Mycologia, 106(4), 2014, pp. 785–796. DOI: 10.3852/13-312 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

Cercopemyces crocodilinus, a new genus and species related to Ripartitella, is described from North America Timothy J. Baroni1

Smith (1978). A relatively limited number of papers on macrofungi have been published for the extensive dry areas of the interior western states that are covered by plant communities such as pinyon-juniper, scrub oak or mountain mahogany woodlands (Southworth and Frank 2011, Kropp et al. 2012). The unpredictable precipitation patterns in these ecosystems makes fieldwork challenging, and as a consequence these areas often are ignored by macrofungal systematists. Yet, the fungal communities are diverse (Kropp pers obs). We describe here an unusual agaric species from one of these ecosystems that does not fit into any known genus of mushroom. The large basidiomata of this species are conspicuous in the field but have been collected rarely in the past decade. Thus far it appears that this taxon occurs only in association with mountain mahogany (Cercocarpus species) woodlands. Populations of this white-spored agaric now are known from Utah and Colorado. We also present a phylogenetic analysis that provides an understanding of its position within the Agaricales. Finally, we provide morphological and molecular evidence to make a new combination of a second rarely encountered agaric (Ripartitella ponderosa [A.H. Sm. & Singer] Franco-Mol.) into this new genus.

Department of Biological Sciences, 340 Bowers Hall, Graham Ave., PO Box 2000, State University of New York – College at Cortland, Cortland, New York 13045

Bradley R. Kropp Biology Department, Utah State University, 5305 Old Main Hill, Logan, Utah 84322

Vera S. Evenson Sam Mitchel Herbarium of Fungi, Denver Botanical Gardens, 909 York Street, Denver, Colorado 80206

Markus Wilhelm Felsenweg 66, CH-4123 Allschwil, Switzerland

Abstract: Cercopemyces is described as a new genus based on collections of a newly discovered agaricoid species from the arid Cercocarpus forests of Utah and Colorado. The new genus is near Ripartitella and Cystodermella based on nLSU, rpb1 and ITS molecular sequences but distinctly different from these taxa. The ornamented basidiospores and inflated cells in the scales of the pileus surface of Cercopemyces indicate a close relationship to some species of Ripartitella, and the molecular analyses support the sister group relationship. Morphologically Cercopemyces crocodilinus is reminiscent of the genus Amanita in the field, but the inamyloid, cyanophilic, ornamented basidiospores and lack of acrophysalidic hyphae in the trama indicate otherwise. A rare eastern USA species, Ripartitella ponderosa, is transferred to Cercopemyces based on morphological and molecular data. Key words: Cercocarpus, Cystoderma, Cystodermella, Great Basin, ITS, new combination, nLSU, phylogenetics, rpb1

MATERIALS AND METHODS Field notes on dimensions and colors were obtained from fresh specimens within a few hours of collection. Specimens were dried with food dehydrators. Microscopic features were studied in 3% KOH, 10% NH4OH, Congo red in ammonia, cotton blue in lactic acid and Melzer’s reagent. For basidiospore measurements, the hilar appendix or apiculus was excluded. In those measurements Q refers to the length divided by the width of an individual spore. The notation n 5 40/2, indicates that 40 individual basidiospores were measured from two collections. Means for length and width are given with their standard deviations, as is the mean of Q values, with the mean of the length divided by the width of all basidiospores designated Qm. All light microscopic digital images were made with an Olympus BX 50 transmitted light microscope using DIC or bright field optics and captured with a Diagnostic Instruments Inc. Insight Spot three-shot color digital camera. Line drawings were traced with the aid of a drawing tube attached to the light microscope, and the finished illustrations were completed with Adobe Illustrator on a Wacom Intuos drawing tablet. For scanning electron

INTRODUCTION Most of the macrofungal taxonomic work in the western United States has focused on the biologically diverse coniferous forests along the coast or on similar mesophytic forested ecosystems in the interior mountain ranges, for example: Kauffman (1921, 1926, 1930); Smith (1937, 1941, 1944); Smith and Smith and Stuntz (1950); Solheim (1953); Bigelow and Smith (1962); Smith et al. (1965); Mitchel and Submitted 26 Sep 2013; accepted for publication 30 Jan 2012. 1 Corresponding author. E-mail: [email protected]

