likely much higher (Tedersoo et al. 2010) due to infrequent production Abstract: Species of the genus Geopora are important of sporocarps or the production of hypogeous or small ectomycorrhizal associates that can dominate the nondescript fruiting bodies by some EcM fungi. communities of some plant taxa, such as pinyon pine Despite the apparently well documented hypoge- (Pinus edulis), a widespread tree of the western ous habit of some EcM-forming fungi, new species United States. Several members of the genus Geopora descriptions are still common from either sporocarps are known only from ectomycorrhizal root tips and or from mycorrhiza (e.g. Guevara et al. 2008, 2013; thus have not been described formally. The sporo- Guevara-Guerrero et al. 2012; Lantieri et al. 2012). carps of some Geopora species occur infrequently The advantage of characterizing undescribed species because they depend on wet years for sporulation. In directly from the mycorrhiza allows the assessment of addition, Geopora sporocarps can be small and may be their symbiotic interaction. However, there are a hypogeous at some developmental stage, limiting the substantial number of ectomycorrhizal fungal species opportunities for describing their morphology. Using characterized by molecular data, which do not match molecular and morphological data, we have described sporocarp collections from the same site or any a new species of fungus, Geopora pinyonensis, which vouchered specimen in GenBank (Southworth and produced ascocarps after unusually high precipitation Frank 2011). These EcM may represent taxa that at a northern Arizona site in summer 2012. Based on sporulate rarely, or not at all, and taxa for which the analysis of the ITS and nuLSU regions of the rDNA, sporocarp is difficult to find or to describe. For G. pinyonensis is a new species of Geopora. It has small example, the mycobionts of EcMs of Geopora spp., sporocarps and ascospores relative to other members (Pyronemataceae), identified with molecular meth- of the genus; however, these morphological features ods, are not often reported associated with hypogeous overlap with other species. Using rDNA data from sporocarps and ectomycorrhizal root tips, we show fruiting bodies and are rarely classified to species that the sporocarps correspond to an abundant (Gehring et al. 1998, Fujimura et al. 2005, Tedersoo species of ectomycorrhizal fungus associated with et al. 2006, Hrynkiewicz et al. 2009, Ishida et al. 2009, pinyon pines that is increasing in abundance in Sthultz et al. 2009, Wei et al. 2010, Gordon and drought-affected landscapes and may promote Gehring 2011). Moreover, species delimitation in drought tolerance. Geopora is complicated because of the small number Key words: earth pore, ectomycorrhiza, hypoge- of differentiating morphological characters, values of ous fungi, Pyronemataceae, Sunset Crater which tend to overlap among species. Classification of Geopora species has relied mainly on the size and shape of the ascospores, position of the apothecia INTRODUCTION in the ground and the length of excipular hairs Ectomycorrhizal (EcM) fungi are root symbionts that (filamentous hyphae on the outside surface of the promote host plant growth by increasing plant acqui- cup). However, molecular analyses have shown that sition of soil nutrients and improving host tolerance to well supported clades do not correspond to species environmental stresses (Smith and Read 1997). They concepts based on morphological characters (Tamm are important both economically and ecologically et al. 2010). Therefore using molecular data in because they form symbiotic interactions with a range combination with morphological information is con- sidered a more reliable approach to link the EcM Submitted 16 Aug 2013; accepted for publication 25 Nov 2013. to sporocarps and define species (Tamm et al. 2010, 1 Corresponding author. E-mail: [email protected] Southworth and Frank 2011). 