Mycologia, 95(3), 2003, pp. 480±487. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897 Taxonomy of the Rhizopogon vinicolor species complex based on analysis of ITS sequences and microsatellite loci Annette M. Kretzer1 mushroom genera Suillus, Gomphidius and Chroogom- S.U.N.Y. College of Environmental Science and phus and is thought to be derived from an epigeous Forestry, Faculty of Environmental and Forest Biology, (possibly Suillus-like) ancestor through loss of com- 350 Illick Hall, Syracuse, New York 13210-2788 plex morphological characters, such as a stipe, verti- Daniel L. Luoma cally oriented tubes and forcible spore discharge Department of Forest Science, Oregon State University, (Bruns et al 1989). Because of its reduced morphol- 154 Peavy Hall, Corvallis, Oregon 97331-7501 ogy, Rhizopogon is a taxonomically dif®cult genus and Randy Molina the current taxonomy has largely been shaped by the U.S.D.A. Forest Service, Paci®c Northwest Research in¯uential work of Smith and Zeller (1966). In ad- Station, 2300 SW Jefferson Way, Corvallis, Oregon dition to describing numerous new species, Smith 97331 and Zeller (1966) also established a detailed subge- neric classi®cation with two subgenera (Rhizopogonel- Joseph W. Spatafora la and Rhizopogon) that comprise two sections (Rhi- Department of Botany and Plant Pathology, Oregon State University, 2082 Cordley Hall, Corvallis, Oregon zopogonella, Fibulatae) and four sections (Rhizopogon, 97331-2902 Amylopogon, Fulviglebae, Villosuli), respectively. Spe- cies in the subgenus Rhizopogonella later have been transferred to the genus Alpova (Trappe 1975), and Abstract: We are re-addressing species concepts in what we recognize as genus Rhizopogon today is es- the Rhizopogon vinicolor species complex (Boletales, sentially Smith and Zeller's subgenus Rhizopogon plus Basidiomycota) using sequence data from the inter- Alpova olivaceotinctus (from section Rhizopogonella) nal-transcribed spacer (ITS) region of the nuclear ri- which has been transferred back to the genus Rhi- bosomal repeat, as well as genotypic data from ®ve zopogon by Grubisha et al (2001); it remains to be microsatellite loci. The R. vinicolor species complex determined in future studies, however, if other spe- by our de®nition includes, but is not limited to, col- cies from Alpova section Rhizopogonella will have to lections referred to as R. vinicolor Smith, R. diabolicus be transferred back to genus Rhizopogon as well. Smith, R. ochraceisporus Smith, R. parvulus Smith or Grubisha et al (2002) recently conducted an ITS R. vesiculosus Smith. Holo- and/or paratype material sequence-based phylogenetic study of the genus Rhi- for the named species is included. Analyses of both zopogon. They found that Smith's and Zeller's (1966) ITS sequences and microsatellite loci separate collec- section Amylopogon was monophyletic, section Rhizo- tions of the R. vinicolor species complex into two dis- pogon was polyphyletic and section Fulviglebae likely tinct clades or clusters, suggestive of two biological would have been polyphyletic, if more species had species that subsequently are referred to as R. vini- been included; those species from section Fulviglebae color sensu Kretzer et al and R. vesiculosus sensu that were included in the study formed a monophy- Kretzer et al. Choice of the latter names, as well as letic clade nested within a paraphyletic section Villo- morphological characters, are discussed. suli. Based on these results, Grubisha et al (2002) Key words: fungal species concepts, internal have proposed a number of changes to the subge- transcribed spacers, microsatellite markers, Rhizopo- neric classi®cation of Rhizopogon that include reinsti- gon tution of an emended subgenus Rhizopogon, eleva- tion of sections Amylopogon and Villosuli to subgenus level and creation of two new subgenera, Roseoli and INTRODUCTION Versicolores (for more details see Grubisha et al 2002). Rhizopogon is an ectomycorrhizal genus in the Bole- Those species of section Fulviglebae that group with tales (Basidiomycota) that forms hypogeous, truf¯e- former section Villosuli were transferred to the new like fruit bodies. It is closely related to the epigeous subgenus Villosuli. Subgenus Villosuli is a strongly supported, monophyletic clade that will be the focus Accepted for publication February 5, 2003. of the study presented below. Members of the sub- 1 Corresponding author. E-mail: [email protected] genus Villosuli share a high level of host speci®city 480 KRETZER ET AL: RHIZOPOGON VINICOLOR SPECIES COMPLEX 481 and are known to associate only with Pseudotsuga spp. They lend themselves to population genetic work be- (Massicotte et al 1994, Molina et al 1997). Another cause they are large (up to several cm across) and detailed phylogenetic study of North American col- are encountered more frequently than fruit bodies, lections of subgenus Amylopogon recently has been making them relatively easy to sample in the ®eld. published by Bidartondo and Bruns (2002). Spurred