Rep. Tottori Mycol. Inst. 45 : 1Ð57, 2007.

Megacollybia (Agaricales)

KAREN W. HUGHES1, RONALD H. PETERSEN1, JUAN LUIS MATA2,

NADEZHDA V. PSURTSEVA3, ALEXANDER E. KOVALENKO3, OLGA V. MOROZOVA3,

EDGAR B. LICKEY 4, JOAQUIN CIFUENTES BLANCO5, DAVID P. LEWIS6,

EIJI NAGASAWA7, ROY E. HALLING8, SEIJI TAKEHASHI9,

M. CATHERINE AIME10, TOLGOR BAU11, TERRY HENKEL12

Abstract

The genus Megacollybia, originally proposed for M. (Collybia) platyphylla, has traditional- ly been treated as monotaxic. A phylogenetic reconstruction based on ITS rDNA sequences indicates that several species are involved, with strong phylogeographic signal. Although morphological characters are largely qualitative, examination of basidiomata suggests that specimens included in discrete clades can be distinguished at the species level. On these bases (phylogenetic, morphological), several new taxa are proposed: M. clitocyboidea, M. texensis, M. fusca, M. subfurfuracea, M. rodmani (with f. murina) and M. marginata. Tricholomopsis fallax is transferred to Megacollybia; M. platyphylla remains the type species of the genus but appears to be restricted to Europe, Scandinavia and western and central Russia.

Key words: Taxonomy, systematics, biogeography, phylogeny, phylogeography

1 Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37996-1100 USA. 2 Department of Biology, University of South Alabama, Mobile, AL 36688 USA 3 Komarov Botanical Institute, 2 Prof. Popov Street, St Petersburg, 197376 Russia 4 Department of Biology, Bridgewater College, Bridgewater, VA 22812 USA 5 Herbario FCME, Facultad de Ciencias UNAM, AP 86-95, C.P. 14391, Tlalpan, Mexico D.F., Mexico 6 262 CR 3062, Newton, TX 75966-7003 USA 7 Tottori Mycological Institute, 211 Kokoge, Tottori 689-1125, Japan 8 New York Botanical Garden, Bronx, NY 10458-5126, USA 9 1-3-10-3 Kanayama, Teine-ku, Sapporo, Hokkaido 006-0041, Japan 10 Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 USA 11 Institute of Mycology, Jilin Agricultural University, 2888 Xincheng St., Changhun City, Peoples’ Republic of China 12 Department of Biological Sciences, Humboldt State University, Arcata, CA 95521 USA

1 K.H. HUGHES et al.

Introduction Moncalvo, et al. (2002), in which Tricholomop- sis rutilans is found in a poorly supported clade By the time Kotlaba and Pouzar (1972) (24, /tricholomopsis) with Clavaria fusiformis, segregated Megacollybia, Collybia (Tricho- Marasmius rhyssophyllus and Collybia aurea, of lomopsis, Oudemansiella, Megacollybia) which all of the latter three can find more com- platyphylla had become a popular name in use fortable morphogeneric placements. Mega- throughout Europe and eastern North collybia platyphylla, conversely, is in a clade America. As long as workers from these (27, /hydropoid) with three Gerronema taxa, regions did not seriously collect in each oth- Clitocybula oculus, two Hydropus taxa and a ers’ territory this single name seemed applica- Porotheleum and these findings have recently ble for what was considered to be a been confirmed by the six-locus analyses of transAtlantic organism. The name was also Matheny et al. (2006). Tricholomopsis fallax applied to far east-Asian basidiomata which ITS sequences are most closely related to seemed to resemble those from Europe. Megacollybia platyphylla by blast search, then Comparing photos by Phillips (1981: 44, as to Clitocybula and Gerronema but not to Tricholomopsis platyphylla) of the mushroom Tricholomopsis sensu stricto. from Europe, by Phillips (1991: 46 under the Petersen and Gordon (1994) reported that same name) for the mushroom in North eastern North American Megacollybia had not America, and Halling and Mueller (2005: 151, yielded to monokaryon culture, while spores as M. platyphylla) for the mushroom in Costa from EuroScandinavian collections not only Rica, it is a wonder that the same epithet has underwent in vitro spore germination, but been applied for such disparate morphological crossed in culture and exhibited a tetrapolar forms. The photo from Huffman et al. (1989: mating system. Combining these data with Fig. 93) may show yet another taxon from the morphology, it was suggested that a more thor- midsection of North America. ough investigation of Megacollybia sensu lato Singer (1986) and Smith (1960) considered could be profitable. Any study of Megacolly- that Tricholomopsis sheltered a complex of bia, whether morphological or molecular, must about 30 species [T. rutilans (typus generis), T. start with knowledge of western European decora, etc.] of brightly colored, centrally stipi- Megacollybia platyphylla since the epithet was tate agarics fruiting on wood, but included, adopted from Persoon. Material from west-cen- sometimes as a separate section Platyphyllae tral Germany should serve as the foundation for (Singer, 1986: 253), T. platyphylla (typus sec- comparison of other collections. In the litera- tionis). Singer (1986) listed several names ture, good descriptions of the western European under sect. Platyphyllae, most of which he con- organism have been furnished by Boekhout (in sidered to belong in other genera or were Bas et al., 1999), Breitenbach and Kränzlin unpublished. Oblique reference to South (1991) and Gulden (in Hansen and Knudsen, American species of Platyphyllae, “...confirm- 1992). ing the position of the whole group within Our project had the following objectives: 1) Tricholomopsis” (Singer 1986: 252, ftnt) fur- to investigate whether a single species epithet nished no data. could accommodate many collections of The conjecture that Tricholomopsis and Megacollybia platyphylla from across its pur- Megacollybia are separate entities has been sup- ported range; 2) to determine infrageneric ported by molecular data, most convincingly by clades and their relationships within

2 Megacollybia (Agaricales)

Megacollybia; 3) to explore whether phylogeo- Other herbarium acronyms are from Index graphic signal was present in a phylogenetic Herbariorum (http://sciweb.nybg.org/sci- reconstruction of the genus; 4) to propose and ence2/IndexHerbariorum.asp). TFB = number describe whatever additional taxa might be from Tennessee fieldbooks assigned to speci- necessary as a result of the study; and 5) to mens for documentation (i.e. notes, photos, explore the relationship of Megacollybia with etc.), supplanted by TENN numbers when Tricholomopsis fallax , a closely related accessioned into TENN herbarium. GSMNP = species reported from the North American Great Smoky Mountains National Park, strad- temperate western coast (British Columbia, dling the border between Tennessee and North Washington, Idaho, Oregon) and the dry Carolina, USA. In species descriptions, char- southwest (Arizona) . acters in Italics are considered diagnostic for Macro- and micromorphology were com- the taxon. bined with an ITS-based phylogenetic recon- Abbreviations of basidiospore statistics are struction to investigate collections from eastern as follows: x = number of spores measured; Q and western North America, Scandinavia, west- = spore length divided by spore width; Qm = ern and eastern Europe, western, central and far median Q value over the spore population; Lm = eastern Russia, east Asia, Central America and median spore length over the spore population. northern South America. Using the western Collections from which DNA sequences European organism as prototype, results were obtained are listed in Table 1. Specimens include: 1) the eastern North American organ- examined morphologically are listed under ism requires a new species epithet (M. each taxon in “Specimens examined.” rodmani); 2) temperate east Asia shelters a Basidiomata used for both purposes appear in species (M. marginata) closely related to but both places. distinct from the northern and western European organism; 3) northern South America Molecular protocols produces yet another species (M. fusca); and 4) Procedures for DNA extractions were there are at least three lineages represented described in Mata et al. (2007). PCR amplifica- across North America. Tricholomopsis fallax tion of the ribosomal ITS1-5.8S-ITS2 region falls within Megacollybia and transfer to was carried out with primers ITS1F and ITS4. Megacollybia is proposed. Thus, reflected in DNA sequencing followed manufacturer’s the ITS-based phylogeny, Megacollybia must directions for Big Dye Terminator mix (ABI) include at least seven species, with more to with forward primer ITS5 and reverse primer come as additional geographic areas are ITS4. Intermediate sequencing primers ITS2 explored. and ITS3 were used as necessary. Primers ITS2, ITS3, ITS4 and ITS5 are described in White et Materials and Methods al. (1990); primer ITS1F is described in Gardes and Bruns (1993). Cloning was accomplished Color notations in quotation marks are from with a Promega pGem-T vector and JM109 Ridgway (1912); those cited alphanumerically competent cells (Promega Corp., Madison, WI) (i.e. 7B3) are from Kornerup and Wanscher following manufacturer’s instructions when (1967). BF = bright field microscopy; PhC = direct sequencing did not resolve a sequence. phase contrast microscopy. TENN = herbarium Five clones per collection were sequenced. of the University of Tennessee, Knoxville. Restriction sites were determined by the GCG

3 K.H. HUGHES et al. Table 1 — GenBank Numbers for collections used in this study

4 Megacollybia (Agaricales)

5 K.H. HUGHES et al.

6 Megacollybia (Agaricales)

7 K.H. HUGHES et al. efers to clone number; ss refers to single spore isolate number; h refers to a haplotype number; GSMNP = Great Smoky Mountains National Park, USA; Kedrovaya Pad= Kedrovaya Pad Nature Reserve, Khasansky Dist., Primorsky Territory, Russia. Sequences not generated by authors M. Catherine Aime, Louisiana State University, DeptA. of Voikt, Plant P.O. Pathology Box & 2312, Crop RR#1, Physiology, Corner Baton Brook, Rouge, NL LA A2H 70803 2N2, Canada cr 1 2 3

8 Megacollybia (Agaricales) map program (GCG 2000). Restriction diges- using Mr. Bayes (Huelsenbeck and Ronquist tions with Nsi I were carried out according to 2000) with the following settings; nst = 6, manufacturer’s directions (Promega Corp., basefreq = estimate. The MCMC search was Madison, WI). run with 4 chains for 500,000 generations with sampling every 100 generations. The first Molecular Data Analyses 1,000 trees were discarded based on prelimi- Initial sequence alignments were carried out nary analyses showing that likelihood values using the “pileup” program in GCG which per- had reached stability with the first 1000 trees. forms UPGMA, followed by manual adjust- Posterior probabilities were estimated by sam- ments to the alignment. Gerronema and pling trees generated after likelihood values Clitocybula were selected as outgroups based diverged. For Bayesian analysis, the ca. 90bp on Moncalvo et al. (2002), and on blast similar- ITS1 insertion was included in the analysis. ity in GenBank. There was a large ca. 90bp variable insertion in the ribosomal ITS1 area Results that separated New World and Old World Megacollybia and data were analyzed with and Molecular analyses without this area by treating gaps as missing or Heterozygosity for indels was common in as a 5th base in parsimony analysis. No gap Megacollybia collections and some sequences coding was attempted. could not be resolved without cloning, especially Both Parsimony and Bayesian analyses were in collections from Texas, Arkansas and performed. Parsimony relies on the concept Kedrovaya Pad Nature Reserve in Russia. that the tree needing the fewest changes in state Heterozygosity was usually due to 1-2bp indels, along its branches is the best. Bayesian analysis often an increase or decrease in the number of searches for trees that are consistent with both base pairs in runs of a single base. Apparently, the data and with a selected model of evolution. there is considerable genetic diversity within this Maximum parsimony was performed using genus and hybridization of within-clade haplo- PAUP* (Swofford 2002). Characters were types is common. The average sequence diver- unordered and weighted equally. The number gence between clones of the same individual in of trees retained was limited to 1000; multistate Megacollybia was 0.423% (STD = 0.206). The taxa were interpreted as uncertainty; starting frequency of bp cloning errors was approximately trees were obtained via stepwise addition; addi- 0.15%. Haplotypes were always found in the tion sequence = furthest; the number of trees same clade in phylogenetic analyses. Cloning held at each step during stepwise addition = 1; was also necessary when specimens were conta- the branch-swapping algorithm was a tree- minated with a second fungus such as Candida bisection-reconnection (TBR); the steepest sp. (see under M. texensis below) descent option was not in effect. One-thousand Parsimony and Bayesian analyses produced bootstrap replicates were performed. seven major clades (Figs. 1a, b, 2). One of these Modeltest was used to determine the appropri- clades is morphologically and genetically subdi- ate model of evolution for Bayesian analysis vided into two additional clades (clades 8, 9, 10). (Posada and Crandall 1998). The model select- Species comprising each clade are described ed by Model Test was a general time reversible and named below. Parsimony analysis with the model with rate variation among sites ca. 90bp ITS1 insertion region included or (GTR+G). Bayesian analysis was performed excluded produced the same terminal taxa, as

9 K.H. HUGHES et al.

FIG 1a, b. One of 1000 most parsimonious trees of length 1230 steps. Boot strap support is given to the left of the node, followed by the Bayesian posterior probability. Of 914 total characters:All characters were unordered and of equal weight; 322 characters were constant, 93 variable characters were parsimony-uninformative, 499 characters were parsimony-infor- mative. Gaps were treated as “fifth base.” Numbers adjacent to clades refer to numbers in manuscript under “results.”

10 Megacollybia (Agaricales)

did Bayesian analysis with the insertion region (Fig. 1b) and grouping with Old World taxa included. The position of Megacollybia fallax, when the insertion was excluded in parsimony however, was unstable depending on the analy- analysis, or included in Bayesian analysis (Fig. sis, sometimes grouping with other New World 2). While the complex nature of gaps in this taxa when the insertion was included in analyses data set made gap-coding unfeasible, exemplars

11 K.H. HUGHES et al.

FIG. 2. Bayesian analysis using a GST+G model. Posterior probabilities are given to the left of the node.

of M. fallax, M. clitocyboidea and M. rodmani sequence homology) than to M. rodmani were extensively gap-coded and compared for (90.66% sequence homology). Given the sequence homology. These data suggest that M. sequence divergence for the ca. 90bp insertion fallax is closer to M. clitocyboidea (94.7% between M. fallax and other New World taxa

12 Megacollybia (Agaricales) and extensive small indels, the ambiguity is character, little else is unique or startling. Some understandable and sequences from other genes quantitative differences can be found, however, will ultimately be required to resolve the place- and these can be summarized as follows. ment of M. fallax relative to other Basidiome size and stature: Generally Megacollybia taxa. speaking, basidiomata across the genus are In temperate Asia, two taxa were identified: collybioid (M. platyphylla, M. marginata, M. M. clitocyboidea and M. marginata. texensis) or clitocyboid (M. clitocyboidea). Otherwise, Eurasia seems represented by one Three species are characterized by stouter, species (M. platyphylla). The New World sup- somewhat tricholomoid or russuloid basidioma- ports at least five species (M. fallax, M. texen- ta (M. fusca, M. rodmani f. murina, M. fallax). sis, M. fusca, M. subfurfuracea, M. rodmani), Pileus surface: Pileus surface of almost all with the likelihood of more to be revealed. basidiomata is characterized as fibrillose, deli- There is a possibility of further cryptic species cately radially streaked, or, in age, rimose. This in both M. platyphylla and M. rodmani based texture, when seen on juvenile basidiomata or on sequence divergence and morphology. the disc of mature basidiomata, may be so con- gested with pileipellis terminal cells as to Taxonomic characters appear minutely scabrous (especially M. clito- Rexer and Kost (1989a, b) carefully reported cyboidea, and in /costaricensis), or, if interrupt- and illustrated the various tissues of M. platy- ed, as minutely squamulose. In all cases, how- phylla. All the material studied was from ever, the pileus surface attains its radial streak- Germany, and there is no reason to doubt their ing over the limb. This streaking may become species identification. Their observations were somewhat diffuse toward the pileus margin, made in the context of what they considered a and can be mistaken for a striate margin. taxonomic alliance [M. platyphylla, In rain, the radial streaks may be somewhat Oudemansiella mucida, Xerula radicata (as detersile, and when interrupted, can appear squa- Oudemansiella) and Strobilurus esculentus mulose. Such secondary phenomena should not based, in part, on Redhead’s (1987) placement be mistaken for a common, truly squamulose of these genera]. The present paper considers appearance of the pileus disc. several putative taxa within Megacollybia, The radial streaks are produced by pigmented, however, and because distinguishing characters superficial, radially oriented surface hyphae. among taxa are more quantitative than qualita- These pigmented hyphae (ranging microscopi- tive, we consider it important to review taxo- cally from weakly olivaceous gray to dark oli- nomic characters in the genus, largely as a vaceous brown and macroscopically from guide to future considerations in identifying or olive-gray to olive-black) form differentiated describing specimens. terminal cells also oriented radially. These cells General considerations: The accompany- are consistently clavate, conspicuously ing phylogenetic reconstruction has been used clamped, occasionally transversely septate and as a guide to taxonomic delimitations. The can be quite thick-walled. task for morphology, therefore, has been to Intriguingly, the delicate radial streaks are attempt to separate the major (and sometimes often interrupted by small lenticular openings minor) clades into recognizable morphotaxa. (approx. 1Ð2 × 0.3Ð0.5 mm) revealing the white For the most part, this is very difficult, for tissue of the outer pileus trama. In numerous once geographic location has been dismissed as a specimens from worldwide locations, these

