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Biology of the ectomycorrhizal genus Rhizopogon. VI. Re-examination of infrageneric relationships inferred from phylogenetic analyses of ITS sequences

     Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331-2902

James M. Trappe

     Department of Forest Science, Oregon State University, Corvallis, Oregon 97331

Randy Molina

     U. S. Department of Agriculture, Forest Service, Pacific Northwest Research Station, Forestry Science Laboratory, 3200 Jefferson Way, Corvallis, Oregon 97331

Joseph W. Spatafora

     Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331-2902

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 

Rhizopogon (Basidiomycota, Boletales) is a genus of hypogeous fungi that form ectomycorrhizal associations mostly with members of the Pinaceae. This genus comprises an estimated 100⁺ species, with the greatest diversity found in coniferous forests of the Pacific northwestern United States. Maximum parsimony analyses of 54 nuclear ribosomal DNA internal transcribed spacer (ITS) sequences including 27 Rhizopogon and 10 Suillus species were conducted to test sectional relationships in Rhizopogon and examine phylogenetic relationships with the closely related epigeous genus, Suillus. Sequences from 10 Rhizopogon type collections were included in these analyses. Rhizopogon and Suillus were both monophyletic. Rhizopogon section Rhizopogon is not monophyletic and comprised two clades, one of which consisted of two well supported lineages characterized by several long insertions. Rhizopogon sections Amylopogon and Villosuli formed well supported groups, but certain species concepts within these sections were unresolved. Four species from section Fulviglebae formed a strongly supported clade within section Villosuli. Subgeneric taxonomic revisions are presented.

Key words: Boletales, indels, phylogeny, Rhizopogonaceae, Suillus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
Rhizopogon Fries (Basidiomycota, Rhizopogonaceae) contains more than 100 species (Martín 1996). It is ectomycorrhizal mostly with Pinaceae and its worldwide distribution correlates with natural and exotic Pinaceae forests (Molina et al 1999). Despite this cosmopolitan range, most species are found in pine (Pinus L.) and Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] forests of the Pacific northwestern United States (Smith 1964, Smith and Zeller 1966). Rhizopogon is a common ectomycorrhizal fungus in these coniferous forests and thus an important component of the forest ecosystem.

The systematics of Rhizopogon remains in a state of flux that dates to the early 19th century, when notes on fresh characters were scanty and only gross morphological characters were used to describe species (Lange 1956, Smith 1971, Smith and Zeller 1966). Current understanding of Rhizopogon taxonomy is based primarily on a landmark publication by Smith and Zeller (1966) who increased the number of described North American species from 17 to 110, and included redescribed "European" species found in North America. Smith and Zeller (1966) divided the genus into two subgenera, Rhizopogonella and Rhizopogon. Species in subgenus Rhizopogonella were subsequently moved to Alpova (Trappe 1975). Subgenus Rhizopogon was divided into four sections, Amylopogon, Fulviglebae, Rhizopogon, and Villosuli, based on macroscopic and microscopic sporocarp characters and color changes on the peridium from chemical reactions and bruising of the sporocarp (Smith 1964, Smith and Zeller 1966).

Although this work is an important contribution to the systematics of Rhizopogon, several unresolved issues remain regarding placement of species in sections Fulviglebae and Rhizopogon. In Smith and Zeller (1966), R. section Fulviglebae comprises twenty-two species, of which six are identified as unusual species (e.g., R. hysterangioides, R. lowii, and R. pannosus), including the type for the section, R. exiguus. Ten species in this section share morphological and ecological affinities with section Villosuli, e.g., Rhizopogon vinicolor, R. clavitisporus, etc., but are tied to species in section Fulviglebae only by possessing truncated spores (Smith and Zeller 1966, Molina and Trappe 1994, J. Trappe unpubl).