by our interest in population genetics of In addition to subgeneric classi®cation schemes, this group, we are re-addressing taxonomic issues and there has been controversy about species delinea- species delineations within the R. vinicolor species tions in Rhizopogon. Most species currently recog- complex using both ITS sequences and microsatellite nized in North America date back to Zeller and markers. Characterization of six polymorphic loci Dodge (1918), Zeller (1941) and Smith and Zeller with trinucleotide repeat motifs from R. vinicolor was (1966), with subsequent additions by Smith (1966, reported earlier (Kretzer et al 2000). Included in this 1968), Harrison and Smith (1968), Trappe and Guz- study are several Rhizopogon spp. type collections maÂn (1971), Hosford (1975), CaÂzares et al (1992) from which DNA has not been extracted and ana- and Allen et al (1999). However, many species mor- lyzed before. phologically are very similar and described differenc- es might represent morphological and/or ontoge- netic variation within biological species. A number of MATERIALS AND METHODS synonymies therefore have been proposed (MartõÂn et Type collections were obtained from The University of al 1998). Grubisha et al (2002) also reported a num- Michigan Fungus Collection. When collections consisted of ber of irregularities with species delineations and several sporocarps, one well-preserved sporocarp was cho- identi®cations. Of particular interest in the context sen for DNA extraction and ampli®cation. Tuberculate EM of this study was the fact that several collections of R. analyzed in this study were collected in the spring of 1998 vinicolor were not monophyletic but appeared to in two plots, one located at Mary's Peak (448 31.869 N and form a paraphyletic grade. Collections of R. diaboli- 1238 32.869 W) in the Oregon Coast Range and the other cus, R. ochraceisporus and R. parvulus were derived at Mill Creek (448 12.179 N and 1228 13.959 W) in the from within the paraphyletic R. vinicolor grade. We Oregon Cascade Range. Plots were within 40±80- and 40± 50-year-old second-growth forests, respectively, dominated collectively will refer to these taxa as the R. vinicolor by Douglas ®r (Pseudotsuga menziesii), western hemlock species complex. R. vinicolor and R. ochraceisporus (Tsuga heterophylla) and western red cedar (Thuja plicata). can be differentiated primarily by subtle color differ- The plots were 10 m 3 10 m, and an 11 3 11 point grid ences in the reaction of the peridium to KOH as well was established with 1 m point intervals. Soil cores (5 cm as by an olive-brown versus rusty-brown (``russet'') wide and approx. 30 cm deep) were taken at each grid gleba at maturity, according to Smith and Zeller point (121 cores per plot), and soils were sifted through a (1966). In R. diabolicus, the mature gleba is ``russet,'' W. S. Tyler Company 2 mm sieve. In total, 30 independent as in the case of R. ochraceisporus, but maintains a collections of tuberculate EM were obtained, 15 from bright rusty-cinnamon color after drying. R. parvulus Mary's Peak and 15 from Mill Creek. Tuberculate EM col- is distinguished by some irregularly shaped spores. lections were freeze-dried before molecular analysis and will Finally, we also include R. vesiculosus in the R. vini- be identi®ed as MP1-98 (Mary's Peak) and MC1-98 (Mill Creek) samples, respectively. Finally, cultures of R. vinicolor color species complex because Smith calls it ``scarcely T20787 and T20874 were kindly provided by Dr. Ari Jump- distinguishable'' from R. vinicolor in the dried stage; ponen (now at Kansas State University). Voucher collections when fresh, R. vesiculosus is distinguished by yellow- of sporocarps from which these cultures were derived are brown in¯ated cells in the epicutis (Smith and Zeller housed in the Fungus Collection of the Oregon State Uni- 1966). versity Herbarium (OSC#61147 and OSC#61148). Despite known dif®culties with species delineations Genomic DNA was extracted from dried material, as de- in the R. vinicolor species complex, we have chosen scribed in Kretzer et al (2000). The ITS region (comprising to work with R. vinicolor as a model taxon to study ITS-I, the 5.8S rRNA gene, and ITS-II) was PCR-ampli®ed the population structure of hypogeous basidiomy- with primers ITS1f and ITS4. When ampli®cation of the cetes (false-truf¯es). Rhizopogon vinicolor is a pre- entire region was unsuccessful, only ITS-I was ampli®ed with dominantly spring-fruiting species (Luoma et al primers ITS1f and ITS2. For primer sequences, see White et al (1990) and Gardes and Bruns (1993). PCR reactions 1991) that is very abundant in the Paci®c Northwest. contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 C), Most important, it forms morphologically distinct ec- 2.5 mM MgCl2, 0.1% Tritont X-100, 0.2 mM of each dNTP, tomycorrhizae (EM) on Douglas ®r (Pseudotsuga 0.5 mM of each primer, 50 U/mL Taq DNA polymerase, and menziesii) known as tuberculate EM (Zak 1971, Mas- empirical amounts of template DNA.