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microscopy small pieces of dried lamella tissue, approximately 1–2 mm squared, were excised from basidiomata and rehydrated in 95% ETOH for 1 min, transferred to 3% KOH for 1 min, then washed two times in dH2O for 1 min each. The samples then were sandwiched between Whatman No. 4 filter paper in modified BEEM capsules and placed immediately into 10% ETOH. Dehydration in an ethanol series (10%, 30%, 50%, 70%, 80%, 90%, 95%, 100% for 2 min each at room temperature, then 2 min in100% ice-cold ETOH) was followed by critical point drying of the samples in a Tousimis Samdri PVT-3B in liquid CO2. The dried tissues were mounted on aluminum stubs with carbonimpregnated sticky tape and coated with approximately 350 A˚ gold in a Polaron SC502 sputter coater. Digital images were obtained with Orion 6.0 software on an ISI DS130C SEM at 10 mm working distance and 15 Kv accelerating voltage. Standard protocols were used to extract DNA from Cercopemyces crocodilinus, Smithiomyces mexicanus, and Ripartitella brasiliensis and to amplify the target sequences to be used in the phylogenetic analyses (White et al. 1990, Kropp et al. 1996). Amplified PCR products were obtained for the portion of the nuclear large ribosomal subunit (nLSU) between primers LROR and LR5 (Vilgalys and Hester 1990, Moncalvo et al. 2000) and for the internal transcribed spacer (ITS) with primers ITS4 and ITS5 (White et al. 1990). In addition, sequences were obtained for rpb1 (largest subunit of RNA Polymerase II) with primers g-rpb1A for and a-rpb1-B rev (Matheny et al. 2002). Sequences of the purified PCR products were obtained with dye terminator methodology with an ABI 3730 DNA analyzer and the sequences were deposited in GenBank (TABLE I). The specimens used to generate new DNA sequences are deposited at the Intermountain Herbarium (UTC) and the Herbarium at the State University of New York – College at Cortland (CORT) with accession numbers Cercopemyces crocodilinus (UTC258260 and CORT-BK28-June-97), Smithiomyces mexicanus (UTC258259), Ripartitella brasiliensis. (UTC259520 and CORT-BOS-340 (BZ-1689). An initial BLAST query with the new nLSU sequence indicated that C. crocodilinus had affinities to species in Ripartitella and Cystodermella and that it belonged to the agaricoid clade in the sense of Matheny et al. (2006). Based on information from the BLAST query, taxon sampling for the phylogenetic analyses focused on the agaricoid clade and members of the Agaricaceae were used to root the trees. For the nLSU-rpb1 analysis the nLSU and rpb1 sequences for each of the sampled taxa were concatenated and aligned with Clustal W (Thompson et al. 1994). For the nLSU-ITS analysis the sequences were concatenated and aligned in the same way. Unalignable regions were removed and minor adjustments were made by hand with BioEdit (Hall 1999). For each of the concatenated alignments, care was taken to select sequences derived from the same specimen based on the voucher or specimen numbers in GenBank. For the nLSU-rpb1 tree, a total of 2258 characters was used in the analysis whereas 1666 characters were used in the nLSU-ITS analysis. The ITS sequence for R. ponderosa (HQ179669) available in GenBank was used to analyze its relationship with C. crocodilinus. For this analysis, taxon

sampling again was focused on the agaricoid clade. ITS sequences for most of the taxa that had been used in the nLSU-ITS analysis again were used to test the relationship between R. ponderosa and C crocodilinus. However, additional species from the tricholomatoid clade of Matheny et al. (2006) were added to this analysis to root the tree. The alignments are available at TreeBASE (S13465, S13466, S15119). Mr Bayes 3.1 was used to analyze the three aligned datasets (Ronquist and Huelsenbeck 2003). Tree searches were performed with a general time reversible model of evolution (GTR) with six substitution types assuming a gamma distribution. The runs were conducted with eight active Markov chain Monte Carlo (MCMC) chains heated at 0.2, and posterior probabilities were approximated by sampling every 100 trees simulated with MCMC. The simulations were iterated for 1 000 000, generations and a majority rule consensus tree was calculated from the last 7500 trees. TreeView (Page 1996) was used to visualize the output from each analysis. Measures for nodes with less than 80% support are not illustrated

