Mycologia, 102(3), 2010, pp. 605–612. DOI: 10.3852/09-128 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897 Ramophialophora humicola and Fibulochlamys chilensis, two new microfungi from soil H. Madrid J. Cano A. Stchigel J. Gené1 J. Guarro only member of the basidiomycetous anamorphic genus Fibulochlamys Romero & Cabral, Fibulochlamys ferruginosa Romero & Cabral (Romero et al. 1989). Both fungi show distinctive morphological features that differentiate them from other species of the respective genera and therefore are proposed as new species. To confirm our proposal and to infer the phylogenetic relationships among the apparently new fungi and other related taxa we compared their sequences of different nuclear ribosomal DNA regions. Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, C/ Sant Llorenç 21, 43201 Reus, Tarragona, Spain Abstract: In a study on soil microfungi from different countries two new hyphomycetes were found. The first one, Ramophialophora humicola, isolated from a soil sample collected in Ronda (Spain), is characterized by producing profusely branched conidiophores ending in sterile, sometimes swollen apices, and subhyaline, dacryoid conidia borne from both integrated and discrete phialides with conspicuous collaretes. ITS sequence data reveal its relationships with members of the Sordariales and its genetic differences with other fungi morphologically close, such as Cladorrhinum spp. The second species, Fibulochlamys chilensis, isolated from a soil sample collected in La Junta (Chile), is characterized by micronematous, clamped, mostly branched conidiophores producing thallic, one-celled, thick-walled conidia that exhibit strongly wrinkled surfaces in age. The analysis of partial sequences of the ITS region and 28S rRNA gene reveal that this fungus is close to members of the gilled Agaricales. Key words: Agaricales, anamorphic fungi, phylogeny, Sordariales, taxonomy MATERIALS AND METHODS Sampling and fungal isolation.— Soil samples rich in organic material (humus) were collected in areas of Ronda, Málaga Province, Spain, and La Junta, Aisén Province, Chile. The vegetation of Ronda is composed mainly of different species of Quercus L., Pinus L., Populus L. and Fraxinus L. This area is characterized by a Mediterranean climate with average temperature of 15 C, and average annual rainfall above 607 mm. The soil is sandy and rich in dolomite and limestone. The vegetation of La Junta is composed mainly of dense rainforests, dominated by Nothofagus dombeyi (Mirb.) Oerst., Saxegothea conspicua Lindl. and Laureliopsis philippiana (Looser) Schodde. This area has a cold oceanic climate with average temperatures of 8–9 C and average annual rainfall of 3000–4000 mm. The soil contains volcanic material and has been enriched with fluvial, fluvoglacial and coluvial sediments. Soil samples were placed in sterilized polyethylene bags. At the laboratory the material was stored at 4–7 C until processed. Fungi were isolated with a wood bait technique and by inoculating soil dilutions in sterile water onto dichloran rose-bengal chloramphenicol (DRBC) agar (Oxoid, United Kingdom) plates supplemented with benomyl at a final concentration of 10 mg/mL, as described respectively in Calduch et al. (2004) and Gilgado et al. (2005). The plates were incubated at 25 6 1 C and examined weekly 2 mo. To achieve pure cultures a sterile dissection needle was used to transfer conidia from primary cultures to Petri dishes containing potato carrot agar (PCA; 20 g potatoes, 20 g carrots, 20 g agar, 1 L distilled water) and oatmeal agar (OA; 30 g filtered oat flakes, 20 g agar, 1 L distilled water), which were incubated at 25 6 1 C for 21 d in the dark. INTRODUCTION Two interesting taxa were found during a continued survey of filamentous microfungi from different geographic regions and habitats. The first one is a dematiaceous hyphomycete isolated from a Spanish soil sample whose morphological features fit into Ramophialophora Calduch, Stchigel, Gené & Guarro, a recently described anamorphic genus that until now included only the species Ramophialophora vesiculosa Calduch, Stchigel, Gené & Guarro (Calduch et al. 2004). The second interesting fungus, isolated from a Chilean soil sample, shows clamped hyaline mycelium with a conidiogenous apparatus similar to that of the Morphological and physiological studies.