MYCOLOGICAL RESEARCH I I0 (2006) 57 5-582 Gymnomyces xerophilus sp. nov. (sequestrate Russulaceae), an ectomycorrhizal associate of Quercus in California Matthew E. SMITHa1*,James M. TRAPPE~,Dauid M. RIZZOa, Steven I. MILLERC 'Department of Plant Pathology, University of California at Davis, Davis CA 95616, USA b~epartmentof Forest Science, Oregon State University, Camallis, OR 97331-5752, USA CDeparhnentof Botany, University of Wyoming, Laramie, WY 82071, USA ARTICLE INFO ABSTRACT Article history: Gymnomyces xerophilus sp. nov., a sequestrate species in the Russulaceae, is characterized Received 30 August 2005 and descn'bed morphologically as a new species from Quercus-dominated woodlands in Accepted 14 February 2006 California. ITS sequences recovered from healthy, ectomycorrhizal roots of Quercus dougla- Corresponding Editor: Michael Weiss sii and Q. wislizeni matched those of G. xerophfius basidiomata, confirming the ectomycor- -- rhizal status of this fungus. Phylogenetic analysis of the ITS region places G. xerophilus in Keywords: a clade with both agaricoid (Russula in the section Polychromae) and sequestrate (Gymno- Basidiomycota myces, Cystangium) relatives. We include a dichotomous key to the species of Gymnomyces Hypogeous fungi associated with Quercus. ITS O 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Molecular phylogeny Russula Introduction mycelium, potentially reducing drought stress (Duddridge et al. 1980; Parke et al. 1983). Quercus-dominated ecosystems cover about one third of dali- Although the EM fungi associated with Quercus in California fornia's 404,000bm2(Pavlik et al. 1991). Quercus spp. are well have not been studied extensively, Thiers (1984) and Trappe adapted to the state's extensive areas of dry, Mediterranean and Claridge (2005) suggest that seasonally dry climates exert climate, with at least 7 species considered endemic (Nixon a selection pressure towards a sequestrate fruiting habit in 2002). Quercus, like other members of the Fagaceae, form ecto- EM fungi. Evidence from seasonally dry locations that have mycorrhizae (EM) with diverse Ascomycota, Basidiomycota, and been extensively studied (e.g., coniferous forests in California Zygornycota, including members of the Russulaceae (Trappe and Oregon, eucalypt communities in Australia) indicates 1962; Gerdemann & Trappe 1974; Froidevaux & Schwarzel that sequestrate species make up a significant portion of the 1977; North 2002; Avis et al. 2003; Walker et al. 2005). Quercus EM taxa (Johnson 1994; Waters et al. 1997; North 2002). Seques- spp. depend on formation of EM for normal function and sur- trate fungi have been shown to produce large amounts of bio- vival (Frank 1885; Smith & Read 1997). EM fungi are thus vital mass (Luoma et al. 1991; Smith et al. 2002) and provide symbionts for host plants in idea1 and harsh environments important food resources for animals in several temperate for- alike (Allen 1991). In xeric habitats the EM symbiosis provides ests (Maser et al. 1978; Johnson 1994; Trappe & Claridge 2005). the host plant access to water reserves via the fungal Though relatively few groups have been studied in detail, * Corresponding author. E-mail address: mesmithQucdavis.edu. 0953-7562/$- see front matter O 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.03.001 576 M. E. Smith et al. most sequestrate fungi form EM with multiple plant hosts MorphologicaI examination of basidiomata (Miller 1983; Tragpe & Castellano 1986; Molina et al. 1999). The Russulaceae, including sequestrate species, are domi- Macroscopic characters were described from fresh specimens. nant and diverse EM root symbionts in many forest ecosystems Basidiomata colors are designated by ISCC-NBS terminology (Horton & Bruns 2001). For example, in a Minnesota oak sa- (Kelly & Judd 1955). Microscopic characters were determined vanna, Russula spp. and other unidentified Russulaceae were from two types of mounts: (1) temporary mounts hand-sec- among the most abundant EM symbionts encountered on roots tioned with a razor blade on the vertical axis of the basidio- of mature Quercus (Avis et al. 2003). Similarly, a study of EM mata and mounted in 3 % KOH and Melzer's reagent, roots of Quercus seedlings in southern Appalachia revealed 17 respectively; and (2) paraffin-embedded specimens sectioned Russulaceae species among the 75 taxa encountered (Walker with a sliding microtome to a thickness of S10 m, stained et al. 2005). Currently, scant data is available regarding the EM in safranin-fast green, and made into permanent mounts. fungi on roots of Quercus in California. However, EMroots of an- The basic fuchsin reation (Romagnesi 1967) was used to other California angiosperm, Arctostaphylos glandulosa, were