RESULTS Phylogenetics.—The Bayesian analysis of the combined nLSU-rpb1 sequences (FIG. 1) indicates that Cercopemyces crocodilinus has an affinity to the Agaricoid clade of Matheny et al. (2006) and that within this clade it falls into a well-supported cluster that includes species of both Cystodermella and Ripartitella. In addition, Cercopemyces is supported by both the nLSU-rpb1 and nLSU-ITS analyses as being sister to Ripartitella but phylogenetically distinct from it. The analysis of the ITS sequences (FIG. 3) indicates that there is a close relationship between C. crocodilinus and R. ponderosa and supports the inclusion of both species in the new genus Cercopemyces. Although Cercopemyces crocodilinus originally was thought to be a species of Smithiomyces based mainly on basidiospore morphology, the nLSU-rpb1 (FIG. 1) and nLSU-ITS analysis (FIG. 2), both show that Smithiomyces mexicanus is unrelated to Cercopemyces. The results presented here are also consistent with the results of Saar et al. (2009) in showing that Cystodermella and Ripartitella are separate phylogenetically from Cystoderma. Taxonomy.—New Taxa. Cercopemyces T.J. Baroni, Kropp and V.S. Evenson, gen. nov. FIGS. 4–8 MycoBank MB805634 Distinctive by possessing large fleshy amanitoid basidiomata with a squarrose-scaly pileus, evanescent annular ring at the base of the stipe, small punctate ornamented, inamyloid, cyanophilic basidiospores, adnexed, sinuate or adnate lamellae with parallel or

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GenBank numbers for the species sampled for the phylogenetic analyses (FIGS. 1–3) Taxon

nLSU

ITS

rpb1

Agrocybe praecox (Pers.) Fayod Calocybe gambosa (Fr.) Singer Cercopemyces crocodilinus T.J.Baroni, Kropp & V.S.Evenson Cercopemyces ponderosus a (A.H. Smith & Singer) T.J.Baroni, Kropp & V.S.Evenson Chlorophyllum agaricoides (Czern.) Vellinga Coprinus comatus (O.F. Mu¨ll.) Pers. Cortinarius iodes Berk. & M.A. Curtis Cortinarius violaceus (L.) Gray Cystoderma amianthinum (Scop.) Fayod Cystoderma amianthinum (Scop.) Fayod Cystodermella adnatifolia (Peck) Harmaja Cystodermella ambrosii (Bres.) Harmaja Cystodermella cinnabarina (Alb. & Schwein.) Harmaja Cystodermella granulosa (Batsch) Harmaja Cystodermella papallactae (I. Saar & Læssøe) Vizzini Flammulaster muricatus (Fr.) Watling Galerina marginata (Batsch) Ku¨hner Galerina semilanceata (Peck) A.H. Smith & Singer Laccaria bicolor (Maire) P.D. Orton Lacrymaria velutina (Pers.) Konrad & Maubl. Lepiota cristata (Bolton) P. Kumm. Leucoagaricus leucothites (Vittad.) Wasser Lyophyllum sp. Macrolepiota mastoidea (Fr.) Singer Mythicomyces corneipes (Fr.) Redhead. & A.H. Sm Nolanea sericea (Que´l.) P.D. Horton Phaeomarasmius curcuma (Berk. & M.A. Curtis) Singer Phaeomarasmius proximans (A.H. Sm. & Hesler) Singer Pholiota squarrosa (Vahl) P. Kumm. Psathyrella candolleana (Fr.) Maire Ripartitella alba Halling & Franco-Mol. Ripartitella brasiliensis (Speg.) Singer Ripartitella sp. Smithiomyces mexicanus (Murrill) Singer Stropharia ambigua (Peck) Zeller Termitomyces sp. Tubaria confragosa (Fr.) Ku¨hner

AY646101.1 DQ071716.2 JX409897

— — JX409899

DQ516069.1 — JX409898

— AY700187.1 AY207179.1 AY702013.1 DQ071705.2 DQ071703.2 DQ154108.1 AM946421.1 AM946422.1 AM946429.1 AM946431.1 AM946439.1 DQ071740.2 DQ457669.1 AY038309.2 DQ071702.2 AY700198.1 JN940284.1 JN940292.1 — JN940271.1 AY745707.1 — AY038329.1 AY380410.1 DQ470818.1 — AM946464.1 AM946465.1 JX462553 JX409900 AY646102.1 — AY700190.1

HQ179669 DQ200928.1 — — — — — AM946510.1 — AM946512.1 AM946518.1 — — — — — DQ490639.1 JN944090.1 JN944084.1 DQ182502.1 HM125530.1 DQ404393.1 DQ367430.1 — — — DQ494689.1 AM946525.1 AM946524.1 JX462554 JX409901 — DQ494698.1 —

— — — AY857984.1 DQ067972.1 — DQ516073.1 — — — — — DQ068012.1 DQ447901.1 AF389531.1 DQ067942.1 — JN987894.1 JN987891.1 — JN987898.1 DQ447929.1 — AF389551.1 AY333307.1 DQ447931.1 — — — JX409896 — DQ447941.1 — DQ447944.1

a

Cystoderma ponderosum in GenBank.

interwoven hyphae in the lamella trama, inflated cells in the squamules of the pileus surface and by the abundant clamp connections in all tissues. Tissues of the stipe context lack acrophysalidic hyphae. Holotype: Cercopemyces crocodilinus T.J. Baroni, Kropp and V.S. Evenson. Etymology: In Greek mythology the Cercopes were mischievous forest creatures who might turn up wherever trouble was brewing. This taxon was mischievously difficult to recognize and thus has earned its genus name.