Species placed in Rhizopogon section Rhizopogon lacked characters that defined the other three sections (Smith and Zeller 1966). Divisions within Rhizopogon section Rhizopogon were based on spore width and colors of the sporocarp when bruised (Smith and Zeller 1966, Table I). Section Rhizopogon contained an estimated 60 species at the writing of Smith and Zeller (1966) making it the largest section in the genus. Since section Rhizopogon was not based on common morphological or ecological features of these species, analysis of sequence data provides a way to test the validity of this taxonomic group. Smith and Zeller (1966) emphasized that this major taxonomic work was based on techniques available at the time and future revision was expected. A current review of the status of Rhizopogon taxonomy is found in Martín (1996).

As part of a continuing series of studies into the systematics of Rhizopogon and related fungi, maximum parsimony analyses were performed on nucleotide data from the nuclear ribosomal DNA internal transcribed spacer regions 1 and 2 and the 5.8S subunit. The major objectives of this study were to: i) categorize infrageneric sectional relationships in Rhizopogon, and ii) further qualify the phylogenetic relationship between Rhizopogon and Suillus.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
Fungal specimens – Species representing the sections Amylopogon, Fulviglebae, Rhizopogon, and Villosuli of the genus Rhizopogon were selected for phylogenetic analysis of nucleotide data (Table II). Forty collections used for DNA extraction were from the University of Michigan Herbarium (MICH) and the Mycological Collection of the Oregon State University Herbarium (OSC). Pieces of ten of these were donated from type collections by MICH. Specimens of Alpova trappei, Boletus edulis, and Chalciporus piperatus were also included (Table II). GenBank numbers are given in Table II for sequences from Chroogomphus vinicolor, Gomphidius glutinosus, Rhizopogon subcaerulescens, 10 Suillus spp., and Truncocolumella citrina.

Choice of outgroup – Complete ITS sequences were obtained from Alpova trappei, Boletus edulis, and Chalciporus (Boletus) piperatus for use as an outgroup. Using only Alpova trappei as the outgroup, ambiguous and difficult alignment regions were excluded from the A. trappei sequence and replaced with missing character states (-) while ingroup characters were retained (Nixon and Carpenter 1993). Once the polarity of the topology was determined, the most basal taxa were designated the outgroup and the Alpova trappei sequence was removed from the data set used for phylogenetic analyses.

Phylogenetic analysis – Using Chroogomphus and Gomphidius as an outgroup, an alignment of 892 nucleotide bases representing the ITS1, ITS2, and 5.8S subunit was analyzed. Alignment gaps were treated as follows: 1) ALL SET—all characters were included and gaps treated as missing data; 2) CULLED SET—multiple-base insertion/deletion events (indels) and areas of ambiguous alignment were excluded, remaining gaps treated as missing data; 3) I-GAP SET—a new character "I" was inserted to indels, ambiguous areas deleted, and remaining gaps treated as missing data; and 4) BINARY SET—indels were excluded and re-coded as presence/absence (0,1) in the data matrix at the end of the alignment, remaining single-base gaps treated as missing, and ambiguous areas of the alignment were excluded. The alignment is available in TreeBASE as S689. Maximum parsimony analyses were performed using PAUP* version 4.0 (Swofford 1999). Uninformative characters were excluded from all phylogenetic analyses. One hundred heuristic searches were conducted with random sequence addition and tree bisection-reconnection (TBR) branch-swapping algorithms, collapsing zero-length branches and saving all minimal length trees (MulTrees). To measure relative support for the resulting clades, 1000 bootstrap replications (Felsenstein 1985) were performed only on phylogenetically informative characters with the following parameters: 10 random sequence additions, TBR, and MulTrees off. Because the alignment revealed several indels that did not align across all species and may have resulted in loss of resolution within sections, unrooted branch and bound searches from section-specific alignments of sections Amylopogon, Rhizopogon, and Villosuli were performed. Bootstrap analyses were conducted as described above, with MulTrees option in effect.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
Choice of outgroup – We wanted to use an outgroup that was outside the suilloid group and obtained ITS sequences of Alpova trappei, Boletus edulis, and Chalciporus (Boletus) piperatus. However, sequences from these species were highly divergent and simply too difficult to align with the ingroup. Introduction of excessive and ambiguous alignment gaps was necessary and lead to problems in homology assessment. The 5.8S region was the only area that aligned with confidence across all species and since there were only 33 phylogenetically informative characters in the 5.8S region it was considered an unsuitable region to base the root of the tree. An unrooted tree is presented in Fig. 1. The placement of the root with Alpova trappei as the outgroup is indicated. When analyses were run with A. trappei as the outgroup, there was only bootstrap support for the sections within Rhizopogon and for Suillus as monophyletic. To test infrageneric relationships in Rhizopogon, further analyses were conducted using Chroogomphus and Gomphidius as the outgroup, which were basal in the Alpova-rooted tree.