test for the presence of dermatocystidia and sulfovanillin heavily colonized by Russulaceae (Horton et al. 1999). was used to test for the presence of encrusted hyphae on Despite the apparent dominance of RussuIaceae in many EM the peridial surface (Singer 1986). fungal communites, only three sequestrate species have been All measurements were with the oil-immersion micro- conclusively shown to form EM. Arcangeliella borziana with scope objective at x 1000 magnification. Spore measurements Picea abies (Peter et a!. 2001), Gymnomyces medlockii (syn. Martel- included the largest and smallest spores and at least 20 addi- lia medlockii) with Pinus contorta and Tsuga heterophylla (Trappe tional, randomly selected spores from each specimen; spore & Castellano 1986), and an unidentified Gymnomyces sp. with dimensions excluding ornamentation are reported in this pa- Pinus ponderosa (Stendel et al. 1999). per. Because spores of sequestrate Basidiomycota are usually During a study of the EM fungal community associated with statismosporic, spore prints cannot normally be obtained Quercus in a xeric woodland savannain northern California, we from their basidiomata. Consequently, to best insure that ma- encountered basidiomata of a previously undescribed Gymno- ture spores and their ornamentation are being observed, basi- myces sp. (Russulaceae).In this paper we describe it as Gymno- diomata with spore masses so abundant as to obscure the myces xerophilus sp. nov. and use phylogenetic analysis of the hymenia should be selected. Because spores in the Russulaceae ITS region to place it in a clade of the genus Russula containing are smooth in youth and the ornamentation builds on the sur- both agaricoid and sequestrate taxa. In addition we compare face of the spores as they mature, spores with the tallest and G. xerophilus with other known Gymnomyces spp. associated most strongly developed ornamentation should be measured. with Quercus and provide a dichotomous key to these taxa. Molecular techniques DNA sequences for basidiomata were generated as in Miller Materials and methods and Buyck (2002). For EM, lyophilized root tips were ground with a micropestle and DNA extracted by a modified CTAB Sampling of basidiomata and ectomycorrhizas method (Gardes & Bruns 1993) followed by purification with a MO-BIO Soil DNA kit (MO-BIO Laboratories, Solana Beach, Basidiomata and EM roots were sampled at the Koch Natural CA, USA). PCR was then performed with primers ITSlf (Gardes Area of the University of California Sierra Foothill Research & Bruns 1993) and LR3 (Hopple & Vilgalys 1994). The reaction and Extension Center, in Yuba County, California. The terrain protocol began with initial denaturation of 94 OCfor 5 min fol- consists of low hills 50-650 m above sea level, Overstory vege- lowedby20cycles of 1min. (94 OC); lrnin. (55 "C);4min. (72 OC) tation is dominated by three species; Quercus douglasii, Q. wisli- followedby a final extension of 72 OCfor 7 min. FreshPCRprod- zeni, and Pinus sabiniana Douglas. The Mediterranean climate ucts were cloned with a TOPO-TAkit (Invitrogen,Carlsbad, CA, is characterized by cool, wet winters and hot, dry summers. USA). At least 48 successful clones per reaction were grown Precipitation generally occurs between October and May (an- overnight in LB media amended with 100 g/ml of ampicfin. nual mean 71 cm, range 23-132 cm) and temperature varies Cloned fragments were re-amplified in a PCR reaction using seasonally (mean 17.8 OC, range 10 "C-43 "C) (UCSFREC online: approximately 0.5 pl of the bacterial suspension as template. http://danrrec.ucdavis.edu). Amplicons were digested with the restriction enzymes ALUl Basidiomata of EM fungi were collected under Q. douglasii and Hinfl and electrophoresed through a 1.5 % agarose gel and and Q.wislizeni between December 2000 and January 2005. A stained with SYBR Green I (MolecularRobes, Eugene, OR, USA). garden cultivator was used to carefully remove litter and soil One to four representative clones of each restriction fragment at random locations beneath mature host canopies. Basidio- length polymorphism (RFLP) type were sequenced with ITSlF mata were described, photographed, and taken to the labora- and LR3 and/or ITS4 with the ABI Big Dye Terminator Sequenc- tory for tissue sampling and drying on the same day. ing Kit (v3.1).Sequences were read using an AB11373Oxl capillary For EM, litter was removed and soil cores of ca 900 cm3 sequencer (AppliedBiosystems, Foster City, CA, USA) at the Col- were collected under canopies of Q. douglasii and Q.wislizeni, lege of Agricultural and Environmental Sciences Genomics Fa- stored at 4 OC and processed within 15 d of sampling. Soil cility, University of California at Davis and