Cercopemyces crocodilinus T.J. Baroni, Kropp and V.S. Evenson, sp. nov. FIGS. 4–8 MycoBank MB805635

Distinctive from the other species in the genus by the white pileus and stipe, the thick pyramidal scales or conical warts covering the pileus, the pale cream sinuate or adnexed, close, narrow lamellae, the short equal stipe with bulbous-napiform base with an ephemeral ring of conical-shaped floccose warts adhering to the top of the bulb and larger basidispores (4.8–6.4 (3.5–5.5 mm) than found in Cercopemyces ponderosus (3.6–4(–5) 3 2.7–3 mm, vide Franco-Molano 1993), the only other taxon in the genus (see below). Holotype: USA, Utah, Boxelder County, Sawtooth National Forest, Raft River Mountains, about 2.4 km (1K miles) upstream of the Clear Creek Campground, approx. 41u56950.900N, 113u21909.020W,

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FIG. 1. Phylogram resulting from a Bayesian analysis of a combined nLSU-rpb1 dataset shows placement of Cercopemyces crocodilinus. Support measures shown for nodes with 79 percent or greater posterior probability support. GenBank numbers represent nLSU sequences.

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FIG. 2. Phylogram resulting from a Bayesian analysis of a combined nLSU-ITS dataset showing placement of Cercopemyces crocodilinus. Support measures are shown for nodes with 80 percent or greater posterior probability support. GenBank numbers are for nLSU sequences.

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FIG. 3. Phylogram resulting from a Bayesian analysis of ITS sequences including both species of Cercopemyces. GenBank numbers are for the ITS sequences.

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FIG. 6. Cercopemyces crocodilinus (Holotype). DIC light microscopic image of basidiospores shows obscure ornamentation. Bar 5 5 mm. FIG. 4. Cercopemyces crocodilinus (DBG 24386). Basidiomata. Bar 5 10 mm. Image by Ed Lubow.

2120 m, 28 Jun 1997, leg. B.R. Kropp, BK28-June-97-4 (HOLOTYPE, UTC-258260; ISOTYPE, CORT-BK28June-97-4). Etymology: The specific epithet, crocodilinus, refers to the erect, thick pyramidal scales on the pileus surface, resembling the dorsal surface of a crocodile (Latin crocodillus).

Pileus white but with pale buff brown or pale earthen brown especially over the disk (10YR9/4–8/6 Munsell), 40–100 mm broad, convex, becoming broadly and shallowly depressed with expansion; surface dry, becoming subareolate, densely covered with fleshy conical firmly attached pyramidal scales that are 1–4 mm across and white or concolorous with earthen or with pale yellow hues mixed in; near the margin small floccose scales form an appendiculate veil along the margin; margin inrolled, remaining incurved with expansion. Flesh features not recorded when fresh, but as dried uniformly white, thick (10 mm). Lamellae white when fresh, becoming pale

FIG. 5. Cercopemyces crocodilinus (DGB 24386). Line drawings of A. Cystidiate end cells from appendiculate tissue on pileus margin. B. Basidiospores. C. Cystidiate end cells from annular ring on stipe basal bulb. Bars: A, C 5 10 mm; B 5 5 mm.

cream yellow or cream olive buff when dried, sinuate or finely adnexed, not free, close or almost crowded, with numerous short lamellulae, narrow (5 mm in dried state), edges more or less even, concolorous. Stipe white with pale buff earthen brown as in the pileus, 42–62 mm long, 15–27 mm broad at the apex, 35–50 mm broad at the base, more or less equal with a bulbous and napiform base on some, bulbous portion similar in length to upper equal portion making the stipe seem short; surface dry, glabrous but with a ring of ephemeral, white conical floccose rounded scales adhering to the top of the basal bulb in a collar-like ring; this tissue similar to that of the appendiculate margin of the pileus indicating a universal or peronate veil was present in button stages; flesh

FIG. 7. Cercopemyces crocodilinus (Holotype). Scanning electron image of basidiospores. Bar 5 1 mm.