—Colony morphology and growth rates were studied on OA, PCA and potato dextrose agar (PDA; Difco) incubated at 25 6 1 C for 14 or 21 d in the dark. The ability of the isolates to grow at 30, 37 and 42 C was tested on PDA. Color notations in parentheses are from Kornerup and Wanscher (1978). In addition the Submitted 3 Jun 2009; accepted for publication 27 Sep 2009. 1 Corresponding author. E-mail: josepa.gene@urv.cat 605 606 MYCOLOGIA isolates were grown on sterilized wood 3 wk at 25 6 1 C to study microscopic morphology. Microscopic features were studied in lactic acid. Photomicrographs were obtained with an Axio Imager M1 light microscope and a Cannon Ixus 60 digital camera. Molecular study.—DNA extraction was performed directly from fungal colonies by following the Fast DNA Kit protocol (Bio 101 Inc., Vista, California), with the homogenization step repeated five times. Amplification of the ITS region and D1/D2 locus of the 28S rRNA gene was performed with primer pairs ITS5/ITS4 and NL1/NL4 respectively as described by Cano et al. (2004) and Gilgado et al. (2005). PCR products were purified with a GFXTM PCR DNA kit (Pharmacia Biotech, Cerdanyola, Spain) and were stored at 220 C until PCR products were sequenced with the same primers employed for amplification and following the Taq DyeDeoxy Terminator cycle sequencing kit protocol (Applied Biosystems, Gouda, Netherlands). DNA sequencing reaction mixtures were analyzed on a 310 DNA sequencer (Applied Biosystems). The program Autoassembler 1.40 (Applied Biosystems) was used to obtain consensus sequences from the complementary sequences of each isolate. BLAST sequence homology analyses were performed to compare data of our isolates with those of other fungi deposited in GenBank. Nucleotide sequence alignments were performed with Clustal X 1.81 (Thompson et al. 1997), followed by manual adjustments with a text editor. The ITS sequence of the Ramophialophora isolate was aligned with those of other morphologically similar or related fungi retrieved from GenBank or generated in this study (TABLE I). The D1/D2 region sequence of the Fibulochlamys isolate was aligned with those of other morphologically similar or related fungi available in GenBank (TABLE I). This locus, instead of the ITS region, was chosen for the phylogenetic study because the latter was excessively variable and generated numerous regions with ambiguous alignments. Phylogenetic trees were constructed with neighbor joining (Saitou and Nei 1987), excluding ambiguously aligned regions, and with the Kimura-2parameter substitution model with pair-wise deletion of gaps, as implemented in the MEGA 3.1 program (Kumar et al. 2004). The robustness of branches was assessed by bootstrap analysis of 1000 replicates. The alignments used in both phylogenetic analyses were deposited in TreeBASE (www.treebase.org). TAXONOMY Ramophialophora humicola Madrid, Stchigel, Gené & Guarro, sp. nov. FIG. 1 MycoBank MB 513292 Coloniae in ligno sterili effusae, pilosae, cinerascentes virides. Hyphae 1–3 mm latae. Conidiophora cylindrica, septata, ramosa, pallide olivaceo-brunnea vel atrobrunnea, pallidiora ad apicem, longitudine indeterminata, 2–5 mm lata, laevia vel asperulata, saepe crassitunicata, ad apicem 1.5–4 mm lata in parte latissima. Cellulae