13 K.H. HUGHES et al. small openings were covered by small, deli- Interlamellar areas are usually smooth and cate, white cocoons, sometimes still containing hymenial, but occasionally can be transversely the larvae of an insect. The identity of the ridged or anastomosed. insect(s) is unknown, but the phenomenon rais- Two instances of marginate lamellar edges es the question of whether it is the insect larvae have been encountered. In one instance (M. which cause the small lacunae originally, or marginata), lamellae are delicately and abrupt- whether they merely take advantage of the ly marginate with a deep olive-brown or olive- opening (with the possibility of moisture and/or black edge. This is caused by strongly pigment- nutrients) for pupation. ed cheilocystidia. In the other instance (M. Pileus trama: Invariably, the pileus trama is platyphylla form A), the delicate lamellar edge white, remaining so on drying. Upon drying, it is purple (in dried material). Cheilocystidia is friable, not tough or woody or even compact. appear hyaline (BF), and the marginate appear- This texture is caused by the greatly inflated ance may be the result of exposure to frost or tramal cells (sometimes over 50 µm diam), perhaps contact. This condition, while distin- which can be thick-walled and appear orna- guishing a small clade, is probably not indica- mented on the inner wall surface. These hyphae tive of taxonomic rank. are rarely branched, are long-celled (cells often Basidia: Across the genus, basidial form and over 750 µm long) and only obscurely dimensions are quite uniform. Basidia fit gener- clamped. Interwoven with these inflated cells ally within 35Ð43 × 8–11 µm and although a are slender (2.5Ð5 µm diam), frequently few collections seemed to form long basidia, branched, frequently clamped, thin- to firm- these collections invariably were included in walled hyphae. This mixture of hyphal types clades of which the other members were more has been judged “sarcodimitic” (Corner, 1966) typical. All basidia are clavate, clamped, thin- a term whose meaning has been altered over walled, hyaline, 4-spored, with usually a few, the years (see Corner, 1991; Redhead, 1987; scattered, small, refringent guttules. Bas et al., 1990: 65Ð66). Basidioles: Dominating the hymenium Lamellae: Basidiomata of all taxa ordinarily together with scattered mature basidia, basidi- exhibit adnexed to adnate lamellae, usually oles are consistently digitate, sometimes with a significant tooth merging into the longi- gnarled, lobed or otherwise contorted, but never tudinal ridges of the stipe. Lamellae are subdis- mucronate or torpedo-shaped. tant, usually broad (up to 20 mm in some Basidiospores: Basidiospores throughout cases), but not remarkably thick. Although usu- the genus present little variation in size and ally described as white, lamellae are often shape, as descriptions of individual taxa will slightly tinted yellow-olive to pallid olive-gray. attest. All fit within a narrow range of dimen- Typically, lamellae are transversely streaked sions, 6Ð10 × 5Ð7 µm with similar Q and Lm with paler colors, and in drying, the outer por- values. Spores are thin-walled, ellipsoid to tion of lamellae turns taffy colored or light somewhat ovate, always somewhat flattened ochraceous buff, while the area closest to the adaxially, with a significant hilar appendage pileus attachment remains off-white with deli- and consistently inamyloid. Contents of cate streaks into the more mellow color. The mature spores show 1-few refringent guttules transverse streaking is sometimes pronounced which, in fresh material, can obscure accurate and accompanied by some relief as delicate observation of the spore wall. transverse ridges. Basidiospores left overnight in KOH swell

14 Megacollybia (Agaricales) significantly over the same spores freshly ously clamped and hyaline (BF; pigmented in mounted; they are also without guttules, and M. marginata). In some collections, cheilocys- decidedly more plump (subglobose rather than tidia were observed as transversely septate, but, ellipsoid to ovate). while a diminutive clamp connection was only Lamellar trama: In all examined basid- rarely seen at this septum, it is probably present iomata, the lamellar trama exhibits large areas and the cheilocystidial terminal cell is sessile of intricately interwoven hyphae, usually (not pedicellate) on the penultimate cell. Under termed “regular” (see Bas et al., 1999: 172). PhC, cheilocystidial contents appear somewhat Singer’s (1986: 251) report of “hymnophoral darker than basidial contents, but this may be a trama regular or subregular, consisting of rather function of condensed protoplasm; under BF long, sometimes even voluminous hyphae this feature is not visible, and cheilocystidia which are strongly interwoven to subparallel” (except those of M. marginata) are hyaline. appears as part of his description of Clamp connections: Previous literature Tricholomopsis and is not easily teased apart (Singer, 1986) has pointed out that clamp con- with recognition of Megacollybia. In at least M. nections in this complex (including, for exam- clitocyboidea, however, a distinct central plate ple, Clitocybula and Gerronema) are conspicu- or strand is differentiated, with intricately inter- ous. In some traditions, they would be known woven tissue between the central strand and as “medallion clamps,” for often a visible aper- subhymenium. The central stratum is broader ture can be seen in the crook of the “hook cell” toward the pileus context and narrower toward of the clamp. This feature is especially notice- the gill edge, often disappearing there. able when searching for subbasidial (or subhy- Vascular hyphae: Also known as gloeo- menial) clamp connections, where they are eas- plerous hyphae, these hyphae are somewhat ily demonstrated. They are also obvious on the refringent under PhC, are common in pileus slender hyphae of tramal tissue, but the inflated and lamellar tramae, and can be abundant. hyphae of these tissues exhibit smaller, more They are seen as meandering through otherwise obscure clamp connections on the constricted differentiated tissue (i.e. through the lamellar septa. These demonstrations, fortunately, are trama), and range from almost unbranched to unneeded, for all collections examined exhibit- frequently branched, but rarely septate and ed similar, conspicuous clamps. apparently rarely clamped (or without clamps). In examining for pileipellis terminal cells or These gloeoplerous hyphae are strongly cheilocystidia, the basal clamp is often disartic- cyanophilous, although cotton blue-lactic acid ulated, but the telltale angular base on these mounting medium is of little use for observing structures reflects that a clamp was present. diagnostic microstructures of basidiomata. Stipe surface: Hyphae of the stipe surface Cheilocystidia: Cheilocystidia were plenti- can be seen as two types: 1) a superficial net- ful in all basidiomata examined. Occasionally, work producing differentiated terminal cells; cheilocystidia protrude beyond basidia on the and 2) true surface hyphae. The superficial lamellar edge, and while noteworthy, this fea- network varies from an arachnoid reticulum to ture proved unreliable as a distinguishing char- a considerable (up to 25 µm thick) layer. In all acter. Cheilocystidia were typically broadly instances, the tissue comprises slender (2.5Ð5 clavate, variable in length and width, firm- to µm diam), thin- to firm-walled, conspicuously thick-walled (wall up to 1.0 µm thick, and then clamped, rarely branched, hyaline hyphae. usually irregular on inner surface), conspicu- The stipe cortical tissue is often coherent (disar-

15 K.H. HUGHES et al.

FIG. 3. Restriction digestions with Nsi I for collections from the Kedrovaya Pad Reserve in the Russian Primorsky dis- trict. M= Phi X Hae III digestion; Megacollybia marginata lanes 1 = 11859, 3 = 11860, 5 = 11861, 7 = 11862, 8 = 11869, 9 = 11888; Megacollybia clitocyboidea lanes 2 = 11832, 4 = 11864, 6 = 11868, 10 = 11884, 11 = 11887, 12 = 11893. Weak bands have been photographically enhanced.

ticulating as sheets of hyphal parts rather than as (1973) summarized the genus, with emphasis free hyphae), and composed of two hyphal on North America. types: 1) somewhat inflated, usually thick- walled hyphae never as inflated as those of 2. Megacollybia clitocyboidea [Figs. 1A, 2 Ð pileus or lamella trama; and 2) slender, more clade 2, Figs. 4Ð7] frequently branched, longitudinal hyphae. Little Several collections of putative M. platyphylla variation was seen in this tissue. were gathered recently from Kedrovaya Pad The terminal cells produced by the superficial Nature Reserve south of Vladivostok in far external layer are repent to somewhat bent out- eastern Russia. ITS sequences showed that ward, and range from narrowly to broadly basidiomata belonged to two distinct clades: clavate. They could be termed caulocystidia and, one related to EuroScandinavian M. platyphylla except for some differences in dimensions, are (viz. M. marginata), the other to a haplotype considered hardly worthy of taxonomic rank. unique to temperate eastern Asia (viz. M. clito- cyboidea). The latter clade is sister to all other Discussion and naming of taxa Megacollybia taxa and would seem to be basal in the genus. Restriction digests of the ITS 1. Outgroups [Figs. 1A, 2 Ð clade 1] region with Nsi I, were carried out on all Species of Clitocybula and Gerronema were Kedrovayan collections and conclusively used as outgroups in this study. A recent paper showed that there was no interbreeding by Barrasa et al. (2006) furnishes a key to between the two clade genotypes (Fig. 3). species of Clitocybula in Europe and North Thus, M. marginata and M. clitocyboidea America, and the contribution by Bigelow appear to be two distinct biological species.

16 Megacollybia (Agaricales)

Because basidiomata are thought to resemble ored but increasingly radially streaked with those of Clitocybe, one of the Asian species has olive-brown to olive-black pileipellis hyphae been named M. clitocyboidea. on the yellowish background so that at pileus margin there is no evidence of yellow pig- Megacollybia clitocyboidea R.H. Petersen, ments; margin inrolled when young, becoming S. Takehashi & Nagas. sp. nov. extended with age, not striate, concolorous Pileus 35Ð108 mm latus, convexus ad planus with limb (Ð6D4), often eroded, becoming vel depressus, brunneus ad fusco-brunneus, fib- inrolled upon drying. Pileus flesh thin, up to 4 rillosus ad subrimosus. Lamellae adnatae, unci- mm thick over stipe, off-white, almost absent natae ad subdecurrentes, latae, lacteae ad outward, soft, becoming friable upon drying. eburneae. Stipes 50Ð130 × 3Ð12 mm, equalus Lamellae adnexed to adnate to shallowly ad sursum decrescens, brunneus ad olivaceo- decurrent, subdistant to distant, thin (and brunneus. Pileocystidia 33Ð136 × 6–22 µm, parchment-like when dry), ivory colored, “pale clavata, olivacea, fibulata, levis. Sporae 6Ð9.5 × olive buff” (3B2), by maturity “pale cinnamon 5–7.5 µm (n = 167; Q = (1.00Ð)1.07Ð1.60; Qm = pink” (5A2) to “pearl gray” (23B2), ventricose, 1.24; Lm = 7.28 µm), lato-ellipsoideae ad lato- up to 12 mm deep, on drying becoming light ovatae, levae, tenuitunicatae, hyalinae. Basidia ochraceous buff, very brittle and delicately 28Ð50 × 7–11 µm, clavata, tenuitunicata, fibu- transversely streaked as though hygrophanous lata, tetraspora. Pleurocystidia nulla. (but not so); interlamellar surface often intri- Cheilocystidia 20Ð65 × (6–)9–17 µm, angusto- cate cross-ribbed outward, mostly translucent, ad lato-clavata, fibulata, tenuitunicata. showing color of pileus surface; lamellar edge Caulocystidia 30Ð158 × 10–15 µm, pedicellata not marginate. Stipe central, 50Ð130 mm long, ad elongate capitulata, pallidobrunnea. Asia 3Ð12 mm broad apically, 4Ð20 mm broad near occidentalis, temperata. base, smooth to silky-shining, or at times pru- Holotype: JAPAN, Hokkaido, Sorachi inose-scaly downward, off-white apically, sor- Prov., Iwamizawa-shi, Tonebetu, 30.VIII.2005, did pallid fuscous downward, “buffy brown” coll. S & C Takehashi, ST 3 (TENN 62231). (6D4) when young, paler by maturity [“pale Basidiomata (Figs. 4Ð5) collybioid to cli- vinaceous fawn” (17A2), downward near tocyboid, occasionally large and tricholoma- “light drab” (17B2), “pale pinkish buff”, toid. Pileus 35Ð108 mm broad, convex when “deep olive buff” (3C3)], occasionally with young, expanding to plane and shallowly to reluctant reddish-brown staining; base always deeply depressed, at times weakly or distinctly somewhat enlarged, sometimes geniculate, papillate-umbonate,”mummy brown” (6F8) to with very tight, thin whitish tomentum; rhi- “clove brown” (6F5) at first, later discoloring zomorphs occasional, white, ropy. Taste and to grayish brown to yellowish brown (“buffy odor not distinctive. brown;” 6D4-5D3-4) with center often remain- Habitat: China, rotten wood, mixed forest; ing dark brown to blackish; disc surface Japan, in hardwood forests or rarely in bamboo minutely scabrous or furfuraceous with minute stands, on and around rotten stumps, or on the olive-black, olive-brown to fuscous brown ground on rotten wood or roots; Russia, very scabers almost solid in center, becoming less rotten hardwood logs. frequent outward and less discrete and nearly Pileipellis a repent layer of slender, radial obscure over outer limb; pileus limb surface hyphae; hyphae 3Ð4.5(–8) µm diam, smooth becoming smooth outward, basically ivory col- (no evidence of incrustation in KOH), firm-

17 K.H. HUGHES et al.

FIGS. 4, 5. Megacollybia clitocyboidea, basidiomata. Fig. 4. TAK 3. Fig. 5. TAK 1. Fig. 4 = 1.8×. Fig. 5 = 1×. walled (wall up to 0.4 µm thick), conspicuously µm, subflagelliform, digitate, clavate, inequilat- and frequently clamped, often (but not invari- erally clavate, vermiform to subcapitulate, ably) cytoplasmically pigmented (olive, dull often curved, conspicuously clamped, cytoplas- brown or yellowish brown; KOH, BF), often mically pigmented (olive; usually strongly), producing upturned, inflated terminal (and often smooth, firm-walled, occasionally hyaline and subterminal) cells; pileipellis terminal cells in rare instances inflated up to 38 µm broad, (pileocystidia; Fig. 6A, B) 33Ð126 × 6Ð22(Ð31) broadly clavate. Pileus trama constructed of

18 Megacollybia (Agaricales)

FIG. 6. Megacollybia clitocyboidea. Pileipellis terminal FIG. 7. Megacollybia clitocyboidea. A. Basidia. B. cells. A. TAK 3. B. TAK 2. Standard bar = 20 µm. Basidiospores. C, D. Cheilocystidia. A, B, D. BAU 5343. C. Tak 3. Standard bar = 20 µm. two hyphal types: 1) inflated up to 45 µm diam, neous, occasionally with 1Ð3 amorphous (but thin- to thick-walled (wall up to 1.0 µm thick, well-defined) dark inclusions (PhC). Stipe sur- irregularly thickened and appearing mottled or face near apex an arachnoid layer of slender banded; PhC), long-celled, constricted at septa, [2.5–4(–7) µm diam] hyphae producing repent inconspicuously clamped, hyaline; and 2) slen- or out-turned terminal cells (caulocystidia) der (3–8 µm diam), firm-walled, hyaline, fre- 30Ðmore than 158 × 10–15 µm with narrowly quently branched, frequently clamped, smooth, rounded to subcapitate ends. Inner tissue (per- not mottled. Basidia (Fig. 7A) 28Ð50 × 7Ð11 haps 50–100 µm deep): hyphae similar, µm, clavate, thin-walled, conspicuously 7–13(–24) µm diam, hyaline, coherent, clamped. clamped, 4-spored (perhaps rarely 2-spored), Stipe flesh constructed of two hyphal types, hyaline; contents homogeneous or 1-few-gut- both hyaline, both clamped, both longitudinally tulate; basidioles 25Ð45 × 5Ð10 µm, clavate. arranged, both free (not adherent): 1) inflated Basidiospores (Fig. 7B) 6Ð9.5 × 5–7.5 µm [n = hyphae 8–more than 30 µm diam, thick-walled 167; Q = (1.00Ð)1.07Ð1.60; Qm = 1.24; Lm = 7.3 (wall up to 1.5 µm thick, long-celled, constricted µm]), broadly ellipsoid to broadly ovate, flat- at septa, irregular in outline and somewhat mot- tened adaxially, smooth, thin-walled, hyaline; tled, especially in wider individuals); and 2) contents homogeneous to 1-few-guttulate. slender, 6–13 µm diam, frequently branched and Cheilocystidia (Figs. 7C, D) not or rarely pro- frequently clamped, firm-walled. truding beyond basidia, 20Ð65 × (6–) 9–17 µm, Commentary: Characters which separate digitate, narrowly or broadly clavate or this Asian taxon from others include: 1) grayish sphaeropedunculate (rarely with short apical brown, dark olive-brown to olive-black pileus outgrowths), occasionally transversely septate, (at least pileus disc); 2) minutely scabrous or conspicuously clamped (but often disarticulated furfuraceous pileus disc; and 3) cheilocystidia from clamp), thin-walled; contents homoge- with thin walls (not firm-walled). The other

19 K.H. HUGHES et al.

Asian taxon known to us is M. marginata, Antu Co., Beihe, forest behind fire tower, immediately recognizable by its delicately mar- 14.VIII.1988, coll. R.H. Petersen (as ginate lamellae. Megacollybia sp.), TFB 1451 (TENN 48647); In M. clitocyboidea, cheilocystidia are often Jilin Prov., Fusong Co., Lushuihe, 26.VII.2006, difficult to distinguish from basidia. Under coll. & det. T. Bau (as Oudemansiella platy- PhC, basidia are usually paler and include phylla), Bau 5343 (TENN 62229); same loca- small guttules, while cheilocystidia appear tion, 28.VI.2005, coll. & det. T. Bau, HMJAU empty and are usually uniformly dark. 4024 (TENN 62230). JAPAN, Honshu, Tottori The aquarelle in Imazeki and Hongo (1965: Pref., Tottori-shi, Koyama, 16.VI.2007, coll. M Fig. 33, as Tricholomopsis) and the color pho- Yoshimura, det. E Nagasawa (as M. tographs in Imazeki et al. (1988: 117, as platyphylla), EN 07-27NR (TMI); Tottori-shi, Oudemansiella) seem to represent M. clitocy- Hamasaka (near Tanegaike Pond), 26.VI.2007, boidea both in color (dull neutral brown pileus, coll. M Kanbe, det. E Nagasawa (as M. platy- stipe off-white apically, sordid pallid fuscous phylla), EN 07-30 (TMI); Tottori-shi, downward), and stature (depressed pileus disc). Tatsurami, 9.IX.2007, coll. Yukihiro Nishio, DNA of TENN 60766 showed it to be the det. E Nagasawa (as M. platyphylla), EN 07- product of two haplotypes, differing in a few base 27NR (TMI); Hokkaido, Ishikari Prov., pairs. One of the haplotypes was identical to that Sapporo-shi, Mt. Moiwa, 31.VII.2005, coll. T. of TENN 62230, while the other did not match Takazawa, ST 2 (TENN 62232); Hokkaido, any other haplotype of M. clitocyboidea. Sorachi Prov., Iwamizawa-shi, Tonebetu, Likewise, TENN 62232 was the product of two 30.VIII.2005, coll. S & C Takehashi, ST 3 haplotypes, one identical to several other homozy- (TENN 62231, holotype); same location, gous collections, but the other unique. TENN 30.VIII.2005, coll. S & C Takehashi, ST 1 62232 and TENN 62229 also were heterozygous, (TENN 62233). RUSSIA, Primorsky Territory, with similar patterns. Such results strongly indi- Khasansky Dist., Kedrovaya Pad Nature cate that hybridization is occurring among popula- Preserve, N 43û 05.869’, E 131˚ 33.403’, tions of M. clitocyboidea. Conversely, there is no 17.VIII.2005, coll. RHP, TFB 11832 (TENN evidence of hybrids between M. clitocyboidea and 60717); same location, N 43û 06.074’, E 131û M. marginata. 33.125’, 20.VIII.2005, coll. R.H. Petersen (as Petersen and Gordon (1994) noted that M. platyphylla), TFB 11864 (TENN 60748); European M. platyphylla emitted a pleasant, floral same location, N43û 07’ 01”, E 131˚ 29’ 14”, perfume in culture. TENN 60766 (M. clitocy- 22.VIII.2005, coll. O. Morozova, TFB 11884 boidea, Russia) produced a pleasant, citrus-like (TENN 60766); same location, 22.VIII.2005, odor in culture, absent from the few other cultures coll. A. Kovalenko (as M. platyphylla), TFB of Asian Megacollybia collections. 11893 (TENN 60774). Vassilyeva (1973: 126) reported Oudemansiella platyphylla from the Primorsky Region of far east- 3. Megacollybia marginata [Figs. 1A, 2 Ð ern Russia, but which of the taxa in the area (or clade 3, Figs. 8, 9] perhaps more than one) was described remains Several collections of solitary Megacollybia open to question. With pileus 8Ð14 cm broad and basidiomata were gathered south of stipe 8Ð10 × 1Ð2 cm, perhaps larger basidiomata Vladivostok, Russia, in 2005. The only separat- were intended (i.e. EN 07-27NR; TAK 1). ing character in the field was the delicately Specimens examined: CHINA, Jilin Prov., marginate lamellae of one group of collections,