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FIG. 8. Cercopemyces crocodilinus (DBG 24386). DIC light microscope image of inflated end cells in the pyramidal scales of the pileus. Bar 5 10 mm.

white, solid as in pileus, unchanging when exposed; basal mycelium white, tightly binding soil and humus. Odor fungoid. Taste not determined. Basidiospores (FIGS. 5B, 6, 7) hyaline under compound light microscope, 4.8–6.4 3 3.5–5.5 mm (n 5 40/2, x 5 5.44 6 0.40 3 4.0 6 0.40 mm, Q 5 1.02–1.6, Qm 5 1.37 6 0.13), ellipsoid, broadly ellipsoid or some globose (Q 5 1.02), round in polar view; walls inamyloid, cyanophilic with a delicately punctate roughened ornamentation that can be observed in Melzer’s reagent or cotton blue in lactic acid with the aid of DIC optics, but best observed with SEM. Basidia (1–)4-sterigmate, long, narrowly clavate, 27–36 3 6.4– 8(–9) mm, with long, tapered sterigmata, 4–5.6 mm long and 1.6 mm wide at the base, lacking cyanophilic bodies in the basidia. Hymenial cystidia absent. Lamella trama hyaline, parallel, cylindrical or slightly inflated hyphae, 4–12 mm diam; cells often short (20– 60 mm long). Pileipellis a hyaline or a sordid, yellowish tan layer of loosely interwoven cylindrical hyphae, 4– 8(–10) mm diam, not well differentiated from the context, not encrusted; the pyramidal scales composed of two layers, an upper yellowish brown compact layer, 160–240 mm deep, often with abundant cystidiate inflated end cells present, overlying a deeper (800–1000 mm) hyaline, loosely entangled layer of cylindrical or slightly inflated hyphae, 5–10 mm diam, this hyaline layer also with some scattered inflated cystidiate end cells in the upper regions near

the yellowish brown compact layer; cystidiate end cells, 28–62 3 12–18(–24) mm, variable in shape: clavate, capitate, fusiform, sphaeropedunculate, broadly lageniform, utriform, occasionally septate (FIGS. 5A, 7). Pileus context hardly differentiated from the pellis, composed of hyaline, loosely or densely interwoven, hyphae that are mostly cylindrical, some slightly inflated, 4–16 mm diam. Stipitipellis a hyaline layer of repent, interwoven, narrowly cylindrical hyphae, 3.2–5.6 mm diam, not encrusted; the conical floccose warts making up the ring on the surface of basal bulb composed of hyaline, interwoven, cylindrical hyphae, 4–16 mm diam, with many swollen inflated cystidiate end cells similar in construction and size to the cystidiate end cells found on the pileus margin and in the upper layers of the pyramidal warts on the pileus surface. Stipe context a hyaline compact layer of parallel or somewhat interwoven, cylindrical or slightly inflated hyphae, 6–15 mm diam, thin-walled, mostly filled with refractive bodies of various dimensions. Clamp connections present in all tissues. Habit and habitat: Solitary, gregarious, caespitose, or scattered, on the ground, under Cercocarpus ledifolius with Pinus monophylla present (Raft River Mountains, Utah) or associated with Cercocarpus montanus (east slope of the Colorado Rocky Mountains). June. Additional material examined: Cercopemyces crocodilinus. USA, Colorado, Larimer County, Soapstone Natural Area, Towhee Trail, 2042 m, 26 Jun 2009, leg. Jack Jones, (DBG 24386). Ripartitella brasiliensis Belize, Orange Walk District, La Milpa Station, Lagunita Trail, approx. 17u50930.00N, 89u01909.40W, 142 m, 10 Oct. 2002, leg. Beatrice Ortiz Santana BOS340 (BZ-1689) (CORT). Smithiomyces mexicanus Switzerland, Zurich, Zurich Zoo, Masoala Hall, rainforest exhibit, 600 m, 9 Aug 2011, leg. Markus Wilhelm (UTC-00259587). Commentary: Cercopemyces belongs to the Agaricoid clade (Matheny et al. 2006) but rests outside the Agaricaceae based on our molecular analyses (nLSU, ITS and rpb1 datasets; FIGS. 1, 2). Our results also are consistent with those of Saar et al. (2009) in showing that the Cystodermateae sensu Singer (1986) is not monophyletic. Cystodermella, Ripartitella and Cercopemyces cluster together with solid bootstrap support, but their relationship to other agarics remains somewhat unclear at this time. Cercopemyces is phylogenetically related to both Cystodermella and Ripartitella and is obviously a sister taxon of Ripartitella (FIGS. 1, 2). However, because Ripartitella forms a strongly supported monophyletic branch in both our analyses and those branch lengths separating Ripartitella and Cystodermella from Cercopemyces point to substantial character changes be-

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FIG. 9. Ripartitella brasiliensis (BOS 340). DIC light microscope image of basidiospores. Bar 5 5 mm.