View larger version (24K): [in this window] [in a new window]    FIG. 1. Unrooted tree based on the CULLED SET analyses to show outgroup rooting with Alpova trappei. Rhizopogon luteolus is the type species for the genus Rhizopogon. Placement of species in sections of genus Rhizopogon is according to Smith and Zeller (1966)

View larger version (48K): [in this window] [in a new window]    FIG. 2. One of 360 equally parsimonious trees of 585 steps based on ITS1, ITS2, and 5.8S subunit nrDNA sequences resulting from the CULLED SET of analyses. Bootstrap values are indicated at the respective internode. CI = 0.512, RI = 0.801. Placement of species in sections of genus Rhizopogon is according to Smith and Zeller (1966). Sections appear to be associated with either pines or Douglas-fir (Pseudotsuga menziesii), with the exception of subgenus Amylopogon that is associated with a broad range of hosts. Host information is based on collection and ecological data, and pure culture synthesis studies (Molina et al 1999). Proposed taxonomic revisions at the subgeneric level, and sectional level in subgenus Villosuli, are provided in bold print next to sectional classification of Smith and Zeller (1966)

View larger version (24K): [in this window] [in a new window]    FIG. 3. Unrooted section-specific trees resulting from branch and bound searches based on alignments of Rhizopogon sequences from A. section Amylopogon, B. sections Fulviglebae and Villosuli, and C. section Rhizopogon. Placement of species in sections of genus Rhizopogon is according to Smith and Zeller (1966). Bootstrap values are in bold noted above the respective internode. The number of changes is in smaller font below internodes

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

Examination of infrageneric relationships in Rhizopogon – Although many sectional relationships as defined by Smith (Smith 1964, Smith and Zeller 1966) are well supported, many lower taxonomic groupings, e.g., subsections, series, stirps, are polyphyletic. Section Amylopogon is strongly supported as monophyletic with a bootstrap value of 99. Section Rhizopogon is not monophyletic and forms three well supported clades with high bootstrap values of 100, 93, and 95 (clades A, B, C; Fig. 2). The type of the genus, R. luteolus, is present in the Rhizopogon section Rhizopogon clade A. Section Villosuli is well supported, but the species sampled from section Fulviglebae are found within section Villosuli, and form a strongly supported group with a bootstrap value of 99. Although Rhizopogon section Rhizopogon clades A and B appear to form a sister-group to species sampled from the other sections in Rhizopogon, which form another monophyletic group, bootstrap support for this placement is moderate at best. Rhizopogon sections Amylopogon, Rhizopogon clade C, and Villosuli are well supported as separate groups and distinct from the Rhizopogon section Rhizopogon clade A and B, but the relationships among these groups are not resolved.

