Rediscovery of the Vesicles That Characterized Rhizopogon Vesiculosus
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Mycologia, 103(5), 2011, pp. 1074–1079. DOI: 10.3852/10-347 # 2011 by The Mycological Society of America, Lawrence, KS 66044-8897 Rediscovery of the vesicles that characterized Rhizopogon vesiculosus D.L. Luoma1 were placed in the Fulviglebae based on gleba color Department of Forest Ecosystems and Society, and spore shape (Smith and Zeller 1966). Peavy Hall 154, Oregon State University, Corvallis, Section Villosuli was distinguished by a loosely Oregon 97331-7501 interwoven epicutis of brown-walled hyphae with D.M. Durall somewhat thickened walls that become dark brown University of British Columbia Okanagan, Institute for in KOH (Smith 1964, Smith and Zeller 1966, Smith et Species at Risk and Habitat Studies, Biology and al. 1981). A corollary character was the occasional to Physical Geography, 3333 University Way, Kelowna, frequent presence of brown, thick-walled, inflated, British Columbia, V1X 1B7 Canada and irregularly to vesiculose-shaped cells in the J.L. Eberhart epicutis (Smith and Zeller 1966 p 167). The presence Department of Forest Ecosystems and Society, of these distinctive elements is greatly restricted in R. Peavy Hall 154, Oregon State University, Corvallis, vinicolor and R. vesiculosus, which largely led Smith to Oregon 97331-7501 reject placement of those species into section Villosuli K. Sidlar (Smith and Zeller 1966). University of British Columbia Okanagan, Institute for Grubisha et al. (2002) proposed substantial chang- Species at Risk and Habitat Studies, Biology and es to the subgeneric classification of Rhizopogon that Physical Geography, 3333 University Way, Kelowna, included elevation of section Villosuli to subgenus British Columbia, V1X 1B7 Canada rank. They discovered that R. vinicolor and closely related taxa grouped (based on ITS data) with members of the Villosuli but had a distinct, mono- Abstract: Molecular distinction between Rhizopogon phyletic linage. Therefore they created section vinicolor and R. vesiculosus has been made recently, Vinicolores to accommodate that linage. Rhizopogon but the diagnostic ‘‘yellow-brown (fresh) inflated vinicolor was transferred to the new subgenus Villosuli cells’’ of R. vesiculosus, originally described by AH and the new section Vinicolores (Grubisha et al. 2002), Smith, were not observed. These distinctive hyphal and R. vesiculosus has been shown to belong there too cells (vesicles) have not been reported since the type (Kretzer et al. 2003). description. In that description they were said to Members of section Villosuli possess the above- collapse upon drying and described as being difficult mentioned distinctive epicutis and non-truncate to find. Here we report the rediscovery of these spores, while members of section Vinicolores have vesicles and describe their specific location on truncate spores and exhibit little development of the sporocarps of R. vesiculosus.Wealsoreportan distinctive epicutis. In this paper we confirm the original discovery that coiled, dark-walled hyphae on presence of and describe the location of the the sporocarps and in the mycorrhizae of R. vinicolor distinctive epicuticular elements for members of are of taxonomic value. The coiled hyphae, combined section Vinicolores.Wealsopostulatethatthese with the presence or absence of the elusive vesicles, elements derive from hyphae of the rhizomorph allow R. vesiculosus and R. vinicolor to be morpho- and ultimately the species’ mycorrhizae. logically distinguished with increased accuracy. Rhizopogon vinicolor and R. vesiculosus are ectomy- Key words: hyphal morphology, sporocarp taxon- corrhizal fungi that produce durable tuberculate omy, tuberculate ectomycorrhiza ectomycorrhizae and form relatively long rhizo- morphs (Trappe 1965, Zak 1971, Kretzer et al. 2004). The rhizomorphs can be important for INTRODUCTION translocating nutrients and water through mycelial Rhizopogon was partitioned into subgenera and networks (Brownlee et al. 1983, Egerton-Warburton et sections by Smith (1964). Within subgenus Rhizopo- al. 2007). Both species have been observed (as gon he established four sections, Rhizopogon, Amylo- mycorrhizae and sporocarps) across a chronose- pogon, Fulviglebae and Villosuli. Rhizopogon vinicolor quence of 5, 25, 65 and 100 y old sites and have A.H. Smith and R. vesiculosus A.H. Smith initially been shown to form robust mycorrhizal networks that might play a foundational role in aiding regeneration Submitted 12 Nov 2010; accepted for publication 18 Feb 2011. of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) 1 Corresponding author. E-mail: [email protected]; telephone: seedlings (Twieg et al. 2007, Beiler et al. 2010). These (541) 737-8595 Rhizopogon species have been reported as dominant 1074 LUOMA ET AL.: REDISCOVERY OF VESICLES 1075 members of several Douglas-fir ectomycorrhiza com- Laboratory.