553 554 MYCOLOGIA The difficulties in correctly identifying species of formed by other taxonomic groups (Gehring et al. Geopora are particularly evident in southwestern USA 2013, Gehring et al. 2014). where arid conditions may not only reduce sporocarp After unusually high precipitation at Sunset production but also favor the evolution of hypogeous Crater, northern Arizona, during the summer mon- sporocarps that reduce the risk of desiccation (Thiers soon in 2012, we detected sporocarps whose mor- 1984). For example, in Sunset Crater, Arizona, in a phology was consistent with that of Geopora but drought-affected pinyon-juniper woodland where a distinct from G. cooperi, the only member of this long-term study (more than 20 y) has been conduct- genus recorded at the study site. In this study we ed, sporocarps have been detected infrequently evaluated whether these sporocarps were related to (Gehring et al. 1998). The study of the effect of the mycobionts reported in P. edulis by using both EcM on trees in this area is relevant because they have DNA sequences and morphological characters of experienced more than 10 y of drought including 2 y ectomycorrhiza and sporocarps. Herein we describe of exceptionally dry conditions (1996, 2002) that this new species of Geopora on the basis of resulted in widespread mature pinyon pine (Pinus morphological and molecular characteristics of both edulis) mortality (Breshears et al. 2005, Mueller et al. mycorrhiza and sporocarps. Furthermore we evalu- 2005). However, the EcM fungi associated with P. ated the phylogenetic relationships of this new edulis, which is a dominant species of this region, have Geopora species in relation to the currently accepted been well documented (Gehring et al. 1998, Mueller species for the genus based on the recent phyloge- and Gehring 2006, McHugh and Gehring 2006, netic inferences using ITS (e.g. Tamm et al. 2010, Hubert and Gehring 2008, Sthultz et al. 2009). Southworth and Frank 2011, Guevara-Guerrero et al. Unidentified members of the Pezizales have been 2012) and nuLSU (Perry et al. 2007). observed in almost all studies of pinyon EcM (Gehring et al. 1998, Mueller and Gehring 2006, MATERIALS AND METHODS Sthultz et al. 2009, Gordon and Gehring 2011, Gehring et al. 2013) and in two studies of P. ponderosa Study sites and collection material.—Ten sporocarp speci- EcM (Fujimura et al. 2005, Hubert and Gehring mens were collected 13 Sep 2012 at Sunset Crater, Arizona 2008). Many of these EcM have been identified as (35.397689 N, 2111.425058 W, 1900 m). This area has a Geopora, but only the sporocarps of G. cooperi,an mean annual air temperature of 12 C, mean annual infrequent member of the P. edulis community, have precipitation of 328 mm; the soil is a coarse cinder, been observed (Gordon and Gehring 2011). Repeat- classified as a Typic Ustorthent (Selmants and Hart 2008). Dominant vegetation consists of trees and shrubs and is ed sampling of varying sites and individual trees characterized by Pinus edulis, Juniperus monosperma, Rhus within a site before and during drought have shown trilobata, Fallugia paradoxa, and Ephedra viridis.Onlytwo dramatic increases in the relative abundance of sporocarps were exposed on the surface while the rest Geopora mycorrhizas with drought (Gordon and were buried under the basaltic soil at different develop- Gehring 2011, Gehring et al. 2014). Gordon and mental stages including maturity. The sporocarps were Gehring (2011) used molecular data to identify located in mostly open space between trees on exposed several unknown, genetically distinct Geopora EcM soil without litter. The mosses Ceratadon purpurea and types, which did not match any vouchered specimens Bryum argentum occurred near the sporocarps. Sporocarp in GenBank. These EcM types were morphologically collections were deposited in the Sam Mitchel Herbarium similar when observed as EcM root tips, but they had of Fungi (Denver Botanic Garden) (DBG), voucher different restriction fragment length polymorphism catalog DGB 27586. (RFLP) patterns and ITS sequences, representing DNA extraction and sequencing.—DNA was extracted from potential novel species. Sporocarps of these Geopora two sporocarps with a DNA easy mini plant kit (QIAGEN, taxa had never been observed, limiting their mor- Valencia, California), according to the manufacturer’s phological characterization to indistinguishable EcM instructions. Molecular data were obtained by amplifying root tips. Further characterization of these taxa is the internal transcribed spacer (ITS) region, including particularly important because Geopora spp. and other ITS1, the 5.8S ribosomal DNA gene and ITS2, with forward