20 Megacollybia (Agaricales) versus the non-marginate lamellae of the other. 20.VIII.2005, coll. O Morozova (as M. platy- Since that time, additional collections of the phylla), TFB 11869 (TENN 60752). non-marginate taxon have been examined and Basidiomata collybioid or clitocyboid. sequenced (see under M. clitocyboidea), but the Pileus 35Ð85 mm broad, plane to shallowly or marginate-gilled taxon remains known only strongly depressed, occasionally with small from the original collections. Both taxa appear umbo; disc dark fuscous brown (“sepia,” 4F8, to be new. “bister,” 5F8, “clove brown,” 6F5), usually The extent of distribution of M. marginata is without scabers or squamules (but occasionally unknown but Imai (1938: 116) reported under minutely scabrous or minutely hispid), becom- the name Collybia platyphylla a fungus with ing glabrous-shining and “olive brown” (5E5) “lamellae ..., edge wavy or subserrate and tinged upon drying, densely radially streaked; limb with dark-brown color” from Hokkaido and somewhat lighter (5D3) in color, sometimes Kuriles (Kunashiri Is.). These phylogeographical with small lenticular scars revealing white data resemble findings by Aanen et al. (2000) in flesh; margin thin, not striate, usually down- European and American Hebeloma velutipes, turned to gently inrolled. Lamellae adnate with Matsumoto et al (2005) with Pleurocybella por- insignificant tooth, ventricose, close to subdis- rigens and Terashima et al. (2006) with tant, up to 13 mm broad, distinctly olivaceous Armillaria mellea subsp. nipponica. when young (“deep olive buff”), becoming off- white by maturity (“pale pinkish cinnamon”), Megacollybia marginata R.H. Petersen, ochraceous buff with some delicately trans- Morozova & J.L. Mata, sp. nov. verse streaking of pale ochraceous buff, mostly Pileus 35Ð85 mm latus, planus ad leviter curled upon drying, obscuring lamellar edge; depressus, atrofuscus ad atrobrunneus, radio- lamellar edge delicately but distinctly mar- fibrillosus, siccus. Lamellae adnatae, ventri- ginate to dark brown. Stipe 50Ð100 mm long, cosae, ad 13 mm latae, albo-olivaceae ad 3Ð6 mm broad in midsection, somewhat eburneae, margine delicate atrobrunneo. Stipes expanded apically and basally (up to 12 mm 50Ð80 × 10Ð12 mm, equalus ad sursum broad) and there off-white, terete, pallid oliva- decrescens, olivaceoalbus ad pallide griseus. ceous to drab in midsection (“deep olive buff”), Pileocystidia 35Ð96 × 7–15(–25) µm, elongate- significantly lined or ridged, occasionally stain- clavata, olivacea ad atro-olivacea, tenui- ad ing rufous in the field, drying to dark neutral crassitunicatae, fibulata. Sporae 6.5Ð10 × 5Ð7 brown (upward “olive brown,” downward µm (Q = 1.11Ð1.54, Qm = 1.30; Lm = 8.53 µm), somewhat paler, “tawny olive”, 5C5). subglobosae ad late ellipsoideae ad ovatae, lae- Rhizomorphs occasional, white, ropy. Odor vae, tenuitunicatae, hyalinae. Basidia 30Ð41 × and taste none. 7–10 µm, clavata, tenuitunicata, fibulata, Habitat: Apparently endemic to far eastern tetraspora. Pleurocystidia nulla. Cheilocystidia Russia; on or near very rotten wood of Alnus (?). 31Ð65 × 7–15 µm, clavata ad late clavata, fibu- Pileipellis terminal cells (Fig. 8A) 35Ð96 × lata, tenui- ad crassitunicata. Caulocystidia 7–15(–25) µm, mostly long-clavate, prostrate to (20Ð)35Ð80 × 6–14 µm, clavata ad subventri- semi-erect, single or in fascicles, thin- to firm- cosa, prope hyalina. Rossica orientalis. walled, conspicuously clamped, moderately to Holotype: RUSSIA, Primorsky Reg., strongly pigmented (especially distally); con- Khasansky Dist., Kedrovaya Pad Nature tents homogeneous. Pileus outer flesh con- Reserve, N 43˚ 06’ 37”, E 131˚ 31’ 31”, structed of two hyphal types: 1) 13–65 µm

21 K.H. HUGHES et al.

FIG. 8. Megacollybia marginata, TENN 60752. FIG. 9. Megacollybia marginata, TENN 60752. A. A. Pileipellis terminal cells. B. Basidia. Standard bar Basidiospores. B. Cheilocystidia. C. Caulocystidia. = 20 µm. Standard bar = 20 µm. diam, irregularly thick-walled (wall up to 1.5 hyaline, constricted somewhat at septa in µm thick), obscurely blotched or perhaps orna- broader hyphae; wall up to 2 µm thick. mented, hyaline, constricted at septa, obscurely Caulocystidia (Fig. 9C) (20Ð) 35Ð more than clamped; and 2) 3.5Ð6 µm diam, thin-walled, 80 × 6–14 µm, pedicellate, narrowly clavate to frequently branched, infrequently but conspicu- subsphaeropedunculate, uncommonly with api- ously clamped, interwoven among wide cal mammiform or strangulate outgrowth, firm- hyphae. Basidia (Fig. 8B) 30Ð41 × 7–10 µm, 4- to thick-walled (wall never more than 0.7 µm spored, clamped, thin-walled; contents with a thick), conspicuously clamped, subhyaline; few scattered guttules; basidioles similarly contents homogeneous. shaped as basidia, not mucronate. Basidiospores Commentary: Phylogenetically, the temper- (Fig. 9A) 6.5Ð10 × 5–7 µm (n = 65, Q = ate Asian marginate (M. marginata) and non- 1.11Ð1.54, Qm = 1.30; Lm = 8.53 µm), broadly marginate (M. clitocyboidea) forms are only ellipsoid, rarely subglobose, flattened adaxially, distantly related, with the marginate form thin-walled, smooth, hyaline in KOH, inamy- closely related to the typical European M. loid; contents 1-few guttulate. Cheilocystidia platyphylla. Nonetheless, the two temperate (Fig. 9B) abundant, protruding beyond basidia Asian taxa covered here are almost indistin- singly or in 2-3s, 31Ð65 × 7–15 µm, nearly ses- guishable morphologically except for the very sile (rarely) to long-pedicellate, clavate to delicate brown or black lamellar edge. Even broadly clavate, rarely with 1-2 apical out- then, basidiomata of TENN 60769, belonging growths, subhyaline singly, pale pinkish to pale in the clade of marginate collections, is not so, tan (100×, BF) in mass, conspicuously and is thus virtually indistinguishable from M. clamped, thin- to firm-walled. Stipe tissues clitocyboidea. Whether this constitutes evi- composed of parallel hyphae, 4–20 µm diam, dence of hybridization between the two forms

22 Megacollybia (Agaricales) or population variability remains unknown. in placing the species. Also implied in the Cheilocystidia are clearly pigmented in list are differing generic concepts by various mass, causing the marginate lamellar edge. authors, often stretched to accommodate this Microscopically, however, the cytoplasmic pig- species. Placement is especially anachronis- ment is immediately bleached by KOH (not as tic in Oudemansiella and Hydropus, both drastically in H2O), and if not viewed within a characterized by prominent cystidia not few seconds, appears almost totally hyaline. found in Megacollybia. Singer’s (1939, Two specimens currently listed in M. 1986) and Smith’s (1960; Smith et al., 1979) platyphylla form B, a collection from the concept of Tricholomopsis co-opted T. platy- Czech Republic (TFB7206/TENN58493) and phylla, but Malençon and Bertault’s (1975) one from Terebda (LE202274) have sequences placement most closely approximates the related to (but not the same as) M. marginata phylogenetic placement of M. platyphylla by but lack marginate gills. These collections may Moncalvo et al. (2002).

represent an intermediate taxon between M. marginata and M. platyphylla. Megacollybia platyphylla＾ (Pers.: Fr.) Kotl. Specimens examined: RUSSIA, Primorsky & Pouzar. 1972. Ceska Mykol. 26: 220. Region, Khasansky Dist., Kedrovaya Pad Basionym: Agaricus platyphyllus Persoon: Nature Reserve, N side of Kedrovaya River, N Fries. 1821. Systema Mycologicum 1: 117. 43˚ 05.869’, E 131˚ 33.418’, 19.VIII.2005, [≡ Agaricus platyphyllus Persoon. 1796. coll. A. Kovalenko, TFB 11859 (TENN Observ. Mycol. 1: 47; pre-validation date] 60743); same data, coll. A. Kovalenko, TFB ≡ Collybia platyphylla (Pers.: Fr.) P. Kumm. 11860 (TENN 60744); same location, N 43û 1871. Führ. Pilzk. 117. 06.074’, E 131˚ 33.125’, 20.VIII.2005, coll. ≡ Tricholomopsis platyphylla (Pers.: Fr.) R.H. Petersen (as M. platyphylla), TFB 11861 Singer. 1939. Schweiz. Zeitschr. Pilzk. 17: 13. (TENN 60745); same data, coll. R.H. Petersen, ≡ Clitocybula platyphylla (Pers.: Fr.) TFB 11862 (TENN 60746); same data, coll. Malençon & Bertault. 1975. Trav. Inst. Sci. R.H. Petersen (as M. platyphylla), TFB 11869 Cherifien, ser. bot. 33: 398 (nom. inval.); C. (TENN 60752, holotype); same location, S side platyphylla (Pers.: Fr.) E. Ludwig. 2001. in of Kedrovaya River, N 43˚ 07.01’, E 131˚ Pilzkompendium. Bd. 1: 58. 29.14’, 22.VIII.2005, coll. O. Morozova, TFB ≡ Hydropus platyphyllus (Pers.: Fr.) Kühner. 11888 (TENN 60769). 1980. Bull. mens. Soc. Linn. Lyon, num. spec. 49: 895. 4. Megacollybia platyphylla [Figs. 1A, 2 Ð ≡ Oudemansiella platyphylla (Pers.: Fr.) M. clade 4, Figs. 10-14] M. Moser. 1983. in Gams, Kleine Collections conforming to the original con- Kryptogamenfl., rev. ed. 5 IIb/2: 156. cept of M. platyphylla seem distributed through- Exemplar: GERMANY, Mecklenberg- out Europe, Scandinavia and even to south- Vorpomme, Mecklenburg-Strelitz, Burg western Siberia (Novosibirsk Region and Altai Stargar, N 53˚ 31’ 7.6”, E 13˚ 18’ 56.5”, Republic). Of all the major infrageneric clades, 24.V.1999, coll. R. H. Petersen & K. W. /platyphylla seems the most homogeneous Hughes, TFB 10315 (TENN 57963). morphologically as well as phylogenetically. Basidiomata (Figs. 10, 11) collybioid. A summary of typonyms for Agaricus Pileus 40Ð85 mm broad, campanulate when platyphyllus reveals the historical difficulties young, becoming plane to slightly raised over

23 K.H. HUGHES et al.

FIGS. 10, 11. Megacollybia platyphylla, basidiomata. Fig. 10. TFB 11637. Fig. 11. TFB 11570. Numerals = 4 mm.

24 Megacollybia (Agaricales) the disc when mature, radially fibrillose to deli- cately radially rivulose; disc usually minutely scabrous, furfuraceous, minutely squamulose or densely radially streaked, “buffy brown” (6D4), “deep olive” (4E6), “dark olive” (4F5), “olive brown” (5E5), “hair brown” (6E3), out- ward radially fibrillose to radially streaked, 5B3 to 5C4 with olive tint, “deep olive buff” (3C3), “olive buff” (3B3), “tawny olive” (5C5) to “sayal brown” (6C5), occasionally signifi- cantly paler and without scabrous or furfura- ceous disc (i.e. TENN 59308); flesh solid, off- white, sometimes with hints of “wood brown” (7C4) where bruised; margin not striate, thin, often eroded, becoming inrolled upon drying. Lamellae adnate to adnexed, thick, subdistant, ventricose (Ð13 mm broad), segmentiform, white when fresh, in age “tilleul buff” (7B2), “vinaceous buff” (9B2), “pale olive buff” (3B2), “pale ochraceous buff” (4A- B2), FIG. 12. Megacollybia platyphylla, TENN 57963. A, “cream color” (3A3), “pale pinkish cinnamon” pileipellis terminal cells. B. basidia. Standard bar = 20 µm. (6A2), drying very brittle; lamellar edge not marginate, sometimes slightly paler than lamel- lar face. Stipe 45-80 mm long, 5-8 mm broad producing repent to upturned terminal cells; apically, more or less equal, terete, usually terminal cells (Fig. 12A) 44Ð128 × 8Ð21 µm somewhat enlarged downward, lined or “mus- (at widest point), narrowly clavate, clavate to cled” (i.e. russuloid), expanded apically and broadly clavate, occasionally sphaeropeduncu- expanded at base, terete, off-white to “pale late, rarely apically mucronate or apically olive buff” (3B2) apically, downward “deep lobed, rarely transversely septate, firm-walled, olive buff” (3C3), “dark olive buff” (3C5), conspicuously clamped, cytoplasmically pig- 4B2, “pale ochraceous buff” (4A2) to “light mented olive. Pileipellis near margin of ochraceous buff” (5A4) where handled. Odor mature basidiome similar to disc; terminal cells and taste not distinctive. Rhizomorphs white, (48Ð) 68Ð151(Ð168) × 7–19 µm (at widest round in cross-section, only occasionally point), thickly imbricate to scattered, elongate- branched, with small resupinate attachments to digitate, narrowly clavate to clavate, rarely substrate evident. transversely secondarily septate, firm-walled, Habitat: Europe, Scandinavia, western mostly olive, but some hyaline, conspicuously and central Russia; on well-rotted wood, clamped; contents weakly to moderately pig- including Fagus. mented with some hyaline individuals with Pileipellis over pileus disc a superficial subtly mottled appearance (PhC). Pileus trama layer of intricately, tightly interwoven, slender hyphae of two types: 1) inflated, long-celled (2–4 µm diam), thin-walled, conspicuously hyphae 13–28 µm diam, firm- to thick-walled clamped, weakly to strongly pigmented hyphae (wall up to 1.0 µm thick, and then irregular on

25 K.H. HUGHES et al.

× (4–)5.2–7(–9) µm [n = 310, Q = (1.00Ð)1.05Ð1.67(Ð1.73), Qm = 1.26, Lm = 8.16 µm], subglobose, broadly ellipsoid to ovate, somewhat flattened adaxially, hyaline, inamy- loid, acyanophilous, often in small clumps of - 6 individuals; wall thin, smooth; contents uni- or multiguttulate. Lamellar edge fertile, with scattered cheilocystidia often protruding beyond basidia; cheilocystidia (Fig. 13B) (20Ð)30Ð75(Ð86) × 6Ð22(Ð28) µm, pedicellate to long-pedicellate, clavate to sphaeropeduncu- late, rarely fusoid-ventricose or with slender, digitate apical outgrowths, conspicuously clamped, occasionally transversely septate toward base (with or without clamp connec- tion), thin- to occasionally firm-walled; con- tents homogeneous, hyaline (BF); abundant FIG. 13. Megacollybia platyphylla, TENN 57963. A. gnarled, often branched cystidioles ca 3 µm Basidiospores. B. Cheilocystidia. C. Caulocystidia. diam, conspicuously clamped occasionally pre- Standard bar = 20 µm. sent. Occasional cheilocystidia produced up to 100 µm away from lamellar edge. Stipe sur- face hyphae near stipe apex superficial, slender inner surface), obscurely clamped, hyaline; and (2.5–5 µm diam), interwoven but longitudinal, 2) 4–7 µm diam, frequently clamped, frequent- producing caulocystidia (Fig. 13C) ly branched, interwoven among inflated (16Ð)32Ð147 × 6–12(–16) µm (at widest point), hyphae. Lamellar trama irregular, apparent- clavate to subsphaeropedunculate, slender, ly sarcodimitic, of two hyphal types: 1) repent to out-turned, hyaline, firm-walled, con- inflated up to 27 µm diam, long-celled, thin- spicuously clamped. Stipe trama “sar- walled, obscurely clamped, hyaline, often codimitic,” of two hyphal types: 1) up to 28 µm with inner surface of wall irregularly thick- diam, firm- to thick-walled (wall up to 2 µm ened; and 2) 3–4 µm diam, sinuate to tortu- thick), obscurely clamped, long-celled; and 2) ous, interweaving hyphae, conspicuously and 3–5 µm diam, frequently branched, conspicu- frequently clamped, hyaline, thin-walled. ously clamped, thin-walled, hyaline, interwo- Numerous subrefringent vascular hyphae ven. Scattered vascular hyphae, 5–8 µm diam, apparently modified from the slender tra- thin-walled, subrefringent, aseptate, meander- mals, with no clamps observed. Hymenium ing (not strictly longitudinal), strongly composed of basidia and basidioles; basidia cyanophilous. Rhizomorphs constructed of two (Fig. 12B) (28Ð)32Ð48(Ð55) × (6–)8–12 µm, types of hyphae, tightly packed, longitudinal, clavate, four-spored; contents with a few free (not coherent): 1) generative, 5Ð11 µm scattered, small, refringent (PhC) guttules; diam, thin-walled, hyaline, frequently septate, basidioles 32Ð44 × 6–8 µm, digitate to nar- ?clamped, interwoven; and 2) skeletal, 1.5–3 µm rowly clavate, slender and often sinuate. diam, thick-walled (wall 1 µm thick to obscuring Basidiospores (Fig. 13A) (6.0Ð)6.8Ð9.5(Ð10) cell lumen), not cyanophilous, ?aseptate or