—The sporocarps (fresh and dried) and tubercles munities (Jones et al. 1997, Luoma et al. 2006, Twieg (dried) were examined with binocular and compound et al. 2007) and have been shown to increase seedling microscopy. For sporocarps, wefocusedourobservationson growth and resistance to drought (Parke et al. 1983, the epicutis in the vicinity of the rhizomorph attachment (FIG.1a–c)becauseforspeciesinsectionVinicolores it is that Molina et al. 1999). Rhizopogon sporocarps are area in which are found the brown-walled hyphal elements important items in the diets of small mammals (including vesicles) characteristic of the subgenus Villosuli. (Luoma et al. 2003, Jacobs and Luoma 2008). Given The same dark brown hyphae were examined in the peridium- the ecological importance of these two species and like sheath from tuberculate ectomycorrhizae of both Rhizo- their frequency of encounter across various studies, pogon species. Microscope mounts were made in 5% KOH. We further investigation of morphological characters that assigned sporocarps and tubercles to either R. vesiculosus or R. might distinguish between them was undertaken. vinicolor with molecular methods (Kretzer et al. 2003). Kretzer et al. (2003) molecularly distinguished Molecular analysis.—DNA was extracted from both fruit Rhizopogon vinicolor and R. vesiculosus with sequence bodies and tubercles in 400 mL QIAGEN AP1 lysis buffer data from the internal transcribed spacer (ITS) mixed with 2 mL anti-foam Y-30 emulsion (Sigma-Aldrich region of the nuclear ribosomal repeat and data from Canada Ltd., Oakville, Ontario) with a ceramic bead and five microsatellite loci but expressed frustration at shaking extractor (Fast Prep FP120, Holbrook, New York) for being unable to observe the ‘‘yellow-brown (fresh) 45 s at 6.5 m/s to pulverize the sample. DNA was isolated with inflated cells similar in size and shape to those found the QIAGEN DNeasy 96 Plant Extraction Kit protocol in many species of sect. Villosuli (up to 50 m diam)’’ (QIAGEN, Valencia, California) and then diluted 10-fold in that were used to characterize R. vesiculosus and ultrapure deionized water. The ITS region, located between the nuclear small and nuclear large rDNA, was amplified distinguish it from R. vinicolor (Smith and Zeller using PCR with the standard fungal specific primers ITS-1F (1966). These distinctive hyphal cells (vesicles) have and ITS-4 (Gardes and Bruns 1993). Cycling conditions not been reported in the peridium of R. vesiculosus included an initial denaturation at 94 C for 30 s followed by sporocarps since the type description. In that descrip- 35 PCR cycles (93 C, 35 s; 55 C, 53 s; 72 C, 30 + 05 s per cycle). tion the vesiculate hyphae were noted to collapse PCR products were viewed via electrophoresis in 2% agarose upon drying and as being difficult to find afterward gels made with SYBR-Safe stain (Invitrogen Canada Inc., (Smith and Zeller 1966). Kretzer et al. (2003) did not Burlington, Ontario). For all sporocarps and some tubercles conduct studies of this character in fresh material of restriction digests were performed on PCR products (ITS R. vesiculosus. region) with AluI to produce restriction fragment-length polymorphisms (RFLPs) that differentiated between R. The objectives of this study were to observe vesicles vesiculosus and R. vinicolor (Kretzer et al. 2003). Tubercles associated with either fruiting bodies or mycorrhizae, also were determined as R. vesiculosus or R. vinicolor with to determine their usefulness for distinguishing microsatellite DNA analysis with primers Rve2.10 and Rve2.14 between R. vesiculosus and R. vinicolor, and to evaluate (Kretzer et al. 2004) in a multiplex PCR reaction per Beiler other potentially useful diagnostic characters. et al. (2010). Microsatellite loci were analyzed and genotyped with a 3130XL genetic analyzer and Gene Mapper 4.0 software (both Applied Biosystems, Foster City, California). MATERIALS AND METHODS For each Rhizopogon species, representative sporocarp Field sites.—Sporocarps belonging to Rhizopogon species in samples producing a single PCR product were cleaned with section Vinicolores were collected from mixed conifer/ a Charge Switch PCR Clean-up Kit (Invitrogen), and cycle hardwood forests in the vicinity of Enderby, British sequencing was performed by the Center for Genome Columbia, Canada. The study sites were in the Interior Research and Biocomputing Core Laboratory at Oregon State Cedar-Hemlock (ICH) biogeoclimatic zone of southern University with a Big Dye kit (Applied Biosystems, Foster City, interior British Columbia (Lloyd et al. 1990). In this zone California) using forward and reverse (EM only) primers forests regenerate after wildfire or clear-cutting in a single