Section Rhizopogon. Smith and Zeller (1966) divided Rhizopogon section Rhizopogon into two subsections, Angustispori and Rhizopogon, two series and 11 stirps. We sampled 15 sequences from 12 species representing both subsections. The subsections are separated by spore width and whether or not the peridium stains red and/or yellow at some stage of development (Table I). Our results show that spore width, the presence or absence of yellow in the peridium, or the pink-red staining reaction are not phylogenetically informative at the sectional (subgeneric, see taxonomic revision below) level, inasmuch as these characters occur in both Rhizopogon section Rhizopogon clades A and C. However, the absence of yellow does seem to be important for Rhizopogon clade B. Combined with other characters, the type of peridium appears to be a meaningful phylogenetic character, although this was not included in Smith's keys, e.g., a peridium of interwoven hyphal strands (Rhizopogon clade A) or interwoven hyphae (Rhizopogon clades B, C). Subsections Angustispori and Rhizopogon appear to be polyphyletic based on our results.

Rhizopogon section Rhizopogon clade A comprises R. fuscorubens, R. luteolus, R. occidentalis, R. ochraceorubens, and R. succosus. Rhizopogon succosus and R. luteolus share several morphological characters but are distinct species (Miller 1986, Hosford and Trappe 1988). Based on peridium coloration, microscopic characters, and the glass-hard consistency of the dried gleba, Miller (1986) suggested that a better placement of R. succosus is in stirps Luteolus. These observations are supported by the data presented here. The relationship between these two species is supported by a bootstrap of 100 in these analyses. In addition to being morphologically similar, they share similar long insertions in the ITS1 sequences and are both associated with Pinus spp.

The two holotypes from MICH that were sampled from Rhizopogon section Rhizopogon, R. ochraceorubens and R. evadens, are from subsection Angustispori, series Lutei and Versicolores respectively. Rhizopogon ochraceorubens and R. fuscorubens are closely related and placed in stirps Ochraceorubens. Smith indicates that the major difference between these two is that the rhizomorphs on the peridium of R. fuscorubens dry black and the peridium dries yellow, whereas the rhizomorphs on the peridium of R. ochraceorubens do not dry black and the peridium dries red. When rehydrated in KOH, the sectioned peridium is bright red for both species and very prominent in the holotype specimen. Rhizopogon occidentalis, originally placed in stirps Rubescens, appears to be closely related to both R. ochraceorubens and R. fuscorubens, although the sectioned peridium lacks the bright red reaction to KOH. All three species fruit in association with pines and generally form ectomycorrhizae only with pines in pure culture syntheses (Molina and Trappe 1982, 1994). Rhizopogon occidentalis will form mycorrhizae with Arctostaphylos and Arbutus spp. if pines are present as the primary host (Molina et al 1997).

Two species were sampled from series Versicolores, R. subsalmonius and R. evadens, and belong to stirps Subsalmonius and Evadens respectively. These species form Rhizopogon section Rhizopogon clade B (Fig. 2). Rhizopogon subsalmonius does not stain red when cut. Rhizopogon evadens stains red, but the peridium is white and lacks yellow coloration. The peridium does not stain bright red when sections are treated with KOH. Both have a peridum of interwoven hyphae and lack yellow coloration/staining. Rhizopogon subsalmonius is found with Abies spp. while R. evadens is associated with Pinus spp. These species form a clade distinct from Rhizopogon section Rhizopogon clade A.

Rhizopogon burlinghamii, R. roseolus, and R. vulgaris form Rhizopogon section Rhizopogon clade C. Smith and Zeller (1966) placed R. vulgaris in subsection Angustispori, stirps Vulgaris because it has narrow spores, stains red, and is yellow at some point during its development. Smith recognized the similarity of species in stirps Vulgaris with those in stirps Rubescens and mentions that stirps Vulgaris is a continuation of stirps Rubescens into the narrow spored species. Smith's descriptions of R. roseolus and R. vulgaris included in Smith and Zeller (1966) are based on examinations of North American collections. These two species were originally described from Europe in the nineteenth century (Smith and Zeller 1966). This study supports the close relationship of these species, sensu A. H. Smith. Rhizopogon roseolus (rubescens), R. vulgaris, and R. burlinghamii, form a distinct clade (B) separate from the other species sampled from section Rhizopogon (clades A and B). These species also lack several large indels present in species found in the other section Rhizopogon clades. These three species all associate with Pinus spp. These results and the morphological similarities of these species support their separation from Rhizopogon section Rhizopogon.

