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Two new species of Gomphidius from the Western United States and Eastern Siberia

     Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA 24061

Mary Catherine Aime

     Department of Plant Sciences, South Parks Rd., University of Oxford, OX1 3RB UK

Francisco J. Camacho

     Department of Plant and Microbial Biology, University of California, Berkeley, California, USA 94720

Ursula Peintner

     Institute of Microbiology, University of Innsbruck, Technikerstr 25, A 6020 Innsbruck, Austria

	ABSTRACT

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Study of the genus Gomphidius from recent material from Asia and North America has been carried out using traditional taxonomy combined with molecular systematics. Two new species of Gomphidius (G. borealis and G. pseudoflavipes) are described, one from Eastern Siberia and a second from rarely collected habitats in the Western United States. One taxon has the longest spores reported for the genus and the second species appears to be associated with a Siberian larch.

Key words: Basidiomycetes, Gomphidiaceae, ITS sequence data, North America, Russia

	INTRODUCTION

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The Gomphidiaceae has been treated recently by Miller and Aime (2001), previously by Miller (1963, 1964, 1966, 1971), and Miller and Trappe (1970). Two ballistosporic genera have been described, Gomphidius Fr. and Chroogomphus (Sing.) O.K. Mill. (Miller 1964) and two statismosporic genera, Brauniellula and Gomphogaster (Smith and Singer 1958, Miller 1973). More recently, new collections of Gomphidius and Chroogomphus have been made in Eastern Siberia, Nepal, the Republic of Korea, Japan, the Dominican Republic and in rarely collected habitats in the Western United States, warranting a re-examination of the family. Intensive morphological and taxonomic study of these collections has taken place. In addition, sequences of the internal transcribed spacer region of the rDNA (ITS) have been analyzed for as many species as possible. Based on these data, revision of the genus Chroogomphus was reported in Miller and Aime (2001). In this paper we report two new taxa in the genus Gomphidius and present a revised phylogeny based on ITS data for the genus.

	MATERIALS AND METHODS

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Color comparisons were made using Kornerup and Wanscher (1967) and designated Met 6E-4 which indicates the plate, row, and color block. Chemical reagents used include Melzer's solution for determining amyloid reactions of the tramal tissues and 3% KOH as a mounting medium. Drawings of the illustrated taxa were made from mounts in 3% KOH and Melzer's solution using a camera lucida mounted on a Leitz Ortholux microscope. Smell and taste were recorded when possible from fresh material. The E value is the length divided by the width of the basidiospore and the Em value is the mean E of N = 31 spores.

DNA was isolated from dried herbarium specimens using a 2x CTAB lysis buffer with 0.2% ß-mercaptoethanol added and precipitation with cold isopropanol, and otherwise following the methods of Bruns et al (1990). For many samples, the resulting DNA was further cleaned using the Geneclean II kit and protocol (Bio 101). Primer pairs ITS1 and ITS4 (White et al 1990), or ITS1-F and ITS4-B (Gardes and Bruns 1993) were used to both amplify and sequence the entire ITS region including the 5.8S gene. In some instances primer pairs ITS1-F and 5.8SR, and ITS4-B and 5.8S (see Vilgalys and Hester 1990 for primer sequences of 5.8SR and 5.8S) were used to separately amplify the ITS-1 and ITS-2 regions. Reaction conditions for amplification follow Vilgalys and Hester (1990). The PCR products were purified with Ultra-free-MC filters (Millipore) and sequenced using fluorescent dye terminator chemistries (Perkin Elmer) on an automated sequencer (ABI 373 or ABI 377).