conidiogenae discretae et integratae, pallide olivaceae vel brunneae, laeves. Cellulae conidiogenae discretae terminales et laterales, plerumque lageniformes, 5–13 mm longae, 2– 4 mm latae in parte maxima. Cellulae conidiogenae integratae intercalares cylindricae, 9–14 mm longae, 2–3 mm latae, collulo phialidico laterali, 2.5–4 mm longae. Conidia unicellularia, subhyalina, laevia, dacryoidea, 2.5–4 mm longa, 2–3 mm lata, cum hilo basilari cylindrico, in capitulis mucidis aggregata. Teleomorphosis ignota. Colonies on sterilized wood effuse, hairy, dull green (30D4). Vegetative hyphae hyaline to subhyaline, septate, branched, 1–3 mm wide. Conidiophores macronematous, mononematous, cylindrical, regularly septate, profusely branched, pale olivaceous brown to dark brown, becoming paler toward the apex, 2– 5 mm wide, smooth to asperulate and thick-walled. Conidiophore axis and branches mostly ending in a sterile, subhyaline to brown, obtuse, sometimes slightly swollen apex, 1.5–4 mm wide at the broadest part. Conidiogenous cells monophialidic or polyphialidic, discrete and integrated, pale olive to brown, smooth-walled, with conspicuous collarettes up to 2 mm wide. Discrete phialides terminal or lateral, mostly lageniform, but also subcylindrical or centrally swollen, often sinuous, 5–13 mm long, 2–4 mm wide at the broadest part. Integrated phialides intercalary, cylindrical, 9–14 mm long, 2–3 mm wide, with a lateral neck 2.5–4 mm long. Conidia one-celled, subhyaline, smooth-walled, dacryoid, 2.5–4 3 2–3 mm, guttulate, often with a cylindrical hilum, accumulating in slimy masses. Teleomorph not observed. Colonies on OA attaining 55–59 mm diam in 21 d at 25 6 1 C, composed mostly of immersed mycelium, almost glabrous, yellowish green (30B8), with concentric dull green (30D4) conidial tufts and slightly irregular margin; reverse yellowish green (30B8). Colonies on PCA attaining 25–39 mm diam in 21 d at 25 6 1 C, almost glabrous, cream (4A3) at the center, yellow (3A6) toward the periphery. Colonies on PDA at 25 6 1 C attaining 27–28 mm diam in 21 d, glabrous, irregularly convoluted at the center, cream (4A3), with fimbriate margins. Best sporulation was obtained on OA. The fungus grew well on PDA at 30 C, attaining 19–25 mm diam in 21 d but did not sporulate. No growth was observed at 37 C. Holotype. SPAIN. MÁLAGA PROVINCE: Ronda, ca. 36u449140N, 5u099530W, ca. 739 m, from forest soil, Nov 2006, C. Silvera & A. Mercado (HOLOTYPE: IMI 397094, ex-type cultures CBS 124563 and FMR 9523). Comments. This fungus, isolated with a wood bait technique, resembles the type species of the genus, R. vesiculosa, in producing well developed, strongly pigmented, branched conidiophores ending in sterile, setiform apices. Conidial shape and size of the new taxon are also similar to those of R. vesiculosa. However the latter fungus possesses typically wider conidiophore apices (vesicles 3.5–6.5 mm wide), has MADRID ET AL.: NEW SOIL MICROFUNGI TABLE I. 607 Strains, sources and sequences used in the analyses Ascomycota Sordariales Xylariales Basidiomycota Agaricales Polyporales EMBL accession No. Species Collection number Origin Cercophora caudata Cercophora samala Cercophora sparsa Cercophora sulphurella Cladorrhinum brunnescens CBS 606.72 CBS 109.93 JF00229 SMH2531 CBS 643.75A (T) AY999135a AY999134 a AY587912 a AY587913 a FM955446 a Cladorrhinum bulbillosum Cladorrhinum foecundissimum Cladorrhinum phialophoroides Cladorrhinum samala Gelasinospora tetrasperma Neurospora tetrasperma Podospora appendiculata Podospora comata Podospora didyma Podospora fimiseda Podospora pauciseta Ramophialophora humicola CBS 304.90 (T) CBS 180.66 (NT) Soil, Netherlands Dung, Japan France USA Fibers of Cocos nucifera, Netherlands Desert sand, Egypt Agricultural soil, Netherlands FM955448 FM955445 a CBS 301.90 (T) Desert soil, Egypt FM955444 a CBS 303.90 CBS 178.33 NITE 32011 NITE 8549 NA CBS 232.78 Wang 9727 B s. n. CBS 124563, FMR 9523 CBS 110629 Desert soil, Egypt Dung, Canada Burnt soil Dung, Japan NA Dung, Canada NA Horse dung, France Forest soil, Spain FM955447 a AY681178 a AY681194 a AY999126 a AF443849 a AY999127 a EF197077 a AY525771 a FM955449 a Forest soil, Spain AJ579563 a CBS 255.71 CBS 230.78 NITE 32904 BRIP 25468 Dung, Africa Dung, Canada Soil Garcinia mangostana, Australia AY999133 AY999132 AY999130 AF409993 a CBS 170.86 CBS 683.82 CBS 123018, FMR 9694 GLM 45921 TENN5616 Lc42T5P BAYER G 314 NA CBS 451.87 BSI 92.245 HKI ST 27321 DAOM225481 NA CBS 363.65 Germany NA Soil, Chile AF223190b AF223191b FM955450a FM955451b AY207200b AF261394b AF357078b AY207229b AF042584b AF223215b AF223195b AY207274b AF261507b AM946475b AB359433b Ramophialophora vesiculosa Zopfiella erostrata Zopfiella tabulata Zopfiella tetraspora Pestalotiopsis versicolor Asterophora lycoperdoides Asterophora parasitica Fibulochlamys chilensis Flammulina velutipes Leucopaxillus gentianeus Lyophyllum decastes Lyophyllum fumosum Lyophyllum ulmarium Tephrocybe ambusta Tephrocybe tylicolor Pholiota aurivella Squamanita odorata Xerula radicata Phanerochaete chrysosporium NA USA NA Germany NA France NA NA NA Estonia Soil, India a a a a Sequences generated during this study appear in bold type. T, type strain; NT, neotype strain. ITS/5.8S rDNA. b 28S rDNA; NA, not available; BAYER, Bayer Healthcare, Wupertal, Germany; BRIP, Queensland Department of Primary Industries Plant Pathology Herbarium, Australia; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; DAOM, National Mycological Herbarium, Ottawa, Canada; FMR, Faculty of Medicine collection, Reus, Spain; HKI, Hans-KnöllInstitute, Jena, Germany; JF, Jacques Fourier, Las Muros; NITE, Nacional Institute of Technology and Evaluation, Tsukuba, Japan; SMH, Sabine M. Huhndorf, Field Museum, Chicago, USA; TENN, University of Tennessee, USA; Wang, Yei Zei-Zeng Wang, Hong Kong University, China. a 608 MYCOLOGIA FIG. 1. Ramophialophora humicola (CBS 124563). A. Conidiophore sketch. B. Conidiophore detail. C. Detail of the sterile terminal cells of the conidiophores (upper arrow, noninflated; lower arrow, inflated). D. Detail of conidiogenous cells (discrete and integrated). Bars 5 10 mm. brown conidia and does not produce intercalary phialides (Calduch et al. 2004). R. humicola also is comparable to species of Cladorrhinum Sacc. & Marchal, an anamorphic genus of Sordariales related to Cercophora Fuckel, Podospora Ces. and Apiosordaria Arx & Gams teleomorphs. Cladorrhinum anamorphs are characterized by producing tufted aggregations of conidiophores, usually bearing intercalary phialides with lateral openings and collaretes, and one-celled conidia aggregated in slimy masses (Mouchacca and Gams 1993). In contrast to Cladorrhinum species our isolate shows predominantly discrete conidiogenous cells and produces well developed, branched, dark brown conidiophores on wood. Numerous Podospora, Cercophora and Lasiosphaeria Ces. & de Not. species produce Phialophora-like asexual states with conidia similar to those of Ramophialophora, but these anamorphs have undifferentiated or poorly differentiated conidiophores (Mirza and Cain 1969; Lundqvist 1972; Gams and Holubová-Jechová 1976; Udagawa and Muroi 1979; Bell and Mahoney 1995; Lundqvist et al. 1999; Gams 2000; Miller and Huhndorf 2001, 2004; Miller et al. 2007). A recently described ascomycete, Jattaea prunicola Damm & Crous (Calosphaeriales), has a phialidic anamorph with discrete and integrated phialides, and branched conidiophores that often end in sterile inflated cells. This fungus is easily distinguished from Ramophialophora species by the shape of its conidia, which are cylindrical to allantoid. Furthermore the