Section Amylopogon. Section Amylopogon is monophyletic and forms a well-supported clade with a bootstrap value of 99. The HOLOTYPE of R. ellenae and a PARATYPE of R. subpurpurascens were sampled. Martín (1996) moved R. ellenae to section Rhizopogon because dried specimens did not have amyloid spores. In our results, the holotype of R. ellenae is found in the strongly supported section Amylopogon clade. Amyloid spores seem to be an important character for taxonomic and phylogenetic studies in Rhizopogon. The fact that this character may not be detected in dried herbarium specimens may lead to misidentifications and should be considered in future studies of herbarium specimens of section Amylopogon. Smith and Zeller (1966) stated that although not all species in section Amylopogon have amyloid spores, all Rhizopogon species with amyloid spores are placed in this section. Section Amylopogon is supported by anatomy, the olive to green, blue, pink, or red reaction of the peridium to KOH, and, when present, amyloid spores. Species in section Amylopogon are the most broad-ranging in the genus in terms of mycorrhizal hosts, but they typically occur in conifer forests with pines and true firs (Abies Mill). Rhizopogon subcaerulescens forms ectomycorrhizae with Douglas-fir in laboratory conditions (Massicotte et al 1994).

Section Fulviglebae. The four species sampled from section Fulviglebae (R. diabolicus, R. ochraceisporus, R. parvulus, and R. vinicolor) were selected because they shared some peridial characters with section Villosuli and, as with the Villosuli, are associated with Douglas-fir. They form a well supported clade with a bootstrap value of 99, that is placed within section Villosuli. Although Rhizopogon parvulus and R. diabolicus are closely related species, both morphologically (Smith and Zeller 1966) and based on our data, their relationship to R. vinicolor and R. ochraceisporus is unclear and currently under investigation (A. Kretzer pers comm).

Species in stirps Vinicolor (e.g., R. diabolicus, R. parvulus, and R. vinicolor) and R. ochraceisporus (stirps Thaxteri) in section Fulviglebae are morphologically similar. Although Smith and Zeller (1966) mention that within stirps Vinicolor there is a trend towards brown-walled hyphae in the peridium, a characteristic of species in section Villosuli, descriptions of brown-walled hyphae are not included in species descriptions for stirps Vinicolor. The species in stirps Vinicolor and R. ochraceisporus also associate with Douglas-fir. Rhizopogon vinicolor and R. ochraceisporus may be ontogenetic stages of a single species, because except for glebal color, these two species are very similar morphologically.

Section Villosuli. Smith (1964) recognized twenty-one species of Rhizopogon in section Villosuli. These are separated from the other three sections by having brown-walled hyphae that form a distinct epicutis in the peridium and nontruncate, nonamyloid spores. Based on the findings presented here, R. colossus, R. villosulus, R. rogersii, R. hawkerae, and R. villescens could be a single species that shows variation, or several very closely related species. Martín et al (1998) synonomized R. colossus var. colossus, R. hawkerae, R. parksii, R. reticulatus, R. subareolatus, and R. villosulus to R. villosulus based on the lack of polymorphic bands in Restriction Fragment Length Polymorphism (RFLP) analyses of ITS rDNA. However, their findings do not entirely agree with those presented here. The two vouchers of Rhizopogon parksii always group as a pair and are distinct from the R. colossus, R. hawkerae, and R. villosulus in these analyses. In addition, some years after publication by Smith and Zeller (1966), Smith concluded from additional collecting that R. colossus was a developmental stage of R. villosulus (pers comm to J. M. Trappe), and we agree based on morphological and molecular evidence. In order to address this question of conspecificity, the two R. villosulus vouchers included in this study were re-examined macroscopically and microscopically. Rhizopogon villosulus AHS 59143 does not entirely match with the descriptive features. It lacks flagellate hyphae or any suggestion of pink blush, so it also does not fit R. hawkerae, and microscopically the best match is with R. viridis. Although we have some doubts about the identity of R. villosulus AHS 59143, we feel quite certain of the identification of R. villosulus JMT 19466. This exemplifies the need for additional critical studies of the species in this section.