Contiguous sequences were assembled in SEQUENCHER 3.0 (Gene Codes Corp.), and manually aligned in Paup* ver. 4.0b8 (Swofford 2001). The data matrix included a total of 875 characters, 327 of which were excluded due to ambiguous alignment; of the remaining characters, 75 were parsimony-informative. Sequences were obtained for 17 collections of Gomphidiaceae, including all species of Gomphidius except for G. oregonensis Pk., G. largus O.K. Mill., and G. pseudomaculatus O.K. Mill., for which we were unable to obtain recent collections. Chroogomphus rutilus, C. sibericus, C. ochraceus, and C. asiaticus were selected as outgroup taxa for rooting purposes as the genus Chroogomphus has been shown to be the sister taxon to Gomphidius (Miller and Aime 2001). Taxa analyzed are given in Table I. Sequences are deposited in GenBank; voucher collections are deposited at VPI. All analyses were conducted in Paup* ver. 4.0b8. Parsimony analyses were performed using heuristic search algorithms with multiple (10) random sequence additions to generate starting trees, and tree-bisection-reconnection (TBR) branch-swapping algorithm. These yielded three most parsimonious trees of length 216, with a consistency index of 0.6174 and retention index of 0.7503. Bootstrapping frequencies were calculated using TBR branch swapping with 1000 replicates of ten random addition sequences.

	TAXONOMY

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Gomphidius pseudoflavipes O.K. Mill. et Camacho, sp. nov. Figs. 1–4, 9

View larger version (21K): [in this window] [in a new window]    FIGS. 1–4. Gomphidius pseudoflavipes. Fig. 1. Basidiomes. Fig. 2. Basidiospores. Fig. 3. Basidium. Fig. 4. Pleurocystidium. Upper bar = 1 cm. Lower bar = 10 µm

View larger version (137K): [in this window] [in a new window]   FIGS. 9–10. Fig. 9. Gomphidius pseudoflavipes. Fig. 10. Gomphidius borealis. Lower & upper bars = 1 cm.

Pileus to 4 cm broad, convex, dry, radially arranged dark brown fibrils with orange brown ground color (MET 7B4-5, MET 8B4-5). Lamellae short decurrent, subdistant, broad, gray-brown with one tier of lamellulae. Stipe 5–8 cm long, up to 2 cm wide, equal or tapering at the base, dry, surface with scattered brown fibrils, dull yellowish base. Partial veil densely fibrous in very young cap leaving only scattered fibrils in a superior, thin annular zone. Context firm, white, lower one-fourth of stipe bright yellow (MET 4A5).

Pileipellis of erect to mostly decumbent hyphae (3–) 4–10 µm diam, clearly distinct from the pileitrama, dark brown in 3% KOH to brown or reddish brown in Melzer's solution. Pileitrama of interwoven, filamentous, thin-walled hyphae 4.5–11 (–17) µm diam, hyaline throughout in 3% KOH, hyaline with scattered weakly amyloid cells in Melzer's solution. Lamellar trama of thin-walled, filamentous to occasionally ventricose, interwoven hyphae 4.5–12 µm diam, hyaline in 3% KOH, light orange-brown with shiny orange oleiferous hyphae in the center in Melzer's solution. Mycelium adhering to stipe base of filamentous, thin-walled hyphae 2.2–5.4 µm diam, simple septate hyaline or with yellow-brown contents, orange to orange-red lipid drops and incrustations adhering to some cells. Pleuro- and Cheilocystidia 116–123 x 13–19 µm, narrowly clavate to subfusiform, thin-walled, hyaline or with yellow-brown contents and some brown incrustations in 3% KOH and Melzer's solution. Basidia 50–68 x 9.5–12.5 µm, sterigmata 10–12 µm long, clavate, thin walled, 4-spored, hyaline in 3% KOH and Melzer's solution. Basidiospores 18–33 (–40) x 6–8 (–9.5) µm (Em = 3.75:E = 2.42–5.00) broadly fusiform, subfusiform to nearly cylindric, slightly thick-walled, yellow-brown in 3% KOH, weakly dextrinoid in Melzer's solution.

Specimen examined. USA. CALIFORNIA: Fresno Co., Dinkey Creek area. Under Abies and Pinus, 4 Aug 2000, Coll: Francisco J. Camacho, Lisa Grubisha, and Erik Lilleskow, ANK 1799 (HOLOTYPE B, lectotype VPI).

Etymology. Pseudoflavipes, Latin = near flavipes. Referring to the similarity of the two species.

