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 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 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- (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 ITS1F and ITS4. Forward and reverse sequences were aligned cohort, with Douglas-fir, paper birch (Betula papyrifera in Sequencher 4.2 (Gene Codes Corp., Ann Arbor, Michigan). Marsh.) and lodgepole (Pinus contorta Douglas ex The resulting consensus sequences then were corrected and Louden var. latifolia Engelm. ex S. Watson.), forming the trimmed manually. Each sequence was subjected BLAST canopy during the first 100 y. queries through the NCBI-linked database (Altschul et al. Tuberculate ectomycorrhizae (EM ‘‘tubercles’’) were 1997). A functional species-level match criterion was set at % collected from the dry interior Douglas-fir biogeoclimatic 98 sequence similarity over at least 500 base pairs. Our new sequences were submitted to GenBank (TABLE zone near Kamloops, British Columbia (Lloyd et al. 1990). I). These old-growth forests regenerate into multi-cohort stands after natural disturbances and have a predominantly RESULTS Douglas-fir overstory and pinegrass (Calamagrostis rubescens Buckley) understory. The sporocarp and sam- Sporocarps.—Vesicles (FIG. 1d) were found on 75% pling areas were about 85 km apart. (12/16) of the sporocarps examined that had been 1076 MYCOLOGIA

FIG. 1. Morphological aspects of Rhizopogon vinicolor and R. vesiculosus sporocarps. a. Trama and peridium (fresh) of R. vinicolor sporocarp halves (coll. FS208); R 5 location of rhizomorph attachment. b. Detail of R. vesiculosus rhizomorph attachment on dried sporocarp (coll. FS62); R 5 rhizomorph, E 5 adjacent epicutis of peridium. This small area of well developed epicutis contains dispersive hyphae from the rhizomorph and vesiculate hyphae. c. Detail of R. vinicolor rhizomorph attachment on a fresh sporocarp (coll. FS181); note rhizomorph hyphae dispersing into the peridium without buildup of an epicutis. d. Vesiculate hyphal cells in the epicutis of R. vesiculosus (coll. FS95). e. Hyphal coils (HC) in the dispersive brown-walled hyphae associated with the rhizomorph attachment of R. vinicolor (coll. FS181). f. Non-coiled dispersive brown-walled hyphae associated with the rhizomorph attachment of R. vesiculosus (coll. FS161). LUOMA ET AL.: REDISCOVERY OF VESICLES 1077

TABLE I. Reference sporocarps sequenced for this study (ITS, 5.8S) and GenBank accession numbers

Species Collection IDa GenBank accession number

Rhizopogon vesiculosus FS-151, OSC-129170 HQ385849 Rhizopogon vesiculosus FS-164, OSC-129171 HQ385850 Rhizopogon vesiculosus FS-225, OSC-129175 HQ385854 Rhizopogon vinicolor FS-91, OSC-129176 HQ385847 Rhizopogon vinicolor FS-95, OSC-129177 HQ385848 Rhizopogon vinicolor FS-208, OSC-129172 HQ385851 Rhizopogon vinicolor FS-210, OSC-129173 HQ385852 Rhizopogon vinicolor FS-214, OSC-129174 HQ385853

a Study collection number followed by OSC Herbarium accession number.