sterile terminal cells of conidiophores differ from those of Ramophialophora in having an irregular shape, often MADRID ET AL.: NEW SOIL MICROFUNGI 609 Fibulochlamys chilensis Madrid, Stchigel, Gené & Guarro, sp. nov. FIG. 3 MycoBank MB 513296 Coloniae effusae, farinaceae, cremeae. Hyphae hyalinae, septatae, ramosae, fibulatae, 1–6 mm latae. Conidiophora micronematosa, mononematosa, cylindrica vel subcylindrica, hyalina, septata, plerumque ramosa, fibulata. Cellulae conidiogenae terminales et intercalares, plerumque cylindricae. Conidia holothallica, unicellularia, pallida flava vel flava, crassitunicata, globosa vel subglobosa vel obovoidea, 9–20 mm longa, 8–13 mm lata, interdum pedicellata, plerumque catenata. Conidia matura cum superficie corrugatissima. Teleomorphosis ignota. FIG. 2. Neighbor joining tree constructed with sequences of the 5.8S rRNA gene and partial ITS region. Branch lengths are proportional to distance. Bootstrap values above 70% are indicated in the internodes. Pestalotiopsis versicolor was used as outgroup. appearing as phialides without openings (Damm et al. 2008). A BLAST sequence homology analysis revealed that the ITS sequence of our isolate showed a high level of identity with different members of the Sordariales. However, the percentages of sequence identities to the closest taxa did not exceed 93%. In the phylogenetic study we included the ITS sequences of morphologically similar species of Cladorrhinum and Ramophialophora and those of some teleomorphic taxa belonging to different lineages of the Sordariales (FIG. 2). The new species grouped into a well supported clade that also included the type species of Ramophialophora as well as Cercophora sparsa (Sacc. & Fairm.) R. Hilber, Podospora appendiculata (Auersw. ex Niessl) Niessl, Podospora didyma J.H. Mirza & Cain, Zopfiella tabulata (Zopf) G. Winter and Cercophora sulphurella (Sacc.) R Hilber, five species that lack known anamorphs. In Cai et al. (2006) a phylogeny reconstruction of the Sordariales based on sequences of the ITS region, 28S rRNA and b-tubulin genes, placed the latter four species in a strongly supported clade distant from that grouping species with Cladorrhinum anamorphs. Our phylogeny also agrees with that study in revealing that Podospora, Cercophora and Zopfiella are polyphyletic genera that need to be recircumscribed. The type strain of R. vesiculosa also was isolated from forest soil with a wood bait technique, but from a sample collected in the Asturias Province, Spain (Calduch et al. 2004). Because no additional reports of the fungus exist, the genus Ramophialophora is known thus far only from Spain. Colonies on sterile wood effuse, farinose, cream (4A3). Hyphae hyaline, septate, branched, clamped, 1– 6 mm wide. Conidiophores micronematous, mononematous, cylindrical to subcylindrical, hyaline, septate, mostly branched. Conidiogenous cells mostly cylindrical, terminal or intercalary, generally associated with a basal clamp. Conidia holothallic, one-celled, paleyellow to yellow, globose, subglobose to obovoidal, 9– 20 3 8–13 mm, sometimes pedicellate, commonly forming simple chains of up to five cells, with thick walls that become strongly wrinkled in age. Conidial secession rhexolytic. Teleomorph not observed. Colonies on OA at 25 6 1 C attaining 60–66 mm diam in 14 d, composed mostly of submerged hyphae, yellowish white (4A2). Colonies on PCA at 25 6 1 C attaining 70–77 mm in 14 d, flat, powdery, cream (4A3) with light yellow (4A5) reverse. Colonies on PDA at 25 6 1 C attaining 71–74 mm diam in 14 d, cerebriform, yellowish white (4A2), concolorous on reverse. Good sporulation was obtained in the three culture media used. The fungus attained 8 mm diam at 42 C in 14 d, but no growth was observed at 45 C. Holotype. CHILE. AISÉN: La Junta, ca. 43u589260S, 72u249170W, ca. 