FIG. 10. Ripartitella brasiliensis (BOS 340). Scanning electron micrograph of basidiospores. Bar 5 1 mm.

tween them, Cercopemyces is considered an independent genus. Morphological and ecological evidence also support this conclusion. Cercopemyces differs from Ripartitella by three major features. Cercopemyces produces robust basidiomata (FIG. 4) that are found on soil rather than the typical medium- to small-sized and thin fleshed basidiomata of Ripartitella that occur on decaying wood or woody debris (Ripartitella brasiliensis [Speg.] Singer, Ripartitella alba Halling & Franco-Mol., Ripartitella sipariana [Dennis] Dennis, and the type of the genus Ripartitella squamosidisca [Murrill] Singer). A third and distinctive feature is that Ripartitella species possess a Melanoleuca-type pleurocystidium that is lageniform or ampullaceous and frequently crystalline encrusted (FIG. 11) (also see Singer 1986, Ovrebo 1988, Halling and Franco-Molano 1996, Capellari and Asai 2009), whereas Cercopemyces lacks any type of hymenial cystidia. The basidiospores of Cercopemyces are obscurely punctate ornamented under the light microscope (FIG. 6), although these ornamentations are obvious with scanning electron microscopy (FIG. 7). The ornamented basidiospores of Ripartitella species have been described as echinulate, and the ornamentation is easily visible with transmitted light and scanning electron microscopy (FIGS. 9–11). As one might expect, based on the molecular evidence, several features indicate a morphological relatedness of the temperate Cercopemyces to the tropical/subtropical Ripartitella species. The erect squamules on the pileus bearing scattered inflated cystidiate end cells (FIGS. 5A, 8), the ornamented basidiospores with cyanophilic but inamyloid walls, the regularly arranged cylindrical hyphae in the

lamellar trama, the ephemeral partial veil on the stipe and the abundant clamp connections are features that are shared by and appear to connect Ripartitella and Cercopemyces. In a final note on taxonomy, the macromorphology of C. crocodilinus appears much like that of a species of Amanita in the field (FIG. 4). However, in addition to the fleshy pyramidal scales being firmly attached and not obviously part of a universal veil, several microscopic features confirmed that C. crocodilinus was not a member of the genus Amanita. The small punctate-roughened basidiospore is a character not found in species of Amanita; their basidiospores are smooth. The parallel, not divergent, hyphae in the lamellar trama and the lack of acrophysalidic hyphae in the stipe context of C. crocodilinus confirmed it is not related to species of Amanita (see Tulloss 2012). Also, despite the robust habit of our material, we initially considered the rarely collected genus Smithiomyces to be a potential match based on some microanatomical characters. However, our phylogenetic analysis indicated that Cercopemyces and Smithiomyces are not closely related (FIGS. 1, 2). Thus far Cercopemyces crocodilinus has been found only once in Utah and once in Colorado. We are unaware of any additional collections of this taxon, although, based on the geographical distance between the two known collections, this species is potentially much more widely distributed. The arid, high elevation environments that it inhabits are not conducive to basidiome production. Because of this limitation, detailed ecological studies of the fungus are challenging. Except for the fact that both collections of C. crocodilinus were found in humus

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FIG. 11. Ripartitella brasiliensis (BOS 340). Scanning electron micrograph of a pleurocystidium and basidiospores. Note characteristic Melanoleuca type encrustations on the tapered apex of the pleurocystidium. Bar 5 1 mm.

in association with Cercocarpus species, almost nothing else is known about its ecology. Cercocarpus is a small but interesting genus of shrubs and small trees in the Rosaceae and is restricted to semiarid areas of western North America. Cercocarpus finds its niche in harsh environments and has been known to form both actinorrhizal and ectomycorrhizal symbioses (Trappe 1962, Baker 1990). McDonald et al. (2010) published further evidence to support the ectomycorrhizal status of Cercocarpus ledifolius by examining fungal DNA from roots, and Southworth and Frank (2011) published a new species of Geopora as a sequestrate ectotrophic mycorrhizal partner with Cercocarpus ledifolius. A number of other ectomycorrhizal fungi also are found with this host, including Cenococcum geophilum Fr., Inocybe cercocarpi Kropp, Matheny & Hutchinson (Trappe 1962, Kropp et al. 2013), and an undescribed species of Hebeloma (Kropp and Hutchison unpubl). If robustness of basidiomata can be considered an indicator of an ectotrophic mycorrhizal nutrition mode, it may be that Cercopemyces crocodilinus is also a mutualist with Cercocarpus. However, that relationship has yet to be confirmed. New combination. Ripartitella ponderosa recently placed in Ripartitella (Franco-Molano 1993) is a robust species (pileus 4–11 cm broad) with a squarrose-squamulose pileus that lacks hymenial