26 Megacollybia (Agaricales) arising from a clamp connection. Outer surface contained multiple alleles. The collections used of rhizomorphs a tomentum of tightly interwo- in those intercollection pairings included TFB ven, pseudoparenchymatous cells involving 11569, which, although intercompatible with bits of substratum. four other Finnish specimens, is somewhat dif- Commentary: Literature indicates that pleuro- ferent molecularly. cystidia are rare or absent, and we have observed In a separate set of pairings, four collections cheilocystidia (with similar form to lamellar edge from Euroscandia (TFB 4252, Sweden; TFB structures) formed some distance away from the 1147, Austria; TFB 10813, TFB 10819, both lamellar edge. Boekhout (in Bas et al., 1999) Russia) were found to be totally or partially reported “pleurocystidia absent;” his description intercompatible. and illustration of cheilocystidia match the struc- In addition, Petersen and Gordon (1994) tures we have seen. Gulden (in Hansen and reported that European cultures emitted a pleas- Knudsen, 1992) wrote “Cheilocystidia prominent ant, floral perfume, while the North American ... lageniform ... pleurocystidia few or absent.” cultures were without odor, a further indication The illustration matches the structures we have that the two taxa were distinguishable. seen. Rexer and Kost (1989a) did not report the Basidiomatal differences, while qualitative, presence of pleurocystidia. were also noted. It appears that fertile basidia collapse as soon Numerous additional German specimens as spores are discharged and so are hardly visi- were examined by Rexer and Kost (1989a). ble thereafter. Scarcity of fertile basidia may be Specimens examined: AUSTRIA. Lower reflected in the often rare basidiospores. Austria, Waldviertel, Forest vic. Dobrasperre, 48û Except for the preponderance of immature 38’ N, 15˚ 48’ E, 24.IX.2001, col. R.H. Petersen hymenium, TENN 57963 can act as an exem- & H. Vogelmayr TFB 11447 (TENN 59308); plar for the true (European) concept of M. Lower Austria, Vienna, 23rd District, platyphylla. It comes from generally the right Mauerwald, N 48˚ 9.004’, E 16˚ 15.087’, place, and has basidiomata of most ages plus 28.IX.2001, coll. E. Grand, TFB 11471 (TENN well-developed rhizomorphs. It conforms well 59338). FINLAND, Etelä-Häme, Liesjärvi to the European literature. Basidiomata are usu- National Park, trail from Korteniemi, 6.VIII.2002, ally smaller than those of eastern North coll. S.A. Redhead, TFB 11572 (TENN 59523); America, which also have a somewhat different same location, 6.VIII.2002, coll. E. Grand, TFB color scheme. 11570 (TENN 59524 PHOTO). GERMANY, Petersen and Gordon (1994) described early Mecklenburg-Vorpomme, Mecklenburg-Strelitz, attempts to culture M. platyphylla from single Carpin, Müritz Nat. Park, N 53˚ 20’ 35.7”, E 13˚ basidiospores, reporting that the European repre- 11’f 40.5”, 25.V.1999, coll. K.W. Hughes, TFB sentatives cultured easily, but that the eastern 10319 (TENN 57967); same location, 25.V.1999, North American entity, while germinating in coll. D. Krüger, TFB 10512 (TENN 57442); vitro, did not progress to a mycelial state, and Mecklenberg-Vorpomme, Mecklenburg-Strelitz, therefore could not be self-crossed (but see Burg Stargar, N 53˚ 31’ 7.6”, E 13˚ 18’ 56.5”, under M. rodmani). J. L. Mata (unpubl. data) 24.V.1999, coll. R.H. Petersen & K.W. Hughes, was able to successfully show sexual intercom- TFB 10315 (TENN 57963; exemplar); Baden patibility among several Finnish collections, Würtenberg, Tübingen Stadt, Tübingen thus implying that not only was a tetrapolar mat- Hagelloch, Schönbuch Natupark, Bettelbach ing system present, but that the mating genes Valley, 48˚ 32’ 49” N, 9˚ 01’59” E, 355 m,

27 K.H. HUGHES et al.

31.V.1999, col. D. Krüger, TFB 10522 (TENN three clusters of collections which differ 57752). RUSSIA, Leningrad, Lodeynove-Pole. somewhat morphologically and phylogenetical- Dist., Kut-Lakhta, Nyzhnesvirsky Nature ly, although none are considered sufficiently Reserve, road to Gumbaritsky Ornithological distinguishable to warrant taxonomic rank. One Station, 60˚36’ N, 33˚ 07’ E, 29.VIII.1999, col. group (form A) occurs across a considerable N. Psurtseva & R.H. Petersen TFB 10819 distance of southern Russia, a second group (TENN 58227); Leningrad Region, Lodeynoye (form B) was seen from the Caucasus and Pole Dist., Nyzhnesvirsky Nature Reserve, Czech Republic, and the third group (form C) bank of River Pel’fchuzhnaya, N 60˚ 36.775’, was gathered in Finland. E 33˚ 07.628’, 28.VIII.1999, col. N. Psurtseva, TFB 10813 (TENN 58222); Novgorod Region, Megacollybia platyphylla form A Valdai Dist., Valdaisky coll. R.H. Petersen (as Basidiomata collybioid to xeruloid. Pileus M. platyphylla), TFB 11637 (TENN 59600); (mature) 45Ð95 mm broad, plane to shallowly Novgorod Region, Valdai District, Road to convex with somewhat depressed disc; disc uni- Valdaisky National Park, 57˚ 57’ N, 33˚19’E, formly pigmented, delicately squamulose or 20.VIII.2003, col. N. Psurtseva TFB 11617 minutely furfuraceous, almost olive-black; limb (TENN 59580 PHOTO); Novgorod Region, distinctly to hardly radially streaked (but streaks Valdai District, Kasnaya Gorka, 58˚ 06’ N, 33˚ may be partially detersile), almost olive-black 13’ E, 21.VIII.2003, col. R.H. Petersen et al. against off-white background, becoming obscure TFB 11627 (TENN 59590 PHOTO); Samara so that margin is uniformly olive-brown; margin Region, vic Bakhilovo, Zhigulevsky Nature extended when fresh, tightly inrolled upon dry- Reserve, top of Big Bakhilovo Mt., N 53û ing, obscurely or not striate, thin, eroded. 26.012’, E 49˚ 41.350’, 10146 ft elev., coll. Lamellae adnexed to adnate to shortly decurrent R.H. Petersen (as M. platyphylla), TFB 12147 (or significant tooth), ventricose or not appear- (TENN 60086); Novosibirsk Region, ing so, Ð12 mm broad, thin, subdistant, white to Akademgorodok, vic. Novosibirsk State off-white when fresh, uniformly dull ochraceous University, VIII.2006, coll. and det. A. buff upon drying, with delicate transverse Gorbunova, no. LE212076; Toguchinsky Dist., streaking remaining off-white; lamellar edge not vic Kotorovo, 10.VIII.1973, leg & det. N.V. marginate or very delicately marginate to dark Perova, LE212074; same location, 6.VII.1974, purple, delicately eroded. Stipe 70Ð90 mm long, leg & det. NV Perova, LE212068 ; Samara perhaps 5Ð8 mm broad apically, 4Ð6 mm broad Region, Bakhilovo Co., vic. Bakhilovo, old in midsection, gradually expanded downward to Volga Riverbed forest, N 53˚ 23.989’, E 9Ð10(Ð17) mm broad at base, off-white when 49˚54.958’, 86 m elev., 16.VIII.2004, coll. fresh, uniformly dull tan on drying, perhaps R.H. Petersen, TFB 12163 (TENN 60102). slightly discolored with handling, significantly SWEDEN, vic Trollhättaän, village Rommele, lined or ridged; context stuffed, white. c. 1 km S Tokebäcken, 21.IX.1991, coll. L. & Rhizomorphic strands not included. Odor and A. Stridvall, TFB 4252 (TENN 50588). taste not recorded. Pileipellis terminal cells (disc) densely Megacollybia platyphylla: infraspecific scattered or in small fascicles, 33Ð96(Ð more forms than 210) × (8–)14–37 µm, vermiform, narrow- Within the larger clade comprising specimens ly to broadly clavate, firm- to thick-walled of M. platyphylla sensu stricto, there occur. (wall never more than 1.5 µm thick, and then

28 Megacollybia (Agaricales) irregular over inner surface), smooth, conspicu- pigmented pileipellis terminal cells. Unlike most ously clamped, commonly transversely septate; collections of M. platyphylla, these cells are contents homogeneous, strongly cytoplasmical- commonly transversely septate, but whether ly pigmented but with common hyaline indi- this feature has taxonomic value is unknown. viduals. Basidia 30Ð40(Ð53) × 8–12 µm, Caulocystidia are sometimes poorly devel- clavate, clamped, thin-walled, 4-spored, con- oped and often seem to be in small fascicles. The spicuously clamped; contents with only a very superficial “arachnoid network” on stipe surface few scattered, small, refringent guttules. is rather variable in thickness, which might indi- Basidioles often with gnarled apical outgrowths, cate that it is under some edaphic control. but usually digitate. Basidiospores 6.5Ð10 × 5Ð7 The delicate purple margin in LE 212073 on µm [n = 39; Q = 1.08Ð1.64; Qm = 1.31; Lm = 8.0 the only fragment of exposed lamella seems to µm], ellipsoid to ovate, flattened adaxially, thin- link this collection to LE 212075, also from walled, hyaline; contents aguttulate to 1Ð3-gut- Altai Republic. DNA from LE 212075 matches tulate. Cheilocystidia arising from basidial that of two other Russian collections from cymes, not protruding beyond basidia or pro- Rostov Region (between and north of Black truding up to 15 µm singly or in 2–3s, 32–94 × Sea and Caspian Sea, near the border with 13–24 µm, narrowly to broadly clavate, rarely to Ukraine). Thus the four collections seem to commonly transversely septate, firm-walled, span considerable distance in the Russian conspicuously clamped; contents homogeneous, south, clustered around the N 50th parallel. If hyaline. Stipe outer trama coherent, strictly par- true, then similar forms should be found in allel, longitudinal, composed of two hyphal Kazakhstan and perhaps the Ukraine. types: 1) up to 14 µm diam, thick-walled (wall Specimens examined: RUSSIA, Altai up to 1.0 µm thick), hyaline, speckled (as though Republic, southern shore of Teletskoye Lake, ornamented); and 2) 3–4 µm diam, hardly embouchure of river Chulyshman, 13.VIII.1996, branched, very rarely septate, obscurely leg & det. I.A. Gorbunova, LE 212075; Altai clamped. Stipe surface an arachnoid to relatively Republic, Shebalinsky Dist., vic village UstÅf- thick network of slender (2.5–4 µm diam) Sema (~N 50û 8’ E 85˚ 8’) 10.VIII.1997, leg & hyphae, producing relatively few to plentiful dif- det. I.A. Gorbunova, LE 212073; Rostov ferentiated terminal cells. Terminal cells (caulo- Region, vic. Veshenskaya, village Kalininsky, cystidia) 24Ð74(Ð more than 180) × 4Ð15(Ð30) gully Semenovskaya, N 49û 31’ 36.1”, E 41˚ 50’ µm, digitate to clavate, firm- to thick-walled 28.0”, 9.X.2006, leg & det. N.V. Psurtseva, (wall up to 0.7 µm thick), conspicuously NVP 47-06 (LE 212070); same data, NVP 49-06 clamped, thin- to firm-walled. (LE 212071). Commentary: Three of four basidiomata have delicately marginate gills, but not to dark brown Megacollybia platyphylla form B or black (as expected with pigmented cheilocys- Basidiomata collybioid. Pileus Ð100 mm tidia and seen in M. marginata), but to dark pur- broad, convex, innately radially fibrillose; disc ple (25×, even after drying). Cheilocystidia are “fuscous” (6E4; pallid sordid gray with very hyaline, however, which might indicate that the weak yellowish tint in TENN 58493), drying marginate lamellar edge is caused by weather glabrous-shining (not scabrous or furfuraceous); conditions or perhaps by handling, but not by nat- limb weakly to strongly radially streaked, ural marginate condition. “buffy brown” (6D4), drying glabrous-shining; All four collections exhibit large, strongly margin thin, lacerate, not striate, becoming

29 K.H. HUGHES et al. tightly inrolled on drying (not so in TENN but this small group seems somewhat internally 58493). Lamellae thin, somewhat ventricose, incongruent, and hardly separable morphologi- adnexed to adnate-decurrent by a tooth, “mar- cally from typical M. platyphylla. When single- guerite yellow” (30A2), to white (in TENN basidiospore isolates of TFB 7206 were paired 58493 taking on a ruddy orange tint on drying), with Finnish isolates, total interINcompatibility easily heavily eroded, upon drying with very was found (J. L. Mata, unpubl. data). As noted delicate transverse streaking of paler color, under M. marginata, these collections have close (not at all subdistant), perhaps Ð8 mm ITS sequences that are close to but not the broad; lamellar edge not marginate. Stipe Ð85 same as M. marginata. Thus, these collections mm long, slender, lined, white with “buffy may represent one or more transitional forms or brown” (6D4) vesture, expanded somewhat at introgressive hybridization (see also commen- base. Odor and taste none. tary under M. marginata). Habitat: Caucasus area of Russia, in Betula- Pileipellis terminal cells may be unusually Populus-Abies forest; Czech Republic, on rot- long (often with no visible basal ten hardwood, including Fagus. septum/clamp), strongly pigmented in the Pileipellis terminal cells 26Ð more than 183 Russian specimens, but significantly paler in × 7–31 µm, digitate to clavate to broadly the Czech basidiome. Pileipellis terminal cells clavate, often with mammiform tip on larger may be unusual in the high number of trans- individuals, strongly pigmented (almost hya- versely septate individuals. line in TENN 58493) but with common hya- Specimens examined: CZECH REPUBLIC, line, thick-walled individuals (wall up to 1.0 vic. Prague, vic. Jevany and Cerné Voderady, 30 µm thick, and then usually irregular on inner km ESE of Prague, 8.VII.1994, coll. R.H. surface), firm-walled, conspicuously clamped, Petersen, TFB 7206 (TENN 58493). RUSSIA, commonly transversely septate. Basidia 37Ð50 Republic of Karachaevo — Cherkessia, × 9–11 µm, clavate to pedicellate-clavate, thin- Northern Caucasus Mts., Teberda State walled, clamped, 4-spored; contents with scat- Biosphere Reserve, Malaya Khatipara, N ~43û tered refringent guttules. Basidiospores 7.5Ð10 40’, E ~41˚ 40’, 2.VII.1998, coll. & det. R. × 5.5–7.5 µm [n = 34, Q = 1.14Ð1.64; Qm = Botashev (as M. platyphylla) , no. LE 202274; 1.34; Lm = 8.9 µm], ellipsoid to subovate, flat- Krasnodar Region, Caucasus Nature Biosphere tened somewhat adaxially, thin-walled, inamy- Reserve, N 43˚ 50.546’, E 39˚ 45.852’, loid, hyaline; contents uni- several-guttulate. 12.IX.1996, coll. RHP (as M. platyphylla), TFB Cheilocystidia protruding beyond basidia only 8906, (TENN 55526). in 1Ð2s, but then sometimes significantly, 26Ð57 × 9Ð22 µm, sessile to clavate or broadly Megacollybia platyphylla form C [Fig. 14] clavate to nearly sphaeropedunculate, firm- Basidiomata (Fig. 14) collybioid. Pileus walled, conspicuously clamped; contents 50Ð65 mm broad, nearly hemispherical when homogeneous, hyaline. immature, expanding to shallowly convex with Commentary: Within M. platyphylla, a shallow umbo and uplifted margin, radially fib- small clade segregates, comprising two rillose with scurfy center; disc “fuscous black” sequenced specimens, one from the Caucasus (6F4), “deep olive” (4E6) to “dark olive” (LE202274), the other from Czech Republic (4F5), minutely broken-furfuraceous, outward (TFB7206). One additional specimen from the “chaetura drab” (2F2) fading to “hair brown” Caucasus can be added based on morphology, (6E3) “citrine drab” (4D5) alternating with

30 Megacollybia (Agaricales)

FIG. 14. Megacollybia platyphylla form C, basidiomata. TFB 11569. Numerals = 4 mm.