904 m, from soil, Dec 1995, L. Zaror (HOLOTYPE: IMI 397095, ex-type cultures CBS 123018 and FMR 9694). Comments. The fungus, isolated with DRBC agar and benomyl, was placed in genus Fibulochlamys because of the presence of hyaline, clamped mycelium and conidiophores producing holothallic, unicellular, thick-walled, yellowish conidia. We examined the type material of the only previously known species of the genus, F. ferruginosa, BAFC 30826, consisting of dry colonies on bark and wood of Eucalyptus viminalis Labill. This species differs from our isolate in producing mostly unbranched conidiophores and smooth conidia (Romero et al. 1989). Unfortunately no strain or DNA sequence of F. ferruginosa is available and no teleomorph has been described for this taxon. Attempts to culture the fungus from the holotype were unsuccessful, and DNA extraction could not be performed directly from the holotype due to contamination with other fungi. 610 MYCOLOGIA FIG. 3. Fibulochlamys chilensis (CBS 123018). A. Branched conidiophore and conidia. B. Released mature conidia. C. Detail of a hypha showing a clamp connection. D. Branched conidiophore. E. Conidia in a short chain. F. Group of young and mature conidia. G. Rhexolytic conidial secession (the arrow shows the subterminal cell in lytic process). H. Released conidium. Bars 5 10 mm. BLAST analyses with the ITS and D1/D2 sequences both revealed the genetic relationship of F. chilensis with different members of the Tricholomataceae (Agaricales), but only a maximum of 96% identity was obtained with the D1/D2 sequences of Tephrocybe ambusta (Fr.) Donk (AF223215), Lyophyllum decastes (AF357078), and Tephrocybe atrata (Fr.) Donk (AF223210) as closest matches. A cladogram built with the D1/D2 sequences of F. chilensis, some Tricholomataceae and basidiomycetes of other fami- lies producing anamorphs with thick-walled propagules, such as Pholiota aurivella (Batsch ex Fr.) Kumm. (Strophariaceae), Flammulina velutipes (Curtis) Singer, Xerula radicata (Relhan) Dörfelt (Physalacriaceae) and Phanerochaete chrysosporium Burds. (Phanerochaetaceae) (Kendrick and Watling, 1979), showed a clade with 100% bootstrap support that includes our isolate, Lyophyllum fumosum (Pers.) P.D. Orton, T. ambusta and L. decastes (FIG. 4). F. chilensis and L. decastes appeared as neighbors and grouped MADRID ET AL.: NEW SOIL MICROFUNGI 611 and basidiomycetes in the orders Geastrales, Hymenochaetales and Polyporales produce thick-walled, one-celled conidia in culture or in nature (Lamoure 1954, Nobles 1964, Bas 1965, Lentz and McKay 1976, Kendrick and Watling 1979, Pantidou et al. 1983, Jacobsson 1989, Klán et al. 1989, Botha and Eicker 1991, Sede and Lopez 1999, Stalpers 2000, Stoytchev et al. 2001, Fausto-Guerra et al. 2002, Kobayashi and Yamada 2003, Baroni et al. 2007, Vizzini et al. 2007), but none of those anamorphs shows the combination of conidial ornamentation, secession mode and conidiophore structure observed in F. chilensis. FIG. 4. Neighbor joining tree constructed with partial sequences of the 28S rRNA gene (D1–D2 domains). Branch lengths are proportional to distance. Bootstrap values above 70% are indicated in the internodes. Phanerochaete chrysosporium was used as outgroup. into a statistically well supported subclade. Species of Lyophyllum P. Karst. and Tephrocybe Donk were included within two main clades with high bootstrap supports, one of them containing a subclade grouping two species of Asterophora Ditmar. Our phylogenetic analysis suggests that Lyophyllum and Tephrocybe are polyphyletic. Species genetically related to F. chilensis, such as T. ambusta, L. decastes and L. fumosum, do not produce conidia (Clémençon and Moncalvo 1990, Moncalvo et al. 1993, Walther et al. 2005). However anamorphs have been reported in other species of these