cystidia and was found on soil over rocky ground in the southern Appalachian Mountains (Mount Le Conte, Great Smoky Mountains National Park, Tennessee) (Smith and Singer 1945). Franco-Molano (1993) illustrated with scanning electron microscopy the low-tuberculate type of basidiospore ornamentation for R. ponderosa that was missed in the original description because, as in Cercopemyces crocodilinus, the basidiospores appear finely ornamented under light microscopy and thus the ornamentation is easily overlooked. As revealed by SEM imaging (FrancoMolano 1993), the ornamentation of the basidiospores is more highly developed in R. ponderosa than in C. crocodilinus, but the type of ornamentation is similar. Based on the morphology of R. ponderosa (i.e. the robust basidiomata with squarrose scaly pileus, small finely ornamented inamyloid basidiospores, lack of Melanoleuca-type pleurocystidia, the inflated elements in the pileipellis [Franco-Molano 1993] and fruiting from soil) R. ponderosa fits nicely in Cercopemyces along with C. crocodilinus. The analysis of the ITS sequence for R. ponderosa, and other closely related fungi including C. crocodilinus supports the inclusion of both taxa in Cercopemyces (FIG 3). Therefore we propose this new combination: Cercopemyces ponderosus (A.H. Sm. & Singer) T.J. Baroni, Kropp & V.S. Evenson, comb. nov. (Basionym: Cystoderma ponderosum A.H. Sm. & Singer, Pap Mich Acad Sci 30:92. 1945)

MycoBank MB805636 It appears that Cercopemyces ponderosus is a rare species that has been collected only two other times in North America since the type collection in 1942 (Mycoportal.org), and those collections were compiled 1942–1956 by the original collector of the species, L.R. Hesler. From online records it appears that Hesler likely obtained all three collections at the same location. One additional collection by Locquin in 1944 is cited from France and is deposited in the MICH Herbarium (Mycoportal.org). We have not attempted to confirm that identification. ACKNOWLEDGMENTS We thank the Utah Agricultural Experiment Station for financial support. This is Utah Agricultural Experiment Station paper 8480. We also thank Dr Beatriz Ortiz Santana, USDA Forest Service Northern Research Station, for providing a collection of Ripartitella brasiliensis that came from research supported by the National Science Foundation, Biodiversity Surveys and Inventories Program (NSF Awards DEB-0103621, DEB-9525902) to TJB and D.J. Lodge (Research Foundation of the State University of New York,

BARONI ET AL.: CERCOPEMYCES, College at Cortland, in collaboration with the USDA Forest Service, Center for Forest Mycology Research, Forest Products Laboratory, Madison, Wisconsin). Mr Jack Jones found the Colorado collection and we gratefully acknowledge his contribution to the discovery of this new taxon. The image of the fresh specimens was taken by Mr Ed Lubow of Colorado and he is gratefully acknowledged for this contribution. Two anonymous reviewers also provided important helpful suggestions for improving the manuscript and we do thank them.

LITERATURE CITED Baker D. 1990. Methods for the isolation, culture and characterization of the Frankiaceae: soil actinomycetes and symbionts of actinorhizal plants. In: Labeda DP, ed. Isolation of biotechnological organisms from nature. New York: McGraw-Hill. p 213–236. Bigelow HE, Smith AH. 1962. Clitocybe species from the western United States. Mycologia 54: 498– 515, doi:10.2307/3756319 Capelari M, Asai T. 2009. Cystoderma, Cystodermella and Ripartitella in Atlantic Forest, Sa˜o Paulo State, Brazil. Hoehnea 36: 339–348, doi:10.1590/S2236-89062009000200011 Franco-Molano AE. 1993. Studies on Cystoderma: a new species and a new combination. Mycologia 85:672–676, doi:10.2307/3760512 Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98. Halling RE, Franco-Molano AE. 1996. Agaricales from Costa Rica: new taxa with ornamented spores. Mycologia 88: 666–670, doi:10.2307/3761165 Kauffman CH. 1921. The mycological flora of the higher Rockies of Colorado. Pap Mich Acad Sci 1:101–150. ———. 1926. The fungus flora of Mt. Hood, with some new species. Pap Mich Acad Sci 5:115–148. ———. 1930. The fungus flora of the Siskiyou Mountains in southern Oregon. Pap Mich Acad Sci 11:151–210. Kropp BR, Albee-Scott S, Castellano M, Trappe JM. 2012. Cryptolepiota, a new sequestrate genus in the Agaricaceae with evidence for adaptive radiation in western North America. Mycologia 104:164–174, doi:10.3852/11-046 ———, Hansen D, Flint KM, Thomson SV. 1996. Artificial inoculation and colonization of Dyer’s Woad (Isatis tinctoria) by the systemic fungus Puccinia thlaspeos. Phytopathology 86:891–896, doi:10.1094/Phyto-86-891 ———, Matheny PB, Hutchison LJ. 2013. Inocybe section Rimosae in Utah: phylogenetic affinities and new species. Mycologia 105:728–747, doi:10.3852/12-185 Matheny PB, Curtis JM, Hofstetter V, Aime MC, Montalvo J-M, Ge Z-W, Yang Z-L, Slot JC, Binder M, Ammirati JF, Baroni TJ, Bougher NL, Hughes KW, Lodge DJ, Kerrigan RW, Seidl MT, Aanen DK, DeNitis M, Daniele GM, Desjardin DE, Kropp BR, Norvell LL, Parker A, Vellinga EC, Vilgalys R, Hibbett DS. 2006. Six major clades of Agaricales: a multilocus phylogenetic overview. Mycologia 98:982–995, doi:10.3852/mycologia.98.6.982 ———, Liu YJ, Ammirati JF, Hall BD. 2002. Using RPB1 sequences to improve phylogenetic inference among