“deep olive buff” (3C3) when expanded, here Habitat: Southern Finland, on very and there split to reveal “olive buff” (3B3) rotten hardwood. pileus trama; margin fluted, concolorous with Pileipellis terminal cells (disc) not in disc, downturned to inrolled in dried condi- scabers but densely scattered over surface, tion. Lamellae adnate, up to 10 mm deep, 36Ð160 × 11–32 µm, conspicuously clamped, ventricose, subdistant, in 3 ranks, subsinuate, clavate to broadly clavate, often with an apical “tilleul buff” (7B2) when young, mellowing outgrowth or mamma, often somewhat thick- toward “vinaceous buff” (9B2) toward maturi- walled, and often transversely septate near ty (never white); margin eroded. Stipe 53Ð65 base; contents densely pigmented (olive- × 3Ð4 mm, more or less equal, straight but brown); common hyaline individuals scattered curved at base, lined, hardly twisted, near among others, with thick walls (wall up to 1 “deep olive buff” (3C3), slightly expanded µm thick, but very irregular in inner wall sur- apically and there white; base white, with face); contents (in just the right plane of view) white, tight tomentum; context narrowly hol- appearing mottled or very subtly banded (PhC). low to moderately stuffed. Odor and taste Subpellis a thin, tightly interwoven tissue of none. Rhizomorphs white, less than 1 mm slender (1.5–3 µm diam), hyaline, conspicuous- diam, webbed, resupinate where attached to ly clamped hyphae. Pileus flesh “sarcodimitic.” wood, flattened throughout, branched, some- Basidia 36Ð57 × 8–12 µm, narrowly clavate to what mealy. clavate, (2Ð3-)4-spored, clamped; contents with

31 K.H. HUGHES et al. a few scattered, small guttules; basidioles pileipellis terminal cells seem to distinguish 26Ð34 × 4–9 µm, clavate, rarely cylindrical or these two Finnish collections from typical lobed. Basidiospores 6.8Ð7.6(Ð10) × European forms. Likewise, the mixture of hya- 5.5–6.4(–8) µm [n = 20, Q = 1.06Ð1.55, Qm = line pileipellis terminal cells as well as heavily 1.27, Lm = 7.8 µm], broadly ellipsoid to subglo- pigmented, mottled, occasional transversely bose, somewhat flattened adaxially, hyaline, septate individuals also seems unique. Whether inamyloid, thin-walled, smooth; contents uni- rhizomorph appearance and structure is distin- to few-guttulate. Cheilocystidia sparse, locally guishable from that of the typical European common, occasionally protruding beyond taxon is questionable until more specimens are basidia, 29Ð65(Ð88) × 6–20 µm, clavate, broad- examined. The European entity seems to pro- ly clavate to pedicellate-bulbous, rarely apical- duce rhizomorphs that are round in cross-sec- ly diverticulate or knobbed, thin-walled, con- tion, often more than a millimeter diam, while spicuously clamped, hyaline (occasional indi- these Finnish collections exhibit more slender, viduals appear empty); contents homogeneous, flattened rhizomorphs. One of the specimens hyaline. Stipe surface of strictly parallel, slen- (TENN 59541) was successfully sequenced, der (3Ð7 µm diam), long-celled hyphae, occa- and the ITS sequence also is somewhat unique. sionally producing clavate side branches or ter- Specimens examined: FINLAND, Etelä- minal cells (caulocystidia); caulocystidia nar- Hame, Tammelo, Torronsuo Nat. Park, map rowly clavate to clavate, 6Ð13 µm diam, grid 6740: 314, 8.VIII.2002, coll. RHP, TFB smooth, hyaline, repent to rarely recurved. 11577 (TENN 59530); Etelä-Häme, Liesjärvi Stipe flesh of two hyphal types, both hyaline; National Park, trail from Korteniemi, N 61û 1) 18Ðmore than 40 µm diam, thick-walled 00’, E 24˚ 30’, 6.VIII.2002, coll. E Grand, TFB (wall never more than 1 µm thick, irregular in 11569 (TENN 59541). outline, arising from slender hyphae, constrict- ed at septa, conspicuously clamped; and 2) 3Ð8 5. Megacollybia fallax [Figs. 1B, 2 Ð clade 5, µm diam, thin-walled (diaphanous and often Figs. 15-18] semi-collapsed), frequently branched, frequent- A small but discrete clade appears basal to ly clamped, interwoven with inflated hyphae. other North American collections and includes Slender hyphae occasionally ending blindly, the type specimen of Tricholomopsis fallax elongate-clavate (“endocystidia”), 80Ð more A.H. Smith. Sequences were obtained from than 170 × 9Ð24 µm, thin-walled, arising from specimens from Arizona, Idaho and a clamp connection, hyaline. Rhizomorph cor- Washington, indicating an extended range for a tex composed of hyaline, thin-walled hyphae species reported by Smith as “known only from 2Ð11 µm diam, with little differentiation into central Idaho.” Singer (1986) accepted taxa diameter classes, conspicuously clamped, more surrounding M. platyphylla as Tricholomopsis frequently on slender hyphae than on wider Singer (1939. Schweiz. Zeitschr. Pilzk. 17: 13) (not strictly sarcodimitic); rhizomorph medulla sect. Platyphyllae Singer (1943. Ann. Mycol. of “skeletal hyphae,” 1.5–3 µm diam, apparent- 41: 68.). Smith (1960. Brittonia 12: 45) emend- ly aseptate, thick-walled (wall up to 0.5 µm ed the diagnosis of the section, but all these thick or occluding cell lumen), hyaline, non- proposals preceded Kotlaba and Pouzar’s cyanophilous, flexuous. (1972) proposal of Megacollybia. Commentary: Somewhat darker pileus col- Mata (unpubl. data) found low-percentage ors, caused by darker and more congested intercompatibility between TFB 11560 and

32 Megacollybia (Agaricales)

FIGS. 15, 16. Megacollybia fallax, basidiomata. Fig. 15. TENN 59446, 0.7×. Fig. 16. TENN 59447, 0.7×.

33 K.H. HUGHES et al.

TFB 11561. These collections, however, when paired with European isolates, were totally interincompatible. The western United States organism, therefore, seems sexually isolated from the European M. platyphylla. Altogether, T. fallax surely belongs with other taxa in Megacollybia, as confirmed by phylogenetic placement of DNA sequences. A nomenclatural transfer is necessary, as follows:

Megacollybia fallax (A.H. Sm.) R.H. Petersen & J.L. Mata, comb. nov. Basionym: Tricholomopsis fallax A.H. Smith. 1960. Brittonia 12: 48. Holotype: UNITED STATES. Idaho, Valley FIG. 17. Megacollybia fallax, TENN 59447. A. Co., near McCall, Lake Fork Creek, Payette Pileipellis terminal cells. B. Basidia. Standard bar = 20 µm. National Forest, 44˚ 52.48” N, 116˚ 2.75” W, 28.VII.1954, on conifer log, col. AH Smith, no. 45432 (MICH). Habitat: Western North America (Arizona, Basidiomata tricholomoid (Figs. 15, 16). Pacific Northwest); solitary to gregarious on Pileus 40Ð70(Ð120) mm broad, 30Ð50 cm high, rotten conifer logs or lignous soil. obtuse when young, the margin incurved, Pileus cuticle not differentiated as such but the remaining unexpanded or becoming broadly surface with numerous pileipellis terminal cells convex to nearly plane, dry, dark gray-brown (Fig. 17A) 28Ð99 × 8–20 µm, narrowly clavate to on disc, outward streaked with gray to sordid clavate, firm- to thick-walled (wall never more deep gray-olive fibrils, soon radially rimose than 1 µm thick and then irregular over inner sur- with wide lenticular lacunae exposing context; face), moderately cytoplasmically pigmented, margin lobed, soon lacerate in age, thin, occasionally transversely septate (with no clamp), whitish; context white, thin but fairly tough, conspicuously clamped, rarely with some minute unchanging on bruising. Lamellae adnate, residual debris or slimy sheath. Outer pileus flesh close, ventricose (up to 20 mm broad), off- constructed of two hyaline, clamped hyphal white when young, becoming pale yellow types: 1) inflated up to 25 µm diam, thick-walled before maturation, finally creamy ochraceous, (wall up to 1.0 µm thick, irregular on inner sur- not marginate, transversely veined, occasional- face), constricted at septa, obscurely clamped; ly intervenose; edges even to slightly eroded, and 2) slender (3–4.5 µm diam), thin-walled, fre- not changing color when bruised. Stipe 40Ð110 quently branched, frequently clamped, interwo- mm long, 10Ð17(Ð30) mm thick, equal or taper- ven amongst inflated hyphae. Basidia (Fig. 17B) ing slightly downward, solid, yellowish in the (30Ð)36Ð47(Ð52) × (6–)8–11 µm, clavate, thin- cortex, the pith paler; surface white to whitish walled, (2-)4-spored; basidioles 28Ð48 × 4–10 µm, yellow but paler than the gills, naked except for clavate; contents with a few, small, refringent gut- fibrillose striations; rhizomorphs sparse, thick tules. Basidiospores (Fig. 18A) (5.6Ð)6Ð8.5(Ð10) × (up to 4 mm diam), white, round in cross-sec- (4.0–)5–7(–8.0) µm [n = 83; Q = (1.00Ð)1.12Ð1.57; tion. Odor and taste not distinctive. Qm = 1.36; Lm = 7.6 µm], subglobose, ellipsoid to

34 Megacollybia (Agaricales)

1960) are difficult to ascertain. Smith (1960. Brittonia 12: 48–49) wrote: “The yellow pig- ment in the pilocystidia, the lack of diversity in the shape of the cheilocystidia, and the yellow pigment in the gills and cortex of the stipe dis- tinguish this species from T. platyphylla. The habitat on wood of conifers is also an important field character ...” Under T. platyphylla, Smith cited specimens from eastern North America as well as specimens from California, Montana, New Mexico, Oregon and Washington. To our knowledge, T. fallax is found from Arizona north to British Columbia, and M. rodmani (T. platyphylla of Smith, 1960, pro parte) only in the eastern parts of North America. Cheilocystidia of T. fallax are just as vari- able as those of other taxa, although perhaps

FIG. 18. Megacollybia fallax, TENN 59447. A. more commonly transversely septate, so Basidiospores. B. Cheilocystidia. C. Caulocystidia. Smith’s report of “lack of diversity in the shape Standard bar = 20 µm. of the cheilocystidia” is not a distinguishing character, but merely contributes to placement of T. fallax within the same complex as taxa narrowly ovate, flattened adaxially, thin-walled, regarded here as Megacollybia. inamyloid; contents aguttulate or 1-few guttulate. Some quantitative characters can be applied Central strand of lamellar trama subparallel-inter- to these western North American collections to woven; hyphae 4–12 µm diam, thin- to firm- help distinguish them. 1) Basidia of T. fallax are walled, flanked on either side by a narrow strip of generally somewhat longer (35Ð43 × 7–10 µm) parallel hyphae 1.5–3 µm diam. Cheilocystidia than those of other taxa (for M. rodmani: 25Ð38 (Fig. 18B) occasionally protruding beyond × 7–10 µm), attributable to a cylindrical stalk- basidia (but not generally), 25Ð55(Ð80) × like base. 2) Pileus color in T. fallax (Smith, (6–)9–16 µm, clavate to broadly clavate, occa- 1960: “more or less streaked with gray fibrils as sionally with small apical outgrowths, firm- in T. platyphylla”) (notes with Parker speci- walled, occasionally secondarily transversely sep- mens: “grayish brown,” “medium grayish tate, conspicuously clamped, hyaline; contents brown”) somewhat paler than in M. rodmani and homogeneous. Upper stipe delicately silky, white; with radial streaks more widely separated, surface a layer (–50 µm thick) of slender (3–4.5 revealing white underlying pileipellis. 3) In T. µm diam), thin-walled, frequently clamped, com- fallax, caulocystidia from near stipe apex are monly branched, hyaline hyphae, producing hardly inflated (3–8 µm diam at widest point) rounded terminal cells (caulocystidia) (Fig. 18C) while they are inflated and subbulbous (5Ð18 26Ð88 × 3–8 µm diam (at widest point), clavate, µm diam) in M. rodmani. 4) In our experience, conspicuously clamped, hardly inflated, elongate- T. fallax is limited to western North America, digitate to hardly clavate. while M. rodmani seems distributed in eastern Commentary: Diagnostic characters (Smith, North America. 5) Reflecting its distribution,

35 K.H. HUGHES et al.

T. fallax is reported only from rotting conifer ern Texas, may be found as sister to some wood and debris, while M. rodmani prefers hard- specimens from Guyana and Colombia and is wood (in spite of Smith’s report to the contrary). genetically distinct from M. rodmani. Mata (unpubl. data) found low-percentage Collections were contaminated with a species intercompatibility between TFB 11560 and of Candida related to a GenBank sequence TFB 11561. These collections, however, when deposited as C. fungicola. From cloned ITS paired with European isolates, were totally sequences, the Texas collections are heterozy- interINcompatible. The western United States gous for different but related haplotypes. organism, therefore, is sexually isolated from Haplotypes of TENN 62058 (DPL7405) dif- the European M. platyphylla. fered by only a few base pairs, but the two hap- Specimens examined: UNITED STATES; lotypes from TENN 59935 (TFB 11676) are Arizona, Pima Co., Mt. Bigelow, Santa found in two different minor clades, one of Catalina Mots., Coronado National Forest, which also includes one of the haplotypes from 25.VII.1972, col. K. Muetin (as Collybia platy- TENN 62059 (DPL7682). TENN 62059 pro- phylla), KM 214 (011591; AN); Pima Co., Mt. duced four haplotypes, distributed over the Lemon, 8.V.1959, col. Leathers & Keener (as entire /texensis clade. The maximum within- Megacollybia platyphylla), PH 3503 (011589, clade divergence for this clade is 0.85% con- AN). Idaho (all as Tricholomopsis fallax), sidering all base pairs or 0.71% considering Valley Co., Lake Fork creek, Payette lakes, on indels as a single event. Parental strains, conifer log, 14.VII.1954, col. A.H. Smith, therefore, remain unclear, but for its hybrid 44965 (MICH; paratype); same location, state, morphological characters of all these 18.VII.1954, col. AH Smith, 45133 (MICH; specimens are remarkably similar. paratype); same location, 1954, col. A.H. We conclude that the Texas material repre- Smith, 45389 (MICH; paratype); same loca- sents a separate and new species, as follows: tion, 29.VI.1958, col. C.S. Lowthry & A.H. Smith, no. 58401 (MICH; paratype); same Megacollybia texensis R.H. Petersen & location, 15.VII.1958, col. T Westerdale no. David P. Lewis, sp. nov. 58870 (MICH; paratype); same location, Pileus 40Ð85 latus, planus ad parum depres- 14.VII.1954, col. H.E. Bigelow, 44955 (MICH; sus, pallide griseo-brunneus, fibrillosus ad sub- paratype); same location, 16.VII.1954, col. rimosus, radio-fibrillosus, siccus. Lamellae A.H. Smith, 45002 (MICH; paratype). adnatae ad subdecurrentae, ventricosae, lactea Washington, Pond Oreille Co., vic Metalline ad eburneae, non-marginatae. Stipes 50Ð145 × Falls, 2 mi S of Slate Creek, W of highway 31, 3Ð8 mm, equalus ad sursum decrescens, brun- 48˚ 54.226’ N, 117˚ 19.934’ W, 2500’ elev, neus ad olivaceobrunneus. Pileocystidia 30.V.2002, coll. D. Parker, TFB 11560 (TENN 29Ð125 × 7–15 µm, clavata, olivacea, tenui- ad 59446); Pond Oreille Co., Lake Lucerne, high- crassitunicatae, fibulata, levis. Sporae 6.5Ð10 × way 31, 2 mi from Canadian border, 5–7.5 µm (Q = 1.07-1.60; Qm = 1.28; Lm = 7.9 28.VI.2002, coll. D. Parker, TFB 11561 µm), ellipsoideae ad ovatae, levae, tenuituni- (TENN 59447). catae, hyalinae. Basidia 26Ð36 × 7–10(–12) µm, clavata, tenuitunicata, fibulata, tetraspora. 6. Megacollybia texensis [Figs. 1B, 2 Ð clade Pleurocystidia nulla. Cheilocystidia 25-67 × 6, Figs. 19-21] (11–)19–22 µm, lato-clavata, fibulata, tenui- ad A small clade of specimens, all from east- crassitunicata. Caulocystidia (24Ð)47Ð150 ×

36 Megacollybia (Agaricales)

FIG. 19. Megacollybia texensis, basidiomata. DPL 7863. 0.7×.

7–20 µm, pedicellata ad elongate clavata, hyali- mm deep, subdistant, in 3Ð4 ranks, thin to na, tenui- ad crassitunicata, fibulata. America somewhat thick, white to off-white when fresh, septentrionalis; austro-centralis. drying to light ochraceous buff, not marginate, Holotype: UNITED STATES, Texas, often locally transversely striped as though Hardin Co., Lance Rosier Unit, Big Thicket hygrophanous; interlamellar areas smooth, National Preserve, Teel Rd., N 30˚ 16. 629’, often without hymenium; lamellar edge often W 94û 31. 532’ 11.XI.2006, coll. D.P. Lewis, deeply eroded. Stipe 50Ð145 mm long to sub- DPL 7682 (TENN 62059). stratum, 3Ð8 mm broad apically, terete, equal Basidiomata (Fig. 19) collybioid to xeru- to gradually expanded downward to 4Ð9 mm loid, gracile. Pileus 40Ð85 mm broad, plane to broad, delicately lined, perhaps minutely silky, slightly depressed, and then occasionally with hardly twisted, neutral pale drab downward small, shallow umbo, medium gray-brown to where handled (nearly concolorous with pallid gray-tan, closely to sparsely, delicately pileus), often abruptly expanded at base into a radially streaked (sometimes almost invisibly irregular knot or sometimes extended as a stout so), smooth, not drying glabrous; margin pseudorhiza; rhizomorphic strand presence or inturned to plane or delicately uplifted at matu- abundance unknown. rity, often ragged to lacerate, thin, not striate; Habitat: Eastern Texas; on soil in hardwood flesh thin throughout, drying fragile and parch- or mixed hardwood-pine forests. ment-like. Lamellae adnate with small to con- Pileipellis an arachnoid network (too thin to siderable decurrent tooth, ventricose, up to 12 be a layer) of slender (3Ð5 µm diam), radially