Comments. Phylogenetically, Gomphidius pseudoflavipes appears very closely related to G. flavipes Peck (Fig. 11). Gomphidius flavipes Peck has the second largest spores in genus (18–29 x 6–8.5 µm), a thin fibrillose veil, white context in the pileus and upper stipe, while the lower two-thirds of the stipe is bright yellow. It is found in NE and SE U.S. in bogs under Picea, Tsuga, and Larix. It has recently been associated with red spruce (Picea rubens Sarg.) in high mountain bogs of North Carolina, and with black spruce (Picea meriana Mill.) in northern boreal forests in old glacial pot holes. In contrast, G. pseudoflavipes has the largest spores in the genus (18–40 x 6–9.5 µm) (Fig. 2), the pileus is brown to orange-brown, the partial veil densely fibrous but soon gone, dry, and with white context becoming yellow solely at the base of the stipe (Fig. 9). It is also more robust than G. flavipes with the stipe reaching to 80 mm long and up to 20 mm in diameter. It is associated with different hosts, being found under mixed conifers including Abies and Pinus. At the present it is known only from Fresno Co., California. It is interesting to note that a species of Gastrosuillus Thiers was growing very close to G. pseudoflavipes. In 1964 Miller placed G. flavipes in Chroogomphus since it has scattered amyloid tramal cells (Miller 1964) but it also has the white pileus context and the yellow stipe base typical of species of Gomphidius. However, phylogenetic analysis carried out by Miller and Aime (2001) clearly shows it to be a member of Gomphidius, where it is now placed.

View larger version (25K): [in this window] [in a new window]    FIG. 11. Phylogenetic analysis of the genus Gomphidius based on Internal Transcribed Spacer (ITS) sequence data. The first of three most parsimonius trees is depicted. Branches with strong statistical support are indicated by a bold line. Bootstrapping values are given above supported branches. Superscripts distinguish redundant species sequences for more than one collection and refer to the collection as listed in Table I

Gomphidius borealis O.K. Mill., Aime et Peintner, sp. nov. Figs. 5–8, 10

View larger version (21K): [in this window] [in a new window]    FIGS. 5–8. Gomphidius borealis. Fig. 5. Basidiomes. Fig. 6. Basidiospores. Fig. 7. Basidium. Fig. 8. Pleurocystidia. Upper bar = 1 cm. Lower bar = 10 µm

Pileus 1.5–3 cm broad, convex, broadly convex in age, viscid, glabrous, red at first to orange-red, in age. Lamellae subdistant, 2–3 mm between gills, short decurrent, thick, smoky gray. Stipe 6–9 cm long, 3–4 mm broad, tapering somewhat toward the base, surface white over upper half with a thin, superior, fibrous partial veil, light orange below with irregular black staining over the lower half. Context buff in the pileus to orange-buff becoming orange over the base. Pileipellis a thick ixomixocutis, of hyphae 1.7–3.5 µm diam, nearly hyaline in 3% KOH, light orange-brown in Melzer's reagent with some incrusted material in a hyaline gelatinous matrix. Pileitrama of interwoven hyphae 3.4–11.5 (–25) µm diam, with dense orange-brown incrusted material in 3% KOH and Melzer's solution. Lamellar trama of densely interwoven, thin-walled hyphae 2.5–6.0 µm diam, hyaline in 3% KOH, orange-brown in Melzer's solution. Pleuro- and Cheilocystidia 102–166 x 12–18 µm, narrowly clavate to nearly cylindric, thin-walled, hyaline in 3% KOH and Melzer's solution. Basidia 40–51 x 7–11 µm, narrowly clavate, thin-walled, 4-spored, hyaline in 3% KOH and Melzer's solution. Basidiospores (15–)16–19 (–20.5) x 6–7 µm, [Em = 2.71:E = 2.28–3.00] N = 20, subfusiform in profile and face view, orange-brown in 3% KOH and Melzer's solution.