Host specificity and evolution – Rhizopogon spp. show a great deal of host specificity with members of the Pinaceae (Smith 1964, Smith and Zeller 1966, Molina et al 1992). Smith and Zeller (1966) noted that the greatest species diversity occurs in the coniferous forests of the Pacific Northwest of the United States; however, Pseudotsuga forests in Asia and Mexico have not been extensively searched. In general, sections of Rhizopogon show a certain degree of specificity for particular genera of Pinaceae and some species show specificity with either Pinus spp. or Douglas-fir (Molina et al 1999). For several Rhizopogon species host specificity was supported by pure culture synthesis (Molina and Trappe 1982, 1994) and spore inoculation studies (Massicotte et al 1994, Molina et al 1997). These ecological data offer further support to Smith's sectional hypotheses (Smith 1964, Smith and Zeller 1966) (Fig. 4). Molina and Trappe (1994) and Molina et al (1999) suggest that because of its diversity and quantity of Pinaceae hosts, the Pacific Northwestern United States has been a major area for the evolution and speciation of Rhizopogon and their conifer hosts.

Evolutionary relationships at the generic level of the Pinaceae are not strongly supported in phylogenetic studies (Prager et al 1976, Price et al 1987, Chaw et al 1997, Stefanovic et al 1998). Hart's (1987) cladistic analysis of morphological characters includes the genera Larix Adans., Pseudotsuga, Pinus, Abies, Picea A. Dietr., and Tsuga Carr., but provides no measure of support for the resulting clades. In that study, Pinus appeared to be the ancestral host genus, while the pairs Pseudotsuga/Larix and Abies/Tsuga formed a sister group. Based on comparison to Suillus (Kretzer et al 1996), it appears that Rhizopogon clades A, B, and C have retained the plesimorphic association with Pinus. Conversely, the monophyly of the Rhizopogon associates of Pseudotsuga (R. section Villosuli and the isolates sampled from R. section Fulviglebae) suggests a single origin of the Pseudotsuga mycorrhizal association within Rhizopogon.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
We propose revision at the subgeneric levels within Rhizopogon. Detailed examination of species-level taxonomic relationships is beyond the scope of this study and is reserved for a future publication. Species sampled from this study are listed below in the proposed revisions. The disposition of species not included in this study must await reexamination of the types to insure accuracy of their placement.

Rhizopogon Fries in Symb. Gast. 1:5. 1817.

Type: Rhizopogon luteolus Fr.

Rhizopogon subgen. Rhizopogon

sensu A. H. Smith emend. Grubisha & Trappe

Peridium of interwoven hyphal strands, not producing a green to olive, blue or black reaction to KOH, the strands yellow to red, reddish brown or black, lacking brown-walled hyphae on the surface of peridium or rhizomorphs, the pigments in KOH mounts not blue. Gleba not reacting to Melzer's reagent in shades of gray to purple, blue or black. Spores neither truncate nor amyloid. Forming mycorrhizae with Pinus spp. Type species: Rhizopogon luteolus Fr.

Commentary. Subgenus Rhizopogon forms a cohesive group of species with peridia formed of interwoven, cable-like mycelial strands and rhizomorphs but with nonamyloid spores. Species from this study are: R. fuscorubens, R. luteolus, R. occidentalis, R. ochraceorubens, and R. succosus.

Rhizopogon subgen. Amylopogon

(A. H. Smith) Grubisha & Trappe, stat. nov. Basionym: Rhizopogon subgen. Rhizopogon sect. Amylopogon A. H. Smith, Mich. Botanist 3:17. 1964.