molecularly determined to be R. vesiculosus. Several DISCUSSION immature sporocarps of R. vesiculosus had not ‘‘The shortest road to the evaluation of a new set of developed epicuticular hyphae in the vicinity of the characters is to set up a classification based on them rhizomorph attachment and did not exhibit vesicles. and then let your colleagues try to find fault with it,’’ However, vesicles were not exclusively associated with said Smith in Smith and Zeller (1966, p 2). In this sporocarps that had been determined to be R. research we determined that Smith’s use of the vesiculosus. Vesicles were found in 19% (4/21) of presence of vesicles as a defining character in the sporocarps that had been identified as R. vinicolor species concept of R. vesiculosus was generally with molecular methods. Three of the four R. warranted and yet contained an element of serendip- vinicolor sporocarps that exhibited vesicles were ity in that the material chosen for the type was not the sequenced to confirm the RFLP results. Vesicles did occasional R. vinicolor collection that exhibits vesicles. not collapse upon drying and were observed readily Conversely, Kretzer et al. (2003) determined that near the rhizomorph attachment when present. some of the paratype material of R. vinicolor was We observed coiled hyphae (FIG. 1e) dispersing actually R. vesiculosus. Those original identifications from the rhizomorph on the surface of R. vinicolor (Smith and Zeller 1966) were likely the result of sporocarps (FIG. 1c). However, on R. vesiculosus incomplete knowledge about the restricted location sporocarps the brown-walled dispersive hyphae were of vesicle development. Instead of being scattered not coiled (FIG. 1f) but were similar to the flagellate through the epicutis and collapsing on drying, we hyphae depicted in Smith and Zeller (1966, FIG. 88). have shown that the vesicles are highly localized in the The peridium immediately adjacent to the rhizo- epitcutis among the dispersive hyphae of the rhizo- morph attachment was the only location where dark- morph (FIG. 1b) and do not collapse upon drying. walled vesicles, flagellate hyphae or coiled hyphae In the absence of vesicles, Kretzer et al. (2003) were found on these two species. found that the most reliable morphological charac- Tuberculate mycorrhizae.—Vesiculate hyphae were not ters for distinguishing sporocarps of R. vesiculosus found associated with the dried tuberculate EM of and R. vinicolor were aspects of peridium and gleba either species. However, a consistent morphological color. Only R. vinicolor was noted to pass through a difference was found among the macroscopically developmental stage that may exhibit bright yellow similar tubercles (FIG. 2a, b). Coiled hyphae (FIG. 2c), patches on the peridium. In addition, the mature similar to those associated with the dispersive hyphae gleba of R. vinicolor was noted to be dark greenish on sporocarps, were found on dried R. vinicolor brown, brown or reddish brown as opposed to tuberculate EM, particularly at the interface of the ‘‘blackish brown’’ for R. vesiculosus (Kretzer et al. ‘‘peridium’’ and the mycorrhiza mantle of the outer 2003). Nevertheless, use of these characters for roots of the tubercle. However, coiled hyphae were diagnosis depends on the investigator obtaining not found with dried EM of R. vesiculosus (FIG. 2d) in specific developmental stages in a single collection. that location. In a blind test of 10 dried tubercles Kretzer et al. (2003) also described subtle differ- identified with molecular methods, one of us (DL) ences in the structure and morphology of the correctly classified all the test tubercles based on the tubercles of the two Rhizopogon species. They noted presence or absence of the dark-walled, coiled hyphae that the peridium-like rind that encases the tubercu- at the interface of the ‘‘peridium’’ and the mycorrhi- late EM of R. vinicolor is often loosely tomentose but za mantle (the inner layer of ‘‘P’’, FIG. 2a, b). molded to the enclosed roots such that it is not 1078 MYCOLOGIA

FIG. 2. Morphological aspects of Rhizopogon vinicolor and R. vesiculosus ectomycorrhizae. a. Tuberculate mycorrhiza of Rhizopogon vinicolor with peridium peeled back to expose the tightly clustered mycorrhizal root tips; P 5 peridium, M 5 mycorrhiza. b. Cross section through the tightly clustered root tips of a tuberculate mycorrhiza of R. cf. vinicolor;P5 peridium, M 5 mycorrhiza. c. Hyphal coils (HC) in the inner peridium of a R. vinicolor tuberculate mycorrhiza (coll. M457N1). d. Noncoiled hyphae in the inner peridium of a R. vesiculosus tuberculate mycorrhiza (coll. M456S1). readily peeled back. In contrast, the ‘‘peridium’’ of R. hyphae that are characteristic of subgenus Villosuli. vesiculosus tubercles was noted to be appressed often Therefore the possibility of their presence should not but readily peeled back in patches. Subsequent tests be ruled out. of this character set have produced variable results. Our results show that the presence of vesicles is a Rhizopogon vesiculosus mostly was readily peeled, but good first approximation for identification of R. R. vinicolor was inconsistently tightly bound (K. Beiler vesiculosus sporocarps. The presence of dark brown- pers comm). walled, coiled, dispersive hyphae provides researchers Our finding of coiled epicuticular hyphae in R. an additional character of hyphal morphology to vinicolor versus flagellate hyphae in R. vesiculosus assign sporocarps and tuberculate mycorrhizae to R. would seem to parallel the tomentose vs. appressed vinicolor instead of to R. vesiculosus.Molecular tubercle epicutis morphology noted by Kretzer et al. methods such as RFLP or microsatellite DNA analysis (2003). We speculate that presence of coiled hyphae provide definitive separation of R. vesiculosus and R. (observable throughout the felt-like peridium of vinicolor but may not be necessary or practical in tubercles) contributes to that tomentose appearance certain contexts. and tendency for tighter binding to the roots within R. vinicolor tubercles. Although we did not detect vesiculate hyphae in ACKNOWLEDGMENTS tuberculate EM, Trappe (1965, FIG. 5) appears to Kevin Beiler provided ectomycorrhizae, microsatellite re- illustrate vesicles formed by the dark brown-walled sults and valuable comments on the manuscript. Brendon LUOMA ET AL.: REDISCOVERY OF VESICLES 1079