Specimens examined. RUSSIA. SAKHA: two km southeast of the Magan Airport, 62°05' N, 1290°35'E. Several together, in a dry Larix gmelini Turczaninow forest with Betula platyphylla Sukatchev, Pinus silvestris L., Populus tremula L., and Alnus fruticosus Rupr., 4 Aug 1999, leg. Ursula Peintner, IB1999/0532 (HOLOTYPE: IN; LECTOTYPE: VPI). RUSSIA, SAKHA, Khangalasskiy Ulus, Sasabyt River, 2 km NW of Bestuakh, 61°05'N, 128°52'E. In a ravine in a Larix gmelini stand with Picea obovata, Alnus fruiticosa, and Salix bebbiana. 21 Aug. 1999, leg. Ursula Peintner IB1999/0843. SAKHA, Yakutsk, Viljujsk road by Kengkeme River, 62°03'N, 128°56'E. In a river forest with old growth L. gmelini, Betula plathyphylla, and S. bebbania. 16 Aug. 1999. leg. Ursula Peintner IB1999/0726.

Etymology. Borealis, Latin = northern. Referring to the habitat and distribution.

Comments. Gomphidius borealis has spores 15–20 x 6–7 µm, is not caespitose, and has a thin superior white fibrillose veil (Fig. 10). The stipe is light orange at the base inside and out and the lower surface blackens especially on handling. It appears to be associated with Larix gmelini in eastern Russia. In phylogenetic analysis of the ITS region (Fig. 11), G. borealis is most closely related to G. maculatus, both of which are putative mycorrhizal associates of species of Larix, and both display characteristic blackening of the stipe when handled. However, the presence of a partial veil and the pileus and context coloration are distinctive for G. borealis.

	ACKNOWLEDGMENTS

 
We wish to thank Ms. Patricia Eckel of the Buffalo Museum of Science for providing Latin diagnoses for the new species. Hope Miller read and corrected the manuscript. Funding for the molecular work was provided by an Aspires Grant to the first and second authors from Virginia Tech.

	FOOTNOTES

 
¹ Corresponding author, Orsonk{at}frontiernet.net

Accepted for publication July 9, 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY LITERATURE CITED

 
Bruns TD, Fogel R, Taylor JW., 1990 Amplification and sequencing of DNA from fungal herbarium specimens. Mycologia 82:175-184

Gardes M, Bruns TD., 1993 ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113-118[Medline]

Kornerup A, Wanscher JH., 1967 Methuen Handbook of Color. 2nd ed. London: Methuen and Co., Ltd

Miller OK., 1963 The Gomphidiaceae: a monograph of the genera and species and their world distribution [PhD Dissertation]. Michigan: Rackham School of Graduate Study, University of Michigan. 136 p

———. 1964 Monograph of Chroogomphus (Gomphidiaceae). Mycologia 56:526-549

———. 1966 A new western species of Chroogomphus. Mycologia 58:855-861

———. 1971 The genus Gomphidius with a revised description of the Gomphidiaceae and a key to the genera. Mycologia 63:1129-1163

———. 1973 A new gastroid genus related to Gomphidius. Mycologia 65:226-229

———, Aime MC., 2001 Systematics, ecology and world distribution in the genus Chroogomphus (Gomphidiaceae). In: Misra JK, Horn BW, eds. Trichomycetes and other fungal groups: Robert W. Lichtwardt Commemoration Vol. Enfield, New Hampshire: Science Publishers Inc. p 315–333

———, Trappe JM., 1970 A new Chroogomphus with a loculate hymenium and a revised key to section Floccigomphus. Mycologia 52:831-836

Smith AH, Singer R., 1958 Studies on secotiaceous fungi VIII. A new genus in the Secotiaceae related to Gomphidius. Mycologia 50:927-938

Swofford DL., 2001 PAUP*: phylogenetic analysis using parsimony (*and other methods), beta version 4.0b8. Sunderland, Massachusetts: Sinauer Associates

Vilgalys R, Hester M., 1990 Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238-4246[Abstract/Free Full Text]

White TJ, Bruns T, Lee S, Taylor JW., 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. New York: Academic Press, Inc. p 315–322

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