A NEW

AGARICOMYCETE

795

mushrooms (Inocybe, Agaricales). Am J Bot 89:688–698, doi:10.3732/ajb.89.4.688 McDonald KR, Pennell J, Frank JL, Southworth D. 2010. Ectomycorrhizas of Cercocarpus ledifolius (Rosaceae). Am J Bot 97:1867–1872, doi:10.3732/ajb.0900357 Mitchel DH, Smith AH. 1978. Notes on Colorado fungi III: new and interesting mushrooms from the aspen zone. Mycologia 70:1040–1063, doi:10.2307/3759137 Moncalvo J-M, Lutzoni FM, Rehner SA, Johnson J, Vilgalys R. 2000. Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Syst Biol 49:278–305, doi:10.1093/sysbio/ 49.2.278 Ovrebo CL. 1988. Notes on cultural characters, morphology and distribution of Ripartitella brasiliensis. Mycotaxon 31:229–237. Page RDM. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358. Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574, doi:10.1093/bioinformatics/ btg180 Saar I, Po˜ldmaa K, Ko˜ljalg U. 2009. The phylogeny and taxonomy of genera Cystoderma and Cystodermella (Agaricales) based on nuclear ITS and LSU sequences. Mycol Prog 8:59–73, doi:10.1007/s11557-008-0578-9 Singer R. 1986. The Agaricales in modern taxonomy. 4th ed. Koenigstein, Germany: Koeltz Scientific Books. 981 p. Smith AH. 1937. New and unusual agarics from the western United States. Mycologia 29:45–59, doi:10.2307/ 3754199 ———. 1941. Studies of North American agarics I. Univ Mich Herbarium 5:1–73. 32 plates. ———. 1944. New North American agarics. Mycologia 36: 242–262, doi:10.2307/3754821 ———, Singer R. 1945. A monograph of the genus Cystoderma. Pap Mich Acad Sci 30:71–124. ———, Solheim WG. 1953. New and unusual fleshy fungi from Wyoming. Madron ˜ o 12:103–109. ———, Stuntz DE. 1950. New or noteworthy fungi from Mt Rainier National Park. Mycologia 42:80–134, doi:10.2307/3755245 ———, Thiers HD, Miller OK. 1965. The species of Suillus and Fuscoboletinus of the Priest River Experimental Forest and vicinity, Priest River, Idaho. Llyodia 28:120– 138. Southworth D, Frank JL. 2011. Linking mycorrhizas to sporocarps: a new species, Geopora cercocarpi, on Cercocarpus ledifolius (Rosaceae). Mycologia 103: 1194–1200, doi:10.3852/11-053 Thompson JD, Higgins DG, Gibson TJ. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680, doi:10.1093/nar/ 22.22.4673 Trappe JM. 1962. Fungus associates of ectotrophic mycorrhizae. Bot Rev 28:538–606, doi:10.1007/BF02868758

796

MYCOLOGIA

Tulloss RE. (http://www.amanitaceae.org/?About+Amani taceae at www.Amanitaceae.org) Downloaded 6 Sep 2012. Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal RNA from several Cryptococcus species. J Bacteriol 172:4238–4246.

White TJ, Bruns T, Lee S, Taylor J. 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. p 315–322.