37 K.H. HUGHES et al.

FIG. 20. Megacollybia texensis. TENN 62059. A. FIG. 21. Megacollybia texensis. TENN 62059. A. Pileipellis terminal cells. B. Basidia. Standard bar = Basidiospores. B. Cheilocystidia. C. Caulocystidia. 20 µm. Standard bar = 20 µm. oriented, firm-walled, hyaline, frequently con- line; contents with several refringent guttules spicuously clamped, occasionally branched scattered throughout, but often with a large api- hyphae producing sparse terminal cells over cal guttule just before maturity. Basidiospores disc; terminal cells (Fig. 20A) 29Ð125 × 7Ð15 (Fig. 21A) 6.5Ð10 × 5–7.5 µm [n = 128; Q = µm, elongate-clavate, narrowly clavate to 1.07-1.60; Qm = 1.28; Lm = 7.9 µm], ellipsoid to clavate, firm- to thick-walled (wall never more ovate, flattened adaxially, thin-walled, hyaline, than 0.7 µm thick), conspicuously clamped, inamyloid; contents 1-several-guttulate. weakly pigmented when part of a pigmented Lamellar edge sterile, usually extending in radial streak, to subhyaline when between radial KOH up to 25 µmm beyond hymenium. streaks. Pileus outer context constructed of Cheilocystidia (Fig. 21B) 25Ð67 × (11Ð)19Ð22 two hyaline hyphal types: 1) inflated up to 35 µm, broadly clavate, pedicellate to nearly ses- µm diam, firm- to thick-walled (wall never sile, firm- to thick-walled (wall never more more than 0.7 µm thick, irregularly thickened than 0.5 µm thick, irregular over inner surface), on inner surface), constricted somewhat at hyaline, conspicuously clamped, rarely trans- septa, occasionally adventitiously branched; versely septate in lower pedicel; contents and 2) slender (2.5–4.5 µm diam), frequently homogeneous. Upper stipe with closely lined branched, frequently clamped hyphae similar to ridges sometimes covered with basidia and those of the superficial hyphae of the pileus cheilocystidia. Downward, stipe surface with surface, interwoven among inflated hyphae. intricate layer (arachnoid to 20–25 µm thick) of Pleurocystidia absent or as cheilocystidia pro- slender (3–4.5 µm diam), thin-walled, frequent- duced a short distance from lamellar edge. ly clamped, rarely branched hyphae, producing Basidia (Fig. 20B) 26Ð36 × 7–10(–2) µm, copious out-turned caulocystidia; caulocystidia clavate, thin-walled, clamped, 4-spored, hya- (Fig. 21C) (24Ð)47Ð more than 150 × 7–20 µm,

38 Megacollybia (Agaricales) elongate-clavate, clavate to various contortions are especially abundant. Variation amongst of clavate, firm- to thick-walled (wall never caulocystidia is great, both in length, apical more than 1 µm thick, often irregular on inner shape and wall thickness. surface when thickened), hyaline, conspicuous- The similarity among pileipellis terminal ly clamped; contents homogeneous, appearing cells, cheilocystidia and caulocystidia is strik- “empty” in bulb but more densely cytoplasmic ing throughout the genus. Shape and origin (dark under PhC) in pedicel. Outer stipe flesh does not differ, but extent of pigmentation constructed of somewhat inflated (up to 13 µm (especially of pileipellis terminal cells in M. diam), thick-walled (wall up to 1.5 µm thick), clitocyboidea and M. rodmani f. murina), abun- coherent (shearing off as plates, not as liberated dance (pileipellis terminal cells in scabers or hyphae), long-celled, rarely clamped, hyaline patches in Asiatic species; caulocystidia in M. hyphae strictly parallel and longitudinal. texensis), and prominence of cheilocystidia Commentary: Diagnostic microscopic char- (protruding beyond basidia in M. rodmani f. acters are difficult to ascertain unless compared murina) can be used as separating characters. to those of other taxa. Based on herbarium Specimens examined: UNITED STATES specimens with minimal accompanying notes, (all as Megacollybia platyphylla), Texas, the following separating characters can be Hardin Co., Lance Rosier Unit, Big Thicket offered: 1) basidiome stature gracile, collybioid National Preserve, Teel Rd., N 30˚ 16. 629’, W or xeruloid; 2) stipe base often (?always) 94˚ 31. 532’, 11.XI.2006, coll. D.P. Lewis, abruptly expanded; 3) pileus pallid grey-tan DPL 7682 (TENN 62059); same location, with very delicate radial streaks (20×); 4) small 17.VI.2006, coll. D.P. Lewis, DPL 7405 basidia as compared to those of other taxa; 5) (TENN 62058); Lumberton, Village Creek outer stipe flesh of coherent, hardly inflated State Park, 28.VI.2003, coll. D.P. Lewis, DPL hyphae; 6) upper stipe with superficial layer of 6711, TFB 11676 (TENN 59935); Henderson tangled, slender hyphae; and 7) caulocystidia Co., Athens, Athens Botanical Garden, plentiful and variable. 31.X.2004, coll. D.P. Lewis, DPL 7081 (TENN Basidial stature is closer to that found in the 62057) rest of the genus, not that of M. rodmani f. murina, i.e. large pileus, thin flesh, thin, eroded 7. Megacollybia fusca [Figs. 1B, 2 Ð clade 7; pileus margin, ventricose lamellae and some- Figs. 22-25] what slender stipe that is longer than pileus This clade is composed of sequences from diameter. two specimens from Colombia and Guyana in Lamellar edge in some specimens is unique South America. ITS Sequences from Guyana in extending in KOH some 20Ð25 µm beyond and from Colombia are sufficiently different hymenium (but not gelatinous as seen in (4.05% divergence if gaps are coded as a single Xerula). Cheilocystidia, therefore, are not gen- event) that these collections may well represent erally mixed with basidia, but are produced by two species, but with only a single collection a separate hyphal system. Conversely, cheilo- from each region, we are taking a conservative cystidia show less variation than in other taxa approach at this time and placing them under a of Megacollybia. single species epithet. Phylogenetically, collec- The superficial layer of intricate hyphae pro- tions from Guyana and Colombia are separate ducing caulocystidia is somewhat more luxuri- from their sister clade, Megacollybia texensis. ant than typical of the genus, and caulocystidia A new species is proposed:

39 K.H. HUGHES et al.

FIGS. 22, 23. Megacollybia fusca, MCA 1179. Fig. 22. Basidioma in the habitat, 0.8×. Fig. 23. Field sketch, 0.8×.

Pleurocystidia nulla. Cheilocystidia 28Ð60 × Megacollybia fusca J.L. Mata, M.C. Aime, 11–25 µm, lato-clavata, fibulata, tenuitunicata. & T. W. Henkel, sp. nov. Caulocystidia 28Ð90 × 8–20 µm, elongato-pedi- Pileus 110Ð150 mm latus, convexus juve- cellata, clavata ad subventricosa, pallidobrun- nilus, planus ad depressus, luteobrunneus ad nea. America Australis; septentrionalis. atroluteobrunneus, radio-fibrillosus, siccus. Holotype: GUYANA. REGION 8 Lamellae adnatae ad subdecurrentae, ventri- POTARO-SIPARUNI: Pakaraima Mountains, cosae, lacteae, delicate marginatae. Stipes Upper Potaro River, 20 km east of Mt. 60Ð170 × 10Ð18 mm, equalus ad sursum Ayanganna, near confluence of Potaro River and decrescens, brunneus ad luteobrunneus. Alukyadongbaru Creek, within 2 km of a perma- Pileocystidia 48Ð112 × 10–25 µm, clavata, ven- nent base camp at 5˚ 18’ 04.8” N; 59˚ 54’ 40.4” tricosa ad subfusoidea, brunnea, tenuitunicata, W; elevation 710m, Dicymbe corymbosa domi- fibulata, levis. Sporae 5.6Ð10 × 4.4–7.2 µm (n= nated forest, , 10.VI.2000, coll. Karol, MCA 50, Q = 1.07Ð1.44, Qm = 1.28, Lm = 7.6 µm), 1179 (BRG, Isotype LSU). latae ellipsoideae ad ovatae, levae, tenuituni- Basidiomata (Figs. 22, 23) collybioid to catae, hyalinae. Basidia 25Ð30(Ð41) × 6–11 µm, xeruloid. Pileus 110Ð150 mm in diameter, clavata, tenuitunicata, fibulata, tetraspora. 30Ð50 mm deep, convex when young, later

40 Megacollybia (Agaricales) broadly plane to deeply depressed or everted, with an irregular orbicular outline; surface radi- ally fibrillose, fibrils most concentrated over disc, becoming flat appressed squamulose in some toward margin, taupe (4E5) overall or yellow-brown (5F5), remaining darker at disk; margin uplifted, with broad tears in some, par- tially eroded. Context up to 10 mm thick at the center, to very thin outwards, watery white, unchanging. Lamellae adnate to sinuate, 7Ð15 mm broad, subdistant, white, appearing hygro- phanous; margin smooth, finely stippled taupe to yellow brown; lamellulae in two tiers of dif- ferent lengths, frequently anastomosing and with intervenose projections present. Stipe FIG. 24. Megacollybia fusca. A, B. Pileipellis terminal 60Ð170 × 10Ð14 mm , with a subbulbous base cells. C, D. Basidia. A, C. MCA 1179. B, D. AEFM 1892. up to 18 mm diam, tip of base sometimes Standard bar = 20 µm. tapered and abruptly radicating; surface appressed fibrillose, appearing longitudinally streaked, brown (4D4) to yellow brown (5F5), Hymenium composed of basidia (Figs. 24C, paler and pruinose toward the apex; interior D) 25Ð30(Ð41) × 6–11 µm, typically clavate, hollow at center, white, thick and tough; con- four-spored; and basidioles 20Ð26(Ð40) × text fibrous to cottony. Rhizomorphic strand 5Ð6(–10) µm, digitate to narrowly clavate, present, white. Colors unchanging on bruising. occasionally cylindrical; wall thin. Odor faint; taste none. Basidiospores (Figs. 25A, B) 5.6Ð10 × 4.4Ð7.2 Habitat: Colombia, in Quercus humboldtii µm (n = 50, Q = 1.07Ð.44, Qm = 1.28, Lm = 7.6 forests; Guyana, in forests dominated by the µm), broadly ellipsoid to subglobose in side leguminous, ectomycorrhizal tree Dicymbe view and profile, adaxially flattened, hyaline, corymbosa. inamyloid, acyanophilous, wall thin, smooth, Pileipellis a parallelocutis; suprapellis com- typically uniguttulate. Pleurocystidia none. posed of terminal cells (Figs. 24A, B), 48Ð112 Cheilocystidia (Figs. 25C, D) abundant, 28Ð60 × 10–25 µm, mostly long to short clavate, or × 11–25 µm, mostly clavate-inflated to broadly ventricose to subfusoid, some mucronate, semi- clavate, sometimes pedicellate, or sphaero- prostrate, ± intertwined, single or more often in pedunculate, rarely fusoid-ventricose or sub- fascicles, with brown-pigmented vacuolar con- mucronate, sometimes transversely septate tents, thin-walled; subpellis ± 40 µm thick, towards base (but not clamped), thin-walled, composed of filamentous hyphae, 2Ð8 µm conspicuously clamped. Stipe tissues parallel, diam, ± radially oriented. Pileus trama undif- of two hyphal types: 1) up to 32 µm diam, ferentiated to loosely interwoven; hyphae nar- thick-walled (wall up to 2 µm thick); and 2) row, less than 6 µm diam, or broad up to 30 µm 4Ð6 µm diam, frequently branched, frequently diam, filamentous, hyaline singly; wall thin to clamped. Caulocystidia (Figs. 25E, F) com- slightly thickened. Lamellar trama irregular; mon on upper stipe, scattered and uncommon hyphae 6–20(–30) µm diam, hyaline; wall thin. to rare downwards, 28Ð90 × 8–20 µm, typically

41 K.H. HUGHES et al.

SIPARUNI: Pakaraima Mountains, Upper Potaro River, 20 km east of Mt. Ayanganna, near confluence of Potaro River and Alukyadongbaru Creek, within 2 km of a per- manent base camp at 5˚ 18’ 04.8” N; 59˚ 54’ 40” W; elevation 710m, Dicymbe corymbosa dominated forest, , 10.VI.2000, coll. Karol, MCA 1179 (BRG, Isotype LSU).

8. Megacollybia rodmani [Figs. 1B, 2 Ð clade 8; Figs. 26-31] Phylogenetic information, morphological observations and a few sexual compatibility pairings all suggest that the eastern North American representative of Megacollybia dif- FIG. 25. Megacollybia fusca. A, B. Basidiospores. C, fers significantly from European M. D. Cheilocystidia. E, F. Caulocystidia. A, C, F. MCA 1179. platyphylla. Although two poorly differentiated B, D, E. AEFM 1892. Standard bar = 20 µm. forms seem to occur (here named), only a sin- gle species is accepted at this time. The species epithet intends to recognize long-pedicellate, clavate to short ventricose, or sub- James Rodman, long a Program Coordinator in mucronate, with brownish vacuolar pigmentation. the Biological Sciences Program of the Commentary: Overall macroscopic and National Science Foundation (USA), in appre- microscopic characteristics of M. fusca are sim- ciation of his service to biological systematics. ilar to other species in Megacollybia, differing only in smaller details. A brown-gray pileus Megacollybia rodmani R.H. Petersen, K.W. coloration of M. fusca is similar to all other Hughes & E.B. Lickey, sp. nov. species but the shape of pileipellis terminal Pileus plerumque ad 100 mm latus, con- cells seems to be more variable. Cheilocystidia vexus juvenilus, planus ad leviter convexus, are numerous but in general they seem not to olivaceo-griseus ad olivaceo-brunneus, brun- achieve long dimensions and appear more nescens, radio-fibrillosus, siccus. Lamellae inflated when compared to other American adnatae ad adnexae, ventricosae, lacteae ad species such in M. rodmani and M. texensis. eburneae, non-marginatae. Stipes Ð80 × 10Ð25 Ecology and distribution of M. fusca is unique mm, equalus ad sursum decrescens, albus ad as it appears to be associated with Dicymbe pallide griseus. Pileocystidia 23Ð74 × 6–16 µm, forests from the Pakaraima Mountains of west- clavata, ventricosa ad subfusoidea, subhyalina, ern Guyana and oak-dominated forests of tenui- ad crassitunicatae, fibulata. Sporae 6Ð10 southwestern Colombia. × 5–7.5 µm (Q = 1.07-1.67; Qm = 1.33; Lm = 7.9 Specimens examined: COLOMBIA. µm), subglobosae ad latae ellipsoideae ad Departamento de Antioquia, Municipio de ovatae, laevae, tenuitunicatae, hyalinae. Támesis, vereda Rio Frío, Quercus humboldtii, Basidia 25Ð38 × 7–10 µm, clavata, tenuitunica- 23.VI.1992, col A.E. Franco-Molano AEFM ta, fibulata, tetraspora. Pleurocystidia nulla. 1012 (NY). GUYANA, REGION 8 POTARO- Cheilocystidia 28Ð65 × 7Ð18 µm, clavata ad

42 Megacollybia (Agaricales) late clavata, fibulata, tenui- ad crassitunicata. Caulocystidia 25Ð72 × 5–18 µm, clavata ad subventricosa, prope hyalina. America Septentrionalis; orientalis. Holotype: UNITED STATES, Tennessee, Sevier Co., vic. Gatlinburg, GSMNP, Greenbrier, Porter’s Creek Rd., 5.V.2002, coll. R.H. Petersen & K.W. Hughes, TFB 11485 (TENN 59430). Basidiomata stout, fleshy, tricholomatoid to collybioid. Pileus often 100 mm broad or more, convex when young, soon shallowly depressed over disc and there darkly pigment- ed, smooth, outward over limb and to margin obscurely, delicately streaked in dark colors from dark brown with olive tints to almost FIG. 26. Megacollybia rodmani f. rodmani. TENN black with olive tints, in the typical form 59430. A. Pileipellis terminal cells. B. Basidia. Standard changing to neutral brown on drying; margin bar = 20 µm. inturned when immature, by maturity plane or uplifted; flesh white to off-white, soft when fresh, punky when dried. Lamellae adnexed to debris, then probably attached to buried wood adnate, usually with small decurrent tooth, ven- in hardwood forests. tricose, subdistant, thick to thin, up to 15 mm Pileipellis a repent layer of slender (3–6 µm deep, off-white to pallid olive or pallid yellow- diam), radially oriented hyphae, either moder- ish shades, in f. murina changing to ochraceous ately cytoplasmically pigmented (composing buff with pale buff inner surfaces, in 3-5 ranks, dark radial streaks) or subhyaline (in areas delicately transversely ridged and/or streaked between dark streaks); pileipellis terminal as though hygrophanous (but not so), often cells (Fig. 26A) never in scabers, never in eroded to irregularly serrate, brittle when dried patches, 23Ð74 × 6–16 µm, nearly sessile to and then crisped; interlamellar surface rugose, elongate-clavate, firm-walled, weakly pigment- ridged to buttressed. Stipe up to 80 mm long, ed, conspicuously clamped, without hyaline 10Ð25 mm broad, smooth, minutely lined, usu- thick-walled individuals; contents homoge- ally not twisted, apically concolorous with neous, subhyaline. Pileus flesh over stipe up to lamellae, downward off-white to pallid olive, 15 mm thick, constructed of two hyphal types, slowly discoloring to gray or brownish in han- both hyaline, both clamped; 1) inflated, 10Ð50 dling; base not or gradually expanded, often µm diam, somewhat thick-walled (wall up to 1 attached to ropy, white rhizomorphic strands; µm thick, irregular on inner surface), appearing flesh white, stuffed to hollow (especially when obscurely mottled, including small circular wall mature) with thick rind. Odor none; taste not structures, constricted at septa, conspicuously distinctive. clamped; and 2) 3–10 µm diam, thick-walled Habitat: Eastern North America and Central (wall up to 0.7 µm thick), frequently branched, America (Costa Rica to Newfoundland); on or frequently conspicuously clamped, smooth (on juxtaposed to very rotten wood; when on forest both wall surfaces). Pleurocystidia absent, or