Peridium of usually white or, often at the surface, brown, interwoven hyphal strands with extracellular pigment deposits that in KOH mounts show pink to olive or blue pigments that form orange to red or brown pigment globules in Melzer's reagent; peridium mostly becoming dark brown to black when dried. Spores hyaline to weakly or strongly amyloid (gray, blue or purple) in Melzer's reagent mounts, if hyaline or weakly amyloid, then fresh gleba reacting to a drop of Melzer's reagent by turning gray to purple or black. Forming mycorrhizae with various genera of the Pinaceae. Type species: Rhizopogon subpurpurascens A. H. Smith

Commentary. Subgen. Amylopogon remains as originally described as a section by Smith (1964). Smith did not mention the striking peridial structure characteristic of the group: strongly interwoven, cable-like hyphal strands. Some species, e.g., R. rudus A. H. Smith, seem more closely related to subgen. Villosuli, and preliminary sequence data supports this relationship (M. Bidartondo pers comm). Species in this study included in R. subgenus Amylopogon are: R. ellenae, R. semireticulatus, R. subcaerulescens, R. subgelatinosus, and R. subpurpurascens.

Rhizopogon subgen. Roseoli

Grubisha & Trappe, subgen. nov.

Peridium hyphis intertextis, mox lutescens vel luteobrunescens, noxis rubescens, in KOH non viridescens, olivascens, cyanescens vel nigrescens, sine hyphis brunneis in paginis peridiorum vel rhizomorphorum. Gleba solutione Melzeri non canescens, purpurascens, cyanescens vel nigrescens. Sporae truncatae vel non truncatae, nonamyloideae.

Peridium of interwoven hyphae, becoming yellow to yellowish brown early in development, often staining pink to salmon or red where cut or bruised, not producing a green to olive, blue or black reaction to KOH, lacking brown-walled hyphae on the surface of peridium or rhizomorphs, lacking blue pigments in KOH mounts. Gleba not reacting to Melzer's reagent in shades of gray to purple, blue or black. Spores truncate or not, not amyloid. Type species here designated: Rhizopogon roseolus Corda.

Rhizopogon subgen. Roseoli sect. Roseoli

As in subgen. Roseoli except spores not truncate. Type species here designated: Rhizopogon roseolus Corda.

Species in this study in sect. Roseoli are: R. burlinghamii, R. roseolus, and R. vulgaris.

Rhizopogon subgen. Roseoli sect. Fulviglebi

A. H. Smith emend. Grubisha & Trappe

As in subgen. Roseoli except spores truncate. Type species; Rhizopogon exiguus Zeller.

Commentary. Subgenus Roseoli includes species placed by Smith and Zeller (1966) in stirps Rubescens and Vulgaris. Based on mophological, ecological, and sequence data, species in stirps Vinicolores (R. diabolicus, R. parvulus, R. vinicolor, etc.), R. ochraceosporus, R. clavitisporus and R. subclavitisporus placed by Smith in his section Fulviglebae are reassigned to Rhizopogon subgen. Villosuli sect. Vinicolores (this study, Smith and Zeller 1966, Molina and Trappe 1994). The remaining species from Smith's descriptions of section Fulviglebae appear to fit in our subgenus Roseoli, so we are transferring the rest of section Fulviglebi, including the type for section Fulviglebae R. exiguus, as we have emended it to subgenus Roseoli. As more data on these species accrue, further species reassignments will likely be appropriate.

Rhizopogon subgen. Versicolores

(A. H. Smith) Grubisha & Trappe stat. nov. Basionym: Rhizopogon subgen. Rhizopogon sect. Rhizopogon subsect. Angustispori ser. Versicolores A. H. Smith in Smith & Zeller, Mem. New York Bot Gard. 14 (2): 141. 1966.

Peridium lacking yellow colors in all stages of development, of interwoven hyphae rather than hyphal strands, in some species staining pink to red where bruised or cut. Type species: Rhizopogon evadens A. H. Smith.