genera, that is Lyophyllum leucopaxilloides (H.E. Bigelow & A.H. Smith) Clémençon, Lyophyllum suburens Clémençon, Lyophyllum ulmarium (Bull.) Kühner, Tephrocybe palustris (Peck) Donk and Tephrocybe tylicolor (Fr.) M.M. Moser. The anamorphs of the two former species differ from F. chilensis in producing smaller conidia and in forming synnema-like structures (Clémençon 1968). The anamorphs of L. ulmarium and T. tylicolor are characterized by forming both rhexolytically seceding ‘‘chlamydospores’’ and apparently schizolytic arthroconidia (Nagasawa and Arita 1988, Moncalvo et al. 1993). The anamorph of T. palustris forms schizolytic conidia by simple fragmentation of undifferentiated conidiogenous hyphae (Walther et al. 2005). Other members of the Tricholomataceae, such as Calocybe fallax (Sacc.) Redhead & Singer, Leucopaxillus gentianeus (Quél.) Kotl., Leucopaxillus giganteus (Sowerby) Singer, Hypsizygus marmoreus (Peck) H.E. Bigelow and Asterophora spp., produce anamorphs with rhexolytic conidial dehiscence in culture (Brefeld 1889, Pantidou et al. 1983, Buchalo 1988, Brunner and Miller 1988, Nagasawa and Arita 1988), but none of them shows the conidial ornamentation of F. chilensis. In addition, numerous other Agaricales ACKNOWLEDGMENTS Dr Andrea Romero and Ms Susana Pereira are thanked for providing the type material of F. ferruginosa. We are indebted to the curators of the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands) for supplying some of the isolates studied. This work was supported by the Spanish Ministerio de Educación y Ciencia grant CGL2008-04226/BOS. LITERATURE CITED Baroni TJ, Franco-Moleano AE, Lodge DJ, Lindner DL, Horak E, Hofstetter V. 2007. Arthromyces and Blastosporella, two new genera of conidia-producing lyophylloid agarics (Agaricales, Basidiomycota) from the Neotropics. Mycol Res 111:572–580. Bas C. 1965. The genus Squamanita. Persoonia 3:331–359. Bell A, Mahoney DP. 1995. Coprophilous fungi in New Zealand I. Podospora species with swollen agglutinated perithecial hairs. Mycologia 87:375–396. Botha WJ, Eicker A. 1991. Cultural studies on the genus Termitomyces in South Africa I. Macro- and microscopic characters of basidiome context cultures. Mycol Res 95: 435–443. Brefeld O. 1889. Untersuchungen aus dem Gesammtgebiete der Mykologie VIII. Heft: Basidiomyceten III. Autobasidiomyceten. Leipzig: Arthur Felix. 305 p. Brunner IL, Miller OK Jr. 1988. Calocybe fallax: its ecology and cultural behavior. Mycotaxon 33:41–50. Buchalo AS. 1988. Higher edible basidiomycetes in pure culture. Kiev, Ukraine: Naukova Dumka. 144 p. Cai L, Jeewon R, Hyde KD. 2006. Molecular systematics of Zopfiella and allied genera: evidence from multi-gene sequence analyses. Mycol Res 110:359–368. Calduch M, Gené J, Stchigel AM, Cano J, Guarro J. 2004. Ramophialophora, a new anamorphic genus of Sordariales. Stud Mycol 50:83–88. Cano J, Guarro J, Gené J. 2004. Molecular and morphological identification of Colletotrichum species of clinical interest. J Clin Microbiol 42:2450–2454. Clémençon H. 1968. Contributions to the knowledge of the genera Lyophyllum and Calocybe (Agaricales, Basidiomycetes) III. Two chlamydospore forming Lyophyllum species. Nova Hedwig 16:417–427. 612 MYCOLOGIA ———, Moncalvo JM. 1990. Taxonomic analysis of cultural characters in the group Lyophyllum shimeji (Agaricales, Basidiomycetes) from Japan. Trans Mycol Soc Japan 31: 479–488. Damm U, Crous PW, Fourie PH. 2008. A fissitunicate ascus mechanism in the Calosphaeriaceae, and novel species of Jattaea and Calosphaeria on Prunus wood. Persoonia 20:39–52. Fausto-Guerra S, Guzmán-Dávalos L, Velásquez-Hueso JC. 2002. Cultural studies of Gymnopilus species (Cortinariaceae, Agaricales). Mycotaxon 84:429–444. Gams W. 2000. Phialophora and some similar morphologically little-differentiated anamorphs of divergent ascomycetes. Stud Mycol 45:187–199. ———, Holubová-Jechová V. 1976. Chloridium and some other dematiaceous hyphomycetes growing on decaying wood. Stud Mycol 13:1–99. Gilgado F, Cano J, Gené J, Guarro J. 2005. Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species. J Clin Microbiol 43: 4930–4942. Jacobsson S. 1989. Studies on Pholiota in culture. Mycotaxon 36:95–145. Kendrick B, Watling R. 1979. Mitospores in basidiomycetes. In: Kendrick B, ed. The Whole Fungus. Ottawa: National Museum of Natural Sciences. p 473–545. Klán J, Baudišová D, Rulfová I. 1989. Cultural, enzymatic and cytological studies in the genus Pholiota. Mycotaxon 36:249–271. Kobayashi H, Yamada A. 2003. Chlamydospore formation of Entoloma clypeatum f. hybridum on mycorrhizas and rhizomorphs associated with Rosa multiflora. Mycoscience 44:61–62. Kornerup A, Wanscher JH. 1978. Methuen handbook of colour. 3rd ed. London: Eyre Methuen. 252 p. Kumar S, Tamura K, Nei M. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163. Lamoure D. 1954. Contribution à l’étude cytologique des germinations et du mycélium de quelques Agaricales, en particulier des Coprins de la Section Setulosi (Lange). Annal Univ Lyon, Sect C, Sci Nat 8:21–55. Lentz PL, McKay HH. 1976. Basidiocarp and culture descriptions of Hyphoderma and Hyphodontia (Corticiaceae) in the Mississippi Delta region. Mem NY Bot Garden 28:141–162. Lundqvist N. 1972. Nordic Sordariaceae s. lat. Symb Bot Upsal 20:1–374. ———, Mahoney DP, Bell A, Lorenzo LE. 1999. Podospora austrohemisphaerica, a new heterothallic ascomycete from dung. Mycologia 91:405–415. Miller AN, Huhndorf SM. 2001. Neotropical ascomycetes 10. New and interesting Cercophora species. Sydowia 53: 211–226. ———, ———. 2004. A natural classification of Lasiosphaeria based on nuclear LSU rDNA sequences. Mycol Res 108:26–34. ———, Mugambi GK, Huhndorf SM. 2007. Cercophora rubrotuberculata sp. nov., a new pyrenomycete from the Great Smoky Mountains National Park. Mycologia 99: 488–491. Mirza JH, Cain RF. 1969. Revision of the genus Podospora. Can J Bot 47:1999–2048. Moncalvo JM, Rehner SA, Vilgalys R. 1993. Systematics of Lyophyllum section Difformia based on evidence from culture studies and ribosomal DNA sequences. Mycologia 85:788–794. Mouchacca J, Gams W. 1993. The hyphomycete genus Cladorrhinum and its teleomorph connections. Mycotaxon 48:415–440. Nagasawa E, Arita I. 1988. A note on Hypsizygus ulmarius and Hypsizygus marmoreus in Japan. Rep Tottori Mycol Inst 26:71–78. Nobles MK. 1964. Identification of cultures of woodinhabiting hymenomycetes. Can J Bot 43:1097–1139. Pantidou M, Watling R, Gonou Z. 1983. Mycelial characters, anamorphs, and teleomorphs in genera and species of various families of Agaricales in culture. Mycotaxon 17: 409–432. Romero AI, Cabral D, Lopez SE. 1989. Studies on xylophilous fungi from Argentina IV. Anamorphs of basidiomycetes on Eucalyptus viminalis (Myrtaceae). Mycotaxon 34:429–440. Saitou N, Nei M. 1987. The neighbor joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. Sede SM, Lopez SE. 1999. Cultural studies of Agrocybe cylindrica, Gymnopilus pampeanus and Oudemansiella canarii (Agaricales) isolated from urban trees. Mycotaxon 70:377–386. Stalpers JA. 2000. The genus Ptychogaster. Karstenia 40:167– 180. Stoytchev I, Homolka L, Nerud F. 2001. Geastrum pouzarii Staněk in culture. Antonie van Leeuwenhoek 79:363– 364. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876–4882. Udagawa S, Muroi T. 1979. Coprophilous Pyrenomycetes from Japan V. Trans Mycol Soc Japan 20:453–468. Vizzini A, Antonı́n V, Noorderloos M. 2007. Crinipellis pedemontana sp. nov. (Agaricomycetes), a new basidiomycete from Italy. Mycologia 99:786–791. Walther G, Garnica S, Weiß M. 2005. The systematic relevance of conidiogenesis modes in the gilled Agaricales. Mycol Res 109:525–544.