43 K.H. HUGHES et al. as cheilocystidia produced a few micrometers from lamellar edge. Subhymenium a tightly interwoven layer of intricately tortuous, slender (2.5–4 µm diam), frequently clamped hyphae producing basidia and often coralloid, branched or lobed hymenial elements. Basidia (Fig. 26B) 25Ð38 × 7–10 µm, clavate, thin-walled, 4- spored, clamped; contents with a few scattered, refringent (PhC) guttules; sterile hyphal tips (?young basidioles) gnarled, often branched, filamentous, 2.5–3.5 µm diam, hyaline, thin- walled, clamped. Basidiospores (Fig. 27A) 6Ð10 × 5–7.5 µm (Q = 1.33; Lm = 7.9 µm), sub- globose, ovate or ellipsoid, flattened somewhat adaxially, thin-walled, inamyloid; contents 1- few guttulate, refringent (PhC). Lamellar FIG. 27. Megacollybia rodmani f. rodmani. TENN trama composed of two hyaline, firm- to thick- 59430. A. Basidiospores. B. Cheilocystidia. C. walled, clamped, hyphal types: 1) inflated up to Caulocystidia. Standard bar = 20 µm.. 35 µm diam, firm- to thick-walled (wall never more than 0.5 µm thick), constricted at septa, obscurely clamped; and 2) slender, 2.5–4 µm celled, rarely clamped hyphae not interwoven diam, frequently branched, frequently clamped, with inflated hyphae. Rhizomorphic strands thin-walled, interwoven among inflated usually thick, terete, prominent, extensive, hyphae. Lamellar edge composed of loosely sometimes extending for a meter or more in rot- tangled slender (2.5–4.5 µm diam), tortuous, ten wood, white; cortical tomentum of tightly frequently septate, frequently clamped hyphae interwoven, irregular, thick-walled, clamped producing cheilocystidia. Cheilocystidia (Fig. hyphae; medullary tissue of skeletal hyphae, 27B) usually plentiful, prominent or not so, slender (1.5–2 µm diam), thick-walled (wall up clavate, broadly clavate to subsphaeropeduncu- to 0.5 µm thick, often obscuring cell lumen), late, firm- to thick-walled (wall never more inamyloid, non-cyanophilous, rarely clamped. than 0.7 µm thick), hyaline; contents homoge- Commentary: In Megacollybia, distinguish- neous. Stipe surface a repent, arachnoid net- ing morphological characters are difficult to work or layer (–30 µm thick) of slender (3–5.5 find and even more difficult to describe. It is µm diam), conspicuously clamped hyphae pro- necessary to examine numerous specimens ducing pedicellate terminal cells (caulocystidia) from scattered locations to estimate characters (Fig. 27C) 25Ð72 × 5–18 µm, firm- to thick- which could be used in the field or with the aid walled (wall never more than 0.7 µm thick), of a microscope. In this case, a molecular phy- clavate to subbulbous, out-turned; outer stipe logenetic reconstruction provides a guide, and flesh a thick layer of strictly parallel, tightly the task is to find characters which bind collec- packed, often coherent hyphae of two types: 1) tions within a clade, and diagnostic characters inflated up to 25 µm diam, usually thick-walled, which separate clades. Thus, the eastern North hyaline, long-celled, obscurely clamped; and 2) American taxon, here proposed as M. rodmani, slender (3–5.5 µm diam), straight, stiff, long- forms a distinct clade. Problems arise when

44 Megacollybia (Agaricales) two forms can be identified within the clade: depressed, “Saccardo’s umber” (5E8), “dark they must be summarized together, and then olive buff” (3C5), “tawny olive” (5C5) to separated. It is possible that they form two “buffy brown” (6D4), outward minutely fibril- “cryptic species,” insufficiently separated by lose, delicately shining when dried, delicately ITS sequences. Additional molecular work may radially streaked, “tawny olive” (5C5), “deep solve this problem. olive buff” (3C3) and “tilleul buff” (5B2); mar- Two collections from Mexico [Hidalgo, gin thin, minutely lobed, not striate, inturned Pachuca-Laredo, km 163, 31.VIII.2002, coll. J. when immature, later sometimes uplifted, often Cifuentes (as M. platyphylla), TFB 10989 lacerate in age; flesh thin except over stipe, (FCME 19183); Guerrero, km 0Ð2 desv. al white, changing to “light ochraceous buff”when Cerro del Huizteco, 29.VI.1981, coll. R.M. dried. Lamellae adnexed to adnate with small Villegas Rios (as Oudemansiella platyphylla), decurrent tooth, subdistant, ventricose, -19 mm no. 13275 (FCME)] yielded ITS sequences typ- deep, thin, white, “cartridge buff,” “cream ical of M. rodmani. The basidiomata could not buff”(4A4), “pale olive buff” (3B2) usually be accurately attributed to either infraspecific eroded to sublacerate in age, in 4-5 ranks, form, however. This situation extends the dis- sometimes diffuse-stained to tan (“sayal tribution of the species into the transvolcanic brown” 6C5) , and changing so on drying, not axis of central Mexico, where fresh material marginate; interlamellar surface rugulose, as should be sought. though interveined. Stipe up to 80 mm long, 4- With several specimens of M. rodmani there 7 mm broad, hardly twisted, unlined to deli- are notes that spores germinated on agar starting cately lined, “cream buff” (4A4) apically, “car- at 18 hours, and attained maximum percentages tridge buff” to “ivory yellow” downward, around 24Ð36 hours. Our observations, however, loosely stuffed to hollow; base occasionally indicate that spore germination rarely succeeds in expanded. Rhizomorphic strands occasional. producing viable hyphae, and it was long consid- Taste none; odor weakly musky. ered impractical to grow enough strains to per- Basidiospores 6Ð10 × 5–7 µm (n = 238; Q form a self-cross. Mata (unpubl. data) succeeded = 1.07Ð1.67; Qm = 1.29; Lm = 7.8 µm), ellipsoid in performing intercollection pairings using four to ovate, flattened adaxially, thin-walled; con- collections from Eastern United States, conclud- tents 1-few-guttulate. Cheilocystidia ing that low-percentage intercompatibility was 20Ð58(Ð86) × 8–25 µm, not protruding beyond demonstrated among the four. Conversely, two basidia, obscure but plentiful, pedicellate to collections from western United States, while nearly sessile, clavate to subbulbous, clamped, partially intercompatible, were totally firm- to (rarely) thick-walled (wall never more interINcompatible with eastern United States col- than 0.7 µm thick), hyaline, occasionally trans- lections. In light of results in this paper, such versely septate, hyaline; contents homoge- interINcompatibility is to be expected. neous. Caulocystidia sparse, 32Ð more than 100 × 5–12 µm (at widest point), elongate-digi- 8.1 Megacollybia rodmani forma rodmani tate to subbulbous or clavate, firm-walled, (autonym; forma typica). occasionally transversely septate, conspicuous- Basidiomata collybioid. Pileus shallowly ly clamped, hyaline; contents homogeneous. convex, often shallowly depressed at center; Stipe flesh constructed of two hyphal types: 1) disc smooth, glabrous-shining when dry, not inflated up to 25 µm diam, thick-walled (wall furfuraceous or scabrous, usually shallowly up to 1.0 µm thick), irregular in outline,

45 K.H. HUGHES et al. obscurely sculptured on inside surface, constrict- (TENN 59430; holotype); same data, TFB ed somewhat at septa, obscurely clamped; and 11487 (TENN 59432). 2) slender, 3Ð5 µm diam, firm-walled, rather stiff, straight, rarely branched, even more rarely 8.2 Megacollybia rodmani forma murina clamped, hyaline. Lower stipe covered with the R.H. Petersen & K.W. Hughes, f. nov. [Figs. 28-31] same arachnoid layer of slender hyphae, which Similis forma rodmani, vel pileus atrobrun- seem not to produce caulocystidia but lie repent neus, atro-olivaceus ad atrofuscus; lamellae on the stipe surface. Stipe flesh of coherent, pallide eburneus ad pallide olivaceus. America thick-walled (wall up to 1.5 µm thick), some- Septentrionalis; occidentalis. what inflated (up to 24 µm diam), strictly longi- Holotype: UNITED STATES, Tennessee, tudinal hyphae. Sevier Co., vic. Gatlinburg, GSMNP, Commentary: Pileipellis terminal cells are Greenbrier, Porter’s Creek Rd., 5.V.2002, coll. bland — weakly pigmented, firm-walled, R.H.Petersen & K.W. Hughes, TFB 11488 clamped, clavate — typical of the genus except (TENN 59433). generally shorter and less pigmented. Pileus Basidiomata (Figs. 28, 29) tricholomoid or color (with olive shades) and obscure cheilocys- russuloid, stout, solid. Pileus commonly 80 tidia seem characteristic of f. rodmani. The lax mm broad or more, convex, smooth, shallowly pileus and thin margin also point in that direc- depressed over disc, “chaetura drab” (2F2), tion. Moreover, not only do pilei of f. rodmani “chaetura black” (2F3), “dark grayish olive” dry very brittle and fragile, but stipes seem not (2F4) to “fuscous” (6E4), radially fibrillose, nearly as stout as when fresh, unlike f. murina, becoming glabrous-shining on drying; margin where stout stature and basidiome colors remain inturned (especially in drying). Lamellae more or less intact upon drying. adnexed to adnate with small tooth, ventricose, Cheilocystidia shape and size in f. rodmani up to 13 mm deep, in 4 ranks, “pale olive buff” are not remarkable, quite like those of other (3B2) to “cartridge buff,” not marginate. Stipe species of Megacollybia and allies, but unlike 60Ð90 × 15Ð25 mm, fleshy, silky apically, those of f. murina, are not prominent (rarely white to “pale olive buff” (3B2), usually dark- protruding beyond basidia). ening somewhat on handling, fibrous, enlarged Specimens examined (f. rodmani): at base, sometimes twisted, fibrous, stuffed. CANADA, Quebec, vic. Quebec City, Rhizomorphic strands common, usually 29.VII.2006, coll. J.L. Mata, TFB 13160 extensive, white, terete; context “dimitic” with (TENN 61269). UNITED STATES, Arkansas, slender (1–2 µm diam), thick-walled, non- Newton Co., vic. Salus, Ozark Highlands Trail at cyanophilous skeletal hyphae. Taste not dis- Moonhull Rd, N 35˚44.474’, W 93˚ 27.734’ tinctive; odor none. 29.V.2003, coll. R.H. Petersen (as M. Pileus outermost layer a tangled arachnoid platyphylla), TFB 11083 (TENN 56566); layer of slender (3–-5 µm diam), firm-walled, Tennessee, Cocke Co., GSMNP, Cosby frequently clamped, moderately pigmented Campground, Nature Loop Trail, 13.V.2002, hyphae; pileipellis terminal cells (Fig. 30A) coll. J.L. Mata, TFB 11492 (TENN 59436); 25Ð more than 150 × 7–15 µm, sessile to elon- same data, coll. E. Grand, TFB 11491 (TENN gate-pedicellate, clavate, often tortuous, often 59435); Sevier Co., vic. Gatlinburg, GSMNP, with small outgrowths (like false clamps), con- Greenbrier, Porter’s Creek Rd., 5.V.2002, coll. spicuously clamped, firm-walled, moderately R.H. Petersen & K.W. Hughes, TFB 11485 to strongly pigmented; contents homogeneous.

46 Megacollybia (Agaricales)

FIGS. 28, 29. Megacollybia rodmani f. murina. Basidiomata. Fig. 28. TFB 11484. Fig. 29. TFB 11488. Numerals = 4 mm.

47 K.H. HUGHES et al.

FIG. 30. Megacollybia rodmani f. murina. TENN FIG. 31. Megacollybia rodmani f. murina. TENN 59433. A. Pileipellis terminal cells. B. Basidia. Standard bar 59433. A. Basidiospores. B. Cheilocystidia. C. Caulocys- = 20 µm. tidia. Standard bar = 20 µm.

Basidia (Fig. 30B) 26Ð43 × 7–11 µm, clavate, firm- to thick-walled (wall up to 1.0 µm thick), thin-walled, 4-spored, clamped; contents with hyaline. Stipe apex (c 10 mm from gills attach- scattered small, refringent guttules. Gnarled ment) delicately ridged and lined, between basidioles absent, but subhymenium a tightly, ridges is white, silky. Stipe surface with a tis- freely (not coherent) interwoven layer of sue (perhaps 20–30 µm thick) of slender (3–5.5 gnarled hyphae frequently branched, very fre- µm diam), branched, conspicuously clamped, quently and prominently clamped. firm-walled, hyaline hyphae which produce Basidiospores (Fig. 31A) 7Ð10 × 5.5–7.5 µm somewhat inflated terminal cells (caulocys- (n = 174; Q = 1.00Ð1.67; Qm = 1.25; Lm = 7.7 tidia). Midstipe caulocystidia 25Ð more than µm), globose (rarely), ellipsoid to ovate, flat- 115 × 6–11 µm (at widest point), obscure, tened adaxially, thin-walled, hyaline; contents repent, as terminal cells or as side branches, obscurely uniguttulate. Cheilocystidia (Fig. usually slightly curved abaxially, usually long- 31B) 24Ð79 × 7–19 µm, densely scattered pedicellate, subbulbous, firm-walled; contents (lamellar edge nearly sterile), protruding homogeneous. beyond basidia up to 15 µm, short- to long- Commentary: In f. murina, pileus color pedicellate, clavate to broadly clavate, firm- to remains dark upon drying and stipe remains thick-walled (wall never more than 0.5 µm stout. In f. rodmani, pileus color changes to thick), conspicuously clamped, hyaline; con- neutral tan and stipe appears much more slen- tents homogeneous. Caulocystidia (Fig. 31C) der when dried. from near stipe apex 31Ð115 × 6–10 µm, For TENN 59429, pileus color and convexi- clavate to subbulbous, conspicuously clamped, ty and large, protruding cheilocystidia all

48 Megacollybia (Agaricales) denote f. murina, but lamellae were shallow, Megacollybia subfurfuracea R.H. Petersen, adnexed (not adnate), and seem to have changed sp. nov. on drying color to ruddy (as in f. rodmani). In Pileus 70Ð110 mm latus, planus ad leviter the herbarium specimen, the stipe seems slender, convexus, pallide olivaceo-griseus ad pallide not stout as in f. murina, all of which may indi- flavo-olivaceus, brunnescens, radio-fibrillosus, cate that the two forms interbreed. siccus. Lamellae adnatae ad adnexae, subven- In f. murina, cheilocystidia in all collections tricosae, eburneae, delicati-marginatae. Stipes were prominent, protrude beyond basidia and 45Ð72 × 5Ð17 mm, equalus ad sursum are very plentiful. Moreover, in f. murina, decrescens, brunneus ad luteobrunneus. cheilocystidia seem to be formed some Pileocystidia 40Ð113 × 9–47 µm, scabrosa as micrometers away from the lamellar edge and squamulosa, clavata, ventricosa ad subfusoidea, therefore could be counted as pleurocystidia. saepe submammiformea, brunnea, tenui- ad Specimens examined (f. murina): CANADA, crassitunicatae, fibulata. Sporae 6.5Ð9(Ð12.5) × Quebec, vic Quebec City, 29.VII.2006, coll. 5.5–7.5(–8) µm (Q = 1.07Ð1.67; Qm = 1.26; Lm = unknown (MSA foray participant), TFB 13159 7.90 µm), latae ellipsoideae ad ovatae, laevae, (TENN 61268). UNITED STATES, Tennessee, tenuitunicatae, hyalinae. Basidia 27Ð43 × 8Ð9 Sevier Co., vic. Gatlinburg, GSMNP, Greenbrier, µm, clavata, tenuitunicata, fibulata, tetraspora. Porter’s Creek Rd., 5.V.2002, coll. R.H. Petersen Pleurocystidia nulla. Cheilocystidia 28Ð65 × & K.W. Hughes, TFB 11488 (TENN 59433, 7–18 µm, clavata, fibulata, tenuitunicata. holotype); same data, TFB 11586 (TENN Caulocystidia 20Ð210 × 6–23 µm, clavata ad 59431); same data, TFB 11484 (TENN 59429). subventricosa, prope hyalina. America Septentrionalis; austro-centralis. 9. Megacollybia subfurfuracea [Figs. 1B, 2 Ð Holotype: UNITED STATES, Arkansas, clade 9, Figs. 32, 33] Faulkner Co.,vic. Conway, Woody Hollow A small clade comprising two specimens, State Park, N 35˚ 17.221’, W 92˚ 17.220’, one from Arkansas, the other from eastern 27.V.2003, coll. K.W. Hughes (as M. platy- Tennessee, can be identified as sister to /rod- phylla), TFB 11075 (TENN 59558). mani, the dominant assemblage of eastern Basidiome collybioid. Pileus (mature) North America. The following morphological 70Ð110 mm broad, plane to shallowly convex, characters diagnose /subfurfuracea: 1) the tex- shallowly depressed over disc, pallid oliva- ture of the pileus disc is minutely furfuraceous, ceous gray (4B-D2), greenish olive (“Isabella with pileipellis terminal cells apparently erect color,” 4D6), “citrine drab” (4D5), “buffy cit- and gathered into minute scabers; 2) pileipellis rine” (3D5) to dull grayish brown (“buffy terminal cells unusually large and often mam- brown,” 6D4), becoming neutral brown (“sayal miform; 3) pileus colors with distinct oliva- brown” 6C5) “tawny olive” (5C5) upon drying; ceous tint, becoming neutral brown upon dry- disc minutely furfuraceous (30×), not streaked, ing; and 4) friable, fragile condition of pileus somewhat darker than limb or margin; inner and lamellar tissues upon drying. There is little limb and limb (10Ð11 mm from center and out- genetic separation based on ITS sequences ward) obscurely, densely streaked in oliva- between this taxon and M. rodmani but because ceous brown; margin downturned, thin, entire, morphocharacters do not match any other taxon eroded in age, not striate; pileus flesh snow in Megacollybia, a new species is proposed. white, friable. Lamellae adnexed to adnate, with or without tooth, subsinuate, ventricose, at

49 K.H. HUGHES et al. least 11 mm deep “cartridge buff” to “cream buff” (deteriorated), close to subdistant, thin, in 3–4 ranks, on drying becoming “ochraceous buff” with delicate transverse streaks of “light ochraceous buff.” Stipe 45Ð72 mm long, 5Ð7 mm broad apically, 6Ð9 mm broad in midsec- tion, expanded somewhat (6Ð15 mm broad) at base, off-white to “cartridge buff” (pale cream color), minutely lined to delicately ridged, not twisted, expanded slightly at base, reluctantly discolored to grayish where handled. Rhizomorphs absent from in-hand material. Taste and odor not recorded. Pileipellis terminal cells (Fig. 32A) over disc nearly erect, in rough, minute patches (scabers or furfuraceous), 40Ð113 × 9–47 µm, thin- firm- to thick-walled (wall never more FIG. 32. Megacollybia subfurfuracea. TENN 59558. A. than 1.0 µm thick, and then irregular on inner Pileipellis terminal cells. B. Basidia. Standard bar = 20 µm. surface), with pedicel easily obscured by col- lapsed surrounding tissue, narrowly clavate to broadly clavate, occasionally to commonly with submammiform tip, conspicuously clamped, moderately to weakly pigmented. Basidia (Fig. 32B) 27Ð43 × 8–9 µm, clavate, clamped, thin-walled, 4-spored; contents with a few very small refringent guttules (PhC). Basidiospores (Fig. 33A) 6.5Ð9(Ð12.5) × 5.5–7.5(–8) µm (n = 38; Q = 1.07Ð1.67; Qm = 1.26; Lm = 7.9 µm), broadly ellipsoid to broad- ly ovate, flattened adaxially, thin-walled; con- tents 1Ðfew-guttulate, refringent (PhC). Cheilocystidia (Fig. 33B) protruding beyond basidia singly or in 2Ð3s, 28Ð65 × 7–18 µm, pedicellate, narrowly to broadly clavate, rarely with irregular apical outgrowths, thin- to firm-walled (wall never more than 0.5 µm FIG. 33. Megacollybia subfurfuracea. TENN 59558. A. thick), conspicuously clamped, hyaline: con- Basidiospores. B. Cheilocystidia. C. Caulocystidia. tents homogeneous; numerous filamentous Standard bar = 20 µm. (2.5–3.5 µm diam), subtly capitulate (capitu- lum 3.5–4 µm), conspicuously clamped, thin- walled, hyaline, hyphal tips amongst cheilo- 33C) 20Ð more than 210 × 6–23 µm, digitate, cystidia. Upper stem ridged, silky between narrowly clavate to clavate, firm- to thick- ridges. Caulocystidial terminal cells (Fig. walled (wall never more than 0.7 µm thick),