Commentary. Subgenus Versicolores is phylogenetically close to subgenus Rhizopogon but differs strikingly from the latter in peridial structure. Morphologically it rather more closely resembles subgenus Roseoli, but lacks the yellow color of the latter. The molecular data indicate that this difference in peridial coloration is phylogenetically meaningful. Species in this study included in subgen. Versicolores are: R. evadens and R. subsalmonius.

Rhizopogon subgen. Villosuli

(A. H. Smith) Grubisha & Trappe, stat. nov. Basionym: Rhizopogon subgen. Rhizopogon sect. Villosuli A. H. Smith, Mich. Botanist 3: 17, 1964.

With brown-walled, often versiform or flagellate hyphae thinly to thickly covering the surface of the peridium or rhizomorphs; inner peridium and adjacent gleba often with black granules in H₂O mounts, these dissolving into a green to olive pigment in KOH mounts. Spores truncate or not. Forming mycorrhizae with Pseudotsuga spp. Type species: Rhizopogon villosulus Zeller.

Rhizopogon subgen. Villosuli sect. Villosuli

A. H. Smith

Peridial and rhizomorph surfaces covered thinly to thickly with brown-walled, often versiform or flagellate hyphae; spores not truncate. Type species: Rhizopogon villosulus Zeller

Species from this study included in subgen. Villosuli sect. Villosuli: R. colossus, R. hawkerae, R. parksii, R. villescens, R. villosulus, and R. zelleri.

Rhizopogon subgen. Villosuli sect. Vinicolores

Grubisha & Trappe, sect. nov.

[ Rhizopogon subgen. Rhizopogon sect. Fulviglebae subsect. Fulviglebae stirps Vinicolor A. H. Smith nom. nud., Mem. NY Bot. Gard. 14(2):50.]

A sectione Villosuli sporis truncatis vel subtruncatis et hyphis brunneis paucis vel nullis in pagina peridii differt.

Differing from Section Villosuli by the truncate to subtruncate spores and few or no brown hyphae on the spore surface (but such hyphae on surface of rhizomorphs). Type species: Rhizopogon vinicolor A. H. Smith

Commentary. The brown-walled hyphae on surfaces of peridia and/or rhizomorphs plus the evidently obligate mycorrhizal association with Pseudotsuga spp. distinguish subgen. Villosuli from the other subgenera of the genus, and the nrDNA ITS sequence data confirm its cohesiveness, once the related species from Smith's original sect. Fulviglebae are included. Species from this study that are transferred from Rhizopogon section Fulviglebae Smith to subgen. Villosuli sect. Vinicolores are: R. vinicolor, R. diabolicus, R. ochraceisporus, and R. parvulus. The remaining species from section Fulviglebae stirps Vinicolores (R. inquinatus, R. olivaceofuscus, R. subcinnamomeus, and R. vesiculosus) and stirps Clavitisporus (R. clavitisporus and R. subclavitisporus) (Smith and Zeller 1966), are also transferred to subgen. Villosuli sect. Vinicolores based on the morphological and ecological evidence mentioned above.

	ACKNOWLEDGMENTS

 
This study was funded by the USDA Forest Service Pacific Northwest Research Station (PNW 96-5021-1-CA). Additional support came from the Mycological Society of America's Alexander H. and Helen V. Smith Research Fund and the Hardman Award for Native Plant Research. We are grateful to Robert Fogel and the University of Michigan Herbarium for donations of pieces from type collections of Rhizopogon for DNA extractions and sequencing. Francisco Camacho donated Boletus and Chalciporus specimens and Eric Danell donated the R. luteolus specimen. This paper was derived in part from a thesis submitted by LCG in partial fulfillment of a Master of Science degree, Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon.

	FOOTNOTES

 
¹ Corresponding author, current address: Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720-3102; grubishl{at}nature.berkeley.edu

Accepted for publication December 25, 2001.

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