Twieg provided much appreciated logistical support with tuberculate on Douglas-fir. New Phytol the field work. Jen Walker, Jenna Benson, Matthew Weiss, 161:313–320, doi:10.1046/j.1469-8137.2003.00915.x Walter Garrison, Adam Collins, Natasha Lukey, and Cathy, ———, ———, ———, ———. 2005. Patterns of vegetative Quentin, and Cody Wyne all assisted with sporocarp growth and gene flow in Rhizopogon vinicolor and R. collection. Dan Salloum contributed in the field and vesiculosus (, ). Mol Ecol 14: laboratory. We thank Dr Jane Smith and the USDA Forest 2259–2268, doi:10.1111/j.1365-294X.2005.02547.x Service, PNW Research Station, for helping with office and ———, Luoma DL, Molina R, Spatafora JW. 2003. laboratory space. Doni McKay provided valuable advice with of the Rhizopogon vinicolor species complex aspects of the lab work. Two anonymous reviewers made based on analysis of ITS sequences and microsatellite greatly appreciated comments on the manuscript. We loci. Mycologia 95:480–487, doi:10.2307/3761890 acknowledge the financial support of NSERC, Canada, Lloyd DA, Angove D, Hope G, Thompson C. 1990. A guide and FSP of British Columbia for this study. We also are to site identification and interpretation for the Kam- grateful for the infrastructure support from Canadian loops forest region. Victoria, British Columbia: British Foundation for Innovation granted to the Institute for Columbia Ministry of Forests. 399 p. Species at Risk and Habitat Studies at UBC Okanagan. Luoma DL, Stockdale CA, Molina R, Eberhart JL. 2006. The spatial influence of Douglas-fir retention trees on ectomycorrhiza diversity. Can J For Res 36:2561–2573, LITERATURE CITED doi:10.1139/x06-143 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, ———, Trappe JM, Claridge AW, Jacobs KM, Cazares E. Miller W, Lipman DJ. 1997. Gapped BLAST and PSI- 2003. Chapter 10. Relationships among fungi and small BLAST: a new generation of protein database search mammals in forested ecosystems. In: Zable CJ, Anthony programs. Nucleic Acids Res 25:3389–3402, doi:10.1093/ RG, eds. Mammal community dynamics: Management nar/25.17.3389 and conservation in the coniferous forests of western Beiler KJ, Durall DM, Simard SW, Maxwell SA, Kretzer AM. North America. Cambridge, UK: Cambridge Univ 2010. Architecture of the wood-wide web: Rhizopogon Press. p 343–373. spp. genets link multiple Douglas-fir cohorts. New Molina R, Trappe JM, Grubisha LC, Spatafora JW. 1999. Phytol 185:543–553, doi:10.1111/j.1469-8137.2009. Rhizopogon.In:CairneyWG,ChambersSM,eds. 03069.x Ectomycorrhizal fungi: key genera in profile. Berlin: Brownlee C, Duddridge JA, Malibari A, Read DJ. 1983. The Springer-Verlag. p 129–160. structure and function of mycelial systems of ectomy- Parke JL, Linderman RG, Black CH. 1983. The role of corrhizal roots with special reference to their role in ectomycorrhizas in drought tolerance of Douglas-fir forming inter-plant connections and providing path- seedlings. New Phytol 95:83–95, doi:10.1111/j.1469-8137. ways for assimilate and water transport. Plant Soil 71: 1983.tb03471.x 433–443, doi:10.1007/BF02182684 Smith AH. 1964. Rhizopogon, a curious genus of false Egerton-Warburton LM, Querejeta JI, Allen MF. 2007. truffles. Mich Botanist 3:13–19. Common mycorrhizal networks provide a potential ———, Smith HV, Weber NS. 1981. How to know the non- pathway for the transfer of hydraulically lifted water gilled mushrooms. 2nd ed. Dubuque, Iowa: Wm. C. between plants. J Exp Bot 58:1473–1483, doi:10.1093/ Brown Co. jxb/erm009 ———, Zeller SM. 1966. A preliminary account of the North Jacobs KM, Luoma DL. 2008. Small mammal mycophagy American species of Rhizopogon. Mem New York Bot response to variations in green-tree retention. J Wildlife Garden 14:1–178. Manage 72:1747–1755, doi:10.2193/2007-341 Trappe JM. 1965. Tuberculate mycorrhizae of Douglas-fir. Jones MD, Durall DM, Harniman SMK, Classen DC, Simard For Sci 11:27–32. SW. 1997. Ectomycorrhizal diversity on Betula papyr- Twieg B, Durall DM, Simard SW. 2007. Ectomycorrhizal ifera and Pseudotsuga menziesii seedlings grown in the fungal succession in mixed temperate forests. New greenhouse or out-planted in single-species and mixed Phytol 176:437–447, doi:10.1111/j.1469-8137.2007. plots in southern British Columbia. Can J For Res 27: 02173.x 1872–1889, doi:10.1139/x97-160 Zak B. 1971. Characterization and classification of mycor- Kretzer AM, Dunham S, Molina R, Spatafora JW. 2004. rhizae of II. Pseudotsuga menziesii + Microsatellite markers reveal the belowground distri- Rhizopogon vinicolor.CanJBot49:1079–1084, bution of genets in two species of Rhizopogon forming doi:10.1139/b71-154