50 Megacollybia (Agaricales) smooth, conspicuously clamped, weakly walled (wall never more than 0.3 µm thick), pigmented (off-hyaline). conspicuously clamped, occasionally trans- Commentary: Pileipellis terminal cells are versely septate (without clamp), moderately the largest thus far seen in the genus, and com- pigmented or hyaline; contents homogeneous. monly (at least half of the larger individuals) Cheilocystidia mostly clavate-inflated, some- mammiform. Their position on the pileus disc times uneven in shape and lobed, frequently with (erect to semi-erect, in minute scabers or a secondary septum; wall thin. Basidiospores roughly furfuraceous) is unlike that of other not found. North American taxa, but reminiscent of Exemplar: COSTA RICA, Prov. San José, species of temperate Asia. Bosque Experimental de Villa Mills, Cheilocystidia seem somewhat larger than 22.VI.1995, coll. R.E. Halling, TFB 7903 usual in the genus, but this would not be reflect- (TENN 53803). ed merely in measurements. The filamentous Commentary: Although this specimen hyphal tips among cheilocystidia is novel. occurs within a small clade with Arkansas and Specimens examined: UNITED STATES, Tennessee collections, it is surely not the same Arkansas, Faulkner Co.,vic. Conway, Woody organism. Key differentiating character Hollow State Park, N 35˚ 17.221’, W 92˚ include: 1) pileus hemispherical; 2) pileus 17.220’, 27.V.2003, coll. K.W. Hughes (as M. almost black; 3) pileipellis terminal cells nar- platyphylla), TFB 11075 (TENN 59558, holo- rower than most others; 4) in a squash, copious type); Tennessee, Sevier Co., Strawberry oily or viscid residue leaches from the pileus Plains, vic Hickory Township, 19.IX.2004, flesh (apparently not from the pileipellis). coll. E.B. Lickey (as M. platyphylla), TFB 12095 (TENN 60343). Discussion

10. Clade costaricensis [Figs. 1B, 2 Ð clade The micromorphological characters here 10.] considered informative differ little from the This small clade, represented by a single, morphological analysis of M. platyphylla immature specimen, not only segregates based offered by Rexer and Kost (1989a). They not on ITS sequence, but also by macromorpholo- only depicted these structures, but described gy. It cannot be formally proposed, since a con- how each was produced and the tissues from vincing holotype specimen is not yet available. which they arose. Their analysis, however, was Two photographs show the organism: Halling limited to a single species (M. platyphylla) and and Mueller, 2005; Mata, 1999. Both show a compared its micro-characters to those of taxa tricholomoid or russuloid mushroom with thought related through presence of “sar- pileus almost black, strongly convex with codimitic” tissue construction. Redhead’s inturned margin, strongly radially fibrillose; (1987) elucidation of the Xerulaceae, based lamellae off-white, subdistant, perhaps ventri- essentially on the same premise, included all cose; stipe stout, white. of the genera studied by Rexer and Kost Pileipellis terminal cells semi-erect, (1989a,b) (plus others), but Corner’s rejoinder appearing minutely (30×) scurfy, furfuraceous (1991) was not yet available. The subsequent or roughened, almost black (“chaetura black”), phylogenetic reconstruction by Moncalvo et al. 31Ð81 × 7–2 µm, digitate, narrowly clavate to (2002) showed that the taxa included by clavate (never broadly clavate), thin- to firm- Redhead (1987) and by Rexer and Kost

51 K.H. HUGHES et al.

(1989a) were distributed over at least two arrived in their present geographic locations clades: Oudemansiella (inclusive of Xerula for after migrating from separate refugia follow- Rexer and Kost) and Strobilurus in the ing the glacial maximum in North America. /physalachrioid clade and Megacollybia in We, and others (see Halling, 2001; Redhead, /hydropoid. Similarities in microstructure rep- 1989), have repeatedly found connections resented across all of these organisms appeared between Central America and eastern North to be convergent rather than synapomorphic. America and we have speculated that Central Phylogenetic reconstruction, when com- America was a refugium during periods of bined with micromorphological analyses, glaciation but other refugia, notably along the demonstrates that the most discriminating taxo- Mississippi River, surely existed (Delcourt and nomic character in Megacollybia is geography. Delcourt, 1984). There are few discriminating morphological The partitioning of eastern North American characters in Megacollybia suggesting morpho- Megacollybia taxa into small endemic clades in logical stasis during sequence divergence and Texas and Arkansas could reflect speciation establishment of reproductively isolated popu- events that occurred in glacial refugia or during lations. Similar morphological stasis in the face subsequent migrations, but it is possible that of sequence divergence was seen in studies by migration patterns of opportunistic saprophytic Mueller et al (2001) comparing macrofungi fungi may have differed from those of mycor- from China and Eastern North America. rhizal fungi, which may be more dependent on The clear separation of Old and New World co-migration with their host taxa (see Halling, Megacollybia into two major clades and the 2001; Horak 1983). In Mexico, additional finding that there is considerable unique genet- taxa may be found due to complex physio- ic variability within them suggests that the graphic events including the east-west volcanic major clades are not recent. Similar New belt capping the tropical MesoAmerican World-Old World north-temperate disjuncts region (Morrone, 2006; Morrone, et al., 2002) within macrofungal genera have been observed and additional sampling. in other studies (Vilgalys, 1986, 1991; Vilgalys One potential biogeographic problem con- and Miller, 1983, 1987a, 1987b, Zervakis et al., cerns the distribution of M. platyphylla in 1994, 2004; Gordon, 1994; Gordon and Russia. Megacollybia clitocyboidea appears Petersen, 1997, 1998; Shen et al. 2002). basal to the rest of the genus, and (to this time) Surprisingly, eastern North American its collections are limited to temperate Asia. Megacollybia is divided into two distinct and Megacollybia marginata and M. platyphylla are apparently non-interbreeding clades, a clade closely related, spanning Eurasia and there is a from Texas (M. texensis) that is related to col- possible additional taxon (M. platyphylla form lections from South America and a much larger B) found in the Czech Republic and the northeastern clade which extends from Costa Caucasus that appears transitional between M. Rica through Nova Scotia and includes M. rod- marginata and M. platyphylla. Morphology mani, M. subfurfuracea and /costaricensis, the and phylogeny concluded that the Megacollybia latter two differentiated from M. rodmani on taxon in western Russia (i.e. west of the Ural the basis of non-overlapping haplotypes and Mountain range) was the same as that in west- morphology. A number of studies (Lickey et ern Europe and Scandinavia, namely typical M. al., 2002, 2003; Mata et al., 2007) have indicat- platyphylla.Along the eastern coast of Russia, ed that eastern North American taxa may have however, at least two other taxa (M. marginata,

52 Megacollybia (Agaricales)

M. clitocyboidea) occurred sympatrically. Care Hughes et al. 1999; Jin et al. 1998) was taken, therefore, to examine and sequence The finding that a presumed globally distrib- material from middle Russia. The results are uted species was in fact several distinct taxa, not conclusive, but M. platyphylla extends as defined by morphology, sequence divergence far east as the Altai Republic, considerably east and in some cases inferred ability to intercross, of the Urals. Thus the Urals are not acting as a is not surprising. Increasingly, global partition- barrier to distribution of M. platyphylla. It ing is being revealed in fungi, even in the face seems more likely that a combination of the of strong balancing selection for mating genes Siberian Boreal Forest and the Russia Steppe in fungi and conservation of ability to inter- may present the barrier. Concomitantly, the far cross (Vilgalys, 1986, 1991; Vilgalys and eastern Russian taxa may be limited to the Miller, 1983, 1987a, 1987b, Zervakis et al., ameliorated climate adjacent to the Sea of 1994, Gordon, 1994; Gordon and Petersen, Japan. If this is the case, these taxa may be 1997, 1998; James, et al. 2001). added to others endemic along the Pacific Rim Except for M. fusca (and /costaricensis), all (Hughes and Petersen, 2004; Petersen and Megacollybia taxa appear to be North Temperate Hughes, 2002, 2005, 2007). The intimate sym- in distribution, perhaps extending into tropical patry of two reproductively isolated species in areas such as Costa Rica. This is illusory, howev- Kedrovaya Pad (M. marginata, M. clitocy- er, for this paper covers only taxa of which we boidea) raises the question of whether these have seen material, especially if DNA sequences species evolved allopatrically and then migrat- could be derived. There may be taxa in Southeast ed into common territory or whether this is an Asia, most of China and other areas, with which example of sympatric speciation. Our current we are not familiar, and which await elucidation. studies cannot answer that question. For example, Pegler (1986) included two species Megacollybia fallax is a western North under Tricholomopsis, T. nigra (Petch) Pegler (≡ American species geographically extending Clitocybe nigra Petch) and T. crocobapha (Berk. from Arizona to Washington and Idaho. & Broome) Pegler (≡ Agaricus crocobapha Interestingly, an environmental sample recov- Berk. & Broome), descriptions of which could ered from an orchid inflorescence collected on apply to Megacollybia. Tricholomopsis is diag- Vancouver Island (GenBank EU218887) is M. nosed as lignicolous, but this could include fallax by ITS sequence. Megacollybia fallax is buried wood or woody debris. No placement in not known to be either mycorrhizal or endo- an infrageneric group was furnished, and no phytic and this may be accidental contamina- taxon in this paper answers to the descriptions of tion from a spore rain. Alternately, this finding these taxa. could represent a new role for M. fallax. The unresolved position of M. fallax makes biogeo- Acknowledgements: The authors (RHP, graphical conclusions difficult but its some- AEK, OVM, NVP, REH) thank the administra- what closer sequence homology with Asian M. tions of Kedrovaya Pad, Zhigulevsky Nature clitocyboidea raises the possibility that M. fal- Reserves and Valdaisky National Park who lax represents an ancient migrant across the arranged use of facilities during fieldwork in Bering Strait which has since significantly Russia and I.A. Gorbunova, who provided diverged. In other genera, notably Flammulina, some specimens for this study. We also thank Panellus and Amanita muscaria, there is evi- the administration of the Far Eastern Branch, dence for such migrations (Geml et al. 2006; Russian Academy of Sciences, Vladivostok,

53 K.H. HUGHES et al. which arranged use of facilities at Kedrovaya San Diego, California, Accelrys Inc. Pad Nature Reserve. Collections from the Geml, J., Laursen, K., O’Neill, K., Nusbaum, Great Smoky Mountains National Park were C. andTaylor, D.L. 2006. Beringian origins supported by NSF DEB-0338699. We also of cryptic speciation events in the fly agaric thank students Shawn Robertson and David (Amanita muscaria). Mol. Ecol. 15: 225-239. Mather for help with sequencing and cloning. Gordon, S.A. 1994. Infraspecific variation within three species of Marasmius (Tricholoma- References taceae, Agaricales, Basidiomycotina). Ph.D. Dissertation, ined. Univ. Tennessee, Knoxville. Aanen, D.K., Kuyper, T.W., Boekhout, T., and Gordon, S.A. and Petersen, R.H. 1997. Hoekstra, R.F. 2000. Phylogenetic relation- Infraspecific variation among geographical- ships in the genus Hebeloma based on ITS1 ly separated collections of Marasmius and 2 sequences, with special emphasis on androsaceus. Mycol. Res. 101: 365-371. the Hebeloma crustuliniforme complex. Gordon, S.A. and Petersen, R.H. 1998. Mycologia 92: 269-281. Infraspecific variation among geographical- Barrasa, J.M., Esteve-Raventós, F. and ly separated collections of Marasmius Dähnke, R.M. 2006. Clitocybula canariensis scorodonius. Mycotaxon 69: 453-466. (Tricholomataceae), a new brown-rot fungus Halling, R.E. 2001. Ectomycorrhizae: co-evo- from the Canary Islands (Spain). Fungal lution, significance, and biogeography. Ann. Diversity 22: 1-11. Missouri. Bot. Gard. 88: 5-13. Bas, C., Kuyper, T.W., Noordeloos, M.E. and Halling, R.E., and Mueller, G.M. 2005. Vellinga, E.C. 1990. Flora Agaricina Common mushrooms of the Talamanca Neerlandica. Vol. 2. 137 pp. Rotterdam. Mountains, Costa Rica. Mem. N.Y. Bot. Bas, C., Kuyper, T.W., Noordeloos, M.E. and Gard. 90: 1-195. Vellinga, E.C. 1999. Flora Agaricina Hansen, L., and Knudsen, H. 1992. Nordic Neerlandica. Vol. 4. 191 pp. Rotterdam. macromycetes, vol. 2. 474 pp. Helsinki. Bigelow, H.E. 1973. The genus Clitocybula. Horak, E. 1983. Mycogeography in the South Mycologia 65: 1101-1116. Pacific region: Agaricales, Boletales. Breitenbach & Kränzlin. 1991. Fungi of Austral. J. Bot., Supple. Ser. 10: 1-41. Switzerland: Boletes and Agarics Vol. 3. Huelsenbeck, J., and Ronquist, F. 2000. Mr Bayes: pt1. Sticher Printing AG, Lucerme. 361pp. Bayesian Inferences of Phylogeny (software), Corner, E.J.H. 1966. A monograph of can- University of California, San Diego. tharelloid fungi. Ann. Bot. Mem. 2. Oxford Huffman, D.M., Tiffany, L.H. and Knaphus, G. Univ. Press, London. 1989. Mushrooms & other fungi of the mid- Corner, E.J.H. 1991. Trogia (Basidiomycetes). continental United States. Iowa State Univ. Gard. Bull (Singapore), supple. 2: 1-100. Press, Ames. 326 pp. Delcourt, H. R. and Delcourt, P.A. 1984. Ice age Hughes, K. W., McGhee, L. L., Methven, A.S., haven for hardwoods. Nat. Hist. 9: 22-28. Johnson, J.E., and Petersen, R.H. 1999. Gardes, M., and Bruns, T.D. 1993. ITS primers Patterns of geographic speciation in the genus with enhanced specificity for basid- Flammulina based on sequences of the ribo- iomycetes—application to the identification of somal ITS1-5.8S-ITS2 area. Mycologia 91: mycorrhizae and rusts. Mol. Ecol. 2: 113-118. 978-986. GCG. 2000. Wisconsin Package, Version 10.3. Hughes, K.W., and Petersen, R.H. 2004.

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Kotlaba, F., and Pouzar, Z. 1972. Taxonomic Clemençon, H., and O.K. Miller. 2002. One and nomenclatural＾ notes on some hundred and seventeen clades of euagarics. macromycetes. Ceska Mykol. 26: 217-222. Molec. Phylog. Evol. 23: 357-400. Lickey, E.B., Hughes, K.W., and Petersen, Morrone, J.J. 2006. Hacia una sintesis biogeo- R.H. 2002. Biogeographical patterns in graphica de Mexico. Rev. Mex. Biodivers. Artomyces pyxidatus. Mycologia 94: 461- 76: 207-252. 471. Morrone, J.J., Oragnista, D.E., and Bousquets, Lickey, E.B., Hughes, K.W., and Petersen, J.L. 2002. Mexican biogeographic R.H. 2003. Phylogenetic and taxonomic provinces: preliminary scheme, general studies in Artomyces and Clavicorona characterizations, and synonymies. Acta (Homobasidiomyetes: Auriscalpiaceae). Zool. Mex. (n.s.) 85: 83-108. Sydowia 55: 181-253. Mueller, G. M., Wu, Q-X., Huang, Y-Q., Guo, Malençon, G., and Bertault, R. 1975. Flore des S-Y., Aldana-Gomez, R., and Vilgalys, R. Champignons superieurs du Maroc. Vol. 2. 2001. Assessing biogeographic relationships Trav. Inst. Sci. Cherifien, ser. Bot. boil. betwen North American and Chinese macro- Veget. 33: 1-541. fungi. J. Biogeog. 28: 271-281. Mata, J.L.,Hughes, K.W., and Petersen, R.H. Pegler, D.N. 1986. Agaric flora of Sri. Lanka. 2007. An investigation of /omphalotaceae Kew Bull., addit. Ser. 12: 1-519.

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摘 要

ヒロヒダタケ属（ハラタケ目）

K. W. Hughes ・ R. H. Petersen ・ J. L. Mata,・ N. V. Psurtseva ・ A. E. Kovalenko O. V. Morozova ・ E. B. Lickey ・ J. Cifuentes-Blanco.・ D. P. Lewis ・長沢栄史 R. E. Halling ・竹橋誠司・ M. C. Aime ・ T. Bau ・ T. Henkel

ヒロヒダタケ属 Megacollybia は，ヒロヒダタケ M. (Collybia) platyphylla に基づいて提案され，従来１ 属１種からなる属として取り扱われてきた。今回，rDNA ITS 領域の塩基配列に基づいて本属の系統学 的再検討を行ったところ，広義のヒロヒダタケは地理的分布と密接な関連性を示す数種を含むとの結 果が得られた。形態学的な特徴は大部分において質的なものであったが，各クレードの標本（子実体） は形態学的特徴に基づいて種レベルで識別可能であった。分子系統学的および形態学的研究結果に基 づいて，M. clitocyboidea, M. texensis, M. fusca, M. subfurfuracea, M. rodmani （新品種 f. murina を含む) および M. marginata を新種および新品種として報告した。また，Tricholomopsis fallax をヒロヒダタ ケ属に転属した。ヒロヒダタケはヒロヒダタケ属の基準種として残るが，今のところその分布はヨ ーロッパ，スカンジナビア，および西および中央ロシアに限られているようである。

57